Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

PubMed

Bruzzone, Bianca; Saladini, Francesco; Sticchi, Laura; Mayinda Mboungou, Franc A; Barresi, Renata; Caligiuri, Patrizia; Calzi, Anna; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio; Bisio, Francesca

2015-08-01

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy.

PubMed

Crawford, Keith W; Njeru, Dorothy; Maswai, Jonah; Omondi, Milton; Apollo, Duncan; Kimetto, Jane; Gitonga, Lawrence; Munyao, James; Langat, Raphael; Aoko, Appolonia; Tarus, Jemutai; Khamadi, Samoel; Hamm, Tiffany E

2014-01-28

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

Lower genetic variability of HIV-1 and antiretroviral drug resistance in pregnant women from the state of Pará, Brazil.

PubMed

Machado, Luiz Fernando Almeida; Costa, Iran Barros; Folha, Maria Nazaré; da Luz, Anderson Levy Bessa; Vallinoto, Antonio Carlos Rosário; Ishak, Ricardo; Ishak, Marluisa Oliveira Guimarães

2017-04-12

The present study aimed to describe the genetic diversity of HIV-1, as well as the resistance profile of the viruses identified in HIV-1 infected pregnant women under antiretroviral therapy in the state of Pará, Northern Brazil. Blood samples were collected from 45 HIV-1 infected pregnant to determine the virus subtypes according to the HIV-1 protease (PR) gene and part of the HIV-1 reverse transcriptase (RT) gene by sequencing the nucleotides of these regions. Drug resistance mutations and susceptibility to antiretroviral drugs were analyzed by the Stanford HIV Drug Resistance Database. Out of 45 samples, only 34 could be amplified for PR and 30 for RT. Regarding the PR gene, subtypes B (97.1%) and C (2.9%) were identified; for the RT gene, subtypes B (90.0%), F (6.7%), and C (3.3%) were detected. Resistance to protease inhibitors (PI) was identified in 5.8% of the pregnant, and mutations conferring resistance to nucleoside reverse transcriptase inhibitors were found in 3.3%, while mutations conferring resistance to non-nucleoside reverse transcriptase inhibitors were found in 3.3%. These results showed a low frequency of strains resistant to antiretroviral drugs, the prevalence of subtypes B and F, and the persistent low transmission of subtype C in pregnant of the state of Pará, Brazil.

Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K; Hong, Zhi

2006-08-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

PubMed Central

Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K.; Hong, Zhi

2006-01-01

Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies. PMID:16870771

Drug Susceptibility and Resistance Mutations After First-Line Failure in Resource Limited Settings

PubMed Central

Wallis, Carole L.; Aga, Evgenia; Ribaudo, Heather; Saravanan, Shanmugam; Norton, Michael; Stevens, Wendy; Kumarasamy, Nagalingeswaran; Bartlett, John; Katzenstein, David

2014-01-01

Background. The development of drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been associated with baseline human immunodeficiency virus (HIV)-1 RNA level (VL), CD4 cell counts (CD4), subtype, and treatment failure duration. This study describes drug resistance and levels of susceptibility after first-line virologic failure in individuals from Thailand, South Africa, India, Malawi, Tanzania. Methods. CD4 and VL were captured at AIDs Clinical Trial Group (ACTG) A5230 study entry, a study of lopinavir/ritonavir (LPV/r) monotherapy after first-line virologic failure on an NNRTI regimen. HIV drug-resistance mutation associations with subtype, site, study entry VL, and CD4 were evaluated using Fisher exact and Kruskall–Wallis tests. Results. Of the 207 individuals who were screened for A5230, sequence data were available for 148 individuals. Subtypes observed: subtype C (n = 97, 66%) AE (n = 27, 18%), A1 (n = 12, 8%), and D (n = 10, 7%). Of the 148 individuals, 93% (n = 138) and 96% (n = 142) had at least 1 reverse transcriptase (RT) mutation associated with NRTI and NNRTI resistance, respectively. The number of NRTI mutations was significantly associated with a higher study screening VL and lower study screening CD4 (P < .001). Differences in drug-resistance patterns in both NRTI and NNRTI were observed by site. Conclusions. The degree of NNRTI and NRTI resistance after first-line virologic failure was associated with higher VL at study entry. Thirty-two percent of individuals remained fully susceptible to etravirine and rilpivirine, protease inhibitor resistance was rare. Some level of susceptibility to NRTI remained; however, VL monitoring and earlier virologic failure detection may result in lower NRTI resistance. PMID:24795328

Genetic characterization and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China.

PubMed

Guo, Jinlei; Yan, Yong; Zhang, Jiafeng; Ji, Jimei; Ge, Zhijian; Ge, Rui; Zhang, Xiaofei; Wang, Henghui; Chen, Zhongwen; Luo, Jianyong

2017-03-14

The aim of this study was to characterize HIV-1 genotypes and antiretroviral resistance mutations among treatment-naive HIV-infected individuals in Jiaxing, China. The HIV-1 partial polymerase (pol) genes in 93 of the 99 plasma samples were successfully amplified and analyzed. Phylogenetic analysis revealed the existence of five HIV-1 genotypes, of which the most prevalent genotype was CRF01_AE (38.7%), followed by CRF07_BC (34.4%), CRF08_BC (16.1%), subtype B/B' (5.4%), and CRF55_01B (2.1%). Besides, three types of unique recombination forms (URFs) were also observed, including C/F2/A1, CRF01_AE/B, and CRF08_BC/CRF07_BC. Among 93 amplicons, 46.2% had drug resistance-associated mutations, including 23.7% for protease inhibitors (PIs) mutations, 1.1% for nucleoside reverse transcriptase inhibitors (NRTIs) mutations, and 20.4% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations. Six (6.5%) out of 93 treatment-naive subjects were identified to be resistant to one or more NNRTIs, while resistance to NRTIs or PIs was not observed. Our study showed the genetic diversity of HIV-1 strains circulating in Jiaxing and a relative high proportion of antiretroviral resistance mutations among treatment-naive patients, indicating a serious challenge for HIV prevention and treatment program.

2004: which HIV-1 drug resistance mutations are common in clinical practice?

PubMed

Cheung, Peter K; Wynhoven, Brian; Harrigan, P Richard

2004-01-01

The emergence of drug resistance remains a major problem for the treatment of HIV-infected patients. However, the variety of mutational patterns that evolve in clinical practice have made the application of resistance data to clinical decision-making challenging. Despite (or because of) an abundance of drug-resistance data from disparate sources, there is only limited information available describing the patterns of drug resistance which usually appear in the clinic. Here we attempt to address this issue by reviewing HIV drug resistance in the population of patients failing antiretroviral therapy in British Columbia, Canada from June 1996 to December 2003 as an example. Our findings suggest that, although hundreds of mutations have been associated with resistance, relatively few key mutations occur at a high frequency. For example, only the nucleoside reverse transcriptase inhibitor (NRTI) mutations M184V, M41L T215Y, D67N, K70R and L210W, non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and Y181C, and protease inhibitor (PI) mutation L90M, occur in more than 10% of samples tested for resistance in this population. The introduction of new drugs allows for the selection of new mutations. Trends in the prevalence of resistance-associated mutations have generally followed trends in drug usage, but have not always mirrored them. The phenomenon of cross-resistance can play an important role in the efficacy of new antiretroviral agents, even before they become available. The extent of this cross-resistance depends in part on the prevalence of specific mutations in the population of individuals who have previously received antiretroviral therapy. Hence there is a need to determine which mutations are prevalent in the treated population. The tremendous capacity of HIV to adapt means that common resistance pathways are likely to change over time, and new pathways to resistance are likely to continue to be discovered in the future.

A Novel Point Mutation at Position 156 of Reverse Transcriptase from Feline Immunodeficiency Virus Confers Resistance to the Combination of (−)-β-2′,3′-Dideoxy-3′-Thiacytidine and 3′-Azido-3′-Deoxythymidine

PubMed Central

Smith, Robert A.; Remington, Kathryn M.; Preston, Bradley D.; Schinazi, Raymond F.; North, Thomas W.

1998-01-01

Mutants of feline immunodeficiency virus (FIV) resistant to (−)-β-2′,3′-dideoxy-3′-thiacytidine (3TC) were selected by culturing virus in the presence of increasing stepwise concentrations of 3TC. Two plaque-purified variants were isolated from the original mutant population, and both of these mutants were resistant to 3TC. Surprisingly, these mutants were also phenotypically resistant to 3′-azido-3′-deoxythymidine (AZT) and to the combination of 3TC and AZT. Purified reverse transcriptase (RT) from one of these plaque-purified mutants was resistant to the 5′-triphosphates of 3TC and AZT. DNA sequence analysis of the RT-encoding region of the pol gene amplified from the plaque-purified mutants revealed a Pro-to-Ser mutation at position 156 of RT. A site-directed mutant of FIV engineered to contain this Pro-156-Ser mutation was resistant to 3TC, AZT, and the combination of 3TC and AZT, confirming the role of the Pro-156-Ser mutation in the resistance of FIV to these two nucleoside analogs. This represents the first report of a lentiviral mutant resistant to the combination of AZT and 3TC due to a single, unique point mutation. PMID:9499094

Low prevalence of primary HIV resistance in western Massachusetts.

PubMed

Iarikov, Dmitri E; Irizarry-Acosta, Melina; Martorell, Claudia; Hoffman, Robert P; Skiest, Daniel J

2010-01-01

Most studies of primary antiretroviral (ARV) resistance have been conducted in large metropolitan areas with reported rates of 8% to 25%. We collected data on 99 HIV-1-infected antiretroviral-naive patients from several sites in Springfield, MA, who underwent genotypic resistance assay between 2004 and 2008. Only major resistance mutations per International AIDS Society-USA (IAS-USA) drug resistance mutations list were considered. The prevalence of resistance was 5% (5 of 99). Three patients had one nonnucleoside reverse transcriptase inhibitor (NNRTI) mutation: 103N, 103N, and 190A, 1 patient had a protease inhibitor (PI) mutation: 90M; and 1 patient had 3-class resistance with NNRTI: 181C, 190A, PI: 90M, and nucleoside analogue reverse transcriptase inhibitor (NRTI): 41L, 210W. Mean time from HIV diagnosis to resistance testing was shorter in patients with resistance versus those without: 9 (range 0.3-42 months) versus 27 (range 0.1-418 months), P = .11. There was a trend to lower mean CD4 count in those with resistance, 170 versus 318 cells/mm(3), P = .06. No differences were noted in gender, age, HIV risk category, or HIV RNA level. The low prevalence of primary resistance may be explained by differences in demographic and risk factors or may reflect the time from infection to resistance testing. Our findings emphasize the importance of continued resistance surveillance.

Molecular Characterization of the Human Immunodeficiency Virus Type 1 in Women and Their Vertically Infected Children.

PubMed

Vaz, Sara Nunes; Giovanetti, Marta; Rego, Filipe Ferreira de Almeida; Oliveira, Tulio de; Danaviah, Siva; Gonçalves, Maria Luiza Freire; Alcantara, Luiz Carlos Junior; Brites, Carlos

2015-10-01

Approximately 35 million people worldwide are infected with human immunodeficiency virus (HIV) around 3.2 million of whom are children under 15 years. Mother-to-child-transmission (MTCT) of HIV-1 accounts for 90% of all infections in children. Despite great advances in the prevention of MTCT in Brazil, children are still becoming infected. Samples from 19 HIV-1-infected families were collected. DNA was extracted and fragments from gag, pol, and env were amplified and sequenced directly. Phylogenetic reconstruction was performed. Drug resistance analyses were performed in pol and env sequences. We found 82.1% of subtype B and 17.9% of BF recombinants. A prevalence of 43.9% drug resistance-associated mutations in pol sequences was identified. Of the drug-naive children 33.3% presented at least one mutation related to protease inhibitor/nucleoside reverse transcriptase inhibitor/nonnucleoside reverse transcriptase inhibitor (PI/NRTI/NNRTI) resistance. The prevalence of transmitted drug resistance mutations was 4.9%. On env we found a low prevalence of HR1 (4.9%) and HR2 (14.6%) mutations.

[Mutations of resistance of HIV-1 in previously untreated patients at penitentiary centers of the Autonomous Community of Valencia, Spain. REPRICOVA study].

PubMed

García-Guerrero, Julio; Herrero, Agustín; Vera, Enrique; Almenara, José M; Araújo, Rosa; Saurí, Vicente V; Castellano, Juan C; Fernández-Clemente, Luis; Bedia, Miguel; Llorente, María I; González-Morán, Francisco

2002-03-02

Our purpose was to determine the prevalence of mutations of resistance to nucleoside inhibitors of reverse transcriptase (NIRT) and protease inhibitors (PI) in the HIV-1 genotype of naïve infected subjects in the prisons of the Autonomous Community of Valencia, Spain. Multicentric, descriptive, cross-sectional study of prevalence including a systematic stratified and randomised sampling by centres. Demographic, clinical, virological and immunological data were collected. The HIV gene of protease and transcriptase was studied in peripheral blood plasma samples by means of double PCR amplification and subsequent automatic sequence. Reference: wild strain HXB2. Plasma was obtained from 133 individuals (119 men and 14 women). 117 samples were selected and the rest did not have enough copies for transcription. With regard to NIRT, 7 samples (5.2% of total) showed some mutation of resistance: M41L, D67N, L210W and K219Q, all them secondary to and associated with resistance to zidovudine, abacavir as well as group B multinucleoside-resistance. With regard to PI, only one sample showed a primary mutation, M46I, which was associated with resistance to indinavir. Moreover, a further 41 samples were found to express some secondary mutation. In our series, there was a low number of primary mutations of resistance. These results allow us to exclude the systematic use of resistance tests before an initiation antiretroviral therapy.

HIV-1 transmitted drug resistance and genetic diversity among patients from Piauí State, Northeast Brazil.

PubMed

Moura, Maria Edileuza Soares; da Guarda Reis, Mônica Nogueira; Lima, Yanna Andressa Ramos; Eulálio, Kelsen Dantas; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2015-05-01

HIV-1 transmitted-drug-resistance and genetic diversity are dynamic and may differ in distinct locations/risk groups. In Brazil, increased AIDS incidence and related mortality have been detected in the Northeast region, differently from the epicenter in the Southeast. This cross-sectional study describes transmitted-dru- resistance and HIV-1 subtypes in protease/PR and reverse transcriptase/RT regions among antiretroviral naïve patients from Piauí State, Northeast Brazil. Among 96 patients recruited 89 (92.7%) had HIV-1 PR/RT regions sequenced: 44 females and 45 males, 22 self-declared as men who have sex with men. Transmitted-drug-resistance was investigated by CPR tool (Stanford HIV-1 Drug Resistance/SDRM). HIV-1 subtypes were assigned by REGA and phylogenetic inference. Overall, transmitted-drug-resistance rate was 11.2% (10/89; CI 95%: 5.8-19.1%); 22.7% among men who have sex with men (5/22; CI 95%: 8.8-43.4%), 10% in heterosexual men (2/20; CI 95%: 1.7-29.3%) and 6.8% in women (3/44; CI 95%: 1.8-17.4%). Singleton mutations to protease-inhibitor/PI, nucleoside-reverse-transcriptase-inhibitor/NRTI or non-nucleoside-reverse-transcriptase-inhibitor/NNRTI predominated (8/10): PI mutations (M46L, V82F, L90M); NRTI mutations (M41L, D67N) and NNRTI mutations (K103N/S). Dual class resistance mutations to NRTI and NNRTI were observed: T215L (NRTI), Y188L (NNRTI) and T215N (NRTI), F227L (NNRTI). Subtype B prevailed (86.6%; 77/89), followed by subtype F1 (1.1%, 1/89) and subtype C (1.1%, 1/89). B/F1 and B/C intersubtype recombinants represented 11.2% (10/89). In Piauí State extensive testing of incidence and transmitted-drug-resistance in all populations with risk behaviors may help control AIDS epidemic locally. © 2015 Wiley Periodicals, Inc.

Standardized comparison of the relative impacts of HIV-1 reverse transcriptase (RT) mutations on nucleoside RT inhibitor susceptibility.

PubMed

Melikian, George L; Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia-Cancio, Paolo V; Zolopa, Andrew; Robbins, Gregory K; Kagan, Ron; Israelski, Dennis; Shafer, Robert W

2012-05-01

Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda

PubMed Central

Eshleman, Susan H.; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C.; Serwadda, David; Reynolds, Steven J.; Kiwanuka, Noah; Quinn, Thomas C.; Gray, Ronald; Wawer, Maria

2009-01-01

Objective To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Methods Samples obtained at the time of HIV seroconversion (1998–2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Results Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hyper-susceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Conclusion Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection. PMID:19276794

Antiretroviral drug susceptibility among drug-naive adults with recent HIV infection in Rakai, Uganda.

PubMed

Eshleman, Susan H; Laeyendecker, Oliver; Parkin, Neil; Huang, Wei; Chappey, Colombe; Paquet, Agnes C; Serwadda, David; Reynolds, Steven J; Kiwanuka, Noah; Quinn, Thomas C; Gray, Ronald; Wawer, Maria

2009-04-27

To analyze antiretroviral drug susceptibility in HIV from recently infected adults in Rakai, Uganda, prior to the availability of antiretroviral drug treatment. Samples obtained at the time of HIV seroconversion (1998-2003) were analyzed using the GeneSeq HIV and PhenoSense HIV assays (Monogram Biosciences, Inc., South San Francisco, California, USA). Test results were obtained for 104 samples (subtypes: 26A, 1C, 66D, 9A/D, 1C/D, 1 intersubtype recombinant). Mutations used for genotypic surveillance of transmitted antiretroviral drug resistance were identified in six samples: three had nucleoside reverse transcriptase inhibitor (NRTI) surveillance mutations (two had M41L, one had K219R), and three had protease inhibitor surveillance mutations (I47V, F53L, N88D); none had nonnucleoside reverse transcriptase inhibitor (NNRTI) surveillance mutations. Other resistance-associated mutations were identified in some samples. However, none of the samples had a sufficient number of mutations to predict reduced antiretroviral drug susceptibility. Ten (9.6%) of the samples had reduced phenotypic susceptibility to at least one drug (one had partial susceptibility to didanosine, one had nevirapine resistance, and eight had resistance or partial susceptibility to at least one protease inhibitor). Fifty-three (51%) of the samples had hypersusceptibility to at least one drug (seven had zidovudine hypersusceptibility, 28 had NNRTI hypersusceptibility, 34 had protease inhibitor hypersusceptibility). Delavirdine hypersusceptibility was more frequent in subtype A than D. In subtype D, efavirenz hypersusceptibility was associated with substitutions at codon 11 in HIV-reverse transcriptase. Phenotyping detected reduced antiretroviral drug susceptibility and hypersusceptibility in HIV from some antiretroviral-naive Ugandan adults that was not predicted by genotyping. Phenotyping may complement genotyping for analysis of antiretroviral drug susceptibility in populations with nonsubtype B HIV infection.

Tenofovir-based regimens associated with less drug resistance in HIV-1-infected Nigerians failing first-line antiretroviral therapy.

PubMed

Etiebet, Mary-Ann A; Shepherd, James; Nowak, Rebecca G; Charurat, Man; Chang, Harry; Ajayi, Samuel; Elegba, Olufunmilayo; Ndembi, Nicaise; Abimiku, Alashle; Carr, Jean K; Eyzaguirre, Lindsay M; Blattner, William A

2013-02-20

In resource-limited settings, HIV-1 drug resistance testing to guide antiretroviral therapy (ART) selection is unavailable. We retrospectively conducted genotypic analysis on archived samples from Nigerian patients who received targeted viral load testing to confirm treatment failure and report their drug resistance mutation patterns. Stored plasma from 349 adult patients on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens was assayed for HIV-1 RNA viral load, and samples with more than 1000 copies/ml were sequenced in the pol gene. Analysis for resistance mutations utilized the IAS-US 2011 Drug Resistance Mutation list. One hundred and seventy-five samples were genotyped; the majority of the subtypes were G (42.9%) and CRF02_AG (33.7%). Patients were on ART for a median of 27 months. 90% had the M184V/I mutation, 62% had at least one thymidine analog mutation, and 14% had the K65R mutation. 97% had an NNRTI resistance mutation and 47% had at least two etravirine-associated mutations. In multivariate analysis tenofovir-based regimens were less likely to have at least three nucleoside reverse transcriptase inhibitor (NRTI) mutations after adjusting for subtype, previous ART, CD4, and HIV viral load [P < 0.001, odds ratio (OR) 0.04]. 70% of patients on tenofovir-based regimens had at least two susceptible NRTIs to include in a second-line regimen compared with 40% on zidovudine-based regimens (P = 0.04, OR = 3.4). At recognition of treatment failure, patients on tenofovir-based first-line regimens had fewer NRTI drug-resistant mutations and more active NRTI drugs available for second-line regimens. These findings can inform strategies for ART regimen sequencing to optimize long-term HIV treatment outcomes in low-resource settings.

Differences in resistance mutations among HIV-1 non-subtype B infections: a systematic review of evidence (1996–2008)

PubMed Central

2009-01-01

Ninety percent of HIV-1-infected people worldwide harbour non-subtype B variants of HIV-1. Yet knowledge of resistance mutations in non-B HIV-1 and their clinical relevance is limited. Although a few reviews, editorials and perspectives have been published alluding to this lack of data among non-B subtypes, no systematic review has been performed to date. With this in mind, we conducted a systematic review (1996–2008) of all published studies performed on the basis of non-subtype B HIV-1 infections treated with antiretroviral drugs that reported genotype resistance tests. Using an established search string, 50 studies were deemed relevant for this review. These studies reported genotyping data from non-B HIV-1 infections that had been treated with either reverse transcriptase inhibitors or protease inhibitors. While most major resistance mutations in subtype B were also found in non-B subtypes, a few novel mutations in non-B subtypes were recognized. The main differences are reflected in the discoveries that: (i) the non-nucleoside reverse transcriptase inhibitor resistance mutation, V106M, has been seen in subtype C and CRF01_AE, but not in subtype B, (ii) the protease inhibitor mutations L89I/V have been reported in C, F and G subtypes, but not in B, (iii) a nelfinavir selected non-D30N containing pathway predominated in CRF01_AE and CRF02_AG, while the emergence of D30N is favoured in subtypes B and D, (iv) studies on thymidine analog-treated subtype C infections from South Africa, Botswana and Malawi have reported a higher frequency of the K65R resistance mutation than that typically seen with subtype B. Additionally, some substitutions that seem to impact non-B viruses differentially are: reverse transcriptase mutations G196E, A98G/S, and V75M; and protease mutations M89I/V and I93L. Polymorphisms that were common in non-B subtypes and that may contribute to resistance tended to persist or become more frequent after drug exposure. Some, but not all, are recognized as minor resistance mutations in B subtypes. These observed differences in resistance pathways may impact cross-resistance and the selection of second-line regimens with protease inhibitors. Attention to newer drug combinations, as well as baseline genotyping of non-B isolates, in well-designed longitudinal studies with long duration of follow up are needed. PMID:19566959

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE PAGES

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling; ...

2018-02-21

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

Emergence of resistance mutations in simian immunodeficiency virus (SIV)-infected rhesus macaques receiving non-suppressive antiretroviral therapy (ART)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Policicchio, Benjamin Bruno; Sette, Paola; Xu, Cuiling

Two SIVmac251-infected rhesus macaques received tenofovir/emtricitabine with raltegravir intensification. Viral rebound occurred during treatment and sequencing of reverse transcriptase and integrase genes identified multiple resistance mutations. Similar to HIV infection, antiretroviral-resistance mutations may occur in SIV-infected nonhuman primates receiving nonsuppressive ART. As ART administration to nonhuman primates is currently dramatically expanding, fueled by both cure research and the study of HIV-related comorbidities, viral resistance should be factored in the study design and data interpretation

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

PubMed

Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Short communication: high prevalence of drug resistance in HIV type 1-infected children born in Honduras and Belize 2001 to 2004.

PubMed

Parham, Leda; de Rivera, Ivette Lorenzana; Murillo, Wendy; Naver, Lars; Largaespada, Natalia; Albert, Jan; Karlsson, Annika C

2011-10-01

Antiretroviral therapy has had a great impact on the prevention of mother-to-child transmission (MTCT) of HIV-1. However, development of drug resistance, which could be subsequently transmitted to the child, is a major concern. In Honduras and Belize the prevalence of drug resistance among HIV-1-infected children remains unknown. A total of 95 dried blood spot samples was obtained from HIV-1-infected, untreated children in Honduras and Belize born during 2001 to 2004, when preventive antiretroviral therapy was often suboptimal and consisted of monotherapy with nevirapine or zidovudine. Partial HIV-1 pol gene sequences were successfully obtained from 66 children (Honduras n=55; Belize n=11). Mutations associated with drug resistance were detected in 13% of the Honduran and 27% of the Belizean children. Most of the mutations detected in Honduras (43%) and all mutations detected in Belize were associated with resistance to nonnucleoside reverse transcriptase inhibitors, which was expected from the wide use of nevirapine to prevent MTCT during the study period. In addition, although several mothers reported that they had not received antiretroviral therapy, mutations associated with resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors were found in Honduras. This suggests prior and unreported use of these drugs, or that these women had been infected with resistant virus. The present study demonstrates, for the first time, the presence of drug resistance-associated mutations in HIV-1-infected Honduran and Belizean children.

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

PubMed Central

2012-01-01

Abstract In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. Patients and methods A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Results Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. Conclusion The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973 PMID:23044036

HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients.

PubMed

Elmi Abar, Aden; Jlizi, Asma; Darar, Houssein Youssouf; Kacem, Mohamed Ali Ben Hadj; Slim, Amine

2012-10-08

In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973.

A Novel Nonnucleoside Analogue That Inhibits Human Immunodeficiency Virus Type 1 Isolates Resistant to Current Nonnucleoside Reverse Transcriptase Inhibitors▿

PubMed Central

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K.; Hong, Zhi

2007-01-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients. PMID:17116677

A novel nonnucleoside analogue that inhibits human immunodeficiency virus type 1 isolates resistant to current nonnucleoside reverse transcriptase inhibitors.

PubMed

Zhang, Zhijun; Xu, Wen; Koh, Yung-Hyo; Shim, Jae Hoon; Girardet, Jean-Luc; Yeh, Li-Tain; Hamatake, Robert K; Hong, Zhi

2007-02-01

Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance, and serious side effects can compromise the benefits of the two current drugs in this class (efavirenz and nevirapine). In this study, we report a novel and potent NNRTI, VRX-480773, that inhibits viruses from efavirenz-resistant molecular clones and most NNRTI-resistant clinical HIV-1 isolates tested. In vitro mutation selection experiments revealed that longer times were required for viruses to develop resistance to VRX-480773 than to efavirenz. RT mutations selected by VRX-480773 after 3 months of cell culture in the presence of 1 nM VRX-480773 carried the Y181C mutation, resulting in a less-than-twofold increase in resistance to the compound. A virus containing the double mutation V106I-Y181C emerged after 4 months, causing a sixfold increase in resistance. Viruses containing additional mutations of D123G, F227L, and T369I emerged when the cultures were incubated with increasing concentrations of VRX-480773. Most of the resistant viruses selected by VRX-480773 are susceptible to efavirenz. Oral administration of VRX-480773 to dogs resulted in plasma concentrations that were significantly higher than those required for the inhibition of wild-type and mutant viruses. These results warrant further clinical development of VRX-480773 for the treatment of HIV infection in both NNRTI-naive and -experienced patients.

Nonnucleoside reverse transcriptase inhibitor phenotypic hypersusceptibility can be demonstrated in different assays.

PubMed

Shulman, Nancy S; Delgado, Jamael; Bosch, Ronald J; Winters, Mark A; Johnston, Elizabeth; Shafer, Robert W; Katzenstein, David A; Merigan, Thomas C

2005-05-01

HIV-1 isolates harboring multiple nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations are more susceptible ("hypersusceptible") to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) than isolates lacking NRTI resistance mutations, but this has only been reported with a single-cycle replication phenotypic assay. In fact, there was a report that a commercial multicycle assay did not readily detect hypersusceptibility. To see whether NNRTI hypersusceptibility can be demonstrated in other types of phenotypic assays, including multicycle assays and enzyme inhibition assays. The susceptibility of HIV-1 clones derived from different patients in multicycle assays was tested in peripheral blood mononuclear cells (PBMCs) and in an established cell line. In addition, the reverse transcriptase (RT) of many of these clones was expressed and their susceptibility tested in an RT inhibition assay. Nevirapine and efavirenz susceptibilities were tested and compared with a control wild-type virus or RT. Hypersusceptibility to nevirapine and efavirenz was detected using each of the methods described above. R values correlating the other methods with single-cycle assay values were between 0.66 and 0.96. In addition to the high correlations, the different methods gave similar numeric results. NNRTI hypersusceptibility is readily seen in multicycle susceptibility assays and in enzyme inhibition assays.

Transmitted Antiretroviral Drug Resistance in Newly HIV-Infected and Untreated Patients in Ségou and Bamako, Mali

PubMed Central

Fofana, Djeneba Bocar; Maiga, Aichatou Chehy; Diallo, Fodie; Ait-Arkoub, Zaina; Daou, Fatoumata; Cisse, Mamadou; Sarro, Yaya dit Sadio; Oumar, Aboubacar Alassane; Sylla, Aliou; Katlama, Christine; Taiwo, Babafemi; Murphy, Robert; Tounkara, Anatole; Marcelin, Anne-Genevieve; Calvez, Vincent

2013-01-01

Abstract The WHO recommends regular surveillance for transmitted antiretroviral drug-resistant viruses in HIV antiretroviral treatment (ART)-naive patients in resource-limited settings. This study aimed to assess the prevalence of mutations associated with resistance in ART-naive patients newly diagnosed with HIV in Bamako and Ségou in Mali. HIV-positive patients who never received ART were recruited in Bamako and Ségou, Mali. The reverse transcriptase (RT) and protease (PR) genes of these patients were sequenced by the “ViroSeq” method. Analysis and interpretation of the resistance were made according to the WHO 2009 list of drug resistance mutations. In all, 51/54 (94.4%) sample patients were sequenced. The median age (IQR) of our patients was 24 (22–27) years and the median CD4 count was 380 (340–456) cells/mm3. The predominant subtype was recombinant HIV-1 CRF02_AG (66.7%) followed by CRF06_cpx (12%) and CRF09_cpx (4%). Four patients had mutations associated with resistance, giving an overall prevalence of resistance estimated at 7.9%. There were two (4%) patients with nucleoside reverse transcriptase inhibitor (NRTI) mutations (one M184V and one T215Y), two (4%) with non-NRTI mutations (two K103N), and one (2%) with a protease inhibitor mutation (one I54V). The prevalence of primary resistance in newly infected patients in Mali is moderate (7.9%). This indicates that the standard NNRTI-based first-line regimen used in Mali is suboptimal for some patients. This study should be done regularly to inform clinical practice. PMID:22823755

Drug resistance mutations in HIV type 1 isolates from naive patients eligible for first line antiretroviral therapy in JJ Hospital, Mumbai, India.

PubMed

Deshpande, Alake; Karki, Surendra; Recordon-Pinson, Patricia; Fleury, Herve J

2011-12-01

More than 50 HIV-1-infected patients, naive of antiretroviral therapy (ART) but eligible for first line ART in JJ Hospital, Mumbai, India were investigated for surveillance drug resistance mutations (SDRMs); all but one virus belonged to subtype C; we could observe SDRMs to nonnucleoside reverse transcriptase inhibitors and protease inhibitors in 9.6% of the patients.

Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

PubMed

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-04-01

The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

Global Comparison of Drug Resistance Mutations After First-Line Antiretroviral Therapy Across Human Immunodeficiency Virus-1 Subtypes

PubMed Central

Huang, Austin; Hogan, Joseph W.; Luo, Xi; DeLong, Allison; Saravanan, Shanmugam; Wu, Yasong; Sirivichayakul, Sunee; Kumarasamy, Nagalingeswaran; Zhang, Fujie; Phanuphak, Praphan; Diero, Lameck; Buziba, Nathan; Istrail, Sorin; Katzenstein, David A.; Kantor, Rami

2016-01-01

Background. Human immunodeficiency virus (HIV)-1 drug resistance mutations (DRMs) often accompany treatment failure. Although subtype differences are widely studied, DRM comparisons between subtypes either focus on specific geographic regions or include populations with heterogeneous treatments. Methods. We characterized DRM patterns following first-line failure and their impact on future treatment in a global, multi-subtype reverse-transcriptase sequence dataset. We developed a hierarchical modeling approach to address the high-dimensional challenge of modeling and comparing frequencies of multiple DRMs in varying first-line regimens, durations, and subtypes. Drug resistance mutation co-occurrence was characterized using a novel application of a statistical network model. Results. In 1425 sequences, 202 subtype B, 696 C, 44 G, 351 circulating recombinant forms (CRF)01_AE, 58 CRF02_AG, and 74 from other subtypes mutation frequencies were higher in subtypes C and CRF01_AE compared with B overall. Mutation frequency increased by 9%–20% at reverse transcriptase positions 41, 67, 70, 184, 215, and 219 in subtype C and CRF01_AE vs B. Subtype C and CRF01_AE exhibited higher predicted cross-resistance (+12%–18%) to future therapy options compared with subtype B. Topologies of subtype mutation networks were mostly similar. Conclusions. We find clear differences in DRM outcomes following first-line failure, suggesting subtype-specific ecological or biological factors that determine DRM patterns. PMID:27419147

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

PubMed

Javanbakht, H; Ptak, R G; Chow, E; Yan, J M; Russell, J D; Mankowski, M K; Hogan, P A; Hogg, J H; Vora, H; Hang, J Q; Li, Y; Su, G; Paul, A; Cammack, N; Klumpp, K; Heilek, G

2010-05-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

Characterization of Nucleoside Reverse Transcriptase Inhibitor-Associated Mutations in the RNase H Region of HIV-1 Subtype C Infected Individuals.

PubMed

Ngcapu, Sinaye; Theys, Kristof; Libin, Pieter; Marconi, Vincent C; Sunpath, Henry; Ndung'u, Thumbi; Gordon, Michelle L

2017-11-08

The South African national treatment programme includes nucleoside reverse transcriptase inhibitors (NRTIs) in both first and second line highly active antiretroviral therapy regimens. Mutations in the RNase H domain have been associated with resistance to NRTIs but primarily in HIV-1 subtype B studies. Here, we investigated the prevalence and association of RNase H mutations with NRTI resistance in sequences from HIV-1 subtype C infected individuals. RNase H sequences from 112 NRTI treated but virologically failing individuals and 28 antiretroviral therapy (ART)-naive individuals were generated and analysed. In addition, sequences from 359 subtype C ART-naive sequences were downloaded from Los Alamos database to give a total of 387 sequences from ART-naive individuals for the analysis. Fisher's exact test was used to identify mutations and Bayesian network learning was applied to identify novel NRTI resistance mutation pathways in RNase H domain. The mutations A435L, S468A, T470S, L484I, A508S, Q509L, L517I, Q524E and E529D were more prevalent in sequences from treatment-experienced compared to antiretroviral treatment naive individuals, however, only the E529D mutation remained significant after correction for multiple comparison. Our findings suggest a potential interaction between E529D and NRTI-treatment; however, site-directed mutagenesis is needed to understand the impact of this RNase H mutation.

Prevalence of Drug-Resistance Mutations and Non–Subtype B Strains Among HIV-Infected Infants From New York State

PubMed Central

Karchava, Marine; Pulver, Wendy; Smith, Lou; Philpott, Sean; Sullivan, Timothy J.; Wethers, Judith; Parker, Monica M.

2010-01-01

Summary Prevalence studies indicate that transmission of drug-resistant HIV has been rising in the adult population, but data from the perinatally infected pediatric population are limited. In this retrospective study, we sequenced the pol region of HIV from perinatally infected infants diagnosed in New York State in 2001–2002. Analyses of drug resistance, subtype diversity, and perinatal antiretroviral exposure were conducted, and the results were compared with those from a previous study of HIV-infected infants identified in 1998–1999. Eight of 42 infants (19.1%) had provirus carrying at least 1 drug-resistance mutation, an increase of 58% over the 1998–1999 results. Mutations conferring resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 7.1%, 11.9%, and 2.4% of specimens, respectively. Consistent with previous results, perinatal antiretroviral exposure was not associated with drug resistance (P = 0.70). Phylogenetic analysis indicated that 16.7% of infants were infected with a non–subtype B strain of HIV. It seems that drug-resistant and non–subtype B strains of HIV are becoming increasingly common in the perinatally infected population. Our results highlight the value of resistance testing for all HIV-infected infants upon diagnosis and the need to consider subtype diversity in diagnostic and treatment strategies. PMID:16868498

Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

PubMed

Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

2016-03-01

Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe.

PubMed

Hofstra, L Marije; Sauvageot, Nicolas; Albert, Jan; Alexiev, Ivailo; Garcia, Federico; Struck, Daniel; Van de Vijver, David A M C; Åsjö, Birgitta; Beshkov, Danail; Coughlan, Suzie; Descamps, Diane; Griskevicius, Algirdas; Hamouda, Osamah; Horban, Andrzej; Van Kasteren, Marjo; Kolupajeva, Tatjana; Kostrikis, Leondios G; Liitsola, Kirsi; Linka, Marek; Mor, Orna; Nielsen, Claus; Otelea, Dan; Paraskevis, Dimitrios; Paredes, Roger; Poljak, Mario; Puchhammer-Stöckl, Elisabeth; Sönnerborg, Anders; Staneková, Danica; Stanojevic, Maja; Van Laethem, Kristel; Zazzi, Maurizio; Zidovec Lepej, Snjezana; Boucher, Charles A B; Schmit, Jean-Claude; Wensing, Annemarie M J; Puchhammer-Stockl, E; Sarcletti, M; Schmied, B; Geit, M; Balluch, G; Vandamme, A-M; Vercauteren, J; Derdelinckx, I; Sasse, A; Bogaert, M; Ceunen, H; De Roo, A; De Wit, S; Echahidi, F; Fransen, K; Goffard, J-C; Goubau, P; Goudeseune, E; Yombi, J-C; Lacor, P; Liesnard, C; Moutschen, M; Pierard, D; Rens, R; Schrooten, Y; Vaira, D; Vandekerckhove, L P R; Van den Heuvel, A; Van Der Gucht, B; Van Ranst, M; Van Wijngaerden, E; Vandercam, B; Vekemans, M; Verhofstede, C; Clumeck, N; Van Laethem, K; Beshkov, D; Alexiev, I; Lepej, S Zidovec; Begovac, J; Kostrikis, L; Demetriades, I; Kousiappa, I; Demetriou, V; Hezka, J; Linka, M; Maly, M; Machala, L; Nielsen, C; Jørgensen, L B; Gerstoft, J; Mathiesen, L; Pedersen, C; Nielsen, H; Laursen, A; Kvinesdal, B; Liitsola, K; Ristola, M; Suni, J; Sutinen, J; Descamps, D; Assoumou, L; Castor, G; Grude, M; Flandre, P; Storto, A; Hamouda, O; Kücherer, C; Berg, T; Braun, P; Poggensee, G; Däumer, M; Eberle, J; Heiken, H; Kaiser, R; Knechten, H; Korn, K; Müller, H; Neifer, S; Schmidt, B; Walter, H; Gunsenheimer-Bartmeyer, B; Harrer, T; Paraskevis, D; Hatzakis, A; Zavitsanou, A; Vassilakis, A; Lazanas, M; Chini, M; Lioni, A; Sakka, V; Kourkounti, S; Paparizos, V; Antoniadou, A; Papadopoulos, A; Poulakou, G; Katsarolis, I; Protopapas, K; Chryssos, G; Drimis, S; Gargalianos, P; Xylomenos, G; Lourida, G; Psichogiou, M; Daikos, G L; Sipsas, N V; Kontos, A; Gamaletsou, M N; Koratzanis, G; Sambatakou, H; Mariolis, H; Skoutelis, A; Papastamopoulos, V; Georgiou, O; Panagopoulos, P; Maltezos, E; Coughlan, S; De Gascun, C; Byrne, C; Duffy, M; Bergin, C; Reidy, D; Farrell, G; Lambert, J; O'Connor, E; Rochford, A; Low, J; Coakely, P; O'Dea, S; Hall, W; Mor, O; Levi, I; Chemtob, D; Grossman, Z; Zazzi, M; de Luca, A; Balotta, C; Riva, C; Mussini, C; Caramma, I; Capetti, A; Colombo, M C; Rossi, C; Prati, F; Tramuto, F; Vitale, F; Ciccozzi, M; Angarano, G; Rezza, G; Kolupajeva, T; Vasins, O; Griskevicius, A; Lipnickiene, V; Schmit, J C; Struck, D; Sauvageot, N; Hemmer, R; Arendt, V; Michaux, C; Staub, T; Sequin-Devaux, C; Wensing, A M J; Boucher, C A B; van de Vijver, D A M C; van Kessel, A; van Bentum, P H M; Brinkman, K; Connell, B J; van der Ende, M E; Hoepelman, I M; van Kasteren, M; Kuipers, M; Langebeek, N; Richter, C; Santegoets, R M W J; Schrijnders-Gudde, L; Schuurman, R; van de Ven, B J M; Åsjö, B; Kran, A-M Bakken; Ormaasen, V; Aavitsland, P; Horban, A; Stanczak, J J; Stanczak, G P; Firlag-Burkacka, E; Wiercinska-Drapalo, A; Jablonowska, E; Maolepsza, E; Leszczyszyn-Pynka, M; Szata, W; Camacho, R; Palma, C; Borges, F; Paixão, T; Duque, V; Araújo, F; Otelea, D; Paraschiv, S; Tudor, A M; Cernat, R; Chiriac, C; Dumitrescu, F; Prisecariu, L J; Stanojevic, M; Jevtovic, Dj; Salemovic, D; Stanekova, D; Habekova, M; Chabadová, Z; Drobkova, T; Bukovinova, P; Shunnar, A; Truska, P; Poljak, M; Lunar, M; Babic, D; Tomazic, J; Vidmar, L; Vovko, T; Karner, P; Garcia, F; Paredes, R; Monge, S; Moreno, S; Del Amo, J; Asensi, V; Sirvent, J L; de Mendoza, C; Delgado, R; Gutiérrez, F; Berenguer, J; Garcia-Bujalance, S; Stella, N; de Los Santos, I; Blanco, J R; Dalmau, D; Rivero, M; Segura, F; Elías, M J Pérez; Alvarez, M; Chueca, N; Rodríguez-Martín, C; Vidal, C; Palomares, J C; Viciana, I; Viciana, P; Cordoba, J; Aguilera, A; Domingo, P; Galindo, M J; Miralles, C; Del Pozo, M A; Ribera, E; Iribarren, J A; Ruiz, L; de la Torre, J; Vidal, F; Clotet, B; Albert, J; Heidarian, A; Aperia-Peipke, K; Axelsson, M; Mild, M; Karlsson, A; Sönnerborg, A; Thalme, A; Navér, L; Bratt, G; Karlsson, A; Blaxhult, A; Gisslén, M; Svennerholm, B; Bergbrant, I; Björkman, P; Säll, C; Mellgren, Å; Lindholm, A; Kuylenstierna, N; Montelius, R; Azimi, F; Johansson, B; Carlsson, M; Johansson, E; Ljungberg, B; Ekvall, H; Strand, A; Mäkitalo, S; Öberg, S; Holmblad, P; Höfer, M; Holmberg, H; Josefson, P; Ryding, U

2016-03-01

Numerous studies have shown that baseline drug resistance patterns may influence the outcome of antiretroviral therapy. Therefore, guidelines recommend drug resistance testing to guide the choice of initial regimen. In addition to optimizing individual patient management, these baseline resistance data enable transmitted drug resistance (TDR) to be surveyed for public health purposes. The SPREAD program systematically collects data to gain insight into TDR occurring in Europe since 2001. Demographic, clinical, and virological data from 4140 antiretroviral-naive human immunodeficiency virus (HIV)-infected individuals from 26 countries who were newly diagnosed between 2008 and 2010 were analyzed. Evidence of TDR was defined using the WHO list for surveillance of drug resistance mutations. Prevalence of TDR was assessed over time by comparing the results to SPREAD data from 2002 to 2007. Baseline susceptibility to antiretroviral drugs was predicted using the Stanford HIVdb program version 7.0. The overall prevalence of TDR did not change significantly over time and was 8.3% (95% confidence interval, 7.2%-9.5%) in 2008-2010. The most frequent indicators of TDR were nucleoside reverse transcriptase inhibitor (NRTI) mutations (4.5%), followed by nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations (2.9%) and protease inhibitor mutations (2.0%). Baseline mutations were most predictive of reduced susceptibility to initial NNRTI-based regimens: 4.5% and 6.5% of patient isolates were predicted to have resistance to regimens containing efavirenz or rilpivirine, respectively, independent of current NRTI backbones. Although TDR was highest for NRTIs, the impact of baseline drug resistance patterns on susceptibility was largest for NNRTIs. The prevalence of TDR assessed by epidemiological surveys does not clearly indicate to what degree susceptibility to different drug classes is affected. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America.

Clinical and virologic follow-up in perinatally HIV-1-infected children and adolescents in Madrid with triple-class antiretroviral drug-resistant viruses.

PubMed

Rojas Sánchez, P; de Mulder, M; Fernandez-Cooke, E; Prieto, L; Rojo, P; Jiménez de Ory, S; José Mellado, M; Navarro, M; Tomas Ramos, J; Holguín, Á

2015-06-01

Drug resistance mutations compromise the success of antiretroviral treatment in human immunodeficiency virus type 1 (HIV-1)-infected children. We report the virologic and clinical follow-up of the Madrid cohort of perinatally HIV-infected children and adolescents after the selection of triple-class drug-resistant mutations (TC-DRM). We identified patients from the cohort carrying HIV-1 variants with TC-DRM to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors according to IAS-USA-2013. We recovered pol sequences or resistance profiles from 2000 to 2011 and clinical-immunologic-virologic data from the moment of TC-DRM detection until December 2013. Viruses harbouring TC-DRM were observed in 48 (9%) of the 534 children and adolescents from 2000 to 2011, rising to 24.4% among those 197 with resistance data. Among them, 95.8% were diagnosed before 2003, 91.7% were Spaniards, 89.6% carried HIV-1-subtype B and 75% received mono/dual therapy as first regimen. The most common TC-DRM present in ≥50% of them were D67NME, T215FVY, M41L and K103N (retrotranscriptase) and L90M (protease). The susceptibility to darunavir, tipranavir, etravirine and rilpivirine was 67.7%, 43.7%, 33.3% and 33.3%, respectively, and all reported high resistance to didanosine, abacavir and nelfinavir. Despite the presence of HIV-1 resistance mutations to the three main antiretroviral families in our paediatric cohort, some drugs maintained their susceptibility, mainly the new protease inhibitors (tipranavir and darunavir) and nonnucleoside reverse transcriptase inhibitors (etravirine and rilpivirine). These data will help to improve the clinical management of HIV-infected children with triple resistance in Spain. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Finding Relational Associations in HIV Resistance Mutation Data

NASA Astrophysics Data System (ADS)

Richter, Lothar; Augustin, Regina; Kramer, Stefan

HIV therapy optimization is a hard task due to rapidly evolving mutations leading to drug resistance. Over the past five years, several machine learning approaches have been developed for decision support, mostly to predict therapy failure from the genotypic sequence of viral proteins and additional factors. In this paper, we define a relational representation for an important part of the data, namely the sequences of a viral protein (reverse transcriptase), their mutations, and the drug resistance(s) associated with those mutations. The data were retrieved from the Los Alamos National Laboratories' (LANL) HIV databases. In contrast to existing work in this area, we do not aim directly for predictive modeling, but take one step back and apply descriptive mining methods to develop a better understanding of the correlations and associations between mutations and resistances. In our particular application, we use the Warmr algorithm to detect non-trivial patterns connecting mutations and resistances. Our findings suggest that well-known facts can be rediscovered, but also hint at the potential of discovering yet unknown associations.

Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

PubMed Central

Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P. Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

2005-01-01

Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

Mutations Related to Antiretroviral Resistance Identified by Ultra-Deep Sequencing in HIV-1 Infected Children under Structured Interruptions of HAART

PubMed Central

Vazquez-Guillen, Jose Manuel; Palacios-Saucedo, Gerardo C.; Rivera-Morales, Lydia G.; Garcia-Campos, Jorge; Ortiz-Lopez, Rocio; Noguera-Julian, Marc; Paredes, Roger; Vielma-Ramirez, Herlinda J.; Ramirez, Teresa J.; Chavez-Garcia, Marcelino; Lopez-Guillen, Paulo; Briones-Lara, Evangelina; Sanchez-Sanchez, Luz M.; Vazquez-Martinez, Carlos A.; Rodriguez-Padilla, Cristina

2016-01-01

Although Structured Treatment Interruptions (STI) are currently not considered an alternative strategy for antiretroviral treatment, their true benefits and limitations have not been fully established. Some studies suggest the possibility of improving the quality of life of patients with this strategy; however, the information that has been obtained corresponds mostly to studies conducted in adults, with a lack of knowledge about its impact on children. Furthermore, mutations associated with antiretroviral resistance could be selected due to sub-therapeutic levels of HAART at each interruption period. Genotyping methods to determine the resistance profiles of the infecting viruses have become increasingly important for the management of patients under STI, thus low-abundance antiretroviral drug-resistant mutations (DRM’s) at levels under limit of detection of conventional genotyping (<20% of quasispecies) could increase the risk of virologic failure. In this work, we analyzed the protease and reverse transcriptase regions of the pol gene by ultra-deep sequencing in pediatric patients under STI with the aim of determining the presence of high- and low-abundance DRM’s in the viral rebounds generated by the STI. High-abundance mutations in protease and high- and low-abundance mutations in reverse transcriptase were detected but no one of these are directly associated with resistance to antiretroviral drugs. The results could suggest that the evaluated STI program is virologically safe, but strict and carefully planned studies, with greater numbers of patients and interruption/restart cycles, are still needed to evaluate the selection of DRM’s during STI. PMID:26807922

Connection Subdomain Mutations in HIV-1 Subtype-C Treatment-Experienced Patients Enhance NRTI and NNRTI Drug Resistance

PubMed Central

Delviks-Frankenberry, Krista A.; Lengruber, Renan B.; Santos, Andre F.; Silveira, Jussara M.; Soares, Marcelo A.; Kearney, Mary F.; Maldarelli, Frank; Pathak, Vinay K.

2012-01-01

Mutations in the connection subdomain (CN) and RNase H domain (RH) of HIV-1 reverse transcriptase (RT) from subtype B-infected patients enhance nucleoside and nonnucleoside RT inhibitor (NRTI and NNRTI) resistance by affecting the balance between polymerization and RNase H activity. To determine whether CN mutations in subtype C influence drug sensitivity, single genome sequencing was performed on Brazilian subtype C-infected patients failing RTI therapy. CN mutations identified were similar to subtype B, including A376S, A400T, Q334D, G335D, N348I, and A371V, and increased AZT resistance in the presence of thymidine analog mutations. CN mutations also enhanced NNRTI resistance in the presence of classical NNRTI mutations: etravirine resistance was enhanced 6- to 11-fold in the presence of L100I/K103N/Y181C. These results indicate that selection of CN mutations in treatment-experienced patients also occurs in subtype-C-infected patients and are likely to provide valuable information in predicting clinical RTI resistance. PMID:23068886

Time trends in drug resistant HIV-1 infections in the United Kingdom up to 2009: multicentre observational study.

PubMed

Dolling, David; Sabin, Caroline; Delpech, Valerie; Smit, Erasmus; Pozniak, Anton; Asboe, David; Brown, Andrew Leigh; Churchill, Duncan; Williams, Ian; Geretti, Anna Maria; Phillips, Andrew; Mackie, Nicola; Murphy, Gary; Castro, Hannah; Pillay, Deenan; Cane, Patricia; Dunn, David; Dolling, David

2012-08-21

To evaluate whether the prevalence of HIV-1 transmitted drug resistance has continued to decline in infections probably acquired within the United Kingdom. Multicentre observational study. All UK public laboratories conducting tests for genotypic HIV resistance as a part of routine care. 14,584 patients infected with HIV-1 subtype B virus, who were first tested for resistance before receiving antiretroviral therapy between January 2002 and December 2009. Prevalence of transmitted drug resistance, defined as one or more resistance mutations from the surveillance list recommended by the World Health Organization. 1654 (11.3%, 95% confidence interval 10.8% to 11.9%) patients had one or more mutations associated with transmitted HIV-1 drug resistance; prevalence was found to decline from 15.5% in 2002 to 9.6% in 2007, followed by a slight increase to 10.9% in 2009 (P=0.21). This later rise was mainly a result of increases in resistance to nucleos(t)ide reverse transcriptase inhibitors (from 5.4% in 2007 to 6.6% in 2009, P=0.24) and protease inhibitors (1.5% to 2.1%, P=0.12). Thymidine analogue mutations, including T215 revertants, remained the most frequent mutations associated with nucleos(t)ide reverse transcriptase inhibitors, despite a considerable fall in stavudine and zidovudine use between 2002 and 2009 (from 29.4% of drug regimens in 2002 to 0.8% in 2009, from 47.9% to 8.8%, respectively). The previously observed decline in the prevalence of transmitted drug resistance in HIV-1 infections probably acquired in the UK seems to have stabilised. The continued high prevalence of thymidine analogue mutations suggests that the source of this resistance may be increasingly from patients who have not undergone antiretroviral therapy and who harbour resistant viruses. Testing of all newly diagnosed HIV-1 positive people should be continued.

Trends in Drug Resistance Prevalence, HIV-1 Variants and Clinical Status in HIV-1-infected Pediatric Population in Madrid: 1993 to 2015 Analysis.

PubMed

Rojas Sánchez, Patricia; Domínguez, Sara; Jiménez De Ory, Santiago; Prieto, Luis; Rojo, Pablo; Mellado, Pepa; Navarro, Marisa; Delgado, Rafael; Ramos, José Tomas; Holguín, África

2018-03-01

The expanded use of long-term antiretroviral treatments in infected children may exacerbate the problem of drug resistance mutations selection, which can compromise treatment efficiency. We describe the temporal trends of HIV drug resistance mutations and the HIV-1 variants during 23 years (1993 to March 2016) in the Madrid cohort of HIV-infected children and adolescents. We selected patients with at least one available HIV-1 pol sequence/genotypic resistance profile, establishing different groups according to the sampling year of first resistance data. We determined the prevalence of transmitted drug resistance mutations or acquired drug resistance mutations (DRM), the drug susceptibility among resistant viruses and HIV-1 variants characterized by phylogeny across time. A total of 245 pediatric patients were selected, being mainly female, Spanish native, perinatally infected and carrying HIV-1 subtype B. At first sampling, most pediatric patients were on antiretroviral therapy and heavily pretreated. During 1993 to 2016, transmitted drug resistance mutations was found in 13 (26%) of 50 naive children [non-nucleoside reverse transcriptase inhibitors (NNRTI), 14.6%; nucleoside reverse transcriptase inhibitors (NRTI), 10.4%; protease inhibitors, 8.7%]. DRM appeared in 139 (73.2%) of 190 pretreated patients (NRTI, 64.5%; NNRTI, 36%; protease inhibitors, 35.1%). DRM to NNRTI was higher in last 5 years. Non-B variants infected 14.5% of children and adolescents of the Madrid Cohort, being mainly intersubtype recombinants (76.5%), including complex unique recombinant strains. They caused 3.4% infections before 2000, rising to 85.7% during 2011 to 2016. Periodic surveillance resistance and molecular epidemiology studies in long-term pretreated HIV-infected pediatric populations are required to optimize treatment regimens. Results will permit a better understanding of long-time dynamics of viral resistance and HIV-1 variants in Spain.

Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

PubMed Central

Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

1992-01-01

To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

An overview of the molecular and epidemiological features of HIV-1 infection in two major cities of Bahia state, Brazil.

PubMed

Amaral, Amanda Gm; Oliveira, Isabele B; Carneiro, Diego C; Alcantara, Luiz Cj; Monteiro-Cunha, Joana P

2017-06-01

The high mutation rate of the human immunodeficiency virus (HIV) has created a public health challenge because the use of antiretroviral drugs can generate selective pressure that drives resistance in these viruses. The aim of this work was to characterise the molecular and epidemiological profile of HIV in Bahia, Brazil. DNA sequences from regions of HIV gag, pol, and env genes were obtained from previous studies performed in this area between 2002 and 2012. Their genotype and drug-resistance mutations were identified using bioinformatics tools. Clinical and epidemiological data were analysed. Among 263 individuals (46.4% male), 97.5% were asymptomatic and 49.1% were receiving treatment. Most of the individuals were 31 to 40 years old (36.9%) and infected through heterosexual contact (40.7%). The predominant genotype was B (68.1%) followed by BF recombinants (18.6%). Among the individuals infected with either F or BF genotypes, 68.4% were women and 76.8% were infected through heterosexual transmission. The prevalence of associated mutations conferring antiretroviral resistance was 14.2%, with 3.8% of all mutations conferring resistance to protease inhibitors, 9.43% to nucleoside reverse transcriptase inhibitors, and 8.5% to non-nucleoside reverse transcriptase inhibitors. Drug resistance was higher in individuals receiving treatment (26.1%) than in the drug-naïve (4.3%) individuals. This study will contribute to the understanding and monitoring of HIV epidemic in this Brazilian region.

Etravirine and rilpivirine resistance in HIV-1 subtype CRF01_AE-infected adults failing non-nucleoside reverse transcriptase inhibitor-based regimens.

PubMed

Bunupuradah, Torsak; Ananworanich, Jintanat; Chetchotisakd, Ploenchan; Kantipong, Pacharee; Jirajariyavej, Supunnee; Sirivichayakul, Sunee; Munsakul, Warangkana; Prasithsirikul, Wisit; Sungkanuparph, Somnuek; Bowonwattanuwong, Chureeratana; Klinbuayaem, Virat; Petoumenos, Kathy; Hirschel, Bernard; Bhakeecheep, Sorakij; Ruxrungtham, Kiat

2011-01-01

We studied prevalence of etravirine (ETR) and rilpivirine (RPV) resistance in HIV-1 subtype CRF01_AE infection with first-line non-nucleoside reverse transcriptase inhibitor (NNRTI) failure. A total of 225 adults failing two nucleoside reverse transcriptase inhibitors (NRTIs) plus 1 NNRTI in Thailand with HIV RNA>1,000 copies/ml were included. Genotypic resistance results and HIV-1 subtype were interpreted by Stanford DR database. ETR resistance was calculated by the new Monogram weighted score (Monogram WS; ≥ 4 indicating high-level ETR resistance) and by DUET weighted score (DUET WS; 2.5-3.5 and ≥ 4 resulted in intermediate and reduce ETR response, respectively). RPV resistance interpretation was based on previous reports. Median (IQR) age was 38 (34-42) years, 41% were female and CDC A:B:C were 22%:21%:57%. HIV subtypes were 96% CRF01_AE and 4% B. Antiretrovirals at failure were lamivudine (100%), stavudine (93%), nevirapine (90%) and efavirenz (10%) with a median (IQR) duration of 3.4 (1.8-4.5) years. Median (IQR) CD4(+) T-cell count and HIV RNA were 194 (121-280) cells/mm³ and 4.1 (3.6-4.6) log₁₀ copies/ml, respectively. The common NNRTI mutations were Y181C (41%), G190A (22%) and K103N (19%). The proportion of patients with Monogram WS score ≥ 4 was 61.3%. By DUET WS, 49.8% and 7.5% of patients were scored 2.5-3.5 and ≥4, respectively. Only HIV RNA ≥ 4 log₁₀ copies/ml at failure was associated with both Monogram WS ≥ 4 (OR 2.3, 95% CI 1.3-3.9; P=0.003) and DUET WS ≥ 2.5 (OR 1.9, 95% CI 1.1-3.3; P=0.02). The RVP resistance-associated mutations (RAMs) detected were K101P (1.8%), Y181I (2.7%) and Y181V (3.6%). All patients with RPV mutation had ETR resistance. No E138R/E138K mutations were detected. Approximately 60% of patients had high-level ETR resistance. The role of ETR in second-line therapy is limited in late NNRTI failure settings. RVP RAMs were uncommon, but cross-resistance between ETR and RVP was high.

Detection of drug resistance mutations at low plasma HIV-1 RNA load in a European multicentre cohort study.

PubMed

Prosperi, Mattia C F; Mackie, Nicola; Di Giambenedetto, Simona; Zazzi, Maurizio; Camacho, Ricardo; Fanti, Iuri; Torti, Carlo; Sönnerborg, Anders; Kaiser, Rolf; Codoñer, Francisco M; Van Laethem, Kristel; Bansi, Loveleen; van de Vijver, David A M C; Geretti, Anna Maria; De Luca, Andrea

2011-08-01

Guidelines indicate a plasma HIV-1 RNA load of 500-1000 copies/mL as the minimal threshold for antiretroviral drug resistance testing. Resistance testing at lower viral load levels may be useful to guide timely treatment switches, although data on the clinical utility of this remain limited. We report here the influence of viral load levels on the probability of detecting drug resistance mutations (DRMs) and other mutations by routine genotypic testing in a large multicentre European cohort, with a focus on tests performed at a viral load <1000 copies/mL. A total of 16 511 HIV-1 reverse transcriptase and protease sequences from 11 492 treatment-experienced patients were identified, and linked to clinical data on viral load, CD4 T cell counts and antiretroviral treatment history. Test results from 3162 treatment-naive patients served as controls. Multivariable analysis was employed to identify predictors of reverse transcriptase and protease DRMs. Overall, 2500/16 511 (15.14%) test results were obtained at a viral load <1000 copies/mL. Individuals with viral load levels of 1000-10000 copies/mL showed the highest probability of drug resistance to any drug class. Independently from other measurable confounders, treatment-experienced patients showed a trend for DRMs and other mutations to decrease at viral load levels <500 copies/mL. Genotypic testing at low viral load may identify emerging antiretroviral drug resistance at an early stage, and thus might be successfully employed in guiding prompt management strategies that may reduce the accumulation of resistance and cross-resistance, viral adaptive changes, and larger viral load increases.

HIV-1 drug resistance in antiretroviral-naive individuals with HIV-1-associated tuberculous meningitis initiating antiretroviral therapy in Vietnam.

PubMed

Thao, Vu P; Le, Thuy; Török, Estee M; Yen, Nguyen T B; Chau, Tran T H; Jurriaans, Suzanne; van Doorn, H Rogier; van Doorn, Rogier H; de Jong, Menno D; Farrar, Jeremy J; Dunstan, Sarah J

2012-01-01

Access to antiretroviral therapy (ART) for HIV-infected individuals in Vietnam is rapidly expanding, but there are limited data on HIV drug resistance (HIVDR) to guide ART strategies. We retrospectively conducted HIVDR testing in 220 ART-naive individuals recruited to a randomized controlled trial of immediate versus deferred ART in individuals with HIV-associated tuberculous meningitis in Ho Chi Minh City (HCMC) from 2005-2008. HIVDR mutations were identified by population sequencing of the HIV pol gene and were defined based on 2009 WHO surveillance drug resistance mutations (SDRMs). We successfully sequenced 219/220 plasma samples of subjects prior to ART; 218 were subtype CRF01_AE and 1 was subtype B. SDRMs were identified in 14/219 (6.4%) subjects; 8/14 were resistant to nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs; T69D, L74V, V75M, M184V/I and K219R), 5/14 to non-nucleoside reverse transcriptase inhibitors (NNRTIs; K103N, V106M, Y181C, Y188C and G190A), 1/14 to both NRTIs and NNRTIs (D67N and Y181C) and none to protease inhibitors. After 6 months of ART, eight subjects developed protocol-defined virological failure. HIVDR mutations were identified in 5/8 subjects. All five had mutations with high-level resistance to NNRTIs and three had mutations with high-level resistance to NRTIs. Due to a high early mortality rate (58%), the effect of pre-existing HIVDR mutations on treatment outcome could not be accurately assessed. The prevalence of WHO SDRMs in ART-naive individuals with HIV-associated tuberculous meningitis in HCMC from 2005-2008 is 6.4%. The SDRMs identified conferred resistance to NRTIs and/or NNRTIs, reflecting the standard first-line ART regimens in Vietnam.

Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

PubMed Central

Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

2015-01-01

Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

Predicting human immunodeficiency virus inhibitors using multi-dimensional Bayesian network classifiers.

PubMed

Borchani, Hanen; Bielza, Concha; Toro, Carlos; Larrañaga, Pedro

2013-03-01

Our aim is to use multi-dimensional Bayesian network classifiers in order to predict the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease inhibitors given an input set of respective resistance mutations that an HIV patient carries. Multi-dimensional Bayesian network classifiers (MBCs) are probabilistic graphical models especially designed to solve multi-dimensional classification problems, where each input instance in the data set has to be assigned simultaneously to multiple output class variables that are not necessarily binary. In this paper, we introduce a new method, named MB-MBC, for learning MBCs from data by determining the Markov blanket around each class variable using the HITON algorithm. Our method is applied to both reverse transcriptase and protease data sets obtained from the Stanford HIV-1 database. Regarding the prediction of antiretroviral combination therapies, the experimental study shows promising results in terms of classification accuracy compared with state-of-the-art MBC learning algorithms. For reverse transcriptase inhibitors, we get 71% and 11% in mean and global accuracy, respectively; while for protease inhibitors, we get more than 84% and 31% in mean and global accuracy, respectively. In addition, the analysis of MBC graphical structures lets us gain insight into both known and novel interactions between reverse transcriptase and protease inhibitors and their respective resistance mutations. MB-MBC algorithm is a valuable tool to analyze the HIV-1 reverse transcriptase and protease inhibitors prediction problem and to discover interactions within and between these two classes of inhibitors. Copyright © 2012 Elsevier B.V. All rights reserved.

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil.

PubMed

Ferreira, Ana Cristina G; Coelho, Lara E; Grinsztejn, Eduarda; Jesus, Carlos S de; Guimarães, Monick L; Veloso, Valdiléa G; Grinsztejn, Beatriz; Cardoso, Sandra W

The widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. Patients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal-Wallis and Chi-square tests. Forty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1-30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1-24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. The high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population. Copyright © 2017 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

HIV Resistance Prediction to Reverse Transcriptase Inhibitors: Focus on Open Data.

PubMed

Tarasova, Olga; Poroikov, Vladimir

2018-04-19

Research and development of new antiretroviral agents are in great demand due to issues with safety and efficacy of the antiretroviral drugs. HIV reverse transcriptase (RT) is an important target for HIV treatment. RT inhibitors targeting early stages of the virus-host interaction are of great interest for researchers. There are a lot of clinical and biochemical data on relationships between the occurring of the single point mutations and their combinations in the pol gene of HIV and resistance of the particular variants of HIV to nucleoside and non-nucleoside reverse transcriptase inhibitors. The experimental data stored in the databases of HIV sequences can be used for development of methods that are able to predict HIV resistance based on amino acid or nucleotide sequences. The data on HIV sequences resistance can be further used for (1) development of new antiretroviral agents with high potential for HIV inhibition and elimination and (2) optimization of antiretroviral therapy. In our communication, we focus on the data on the RT sequences and HIV resistance, which are available on the Internet. The experimental methods, which are applied to produce the data on HIV-1 resistance, the known data on their concordance, are also discussed.

High Levels of Dual-Class Drug Resistance in HIV-Infected Children Failing First-Line Antiretroviral Therapy in Southern Ethiopia

PubMed Central

Kinloch, Natalie N.; Lapointe, Hope R.; Cobarrubias, Kyle D.; Foster, Byron A.; Jerene, Degu; Makonnen, Eyasu; Brumme, Zabrina L.

2018-01-01

Clinical monitoring of pediatric HIV treatment remains a major challenge in settings where drug resistance genotyping is not routinely available. As a result, our understanding of drug resistance, and its impact on subsequent therapeutic regimens available in these settings, remains limited. We investigate the prevalence and correlates of HIV-1 drug resistance among 94 participants of the Ethiopia Pediatric HIV Cohort failing first-line combination antiretroviral therapy (cART) using dried blood spot-based genotyping. Overall, 81% (73/90) of successfully genotyped participants harbored resistance mutations, including 69% (62/90) who harbored resistance to both Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs). Strikingly, 42% of resistant participants harbored resistance to all four NRTIs recommended for second-line use in this setting, meaning that there are effectively no remaining cART options for these children. Longer cART duration and prior regimen changes were significantly associated with detection of drug resistance mutations. Replicate genotyping increased the breadth of drug resistance detected in 34% of cases, and thus is recommended for consideration when typing from blood spots. Implementation of timely drug resistance testing and access to newer antiretrovirals and drug classes are urgently needed to guide clinical decision-making and improve outcomes for HIV-infected children on first-line cART in Ethiopia. PMID:29389912

High Levels of Transmitted HIV Drug Resistance in a Study in Papua New Guinea.

PubMed

Lavu, Evelyn; Kave, Ellan; Mosoro, Euodia; Markby, Jessica; Aleksic, Eman; Gare, Janet; Elsum, Imogen A; Nano, Gideon; Kaima, Petronia; Dala, Nick; Gurung, Anup; Bertagnolio, Silvia; Crowe, Suzanne M; Myatt, Mark; Hearps, Anna C; Jordan, Michael R

2017-01-01

Papua New Guinea is a Pacific Island nation of 7.3 million people with an estimated HIV prevalence of 0.8%. ART initiation and monitoring are guided by clinical staging and CD4 cell counts, when available. Little is known about levels of transmitted HIV drug resistance in recently infected individuals in Papua New Guinea. Surveillance of transmitted HIV drug resistance in a total of 123 individuals recently infected with HIV and aged less than 30 years was implemented in Port Moresby (n = 62) and Mount Hagen (n = 61) during the period May 2013-April 2014. HIV drug resistance testing was performed using dried blood spots. Transmitted HIV drug resistance was defined by the presence of one or more drug resistance mutations as defined by the World Health Organization surveillance drug resistance mutations list. The prevalence of non-nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 16.1% (95% CI 8.8%-27.4%) and 8.2% (95% CI 3.2%-18.2%) in Port Moresby and Mount Hagen, respectively. The prevalence of nucleoside reverse transcriptase inhibitor transmitted HIV drug resistance was 3.2% (95% CI 0.2%-11.7%) and 3.3% (95% CI 0.2%-11.8%) in Port Moresby and Mount Hagen, respectively. No protease inhibitor transmitted HIV drug resistance was observed. The level of non-nucleoside reverse transcriptase inhibitor drug resistance in antiretroviral drug naïve individuals recently infected with HIV in Port Moresby is amongst the highest reported globally. This alarming level of transmitted HIV drug resistance in a young sexually active population threatens to limit the on-going effective use of NNRTIs as a component of first-line ART in Papua New Guinea. To support the choice of nationally recommended first-line antiretroviral therapy, representative surveillance of HIV drug resistance among antiretroviral therapy initiators in Papua New Guinea should be urgently implemented.

HIV-1 reverse transcriptase and antiviral drug resistance. Part 2.

PubMed

Das, Kalyan; Arnold, Eddy

2013-04-01

Structures of RT and its complexes combined with biochemical and clinical data help in illuminating the molecular mechanisms of different drug-resistance mutations. The NRTI drugs that are used in combinations have different primary mutation sites. RT mutations that confer resistance to one drug can be hypersensitive to another RT drug. Structure of an RT-DNA-nevirapine complex revealed how NNRTI binding forbids RT from forming a polymerase competent complex. Collective knowledge about various mechanisms of drug resistance by RT has broader implications for understanding and targeting drug resistance in general. In Part 1, we discussed the role of RT in developing HIV-1 drug resistance, structural and functional states of RT, and the nucleoside/nucleotide analog (NRTI) and non-nucleoside (NNRTI) drugs used in treating HIV-1 infections. In this part, we discuss structural understanding of various mechanisms by which RT confers antiviral drug resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

Risk factors and outcomes for the Q151M and T69 insertion HIV-1 resistance mutations in historic UK data.

PubMed

Stirrup, Oliver T; Dunn, David T; Tostevin, Anna; Sabin, Caroline A; Pozniak, Anton; Asboe, David; Cox, Alison; Orkin, Chloe; Martin, Fabiola; Cane, Patricia

2018-04-16

The prevalence of HIV-1 resistance to antiretroviral therapies (ART) has declined in high-income countries over recent years, but drug resistance remains a substantial concern in many low and middle-income countries. The Q151M and T69 insertion (T69i) resistance mutations in the viral reverse transcriptase gene can reduce susceptibility to all nucleoside/tide analogue reverse transcriptase inhibitors, motivating the present study to investigate the risk factors and outcomes associated with these mutations. We considered all data in the UK HIV Drug Resistance Database for blood samples obtained in the period 1997-2014. Where available, treatment history and patient outcomes were obtained through linkage to the UK Collaborative HIV Cohort study. A matched case-control approach was used to assess risk factors associated with the appearance of each of the mutations in ART-experienced patients, and survival analysis was used to investigate factors associated with viral suppression. A further analysis using matched controls was performed to investigate the impact of each mutation on survival. A total of 180 patients with Q151M mutation and 85 with T69i mutation were identified, almost entirely from before 2006. Occurrence of both the Q151M and T69i mutations was strongly associated with cumulative period of virological failure while on ART, and for Q151M there was a particular positive association with use of stavudine and negative association with use of boosted-protease inhibitors. Subsequent viral suppression was negatively associated with viral load at sequencing for both mutations, and for Q151M we found a negative association with didanosine use but a positive association with boosted-protease inhibitor use. The results obtained in these analyses were also consistent with potentially large associations with other drugs. Analyses were inconclusive regarding associations between the mutations and mortality, but mortality was high for patients with low CD4 at detection. The Q151M and T69i resistance mutations are now very rare in the UK. Our results suggest that good outcomes are possible for people with these mutations. However, in this historic sample, viral load and CD4 at detection were important factors in determining prognosis.

Identification of the critical sites of NNRTI-resistance in reverse transcriptase of HIV-1 CRF_BC strains.

PubMed

Huang, Yang; Li, Zhenpeng; Xing, Hui; Jiao, Yang; Ouyang, Yabo; Liao, Lingjie; Jiang, Shibo; Armstrong, Rebecca; Shao, Yiming; Ma, Liying

2014-01-01

The polymorphisms involved in drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1 CRF_BC, the most prevalent HIV-1 strain in China, have been poorly characterized. To reveal the drug resistance mutations, we compared the gene sequences of pol region of HIV-1 CRF_BC from 631 treatment-naïve and 363 treatment-experienced patients using the selection pressure-based method. We calculated an individual Ka/Ks value for each specific amino acid mutation. Result showed that eight polymorphic mutations (W88C, K101Q, I132L, R135L, T139K/R, H221Y and L228R) in RT for treatment-experienced patients were identified, while they, except for R135L, were completely absent in those from treatment-naïve patients. The I132L and T139K/R mutants exhibited high-level resistance to DLV and NVP and moderate resistance to TMC-125 and EFV, while the K101Q and H221Y mutants exhibited an increased resistance to all four NNRTIs tested. The W88C, R135L, and L228R may be RTI-induced adaptive mutations. Y181C+K101Q mutant showed a 2.5-, 4.4-, and 4.7-fold higher resistance to TMC-125, NVP and EFV, respectively, than Y181C alone mutant, while Y181C+H221Y or K103N+H221Y mutants had significantly higher resistance to all four NNRTIs than Y181C or K103N mutants. K103N+T139K and G190A+T139K mutant induce higher resistance (2.0∼14.2-fold and 1.5∼7.2-fold, respectively) to all four NNRTIs than K103N or G190A alone mutation. I132L and T139K/R are rare but critical mutations associated with NNRTI-resistance for some NNRTIs. K101Q, H221Y and T139K can enhance K103N/Y181C/G190A-assocated NNRTI-resistance. Monitoring these mutations will provide useful information for rational design of the NNRTI-based antiretroviral regimen for HIV-1 CRF_BC-infected patients.

Follow-up on long-term antiretroviral therapy for cats infected with feline immunodeficiency virus.

PubMed

Medeiros, Sheila de Oliveira; Abreu, Celina Monteiro; Delvecchio, Rodrigo; Ribeiro, Anísia Praxedes; Vasconcelos, Zilton; Brindeiro, Rodrigo de Moraes; Tanuri, Amilcar

2016-04-01

Feline immunodeficiency virus (FIV) is a lentivirus that induces AIDS-like disease in cats. Some of the antiretroviral drugs available to treat patients with HIV type 1 are used to treat FIV-infected cats; however, antiretroviral therapy (ART) is not used in cats as a long-term treatment. In this study, the effects of long-term ART were evaluated in domestic cats treated initially with the nucleoside transcriptase reverse inhibitor (NTRI) zidovudine (AZT) over a period ranging from 5-6 years, followed by a regimen of the NTRI lamivudine (3TC) plus AZT over 3 years. Viral load, sequencing of pol (reverse transcriptase [RT]) region and CD4:CD8 lymphocyte ratio were evaluated during and after treatment. Untreated cats were evaluated as a control group. CD4:CD8 ratios were lower, and uncharacterized resistance mutations were found in the RT region in the group of treated cats. A slight increase in viral load was observed in some cats after discontinuing treatment. The data strongly suggest that treated cats were resistant to therapy, and uncharacterized resistance mutations in the RT gene of FIV were selected for by AZT. Few studies have been conducted to evaluate the effect of long-term antiretroviral therapy in cats. To date, resistance mutations have not been described in vivo. © ISFM and AAFP 2015.

