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Sample records for chloroquine-susceptible pfcrt k76

  1. In vitro chloroquine susceptibility and PCR analysis of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from Senegal.

    PubMed

    Thomas, Susan M; Ndir, Omar; Dieng, Therese; Mboup, Souleymane; Wypij, David; Maguire, James H; Wirth, Dyann F

    2002-05-01

    Chloroquine resistance has been linked to mutations in the pfcrt and pfmdr1 genes of Plasmodium falciparum. To estimate the prevalence of the pfcrt K76T, pfmdr1 N86Y, and pfmdr1 D1246Y polymorphisms, isolates of P. falciparum from Senegal, West Africa, were analyzed, and the results were compared to in vitro chloroquine susceptibility. By the in vitro DELI test, 31% of these samples were resistant to chloroquine. Polymerase chain reaction-based assays and confirmatory sequencing demonstrated the pfcrt T76, pfmdr1 Y86, and pfmdr1 Y1246 alleles in 79%, 31%, and 2% of the isolates, respectively. All three mutant alleles were present in both in vitro susceptible and resistant isolates. On the basis of these findings, it appears that these molecular markers are not consistently predictive of in vitro chloroquine resistance in Senegal.

  2. Plasmodium falciparum K76T pfcrt Gene Mutations and Parasite Population Structure, Haiti, 2006-2009.

    PubMed

    Charles, Macarthur; Das, Sanchita; Daniels, Rachel; Kirkman, Laura; Delva, Glavdia G; Destine, Rodney; Escalante, Ananias; Villegas, Leopoldo; Daniels, Noah M; Shigyo, Kristi; Volkman, Sarah K; Pape, Jean W; Golightly, Linnie M

    2016-05-01

    Hispaniola is the only Caribbean island to which Plasmodium falciparum malaria remains endemic. Resistance to the antimalarial drug chloroquine has rarely been reported in Haiti, which is located on Hispaniola, but the K76T pfcrt (P. falciparum chloroquine resistance transporter) gene mutation that confers chloroquine resistance has been detected intermittently. We analyzed 901 patient samples collected during 2006-2009 and found 2 samples showed possible mixed parasite infections of genetically chloroquine-resistant and -sensitive parasites. Direct sequencing of the pfcrt resistance locus and single-nucleotide polymorphism barcoding did not definitively identify a resistant population, suggesting that sustained propagation of chloroquine-resistant parasites was not occurring in Haiti during the study period. Comparison of parasites from Haiti with those from Colombia, Panama, and Venezuela reveals a geographically distinct population with highly related parasites. Our findings indicate low genetic diversity in the parasite population and low levels of chloroquine resistance in Haiti, raising the possibility that reported cases may be of exogenous origin.

  3. Plasmodium falciparum K76T pfcrt Gene Mutations and Parasite Population Structure, Haiti, 2006–2009

    PubMed Central

    Charles, Macarthur; Das, Sanchita; Daniels, Rachel; Kirkman, Laura; Delva, Glavdia G.; Destine, Rodney; Escalante, Ananias; Villegas, Leopoldo; Daniels, Noah M.; Shigyo, Kristi; Volkman, Sarah K.; Pape, Jean W.

    2016-01-01

    Hispaniola is the only Caribbean island to which Plasmodium falciparum malaria remains endemic. Resistance to the antimalarial drug chloroquine has rarely been reported in Haiti, which is located on Hispaniola, but the K76T pfcrt (P. falciparum chloroquine resistance transporter) gene mutation that confers chloroquine resistance has been detected intermittently. We analyzed 901 patient samples collected during 2006–2009 and found 2 samples showed possible mixed parasite infections of genetically chloroquine-resistant and -sensitive parasites. Direct sequencing of the pfcrt resistance locus and single-nucleotide polymorphism barcoding did not definitively identify a resistant population, suggesting that sustained propagation of chloroquine-resistant parasites was not occurring in Haiti during the study period. Comparison of parasites from Haiti with those from Colombia, Panama, and Venezuela reveals a geographically distinct population with highly related parasites. Our findings indicate low genetic diversity in the parasite population and low levels of chloroquine resistance in Haiti, raising the possibility that reported cases may be of exogenous origin. PMID:27089479

  4. Prevalence of the K76T mutation in the pfcrt gene of Plasmodium falciparum among chloroquine responders in India.

    PubMed

    Vinayak, Sumiti; Biswas, Sukla; Dev, Vas; Kumar, Ashwani; Ansari, M A; Sharma, Y D