Herpes Simplex Virus Type 2 Suppressive Therapy with Acyclovir or Valacyclovir Does Not Select for Specific HIV-1 Resistance in HIV-1/HSV-2 Dually Infected Persons

PubMed Central

Lingappa, Jairam; Beck, Ingrid; Frenkel, Lisa M.; Pepper, Gregory; Celum, Connie; Wald, Anna; Fife, Kenneth H.; Were, Edwin; Mugo, Nelly; Sanchez, Jorge; Essex, Myron; Makhema, Joseph; Kiarie, James; Farquhar, Carey; Corey, Lawrence

2011-01-01

Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1–infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%–2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance. PMID:21148504

High prevalence of bevirimat resistance mutations in protease inhibitor-resistant HIV isolates.

PubMed

Verheyen, Jens; Verhofstede, Chris; Knops, Elena; Vandekerckhove, Linos; Fun, Axel; Brunen, Diede; Dauwe, Kenny; Wensing, Annemarie M J; Pfister, Herbert; Kaiser, Rolf; Nijhuis, Monique

2010-03-13

Bevirimat is the first drug of a new class of antivirals that hamper the maturation of HIV. The objective of this study was to evaluate the sequence variability of the gag region targeted by bevirimat in HIV subtype-B isolates. Of 484 HIV subtype-B isolates, the gag region comprising amino acids 357-382 was sequenced. Of the patients included, 270 were treatment naive and 214 were treatment experienced. In the latter group, 48 HIV isolates harboured mutations associated with reverse transcriptase inhibitor resistance only, and 166 HIV isolates carried mutations associated with protease inhibitor resistance. In the treatment-naive patient population, approximately 30% harboured an HIV isolate with at least one mutation associated with a reduced susceptibility to bevirimat (H358Y, L363M, Q369H, V370A/M/del and T371del). In HIV isolates with protease inhibitor resistance, the prevalence of bevirimat resistance mutations increased to 45%. Accumulation of mutations at four positions in the bevirimat target region, S368C, Q369H, V370A and S373P, was significantly observed. Mutations associated with bevirimat resistance were detected more frequently in HIV isolates with three or more protease inhibitor resistance mutations than in those with less than three protease inhibitor mutations. Reduced bevirimat activity can be expected in one-third of treatment-naive HIV subtype-B isolates and significantly more in protease inhibitor-resistant HIV. These data indicate that screening for bevirimat resistance mutations before administration of the drug is essential.

The effect of transmitted HIV-1 drug resistance on pre-therapy viral load.

PubMed

Harrison, Linda; Castro, Hannah; Cane, Patricia; Pillay, Deenan; Booth, Clare; Phillips, Andrew; Geretti, Anna Maria; Dunn, David

2010-07-31

Reduced replication capacity of viruses expressing drug resistant mutations implies that patients with transmitted drug resistance (TDR) could have lower HIV RNA viral load than those infected with wild-type virus. We performed analysis using data from the UK HIV Drug Resistance Database and the UK CHIC study. Eligible patients had a resistance test performed between 1997 and 2007 while naive to antiretroviral therapy, were 16 years or older, and had a viral load and CD4 cell count measurement within 6 months of this test. Models were adjusted for CD4 cell count, viral subtype, ethnicity, risk group, sex, age, calendar year, clinical centre, and viral load assay. Of a total of 7994 patients included, 709 (9%) had TDR: 604 (85%) had resistance to one drug class only [350 nucleos(t)ide reverse transcriptase inhibitors (NRTIs), 164 non-nucleos(t)ide reverse transcriptase inhibitors (NNRTIs), 90 protease inhibitors (PIs)], 77 (11%) to two classes (42 NRTIs/NNRTIs, 31 NRTIs/PIs, 4 NNRTIs/PIs), and 28 (4%) had resistance to all three classes. The overall mean (SD) viral load at the time of resistance testing was 4.60 (0.82) log(10) copies/ml, and did not differ by class of TDR. However, patients harbouring M184V/I (n = 61) had a significantly lower viral load [adjusted mean difference -0.33 log10 copies/ml (95% CI -0.54 to -0.11), 53% lower (95% CI 22 to 71%), P = 0.002] compared to wild-type virus. Our study provides clear evidence of an in-vivo fitness cost associated with the M184V/I mutation independent of drug effects which select for this mutation. This was not observed for any other mutation, but true effects may have been obscured by reversion of initially resistant viruses to wild-type.

Long-term persistence of primary genotypic resistance after HIV-1 seroconversion.

PubMed

Pao, David; Andrady, Ushan; Clarke, Janette; Dean, Gillian; Drake, Susan; Fisher, Martin; Green, Tanya; Kumar, Siva; Murphy, Maurice; Tang, Alan; Taylor, Stephen; White, David; Underhill, Gillian; Pillay, Deenan; Cane, Patricia

2004-12-15

Primary infection with drug-resistant HIV-1 is well documented. We have followed up patients infected with such viruses to determine the stability of resistance-associated mutations. Fourteen patients who experienced primary infection with genotypic evidence of resistance were followed for up to 3 years. Drug resistance-associated mutations persisted over time in most patients studied. In particular, M41L, T69N, K103N, and T215 variants within reverse transcriptase (RT) and multidrug resistance demonstrated little reversion to wild-type virus. By contrast, Y181C and K219Q in RT, occurring alone, disappeared within 25 and 9 months, respectively. Multidrug resistance in 2 patients was found to be stable for up to 18 months, the maximum period studied. We conclude that certain resistance-associated mutations are highly stable and these data support the recommendation that all new HIV diagnoses in areas where primary resistance may occur should undergo genotyping irrespective of whether the date of seroconversion is known.

Drug resistance-related mutations T369V/I in the connection subdomain of HIV-1 reverse transcriptase severely impair viral fitness.

PubMed

Wang, Zheng; Zhang, Junli; Li, Fan; Ji, Xiaolin; Liao, Lingjie; Ma, Liying; Xing, Hui; Feng, Yi; Li, Dan; Shao, Yiming

2017-04-02

Fitness is a key parameter in the measurement of transmission capacity of individual drug-resistant HIV. Drug-resistance related mutations (DRMs) T369V/I and A371V in the connection subdomain (CN) of reverse transcriptase (RT) occur at higher frequencies in the individuals experiencing antiretroviral therapy failure. Here, we evaluated the effects of T369V/I and A371V on viral fitness, in the presence or in the absence of thymidine analogue resistance-associated mutations (TAMs) and assessed the effect of potential RT structure-related mechanism on change in viral fitness. Mutations T369V/I, A371V, alone or in combination with TAMs were introduced into a modified HIV-1 infectious clone AT1 by site-directed mutagenesis. Then, experiments on mutant and wild-type virus AT2 were performed separately using a growth-competition assay, and then the relative fitness was calculated. Structural analysis of RT was conducted using Pymol software. Results showed that T369V/I severely impaired the relative virus fitness, and A371V compensated for the viral fitness reduction caused by TAMs. Structural modeling of RT suggests that T369V/I substitutions disrupt powerful hydrogen bonds formed by T369 and V365 in p51 and p66. This study indicates that the secondary DRMs within CN might efficiently damage viral fitness, and provides valuable information for clinical surveillance and prevention of HIV-1 strains carrying these DRMs. Copyright © 2017 Elsevier B.V. All rights reserved.

Insights about minority HIV-1 strains in transmitted drug resistance mutation dynamics and disease progression.

PubMed

Leda, Ana Rachel; Hunter, James; Oliveira, Ursula Castro; Azevedo, Inacio Junqueira; Sucupira, Maria Cecilia Araripe; Diaz, Ricardo Sobhie

2018-04-19

The presence of minority transmitted drug resistance mutations was assessed using ultra-deep sequencing and correlated with disease progression among recently HIV-1-infected individuals from Brazil. Samples at baseline during recent infection and 1 year after the establishment of the infection were analysed. Viral RNA and proviral DNA from 25 individuals were subjected to ultra-deep sequencing of the reverse transcriptase and protease regions of HIV-1. Viral strains carrying transmitted drug resistance mutations were detected in 9 out of the 25 patients, for all major antiretroviral classes, ranging from one to five mutations per patient. Ultra-deep sequencing detected strains with frequencies as low as 1.6% and only strains with frequencies >20% were detected by population plasma sequencing (three patients). Transmitted drug resistance strains with frequencies <14.8% did not persist upon established infection. The presence of transmitted drug resistance mutations was negatively correlated with the viral load and with CD4+ T cell count decay. Transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion.

Prevalence of antiretroviral drug resistance and resistance-associated mutations in antiretroviral therapy-naïve HIV-infected individuals from 40 United States cities.

PubMed

Ross, Lisa; Lim, Michael L; Liao, Qiming; Wine, Brian; Rodriguez, Allan E; Weinberg, Winkler; Shaefer, Mark

2007-01-01

Transmission of drug-resistant HIV strains to antiretroviral therapy (ART)-naïve subjects can negatively impact therapy response. As treatment strategies and utilization of antiretroviral drugs evolve, patterns of transmitted mutations may shift. Paired genotypic and phenotypic susceptibility data were retrospectively analyzed for 317 ART-naïve, HIV-infected subjects from 40 small and major metropolitan cities in the Northeastern, Midwestern, Southern, Southwestern, and Northwestern United States during 2003. Using current (January 2007) PhenoSense cutoffs, HIV-from 8% of subjects had reduced susceptibility to > or = 1 drug. By class, < 1% had reduced susceptibility to protease inhibitors (PIs), and 1% had reduced susceptibility to nucleoside reverse transcriptase inhibitors (NRTIs); reduced susceptibility to > or = 1 non-nucleoside reverse transcriptase inhibitor (NNRTIs) was seen in 7% of subjects, with 4% of all subjects having reduced susceptibility to all NNRTIs. IAS-USA-defined NRTI, NNRTI, and/or major PI HIV-drug resistance-associated mutations were detected for 0% of the subjects. HIV risk factors included homosexual contact (74%), heterosexual contact (28%), and injectable drug use/transfusion/other (7%). Reduced susceptibility to > or = 1 drug was significantly higher (p = .034) for white subjects than African Americans and Hispanics/others. The high prevalence of drug resistance in these ART-naïve subjects suggests that transmitted resistance is occurring widely within the United States. HIV genotyping and/or phenotyping for antiretroviral-naïve patients seeking treatment should be considered, especially if the therapy will include an NNRTI.

Mutation covariation of HIV-1 CRF07_BC reverse transcriptase during antiretroviral therapy.

PubMed

Li, Zhenpeng; Huang, Yang; Ouyang, Yabo; Xing, Hui; Liao, Lingjie; Jiang, Shibo; Shao, Yiming; Ma, Liying

2013-11-01

To understand the effect of HIV-1 drug resistance mutations in the context of antiretroviral therapy (ART), we compared the prevalence of protease (PR) and reverse transcriptase (RT) mutations in HIV-1 CRF07_BC sequences from blood samples of treatment-naive and ART-treated patients. Mutation covariation in the RT and PR of HIV-1 CRF07_BC viruses from 542 treatment-naive patients and 261 patients treated with lamivudine/zidovudine/nevirapine or lamivudine/zidovudine/efavirenz was analysed. Stratified networks were used to display the mutation covariation. Based on the comparison between treatment-naive and ART-treated patients, three types of featured mutations for RT and PR were initially identified: treatment-associated mutations, treatment-agonistic mutations and overlapping polymorphisms. Twelve significant covariation pairs were found between five treatment-associated mutations (K103N, M184V, Q197K, G190A and Y181C) and nine overlapping polymorphisms (A36E, D39N, Y121H, D123E, R135I, T200A, R277K, L283I and D291E). Meanwhile, three covariation pairs between three treatment-associated mutations (I132L and M184V for RT and I15V for PR) and three overlapping polymorphisms (L10I, L36M and A71V) for PR were also detected. Finally, the overlapping polymorphisms for RT and PR were both found to have significant correlations with treatment-associated mutations, indicating a possible association between polymorphisms and drug resistance. When compared with HIV-1 subtype B under the same regimens as CRF07_BC, the mutation covariations of CRF07_BC showed a distinct pattern of RT and PR mutation covariation. The role of polymorphisms in the development of drug resistance has been widely reported. Here, we found a significant correlation between overlapping polymorphisms for RT and PR and treatment-associated mutations, indicating that polymorphisms exert a global influence on treatment-associated mutations. Polymorphism mutations might therefore be considered before initiating ART to improve the efficacy of drug combinations.

Dynamics of drug resistance-associated mutations in HIV-1 DNA reverse transcriptase sequence during effective ART.

PubMed

Nouchi, A; Nguyen, T; Valantin, M A; Simon, A; Sayon, S; Agher, R; Calvez, V; Katlama, C; Marcelin, A G; Soulie, C

2018-05-29

To investigate the dynamics of HIV-1 variants archived in cells harbouring drug resistance-associated mutations (DRAMs) to lamivudine/emtricitabine, etravirine and rilpivirine in patients under effective ART free from selective pressure on these DRAMs, in order to assess the possibility of recycling molecules with resistance history. We studied 25 patients with at least one DRAM to lamivudine/emtricitabine, etravirine and/or rilpivirine identified on an RNA sequence in their history and with virological control for at least 5 years under a regimen excluding all drugs from the resistant class. Longitudinal ultra-deep sequencing (UDS) and Sanger sequencing of the reverse transcriptase region were performed on cell-associated HIV-1 DNA samples taken over the 5 years of follow-up. Viral variants harbouring the analysed DRAMs were no longer detected by UDS over the 5 years in 72% of patients, with viruses susceptible to the molecules of interest found after 5 years in 80% of patients with UDS and in 88% of patients with Sanger. Residual viraemia with <50 copies/mL was detected in 52% of patients. The median HIV DNA level remained stable (2.4 at baseline versus 2.1 log10 copies/106 cells 5 years later). These results show a clear trend towards clearance of archived DRAMs to reverse transcriptase inhibitors in cell-associated HIV-1 DNA after a long period of virological control, free from therapeutic selective pressure on these DRAMs, reflecting probable residual replication in some reservoirs of the fittest viruses and leading to persistent evolution of the archived HIV-1 DNA resistance profile.

Characterization of potential antiviral resistance mutations in hepatitis B virus reverse transcriptase sequences in treatment-naïve Chinese patients.

PubMed

Liu, Bao-Ming; Li, Tong; Xu, Jie; Li, Xiao-Guang; Dong, Jian-Ping; Yan, Ping; Yang, Jing-Xian; Yan, Ling; Gao, Zhi-Yong; Li, Wen-Peng; Sun, Xie-Wen; Wang, Yu-Hua; Jiao, Xiu-Juan; Hou, Chun-Sheng; Zhuang, Hui

2010-03-01

Full-length hepatitis B virus (HBV) reverse transcriptase (RT) sequences were amplified and sequenced among 192 nucleos(t)ide analogue (NA)-naïve Chinese patients with chronic hepatitis B. Deduced amino acids (AAs) at 42 previously reported potential NA resistance (NAr) mutation positions in RT region were analyzed. Patients were found with either B-genotype (28.65%) or C-genotype (71.35%) infections. Rt53, rt91, rt124, rt134, rt221, rt224, rt238 and rt256 were identified as B- and C-genotype-dependent polymorphic AA positions. AA substitutions at 11 classical NAr mutation positions, i.e. rt80, rt169, rt173, rt180, rt181, rt184, rt194, rt202, rt204, rt236 and rt250, were not detected. However, potential NAr mutations were found in 30.73% (59/192) isolates, which involved 18 positions including rt53, rt207, rt229, rt238 and rt256, etc. The concomitant AA changes of HBsAg occurred in 16.67% (32/192) isolates including sG145R mutation. One-third of mutation positions were located in functional RT domains (e.g. rt207 and rt233), A-B interdomains (overlapping HBsAg 'a' determinant and showing most concomitant immune-associated mutations) and non-A-B interdomains (e.g. rt191 and rt213), respectively. Genotypes B and C each showed several preferred positions to mutate. These results might provide insights into understanding the evolution and selection basis of NAr HBV strains under antiviral therapy.

High Potency of Indolyl Aryl Sulfone Nonnucleoside Inhibitors towards Drug-Resistant Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutants Is Due to Selective Targeting of Different Mechanistic Forms of the Enzyme

PubMed Central

Cancio, Reynel; Silvestri, Romano; Ragno, Rino; Artico, Marino; De Martino, Gabriella; La Regina, Giuseppe; Crespan, Emmanuele; Zanoli, Samantha; Hübscher, Ulrich; Spadari, Silvio; Maga, Giovanni

2005-01-01

Indolyl aryl sulfone (IAS) nonnucleoside inhibitors have been shown to potently inhibit the growth of wild-type and drug-resistant human immunodeficiency virus type 1 (HIV-1), but their exact mechanism of action has not been elucidated yet. Here, we describe the mechanism of inhibition of HIV-1 reverse transcriptase (RT) by selected IAS derivatives. Our results showed that, depending on the substitutions introduced in the IAS common pharmacophore, these compounds can be made selective for different enzyme-substrate complexes. Moreover, we showed that the molecular basis for this selectivity was a different association rate of the drug to a particular enzymatic form along the reaction pathway. By comparing the activities of the different compounds against wild-type RT and the nonnucleoside reverse transcriptase inhibitor-resistant mutant Lys103Asn, it was possible to hypothesize, on the basis of their mechanism of action, a rationale for the design of drugs which could overcome the steric barrier imposed by the Lys103Asn mutation. PMID:16251294

The HEPT Analogue WPR-6 Is Active against a Broad Spectrum of Nonnucleoside Reverse Transcriptase Drug-Resistant HIV-1 Strains of Different Serotypes.

PubMed

Xu, Weisi; Zhao, Jianxiong; Sun, Jianping; Yin, Qianqian; Wang, Yan; Jiao, Yang; Liu, Junyi; Jiang, Shibo; Shao, Yiming; Wang, Xiaowei; Ma, Liying

2015-08-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of the highly active antiretroviral therapy (HAART) used to treat human immunodeficiency type 1 virus (HIV-1). However, because of the emergence of drug resistance and the adverse effects of current anti-HIV drugs, it is essential to develop novel NNRTIs with an excellent safety profile, improved activity against NNRTI-resistant viruses, and enhanced activity against clinical isolates of different subtypes. Here, we have identified 1-[(benzyloxy)methyl]-6-(3,5-dimethylbenzyl)-5-iodopyrimidine-2,4(1H,3H)-dione (WPR-6), a novel NNRTI with a 50% effective concentration (EC50) of 2 to 4 nM against laboratory-adapted HIV-1 strain SF33 and an EC50 of 7 to 14 nM against nucleoside reverse transcriptase inhibitor-resistant HIV-1 strain 7391 with a therapeutic index of >1 × 10(4). A panel of five representative clinical virus isolates of different subtypes circulating predominantly in China was highly sensitive to WPR-6, with EC50s ranging from 1 to 6 nM. In addition, WPR-6 showed excellent antiviral potency against the most prevalent NNRTI-resistant viruses containing the K103N and Y181C mutations. To determine whether WPR-6 selects for novel resistant mutants, in vitro resistance selection was conducted with laboratory-adapted HIV-1 strain SF33 on MT-4 cells. The results demonstrated that V106I and Y188L were the two dominant NNRTI-associated resistance mutations detected in the breakthrough viruses. Taken together, these in vitro data indicate that WPR-6 has greater efficacy than the reference HEPT analogue TNK651 and the marketed drug nevirapine against HIV-1. However, to develop it as a new NNRTI, further improvement of its pharmacological properties is warranted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

HIV drug resistance in infants increases with changing prevention of mother-to-child transmission regimens.

PubMed

Poppe, Lisa K; Chunda-Liyoka, Catherine; Kwon, Eun H; Gondwe, Clement; West, John T; Kankasa, Chipepo; Ndongmo, Clement B; Wood, Charles

2017-08-24

The objectives of this study were to determine HIV drug resistance (HIVDR) prevalence in Zambian infants upon diagnosis, and to determine how changing prevention of mother-to-child transmission (PMTCT) drug regimens affect drug resistance. Dried blood spot (DBS) samples from infants in the Lusaka District of Zambia, obtained during routine diagnostic screening, were collected during four different years representing three different PMTCT drug treatment regimens. DNA extracted from dried blood spot samples was used to sequence a 1493 bp region of the reverse transcriptase gene. Sequences were analyzed via the Stanford HIVDRdatabase (http://hivdb.standford.edu) to screen for resistance mutations. HIVDR in infants increased from 21.5 in 2007/2009 to 40.2% in 2014. Nonnucleoside reverse transcriptase inhibitor resistance increased steadily over the sampling period, whereas nucleoside reverse transcriptase inhibitor resistance and dual class resistance both increased more than threefold in 2014. Analysis of drug resistance scores in each group revealed increasing strength of resistance over time. In 2014, children with reported PMTCT exposure, defined as infant prophylaxis and/or maternal treatment, showed a higher prevalence and strength of resistance compared to those with no reported exposure. HIVDR is on the rise in Zambia and presents a serious problem for the successful lifelong treatment of HIV-infected children. PMTCT affects both the prevalence and strength of resistance and further research is needed to determine how to mitigate its role leading to resistance.

Genotypic evaluation of etravirine sensitivity of clinical human immunodeficiency virus type 1 (HIV-1) isolates carrying resistance mutations to nevirapine and efavirenz.

PubMed

Oumar, A A; Jnaoui, K; Kabamba-Mukadi, B; Yombi, J C; Vandercam, B; Goubau, P; Ruelle, J

2010-01-01

Etravirine is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) with a pattern of resistance mutations quite distinct from the current NNRTIs. We collected all routine samples of HIV-1 patients followed in the AIDS reference laboratory of UCLouvain (in 2006 and 2007) carrying resistance-associated mutations to nevirapine (NVP) or efavirenz (EFV). The sensitivity to Etravirine was estimated using three different drug resistance algorithms: ANRS (July 2008), IAS (December 2008) and Stanford (November 2008). We also verified whether the mutations described as resistance mutations are not due to virus polymorphisms by the study of 58 genotypes of NNRTI-naive patients. Sixty one samples harboured resistance to NVP and EFV: 41/61 had at least one resistance mutation to Etravirine according to ANRS-IAS algorithms; 42/61 samples had at least one resistance mutation to Etravirine according to the Stanford algorithm. 48 and 53 cases were fully sensitive to Etravirine according to ANRS-IAS and Stanford algorithms, respectively. Three cases harboured more than three mutations and presented a pattern of high-degree resistance to Etravirine according to ANRS-IAS algorithm, while one case harboured more than three mutations and presented high degree resistance to Etravirine according to the Stanford algorithm. The V1061 and V179D mutations were more frequent in the ARV-naive group than in the NNRTI-experienced one. According to the currently available algorithms, Etravirine can still be used in the majority of patients with virus showing resistance to NVP and/or EFV, if a combination of other active drugs is included.

Etravirine and Rilpivirine Drug Resistance Among HIV-1 Subtype C Infected Children Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Regimens in South India.

PubMed

Saravanan, Shanmugam; Kausalya, Bagavathi; Gomathi, Selvamurthi; Sivamalar, Sathasivam; Pachamuthu, Balakrishnan; Selvamuthu, Poongulali; Pradeep, Amrose; Sunil, Solomon; Mothi, Sarvode N; Smith, Davey M; Kantor, Rami

2017-06-01

We have analyzed reverse transcriptase (RT) region of HIV-1 pol gene from 97 HIV-infected children who were identified as failing first-line therapy that included first-generation non-nucleoside RT inhibitors (Nevirapine and Efavirenz) for at least 6 months. We found that 54% and 65% of the children had genotypically predicted resistance to second-generation non-nucleoside RT inhibitors drugs Etravirine (ETR) and Rilpivirine, respectively. These cross-resistance mutations may compromise future NNRTI-based regimens, especially in resource-limited settings. To complement these investigations, we also analyzed the sequences in Stanford database, Monogram weighted score, and DUET weighted score algorithms for ETR susceptibility and found almost perfect agreement between the three algorithms in predicting ETR susceptibility from genotypic data.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

PubMed

Yang, Fengyuan; Zheng, Guoxun; Fu, Tingting; Li, Xiaofeng; Tu, Gao; Li, Ying Hong; Yao, Xiaojun; Xue, Weiwei; Zhu, Feng

2018-06-27

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Antiretroviral Drug Susceptibility Among HIV-Infected Adults Failing Antiretroviral Therapy in Rakai, Uganda

PubMed Central

Laeyendecker, Oliver; Nakigozi, Gertrude; Gallant, Joel E.; Huang, Wei; Hudelson, Sarah E.; Quinn, Thomas C.; Newell, Kevin; Serwadda, David; Gray, Ronald H.; Wawer, Maria J.; Eshleman, Susan H.

2012-01-01

Abstract We analyzed antiretroviral drug susceptibility in HIV-infected adults failing first- and second-line antiretroviral treatment (ART) in Rakai, Uganda. Samples obtained from participants at baseline (pretreatment) and at the time of failure on first-line ART and second-line ART were analyzed using genotypic and phenotypic assays for antiretroviral drug resistance. Test results were obtained from 73 samples from 38 individuals (31 baseline samples, 36 first-line failure samples, and six second-line failure samples). Four (13%) of the 31 baseline samples had mutations associated with resistance to nucleoside or nonnucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs, respectively). Among the 36 first-line failure samples, 31 (86%) had NNRTI resistance mutations and 29 (81%) had lamivudine resistance mutations; only eight (22%) had other NRTI resistance mutations. None of the six individuals failing a second-line protease inhibitor (PI)-based regimen had PI resistance mutations. Six (16%) of the participants had discordant genotypic and phenotypic test results. Genotypic resistance to drugs included in first-line ART regimens was detected prior to treatment and among participants failing first-line ART. PI resistance was not detected in individuals failing second-line ART. Surveillance for transmitted and acquired drug resistance remains a priority for scale-up of ART. PMID:22443282

[HIV-1 resistance to antiretroviral drugs in pregnant women from Buenos Aires metropolitan area].

PubMed

Zapiola, Inés; Cecchini, Diego; Fernández Giuliano, Silvina; Martínez, Marina; Rodríguez, Claudia; Bouzas, María Belén

The study aimed to determine the prevalence of antiretroviral resistance associated mutations in HIV-1 infected pregnant woman treated in Buenos Aires metropolitan area (period 2008-2014). A total of 136 women with viral load = 500 copies/ml were included: 77 (56.6%) were treatment-naïve and 59 (43.4%) were antiretroviral-experienced patients either with current (n: 24) or previous (n = 35) antiretroviral therapy. Genotypic baseline resistance was investigated in plasma of antiretroviral-naïve patients and antiretroviral-experienced patients. The resistance mutations were identified according to the lists of the World Health Organization and the International Antiviral Society, respectively. Frequencies of resistance associated mutations detected in 2008-2011 and 2012-2014 were compared. A total of 37 (27.2%) women presented at least one resistance associated mutation: 25/94 (26.5%) in 2008-2011 and 12/42 (28.5%) in 2012-2014 (p > 0.05). Among naïves, 15 (19.5%) had at least one mutation: 10/49 (20.4%) in the period 2008-2011 and 5/28 (17.8%) in 2012-2014 (p > 0.05). The resistance mutations detected in naïves were associated with non nucleoside reverse transcriptase inhibitors, being K103N the most common mutation in both periods. In antiretroviral experienced patients, 22/59 (37.3%) had at least one resistance mutation. This study demonstrates a high frequency of resistance associated mutations which remained stable in the period analyzed. These levels suggest an increased circulation of HIV-1 antiretroviral resistant strains in our setting compared to previous reports from Argentina.

Limited clinical benefit of minority K103N and Y181C-variant detection in addition to routine genotypic resistance testing in antiretroviral therapy-naive patients.

PubMed

Metzner, Karin J; Scherrer, Alexandra U; von Wyl, Viktor; Böni, Jürg; Yerly, Sabine; Klimkait, Thomas; Aubert, Vincent; Furrer, Hansjakob; Hirsch, Hans H; Vernazza, Pietro L; Cavassini, Matthias; Calmy, Alexandra; Bernasconi, Enos; Weber, Rainer; Günthard, Huldrych F

2014-09-24

The presence of minority nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 variants prior to antiretroviral therapy (ART) has been linked to virologic failure in treatment-naive patients. We performed a large retrospective study to determine the number of treatment failures that could have been prevented by implementing minority drug-resistant HIV-1 variant analyses in ART-naïve patients in whom no NNRTI resistance mutations were detected by routine resistance testing. Of 1608 patients in the Swiss HIV Cohort Study, who have initiated first-line ART with two nucleoside reverse transcriptase inhibitors (NRTIs) and one NNRTI before July 2008, 519 patients were eligible by means of HIV-1 subtype, viral load and sample availability. Key NNRTI drug resistance mutations K103N and Y181C were measured by allele-specific PCR in 208 of 519 randomly chosen patients. Minority K103N and Y181C drug resistance mutations were detected in five out of 190 (2.6%) and 10 out of 201 (5%) patients, respectively. Focusing on 183 patients for whom virologic success or failure could be examined, virologic failure occurred in seven out of 183 (3.8%) patients; minority K103N and/or Y181C variants were present prior to ART initiation in only two of those patients. The NNRTI-containing, first-line ART was effective in 10 patients with preexisting minority NNRTI-resistant HIV-1 variant. As revealed in settings of case-control studies, minority NNRTI-resistant HIV-1 variants can have an impact on ART. However, the implementation of minority NNRTI-resistant HIV-1 variant analysis in addition to genotypic resistance testing (GRT) cannot be recommended in routine clinical settings. Additional associated risk factors need to be discovered.

Prevalence and patterns of HIV transmitted drug resistance in Guatemala.

PubMed

Avila-Ríos, Santiago; Mejía-Villatoro, Carlos R; García-Morales, Claudia; Soto-Nava, Maribel; Escobar, Ingrid; Mendizabal, Ricardo; Girón, Amalia; García, Leticia; Reyes-Terán, Gustavo

2011-12-01

To assess human immunodeficiency virus (HIV) diversity and the prevalence of transmitted drug resistance (TDR) in Guatemala. One hundred forty-five antiretroviral treatment-naïve patients referred to the Roosevelt Hospital in Guatemala City were enrolled from October 2010 to March 2011. Plasma HIV pol sequences were obtained and TDR was assessed with the Stanford algorithm and the World Health Organization (WHO) TDR surveillance mutation list. HIV subtype B was highly prevalent in Guatemala (96.6%, 140/145), and a 2.8% (4/145) prevalence of BF1 recombinants and 0.7% (1/145) prevalence of subtype C viruses were found. TDR prevalence for the study period was 8.3% (12/145) with the Stanford database algorithm (score > 15) and the WHO TDR surveillance mutation list. Most TDR cases were associated with non-nucleoside reverse transcriptase inhibitors (NNRTIs) (83.3%, 10/12); a low prevalence of nucleoside reverse transcriptase inhibitors and protease inhibitors was observed in the cohort (< 1% for both families). Low selection of antiretroviral drug resistance mutations was found, except for NNRTI-associated mutations. Major NNRTI mutations such as K101E, K103N, and E138K showed higher frequencies than expected in ART-naïve populations. Higher literacy was associated with a greater risk of TDR (odds ratio 4.14, P = 0.0264). This study represents one of the first efforts to describe HIV diversity and TDR prevalence and trends in Guatemala. TDR prevalence in Guatemala was at the intermediate level. Most TDR cases were associated with NNRTIs. Further and continuous TDR surveillance is necessary to gain more indepth knowledge about TDR spread and trends in Guatemala and to optimize treatment outcomes in the country.

Selection and Characterization of Drug-Resistant Variants of Human Immunodeficiency Virus (AIDS).

DTIC Science & Technology

1995-10-01

on Antiviral Reserach, Santa Fe, New Mexico , 1995. Page 18 APPENDIX Page 19 p - FACTFILE Mutations in HIV-1 Reverse Transcriptase and Protease...including herpes simplex viruses, varicella -zoster Resistance of clinical HIV-1 isolates to foscarnet has not virus, cytomegalovirus (CMV), hepatitis B...This effect of the Tyr-208 substitution was not ob- reported previously for herpes simplex viruses, varicella -zoster served in MT-2 cells, however. virus

The impact of HIV-1 reverse transcriptase polymorphisms on responses to first-line nonnucleoside reverse transcriptase inhibitor-based therapy in HIV-1-infected adults.

PubMed

Mackie, Nicola E; Dunn, David T; Dolling, David; Garvey, Lucy; Harrison, Linda; Fearnhill, Esther; Tilston, Peter; Sabin, Caroline; Geretti, Anna M

2013-09-10

HIV-1 genetic variability may influence antiretroviral therapy (ART) outcomes. The study aim was to determine the impact of polymorphisms in regions known to harbor major nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations (codons 90-108, 135-138, 179-190, 225-348) on virologic responses to first-line NNRTI-based ART. Reverse transcriptase sequences from ART-naive individuals who commenced efavirenz (EFV) or nevirapine (NVP) with at least two nucleos(t)ide reverse transcriptase inhibitors (NRTIs) without major drug resistance mutations were analyzed. The impact of polymorphisms on week 4 viral load decrease and time to virologic failure was measured over a median 97 weeks. Among 4528 patients, most were infected with HIV-1 subtype B (67%) and commenced EFV-based ART (84%). Overall, 2598 (57%) had at least one polymorphism, most frequently at codons 90, 98, 101, 103, 106, 135, 138, 179, and 238. Virologic failure rates were increased in patients with two (n = 597) or more than two (n = 72) polymorphisms [adjusted hazard ratio 1.43; 95% confidence interval (CI) 1.07-1.92; P = 0.016]. Polymorphisms associated with virologic failure occurred at codons 90 (mostly V90I), 98 (mostly A98S), and 103 (mostly K103R), with adjusted hazard ratios of 1.78 (1.15-2.73; P = 0.009), 1.55 (1.16-2.08; P = 0.003), and 1.75 (1.00-3.05: P = 0.049), respectively. Polymorphisms at codon 179, especially V179D/E/T, predicted reduced week 4 responses (P = 0.001) but not virologic failure. The occurrence of multiple polymorphisms, though uncommon, was associated with a small increase in the risk of NNRTI treatment failure; significant effects were seen with polymorphisms at codon 90, 98, and 103. The mechanisms underlying the slower suppression seen with V179D/E/T deserve further investigation.

Analysis of drug resistance during HIV RNA viraemia in the MONET trial of darunavir/ritonavir monotherapy.

PubMed

Pulido, Federico; Arribas, José R; Hill, Andrew; Van Delft, Yvon; Moecklinghoff, Christiane

2011-01-01

When patients have HIV RNA suppressed to <50 copies/ml on current treatment, switching to darunavir (DRV)/ritonavir (DRV/r) monotherapy could prevent the development of resistance to other drug classes. In the MONET trial, 256 patients with HIV RNA<50 copies/ml on current highly active antiretroviral therapy (57% with protease inhibitors [PIs] and 43% with non-nucleoside reverse transcriptase inhibitors) and no history of virological failure were randomized to DRV/r 800/100 mg once daily, either as monotherapy (monotherapy arm) or with two nucleoside reverse transcriptase inhibitors (NRTIs; triple therapy arm). All samples with HIV RNA ≥ 50 copies/ml were genotyped, and a virtual phenotype was calculated (VircoType HIV-1 assays; Virco BVBA, Mechelen, Belgium). A total of 63 patients had ≥ 1 HIV RNA result ≥ 50 copies/ml, of whom 38 were successfully genotyped. Most HIV RNA increases were transient and in the range of 50-200 copies/ml. Overall, 36 of the 38 (95%) successfully genotyped patients showed no International AIDS Society-USA major PI mutations, DRV mutations or NRTI mutations. Two patients showed some evidence of PI resistance during transient HIV RNA elevations: one patient in the monotherapy arm had a single DRV mutation (L33F) when HIV RNA was 63 copies/ml (the virus was phenotypically sensitive to DRV [fold change 0.8]) and one PI pretreated patient taking tenofovir disoproxil fumarate/emtricitabine/DRV/r had re-emergence of pre-existing NRTI (M184V) and PI (V82I and L90M) mutations after a short treatment interruption (this virus remained phenotypically sensitive to DRV/r). Both patients showed sustained HIV RNA suppression to week 48 remaining with the same treatment. Emergence of drug resistance after changing a suppressive triple antiretroviral therapy to DRV/r with or without nucleoside analogues is uncommon.

Deep sequencing analysis of HIV-1 reverse transcriptase at baseline and time of failure in patients receiving rilpivirine in the phase III studies ECHO and THRIVE.

PubMed

Van Eygen, Veerle; Thys, Kim; Van Hove, Carl; Rimsky, Laurence T; De Meyer, Sandra; Aerssens, Jeroen; Picchio, Gaston; Vingerhoets, Johan

2016-05-01

Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS. © 2015 Wiley Periodicals, Inc.

Prevalence of HIV drug resistance mutation in the northern Indian population after failure of the first line antiretroviral therapy.

PubMed

Sinha, S; Shekhar, R C; Ahmad, H; Kumar, N; Samantaray, J C; Sreenivas, V; Khan, N H; Mitsuyasu, R T

2012-09-01

There is limited information available about the prevalence and pattern of human immunodeficiency virus (HIV) drug resistance mutations (DRMs) among antiretroviral therapy (ART) experienced patients from northern India. Results of genotypic drug resistance testing were obtained from plasma samples of 128 patients, who had presented with clinical or immunological failure to treatment after at least six months of ART. Major DRMs associated with any of the three classes of antiretroviral (ARV) drugs, nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI), were seen in 120 out of 128 patients (93.8% prevalence). NRTI and NNRTI DRMs were each seen in 115/128 (89.8%) patients, with M184V, M41L, D67N and T215Y being the most frequent among NRTI associated mutations, and K103N, G190A, Y181C and A98G among NNRTI associated ones. PI DRMs were observed in 14/128 (10.9%) patients, with L10I, V82A and L89V being the commonest. These results present a high prevalence of DRMs among ART experienced patients from northern India with clinical or immunological failure of therapy. It emphasizes the need for regular testing of plasma samples of such patients for DRMs in order to detect and replace a failing regimen early, and also the use of HIV drug resistance genotyping of ART naive individuals prior to initiating first line ART for possible transmitted resistance. It is very important to enhance the access of patients to ARV drugs so that their compliance could be improved and hence development of DRMs be minimized.

Prevalence of drug-resistant mutation among drug-treated HIV/AIDS inpatient in Airlangga University teaching hospital, Surabaya, Indonesia

NASA Astrophysics Data System (ADS)

Rachman, B. E.; Khairunisa, S. Q.; Witaningrum, A. M.; Yunifiar, M. Q.; Widiyanti, P.; Nasronudin

2018-03-01

Increased use of antiretroviral therapy did not completely reduce the incidence of HIV/AIDShospitalization. Various factors can be involved. The aim of this study is to examine HIV-1 drug resistance mutations profile in drug-treated HIV/AIDS patients who underwent hospitalization. HIV/AIDS patients who are admitted to hospital who had received ART are included in the study and then examined for the presence of drug resistance-associated mutations. A total of 17 samples were included in the study, but only 11 samples that could be sequence analyzed. On the mutation examination of drug resistance in reverse transcriptase gene, it werefound a major mutation in K103N (9%) and G190A (9%). Most minor mutations were found in A98S (18.1%), followed by M41L, M184V, L210W, T215Y, V108l, Y181C and H221Y at 9% each. Whereas, on examination of drug resistance mutations in protease genes, there is a major mutation in I84V of 9%. Most minor mutations on M36I (45.4%), followed by L10I (36.3%), H69K (36.3%), I93L (27.2%), G16E, L89M, K20R 18.1%, L64V and V771I 9% respectively.A large number of mutated samples pose a challenge in long-term antiretroviral treatment, so a breakthrough policy is needed to minimize the impact.

Prevalence of drug resistance and importance of viral load measurements in Honduran HIV-infected patients failing antiretroviral treatment.

PubMed

Murillo, Wendy; de Rivera, I L; Parham, L; Jovel, E; Palou, E; Karlsson, A C; Albert, J

2010-02-01

The Honduran HIV/AIDS Program began to scale up access to HIV therapy in 2002. Up to May 2008, more than 6000 patients received combination antiretroviral therapy (cART). As HIV drug resistance is the major obstacle for effective treatment, the purpose of this study was to assess the prevalence of antiretroviral drug resistance in Honduran HIV-1-infected individuals. We collected samples from 138 individuals (97 adults and 41 children) on cART with virological, immunological or clinical signs of treatment failure. HIV-1 pol sequences were obtained using an in-house method. Resistance mutations were identified according to the 2007 International AIDS Society (IAS)-USA list and predicted susceptibility to cART was scored using the ANRS algorithm. Resistance mutations were detected in 112 patients (81%), 74% in adults and 98% in children. Triple-, dual- and single-class drug resistance was documented in 27%, 43% and 11% of the study subjects, respectively. Multiple logistic regression showed that resistance was independently associated with type of treatment failure [virological failure (odds ratio (OR) = 1) vs. immunological failure (OR = 0.11; 95% confidence interval (CI) 0.030-0.43) vs. clinical failure (OR = 0.037; 95% CI 0.0063-0.22)], route of transmission (OR = 42.8; 95% CI 3.73-491), and years on therapy (OR = 1.81; 95% CI 1.11-2.93). The prevalence of antiretroviral resistance was high in Honduran HIV-infected patients with signs of treatment failure. A majority of study subjects showed dual- or triple-class resistance to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors. Virologically defined treatment failure was a strong predictor of resistance, indicating that viral load testing is needed to correctly identify patients with treatment failure attributable to resistance.

Nucleoside reverse transcriptase inhibitor resistance mutations associated with first-line stavudine-containing antiretroviral therapy: programmatic implications for countries phasing out stavudine.

PubMed

Tang, Michele W; Rhee, Soo-Yon; Bertagnolio, Silvia; Ford, Nathan; Holmes, Susan; Sigaloff, Kim C; Hamers, Raph L; de Wit, Tobias F Rinke; Fleury, Herve J; Kanki, Phyllis J; Ruxrungtham, Kiat; Hawkins, Claudia A; Wallis, Carole L; Stevens, Wendy; van Zyl, Gert U; Manosuthi, Weerawat; Hosseinipour, Mina C; Ngo-Giang-Huong, Nicole; Belec, Laurent; Peeters, Martine; Aghokeng, Avelin; Bunupuradah, Torsak; Burda, Sherri; Cane, Patricia; Cappelli, Giulia; Charpentier, Charlotte; Dagnra, Anoumou Y; Deshpande, Alaka K; El-Katib, Ziad; Eshleman, Susan H; Fokam, Joseph; Gody, Jean-Chrysostome; Katzenstein, David; Koyalta, Donato D; Kumwenda, Johnstone J; Lallemant, Marc; Lynen, Lutgarde; Marconi, Vincent C; Margot, Nicolas A; Moussa, Sandrine; Ndung'u, Thumbi; Nyambi, Phillipe N; Orrell, Catherine; Schapiro, Jonathan M; Schuurman, Rob; Sirivichayakul, Sunee; Smith, Davey; Zolfo, Maria; Jordan, Michael R; Shafer, Robert W

2013-06-15

The World Health Organization Antiretroviral Treatment Guidelines recommend phasing-out stavudine because of its risk of long-term toxicity. There are two mutational pathways of stavudine resistance with different implications for zidovudine and tenofovir cross-resistance, the primary candidates for replacing stavudine. However, because resistance testing is rarely available in resource-limited settings, it is critical to identify the cross-resistance patterns associated with first-line stavudine failure. We analyzed HIV-1 resistance mutations following first-line stavudine failure from 35 publications comprising 1,825 individuals. We also assessed the influence of concomitant nevirapine vs. efavirenz, therapy duration, and HIV-1 subtype on the proportions of mutations associated with zidovudine vs. tenofovir cross-resistance. Mutations with preferential zidovudine activity, K65R or K70E, occurred in 5.3% of individuals. Mutations with preferential tenofovir activity, ≥ two thymidine analog mutations (TAMs) or Q151M, occurred in 22% of individuals. Nevirapine increased the risk of TAMs, K65R, and Q151M. Longer therapy increased the risk of TAMs and Q151M but not K65R. Subtype C and CRF01_AE increased the risk of K65R, but only CRF01_AE increased the risk of K65R without Q151M. Regardless of concomitant nevirapine vs. efavirenz, therapy duration, or subtype, tenofovir was more likely than zidovudine to retain antiviral activity following first-line d4T therapy.

Coexistence of BRAF V600E and TERT Promoter Mutations in Low-grade Serous Carcinoma of Ovary Recurring as Carcinosarcoma in a Lymph Node: Report of a Case.

PubMed

Tavallaee, Mahkam; Steiner, David F; Zehnder, James L; Folkins, Ann K; Karam, Amer K

2018-04-03

Low-grade serous carcinomas only rarely coexist with or progress to high-grade tumors. We present a case of low-grade serous carcinoma with transformation to carcinosarcoma on recurrence in the lymph node. Identical BRAF V600E and telomerase reverse transcriptase promoter mutations were identified in both the original and recurrent tumor. Given that telomerase reverse transcriptase promotor mutations are thought to play a role in progression of other tumor types, the function of telomerase reverse transcriptase mutations in BRAF mutated low-grade serous carcinoma deserves investigation.

Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

PubMed Central

Frenkel, L M; Wagner, L E; Atwood, S M; Cummins, T J; Dewhurst, S

1995-01-01

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV. PMID:7714190

Molecular epidemiological analysis of env and pol sequences in newly diagnosed HIV type 1-infected, untreated patients in Hungary.

PubMed

Mezei, Mária; Ay, Eva; Koroknai, Anita; Tóth, Renáta; Balázs, Andrea; Bakos, Agnes; Gyori, Zoltán; Bánáti, Ferenc; Marschalkó, Márta; Kárpáti, Sarolta; Minárovits, János

2011-11-01

The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008-2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classification of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

HIV Drug Resistance Testing in a Resource Limited Setting with High Viral Diversity: The First Twenty Eight Months Experience.

PubMed

Ngo-Malabo, Elodie Teclaire; Ngoupo, Paul Alain; Sadeuh-Mba, Serge Alain; Akongnwi, Emmanuel; Banaï, Robert; Ngono, Laure; Bilong-Bilong, Charles Felix; Kfutwah, Anfumbom; Njouom, Richard

2017-01-01

First line antiretroviral therapy in a resource-limited setting consists of nucleotide and non-nucleotide reverse transcriptase inhibitors. Protease inhibitors are the hub of second line therapy. The decision to change antiretroviral therapy for a patient is frequently presumptive because of the lack of genotypic resistance tests in routine follow-up. We describe here the resistance profiles observed in patients with varying terms of antiretroviral therapy in Cameroon after implementation of HIV genotypic resistance testing in routine practice. HIV genotypic resistance testing was carried out on consecutive samples received between August 2013 and November 2015. Protease (Prot) and reverse transcriptase (Rt) genes of the HIV genome were amplified, sequenced and analyzed for drug resistance mutations following the algorithm set up by the French National Agency for research on HIV/AIDS and viral hepatitis. Specimens from a total of 167 patients infected with non-B HIV subtypes were received during the study period. Overall 61.7% patients had viral loads of more than 3log copies/ml, suggesting treatment failure. Among the 72 patients on first line, 56 (77.8%) were resistant to Lamivudine, 57 (79.1%) to Efavirenz and 58 (80.6%) to Nevirapine. Overall, more patients (75.0%) on first line antiretroviral therapy harbored multi-drug resistance compared to their counterparts on second line (25.8%). This study revealed that a group of patients with antiretroviral therapy failure harbored multi-drug resistance mutations related to the majority of drugs in the first line regimen. Therefore, HIV resistance testing could be a useful tool to improve HIV care in resource limited settings like Cameroon where treatment options are limited. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATPmore » (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.« less

Cytotoxic chemotherapy and the evolution of cellular and viral resistance to antiretroviral therapy in HIV- infected individuals with lymphoma.

PubMed

McFaul, Katie; Liptrott, Neill; Cox, Alison; Martin, Phillip; Egan, Deirdre; Owen, Andrew; Kelly, Sarah; Karolia, Zeenat; Shaw, Kate; Bower, Mark; Boffito, Marta

2016-09-01

The use of combination antiretroviral therapy (cART) and cytotoxic chemotherapy for HIV-associated lymphoma runs the risks of inducing HIV drug resistance. This study examined two possible mechanisms: altered expression of membrane drug transporter protein (MTP) and acquisition of mutations in pro-viral DNA. Expression levels of MTP and pro-viral DNA resistance mutation analysis were performed on peripheral blood mononuclear cells (PBMC) before, during, and after chemotherapy. Twenty nine patients completed the three time point estimations. There were no significant variations before, during, and after chemotherapy in the expression of four MTPs: ABCB1, ABCC1, ABCC2, and SLCO3A1 (OATP3A1). Pro-viral DNA sequencing revealed that only one patient developed a new nucleos/tide reverse transcriptase inhibitor-associated mutation (184V) during the course of the study, giving a mutation rate of 0.0027 per person per year. In conclusion, concomitant administration of cytotoxic chemotherapy and cART does not induce expression of MTP. Furthermore, no significant changes in viral resistance were observed pre- and post-chemotherapy, suggesting mutagenic cytotoxic chemotherapy seems not to induce mutations in HIV pro-viral DNA.

Prevalence and Mutation Patterns of HIV Drug Resistance from 2010 to 2011 among ART-Failure Individuals in the Yunnan Province, China

PubMed Central

Guo, Wei; Zhuang, Daomin; Li, Lin; Liu, Yongjian; Bao, Zuoyi; Liu, Siyang; Wang, Xiaolin; Li, Tianyi; Yang, Shaomin; Li, Jingyun

2013-01-01

Background Assessing the prevalence of HIV-1 drug-resistance and the mutation patterns associated with resistance in the geographical regions implementing free antiretroviral therapy (ART) in China is necessary for preventing the spread of resistant strains and designing the regimens for the subsequent therapies with limited resources. Methods Plasma samples in different cities/prefectures were collected at Yunnan Provincial Hospital of Infectious Disease from January 2010 to December 2011. Genotyping of drug-resistant individuals was conducted using an in-house assay on plasma samples. Viral load, CD4 T cell counts and demographic data were obtained from medical records and an administered questionnaire. Results A total of 609 pol sequences (515 ART-failure and 94 therapy-naïve individuals) derived from 664 samples were obtained. The prevalence of drug-resistance was 45.1% in the ART-failure individuals. Of these, 26.8% harbored HIV strains dually resistant to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors, and 14.8% harbored HIV strains resistant to only one drug category. Mutations such as M184V/I, K103N, V106A, Y181C and G190A were common among the ART-failure individuals, and the frequencies of M184V/I, K103N and V106A were 28.2%, 19.2%, and 22.1%, respectively. The percentages of individuals exhibiting intermediate or high-level resistance to 3TC, FTC, EFV and NVP drugs were 28.4%, 28.2%, 37.3%, and 37.5%, respectively. Factors such as ethnicity, transmission route, CD4 counts, viral load and the duration of ART were significantly correlated with development of drug resistance in the ART-failure individuals. Conclusions The high prevalence of HIV drug-resistance observed among the ART-failure individuals from 2010 to 2011 in Yunnan province should be of increasing concern in regions where the implementation of ART is widespread. Education about the risk factors associated with HIV drug resistance is important for preventing and controlling the spread of HIV drug-resistant strains. PMID:24009694

In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

PubMed Central

La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

2001-01-01

We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

Recent trends and patterns in HIV-1 transmitted drug resistance in the United Kingdom.

PubMed

Tostevin, A; White, E; Dunn, D; Croxford, S; Delpech, V; Williams, I; Asboe, D; Pozniak, A; Churchill, D; Geretti, A M; Pillay, D; Sabin, C; Leigh-Brown, A; Smit, E

2017-03-01

Transmission of drug-resistant HIV-1 has decreased in the UK since the early 2000s. This analysis reports recent trends and characteristics of transmitted drug resistance (TDR) in the UK from 2010 to 2013. Resistance tests conducted in antiretroviral treatment (ART)-naïve individuals between 2010 and 2013 were analysed for the presence of transmitted drug resistance mutations (TDRMs), defined as any mutations from a modified 2009 World Health Organization surveillance list, or a modified 2013 International Antiviral Society-USA list for integrase tests. Logistic regression was used to examine associations between demographics and the prevalence of TDRMs. TDRMs were observed in 1223 (7.5%) of 16 425 individuals; prevalence declined from 8.1% in 2010 to 6.6% in 2013 (P = 0.02). The prevalence of TDRMs was higher among men who have sex with men (MSM) compared with heterosexual men and women (8.7% versus 6.4%, respectively) with a trend for decreasing TDRMs among MSM (P = 0.008) driven by a reduction in nucleoside reverse transcriptase inhibitor (NRTI)-related mutations. The most frequently detected TDRMs were K103N (2.2%), T215 revertants (1.6%), M41L (0.9%) and L90M (0.7%). Predicted phenotypic resistance to first-line ART was highest to the nonnucleoside reverse transcriptase inhibitors (NNRTIs) rilpivirine and efavirenz (6.2% and 3.4%, respectively) but minimal to NRTIs, including tenofovir, and protease inhibitors (PIs). No major integrase TDRMs were detected among 101 individuals tested while ART-naïve. We observed a decrease in TDRMs in recent years. However, this was confined to the MSM population and rates remained stable in those with heterosexually acquired HIV infection. Resistance to currently recommended first-line ART, including integrase inhibitors, remained reassuringly low. © 2016 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.

Viremia and HIV-1 Drug Resistance Mutations Among Patients Receiving Second-Line Highly Active Antiretroviral Therapy in Chennai, Southern India

PubMed Central

Vidya, Madhavan; Balakrishnan, Pachamuthu; Kantor, Rami; Solomon, Sunil S.; Katzenstein, David; Kumarasamy, Nagalingeswaran; Yeptomi, Tokugha; Sivamalar, Sathasivam; Rifkin, Samara; Mayer, Kenneth H.; Solomon, Suniti

2012-01-01

Background. A cross-sectional study among individuals receiving second-line antiretroviral treatment was conducted to report on the level of detectable viremia and the types of drug resistance mutations among those with detectable human immunodeficiency virus (HIV) type 1 plasma viral loads (PVLs). Methods. PVLs were measured using Abbott m2000rt real-time polymerase chain reaction, and genotyping was performed with the ViroSeq genotyping system, version 2.0, and ViroSeq analysis software, version 2.8. Results. Of 107 patient plasma specimens consecutively analyzed, 30 (28%) had undetectable PVLs (<150 copies/mL), and 77 (72%) were viremic with a median PVL of 5450 copies/mL (interquartile range, 169–1 997 967). Sequencing was done for 107 samples with PVLs >2000 copies/mL: 33 patients (73%) had 1 of the protease (PR) inhibitor mutations; 41 (91%) had nucleoside reverse-transcriptase inhibitor (NRTI) mutations; 33 (73%) had non-NRTI (NNRTI) mutations; and 30 (66.7%) had both NRTI and NNRTI mutations. Triple-class resistance to NRTIs, NNRTIs, and PR inhibitors was observed in 24 (53%) patients. Based on the mutational profiles observed, all 45 sequences were susceptible to darunavir and tipranavir, whereas 47% showed resistance to lopinavir, 58% showed resistance to atazanavir, and >60% showed resistance to saquinavir, indinavir, nelfinavir, and fosamprenavir. Conclusions. The results of the study showed that the majority of patients receiving second-line antiretroviral therapy started to accumulate PR resistance mutations, and the mutation profiles suggest that darunavir might be the drug of choice for third-line regimens in India. PMID:22323567

One-, two-, and three-class resistance among HIV-infected patients on antiretroviral therapy in private care clinics: Mumbai, India.

PubMed

Gupta, Amita; Saple, Dattaray G; Nadkarni, Girish; Shah, Bijal; Vaidya, Satish; Hingankar, Nitin; Chaturbhuj, Devidas; Deshmukh, Praveen; Walshe, Louise; Hudelson, Sarah E; James, Maria; Paranjape, Ramesh S; Eshleman, Susan H; Tripathy, Srikanth

2010-01-01

HIV-infected patients receiving antiretroviral (ARV) therapy (ART) in India are not all adequately virally suppressed. We analyzed ARV drug resistance in adults receiving ART in three private clinics in Mumbai, India. HIV viral load was measured in 200 patients with the Roche AMPLICOR HIV-1 Monitor Test, v1.5. HIV genotyping was performed with the ViroSeq HIV-1 Genotyping System for 61 participants who had HIV-1 RNA >1000 copies/ml. Genotyping results were obtained for 51 samples. The participants with resistance results were on ART for a median of 24 months and were on their current regimen for a median of 12 months (median CD4 cell count: 217 cells/mm(3); median HIV viral load: 28,200 copies/ml). ARV regimens included nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (n = 27), dual nucleoside reverse transcriptase inhibitors (NRTIs, n = 19), protease inhibitor (PI)-based regimens (n = 3), and other regimens (n = 2). Twenty-six participants (51.0%) were on their first ARV regimen and 24 (47%) reported >95% adherence. Forty-nine participants (96.1%) had resistance to at least one ARV drug; 47 (92.2%) had NRTI resistance, 32 (62.7%) had NNRTI resistance, and four (7.8%) had PI resistance. Thirty (58.8%) had two-class resistance and three (5.9%) had three-class resistance. Four (8%) had three or more resistance mutations associated with etravirine resistance and two (4%) had two mutations associated with reduced darunavir susceptibility. Almost all patients with HIV-1 RNA >1000 copies/ml had NRTI resistance and nearly two-thirds had NNRTI resistance; PI resistance was uncommon. Nearly 60% and 6% had two- and three-class resistance, respectively. This emphasizes the need for greater viral load and resistance monitoring, use of optimal ART combinations, and increased availability of second- and third-line agents for patients with ARV resistance.

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

PubMed Central

Porter, Danielle P.; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D.; White, Kirsten L.

2015-01-01

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

PubMed

Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

2015-12-07

At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study.

HIV type 1 diversity in the Seychelles.

PubMed

Razafindratsimandresy, Richter; Hollanda, Justina; Soares, Jean-Louis; Rousset, Dominique; Chetty, Agnes P; Reynes, Jean-Marc

2007-06-01

Subtype determination and drug resistance-associated mutations (DRM) detection were performed on 40 HIV-1 Western blot-positive sera detected, obtained from consecutive patients resident in the Seychelles and consulting the Communicable Disease Control Unit, HIV reference center, in Victoria Hospital (Mahe) from October 2005 to June 2006. Amplification and sequencing of at least two of the partial reverse transcriptase, protease, and partial envelope genes were successful for all strains. All three genes sequences were obtained for 39 strains. A high degree of subtype or circulating recombinant forms (CRF) was observed for these 39 strains: A-A1 (17 cases), C (10 cases), B (8 cases), CRF02_AG (2 cases), D (1 case) and CRF01_AE (1 case). According to the ANRS 2006 DRM list and algorithm, none of the 40 isolates was found to be resistant to any protease or reverse transcriptase inhibitors.

Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †

PubMed Central

Mitsuya, Yumi; Varghese, Vici; Wang, Chunlin; Liu, Tommy F.; Holmes, Susan P.; Jayakumar, Prerana; Gharizadeh, Baback; Ronaghi, Mostafa; Klein, Daniel; Fessel, W. Jeffrey; Shafer, Robert W.

2008-01-01

T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants. PMID:18715933

Geographic and temporal trends in the molecular epidemiology and genetic mechanisms of transmitted HIV-1 drug resistance: an individual-patient- and sequence-level meta-analysis.

PubMed

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L; Bertagnolio, Silvia; Rinke de Wit, Tobias F; Aghokeng, Avelin F; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O; Brooks, James I; Boucher, Charles A B; Brumme, Zabrina L; Busch, Michael P; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D'Aquin, Toni T; De Gascun, Cillian F; Derache, Anne; Descamps, Diane; Deshpande, Alaka K; Djoko, Cyrille F; Eshleman, Susan H; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; Ng, Kee Peng; Paranjape, Ramesh S; Peeters, Martine; Poljak, Mario; Price, Matt A; Ragonnet-Cronin, Manon L; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M; Soares, Marcelo A; Soriano, Vincent V; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M; van de Vijver, David A M C; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P A; Vandamme, Anne-Mieke; Shafer, Robert W

2015-04-01

Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05-1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06-1.25), North America (OR = 1.19; 95% CI: 1.12-1.26), Europe (OR = 1.07; 95% CI: 1.01-1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12-1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92-1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

PubMed Central

Rhee, Soo-Yon; Blanco, Jose Luis; Jordan, Michael R.; Taylor, Jonathan; Lemey, Philippe; Varghese, Vici; Hamers, Raph L.; Bertagnolio, Silvia; de Wit, Tobias F. Rinke; Aghokeng, Avelin F.; Albert, Jan; Avi, Radko; Avila-Rios, Santiago; Bessong, Pascal O.; Brooks, James I.; Boucher, Charles A. B.; Brumme, Zabrina L.; Busch, Michael P.; Bussmann, Hermann; Chaix, Marie-Laure; Chin, Bum Sik; D’Aquin, Toni T.; De Gascun, Cillian F.; Derache, Anne; Descamps, Diane; Deshpande, Alaka K.; Djoko, Cyrille F.; Eshleman, Susan H.; Fleury, Herve; Frange, Pierre; Fujisaki, Seiichiro; Harrigan, P. Richard; Hattori, Junko; Holguin, Africa; Hunt, Gillian M.; Ichimura, Hiroshi; Kaleebu, Pontiano; Katzenstein, David; Kiertiburanakul, Sasisopin; Kim, Jerome H.; Kim, Sung Soon; Li, Yanpeng; Lutsar, Irja; Morris, Lynn; Ndembi, Nicaise; NG, Kee Peng; Paranjape, Ramesh S.; Peeters, Martine; Poljak, Mario; Price, Matt A.; Ragonnet-Cronin, Manon L.; Reyes-Terán, Gustavo; Rolland, Morgane; Sirivichayakul, Sunee; Smith, Davey M.; Soares, Marcelo A.; Soriano, Vincent V.; Ssemwanga, Deogratius; Stanojevic, Maja; Stefani, Mariane A.; Sugiura, Wataru; Sungkanuparph, Somnuek; Tanuri, Amilcar; Tee, Kok Keng; Truong, Hong-Ha M.; van de Vijver, David A. M. C.; Vidal, Nicole; Yang, Chunfu; Yang, Rongge; Yebra, Gonzalo; Ioannidis, John P. A.; Vandamme, Anne-Mieke; Shafer, Robert W.

2015-01-01

Background Regional and subtype-specific mutational patterns of HIV-1 transmitted drug resistance (TDR) are essential for informing first-line antiretroviral (ARV) therapy guidelines and designing diagnostic assays for use in regions where standard genotypic resistance testing is not affordable. We sought to understand the molecular epidemiology of TDR and to identify the HIV-1 drug-resistance mutations responsible for TDR in different regions and virus subtypes. Methods and Findings We reviewed all GenBank submissions of HIV-1 reverse transcriptase sequences with or without protease and identified 287 studies published between March 1, 2000, and December 31, 2013, with more than 25 recently or chronically infected ARV-naïve individuals. These studies comprised 50,870 individuals from 111 countries. Each set of study sequences was analyzed for phylogenetic clustering and the presence of 93 surveillance drug-resistance mutations (SDRMs). The median overall TDR prevalence in sub-Saharan Africa (SSA), south/southeast Asia (SSEA), upper-income Asian countries, Latin America/Caribbean, Europe, and North America was 2.8%, 2.9%, 5.6%, 7.6%, 9.4%, and 11.5%, respectively. In SSA, there was a yearly 1.09-fold (95% CI: 1.05–1.14) increase in odds of TDR since national ARV scale-up attributable to an increase in non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance. The odds of NNRTI-associated TDR also increased in Latin America/Caribbean (odds ratio [OR] = 1.16; 95% CI: 1.06–1.25), North America (OR = 1.19; 95% CI: 1.12–1.26), Europe (OR = 1.07; 95% CI: 1.01–1.13), and upper-income Asian countries (OR = 1.33; 95% CI: 1.12–1.55). In SSEA, there was no significant change in the odds of TDR since national ARV scale-up (OR = 0.97; 95% CI: 0.92–1.02). An analysis limited to sequences with mixtures at less than 0.5% of their nucleotide positions—a proxy for recent infection—yielded trends comparable to those obtained using the complete dataset. Four NNRTI SDRMs—K101E, K103N, Y181C, and G190A—accounted for >80% of NNRTI-associated TDR in all regions and subtypes. Sixteen nucleoside reverse transcriptase inhibitor (NRTI) SDRMs accounted for >69% of NRTI-associated TDR in all regions and subtypes. In SSA and SSEA, 89% of NNRTI SDRMs were associated with high-level resistance to nevirapine or efavirenz, whereas only 27% of NRTI SDRMs were associated with high-level resistance to zidovudine, lamivudine, tenofovir, or abacavir. Of 763 viruses with TDR in SSA and SSEA, 725 (95%) were genetically dissimilar; 38 (5%) formed 19 sequence pairs. Inherent limitations of this study are that some cohorts may not represent the broader regional population and that studies were heterogeneous with respect to duration of infection prior to sampling. Conclusions Most TDR strains in SSA and SSEA arose independently, suggesting that ARV regimens with a high genetic barrier to resistance combined with improved patient adherence may mitigate TDR increases by reducing the generation of new ARV-resistant strains. A small number of NNRTI-resistance mutations were responsible for most cases of high-level resistance, suggesting that inexpensive point-mutation assays to detect these mutations may be useful for pre-therapy screening in regions with high levels of TDR. In the context of a public health approach to ARV therapy, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a protease-inhibitor-containing regimen. PMID:25849352

HIV drug resistance tendencies in Latvia.

PubMed

Kolupajeva, Tatjana; Aldins, Pauls; Guseva, Ludmila; Dusacka, Diana; Sondore, Valentina; Viksna, Ludmila; Rozentale, Baiba

2008-09-01

The treatment of HIV infection in Latvia by using highly active antiretroviral therapy (HAART) was started in 1996. The prevalence and tendencies of HIV drug resistance among treated and treatment-naive patients in Latvia in the years 2006-2007 were evaluated in this study. Data of HIV genotyping, performed in 132 HIV-1 infected during years 2006-2007 by TRUGENE HIV-1 genotyping assay (BayerHealthCare-diagnostics) are included in the study. Analysis of data showed that in the group of treatment-naive individuals majority carried wild type virus. Prevalence of resistance-associated mutations (RAMs) in the treatment-naive group according to IAS list was 28%. In most cases it was NRTI mutation A62V that is associated with multinucleoside resistance caused by Q151M, its effect in the absence of Q151M is not known. By many authors A62V is supposed to be a result of polymorphism in RT gene and is excluded from the list of resistance mutations. High prevalence of A62V is typical for HIV-1 subtype A. As majority of treatment-naive cases (89%) in this study were with HIV-1 subtypes A or AE, we excluded A62V mutation and estimated RAMs prevalence in group of treatment-naive HIV-infected individuals as 7%. Minor PI mutations were not included in analyses. In Europe published rates generally very between 5% and 15%. In the group of treatment-experienced HIV infected people 25/75 were with HIV-1 subtype B, the rest part--with non-B subtypes: A/AE (35/75), CRF-01AE (7/75), B/AE (4/75) and others. In treatment-experienced patients RAMs prevalence was estimated as 58.6%. Most frequently RAMs were found for nucleoside reverse transcriptase inhibitors (NRTI) (49.3%) followed by non-nucleoside reverse transcriptase inhibitors (NNRTI) (22.6%) and protease inhibitors (PI) (16%). In the group of NRTI mutations M184V (26/75; 34.6%), A62V (12/75; 16.0%) and T215Y (8/75; 10.6%), in NNRTI mutations K103N (10/75; 13.3%), G190S (6/75; 8.0%), in PI group mutations L90M (6/75; 8.0%) and M461/L (6/75; 8.0%) occurred most frequently. The following drug susceptibility was predicted according to the Trugen expert interpretations: in 33/75 (44%) patients no evidence of resistance, in 21/75 (28%) patients resistance to 1 drug class (NRTI--16/75, NNRTI--4/75, PI--1/75), in 17 patients (22.6%) resistance to 2 drug classes (NRTI+NNRTI--9/75, NRTI+PI--7/75, NNRTI+PI--1/75) and in 3/75 (4%) patients resistance to all 3 classes of drugs (NRTI+NNRTI+PI). We conclude, that prevalence of RAMs in treatment-naive HIV infected persons in Latvia is comparable with prevalence in Europe. The origin of predominated mutation A62V associated with NRTI at present is not clear. In more than half of treated HIV infected patients HIV resistance to at least one HAART class was predicted.

Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus

PubMed Central

Wong, Danny Ka-Ho; Tsoi, Ottilia; Huang, Fung-Yu; Seto, Wai-Kay; Fung, James; Lai, Ching-Lung

2014-01-01

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modified co-amplification at lower denaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P = 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P < 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations. PMID:24951803

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Heeres, Jan; De Winter, Hans; Heyndrickx, Leo; Augustyns, Koen; Vanham, Guido

2016-05-01

The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Molecular Dynamics Study of HIV-1 RT-DNA-Nevirapine Complexes Explains NNRTI Inhibition, and Resistance by Connection Mutations

PubMed Central

Vijayan, R.S.K.; Arnold, Eddy; Das, Kalyan

2015-01-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. PMID:24174331

Pyrosequencing for detection of lamivudine-resistant hepatitis B virus.

PubMed

Lindström, Anna; Odeberg, Jacob; Albert, Jan

2004-10-01

Chronic hepatitis B virus (HBV) infection can cause severe liver disease, including cirrhosis and hepatocellular carcinoma. Lamivudine is a relatively recent alternative to alpha interferon for the treatment of HBV infection, but unfortunately, resistance to lamivudine commonly develops during monotherapy. Lamivudine-resistant HBV mutants display specific mutations in the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the viral polymerase (reverse transcriptase [rt]), which is the catalytic site of the enzyme, i.e., methionine 204 to isoleucine (rtM204I) or valine (rtM204V). The latter mutation is often accompanied by a compensatory leucine-to-methionine change at codon 180 (rtL180M). In the present study, a novel sequencing method, pyrosequencing, was applied to the detection of lamivudine resistance mutations and was compared with direct Sanger sequencing. The new pyrosequencing method had advantages in terms of throughput. Experiments with mixtures of wild-type and resistant viruses indicated that pyrosequencing can detect minor sequence variants in heterogeneous virus populations. The new pyrosequencing method was evaluated with a small number of patient samples, and the results showed that the method could be a useful tool for the detection of lamivudine resistance in the clinical setting.

Prevalence of Transmitted HIV Drug Resistance Among Recently Infected Persons in San Diego, CA 1996-2013.

PubMed

Panichsillapakit, Theppharit; Smith, Davey M; Wertheim, Joel O; Richman, Douglas D; Little, Susan J; Mehta, Sanjay R

2016-02-01

Transmitted drug resistance (TDR) remains an important concern when initiating antiretroviral therapy (ART). Here, we describe the prevalence and phylogenetic relationships of TDR among ART-naive, HIV-infected individuals in San Diego from 1996 to 2013. Data were analyzed from 496 participants of the San Diego Primary Infection Cohort who underwent genotypic resistance testing before initiating therapy. Mutations associated with drug resistance were identified according to the WHO-2009 surveillance list. Network and phylogenetic analyses of the HIV-1 pol sequences were used to evaluate the relationships of TDR within the context of the entire cohort. The overall prevalence of TDR was 13.5% (67/496), with an increasing trend over the study period (P = 0.005). TDR was predominantly toward nonnucleoside reverse transcriptase inhibitors (NNRTIs) [8.5% (42/496)], also increasing over the study period (P = 0.005). By contrast, TDR to protease inhibitors and nucleos(t)ide reverse transcriptase inhibitors were 4.4% (22/496) and 3.8% (19/496), respectively, and did not vary with time. TDR prevalence did not differ by age, gender, race/ethnicity, or risk factors. Using phylogenetic analysis, we identified 52 transmission clusters, including 8 with at least 2 individuals sharing the same mutation, accounting for 23.8% (16/67) of the individuals with TDR. Between 1996 and 2013, the prevalence of TDR significantly increased among recently infected ART-naive individuals in San Diego. Around one-fourth of TDR occurred within clusters of recently infected individuals. These findings highlight the importance of baseline resistance testing to guide selection of ART and for public health monitoring.

Chemical system biology based molecular interactions to identify inhibitors against Q151M mutant of HIV-1 reverse transcriptase.

PubMed

Pandey, Rajan Kumar; Sharma, Drista; Ojha, Rupal; Bhatt, Tarun Kumar; Prajapati, Vijay Kumar

2018-05-09

The emergence of mutations leading to drug resistance is the main cause of therapeutic failure in the human HIV infection. Chemical system biology approach has drawn great attention to discover new antiretroviral hits with high efficacy and negligible toxicity, which can be used as a prerequisite for HIV drug resistance global action plan 2017-21. To discover potential hits, we docked 49 antiretroviral analogs (n = 6294) against HIV-1 reverse transcriptase Q151M mutant & its wild-type form and narrow downed their number in three sequential modes of docking using Schrödinger suite. Later on, 80 ligands having better docking score than reference ligands (tenofovir and lamivudine) were screened for ADME, toxicity prediction, and binding energy estimation. Simultaneously, the area under the curve (AUC) was estimated using receiver operating characteristics (ROC) curve analysis to validate docking protocols. Finally, single point energy and molecular dynamics simulation approaches were performed for best two ligands (L3 and L14). This study reveals the antiretroviral efficacy of obtained two best ligands and delivers the hits against HIV-1 reverse transcriptase Q151M mutant. Copyright © 2018 Elsevier B.V. All rights reserved.

Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

NASA Astrophysics Data System (ADS)

Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

2013-03-01

The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase (RT), which is an essential enzyme for the replication of HIV. These changes allow it to retain potency against mutations that otherwise would render the enzyme resistant. Here we report that water molecules play an essential role in this binding process. Femtosecond experiments and theory expose the molecular level dynamics of rilpivirine bound to HIV-1 RT. Two nitrile substituents, one on each arm of the drug, are used as vibrational probes of the structural dynamics within the binding pocket. Two-dimensional vibrational echo spectroscopy reveals that one nitrile group is unexpectedly hydrogen-bonded to a mobile water molecule, not identified in previous X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure also reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond may help retain the potency of rilpivirine against pocket mutations despite the structural variations they cause.

Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki, E-mail: y-yasutake@aist.go.jp

The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site,more » confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.« less

High prevalence of antiretroviral drug resistance among HIV-1-untreated patients in Guinea-Conakry and in Niger.

PubMed

Charpentier, Charlotte; Bellecave, Pantxika; Cisse, Mohamed; Mamadou, Saidou; Diakite, Mandiou; Peytavin, Gilles; Tchiombiano, Stéphanie; Teisseire, Pierre; Pizarro, Louis; Storto, Alexandre; Brun-Vézinet, Françoise; Katlama, Christine; Calvez, Vincent; Marcelin, Anne-Geneviève; Masquelier, Bernard; Descamps, Diane

2011-01-01

The aim of the study was to assess the prevalence of antiretroviral drug resistance mutations in HIV-1 from recently diagnosed and untreated patients living in Conakry, Guinea-Conakry and in Niamey, Niger. The study was performed in two countries of Western Africa - Guinea-Conakry and Niger - using the same survey method in both sites. All newly HIV-1 diagnosed patients, naive of antiretroviral drugs, were consecutively included during September 2009 in each of the two sites. Protease and reverse transcriptase sequencing was performed using the ANRS procedures. Drug resistance mutations were identified according to the 2009 update surveillance drug resistance mutations. In Conakry, 99 patients were included, most of whom (89%) were infected with CRF02_AG recombinant virus. Resistance analysis among the 93 samples showed that ≥1 drug resistance mutation was observed in 8 samples, leading to a prevalence of primary resistance of 8.6% (95% CI 2.91-14.29%). In Niamey, 96 patients were included; a high diversity in HIV-1 subtypes was observed with 47 (51%) patients infected with CRF02_AG. Resistance analysis performed among the 92 samples with successful genotypic resistance test showed that ≥1 drug resistance mutation was observed in 6 samples, leading to a prevalence of primary resistance of 6.5% (95% CI 1.50-11.50%). We reported the first antiretroviral drug resistance survey studies in antiretroviral-naive patients living in Guinea-Conakry and in Niger. The prevalence of resistance was between 6% and 9% in both sites, which is higher than most of the other countries from Western Africa region.

Appearance of drug resistance mutations among the dominant HIV-1 subtype, CRF01_AE in Maumere, Indonesia.

PubMed

Indriati, Dwi Wahyu; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Witaningrum, Adiana Mutamsari; Matondang, Muhammad Qushai Yunifiar; Ueda, Shuhei; Nasronudin; Purnama, Asep; Kurniawan, Dwi; Kameoka, Masanori

2018-05-01

Human Immunodeficiency Virus (HIV) is still a major health issue in Indonesia. In recent years, the appearance of drug resistance-associated mutations has reduced the effectiveness of antiretroviral therapy (ART). We conducted genotypic studies, including the detection of drug resistance-associated mutations (from first-line regimen drugs), on HIV-1 genes derived from infected individuals in Maumere, West Nusa Tenggara. Maumere, a transit city in West Nusa Tenggara, which has a high HIV-1 transmission rate. We collected 60 peripheral blood samples from 53 ART-experienced and 7 ART-naive individuals at TC Hillers Hospital, Maumere between 2014 and 2015. The amplification and a sequencing analysis of pol genes encoding protease (the PR gene) and reverse transcriptase (the RT gene) as well as the viral env and gag genes were performed. HIV-1 subtyping and the detection of drug resistance-associated mutations were then conducted. Among 60 samples, 46 PR, 31 RT, 30 env, and 20 gag genes were successfully sequenced. The dominant HIV-1 subtype circulating in Maumere was CRF01_AE. Subtype B and recombinant viruses containing gene fragments of CRF01_AE, subtypes A, B, C, and/or G were also identified as minor populations. The major drug resistance-associated mutations, M184V, K103N, Y188L, and M230I, were found in the RT genes. However, no major drug resistance-associated mutations were detected in the PR genes. CRF01_AE was the major HIV-1 subtype prevalent in Maumere. The appearance of drug resistance-associated mutations found in the present study supports the necessity of monitoring the effectiveness of ART in Maumere. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

HIV genotype resistance testing in antiretroviral (ART) exposed Indian children--a need of the hour.

PubMed

Shah, Ira; Parikh, Shefali

2013-04-01

Development of drug resistance in HIV infected children with treatment failure is a major impediment to selection of appropriate therapy. HIV genotype resistance assays predict drug resistance on the basis of mutations in the viral genome. However, their clinical utility, especially in a resource limited setting is still a subject of debate. The authors report two cases in which both the children suffered from treatment failure of various antiretroviral therapy regimes. In both the cases, Genotype Resistance Testing (GRT) prompted a radical change from proposed failure therapy as per existing guidelines. GRT was specifically important for the selection of a new dual Nucleoside reverse transcriptase inhibitors (NRTI) component of failure regimen by identifying TAMS and M184V mutations in the HIV genome. These case reports highlight the importance of GRT in children failing multiple antiretroviral regimes; and emphasizes the need to recognize situations where GRT is absolutely essential to guide appropriate therapy, even in a resource limited setting.

Short communication. Antiretroviral drug resistance among drug-naive HIV-1-infected individuals in Djibouti (Horn of Africa).

PubMed

Maslin, Jérôme; Rogier, Christophe; Caron, Melanie; Grandadam, Marc; Koeck, Jean-Louis; Nicand, Elisabeth

2005-01-01

To survey the frequency of genotypic antiretroviral resistance in drug-naive HIV-1-infected Djiboutians. A national study was conducted in the general population of Djibouti in March 2002 to determine HIV-1 seroprevalence. Blood samples were collected anonymously and plasma samples scoring positive for HIV-1 antibodies were tested for viral load. Genotypic studies were performed with viral RNA from plasma using the consensus technique of the Agence Nationale de Recherche sur le SIDA (www.hivfrenchresistance.org). Mutations were identified using the International AIDS Society-USA resistance panel and resistant virus was defined according to the ANRS algorithm. A panel of 2423 individuals representing the general population of Djibouti was included. Antibodies were detected in 53 of 2423 samples tested. The HIV-1 seroprevalence in the general population was 2.2%. Genotype C was the most prevalent, and the other isolates were CRF_02 AG, or subtype A or D. Forty-seven of the 53 samples were tested for genotypic resistance, and mutations concerning all three classes of antiretrovirals were found. The most frequent were secondary mutations associated with protease inhibitors (PIs): M36I, R41K and K20I/R. A few strains displayed primary mutations (the non-nucleoside reverse transcriptase inhibitor [NNRTI]-associated mutations K101E, K103T, L100I and G190V; the PI-associated mutation N88D; and the NRTI-associated mutation K65R). The presence of these mutations may be due to the transmission of strains from treated patients. Substantial polymorphism and a few primary mutations are found in HIV-1 non-B subtype isolates from Djiboutian antiretroviral-drug-naive individuals. This needs to be taken into account to adapt antiretroviral regimens and prophylactic schedules locally.

Prevalence and predictors of antiretroviral drug resistance in newly diagnosed HIV-1 infection.

PubMed

Booth, Clare L; Garcia-Diaz, Ana M; Youle, Michael S; Johnson, Margaret A; Phillips, Andrew; Geretti, Anna Maria

2007-03-01

To determine prevalence and predictors of antiretroviral drug resistance in newly diagnosed individuals with HIV-1 infection, using a systematic approach to avoid selection bias. Plasma samples from all persons diagnosed HIV-1 seropositive at a large London centre between April 2004 and February 2006 underwent sequencing of HIV-1 reverse transcriptase (RT) and protease genes. Subtype was assigned by phylogenetic analysis. Resistance was scored according to the IAS-USA list (2005) modified to include T215revertants and exclude isolated E44D or V118I and minor protease mutations. Recent seroconversion was identified by HIV antibody avidity testing. The cohort of 239 included 169 (70.7%) males, 126 (52.7%) homosexuals, 118 (49.5%) persons of white ethnicity and 144 (60.0%) persons born outside the UK. Subtypes included B 134 (56.1%), C 46 (19.2%), A 17 (7.1%), other non-B 42 (17.6%). The prevalence of resistance mutations was 17/239 (7.1%; 95% confidence interval 4.5-11.1%), comprising 10/239 (4.2%) nucleoside/nucleotide RT inhibitor (NRTI); 4/239 (1.7%) non-nucleoside RT inhibitor (NNRTI) and 4/239 (1.7%) protease inhibitor (PI) associated mutations. Dual-class (NRTI + PI) resistance mutations were detected in 1/239 (0.4%) person. The prevalence of resistance mutations was 7/85 (8.2%) and 10/154 (6.5%) in persons with recent and established infection, respectively. In multivariate analysis, having been born in the UK and high CD4 count, but not gender, age, risk group, ethnicity or subtype, were independent predictors of resistance. In an unselected UK cohort, subtypes other than B accounted for 43.9% of new HIV-1 diagnoses. The prevalence of resistance mutations was 7.1% and highest in those born in the UK.

Transmitted drug resistance and type of infection in newly diagnosed HIV-1 individuals in Honduras.

PubMed

Murillo, Wendy; Paz-Bailey, Gabriela; Morales, Sonia; Monterroso, Edgar; Paredes, Mayte; Dobbs, Trudy; Parekh, Bharat S; Albert, Jan; Rivera, Ivette Lorenzana de

2010-12-01

Transmitted drug resistance (TDR) reduces the efficacy of antiretroviral treatment and is a public health concern. To gain insight in the epidemiology of TDR in Honduras by evaluating the amount of TDR in a representative sample of newly diagnosed individuals and by determining whether these are recent or established infections. Two hundred treatment-naïve, newly diagnosed HIV-positive individuals representing different population groups (general population, Garifunas ethnic group, female sex workers and men who have sex with men) and different geographic regions were enrolled during April 2004-April 2007. The HIV-1 pol gene was sequenced to identify drug-resistant mutations and TDR was scored as recommended by the WHO. Specimens were classified as recent or established infections using the BED assay. Among 200 samples analyzed from Honduran patients the prevalence of TDR was 7% (95% CI: 3.9-11.5%), 5% for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3% for nucleoside reverse transcriptase inhibitors (NRTIs) and 0.5% for protease inhibitors (PIs). Testing of these samples with the BED assay revealed that 12% of the specimens were associated with recent infections. TDR was significantly more common in specimens with recent infection (21%) than established infection (5%) (p=0.016). The prevalence of TDR in Honduras was moderate (7%). The percentage of specimens who were recently infected was low (12%), suggesting that late HIV diagnosis is common. The TDR prevalence was higher in recent than in established infections, which may indicate that TDR is increasing over time. The higher prevalence of NNRTI and NRTI mutations as compared to PI mutations is probably due to a broader and longer use of these drugs in Honduras. Copyright © 2010 Elsevier B.V. All rights reserved.

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Breast-milk shedding of drug-resistant HIV-1 subtype C in women exposed to single-dose nevirapine.

PubMed

Lee, Esther J; Kantor, Rami; Zijenah, Lynn; Sheldon, Wayne; Emel, Lynda; Mateta, Patrick; Johnston, Elizabeth; Wells, Jennifer; Shetty, Avinash K; Coovadia, Hoosen; Maldonado, Yvonne; Jones, Samuel Adeniyi; Mofenson, Lynne M; Contag, Christopher H; Bassett, Mary; Katzenstein, David A

2005-10-01

Single-dose nevirapine reduces intrapartum human immunodeficiency virus 1 type (HIV-1) transmission but may also select for nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance in breast milk (BM) and plasma. Among 32 Zimbabwean women, median 8-week postpartum plasma and BM HIV-1 RNA levels were 4.57 and 2.13 log(10) copies/mL, respectively. BM samples from women with laboratory-diagnosed mastitis (defined as elevated BM Na(+) levels) were 5.4-fold more likely to have HIV-1 RNA levels above the median. BM RT sequences were not obtained for 12 women with BM HIV-1 RNA levels below the lower limit of detection of the assay used. In 20 paired BM and plasma samples, 65% of BM and 50% of plasma RT sequences had NNRTI-resistance mutations, with divergent mutation patterns.

Molecular dynamics study of HIV-1 RT-DNA-nevirapine complexes explains NNRTI inhibition and resistance by connection mutations.

PubMed

Vijayan, R S K; Arnold, Eddy; Das, Kalyan

2014-05-01

HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of several highly active antiretroviral therapy regimens. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns molecular dynamics (MD) simulations, followed by essential dynamics, free-energy landscape analyses, and network analyses of RT-DNA, RT-DNA-nevirapine (NVP), and N348I/T369I mutant RT-DNA-NVP complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon NVP binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-NVP complex suggesting enhanced rigidity of RT upon NVP binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations. Copyright © 2013 Wiley Periodicals, Inc.

HIV-1 drug-resistance surveillance among treatment-experienced and -naïve patients after the implementation of antiretroviral therapy in Ghana.

PubMed

Nii-Trebi, Nicholas I; Ibe, Shiro; Barnor, Jacob S; Ishikawa, Koichi; Brandful, James A M; Ofori, Sampson B; Yamaoka, Shoji; Ampofo, William K; Sugiura, Wataru

2013-01-01

Limited HIV-1 drug-resistance surveillance has been carried out in Ghana since the implementation of antiretroviral therapy (ART). This study sought to provide data on the profile of HIV-1 drug resistance in ART-experienced and newly diagnosed individuals in Ghana. Samples were collected from 101 HIV-1-infected patients (32 ART-experienced cases with virological failure and 69 newly diagnosed ART-naïve cases, including 11 children), in Koforidua, Eastern region of Ghana, from February 2009 to January 2010. The pol gene sequences were analyzed by in-house HIV-1 drug-resistance testing. The most prevalent HIV-1 subtype was CRF02_AG (66.3%, 67/101) followed by unique recombinant forms (25.7%, 26/101). Among 31 ART-experienced adults, 22 (71.0%) possessed at least one drug-resistance mutation, and 14 (45.2%) had two-class-resistance to nucleoside and non-nucleoside reverse-transcriptase inhibitors used in their first ART regimen. Importantly, the number of accumulated mutations clearly correlated with the duration of ART. The most prevalent mutation was lamivudine-resistance M184V (n = 12, 38.7%) followed by efavirenz/nevirapine-resistance K103N (n = 9, 29.0%), and zidovudine/stavudine-resistance T215Y/F (n = 6, 19.4%). Within the viral protease, the major nelfinavir-resistance mutation L90M was found in one case. No transmitted HIV-1 drug-resistance mutation was found in 59 ART-naïve adults, but K103N and G190S mutations were observed in one ART-naïve child. Despite expanding accessibility to ART in Eastern Ghana, the prevalence of transmitted HIV-1 drug resistance presently appears to be low. As ART provision with limited options is scaled up nationwide in Ghana, careful monitoring of transmitted HIV-1 drug resistance is necessary.

Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa.

PubMed

Penrose, Kerri J; Wallis, Carole L; Brumme, Chanson J; Hamanishi, Kristen A; Gordon, Kelley C; Viana, Raquel V; Harrigan, P Richard; Mellors, John W; Parikh, Urvi M

2017-02-01

A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1 LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC 50 ) from 12 recombinant subtype C HIV-1 LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC 50 s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC 50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring. Copyright © 2017 American Society for Microbiology.

Frequent Cross-Resistance to Dapivirine in HIV-1 Subtype C-Infected Individuals after First-Line Antiretroviral Therapy Failure in South Africa

PubMed Central

Penrose, Kerri J.; Wallis, Carole L.; Brumme, Chanson J.; Hamanishi, Kristen A.; Gordon, Kelley C.; Viana, Raquel V.; Harrigan, P. Richard; Mellors, John W.

2016-01-01

ABSTRACT A vaginal ring containing dapivirine (DPV) has shown moderate protective efficacy against HIV-1 acquisition, but the activity of DPV against efavirenz (EFV)- and nevirapine (NVP)-resistant viruses that could be transmitted is not well defined. We investigated DPV cross-resistance of subtype C HIV-1 from individuals on failing NVP- or EFV-containing antiretroviral therapy (ART) in South Africa. Plasma samples were obtained from individuals with >10,000 copies of HIV RNA/ml and with HIV-1 containing at least one non-nucleoside reverse transcriptase (NNRTI) mutation. Susceptibility to NVP, EFV, and DPV in TZM-bl cells was determined for recombinant HIV-1LAI containing bulk-amplified, plasma-derived, full-length reverse transcriptase sequences. Fold change (FC) values were calculated compared with a composite 50% inhibitory concentration (IC50) from 12 recombinant subtype C HIV-1LAI plasma-derived viruses from treatment-naive individuals in South Africa. A total of 25/100 (25%) samples showed >500-FCs to DPV compared to treatment-naive samples with IC50s exceeding the maximum DPV concentration tested (132 ng/ml). A total of 66/100 (66%) samples displayed 3- to 306-FCs, with a median IC50 of 17.6 ng/ml. Only 9/100 (9%) samples were susceptible to DPV (FC < 3). Mutations L100I and K103N were significantly more frequent in samples with >500-fold resistance to DPV compared to samples with a ≤500-fold resistance. A total of 91% of samples with NNRTI-resistant HIV-1 from individuals on failing first-line ART in South Africa exhibited ≥3-fold cross-resistance to DPV. This level of resistance exceeds expected plasma concentrations, but very high genital tract DPV concentrations from DPV ring use could block viral replication. It is critically important to assess the frequency of transmitted and selected DPV resistance in individuals using the DPV ring. PMID:27895013

Selection Pressure in CD8+ T-cell Epitopes in the pol Gene of HIV-1 Infected Individuals in Colombia. A Bioinformatic Approach

PubMed Central

Acevedo-Sáenz, Liliana; Ochoa, Rodrigo; Rugeles, Maria Teresa; Olaya-García, Patricia; Velilla-Hernández, Paula Andrea; Diaz, Francisco J.

2015-01-01

One of the main characteristics of the human immunodeficiency virus is its genetic variability and rapid adaptation to changing environmental conditions. This variability, resulting from the lack of proofreading activity of the viral reverse transcriptase, generates mutations that could be fixed either by random genetic drift or by positive selection. Among the forces driving positive selection are antiretroviral therapy and CD8+ T-cells, the most important immune mechanism involved in viral control. Here, we describe mutations induced by these selective forces acting on the pol gene of HIV in a group of infected individuals. We used Maximum Likelihood analyses of the ratio of non-synonymous to synonymous mutations per site (dN/dS) to study the extent of positive selection in the protease and the reverse transcriptase, using 614 viral sequences from Colombian patients. We also performed computational approaches, docking and algorithmic analyses, to assess whether the positively selected mutations affected binding to the HLA molecules. We found 19 positively-selected codons in drug resistance-associated sites and 22 located within CD8+ T-cell epitopes. A high percentage of mutations in these epitopes has not been previously reported. According to the docking analyses only one of those mutations affected HLA binding. However, algorithmic methods predicted a decrease in the affinity for the HLA molecule in seven mutated peptides. The bioinformatics strategies described here are useful to identify putative positively selected mutations associated with immune escape but should be complemented with an experimental approach to define the impact of these mutations on the functional profile of the CD8+ T-cells. PMID:25803098

The evolution of HIV-1 group M genetic variability in Southern Cameroon is characterized by several emerging recombinant forms of CRF02_AG and viruses with drug resistance mutations.

PubMed

Agyingi, Lucy; Mayr, Luzia M; Kinge, Thompson; Orock, George Enow; Ngai, Johnson; Asaah, Bladine; Mpoame, Mbida; Hewlett, Indira; Nyambi, Phillipe

2014-03-01

The HIV epidemic in Cameroon is marked by a broad genetic diversity dominated by circulating recombinant forms (CRFs). Studies performed more than a decade ago in urban settings of Southern Cameroon revealed a dominance of the CRF02_AG and clade A variants in >90% of the infected subjects; however, little is known about the evolving viral variants circulating in this region. To document circulating HIV viral diversity, four regions of the viral genome (gag, PR, reverse transcriptase, env) in 116 HIV-1 positive individuals in Limbe, Southern Cameroon, were PCR-amplified. Sequences obtained at the RT and protease regions were analyzed for mutations that conferred drug resistance using the Stanford Drug Resistance Database. The present study reveals a broad genetic diversity characterized by several unique recombinant forms (URF) accounting for 36% of infections, 48.6% of patients infected with CRF02_AG, and the emergence of CRF22_01A1 in 7.2% of patients. Three out of 15 (20%) treated patients and 13 out of 93 (13.9%) drug naïve patients harbor drug resistance mutations to RT inhibitors, while 3.2% of drug naïve patients harbor drug resistance mutations associated with protease inhibitors. The high proportion (13.9%) of drug resistance mutations among the drug naïve patients reveals the ongoing transmission of these viruses in this region of Cameroon and highlights the need for drug resistance testing before starting treatment for patients infected with HIV-1. © 2013 Wiley Periodicals, Inc.

Polymorphisms of HIV RT Gene Among the ART Naïve Native Drug Exposed Rural PLHA.

PubMed

Krishnan, K Mohana; Amsavathani, Sk

2012-04-01

The number of people living with human immunodeficiency virus (HIV) is increasing day by day in India. The disease has now spread from urban areas to rural areas. The proof reading of the reverse transcriptase enzyme is poor, which may lead to genetic diversity within the HIV strains, which in turn leads to problems like failure or resistance in antiretroviral treatment. This study is designed to find out the polymorphisms of the reverse transcriptase gene of HIV, after the native drug pressure among antiretroviral therapy (ART) naïve rural people living with HIV/AIDS (RPLHA). A total of 207 HIV-Reactive patients were allowed to take native drugs from the local area and were advised to attend the center for HIV after six months for a follow-up. At the time of the follow-up visit, a second blood sample was taken from 20 reactive native-drug exposed ART-naïve patients. The plasma was separated and transported at 20°C to the YRG Care Center for genotyping. Among the 20 HIV-reactive samples processed for gene sequencing analysis to detect the genotypic variations, only one sample (5%) showed high-level mutational resistance variations and the predominant polymorphisms detected were V35T (100%), K122E (94.44%), and V60I (88.88%). The presence of drug-resistance mutations, although minimal, was important, as the drug-resistant strains could spread among the RPLHA and to their sexual partners. There was a definite need to generate a drug resistance database and the polymorphic pattern of Indian strains concern to the future clinical management of the disease, and a vaccine design to contain the disease.

Transmitted drug resistance is still low in newly diagnosed human immunodeficiency virus type 1 CRF06_cpx-infected patients in Estonia in 2010.

PubMed

Avi, Radko; Huik, Kristi; Pauskar, Merit; Ustina, Valentina; Karki, Tõnis; Kallas, Eveli; Jõgeda, Ene-Ly; Krispin, Tõnu; Lutsar, Irja

2014-03-01

The presence of transmitted drug resistance (TDR) in treatment-naive HIV-1-positive subjects is of concern, especially in the countries of the former Soviet Union in which the number of subjects exposed to antiretrovirals (ARV) has exponentially increased during the past decade. We assessed the rate of TDR among newly diagnosed subjects in Estonia in 2010 and compared it to that in 2008. The study included 325 subjects (87% of all subjects tested HIV positive from January 1 to December 31, 2010). Of the 244 sequenced viral genomic RNA in the reverse transcriptase (RT) region 214 were CRF06_cpx, nine were subtype A1, three (one each) were subtype B and subtype C, CRF02_AG, and CRF03_AB; 15 viruses remained unclassified as putative recombinant forms between CRF06_cpx and subtype A1. HIV-1 TDR mutations in 2010 and 2008 (n=145) occurred at similar frequency in 4.5% (95% CI 2.45; 7.98) and 5.5% (95% CI 1.8; 9.24) of the patients, respectively. In 2010, 2.5% (6/244) of the sequences harbored nonnucleoside reverse transcriptase inhibitor (NNRTI) (K103N and K101E), 1.6% (4/244) nucleoside reverse transcriptase inhibitor (NRTI) (M41L, M184I, and K219E), and 0.4% (1/244) protease inhibitor (PI) (V82A) mutations. Our findings indicate that in spite of the increased consumption of ARVs the rate of TDR in Estonia has remained unchanged over the past 3 years. Similar stabilizing or even decreasing trends have been described in Western Europe and North America albeit at higher levels and in different socioeconomic backgrounds.

Transmitted HIV Drug Resistance Is High and Longstanding in Metropolitan Washington, DC

PubMed Central

Kassaye, Seble G.; Grossman, Zehava; Balamane, Maya; Johnston-White, Betsy; Liu, Chenglong; Kumar, Princy; Young, Mary; Sneller, Michael C.; Sereti, Irini; Dewar, Robin; Rehm, Catherine; Meyer, William; Shafer, Robert; Katzenstein, David; Maldarelli, Frank

2016-01-01

Background. Washington, DC, has 2.5% human immunodeficiency virus (HIV) prevalence, 3.9% among African Americans. Antiretrovirals (ARTs) are the cornerstone for treatment and prevention. Monitoring changes in transmitted drug resistance (TDR) is critical for effective HIV care. Methods. HIV genotype data for individuals enrolled in research studies in metropolitan Washington, D.C., were used to identify TDR using the World Health Organization mutation list [Bennett DE, Camacho RJ, Otelea D, et al. Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PloS One 2009; 4:e4724]. HIV phylogenies were reconstructed using maximum likelihood and Bayesian methods. HIV transmission clusters were supported by 1000 bootstrap values >0.70 and posterior probability >0.95 of having a common ancestor. Results. Among 710 individuals enrolled in 1994–2013, the median age was 38.6 years, 46.2% were female, and 53.3% were African-American. TDR was 22.5% among 566 treatment-naive individuals; 15.8% had nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) resistance, 9.8% had nonnucleoside reverse-transcriptase inhibitor (NNRTI) resistance, and 4.2% had protease inhibitor (PI) resistance. Single class TDR was 10.0%, 5.1%, and 1.6% to NRTIs, NNRTIs, and PIs. Dual TDR to PI and NRTI was seen in 1.6%, NRTI and NNRTI in 3.4%, and triple class TDR in 0.9%. TDR frequency decreased from 1994–2006 (27.1%) to 2007–2013 (19.4%; P = .02). Only 6/79 (7.6%) individuals within transmission clusters had evidence of TDR. Discussions. We identified high prevalence of TDR among HIV-infected individuals in metropolitan Washington, DC, regardless of gender. Active surveillance for TDR is needed to guide ART usage and analyses of risk group contributions to HIV transmission and resistance. PMID:27307507

HIV-1 Drug Resistance and Second-Line Treatment in Children Randomized to Switch at Low Versus Higher RNA Thresholds.

PubMed

Harrison, Linda; Melvin, Ann; Fiscus, Susan; Saidi, Yacine; Nastouli, Eleni; Harper, Lynda; Compagnucci, Alexandra; Babiker, Abdel; McKinney, Ross; Gibb, Diana; Tudor-Williams, Gareth

2015-09-01

The PENPACT-1 trial compared virologic thresholds to determine when to switch to second-line antiretroviral therapy (ART). Using PENPACT-1 data, we aimed to describe HIV-1 drug resistance accumulation on first-line ART by virologic threshold. PENPACT-1 had a 2 × 2 factorial design, randomizing HIV-infected children to start protease inhibitor (PI) versus nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART, and switch at a 1000 copies/mL versus 30,000 copies/mL threshold. Switch criteria were not achieving the threshold by week 24, confirmed rebound above the threshold thereafter, or Center for Disease Control and Prevention stage C event. Resistance tests were performed on samples ≥1000 copies/mL before switch, resuppression, and at 4-years/trial end. Sixty-seven children started PI-based ART and were randomized to switch at 1000 copies/mL (PI-1000), 64 PIs and 30,000 copies/mL (PI-30,000), 67 NNRTIs and 1000 copies/mL (NNRTI-1000), and 65 NNRTI and 30,000 copies/mL (NNRTI-30,000). Ninety-four (36%) children reached the 1000 copies/mL switch criteria during 5-year follow-up. In 30,000 copies/mL threshold arms, median time from 1000 to 30,000 copies/mL switch criteria was 58 (PI) versus 80 (NNRTI) weeks (P = 0.81). In NNRTI-30,000, more nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations accumulated than other groups. NNRTI mutations were selected before switching at 1000 copies/mL (23% NNRTI-1000, 27% NNRTI-30,000). Sixty-two children started abacavir + lamivudine, 166 lamivudine + zidovudine or stavudine, and 35 other NRTIs. The abacavir + lamivudine group acquired fewest NRTI mutations. Of 60 switched to second-line, 79% PI-1000, 63% PI-30,000, 64% NNRTI-1000, and 100% NNRTI-30,000 were <400 copies/mL 24 weeks later. Children on first-line NNRTI-based ART who were randomized to switch at a higher virologic threshold developed the most resistance, yet resuppressed on second-line. An abacavir + lamivudine NRTI combination seemed protective against development of NRTI resistance.

Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study.

PubMed

Porter, Danielle P; Toma, Jonathan; Tan, Yuping; Solberg, Owen; Cai, Suqin; Kulkarni, Rima; Andreatta, Kristen; Lie, Yolanda; Chuck, Susan K; Palella, Frank; Miller, Michael D; White, Kirsten L

2016-02-01

Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance data are unavailable.

[Prevalence of transmission of zidovudine-resistant viruses in Switzerland. l'Etude suisse de cohorte VIH].

PubMed

Yerly, S; Rakik, A; Kinloch-de-Loes, S; Erb, P; Vernazza, P; Hirschel, B; Perrin, L

1996-10-26

Zidovudine (ZDV) was the most widely used anti-HIV drug between 1987 and 1995, and, as already reported, transmission of ZDV-resistant viruses occurs. Several mutations of the reverse transcriptase gene have been identified; one of them affects the 215 codon and is associated with a high degree of resistance. We have determined, using selective PCR, the prevalence of transmission of 215 mutant isolates in 134 patients with primary HIV infection (PHI) and have identified 8 patients with 215 mutant virus between 1989 and 1995 in Switzerland. Mutant resistant viruses have been isolated from patients treated with most antiviral drugs. A systematic search for mutant viruses may provide useful information for the adaptation of treatment strategies.

Low Prevalence of Antiretroviral Resistance Among HIV Type 1-Positive Prisoners in the Southeast United States

PubMed Central

Rosen, David; Wohl, David A.; Kiziah, Nichole; Sebastian, Joseph; Eron, Joseph J.; White, Becky

2013-01-01

Abstract Drug-resistant HIV complicates management of HIV infection. Although an estimated 14% of all HIV-positive persons pass through a prison or jail in the United States each year, little is known about the overall prevalence of antiretroviral (ARV) resistance in incarcerated persons. All genotypic sequence data on HIV-positive prisoners in the North Carolina (NC) Department of Corrections (DOC) were obtained from LabCorp. Screening for major resistance mutations in protease (PI) and reverse transcriptase (NRTI and NNRTI) was done using Genosure and the Stanford HIV Database. For subjects with multiple genotype reports, each mutation was counted only once and considered present on all subsequent genotypes. Between October 2006 and February 2010, the NC DOC incarcerated 1,911 HIV+ individuals of whom 19.2% (n=367) had at least one genotype performed. The overall prevalence of a major resistance mutation was 28.3% (95% CI 23.7, 33.0). Among prisoners ever exposed to an ARV during incarceration (n=329) prevalence of a major resistance mutation was 29.8% (95% CI 24.9, 34.7); resistance by class was 20.4% (95% CI 16.0, 24.7) for NRTIs, 19.8% (95% CI 15.5, 24.1) for NNRTIs, and 8.8% (95% CI 5.8,11.9) for PIs. Single class drug resistance was most prevalent at 14.2% (10.2,17.7) followed by dual 12.5% (I8.9,16.0) and triple class 3.3% (1.4,5.3) resistance. The three most prevalent mutations were K103N 15.8% (12.0, 20.2), M184V 14.3% (10.7,18.5), and M41L 4.9% (2.8,7.8). In the NC DOC ARV resistance prevalence, dual and triple class drug resistance was moderate over the study period. Resistance to PIs was lower than NNRTIs and NRTIs, likely reflecting higher usage of these two classes or a lower barrier to resistance. PMID:22966822

Epidemiological Surveillance of HIV-1 Transmitted Drug Resistance in Spain in 2004-2012: Relevance of Transmission Clusters in the Propagation of Resistance Mutations.

PubMed

Vega, Yolanda; Delgado, Elena; Fernández-García, Aurora; Cuevas, Maria Teresa; Thomson, Michael M; Montero, Vanessa; Sánchez, Monica; Sánchez, Ana Maria; Pérez-Álvarez, Lucia

2015-01-01

Our objectives were to carry out an epidemiological surveillance study on transmitted drug resistance (TDR) among individuals newly diagnosed of HIV-1 infection during a nine year period in Spain and to assess the role of transmission clusters (TC) in the propagation of resistant strains. An overall of 1614 newly diagnosed individuals were included in the study from January 2004 through December 2012. Individuals come from two different Spanish regions: Galicia and the Basque Country. Resistance mutations to reverse transcriptase inhibitors (RTI) and protease inhibitors (PI) were analyzed according to mutations included in the surveillance drug-resistance mutations list updated in 2009. TC were defined as those comprising viruses from five or more individuals whose sequences clustered in maximum likelihood phylogenetic trees with a bootstrap value ≥90%. The overall prevalence of TDR to any drug was 9.9%: 4.9% to nucleoside RTIs (NRTIs), 3.6% to non-nucleoside RTIs (NNRTIs), and 2.7% to PIs. A significant decrease of TDR to NRTIs over time was observed [from 10% in 2004 to 2% in 2012 (p=0.01)]. Sixty eight (42.2%) of 161 sequences with TDR were included in 25 TC composed of 5 or more individuals. Of them, 9 clusters harbored TDR associated with high level resistance to antiretroviral drugs. T215D revertant mutation was transmitted in a large cluster comprising 25 individuals. The impact of epidemiological networks on TDR frequency may explain its persistence in newly diagnosed individuals. The knowledge of the populations involved in TC would facilitate the design of prevention programs and public health interventions.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

PubMed

Mulu, Andargachew; Maier, Melanie; Liebert, Uwe Gerd

2015-12-01

Although biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs. Plasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database ( http://hivdb.stanford.edu ) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates. All subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions. Although the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors

PubMed Central

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-01-01

Motivation Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. Results We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. Availability A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm. PMID:21234299

Computational Analysis of Molecular Interaction Networks Underlying Change of HIV-1 Resistance to Selected Reverse Transcriptase Inhibitors.

PubMed

Kierczak, Marcin; Dramiński, Michał; Koronacki, Jacek; Komorowski, Jan

2010-12-12

Despite more than two decades of research, HIV resistance to drugs remains a serious obstacle in developing efficient AIDS treatments. Several computational methods have been developed to predict resistance level from the sequence of viral proteins such as reverse transcriptase (RT) or protease. These methods, while powerful and accurate, give very little insight into the molecular interactions that underly acquisition of drug resistance/hypersusceptibility. Here, we attempt at filling this gap by using our Monte Carlo feature selection and interdependency discovery method (MCFS-ID) to elucidate molecular interaction networks that characterize viral strains with altered drug resistance levels. We analyzed a number of HIV-1 RT sequences annotated with drug resistance level using the MCFS-ID method. This let us expound interdependency networks that characterize change of drug resistance to six selected RT inhibitors: Abacavir, Lamivudine, Stavudine, Zidovudine, Tenofovir and Nevirapine. The networks consider interdependencies at the level of physicochemical properties of mutating amino acids, eg,: polarity. We mapped each network on the 3D structure of RT in attempt to understand the molecular meaning of interacting pairs. The discovered interactions describe several known drug resistance mechanisms and, importantly, some previously unidentified ones. Our approach can be easily applied to a whole range of problems from the domain of protein engineering. A portable Java implementation of our MCFS-ID method is freely available for academic users and can be obtained at: http://www.ipipan.eu/staff/m.draminski/software.htm.

[Genetic analysis of the mutations in HIV-1 infected population in Ecuador].

PubMed

González-González, Manuel; Correa-Sierra, Consuelo; Hermida-Álava, Katherine; Machado-Díaz, Ana; Gómez-Andrade, L Fernando; Castillo-Segovia, Martha; Pérez-Santos, C Lissette; Kourí-Cardellá, Vivian

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

Reverse Transcriptase Inhibitors as Potential Colorectal Microbicides▿ †

PubMed Central

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J.

2009-01-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides. PMID:19258271

Reverse transcriptase inhibitors as potential colorectal microbicides.

PubMed

Herrera, Carolina; Cranage, Martin; McGowan, Ian; Anton, Peter; Shattock, Robin J

2009-05-01

We investigated whether reverse transcriptase (RT) inhibitors (RTI) can be combined to inhibit human immunodeficiency virus type 1 (HIV-1) infection of colorectal tissue ex vivo as part of a strategy to develop an effective rectal microbicide. The nucleotide RTI (NRTI) PMPA (tenofovir) and two nonnucleoside RTI (NNRTI), UC-781 and TMC120 (dapivirine), were evaluated. Each compound inhibited the replication of the HIV isolates tested in TZM-bl cells, peripheral blood mononuclear cells, and colorectal explants. Dual combinations of the three compounds, either NRTI-NNRTI or NNRTI-NNRTI combinations, were more active than any of the individual compounds in both cellular and tissue models. Combinations were key to inhibiting infection by NRTI- and NNRTI-resistant isolates in all models tested. Moreover, we found that the replication capacities of HIV-1 isolates in colorectal explants were affected by single point mutations in RT that confer resistance to RTI. These data demonstrate that colorectal explants can be used to screen compounds for potential efficacy as part of a combination microbicide and to determine the mucosal fitness of RTI-resistant isolates. These findings may have important implications for the rational design of effective rectal microbicides.

Recent advances in the development of next generation non-nucleoside reverse transcriptase inhibitors.

PubMed

Tarby, Christine M

2004-01-01

Since their discovery, non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become one of the cornerstones of highly active anti-retroviral therapy (HAART). Currently, three NNRTI agents, efavirenz, nevirapine and delavirdine are commercially available. Efavirenz and nevirapine, used in combination with nucleoside reverse transcriptase inhibitors (NRTIs), provide durable regimens with efficacy comparable to protease inhibitor (PI) containing therapies. When virological failure occurs following treatment with an NNRTI, the resistance mutations can confer reduced sensitivity to the entire agent class. Therefore, the strategy for the development of next generation NNRTIs has been to focus on compounds which have improved potencies against the clinically relevant viral mutants. Agents with improved virological profiles and which maintain the ease of administration and favorable safety profiles of the current agents should find use in anti-retroviral naïve patients as well as in components of salvage regimens in the anti-retroviral experienced patient. This review summarizes the recent developments with compounds in clinical trials as of January 2002 as well as to summarize information on new agents appearing in the primary and patent literature between January 2001 and December 2002.

Broad advances in understanding HIV resistance to antiretrovirals: report on the XVII International HIV Drug Resistance Workshop.

PubMed

Mascolini, Mark; Larder, Brendan A; Boucher, Charles A B; Richman, Douglas D; Mellors, John W

2008-01-01

The 2008 International HIV Drug Resistance Workshop explored six topics on viral resistance: new antiretrovirals; clinical implications; epidemiology; new technologies and interpretations; HIV pathogenesis, fitness, and resistance; and mechanisms of resistance. The last of these topics provided a forum for new work on resistance of hepatitis B and C viruses, which were also explored in two poster sessions. Much work focused on resistance to the two most recent antiretroviral classes (integrase inhibitors and CCR5 antagonists), a new set of entry inhibitor candidates and one new class represented by the maturation inhibitor bevirimat. Other research explored two novel non-nucleoside reverse transcriptase inhibitors, etravirine and IDX899. Epidemiological work analysed rates of transmitted resistant virus, multiclass resistance in antiretroviral-experienced patients and a heightened resistance risk in injecting drug users regardless of adherence. New research on resistance technologies involved an enhanced assay for HIV-1 coreceptor determination and improved gene-based tools for predicting coreceptor use. In the pathogenesis arena, a small study of intensification shed light on the likely source of residual viraemia in patients on successful antiretroviral therapy. A large study in Mozambique correlated the timing of infant infection with selection, transmission and persistence of nevirapine resistance mutations. Mechanistic research explored resistance to the integrase inhibitor raltegravir, K65R-mediated resistance to tenofovir and the role of connection domain mutations in resistance to zidovudine.

Prevalence of HIV Antiretroviral Drug Resistance and Its Impacts on HIV-1 Virological Failures in Jiangsu, China: A Cross-Sectional Study.

PubMed

Zhou, Ying; Lu, Jing; Wang, Jinge; Yan, Hongjing; Li, Jianjun; Xu, Xiaoqin; Zhang, Zhi; Qiu, Tao; Ding, Ping; Fu, Gengfeng; Huan, Xiping; Hu, Haiyang

2016-01-01

Antiretroviral therapy (ART) has been shown to improve survival of patients with Human Immunodeficiency Virus (HIV) infection and to reduce HIV-1 transmission. Therefore, the Chinese central government initiated a national program to provide ART free of charge to HIV-1 patients. We conducted a cross-sectional survey in Jiangsu province to determine the level of drug resistance (DR) in HIV-1 infected patients and the correlates of DR in virological failures in 2012. Approximately 10.4% of the HIV-1 patients in the study experienced virological failure after one year of ART and were divided into drug sensitive and drug resistant groups based on genotype determination. The viral loads (VLs) in the drug resistant group were significantly lower than the drug sensitive group. There were two independent predictors of virological failure: male gender and increasing duration of treatment. The primary mutations observed in the study were against nucleoside reverse transcriptase inhibitors (NRTIs) which were M184V (79.45%) and K103N (33.70%) in nonnucleoside reverse transcriptase inhibitors (NNRTIs). The overall rate of DR in Jiangsu province is still relatively low among treated patients. However, close monitoring of drug resistance in male patients in the early stages of treatment is vital to maintaining and increasing the benefits of HIV ART achieved to date.

Connection Domain Mutations in HIV-1 Reverse Transcriptase Do Not Impact Etravirine Susceptibility and Virologic Responses to Etravirine-Containing Regimens▿†

PubMed Central

Gupta, Soumi; Vingerhoets, Johan; Fransen, Signe; Tambuyzer, Lotke; Azijn, Hilde; Frantzell, Arne; Paredes, Roger; Coakley, Eoin; Nijs, Steven; Clotet, Bonaventura; Petropoulos, Christos J.; Schapiro, Jonathan; Huang, Wei; Picchio, Gaston

2011-01-01

Connection domain mutations (CDMs) in HIV-1 reverse transcriptase (RT) alter susceptibility to some nucleoside/nonnucleoside RT inhibitors (NRTIs/NNRTIs). Their effects on susceptibility and virologic responses to etravirine were analyzed. Seventeen CDMs were evaluated: L283I, E312Q, G333D, G333E, G335C, G335D, N348I, A360I, A360T, A360V, V365I, T369I, A371V, A376S, I393L, E399D, and E399G. CDM prevalence and effects on virologic responses were analyzed retrospectively using clinical data. The effects on etravirine susceptibility were assessed in clinical samples and confirmed using site-directed mutants. The most prevalent CDMs (>10%) were A371V, E399D, A376S, N348I, A360T, G333E, and L283I. CDM presence was positively correlated with thymidine analogue-associated mutations, but not with NNRTI resistance-associated mutations (RAMs). The presence or number of CDMs did not significantly reduce etravirine susceptibility, although small reductions were seen in samples with G333D, N348I, A360V, T369I, and A376S. N348I, E399G, and N348I/T369I were associated with reduced etravirine susceptibility when present with K103N, L100I, or Y181C. N348I or T369I was associated with reduced etravirine susceptibility when present with K101P or K103R/V179D. Virologic responses to an etravirine-containing regimen were slightly diminished when G333D, G335D, or A376S was present, but this was not confirmed in subgroups with higher baseline resistance or without etravirine RAMs. CDMs alone do not confer substantial reductions in etravirine susceptibility but can further reduce etravirine susceptibility in combination with certain NNRTI mutations. Since virologic responses to etravirine were not affected by CDMs, the clinical impacts of these mutations on etravirine susceptibility appear to be minimal. PMID:21464253

HIV-1 genetic diversity and primary drug resistance mutations before large-scale access to antiretroviral therapy, Republic of Congo.

PubMed

Niama, Fabien Roch; Vidal, Nicole; Diop-Ndiaye, Halimatou; Nguimbi, Etienne; Ahombo, Gabriel; Diakabana, Philippe; Bayonne Kombo, Édith Sophie; Mayengue, Pembe Issamou; Kobawila, Simon-Charles; Parra, Henri Joseph; Toure-Kane, Coumba

2017-07-05

In this work, we investigated the genetic diversity of HIV-1 and the presence of mutations conferring antiretroviral drug resistance in 50 drug-naïve infected persons in the Republic of Congo (RoC). Samples were obtained before large-scale access to HAART in 2002 and 2004. To assess the HIV-1 genetic recombination, the sequencing of the pol gene encoding a protease and partial reverse transcriptase was performed and analyzed with updated references, including newly characterized CRFs. The assessment of drug resistance was conducted according to the WHO protocol. Among the 50 samples analyzed for the pol gene, 50% were classified as intersubtype recombinants, charring complex structures inside the pol fragment. Five samples could not be classified (noted U). The most prevalent subtypes were G with 10 isolates and D with 11 isolates. One isolate of A, J, H, CRF05, CRF18 and CRF37 were also found. Two samples (4%) harboring the mutations M230L and Y181C associated with the TAMs M41L and T215Y, respectively, were found. This first study in the RoC, based on WHO classification, shows that the threshold of transmitted drug resistance before large-scale access to antiretroviral therapy is 4%.

Effect of dolutegravir in combination with Nucleoside Reverse Transcriptase Inhibitors (NRTIs) on people living with HIV who have pre-existing NRTI mutations.

PubMed

Sörstedt, Erik; Carlander, Christina; Flamholc, Leo; Hejdeman, Bo; Svedhem, Veronica; Sönnerborg, Anders; Gisslén, Magnus; Yilmaz, Aylin

2018-05-01

Until the introduction of dolutegravir (DTG), people living with HIV (PLWH) who have developed nucleoside reverse transcriptase inhibitor (NRTI) mutations have had few other treatment options outside of regimens based on ritonavir-boosted protease inhibitors (PI/r). Here we report treatment results among PLWH in Sweden with pre-existing NRTI mutations on antiretroviral treatment (ART) with DTG and one to two NRTIs. All PLWH on ART with DTG and one to two NRTIs with pre-existing NRTI mutations were retrospectively identified from the National InfCare HIV database. As controls, PLWH on PI/r and one to two NRTIs, matched according to Genotypic Susceptibility Score and observation time, were included. Data were collected as long as the study population was on treatment with DTG; controls were monitored for the same interval. Outcome was classified as either treatment success or failure. In total, 244 participants (122 individuals treated with DTG and 122 individuals treated with PI/r) were included. Median observation time was 78 weeks (interquartile range 50-98 weeks) for participants on DTG and 75 weeks (50-101 weeks) for individuals on PI/r. Viral failure was detected in four individuals treated with DTG and three individuals treated with PI/r, resulting in similar success rates of 96.7% and 97.5%, respectively. No new mutations were found among participants with treatment failure. DTG in combination with one to two NRTIs was as efficient as PI/r in individuals with pre-existing NRTI mutations in this setting. It may be considered an alternative to PI/r-based ART even in the presence of NRTI resistance. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

Low prevalence of transmitted K65R and other tenofovir resistance mutations across different HIV-1 subtypes: implications for pre-exposure prophylaxis.

PubMed

Chan, Philip A; Huang, Austin; Kantor, Rami

2012-10-15

Tenofovir-containing regimens have demonstrated potential efficacy as pre-exposure prophylaxis (PrEP) in preventing HIV-1 infection. Transmitted drug resistance mutations associated with tenofovir, specifically the reverse transcriptase (RT) mutation K65R, may impact the effectiveness of PrEP. The worldwide prevalence of transmitted tenofovir resistance in different HIV-1 subtypes is unknown. Sequences from treatment-naïve studies and databases were aggregated and analyzed by Stanford Database tools and as per the International AIDS Society (IAS-USA) resistance criteria. RT sequences were collected from GenBank, the Stanford HIV Sequence Database and the Los Alamos HIV Sequence Database. Sequences underwent rigorous quality control measures. Tenofovir-associated resistance mutations included K65R, K70E, T69-insertion and ≥3 thymidine analogue mutations (TAMs), inclusive of M41L or L210W. A total of 19,823 sequences were evaluated across diverse HIV-1 subtypes (Subtype A: 1549 sequences, B: 9783, C: 3198, D: 483, F: 372, G: 594, H: 41, J: 69, K: 239, CRF01_AE: 1797 and CRF02_AG: 1698). Overall, tenofovir resistance prevalence was 0.4% (n=77/19,823, 95% confidence interval or CI: 0.3 to 0.5). K65R was found in 20 sequences (0.1%, 95% CI: 0.06 to 0.15). Differences in the prevalence of K65R between HIV-1 subtypes were not statistically significant. K70E and ≥3 TAMs were found in 0.015% (95% CI: 0.004 to 0.04) and 0.27% (95% CI: 0.2 to 0.4) of sequences, respectively. Prevalence of transmitted K65R and other tenofovir resistance mutations across diverse HIV-1 subtypes and recombinants is low, suggesting minimal effect on tenofovir-containing PrEP regimens.

Development and customization of a color-coded microbeads-based assay for drug resistance in HIV-1 reverse transcriptase.

PubMed

Gu, Lijun; Kawana-Tachikawa, Ai; Shiino, Teiichiro; Nakamura, Hitomi; Koga, Michiko; Kikuchi, Tadashi; Adachi, Eisuke; Koibuchi, Tomohiko; Ishida, Takaomi; Gao, George F; Matsushita, Masaki; Sugiura, Wataru; Iwamoto, Aikichi; Hosoya, Noriaki

2014-01-01

Drug resistance (DR) of HIV-1 can be examined genotypically or phenotypically. Although sequencing is the gold standard of the genotypic resistance testing (GRT), high-throughput GRT targeted to the codons responsible for DR may be more appropriate for epidemiological studies and public health research. We used a Japanese database to design and synthesize sequence-specific oligonucleotide probes (SSOP) for the detection of wild-type sequences and 6 DR mutations in the clade B HIV-1 reverse transcriptase region. We coupled SSOP to microbeads of the Luminex 100 xMAP system and developed a GRT based on the polymerase chain reaction (PCR)-SSOP-Luminex method. Sixteen oligoprobes for discriminating DR mutations from wild-type sequences at 6 loci were designed and synthesized, and their sensitivity and specificity were confirmed using isogenic plasmids. The PCR-SSOP-Luminex DR assay was then compared to direct sequencing using 74 plasma specimens from treatment-naïve patients or those on failing treatment. In the majority of specimens, the results of the PCR-SSOP-Luminex DR assay were concordant with sequencing results: 62/74 (83.8%) for M41, 43/74 (58.1%) for K65, 70/74 (94.6%) for K70, 55/73 (75.3%) for K103, 63/73 (86.3%) for M184 and 68/73 (93.2%) for T215. There were a number of specimens without any positive signals, especially for K65. The nucleotide position of A2723G, A2747G and C2750T were frequent polymorphisms for the wild-type amino acids K65, K66 and D67, respectively, and 14 specimens had the D67N mutation encoded by G2748A. We synthesized 14 additional oligoprobes for K65, and the sensitivity for K65 loci improved from 43/74 (58.1%) to 68/74 (91.9%). We developed a rapid high-throughput assay for clade B HIV-1 DR mutations, which could be customized by synthesizing oligoprobes suitable for the circulating viruses. The assay could be a useful tool especially for public health research in both resource-rich and resource-limited settings.

Mutants of feline immunodeficiency virus resistant to 2',3'-dideoxy-2',3'-didehydrothymidine.

PubMed Central

Zhu, Y Q; Remington, K M; North, T W

1996-01-01

We selected mutants of feline immunodeficiency virus (FIV) that are resistant to 2',3'-dideoxy-2',3'-didehydrothymidine (d4T). Two mutants were selected in cultured cells with a stepwise increase in d4T concentration, resulting in mutants able to replicate in 100 microM d4T. These mutants were three- to sixfold more resistant to d4T than wild-type FIV. They were also cross-resistant to 3'-azido-3'-deoxythymidine (AZT), 3'-fluoro-2',3'-dideoxythymidine, 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and 9-(2-phosphonylmethoxyethyl)adenine, and they were highly resistant to phosphonoformic acid (PFA). Plaque-purified mutants were isolated from each of the mutant populations. The mutant phenotype was stable, because both of the plaque-purified mutants remained d4T resistant even after three passages in the absence of d4T. One of the plaque-purified mutants, designated D4R-3c, was further characterized. Compared with wild-type reverse transcriptase (RT), RT purified from D4R-3c was 3-fold resistant to inhibition by the 5'-triphosphate of d4T, 10-fold resistant to inhibition by the 5'-triphosphate of AZT, and 6-fold resistant to PFA. D4R-3c had a single point mutation in the RT-encoding region of the pol gene at position 2474, resulting in a Val to Ile mutation at codon 47 of the FIV RT. The role of this mutation in d4T resistance was confirmed by site-directed mutagenesis. PMID:8878567

Meta-analysis and time series modeling allow a systematic review of primary HIV-1 drug-resistant prevalence in Latin America and Caribbean.

PubMed

Coelho, Antonio Victor Campos; De Moura, Ronald Rodrigues; Da Silva, Ronaldo Celerino; Kamada, Anselmo Jiro; Guimarães, Rafael Lima; Brandão, Lucas André Cavalcanti; Coelho, Hemílio Fernandes Campos; Crovella, Sergio

2015-01-01

Here we review the prevalence of HIV-1 primary drug resistance in Latin America and Caribbean using meta-analysis as well as time-series modeling. We also discuss whether there could be a drawback to HIV/AIDS programs due to drug resistance in Latin America and Caribbean in the next years. We observed that, although some studies report low or moderate primary drug resistance prevalence in Caribbean countries, this evidence needs to be updated. In other countries, such as Brazil and Argentina, the prevalence of drug resistance appears to be rising. Mutations conferring resistance against reverse transcriptase inhibitors were the most frequent in the analyzed populations (70% of all mutational events). HIV-1 subtype B was the most prevalent in Latin America and the Caribbean, although subtype C and B/F recombinants have significant contributions in Argentina and Brazil. Thus, we suggest that primary drug resistance in Latin America and the Caribbean could have been underestimated. Clinical monitoring should be improved to offer better therapy, reducing the risk for HIV-1 resistance emergence and spread, principally in vulnerable populations, such as men who have sex with men transmission group, sex workers and intravenous drug users.

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

PubMed

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or <0.5% or >15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Polymorphisms in the HIV-1 gp41 env gene, natural resistance to enfuvirtide (T-20) and pol resistance among pregnant Brazilian women.

PubMed

Reis, Mônica Nogueira da Guarda; de Alcântara, Keila Correa; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

2014-01-01

The selective pressure of antiretroviral drugs (ARVs) targeting HIV-1 pol can promote drug resistance mutations in other genomic regions, such as env. Drug resistance among women should be monitored to avoid horizontal and mother-to-child transmission. To describe natural resistance to T-20 (enfuvirtide), gp41 env polymorphisms, mutations in pol and HIV-1 subtypes, 124 pregnant women were recruited. For 98 patients, the gp41 env, protease (PR) and reverse transcriptase (RT) fragments were sequenced. The patients were ARV naïve (n = 30), taking mother-to-child transmission prophylaxis (n = 50), or being treated with highly active ARV therapy/HAART (n = 18). The Stanford and IAS/USA databases and other sources were used to analyze PR/RT, gp41 env resistance mutations. The HIV-1 genetic diversity was analyzed by REGA/phylogenetic analyses. The patients' median age was 25 years (range, 16-42), 18.4% had AIDS. The frequency of natural resistance to T-20 (N42D, L44M, and R46M-low-impact mutations) was 6.1% (6/98); 20.4% (20/98) had compensatory mutations in HR2. The prevalence of transmitted drug resistance in the pol was 13.3% (4/30), and the prevalence of secondary drug resistance was 33.3% (6/18). Two patients were infected with multidrug resistant/MDR viruses. The analysis of HIV-1 subtypes (PR/RT/gp41) revealed that 61.2% (60/98) were subtype B, 12.2% (12/98) were subtype C, 4.1% (4/98) were subtype F1, and 22.4% (22/98) were possible recombinants (BF1 = 20.4%; BC = 2%). Natural resistance to T-20 was not associated with pol resistance or previous ARV use. The high rate of secondary resistance, including MDR, indicates that the number of women that may need T-20 salvage therapy may be higher than anticipated. © 2013 Wiley Periodicals, Inc.

Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Smith, Robert A; Anderson, Donovan J; Preston, Bradley D

2006-07-01

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.

HIV-1 drug resistance in recently HIV-infected pregnant mother's naïve to antiretroviral therapy in Dodoma urban, Tanzania.

PubMed

Vairo, Francesco; Nicastri, Emanuele; Liuzzi, Giuseppina; Chaula, Zainab; Nguhuni, Boniface; Bevilacqua, Nazario; Forbici, Federica; Amendola, Alessandra; Fabeni, Lavinia; De Nardo, Pasquale; Perno, Carlo Federico; Cannas, Angela; Sakhoo, Calistus; Capobianchi, Maria Rosaria; Ippolito, Giuseppe

2013-09-21

HIV resistance affects virological response to therapy and efficacy of prophylaxis in mother-to-child-transmission. The study aims to assess the prevalence of HIV primary resistance in pregnant women naïve to antiretrovirals. Cross sectional baseline analysis of a cohort of HIV + pregnant women (HPW) enrolled in the study entitled Antiretroviral Management of Antenatal and Natal HIV Infection (AMANI, peace in Kiswahili language). The AMANI study began in May 2010 in Dodoma, Tanzania. In this observational cohort, antiretroviral treatment was provided to all women from the 28th week of gestation until the end of the breastfeeding period. Baseline CD4 cell count, viral load and HIV drug-resistance genotype were collected. Drug-resistance analysis was performed on 97 naïve infected-mothers. The prevalence of all primary drug resistance and primary non-nucleoside reverse-transcriptase inhibitors resistance was 11.9% and 7.5%, respectively. K103S was found in two women with no M184V detection. HIV-1 subtype A was the most commonly identified, with a high prevalence of subtype A1, followed by C, D, C/D recombinant, A/C recombinant and A/D recombinant. HIV drug- resistance mutations were detected in A1 and C subtypes. Our study reports an 11.9% prevalence rate of primary drug resistance in naïve HIV-infected pregnant women from a remote area of Tanzania. Considering that the non-nucleoside reverse-transcriptase inhibitors are part of the first-line antiretroviral regimen in Tanzania and all of Africa, resistance surveys should be prioritized in settings where antiretroviral therapy programs are scaled up.

Antiretroviral treatment sequencing strategies to overcome HIV type 1 drug resistance in adolescents and adults in low-middle-income countries.

PubMed

De Luca, Andrea; Hamers, Raphael L; Schapiro, Jonathan M

2013-06-15

Antiretroviral treatment (ART) is expanding to human immunodeficiency virus type 1 (HIV-1)-infected persons in low-middle income countries, thanks to a public health approach. With 3 available drug classes, 2 ART sequencing lines are programmatically foreseen. The emergence and transmission of viral drug resistance represents a challenge to the efficacy of ART. Knowledge of HIV-1 drug resistance selection associated with specific drugs and regimens and the consequent activity of residual drug options are essential in programming ART sequencing options aimed at preserving ART efficacy for as long as possible. This article determines optimal ART sequencing options for overcoming HIV-1 drug resistance in resource-limited settings, using currently available drugs and treatment monitoring opportunities. From the perspective of drug resistance and on the basis of limited virologic monitoring data, optimal sequencing seems to involve use of a tenofovir-containing nonnucleoside reverse-transcriptase inhibitor-based first-line regimen, followed by a zidovudine-containing, protease inhibitor (PI)-based second-line regimen. Other options and their consequences are explored by considering within-class and between-class sequencing opportunities, including boosted PI monotherapies and future options with integrase inhibitors. Nucleoside reverse-transcriptase inhibitor resistance pathways in HIV-1 subtype C suggest an additional reason for accelerating stavudine phase out. Viral load monitoring avoids the accumulation of resistance mutations that significantly reduce the activity of next-line options. Rational use of resources, including broader access to viral load monitoring, will help ensure 3 lines of fully active treatment options, thereby increasing the duration of ART success.

Early selection of resistance-associated mutations in HIV-1 RT C-terminal domains across different subtypes: role of the genetic barrier to resistance.

PubMed

Muniz, Cláudia P; Soares, Marcelo A; Santos, André F

2014-10-01

Interpretation of drug resistance mutation (DRM) has been based solely on HIV-1 subtype B. Reverse transcriptase (RT) C-terminal domains have been disregarded in resistance interpretation, as their clinical relevance is still controversial. We determined the emergence of DRM in RT C-terminal domains of different HIV-1 subtypes, the genetic barrier for the acquisition of these DRM and their temporal appearance with 'classical' RT inhibitor (RTI) mutations. HIV-1 RT sequences were obtained from information from 6087 treatment-naive and 3795 RTI-treated patients deposited in the Stanford HIV Resistance Database, including all major subtypes. DRM emergence was evaluated for subtype B, and was correlated with the number of DRM in the polymerase domain. Genetic barrier was calculated for each DRM studied and in each subtype. N348I, T369I and A360V were found at low prevalence in treatment-naive isolates of all subtypes. A371V was common to treatment-naive isolates. N348I was observed in all subtypes, while T369I was only selected in subtype C. A360V and T369V were selected by RTI treatment in several subtypes. A371V was selected in subtypes B and C, but is a signature in subtype A. RT C-terminal mutations were correlated with early drug resistance in subtype B. All subtypes have a low calculated genetic barrier towards C-terminal DRM acquisition, despite a few disparities having been observed. C-terminal mutations were selected in all HIV-1 subtypes, while some represent subtype-specific signatures. The selection of C-terminal DRMs occurs early in RTI resistance failure in subtype B. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Short communication prevalence of susceptibility to etravirine by genotype and phenotype in samples received for routine HIV type 1 resistance testing in the United States.

PubMed

Picchio, Gaston; Vingerhoets, Johan; Tambuyzer, Lotke; Coakley, Eoin; Haddad, Mojgan; Witek, James

2011-12-01

Abstract The prevalence of susceptibility to etravirine was investigated among clinical samples submitted for routine clinical testing in the United States using two separate weighted genotypic scoring systems. The presence of etravirine mutations and susceptibility to etravirine by phenotype of clinical samples from HIV-1-infected patients, submitted to Monogram Biosciences for routine resistance testing between June 2008 and June 2009, were analyzed. Susceptibility by genotype was determined using the Monogram and Tibotec etravirine-weighted genotypic scoring systems, with scores of ≤3 and ≤2, respectively, indicating full susceptibility. Susceptibility by phenotype was determined using the PhenoSense HIV assay, with lower and higher clinical cut-offs of 2.9 and 10, respectively. The frequency of individual etravirine mutations and the impact of the K103N mutation on susceptibility to etravirine by genotype were also determined. Among the 5482 samples with ≥1 defined nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations associated with resistance, 67% were classed as susceptible to etravirine by genotype by both scoring systems. Susceptibility to etravirine by phenotype was higher (76%). The proportion of first-generation NNRTI-resistant samples with (n=3598) and without (n=1884) K103N with susceptibility to etravirine by genotype was 77% and 49%, respectively. Among samples susceptible to first-generation NNRTIs (n=9458), >99% of samples were susceptible to etravirine by phenotype (FC <2.9); the remaining samples had FC ≥2.9-10. In summary, among samples submitted for routine clinical testing in the United States, a high proportion of samples with first-generation NNRTI resistance was susceptible to etravirine by genotype and phenotype. A higher proportion of NNRTI-resistant samples with K103N than without was susceptible to etravirine.

Pharmacokinetics of antiretroviral drugs in anatomical sanctuary sites: the male and female genital tract.

PubMed

Else, Laura J; Taylor, Stephen; Back, David J; Khoo, Saye H

2011-01-01

HIV resides within anatomical 'sanctuary sites', where local drug exposure and viral dynamics may differ significantly from the systemic compartment. Suboptimal antiretroviral concentrations in the genital tract may result in compartmentalized viral replication, selection of resistant mutations and possible re-entry of wild-type/resistant virus into the systemic circulation. Therefore, achieving adequate antiretroviral exposure in the genital tract has implications for the prevention of sexual and vertical transmission of HIV. Penetration of antiretrovirals in the genital tract is expressed by accumulation ratios derived from the measurement of drug concentrations in time-matched seminal plasma/cervicovaginal fluid and plasma samples. Penetration varies by gender and may be drug (as opposed to class) specific with high interindividual variability. Concentrations in seminal plasma are highest for nucleoside analogues and lowest for protease inhibitors and efavirenz. Seminal accumulation of newer agents, raltegravir and maraviroc, is moderate (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitors [lamivudine/zidovudine/tenofovir/didanosine > stavudine/abacavir] > raltegravir > indinavir/maraviroc/nevirapine > efavirenz/protease inhibitors [amprenavir/atazanavir/darunavir > lopinavir/ritonavir > saquinavir] > enfuvirtide). In the female genital tract, the nucleoside analogues exhibit high accumulation ratios, whereas protease inhibitors have limited penetration; however, substantial variability exists between individuals and study centres. Second generation non-nucleoside reverse transcriptase inhibitor etravirine, and maraviroc and raltegravir, demonstrate effective accumulation in cervicovaginal secretions (rank order of accumulation is nucleoside/nucleotide reverse transcriptase inhibitor [zidovudine/lamivudine/didanosine > emtricitabine/tenofovir] > indinavir > maraviroc/raltegravir/darunavir/etravirine > nevirapine/abacavir > protease inhibitors [amprenavir/atazanavir/ritonavir] > lopinavir/stavudine/efavirenz > saquinavir).

Increase of Transmitted Drug Resistance among HIV-Infected Sub-Saharan Africans Residing in Spain in Contrast to the Native Population

PubMed Central

Yebra, Gonzalo; de Mulder, Miguel; Pérez-Elías, María Jesús; Pérez-Molina, José Antonio; Galán, Juan Carlos; Llenas-García, Jara; Moreno, Santiago; Holguín, África

2011-01-01

Background The prevalence of transmitted HIV drug resistance (TDR) is stabilizing or decreasing in developed countries. However, this trend is not specifically evaluated among immigrants from regions without well-implemented antiretroviral strategies. Methods TDR trends during 1996–2010 were analyzed among naïve HIV-infected patients in Spain, considering their origin and other factors. TDR mutations were defined according to the World Health Organization list. Results Pol sequence was available for 732 HIV-infected patients: 292 native Spanish, 226 sub-Saharan Africans (SSA), 114 Central-South Americans (CSA) and 100 from other regions. Global TDR prevalence was 9.7% (10.6% for Spanish, 8.4% for SSA and 7.9% for CSA). The highest prevalences were found for protease inhibitors (PI) in Spanish (3.1%), for non-nucleoside reverse transcriptase inhibitors (NNRTI) in SSA (6.5%) and for nucleoside reverse transcriptase inhibitors (NRTI) in both Spanish and SSA (6.5%). The global TDR rate decreased from 11.3% in 2004–2006 to 8.4% in 2007–2010. Characteristics related to a decreasing TDR trend in 2007-10 were Spanish and CSA origin, NRTI- and NNRTI-resistance, HIV-1 subtype B, male sex and infection through injection drug use. TDR remained stable for PI-resistance, in patients infected through sexual intercourse and in those carrying non-B variants. However, TDR increased among SSA and females. K103N was the predominant mutation in all groups and periods. Conclusion TDR prevalence tended to decrease among HIV-infected native Spanish and Central-South Americans, but it increased up to 13% in sub-Saharan immigrants in 2007–2010. These results highlight the importance of a specific TDR surveillance among immigrants to prevent future therapeutic failures, especially when administering NNRTIs. PMID:22046345

HIV-1 transmission networks across Cyprus (2010-2012).

PubMed

Kostrikis, Leondios G; Hezka, Johana; Stylianou, Dora C; Kostaki, Evangelia; Andreou, Maria; Kousiappa, Ioanna; Paraskevis, Dimitrios; Demetriades, Ioannis

2018-01-01

A molecular epidemiology study of HIV-1 infection was conducted in one hundred diagnosed and untreated HIV-1-infected patients in Cyprus between 2010 and 2012, representing 65.4% of all the reported HIV-1 infections in Cyprus in this three-year period, using a previously defined enrolment strategy. Eighty-two patients were newly diagnosed (genotypic drug resistance testing within six months from diagnosis), and eighteen patients were HIV-1 diagnosed for a longer period or the diagnosis date was unknown. Phylogenetic trees of the pol sequences obtained in this study with reference sequences indicated that subtypes B and A1 were the most common subtypes present and accounted for 41.0 and 19.0% respectively, followed by subtype C (7.0%), F1 (8.0%), CRF02_AG (4.0%), A2 (2.0%), other circulating recombinant forms (CRFs) (7.0%) and unknown recombinant forms (URFs) (12%). Most of the newly-diagnosed study subjects were Cypriots (63%), males (78%) with median age 39 (Interquartile Range, IQR 33-48) reporting having sex with other men (MSM) (51%). A high rate of clustered transmission of subtype B drug-sensitive strains to reverse transcriptase and protease inhibitors was observed among MSM, twenty-eight out of forty-one MSM study subjects (68.0%) infected were implicated in five transmission clusters, two of which are sub-subtype A1 and three of which are subtype B strains. The two largest MSM subtype B clusters included nine and eight Cypriot men, respectively, living in all major cities in Cyprus. There were only three newly diagnosed patients with transmitted drug resistant HIV-1 strains, one study subject from the United Kingdom infected with subtype B strain and one from Romania with sub-subtype A2 strain, both with PI drug resistance mutation M46L and one from Greece with sub-subtype A1 with non-nucleoside reverse transcriptase inhibitors (NNRTI) drug resistance mutation K103N.

A model of directional selection applied to the evolution of drug resistance in HIV-1.

PubMed

Seoighe, Cathal; Ketwaroo, Farahnaz; Pillay, Visva; Scheffler, Konrad; Wood, Natasha; Duffet, Rodger; Zvelebil, Marketa; Martinson, Neil; McIntyre, James; Morris, Lynn; Hide, Winston

2007-04-01

Understanding how pathogens acquire resistance to drugs is important for the design of treatment strategies, particularly for rapidly evolving viruses such as HIV-1. Drug treatment can exert strong selective pressures and sites within targeted genes that confer resistance frequently evolve far more rapidly than the neutral rate. Rapid evolution at sites that confer resistance to drugs can be used to help elucidate the mechanisms of evolution of drug resistance and to discover or corroborate novel resistance mutations. We have implemented standard maximum likelihood methods that are used to detect diversifying selection and adapted them for use with serially sampled reverse transcriptase (RT) coding sequences isolated from a group of 300 HIV-1 subtype C-infected women before and after single-dose nevirapine (sdNVP) to prevent mother-to-child transmission. We have also extended the standard models of codon evolution for application to the detection of directional selection. Through simulation, we show that the directional selection model can provide a substantial improvement in sensitivity over models of diversifying selection. Five of the sites within the RT gene that are known to harbor mutations that confer resistance to nevirapine (NVP) strongly supported the directional selection model. There was no evidence that other mutations that are known to confer NVP resistance were selected in this cohort. The directional selection model, applied to serially sampled sequences, also had more power than the diversifying selection model to detect selection resulting from factors other than drug resistance. Because inference of selection from serial samples is unlikely to be adversely affected by recombination, the methods we describe may have general applicability to the analysis of positive selection affecting recombining coding sequences when serially sampled data are available.

The impact of transmission clusters on primary drug resistance in newly diagnosed HIV-1 infection.

PubMed

Yerly, Sabine; Junier, Thomas; Gayet-Ageron, Angèle; Amari, Emmanuelle Boffi El; von Wyl, Viktor; Günthard, Huldrych F; Hirschel, Bernard; Zdobnov, Evgeny; Kaiser, Laurent

2009-07-17

To monitor HIV-1 transmitted drug resistance (TDR) in a well defined urban area with large access to antiretroviral therapy and to assess the potential source of infection of newly diagnosed HIV individuals. All individuals resident in Geneva, Switzerland, with a newly diagnosed HIV infection between 2000 and 2008 were screened for HIV resistance. An infection was considered as recent when the positive test followed a negative screening test within less than 1 year. Phylogenetic analyses were performed by using the maximum likelihood method on pol sequences including 1058 individuals with chronic infection living in Geneva. Of 637 individuals with newly diagnosed HIV infection, 20% had a recent infection. Mutations associated with resistance to at least one drug class were detected in 8.5% [nucleoside reverse transcriptase inhibitors (NRTIs), 6.3%; non-nucleoside reverse transcriptase inhibitors (NNRTIs), 3.5%; protease inhibitors, 1.9%]. TDR (P-trend = 0.015) and, in particular, NNRTI resistance (P = 0.002) increased from 2000 to 2008. Phylogenetic analyses revealed that 34.9% of newly diagnosed individuals, and 52.7% of those with recent infection were linked to transmission clusters. Clusters were more frequent in individuals with TDR than in those with sensitive strains (59.3 vs. 32.6%, respectively; P < 0.0001). Moreover, 84% of newly diagnosed individuals with TDR were part of clusters composed of only newly diagnosed individuals. Reconstruction of the HIV transmission networks using phylogenetic analysis shows that newly diagnosed HIV infections are a significant source of onward transmission, particularly of resistant strains, thus suggesting an important self-fueling mechanism for TDR.

Global epidemiology of drug resistance after failure of WHO recommended first-line regimens for adult HIV-1 infection: a multicentre retrospective cohort study

PubMed Central

2016-01-01

Summary Background Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART. Methods The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase (RT) gene. Findings We included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count <100 cells per μL). Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions (OR 1·48, 95% CI 1·20–1·82). Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma viral load at virological failure was similar in individuals with and without tenofovir resistance (145 700 copies per mL [SE 12 480] versus 133 900 copies per mL [SE 16 650; p=0·626]). Interpretation We recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial. Funding The Wellcome Trust. PMID:26831472

Impact of Human Immunodeficiency Virus Type 1 Minority Variants on the Virus Response to a Rilpivirine-Based First-line Regimen.

PubMed

Raymond, Stéphanie; Nicot, Florence; Pallier, Coralie; Bellecave, Pantxika; Maillard, Anne; Trabaud, Mary Anne; Morand-Joubert, Laurence; Rodallec, Audrey; Amiel, Corinne; Mourez, Thomas; Bocket, Laurence; Beby-Defaux, Agnès; Bouvier-Alias, Magali; Lambert-Niclot, Sidonie; Charpentier, Charlotte; Malve, Brice; Mirand, Audrey; Dina, Julia; Le Guillou-Guillemette, Hélène; Marque-Juillet, Stéphanie; Signori-Schmuck, Anne; Barin, Francis; Si-Mohamed, Ali; Avettand Fenoel, Véronique; Roussel, Catherine; Calvez, Vincent; Saune, Karine; Marcelin, Anne Geneviève; Rodriguez, Christophe; Descamps, Diane; Izopet, Jacques

2018-05-02

Minority resistant variants of human immunodeficiency virus type 1 (HIV-1) could influence the virological response to treatment based on nonnucleoside reverse transcriptase inhibitors (NNRTIs). Data on minority rilpivirine-resistant variants are scarce. This study used next-generation sequencing (NGS) to identify patients harboring minority resistant variants to nucleos(t)ide reverse transcriptase inhibitors and NNRTIs and to assess their influence on the virological response (VR). All the subjects, 541 HIV-1-infected patients started a first-line regimen containing rilpivirine. VR was defined as a HIV-1 RNA load <50 copies/mL at month 6 with continued suppression at month 12. NGS was performed at baseline (retrospectively) on the 454 GS-FLX platform (Roche). NGS revealed resistance-associated mutations accounting for 1% to <5% of variants in 17.2% of samples, for 5%-20% in 5.7% of samples, and for >20% in 29% of samples. We identified 43 (8.8%) and 36 (7.4%) patients who harbored rilpivirine-resistant variants with a 1% sensitivity threshold according to the French National Agency for Research on AIDS and Viral Hepatitis and Stanford algorithms, respectively. The VR was 96.9% at month 12. Detection of minority rilpivirine resistant variants was not associated with virological failure (VF). Multivariate analysis indicated that VF at month 12 was associated with a CD4 count <250 cells/µL at baseline, a slower decrease in viral load at month 3, and rilpivirine resistance at baseline using the Stanford algorithm with a 20% threshold. Minority resistant variants had no impact on the VR of treatment-naive patients to a rilpivirine-based regimen.

The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

PubMed Central

Wainberg, Mark A.

2012-01-01

The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

Promises and pitfalls of Illumina sequencing for HIV resistance genotyping.

PubMed

Brumme, Chanson J; Poon, Art F Y

2017-07-15

Genetic sequencing ("genotyping") plays a critical role in the modern clinical management of HIV infection. This virus evolves rapidly within patients because of its error-prone reverse transcriptase and short generation time. Consequently, HIV variants with mutations that confer resistance to one or more antiretroviral drugs can emerge during sub-optimal treatment. There are now multiple HIV drug resistance interpretation algorithms that take the region of the HIV genome encoding the major drug targets as inputs; expert use of these algorithms can significantly improve to clinical outcomes in HIV treatment. Next-generation sequencing has the potential to revolutionize HIV resistance genotyping by lowering the threshold that rare but clinically significant HIV variants can be detected reproducibly, and by conferring improved cost-effectiveness in high-throughput scenarios. In this review, we discuss the relative merits and challenges of deploying the Illumina MiSeq instrument for clinical HIV genotyping. Copyright © 2016 Elsevier B.V. All rights reserved.

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

PubMed

Tamalet, Catherine; Tissot-Dupont, Hervé; Motte, Anne; Tourrès, Christian; Dhiver, Catherine; Ravaux, Isabelle; Poizot-Martin, Isabelle; Dieng, Thérèse; Tomei, Christelle; Bregigeon, Sylvie; Zaegel-Faucher, Olivia; Laroche, Hélène; Aherfi, Sarah; Mokhtari, Saadia; Chaudet, Hervé; Ménard, Amelie; Brouqui, Philippe; Stein, Andreas; Colson, Philippe

2018-05-24

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies. © 2018 Wiley Periodicals, Inc.

Hereditary vitamin D resistant rickets: identification of a novel splice site mutation in the vitamin D receptor gene and successful treatment with oral calcium therapy.

PubMed

Ma, Nina S; Malloy, Peter J; Pitukcheewanont, Pisit; Dreimane, Daina; Geffner, Mitchell E; Feldman, David

2009-10-01

To study the vitamin D receptor (VDR) gene in a young girl with severe rickets and clinical features of hereditary vitamin D resistant rickets, including hypocalcemia, hypophosphatemia, partial alopecia, and elevated serum levels of 1,25-dihydroxyvitamin D. We amplified and sequenced DNA samples from blood from the patient, her mother, and the patient's two siblings. We also amplified and sequenced the VDR cDNA from RNA isolated from the patient's blood. DNA sequence analyses of the VDR gene showed that the patient was homozygous for a novel guanine to thymine substitution in the 5'-splice site in the exon 8-intron J junction. Analysis of the VDR cDNA using reverse transcriptase-polymerase chain reaction showed that exons 7 and 9 were fused, and that exon 8 was skipped. The mother was heterozygous for the mutation and the two siblings were unaffected. A novel splice site mutation was identified in the VDR gene that caused exon 8 to be skipped. The mutation deleted amino acids 303-341 in the VDR ligand-binding domain, which is expected to render the VDR non-functional. Nevertheless, successful outpatient treatment was achieved with frequent high doses of oral calcium.