    2003-07-01

    Chloroquine-resistant Plasmodium falciparum needs to be monitored in the field for effective malaria control strategies. A point mutation K76T in the P. falciparum chloroquine resistance transporter (Pfcrt) protein has recently been proposed as a molecular marker for the faster detection of chloroquine-resistant falciparum malaria in field. We describe here the evaluation of this marker in Indian P. falciparum isolates. A total of 274 Indian P. falciparum isolates were analyzed for the K76T mutation. This mutation was detected in all the clinical isolates obtained from the in vivo chloroquine non-responders. But majority of the clinical isolates from chloroquine responders (71 of 74 patients, i.e. 96%) also harbored this mutation. The K76T mutation was indeed highly prevalent (91%) among 213 clinical isolates. There was a significant association between K76T mutation and the in vitro chloroquine response (P<0.05) but six isolates showed discordant results. In conclusion, the K76T mutation fails to differentiate majority of the chloroquine responders from that of the non-responders and thus will be of limited use in the field in India.

  5. Mutations in PFCRT K76T do not correlate with sulfadoxine-pyrimethamine-amodiaquine failure in Pikine, Senegal.

    PubMed

    Sarr, Ousmane; Ahouidi, Ambroise D; Ly, Omar; Daily, Johanna P; Ndiaye, Daouda; Ndir, Omar; Mboup, Souleymane; Wirth, Dyann F

    2008-09-01

    In 2003, the high level of chloroquine (CQ) treatment failure for uncomplicated Plasmodium falciparum malaria cases has led Senegal to adopt a new combination therapy with sulfadoxine-pyrimethamine and amodiaquine (SP-AQ). From September through November 2004, we used the 14-day World Health Organization follow-up protocol to assess the therapeutic response in patients with uncomplicated P. falciparum malaria in an area of high prevalence of pfcrt T76 mutant allele and SP resistance mutations. Of the 82 patients who were recruited, 68 (82.9%) completed follow-up. The response of the patients to treatment was adequate clinical response for 63 out of 68 patients (92.6%), while five (7.4%) clinical failures were recorded, four early treatment failures, and one late treatment failure. The prevalence of the pfcrt T76 allele at day 0 was 59.5%. The two-sided Fisher's exact test did not show an association between pfcrt T76 allele and treatment failure (p=0.167). The transitory treatment is effective and safe. However, the presence of high levels of mutant alleles points out the need to closely monitor the new therapeutic regimen.

  6. Combinatorial Genetic Modeling of pfcrt-Mediated Drug Resistance Evolution in Plasmodium falciparum.

    PubMed

    Gabryszewski, Stanislaw J; Modchang, Charin; Musset, Lise; Chookajorn, Thanat; Fidock, David A

    2016-06-01

    The emergence of drug resistance continuously threatens global control of infectious diseases, including malaria caused by the protozoan parasite Plasmodium falciparum A critical parasite determinant is the P. falciparum chloroquine resistance transporter (PfCRT), the primary mediator of chloroquine (CQ) resistance (CQR), and a pleiotropic modulator of susceptibility to several first-line artemisinin-based combination therapy partner drugs. Aside from the validated CQR molecular marker K76T, P. falciparum parasites have acquired at least three additional pfcrt mutations, whose contributions to resistance and fitness have been heretofore unclear. Focusing on the quadruple-mutant Ecuadorian PfCRT haplotype Ecu1110 (K76T/A220S/N326D/I356L), we genetically modified the pfcrt locus of isogenic, asexual blood stage P. falciparum parasites using zinc-finger nucleases, producing all possible combinations of intermediate pfcrt alleles. Our analysis included the related quintuple-mutant PfCRT haplotype 7G8 (Ecu1110 + C72S) that is widespread throughout South America and the Western Pacific. Drug susceptibilities and in vitro growth profiles of our combinatorial pfcrt-modified parasites were used to simulate the mutational trajectories accessible to parasites as they evolved CQR. Our results uncover unique contributions to parasite drug resistance and growth for mutations beyond K76T and predict critical roles for the CQ metabolite monodesethyl-CQ and the related quinoline-type drug amodiaquine in driving mutant pfcrt evolution. Modeling outputs further highlight the influence of parasite proliferation rates alongside gains in drug resistance in dictating successful trajectories. Our findings suggest that P. falciparum parasites have navigated constrained pfcrt adaptive landscapes by means of probabilistically rare mutational bursts that led to the infrequent emergence of pfcrt alleles in the field.

  7. Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India.

    PubMed

    Sutar, Sasmita Kumari Das; Gupta, Bhavna; Ranjit, Manoranjan; Kar, Shantanu Kumar; Das, Aparup

    2011-02-01

    The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

  8. PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

    PubMed Central

    Bray, Patrick G.; Mungthin, Mathirut; Hastings, Ian M.; Biagini, Giancarlo A.; Saidu, Dauda K.; Lakshmanan, Viswanathan; Johnson, David J.; Hughes, Ruth H.; Stocks, Paul A.; O'Neill, Paul M.; Fidock, David A.; Warhurst, David C.; Ward, Stephen A.