First Line Treatment Response in Patients with Transmitted HIV Drug Resistance and Well Defined Time Point of HIV Infection: Updated Results from the German HIV-1 Seroconverter Study

PubMed Central

zu Knyphausen, Fabia; Scheufele, Ramona; Kücherer, Claudia; Jansen, Klaus; Somogyi, Sybille; Dupke, Stephan; Jessen, Heiko; Schürmann, Dirk; Hamouda, Osamah; Meixenberger, Karolin; Bartmeyer, Barbara

2014-01-01

Background Transmission of drug-resistant HIV-1 (TDR) can impair the virologic response to antiretroviral combination therapy. Aim of the study was to assess the impact of TDR on treatment success of resistance test-guided first-line therapy in the German HIV-1 Seroconverter Cohort for patients infected with HIV between 1996 and 2010. An update of the prevalence of TDR and trend over time was performed. Methods Data of 1,667 HIV-infected individuals who seroconverted between 1996 and 2010 were analysed. The WHO drug resistance mutations list was used to identify resistance-associated HIV mutations in drug-naïve patients for epidemiological analysis. For treatment success analysis the Stanford algorithm was used to classify a subset of 323 drug-naïve genotyped patients who received a first-line cART into three resistance groups: patients without TDR, patients with TDR and fully active cART and patients with TDR and non-fully active cART. The frequency of virologic failure 5 to 12 months after treatment initiation was determined. Results Prevalence of TDR was stable at a high mean level of 11.9% (198/1,667) in the HIV-1 Seroconverter Cohort without significant trend over time. Nucleotide reverse transcriptase inhibitor resistance was predominant (6.0%) and decreased significantly over time (OR = 0.92, CI = 0.87–0.98, p = 0.01). Non-nucleoside reverse transcriptase inhibitor (2.4%; OR = 1.00, CI = 0.92–1.09, p = 0.96) and protease inhibitor resistance (2.0%; OR = 0.94, CI = 0.861.03, p = 0.17) remained stable. Virologic failure was observed in 6.5% of patients with TDR receiving fully active cART, 5,6% of patients with TDR receiving non-fully active cART and 3.2% of patients without TDR. The difference between the three groups was not significant (p = 0.41). Conclusion Overall prevalence of TDR remained stable at a rather high level. No significant differences in the frequency of virologic failure were identified during first-line cART between patients with TDR and fully-active cART, patients with TDR and non-fully active cART and patients without TDR. PMID:24788613

Anti-human immunodeficiency virus (HIV) activities of halogenated gomisin J derivatives, new nonnucleoside inhibitors of HIV type 1 reverse transcriptase.

PubMed

Fujihashi, T; Hara, H; Sakata, T; Mori, K; Higuchi, H; Tanaka, A; Kaji, H; Kaji, A

1995-09-01

Halogenated gomisin J (a derivative of lignan compound), represented by the bromine derivative 1506 [(6R, 7S, S-biar)-4,9-dibromo-3,10-dihydroxy-1,2,11,12-tetramethoxy-6, 7-dimethyl-5,6,7,8- tetrahydrodibenzo[a,c]cyclo-octene], was found to be a potent inhibitor of the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) on MT-4 human T cells (50% effective dose, 0.1 to 0.5 microM). Gomisin J derivatives were active in preventing p24 production from acutely HIV-1-infected H9 cells. The selective indices (toxic dose/effective dose) of these compounds were as high as > 300 in some systems. 1506 was active against 3'-azido-3'-deoxythymidine-resistant HIV-1 and acted synergistically with AZT and 2',3'-ddC. 1506 inhibited HIV-1 reverse transcriptase (RT) in vitro but not HIV-1 protease. From the time-of-addition experiment, 1506 was found to inhibit the early phase of the HIV life cycle. A 1506-resistant HIV mutant was selected and shown to possess a mutation within the RT-coding region (at position 188 [Tyr to Leu]). The mutant RT expressed in Escherichia coli was resistant to 1506 in the in vitro RT assay. Some of the HIV strains resistant to other nonnucleoside HIV-1 RT inhibitors were also resistant to 1506. Comparison of various gomisin J derivatives with gomisin J showed that iodine, bromine, and chlorine in the fourth and ninth positions increased RT inhibitory activity as well as cytoprotective activity.

Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus.

PubMed

de Ronde, A; van Dooren, M; van Der Hoek, L; Bouwhuis, D; de Rooij, E; van Gemen, B; de Boer, R; Goudsmit, J

2001-01-01

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.

Epistasis and Pleiotropy Affect the Modularity of the Genotype-Phenotype Map of Cross-Resistance in HIV-1.

PubMed

Polster, Robert; Petropoulos, Christos J; Bonhoeffer, Sebastian; Guillaume, Frédéric

2016-12-01

The genotype-phenotype (GP) map is a central concept in evolutionary biology as it describes the mapping of molecular genetic variation onto phenotypic trait variation. Our understanding of that mapping remains partial, especially when trying to link functional clustering of pleiotropic gene effects with patterns of phenotypic trait co-variation. Only on rare occasions have studies been able to fully explore that link and tend to show poor correspondence between modular structures within the GP map and among phenotypes. By dissecting the structure of the GP map of the replicative capacity of HIV-1 in 15 drug environments, we provide a detailed view of that mapping from mutational pleiotropic variation to phenotypic co-variation, including epistatic effects of a set of amino-acid substitutions in the reverse transcriptase and protease genes. We show that epistasis increases the pleiotropic degree of single mutations and provides modularity to the GP map of drug resistance in HIV-1. Moreover, modules of epistatic pleiotropic effects within the GP map match the phenotypic modules of correlated replicative capacity among drug classes. Epistasis thus increases the evolvability of cross-resistance in HIV by providing more drug- and class-specific pleiotropic profiles to the main effects of the mutations. We discuss the implications for the evolution of cross-resistance in HIV. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Transmitted Drug Resistance Among Recently Diagnosed Adults and Children in São Paulo, Brazil.

PubMed

Guimarães, Paula Morena de Souza; Ferreira, João Leandro de Paula; Coelho, Luana Portes Ozório; Cavalcanti, Jaqueline de Souza; Lopes, Giselle Ibette Silva Lopez; Matsuda, Elaine Monteiro; Almeida, Flávia Jacqueline; Almeida, Valéria Correia; Campeas, Alexandre Ely; Junior, Luiz Carlos Pereira; Brígido, Luís Fernando de Macedo

2015-12-01

Transmitted drug resistance mutations (TDRM) have been a constant threat to treatment efficacy. We evaluated TDRM in plasma RNA of 217 antiretroviral therapy-naive patients from sites in the São Paulo metropolitan area, collected from 2012 to 2014. The partial HIV-1 polymerase region was sequenced using Big Dye terminators at an ABI 3130 Genetic Analyzer. TDRM was defined according to the Stanford database calibrated population resistance (CPR v.6.0), but other drug resistance mutations (DRM) considered at the IAS list (IAS, 2014) and at the Stanford HIV Database Genotyping Resistance Interpretation (GRI-HIVdb) were also described. Out of 78% (170/217) of patients with information on the time of diagnosis, most (83%, 141/170) had been recently diagnosed, with the first positive HIV serology at a median of 58 days (IQR 18-184). Subtype B predominated (70%), followed by subtype F (10%), BF (7.5%), C (7.5%), and BC (5%). TDRMs were observed in 9.2% (20/217, CI 95% 5.9% to 13.6%), mostly (5.2%) to nonnucleoside reverse transcriptase inhibitor (NNRTI) antiretroviral class. Among children and adolescents, only a single patient showed TDRMs. Additional non-CPR mutations were observed: 11.5% (25/217) according to IAS or 4.6% (10/217) according to GRI-HIVdb. Overall, 23.5% (51/217) of the cases had one or more DRM identified. TDRM prevalence differed significantly among some sites. These trends deserve continuous and systematic surveillance, especially with the new policies of treatment as prevention being implemented in the country.

Selection of HIV resistance associated with antiretroviral therapy initiated due to pregnancy and suspended postpartum.

PubMed

Ellis, Giovanina M; Huang, Sharon; Hitti, Jane; Frenkel, Lisa M

2011-11-01

Compare the risk of HIV drug resistance in women stopping suppressive nelfinavir (NFV)-based or Nevirapine (NVP)-based antiretroviral therapy (ART) after pregnancy. Specimens collected after stopping ART were tested for drug resistance by an oligonucleotide ligation assay and consensus sequencing. When postpartum drug resistance was detected, specimens obtained at study entry and during ART were evaluated. Sixteen of 38 women with ART-induced suppression of viral replication suspended ART postpartum. Resistance mutations were detected in 75% who stopped NFV-ART and in 50% who stopped NVP-ART. M184V, associated with Lamivudine resistance, was more frequent among those randomized to NFV-ART compared with NVP-ART (6 of 8 versus 1 of 8; P = 0.04), and nonnucleoside reverse transcriptase inhibitor resistance was detected in 4 of 8 stopping NVP-ART. HIV drug resistance was frequently observed among women who stopped suppressive NVP-ART or NFV-ART postpartum. This suggests that NFV-ART may have suboptimal potency, that staggering discontinuation of NVP-ART may be warranted, and/or ART adherence may be lax in women who choose to stop ART postpartum.

New and investigational antiretroviral drugs for HIV infection: mechanisms of action and early research findings.

PubMed

Saag, Michael S

2012-12-01

Numerous investigational antiretroviral agents are in clinical development. Among them are festinavir (BMS986001), a thymidine analogue similar to stavudine with reduced potential for toxicity; GS-7340, a prodrug of tenofovir that achieves greater intracellular concentrations; MK-1439, a nonnucleoside analogue reverse transcriptase inhibitor (NNRTI) that retains activity against common NNRTI-associated resistance mutations; and albuvirtide, a long-acting parenteral fusion inhibitor. Investigational integrase strand transfer inhibitors (InSTIs) include elvitegravir, recently approved by the US Food and Drug Administration (FDA) as part of a once-daily, single-tablet formulation with cobicistat/tenofovir/emtricitabine; dolutegravir, which maintains some activity against raltegravir- and elvitegravir-resistant mutants; and S/GSK1265744, which also maintains some activity against resistance mutations in the integrase gene and is being developed as a long-lasting parenteral agent. Novel 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs), agents that were originally thought to inhibit the interaction of integrase with its cofactor lens epithelium-derived growth factor p75 (LEDGF/p75), be active against InSTI-resistant mutants and to have additive activity when combined with InSTIs. This article summarizes a presentation by Michael S. Saag, MD, at the IAS-USA live Improving the Management of HCV Disease continuing medical education program held in New York in October 2012.

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

PubMed Central

Mizrahi, V; Usdin, M T; Harington, A; Dudding, L R

1990-01-01

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity. Images PMID:1699202

Impact of lopinavir/ritonavir use on antiretroviral resistance in recent clinical practice.

PubMed

Lambert-Niclot, Sidonie; Masquelier, Bernard; Cohen Codar, Isabelle; Soulie, Cathia; Delaugerre, Constance; Morand-Joubert, Laurence; Charpentier, Charlotte; Ferre, Virginie; Plantier, Jean-Christophe; Montes, Brigitte; Carret, Sophie; Perrot, Valerie; Peytavin, Gilles; Costagliola, Dominique; Calvez, Vincent; Marcelin, Anne-Geneviève

2012-10-01

This observational study was requested by French health authorities to determine the impact of lopinavir/ritonavir (Kaletra(®)) on antiretroviral resistance in clinical practice. Virological failures of lopinavir/ritonavir and their effects on the resistance to protease inhibitors and reverse transcriptase inhibitors were evaluated in protease inhibitor-experienced patients. Virological failure was defined as an HIV-1 plasma viral load >50 copies/mL after at least 3 months of lopinavir/ritonavir-containing antiretroviral therapy. For all patients, a resistance genotypic test was available at failure and before lopinavir/ritonavir treatment. Data from 72 patients with inclusion criteria were studied. The mean viral load at baseline was 4 log(10) copies/mL (1.6-6.5). Mutations in the protease gene significantly selected between baseline and failure were L10V, K20R, L33F, M36I, I47V, I54V, A71V and I85V (P < 0.05). Patients who had more than seven protease inhibitor mutations at baseline showed a significantly increased risk of occurrence of protease inhibitor mutations. The proportion of viruses susceptible to atazanavir, fosamprenavir and darunavir decreased significantly between baseline and failure (P < 0.05). Among patients with a virus susceptible to atazanavir at day 0, 26% (n = 14) exhibited a virus resistant or possibly resistant at the time of failure. This proportion was 32% (n = 16) for fosamprenavir and 16% (n = 7) for darunavir. A darunavir-based regimen appears to be a sequential option in the case of lopinavir/ritonavir failure. To compare and determine the best treatment sequencing, similar studies should be performed for darunavir/ritonavir and atazanavir/ritonavir.

Unnecessary antiretroviral treatment switches and accumulation of HIV resistance mutations; two arguments for viral load monitoring in Africa.

PubMed

Sigaloff, Kim C E; Hamers, Raph L; Wallis, Carole L; Kityo, Cissy; Siwale, Margaret; Ive, Prudence; Botes, Mariette E; Mandaliya, Kishor; Wellington, Maureen; Osibogun, Akin; Stevens, Wendy S; van Vugt, Michèle; de Wit, Tobias F Rinke

2011-09-01

This study aimed to investigate the consequences of using clinicoimmunological criteria to detect antiretroviral treatment (ART) failure and guide regimen switches in HIV-infected adults in sub-Saharan Africa. Frequencies of unnecessary switches, patterns of HIV drug resistance, and risk factors for the accumulation of nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations were evaluated. Cross-sectional analysis of adults switching ART regimens at 13 clinical sites in 6 African countries was performed. Two types of failure identification were compared: diagnosis of clinicoimmunological failure without viral load testing (CIF only) or CIF with local targeted viral load testing (targeted VL). After study enrollment, reference HIV RNA and genotype were determined retrospectively. Logistic regression assessed factors associated with multiple thymidine analogue mutations (TAMs) and NRTI cross-resistance (≥2 TAMs or Q151M or K65R/K70E). Of 250 patients with CIF switching to second-line ART, targeted VL was performed in 186. Unnecessary switch at reference HIV RNA <1000 copies per milliliter occurred in 46.9% of CIF only patients versus 12.4% of patients with targeted VL (P < 0.001). NRTI cross-resistance was observed in 48.0% of 183 specimens available for genotypic analysis, comprising ≥2 TAMs (37.7%), K65R (7.1%), K70E (3.3%), or Q151M (3.3%). The presence of NRTI cross-resistance was associated with the duration of ART exposure and zidovudine use. Clinicoimmunological monitoring without viral load testing resulted in frequent unnecessary regimen switches. Prolonged treatment failure was indicated by extensive NRTI cross-resistance. Access to virological monitoring should be expanded to prevent inappropriate switches, enable early failure detection and preserve second-line treatment options in Africa.

Modelling Hepatitis B Virus Antiviral Therapy and Drug Resistant Mutant Strains

NASA Astrophysics Data System (ADS)

Bernal, Julie; Dix, Trevor; Allison, Lloyd; Bartholomeusz, Angeline; Yuen, Lilly

Despite the existence of vaccines, the Hepatitis B virus (HBV) is still a serious global health concern. HBV targets liver cells. It has an unusual replication process involving an RNA pre-genome that the reverse transcriptase domain of the viral polymerase protein translates into viral DNA. The reverse transcription process is error prone and together with the high replication rates of the virus, allows the virus to exist as a heterogeneous population of mutants, known as a quasispecies, that can adapt and become resistant to antiviral therapy. This study presents an individual-based model of HBV inside an artificial liver, and associated blood serum, undergoing antiviral therapy. This model aims to provide insights into the evolution of the HBV quasispecies and the individual contribution of HBV mutations in the outcome of therapy.

Preliminary investigation of bottlenose dolphins (Tursiops truncatus) for hfe gene-related hemochromatosis.

PubMed

Phillips, Brianne E; Venn-Watson, Stephanie; Archer, Linda L; Nollens, Hendrik H; Wellehan, James F X

2014-10-01

Hemochromatosis (iron storage disease) has been reported in diverse mammals including bottlenose dolphins (Tursiops truncatus). The primary cause of excessive iron storage in humans is hereditary hemochromatosis. Most human hereditary hemochromatosis cases (up to 90%) are caused by a point mutation in the hfe gene, resulting in a C282Y substitution leading to iron accumulation. To evaluate the possibility of a hereditary hemochromatosis-like genetic predisposition in dolphins, we sequenced the bottlenose dolphin hfe gene, using reverse transcriptase-PCR and hfe primers designed from the dolphin genome, from liver of affected and healthy control dolphins. Sample size included two case animals and five control animals. Although isotype diversity was evident, no coding differences were identified in the hfe gene between any of the animals examined. Because our sample size was small, we cannot exclude the possibility that hemochromatosis in dolphins is due to a coding mutation in the hfe gene. Other potential causes of hemochromatosis, including mutations in different genes, diet, primary liver disease, and insulin resistance, should be evaluated.

Prevalence of etravirine-associated mutations in clinical samples with resistance to nevirapine and efavirenz.

PubMed

Llibre, J M; Santos, J R; Puig, T; Moltó, J; Ruiz, L; Paredes, R; Clotet, B

2008-11-01

To evaluate the expected activity of etravirine in clinical samples, according to mutational patterns associated with decreased virological response (VR). We identified 1586 routine clinical samples with resistance-associated mutations (RAMs) to nevirapine and efavirenz (K103N 60%, Y181C 37%, G190A 27%, V108I 13%). Concerning in vitro identified etravirine mutations, samples with F227C, Y181I, M230L or L100I plus K103N plus Y181C were considered highly resistant. Samples with two RAMs plus Y181C or V179D or K101E or Y188L were considered intermediate. The prevalence of 13 RAMs recently associated with decreased VR to etravirine in the DUET clinical trials was also investigated. Most samples (69%) harboured more than one IAS-USA RAM to first-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs): 42% harboured two RAMs, 21% three RAMs and 6% four or more RAMs. The prevalence of 13 specific etravirine RAMs was V179F 0.12%, G190S 3.9%, Y181V 0.1%, V106I 2.6%, V179D 1.6%, K101P 2.0%, K101E 10.1%, Y181C 36.9%, A98G 5.9%, V90I 6.9%, Y181I 3.6%, G190A 27% and L100I 9.1%. The five RAMs with the most impact on VR (V179F/D, G190S, Y181V and V106I) occurred less often. Overall, 8.2% of the samples had three or more etravirine RAMs and only 1.1% had four or more. In addition, patterns of RAMs previously associated with intermediate etravirine resistance were present in 26.2% of the samples, whereas 4.85% displayed patterns of high-degree resistance. For RAMs associated with decreased VR, etravirine resistance in routine clinical samples was lower than previously reported. High-degree resistance was uncommon, even in patients with resistance to first-generation NNRTIs, whereas low-to-intermediate etravirine resistance was more common.

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Therapy-Naive Individuals Residing in Sorong, West Papua.

PubMed

Witaningrum, Adiana Mutamsari; Kotaki, Tomohiro; Khairunisa, Siti Qamariyah; Yunifiar M, Muhammad Qushai; Indriati, Dwi Wahyu; Bramanthi, Rendra; Nasronudin; Kameoka, Masanori

2016-08-01

Papua and West Papua provinces have the highest prevalence rate of human immunodeficiency virus type 1 (HIV-1) infection in Indonesia; however, data on the molecular epidemiology of HIV-1 are limited. We conducted a genotypic study on HIV-1 genes derived from antiretroviral therapy-naive individuals residing in Sorong, West Papua. HIV-1 genomic fragments were amplified from 43 peripheral blood samples, and sequencing analysis of the genes was carried out. Of the 43 samples, 41 protease (PR), 31 reverse transcriptase (RT), 26 gag, and 25 env genes were sequenced. HIV-1 subtyping revealed that CRF01_AE (48.8%, 21/43) and subtype B (41.9%, 18/43) were the major subtypes prevalent in the region, whereas other recombinant forms were also detected. Major drug resistance-associated mutations for PR inhibitors were not detected; however, mutations for the RT inhibitors, A62V and E138A, appeared in a few samples, indicating the possible emergence of transmitted HIV-1 drug resistance in Sorong, West Papua.

Intrapatient Evolutionary Dynamics of Human Immunodeficiency Virus Type 1 in Individuals Undergoing Alternative Treatment Strategies with Reverse Transcriptase Inhibitors.

PubMed

Kayondo, Jonathan K; Ndembi, Nicaise; Parry, Chris M; Cane, Patricia A; Hué, Stephane; Goodall, Ruth; Dunn, David T; Kaleebu, Pontiano; Pillay, Deenan; Mbisa, Jean L

2015-07-01

Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies.

Phenotype, Genotype, and Drug Resistance in Subtype C HIV-1 Infection.

PubMed

Derache, Anne; Wallis, Carole L; Vardhanabhuti, Saran; Bartlett, John; Kumarasamy, Nagalingeswaran; Katzenstein, David

2016-01-15

Virologic failure in subtype C is characterized by high resistance to first-line antiretroviral (ARV) drugs, including efavirenz, nevirapine, and lamivudine, with nucleoside resistance including type 2 thymidine analog mutations, K65R, a T69del, and M184V. However, genotypic algorithms predicting resistance are mainly based on subtype B viruses and may under- or overestimate drug resistance in non-B subtypes. To explore potential treatment strategies after first-line failure, we compared genotypic and phenotypic susceptibility of subtype C human immunodeficiency virus 1 (HIV-1) following first-line ARV failure. AIDS Clinical Trials Group 5230 evaluated patients failing an initial nonnucleoside reverse-transcriptase inhibitor (NNRTI) regimen in Africa and Asia, comparing the genotypic drug resistance and phenotypic profile from the PhenoSense (Monogram). Site-directed mutagenesis studies of K65R and T69del assessed the phenotypic impact of these mutations. Genotypic algorithms overestimated resistance to etravirine and rilpivirine, misclassifying 28% and 32%, respectively. Despite K65R with the T69del in 9 samples, tenofovir retained activity in >60%. Reversion of the K65R increased susceptibility to tenofovir and other nucleosides, while reversion of the T69del showed increased resistance to zidovudine, with little impact on other NRTI. Although genotype and phenotype were largely concordant for first-line drugs, estimates of genotypic resistance to etravirine and rilpivirine may misclassify subtype C isolates compared to phenotype. © The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Perinatal acquisition of drug-resistant HIV-1 infection: mechanisms and long-term outcome

PubMed Central

Delaugerre, Constance; Chaix, Marie-Laure; Blanche, Stephane; Warszawski, Josiane; Cornet, Dorine; Dollfus, Catherine; Schneider, Veronique; Burgard, Marianne; Faye, Albert; Mandelbrot, Laurent; Tubiana, Roland; Rouzioux, Christine

2009-01-01

Background Primary-HIV-1-infection in newborns that occurs under antiretroviral prophylaxis that is a high risk of drug-resistance acquisition. We examine the frequency and the mechanisms of resistance acquisition at the time of infection in newborns. Patients and Methods We studied HIV-1-infected infants born between 01 January 1997 and 31 December 2004 and enrolled in the ANRS-EPF cohort. HIV-1-RNA and HIV-1-DNA samples obtained perinatally from the newborn and mother were subjected to population-based and clonal analyses of drug resistance. If positive, serial samples were obtained from the child for resistance testing. Results Ninety-two HIV-1-infected infants were born during the study period. Samples were obtained from 32 mother-child pairs and from another 28 newborns. Drug resistance was detected in 12 newborns (20%): drug resistance to nucleoside reverse transcriptase inhibitors was seen in 10 cases, non-nucleoside reverse transcriptase inhibitors in two cases, and protease inhibitors in one case. For 9 children, the detection of the same resistance mutations in mothers' samples (6 among 10 available) and in newborn lymphocytes (6/8) suggests that the newborn was initially infected by a drug-resistant strain. Resistance variants were either transmitted from mother-to-child or selected during subsequent temporal exposure under suboptimal perinatal prophylaxis. Follow-up studies of the infants showed that the resistance pattern remained stable over time, regardless of antiretroviral therapy, suggesting the early cellular archiving of resistant viruses. The absence of resistance in the mother of the other three children (3/10) and neonatal lymphocytes (2/8) suggests that the newborns were infected by a wild-type strain without long-term persistence of resistance when suboptimal prophylaxis was stopped. Conclusion This study confirms the importance of early resistance genotyping of HIV-1-infected newborns. In most cases (75%), drug resistance was archived in the cellular reservoir and persisted during infancy, with or without antiretroviral treatment. This finding stresses the need for effective antiretroviral treatment of pregnant women. PMID:19765313

Trends in Transmission of Drug Resistance and Prevalence of Non-B Subtypes in Patients with Acute or Recent HIV-1 Infection in Barcelona in the Last 16 Years (1997-2012).

PubMed

Ambrosioni, Juan; Sued, Omar; Nicolas, David; Parera, Marta; López-Diéguez, María; Romero, Anabel; Agüero, Fernando; Marcos, María Ángeles; Manzardo, Christian; Zamora, Laura; Gómez-Carrillo, Manuel; Gatell, José María; Pumarola, Tomás; Miró, José María

2015-01-01

To evaluate the prevalence of transmitted drug resistance (TDR) and non-B subtypes in patients with acute/recent HIV-1 infection in Barcelona during the period 1997-2012. Patients from the "Hospital Clínic Primary HIV-1 Infection Cohort" with a genotyping test performed within 180 days of infection were included. The 2009 WHO List of Mutations for Surveillance of Transmitted HIV-1 Drug Resistance was used for estimating the prevalence of TDR and phylogenetic analysis for subtype determination. 189 patients with acute/recent HIV-1 infection were analyzed in 4 time periods (1997-2000, n=28; 2001-4, n=42; 2005-8, n=55 and 2009-12, n=64). The proportion of patients with acute/recent HIV-1 infection with respect to the total of newly HIV-diagnosed patients in our center increased over the time and was 2.18%, 3.82%, 4.15% and 4.55% for the 4 periods, respectively (p=0.005). The global prevalence of TDR was 9%, or 17.9%, 9.5%, 3.6% and 9.4% by study period (p=0.2). The increase in the last period was driven by protease-inhibitor and nucleoside-reverse-transcriptase-inhibitor resistance mutations while non-nucleoside-reverse-transcriptase inhibitor TDR and TDR of more than one family decreased. The overall prevalence of non-B subtypes was 11.1%, or 0%, 4.8%, 9.1% and 20.3 by study period (p=0.01). B/F recombinants, B/G recombinants and subtype F emerged in the last period. We also noticed an increase in the number of immigrant patients (p=0.052). The proportion of men-who-have-sex-with-men (MSM) among patients with acute/recent HIV-1 infection increased over the time (p=0.04). The overall prevalence of TDR in patients with acute/recent HIV-1 infection in Barcelona was 9%, and it has stayed relatively stable in recent years. Non-B subtypes and immigrants proportions progressively increased.

HIV-1 genetic diversity and antiretroviral drug resistance among individuals from Roraima state, northern Brazil

PubMed Central

Leão, Renato Augusto Carvalho; Granja, Fabiana; Naveca, Felipe Gomes

2017-01-01

The HIV-1 epidemic in Brazil has spread towards the Northern country region, but little is known about HIV-1 subtypes and prevalence of HIV strains with resistance mutations to antiretrovirals in some of the Northern states. HIV-1 protease (PR) and reverse transcriptase (RT) sequences were obtained from 73 treatment-naive and -experienced subjects followed between 2013 and 2014 at a public health reference unit from Roraima, the northernmost Brazilian state. The most prevalent HIV-1 clade observed in the study population was the subtype B (91%), followed by subtype C (9%). Among 12 HIV-1 strains from treatment-naïve patients, only one had a transmitted drug resistance mutation for NNRTI. Among 59 treatment-experienced patients, 12 (20%) harbored HIV-1 strains with acquired drug resistance mutations (ADRM) that reduce the susceptibility to two classes of antiretroviral drugs (NRTI and NNRTI or NRTI and PI), and five (8%) harbored HIV-1 strains with ADRM that reduced susceptibility to only one class of antiretroviral drugs (NNRTI or PI). No patients harboring HIV strains with reduced susceptibility to all three classes of antiretroviral drugs were detected. A substantial fraction of treatment-experienced patients with (63%) and without (70%) ADRM had undetectable plasma viral loads (<40 copies/ml) at the time of sampling. Among treatment-experienced with plasma viral loads above 2,000 copies/ml, 44% displayed no ADRM. This data showed that the HIV-1 epidemic in Roraima displayed a much lower level of genetic diversity and a lower prevalence of ADRM than that described in other Brazilian states. PMID:28301548

MIV-150 and zinc acetate combination provides potent and broad activity against HIV-1.

PubMed

Mizenina, Olga; Hsu, Mayla; Jean-Pierre, Ninochka; Aravantinou, Meropi; Levendosky, Keith; Paglini, Gabriela; Zydowsky, Thomas M; Robbiani, Melissa; Fernández-Romero, José A

2017-12-01

We previously showed that the combination of the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 with zinc acetate (ZA) formulated in a carrageenan (CG; MZC) gel provided macaques significant protection against vaginal simian-human immunodeficiency virus-RT (SHIV-RT) challenge, better than either MIV-150/CG or ZA/CG. The MZC gel was shown to be safe in a phase 1 clinical trial. Herein, we used in vitro approaches to study the antiviral properties of ZA and the MIV-150/ZA combination, compared to other NNRTIs. Like other NNRTIs, MIV-150 has EC 50 values in the subnanomolar to nanomolar range against wild type and NNRTI or RT-resistant HIVs. While less potent than NNRTIs, ZA was shown to be active in primary cells against laboratory-adapted and primary HIV-1 isolates and HIV-1 isolates/clones with NNRTI and RT resistance mutations, with EC 50 values between 20 and 110 μM. The MIV-150/ZA combination had a potent and broad antiviral activity in primary cells. In vitro resistance selection studies revealed that previously described NNRTI-resistant mutations were selected by MIV-150. ZA-resistant virus retained susceptibility to MIV-150 (and other RTIs) and MIV-150-selected virus remained sensitive to ZA. Notably, resistant virus was not selected when cultured in the presence of both ZA and MIV-150. This underscores the potency and breadth of the MIV-150/ZA combination, supporting preclinical macaque studies and the advancement of MZC microbicides into clinical testing.

Prevalence of etravirine (ETR)-RAMs at NNRTI failure and predictors of resistance to ETR in a large Italian resistance database (ARCA).

PubMed

Rusconi, S; Adorni, F; Bruzzone, B; Di Biagio, A; Meini, G; Callegaro, A; Punzi, G; Boeri, E; Pecorari, M; Monno, L; Gianotti, N; Butini, L; Galli, L; Polilli, E; Galli, M

2013-10-01

The prevalence of drug resistance associated with the failure of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and the predictors of resistance to Etravirine (ETR) were assessed in 2854 subjects: 39 < 18 (paediatric) and 2815 ≥ 18 (adult) years old. These subjects failed to respond to their current NNRTI treatment, were three-class experienced and had been exposed to NNRTI for ≥3 months. A total of 1827 adult (64.9%) and 32 paediatric subjects (82.1%) harboured the virus with at least one ETR mutation. V179I, Y181C and G190A were the most frequent mutations in both groups. A significantly increased risk of ETR resistance with all three algorithms (Monogram (MGR) >3, Tibotec (TBT) >2 and enhanced MGR (ENH) ≥4) emerged in the paediatric population. Multivariate analysis revealed an increased risk of developing TBT >2 for NNRTI exposure, ENH ≥4 for NNRTI and EFV exposure in paediatric subjects; NVP exposure and higher (≥3.5 log10) HIV-RNA values for all three algorithms in adult subjects, whereas CD4 ≥ 200/μL appeared to be protective. The risk of being ETR resistant was more than doubled for paediatric vs. adult subjects, probably due to a more extensive use of NNRTI and an incomplete virological control. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

Prevalence and resistance mutations of non-B HIV-1 subtypes among immigrants in Southern Spain along the decade 2000-2010

PubMed Central

2011-01-01

Background Most of the non-B HIV-1 subtypes are predominant in Sub-Saharan Africa and India although they have been found worldwide. In the last decade, immigration from these areas has increased considerably in Spain. The objective of this study was to evaluate the prevalence of non-B subtypes circulating in a cohort of HIV-1-infected immigrants in Seville, Southern Spain and to identify drug resistance-associated mutations. Methods Complete protease and first 220 codons of the reverse transcriptase coding regions were amplified and sequenced by population sequencing. HIV-1 subtypes were determined using Stanford University Drug Resistance Database, and phylogenetic analysis was performed comparing multiple reported sequences. Drug resistance mutations were defined according to the International AIDS Society-USA. Results From 2000 to 2010 a total of 1,089 newly diagnosed HIV-1-infected patients were enrolled in our cohort. Of these, 121 were immigrants, of which 98 had ethical approval and informed consent to include in our study. Twenty-nine immigrants (29/98, 29.6%) were infected with non-B subtypes, of which 15/29 (51.7%) were CRF02-AG, mostly from Sub-Saharan Africa, and 2/29 (6.9%) were CRF01-AE from Eastern Europe. A, C, F, J and G subtypes from Eastern Europe, Central-South America and Sub-Saharan Africa were also present. Some others harboured recombinant forms CRF02-AG/CRF01-AE, CRF2-AG/G and F/B, B/C, and K/G, in PR and RT-coding regions. Patients infected with non-B subtypes showed a high frequency of minor protease inhibitor resistance mutations, M36I, L63P, and K20R/I. Only one patient, CRF02_AG, showed major resistance mutation L90M. Major RT inhibitor resistance mutations K70R and A98G were present in one patient with subtype G, L100I in one patient with CRF01_AE, and K103N in another patient with CRF01_AE. Three patients had other mutations such as V118I, E138A and V90I. Conclusions The circulation of non-B subtypes has significantly increased in Southern Spain during the last decade, with 29.6% prevalence, in association with demographic changes among immigrants. This could be an issue in the treatment and management of these patients. Resistance mutations have been detected in these patients with a prevalence of 7% among treatment-naïve patients compared with the 21% detected among patients under HAART or during treatment interruption. PMID:21871090

Longitudinal Detection and Persistence of Minority Drug-Resistant Populations and Their Effect on Salvage Therapy

PubMed Central

Nishizawa, Masako; Matsuda, Masakazu; Hattori, Junko; Shiino, Teiichiro; Matano, Tetsuro; Heneine, Walid; Johnson, Jeffrey A.; Sugiura, Wataru

2015-01-01

Background Drug-resistant HIV are more prevalent and persist longer than previously demonstrated by bulk sequencing due to the ability to detect low-frequency variants. To clarify a clinical benefit to monitoring minority-level drug resistance populations as a guide to select active drugs for salvage therapy, we retrospectively analyzed the dynamics of low-frequency drug-resistant population in antiretroviral (ARV)-exposed drug resistant individuals. Materials and Methods Six HIV-infected individuals treated with ARV for more than five years were analyzed. These individuals had difficulty in controlling viremia, and treatment regimens were switched multiple times guided by standard drug resistance testing using bulk sequencing. To detect minority variant populations with drug resistance, we used a highly sensitive allele-specific PCR (AS-PCR) with detection thresholds of 0.3–2%. According to ARV used in these individuals, we focused on the following seven reverse transcriptase inhibitor-resistant mutations: M41L, K65R, K70R, K103N, Y181C, M184V, and T215F/Y. Results of AS-PCR were compared with bulk sequencing data for concordance and presence of additional mutations. To clarify the genetic relationship between low-frequency and high-frequency populations, AS-PCR amplicon sequences were compared with bulk sequences in phylogenetic analysis. Results The use of AS-PCR enabled detection of the drug-resistant mutations, M41L, K103N, Y181C, M184V and T215Y, present as low-frequency populations in five of the six individuals. These drug resistant variants persisted for several years without ARV pressure. Phylogenetic analysis indicated that pre-existing K103N and T215I variants had close genetic relationships with high-frequency K103N and T215I observed during treatment. Discussion and Conclusion Our results demonstrate the long-term persistence of drug-resistant viruses in the absence of drug pressure. The rapid virologic failures with pre-existing mutant viruses detectable by AS-PCR highlight the clinical importance of low-frequency drug-resistant viruses. Thus, our results highlight the usefulness of AS-PCR and support its expanded evaluation in ART clinical management. PMID:26360259

Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay.

PubMed

Huh, Hee Jae; Kim, Ji-Youn; Lee, Myoung-Keun; Lee, Nam Yong; Kim, Jong-Won; Ki, Chang-Seok

2016-12-01

Long-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. We evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. The analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. The limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6-93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67-0.90). The Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficacy and safety of etravirine (TMC125) in patients with highly resistant HIV-1: primary 24-week analysis.

PubMed

Nadler, Jeffrey P; Berger, Daniel S; Blick, Gary; Cimoch, Paul J; Cohen, Calvin J; Greenberg, Richard N; Hicks, Charles B; Hoetelmans, Richard M W; Iveson, Kathy J; Jayaweera, Dushyantha S; Mills, Anthony M; Peeters, Monika P; Ruane, Peter J; Shalit, Peter; Schrader, Shannon R; Smith, Stephen M; Steinhart, Corklin R; Thompson, Melanie; Vingerhoets, Johan H; Voorspoels, Ellen; Ward, Douglas; Woodfall, Brian

2007-03-30

TMC125-C223 is an open-label, partially blinded, randomized clinical trial to evaluate the efficacy and safety of two dosages of etravirine (TMC125), a non-nucleoside reverse transcriptase inhibitor (NNRTI) with activity against wild-type and NNRTI-resistant HIV-1. A total of 199 patients were randomly assigned 2: 2: 1 to twice-daily etravirine 400 mg, 800 mg and control groups, respectively. The primary endpoint was a change in viral load from baseline at week 24 in the intention-to-treat population. Patients had HIV-1 with genotypic resistance to approved NNRTIs and at least three primary protease inhibitor (PI) mutations. Etravirine groups received an optimized background of at least two approved antiretroviral agents [nucleoside reverse transcriptase inhibitors (NRTI) and/or lopinavir/ritonavir and/or enfuvirtide]. Control patients received optimized regimens of at least three antiretroviral agents (NRTIs or PIs and/or enfuvirtide). The mean change from baseline in HIV-1 RNA at week 24 was -1.04, -1.18 and -0.19 log10 copies/ml for etravirine 400 mg twice a day, 800 mg twice a day and the control group, respectively (P < 0.05 for both etravirine groups versus control). Etravirine showed no dose-related effects on safety and tolerability. No consistent pattern of neuropsychiatric symptoms was observed. There were few hepatic adverse events, and rashes were predominantly early onset and mild to moderate in severity. Etravirine plus an optimized background significantly reduced HIV-1-RNA levels from baseline after 24 weeks in patients with substantial NNRTI and PI resistance, and demonstrated a favorable safety profile compared with control.

Linking HIV and antiretroviral drug resistance surveillance in Peru: a model for a third-generation HIV sentinel surveillance.

PubMed

Lama, Javier R; Sanchez, Jorge; Suarez, Luis; Caballero, Patricia; Laguna, Alberto; Sanchez, Jose L; Whittington, William L H; Celum, Connie; Grant, Robert M

2006-08-01

HIV drug resistance surveillance is limited by recruitment and selection bias and by limited information regarding HIV incidence rates, secondary resistance, and treatment prevalence. A second-generation HIV sentinel surveillance among men who have sex with men (MSM), regardless of prior history of HIV screening, serostatus, or treatment, was conducted in Peru in 2002. Recent HIV infection was estimated using sensitive/less sensitive enzyme immunoassay testing. Genotypic resistance testing was performed. HIV prevalence was 13.9% (456 HIV positive of 3280 participants). HIV incidence was estimated to be 5.1 per 100 person-years (95% confidence interval: 3.1-8.3). Among 143 MSM who were aware of their HIV infection before testing, only 20 (14.0%) were receiving antiretrovirals (ARV). Mutations conferring ARV resistance were found in 12 (3.3%) of 359 treatment-naive and 5 (31.3%) of 16 treatment-experienced participants with successful genotyping. One recently infected man from Lima demonstrated 3-class multidrug resistance. The most frequently observed mutations in treatment-naive, chronically infected persons from Lima were M184V (1.7%), D30N (1.3%), L90M (1.3%), and L10I (1.3%). The prevalence of ARV resistance among treatment-naive MSM in Peru is low, reflecting limited access to treatment before 2004, and contrasts with the history of ARV treatment in developed countries, where high levels of nucleoside reverse transcriptase inhibitor resistance occurred before introduction of highly active antiretroviral therapy. Linking ARV resistance and HIV sentinel surveillance in developing settings is feasible and should be considered in third-generation HIV sentinel surveillance programs.

Telomerase reverse transcriptase (TERT) promoter mutation analysis of benign, malignant and reactive urothelial lesions reveals a subpopulation of inverted papilloma with immortalizing genetic change.

PubMed

Cheng, Liang; Davidson, Darrell D; Wang, Mingsheng; Lopez-Beltran, Antonio; Montironi, Rodolfo; Wang, Lisha; Tan, Puay-Hoon; MacLennan, Gregory T; Williamson, Sean R; Zhang, Shaobo

2016-07-01

To understand more clearly the genetic ontogeny of inverted papilloma of urinary bladder, we analysed telomerase reverse transcriptase (TERT) promoter mutation status in a group of 26 inverted papillomas in comparison with the mutation status of urothelial carcinoma with inverted growth (26 cases), conventional urothelial carcinoma (36 Ta non-invasive urothelial carcinoma, 35 T2 invasive urothelial carcinoma) and cystitis glandularis (25 cases). TERT promoter mutations in inverted papilloma, urothelial carcinoma with inverted growth, urothelial carcinoma and cystitis glandularis were found in 15% (four of 26), 58% (15 of 26), 63% (45 of 71) and 0% (none of 25), respectively. C228T mutations were the predominant mutations (97%) found in bladder tumours, while C250T aberrations occurred in approximately 3% of bladder tumours. In the inverted papilloma group, TERT mutation occurred predominantly in female patients (P = 0.006). Among urothelial carcinomas, TERT promoter mutation status did not correlate with gender, histological grade or pathological stage. TERT promoter mutations were found in 15% of inverted papillomas. Our data suggest that there is a subpopulation of inverted papilloma that shares a carcinogenetic pathway with urothelial carcinoma with inverted growth and conventional urothelial carcinomas. Caution is warranted in exploring TERT promoter mutation status as a screening or adjunct diagnostic test for bladder cancer. © 2015 John Wiley & Sons Ltd.

Antiretroviral drug resistance in HIV-1 therapy-naive patients in Cuba.

PubMed

Pérez, Lissette; Kourí, Vivian; Alemán, Yoan; Abrahantes, Yeisel; Correa, Consuelo; Aragonés, Carlos; Martínez, Orlando; Pérez, Jorge; Fonseca, Carlos; Campos, Jorge; Álvarez, Delmis; Schrooten, Yoeri; Dekeersmaeker, Nathalie; Imbrechts, Stijn; Beheydt, Gertjan; Vinken, Lore; Soto, Yudira; Álvarez, Alina; Vandamme, Anne-Mieke; Van Laethem, Kristel

2013-06-01

In Cuba, antiretroviral therapy rollout started in 2001 and antiretroviral therapy coverage has reached almost 40% since then. The objectives of this study were therefore to analyze subtype distribution, and level and patterns of drug resistance in therapy-naive HIV-1 patients. Four hundred and one plasma samples were collected from HIV-1 therapy-naive patients in 2003 and in 2007-2011. HIV-1 drug resistance genotyping was performed in the pol gene and drug resistance was interpreted according to the WHO surveillance drug-resistance mutations list, version 2009. Potential impact on first-line therapy response was estimated using genotypic drug resistance interpretation systems HIVdb version 6.2.0 and Rega version 8.0.2. Phylogenetic analysis was performed using Neighbor-Joining. The majority of patients were male (84.5%), men who have sex with men (78.1%) and from Havana City (73.6%). Subtype B was the most prevalent subtype (39.3%), followed by CRF20-23-24_BG (19.5%), CRF19_cpx (18.0%) and CRF18_cpx (10.3%). Overall, 29 patients (7.2%) had evidence of drug resistance, with 4.0% (CI 1.6%-4.8%) in 2003 versus 12.5% (CI 7.2%-14.5%) in 2007-2011. A significant increase in drug resistance was observed in recently HIV-1 diagnosed patients, i.e. 14.8% (CI 8.0%-17.0%) in 2007-2011 versus 3.8% (CI 0.9%-4.7%) in 2003 (OR 3.9, CI 1.5-17.0, p=0.02). The majority of drug resistance was restricted to a single drug class (75.8%), with 55.2% patients displaying nucleoside reverse transcriptase inhibitor (NRTI), 10.3% non-NRTI (NNRTI) and 10.3% protease inhibitor (PI) resistance mutations. Respectively, 20.7% and 3.4% patients carried viruses containing drug resistance mutations against NRTI+NNRTI and NRTI+NNRTI+PI. The first cases of resistance towards other drug classes than NRTI were only detected from 2008 onwards. The most frequent resistance mutations were T215Y/rev (44.8%), M41L (31.0%), M184V (17.2%) and K103N (13.8%). The median genotypic susceptibility score for the commonly prescribed first-line therapies was 2.5. This analysis emphasizes the need to perform additional surveillance studies to accurately assess the level of transmitted drug resistance in Cuba, as the extent of drug resistance might jeopardize effectiveness of first-line regimens prescribed in Cuba and might necessitate the implementation of baseline drug resistance testing. Copyright © 2013 Elsevier B.V. All rights reserved.

High prevalence of HIV-1 drug resistance among patients on first-line antiretroviral treatment in Lomé, Togo.

PubMed

Dagnra, Anoumou Y; Vidal, Nicole; Mensah, Akovi; Patassi, Akouda; Aho, Komi; Salou, Mounerou; Monleau, Marjorie; Prince-David, Mireille; Singo, Assétina; Pitche, Palokinam; Delaporte, Eric; Peeters, Martine

2011-06-10

With widespread use of antiretroviral (ARV) drugs in Africa, one of the major potential challenges is the risk of emergence of ARV drug-resistant HIV strains. Our objective is to evaluate the virological failure and genotypic drug-resistance mutations in patients receiving first-line highly active antiretroviral therapy (HAART) in routine clinics that use the World Health Organization public health approach to monitor antiretroviral treatment (ART) in Togo. Patients on HAART for one year (10-14 months) were enrolled between April and October 2008 at three sites in Lomé, the capital city of Togo. Plasma viral load was measured with the NucliSENS EasyQ HIV-1 assay (Biomérieux, Lyon, France) and/or a Generic viral load assay (Biocentric, Bandol, France). Genotypic drug-resistance testing was performed with an inhouse assay on plasma samples from patients with viral loads of more than 1000 copies/ml. CD4 cell counts and demographic data were also obtained from medical records. A total of 188 patients receiving first-line antiretroviral treatment were enrolled, and 58 (30.8%) of them experienced virologic failure. Drug-resistance mutations were present in 46 patients, corresponding to 24.5% of all patients enrolled in the study. All 46 patients were resistant to non-nucleoside reverse-transcriptase inhibitors (NNRTIs): of these, 12 were resistant only to NNRTIs, 25 to NNRTIs and lamivudine/emtricitabine, and eight to all three drugs of their ARV regimes. Importantly, eight patients were already predicted to be resistant to etravirine, the new NNRTI, and three patients harboured the K65R mutation, inducing major resistance to tenofovir. In Togo, efforts to provide access to ARV therapy for infected persons have increased since 2003, and scaling up of ART started in 2007. The high number of resistant strains observed in Togo shows clearly that the emergence of HIV drug resistance is of increasing concern in countries where ART is now widely used, and can compromise the long-term success of first- and second-line ART.

Indolyl aryl sulfones as HIV-1 non-nucleoside reverse transcriptase inhibitors: role of two halogen atoms at the indole ring in developing new analogues with improved antiviral activity.

PubMed

Regina, Giuseppe La; Coluccia, Antonio; Piscitelli, Francesco; Bergamini, Alberto; Sinistro, Anna; Cavazza, Antonella; Maga, Giovanni; Samuele, Alberta; Zanoli, Samantha; Novellino, Ettore; Artico, Marino; Silvestri, Romano

2007-10-04

Indolyl aryl sulfones bearing the 4,5-difluoro (10) or 5-chloro-4-fluoro (16) substitution pattern at the indole ring were potent inhibitors of HIV-1 WT and the NNRTI-resistant strains Y181C and K103N-Y181C. These compounds were highly effective against the 112 and the AB1 strains in lymphocytes and inhibited at nanomolar concentration the multiplication of the IIIBBa-L strain in macrophages. Compound 16 was exceptionally potent against RT WT and RTs carrying the K103N, Y181I, and L100I mutations.

Estimating HIV-1 Fitness Characteristics from Cross-Sectional Genotype Data

PubMed Central

Gopalakrishnan, Sathej; Montazeri, Hesam; Menz, Stephan; Beerenwinkel, Niko; Huisinga, Wilhelm

2014-01-01

Despite the success of highly active antiretroviral therapy (HAART) in the management of human immunodeficiency virus (HIV)-1 infection, virological failure due to drug resistance development remains a major challenge. Resistant mutants display reduced drug susceptibilities, but in the absence of drug, they generally have a lower fitness than the wild type, owing to a mutation-incurred cost. The interaction between these fitness costs and drug resistance dictates the appearance of mutants and influences viral suppression and therapeutic success. Assessing in vivo viral fitness is a challenging task and yet one that has significant clinical relevance. Here, we present a new computational modelling approach for estimating viral fitness that relies on common sparse cross-sectional clinical data by combining statistical approaches to learn drug-specific mutational pathways and resistance factors with viral dynamics models to represent the host-virus interaction and actions of drug mechanistically. We estimate in vivo fitness characteristics of mutant genotypes for two antiretroviral drugs, the reverse transcriptase inhibitor zidovudine (ZDV) and the protease inhibitor indinavir (IDV). Well-known features of HIV-1 fitness landscapes are recovered, both in the absence and presence of drugs. We quantify the complex interplay between fitness costs and resistance by computing selective advantages for different mutants. Our approach extends naturally to multiple drugs and we illustrate this by simulating a dual therapy with ZDV and IDV to assess therapy failure. The combined statistical and dynamical modelling approach may help in dissecting the effects of fitness costs and resistance with the ultimate aim of assisting the choice of salvage therapies after treatment failure. PMID:25375675

Epidemiological surveillance of HIV-1 transmitted drug resistance among newly diagnosed individuals in Shijiazhuang, northern China, 2014-2015.

PubMed

Wang, Xianfeng; Liu, Xiaosong; Li, Feng; Zhou, Hong; Li, Jiefang; Wang, Yingying; Liu, Lihua; Liu, Shujun; Feng, Yi; Wang, Ning

2018-01-01

The widespread use of antiretroviral therapy (ART) has led to considerable concerns about the prevalence of transmitted drug resistance (TDR). Sexual contact, particularly men who have sex with men (MSM) was the most prevalent form of HIV transmission in Shijiazhuang. Hence, we conducted an epidemiological surveillance study on TDR among newly diagnosed individuals who infected-HIV through sexual contact in from 2014-2015. Genotypic resistance mutations were defined using the WHO-2009 surveillance list. Potential impact on antiretroviral drug was predicted according to the Stanford HIV db program version 7.0. The role of transmission clusters in drug resistant strains was evaluated by phylogenetic and network analyses. In this study, 589 individuals were recruited and 542 samples were amplified and sequenced successfully. The over prevalence of TDR was 6.1%: 1.8% to nucleoside reverse transcriptase inhibitors (NRTIs), 2.0% to non- NRTIs (NNRTIs) and 2.4% to protease inhibitors (PIs), respectively. We did not find significant differences in the TDR prevalence by demographic and clinical characteristics (p > 0.05). Using network and phylogenetic analysis, almost 60.0% sequences were clustered together. Of these clusters, 2 included at least two individuals carrying the same resistance mutation, accounting for 21.2% (7/33) individuals with TDR. No significant difference was observed in the clustering rate between the individuals with and without TDR. We obtained a moderate level TDR rate in studied region. These findings enhance our understanding of HIV-1 drug resistance prevalence in Shijiazhuang, and may be helpful for the comprehensive prevention and control of HIV-1.

Early Virological Failure in Naive Human Immunodeficiency Virus Patients Receiving Saquinavir (Soft Gel Capsule)-Stavudine-Zalcitabine (MIKADO Trial) Is Not Associated with Mutations Conferring Viral Resistance

PubMed Central

Mouroux, M.; Yvon-Groussin, A.; Peytavin, G.; Delaugerre, C.; Legrand, M.; Bossi, P.; Do, B.; Trylesinski, A.; Diquet, B.; Dohin, E.; Delfraissy, J. F.; Katlama, C.; Calvez, V.

2000-01-01

The MIKADO trial was designed to evaluate the efficacy of stavudine-zalcitabine-saquinavir (soft gel capsule) [d4T-ddC-SQV(SGC)] in 36 naive patients (−3.3 log10 units at week 24 [W24]). Among the 29 patients remaining on d4T-ddC-SQV(SGC) until W24, 10 harbored a virological failure (viral load of >200 copies/ml at W24) (group 1). To determine the reasons for therapeutic failure, genotypic and phenotypic resistance test results and SQV concentrations in plasma were analyzed and compared to those in successfully treated patients (viral load of <200 copies/ml at W24) (group 2). Reverse transcriptase and protease genotypic analyses in group 1 revealed the acquisition of only one SQV-associated mutation (L90M) in only two patients. There was no significant increase in the 50 or 90% inhibitory concentration of SQV in patients with or without the L90M mutation. However, the fact that two patients developed an L90M mutation only 4 weeks after relapse points to the need for genotypic resistance testing in the context of an initial failure of the antiretroviral regimen. At W24, the median SQV concentration in group 1 (71 ng/ml) was significantly lower than in group 2 (475 ng/ml), and the plasma SQV concentration was correlated with the viral load at W24 (r = −0.5; P < 0.05) and with the drop in viral load between day 0 and W24 (r = −0.5; P < 0.01). These results and the fact that the plasma SQV concentrations in the two groups prior to relapse (W12) were not significantly different strongly suggest that the early failure of this combination is not due to viral resistance but to a lack of compliance, pharmacological variability, and drug interactions or a combination of these factors. PMID:10878071

Antiretroviral drug resistance and phylogenetic diversity of HIV-1 in Chile.

PubMed

Ríos, Maritza; Delgado, Elena; Pérez-Alvarez, Lucía; Fernández, Jorge; Gálvez, Paula; de Parga, Elena Vázquez; Yung, Verónica; Thomson, Michael M; Nájera, Rafael

2007-06-01

This study reports the analysis of human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) coding sequences from 136 HIV-1-infected subjects from Chile, 66 (49%) of them under antiretroviral (ARV) treatment. The prevalence of mutations conferring high or intermediate resistance levels to ARVs was 77% among treated patients and 2.5% among drug-naïve subjects. The distribution of resistance prevalence in treated patients by drug class was 61% to nucleoside RT inhibitors, 84% to nonnucleoside RT inhibitors, and 46% to PR inhibitors. Phylogenetic analysis revealed that 115 (85%) subjects were infected with subtype B viruses, 1 with a subtype F1 virus, and 20 (15%) carried BF intersubtype recombinants. Most BF recombinants grouped into two clusters, one related to CRF12_BF, while the other could represent a new circulating recombinant form (CRF). In conclusion, this is the first report analysing the prevalence of ARV resistance which includes patients under HAART from Chile. Additionally, phylogenetic analysis of the PR-RT coding sequences reveals the presence of BF intersubtype recombinants. (c) 2007 Wiley-Liss, Inc.

Viral Suppression and Resistance in a Cohort of Perinatally-HIV Infected (PHIV+) Pregnant Women

PubMed Central

Cruz, Maria Letícia; Santos, Edwiges; Benamor Teixeira, Maria de Lourdes; Poletti, Monica; Sousa, Carolina; Gouvea, Maria Isabel; Nielsen-Saines, Karin; João, Esaú

2016-01-01

Our objective was to describe viral suppression and antiretroviral (ARV) resistance mutations in an ongoing cohort of perinatally-infected HIV+ (PHIV+) pregnant women. Descriptive analysis was performed using SPSS 18.0. From 2011 to 2014, we followed 22 PHIV+ pregnant women. Median age at prenatal entry was 19 years (Interquartile range (IQR) 17.6–21.0); 86% had an AIDS diagnosis; 81% had disclosed their HIV status to partner 11. The median age at HIV diagnosis was 8.3 y (IQR 4.0–13.6), the median age at sexual debut was 16 years (IQR 14–18). At the time of prenatal care initiation, four (18%) were on their first antiretroviral treatment (ART), eight (36%) in their second regimen and nine (41%) in their third regimen or beyond, and one had no data. Seventeen of 22 (77%) had HIV-viral load (VL) > 50 copies/mL at prenatal care entry, 16 had a genotyping exam performed. Seventeen of 22 PHIV+ had VL results near delivery: 7/17 (41%) had VL < 50 copies/mL. Among those who had genotyping at prenatal entry, 11/16 (69%) had mutations associated with ARV resistance. The most frequent major mutations were K103N, M184V, T215, M41L, D67N at reverse transcriptase gene and M46, I54V and V82A at protease gene. No vertical transmissions occurred. Management of pregnancy among PHIV+ is challenging. Individualized ART are needed to achieve viral suppression in a highly ART-exposed subpopulation. PMID:27338425

Viral Suppression and Resistance in a Cohort of Perinatally-HIV Infected (PHIV+) Pregnant Women.

PubMed

Cruz, Maria Letícia; Santos, Edwiges; Benamor Teixeira, Maria de Lourdes; Poletti, Monica; Sousa, Carolina; Gouvea, Maria Isabel; Nielsen-Saines, Karin; João, Esaú

2016-06-07

Our objective was to describe viral suppression and antiretroviral (ARV) resistance mutations in an ongoing cohort of perinatally-infected HIV+ (PHIV+) pregnant women. Descriptive analysis was performed using SPSS 18.0. From 2011 to 2014, we followed 22 PHIV+ pregnant women. Median age at prenatal entry was 19 years (Interquartile range (IQR) 17.6-21.0); 86% had an AIDS diagnosis; 81% had disclosed their HIV status to partner 11. The median age at HIV diagnosis was 8.3 y (IQR 4.0-13.6), the median age at sexual debut was 16 years (IQR 14-18). At the time of prenatal care initiation, four (18%) were on their first antiretroviral treatment (ART), eight (36%) in their second regimen and nine (41%) in their third regimen or beyond, and one had no data. Seventeen of 22 (77%) had HIV-viral load (VL) > 50 copies/mL at prenatal care entry, 16 had a genotyping exam performed. Seventeen of 22 PHIV+ had VL results near delivery: 7/17 (41%) had VL < 50 copies/mL. Among those who had genotyping at prenatal entry, 11/16 (69%) had mutations associated with ARV resistance. The most frequent major mutations were K103N, M184V, T215, M41L, D67N at reverse transcriptase gene and M46, I54V and V82A at protease gene. No vertical transmissions occurred. Management of pregnancy among PHIV+ is challenging. Individualized ART are needed to achieve viral suppression in a highly ART-exposed subpopulation.

Synthesis and evaluation of "AZT-HEPT", "AZT-pyridinone", and "ddC-HEPT" conjugates as inhibitors of HIV reverse transcriptase.

PubMed

Pontikis, R; Dollé, V; Guillaumel, J; Dechaux, E; Note, R; Nguyen, C H; Legraverend, M; Bisagni, E; Aubertin, A M; Grierson, D S; Monneret, C

2000-05-18

To test the concept that HIV reverse transcriptase could be effectively inhibited by "mixed site inhibitors", a series of seven conjugates containing both a nucleoside analogue component (AZT 1, ddC 2) and a nonnucleoside type inhibitor (HEPT analogue 12, pyridinone 27) were synthesized and evaluated for their ability to block HIV replication. The (N-3 and C-5)AZT-HEPT conjugates 15, 22, and 23 displayed 2-5 microM anti-HIV activity, but they had no effect on the replication of HIV-2 or the HIV-1 strain with the Y181C mutation. The (C-5)AZT-pyridinone conjugates 34-37 were found to be inactive. In marked contrast, the ddC-HEPT molecule 26 displayed the same potency (EC(50) = 0.45 microM) against HIV-1 (wild type and the Y181C nevirapine-resistant strain) and HIV-2 in cell culture. No synergistic effect was observed for these bis-substrate inhibitors, suggesting that the two individual inhibitor components in these molecules do not bind simultaneously in their respective sites. Interestingly, however, the results indicate that the AZT-HEPT conjugates and the ddC-HEPT derivative 26 inhibit reverse transcriptase (RT) in an opposite manner. One explanation for this difference is that the former compounds interact preferentially with the hydrophobic pocket in RT, whereas 26 (after supposed triphosphorylation) inhibits RT through binding in the catalytic site.

Crizotinib resistance in acute myeloid leukemia with inv(2)(p23q13)/RAN binding protein 2 (RANBP2) anaplastic lymphoma kinase (ALK) fusion and monosomy 7.

PubMed

Takeoka, Kayo; Okumura, Atsuko; Maesako, Yoshitomo; Akasaka, Takashi; Ohno, Hitoshi

2015-03-01

This is the first report on the development of a p.G1269A mutation within the kinase domain (KD) of ALK after crizotinib treatment in RANBP2-ALK acute myeloid leukemia (AML). An elderly woman with AML with an inv(2)(p23q13)/RANBP2-ALK and monosomy 7 was treated with crizotinib. After a short-term hematological response and the restoration of normal hematopoiesis, she experienced a relapse of AML. Fluorescence in situ hybridization using the ALK break-apart probe confirmed the inv(2)(p23q13), while G-banded karyotyping revealed the deletion of a segment of the short arm of chromosome 1 [del(1)(p13p22)] after crizotinib therapy. The ALK gene carried a heterozygous mutation at the nucleotide position g.716751G>C within exon 25, causing the p.G1269A amino acid substitution within the ALK-KD. Reverse transcriptase PCR revealed that the mutated ALK allele was selectively transcribed and the mutation occurred in the ALK allele rearranged with RANBP2. As both the del(1)(p13p22) at the cytogenetic level and p.G1269A at the nucleotide level newly appeared after crizotinib treatment, it is likely that they were secondarily acquired alterations involved in crizotinib resistance. Although secondary genetic abnormalities in ALK are most frequently described in non-small cell lung cancers harboring an ALK alteration, this report suggests that an ALK-KD mutation can occur independently of the tumor cell type or fusion partner after crizotinib treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinoma.

PubMed

Han, Ji-Youn; Kim, Sun Hye; Lee, Yeon-Su; Lee, Seung-Youn; Hwang, Jung-Ah; Kim, Jin Young; Yoon, Sung Jin; Lee, Geon Kook

2014-08-01

To investigate the clinical utility of targeted next-generation sequencing (NGS) for predicting the responsiveness to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy, we compared the efficacy with conventional sequencing in never-smokers with lung adenocarcinoma (NSLAs). We obtained DNA from 48 NSLAs who received gefitinib or erlotinib for their recurrent disease after surgery. Sanger sequencing and peptide nucleic acid clamp polymerase chain reaction (PCR) were used to analyze EGFR, KRAS, BRAF, and PIK3CA mutations. We analyzed ALK, RET, and ROS1 rearrangements by fluorescent in situ hybridization or reverse transcriptase-PCR and quantitative real-time PCR. After molecular screening, Ion Torrent NGS was performed in 31 cases harboring only EGFR exon 19 deletions (19DEL), an L858R mutation, or none of the above mutations. The 31 samples were divided into four groups: (1) responders to EGFR-TKIs with only 19DEL or L858R (n=15); (2) primary resistance to EGFR-TKI with only 19DEL or L858R (n=4); (3) primary resistance to EGFR-TKI without any mutations (n=8); (4) responders to EGFR-TKI without any mutations (n=4). With NGS, all conventionally detected mutations were confirmed except for one L858R in group 2. Additional uncovered predictive mutations with NGS included one PIK3CA E542K in group 2, two KRAS (G12V and G12D), one PIK3CA E542K, one concomitant PIK3CA and EGFR L858R in group 3, and one EGFR 19DEL in group 4. Targeted NGS provided a more accurate and clinically useful molecular classification of NSLAs. It may improve the efficacy of EGFR-TKI therapy in lung cancer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection

PubMed Central

Giacobbi, Nicholas S.

2017-01-01

ABSTRACT Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. PMID:28507107

Cross-resistance to elvitegravir and dolutegravir in 502 patients failing on raltegravir: a French national study of raltegravir-experienced HIV-1-infected patients.

PubMed

Fourati, Slim; Charpentier, Charlotte; Amiel, Corinne; Morand-Joubert, Laurence; Reigadas, Sandrine; Trabaud, Mary-Anne; Delaugerre, Constance; Nicot, Florence; Rodallec, Audrey; Maillard, Anne; Mirand, Audrey; Jeulin, Hélène; Montès, Brigitte; Barin, Francis; Bettinger, Dominique; Le Guillou-Guillemette, Hélène; Vallet, Sophie; Signori-Schmuck, Anne; Descamps, Diane; Calvez, Vincent; Flandre, Philippe; Marcelin, Anne-Genevieve

2015-05-01

The objectives of this study were to determine the prevalence and patterns of resistance to integrase strand transfer inhibitors (INSTIs) in patients experiencing virological failure on raltegravir-based ART and the impact on susceptibility to INSTIs (raltegravir, elvitegravir and dolutegravir). Data were collected from 502 treatment-experienced patients failing a raltegravir-containing regimen in a multicentre study. Reverse transcriptase, protease and integrase were sequenced at failure for each patient. INSTI resistance-associated mutations investigated were those included in the last ANRS genotypic algorithm (v23). Among the 502 patients, at failure, median baseline HIV-1 RNA (viral load) was 2.9 log10 copies/mL. Patients had been previously exposed to a median of five NRTIs, one NNRTI and three PIs. Seventy-one percent harboured HIV-1 subtype B and the most frequent non-B subtype was CRF02_AG (13.3%). The most frequent mutations observed were N155H/S (19.1%), Q148G/H/K/R (15.4%) and Y143C/G/H/R/S (6.7%). At failure, viruses were considered as fully susceptible to all INSTIs in 61.0% of cases, whilst 38.6% were considered as resistant to raltegravir, 34.9% to elvitegravir and 13.9% to dolutegravir. In the case of resistance to raltegravir, viruses were considered as susceptible to elvitegravir in 11% and to dolutegravir in 64% of cases. High HIV-1 viral load at failure (P < 0.001) and low genotypic sensitivity score of the associated treatment with raltegravir (P < 0.001) were associated with the presence of raltegravir-associated mutations at failure. Q148 mutations were selected more frequently in B subtypes versus non-B subtypes (P = 0.004). This study shows that a high proportion of viruses remain susceptible to dolutegravir in the case of failure on a raltegravir-containing regimen. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Discovery of 3-{5-[(6-Amino-1H-pyrazolo[3,4-b]pyridine-3-yl)methoxy]-2-chlorophenoxy}-5-chlorobenzonitrile (MK-4965): A Potent, Orally Bioavailable HIV-1 Non-Nucleoside Reverse Transcriptase Inhibitor with Improved Potency against Key Mutant Viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tucker, Thomas J.; Sisko, John T.; Tynebor, Robert M.

2009-07-10

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have been shown to be a key component of highly active antiretroviral therapy (HAART). The use of NNRTIs has become part of standard combination antiviral therapies producing clinical outcomes with efficacy comparable to other antiviral regimens. There is, however, a critical issue with the emergence of clinical resistance, and a need has arisen for novel NNRTIs with a broad spectrum of activity against key HIV-1 RT mutations. Using a combination of traditional medicinal chemistry/SAR analyses, crystallography, and molecular modeling, we have designed and synthesized a series of novel, highly potent NNRTIs that possess broad spectrummore » antiviral activity and good pharmacokinetic profiles. Further refinement of key compounds in this series to optimize physical properties and pharmacokinetics has resulted in the identification of 8e (MK-4965), which has high levels of potency against wild-type and key mutant viruses, excellent oral bioavailability and overall pharmacokinetics, and a clean ancillary profile.« less

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

PubMed

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H; Yang, Rui; Killela, Patrick J; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Liang, Junbo; Wang, Sizhen; Jiao, Yuchen; Yan, Hai; Tao, Hou-Quan

2015-05-01

Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dynamic HIV-1 genetic recombination and genotypic drug resistance among treatment-experienced adults in northern Ghana.

PubMed

Nii-Trebi, Nicholas Israel; Brandful, James Ashun Mensah; Ibe, Shiro; Sugiura, Wataru; Barnor, Jacob Samson; Bampoh, Patrick Owiredu; Yamaoka, Shoji; Matano, Tetsuro; Yoshimura, Kazuhisa; Ishikawa, Koichi; Ampofo, William Kwabena

2017-11-01

There have been hardly any reports on the human immunodeficiency virus type 1 (HIV-1) drug-resistance profile from northern Ghana since antiretroviral therapy (ART) was introduced over a decade ago. This study investigated prevailing HIV-1 subtypes and examined the occurrence of drug resistance in ART-experienced patients in Tamale, the capital of the Northern Region of Ghana. A cross-sectional study was carried out on HIV-infected adult patients receiving first-line ART. HIV viral load (VL) and CD4 + T-cell counts were measured. The pol gene sequences were analysed for genotypic resistance by an in-house HIV-1 drug-resistance test; the prevailing HIV-1 subtypes were analysed in detail.Results/Key findings. A total of 33 subjects were studied. Participants comprised 11 males (33.3 %) and 22 (66.7 %) females, with a median age of 34.5 years [interquartile range (IQR) 30.0-40.3]. The median duration on ART was 12 months (IQR 8.0-24). Of the 24 subjects successfully genotyped, 10 (41.7 %) viruses possessed at least one mutation conferring resistance to nucleoside or non-nucleoside reverse-transcriptase inhibitors (NRTIs/NNRTIs). Two-class drug resistance to NRTI and NNRTI was mostly detected (25 %, 6/24). The most frequent mutations were lamivudine-resistance M184V and efavirenz/nevirapine-resistance K103N. HIV-1 subtype CRF02_AG was predominant (79.2 %). Other HIV-1 subtypes detected were G (8.3 %), A3 (4.2 %) and importantly two (8.3 %) unique HIV-1 recombinant forms with CRF02_AG/A3 mosaic. HIV-1 shows high genetic diversity and on-going viral genetic recombination in the study region. Nearly 42 % of the patients studied harboured a drug-resistant virus. The study underscores the need for continued surveillance of HIV-1 subtype diversity; and of drug-resistance patterns to guide selection of second-line regimens in northern Ghana.

Virological Failure and HIV-1 Drug Resistance Mutations among Naive and Antiretroviral Pre-Treated Patients Entering the ESTHER Program of Calmette Hospital in Cambodia

PubMed Central

Limsreng, Setha; Him, Sovanvatey; Nouhin, Janin; Hak, Chanroeurn; Srun, Chanvatey; Viretto, Gerald; Ouk, Vara; Delfraissy, Jean Francois; Ségéral, Olivier

2014-01-01

Introduction In resource limited settings, patients entering an antiretroviral therapy (ART) program comprise ART naive and ART pre-treated patients who may show differential virological outcomes. Methods This retrospective study, conducted in 2010–2012 in the HIV clinic of Calmette Hospital located in Phnom Penh (Cambodia) assessed virological failure (VF) rates and patterns of drug resistance of naive and pre-treated patients. Naive and ART pre-treated patients were included when a Viral Load (VL) was performed during the first year of ART for naive subjects or at the first consultation for pre-treated individuals. Patients showing Virological failure (VF) (>1,000 copies/ml) underwent HIV DR genotyping testing. Interpretation of drug resistance mutations was done according to 2013 version 23 ANRS algorithms. Results On a total of 209 patients, 164 (78.4%) were naive and 45 (21.5%) were ART pre-treated. Their median initial CD4 counts were 74 cells/mm3 (IQR: 30–194) and 279 cells/mm3 (IQR: 103–455) (p<0.001), respectively. Twenty seven patients (12.9%) exhibited VF (95% CI: 8.6–18.2%), including 10 naive (10/164, 6.0%) and 17 pre-treated (17/45, 37.8%) patients (p<0.001). Among these viremic patients, twenty-two (81.4%) were sequenced in reverse transcriptase and protease coding regions. Overall, 19 (86.3%) harbored ≥1 drug resistance mutations (DRMs) whereas 3 (all belonging to pre-treated patients) harbored wild-types viruses. The most frequent DRMs were M184V (86.3%), K103N (45.5%) and thymidine analog mutations (TAMs) (40.9%). Two (13.3%) pre-treated patients harbored viruses that showed a multi-nucleos(t)ide resistance including Q151M, K65R, E33A/D, E44A/D mutations. Conclusion In Cambodia, VF rates were low for naive patients but the emergence of DRMs to NNRTI and 3TC occurred relatively quickly in this subgroup. In pre-treated patients, VF rates were much higher and TAMs were relatively common. HIV genotypic assays before ART initiation and for ART pre-treated patients infection should be considered as well. PMID:25166019

A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template–primer

PubMed Central

Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S.; Rouzaut, Ana; Martinez-Irujo, Juan J.

2007-01-01

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PPi-dependent phosphorolysis catalysed by wild-type and AZT (3′-azido-3′-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template–primer (Kd=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template–primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues. PMID:17355225

A new strategy to inhibit the excision reaction catalysed by HIV-1 reverse transcriptase: compounds that compete with the template-primer.

PubMed

Cruchaga, Carlos; Anso, Elena; Font, María; Martino, Virginia S; Rouzaut, Ana; Martinez-Irujo, Juan J

2007-07-01

Inhibitors of the excision reaction catalysed by HIV-1 RT (reverse transcriptase) represent a promising approach in the fight against HIV, because these molecules would interfere with the main mechanism of resistance of this enzyme towards chain-terminating nucleotides. Only a limited number of compounds have been demonstrated to inhibit this reaction to date, including NNRTIs (non-nucleoside RT inhibitors) and certain pyrophosphate analogues. We have found previously that 2GP (2-O-galloylpunicalin), an antiviral compound extracted from the leaves of Terminalia triflora, was able to inhibit both the RT and the RNase H activities of HIV-1 RT without affecting cell proliferation or viability. In the present study, we show that 2GP also inhibited the ATP- and PP(i)-dependent phosphorolysis catalysed by wild-type and AZT (3'-azido-3'-deoxythymidine)-resistant enzymes at sub-micromolar concentrations. Kinetic and direct-binding analysis showed that 2GP was a non-competitive inhibitor against the nucleotide substrate, whereas it competed with the binding of RT to the template-primer (K(d)=85 nM). As expected from its mechanism of action, 2GP was active against mutations conferring resistance to NNRTIs and AZT. The combination of AZT with 2GP was highly synergistic when tested in the presence of pyrophosphate, indicating that the inhibition of RT-catalysed phosphorolysis was responsible for the synergy found. Although other RT inhibitors that compete with the template-primer have been described, this is the first demonstration that these compounds can be used to block the excision of chain terminating nucleotides, providing a rationale for their combination with nucleoside analogues.

Update on HIV resistance and resistance testing.

PubMed

Sebastian, Joseph; Faruki, Hawazin

2004-01-01

The introduction of highly active antiretroviral therapy, including a combination of antivirals directed at various steps in the viral life cycle, has led to significant decreases in morbidity and mortality associated with human immunodeficiency virus (HIV-1) infections. Despite the availability of numerous antivirals, many extensively treated patients gradually loose the ability to control viral replication because of development of antiviral resistance. Laboratory tests have been developed and validated to assist in recognizing such resistance and to help predict which antivirals may be more likely to control viral replication in a given patient. Both genotypic and phenotypic assays have been developed to assess HIV-1 antiviral resistance. The assay methodologies, including the advantages and disadvantages of each method, as well as the limitations of each method are reviewed. The ability to predict likely drug response from a genotype or a phenotype is continually evolving, and the more recently discovered mutation/drug resistance associations are discussed in terms of their implications for HIV resistance assays. To provide additional options for those who have developed resistance to all currently available drugs, new antivirals, such as the fusion inhibitors, are being developed. These new classes of antivirals block the HIV viral life cycle at sites other than reverse transcriptase and protease. Unique and novel resistance assays are being developed to measure HIV resistance to these new drugs. Copyright 2003 Wiley Periodicals, Inc.

In Vitro Cross-Resistance Profiles of Rilpivirine, Dapivirine, and MIV-150, Nonnucleoside Reverse Transcriptase Inhibitor Microbicides in Clinical Development for the Prevention of HIV-1 Infection.

PubMed

Giacobbi, Nicholas S; Sluis-Cremer, Nicolas

2017-07-01

Rilpivirine (RPV), dapivirine (DPV), and MIV-150 are in development as microbicides. It is not known whether they will block infection of circulating nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant human immunodeficiency virus type 1 (HIV-1) variants. Here, we demonstrate that the activity of DPV and MIV-150 is compromised by many resistant viruses containing single or double substitutions. High DPV genital tract concentrations from DPV ring use may block replication of resistant viruses. However, MIV-150 genital tract concentrations may be insufficient to inhibit many resistant viruses, including those harboring K103N or Y181C. Copyright © 2017 American Society for Microbiology.

Novel Codon Insert in HIV Type 1 Clade B Reverse Transcriptase Associated with Low-Level Viremia During Antiretroviral Therapy

PubMed Central

Gianella, Sara; Vazquez, Homero; Ignacio, Caroline; Zweig, Adam C.; Richman, Douglas D.; Smith, Davey M.

2014-01-01

Abstract We investigated the pol genotype in two phylogenetically and epidemiologically linked partners, who were both experiencing persistent low-level viremia during antiretroviral therapy. In one partner we identified a new residue insertion between codon 248 and 249 of the HIV-1 RNA reverse transcriptase (RT) coding region (HXB2 numbering). We then investigated the potential impact of identified mutations in RT and antiretroviral binding affinity using a novel computational approach. PMID:24020934

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

Suboptimal etravirine activity is common during failure of nevirapine-based combination antiretroviral therapy in a cohort infected with non-B subtype HIV-1.

PubMed

Taiwo, Babafemi; Chaplin, Beth; Penugonda, Sudhir; Meloni, Seema; Akanmu, Sulaimon; Gashau, Wadzani; Idoko, John; Adewole, Isaac; Murphy, Robert; Kanki, Phyllis

2010-04-01

The primary objective of this study was to estimate etravirine activity in a cohort of patients infected with non-B subtype HIV-1 and failing nevirapine-based therapy. Genotypic resistance testing was performed if viral load was >OR= 1,000 copies/ml after receiving at least six months of therapy. Suboptimal response to etravirine was predicted by a score >OR= 2.5 on the Tibotec weighting schema, >OR= 4 in the Monogram schema, or classification as high to low-level resistant by a modification of the Stanford HIVdb algorithm (Version 5.1.2). Bivariate and multivariate analyses were conducted to determine the risk factors for suboptimal etravirine activity. The patients (n=91) were receiving nevirapine and lamivudine plus stavudine (57.1%) or zidovudine (42.9%). Median duration of nevirapine exposure was 53 weeks (IQR 46-101 weeks). The most common etravirine resistance associated mutations were Y181C (42.9%), G190A (25.3%), H221Y (19.8%), A98G (18.7%), K101E (16.5%), and V90I (12.1%). Suboptimal etravirine activity was predicted in 47.3 to 56.0%. There were disparities in mutations listed in Tibotec versus Monogram Schemas. Predicted suboptimal activity was not associated with nucleoside reverse transcriptase inhibitor (NRTI) used, gender, pretreatment or current CD4 cell count or viral load, subtype or NRTI mutations. Etravirine has compromised activity in approximately half of the patients failing nevirapine-based first-line treatment in this cohort, which supports guidelines that caution against using it with NRTIs alone in such patients.

No Substantial Evidence for Sexual Transmission of Minority HIV Drug Resistance Mutations in Men Who Have Sex with Men.

PubMed

Chaillon, Antoine; Nakazawa, Masato; Wertheim, Joel O; Little, Susan J; Smith, Davey M; Mehta, Sanjay R; Gianella, Sara

2017-11-01

During primary HIV infection, the presence of minority drug resistance mutations (DRM) may be a consequence of sexual transmission, de novo mutations, or technical errors in identification. Baseline blood samples were collected from 24 HIV-infected antiretroviral-naive, genetically and epidemiologically linked source and recipient partners shortly after the recipient's estimated date of infection. An additional 32 longitudinal samples were available from 11 recipients. Deep sequencing of HIV reverse transcriptase (RT) was performed (Roche/454), and the sequences were screened for nucleoside and nonnucleoside RT inhibitor DRM. The likelihood of sexual transmission and persistence of DRM was assessed using Bayesian-based statistical modeling. While the majority of DRM (>20%) were consistently transmitted from source to recipient, the probability of detecting a minority DRM in the recipient was not increased when the same minority DRM was detected in the source (Bayes factor [BF] = 6.37). Longitudinal analyses revealed an exponential decay of DRM (BF = 0.05) while genetic diversity increased. Our analysis revealed no substantial evidence for sexual transmission of minority DRM (BF = 0.02). The presence of minority DRM during early infection, followed by a rapid decay, is consistent with the "mutation-selection balance" hypothesis, in which deleterious mutations are more efficiently purged later during HIV infection when the larger effective population size allows more efficient selection. Future studies using more recent sequencing technologies that are less prone to single-base errors should confirm these results by applying a similar Bayesian framework in other clinical settings. IMPORTANCE The advent of sensitive sequencing platforms has led to an increased identification of minority drug resistance mutations (DRM), including among antiretroviral therapy-naive HIV-infected individuals. While transmission of DRM may impact future therapy options for newly infected individuals, the clinical significance of the detection of minority DRM remains controversial. In the present study, we applied deep-sequencing techniques within a Bayesian hierarchical framework to a cohort of 24 transmission pairs to investigate whether minority DRM detected shortly after transmission were the consequence of (i) sexual transmission from the source, (ii) de novo emergence shortly after infection followed by viral selection and evolution, or (iii) technical errors/limitations of deep-sequencing methods. We found no clear evidence to support the sexual transmission of minority resistant variants, and our results suggested that minor resistant variants may emerge de novo shortly after transmission, when the small effective population size limits efficient purge by natural selection. Copyright © 2017 American Society for Microbiology.

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

NASA Astrophysics Data System (ADS)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

High HIV-1 Diversity and Prevalence of Transmitted Drug Resistance Among Antiretroviral-Naive HIV-Infected Pregnant Women from Rio de Janeiro, Brazil.

PubMed

Delatorre, Edson; Silva-de-Jesus, Carlos; Couto-Fernandez, José Carlos; Pilotto, Jose H; Morgado, Mariza G

2017-01-01

Antiretroviral (ARV) resistance mutations in human immunodeficiency virus type 1 (HIV-1) infection may reduce the efficacy of prophylactic therapy to prevent mother-to-child transmission (PMTCT) and future treatment options. This study evaluated the diversity and the prevalence of transmitted drug resistance (TDR) in protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene among 87 ARV-naive HIV-1-infected pregnant women from Rio de Janeiro, Brazil, between 2012 and 2015. The viral diversity comprised HIV-1 subtypes B (67.8%), F1 (17.2%), and C (4.6%); the circulating recombinant forms 12_BF (2.3%), 28/29_BF, 39_BF, 02_AG (1.1% each) and unique recombinants forms (4.5%). The overall prevalence of any TDR was 17.2%, of which 5.7% for nucleoside RT inhibitors, 5.7% for non-nucleoside RT inhibitors, and 8% for PR inhibitors. The TDR prevalence found in this population may affect the virological outcome of the standard PMTCT ARV-regimens, reinforcing the importance of continuous monitoring.

Molecular docking of (5E)-3-(2-aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione on HIV-1 reverse transcriptase: novel drug acting on enzyme.

PubMed

Seniya, Chandrabhan; Yadav, Ajay; Uchadia, Kuldeep; Kumar, Sanjay; Sagar, Nitin; Shrivastava, Priyanka; Shrivastava, Shilpi; Wadhwa, Gulshan

2012-01-01

The study of Human immunodeficiency virus (HIV) in humans and animal models in last 31 years suggested that it is a causative agent of AIDS. This causes serious pandemic public health concern globally. It was reported that the HIV-1 reverse transcriptase (RT) played a critical role in the life cycle of HIV. Therefore, inhibition of HIV-1RT enzyme is one of the major and potential targets in the treatment of AIDS. The enzyme (HIV-1RT) was successfully targeted by non nucleotide reverse transcriptase inhibitors (NNRTIs). But frequent application of NNRTIs led drug resistance mutation on HIV infections. Therefore, there is a need to search new NNRTIs with appropriate pharmacophores. For the purpose, a virtually screened 3D model of unliganded HIV-1RT (1DLO) was explored. The unliganded HIV-1RT (1DLO) was docked with 4-thiazolidinone and its derivatives (ChemBank Database) by using AutoDock4. The best seven docking solutions complex were selected and analyzed by Ligplot. The analysis showed that derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) has maximum potential against unliganded HIV-1RT (1DLO). The analysis was done on the basis of scoring and binding ability. The derivative (5E)-3-(2- aminoethyl)-5-(2- thienylmethylene)-1, 3-thiazolidine-2, 4-dione (CID 3087795) indicated minimum energy score and highest number of interactions with active site residue and could be a promising inhibitor for HIV-1 RT as Drug target.

Transmitted antiretroviral drug resistance in treatment naïve HIV-infected persons in London in 2011 to 2013.

PubMed

McFaul, Katie; Lim, Charlotte; Jones, Rachael; Asboe, David; Pozniak, Anton; Sonecha, Sonali; Boffito, Marta; Nwokolo, Nneka

2014-01-01

Previously published UK data on HIV transmitted drug resistance (TDR) shows that it ranges between 3 and 9.4% [1,2]. However, there are no recent data from populations where HIV transmission rates are increasing. The aim of this study was to assess the prevalence of TDR in untreated HIV-infected individuals attending three HIV specialist clinics under the HIV Directorate, Chelsea and Westminster Hospital and based throughout London - the Kobler Clinic, 56 Dean Street and West London Centre for Sexual Health. We included all patients with a HIV diagnosis, no history of antiretroviral therapy (ART) intake, attending one of the three clinics (Kobler (K), 56 Dean Street (DS) and West London (WL)), between 2011 and 2013 who started antiretrovirals. Reverse transcriptase (RT) and protease region sequencing was performed using Vircotype virtual phenotype resistance analysis. Drug resistance mutations were identified according to Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/). Among 1705 HIV-1-infected patients enrolled in the study, 1252 were males (919 were MSM), 107 were females and 346 had no gender recorded. Ethnicity was 51.1% white British/Irish/other, 6.1% African, 2.1% Caribbean, 2.8% Asian, 1.3% Indian/Pakistani/Bangladeshi, 4.2%, other, 3.2% not stated, and 29.2% unknown. 547 were from K (84.3% males, 48.3% MSM), 826 were from DS (84.3% males, 71.9% MSM), and 109 from WL (87.2% males, 56.0% MSM), 223 from other sites not specified. 77.5% (1321 of 1705) of patients had baseline viral resistance testing performed. Prevalence of primary resistance in those with a baseline viral resistance test was 13.5% overall: 19.3% in K, 14.9% in DS, and 14.7% in WL. The most common mutations detected were: NRTI: 184V, 215F, 41L; NNRTI 103N, 179D, 90I; PI 90M, 46I, and 82A. Among patients who tested with TDR, 79.1% had one single mutation, 18.7% and 2.2% exhibited dual or triple class-resistant viruses, respectively. This study across a large HIV Medicine Directorate reported an overall TDR prevalence which is higher than that previously published and with significant rates of NNRTI resistance at baseline.

HIV-1 Amino Acid Changes Among Participants With Virologic Failure: Associations With First-line Efavirenz or Atazanavir Plus Ritonavir and Disease Status

PubMed Central

Mollan, Katie; Daar, Eric S.; Sax, Paul E.; Balamane, Maya; Collier, Ann C.; Fischl, Margaret A.; Lalama, Christina M.; Bosch, Ronald J.; Tierney, Camlin; Katzenstein, David

2012-01-01

Background. Although specific human immunodeficiency virus type 1 (HIV-1) drug resistance mutations are well studied, little is known about cumulative amino acid changes, or how regimen and participant characteristics influence these changes. Methods. In the AIDS Clinical Trials Group randomized study A5202 of treatment-naive HIV-infected participants, cumulative HIV-1 amino acid changes from pretreatment to virologic failure were evaluated in protease and reverse transcriptase (RT) gene sequences. Results. Among 265 participants with virologic failure, those assigned atazanavir plus ritonavir (ATV/r) did not have significantly more protease changes compared with those assigned efavirenz (EFV) (P ≥ .13). In contrast, participants with virologic failure assigned EFV had more RT changes, including and excluding known resistance codons (P < .001). At pretreatment, lower CD4 cell count, major resistance, more amino acid mixtures (all P < .001), hepatitis C antibody negativity (P = .05), and black race/ethnicity (P = .02) were associated with more HIV-1 amino acid changes. Conclusions. Virologic failure following EFV-containing treatment was associated with more HIV-1 amino acid changes compared to failure of ATV/r-containing treatment. Furthermore, we show that non–drug resistance mutations occurred more frequently among those failing EFV, the clinical relevance of which warrants further investigation. Pretreatment immunologic status may play a role in viral evolution during treatment, as evidenced by increased amino acid changes among those with lower pretreatment CD4 count. Clinical Trials Registration. NCT00118898. PMID:23148287

Characteristics of a group of nonnucleoside reverse transcriptase inhibitors with structural diversity and potent anti-human immunodeficiency virus activity.

PubMed

Yang, S S; Fliakas-Boltz, V; Bader, J P; Buckheit, R W

1995-10-01

Current thrust in controlling the Acquired Immune Deficiency Syndrome (AIDS) focuses on antiviral drug development targeting the infection and replication of the human immunodeficiency virus (HIV), the causative agent of AIDS. To date, treatment of AIDS has relied on nucleoside reverse transcriptase inhibitors such as AZT, ddI, and ddC, which eventually become ineffective upon the emergence of resistant mutants bearing specific nucleotide substitutions. The Anti-AIDS Drug Screening Program of the NCI conducts and coordinates a high-capacity semi-robotic in vitro screening of synthetic or natural compounds submitted by academic, research and pharmaceutical institutions world-wide. About 10,000 synthetic compounds are screened annually for anti-HIV activity. Confirmed active agents are subjected to in-depth studies on range and mechanism of action. Emerging from this intense screening activity were a number of potentially promising categories of nonnucleoside reverse transcriptase inhibitors (NNRTI) with structural diversity but strong and reproducible anti-HIV activity. Over 2500 active compounds were evaluated for their inhibitory activity against a panel of both laboratory and clinical virus isolates in the appropriate established cell line or fresh human peripheral blood leukocyte and macrophage preparations. Out of these, 40 agents could be placed structurally in nine categories with an additional 16 unique compounds that share the characteristics of NNRTI. These NNRTIs were shown to inhibit reverse transcriptase enzymatically using homopolymeric or ribosomal RNA as templates. NNRTIs demonstrated similarity in their inhibitory pattern against the HIV-1 laboratory strains IIIB and RF, and an AZT-resistant strain; all were inactive against HIV-2. These compounds were further tested against NNRTI-resistant HIV-1 isolates. NNRTI-resistant HIV-1 isolates were selected and characterized with respect to the change(s) in the viral reverse transcriptase nucleotide sequence. Also, differential cross-resistance or sensitivity patterns to NNRTIs were studied in detail among NNRTI-resistant mutants. When tested in combination with AZT, all of the NNRTI's uniformly exhibited synergistic inhibition of HIV-1, suggesting that combination antiviral therapy of NNRTIs with AZT may be therapeutically promising for AIDS treatment.

A decade of HIV-1 drug resistance in the United States: trends and characteristics in a large protease/reverse transcriptase and co-receptor tropism database from 2003 to 2012.

PubMed

Paquet, Agnes C; Solberg, Owen D; Napolitano, Laura A; Volpe, Joseph M; Walworth, Charles; Whitcomb, Jeannette M; Petropoulos, Christos J; Haddad, Mojgan

2014-01-01

Drug resistance testing and co-receptor tropism determination are key components of the management of antiretroviral therapy for HIV-1-infected individuals. The purpose of this study was to examine trends of HIV-1 resistance and viral evolution in the past decade by surveying a large commercial patient testing database. Temporal trends of drug resistance, viral fitness and co-receptor usage among samples submitted for routine phenotypic and genotypic resistance testing to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), as well as for tropism determination were investigated. Within 62,397 resistant viruses reported from 2003 to 2012, we observed a decreasing trend in the prevalence of three-class resistance (from 25% to 9%) driven by decreased resistance to PIs (43% to 21%) and NRTIs (79% to 57%), while observing a slight increase in NNRTI resistance (68% to 75%). The prevalence of CXCR4-mediated entry among tropism testing samples (n=52,945) declined over time from 47% in 2007 to 40% in 2012. A higher proportion of CXCR4-tropic viruses was observed within samples with three-class resistance (50%) compared with the group with no resistance (36%). Decreased prevalence of three-class resistance and increased prevalence of one-class resistance was observed within samples reported between 2003 and 2012. The fraction of CXCR4-tropic viruses has decreased over time; however, CXCR4 usage was more prevalent among multi-class-resistant samples, which may be due to the more advanced disease stage of treatment-experienced patients. These trends have important implications for clinical practice and future drug discovery and development.

Prevalence in the USA of rilpivirine resistance-associated mutations in clinical samples and effects on phenotypic susceptibility to rilpivirine and etravirine.

PubMed

Picchio, Gaston R; Rimsky, Laurence T; Van Eygen, Veerle; Haddad, Mojgan; Napolitano, Laura A; Vingerhoets, Johan

2014-01-01

The prevalence of rilpivirine resistance-associated mutations (RAMs) in the USA, and their effect on phenotypic susceptibility to rilpivirine and etravirine, was evaluated in clinical samples from HIV-1-infected patients. In total, 15,991 samples submitted to Monogram Biosciences (South San Francisco, CA, USA) for routine resistance testing between January 2010 and June 2011 were assessed for the presence of known rilpivirine RAMs K101E/P, E138A/G/K/Q/R, V179L, Y181C/I/V, Y188L, H221Y, F227C and M230I/L; non-nucleoside reverse transcriptase inhibitor (NNRTI) RAMs K103N, L100I and L100I+K103N; and the nucleoside reverse transcriptase inhibitor (NRTI) RAMs M184I/V and their combinations with rilpivirine RAMs. Phenotypic susceptibility (PhenoSenseGT(®) assay; Monogram Biosciences) was evaluated, with reduced susceptibility defined as fold change (FC) in 50% inhibitory concentration (IC50)>2.0 for rilpivirine and FC>2.9 for etravirine. Of the 15,991 samples, 17% harboured ≥1 rilpivirine RAMs. The prevalence of most rilpivirine RAMs and combinations of NNRTI RAMs of interest was low (≤3%), except for Y181C (7%). Rilpivirine RAMs were often associated with reduced rilpivirine phenotypic susceptibility. Median FC values >2.0 were observed for clinical isolates with rilpivirine RAMs K101P, E138Q/R, Y181C/I/V, Y188L or M230L, and for the combination of E138K with M184I/V, and K101E with M184I. Most rilpivirine FC values >2.0 were associated with etravirine FC values >2.9 for individual rilpivirine RAMs and those combined with M184I/V. There was no relationship between the presence of K103N and rilpivirine FC. However, the L100I+K103N combination (without rilpivirine RAMs), at <2% prevalence, was associated with a rilpivirine FC>2.0. Based on 15,991 US clinical samples from HIV-1-infected patients, the frequency of most known rilpivirine RAMs apart from Y181C was low.

The role of phenylalanine-119 of the reverse transcriptase of mouse mammary tumour virus in DNA synthesis, ribose selection and drug resistance.

PubMed

Entin-Meer, Michal; Sevilya, Ziv; Hizi, Amnon

2002-10-15

Phe-119 in the reverse transcriptase (RT) of mouse mammary tumour virus (MMTV) is homologous with Tyr-115 in HIV type 1 (HIV-1) RT and to Phe-155 in murine leukaemia virus (MLV) RT. By mutating these residues in HIV-1 and MLV RTs (which are strict DNA polymerases) the enzymes were shown to function also as RNA polymerases. Owing to the uniqueness of MMTV as a type B retrovirus, we have generated a Phe-119-Val mutant of MMTV RT to study the involvement of this residue in affecting the catalytic features of this RT. The data presented here show that the mutant MMTV RT can incorporate both deoxyribonucleosides and ribonucleosides while copying either RNA or DNA. In addition, this mutant RT shows resistance to nucleoside analogues and an enhanced fidelity of DNA synthesis; all relative to the wild-type enzyme. The Phe-119-Val mutant is also different from the wild-type enzyme in its preference for most template primers tested and in its ability to synthesize DNA under non-processive and processive conditions. Overall, it is likely that the aromatic side chain of Phe-119 is located at the dNTP-binding site of MMTV RT and thus might be part of a putative "steric gate" that prevents the incorporation of nucleoside triphosphates. Since the only three-dimensional structures of RTs published so far are those of HIV-1 and MLV, it is likely that MMTV RT folds quite similarly to these RTs.

A computational chemistry perspective on the current status and future direction of hepatitis B antiviral drug discovery.

PubMed

Morgnanesi, Dante; Heinrichs, Eric J; Mele, Anthony R; Wilkinson, Sean; Zhou, Suzanne; Kulp, John L

2015-11-01

Computational chemical biology, applied to research on hepatitis B virus (HBV), has two major branches: bioinformatics (statistical models) and first-principle methods (molecular physics). While bioinformatics focuses on statistical tools and biological databases, molecular physics uses mathematics and chemical theory to study the interactions of biomolecules. Three computational techniques most commonly used in HBV research are homology modeling, molecular docking, and molecular dynamics. Homology modeling is a computational simulation to predict protein structure and has been used to construct conformers of the viral polymerase (reverse transcriptase domain and RNase H domain) and the HBV X protein. Molecular docking is used to predict the most likely orientation of a ligand when it is bound to a protein, as well as determining an energy score of the docked conformation. Molecular dynamics is a simulation that analyzes biomolecule motions and determines conformation and stability patterns. All of these modeling techniques have aided in the understanding of resistance mutations on HBV non-nucleos(t)ide reverse-transcriptase inhibitor binding. Finally, bioinformatics can be used to study the DNA and RNA protein sequences of viruses to both analyze drug resistance and to genotype the viral genomes. Overall, with these techniques, and others, computational chemical biology is becoming more and more necessary in hepatitis B research. This article forms part of a symposium in Antiviral Research on "An unfinished story: from the discovery of the Australia antigen to the development of new curative therapies for hepatitis B." Copyright © 2015 Elsevier B.V. All rights reserved.

A comparison of somatic mutational spectra in healthy study populations from Russia, Sweden and USA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noori, P; Hou, S; Jones, I M

Comparison of mutation spectra at the hypoxanthine-phosphoribosyl transferase (HPRT) gene of peripheral blood T lymphocytes may provide insight into the aetiology of somatic mutation contributing to carcinogenesis and other diseases. To increase knowledge of mutation spectra in healthy people, we have analyzed HPRT mutant T-cells of 50 healthy Russians originally recruited as controls for a study of Chernobyl clean-up workers (Jones et al. Radiation Res. 158, 2002, 424). Reverse transcriptase polymerase chain reactions and DNA sequencing identified 161 independent mutations among 176 thioguanine resistant mutants. Forty (40) mutations affected splicing mechanisms and 27 deletions or insertions of 1 to 60more » nucleotides were identified. Ninety four (94) single base substitutions were identified, including 62 different mutations at 55 different nucleotide positions, of which 19 had not previously been reported in human T-cells. Comparison of this base substitution spectrum with mutation spectra in a USA (Burkhart-Schultz et al. Carcinogenesis 17, 1996, 1871) and two Swedish populations (Podlutsky et al, Carcinogenesis 19, 1998, 557, Podlutsky et al. Mutation Res. 431, 1999, 325) revealed similarity in the type, frequency and distribution of mutations in the four spectra, consistent with aetiologies inherent in human metabolism. There were 15-19 identical mutations in the three pair-wise comparisons of Russian with USA and Swedish spectra. Intriguingly, there were 21 mutations unique to the Russian spectrum, and comparison by the Monte Carlo method of Adams and Skopek (J. Mol. Biol. 194, 1987, 391) indicated that the Russian spectrum was different from both Swedish spectra (P=0.007, 0.002) but not different from the USA spectrum (P=0.07), when Bonferroni correction for multiple comparisons was made (p < 0.008 required for significance). Age and smoking did not account for these differences. Other factors causing mutational differences need to be explored.« less

The molecular basis of resilience to the effect of the Lys103Asn mutation in non-nucleoside HIV-1 reverse transcriptase inhibitors studied by targeted molecular dynamics simulations.

PubMed

Rodríguez-Barrios, Fátima; Balzarini, Jan; Gago, Federico

2005-05-25

A series of targeted molecular dynamics simulations have been carried out in an attempt to assess the effect that the common Lys103Asn mutation in HIV-1 reverse transcriptase (RT) has on the binding of three representative non-nucleoside RT inhibitors (NNRTI), nevirapine, efavirenz, and etravirine. We have shown previously that, in the absence of an incoming inhibitor, creation of the NNRTI binding pocket is hampered due to the existence of a hydrogen bond between the side chains of Asn103 and Tyr188 for which no equivalent exists in the wild-type enzyme. As an extension of this work, we now apply the same methodology to drive the enzyme's conformation from the unbound state to the drug-bound state in the presence of the NNRTI. The location of each drug outside the binding pocket was determined by an automated docking program, and steering into the binding pocket followed a route that is likely to represent the actual entrance pathway. The additional hurdle to inhibitor entry imposed by the extra Asn103-Tyr188 hydrogen bond is seen to affect each NNRTI differently, with the ability to disrupt this interaction increasing in the order etravirine > efavirenz > or = nevirapine, in good accord with the experimental findings. This coherent picture strongly suggests that attempts to overcome resistance through structure-based drug design may be considerably more successful if dynamic structural aspects of the type studied here are considered, particularly in cases where binding energy-based structure-activity relationship methods are unable to provide the required information.

Computational challenges of structure-based approaches applied to HIV.

PubMed

Forli, Stefano; Olson, Arthur J

2015-01-01

Here, we review some of the opportunities and challenges that we face in computational modeling of HIV therapeutic targets and structural biology, both in terms of methodology development and structure-based drug design (SBDD). Computational methods have provided fundamental support to HIV research since the initial structural studies, helping to unravel details of HIV biology. Computational models have proved to be a powerful tool to analyze and understand the impact of mutations and to overcome their structural and functional influence in drug resistance. With the availability of structural data, in silico experiments have been instrumental in exploiting and improving interactions between drugs and viral targets, such as HIV protease, reverse transcriptase, and integrase. Issues such as viral target dynamics and mutational variability, as well as the role of water and estimates of binding free energy in characterizing ligand interactions, are areas of active computational research. Ever-increasing computational resources and theoretical and algorithmic advances have played a significant role in progress to date, and we envision a continually expanding role for computational methods in our understanding of HIV biology and SBDD in the future.

High-Sequence Diversity and Rapid Virus Turnover Contribute to Higher Rates of Coreceptor Switching in Treatment-Experienced Subjects with HIV-1 Viremia.

PubMed

Nedellec, Rebecca; Herbeck, Joshua T; Hunt, Peter W; Deeks, Steven G; Mullins, James I; Anton, Elizabeth D; Reeves, Jacqueline D; Mosier, Donald E

2017-03-01

Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5.

HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

PubMed

de Mulder, M; York, V A; Wiznia, A A; Michaud, H A; Nixon, D F; Holguin, A; Rosenberg, M G

2014-03-01

With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/μL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (< 50 copies/mL) and 40.5% had a CD4 count of > 500 cells/μL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection. © 2013 British HIV Association.

Molecular alterations in endometrial and ovarian clear cell carcinomas: clinical impacts of telomerase reverse transcriptase promoter mutation.

PubMed

Huang, Hsien-Neng; Chiang, Ying-Cheng; Cheng, Wen-Fang; Chen, Chi-An; Lin, Ming-Chieh; Kuo, Kuan-Ting

2015-02-01

Recently, mutations of telomerase reverse transcriptase (TERT) promoter were found in several types of cancer. A few reports demonstrate TERT promoter mutations in ovarian clear cell carcinomas but endometrial clear cell carcinoma has not been studied. The aims of this study were to compare differences of molecular alterations and clinical factors, and identify their prognostic impact in endometrial and ovarian clear cell carcinomas. We evaluated mutations of the TERT promoter and PIK3CA, expression of ARID1A, and other clinicopathological factors in 56 ovarian and 14 endometrial clear cell carcinomas. We found that TERT promoter mutations were present in 21% (3/14) of endometrial clear cell carcinomas and 16% (9/56) of ovarian clear cell carcinomas. Compared with ovarian clear cell carcinomas, endometrial clear cell carcinomas showed older mean patient age (P<0.001), preserved ARID1A immunoreactivity (P=0.017) and infrequent PIK3CA mutation (P=0.025). In ovarian clear cell carcinomas, TERT promoter mutations were correlated with patient age >45 (P=0.045) and preserved ARID1A expression (P=0.003). In cases of endometrial clear cell carcinoma, TERT promoter mutations were not statistically associated with any other clinicopathological factors. In ovarian clear cell carcinoma patients with early FIGO stage (stages I and II), TERT promoter mutation was an independent prognostic factor and correlated with a shorter disease-free survival and overall survival (P=0.015 and 0.009, respectively). In recurrent ovarian clear cell carcinoma patients with early FIGO stage, TERT promoter mutations were associated with early relapse within 6 months (P=0.018). We concluded that TERT promoter mutations were present in endometrial and ovarian clear cell carcinomas. Distinct molecular alteration patterns in endometrial and ovarian clear cell carcinomas implied different processes of tumorigenesis in these morphologically similar tumors. In ovarian clear cell carcinoma of early FIGO stage, patients with TERT promoter mutation require close follow-up during the initial 6 months following chemotherapy.

Novel Acylguanidine-Based Inhibitor of HIV-1

PubMed Central

Mwimanzi, Philip; Tietjen, Ian; Miller, Scott C.; Shahid, Aniqa; Cobarrubias, Kyle; Kinloch, Natalie N.; Baraki, Bemuluyigza; Richard, Jonathan; Finzi, Andrés; Fedida, David; Brumme, Zabrina L.

2016-01-01

ABSTRACT The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24Gag production was unaffected, but virion release (measured as extracellular p24Gag) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1. PMID:27512074

HIV-1 Variants and Drug Resistance in Pregnant Women from Bata (Equatorial Guinea): 2012-2013.

PubMed

Alvarez, Patricia; Fernández McPhee, Carolina; Prieto, Luis; Martín, Leticia; Obiang, Jacinta; Avedillo, Pedro; Vargas, Antonio; Rojo, Pablo; Benito, Agustín; Ramos, José Tomás; Holguín, África

2016-01-01

This is the first study describing drug resistance mutations (DRM) and HIV-1 variants among infected pregnant women in Equatorial Guinea (GQ), a country with high (6.2%) and increasing HIV prevalence. Dried blood spots (DBS) were collected from November 2012 to December 2013 from 69 HIV-1 infected women participating in a prevention of mother-to-child transmission program in the Hospital Regional of Bata and Primary Health Care Centre María Rafols, Bata, GQ. The transmitted (TDR) or acquired (ADR) antiretroviral drug resistance mutations at partial pol sequence among naive or antiretroviral therapy (ART)-exposed women were defined following WHO or IAS USA 2015 lists, respectively. HIV-1 variants were identified by phylogenetic analyses. A total of 38 of 69 HIV-1 specimens were successfully amplified and sequenced. Thirty (79%) belonged to ART-experienced women: 15 exposed to nucleoside reverse transcriptase inhibitors (NRTI) monotherapy, and 15 to combined ART (cART) as first regimen including two NRTI and one non-NRTI (NNRTI) or one protease inhibitor (PI). The TDR rate was only found for PI (3.4%). The ADR rate was 37.5% for NNRTI, 8.7% for NRTI and absent for PI or NRTI+NNRTI. HIV-1 group M non-B variants caused most (97.4%) infections, mainly (78.9%) recombinants: CRF02_AG (55.2%), CRF22_A101 (10.5%), subtype C (10.5%), unique recombinants (5.3%), and A3, D, F2, G, CRF06_cpx and CRF11_cpx (2.6% each). The high rate of ADR to retrotranscriptase inhibitors (mainly to NNRTIs) observed among pretreated pregnant women reinforces the importance of systematic DRM monitoring in GQ to reduce HIV-1 resistance transmission and to optimize first and second-line ART regimens when DRM are present.

Selection of drug-resistant HIV-1 during the early phase of viral decay is uncommon in treatment-naïve patients initiated on a three- or four-drug antiretroviral regimen including lamivudine.

PubMed

Bergroth, Tobias; Ekici, Halime; Gisslén, Magnus; Loes, Sabine Kinloch-de; Goh, Li-Ean; Freedman, Andrew; Lampe, Fiona; Johnson, Margaret A; Sönnerborg, Anders

2009-01-01

Therapy failure due to drug resistance development is a common phenomenon in HIV-infected patients. However, when the drug pressure leads to the earliest selection of drug-resistant HIV-1 populations is still unclear. In this study, the extent to which selection of the HIV-1 reverse transcriptase M184I/V mutations occur during the initial phase of viral decay in treatment-naïve HIV-1 infected patients receiving antiretroviral therapy (ART) was examined. Plasma virus from three cohorts of treatment-naïve patients initiating quadruple (n = 43), triple (n = 14) or dual (n = 15) lamivudine-containing ART were analyzed for M184I/V during the first 6 months of therapy using direct sequencing and a sensitive selective real-time PCR method. Among quadruple ART patients, who all were treated at primary HIV-1 infection, only one patient developed M184V after 6 weeks of therapy, having had wild-type virus at baseline. No mutations were found in chronically infected patients on triple ART. In patients on dual therapy, M184I/V mutants were found frequently. Selection of M184I/V mutants was found to be rare during the initial phase of viral decay after initiation of ART in adherent patients given a three or four-drug combination, in contrast to those receiving a less potent regimen. The results suggest that triple and quadruple lamivudine + PI or PI/r containing ART given to treatment-naïve adherent patients is potent enough to prevent development of resistance during the first months of therapy.

Role of the K101E Substitution in HIV-1 Reverse Transcriptase in Resistance to Rilpivirine and Other Nonnucleoside Reverse Transcriptase Inhibitors

PubMed Central

Xu, Hong-Tao; Colby-Germinario, Susan P.; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J.

2013-01-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV. PMID:24002090

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

PubMed

Xu, Hong-Tao; Colby-Germinario, Susan P; Huang, Wei; Oliveira, Maureen; Han, Yingshan; Quan, Yudong; Petropoulos, Christos J; Wainberg, Mark A

2013-11-01

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Base modifications affecting RNA polymerase and reverse transcriptase fidelity.

PubMed

Potapov, Vladimir; Fu, Xiaoqing; Dai, Nan; Corrêa, Ivan R; Tanner, Nathan A; Ong, Jennifer L

2018-06-20

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Genotypic Characterization of Human Immunodeficiency Virus Type 1 Derived from Antiretroviral Drug-Treated Individuals Residing in Earthquake-Affected Areas in Nepal.

PubMed

Negi, Bharat Singh; Kotaki, Tomohiro; Joshi, Sunil Kumar; Bastola, Anup; Nakazawa, Minato; Kameoka, Masanori

2017-09-01

Molecular epidemiological data on human immunodeficiency virus type 1 (HIV-1) are limited in Nepal and have not been available in areas affected by the April 2015 earthquake. Therefore, we conducted a genotypic study on HIV-1 genes derived from individuals on antiretroviral therapy residing in 14 districts in Nepal highly affected by the earthquake. HIV-1 genomic fragments were amplified from 40 blood samples of HIV treatment-failure individuals, and a sequencing analysis was performed on these genes. In the 40 samples, 29 protease, 32 reverse transcriptase, 25 gag, and 21 env genes were sequenced. HIV-1 subtyping revealed that subtype C (84.2%, 32/38) was the major subtype prevalent in the region, while CRF01_AE (7.9%, 3/38) and other recombinant forms (7.9%, 3/38) were also detected. In addition, major drug resistance mutations were identified in 21.9% (7/32) of samples, indicating the possible emergence of HIV-1 drug resistance in earthquake-affected areas in Nepal.

High-Sequence Diversity and Rapid Virus Turnover Contribute to Higher Rates of Coreceptor Switching in Treatment-Experienced Subjects with HIV-1 Viremia

PubMed Central

Nedellec, Rebecca; Herbeck, Joshua T.; Hunt, Peter W.; Deeks, Steven G.; Mullins, James I.; Anton, Elizabeth D.; Reeves, Jacqueline D.

2017-01-01

Abstract Coreceptor switching from CCR5 to CXCR4 is common during chronic HIV-1 infection, but is even more common in individuals who have failed antiretroviral therapy (ART). Prior studies have suggested rapid mutation and/or recombination of HIV-1 envelope (env) genes during coreceptor switching. We compared the functional and genotypic changes in env of viruses from viremic subjects who had failed ART just before and after coreceptor switching and compared those to viruses from matched subjects without coreceptor switching. Analysis of multiple unique functional env clones from each subject revealed extensive diversity at both sample time points and rapid diversification of sequences during the 4-month interval in viruses from both 9 subjects with coreceptor switching and 15 control subjects. Only two subjects had envs with evidence of recombination. Three findings distinguished env clones from subjects with coreceptor switching from controls: (1) lower entry efficiency via CCR5; (2) longer V1/V2 regions; and (3), lower nadir CD4 T cell counts during prior years of infection. Most of these subjects harbored virus with lower replicative capacity associated with protease (PR) and/or reverse transcriptase inhibitor resistance mutations, and the extensive diversification tended to lead either to improved entry efficiency via CCR5 or the gain of entry function via CXCR4. These results suggest that R5X4 or X4 variants emerge from a diverse, low-fitness landscape shaped by chronic infection, multiple ART resistance mutations, the availability of target cells, and reduced entry efficiency via CCR5. PMID:27604829

KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

PubMed Central

Lin, Yu-Lin; Ou, Da-Liang; Lin, Liang-In; Tseng, Li-Hui; Chang, Yih-Leong; Yeh, Kun-Huei; Cheng, Ann-Lii

2012-01-01

Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation. PMID:23209813

Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs.

PubMed

Ding, Feng; Miao, Xi-Li; Li, Yan-Xia; Dai, Jin-Fen; Yu, Hong-Gang

2016-01-01

The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. This research aimed to determine the clinical and virological features of the rare pattern. A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. The amino acid variability within major hydrophilic region, especially the "a" determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the "a" determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Telomerase reverse transcriptase (TERT) - enhancer of zeste homolog 2 (EZH2) network regulates lipid metabolism and DNA damage responses in glioblastoma.

PubMed

Ahmad, Fahim; Patrick, Shruti; Sheikh, Touseef; Sharma, Vikas; Pathak, Pankaj; Malgulwar, Prit Benny; Kumar, Anupam; Joshi, Shanker Datt; Sarkar, Chitra; Sen, Ellora

2017-12-01

Elevated expression of enhancer of zeste homolog 2 (EZH2), a histone H3K27 methyltransferase, was observed in gliomas harboring telomerase reverse transcriptase (TERT) promoter mutations. Given the known involvement of TERT and EZH2 in glioma progression, the correlation between the two and subsequently its involvement in metabolic programming was investigated. Inhibition of human telomerase reverse transcriptase either pharmacologically or through genetic manipulation not only decreased EZH2 expression, but also (i) abrogated FASN levels, (ii) decreased de novo fatty acid accumulation, and (iii) increased ataxia-telangiectasia-mutated (ATM) phosphorylation levels. Conversely, diminished TERT and FASN levels upon siRNA-mediated EZH2 knockdown indicated a positive correlation between TERT and EZH2. Interestingly, ATM kinase inhibitor rescued TERT inhibition-mediated decrease in FASN and EZH2 levels. Importantly, TERT promoter mutant tumors exhibited greater microsatellite instability, heightened FASN levels and lipid accumulation. Coherent with in vitro findings, pharmacological inhibition of TERT by costunolide decreased lipid accumulation and elevated ATM expression in heterotypic xenograft glioma mouse model. By bringing TERT-EZH2 network at the forefront as driver of dysregulated metabolism, our findings highlight the non-canonical but distinct role of TERT in metabolic reprogramming and DNA damage responses in glioblastoma. © 2017 International Society for Neurochemistry.

The differential importance of mutations within AmpD in cephalosporin resistance of Enterobacter aerogenes and Enterobacter cloacae.

PubMed

Babouee Flury, Baharak; Ellington, Matthew J; Hopkins, Katie L; Turton, Jane F; Doumith, Michel; Woodford, Neil

2016-11-01

Mechanisms leading to carbapenem and cephalosporin resistance were sought in Enterobacter aerogenes isolates that were highly resistant to carbapenems but had no known carbapenemase. Results were compared with recent work examining carbapenem-resistant Enterobacter cloacae. Eighteen carbapenem-resistant E. aerogenes were screened for known β-lactamase and carbapenemase genes, and novel carbapenemases were sought in whole-genome sequencing (WGS) data of the three most resistant isolates. For all isolates, ampC, ampR, ampD and the porin genes omp35 and omp36 were investigated by Sanger sequencing or from available WGS data. Expression of ampC and porin genes was measured in comparison with cephalosporin- and carbapenem-susceptible control strains by reverse transcriptase PCR, with porin translation also detected by SDS-PAGE. Loss of Omp35, primarily due to decreased transcription (up to 250×), was observed in ertapenem-resistant isolates (MICs ≥ 2 mg/L), whereas meropenem resistance (MICs ≥ 4 mg/L) was observed in those isolates also showing decreased or no production of Omp36. Loss of Omp36 was due to combinations of premature translation termination or reduced transcription. In contrast to E. cloacae, cephalosporin resistance in E. aerogenes was not associated with lesions in AmpD. High-level cefepime resistance (MIC = 32 mg/L) was caused by a novel modification in the H-10 helix of AmpC in one isolate. The differential importance of AmpD lesions in cephalosporin resistance in E. cloacae and E. aerogenes underlines the differences between these contrasting members of the Enterobacter genus. Porin loss resulted in high-level carbapenem resistance with gradual loss of Omp36, which led to high-level meropenem resistance. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Molecular epidemiology of co-infection with hepatitis B virus and human immunodeficiency virus (HIV) among adult patients in Harare, Zimbabwe.

PubMed

Baudi, Ian; Iijima, Sayuki; Chin'ombe, Nyasha; Mtapuri-Zinyowera, Sekesai; Murakami, Shuko; Isogawa, Masanori; Hachiya, Atsuko; Iwatani, Yasumasa; Tanaka, Yasuhito

2017-02-01

The objective of this study was to determine the prevalence of co-infection with hepatitis B virus (HBV) and human immunodeficiency virus (HIV) and the genetic characteristics of both viruses among pre-HIV-treatment patients in Harare, Zimbabwe. This cross-sectional survey involved 176 remnant plasma samples collected from consenting HIV patients (median age 35 [18-74]) between June and September 2014. HBV seromarkers were determined by high-sensitivity chemiluminescence assays. Molecular evolutionary analyses were conducted on the basal core promoter/precore (BCP/PC) and S regions of HBV, as well as part of the HIV pol region. Of the 176 participants (65.7% female), 19 (10.8%) were positive for HBsAg (median 0.033 IU/ml (IQR 0.01-415). The HBsAg incidence was higher in men than women (P = 0.009). HBsAg-positive subjects had lower median CD4 counts (P = 0.016). HBV DNA was detectable in 12 HBsAg-positive samples (median 3.36 log cp/ml (2.86-4.51), seven being amplified and sequenced. All isolates were subgenotype A1 without HBV drug resistance mutations but each had at least one BCP/PC mutation. PreS deletion mutants and small S antigen variants M133I/T and D144G were identified. Of the 164 HIV isolates successfully genotyped, 163 (99.4%) were HIV-1 subtype C and only one was HIV-1 subtype F1. Sixteen (9.8%) had at least one drug resistance mutation, predominantly non-nucleoside reverse transcriptase inhibitor-related mutations, observed mostly among female participants. This study shows that co-infection with HBV is present among HIV patients enrolling into HIV care in Zimbabwe, suggesting that HBV screening and monitoring programmes be strengthened in this context. J. Med. Virol. 89:257-266, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

High Rates of Baseline Drug Resistance and Virologic Failure Among ART-naive HIV-infected Children in Mali.

PubMed

Crowell, Claudia S; Maiga, Almoustapha I; Sylla, Mariam; Taiwo, Babafemi; Kone, Niaboula; Oron, Assaf P; Murphy, Robert L; Marcelin, Anne-Geneviève; Traore, Ban; Fofana, Djeneba B; Peytavin, Gilles; Chadwick, Ellen G

2017-11-01

Limited data exist on drug resistance and antiretroviral treatment (ART) outcomes in HIV-1-infected children in West Africa. We determined the prevalence of baseline resistance and correlates of virologic failure (VF) in a cohort of ART-naive HIV-1-infected children <10 years of age initiating ART in Mali. Reverse transcriptase and protease genes were sequenced at baseline (before ART) and at 6 months. Resistance was defined according to the Stanford HIV Genotypic Resistance database. VF was defined as viral load ≥1000 copies/mL after 6 months of ART. Logistic regression was used to evaluate factors associated with VF or death >1 month after enrollment. Post hoc, antiretroviral concentrations were assayed on baseline samples of participants with baseline resistance. One-hundred twenty children with a median age 2.6 years (interquartile range: 1.6-5.0) were included. Eighty-eight percent reported no prevention of mother-to-child transmission exposure. At baseline, 27 (23%), 4 (3%) and none had non-nucleoside reverse transcriptase inhibitor (NNRTI), nucleoside reverse transcriptase inhibitor or protease inhibitor resistance, respectively. Thirty-nine (33%) developed VF and 4 died >1 month post-ART initiation. In multivariable analyses, poor adherence [odds ratio (OR): 6.1, P = 0.001], baseline NNRTI resistance among children receiving NNRTI-based ART (OR: 22.9, P < 0.001) and protease inhibitor-based ART initiation among children without baseline NNRTI resistance (OR: 5.8, P = 0.018) were significantly associated with VF/death. Ten (38%) with baseline resistance had detectable levels of nevirapine or efavirenz at baseline; 7 were currently breastfeeding, but only 2 reported maternal antiretroviral use. Baseline NNRTI resistance was common in children without reported NNRTI exposure and was associated with increased risk of treatment failure. Detectable NNRTI concentrations were present despite few reports of maternal/infant antiretroviral use.

Combination nucleoside/nucleotide reverse transcriptase inhibitors for treatment of HIV infection.

PubMed

Akanbi, Maxwell O; Scarsi, Kimberly K; Scarci, Kimberly; Taiwo, Babafemi; Murphy, Robert L

2012-01-01

The combination of two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs) and a third agent from another antiretroviral class is currently recommended for initial antiretroviral therapy. In general, N(t)RTIs remain relevant in subsequent regimens. There are currently six nucleoside reverse transcriptase inhibitors and one nucleotide reverse transcriptase inhibitor drug entities available, and several formulations that include two or more N(t)RTIs in a fixed-dose combination. These entities have heterogeneous pharmacological and clinical properties. Accordingly, toxicity, pill burden, dosing frequency, potential drug-drug interaction, preexisting antiretroviral drug resistance and comorbid conditions should be considered when constructing a regimen. This approach is critical in order to optimize virologic efficacy and clinical outcomes. This article reviews N(t)RTI combinations used in the treatment of HIV-infected adults. The pharmacological properties of each N(t)RTI, and the clinical trials that have influenced treatment guidelines are discussed. It is likely that N(t)RTIs will continue to dominate the global landscape of HIV treatment and prevention, despite emerging interest in N(t)RTI-free combination therapy. Clinical domains where only few alternatives to N(t)RTIs exist include treatment of HIV/HBV coinfection and HIV-2. There is a need for novel N(t)RTIs with enhanced safety and resistance profiles compared with current N(t)RTIs.

HIV-1 RT Inhibitors with a Novel Mechanism of Action: NNRTIs that Compete with the Nucleotide Substrate

PubMed Central

Maga, Giovanni; Radi, Marco; Gerard, Marie-Aline; Botta, Maurizio; Ennifar, Eric

2010-01-01

HIV-1 reverse transcriptase (RT) inhibitors currently used in antiretroviral therapy can be divided into two classes: (i) nucleoside analog RT inhibitors (NRTIs), which compete with natural nucleoside substrates and act as terminators of proviral DNA synthesis, and (ii) non-nucleoside RT inhibitors (NNRTIs), which bind to a hydrophobic pocket close to the RT active site. In spite of the efficiency of NRTIs and NNRTIs, the rapid emergence of multidrug-resistant mutations requires the development of new RT inhibitors with an alternative mechanism of action. Recently, several studies reported the discovery of novel non-nucleoside inhibitors with a distinct mechanism of action. Unlike classical NNRTIs, they compete with the nucleotide substrate, thus forming a new class of RT inhibitors: nucleotide-competing RT inhibitors (NcRTIs). In this review, we discuss current progress in the understanding of the peculiar behavior of these compounds. PMID:21994659

Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

PubMed

Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

2016-07-01

Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being from Singapore.

High Prevalence of HIV Drug Resistance Among Newly Diagnosed Infants Aged <18 Months: Results From a Nationwide Surveillance in Nigeria.

PubMed

Inzaule, Seth C; Osi, Samuels J; Akinbiyi, Gbenga; Emeka, Asadu; Khamofu, Hadiza; Mpazanje, Rex; Ilesanmi, Oluwafunke; Ndembi, Nicaise; Odafe, Solomon; Sigaloff, Kim C E; Rinke de Wit, Tobias F; Akanmu, Sulaimon

2018-01-01

WHO recommends protease-inhibitor-based first-line regimen in infants because of risk of drug resistance from failed prophylaxis used in prevention of mother-to-child transmission (PMTCT). However, cost and logistics impede implementation in sub-Saharan Africa, and >75% of children still receive nonnucleoside reverse transcriptase inhibitor-based regimen (NNRTI) used in PMTCT. We assessed the national pretreatment drug resistance prevalence of HIV-infected children aged <18 months in Nigeria, using WHO-recommended HIV drug resistance surveillance protocol. We used remnant dried blood spots collected between June 2014 and July 2015 from 15 early infant diagnosis facilities spread across all the 6 geopolitical regions of Nigeria. Sampling was through a probability proportional-to-size approach. HIV drug resistance was determined by population-based sequencing. Overall, in 48% of infants (205 of 430) drug resistance mutations (DRM) were detected, conferring resistance to predominantly NNRTIs (45%). NRTI and multiclass NRTI/NNRTI resistance were present at 22% and 20%, respectively, while resistance to protease inhibitors was at 2%. Among 204 infants with exposure to drugs for PMTCT, 57% had DRMs, conferring NNRTI resistance in 54% and multiclass NRTI/NNRTI resistance in 29%. DRMs were also detected in 34% of 132 PMTCT unexposed infants. A high frequency of PDR, mainly NNRTI-associated, was observed in a nationwide surveillance among newly diagnosed HIV-infected children in Nigeria. PDR prevalence was equally high in PMTCT-unexposed infants. Our results support the use of protease inhibitor-based first-line regimens in HIV-infected young children regardless of PMTCT history and underscore the need to accelerate implementation of the newly disseminated guideline in Nigeria.

Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

PubMed

Taniguchi, Naohiro; Murakami, Hiroshi

2017-01-01

Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.

CROI 2017: Advances in Antiretroviral Therapy

PubMed Central

Jones, Joyce; Taylor, Barbara S.; Tieu, Hong-Van; Wilkin, Timothy J.

2017-01-01

The 2017 Conference on Retroviruses and Opportunistic Infections (CROI) featured exciting preclinical data on investigational antiretroviral agents with good in vitro efficacy and long half-lives. Investigational medications, including bictegravir, demonstrated excellent efficacy and tolerability, as did dual-agent therapy with dolutegravir paired with rilpivirine or with lamivudine. Dolutegravir monotherapy proved inadvisable due to virologic failure and resistance. The gap between high- and low-income settings along the HIV care continuum is narrowing, with Zimbabwe, Malawi, and Zambia approaching the 90-90-90 targets established by the joint United Nations Programme on HIV/AIDS (UNAIDS), whereas communities in the Southern United States are falling behind. Innovative strategies to improve outcomes include 2-way text messaging, home-based HIV testing, peer navigation, and New York City's realignment of services into comprehensive sexual health programs. A high prevalence of resistance was documented in low- and middle-income settings and policy considerations were modeled to address increasing resistance rates. Novel resistance mutations to integrase strand transfer inhibitors and nucleoside analogue reserve transcriptase inhibitors were identified, but the clinical implications are unclear and require further investigation. Several studies provided insights on dosing and safety of antiretroviral therapy to prevent mother-to-child transmission through pharmacokinetic analysis. A special session devoted to Zika virus included a study of its effects on the central nervous system and a promising animal study of a Zika vaccine. PMID:28598790

Difference in drug resistance patterns between minor HIV-1 populations in cerebrospinal fluid and plasma.

PubMed

Bergroth, T; Ekici, H; Gisslén, M; Hagberg, L; Sönnerborg, A

2009-02-01

The aim of the study was to determine to what extent unique drug resistance patterns appear in minor and major HIV-1 quasispecies in cerebrospinal fluid (CSF) as compared with blood. Forty-four plasma and CSF samples from 13 multi-treatment-experienced patients, seven of whom provided longitudinal samples, were included in the study. The subjects had failed antiretroviral therapy including lamivudine. The reverse transcriptase (RT) gene was examined by selective real-time polymerase chain reaction (SPCR), which can detect M184I/V mutants down to 0.2% of the viral population. SPCR revealed differences at amino acid position 184 in the plasma/CSF populations in 12 paired samples from eight patients. One plasma sample was positive by SPCR where direct sequencing showed wild-type M184. The other 11 paired samples showed quantitative differences in the mixed populations of the mutant or wild-type M184 quasispecies. Differences in other resistance-associated mutations between plasma and CSF viruses were also found by direct sequencing. In multi-treatment-experienced patients with therapy failure, differences in drug resistance patterns were found frequently between plasma and CSF in both minor and major viral populations. To what extent this was a true biological phenomenon remains to be established, and the clinical relevance of these findings is yet to be determined.

Antiretroviral resistance at virological failure in the NEAT 001/ANRS 143 trial: raltegravir plus darunavir/ritonavir or tenofovir/emtricitabine plus darunavir/ritonavir as first-line ART.

PubMed

Lambert-Niclot, S; George, E C; Pozniak, A; White, E; Schwimmer, C; Jessen, H; Johnson, M; Dunn, D; Perno, C F; Clotet, B; Plettenberg, A; Blaxhult, A; Palmisano, L; Wittkop, L; Calvez, V; Marcelin, A G; Raffi, F

2016-04-01

To describe the pattern of drug resistance at virological failure in the NEAT001/ANRS143 trial (first-line treatment with ritonavir-boosted darunavir plus either tenofovir/emtricitabine or raltegravir). Genotypic testing was performed at baseline for reverse transcriptase (RT) and protease genes and for RT, protease and integrase (IN) genes for patients with a confirmed viral load (VL) >50 copies/mL or any single VL >500 copies/mL during or after week 32. A resistance test was obtained for 110/805 (13.7%) randomized participants qualifying for resistance analysis (61/401 of participants in the raltegravir arm and 49/404 of participants in the tenofovir/emtricitabine arm). No resistance-associated mutation (RAM) was observed in the tenofovir/emtricitabine plus darunavir/ritonavir arm, and all further analyses were limited to the raltegravir plus darunavir arm. In this group, 15/55 (27.3%) participants had viruses with IN RAMs (12 N155H alone, 1 N155H + Q148R, 1 F121Y and 1 Y143C), 2/53 (3.8%) with nucleotide analogue RT inhibitor RAMs (K65R, M41L) and 1/57 (1.8%) with primary protease RAM (L76V). The frequency of IN mutations at failure was significantly associated with baseline VL: 7.1% for a VL of <100,000 copies/mL, 25.0% for a VL of ≥100,000 copies/mL and <500,000 copies/mL and 53.8% for a VL of ≥500,000 copies/mL (PTREND = 0.007). Of note, 4/15 participants with IN RAM had a VL < 200 copies/mL at time of testing. In the NEAT001/ANRS143 trial, there was no RAM at virological failure in the standard tenofovir/emtricitabine plus darunavir/ritonavir regimen, contrasting with a rate of 29.5% (mostly IN mutations) in the raltegravir plus darunavir/ritonavir NRTI-sparing regimen. The cumulative risk of IN RAM after 96 weeks of follow-up in participants initiating ART with raltegravir plus darunavir/ritonavir was 3.9%. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cancer-associated TERT promoter mutations abrogate telomerase silencing

PubMed Central

Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

2015-01-01

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells. DOI: http://dx.doi.org/10.7554/eLife.07918.001 PMID:26194807

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

Changes from 2000 to 2009 in the Prevalence of HIV-1 Containing Drug Resistance-associated Mutations from Antiretroviral Therapy-naïve, HIV-1-infected Patients in the United States.

PubMed

Ross, Lisa L; Shortino, Denise; Shaefer, Mark S

2018-05-05

Background: Pre-existing HIV drug resistance can jeopardize first-line antiretroviral therapy (ART) success. Changes in the prevalence of drug-resistance-associated mutations (DRMs) were analyzed from HIV-infected, ART-naïve, United States (USA) individuals seeking ART treatment from 2000-2009. Methods: HIV DRM data from 3,829 ART-naïve subjects were analyzed by year of sample collection using International Antiviral Society (IAS-USA) and World Health Organization (WHO) "surveillance" DRM definitions; minor IAS-USA-defined DRMs were excluded. Results: IAS-USA DRM prevalence between 2000-2009 was 14%; beginning with 8% in 2000, 13% in 2009. The greatest incidence was observed in 2007 (17%). Overall, IAS-USA-defined non-nucleoside reverse transcriptase (NNRTI) DRMs were 9.5%; NRTI: 4% and major protease inhibitor (PI):3%. The most frequently detected IAS-USA-defined DRMs by class were NNRTI: K103N/S (4%); NRTI: M41L (1.5%) and PI: L90M (1%). Overall WHO-defined DRM prevalence was 13% (5% in 2000; 13% in 2009). By class, NNRTI prevalence was 6%, NRTI: 6%, and PI: 3.2%. The most frequent WHO-defined DRMs were NRTI: codon T215 (3.0%); NNRTI: K103N/S (4%) and PI: L90M (1%). WHO-defined NNRTI DRMs declined significantly (p=0.0412) from 2007 to 2009. The overall prevalence of HIV-1-containing major IAS-USA or WHO-defined DRMs to ≥2 or ≥3 classes was 2% and <1%, respectively. The prevalence of HIV-1 with WHO-defined dual or triple-class resistance significantly declined (p=0.0461) from 2008 (4%) to 2009 (<1%). Conclusions: In this USA cohort, prevalence of HIV-1 DRMs increased from 2000 onwards, peaked between 2005-2007 and then declined in 2008-2009; the detection of WHO-defined dual or triple-class DRM similarly decreased from 2008 to 2009.

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Role of Ligand Reorganization and Conformational Restraints on the Binding Free Energies of DAPY Non-Nucleoside Inhibitors to HIV Reverse Transcriptase

PubMed Central

Gallicchio, Emilio

2012-01-01

The results of computer simulations of the binding of etravirine (TMC125) and rilpivirine (TMC278) to HIV reverse transcriptase are reported. It is confirmed that consistent binding free energy estimates are obtained with or without the application of torsional restraints when the free energies of imposing the restraints are taken into account. The restraints have a smaller influence on the thermodynamics and apparent kinetics of binding of TMC125 compared to the more flexible TMC278 inhibitor. The concept of the reorganization free energy of binding is useful to understand and categorize these effects. Contrary to expectations, the use of conformational restraints did not consistently enhance convergence of binding free energy estimates due to suppression of binding/unbinding pathways and due to the influence of rotational degrees of freedom not directly controlled by the restraints. Physical insights concerning the thermodynamic driving forces for binding and the role of “jiggling” and “wiggling” motion of the ligands are discussed. Based on these insights we conclude that an ideal inhibitor, if chemically realizable, would possess the electrostatic charge distribution of TMC125, so as to form strong interactions with the receptor, and the larger and more flexible substituents of TMC278, so as to minimize reorganization free energy penalties and the effects of resistance mutations, suitably modified, as in TMC125, so as to disfavor the formation of non-binding competent extended conformations when free in solution. PMID:22708073

Structure of HIV-1 nonnucleoside reverse transcriptase inhibitors derivatives of N-benzyl-benzimidazole with different substituents in position 4

NASA Astrophysics Data System (ADS)

Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

2010-01-01

The constant development of new drugs against HIV-1 is necessary due to global expansion of AIDS and HIV-1 drug resistance. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic drugs in AIDS therapy. The crystal structures of six nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) derivatives of N-benzyl-benzimidazole are reported here. The investigated compounds belong to the group of so called "butterfly like" inhibitors with characteristic two π-electron moieties with an angled orientation. The structural data show the influence of the substituents of the benzimidazole ring on the geometry of the molecule and correlation between the structure of the inhibitor and its biological activity.

Incorporation of deoxyribonucleotides and ribonucleotides by a dNTP-binding cleft mutated reverse transcriptase in hepatitis B virus core particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Hee-Young; Kim, Hye-Young; Jung, Jaesung

2008-01-05

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate {sup 32}P-ribonucleotides, but not HBV coremore » particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.« less

Rilpivirine versus etravirine validity in NNRTI-based treatment failure in Thailand.

PubMed

Teeranaipong, Phairote; Sirivichayakul, Sunee; Mekprasan, Suwanna; Ruxrungtham, Kiat; Putcharoen, Opass

2014-01-01

Etravirine (ETR) and rilpivirine (RPV) are the second-generation non-nucleoside reverse transcriptase inhibitors (NNRTI) for treatment of HIV-1 infection. Etravirine is recommended for patients with virologic failure from first generation NNRTI-based regimen [1]. RPV has profile with similar properties to ETR but this agent is approved for treatment-naïve patients [2]. In Thailand, ETR is approximately 45 times more expensive than RPV. We aimed to study the patterns of genotypic resistance and possibility of using RPV in patients with virologic failure from two common NNRTI-based regimens: efavirenz (EFV)- or nevirapine (NVP)-based regimen. Data of clinical samples with confirmed virologic failure during 2003-2010 were reviewed. We selected the samples from patients who failed EFV- or NVP-based regimen. Resistance-associated mutations (RAMs) were determined by IAS-USA Drug Resistance Mutations. DUET, Monogram scoring system and Stanford Genotypic Resistance Interpretation were applied to determine the susceptibility of ETR and RPV. A total of 2086 samples were analyzed. Samples from 1482 patients with virologic failure from NVP-based regimen treatment failure (NVP group) and 604 patients with virologic failure from EFV-based regimen treatment failure (EFV group) were included. 95% of samples were HIV-1 CRF01_AE subtype. Approximately 80% of samples in each group had one to three NNRTI-RAMs and 20% had four to seven NNRTI-RAMs. 181C mutation was the most common NVP-associated RAM (54.3% vs 14.7%, p<0.01). 103N mutation was the most common EFV-associated RAM (56.5% vs 19.1%, p<0.01). The calculated scores from all three scoring systems were concordant. In NVP group, 165 (11.1%) and 161 (10.9%) patients were susceptible to ETR and RPV, respectively (p=0.81). In EFV group, 195 (32.2%) and 191 (31.6%) patients were susceptible to ETR and RPV, respectively (p=0.81). The proportions of viruses that remained susceptible to ETR and RPV in EFV group were significantly higher than NPV group (ETR susceptibility 32.2% vs 11.1%, p<0.01, RPV susceptibility 31.6% vs 10.9%, p<0.01), respectively. RPV might be a cost saving and reasonable second line NNRTI for patients who failed EFV- or NVP-containing regimens, especially in resource-limited setting because these two agents have comparable susceptibility identified by genotyping. From our study, approximately 30% of patients who failed EFV-based regimens had viruses that remained susceptible to RPV.

The emerging profile of cross-resistance among the nonnucleoside HIV-1 reverse transcriptase inhibitors.

PubMed

Sluis-Cremer, Nicolas

2014-07-31

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance.

The Emerging Profile of Cross-Resistance among the Nonnucleoside HIV-1 Reverse Transcriptase Inhibitors

PubMed Central

Sluis-Cremer, Nicolas

2014-01-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are widely used to treat HIV-1-infected individuals; indeed most first-line antiretroviral therapies typically include one NNRTI in combination with two nucleoside analogs. In 2008, the next-generation NNRTI etravirine was approved for the treatment of HIV-infected antiretroviral therapy-experienced individuals, including those with prior NNRTI exposure. NNRTIs are also increasingly being included in strategies to prevent HIV-1 infection. For example: (1) nevirapine is used to prevent mother-to-child transmission; (2) the ASPIRE (MTN 020) study will test whether a vaginal ring containing dapivirine can prevent HIV-1 infection in women; (3) a microbicide gel formulation containing the urea-PETT derivative MIV-150 is in a phase I study to evaluate safety, pharmacokinetics, pharmacodynamics and acceptability; and (4) a long acting rilpivirine formulation is under-development for pre-exposure prophylaxis. Given their widespread use, particularly in resource-limited settings, as well as their low genetic barriers to resistance, there are concerns about overlapping resistance between the different NNRTIs. Consequently, a better understanding of the resistance and cross-resistance profiles among the NNRTI class is important for predicting response to treatment, and surveillance of transmitted drug-resistance. PMID:25089538

A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

PubMed Central

Bronze, Michelle; Wallis, Carole L.; Stuyver, Lieven; Steegen, Kim; Balinda, Sheila; Kityo, Cissy; Stevens, Wendy; Rinke de Wit, Tobias F.; Schuurman, Rob

2013-01-01

In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples. PMID:23536405

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

PubMed

Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

2018-05-03

Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

Prevalence of etravirine mutations and impact on response to treatment in routine clinical care: the Swiss HIV Cohort Study (SHCS).

PubMed

Scherrer, A U; Hasse, B; von Wyl, V; Yerly, S; Böni, J; Bürgisser, P; Klimkait, T; Bucher, H C; Ledergerber, B; Günthard, H F

2009-11-01

Etravirine (ETV) is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with reduced cross-resistance to first-generation NNRTIs, which has been primarily studied in randomized clinical trials and not in routine clinical settings. ETV resistance-associated mutations (RAMs) were investigated by analysing 6072 genotypic tests. The antiviral activity of ETV was predicted using different interpretation systems: International AIDS Society-USA (IAS-USA), Stanford, Rega and Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS). The prevalence of ETV RAMs was higher in NNRTI-exposed patients [44.9%, 95% confidence interval (CI) 41.0-48.9%] than in treatment-naïve patients (9.6%, 95% CI 8.5-10.7%). ETV RAMs in treatment-naïve patients mainly represent polymorphism, as prevalence estimates in genotypic tests for treatment-naïve patients with documented recent (<1 year) infection, who had acquired HIV before the introduction of NNRTIs, were almost identical (9.8%, 95% CI 3.3-21.4). Discontinuation of NNRTI treatment led to a marked drop in the detection of ETV RAMs, from 51.7% (95% CI 40.8-62.6%) to 34.5% (95% CI 24.6-45.4%, P=0.032). Differences in prevalence among subtypes were found for V90I and V179T (P<0.001). Estimates of restricted virological response to ETV varied among algorithms in patients with exposure to efavirenz (EFV)/nevirapine (NVP), ranging from 3.8% (95% CI 2.5-5.6%) for ANRS to 56.2% (95% CI 52.2-60.1%) for Stanford. The predicted activity of ETV decreased as the sensitivity of potential optimized background regimens decreased. The presence of major IAS-USA mutations (L100I, K101E/H/P and Y181C/I/V) reduced the treatment response at week 24. Most ETV RAMs in drug-naïve patients are polymorphisms rather than transmitted RAMs. Uncertainty regarding predictions of antiviral activity for ETV in NNRTI-treated patients remains high. The lowest activity was predicted for patients harbouring extensive multidrug-resistant viruses, thus limiting ETV use in those who are most in need.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

PubMed

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-07-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex

PubMed Central

Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-01-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1. PMID:21447560

Cost-Effectiveness of the Third-Agent Class in Treatment-Naive Human Immunodeficiency Virus-Infected Patients in Portugal

PubMed Central

Aragão, Filipa; Vera, José; Vaz Pinto, Inês

2012-01-01

Introduction Current Portuguese HIV treatment guidelines recommend initiating antiretroviral therapy with a regimen composed of two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor (2NRTI+NNRTI) or two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor (2NRTI+PI/r). Given the lower daily cost of NNRTI as the third agent when compared to the average daily costs of PI/r, it is relevant to estimate the long term impact of each treatment option in the Portuguese context. Methods We developed a microsimulation discrete events model for cost-effectiveness analysis of HIV treatment, simulating individual paths from ART initiation to death. Four driving forces determine the course of events: CD4+ cell count, viral load, resistance and adherence. Distributions of time to event are conditional to individuals’ characteristics and past history. Time to event was modeled using parametric survival analysis using Stata 11®. Disease progression was structured according to therapy lines and the model was parameterized with cohort Portuguese observational data. All resources were valued at 2009 prices. The National Health Service’s perspective was assumed considering a lifetime horizon and a 5% annual discount rate. Results In this analysis, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor reduces the average number of switches by 17%, saves 19.573€ per individual and increases life expectancy by 1.7 months showing to be a dominant strategy in 57% of the simulations when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor. Conclusion This study suggests that, when clinically valid, initiating therapy with two Nucleoside Reverse Transcriptase Inhibitors plus one Non-nucleoside Reverse Transcriptase Inhibitor is a cost-saving strategy and equally effective when compared to two Nucleoside Reverse Transcriptase Inhibitors plus one boosted protease inhibitor as the first regimen. PMID:23028618

Predictable Phenotypes of Antibiotic Resistance Mutations.

PubMed

Knopp, M; Andersson, D I

2018-05-15

Antibiotic-resistant bacteria represent a major threat to our ability to treat bacterial infections. Two factors that determine the evolutionary success of antibiotic resistance mutations are their impact on resistance level and the fitness cost. Recent studies suggest that resistance mutations commonly show epistatic interactions, which would complicate predictions of their stability in bacterial populations. We analyzed 13 different chromosomal resistance mutations and 10 host strains of Salmonella enterica and Escherichia coli to address two main questions. (i) Are there epistatic interactions between different chromosomal resistance mutations? (ii) How does the strain background and genetic distance influence the effect of chromosomal resistance mutations on resistance and fitness? Our results show that the effects of combined resistance mutations on resistance and fitness are largely predictable and that epistasis remains rare even when up to four mutations were combined. Furthermore, a majority of the mutations, especially target alteration mutations, demonstrate strain-independent phenotypes across different species. This study extends our understanding of epistasis among resistance mutations and shows that interactions between different resistance mutations are often predictable from the characteristics of the individual mutations. IMPORTANCE The spread of antibiotic-resistant bacteria imposes an urgent threat to public health. The ability to forecast the evolutionary success of resistant mutants would help to combat dissemination of antibiotic resistance. Previous studies have shown that the phenotypic effects (fitness and resistance level) of resistance mutations can vary substantially depending on the genetic context in which they occur. We conducted a broad screen using many different resistance mutations and host strains to identify potential epistatic interactions between various types of resistance mutations and to determine the effect of strain background on resistance phenotypes. Combinations of several different mutations showed a large amount of phenotypic predictability, and the majority of the mutations displayed strain-independent phenotypes. However, we also identified a few outliers from these patterns, illustrating that the choice of host organism can be critically important when studying antibiotic resistance mutations. Copyright © 2018 Knopp and Andersson.

High level of HIV-1 drug resistance mutations in patients with unsuppressed viral loads in rural northern South Africa.

PubMed

Etta, Elizabeth M; Mavhandu, Lufuno; Manhaeve, Cecile; McGonigle, Keanan; Jackson, Patrick; Rekosh, David; Hammarskjold, Marie-Louise; Bessong, Pascal O; Tebit, Denis M

2017-07-27

Combination antiretroviral therapy (cART) has significantly reduced HIV morbidity and mortality in both developed and developing countries. However, the sustainability of cART may be compromised by the emergence of viral drug resistance mutations (DRM) and the cellular persistence of proviruses carrying these DRM. This is potentially a more serious problem in resource limited settings. DRM were evaluated in individuals with unsuppressed viral loads after first or multiple lines of cART at two sites in rural Limpopo, South Africa. Seventy-two patients with viral loads of >1000 copies/ml were recruited between March 2014 and December 2015. Complete protease (PR) and partial Reverse Transcriptase (RT) sequences were amplified from both plasma RNA and paired proviral DNA from 35 of these subjects. Amplicons were directly sequenced to determine subtype and DRM using the Stanford HIV Drug Resistance Interpretation algorithm. Among the 72 samples, 69 could be PCR amplified from RNA and 35 from both RNA and DNA. Sixty-five (94.2%) viruses were subtype C, while one was subtype B (1.4%), one recombinant K/C, one recombinant C/B and one unclassified. Fifty-eight (84%) sequences carried at least one DRM, while 11 (15.9%) displayed no DRM. DRM prevalence according to drug class was: NRTI 60.8% NNRTI 65.2%, and PI 5.8%. The most common DRMs were; M184V (51.7%), K103N (50%), V106M (20.6%), D67N (13.3%), K65R (12%). The frequency of the DRM tracked well with the frequency of use of medications to which the mutations were predicted to confer resistance. Interestingly, a significant number of subjects showed predicted resistance to the newer NNRTIs, etravirine (33%) and rilpivirine (42%), both of which are not yet available in this setting. The proportion of DRM in RNA and DNA were mostly similar with the exception of the thymidine analogue mutations (TAMs) D67N, K70R, K219QE; and K103N which were slightly more prevalent in DNA than RNA. Subjects who had received cART for at least 5 years were more likely to harbour >2 DRM (p < 0.05) compared to those treated for a shorter period. DRM were more prevalent in this rural setting compared to a neighbouring urban setting. We found a very high prevalence of NRTI and NNRTI DRM in patients from rural Limpopo settings with different durations of treatment. The prevalence was significantly higher than those reported in urban settings in South Africa. The dominance of NNRTI based mutations late in treatment supports the use of PI based regimens for second line treatment in this setting. The slight dominance of TAMs in DNA from infected PBMCs compared to plasma virus requires further studies that should include cART subjects with suppressed virus. Such studies will improve our understanding of the pattern of drug resistance and dynamics of viral persistence in these rural settings.

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

PubMed

Famiglini, Valeria; La Regina, Giuseppe; Coluccia, Antonio; Pelliccia, Sveva; Brancale, Andrea; Maga, Giovanni; Crespan, Emmanuele; Badia, Roger; Riveira-Muñoz, Eva; Esté, José A; Ferretti, Rosella; Cirilli, Roberto; Zamperini, Claudio; Botta, Maurizio; Schols, Dominique; Limongelli, Vittorio; Agostino, Bruno; Novellino, Ettore; Silvestri, Romano

2014-12-11

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.

High Rates of Transmission of Drug-resistant HIV in Aruba Resulting in Reduced Susceptibility to the WHO Recommended First-line Regimen in Nearly Half of Newly Diagnosed HIV-infected Patients.

PubMed

Hofstra, L Marije; Sánchez Rivas, Elena; Nijhuis, Monique; Bank, Leonie E A; Wilkinson, Eduan; Kelly, Karina; Mudrikova, Tania; Schuurman, Rob; de Oliveira, Tulio; de Kort, Jaclyn; Wensing, Annemarie M J

2017-04-15

In Western countries emergence of human immunodeficiency virus (HIV) drug resistance has tremendously decreased, and transmission of drug resistance has merely stabilized in recent years. However, in many endemic settings with limited resources rates of emerging and transmitted drug resistance are not regularly assessed. We performed a survey including all HIV-infected individuals who received resistance testing in 2010-2015 in Aruba, a highly endemic HIV area in the Caribbean. Transmitted HIV drug resistance was determined using World Health Organization (WHO) criteria. Transmission dynamics were investigated using phylogenetic analyses. In a subset, baseline samples were re-analyzed using next generation sequencing (NGS). Baseline resistance testing was performed in 104 newly diagnosed untreated individuals (54% of all newly diagnosed individuals in 2010-2015): 86% were men, 39% were foreign-born, and 22% had AIDS at diagnosis. And 33% (95% CI: 24-42%) was infected with a drug-resistant HIV variant. The prevalence of resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) reached 45% (95% CI: 27-64%) in 2015, all based on the prevalence of mutation K103N. NGS did not demonstrate additional minority K103N-variants compared to routine resistance testing. K103N-harboring strains were introduced into the therapy-unexposed population via at least 6 independent transmissions epidemiologically linked to the surrounding countries. Virological failure of the WHO-recommended first-line NNRTI-based regimen was higher in the presence of K103N. The prevalence of resistant HIV in Aruba has increased to alarming levels, compromising the WHO-recommended first-line regimen. As adequate surveillance as advocated by the WHO is limited, the Caribbean region could face an unidentified rise of NNRTI-resistant HIV. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018.

PubMed

Bhalla, Amarpreet; Zulfiqar, Muhammad; Bluth, Martin H

2018-06-01

The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenomaecarcinoma sequence, serrated polyp pathway, and microsatellite instability (MSI). The genetic basis for hereditary nonpolyposis colorectal cancer is the detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. Genetic testing for Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in MMR genes. Molecular makers with predictive and prognostic implications include quantitative multigene reverse transcriptase polymerase chain reaction assay and KRAS and BRAF mutation analysis. Mismatch repair-deficient tumors have higher rates of programmed death-ligand 1 expression. Cell-free DNA analysis in fluids are proving beneficial for diagnosis and prognosis in these disease states towards effective patient management. Copyright © 2018 Elsevier Inc. All rights reserved.

Population Genetics Study of Isoniazid Resistance Mutations and Evolution of Multidrug-Resistant Mycobacterium tuberculosis†

PubMed Central

Hazbón, Manzour Hernando; Brimacombe, Michael; Bobadilla del Valle, Miriam; Cavatore, Magali; Guerrero, Marta Inírida; Varma-Basil, Mandira; Billman-Jacobe, Helen; Lavender, Caroline; Fyfe, Janet; García-García, Lourdes; León, Clara Inés; Bose, Mridula; Chaves, Fernando; Murray, Megan; Eisenach, Kathleen D.; Sifuentes-Osornio, José; Cave, M. Donald; Ponce de León, Alfredo; Alland, David

2006-01-01

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes. PMID:16870753

High discordance in blood and genital tract HIV-1 drug resistance in Indian women failing first-line therapy.