    2010-01-01

    Summary It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV. PMID:16956382

  9. Artesunate-Amodiaquine and Artemether-Lumefantrine Therapies and Selection of Pfcrt and Pfmdr1 Alleles in Nanoro, Burkina Faso

    PubMed Central

    Sondo, Paul; Derra, Karim; Diallo Nakanabo, Seydou; Tarnagda, Zekiba; Kazienga, Adama; Zampa, Odile; Valéa, Innocent; Sorgho, Hermann; Owusu-Dabo, Ellis; Ouédraogo, Jean-Bosco; Guiguemdé, Tinga Robert; Tinto, Halidou

    2016-01-01

    The adoption of Artemisinin based combination therapies (ACT) constitutes a basic strategy for malaria control in sub-Saharan Africa. Moreover, since cases of ACT resistance have been reported in South-East Asia, the need to understand P. falciparum resistance mechanism to ACT has become a global research goal. The selective pressure of ACT and the possibility that some specific Pfcrt and Pfmdr1 alleles are associated with treatment failures was assessed in a clinical trial comparing ASAQ to AL in Nanoro. Dried blood spots collected on Day 0 and on the day of recurrent parasitaemia during the 28-day follow-up were analyzed using the restriction fragments length polymorphism (PCR-RFLP) method to detect single nucleotide polymorphisms (SNPs) in Pfcrt (codon76) and Pfmdr1 (codons 86, 184, 1034, 1042, and 1246) genes. Multivariate analysis of the relationship between the presence of Pfcrt and Pfmdr1 alleles and treatment outcome was performed. AL and ASAQ exerted opposite trends in selecting Pfcrt K76T and Pfmdr1-N86Y alleles, raising the potential beneficial effect of using diverse ACT at the same time as first line treatments to reduce the selective pressure by each treatment regimen. No clear association between the presence of Pfcrt and Pfmdr1 alleles carried at baseline and treatment failure was observed. PMID:27031231

  10. Synergism between Pfcrt and Pfmdr1 genes could account for the slow recovery of chloroquine sensitive Plasmodium falciparum strains in Ghana after chloroquine withdrawal.

    PubMed

    Asare, Kwame K; Boampong, Johnson N; Duah, Nancy O; Afoakwah, Richmond; Sehgal, Rakesh; Quashie, Neils B

    Unlike other countries, the chloroquine resistant marker Pfcrt T76 mutant has remained fairly stable in Ghana several years after official disuse of chloroquine. Certain mutations in Pfmdr1 may potentiate Pfcrt T76, offering a possible explanation for this observation. To understand the phenomenon, the co-existence of mutations in Pfmdr1 with Pfcrt T76 in Ghanaian Plasmodium falciparum isolates was studied. The reported presence of parasites with reduced sensitivity to amodiaquine and quinine in the country was also studied. Blood samples collected from confirmed malaria patients presenting at health facilities in two distinct ecological zones were analyzed. The prevalence of Pfcrt K76T and the five point mutations in Pfmdr1 were determined using nested PCR followed by RFLP analysis. The association between genes was determined by chi square analysis, and synergism between the two genes was ascertained using the Jonckheere-Terptra (J-T) test followed by Monte Carlo simulation (MCS). Nearly fifty-four percent (53.7%) of the P. falciparum isolates examined had the Pfcrt T76 gene, out of which 18.3% had both K76 and T76 alleles. Mutations at codon 86, 184, 1034, 1042 and 1246 of the Pfmdr1 gene were detected in 36.0%, 87.9%, 71.0%, 91.6% and 8.4% of the isolates, respectively. The haplotypes of Pfmdr1 present were NFCDD (43.46%), YFCDD (27.57%), NFSDD (7.48%), NYSNY (5.14%) and YFSDD (4.67%). Pfcrt T76 was significantly associated with a double mutation at codon 86 and 184 of Pfmdr1 (YF; χ(2)=18.045, p=0.006). Associations were observed between Pfcrt K76T and Pfmdr1 triple mutation at codons 86, 184 and 1034 (NFC; χ(2)=13.770, p=0.032 and YFC; χ(2)=16.489, p=0.011). The J-T test showed significant synergism between Pfcrt 76 and Pfmdr1 polymorphisms (p<0.0001), which was confirmed by MCS at 99% CI. Synergism between Pfcrt and Pfmdr1 mutant genes could account for the slow recovery of chloroquine sensitive P. falciparum in Ghana. The same phenomenon could explain

  11. Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt.

    PubMed

    Pelleau, Stéphane; Moss, Eli L; Dhingra, Satish K; Volney, Béatrice; Casteras, Jessica; Gabryszewski, Stanislaw J; Volkman, Sarah K; Wirth, Dyann F; Legrand, Eric; Fidock, David A; Neafsey, Daniel E; Musset, Lise

    2015-09-15

    In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.