PubMed

Saravanan, Shanmugam; Gomathi, Selvamurthi; Delong, Allison; Kausalya, Bagavathi; Sivamalar, Sathasivam; Poongulali, Selvamuthu; Brooks, Katherine; Kumarasamy, Nagalingeswaran; Balakrishnan, Pachamuthu; Solomon, Sunil S; Cu-Uvin, Susan; Kantor, Rami

2018-05-24

Examine HIV-1 plasma viral load (PVL) and genital tract (GT) viral load (GVL) and drug resistance in India. At the YRG Centre for AIDS Research and Education, Chennai, we tested: PVL in women on first-line ART for ≥6 months; GVL when PVL >2000 copies/mL; and plasma, genital and proviral reverse transcriptase drug resistance when GVL >2000 copies/mL. Wilcoxon rank-sum and Fisher's exact tests were used to identify failure and resistance associations. Pearson correlations were calculated to evaluate PVL-GVL associations. Inter-compartmental resistance discordance was evaluated using generalized estimating equations. Of 200 women, 37% had detectable (>400 copies/mL) PVL and 31% had PVL >1000 copies/mL. Of women with detectable PVL, 74% had PVL >2000 copies/mL, of which 74% had detectable GVL. Higher PVL was associated with higher GVL. Paired plasma and genital sequences were available for 21 women; mean age of 34 years, median ART duration of 33 months, median CD4 count of 217 cells/mm3, median PVL of 5.4 log10 copies/mL and median GVL of 4.6 log10 copies/mL. Drug resistance was detected in 81%-91% of samples and 67%-76% of samples had dual-class resistance. Complete three-compartment concordance was seen in only 10% of women. GT-proviral discordance was significantly larger than plasma-proviral discordance. GT or proviral mutations discordant from plasma led to clinically relevant resistance in 24% and 30%, respectively. We identified high resistance and high inter-compartmental resistance discordance in Indian women, which might lead to unrecognized resistance transmission and re-emergence compromising treatment outcomes, particularly relevant to countries like India, where sexual HIV transmission is predominant.

HIV multi-drug resistance at first-line antiretroviral failure and subsequent virological response in Asia

PubMed Central

Jiamsakul, Awachana; Sungkanuparph, Somnuek; Law, Matthew; Kantor, Rami; Praparattanapan, Jutarat; Li, Patrick CK; Phanuphak, Praphan; Merati, Tuti; Ratanasuwan, Winai; Lee, Christopher KC; Ditangco, Rossana; Mustafa, Mahiran; Singtoroj, Thida; Kiertiburanakul, Sasisopin

2014-01-01

Introduction First-line antiretroviral therapy (ART) failure often results from the development of resistance-associated mutations (RAMs). Three patterns, including thymidine analogue mutations (TAMs), 69 Insertion (69Ins) and the Q151M complex, are associated with resistance to multiple-nucleoside reverse transcriptase inhibitors (NRTIs) and may compromise treatment options for second-line ART. Methods We investigated patterns and factors associated with multi-NRTI RAMs at first-line failure in patients from The TREAT Asia Studies to Evaluate Resistance – Monitoring study (TASER-M), and evaluated their impact on virological responses at 12 months after switching to second-line ART. RAMs were compared with the IAS-USA 2013 mutations list. We defined multi-NRTI RAMs as the presence of either Q151M; 69Ins; ≥2 TAMs; or M184V+≥1 TAM. Virological suppression was defined as viral load (VL) <400 copies/ml at 12 months from switch to second-line. Logistic regression was used to analyze (1) factors associated with multi-NRTI RAMs at first-line failure and (2) factors associated with virological suppression after 12 months on second-line. Results A total of 105 patients from 10 sites in Thailand, Hong Kong, Indonesia, Malaysia and Philippines were included. There were 97/105 (92%) patients harbouring ≥1 RAMs at first-line failure, 39/105 with multi-NRTI RAMs: six with Q151M; 24 with ≥2 TAMs; and 32 with M184V+≥1 TAM. Factors associated with multi-NRTI RAMs were CD4 ≤200 cells/µL at genotyping (OR=4.43, 95% CI [1.59–12.37], p=0.004) and ART duration >2 years (OR=6.25, 95% CI [2.39–16.36], p<0.001). Among 87/105 patients with available VL at 12 months after switch to second-line ART, virological suppression was achieved in 85%. The median genotypic susceptibility score (GSS) for the second-line regimen was 2.00. Patients with ART adherence ≥95% were more likely to be virologically suppressed (OR=9.33, 95% CI (2.43–35.81), p=0.001). Measures of patient resistance to second-line ART, including the GSS, were not significantly associated with virological outcome. Conclusions Multi-NRTI RAMs at first-line failure were associated with low CD4 level and longer duration of ART. With many patients switching to highly susceptible regimens, good adherence was still crucial in achieving virological response. This emphasizes the importance of continued adherence counselling well into second-line therapy. PMID:25141905

HIV multi-drug resistance at first-line antiretroviral failure and subsequent virological response in Asia.

PubMed

Jiamsakul, Awachana; Sungkanuparph, Somnuek; Law, Matthew; Kantor, Rami; Praparattanapan, Jutarat; Li, Patrick C K; Phanuphak, Praphan; Merati, Tuti; Ratanasuwan, Winai; Lee, Christopher K C; Ditangco, Rossana; Mustafa, Mahiran; Singtoroj, Thida; Kiertiburanakul, Sasisopin

2014-01-01

First-line antiretroviral therapy (ART) failure often results from the development of resistance-associated mutations (RAMs). Three patterns, including thymidine analogue mutations (TAMs), 69 Insertion (69Ins) and the Q151M complex, are associated with resistance to multiple-nucleoside reverse transcriptase inhibitors (NRTIs) and may compromise treatment options for second-line ART. We investigated patterns and factors associated with multi-NRTI RAMs at first-line failure in patients from The TREAT Asia Studies to Evaluate Resistance - Monitoring study (TASER-M), and evaluated their impact on virological responses at 12 months after switching to second-line ART. RAMs were compared with the IAS-USA 2013 mutations list. We defined multi-NRTI RAMs as the presence of either Q151M; 69Ins; ≥ 2 TAMs; or M184V+≥ 1 TAM. Virological suppression was defined as viral load (VL) <400 copies/ml at 12 months from switch to second-line. Logistic regression was used to analyze (1) factors associated with multi-NRTI RAMs at first-line failure and (2) factors associated with virological suppression after 12 months on second-line. A total of 105 patients from 10 sites in Thailand, Hong Kong, Indonesia, Malaysia and Philippines were included. There were 97/105 (92%) patients harbouring ≥ 1 RAMs at first-line failure, 39/105 with multi-NRTI RAMs: six with Q151M; 24 with ≥ 2 TAMs; and 32 with M184V+≥ 1 TAM. Factors associated with multi-NRTI RAMs were CD4 ≤ 200 cells/µL at genotyping (OR=4.43, 95% CI [1.59-12.37], p=0.004) and ART duration >2 years (OR=6.25, 95% CI [2.39-16.36], p<0.001). Among 87/105 patients with available VL at 12 months after switch to second-line ART, virological suppression was achieved in 85%. The median genotypic susceptibility score (GSS) for the second-line regimen was 2.00. Patients with ART adherence ≥ 95% were more likely to be virologically suppressed (OR=9.33, 95% CI (2.43-35.81), p=0.001). Measures of patient resistance to second-line ART, including the GSS, were not significantly associated with virological outcome. Multi-NRTI RAMs at first-line failure were associated with low CD4 level and longer duration of ART. With many patients switching to highly susceptible regimens, good adherence was still crucial in achieving virological response. This emphasizes the importance of continued adherence counselling well into second-line therapy.

Anti-HIV drugs: 25 compounds approved within 25 years after the discovery of HIV.

PubMed

De Clercq, Erik

2009-04-01

In 2008, 25 years after the human immunodeficiency virus (HIV) was discovered as the then tentative aetiological agent of acquired immune deficiency syndrome (AIDS), exactly 25 anti-HIV compounds have been formally approved for clinical use in the treatment of AIDS. These compounds fall into six categories: nucleoside reverse transcriptase inhibitors (NRTIs: zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine); nucleotide reverse transcriptase inhibitors (NtRTIs: tenofovir); non-nucleoside reverse transcriptase inhibitors (NNRTIs: nevirapine, delavirdine, efavirenz and etravirine); protease inhibitors (PIs: saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir); cell entry inhibitors [fusion inhibitors (FIs: enfuvirtide) and co-receptor inhibitors (CRIs: maraviroc)]; and integrase inhibitors (INIs: raltegravir). These compounds should be used in drug combination regimens to achieve the highest possible benefit, tolerability and compliance and to diminish the risk of resistance development.

A second chance for telomerase reverse transcriptase in anticancer immunotherapy.

PubMed

Zanetti, Maurizio

2017-02-01

Telomerase reverse transcriptase (TERT) is a self-antigen that is expressed constitutively in many tumours, and is, therefore, an important target for anticancer immunotherapy. In the past 10 years, trials of immunotherapy with TERT-based vaccines have demonstrated only modest benefits. In this Perspectives, I discuss the possible immunological reasons for this limited antitumour efficacy, and propose that advances in our understanding of the genetics and biology of the involvement of TERT in cancer provides the basis for renewed interest in TERT- based immunotherapy. Telomerase and TERT are expressed in cancer cells at every stage of tumour evolution, from the cancer stem cell to circulating tumour cells and tumour metastases. Many cancer types also harbour cells with mutations in the TERT promoter region, which increase transcriptional activation of this gene. These new findings should spur new interest in the development of TERT-based immunotherapies that are redesigned in line with established immunological considerations and working principles, and are tailored to patients stratified on the basis of TERT-promoter mutations and other underlying tumour characteristics. Thus, despite the disappointment of previous clinical trials, TERT offers the potential for personalized immunotherapy, perhaps in combination with immune-checkpoint inhibition.

Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

PubMed Central

Hachiya, Atsuko; Aizawa-Matsuoka, Saori; Tanaka, Mari; Takahashi, Yukiko; Ida, Setsuko; Gatanaga, Hiroyuki; Hirabayashi, Yoshihiro; Kojima, Asato; Tatsumi, Masashi; Oka, Shinichi

2001-01-01

We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104 copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy. PMID:11158746

The Anti-Human Immunodeficiency Virus Drug Tenofovir, a Reverse Transcriptase Inhibitor, Also Targets the Herpes Simplex Virus DNA Polymerase.

PubMed

Andrei, Graciela; Gillemot, Sarah; Topalis, Dimitrios; Snoeck, Robert

2018-02-14

Genital herpes is an important cofactor for acquisition of human immunodeficiency virus (HIV) infection, and effective prophylaxis is a helpful strategy to halt both HIV and herpes simplex virus (HSV) transmission. The antiretroviral agent tenofovir, formulated as a vaginal microbicide gel, was shown to reduce the risk of HIV and HSV type 2 (HSV-2) acquisition. HSV type 1 (HSV-1) and HSV-2 mutants were selected for resistance to tenofovir and PMEO-DAPy (6-phosphonylmethoxyethoxy-2,4-diaminopyrimidine, an acyclic nucleoside phosphonate with dual anti-HSV and anti-HIV activity) by stepwise dose escalation. Several plaque-purified viruses were characterized phenotypically (drug resistance profiling) and genotypically (sequencing of the viral DNA polymerase gene). Tenofovir resistant and PMEO-DAPy-resistant viruses harbored specific amino acid substitutions associated with resistance not only to tenofovir and PMEO-DAPy but also to acyclovir and foscarnet. These amino acid changes (A719V, S724N, and L802F [HSV-1] and M789T and A724V [HSV-2]) were also found in clinical isolates recovered from patients refractory to acyclovir and/or foscarnet therapy or in laboratory-derived strains. A total of 10 (HSV-1) and 18 (HSV-2) well-characterized DNA polymerase mutants had decreased susceptibility to tenofovir and PMEO-DAPy. Tenofovir and PMEO-DAPy target the HSV DNA polymerase, and clinical isolates with DNA polymerase mutations emerging under acyclovir and/or foscarnet therapy showed cross-resistance to tenofovir and PMEO-DAPy. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Diketo acids derivatives as integrase inhibitors: the war against the acquired immunodeficiency syndrome.

PubMed

Henao-Mejia, Jorge; Góez, Yenny; Patiño, Pablo; Rugeles, Maria T

2006-06-01

Since the human immunodeficiency virus was identified as etiological agent of the acquired immunodeficiency syndrome, great advances have been accomplished in the therapeutic field leading to reduced morbidity and mortality among infected patients. However, the high mutation rate of the viral genome generates strains resistant to multiple drugs, pointing to the importance of finding new therapeutic targets. Among the HIV structural genes, the POL gene codes for three essential enzymes: reverse transcriptase, protease, and integrase; nineteen of the twenty drugs currently approved by the Food and Drug Administration to treat this viral infection, inhibit the reverse transcriptase and the protease. Although intense research has been carried out in this area during the last 10 years, HIV integrase inhibitors are not yet approved for clinical use; however the fact that presence of this enzyme is a sine qua non for a productive HIV life cycle joined to its unique properties makes it a promissory target for anti-HIV therapy. Many compounds have been claimed to inhibit integrase in vitro; however, few of them have proven to have antiviral activity and low cytotoxicity in cell systems. Diketoacid derivatives are the most promising integrase inhibitors so far reported. Initially discovered independently by Shionogi & Co. and the Merck Research Laboratories, these compounds are highly specific for the integrase with potent antiviral activity in vitro and in vivo, and low cytotoxicity in cell cultures. Some of these compounds have recently entered clinical trials. Due to the high relevance of integrase inhibitors, and specifically of diketoacid derivatives, we review the latest findings and patents in this important field of research.

ACTG A5353: A Pilot Study of Dolutegravir Plus Lamivudine for Initial Treatment of Human Immunodeficiency Virus-1 (HIV-1)-infected Participants With HIV-1 RNA <500000 Copies/mL.

PubMed

Taiwo, Babafemi O; Zheng, Lu; Stefanescu, Andrei; Nyaku, Amesika; Bezins, Baiba; Wallis, Carole L; Godfrey, Catherine; Sax, Paul E; Acosta, Edward; Haas, David; Smith, Kimberly Y; Sha, Beverly; Van Dam, Cornelius; Gulick, Roy M

2018-05-17

Limited data exist on initial human immunodeficiency virus type 1 (HIV-1) treatment with dolutegravir plus lamivudine. A5353 is a phase 2, single-arm, pilot study of once-daily dolutegravir (50 mg) plus lamivudine (300 mg) in treatment-naive participants with HIV-1 RNA ≥1000 and <500000 copies/mL. Exclusion criteria included active hepatitis B or major protease, reverse transcriptase, or integrase resistance. The primary efficacy measure was the proportion with HIV-1 RNA <50 copies/mL (FDA [US Food and Drug Administration] Snapshot) at week 24. Virologic failure (VF) was confirmed HIV-1 RNA >400 copies/mL at week 16/20 or >200 copies/mL at or after week 24. Dolutegravir levels and drug resistance testing were performed at VF. One hundred and twenty participants (87% male, median age 30 years, 37 (31%) HIV-1 RNA >100000 copies/mL) initiated study treatment. Median entry HIV-1 RNA and CD4 count were 4.61 log10 copies/mL and 387 cells/mm3. Virologic efficacy at week 24 was 108/120 (90%, confidence interval [83%, 95%]), with comparable results in the >100000 copies/mL and ≤100000 copies/mL strata, that is, 89% (75%, 97%) and 90% (82%, 96%), respectively. Three participants with VF, had undetected plasma dolutegravir at ≥1 time points; the M184V and R263R/K mutations developed in 1 participant. Two participants experienced grade 3 possible/probable treatment-related adverse events; none discontinued treatment due to adverse events. Dolutegravir plus lamivudine demonstrated efficacy in individuals with pretreatment HIV-1 RNA up to 500000 copies/mL in this pilot trial, but a participant developed resistance mutations. NCT02582684.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

PubMed

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.

PubMed

Martín, Verónica; Perales, Celia; Fernández-Algar, María; Dos Santos, Helena G; Garrido, Patricia; Pernas, María; Parro, Víctor; Moreno, Miguel; García-Pérez, Javier; Alcamí, José; Torán, José Luis; Abia, David; Domingo, Esteban; Briones, Carlos

2016-01-01

The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.

The history of antiretrovirals: key discoveries over the past 25 years.

PubMed

De Clercq, Erik

2009-09-01

Within 25 years after zidovudine (3'-azido-2',3'-dideoxythymidine, AZT) was first described as an inhibitor of HIV replication, 25 anti-HIV drugs have been formally approved for clinical use in the treatment of HIV infections: seven nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir and emtricitabine; one nucleotide reverse transcriptase inhibitor (NtRTI): tenofovir [in its oral prodrug form: tenofovir disoproxil fumarate (TDF)]; four non-nucleoside reverse transcriptase inhibitors (NNRTIs): nevirapine, delavirdine, efavirenz and etravirine; ten protease inhibitors (PIs): saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir and darunavir; one fusion inhibitor (FI): enfuvirtide; one co-receptor inhibitor (CRI): maraviroc and one integrase inhibitor (INI): raltegravir. These compounds are used in various drug combination (some at fixed dose) regimens so as to achieve the highest possible benefit and tolerability, and to diminish the risk of virus-drug resistance development. (c) 2009 John Wiley & Sons, Ltd.

Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase.

PubMed

He, Shan; Li, Yangyang; Chen, Yang; Zhu, Yue; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

2016-08-01

Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.

Search for Novel Candidate Mutations for Metronidazole Resistance in Helicobacter pylori Using Next-Generation Sequencing

PubMed Central

Binh, Tran Thanh; Suzuki, Rumiko; Trang, Tran Thi Huyen; Kwon, Dong Hyeon

2015-01-01

Metronidazole resistance is a key factor associated with Helicobacter pylori treatment failure. Although this resistance is mainly associated with mutations in the rdxA and frxA genes, the question of whether metronidazole resistance is caused by the inactivation of frxA alone is still debated. Furthermore, it is unclear whether there are other mutations involved in addition to the two genes that are associated with resistance. A metronidazole-resistant strain was cultured from the metronidazole-susceptible H. pylori strain 26695-1 by exposure to low concentrations of metronidazole. The genome sequences of both susceptible and resistant H. pylori strains were determined by Illumina next-generation sequencing, from which putative candidate resistance mutations were identified. Natural transformation was used to introduce PCR products containing candidate mutations into the susceptible parent strain 26695-1, and the metronidazole MIC was determined for each strain. Mutations in frxA (hp0642), rdxA (hp0954), and rpsU (hp0562) were confirmed by the Sanger method. The mutated sequence in rdxA was successfully transformed into strain 26695-1, and the transformants showed resistance to metronidazole. The transformants containing a single mutation in rdxA showed a low MIC (16 mg/liter), while those containing mutations in both rdxA and frxA showed a higher MIC (48 mg/liter). No transformants containing a single mutation in frxA or rpsU were obtained. Next-generation sequencing was used to identify mutations related to drug resistance. We confirmed that the mutations in rdxA are mainly associated with metronidazole resistance, and mutations in frxA are able to enhance H. pylori resistance only in the presence of rdxA mutations. Moreover, mutations in rpsU may play a role in metronidazole resistance. PMID:25645832

Increase in transmitted drug resistance in migrants from sub-Saharan Africa diagnosed with HIV-1 in Sweden.

PubMed

Andersson, Emmi; Nordquist, Agnes; Esbjörnsson, Joakim; Flamholc, Leo; Gisslén, Magnus; Hejdeman, Bo; Marrone, Gaetano; Norrgren, Hans; Svedhem, Veronica; Wendahl, Suzanne; Albert, Jan; Sönnerborg, Anders

2018-04-24

To study the trends of transmitted drug resistance (TDR) in HIV-1 patients newly diagnosed in Sweden, 2010-2016. Register-based study including all antiretroviral therapy-naive patients ≥18 years diagnosed with HIV-1 in Sweden 2010-2016. Patient data and viral pol sequences were extracted from the national InfCareHIV database. TDR was defined as the presence of surveillance drug resistance mutations (SDRMs). A CD4 T-cell decline trajectory model estimated time of infection. Phylogenetic inference was used for cluster analysis. Chi-square tests and logistic regressions were used to investigate relations between TDR, epidemiological and viral factors. One thousand, seven hundred and thirteen pol sequences were analyzed, corresponding to 71% of patients with a new HIV-1 diagnosis (heterosexuals: 53%; MSM: 34%). The overall prevalence of TDR was 7.1% (95% CI 5.8-8.3%). Nonnucleoside reverse transcriptase inhibitor (NNRTI) TDR increased significantly from 1.5% in 2010 to 6.2% in 2016, and was associated to infection and/or origin in sub-Saharan Africa (SSA). An MSM transmission cluster dating back to the 1990s with the M41L SDRM was identified. Twenty-five (1.5%) patients exhibited TDR to tenofovir (TDF; n = 8), emtricitabine/lamivudine (n = 9) or both (n = 8). NNRTI TDR has increased from 2010 to 2016 in HIV-1-infected migrants from SSA diagnosed in Sweden, mirroring the situation in SSA. TDR to tenofovir/emtricitabine, used in preexposure prophylaxis, confirms the clinical and epidemiological need for resistance testing in newly diagnosed patients.

Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys

PubMed Central

2010-01-01

Background The highly pathogenic H5N1 is a major avian pathogen that crosses species barriers and seriously affects humans as well as some mammals. It mutates in an intensified manner and is considered a potential candidate for the possible next pandemic with all the catastrophic consequences. Methods Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE). Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI) assay on 105 donkey serum samples. Results We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate. Conclusions These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas. PMID:20398268

Global trends in antiretroviral resistance in treatment-naive individuals with HIV after rollout of antiretroviral treatment in resource-limited settings: a global collaborative study and meta-regression analysis.

PubMed

Gupta, Ravindra K; Jordan, Michael R; Sultan, Binta J; Hill, Andrew; Davis, Daniel H J; Gregson, John; Sawyer, Anthony W; Hamers, Raph L; Ndembi, Nicaise; Pillay, Deenan; Bertagnolio, Silvia

2012-10-06

The emergence and spread of high levels of HIV-1 drug resistance in resource-limited settings where combination antiretroviral treatment has been scaled up could compromise the effectiveness of national HIV treatment programmes. We aimed to estimate changes in the prevalence of HIV-1 drug resistance in treatment-naive individuals with HIV since initiation of rollout in resource-limited settings. We did a systematic search for studies and conference abstracts published between January, 2001, and July, 2011, and included additional data from the WHO HIV drug resistance surveillance programme. We assessed the prevalence of drug-resistance mutations in untreated individuals with respect to time since rollout in a series of random-effects meta-regression models. Study-level data were available for 26,102 patients from sub-Saharan Africa, Asia, and Latin America. We recorded no difference between chronic and recent infection on the prevalence of one or more drug-resistance mutations for any region. East Africa had the highest estimated rate of increase at 29% per year (95% CI 15 to 45; p=0·0001) since rollout, with an estimated prevalence of HIV-1 drug resistance at 8 years after rollout of 7·4% (4·3 to 12·7). We recorded an annual increase of 14% (0% to 29%; p=0·054) in southern Africa and a non-significant increase of 3% (-0·9 to 16; p=0·618) in west and central Africa. There was no change in resistance over time in Latin America, and because of much country-level heterogeneity the meta-regression analysis was not appropriate for Asia. With respect to class of antiretroviral, there were substantial increases in resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) in east Africa (36% per year [21 to 52]; p<0·0001) and southern Africa (23% per year [7 to 42]; p=0·0049). No increase was noted for the other drug classes in any region. Our findings suggest a significant increase in prevalence of drug resistance over time since antiretroviral rollout in regions of sub-Saharan Africa; this rise is driven by NNRTI resistance in studies from east and southern Africa. The findings are of concern and draw attention to the need for enhanced surveillance and drug-resistance prevention efforts by national HIV treatment programmes. Nevertheless, estimated levels, although increasing, are not unexpected in view of the large expansion of antiretroviral treatment coverage seen in low-income and middle-income countries--no changes in antiretroviral treatment guidelines are warranted at the moment. Bill & Melinda Gates Foundation and the European Community's Seventh Framework Programme. Copyright © 2012 Elsevier Ltd. All rights reserved.

Frequency and Distribution of Tuberculosis Resistance-Associated Mutations between Mumbai, Moldova, and Eastern Cape

PubMed Central

Seifert, M.; Catanzaro, D.; Garfein, R. S.; Valafar, F.; Crudu, V.; Rodrigues, C.; Victor, T. C.; Catanzaro, A.; Rodwell, T. C.

2016-01-01

Molecular diagnostic assays, with their ability to rapidly detect resistance-associated mutations in bacterial genes, are promising technologies to control the spread of drug-resistant tuberculosis (DR-TB). Sequencing assays provide detailed information for specific gene regions and can help diagnostic assay developers prioritize mutations for inclusion in their assays. We performed pyrosequencing of seven Mycobacterium tuberculosis gene regions (katG, inhA, ahpC, rpoB, gyrA, rrs, and eis) for 1,128 clinical specimens from India, Moldova, and South Africa. We determined the frequencies of each mutation among drug-resistant and -susceptible specimens based on phenotypic drug susceptibility testing results and examined mutation distributions by country. The most common mutation among isoniazid-resistant (INHr) specimens was the katG 315ACC mutation (87%). However, in the Eastern Cape, INHr specimens had a lower frequency of katG mutations (44%) and higher frequencies of inhA (47%) and ahpC (10%) promoter mutations. The most common mutation among rifampin-resistant (RIFr) specimens was the rpoB 531TTG mutation (80%). The mutation was common in RIFr specimens in Mumbai (83%) and Moldova (84%) but not the Eastern Cape (17%), where the 516GTC mutation appeared more frequently (57%). The most common mutation among fluoroquinolone-resistant specimens was the gyrA 94GGC mutation (44%). The rrs 1401G mutation was found in 84%, 84%, and 50% of amikacin-resistant, capreomycin-resistant, and kanamycin (KAN)-resistant (KANr) specimens, respectively. The eis promoter mutation −12T was found in 26% of KANr and 4% of KAN-susceptible (KANs) specimens. Inclusion of the ahpC and eis promoter gene regions was critical for optimal test sensitivity for the detection of INH resistance in the Eastern Cape and KAN resistance in Moldova. (This study has been registered at ClinicalTrials.gov under registration number NCT02170441.) PMID:27090176

Sensitive Deep-Sequencing-Based HIV-1 Genotyping Assay To Simultaneously Determine Susceptibility to Protease, Reverse Transcriptase, Integrase, and Maturation Inhibitors, as Well as HIV-1 Coreceptor Tropism

PubMed Central

Gibson, Richard M.; Meyer, Ashley M.; Winner, Dane; Archer, John; Feyertag, Felix; Ruiz-Mateos, Ezequiel; Leal, Manuel; Robertson, David L.; Schmotzer, Christine L.

2014-01-01

With 29 individual antiretroviral drugs available from six classes that are approved for the treatment of HIV-1 infection, a combination of different phenotypic and genotypic tests is currently needed to monitor HIV-infected individuals. In this study, we developed a novel HIV-1 genotypic assay based on deep sequencing (DeepGen HIV) to simultaneously assess HIV-1 susceptibilities to all drugs targeting the three viral enzymes and to predict HIV-1 coreceptor tropism. Patient-derived gag-p2/NCp7/p1/p6/pol-PR/RT/IN- and env-C2V3 PCR products were sequenced using the Ion Torrent Personal Genome Machine. Reads spanning the 3′ end of the Gag, protease (PR), reverse transcriptase (RT), integrase (IN), and V3 regions were extracted, truncated, translated, and assembled for genotype and HIV-1 coreceptor tropism determination. DeepGen HIV consistently detected both minority drug-resistant viruses and non-R5 HIV-1 variants from clinical specimens with viral loads of ≥1,000 copies/ml and from B and non-B subtypes. Additional mutations associated with resistance to PR, RT, and IN inhibitors, previously undetected by standard (Sanger) population sequencing, were reliably identified at frequencies as low as 1%. DeepGen HIV results correlated with phenotypic (original Trofile, 92%; enhanced-sensitivity Trofile assay [ESTA], 80%; TROCAI, 81%; and VeriTrop, 80%) and genotypic (population sequencing/Geno2Pheno with a 10% false-positive rate [FPR], 84%) HIV-1 tropism test results. DeepGen HIV (83%) and Trofile (85%) showed similar concordances with the clinical response following an 8-day course of maraviroc monotherapy (MCT). In summary, this novel all-inclusive HIV-1 genotypic and coreceptor tropism assay, based on deep sequencing of the PR, RT, IN, and V3 regions, permits simultaneous multiplex detection of low-level drug-resistant and/or non-R5 viruses in up to 96 clinical samples. This comprehensive test, the first of its class, will be instrumental in the development of new antiretroviral drugs and, more importantly, will aid in the treatment and management of HIV-infected individuals. PMID:24468782

Report of Chinese family with severe dermatitis, multiple allergies and metabolic wasting syndrome caused by novel homozygous desmoglein-1 gene mutation.

PubMed

Cheng, Ruhong; Yan, Ming; Ni, Cheng; Zhang, Jia; Li, Ming; Yao, Zhirong

2016-10-01

Recently, homozygous mutations in the desmoglein-1 (DSG1) gene and heterozygous mutation in the desmoplakin (DSP) gene have been demonstrated to be associated with severe dermatitis, multiple allergies and metabolic wasting (SAM) syndrome (Mendelian Inheritance in Man no. 615508). We aim to identify the molecular basis for a Chinese pedigree of SAM syndrome. A Chinese pedigree of SAM syndrome was subjected to mutation detection in the DSG1 gene. Sequence analysis of the DSG1 gene and quantitative reverse transcriptase polymerase chain reaction analysis for gene expression of DSG1 using cDNA derived from the epidermis of patients and controls were both performed. Skin biopsies were also taken from patients for pathological study and transmission electron microscopy observation. Novel homozygous splicing mutation c.1892-1delG in the exon-intron border of the DSG1 gene has been demonstrated to be associated with SAM syndrome. We report a new family of SAM syndrome of Asian decent and expand the spectrum of mutations in the DSG1 gene. © 2016 Japanese Dermatological Association.

NEW TYPE OF STREPTOMYCIN RESISTANCE RESULTING FROM ACTION OF THE EPISOMELIKE MUTATOR FACTOR IN ESCHERICHIA COLI

PubMed Central

Gundersen, Wenche B.

1963-01-01

Gundersen, Wenche B. (Oslo University, Oslo, Norway). New type of streptomycin resistance resulting from action of the episomelike mutator factor in Escherichia coli. J. Bacteriol. 86:510–516. 1963.—Analyses have been performed to elucidate the genetic nature of the streptomycin resistance that results from the action of the previously described episomelike mutator factor in Escherichia coli. This streptomycin resistance has been found to differ from ordinary one-step streptomycin resistance. The new type of streptomycin resistance, “mutator resistance,” can be lost, either spontaneously or by treatment with ultraviolet light and acriflavine. It is more stable in a K-12 strain than in the original E. coli strain 635. Mutator resistance segregates like a chromosomal marker in genetic crosses, and is located near the ordinary streptomycin locus. The locus for mutator resistance is distinct from that of ordinary streptomycin resistance, apparently located further toward the threonine region. Mutator resistance, unlike ordinary one-step streptomycin resistance, appears as a dominant character. The possibilities of its being a suppressor or regulator mutation are discussed. PMID:14066430

Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.

PubMed Central

Kögel, D; Aboud, M; Flügel, R M

1995-01-01

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent. Images PMID:7544460

Drug Resistance Missense Mutations in Cancer Are Subject to Evolutionary Constraints

PubMed Central

Friedman, Ran

2013-01-01

Several tumour types are sensitive to deactivation of just one or very few genes that are constantly active in the cancer cells, a phenomenon that is termed ‘oncogene addiction’. Drugs that target the products of those oncogenes can yield a temporary relief, and even complete remission. Unfortunately, many patients receiving oncogene-targeted therapies relapse on treatment. This often happens due to somatic mutations in the oncogene (‘resistance mutations’). ‘Compound mutations’, which in the context of cancer drug resistance are defined as two or more mutations of the drug target in the same clone may lead to enhanced resistance against the most selective inhibitors. Here, it is shown that the vast majority of the resistance mutations occurring in cancer patients treated with tyrosin kinase inhibitors aimed at three different proteins follow an evolutionary pathway. Using bioinformatic analysis tools, it is found that the drug-resistance mutations in the tyrosine kinase domains of Abl1, ALK and exons 20 and 21 of EGFR favour transformations to residues that can be identified in similar positions in evolutionary related proteins. The results demonstrate that evolutionary pressure shapes the mutational landscape in the case of drug-resistance somatic mutations. The constraints on the mutational landscape suggest that it may be possible to counter single drug-resistance point mutations. The observation of relatively many resistance mutations in Abl1, but not in the other genes, is explained by the fact that mutations in Abl1 tend to be biochemically conservative, whereas mutations in EGFR and ALK tend to be radical. Analysis of Abl1 compound mutations suggests that such mutations are more prevalent than hitherto reported and may be more difficult to counter. This supports the notion that such mutations may provide an escape route for targeted cancer drug resistance. PMID:24376513

Specific patterns of gyrA mutations determine the resistance difference to ciprofloxacin and levofloxacin in Klebsiella pneumoniae and Escherichia coli

PubMed Central

2013-01-01

Background Wide use of ciprofloxacin and levofloxacin has often led to increased resistance. The resistance rate to these two agents varies in different clinical isolates of Enterobacteriaceae. Mutations of GyrA within the quinolone resistance-determining regions have been found to be the main mechanism for quinolone resistance in Enterobacteriaceae. It has been shown that only some of the mutations in the gyrA gene identified from clinical sources were involved in fluoroquinolone resistance. Whether different patterns of gyrA mutation are related to antimicrobial resistance against ciprofloxacin and levofloxacin is unclear. Methods The minimum inhibitory concentration (MIC) of ciprofloxacin and levofloxacin were determined by the agar dilution method followed by PCR amplification and sequencing of the quinolone resistance determining region of gyrA to identify all the mutation types. The correlation between fluoroquinolone resistance and the individual mutation type was analyzed. Results Resistance differences between ciprofloxacin and levofloxacin were found in 327 isolates of K. pneumoniae and E. coli in Harbin, China and in the isolates reported in PubMed publications. GyrA mutations were found in both susceptible and resistant isolates. For the isolates with QRDR mutations, the resistance rates to ciprofloxacin and levofloxacin were also statistically different. Among the 14 patterns of alterations, two single mutations (Ser83Tyr and Ser83Ile), and three double mutations (Ser83Leu+Asp87Asn, Ser83Leu+Asp87Tyr and Ser83Phe+Asp87Asn) were associated with both ciprofloxacin and levofloxacin resistance. Two single mutations (Ser83Phe and Ser83Leu) were related with ciprofloxacin resistance but not to levofloxacin. Resistance difference between ciprofloxacin and levofloxacin in isolates harboring mutation Ser83Leu+Asp87Asn were of statistical significance among all Enterobacteriaceae (P<0.001). Conclusions Resistance rate to ciprofloxacin and levofloxacin were statistically different among clinical isolates of Enterobacteriaceae harboring GyrA mutations. Ser83Leu+Asp87Asn may account for the antimicrobial resistance difference between ciprofloxacin and levofloxacin. PMID:23295059

Clinicopathological characteristics of TERT promoter mutation and telomere length in hepatocellular carcinoma.

PubMed

Lee, Hye Won; Park, Tae In; Jang, Se Young; Park, Soo Young; Park, Won-Jin; Jung, Soo-Jung; Lee, Jae-Ho

2017-02-01

Promoter mutations in telomerase reverse transcriptase (TERT) and telomere length have been studied in various tumors. In the present study, the frequency and clinical characteristics of TERT promoter mutation and telomere length were studied in hepatocellular carcinoma (HCC). TERT promoter mutation and telomere length were analyzed in 162 tumor samples of the patients with HCC by sequencing and real-time PCR, respectively. The TERT promoter mutation rate was 28.8% (46/160) in HCC and was associated with males (P = 0.027). The telomere length was not significantly different in the presence of a TERT promoter mutation but was shorter in high-grade tumor stages (P = 0.048). Survival analyses showed that poor overall survival was associated with longer telomere length (P = 0.013). However, the TERT promoter mutation did not have a prognostic value for HCC. Multivariate survival analyses demonstrated that the telomere length was an independent prognostic marker for poor overall survival (hazard ratio = 1.75, 95% confidence interval: 1.046-2.913, P = 0.033). These data demonstrated that TERT promoter mutation is a frequent event in HCC; however, telomere length, but not the presence of a TERT promoter mutation, might have potential value as a prognostic indicator of HCC.

The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa

PubMed Central

Toll-Riera, Macarena; Heilbron, Karl

2016-01-01

Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings. PMID:26763710

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

PubMed

Qiu, Haixiang; Gong, Jiansen; Butaye, Patrick; Lu, Guangwu; Huang, Ke; Zhu, Guoqiang; Zhang, Jilei; Hathcock, Terri; Cheng, Darong; Wang, Chengming

2018-05-14

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Impact of lopinavir-ritonavir exposure in HIV-1 infected children and adolescents in Madrid, Spain during 2000-2014.

PubMed

Rojas Sánchez, Patricia; Prieto, Luis; Jiménez De Ory, Santiago; Fernández Cooke, Elisa; Navarro, Maria Luisa; Ramos, José Tomas; Holguín, África

2017-01-01

The most-used protease-inhibitor in children is Lopinavir-ritonavir (LPV/r), which provides durable suppression of viral load and increases CD4+T-counts. This study describes the virological outcome of the HIV-1-infected paediatric population exposed to LPV/r during 15 years in Spain. Patients from the Madrid Cohort of HIV-1-infected-children and adolescents exposed to LPV/r as different line therapy during 2000-2014 were selected. The baseline epidemiological-clinical features, viral suppression, changes in CD4+T-CD8+T cell counts and drug susceptibility were recorded before and during LPV/r exposure. Drug resistance mutations (DRM) were identified in viruses from samples collected until 2011. We predicted drug susceptibility to 19 antiretrovirals among those carrying DRM using the Stanford's HIVdb Algorithm. A total of 199 (37.3%) of the 534 patients from the cohort were exposed to LPV/r during 2000-2014 in first (group 1), second (group 2) or more line-therapies (group 3). Patients were mainly Spaniards (81.9%), perinatally infected (96.5%) with subtype-B (65.3%) and HIV-diagnosed before year 2000 (67.8%). The mean age at first LPV/r exposure was 9.7 years. After protease-inhibitor exposure, viral suppression was higher in groups 1 and 2 than in group 3. Viral suppression occurred in 87.5%, 68.6% and 64.8% patients from groups 1, 2 and 3, respectively. Among the 64 patients with available resistance data during LPV/r treatment, 27(42.3%) carried DRM to protease-inhibitor, 28 (58.3%) to reverse-transcriptase-inhibitors and 21 (43.7%) to non-reverse-transcriptase-inhibitors. Darunavir/ritonavir, atazanavir-ritonavir and tipranavir/ritonavir presented the highest susceptibility and nelfinavir the lowest. A better lymphocyte recovering occurred when protease-inhibitor was taken as part of a first-line regimen and a higher number of patients reached viral suppression. The least compromised antiretrovirals for rescue antiretroviral regimens, according to DRM in the LPV/r-exposed-paediatric cohort, were mainly the new protease inhibitors.

Theoretical benefits of mitogen applications for HIV-1 infections.

PubMed

Wimer, B M; Morris, R E

1997-06-01

Ideal treatment of HIV-1 infections should include an agent that can reverse the capacity of the virus to evade destruction by hiding in sanctuaries and by frequently mutating the epitopes it displays. The rapid proliferation of virions during the years of symptomatic quiescence obligates rapid replacement of CD4+ lymphocytes that leads to a gradual attrition of the T lymphocytes needed to control infections. In vitro evidences suggest that, given systematically, certain mitogenic lectins would interfere with HIV-1 invasion of CD4+ cells by blocking gp120 molecules on the viral membrane before activating T lymphocytes subsequent to binding with their Ti/CD3 molecules. The nonspecific nature of antiviral effector cells generated by this activation should circumvent HIV-1 mutations at the same time it reconstitutes depleted T lymphocytes, stimulates myelopoiesis, and reinforces resistance to malignancies and infections prevalent with the immunodeficiency state. Properly coordinating these effects with appropriate combinations of reverse transcriptase and protease inhibitors could theoretically expedite complete elimination of HIV in a timely fashion that shorten the required treatment duration and excludes the detrimental effects of virus mutations. The proper sequence of this treatment should be maximum reduction of the HIV-1 load with drug combinations, control of complicating infection by other means to reduce mitogen-induced tissue necrosis, and addition of systemic PHA-L4 administration regulated to maintain a 5-10 micrograms/mL serum concentration. The antiviral regimen should be continued an undetermined time beyond when HIV-1 is no longer detectable, and systemic L4 administration until satisfactory immunologic and hematologic competences are re-established. Partially-matched mitogen-activated adoptive leukocyte therapy might be additionally helpful.

[Study of human immunodeficiency virus transmission chains in Andalusia: analysis from baseline antiretroviral resistance sequences].

PubMed

Pérez-Parra, Santiago; Chueca-Porcuna, Natalia; Álvarez-Estevez, Marta; Pasquau, Juan; Omar, Mohamed; Collado, Antonio; Vinuesa, David; Lozano, Ana Belen; García-García, Federico

2015-11-01

Protease and reverse transcriptase HIV-1 sequences provide useful information for patient clinical management, as well as information on resistance to antiretrovirals. The aim of this study is to evaluate transmission events, transmitted drug resistance, and to georeference subtypes among newly diagnosed patients referred to our center. A study was conducted on 693 patients diagnosed between 2005 and 2012 in Southern Spain. Protease and reverse transcriptase sequences were obtained for resistance to cART analysis with Trugene(®) HIV Genotyping Kit (Siemens, NAD). MEGA 5.2, Neighbor-Joining, ArcGIS and REGA were used for subsequent analysis. The results showed 298 patients clustered into 77 different transmission events. Most of the clusters were formed by pairs (n=49), of men having sex with men (n=26), Spanish (n=37), and below 45 years of age (73.5%). Urban areas from Granada, and the coastal areas of Almeria and Granada showed the greatest subtype heterogeneity. Five clusters were formed by more than 10 patients, and 15 clusters had transmitted drug resistance. The study data demonstrate how the phylogenetic characterization of transmission clusters is a powerful tool to monitor the spread of HIV, and may contribute to design correct preventive measures to minimize it. Copyright © 2015 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Active methamphetamine use is associated with transmitted drug resistance to non-nucleoside reverse transcriptase inhibitors in individuals with HIV infection of unknown duration.

PubMed

Cachay, Edward R; Moini, Niousha; Kosakovsky Pond, Sergei L; Pesano, Rick; Lie, Yolanda S; Aiem, Heidi; Butler, David M; Letendre, Scott; Mathews, Wm Christopher; Smith, Davey M

2007-01-01

Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Cross-sectional analysis. Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI.

Molecular Characteristics of Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Strains Isolated in Vietnam

PubMed Central

Van Bac, Nguyen; Son, Nguyen Thai; Lien, Vu Thi Kim; Ha, Chu Hoang; Cuong, Nguyen Huu; Mai, Cung Thi Ngoc; Le, Thanh Hoa

2012-01-01

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp “hot-spot” region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests. PMID:22170905

Molecular characteristics of rifampin- and isoniazid-resistant mycobacterium tuberculosis strains isolated in Vietnam.

PubMed

Minh, Nghiem Ngoc; Van Bac, Nguyen; Son, Nguyen Thai; Lien, Vu Thi Kim; Ha, Chu Hoang; Cuong, Nguyen Huu; Mai, Cung Thi Ngoc; Le, Thanh Hoa

2012-03-01

Molecular characterization of the drug resistance of Mycobacterium tuberculosis strains with different origins can generate information that is useful for developing molecular methods. These methods are widely applicable for rapid detection of drug resistance. A total of 166 rifampin (RIF)- and/or isoniazid (INH)-resistant strains of M. tuberculosis have been isolated from different parts of Vietnam; they were screened for mutations associated with resistance to these drugs by sequence analysis investigating genetic mutations associated with RIF and INH resistance. Seventeen different mutations were identified in 74 RIF-resistant strains, 56 of which (approximately 76%) had mutations in the so-called 81-bp "hot-spot" region of the rpoB gene. The most common point mutations were in codons 531 (37.8%), 526 (23%), and 516 (9.46%) of the rpoB gene. Mutations were not found in three strains (4.05%). In the case of INH resistance, five different mutations in the katG genes of 82 resistant strains were detected, among which the nucleotide substitution at codon 315 (76.83%) is the most common mutation. This study provided the first molecular characterization of INH and RIF resistance of M. tuberculosis strains from Vietnam, and detection of the katG and rpoB mutations of the INH and RIF-resistant strains should be useful for rapid detection of the INH- and RIF-resistant strains by molecular tests.

Impact of Antiretroviral Regimens on CSF Viral Escape in a Prospective Multicohort Study of ART-Experienced HIV-1 Infected Adults in the United States.

PubMed

Mukerji, Shibani S; Misra, Vikas; Lorenz, David R; Uno, Hajime; Morgello, Susan; Franklin, Donald; Ellis, Ronald J; Letendre, Scott; Gabuzda, Dana

2018-04-03

Cerebrospinal fluid (CSF) viral escape occurs in 4-20% of HIV-infected adults, yet the impact of antiretroviral therapy (ART) on CSF escape is unclear. Prospective study of 1063 participants with baseline plasma viral load (VL) ≤400 copies/ml between 2005-2016. Odds ratio for ART regimens (PI with nucleoside reverse transcriptase inhibitor [PI+NRTI] versus other ART) and CSF escape was estimated using mixed-effects models. Drug resistance mutation frequencies were calculated. Baseline mean age was 46, median plasma VL, CD4 nadir, and CD4 count were 50 copies/mL, 88 cells/μL, and 424 cells/μL, respectively; 48% on PI+NRTI, 33% on non-NRTI, and 6% on integrase inhibitors. During median follow-up of 4.4 years, CSF escape occurred in 77 participants (7.2%). PI+NRTI use was an independent predictor of CSF escape (OR 3.1 [95% CI 1.8-5.0]) in adjusted analyses and models restricted to plasma VL ≤50 copies/ml (p<0.001). Regimens containing atazanavir (ATV) were a stronger predictor of CSF viral escape than non-ATV PI+NRTI regimens. Plasma and CSF M184V/I combined with thymidine-analog mutations were more frequent in CSF escape versus no escape (23% vs. 2.3%). Genotypic susceptibility score-adjusted CNS penetration-effectiveness (CPE) values were calculated for CSF escape with M184V/I mutations (n=34). Adjusted CPE values were low (<5) for CSF and plasma in 27 (79%) and 13 (38%), respectively, indicating suboptimal CNS drug availability. PI+NRTI regimens are independent predictors of CSF escape in HIV-infected adults. Reduced CNS ART bioavailability may predispose to CSF escape in patients with M184V/I mutations. Optimizing ART regimens may reduce risk of CSF escape.

Allele-specific DNA methylation and its interplay with repressive histone marks at promoter-mutant TERT genes

PubMed Central

Stern, Josh Lewis; Paucek, Richard D.; Huang, Franklin W.; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C.; Cech, Thomas R.

2017-01-01

SUMMARY A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here we find that DNA methylation of the TERT CpG Island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter-mutant cancers. Finally, in several cancers DNA methylation levels at the TERT CGI correlate with altered patient survival. PMID:29281820

Detection of Multidrug Resistance in Mycobacterium tuberculosis▿

PubMed Central

Sekiguchi, Jun-ichiro; Miyoshi-Akiyama, Tohru; Augustynowicz-Kopeć, Ewa; Zwolska, Zofia; Kirikae, Fumiko; Toyota, Emiko; Kobayashi, Intetsu; Morita, Koji; Kudo, Koichiro; Kato, Seiya; Kuratsuji, Tadatoshi; Mori, Toru; Kirikae, Teruo

2007-01-01

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis. PMID:17108078

HIV-1 drug resistance surveillance in antiretroviral treatment-naive individuals from a reference hospital in Guatemala, 2010-2013.

PubMed

Avila-Ríos, Santiago; García-Morales, Claudia; Garrido-Rodríguez, Daniela; Tapia-Trejo, Daniela; Girón-Callejas, Amalia Carolina; Mendizábal-Burastero, Ricardo; Escobar-Urias, Ingrid Yessenia; García-González, Blanca Leticia; Navas-Castillo, Sabrina; Pinzón-Meza, Rodolfo; Mejía-Villatoro, Carlos Rodolfo; Reyes-Terán, Gustavo

2015-04-01

The recent expansion of antiretroviral treatment (ART) coverage in middle/low-income countries has been associated with increasing prevalence of HIV pre-ART drug resistance (PDR). We assessed PDR prevalence, patterns, and trends in Guatemala. Blood samples from 1,084 ART-naive individuals, enrolled from October 2010 to December 2013 at the Roosevelt Hospital in Guatemala City, were obtained. PDR was evaluated using the WHO mutation list for transmitted drug resistance (TDR) surveillance. An overall PDR prevalence of 7.3% (95% CI 5.8-9.0%) was observed for the whole study period. TDR to nonnucleoside reverse transcriptase inhibitors (NNRTI) was the highest (4.9%, p<0.001), followed by nucleoside RT inhibitors (1.8%) and protease inhibitors (1.0%). No significant trends in PDR prevalence were observed during the study period. However, higher NNRTI PDR levels were found in individuals with >500 and 350-500 CD4(+) T cells/μl (7.4% and 8.7%, respectively) compared to individuals with <350 CD4(+) T cells/μl (3.7%; p=0.039 and p=0.007, respectively), as well as a tendency of higher levels of NNRTI transmitted drug resistance (DR) in individuals with recent infection determined by HIV incidence tests (9.7%), suggesting increasing trends in time. Clusters of viruses with NNRTI PDR suggesting complex transmission networks were observed. No associations between PDR and demographic variables were found. PDR in Guatemala remains at an intermediate level. Nevertheless, we have shown evidence suggesting increasing trends in NNRTI PDR, which need to be taken into account in national HIV management policies.

Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing.

PubMed

Tzou, Philip L; Ariyaratne, Pramila; Varghese, Vici; Lee, Charlie; Rakhmanaliev, Elian; Villy, Carolin; Yee, Meiqi; Tan, Kevin; Michel, Gerd; Pinsky, Benjamin A; Shafer, Robert W

2018-06-01

The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy. Copyright © 2018 American Society for Microbiology.

Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone.

PubMed

López-Causapé, Carla; Sommer, Lea Mette; Cabot, Gabriel; Rubio, Rosa; Ocampo-Sosa, Alain A; Johansen, Helle Krogh; Figuerola, Joan; Cantón, Rafael; Kidd, Timothy J; Molin, Soeren; Oliver, Antonio

2017-07-17

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up β-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

The Geogenomic Mutational Atlas of Pathogens (GoMAP) Web System

PubMed Central

Sargeant, David P.; Hedden, Michael W.; Deverasetty, Sandeep; Strong, Christy L.; Alaniz, Izua J.; Bartlett, Alexandria N.; Brandon, Nicholas R.; Brooks, Steven B.; Brown, Frederick A.; Bufi, Flaviona; Chakarova, Monika; David, Roxanne P.; Dobritch, Karlyn M.; Guerra, Horacio P.; Levit, Kelvy S.; Mathew, Kiran R.; Matti, Ray; Maza, Dorothea Q.; Mistry, Sabyasachy; Novakovic, Nemanja; Pomerantz, Austin; Rafalski, Timothy F.; Rathnayake, Viraj; Rezapour, Noura; Ross, Christian A.; Schooler, Steve G.; Songao, Sarah; Tuggle, Sean L.; Wing, Helen J.; Yousif, Sandy; Schiller, Martin R.

2014-01-01

We present a new approach for pathogen surveillance we call Geogenomics. Geogenomics examines the geographic distribution of the genomes of pathogens, with a particular emphasis on those mutations that give rise to drug resistance. We engineered a new web system called Geogenomic Mutational Atlas of Pathogens (GoMAP) that enables investigation of the global distribution of individual drug resistance mutations. As a test case we examined mutations associated with HIV resistance to FDA-approved antiretroviral drugs. GoMAP-HIV makes use of existing public drug resistance and HIV protein sequence data to examine the distribution of 872 drug resistance mutations in ∼502,000 sequences for many countries in the world. We also implemented a broadened classification scheme for HIV drug resistance mutations. Several patterns for geographic distributions of resistance mutations were identified by visual mining using this web tool. GoMAP-HIV is an open access web application available at http://www.bio-toolkit.com/GoMap/project/ PMID:24675726

The Geogenomic Mutational Atlas of Pathogens (GoMAP) web system.

PubMed

Sargeant, David P; Hedden, Michael W; Deverasetty, Sandeep; Strong, Christy L; Alaniz, Izua J; Bartlett, Alexandria N; Brandon, Nicholas R; Brooks, Steven B; Brown, Frederick A; Bufi, Flaviona; Chakarova, Monika; David, Roxanne P; Dobritch, Karlyn M; Guerra, Horacio P; Levit, Kelvy S; Mathew, Kiran R; Matti, Ray; Maza, Dorothea Q; Mistry, Sabyasachy; Novakovic, Nemanja; Pomerantz, Austin; Rafalski, Timothy F; Rathnayake, Viraj; Rezapour, Noura; Ross, Christian A; Schooler, Steve G; Songao, Sarah; Tuggle, Sean L; Wing, Helen J; Yousif, Sandy; Schiller, Martin R

2014-01-01

We present a new approach for pathogen surveillance we call Geogenomics. Geogenomics examines the geographic distribution of the genomes of pathogens, with a particular emphasis on those mutations that give rise to drug resistance. We engineered a new web system called Geogenomic Mutational Atlas of Pathogens (GoMAP) that enables investigation of the global distribution of individual drug resistance mutations. As a test case we examined mutations associated with HIV resistance to FDA-approved antiretroviral drugs. GoMAP-HIV makes use of existing public drug resistance and HIV protein sequence data to examine the distribution of 872 drug resistance mutations in ∼ 502,000 sequences for many countries in the world. We also implemented a broadened classification scheme for HIV drug resistance mutations. Several patterns for geographic distributions of resistance mutations were identified by visual mining using this web tool. GoMAP-HIV is an open access web application available at http://www.bio-toolkit.com/GoMap/project/

Mutations inside rifampicin-resistance determining region of rpoB gene associated with rifampicin-resistance in Mycobacterium tuberculosis.

PubMed

Zaw, Myo T; Emran, Nor A; Lin, Zaw

2018-04-26

Rifampicin (RIF) plays a pivotal role in the treatment of tuberculosis due to its bactericidal effects. Because the action of RIF is on rpoB gene encoding RNA polymerase β subunit, 95% of RIF resistant mutations are present in rpoB gene. The majority of the mutations in rpoB gene are found within an 81bp RIF-resistance determining region (RRDR). Literatures on RIF resistant mutations published between 2010 and 2016 were thoroughly reviewed. The most commonly mutated codons in RRDR of rpoB gene are 531, 526 and 516. The possibilities of absence of mutation in RRDR of rpoB gene in MDR-TB isolates in few studies was due to existence of other rare rpoB mutations outside RRDR or different mechanism of rifampicin resistance. Molecular methods which can identify extensive mutations associated with multiple anti-tuberculous drugs are in urgent need so that the research on drug resistant mutations should be extended. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Low-Cost Ultra-Wide Genotyping Using Roche/454 Pyrosequencing for Surveillance of HIV Drug Resistance

PubMed Central

Dudley, Dawn M.; Chin, Emily N.; Bimber, Benjamin N.; Sanabani, Sabri S.; Tarosso, Leandro F.; Costa, Priscilla R.; Sauer, Mariana M.; Kallas, Esper G.; O.’Connor, David H.

2012-01-01

Background Great efforts have been made to increase accessibility of HIV antiretroviral therapy (ART) in low and middle-income countries. The threat of wide-scale emergence of drug resistance could severely hamper ART scale-up efforts. Population-based surveillance of transmitted HIV drug resistance ensures the use of appropriate first-line regimens to maximize efficacy of ART programs where drug options are limited. However, traditional HIV genotyping is extremely expensive, providing a cost barrier to wide-scale and frequent HIV drug resistance surveillance. Methods/Results We have developed a low-cost laboratory-scale next-generation sequencing-based genotyping method to monitor drug resistance. We designed primers specifically to amplify protease and reverse transcriptase from Brazilian HIV subtypes and developed a multiplexing scheme using multiplex identifier tags to minimize cost while providing more robust data than traditional genotyping techniques. Using this approach, we characterized drug resistance from plasma in 81 HIV infected individuals collected in São Paulo, Brazil. We describe the complexities of analyzing next-generation sequencing data and present a simplified open-source workflow to analyze drug resistance data. From this data, we identified drug resistance mutations in 20% of treatment naïve individuals in our cohort, which is similar to frequencies identified using traditional genotyping in Brazilian patient samples. Conclusion The developed ultra-wide sequencing approach described here allows multiplexing of at least 48 patient samples per sequencing run, 4 times more than the current genotyping method. This method is also 4-fold more sensitive (5% minimal detection frequency vs. 20%) at a cost 3–5× less than the traditional Sanger-based genotyping method. Lastly, by using a benchtop next-generation sequencer (Roche/454 GS Junior), this approach can be more easily implemented in low-resource settings. This data provides proof-of-concept that next-generation HIV drug resistance genotyping is a feasible and low-cost alternative to current genotyping methods and may be particularly beneficial for in-country surveillance of transmitted drug resistance. PMID:22574170

Exploiting Drug-Resistant Enzymes as Tools to Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

PubMed Central

Masaoka, Takashi; Chung, Suhman; Caboni, Pierluigi; Rausch, Jason W.; Wilson, Jennifer A.; Taskent-Sezgin, Humeyra; Beutler, John A.; Tocco, Graziella; Le Grice, Stuart F. J.

2013-01-01

The thienopyrimidinone 5,6-dimethyl-2-(4-nitrophenyl)thieno[2,3-d]pyrimidin-4(3H)-one (DNTP) occupies the interface between the p66 ribonuclease H (RNase H) domain and p51 thumb of human immunodeficiency virus reverse transcriptase (HIV RT), thereby inducing a conformational change incompatible with catalysis. Here, we combined biochemical characterization of 39 DNTP derivatives with antiviral testing of selected compounds. In addition to wild-type HIV-1 RT, derivatives were evaluated with rationally-designed, p66/p51 heterodimers exhibiting high-level DNTP sensitivity or resistance. This strategy identified 3′,4′-dihydroxyphenyl (catechol)-substituted thienopyrimidinones with sub-micromolar in vitro activity against both wild type HIV-1 RT and drug-resistant variants. Thermal shift analysis indicates that, in contrast to active site RNase H inhibitors, these thienopyrimidinones destabilize the enzyme, in some instances reducing the Tm by 5°C. Importantly, catechol-containing thienopyrimidinones also inhibit HIV-1 replication in cells. Our data strengthens the case for allosteric inhibition of HIV RNase H activity, providing a platform for designing improved antagonists for use in combination antiviral therapy. PMID:23631411

Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses.

PubMed

Hu, Yanmei; Zhang, Jiantao; Musharrafieh, Rami Ghassan; Ma, Chunlong; Hau, Raymond; Wang, Jun

2017-09-01

The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals. Copyright © 2017 Elsevier B.V. All rights reserved.

Diaryltriazine non-nucleoside reverse transcriptase inhibitors are potent candidates for pre-exposure prophylaxis in the prevention of sexual HIV transmission.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Abdellati, Saïd; Cuylaerts, Vicky; Crucitti, Tania; Heyndrickx, Leo; Heeres, Jan; Augustyns, Koen; Lewi, Paul J; Vanham, Guido

2013-09-01

Pre-exposure prophylaxis and topical microbicides are important strategies in the prevention of sexual HIV transmission, especially since partial protection has been shown in proof-of-concept studies. In search of new candidate drugs with an improved toxicity profile and with activity against common non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV, we have synthesized and investigated a library of 60 new diaryltriazine analogues. From this library, 15 compounds were evaluated in depth using a broad armamentarium of in vitro assays that are part of a preclinical testing algorithm for microbicide development. Antiviral activity was assessed in a cell line, and in primary human cells, against both subtype B and subtype C HIV-1 and against viruses resistant to therapeutic NNRTIs and the candidate NNRTI microbicide dapivirine. Toxicity towards primary blood-derived cells, cell lines originating from the female reproductive tract and female genital microflora was also studied. We identified several compounds with highly potent antiviral activity and toxicity profiles that are superior to that of dapivirine. In particular, compound UAMC01398 is an interesting new candidate that warrants further investigation because of its superior toxicity profile and potent activity against dapivirine-resistant viruses.

Revealing the drug-resistant mechanism for diarylpyrimidine analogue inhibitors of HIV-1 reverse transcriptase.

PubMed

Zhang, Hao; Qin, Fang; Ye, Wei; Li, Zeng; Ma, Songyao; Xia, Yan; Jiang, Yi; Zhu, Jiayi; Li, Yixue; Zhang, Jian; Chen, Hai-Feng

2011-09-01

Diaryltriazine (DATA) and diarylpyrimidine (DAPY) were two category inhibitors with highly potent activity for wild type (wt) and four principal mutant types (L100I, K103N, Y181C and Y188L) of HIV-1 reverse transcriptase (RT). We had revealed the drug-resistant mechanism of DATA analogue inhibitors with molecular dynamics simulation and three-dimensional quantitative structure-activity relationship (3D-QSAR) methods. In this work, we investigated the drug-resistant mechanism of DAPY analogue inhibitors. It was found that DAPY analogue inhibitors form more hydrogen bonds and hydrophobic contacts with wild type and mutants of HIV-1 RT than DATA inhibitors. This could explain that DAPY analogue inhibitors are more potent than DATA for the wild type and mutants of HIV-1 RT. Then, 3D-QSAR models were constructed for these inhibitors of wild type and four principal mutant types HIV-1 RT and evaluated by test set compounds. These combined models can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 RT inhibitors before resorting to in vitro and in vivo experiment. © 2011 John Wiley & Sons A/S.

Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia.

PubMed

Dang, Kai; Toi, Cheryl S; Lilly, David G; Bu, Wenjun; Doggett, Stephen L

2015-07-01

Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown resistance (kdr) mutations, notably V419L and L925I. Information regarding this kdr-type resistance mechanism is unknown in Australia. This study aims to examine the status of kdr mutations in Australian C. lectularius strains. Several modern field-collected strains and museum-preserved reference collections of Australian C. lectularius were examined. Of the field strains (2007-2013), 96% had the known kdr mutations (L925I or both V419L/L925I). The 'Adelaide' strain (2013) and samples from the preserved reference collections (1994-2002) revealed no known kdr mutations. A novel mutation I936F was apparent in the insecticide-resistant 'Adelaide' strain, one strain from Perth (with L925I) and the majority of the reference collection specimens. The laboratory insecticide-resistant 'Sydney' strain showed a mixture of no kdr mutations (20%) and L925I (80%). The novel mutation I936F may be a kdr mutation but appeared to contribute less resistance to the pyrethroids than the V419L and L925I mutations. The detection of high frequencies of kdr mutations indicates that kdr-type resistance is widespread across Australia. Hence, there should be a reduced reliance on pyrethroid insecticides and an integrated management approach for the control of C. lectularius infestations. © 2014 Society of Chemical Industry.

Characterization of mutations in streptomycin-resistant Mycobacterium tuberculosis isolates in Sichuan, China and the association between Beijing-lineage and dual-mutation in gidB.

PubMed

Sun, Honghu; Zhang, Congcong; Xiang, Ling; Pi, Rui; Guo, Zhen; Zheng, Chao; Li, Song; Zhao, Yuding; Tang, Ke; Luo, Mei; Rastogi, Nalin; Li, Yuqing; Sun, Qun

2016-01-01

Mutations in rpsL, rrs, and gidB are well linked to streptomycin (STR) resistance, some of which are suggested to be potentially associated with Mycobacterium tuberculosis genotypic lineages in certain geographic regions. In this study, we aimed to investigate the mutation characteristics of streptomycin resistance and the relationship between the polymorphism of drug-resistant genes and the lineage of M. tuberculosis isolates in Sichuan, China. A total of 227 M. tuberculosis clinical isolates, including 180 STR-resistant and 47 pan-susceptible isolates, were analyzed for presence of mutations in the rpsL, rrs and gidB loci. Mutation K43R in rpsL was strongly associated with high-level streptomycin resistance (P < 0.01), while mutations in rrs and gidB potentially contributed to low-level resistance (P < 0.05). No general association was exhibited between STR resistance and Beijing genotype, however, in STR-resistant strains, Beijing genotype was significantly correlated with high-level STR resistance, as well as the rpsL mutation K43R (P < 0.01), indicating that Beijing genotype has an evolutionary advantage under streptomycin pressure. Notably, in all isolates of Beijing genotype, a dual mutation E92D (a276c) and A205A (a615g) in gidB was detected, suggesting a highly significant association between this dual mutation and Beijing genotype. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of Mutation Accumulation as Resistance Mechanism Emerging in First-Line Osimertinib Treatment.

PubMed

Uchibori, Ken; Inase, Naohiko; Nishio, Makoto; Fujita, Naoya; Katayama, Ryohei

2018-04-24

The survival of patients with EGFR mutation-positive lung cancer has dramatically improved since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Recently, osimertinib showed significantly prolonged progression-free survival than first-generation EGFR-TKI in first-line treatment, suggesting that a paradigm change that would move osimetinib to first-line treatment is indicated. We performed N-ethyl-N-nitrosourea (ENU) mutagenesis screening to uncover the resistant mechanism in first- and second-line osimertinib treatment. Ba/F3 cells harboring EGFR activating-mutation with or without secondary resistant mutation were exposed to ENU for 24 hours to introduce random mutations and selected with gefitinib, afatinib, or osimertinib. Mutations of emerging resistant cells were assessed. The resistance of T790M and C797S to gefitinib and osimertinib, respectively, was prevalent in the mutagenesis screening with the Ba/F3 cells harboring activating-mutation alone. From C797S/activating-mutation expressing Ba/F3, the additional T790M was a major resistant mechanism in gefitinib and afatinib selection and the additional T854A and L792H were minor resistance mechanisms only in afatinib selection. However, the additional T854A or L792H mediated resistance to all classes of EGFR-TKI. Surprisingly, no resistant clone due to secondary mutation emerged from activating-mutation alone in the gefitinib + osimertinib selection. We showed the resistance mechanism to EGFR-TKI focusing on first- and second-line osimertinib using ENU mutagenesis screening. Additional T854A and L792H on C797S/activating-mutation were found as afatinib resistance and not as gefitinib resistance. Thus, compared to afatinib, the first-generation EGFR-TKI might be preferable as second-line treatment to C797S/activating-mutation emerging after first-line osimertinib treatment. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Mutational Activation of the AmgRS Two-Component System in Aminoglycoside-Resistant Pseudomonas aeruginosa

PubMed Central

Lau, Calvin Ho-Fung; Fraud, Sebastien; Jones, Marcus; Peterson, Scott N.; Poole, Keith

2013-01-01

The amgRS operon encodes a presumed membrane stress-responsive two-component system linked to intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Genome sequencing of a lab isolate showing modest pan-aminoglycoside resistance, strain K2979, revealed a number of mutations, including a substitution in amgS that produced an R182C change in the AmgS sensor kinase product of this gene. Introduction of this mutation into an otherwise wild-type strain recapitulated the resistance phenotype, while correcting the mutation in the resistant mutant abrogated the resistant phenotype, confirming that the amgS mutation is responsible for the aminoglycoside resistance of strain K2979. The amgSR182 mutation promoted an AmgR-dependent, 2- to 3-fold increase in expression of the AmgRS target genes htpX and PA5528, mirroring the impact of aminoglycoside exposure of wild-type cells on htpX and PA5528 expression. This suggests that amgSR182 is a gain-of-function mutation that activates AmgS and the AmgRS two-component system in promoting modest resistance to aminoglycosides. Screening of several pan-aminoglycoside-resistant clinical isolates of P. aeruginosa revealed three that showed elevated htpX and PA5528 expression and harbored single amino acid-altering mutations in amgS (V121G or D106N) and no mutations in amgR. Introduction of the amgSV121G mutation into wild-type P. aeruginosa generated a resistance phenotype reminiscent of the amgSR182 mutant and produced a 2- to 3-fold increase in htpX and PA5528 expression, confirming that it, too, is a gain-of-function aminoglycoside resistance-promoting mutation. These results highlight the contribution of amgS mutations and activation of the AmgRS two-component system to acquired aminoglycoside resistance in lab and clinical isolates of P. aeruginosa. PMID:23459488

Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species

PubMed Central

Yu, Q; Ahmad-Hamdani, M S; Han, H; Christoffers, M J; Powles, S B

2013-01-01

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids. PMID:23047200

Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba.

PubMed

Alemán, Yoan; Vinken, Lore; Kourí, Vivian; Pérez, Lissette; Álvarez, Alina; Abrahantes, Yeissel; Fonseca, Carlos; Pérez, Jorge; Correa, Consuelo; Soto, Yudira; Schrooten, Yoeri; Vandamme, Anne-Mieke; Van Laethem, Kristel

2015-01-01

As commercial human immunodeficiency virus type 1 drug resistance assays are expensive, they are not commonly used in resource-limited settings. Hence, a more affordable in-house procedure was set up taking into account the specific epidemiological and economic circumstances of Cuba. The performance characteristics of the in-house assay were evaluated using clinical samples with various subtypes and resistance patterns. The lower limit of amplification was determined on dilutions series of 20 clinical isolates and ranged from 84 to 529 RNA copies/mL. For the assessment of trueness, 14 clinical samples were analyzed and the ViroSeq HIV-1 Genotyping System v2.0 was used as the reference standard. The mean nucleotide sequence identity between the two assays was 98.7% ± 1.0. Additionally, 99.0% of the amino acids at drug resistance positions were identical. The sensitivity and specificity in detecting drug resistance mutations was respectively 94.1% and 99.5%. Only few discordances in drug resistance interpretation patterns were observed. The repeatability and reproducibility were evaluated using 10 clinical samples with 3 replicates per sample. The in-house test was very precise as nucleotide sequence identity among paired nucleotide sequences ranged from 98.7% to 99.9%. The acceptance criteria were met by the in-house test for all performance characteristics, demonstrating a high degree of accuracy. Subsequently, the applicability in routine clinical practice was evaluated on 380 plasma samples. The amplification success rate was 91% and good quality consensus sequences encoding the entire protease and the first 335 codons in reverse transcriptase could be obtained for 99% of the successful amplicons. The reagent cost per sample using the in-house procedure was around € 80 per genotyping attempt. Overall, the in-house assay provided good results, was feasible with equipment and reagents available in Cuba and was half as expensive as commercial assays.

Longitudinal trends of HIV drug resistance in a large Canadian cohort, 1996-2016.

PubMed

Rocheleau, G; Brumme, C J; Shoveller, J; Lima, V D; Harrigan, P R

2018-02-01

We aim to identify long-term trends in HIV drug resistance before and after combined antiretroviral therapy (cART) initiation. IAS-USA (2015) mutations were identified in 23 271 HIV protease-reverse transcriptase sequences from 6543 treatment naïve adults in British Columbia. Participants who started cART between 1996 and 2014 were followed until April 2016. Equality of proportions test was used to compare the percentage of participants with acquired drug resistance (ADR) or transmitted drug resistance (TDR) in 1996, to those in 2014. Kaplan-Meier was used to estimate time to ADR in four drug resistance categories. Multivariable regression odds ratios (OR) of ADR for select clinical variables were determined by 5-year eras of cART initiation. The proportion of individuals with ADR declined from 39% (51/132) to 3% (8/322) in 1996-2014 (p <0.0001), while the proportion with TDR increased from 12% (16/132) to 18% (59/322) (p 0.14). The estimated proportions of individuals with ADR rose to 29% (NNRTI), 28% (3TC/FTC), 14% (other nRTI), and 7% (PI) after >16 years of therapy. After 5 years on therapy, participants initiating cART in 1996-2000 had 5.5-times more 3TC/FTC ADR, 5.3-times more other nRTI ADR, 4.7-times more NNRTI ADR, and 24-times more PI ADR than those starting in 2011-2014. The individuals with highest odds of developing ADR in 1996-2010 were adherent to regimens at levels between 60% and 80%, which shifted to <40% adherent in 2011-2014. HIV drug resistance transitioned from being primarily selected de-novo to being driven by TDR. Among those who started treatment in the past 5 years, ADR is rare and observed mostly in the lowest adherence strata. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Five Antiretroviral Drug Class-Resistant HIV-1 in a Treatment-Naïve Patient Successfully Suppressed with Optimized Antiretroviral Drug Selection.

PubMed

Volpe, Joseph M; Ward, Douglas J; Napolitano, Laura; Phung, Pham; Toma, Jonathan; Solberg, Owen; Petropoulos, Christos J; Walworth, Charles M

2015-01-01

Transmitted HIV-1 exhibiting reduced susceptibility to protease and reverse transcriptase inhibitors is well documented but limited for integrase inhibitors and enfuvirtide. We describe here a case of transmitted 5 drug class-resistance in an antiretroviral (ARV)-naïve patient who was successfully treated based on the optimized selection of an active ARV drug regimen. The value of baseline resistance testing to determine an optimal ARV treatment regimen is highlighted in this case report. © The Author(s) 2015.

Are BTK and PLCG2 mutations necessary and sufficient for ibrutinib resistance in chronic lymphocytic leukemia?

PubMed

Lampson, Benjamin L; Brown, Jennifer R

2018-03-01

Ibrutinib is the first BTK inhibitor to show efficacy in chronic lymphocytic leukemia (CLL) and is also the first BTK inhibitor to which patients have developed resistance. Mutations in BTK and PLCG2 are found in ≈80% of CLL patients with acquired resistance to ibrutinib, but it remains unclear if these mutations are merely associated with disease relapse or directly cause it. Areas covered: Unique properties of both CLL and ibrutinib that complicate attempts to definitively conclude whether BTK/PLCG2 mutations are passengers or drivers of ibrutinib-resistant disease are reviewed. Characteristics of mutations that drive drug resistance are summarized and whether BTK/PLCG2 mutations possess these is discussed. These characteristics include (1) identification in multiple patients with acquired resistance, (2) in vitro validation of drug-resistant properties, (3) mutual exclusivity with one another, (4) increasing frequency over time on drug, and (5) high frequency at the time and site of clinical relapse. Expert commentary: While BTK/PLCG2 mutations have characteristics suggesting that they can drive ibrutinib resistance, this conclusion remains formally unproven until specific inhibition of such mutations is shown to cause regression of ibrutinib-resistant CLL. Data suggest that alternative mechanisms of resistance do exist in some patients.

Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

PubMed

Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping

2017-06-01

The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

Biological significance of TERT promoter mutation in papillary urothelial neoplasm of low malignant potential.

PubMed

Wang, Chung-Chieh; Huang, Chao-Yuan; Jhuang, Yu-Lin; Chen, Chih-Chi; Jeng, Yung-Ming

2018-04-01

Mutations in FGFR3 and the promoter region of the telomerase reverse transcriptase (TERT) gene have been found frequently in urothelial carcinoma of the urinary bladder. However, related data for papillary urothelial neoplasm of low malignant potential (PUNLMP) are limited. In this study, we investigated the mutation status of the TERT promoter, FGFR3 and HRAS in low-grade papillary urothelial neoplasms and evaluated their prognostic significance. The cases included in this study comprised 21 inverted papillomas, 30 PUNLMPs and 34 low-grade non-invasive papillary urothelial carcinomas (NIPUCs). TERT promoter mutations were observed in 10 (33%) PUNLMPs and 17 (50%) low-grade NIPUCs, but not in any inverted papilloma. FGFR3 mutations were observed more frequently in PUNLMP and low-grade NIPUC than in inverted papillomas (P = 0.009), whereas the opposite trend was noted for HRAS mutations (P < 0.001). Regarding the clinical outcome, TERT promoter mutation was associated with a higher recurrence rate in PUNLMP (P = 0.024) but not in low-grade NIPUC (P = 0.530). Notably, PUNLMP cases with TERT promoter mutations had a similar recurrence rate to that in low-grade NIPUC cases (P = 0.487). Our results suggest that the status of the TERT promoter mutation may serve as a biomarker of prognostic stratification in patients with PUNLMP. © 2017 John Wiley & Sons Ltd.

Switch to Rilpivirine/Emtricitabine/Tenofovir Single-Tablet Regimen of Human Immunodeficiency Virus-1 RNA-Suppressed Patients, Agence Nationale de Recherches sur le SIDA et les Hépatites Virales CO3 Aquitaine Cohort, 2012-2014.

PubMed

Cazanave, Charles; Reigadas, Sandrine; Mazubert, Cyril; Bellecave, Pantxika; Hessamfar, Mojgan; Le Marec, Fabien; Lazaro, Estibaliz; Peytavin, Gilles; Bruyand, Mathias; Fleury, Hervé; Dabis, François; Neau, Didier

2015-01-01

Background.  The purpose of this study was to assess the efficacy and tolerability of combined antiretroviral therapy (cART) in human immunodeficiency virus (HIV)-1 virologically suppressed patients who switched to rilpivirine (RPV)/tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC) as a single-tablet regimen (STR). Methods.  A retrospective multicenter cohort study was performed between September 2012 and February 2014 in Bordeaux University Hospital-affiliated clinics. Patients with a plasma HIV viral load (VL) lower than 50 copies/mL and switching to STR were evaluated at baseline, 3, 6, 9, and 12 months from switch time (M3, M6, M9, M12) for VL and other biological parameters. Change from baseline in CD4 cell counts was evaluated at M6 and M12. Virological failure (VF) was defined as 2 consecutive VL >50 copies/mL. Results.  Three hundred four patients were included in the analysis. Single-tablet regimen switch was proposed to 116 patients with adverse events, mostly efavirenz (EFV)-based (n = 59), and to 224 patients for cART simplification. Thirty of 196 patients with available genotype resistance test results displayed virus with ≥1 drug resistance mutation on reverse-transcriptase gene. After 12 months of follow-up, 93.4% (95.5% confidence interval, 89.9-96.2) of patients remained virologically suppressed. There was no significant change in CD4 cell count. During the study period, 5 patients experienced VF, one of them harboring RPV resistance mutation. Clinical cART tolerability improved in 79 patients overall (29.9%) at M6, especially neurological symptoms related to EFV. Fasting serum lipid profiles improved, but a significant estimated glomerular function rate decrease (-11 mL/min/1.73 m(2); P < 10(-4)) was observed. Conclusions.  Overall, virologic suppression was maintained in patients after switching to RPV/TDF/ FTC. This STR strategy was associated with improved tolerability.

Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation

PubMed Central

Betz-Stablein, B. D.; Töpfer, A.; Littlejohn, M.; Yuen, L.; Colledge, D.; Sozzi, V.; Angus, P.; Thompson, A.; Revill, P.; Beerenwinkel, N.; Warner, N.

2016-01-01

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available. PMID:27252524

Mutational profiling of non-small-cell lung cancer patients resistant to first-generation EGFR tyrosine kinase inhibitors using next generation sequencing

PubMed Central

Jin, Ying; Shao, Yang; Shi, Xun; Lou, Guangyuan; Zhang, Yiping; Wu, Xue; Tong, Xiaoling; Yu, Xinmin

2016-01-01

Patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable genetic alterations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. One of the major resistant mechanisms is secondary EGFR-T790M mutation. Other mechanisms, such as HER2 and MET amplifications, and PIK3CA mutations, were also reported. However, the mechanisms in the remaining patients are still unknown. In this study, we performed mutational profiling in a cohort of 83 NSCLC patients with TKI-sensitizing EGFR mutations at diagnosis and acquired resistance to three different first-generation EGFR TKIs using targeted next generation sequencing (NGS) of 416 cancer-related genes. In total, we identified 322 genetic alterations with a median of 3 mutations per patient. 61% of patients still exhibit TKI-sensitizing EGFR mutations, and 36% of patients acquired EGFR-T790M. Besides other known resistance mechanisms, we identified TET2 mutations in 12% of patients. Interestingly, we also observed SOX2 amplification in EGFR-T790M negative patients, which are restricted to Icotinib treatment resistance, a drug widely used in Chinese NSCLC patients. Our study uncovered mutational profiles of NSCLC patients with first-generation EGFR TKIs resistance with potential therapeutic implications. PMID:27528220

Screening mutations in drug-resistant Mycobacterium tuberculosis strains in Yunnan, China.

PubMed

Li, Daoqun; Song, Yuzhu; Zhang, Cheng-Lin; Li, Xiaofei; Xia, Xueshan; Zhang, A-Mei

Drug-resistant tuberculosis (DR-TB), especially multidrug-resistant tuberculosis (MDR-TB), is a serious medical and societal problem in China. The purpose of this study was to evaluate the mutation characteristics of drug-resistant Mycobacterium tuberculosis (M. tuberculosis) isolates in Yunnan, China. Drug susceptibility testing (DST) was performed in 523 clinical M. tuberculosis isolates. Six drug resistance genes (katG, inhA, rpoB, rpsL, embB, and pncA) were selected to screen for mutations. In total, 54 clinical M. tuberculosis strains were identified as drug-resistant by DST, including 18 single drug-resistant (SDR) strains and 36 multidrug-resistant (MDR) strains. Twenty-four types of mutations in five genes (excluding the inhA gene) were screened in forty-one strains. Six novel mutations were identified in this study, including three missense mutations (p.S302R in katG, p.D78G in embB, and p.M1I in pncA), two frameshift mutations (408 ins A and 538-580 del in pncA), and one mutation in a control region (-6 C>T located upstream of rpsL). The mutation frequencies in the hotspot mutation regions in the katG, rpoB, rpsL, embB, and pncA genes were 92.5%, 44.4%, 54.2%, 52.6%, and 37.5%, respectively. The mutation spectra and frequencies seemed somewhat unique in the Yunnan DR-TB strains. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rifabutin and rifampin resistance levels and associated rpoB mutations in clinical isolates of Mycobacterium tuberculosis complex.

PubMed

Berrada, Zenda L; Lin, Shou-Yean Grace; Rodwell, Timothy C; Nguyen, Duylinh; Schecter, Gisela F; Pham, Lucy; Janda, J Michael; Elmaraachli, Wael; Catanzaro, Antonino; Desmond, Edward

2016-06-01

Cross-resistance in rifamycins has been observed in rifampin (RIF)-resistant Mycobacterium tuberculosis complex isolates; some rpoB mutations do not confer broad in vitro rifamycin resistance. We examined 164 isolates, of which 102 were RIF-resistant, for differential resistance between RIF and rifabutin (RFB). A total of 42 unique single mutations or combinations of mutations were detected. The number of unique mutations identified exceeded that reported in any previous study. RFB and RIF MICs up to 8 μg/mL by MGIT 960 were studied; the cut-off values for susceptibility to RIF and RFB were 1 μg/mL and 0.5 μg/mL, respectively. We identified 31 isolates resistant to RIF but susceptible to RFB with the mutations D516V, D516F, 518 deletion, S522L, H526A, H526C, H526G, H526L, and two dual mutations (S522L + K527R and H526S + K527R). Clinical investigations using RFB to treat multidrug-resistant tuberculosis cases harboring those mutations are recommended. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Clonal hematopoiesis, with and without candidate driver mutations, is common in the elderly

PubMed Central

Zink, Florian; Stacey, Simon N.; Norddahl, Gudmundur L.; Frigge, Michael L.; Magnusson, Olafur T.; Jonsdottir, Ingileif; Thorgeirsson, Thorgeir E.; Sigurdsson, Asgeir; Gudjonsson, Sigurjon A.; Gudmundsson, Julius; Jonasson, Jon G.; Tryggvadottir, Laufey; Jonsson, Thorvaldur; Helgason, Agnar; Gylfason, Arnaldur; Sulem, Patrick; Rafnar, Thorunn; Thorsteinsdottir, Unnur; Gudbjartsson, Daniel F.; Masson, Gisli; Kong, Augustine

2017-01-01

Clonal hematopoiesis (CH) arises when a substantial proportion of mature blood cells is derived from a single dominant hematopoietic stem cell lineage. Somatic mutations in candidate driver (CD) genes are thought to be responsible for at least some cases of CH. Using whole-genome sequencing of 11 262 Icelanders, we found 1403 cases of CH by using barcodes of mosaic somatic mutations in peripheral blood, whether or not they have a mutation in a CD gene. We find that CH is very common in the elderly, trending toward inevitability. We show that somatic mutations in TET2, DNMT3A, ASXL1, and PPM1D are associated with CH at high significance. However, known CD mutations were evident in only a fraction of CH cases. Nevertheless, the highly prevalent CH we detect associates with increased mortality rates, risk for hematological malignancy, smoking behavior, telomere length, Y-chromosome loss, and other phenotypic characteristics. Modeling suggests some CH cases could arise in the absence of CD mutations as a result of neutral drift acting on a small population of active hematopoietic stem cells. Finally, we find a germline deletion in intron 3 of the telomerase reverse transcriptase (TERT) gene that predisposes to CH (rs34002450; P = 7.4 × 10−12; odds ratio, 1.37). PMID:28483762

Heterozygous RTEL1 mutations are associated with familial pulmonary fibrosis.

PubMed

Kannengiesser, Caroline; Borie, Raphael; Ménard, Christelle; Réocreux, Marion; Nitschké, Patrick; Gazal, Steven; Mal, Hervé; Taillé, Camille; Cadranel, Jacques; Nunes, Hilario; Valeyre, Dominique; Cordier, Jean François; Callebaut, Isabelle; Boileau, Catherine; Cottin, Vincent; Grandchamp, Bernard; Revy, Patrick; Crestani, Bruno

2015-08-01

Pulmonary fibrosis is a fatal disease with progressive loss of respiratory function. Defective telomere maintenance leading to telomere shortening is a cause of pulmonary fibrosis, as mutations in the telomerase component genes TERT (reverse transcriptase) and TERC (RNA component) are found in 15% of familial pulmonary fibrosis (FPF) cases. However, so far, about 85% of FPF remain genetically uncharacterised.Here, in order to identify new genetic causes of FPF, we performed whole-exome sequencing, with a candidate-gene approach, of 47 affected subjects from 35 families with FPF without TERT and TERC mutations.We identified heterozygous mutations in regulator of telomere elongation helicase 1 (RTEL1) in four families. RTEL1 is a DNA helicase with roles in DNA replication, genome stability, DNA repair and telomere maintenance. The heterozygous RTEL1 mutations segregated as an autosomal dominant trait in FPF, and were predicted by structural analyses to severely affect the function and/or stability of RTEL1. In agreement with this, RTEL1-mutated patients exhibited short telomeres in comparison with age-matched controls.Our results provide evidence that heterozygous RTEL1 mutations are responsible for FPF and, thereby, extend the clinical spectrum of RTEL1 deficiency. Thus, RTEL1 enlarges the number of telomere-associated genes implicated in FPF. Copyright ©ERS 2015.

Ribosomal mutations promote the evolution of antibiotic resistance in a multidrug environment.

PubMed

Gomez, James E; Kaufmann-Malaga, Benjamin B; Wivagg, Carl N; Kim, Peter B; Silvis, Melanie R; Renedo, Nikolai; Ioerger, Thomas R; Ahmad, Rushdy; Livny, Jonathan; Fishbein, Skye; Sacchettini, James C; Carr, Steven A; Hung, Deborah T

2017-02-21

Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.

Antiretroviral Resistance in HIV/AIDS Patients

NASA Astrophysics Data System (ADS)

Manosuthi, W.; MD

2018-03-01

The higher prevalence of HIV drug resistance was observed in areas with greater ART coverage. The HIV resistance-associated mutations occur when people have inadequate levels of antiretroviral drugs as well as inadequate potency, inadequate adherence, and preexisting resistance. The degree to drug cross-resistance is observed depends on the specific mutations and number of mutation accumulation. In the Southeast Asia region, the challenging of people with treatment failure is the availability and accessibility to subsequent new antiretroviral drugs to construct he second and salvage regimen. Genotypic resistance testing is a useful tool because it can identify the existing drug resistance-associated mutations under the selective drug pressure. Thus, understanding the basic interpretation of HIV drug resistance- associated mutation is useful in guiding clinical decisions for treatment-experienced people living with HIV.

Antibiotic Resistance and Single-Nucleotide Polymorphism Cluster Grouping Type in a Multinational Sample of Resistant Mycobacterium tuberculosis Isolates▿

PubMed Central

Brimacombe, M.; Hazbon, M.; Motiwala, A. S.; Alland, D.

2007-01-01

A single-nucleotide polymorphism-based cluster grouping (SCG) classification system for Mycobacterium tuberculosis was used to examine antibiotic resistance type and resistance mutations in relationship to specific evolutionary lineages. Drug resistance and resistance mutations were seen across all SCGs. SCG-2 had higher proportions of katG codon 315 mutations and resistance to four drugs. PMID:17846140

Multi-country Survey Revealed Prevalent and Novel F1534S Mutation in Voltage-Gated Sodium Channel (VGSC) Gene in Aedes albopictus.

PubMed

Xu, Jiabao; Bonizzoni, Mariangela; Zhong, Daibin; Zhou, Guofa; Cai, Songwu; Li, Yiji; Wang, Xiaoming; Lo, Eugenia; Lee, Rebecca; Sheen, Roger; Duan, Jinhua; Yan, Guiyun; Chen, Xiao-Guang

2016-05-01

Aedes albopictus is an important dengue vector because of its aggressive biting behavior and rapid spread out of its native home range in Southeast Asia. Pyrethroids are widely used for adult mosquito control, and resistance to pyrethroids should be carefully monitored because vector control is the only effective method currently available to prevent dengue transmission. The voltage-gated sodium channel gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). Previous studies reported various mutations in the voltage-gated sodium channel (VGSC) gene, but the spatial distribution of kdr mutations in Ae. albopictus has not been systematically examined, and the association between kdr mutation and phenotypic resistance has not been established. A total of 597 Ae. albopictus individuals from 12 populations across Asia, Africa, America and Europe were examined for mutations in the voltage-gated sodium channel gene. Three domains for a total of 1,107 bp were sequenced for every individual. Two populations from southern China were examined for pyrethroid resistance using the World Health Organization standard tube bioassay, and the association between kdr mutations and phenotypic resistance was tested. A total of 29 synonymous mutations were found across domain II, III and IV of the VGSC gene. Non-synonymous mutations in two codons of the VGSC gene were detected in 5 populations from 4 countries. A novel mutation at 1532 codon (I1532T) was found in Rome, Italy with a frequency of 19.7%. The second novel mutation at codon 1534 (F1534S) was detected in southern China and Florida, USA with a frequency ranging from 9.5-22.6%. The WHO insecticide susceptibility bioassay found 90.1% and 96.1% mortality in the two populations from southern China, suggesting resistance and probable resistance. Positive association between kdr mutations with deltamethrin resistance was established in these two populations. Two novel kdr mutations, I1532T and F1534S were found in Ae. albopictus. This is the first report of I1532T mutations in Italy and F1534S mutation in China and US. Significant association between kdr mutation and protection from deltamethrin raised the possibility that kdr mutation may be a viable biomarker for pyrethroid resistance surveillance in Ae. albopictus. The patchy distribution of kdr mutations in Ae. albopictus mosquitoes calls for developing global surveillance plan for pyrethroid resistance and developing countermeasures to mitigate the spread of resistance.

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

PubMed

Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

2017-08-23

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

PubMed Central

Délye, C; Laigret, F; Corio-Costet, M F

1997-01-01

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol. PMID:9251183

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates.

PubMed

Ibacache-Quiroga, Claudia; Oliveros, Juan C; Couce, Alejandro; Blázquez, Jesus

2018-01-01

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE , and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

PubMed Central

Ibacache-Quiroga, Claudia; Oliveros, Juan C.; Couce, Alejandro; Blázquez, Jesus

2018-01-01

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci. PMID:29615988

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

PubMed

Martin, Lesley-Ann; Ribas, Ricardo; Simigdala, Nikiana; Schuster, Eugene; Pancholi, Sunil; Tenev, Tencho; Gellert, Pascal; Buluwela, Laki; Harrod, Alison; Thornhill, Allan; Nikitorowicz-Buniak, Joanna; Bhamra, Amandeep; Turgeon, Marc-Olivier; Poulogiannis, George; Gao, Qiong; Martins, Vera; Hills, Margaret; Garcia-Murillas, Isaac; Fribbens, Charlotte; Patani, Neill; Li, Zheqi; Sikora, Matthew J; Turner, Nicholas; Zwart, Wilbert; Oesterreich, Steffi; Carroll, Jason; Ali, Simak; Dowsett, Mitch

2017-11-30

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.

RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts

EPA Science Inventory

The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion p...

Active Methamphetamine Use is Associated with Transmitted Drug Resis-tance to Non-Nucleoside Reverse Transcriptase Inhibitors in Individuals with HIV Infection of Unknown Duration

PubMed Central

Cachay, Edward R; Moini, Niousha; Kosakovsky Pond, Sergei L; Pesano, Rick; Lie, Yolanda S; Aiem, Heidi; Butler, David M; Letendre, Scott; Mathews, Wm. Christopher; Smith, Davey M

2007-01-01

Background: Frequent methamphetamine use among recently HIV infected individuals is associated with transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTI); however, the reversion time of TDR to drug susceptible HIV may exceed 3 years. We assessed whether recreational substance use is associated with detectable TDR among individuals newly diagnosed with HIV infection of unknown duration. Design: Cross-sectional analysis. Methods: Subjects were enrolled at the University California, San Diego Early Intervention Program. Demographic, clinical and substance use data were collected using structured interviews. Genotypic resistance testing was performed using GeneSeq™, Monogram Biosciences. We analyzed the association between substance use and TDR using bivariate analyses and the corresponding transmission networks using phylogenetic models. Results: Between April 2004 and July 2006, 115 individuals with genotype data were enrolled. The prevalence of alcohol, marijuana and methamphetamine use were 98%, 71% and 64% respectively. Only active methamphetamine use in the 30 days prior to HIV diagnosis was independently associated with TDR to NNRTI (OR: 6.6; p=0.002). Conclusion: Despite not knowing the duration of their HIV infection, individuals reporting active methamphetamine use in the 30 days prior to HIV diagnosis are at an increased risk of having HIV strains that are resistant to NNRTI. PMID:18923691

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic.

PubMed

Miftahussurur, Muhammad; Cruz, Modesto; Subsomwong, Phawinee; Jiménez Abreu, José A; Hosking, Celso; Nagashima, Hiroyuki; Akada, Junko; Yamaoka, Yoshio

2017-05-01

Abstract Helicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction-based sequencing was used to assess gyrA , gyrB , rdxA , frxA , and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance-associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA , dppB , fdxA , and fdxB , irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA , including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection.

Characterization of rpoB mutations in rifampin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait and Dubai.

PubMed

Ahmad, Suhail; Mokaddas, Eiman; Fares, Esther

2002-11-01

Mutations conferring resistance to rifampin in rifampin-resistant clinical Mycobacterium tuberculosis isolates occur mostly in the 81 bp rifampin-resistance-determining region (RRDR) of the rpoB gene. In this study, 29 rifampin-resistant and 12 -susceptible clinical M. tuberculosis isolates were tested for characterization of mutations in the rpoB gene by line probe (INNO-LiPA Rif. TB) assay and the results were confirmed and extended by DNA sequencing of the PCR amplified target DNA. The line probe assay identified all 12 susceptible strains as rifampin-sensitive and the DNA sequence of RRDR in the amplified rpoB gene from two isolates matched perfectly with the wild-type sequence. The line probe assay identified 28 resistant isolates as rifampin-resistant with specific detection of mutation in 22 isolates including one isolate that exhibited hetro-resistance containing both the wild-type pattern as well as a specific mutation within RRDR while one of the rifampin-resistant strain was identified as rifampin-susceptible. DNA sequencing confirmed these results and, in addition, led to the specific detection of mutations in 5 rifampin-resistant isolates in which specific base changes within RRDR could not be determined by the line probe assay. These analyses identified 8 different mutations within RRDR of the rpoB gene including one novel mutation (S522W) that has not been reported so far. The genotyping performed on the isolates carrying similar mutations showed that majority of these isolates were unique as they exhibited varying DNA banding patterns. Correlating the ethnic origin of the infected TB patients with the occurrence of specific mutations at three main codon positions (516, 526 and 531) in the rpoB gene showed that most patients (11 of 15) from South Asian region contained mutations at codon 526 while majority of isolates from patients (6 of 11) of Middle Eastern origin contained mutations at codon 531.

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome.

PubMed

Warejko, Jillian K; Tan, Weizhen; Daga, Ankana; Schapiro, David; Lawson, Jennifer A; Shril, Shirlee; Lovric, Svjetlana; Ashraf, Shazia; Rao, Jia; Hermle, Tobias; Jobst-Schwan, Tilman; Widmeier, Eugen; Majmundar, Amar J; Schneider, Ronen; Gee, Heon Yung; Schmidt, J Magdalena; Vivante, Asaf; van der Ven, Amelie T; Ityel, Hadas; Chen, Jing; Sadowski, Carolin E; Kohl, Stefan; Pabst, Werner L; Nakayama, Makiko; Somers, Michael J G; Rodig, Nancy M; Daouk, Ghaleb; Baum, Michelle; Stein, Deborah R; Ferguson, Michael A; Traum, Avram Z; Soliman, Neveen A; Kari, Jameela A; El Desoky, Sherif; Fathy, Hanan; Zenker, Martin; Bakkaloglu, Sevcan A; Müller, Dominik; Noyan, Aytul; Ozaltin, Fatih; Cadnapaphornchai, Melissa A; Hashmi, Seema; Hopcian, Jeffrey; Kopp, Jeffrey B; Benador, Nadine; Bockenhauer, Detlef; Bogdanovic, Radovan; Stajić, Nataša; Chernin, Gil; Ettenger, Robert; Fehrenbach, Henry; Kemper, Markus; Munarriz, Reyner Loza; Podracka, Ludmila; Büscher, Rainer; Serdaroglu, Erkin; Tasic, Velibor; Mane, Shrikant; Lifton, Richard P; Braun, Daniela A; Hildebrandt, Friedhelm

2018-01-06

Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1 , PLCE1 , NPHS2 , and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome. Copyright © 2018 by the American Society of Nephrology.

Genetic Mutations Associated with Pesticide Resistance in Rhipicephalus microplus and Haematobia irritans

USDA-ARS?s Scientific Manuscript database

A number of gene mutation in various arthropods have been found to be associated with pesticide resistance. Some of these mutations have been found in the two cattle pests, Rhipicephalus microplus and Haematobia irritans. Sodium channel gene mutations have been associated with pyrethroid resistance ...

Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer.

PubMed

Yoda, Satoshi; Lin, Jessica J; Lawrence, Michael S; Burke, Benjamin J; Friboulet, Luc; Langenbucher, Adam; Dardaei, Leila; Prutisto-Chang, Kylie; Dagogo-Jack, Ibiayi; Timofeevski, Sergei; Hubbeling, Harper; Gainor, Justin F; Ferris, Lorin A; Riley, Amanda K; Kattermann, Krystina E; Timonina, Daria; Heist, Rebecca S; Iafrate, A John; Benes, Cyril H; Lennerz, Jochen K; Mino-Kenudson, Mari; Engelman, Jeffrey A; Johnson, Ted W; Hata, Aaron N; Shaw, Alice T

2018-06-01

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N -ethyl- N -nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies. Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 663 . ©2018 American Association for Cancer Research.

Incidence of Ganciclovir Resistance in CMV-positive Renal Transplant Recipients and its Association with UL97 Gene Mutations.

PubMed

Aslani, Hamid Reza; Ziaie, Shadi; Salamzadeh, Jamshid; Zaheri, Sara; Samadian, Fariba; Mastoor-Tehrani, Shayan

2017-01-01

Human cytomegalovirus (CMV) remains the most common infection affecting organ transplant recipients. Despite advances in the prophylaxis and acute treatment of CMV, it remains an important pathogen affecting the short- and long-term clinical outcome of solid organ transplant recipient. The emergence of CMV resistance in a patient reduces the clinical efficacy of antiviral therapy, complicates therapeutic and clinical management decisions, and in some cases results in loss of the allograft and/or death of the patient. Common mechanisms of CMV resistance to ganciclovir have been described chiefly with the UL97 mutations. Here we evaluate Incidence of ganciclovir resistance in 144 CMV-positive renal transplant recipients and its association with UL97 gene mutations. Active CMV infection was monitored by viral DNA quantification in whole blood, and CMV resistance was assessed by UL97 gene sequencing. Six mutations in six patients were detected. Three patients (2.6%) of 112 patients with history of ganciclovir (GCV) treatment had clinical resistance with single UL97 mutations at loci known to be related to resistance (including mutations at codon 594, codon 460, and codon 520). three patients who were anti-CMV drug naïve had single UL97 mutations (D605E) without clinical resistance. Our results confirm and extend our earlier findings on the specific mutations in the UL97 phosphotransferase gene in loci that have established role in ganciclovir resistance and also indicate that clinical ganciclovir resistance due to UL97 gene mutations is an issue in subjects with history of with ganciclovir treatment. D605E mutations remains a controversial issue that needs further investigations.

Prevalence of macrolide and fluoroquinolone resistance-mediating mutations in Mycoplasma genitalium in five cities in Russia and Estonia

PubMed Central

Shipitsyna, Elena; Rumyantseva, Tatiana; Golparian, Daniel; Khayrullina, Guzel; Lagos, Amaya C.; Edelstein, Inna; Joers, Kai; Jensen, Jörgen S.; Savicheva, Alevtina; Rudneva, Natalia; Sukhanova, Larisa; Kozlov, Roman; Guschin, Alexander

2017-01-01

Background and objective Resistance in the sexually transmitted bacterium Mycoplasma genitalium to all recommended therapeutic antimicrobials have rapidly emerged. However, to date, internationally reported resistance surveillance data for M. genitalium strains circulating in Eastern Europe are entirely lacking. The aim of this study was to estimate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in four cities in Russia and one in Estonia, 2013–2016. Materials and methods Consecutive urogenital samples found positive for M. genitalium during diagnostic testing were retrospectively analyzed for resistance-associated mutations in the 23S rRNA and parC genes using pyrosequencing and conventional Sanger sequencing, respectively. Results In total, 867 M. genitalium positive samples from 2013–2016 were analyzed. Macrolide resistance-associated mutations were detected in 4.6% of the samples from Russia (0.7–6.8% in different cities) and in 10% of the samples from Estonia. The mutations A2059G and A2058G were highly predominating in both Russia and Estonia, accounting together for 90.9% of the cases positive for nucleotide substitutions in the 23S rRNA gene. The rates of possible fluoroquinolone resistance-associated mutations were 6.2% in Russia (2.5–7.6% in different cities) and 5% in Estonia. The mutations S83I and S83N were the most frequent ones in Russia (24.4% each), whereas D87N highly predominated in Estonia (83.3% of all fluoroquinolone resistance-associated mutations). Approximately 1% of the samples in both countries harbored both macrolide and possible fluoroquinolone resistance-associated mutations, with A2058G and S83I being the most frequent combination (37.5%). Conclusions The prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium was 4.6% and 6.2%, respectively, in Russia, and 10% and 5%, respectively, in Estonia. Despite the relatively low rates of macrolide and fluoroquinolone resistance in these countries, antimicrobial resistance surveillance and testing for resistance-associated mutations in M. genitalium positive cases would be valuable. PMID:28407014

Prevalence of macrolide and fluoroquinolone resistance-mediating mutations in Mycoplasma genitalium in five cities in Russia and Estonia.

PubMed

Shipitsyna, Elena; Rumyantseva, Tatiana; Golparian, Daniel; Khayrullina, Guzel; Lagos, Amaya C; Edelstein, Inna; Joers, Kai; Jensen, Jörgen S; Savicheva, Alevtina; Rudneva, Natalia; Sukhanova, Larisa; Kozlov, Roman; Guschin, Alexander; Unemo, Magnus

2017-01-01

Resistance in the sexually transmitted bacterium Mycoplasma genitalium to all recommended therapeutic antimicrobials have rapidly emerged. However, to date, internationally reported resistance surveillance data for M. genitalium strains circulating in Eastern Europe are entirely lacking. The aim of this study was to estimate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in four cities in Russia and one in Estonia, 2013-2016. Consecutive urogenital samples found positive for M. genitalium during diagnostic testing were retrospectively analyzed for resistance-associated mutations in the 23S rRNA and parC genes using pyrosequencing and conventional Sanger sequencing, respectively. In total, 867 M. genitalium positive samples from 2013-2016 were analyzed. Macrolide resistance-associated mutations were detected in 4.6% of the samples from Russia (0.7-6.8% in different cities) and in 10% of the samples from Estonia. The mutations A2059G and A2058G were highly predominating in both Russia and Estonia, accounting together for 90.9% of the cases positive for nucleotide substitutions in the 23S rRNA gene. The rates of possible fluoroquinolone resistance-associated mutations were 6.2% in Russia (2.5-7.6% in different cities) and 5% in Estonia. The mutations S83I and S83N were the most frequent ones in Russia (24.4% each), whereas D87N highly predominated in Estonia (83.3% of all fluoroquinolone resistance-associated mutations). Approximately 1% of the samples in both countries harbored both macrolide and possible fluoroquinolone resistance-associated mutations, with A2058G and S83I being the most frequent combination (37.5%). The prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium was 4.6% and 6.2%, respectively, in Russia, and 10% and 5%, respectively, in Estonia. Despite the relatively low rates of macrolide and fluoroquinolone resistance in these countries, antimicrobial resistance surveillance and testing for resistance-associated mutations in M. genitalium positive cases would be valuable.

Emergence as an outbreak of the HIV-1 CRF19_cpx variant in treatment-naïve patients in southern Spain.

PubMed

González-Domenech, Carmen M; Viciana, Isabel; Delaye, Luis; Mayorga, María Luisa; Palacios, Rosario; de la Torre, Javier; Jarilla, Francisco; Castaño, Manuel; Del Arco, Alfonso; Clavijo, Encarnación; Santos, Jesús

2018-01-01

CRF19_cpx is a complex circulating recombination form (CRF) of HIV-1. We describe the characteristics of an outbreak of the CRF19_cpx variant among treatment-naïve patients in southern Spain. The study was undertaken at the Virgen de la Victoria Hospital, a reference centre for the analysis of HIV-1 genotype in Malaga (Spain). Subtyping was performed through REGA v3.0 and the relationship of our CRF19_cpx sequences, among themselves and regarding other reference sequences from the same variant, was defined by phylogenetic analysis. We used PhyML program to perform a reconstruction of the phylogeny by Maximum Likelihood method as well as further confirmation of the transmission clusters by Bayesian inference. Additionally, we collected demographic, clinical and immunovirological data. Between 2011 and 2016, we detected 57 treatment-naïve patients with the CRF19_cpx variant. Of these, 55 conformed a very well-defined transmission cluster, phylogenetically close to CRF19_cpx sequences from the United Kingdom. The origin of this subtype in Malaga was dated between 2007 and 2010. Over 50% of the patients presented the non-nucleoside reverse transcriptase inhibitor G190A resistance mutation. This variant was mostly represented by young adult Spanish men who had sex with men. Almost half of them were recent seroconverters, though a similar percentage was diagnosed at a late state of HIV infection. Five cases of AIDS and one non-AIDS defined death occurred during follow-up. The majority of patients treated with first-line combination antiretroviral therapy (ART) responded. We report the largest HIV-1 CRF19_cpx cohort of treatment-naïve patients outside Cuba, almost all emerging as an outbreak in the South of Spain. Half the cases had the G190A resistance mutation. Unlike previous studies, the variant from Malaga seems less pathogenic, with few AIDS events and an excellent response to ART.

Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria.

PubMed

Bachir, Meryem; Allem, Rachida; Benejat, Lucie; Tifrit, Abedelkarim; Medjekane, Meriem; Drici, Amine El-Mokhtar; Megraud, Francis; Douidi, Kara Turki

2018-05-11

In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria.

Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains.

PubMed

Lavania, Mallika; Singh, Itu; Turankar, Ravindra P; Gupta, Anuj Kumar; Ahuja, Madhvi; Pathak, Vinay; Sengupta, Utpal

2018-01-01

Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains - comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain - was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB , that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients.

Increased Tuberculosis Patient Mortality Associated with Mycobacterium tuberculosis Mutations Conferring Resistance to Second-Line Antituberculous Drugs

PubMed Central

Seifert, Marva; Garfein, Richard S.; Rodwell, Timothy C.

2017-01-01

ABSTRACT Rapid molecular diagnostics have great potential to limit the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) (M/XDR-TB). These technologies detect mutations in the Mycobacterium tuberculosis genome that confer phenotypic drug resistance. However, there have been few data published regarding the relationships between the detected M. tuberculosis resistance mutations and M/XDR-TB treatment outcomes, limiting our current ability to exploit the full potential of molecular diagnostics. We analyzed clinical, microbiological, and sequencing data for 451 patients and their clinical isolates collected in a multinational, observational cohort study to determine if there was an association between M. tuberculosis resistance mutations and patient mortality. The presence of an rrs 1401G mutation was associated with significantly higher odds of patient mortality (adjusted odds ratio [OR] = 5.72; 95% confidence interval [CI], 1.65 to 19.84]) after adjusting for relevant patient clinical characteristics and all other resistance mutations. Further analysis of mutations, categorized by the associated resistance level, indicated that the detection of mutations associated with high-level fluoroquinolone (OR, 3.99 [95% CI, 1.10 to 14.40]) and kanamycin (OR, 5.47 [95% CI, 1.64 to 18.24]) resistance was also significantly associated with higher odds of patient mortality, even after accounting for clinical site, patient age, reported smoking history, body mass index (BMI), diabetes, HIV, and all other resistance mutations. Specific gyrA and rrs resistance mutations, associated with high-level resistance, were associated with patient mortality as identified in clinical M. tuberculosis isolates from a diverse M/XDR-TB patient population at three high-burden clinical sites. These results have important implications for the interpretation of molecular diagnostics, including identifying patients at increased risk for mortality during treatment. (This study has been registered at ClinicalTrials.gov under registration no. NCT02170441.) PMID:28404672

Increased Tuberculosis Patient Mortality Associated with Mycobacterium tuberculosis Mutations Conferring Resistance to Second-Line Antituberculous Drugs.

PubMed

Georghiou, Sophia B; Seifert, Marva; Catanzaro, Donald G; Garfein, Richard S; Rodwell, Timothy C

2017-06-01

Rapid molecular diagnostics have great potential to limit the spread of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) (M/XDR-TB). These technologies detect mutations in the Mycobacterium tuberculosis genome that confer phenotypic drug resistance. However, there have been few data published regarding the relationships between the detected M. tuberculosis resistance mutations and M/XDR-TB treatment outcomes, limiting our current ability to exploit the full potential of molecular diagnostics. We analyzed clinical, microbiological, and sequencing data for 451 patients and their clinical isolates collected in a multinational, observational cohort study to determine if there was an association between M. tuberculosis resistance mutations and patient mortality. The presence of an rrs 1401G mutation was associated with significantly higher odds of patient mortality (adjusted odds ratio [OR] = 5.72; 95% confidence interval [CI], 1.65 to 19.84]) after adjusting for relevant patient clinical characteristics and all other resistance mutations. Further analysis of mutations, categorized by the associated resistance level, indicated that the detection of mutations associated with high-level fluoroquinolone (OR, 3.99 [95% CI, 1.10 to 14.40]) and kanamycin (OR, 5.47 [95% CI, 1.64 to 18.24]) resistance was also significantly associated with higher odds of patient mortality, even after accounting for clinical site, patient age, reported smoking history, body mass index (BMI), diabetes, HIV, and all other resistance mutations. Specific gyrA and rrs resistance mutations, associated with high-level resistance, were associated with patient mortality as identified in clinical M. tuberculosis isolates from a diverse M/XDR-TB patient population at three high-burden clinical sites. These results have important implications for the interpretation of molecular diagnostics, including identifying patients at increased risk for mortality during treatment. (This study has been registered at ClinicalTrials.gov under registration no. NCT02170441.). Copyright © 2017 American Society for Microbiology.

Comparison of ALS functionality and plant growth in ALS-inhibitor susceptible and resistant Myosoton aquaticum L.

PubMed

Liu, Weitang; Bai, Shuang; Jia, Sisi; Guo, Wenlei; Zhang, Lele; Li, Wei; Wang, Jinxin

2017-10-01

Herbicide target-site resistance mutations may cause pleiotropic effects on plant ecology and physiology. The effect of several known (Pro197Ser, Pro197Leu Pro197Ala, and Pro197Glu) target-site resistance mutations of the ALS gene on both ALS functionality and plant vegetative growth of weed Myosoton aquaticum L. (water chickweed) have been investigated here. The enzyme kinetics of ALS from four purified water chickweed populations that each homozygous for the specific target-site resistance-endowing mutations were characterized and the effect of these mutations on plant growth was assessed via relative growth rate (RGR) analysis. Plants homozygous for Pro197Ser and Pro197Leu exhibited higher extractable ALS activity than susceptible (S) plants, while all ALS mutations with no negative change in ALS kinetics. The Pro197Leu mutation increased ALS sensitivity to isoleucine and valine, and Pro197Glu mutation slightly increased ALS sensitivity to isoleucine. RGR results indicated that none of these ALS resistance mutations impose negative pleiotropic effects on relative growth rate. However, resistant (R) seeds had a lowed germination rate than S seeds. This study provides baseline information on ALS functionality and plant growth characteristics associated with ALS inhibitor resistance-endowing mutations in water chickweed. Copyright © 2017. Published by Elsevier Inc.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

PubMed

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-04-17

The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

PubMed Central

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, HongBin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-01-01

Background The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia. PMID:17439653

ACE: an efficient and sensitive tool to detect insecticide resistance-associated mutations in insect acetylcholinesterase from RNA-Seq data.

PubMed

Guo, Dianhao; Luo, Jiapeng; Zhou, Yuenan; Xiao, Huamei; He, Kang; Yin, Chuanlin; Xu, Jianhua; Li, Fei

2017-07-10

Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%-44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (<5%), but the agricultural pests Chilo suppressalis and Bemisia tabaci had a high resistance frequency. All ace reads in the 30 B. tabaci RNA-Seq data were resistant reads, suggesting that insecticide resistance has spread to very high frequency in B. tabaci. To the best of our knowledge, the ACE pipeline is the first tool to detect resistance mutations from RNA-Seq data, and it facilitates the full utilization of large-scale genetic data obtained by using next-generation sequencing.

Structure-based virtual screening efforts against HIV-1 reverse transcriptase to introduce the new potent non-nucleoside reverse transcriptase inhibitor

NASA Astrophysics Data System (ADS)

Hosseini, Yaser; Mollica, Adriano; Mirzaie, Sako

2016-12-01

The human immunodeficiency virus (HIV) which is strictly related to the development of AIDS, is treated by a cocktail of drugs, but due its high propensity gain drug resistance, the rational development of new medicine is highly desired. Among the different mechanism of action we selected the reverse transcriptase (RT) inhibition, for our studies. With the aim to identify new chemical entities to be used for further rational drug design, a set of 3000 molecules from the Zinc Database have been screened by docking experiments using AutoDock Vina software. The best ranked compounds with respect of the crystallographic inhibitor MK-4965 resulted to be five compounds, and the best among them was further tested by molecular dynamics (MD) simulation. Our results indicate that comp1 has a stronger interaction with the subsite p66 of RT than MK-4965 and that both are able to stabilize specific conformational changes of the RT 3D structure, which may explain their activity as inhibitors. Therefore comp1 could be a good candidate for biological tests and further development.

From multidrug-resistant to extensively drug-resistant tuberculosis in Lisbon, Portugal: the stepwise mode of resistance acquisition.

PubMed

Perdigão, João; Macedo, Rita; Silva, Carla; Machado, Diana; Couto, Isabel; Viveiros, Miguel; Jordao, Luisa; Portugal, Isabel

2013-01-01

The development and transmission of extensively drug-resistant (XDR) tuberculosis (TB) constitutes a serious threat to the effective control of TB in several countries. Here, in an attempt to further elucidate the dynamics of the acquisition of resistance to second-line drugs and investigate an eventual role for eis promoter mutations in aminoglycoside resistance, we have studied a set of multidrug-resistant (MDR)/XDR-TB isolates circulating in Lisbon, Portugal. Forty-four MDR-TB or XDR-TB isolates were genotyped and screened for mutations in genes associated with second-line drug resistance, namely tlyA, gyrA, rrs and eis. The most prevalent mutations found in each gene were Ins755GT in tlyA, A1401G in rrs, G-10A in eis and S91P in gyrA. Additionally, two genetic clusters were found in this study: Lisboa3 and Q1. The characteristic mutational profile found among recent XDR-TB circulating in Lisbon was also found in MDR-TB strains isolated in the 1990s. Also investigated was the resistance level conferred by eis G-10A mutations, revealing that eis G-10A mutations may result in amikacin resistance undetectable by widely used phenotypic assays. The analysis of the distribution of the mutations found by genetic clustering showed that in the Q1 cluster, two mutations, gyrA D94A and rrs A1401G, were enough to ensure development of XDR-TB from an MDR strain. Moreover, in the Lisboa3 cluster it was possible to elaborate a model in which the development of low-level kanamycin resistance was at the origin of the emergence of XDR-TB strains that can be discriminated by tlyA mutations.

Two novel ALK mutations mediate acquired resistance to the next generation ALK inhibitor alectinib

PubMed Central

Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L.; Khan, Tahsin M.; Gainor, Justin F.; Iafrate, A. John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A.; Shaw, Alice T.

2014-01-01

Purpose The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged NSCLC. Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. Experimental Design We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity-relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. Results We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. Conclusions We have identified two novel ALK mutations arising after alectinib exposure which are sensitive to other next generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. PMID:25228534

Two novel ALK mutations mediate acquired resistance to the next-generation ALK inhibitor alectinib.

PubMed

Katayama, Ryohei; Friboulet, Luc; Koike, Sumie; Lockerman, Elizabeth L; Khan, Tahsin M; Gainor, Justin F; Iafrate, A John; Takeuchi, Kengo; Taiji, Makoto; Okuno, Yasushi; Fujita, Naoya; Engelman, Jeffrey A; Shaw, Alice T

2014-11-15

The first-generation ALK tyrosine kinase inhibitor (TKI) crizotinib is a standard therapy for patients with ALK-rearranged non-small cell lung cancer (NSCLC). Several next-generation ALK-TKIs have entered the clinic and have shown promising activity in crizotinib-resistant patients. As patients still relapse even on these next-generation ALK-TKIs, we examined mechanisms of resistance to the next-generation ALK-TKI alectinib and potential strategies to overcome this resistance. We established a cell line model of alectinib resistance, and analyzed a resistant tumor specimen from a patient who had relapsed on alectinib. We developed Ba/F3 models harboring alectinib-resistant ALK mutations and evaluated the potency of other next-generation ALK-TKIs in these models. We tested the antitumor activity of the next-generation ALK-TKI ceritinib in the patient with acquired resistance to alectinib. To elucidate structure-activity relationships of ALK mutations, we performed computational thermodynamic simulation with MP-CAFEE. We identified a novel V1180L gatekeeper mutation from the cell line model and a second novel I1171T mutation from the patient who developed resistance to alectinib. Both ALK mutations conferred resistance to alectinib as well as to crizotinib, but were sensitive to ceritinib and other next-generation ALK-TKIs. Treatment of the patient with ceritinib led to a marked response. Thermodynamics simulation suggests that both mutations lead to distinct structural alterations that decrease the binding affinity with alectinib. We have identified two novel ALK mutations arising after alectinib exposure that are sensitive to other next-generation ALK-TKIs. The ability of ceritinib to overcome alectinib-resistance mutations suggests a potential role for sequential therapy with multiple next-generation ALK-TKIs. ©2014 American Association for Cancer Research.

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic

PubMed Central

Miftahussurur, Muhammad; Cruz, Modesto; Subsomwong, Phawinee; Jiménez Abreu, José A.; Hosking, Celso; Nagashima, Hiroyuki; Akada, Junko; Yamaoka, Yoshio

2017-01-01

Helicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction–based sequencing was used to assess gyrA, gyrB, rdxA, frxA, and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance–associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA, dppB, fdxA, and fdxB, irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA, including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection. PMID:28193745

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

PubMed Central

Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

2011-01-01

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

PubMed

Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

2017-01-01

Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

Expanded-spectrum nonnucleoside reverse transcriptase inhibitors inhibit clinically relevant mutant variants of human immunodeficiency virus type 1.

PubMed

Corbett, J W; Ko, S S; Rodgers, J D; Jeffrey, S; Bacheler, L T; Klabe, R M; Diamond, S; Lai, C M; Rabel, S R; Saye, J A; Adams, S P; Trainor, G L; Anderson, P S; Erickson-Viitanen, S K

1999-12-01

A research program targeted toward the identification of expanded-spectrum nonnucleoside reverse transcriptase inhibitors which possess increased potency toward K103N-containing mutant human immunodeficiency virus (HIV) and which maintain pharmacokinetics consistent with once-a-day dosing has resulted in the identification of the 4-cyclopropylalkynyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 961 and DPC 963 and the 4-cyclopropylalkenyl-4-trifluoromethyl-3, 4-dihydro-2(1H)quinazolinones DPC 082 and DPC 083 for clinical development. DPC 961, DPC 963, DPC 082, and DPC 083 all exhibit low-nanomolar potency toward wild-type virus, K103N and L100I single-mutation variants, and many multiply amino acid-substituted HIV type 1 mutants. This high degree of potency is combined with a high degree of oral bioavailability, as demonstrated in rhesus monkeys and chimpanzees, and with plasma serum protein binding that can result in significant free levels of drug.

Longitudinal whole genome analysis of pre and post drug treatment Mycobacterium tuberculosis isolates reveals progressive steps to drug resistance.

PubMed

Datta, Gargi; Nieto, Luisa M; Davidson, Rebecca M; Mehaffy, Carolina; Pederson, Caroline; Dobos, Karen M; Strong, Michael

2016-05-01

Tuberculosis (TB) is one of the leading causes of death due to an infectious disease in the world. Understanding the mechanisms of drug resistance has become pivotal in the detection and treatment of newly emerging resistant TB cases. We have analyzed three pairs of Mycobacterium tuberculosis strains pre- and post-drug treatment to identify mutations involved in the progression of resistance to the drugs rifampicin and isoniazid. In the rifampicin resistant strain, we confirmed a mutation in rpoB (S450L) that is known to confer resistance to rifampicin. We discovered a novel L101R mutation in the katG gene of an isoniazid resistant strain, which may directly contribute to isoniazid resistance due to the proximity of the mutation to the katG isoniazid-activating site. Another isoniazid resistant strain had a rare mutation in the start codon of katG. We also identified a number of mutations in each longitudinal pair, such as toxin-antitoxin mutations that may influence the progression towards resistance or may play a role in compensatory fitness. These findings improve our knowledge of drug resistance progression during therapy and provide a methodology to monitor longitudinal strains using whole genome sequencing, polymorphism comparison, and functional annotation. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania.

PubMed

Ruesen, Carolien; Riza, Anca Lelia; Florescu, Adriana; Chaidir, Lidya; Editoiu, Cornelia; Aalders, Nicole; Nicolosu, Dragos; Grecu, Victor; Ioana, Mihai; van Crevel, Reinout; van Ingen, Jakko

2018-06-26

Mycobacterium tuberculosis drug resistance poses a major threat to tuberculosis control. Current phenotypic tests for drug susceptibility are time-consuming, technically complex, and expensive. Whole genome sequencing is a promising alternative, though the impact of different drug resistance mutations on the minimum inhibitory concentration (MIC) remains to be investigated. We examined the genomes of 72 phenotypically drug-resistant Mycobacterium tuberculosis isolates from 72 Romanian patients for drug resistance mutations. MICs for first- and second-line drugs were determined using the MycoTB microdilution method. These MICs were compared to macrodilution critical concentration testing by the Mycobacterium Growth Indicator Tube (MGIT) platform and correlated to drug resistance mutations. Sixty-three (87.5%) isolates harboured drug resistance mutations; 48 (66.7%) were genotypically multidrug-resistant. Different drug resistance mutations were associated with different MIC ranges; katG S315T for isoniazid, and rpoB S450L for rifampicin were associated with high MICs. However, several mutations such as in rpoB, rrs and rpsL, or embB were associated with MIC ranges including the critical concentration for rifampicin, aminoglycosides or ethambutol, respectively. Different resistance mutations lead to distinct MICs, some of which may still be overcome by increased dosing. Whole genome sequencing can aid in the timely diagnosis of Mycobacterium tuberculosis drug resistance and guide clinical decision-making.

Protease Inhibitors Drug Resistance Mutations in Turkish Patients with Chronic Hepatitis C.

PubMed

Sargin Altunok, Elif; Sayan, Murat; Akhan, Sila; Aygen, Bilgehan; Yildiz, Orhan; Tekin Koruk, Suda; Mistik, Resit; Demirturk, Nese; Ural, Onur; Kose, Şükran; Aynioglu, Aynur; Korkmaz, Fatime; Ersoz, Gülden; Tuna, Nazan; Ayaz, Celal; Karakecili, Faruk; Keten, Derya; Inan, Dilara; Yazici, Saadet; Koculu, Safiye; Yildirmak, Taner

2016-09-01

Drug resistance development is an expected problem during treatment with protease inhibitors (PIs), this is largely due to the fact that Pls are low-genetic barrier drugs. Resistance-associated variants (RAVs) however may also occur naturally, and prior to treatment with Pls, the clinical impact of this basal resistance remains unknown. In Turkey, there is yet to be an investigation into the hepatitis C (HCV) drug associated resistance to oral antivirals. 178 antiviral-naïve patients infected with HCV genotype 1 were selected from 27 clinical centers of various geographical regions in Turkey and included in the current study. The basal NS3 Pls resistance mutations of these patients were analyzed. In 33 (18.5%) of the patients included in the study, at least one mutation pattern that can cause drug resistance was identified. The most frequently detected mutation pattern was T54S while R109K was the second most frequently detected. Following a more general examination of the patients studied, telaprevir (TVR) resistance in 27 patients (15.2%), boceprevir (BOC) resistance in 26 (14.6%) patients, simeprevir (SMV) resistance in 11 (6.2%) patients and faldaprevir resistance in 13 (7.3%) patients were detected. Our investigation also revealed that rebound developed in the presence of a Q80K mutation and amongst two V55A mutations following treatment with TVR, while no response to treatment was detected in a patient with a R55K mutation. We are of the opinion that drug resistance analyses can be beneficial and necessary in revealing which variants are responsible for pre-treatment natural resistance and which mutations are responsible for the viral breakthrough that may develop during the treatment. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Peruvian and globally reported amino acid substitutions on the Mycobacterium tuberculosis pyrazinamidase suggest a conserved pattern of mutations associated to pyrazinamide resistance

PubMed Central

Zimic, Mirko; Sheen, Patricia; Quiliano, Miguel; Gutierrez, Andrés; Gilman, Robert H.

2010-01-01

Resistance to pyrazinamide in Mycobacterium tuberculosis is usually associated with a reduction of pyrazinamidase activity caused by mutations in pncA, the pyrazinamidase coding gene. Pyrazinamidase is a hydrolase that converts pyrazinamide, the antituberculous drug against the latent stage, to the active compound, pyrazinoic acid. To better understand the relationship between pncA mutations and pyrazinamide-resistance, it is necessary to analyze the distribution of pncA mutations from pyrazinamide resistant strains. We determined the distribution of Peruvian and globally reported pncA missense mutations from M. tuberculosis clinical isolates resistant to pyrazinamide. The distributions of the single amino acid substitutions were compared at the secondary-structure-domains level. The distribution of the Peruvian mutations followed a similar pattern as the mutations reported globally. A consensus clustering of mutations was observed in hot-spot regions located in the metal coordination site and to a lesser extent in the active site of the enzyme. The data was not able to reject the null hypothesis that both distributions are similar, suggesting that pncA mutations associated to pyrazinamide resistance in M. tuberculosis, follow a conserved pattern responsible to impair the pyrazinamidase activity. PMID:19963078

Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients.

PubMed

Yang, Zhe; Yang, Nong; Ou, Qiuxiang; Xiang, Yi; Jiang, Tao; Wu, Xue; Bao, Hua; Tong, Xiaoling; Wang, Xiaonan; Shao, Yang W; Liu, Yunpeng; Wang, Yan; Zhou, Caicun

2018-03-05

Background: The third-generation EGFR tyrosine kinase inhibitor osimertinib is approved to treat patients with EGFR T790M-positive non-small cell lung cancer (NSCLC) who have developed resistance to earlier-generation drugs. Acquired EGFR C797S mutation has been reported to mediate osimertinib resistance in some patients. However, the remaining resistance mechanisms are largely unknown. Methods: We performed mutation profiling using targeted next-generation sequencing (NGS) for 416 cancer-relevant genes on 93 osimertinib-resistant lung cancer patients' samples, mainly cell-free DNAs (cfDNAs), and matched pretreatment samples of 12 patients. In vitro experiments were conducted to functionally study the secondary EGFR mutations identified. Results: EGFR G796/C797, L792, and L718/G719 mutations were identified in 24.7%, 10.8%, and 9.7% of the cases, respectively, with certain mutations coexisting in one patient with different prevalence. L792 and L718 mutants markedly increased the half inhibitory concentration (IC 50 ) of osimertinib in vitro , among which the L718Q mutation conferred the greatest resistance to osimertinib, as well as gefitinib resistance when not coexisting with T790M. Further analysis of the 12 matched pretreatment samples confirmed that these EGFR mutations were acquired during osimertinib treatment. Alterations in parallel or downstream oncogenes such as MET, KRAS , and PIK3CA were also discovered, potentially contributing to the osimertinib-resistance in patients without EGFR secondary mutations. Conclusions: We present comprehensive mutation profiles of a large cohort of osimertinib-resistance lung cancer patients using mainly cfDNA. Besides C797 mutations, novel secondary mutations of EGFR L718 and L792 residues confer osimertinib resistance, both in vitro and in vivo , and are of great clinical and pharmaceutical relevance. Clin Cancer Res; 1-11. ©2018 AACR. ©2018 American Association for Cancer Research.

Indolyl aryl sulfones (IASs): development of highly potent NNRTIs active against wt-HIV-1 and clinically relevant drug resistant mutants.

PubMed

Silvestri, Romano; Artico, Marino

2005-01-01

Indolyl aryl sulfones (IASs) are a potent class of NNRTIs developed from L-737,126, a lead agent discovered by Merck AG. IAS derivatives are endowed with inhibitory activities against wt HIV-1 in the low nanomolar concentration range. Introduction of two methyl groups at positions 3 and 5 of the phenyl ring of the aryl sulfonyl moiety furnished IAS derivatives such as 5-chloro- or 5-bromo-3-[(3,5-dimethylphenyl)sulfonyl]indole-2-carboxyamide, which showed very potent and selective anti-HIV-1 activity against some mutants carrying NNRTI resistant mutations at positions 103 and 181 of the reverse transcriptase. IAS derivatives bearing 2-hydroxyethylcarboxyamide or 2-hydroxyethylcarboxyhydrazide groups at position 2 of the indole nucleus were more active than L-737,126 against the K103N-Y181C double mutant. A great improvement of antiviral activity against wt HIV-1 and resistant mutants was obtained by coupling 1-3 simple amino acids, such as glycine and alanine, in sequence, with the 3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carbonyl moiety. The transformation of the chain terminus into amide or hydrazide, produced short peptides with high selectivity and potent activity against wt HIV-1, and the viral mutants Y181C, K103N-Y181C and EFV(R). IAS having two halogen atoms at the indole showed potent inhibitory activity against the Y181C and the EFV(R) resistant mutant strains. In particular, the introduction of a fluorine atom at position 4 of the indole ring notably contributed to improve the antiviral activities against both wt and the related resistant mutants. 5-Nitro-IASs were highly active against wt HIV-1 and exhibited low cytotoxicity. Experimental data highlighted the class IAS derivatives as promising candidates for clinical trials.

Mutations in a novel gene with transmembrane domains underlie Usher syndrome type 3.

PubMed

Joensuu, T; Hämäläinen, R; Yuan, B; Johnson, C; Tegelberg, S; Gasparini, P; Zelante, L; Pirvola, U; Pakarinen, L; Lehesjoki, A E; de la Chapelle, A; Sankila, E M

2001-10-01

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterized by progressive hearing loss, severe retinal degeneration, and variably present vestibular dysfunction, assigned to 3q21-q25. Here, we report on the positional cloning of the USH3 gene. By haplotype and linkage-disequilibrium analyses in Finnish carriers of a putative founder mutation, the critical region was narrowed to 250 kb, of which we sequenced, assembled, and annotated 207 kb. Two novel genes-NOPAR and UCRP-and one previously identified gene-H963-were excluded as USH3, on the basis of mutational analysis. USH3, the candidate gene that we identified, encodes a 120-amino-acid protein. Fifty-two Finnish patients were homozygous for a termination mutation, Y100X; patients in two Finnish families were compound heterozygous for Y100X and for a missense mutation, M44K, whereas patients in an Italian family were homozygous for a 3-bp deletion leading to an amino acid deletion and substitution. USH3 has two predicted transmembrane domains, and it shows no homology to known genes. As revealed by northern blotting and reverse-transcriptase PCR, it is expressed in many tissues, including the retina.

Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation.

PubMed

Deng, Wei; Yang, Qian; Zhang, Yongzhi; Jiao, Hongtao; Mei, Yu; Li, Xuefeng; Zheng, Mingqi

2017-03-01

Acetolactate synthase (ALS) is the common target of ALS-inhibiting herbicides, and target-site ALS mutations are the main mechanism of resistance to ALS-inhibiting herbicides. In this study, ALS1 and ALS2 genes with full lengths of 2004bp and 1998bp respectively were cloned in individual plants of susceptible (S) or resistant (R) flixweed (Descurainia sophia L.) populations. Two ALS mutations of Pro-197-Thr and/or Trp-574-Leu were identified in plants of three R biotypes (HB24, HB30 and HB42). In order to investigate the function of ALS isozymes in ALS-inhibiting herbicide resistance, pHB24 (a Pro-197-Thr mutation in ALS1 and a wild type ALS2), pHB42 (a Trp-574-Leu mutation in ALS1 and a wild type ALS2) and pHB30 (a Trp-574-Leu mutation in ALS1 and a Pro-197-Thr mutation in ALS2) subpopulations individually homozygous for different ALS mutations were generated. Individuals of pHB30 had mutations in each isozyme of ALS and had higher resistance than pHB24 and pHB42 populations containing mutations in only one ALS isozyme. Moreover, the pHB24 had resistance to SU, TP and SCT herbicides, whereas pHB24 and pHB42 had resistance to these classes of herbicides as well as IMI and PTB herbicides. The sensitivity of isolated ALS enzyme to inhibition by herbicides in these populations correlated with whole plant resistance levels. Therefore, reduced ALS sensitivity resulting from the mutations in ALS was responsible for resistance to ALS-inhibiting herbicides in flixweed. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple Mutations Modulate the Function of Dihydrofolate Reductase in Trimethoprim-Resistant Streptococcus pneumoniae

PubMed Central

Maskell, Jeffrey P.; Sefton, Armine M.; Hall, Lucinda M. C.

2001-01-01

Trimethoprim resistance in Streptococcus pneumoniae can be conferred by a single amino acid substitution (I100-L) in dihydrofolate reductase (DHFR), but resistant clinical isolates usually carry multiple DHFR mutations. DHFR genes from five trimethoprim-resistant isolates from the United Kingdom were compared to susceptible isolates and used to transform a susceptible control strain (CP1015). All trimethoprim-resistant isolates and transformants contained the I100-L mutation. The properties of DHFRs from transformants with different combinations of mutations were compared. In a transformant with only the I100-L mutation (R12/T2) and a D92-A mutation also found in the DHFRs of susceptible isolates, the enzyme was much more resistant to trimethoprim inhibition (50% inhibitory concentration [IC50], 4.2 μM) than was the DHFR from strain CP1015 (IC50, 0.09 μM). However, Km values indicated a lower affinity for the enzyme's natural substrates (Km for dihydrofolate [DHF], 3.1 μM for CP1015 and 27.5 μM for R12/T2) and a twofold decrease in the specificity constant. In transformants with additional mutations in the C-terminal portion of the enzyme, Km values for DHF were reduced (9.2 to 15.2 μM), indicating compensation for the lower affinity generated by I100-L. Additional mutations in the N-terminal portion of the enzyme were associated with up to threefold-increased resistance to trimethoprim (IC50 of up to 13.7 μM). It is postulated that carriage of the mutation M53-I—which, like I100-L, corresponds to a trimethoprim binding site in the Escherichia coli DHFR—is responsible for this increase. This study demonstrates that although the I100-L mutation alone may give rise to trimethoprim resistance, additional mutations serve to enhance resistance and modulate the effects of existing mutations on the affinity of DHFR for its natural substrates. PMID:11257022

Inhibitory effect of aqueous dandelion extract on HIV-1 replication and reverse transcriptase activity

PubMed Central

2011-01-01

Background Acquired immunodeficiency syndrome (AIDS), which is caused by the human immunodeficiency virus (HIV), is an immunosuppressive disease that results in life-threatening opportunistic infections. The general problems in current therapy include the constant emergence of drug-resistant HIV strains, adverse side effects and the unavailability of treatments in developing countries. Natural products from herbs with the abilities to inhibit HIV-1 life cycle at different stages, have served as excellent sources of new anti-HIV-1 drugs. In this study, we aimed to investigate the anti-HIV-1 activity of aqueous dandelion extract. Methods The pseudotyped HIV-1 virus has been utilized to explore the anti-HIV-1 activity of dandelion, the level of HIV-1 replication was assessed by the percentage of GFP-positive cells. The inhibitory effect of the dandelion extract on reverse transcriptase activity was assessed by the reverse transcriptase assay kit. Results Compared to control values obtained from cells infected without treatment, the level of HIV-1 replication and reverse transcriptase activity were decreased in a dose-dependent manner. The data suggest that dandelion extract has a potent inhibitory activity against HIV-1 replication and reverse transcriptase activity. The identification of HIV-1 antiviral compounds from Taraxacum officinale should be pursued. Conclusions The dandelion extract showed strong activity against HIV-1 RT and inhibited both the HIV-1 vector and the hybrid-MoMuLV/MoMuSV retrovirus replication. These findings provide additional support for the potential therapeutic efficacy of Taraxacum officinale. Extracts from this plant may be regarded as another starting point for the development of an antiretroviral therapy with fewer side effects. PMID:22078030

Genetic evolution of HIV in patients remaining on a stable HAART regimen despite insufficient viral suppression.

PubMed

Kristiansen, Thomas B; Pedersen, Anders G; Eugen-Olsen, Jesper; Katzenstein, Terese L; Lundgren, Jens D

2005-01-01

Our objective was to investigate whether steadily increasing resistance levels are inevitable in the course of a failing but unchanged Highly Active Antiretroviral Therapy (HAART) regimen. Patients having an unchanged HAART regimen and a good CD4 response (100 cells/microl above nadir) despite consistent HIV-RNA levels above 200 copies/ml were included in the study. The study period spanned at least 12 months and included 47 plasma samples from 17 patients that were sequenced and analysed with respect to evolutionary changes. At inclusion, the median CD4 count was 300 cells/ml (inter-quartile range (IQR): 231-380) and the median HIV-RNA was 2000 copies/ml (IQR: 1301-6090). Reverse transcription inhibitor (RTI) mutations increased 0.5 mutations per y (STD = 0.8 mutations per y), while major protease inhibitor (PI) resistance mutations increased at a rate of 0.2 mutations per y (STD = 0.8 mutations per y) and minor PI resistance mutations increased at a rate of 0.3 mutations per y (STD = 0.7 mutations per y). The rate at which RTI mutations accumulated decreased during the study period (p = 0.035). Interestingly, the rate of mutation accumulation was not associated with HIV-RNA level. The majority of patients kept accumulating new resistance mutations. However, 3 out of 17 patients with viral failure were caught in an apparent mutational deadlock, thus the development of additional resistance during a failing HAART is not inevitable. We hypothesize that certain patterns of mutations can cause a mutational deadlock where the evolutionary benefit of further resistance mutation is limited if the patient is kept on a stable HAART regimen.

Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes.

PubMed

Stern, Josh Lewis; Paucek, Richard D; Huang, Franklin W; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C; Cech, Thomas R

2017-12-26

A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Molecular perspectives in differentiated thyroid cancer.

PubMed

Buffet, C; Groussin, L

2015-02-01

Progress in understanding the molecular genetics of thyroid cancer in the last 20 years has accelerated recently with the advent of high-throughput sequencing technologies known as Next-Generation Sequencing. Besides classical molecular abnormalities involving the MAPK (Mitogen Activated Protein Kinase) and PI3K (PhosphoInositide 3-Kinase) pathways that play a key role in follicular-derived thyroid tumorigenesis, new molecular abnormalities have been discovered. The major advances in recent years have been the discovery of new somatic driver gene point mutations (such as RASAL1 [RAS protein activator Like 1] mutations in follicular cancer) and/or mutations that have prognostic value (such as TERT [Telomerase reverse transcriptase] promoter mutations); new chromosomal rearrangements, usually having close connection with exposure to ionizing radiation (such as ALK [Anaplastic Lymphoma Kinase] rearrangements); and deregulation of some gene or microRNA expression representing a molecular signature. Progress made in understanding the molecular mechanisms of thyroid cancer offers new perspectives for the diagnosis of the benign or malignant status of a thyroid nodule, to refine prognosis and offer new perspectives of targeted therapy for radioiodine-refractory cancers. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis clinical strains isolated in the Philippines.

PubMed

Herrera, Laura; Valverde, Azucena; Saiz, Pilar; Sáez-Nieto, Juan A; Portero, José L; Jiménez, M Soledad

2004-06-01

The prevalence of mutations in the katG, inhA and oxyR-ahpC genes of isoniazid (INH)-resistant Mycobacterium tuberculosis isolates in the Philippines were determined. Of 306 M. tuberculosis isolates studied, 81 (26.5%) exhibited INH-resistance. Forty-four strains (54.3%) had mutations in the katG gene, eighteen strains (22.2%) had mutations in the putative inhA locus region, seven had mutations in both regions and five strains had mutations in the oxyR-ahpC operon. Only seven strains had no mutations. A total of 71 of the 81 (87.6%) resistant strains and 65 of the 72 (90.3%) INH sensitive randomly selected strains showed amino acid substitution in codon 463 (Arg to Leu) (88.9%). This fact supports the hypothesis that mutations at codon 463 are independent of INH-resistance and are linked to the geographical origins of the strains. Copyright 2004 Elsevier B.V.

[Resistance studies: when are they indicated?].

PubMed

Angeles Marcos, M

2011-12-01

Cytomegalovirus (CMV) resistance to antiviral drugs is an emerging problem and is due to selection of mutations in the viral genome. Although ganciclovir resistance is the most common and widely studied, there is resistance to all antiviral agents. Risk factors for the development of resistance are the absence of preexisting immunity to CMV, lung and pancreas transplantation, high viral loads, intense concomitant immunosuppressive therapy and prolonged exposure to ganciclovir or suboptimal levels of this drug. Antiviral resistance should be suspected when, despite adequate treatment exposure for 2 weeks, an increase in viral load, or persistence or clinical progression of CMV disease are detected. However, failure to respond cannot always be attributed to antiviral resistance nor does resistance always lead to poor clinical outcome. When resistance is suspected, phenotypic and genotypic confirmation is required. The most common mutations are those in the UL97 gene, which confers ganciclovir resistance. However, foscarnet and cidofovir can be used. The UL54 mutation is not uncommon, whether alone or in combination with UL97 mutations. The combination of UL54 and UL97 mutations is associated with high-grade and multiple resistance. Early detection of resistance is essential to prevent unfavorable outcome and the development of multi-drug resistance. In patients with a slow response to treatment and without mutations associated with resistance, plasma ganciclovir levels and specific CMV immunity should be investigated. Copyright © 2011 Elsevier España S.L. All rights reserved.

HIV Resistance Testing

MedlinePlus

... your medications, HIV will multiply more easily. More mutations will occur. Some of them could cause resistance. ... Genotypic resistance: The genetic code of HIV has mutations that are linked to drug resistance. Clinical resistance ...

Emergence of CTNNB1 mutation at acquired resistance to KIT inhibitor in metastatic melanoma.

PubMed

Cho, J; Kim, S Y; Kim, Y J; Sim, M H; Kim, S T; Kim, N K D; Kim, K; Park, W; Kim, J H; Jang, K-T; Lee, J

2017-10-01

The KIT inhibitor, imatinib, has shown promising efficacy in patients with KIT-mutated melanoma; however, acquisition of resistance to imatinib occurs rapidly in the majority of patients. The mechanisms of acquired resistance to imatinib in melanoma remain unclear. We analyzed biopsy samples from paired baseline and post-treatment tumor lesions in one patient with KIT-mutated melanoma who had had an initial objective tumor regression in response to imatinib treatment followed by disease progression 8 months later. Targeted deep sequencing from post-treatment biopsy samples detected an additional mutation in CTNNB1 (S33C) with original KIT L576P mutation. We examined the functional role of the additional CTNNB1 S33C mutation in resistance to imatinib indirectly using the Ba/F3 cell model. Ba/F3 cell lines transfected with both the L576P KIT mutation and the CTNNB1 S33C mutation demonstrated no growth inhibition despite imatinib treatment, whereas growth inhibition was observed in the Ba/F3 cell line transfected with the L576 KIT mutation alone. We report the first identification of the emergence of a CTNNB1 mutation that can confer acquired resistance to imatinib. Further investigation into the causes of acquired resistance to imatinib will be essential to improve the prognosis for patients with KIT-mutated melanoma.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine, MK-8591: a novel HIV-1 reverse transcriptase translocation inhibitor.

PubMed

Markowitz, Martin; Sarafianos, Stefan G

2018-07-01

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside reverse transcriptase inhibitor (NRTI) with a novel mechanism of action, unique structure, and amongst NRTIs, unparalleled anti-HIV-1 activity. We will summarize its structure and function, antiviral activity, resistance profile, and potential as an antiretroviral for use in the treatment and preexposure prophylaxis of HIV-1 infection. EFdA is active against wild-type (EC50 as low as 50 pmol/l) and most highly NRTI-resistant viruses. The active metabolite, EFdA-triphosphate, has been shown to have a prolonged intracellular half-life in human and rhesus (Rh) blood cells. As a result, single drug doses tested in simian immunodeficiency virus mac251-infected Rh macaques and HIV-1-infected individuals exhibited robust antiviral activity of 7-10 days duration. Preclinical studies of EFdA as preexposure prophylaxis in the Rh macaque/simian/human immunodeficiency virus low-dose intrarectal challenge model have shown complete protection when given in clinically relevant doses. EFdA is a novel antiretroviral with activity against both wild-type and NRTI-resistant viruses. As a result of the prolonged intracellular half-life of its active moiety, it is amenable to flexibility in dosing of at least daily to weekly and perhaps longer.

Surveillance of Helicobacter pylori Antibiotic Susceptibility in Indonesia: Different Resistance Types among Regions and with Novel Genetic Mutations

PubMed Central

Miftahussurur, Muhammad; Syam, Ari Fahrial; Nusi, Iswan Abbas; Makmun, Dadang; Waskito, Langgeng Agung; Zein, Lukman Hakim; Akil, Fardah; Uwan, Willy Brodus; Simanjuntak, David; Wibawa, I Dewa Nyoman; Waleleng, Jimmy Bradley; Saudale, Alexander Michael Joseph; Yusuf, Fauzi; Mustika, Syifa; Adi, Pangestu; Maimunah, Ummi; Maulahela, Hasan; Rezkitha, Yudith Annisa Ayu; Subsomwong, Phawinee; Nasronudin; Rahardjo, Dadik; Suzuki, Rumiko; Akada, Junko; Yamaoka, Yoshio

2016-01-01

Information regarding Helicobacter pylori antibiotic resistance in Indonesia was previously inadequate. We assessed antibiotic susceptibility for H. pylori in Indonesia, and determined the association between virulence genes or genetic mutations and antibiotic resistance. We recruited 849 dyspeptic patients who underwent endoscopy in 11 cities in Indonesia. E-test was used to determine the minimum inhibitory concentration of five antibiotics. PCR-based sequencing assessed mutations in 23S rRNA, rdxA, gyrA, gyrB, and virulence genes. Next generation sequencing was used to obtain full-length sequences of 23S rRNA, infB, and rpl22. We cultured 77 strains and identified 9.1% with clarithromycin resistance. Low prevalence was also found for amoxicillin and tetracycline resistance (5.2% and 2.6%, respectively). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were demonstrated. Strains isolated from Sumatera Island had significantly higher metronidazole resistance than those from other locations. Metronidazole resistant strains had highly distributed rdxA amino acid substitutions and the 23S rRNA A2143G mutation was associated with clarithromycin resistance (42.9%). However, one strain with the highest MIC value had a novel mutation in rpl22 without an A2143G mutation. Mutation at Asn-87 and/or Asp-91 of gyrA was associated with levofloxacin-resistance and was related to gyrB mutations. In conclusions, although this is a pilot study for a larger survey, our current data show that Indonesian strains had the high prevalence of metronidazole and levofloxacin resistance with low prevalence of clarithromycin, amoxicillin, and tetracycline resistance. Nevertheless, clarithromycin- or metronidazole-based triple therapy should be administered with caution in some regions of Indonesia. PMID:27906990

Surveillance of Helicobacter pylori Antibiotic Susceptibility in Indonesia: Different Resistance Types among Regions and with Novel Genetic Mutations.

PubMed

Miftahussurur, Muhammad; Syam, Ari Fahrial; Nusi, Iswan Abbas; Makmun, Dadang; Waskito, Langgeng Agung; Zein, Lukman Hakim; Akil, Fardah; Uwan, Willy Brodus; Simanjuntak, David; Wibawa, I Dewa Nyoman; Waleleng, Jimmy Bradley; Saudale, Alexander Michael Joseph; Yusuf, Fauzi; Mustika, Syifa; Adi, Pangestu; Maimunah, Ummi; Maulahela, Hasan; Rezkitha, Yudith Annisa Ayu; Subsomwong, Phawinee; Nasronudin; Rahardjo, Dadik; Suzuki, Rumiko; Akada, Junko; Yamaoka, Yoshio

2016-01-01

Information regarding Helicobacter pylori antibiotic resistance in Indonesia was previously inadequate. We assessed antibiotic susceptibility for H. pylori in Indonesia, and determined the association between virulence genes or genetic mutations and antibiotic resistance. We recruited 849 dyspeptic patients who underwent endoscopy in 11 cities in Indonesia. E-test was used to determine the minimum inhibitory concentration of five antibiotics. PCR-based sequencing assessed mutations in 23S rRNA, rdxA, gyrA, gyrB, and virulence genes. Next generation sequencing was used to obtain full-length sequences of 23S rRNA, infB, and rpl22. We cultured 77 strains and identified 9.1% with clarithromycin resistance. Low prevalence was also found for amoxicillin and tetracycline resistance (5.2% and 2.6%, respectively). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were demonstrated. Strains isolated from Sumatera Island had significantly higher metronidazole resistance than those from other locations. Metronidazole resistant strains had highly distributed rdxA amino acid substitutions and the 23S rRNA A2143G mutation was associated with clarithromycin resistance (42.9%). However, one strain with the highest MIC value had a novel mutation in rpl22 without an A2143G mutation. Mutation at Asn-87 and/or Asp-91 of gyrA was associated with levofloxacin-resistance and was related to gyrB mutations. In conclusions, although this is a pilot study for a larger survey, our current data show that Indonesian strains had the high prevalence of metronidazole and levofloxacin resistance with low prevalence of clarithromycin, amoxicillin, and tetracycline resistance. Nevertheless, clarithromycin- or metronidazole-based triple therapy should be administered with caution in some regions of Indonesia.

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations.

PubMed

Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

2018-04-14

To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori ( H. pylori ) and determine their association with therapeutic failure. PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori ( κ = 0.71). The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori .

Frequent Association between Alteration of the rdxA Gene and Metronidazole Resistance in French and North African Isolates of Helicobacter pylori

PubMed Central

Tankovic, Jacques; Lamarque, Dominique; Delchier, Jean-Charles; Soussy, Claude-James; Labigne, Agnes; Jenks, Peter J.

2000-01-01

Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration in rdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype. PMID:10681326

[Clinical significance of drug resistance-associated mutations in treatment of hepatitis C with direct-acting antiviral agents].

PubMed

Li, Z; Chen, Z W; Ren, H; Hu, P

2017-03-20

Direct-acting antiviral agents (DAAs) achieve a high sustained virologic response rate in the treatment of chronic hepatitis C virus infection. However, drug resistance-associated mutations play an important role in treatment failure and have attracted more and more attention. This article elaborates on the clinical significance of drug resistance-associated mutations from the aspects of their definition, association with genotype, known drug resistance-associated mutations and their prevalence rates, the impact of drug resistance-associated mutations on treatment naive and treatment-experienced patients, and the role of clinical detection, in order to provide a reference for clinical regimens with DAAs and help to achieve higher sustained virologic response rates.

Contribution of putative efflux pump genes to isoniazid resistance in clinical isolates of Mycobacterium tuberculosis.

PubMed

Narang, Anshika; Giri, Astha; Gupta, Shraddha; Garima, Kushal; Bose, Mridula; Varma-Basil, Mandira

2017-01-01

Isoniazid (INH) resistance in Mycobacterium tuberculosis has been mainly attributed to mutations in katG (64%) and inhA (19%). However, 20%-30% resistance to INH cannot be explained by mutations alone. Hence, other mechanisms besides mutations may play a significant role in providing drug resistance. Here, we explored the role of 24 putative efflux pump genes conferring INH-resistance in M. tuberculosis. Real-time expression profiling of the efflux pump genes was performed in five INH-susceptible and six high-level INH-resistant clinical isolates of M. tuberculosis exposed to the drug. Isolates were also analyzed for mutations in katG and inhA. Four high-level INH-resistant isolates (minimum inhibitory concentration [MIC] ≥2.5 mg/L) with mutations at codon 315 (AGC-ACC) of katG showed upregulation of one of the efflux genes Rv1634, Rv0849, efpA, or p55. Another high-level INH-resistant isolate (MIC 1.5 mg/L), with no mutations at katG or inhA overexpressed 8/24 efflux genes, namely, Rv1273c, Rv0194, Rv1634, Rv1250, Rv3823c, Rv0507, jefA, and p55. Five of these, namely, Rv0194, Rv1634, Rv1250, Rv0507, and p55 were induced only in resistant isolates. The high number of efflux genes overexpressed in an INH-resistant isolate with no known INH resistance associated mutations, suggests a role for efflux pumps in resistance to this antituberculous agent, with the role of Rv0194 and Rv0507 in INH resistance being reported for the first time.

A Redox Basis for Metronidazole Resistance in Helicobacter pylori▿

PubMed Central

Kaakoush, N. O.; Asencio, C.; Mégraud, F.; Mendz, G. L.

2009-01-01

Metronidazole resistance in Helicobacter pylori has been attributed to mutations in rdxA or frxA. Insufficient data correlating RdxA and/or FrxA with the resistant phenotype, and the emergence of resistant strains with no mutations in either rdxA or frxA, indicated that the molecular basis of H. pylori resistance to metronidazole required further characterization. The rdxA and frxA genes of four matched pairs of metronidazole-susceptible and -resistant strains were sequenced. The resistant strains had mutations in either rdxA, frxA, neither gene, or both genes. The reduction rates of five substrates suggested that metabolic differences between susceptible and resistant strains cannot be explained only by mutations in rdxA and/or frxA. A more global approach to understanding the resistance phenotype was taken by employing two-dimensional gel electrophoresis combined with tandem mass spectrometry analyses to identify proteins differentially expressed by the matched pair of strains with no mutations in rdxA or frxA. Proteins involved in the oxireduction of ferredoxin were downregulated in the resistant strain. Other redox enzymes, such as thioredoxin reductase, alkyl hydroperoxide reductase, and superoxide dismutase, showed a pI change in the resistant strain. The data suggested that metronidazole resistance involved more complex metabolic changes than specific gene mutations, and they provided evidence of a role for the intracellular redox potential in the development of resistance. PMID:19223619

Resistance to antivirals in human cytomegalovirus: mechanisms and clinical significance.

PubMed

Pérez, J L

1997-09-01

Long term therapies needed for managing human cytomegalovirus (HCMV) infections in immunosupressed patients provided the background for the emergence of the resistance to antivirals active against HCMV. In addition, laboratory selected mutants have also been readily achieved. Both clinical and laboratory resistant strains share the same determinants of resistance. Ganciclovir resistance may be due to a few mutations in the HCMV UL97 gene and/or viral DNA pol gene, the former being responsible for about 70% of clinical resistant isolates. Among them, V464, V594, S595 and F595 are the most frequent mutations. Because of their less extensive clinical use, much less is known about resistance to foscarnet and cidofovir (formerly, HPMPC) but in both cases, it has been associated to mutations in the DNA pol. Ganciclovir resistant strains showing DNA pol mutations are cross-resistant to cidofovir and their corresponding IC50 are normally higher than those from strains harboring only mutations at the UL97 gene. To date, foscarnet resistance seems to be independent of both ganciclovir and cidofovir resistance.

Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum

PubMed Central

Vila-Aiub, Martin M.; Yu, Qin; Han, Heping; Powles, Stephen B.

2015-01-01

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. PMID:26019257

A Novel Environmental Azole Resistance Mutation in Aspergillus fumigatus and a Possible Role of Sexual Reproduction in Its Emergence.

PubMed

Zhang, Jianhua; Snelders, Eveline; Zwaan, Bas J; Schoustra, Sijmen E; Meis, Jacques F; van Dijk, Karin; Hagen, Ferry; van der Beek, Martha T; Kampinga, Greetje A; Zoll, Jan; Melchers, Willem J G; Verweij, Paul E; Debets, Alfons J M

2017-06-27

This study investigated the dynamics of Aspergillus fumigatus azole-resistant phenotypes in two compost heaps with contrasting azole exposures: azole free and azole exposed. After heat shock, to which sexual but not asexual spores are highly resistant, the azole-free compost yielded 98% (49/50) wild-type and 2% (1/50) azole-resistant isolates, whereas the azole-containing compost yielded 9% (4/45) wild-type and 91% (41/45) resistant isolates. From the latter compost, 80% (36/45) of the isolates contained the TR 46 /Y121F/T289A genotype, 2% (1/45) harbored the TR 46 /Y121F/M172I/T289A/G448S genotype, and 9% (4/45) had a novel pan-triazole-resistant mutation (TR 46 3 /Y121F/M172I/T289A/G448S) with a triple 46-bp promoter repeat. Subsequent screening of a representative set of clinical A. fumigatus isolates showed that the novel TR 46 3 mutant was already present in samples from three Dutch medical centers collected since 2012. Furthermore, a second new resistance mutation was found in this set that harbored four TR 46 repeats. Importantly, in the laboratory, we recovered the TR 46 3 mutation from a sexual cross between two TR 46 isolates from the same azole-containing compost, possibly through unequal crossing over between the double tandem repeats (TRs) during meiosis. This possible role of sexual reproduction in the emergence of the mutation was further implicated by the high level of genetic diversity of STR genotypes in the azole-containing compost. Our study confirms that azole resistance mutations continue to emerge in the environment and indicates compost containing azole residues as a possible hot spot. Better insight into the biology of environmental resistance selection is needed to retain the azole class for use in food production and treatment of Aspergillus diseases. IMPORTANCE Composting of organic matter containing azole residues might be important for resistance development and subsequent spread of resistance mutations in Aspergillus fumigatus In this article, we show the dominance of azole-resistant A. fumigatus in azole-exposed compost and the discovery of a new resistance mutation with clinical relevance. Furthermore, our study indicates that current fungicide application is not sustainable as new resistance mutations continue to emerge, thereby threatening the use of triazoles in medicine. We provide evidence that the sexual part of the fungal life cycle may play a role in the emergence of resistance mutations because under laboratory conditions, we reconstructed the resistance mutation through sexual crossing of two azole-resistant A. fumigatus isolates derived from the same compost heap. Understanding the mechanisms of resistance selection in the environment is needed to design strategies against the accumulation of resistance mutations in order to retain the azole class for crop protection and treatment of Aspergillus diseases. Copyright © 2017 Zhang et al.

Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand.

PubMed

Stenhouse, Steven A; Plernsub, Suriya; Yanola, Jintana; Lumjuan, Nongkran; Dantrakool, Anchalee; Choochote, Wej; Somboon, Pradya

2013-08-30

Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin. This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance. The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance. Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.

Abacavir/dolutegravir/lamivudine single-tablet regimen: a review of its use in HIV-1 infection.

PubMed

Greig, Sarah L; Deeks, Emma D

2015-04-01

A fixed-dose, single-tablet regimen comprising the integrase strand transfer inhibitor (INSTI) dolutegravir and the nucleos(t)ide reverse transcriptase inhibitors (NRTIs) abacavir and lamivudine (abacavir/dolutegravir/lamivudine; Triumeq®) is now available for the treatment of HIV-1 infection. In a randomized, double-blind, phase III trial in antiretroviral therapy (ART)-naive adults (SINGLE), once-daily dolutegravir plus abacavir/lamivudine had noninferior efficacy to once-daily efavirenz/tenofovir disoproxil fumarate (tenofovir DF)/emtricitabine with regard to establishing and sustaining virological suppression over 144 weeks, and subsequent superiority testing significantly favoured dolutegravir plus abacavir/lamivudine. This outcome was predominantly driven by more favourable rates of discontinuation due to adverse events versus the efavirenz/tenofovir DF/emtricitabine group. These data were generally supported by findings from other phase III trials in ART-naive adults receiving dolutegravir plus either abacavir/lamivudine or tenofovir DF/emtricitabine (SPRING-2 and FLAMINGO). Dolutegravir plus abacavir/lamivudine is generally well tolerated, with a tolerability profile that appears to be more favourable than efavirenz/tenofovir DF/emtricitabine. In the SINGLE trial, there were no major treatment-emergent INSTI or NRTI resistance-associated mutations in dolutegravir plus abacavir/lamivudine recipients with protocol-defined virological failure, indicating a high genetic barrier to resistance. Thus, triple combination therapy with abacavir, dolutegravir and lamivudine is an effective, generally well tolerated option for the management of HIV-1 infection, with the convenient once-daily fixed-dose tablet providing the first single-tablet regimen option without tenofovir DF.

Analysis of Clinical Isolates of Helicobacter pylori in Pakistan Reveals High Degrees of Pathogenicity and High Frequencies of Antibiotic Resistance

PubMed Central

Rasheed, Faisal; Campbell, Barry James; Alfizah, Hanafiah; Varro, Andrea; Zahra, Rabaab; Yamaoka, Yoshio; Pritchard, David Mark

2014-01-01

Background Antibiotic resistance in Helicobacter pylori contributes to failure in eradicating the infection and is most often due to point and missense mutations in a few key genes. Methods The antibiotic susceptibility profiles of H. pylori isolates from 46 Pakistani patients were determined by Etest. Resistance and pathogenicity genes were amplified, and sequences were analyzed to determine the presence of mutations. Results A high percentage of isolates (73.9%) were resistant to metronidazole (MTZ), with considerable resistance to clarithromycin (CLR; 47.8%) and amoxicillin (AML; 54.3%) also observed. Relatively few isolates were resistant to tetracycline (TET; 4.3%) or to ciprofloxacin (CIP; 13%). However, most isolates (n = 43) exhibited resistance to one or more antibiotics. MTZ-resistant isolates contained missense mutations in oxygen-independent NADPH nitroreductase (RdxA; 8 mutations found) and NADH flavin oxidoreductase (FrxA; 4 mutations found). In the 23S rRNA gene, responsible for CLR resistance, a new point mutation (A2181G) and 4 previously reported mutations were identified. Pathogenicity genes cagA, dupA, and vacA s1a/m1 were detected frequently in isolates which were also found to be resistant to MTZ, CLR, and AML. A high percentage of CagA and VacA seropositivity was also observed in these patients. Phylogenetic analysis of partial sequences showed uniform distribution of the 3′ region of cagA throughout the tree. Conclusions We have identified H. pylori isolates in Pakistan which harbor pathogenicity genes and worrying antibiotic resistance profiles as a result of having acquired multiple point and missense mutations. H. pylori eradication regimens should therefore be reevaluated in this setting. PMID:24827414

Analysis of clinical isolates of Helicobacter pylori in Pakistan reveals high degrees of pathogenicity and high frequencies of antibiotic resistance.

PubMed

Rasheed, Faisal; Campbell, Barry James; Alfizah, Hanafiah; Varro, Andrea; Zahra, Rabaab; Yamaoka, Yoshio; Pritchard, David Mark

2014-10-01

Antibiotic resistance in Helicobacter pylori contributes to failure in eradicating the infection and is most often due to point and missense mutations in a few key genes. The antibiotic susceptibility profiles of H. pylori isolates from 46 Pakistani patients were determined by Etest. Resistance and pathogenicity genes were amplified, and sequences were analyzed to determine the presence of mutations. A high percentage of isolates (73.9%) were resistant to metronidazole (MTZ), with considerable resistance to clarithromycin (CLR; 47.8%) and amoxicillin (AML; 54.3%) also observed. Relatively few isolates were resistant to tetracycline (TET; 4.3%) or to ciprofloxacin (CIP; 13%). However, most isolates (n = 43) exhibited resistance to one or more antibiotics. MTZ-resistant isolates contained missense mutations in oxygen-independent NADPH nitroreductase (RdxA; 8 mutations found) and NADH flavin oxidoreductase (FrxA; 4 mutations found). In the 23S rRNA gene, responsible for CLR resistance, a new point mutation (A2181G) and 4 previously reported mutations were identified. Pathogenicity genes cagA, dupA, and vacA s1a/m1 were detected frequently in isolates which were also found to be resistant to MTZ, CLR, and AML. A high percentage of CagA and VacA seropositivity was also observed in these patients. Phylogenetic analysis of partial sequences showed uniform distribution of the 3' region of cagA throughout the tree. We have identified H. pylori isolates in Pakistan which harbor pathogenicity genes and worrying antibiotic resistance profiles as a result of having acquired multiple point and missense mutations. H. pylori eradication regimens should therefore be reevaluated in this setting. © 2014 John Wiley & Sons Ltd.

Kdr mutations in Triatoma infestans from the Gran Chaco are distributed in two differentiated foci: Implications for pyrethroid resistance management.

PubMed

Sierra, Ivana; Capriotti, Natalia; Fronza, Georgina; Mougabure-Cueto, Gastón; Ons, Sheila

2016-06-01

Point mutations in the voltage-gated sodium channel, the primary target of pyrethroid insecticides, have been associated with the resistance in Triatoma infestans, an important vector of Chagas' disease. Hence, the sustainability of vector control programs requires the implementation of resistance management strategies. We determined the sensitivity of the molecular assays previously designed for early resistance detection to be used in pooled samples from a wide area of the endemic region, and validated them for their routine use in control campaigns for the monitoring of insecticide resistance in T. infestans. Consequently, we used these methods to examine the distribution of resistance-associated mutations in the sodium channel gene in populations of T. infestans from the Argentinean and Bolivian Gran Chaco. The PASA and REA assays tested proved sensitive enough to detect kdr SNPs in pooled samples, indicating these assays are suitable for routine screening in insecticide resistance surveillance. Two geographically differentiated foci were detected in T. infestans populations from the Argentinean and Bolivian Gran Chaco, with populations on the Bolivian-Argentinean border carrying L1014F mutation, and those from the Argentinean Chaco carrying L925I mutation. In all highly resistant populations analyzed, one of both kdr mutations was present, and toxicological assays determined that all pyrethroid resistant populations analyzed herein were sensitive to fenitrothion. The principal cause of pyrethroid resistance in T. infestans from the Gran Chaco ecoregion is kdr mutations in the sodium channel. Different levels of resistance occur in different populations carrying identical mutation, suggesting the existence of contributory mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Changing rates and patterns of drug resistance mutations in antiretroviral-experienced HIV-infected patients.

PubMed

de Mendoza, Carmen; Garrido, Carolina; Corral, Angelica; Ramírez-Olivencia, German; Jiménez-Nacher, Inmaculada; Zahonero, Natalia; Gonzalez-Lahoz, Juan; Soriano, Vincent

2007-07-01

Surveillance of drug resistance mutations in antiretroviral-experienced HIV(+) patients may provide useful information regarding options available for rescue interventions. All resistance tests performed from 1999 to 2005 on antiretroviral-experienced individuals at one reference laboratory in Madrid were examined. Only mutations associated with drug resistance recorded at the September 2006 IAS-USA list were considered. A total of 2137 specimens were analyzed. Overall, 71.1% showed resistance mutations to at least one drug class, 56.1% to at least two, and 21% to all three drug families. Resistance mutations were 65% for NRTI, 44.4% for NNRTI, and 42.5% for PI. Mutations T215Y/F, M184V, and M41L were the most frequent for NRTI. Their rate significantly declined since 1999. K65R significantly increased since 1999 (0.8%) to 2003 (7.3%) but declined up to 3.3% in 2005. For NNRTI, K103N significantly increased from 21.8% in 1999 to 29.5% in 2005 (p < 0.01). The most frequent PI resistance mutations were L90M (24.3%), V82X (19.9%), M46I/L (19.5%), and I54V (17.1%). The presence of five or more was 58.8% in 1999 but declined to 22.2% in 2005. The rate of drug resistance mutations causing NRTI and PI resistance has steadily declined in antiretroviral-experienced patients since 1999. The availability of a large number and/or more convenient NRTI as well as the wide use of ritonavir-boosted PI could explain these observations. However, broad PI cross-resistance was seen in nearly 25% of antiretroviral-experienced patients in 2005. Therefore, there is a still need for new antiretrovirals with different resistance profiles.

Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

PubMed

Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

2016-01-01

Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

PubMed

Zhang, Wei; Hu, Minlu; Shi, Yuan; Gong, Tiantian; Dezzutti, Charlene S; Moncla, Bernard; Sarafianos, Stefan G; Parniak, Michael A; Rohan, Lisa C

2015-09-01

EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV. Assessments of EFdA- and CSIC-loaded films included physicochemical characteristics, in vitro cytotoxicity, epithelia integrity studies, compatibility with the normal vaginal Lactobacillus flora and anti-HIV bioactivity evaluations. No significant change in physicochemical properties or biological activity of the combination films were noted during 3 months storage. In vitro cytotoxicity and bioactivity testing showed that 50% cytotoxic concentration (CC50) of either EFdA or CSIC was several orders of magnitude higher than the 50% effective concentration (EC50) values. Film-formulated EFdA and CSIC combination showed additive inhibitory activity against wild type and drug-resistant variants of HIV. Epithelial integrity studies demonstrated that the combination vaginal film had a much lower toxicity to HEC-1A monolayers compared to that of VCF®, a commercial vaginal film product containing nonoxynol-9. Polarized ectocervical explants showed films with drug alone or in combination were effective at preventing HIV infection. Our data suggest that vaginal microbicide films containing a combination of the NRTI EFdA and the NNRTI CSIC have potential to prevent HIV-1 sexual transmission.

Novel Method for Simultaneous Quantification of Phenotypic Resistance to Maturation, Protease, Reverse Transcriptase, and Integrase HIV Inhibitors Based on 3′Gag(p2/p7/p1/p6)/PR/RT/INT-Recombinant Viruses: a Useful Tool in the Multitarget Era of Antiretroviral Therapy▿†

PubMed Central

Weber, Jan; Vazquez, Ana C.; Winner, Dane; Rose, Justine D.; Wylie, Doug; Rhea, Ariel M.; Henry, Kenneth; Pappas, Jennifer; Wright, Alison; Mohamed, Nizar; Gibson, Richard; Rodriguez, Benigno; Soriano, Vicente; King, Kevin; Arts, Eric J.; Olivo, Paul D.; Quiñones-Mateu, Miguel E.

2011-01-01

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458–467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens. PMID:21628544

Naturally Occurring Mutations in the MPS1 Gene Predispose Cells to Kinase Inhibitor Drug Resistance.

PubMed

Gurden, Mark D; Westwood, Isaac M; Faisal, Amir; Naud, Sébastien; Cheung, Kwai-Ming J; McAndrew, Craig; Wood, Amy; Schmitt, Jessica; Boxall, Kathy; Mak, Grace; Workman, Paul; Burke, Rosemary; Hoelder, Swen; Blagg, Julian; Van Montfort, Rob L M; Linardopoulos, Spiros

2015-08-15

Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. ©2015 American Association for Cancer Research.

Variations in the occurrence of specific rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from patients of different ethnic groups in Kuwait.

PubMed

Ahmad, Suhail; Al-Mutairi, Noura M; Mokaddas, Eiman

2012-05-01

Frequency of resistance-conferring mutations vary among isoniazid- and ethambutol-resistant Mycobacterium tuberculosis isolates obtained from patients of various ethnic groups. This study was aimed to determine the occurrence of specific rpoB mutations in rifampicin-resistant M. tuberculosis isolates from tuberculosis patients of various ethnic groups in Kuwait. Rifampicin-resistant M. tuberculosis isolates (n=119) from South Asian (n=55), Southeast Asian (n=23), Middle Eastern (n=39) and other (n=2) patients and 107 rifampicin-susceptible isolates were tested. Mutations in rpoB were detected by DNA sequencing. Polymorphisms at katG463 and gyrA95 were detected by PCR-RFLP for genetic group assignment. None of rifampicin-susceptible but 116 of 119 rifampicin-resistant isolates showed rpoB mutation(s). Mutations among isolates from South Asian patients were distributed at rpoB516 (20%), rpoB526 (24%) and rpoB531 (27%) while 78 and 51 per cent of isolates from Southeast Asian and Middle Eastern patients, respectively, contained a mutated rpoB531. All isolates with rpoB N-terminal and cluster II mutations were obtained from Middle Eastern and South Asian patients. Most isolates from South Asian (84%) and Southeast Asian (70%) patients belonged to genetic group I while nearly all remaining isolates belonged to genetic group II. Isolates from Middle Eastern patients were distributed among genetic group I (46%), genetic group II (33%) and genetic group III (21%). The occurrence of specific rpoB mutations varied considerably in rifampicin-resistant M. tuberculosis isolates obtained from patients of different ethnic groups within the same country. The present data have important implications for designing region-specific rapid methods for detecting majority of rifampicin-resistant strains.

Frequency of ABL gene mutations in chronic myeloid leukemia patients resistant to imatinib and results of treatment switch to second-generation tyrosine kinase inhibitors.

PubMed

Marcé, Silvia; Zamora, Lurdes; Cabezón, Marta; Xicoy, Blanca; Boqué, Concha; Fernández, Cristalina; Grau, Javier; Navarro, José-Tomás; Fernández de Sevilla, Alberto; Ribera, Josep-Maria; Feliu, Evarist; Millá, Fuensanta

2013-08-04

Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients. Copyright © 2012 Elsevier España, S.L. All rights reserved.

Are We Ready to Use ESR1 Mutations in Clinical Practice?

PubMed

Jeselsohn, Rinath

2017-10-01

The recurrent ligand-binding domain ESR1 mutations are an important mechanism of endocrine resistance in estrogen receptor-positive (ER+) metastatic breast cancer. These mutations evolve under the selective pressure of endocrine treatments and are rarely found in treatment-naïve ER+ breast cancers. Preclinical studies showed that these mutations lead to ligand-independent activity facilitating resistance to aromatase inhibitors and relative resistance to tamoxifen and fulvestrant. Retrospective analyses of ESR1 mutations in baseline plasma circulating tumor DNA from clinical trials suggest that these mutations are prognostic of poor overall survival and predictive of resistance to aromatase inhibitors in metastatic disease. Larger datasets and prospective studies to confirm these results are lacking. In addition, response to other standard treatments for metastatic breast cancer in the presence of the ESR1 mutations is unknown, and studies to determine the optimal treatment combinations for patients with ESR1 mutations are also needed.

Effectiveness of highly active antiretroviral therapy in HIV-positive children: evaluation at 12 months in a routine program in Cambodia.

PubMed

Janssens, Bart; Raleigh, Brian; Soeung, Seithaboth; Akao, Kazumi; Te, Vantha; Gupta, Jitendra; Vun, Mean Chhy; Ford, Nathan; Nouhin, Janin; Nerrienet, Eric

2007-11-01

Increasing access to highly active antiretroviral therapy to reach all those in need in developing countries (scale up) is slowly expanding to HIV-positive children, but documented experience remains limited. We aimed to describe the clinical, immunologic, and virologic outcomes of pediatric patients with >12 months of highly active antiretroviral therapy in 2 routine programs in Cambodia. Between June 2003 and March 2005, 212 children who were younger than 13 years started highly active antiretroviral therapy. Most patients started a standard first-line regimen of lamivudine, stavudine, and nevirapine, using split adult fixed-dosage combinations. CD4 percentage and body weight were monitored routinely. A cross-sectional virologic analysis was conducted in January 2006; genotype resistance testing was performed for patients with a detectable viral load. Mean age of the subjects was 6 years. Median CD4 percentage at baseline was 6. Survival was 92% at 12 months and 91% at 24 months; 13 patients died, and 4 were lost to follow-up. A total of 81% of all patients had an undetectable viral load. Among the patients with a detectable viral load, most mutations were associated with resistance to lamivudine and non-nucleoside reverse-transcriptase inhibitor drugs. Five patients had developed extensive antiretroviral resistance. Being an orphan was found to be a predictor of virologic failure. This study provides additional evidence of the effectiveness of integrating HIV/AIDS care with highly active antiretroviral therapy for children in a routine setting, with good virologic suppression and immunologic recovery achieved by using split adult fixed-dosage combinations. Viral load monitoring and HIV genotyping are valuable tools for the clinical follow-up of the patients. Orphans should receive careful follow-up and extra support.

PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: an in vitro selection study.

PubMed

Dandache, Serge; Coburn, Craig A; Oliveira, Maureen; Allison, Timothy J; Holloway, M Katharine; Wu, Jinzi J; Stranix, Brent R; Panchal, Chandra; Wainberg, Mark A; Vacca, Joseph P

2008-12-01

The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.

MUBII-TB-DB: a database of mutations associated with antibiotic resistance in Mycobacterium tuberculosis.

PubMed

Flandrois, Jean-Pierre; Lina, Gérard; Dumitrescu, Oana

2014-04-14

Tuberculosis is an infectious bacterial disease caused by Mycobacterium tuberculosis. It remains a major health threat, killing over one million people every year worldwide. An early antibiotic therapy is the basis of the treatment, and the emergence and spread of multidrug and extensively drug-resistant mutant strains raise significant challenges. As these bacteria grow very slowly, drug resistance mutations are currently detected using molecular biology techniques. Resistance mutations are identified by sequencing the resistance-linked genes followed by a comparison with the literature data. The only online database is the TB Drug Resistance Mutation database (TBDReaM database); however, it requires mutation detection before use, and its interrogation is complex due to its loose syntax and grammar. The MUBII-TB-DB database is a simple, highly structured text-based database that contains a set of Mycobacterium tuberculosis mutations (DNA and proteins) occurring at seven loci: rpoB, pncA, katG; mabA(fabG1)-inhA, gyrA, gyrB, and rrs. Resistance mutation data were extracted after the systematic review of MEDLINE referenced publications before March 2013. MUBII analyzes the query sequence obtained by PCR-sequencing using two parallel strategies: i) a BLAST search against a set of previously reconstructed mutated sequences and ii) the alignment of the query sequences (DNA and its protein translation) with the wild-type sequences. The post-treatment includes the extraction of the aligned sequences together with their descriptors (position and nature of mutations). The whole procedure is performed using the internet. The results are graphs (alignments) and text (description of the mutation, therapeutic significance). The system is quick and easy to use, even for technicians without bioinformatics training. MUBII-TB-DB is a structured database of the mutations occurring at seven loci of major therapeutic value in tuberculosis management. Moreover, the system provides interpretation of the mutations in biological and therapeutic terms and can evolve by the addition of newly described mutations. Its goal is to provide easy and comprehensive access through a client-server model over the Web to an up-to-date database of mutations that lead to the resistance of M. tuberculosis to antibiotics.

Leprosy Drug Resistance Surveillance in Colombia: The Experience of a Sentinel Country

PubMed Central

Beltrán-Alzate, Camilo; López Díaz, Fernando; Romero-Montoya, Marcela; Sakamuri, Rama; Li, Wei; Kimura, Miyako; Brennan, Patrick

2016-01-01

An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively. PMID:27706165

Mutational Pathway Determines Whether Drug Gradients Accelerate Evolution of Drug-Resistant Cells

NASA Astrophysics Data System (ADS)

Greulich, Philip; Waclaw, Bartłomiej; Allen, Rosalind J.

2012-08-01

Drug gradients are believed to play an important role in the evolution of bacteria resistant to antibiotics and tumors resistant to anticancer drugs. We use a statistical physics model to study the evolution of a population of malignant cells exposed to drug gradients, where drug resistance emerges via a mutational pathway involving multiple mutations. We show that a nonuniform drug distribution has the potential to accelerate the emergence of resistance when the mutational pathway involves a long sequence of mutants with increasing resistance, but if the pathway is short or crosses a fitness valley, the evolution of resistance may actually be slowed down by drug gradients. These predictions can be verified experimentally, and may help to improve strategies for combating the emergence of resistance.

Evolution of high-level resistance during low-level antibiotic exposure.

PubMed

Wistrand-Yuen, Erik; Knopp, Michael; Hjort, Karin; Koskiniemi, Sanna; Berg, Otto G; Andersson, Dan I

2018-04-23

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster.

PubMed

Remnant, Emily J; Morton, Craig J; Daborn, Phillip J; Lumb, Christopher; Yang, Ying Ting; Ng, Hooi Ling; Parker, Michael W; Batterham, Philip

2014-11-01

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala(301) site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impact of Fluoroquinolone Resistance Mutations on Gonococcal Fitness and In Vivo Selection for Compensatory Mutations

PubMed Central

Kunz, Anjali N.; Begum, Afrin A.; Wu, Hong; D'Ambrozio, Jonathan A.; Robinson, James M.; Shafer, William M.; Bash, Margaret C.; Jerse, Ann E.

2012-01-01

Background. Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA91/95 and parC86 mutations on gonococcal fitness. Methods. Mutant gyrA91/95 and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR−79 mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection. Results. In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA91/95 mutation conferred an in vivo fitness benefit to wild-type and mtrR−79 mutant gonococci. The gyrA91/95, parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA91/95, parC86, mtrR−79 mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (CipR) mutant was selected during infection with the gyrA91/95, parC86, mtrR−79 mutant in which the mtrR−79 mutation was repaired and the gyrA91 mutation was altered. This in vivo–selected mutant grew as well as the wild-type strain in vitro. Conclusions. gyrA91/95 mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain CipR. PMID:22492860

Genotypic and phenotypic characteristics of aminoglycoside-resistant Mycobacterium tuberculosis isolates in Latvia.

PubMed

Bauskenieks, Matiss; Pole, Ilva; Skenders, Girts; Jansone, Inta; Broka, Lonija; Nodieva, Anda; Ozere, Iveta; Kalvisa, Adrija; Ranka, Renate; Baumanis, Viesturs

2015-03-01

Mutations causing resistance to aminoglycosides, such as kanamycin (KAN), amikacin (AMK), and streptomycin, are not completely understood. In this study, polymorphisms of aminoglycoside resistance influencing genes such as rrs, eis, rpsL, and gidB in 41 drug-resistant and 17 pan-sensitive Mycobacterium tuberculosis clinical isolates in Latvia were analyzed. Mutation A1400G in rrs gene was detected in 92% isolates with high resistance level to KAN and diverse MIC level to AMK. Mutations in promoter region of eis were detected in 80% isolates with low-level MIC of KAN. The association of K43R mutation in rpsL gene, a mutation in the rrs gene at position 513, and various polymorphisms in gidB gene with distinct genetic lineages of M. tuberculosis was observed. The results of this study suggest that association of different controversial mutations of M. tuberculosis genes to the drug resistance phenotype should be done in respect to genetic lineages. Copyright © 2015 Elsevier Inc. All rights reserved.

Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

PubMed

Mukherjee, Angana; Bopp, Selina; Magistrado, Pamela; Wong, Wesley; Daniels, Rachel; Demas, Allison; Schaffner, Stephen; Amaratunga, Chanaki; Lim, Pharath; Dhorda, Mehul; Miotto, Olivo; Woodrow, Charles; Ashley, Elizabeth A; Dondorp, Arjen M; White, Nicholas J; Wirth, Dyann; Fairhurst, Rick; Volkman, Sarah K

2017-05-12

Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA 0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA 0-3h  = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Clarithromycin Resistance Mutations in Helicobacter pylori in Association with Virulence Factors and Antibiotic Susceptibility of the Strains.

PubMed

Boyanova, Lyudmila; Markovska, Rumyana; Yordanov, Daniel; Gergova, Galina; Mitov, Ivan

2016-04-01

Antibiotic resistance is the major cause for Helicobacter pylori eradication failure. H. pylori clarithromycin resistance mutations were evaluated in 84 (82 phenotypically clarithromycin resistant and 2 intermediately susceptible) strains by allele-specific PCR and 3'-mismatched PCR. Many (57.1%) of these strains were metronidazole resistant. Prevalence of cagA(+), cagE(+), vacA s1a, m1, i1, and i2 strains was 76.2%, 58.0%, 82.1%, 35.7%, 50.0%, and 50.0%, respectively. A2143G, A2142G, A2142C, and A2143G+A2142G mutation rates were 64.3%, 23.8%, 1.2%, and 10.7%, respectively. Strains harboring the A2142G mutation showed 5.3-fold higher clarithromycin MIC50 than those harboring the A2143G mutation. The A2143G mutation alone was 1.7-fold more common in vacA i2 strains compared with vacA i1 strains, while the A2142G mutation alone was 3-fold more frequent in vacA i1 strains than vacA i2 strains and 3.1-fold more common in metronidazole-susceptible compared with metronidazole-resistant strains. Briefly, clarithromycin resistance mutations were significantly linked to vacA i allele and metronidazole susceptibility. This is the first report about associations between the A2143G mutation and less virulent vacA i2 strains, and between the A2142G mutation and more virulent vacA i1 strains. As the 2143G mutation often predicts eradication failure by clarithromycin-based regimens, the results may be linked to the better eradication of more virulent strains compared with the less virulent strains.

[Fluoroquinolone resistance mutations in topoisomerase genes of Salmonella typhimurium isolates].

PubMed

Guo, Yunchang; Pei, Xiaoyan; Liu, Xiumei

2004-09-01

Mutations in topoisomerase genes were main cause of the resistence of Salmonella typhimurium to fluoroquinolone. The MICs of three Salmonella typhimurium isolates X2, X7, X11 to ciprofloxacin were above 32 microg/ml, 0.38 microg/ml and 0.023 microg/ml, respectively. The genetic alterations in four topoisomerase genes, gyrA, gyrB, parC, and parE were detected by multiplex PCR amplimer conformation analysis in these three strains. X2 isolate showed both gyrA mutations (Ser83-->Phe, Asp87-->Asn) and parC mutation (Ser80-->Arg). X7 isolate showed a single gyrA mutation (Ser83-->Phe) and X11 isolate had no changes in all of the four quinolone resistance genes, gyrA, gyrB, parC, and parE. X7 isolate with a single gyrA mutation was less resistant to ciprofloxacin than X2 with double gyrA mutations and an additional parC mutation. GyrA and parC genes play important role of the resistance of Salmonella typhimurium to ciprofloxacin.

Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum.

PubMed

Vila-Aiub, Martin M; Yu, Qin; Han, Heping; Powles, Stephen B

2015-08-01

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Low-abundance HIV drug-resistant viral variants in treatment-experienced persons correlate with historical antiretroviral use.

PubMed

Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L; Dieckhaus, Kevin; Rosen, Marc I; Kozal, Michael J

2009-06-29

It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004-2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85-5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5-74.3, p = 0.0016). Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available.

Low-Abundance HIV Drug-Resistant Viral Variants in Treatment-Experienced Persons Correlate with Historical Antiretroviral Use

PubMed Central

Le, Thuy; Chiarella, Jennifer; Simen, Birgitte B.; Hanczaruk, Bozena; Egholm, Michael; Landry, Marie L.; Dieckhaus, Kevin; Rosen, Marc I.; Kozal, Michael J.

2009-01-01

Background It is largely unknown how frequently low-abundance HIV drug-resistant variants at levels under limit of detection of conventional genotyping (<20% of quasi-species) are present in antiretroviral-experienced persons experiencing virologic failure. Further, the clinical implications of low-abundance drug-resistant variants at time of virologic failure are unknown. Methodology/Principal Findings Plasma samples from 22 antiretroviral-experienced subjects collected at time of virologic failure (viral load 1380 to 304,000 copies/mL) were obtained from a specimen bank (from 2004–2007). The prevalence and profile of drug-resistant mutations were determined using Sanger sequencing and ultra-deep pyrosequencing. Genotypes were interpreted using Stanford HIV database algorithm. Antiretroviral treatment histories were obtained by chart review and correlated with drug-resistant mutations. Low-abundance drug-resistant mutations were detected in all 22 subjects by deep sequencing and only in 3 subjects by Sanger sequencing. In total they accounted for 90 of 247 mutations (36%) detected by deep sequencing; the majority of these (95%) were not detected by standard genotyping. A mean of 4 additional mutations per subject were detected by deep sequencing (p<0.0001, 95%CI: 2.85–5.53). The additional low-abundance drug-resistant mutations increased a subject's genotypic resistance to one or more antiretrovirals in 17 of 22 subjects (77%). When correlated with subjects' antiretroviral treatment histories, the additional low-abundance drug-resistant mutations correlated with the failing antiretroviral drugs in 21% subjects and correlated with historical antiretroviral use in 79% subjects (OR, 13.73; 95% CI, 2.5–74.3, p = 0.0016). Conclusions/Significance Low-abundance HIV drug-resistant mutations in antiretroviral-experienced subjects at time of virologic failure can increase a subject's overall burden of resistance, yet commonly go unrecognized by conventional genotyping. The majority of unrecognized resistant mutations correlate with historical antiretroviral use. Ultra-deep sequencing can provide important historical resistance information for clinicians when planning subsequent antiretroviral regimens for highly treatment-experienced patients, particularly when their prior treatment histories and longitudinal genotypes are not available. PMID:19562031

Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

PubMed

Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

2013-03-01

Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Novel mutations and mutation combinations of ryanodine receptor in a chlorantraniliprole resistant population of Plutella xylostella (L.)

PubMed Central

Guo, Lei; Liang, Pei; Zhou, Xuguo; Gao, Xiwu

2014-01-01

A previous study documented a glycine to glutamic acid mutation (G4946E) in ryanodine receptor (RyR) was highly correlated to diamide insecticide resistance in field populations of Plutella xylostella (Lepidoptera: Plutellidae). In this study, a field population collected in Yunnan province, China, exhibited a 2128-fold resistance to chlorantraniliprole. Sequence comparison between resistant and susceptible P. xylostella revealed three novel mutations including a glutamic acid to valine substitution (E1338D), a glutamine to leucine substitution (Q4594L) and an isoleucine to methionine substitution (I4790M) in highly conserved regions of RyR. Frequency analysis of all four mutations in this field population showed that the three new mutations showed a high frequency of 100%, while the G4946E had a frequency of 20%. Furthermore, the florescent ligand binding assay revealed that the RyR containing multiple mutations displayed a significantly lower affinity to the chlorantraniliprole. The combined results suggested that the co-existence of different combinations of the four mutations was involved in the chlorantraniliprole resistance. An allele-specific PCR based method was developed for the diagnosis of the four mutations in the field populations of P. xylostella. PMID:25377064

Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

PubMed

Sherrard, Laura J; Tai, Anna S; Wee, Bryan A; Ramsay, Kay A; Kidd, Timothy J; Ben Zakour, Nouri L; Whiley, David M; Beatson, Scott A; Bell, Scott C

2017-01-01

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

High rates of virological failure and drug resistance in perinatally HIV-1-infected children and adolescents receiving lifelong antiretroviral therapy in routine clinics in Togo

PubMed Central

Salou, Mounerou; Dagnra, Anoumou Y; Butel, Christelle; Vidal, Nicole; Serrano, Laetitia; Takassi, Elom; Konou, Abla A; Houndenou, Spero; Dapam, Nina; Singo-Tokofaï, Assetina; Pitche, Palokinam; Atakouma, Yao; Prince-David, Mireille; Delaporte, Eric; Peeters, Martine

2016-01-01

Introduction Antiretroviral treatment (ART) has been scaled up over the last decade but compared to adults, children living with HIV are less likely to receive ART. Moreover, children and adolescents are more vulnerable than adults to virological failure (VF) and emergence of drug resistance. In this study we determined virological outcome in perinatally HIV-1-infected children and adolescents receiving ART in Togo. Methods HIV viral load (VL) testing was consecutively proposed to all children and adolescents who were on ART for at least 12 months when attending HIV healthcare services for their routine follow-up visit (June to September 2014). Plasma HIV-1 VL was measured using the m2000 RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA). Genotypic drug resistance was done for all samples with VL>1000 copies/ml. Results and discussion Among 283 perinatally HIV-1-infected children and adolescents included, 167 (59%) were adolescents and 116 (41%) were children. The median duration on ART was 48 months (interquartile range: 28 to 68 months). For 228 (80.6%), the current ART combination consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) (zidovudine and lamivudine) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine or efavirenz). Only 28 (9.9%) were on a protease inhibitor (PI)-based regimen. VL was below the detection limit (i.e. 40 copies/ml) for 102 (36%), between 40 and 1000 copies/ml for 35 (12.4%) and above 1000 copies/ml for 146 (51.6%). Genotypic drug-resistance testing was successful for 125/146 (85.6%); 110/125 (88.0%) were resistant to both NRTIs and NNRTIs, 1/125 (0.8%) to NRTIs only, 4/125 (3.2%) to NNRTIs only and three harboured viruses resistant to reverse transcriptase and PIs. Overall, 86% (108/125) of children and adolescents experiencing VF and successfully genotyped, corresponding thus to at least 38% of the study population, had either no effective ART or had only a single effective drug in their current ART regimen. Conclusions Our study provided important information on virological outcome on lifelong ART in perinatally HIV-1-infected children and adolescents who were still on ART and continued to attend antiretroviral (ARV) clinics for follow-up visits. Actual conditions for scaling up and monitoring lifelong ART in children in resource-limited countries can have dramatic long-term outcomes and illustrate that paediatric ART receives inadequate attention. PMID:27125320

Modulation of Global Transcriptional Regulatory Networks as a Strategy for Increasing Kanamycin Resistance of the Translational Elongation Factor-G Mutants in Escherichia coli

PubMed Central

Mogre, Aalap; Veetil, Reshma T.; Seshasayee, Aswin Sai Narain

2017-01-01

Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics. In our previous work, we used short-term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin, and reported that Escherichia coli develops low cost resistance to kanamycin via different point mutations in the translation Elongation Factor-G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD/cpxA/topA/cyaA. Mutations in three of these genes had been discovered in earlier screens for aminoglycoside resistance. In this work, we expand our understanding of these second site mutations, the goal being to understand how these mutations affect the activities of the mutated gene products to confer resistance. We show that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown role for CyaA and / or cAMP in gene expression. From the transcriptomes of double and single mutants, we describe the epistasis between the mutation in EF-G and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result from increased negative supercoiling in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the misregulated genes in the mutants. PMID:29046437

HBV genotypes and drug resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected patients.

PubMed

Archampong, Timothy Na; Boyce, Ceejay L; Lartey, Margaret; Sagoe, Kwamena W; Obo-Akwa, Adjoa; Kenu, Ernest; Blackard, Jason T; Kwara, Awewura

2017-01-01

The presence of HBV resistance mutations upon initiation or during antiretroviral therapy (ART) in HIV-coinfected patients is an important determinant of treatment response. The main objective of the study was to determine the prevalence of HBV resistance mutations in antiretroviral treatment-naive and treatment-experienced HBV-HIV-coinfected Ghanaian patients with detectable HBV viraemia. HBV-HIV-coinfected patients who were ART-naive or had received at least 9 months of lamivudine (3TC)-containing ART were enrolled in a cross-sectional study. Demographic and clinical data were collected and HBV DNA quantified. Partial HBV sequences were amplified by PCR and sequenced bi-directionally to obtain a 2.1-2.2 kb fragment for phylogenetic analysis of HBV genotypes and evaluation of drug resistance mutations. Of the 100 HBV-HIV-coinfected study patients, 75 were successfully PCR-amplified, and 63 were successfully sequenced. Of these 63 patients, 27 (42.9%) were ART-experienced and 58 (92.1%) had HBV genotype E. No resistance mutations were observed in the 36 ART-naive patients, while 21 (77.8%) of 27 treatment-experienced patients had resistance mutations. All patients with resistance mutations had no tenofovir in their regimens, and 80% of them had HIV RNA <40 copies/ml. The 3TC resistance mutations rtL180M and rtM204V were observed in 10 (47.6%) of the 21 patients, while 5 patients (23.8%) had rtV173L, rtL180M and rtM204V mutations. A high proportion of HBV-HIV-coinfected patients with detectable viraemia on 3TC-containing ART had resistance mutations despite good ART adherence as determined by HIV RNA suppression. This study emphasizes the need for dual therapy as part of a fully suppressive ART in all HBV-HIV-coinfected patients in Ghana.

Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy.

PubMed

De Francesco, Vincenzo; Zullo, Angelo; Giorgio, Floriana; Saracino, Ilaria; Zaccaro, Cristina; Hassan, Cesare; Ierardi, Enzo; Di Leo, Alfredo; Fiorini, Giulia; Castelli, Valentina; Lo Re, Giovanna; Vaira, Dino

2014-03-01

Primary clarithromycin resistance is the main factor affecting the efficacy of Helicobacter pylori therapy. This study aimed: (i) to assess the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in resistant strains; (ii) to search, in the case of disagreement between the methods, for point mutations other than those reported as the most frequent in Europe; and (iii) to compare the MICs associated with the single point mutations. In order to perform real-time PCR, we retrieved biopsies from patients in whom H. pylori infection was successful diagnosed by bacterial culture and clarithromycin resistance was assessed using the Etest. Only patients who had never been previously treated, and with H. pylori strains that were either resistant exclusively to clarithromycin or without any resistance, were included. Biopsies from 82 infected patients were analysed, including 42 strains that were clarithromycin resistant and 40 that were clarithromycin susceptible on culture. On genotypic analysis, at least one of the three most frequently reported point mutations (A2142C, A2142G and A2143G) was detected in only 23 cases (54.8%), with a concordance between the two methods of 0.67. Novel point mutations (A2115G, G2141A and A2144T) were detected in a further 14 out of 19 discordant cases, increasing the resistance detection rate of PCR to 88% (P<0.001; odds ratio 6.1, 95% confidence interval 2-18.6) and the concordance to 0.81. No significant differences in MIC values among different point mutations were observed. This study suggests that: (i) the prevalence of the usually reported point mutations may be decreasing, with a concomitant emergence of new mutations; (ii) PCR-based methods should search for at least six point mutations to achieve good accuracy in detecting clarithromycin resistance; and (iii) none of the tested point mutations is associated with significantly higher MIC values than the others.

Cost-effectiveness of public-health policy options in the presence of pretreatment NNRTI drug resistance in sub-Saharan Africa: a modelling study.

PubMed

Phillips, Andrew N; Cambiano, Valentina; Nakagawa, Fumiyo; Revill, Paul; Jordan, Michael R; Hallett, Timothy B; Doherty, Meg; De Luca, Andrea; Lundgren, Jens D; Mhangara, Mutsa; Apollo, Tsitsi; Mellors, John; Nichols, Brooke; Parikh, Urvi; Pillay, Deenan; Rinke de Wit, Tobias; Sigaloff, Kim; Havlir, Diane; Kuritzkes, Daniel R; Pozniak, Anton; van de Vijver, David; Vitoria, Marco; Wainberg, Mark A; Raizes, Elliot; Bertagnolio, Silvia

2018-03-01

There is concern over increasing prevalence of non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance in people initiating antiretroviral therapy (ART) in low-income and middle-income countries. We assessed the effectiveness and cost-effectiveness of alternative public health responses in countries in sub-Saharan Africa where the prevalence of pretreatment drug resistance to NNRTIs is high. The HIV Synthesis Model is an individual-based simulation model of sexual HIV transmission, progression, and the effect of ART in adults, which is based on extensive published data sources and considers specific drugs and resistance mutations. We used this model to generate multiple setting scenarios mimicking those in sub-Saharan Africa and considered the prevalence of pretreatment NNRTI drug resistance in 2017. We then compared effectiveness and cost-effectiveness of alternative policy options. We took a 20 year time horizon, used a cost effectiveness threshold of US$500 per DALY averted, and discounted DALYs and costs at 3% per year. A transition to use of a dolutegravir as a first-line regimen in all new ART initiators is the option predicted to produce the most health benefits, resulting in a reduction of about 1 death per year per 100 people on ART over the next 20 years in a situation in which more than 10% of ART initiators have NNRTI resistance. The negative effect on population health of postponing the transition to dolutegravir increases substantially with higher prevalence of HIV drug resistance to NNRTI in ART initiators. Because of the reduced risk of resistance acquisition with dolutegravir-based regimens and reduced use of expensive second-line boosted protease inhibitor regimens, this policy option is also predicted to lead to a reduction of overall programme cost. A future transition from first-line regimens containing efavirenz to regimens containing dolutegravir formulations in adult ART initiators is predicted to be effective and cost-effective in low-income settings in sub-Saharan Africa at any prevalence of pre-ART NNRTI resistance. The urgency of the transition will depend largely on the country-specific prevalence of NNRTI resistance. Bill & Melinda Gates Foundation, World Health Organization. Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY IGO 3.0 licence. Published by Elsevier Ltd.. All rights reserved.

Naturally occurring dominant drug resistance mutations occur infrequently in the setting of recently acquired hepatitis C.

PubMed

Applegate, Tanya L; Gaudieri, Silvana; Plauzolles, Anne; Chopra, Abha; Grebely, Jason; Lucas, Michaela; Hellard, Margaret; Luciani, Fabio; Dore, Gregory J; Matthews, Gail V

2015-01-01

Direct-acting antivirals (DAAs) are predicted to transform hepatitis C therapy, yet little is known about the prevalence of naturally occurring resistance mutations in recently acquired HCV. This study aimed to determine the prevalence and frequency of drug resistance mutations in the viral quasispecies among HIV-positive and -negative individuals with recent HCV. The NS3 protease, NS5A and NS5B polymerase genes were amplified from 50 genotype 1a participants of the Australian Trial in Acute Hepatitis C. Amino acid variations at sites known to be associated with possible drug resistance were analysed by ultra-deep pyrosequencing. A total of 12% of individuals harboured dominant resistance mutations, while 36% demonstrated non-dominant resistant variants below that detectable by bulk sequencing (that is, <20%) but above a threshold of 1%. Resistance variants (<1%) were observed at most sites associated with DAA resistance from all classes, with the exception of sofosbuvir. Dominant resistant mutations were uncommonly observed in the setting of recent HCV. However, low-level mutations to all DAA classes were observed by deep sequencing at the majority of sites and in most individuals. The significance of these variants and impact on future treatment options remains to be determined. Clinicaltrials.gov NCT00192569.

Determining Resistance of Toxoplasma gondii Oocysts to UV Disinfection Using Cell Culture and a Mouse Bioassay

USDA-ARS?s Scientific Manuscript database

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated irradiated oocysts by three assays: a SCID mouse biassay, an in vitro T. gondii oocyst plaque assay (TOP-assay), and a quantitative reverse-transcriptase real-t...

Usefulness of detection of clarithromycin-resistant Helicobacter pylori from fecal specimens for young adults treated with eradication therapy.

PubMed

Osaki, Takako; Mabe, Katsuhiro; Zaman, Cynthia; Yonezawa, Hideo; Okuda, Masumi; Amagai, Kenji; Fujieda, Shinji; Goto, Mitsuhide; Shibata, Wataru; Kato, Mototsugu; Kamiya, Shigeru

2017-10-01

To prevent Helicobacter pylori infection in the younger generation, it is necessary to investigate the prevalence of antibiotic-resistant H. pylori. The aim of this study was to evaluate the method of PCR-based sequencing to detect clarithromycin (CAM) resistance-associated mutations using fecal samples as a noninvasive method. DNA extracted from fecal specimens and isolates from gastric biopsy specimens were collected from patients with H. pylori infection. Antibiotic resistance to CAM was analyzed by molecular and culture methods. The detection rates of CAM resistance-associated mutations (A2142C or A2143G) were compared before and after eradication therapy. With CAM resistance of H. pylori evaluated by antibiotic susceptibility test as a gold standard, the sensitivity and the specificity of gene mutation detection from fecal DNA were 80% and 84.8%, respectively. In contrast, using DNA of isolated strains, the sensitivity and the specificity were 80% and 100%. Of the seven cases in which eradication was unsuccessful by triple therapy including CAM, CAM-resistant H. pylori, and resistance-associated mutations were detected in three cases, CAM-resistant H. pylori without the mutation was detected in two patients, and resistance-associated mutation was only detected in one patient. PCR-based sequencing to detect CAM resistance-associated mutations using isolates or fecal samples was useful for finding antibiotic-resistant H. pylori infection. Although the specificity of the detection from fecal samples compared with antibiotic susceptibility testing was lower than that from isolates, this fecal detection method is suitable especially for asymptomatic subjects including children. Further improvement is needed before clinical application. © 2017 John Wiley & Sons Ltd.

TERT promoter mutations in thyroid cancer: a report from a Middle Eastern population.

PubMed

Qasem, Ebtesam; Murugan, Avaniyapuram Kannan; Al-Hindi, Hindi; Xing, Mingzhao; Almohanna, Mai; Alswailem, Meshael; Alzahrani, Ali S

2015-12-01

Telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have recently been described in follicular cell-derived thyroid cancer (TC) in patients from North America and Europe. In this study, we explored whether these findings could be replicated in patients from a different ethnic group. We screened 17 benign thyroid adenomas and 265 TC samples from patients in the Middle East for these mutations by PCR and direct sequencing using DNA isolated from paraffin-embedded tumor tissues. None of the 17 benign adenomas harbored TERT promoter mutations. Of 265 TC, 34 (12.8%) harbored TERT promoter mutations, including 10/153 (6.5%) conventional papillary TC (CPTC), 8/57 (14.0%) follicular variant PTC, 9/30 (30%) tall cell variant PTC, 1/3 (30%) Hurthle cell thyroid cancer (HTC), 1/5 (20%) follicular TC, and 5/13 (38.5%) poorly differentiated TC. C250T mutation was present in only 6/265 (2.3%) cases, while C228T mutation was present in a total of 28/265 (10.6%) cases. These two mutations were mutually exclusive. TERT promoter mutations were significantly more common in older (≥45 years) than younger patients and were associated with larger tumour size, vascular invasion, higher TNM stage (stage III and IV), BRAF(V600E) mutation and persistent/recurrent disease at 6-12 months after initial treatment and at the last follow up. These associations were stronger in non-CPTC. Thus, this study on a large cohort of TC patients from Middle East demonstrates that TERT promoter mutations are relatively common, especially in the non-CPTC, and are associated with more aggressive histopathological features, BRAF(V600E) mutation, and disease persistence/recurrence than the WT TERT. © 2015 Society for Endocrinology.

Homodimerization of the p51 Subunit of HIV-1 Reverse Transcriptase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, X.; Mueller, G; Cuneo, M

2010-01-01

The dimerization of HIV reverse transcriptase (RT), required to obtain the active form of the enzyme, is influenced by mutations, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleotide substrates, Mg ions, temperature, and specifically designed dimerization inhibitors. In this study, we have utilized nuclear magnetic resonance (NMR) spectroscopy of the [methyl-{sup 13}C]methionine-labeled enzyme and small-angle X-ray scattering (SAXS) to investigate how several of these factors influence the dimerization behavior of the p51 subunit. The {sup 1}H-{sup 13}C HSQC spectrum of p51 obtained at micromolar concentrations indicates that a significant fraction of the p51 adopts a 'p66-like' conformation. SAXS data obtained for p51more » samples were used to determine the fractions of monomer and dimer in the sample and to evaluate the conformation of the fingers/thumb subdomain. All of the p51 monomer observed was found to adopt the compact, 'p51C' conformation observed for the p51 subunit in the RT heterodimer. The NMR and SAXS data indicate that the p51 homodimer adopts a structure that is similar to the p66/p51 heterodimer, with one p51C subunit and a second p51 subunit in an extended, 'p51E' conformation that resembles the p66 subunit of the heterodimer. The fractional dimer concentration and the fingers/thumb orientation are found to depend strongly on the experimental conditions and exhibit a qualitative dependence on nevirapine and ionic strength (KCl) that is similar to the behavior reported for the heterodimer and the p66 homodimer. The L289K mutation interferes with p51 homodimer formation as it does with formation of the heterodimer, despite its location far from the dimer interface. This effect is readily interpreted in terms of a conformational selection model, in which p51{sub L289K} has a much greater preference for the compact, p51C conformation. A reduced level of dimer formation then results from the reduced ratio of the p51E{sub L289K} to p51C{sub L289K} monomers.« less

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

PubMed

Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

2017-11-14

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

PubMed

Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-07-01

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations

PubMed Central

Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

2018-01-01

AIM To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori (H. pylori) and determine their association with therapeutic failure. METHODS PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar’s test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. RESULTS 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori (κ = 0.71). CONCLUSION The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori. PMID:29662291

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

PubMed Central

Pauly, Matthew D.; Lyons, Daniel M.; Fitzsimmons, William J.

2017-01-01

ABSTRACT Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. PMID:28815216

EGFR G796D mutation mediates resistance to osimertinib.

PubMed

Zheng, Di; Hu, Min; Bai, Yu; Zhu, Xuehua; Lu, Xuesong; Wu, Chunyan; Wang, Jiying; Liu, Li; Wang, Zheng; Ni, Jian; Yang, Zhenfan; Xu, Jianfang

2017-07-25

Osimertinib is an effective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved in multiple countries and regions for patients with EGFR T790M mutation-positive non-small cell lung cancer (NSCLC). Despite impressive initial tumor responses, development of drug resistance ultimately limits the benefit of this compound. Mechanisms of resistance to osimertinib are just beginning to emerge, such as EGFR C797S and L718Q mutations, BRAF V600E and PIK3CA E545K mutations, as well as ERBB2 and MET amplification. However, a comprehensive view is still missing. In this study, we presented the first case of Chinese NSCLC patient who developed resistance to osimertinib, and discovered de novo EGFR G796D mutation as a potential mechanism. Our findings provided insights into mechanisms of resistance to osimertinib and highlighted tumor heterogeneity and clonal evolution during the development of drug resistance.

EGFR G796D mutation mediates resistance to osimertinib

PubMed Central

Bai, Yu; Zhu, Xuehua; Lu, Xuesong; Wu, Chunyan; Wang, Jiying; Liu, Li; Wang, Zheng; Ni, Jian; Yang, Zhenfan; Xu, Jianfang

2017-01-01

Osimertinib is an effective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) approved in multiple countries and regions for patients with EGFR T790M mutation-positive non-small cell lung cancer (NSCLC). Despite impressive initial tumor responses, development of drug resistance ultimately limits the benefit of this compound. Mechanisms of resistance to osimertinib are just beginning to emerge, such as EGFR C797S and L718Q mutations, BRAF V600E and PIK3CA E545K mutations, as well as ERBB2 and MET amplification. However, a comprehensive view is still missing. In this study, we presented the first case of Chinese NSCLC patient who developed resistance to osimertinib, and discovered de novo EGFR G796D mutation as a potential mechanism. Our findings provided insights into mechanisms of resistance to osimertinib and highlighted tumor heterogeneity and clonal evolution during the development of drug resistance. PMID:28572531

Acquired Resistance to Crizotinib from a Mutation in CD74–ROS1

PubMed Central

Awad, Mark M.; Katayama, Ryohei; McTigue, Michele; Liu, Wei; Deng, Ya-Li; Brooun, Alexei; Friboulet, Luc; Huang, Donghui; Falk, Matthew D.; Timofeevski, Sergei; Wilner, Keith D.; Lockerman, Elizabeth L.; Khan, Tahsin M.; Mahmood, Sidra; Gainor, Justin F.; Digumarthy, Subba R.; Stone, James R.; Mino-Kenudson, Mari; Christensen, James G.; Iafrate, A. John; Engelman, Jeffrey A.; Shaw, Alice T.

2013-01-01

Summary Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74–ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the meta-static sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.) PMID:23724914

Detailed imaging and genetic analysis reveal a secondary BRAF(L505H) resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib.

PubMed

Hoogstraat, Marlous; Gadellaa-van Hooijdonk, Christa G; Ubink, Inge; Besselink, Nicolle J M; Pieterse, Mark; Veldhuis, Wouter; van Stralen, Marijn; Meijer, Eelco F J; Willems, Stefan M; Hadders, Michael A; Kuilman, Thomas; Krijgsman, Oscar; Peeper, Daniel S; Koudijs, Marco J; Cuppen, Edwin; Voest, Emile E; Lolkema, Martijn P

2015-05-01

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three-dimensional imaging. In all patients, only a subset of lesions progressed. Next-generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib-resistant patient-derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

PubMed

Lavania, Mallika; Hena, Abu; Reja, Hasanoor; Nigam, Astha; Biswas, Nibir Kumar; Singh, Itu; Turankar, Ravindra P; Gupta, Ud; Kumar, Senthil; Rewaria, Latika; Patra, Pradip K R; Sengupta, Utpal; Bhattacharya, Basudeb

2016-03-01

Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations

PubMed Central

Tojo, Shigeo; Tanaka, Yukinori

2015-01-01

Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s). PMID:26369962

Global Analysis of the Evolution and Mechanism of Echinocandin Resistance in Candida glabrata

PubMed Central

Singh-Babak, Sheena D.; Babak, Tomas; Diezmann, Stephanie; Hill, Jessica A.; Xie, Jinglin Lucy; Chen, Ying-Lien; Poutanen, Susan M.; Rennie, Robert P.; Heitman, Joseph; Cowen, Leah E.

2012-01-01

The evolution of drug resistance has a profound impact on human health. Candida glabrata is a leading human fungal pathogen that can rapidly evolve resistance to echinocandins, which target cell wall biosynthesis and are front-line therapeutics for Candida infections. Here, we provide the first global analysis of mutations accompanying the evolution of fungal drug resistance in a human host utilizing a series of C. glabrata isolates that evolved echinocandin resistance in a patient treated with the echinocandin caspofungin for recurring bloodstream candidemia. Whole genome sequencing identified a mutation in the drug target, FKS2, accompanying a major resistance increase, and 8 additional non-synonymous mutations. The FKS2-T1987C mutation was sufficient for echinocandin resistance, and associated with a fitness cost that was mitigated with further evolution, observed in vitro and in a murine model of systemic candidemia. A CDC6-A511G(K171E) mutation acquired before FKS2-T1987C(S663P), conferred a small resistance increase. Elevated dosage of CDC55, which acquired a C463T(P155S) mutation after FKS2-T1987C(S663P), ameliorated fitness. To discover strategies to abrogate echinocandin resistance, we focused on the molecular chaperone Hsp90 and downstream effector calcineurin. Genetic or pharmacological compromise of Hsp90 or calcineurin function reduced basal tolerance and resistance. Hsp90 and calcineurin were required for caspofungin-dependent FKS2 induction, providing a mechanism governing echinocandin resistance. A mitochondrial respiration-defective petite mutant in the series revealed that the petite phenotype does not confer echinocandin resistance, but renders strains refractory to synergy between echinocandins and Hsp90 or calcineurin inhibitors. The kidneys of mice infected with the petite mutant were sterile, while those infected with the HSP90-repressible strain had reduced fungal burden. We provide the first global view of mutations accompanying the evolution of fungal drug resistance in a human host, implicate the premier compensatory mutation mitigating the cost of echinocandin resistance, and suggest a new mechanism of echinocandin resistance with broad therapeutic potential. PMID:22615574

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

PubMed Central

Kondrashova, Olga; Nguyen, Minh; Shield-Artin, Kristy; Tinker, Anna V.; Teng, Nelson N.H.; Harrell, Maria I.; Kuiper, Michael J.; Ho, Gwo-Yaw; Barker, Holly; Jasin, Maria; Prakash, Rohit; Kass, Elizabeth M.; Sullivan, Meghan R.; Brunette, Gregory J.; Bernstein, Kara A.; Coleman, Robert L.; Floquet, Anne; Friedlander, Michael; Kichenadasse, Ganessan; O'Malley, David M.; Oza, Amit; Sun, James; Robillard, Liliane; Maloney, Lara; Giordano, Heidi; Wakefield, Matthew J.; Kaufmann, Scott H.; Simmons, Andrew D.; Harding, Thomas C.; Raponi, Mitch; McNeish, Iain A.; Swisher, Elizabeth M.; Lin, Kevin K.; Scott, Clare L.

2017-01-01

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. PMID:28588062

Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco.

PubMed

Oudghiri, Amal; Karimi, Hind; Chetioui, Fouad; Zakham, Fathiah; Bourkadi, Jamal Eddine; Elmessaoudi, My Driss; Laglaoui, Amin; Chaoui, Imane; El Mzibri, Mohammed

2018-02-27

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco. To evaluate pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drug resistance, in 703 clinical isolates from TB patients recruited in Casablanca, and to assess the usefulness of molecular tools in clinical laboratories for better management of M/XDR TB in Morocco. Drug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to rpoB, katG and p-inhA mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of gyrA, gyrB, rrs, tlyA genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs. Among the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within rpoB and katG genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the gyrA gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of eis gene. No mutation was found in gyrB, rrs and tlyA genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes. Most of mutations associated with SLD resistance occurred in gyrA gene (codons 90-94) and eis promoter region. These findings highlight the impact of mutations in gyrA on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.

Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.

PubMed

Nyerges, Ákos; Csörgő, Bálint; Draskovits, Gábor; Kintses, Bálint; Szili, Petra; Ferenc, Györgyi; Révész, Tamás; Ari, Eszter; Nagy, István; Bálint, Balázs; Vásárhelyi, Bálint Márk; Bihari, Péter; Számel, Mónika; Balogh, Dávid; Papp, Henrietta; Kalapis, Dorottya; Papp, Balázs; Pál, Csaba

2018-06-19

Antibiotic development is frequently plagued by the rapid emergence of drug resistance. However, assessing the risk of resistance development in the preclinical stage is difficult. Standard laboratory evolution approaches explore only a small fraction of the sequence space and fail to identify exceedingly rare resistance mutations and combinations thereof. Therefore, new rapid and exhaustive methods are needed to accurately assess the potential of resistance evolution and uncover the underlying mutational mechanisms. Here, we introduce directed evolution with random genomic mutations (DIvERGE), a method that allows an up to million-fold increase in mutation rate along the full lengths of multiple predefined loci in a range of bacterial species. In a single day, DIvERGE generated specific mutation combinations, yielding clinically significant resistance against trimethoprim and ciprofloxacin. Many of these mutations have remained previously undetected or provide resistance in a species-specific manner. These results indicate pathogen-specific resistance mechanisms and the necessity of future narrow-spectrum antibacterial treatments. In contrast to prior claims, we detected the rapid emergence of resistance against gepotidacin, a novel antibiotic currently in clinical trials. Based on these properties, DIvERGE could be applicable to identify less resistance-prone antibiotics at an early stage of drug development. Finally, we discuss potential future applications of DIvERGE in synthetic and evolutionary biology. Copyright © 2018 the Author(s). Published by PNAS.

Mycobacterial interspersed repetitive unit typing and mutational profile for multidrug-resistant and extensively drug-resistant tuberculosis surveillance in Portugal: a 3-year period overview.

PubMed

Silva, Carla; Perdigão, João; Jordão, Luísa; Portugal, Isabel

2014-12-01

Multidrug tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) cases constitute a serious health problem in Portugal, of which the majority of isolates belong to the Lisboa family and the Q1 cluster, highly related to the Lisboa family. Here we sought to investigate the molecular basis of resistant TB as well as to determine the prevalence of specific drug resistance mutations and their association with MDR-TB and/or XDR-TB. In total, 74 Mycobacterium tuberculosis clinical isolates collected in Lisbon Health Region were genotyped by 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR), and the mutational profile associated with first- and second-line drug resistance was studied. Seven new mutations were found, whilst the remaining 28 mutations had been previously associated with drug resistance. None of the mutations was specifically associated with MDR-TB. The mutational patterns observed among isolates belonging to Lisboa3 and Q1 clusters were also observed in isolates with unique MIRU-VNTR patterns but closely related to these strains. Such data suggest that the genotyping technique employed discriminates isolates with the same mutational profile. To establish the most adequate genotyping technique, the discriminatory power of three different MIRU-VNTR sets was analysed. The 15-loci MIRU-VNTR set showed adequate discriminatory power, comparable with the 24-loci set, allowing clustering of 60% and 86% of the MDR-TB and XDR-TB isolates, respectively, the majority of which belonged to the Lisboa3 and Q1 clusters. From an epidemiological standpoint, this study suggests combined mutational and genotyping analysis as a valuable tool for drug resistance surveillance. Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance ▿ †

PubMed Central

Jang, Hyunjung; Kim, Jihyun; Choi, Jae-jin; Son, Yeojin; Park, Heekyung

2010-01-01

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. PMID:20573874

Accumulation of multiple mutations in linezolid-resistant Staphylococcus epidermidis causing bloodstream infections; in silico analysis of L3 amino acid substitutions that might confer high-level linezolid resistance.

PubMed

Ikonomidis, Alexandros; Grapsa, Anastasia; Pavlioglou, Charikleia; Demiri, Antonia; Batarli, Alexandra; Panopoulou, Maria

2016-12-01

Fifty-six Staphylococcus epidermidis clinical isolates, showing high-level linezolid resistance and causing bacteremia in critically ill patients, were studied. All isolates belonged to ST22 clone and carried the T2504A and C2534T mutations in gene coding for 23SrRNA as well as the C189A, G208A, C209T and G384C missense mutations in L3 protein which resulted in Asp159Tyr, Gly152Asp and Leu94Val substitutions. Other silent mutations were also detected in genes coding for ribosomal proteins L3 and L22. In silico analysis of missense mutations showed that although L3 protein retained the sequence of secondary motifs, the tertiary structure was influenced. The observed alteration in L3 protein folding provides an indication on the putative role of L3-coding gene mutations in high-level linezolid resistance. Furthermore, linezolid pressure in health care settings where linezolid consumption is of high rates might lead to the selection of resistant mutants possessing L3 mutations that might confer high-level linezolid resistance.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase.

PubMed

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-17

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth.This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors.

Antitumor effect of combination of the inhibitors of two new oncotargets: proton pumps and reverse transcriptase

PubMed Central

Lugini, Luana; Sciamanna, Ilaria; Federici, Cristina; Iessi, Elisabetta; Spugnini, Enrico Pierluigi; Fais, Stefano

2017-01-01

Tumor therapy needs new approaches in order to improve efficacy and reduce toxicity of the current treatments. The acidic microenvironment and the expression of high levels of endogenous non-telomerase reverse transcriptase (RT) are common features of malignant tumor cells. The anti-acidic proton pump inhibitor Lansoprazole (LAN) and the non-nucleoside RT inhibitor Efavirenz (EFV) have shown independent antitumor efficacy. LAN has shown to counteract drug tumor resistance. We tested the hypothesis that combination of LAN and EFV may improve the overall antitumor effects. We thus pretreated human metastatic melanoma cells with LAN and then with EFV, both in 2D and 3D spheroid models. We evaluated the treatment effect by proliferation and cell death/apoptosis assays in classical and in pulse administration experiments. The action of EFV was negatively affected by the tumor microenvironmental acidity, and LAN pretreatment overcame the problem. LAN potentiated the cytotoxicity of EFV to melanoma cells and, when administered during the drug interruption period, prevented the replacement of tumor cell growth. This study supports the implementation of the current therapies with combination of Proton Pumps and Reverse Transcriptase inhibitors. PMID:27926505

Emerging Helicobacter pylori levofloxacin resistance and novel genetic mutation in Nepal.

PubMed

Miftahussurur, Muhammad; Shrestha, Pradeep Krishna; Subsomwong, Phawinee; Sharma, Rabi Prakash; Yamaoka, Yoshio

2016-11-04

The prevalence of Helicobacter pylori antibiotic susceptibility in the Nepalese strains is untracked. We determined the antibiotic susceptibility for H. pylori and analyzed the presence of genetic mutations associated with antibiotic resistance in Nepalese strains. This study included 146 consecutive patients who underwent gastroduodenal endoscopy in Kathmandu, Nepal. Among 42 isolated H. pylori, there was no resistance to amoxicillin and tetracycline. In contrast, similar with typical South Asian patterns; metronidazole resistance rate in Nepalese strains were extremely high (88.1 %, 37/42). Clarithromycin resistance rate in Nepalese strains were modestly high (21.4 %, 9/42). Most of metronidazole resistant strains had highly distributed rdxA and frxA mutations, but were relative coincidence without a synergistic effect to increase the minimum inhibitory concentration (MIC). Among strains with the high MIC, 63.6 % (7/11) were associated with frameshift mutation at position 18 of frxA with or without rdxA involvement. However, based on next generation sequencing data we found that one strain with the highest MIC value had a novel mutation in the form of amino acid substituted at Ala-212, Gln-382, Ile-485 of dppA and Leu-145, Thr-168, Glu-117, Val-121, Arg-221 in dapF aside from missense mutations in full-length rdxA. Mutations at Asn-87 and/or Asp-91 of the gyrA were predominantly in levofloxacin-resistant strains. The gyrB mutation had steady relationship with the gyrA 87-91 mutations. Although three (44.4 %) and two (22.2 %) of clarithromycin resistant strains had point mutation on A2143G and A2146G, we confirmed the involvement of rpl22 and infB in high MIC strains without an 23SrRNA mutation. The rates of resistance to clarithromycin, metronidazole and levofloxacin were high in Nepalese strains, indicating that these antibiotics-based triple therapies are not useful as first-line treatment in Nepal. Bismuth or non-bismuth-based quadruple regimens, furazolidone-based triple therapy or rifabutin-based triple therapy may become alternative strategy in Nepal.

MTBDRplus and MTBDRsl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis

PubMed Central

Georghiou, Sophia B.; Catanzaro, Donald; Rodrigues, Camilla; Crudu, Valeriu; Victor, Thomas C.; Garfein, Richard S.; Catanzaro, Antonino; Rodwell, Timothy C.

2016-01-01

Accurate identification of drug-resistant Mycobacterium tuberculosis is imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplus and MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results. PMID:26763971

Recombineering reveals a diverse collection of ribosomal proteins L4 and L22 that confer resistance to macrolide antibiotics

PubMed Central

Diner, Elie J.; Hayes, Christopher S.

2009-01-01

Summary Mutations in ribosomal proteins L4 and L22 confer resistance to erythromycin and other macrolide antibiotics in a variety of bacteria. L4 and L22 have elongated loops whose tips converge in the peptide exit tunnel near the macrolide binding site, and resistance mutations typically affect residues within these loops. Here, we use bacteriophage λ Red-mediated recombination, or “recombineering”, to uncover new L4 and L22 alleles that confer macrolide resistance in Escherichia coli. We randomized residues at the tips of the L4 and L22 loops using recombineered oligonucleotide libraries, and selected the mutagenized cells for erythromycin-resistant mutants. These experiments led to the identification of 341 different resistance mutations encoding 278 unique L4 and L22 proteins – the overwhelming majority of which are novel. Many resistance mutations were complex, involving multiple missense mutations, in-frame deletions, and insertions. Transfer of L4 and L22 mutations into wild-type cells by phage P1-mediated transduction demonstrated that each allele was sufficient to confer macrolide resistance. Although L4 and L22 mutants are typically resistant to most macrolides, selections carried out on different antibiotics revealed macrolide-specific resistance mutations. L22 Lys90Trp is one such allele, which confers resistance to erythromycin, but not tylosin or spiramycin. Purified L22 Lys90Trp ribosomes show reduced erythromycin binding, but have the same affinity for tylosin as wild-type ribosomes. Moreover, DMS methylation protection assays demonstrated that L22 Lys90Trp ribosomes bind tylosin more readily than erythromycin in vivo. This work underscores the exceptional functional plasticity of the L4 and L22 proteins, and highlights the utility of Red-mediated recombination in targeted genetic selections. PMID:19150357

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

PubMed

Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

Phenotypic and genotypic evaluation of fluoroquinolone resistance in clinical isolates of Staphylococcus aureus in Tehran

PubMed Central

Aligholi, Marzieh; Mirsalehian, Akbar; Halimi, Shahnaz; Imaneini, Hossein; Taherikalani, Morovat; Jabalameli, Fereshteh; Asadollahi, Parisa; Mohajer, Babak; Abdollahi, Alireza; Emaneini, Mohammad

2011-01-01

Summary Background Fluoroquinolones are broad-spectrum antibiotics widely used in the treatment of bacterial infections such as Staphylococcus aureus isolates. Resistance to these antibiotics is increasing. Material/Methods The occurrence of mutations in the grlA and gyrA loci were evaluated in 69 fluoroquinolone-resistant S. aureus isolates from 2 teaching hospitals of Tehran University of Medical Sciences. Results Out of the 165 S. aureus isolates, 87 (52.7%) were resistant to methicillin and 69 (41.8%) were resistant to fluoroquinolone. Fluoroquinolone-resistant S. aureus isolates had a mutation at codon 80 in the grlA gene and different mutational combinations in the gyrA gene. These mutational combinations included 45 isolates at codons 84 and 86, 23 isolates at codons 84, 86 and 106 and 1 isolate at codons 84, 86 and 90. Fluoroquinolone-resistant S. aureus isolates were clustered into 33 PFGE types. Conclusions The findings of this study show that the fluoroquinolone-resistant S. aureus strains isolated in the teaching hospitals in Tehran had multiple mutations in the QRDRs region of both grlA and gyrA genes. PMID:21873957

Phenotypic and genotypic evaluation of fluoroquinolone resistance in clinical isolates of Staphylococcus aureus in Tehran.

PubMed

Aligholi, Marzieh; Mirsalehian, Akbar; Halimi, Shahnaz; Imaneini, Hossein; Taherikalani, Morovat; Jabalameli, Fereshteh; Asadollahi, Parisa; Mohajer, Babak; Abdollahi, Alireza; Emaneini, Mohammad

2011-09-01

Fluoroquinolones are broad-spectrum antibiotics widely used in the treatment of bacterial infections such as Staphylococcus aureus isolates. Resistance to these antibiotics is increasing. The occurrence of mutations in the grlA and gyrA loci were evaluated in 69 fluoroquinolone-resistant S. aureus isolates from 2 teaching hospitals of Tehran University of Medical Sciences. Out of the 165 S. aureus isolates, 87 (52.7%) were resistant to methicillin and 69 (41.8%) were resistant to fluoroquinolone. Fluoroquinolone-resistant S. aureus isolates had a mutation at codon 80 in the grlA gene and different mutational combinations in the gyrA gene. These mutational combinations included 45 isolates at codons 84 and 86, 23 isolates at codons 84, 86 and 106 and 1 isolate at codons 84, 86 and 90. Fluoroquinolone-resistant S. aureus isolates were clustered into 33 PFGE types. The findings of this study show that the fluoroquinolone-resistant S. aureus strains isolated in the teaching hospitals in Tehran had multiple mutations in the QRDRs region of both grlA and gyrA genes.

Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations.

PubMed

Brammeld, Jonathan S; Petljak, Mia; Martincorena, Inigo; Williams, Steven P; Alonso, Luz Garcia; Dalmases, Alba; Bellosillo, Beatriz; Robles-Espinoza, Carla Daniela; Price, Stacey; Barthorpe, Syd; Tarpey, Patrick; Alifrangis, Constantine; Bignell, Graham; Vidal, Joana; Young, Jamie; Stebbings, Lucy; Beal, Kathryn; Stratton, Michael R; Saez-Rodriguez, Julio; Garnett, Mathew; Montagut, Clara; Iorio, Francesco; McDermott, Ultan

2017-04-01

Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients. © 2017 Brammeld et al.; Published by Cold Spring Harbor Laboratory Press.