  12. PfCRT and its role in antimalarial drug resistance

    PubMed Central

    Ecker, Andrea; Lehane, Adele M.; Clain, Jérôme; Fidock, David A.

    2012-01-01

    Plasmodium falciparum resistance to chloroquine, the former gold standard antimalarial drug, is mediated primarily by mutant forms of the ‘Chloroquine Resistance Transporter’ (PfCRT). These mutations impart upon PfCRT the ability to efflux chloroquine from the intracellular digestive vacuole, the site of drug action. Recent studies reveal that PfCRT variants can also affect parasite fitness, protect immature gametocytes against chloroquine action, and alter P. falciparum susceptibility to current first-line therapies. These results highlight the need to be vigilant in screening for the appearance of novel pfcrt alleles that could contribute to new multi-drug resistance phenotypes. PMID:23020971

  13. Amplification of pfmdr1, pfcrt, pvmdr1, and K13 propeller polymorphisms associated with Plasmodium falciparum and Plasmodium vivax isolates from the China-Myanmar border.

    PubMed

    Feng, Jun; Zhou, Daili; Lin, Yingxue; Xiao, Huihui; Yan, He; Xia, Zhigui

    2015-05-01

    Malaria in the China-Myanmar border region is still severe; local transmission of both falciparum and vivax malaria persists, and there is a risk of geographically expanding antimalarial resistance. In this research, the pfmdr1, pfcrt, pvmdr1, and K13-propeller genotypes were determined in 26 Plasmodium falciparum and 64 Plasmodium vivax isolates from Yingjiang county of Yunnan province. The pfmdr1 (11.5%), pfcrt (34.6%), and pvmdr1 (3.1%) mutations were prevalent at the China-Myanmar border. The indigenous samples exhibited prevalences of 14.3%, 28.6%, and 14.3% for pfmdr1 N86Y, pfcrt K76T, and pfcrt M74I, respectively, whereas the samples from Myanmar showed prevalences of 10.5%, 21.1%, and 5.3%, respectively. The most prevalent genotypes of pfmdr1 and pfcrt were Y86Y184 and M74N75T76, respectively. No pvmdr1 mutation occurred in the indigenous samples but was observed in two cases coming from Myanmar. In addition, we are the first to report on 10 patients (38.5%) with five different K13 point mutations. The F446I allele is predominant (19.2%), and its prevalence was 28.6% in the indigenous samples of Yingjiang county and 15.8% in samples from Myanmar. The present data might be helpful for enrichment of the molecular surveillance of antimalarial resistance and useful for developing and updating guidance for the use of antimalarials in this region.

  14. Wide variation in microsatellite sequences within each Pfcrt mutant haplotype.

    PubMed

    Vinayak, Sumiti; Mittra, Pooja; Sharma, Yagya D

    2006-05-01

    Flanking microsatellites for each of the Pfcrt mutant haplotype of Plasmodium falciparum remain conserved among geographical isolates. We describe here heterogeneity in the intragenic microsatellites among each of the Pfcrt haplotype. There were fourteen different alleles of AT repeats of intron 2 and eight alleles of TA repeats of intron 4 of the pfcrt gene among Indian isolates. This resulted in 33 different two-locus (intron 2 plus intron 4) microsatellite genotypes among 224 isolates. There were 15 different two-locus microsatellite genotypes within the South American Pfcrt haplotype (S72V73M74N75T76S220) and 11 genotypes in the southeast Asian haplotype (C72V73I74E75T76S220) in these isolates. Indian isolates with Pfcrt haplotype C72V73I74E75T76S220 shared one of its two-locus microsatellite genotype with southeast Asian P. falciparum parasite lines from Thailand (K1) and Indochina (Dd2 and W2). Conversely, Indian isolates containing S72V73M74N75T76S220 Pfcrt haplotype did not share any of their two-locus microsatellite genotype with South American parasite line 7G8 from Brazil. Significantly, large number of newer two-locus microsatellite genotypes were detected in a 2-year time period (P<0.05). Microsatellite variation was more prominent in the areas of high malaria transmission. It is concluded that the genetic recombination in the intragenic microsatellites continues in the parasite population even after microsatellites flanking the pfcrt gene had already been fixed. Presence of various Pfcrt haplotypes and a variety of intragenic microsatellites indicates that there is a wide spectrum of chloroquine resistant parasite population in India. This information should be useful for malaria control programs of the country.

  15. Polymorphisms in K13, pfcrt, pfmdr1, pfdhfr, and pfdhps in parasites isolated from symptomatic malaria patients in Burkina Faso

    PubMed Central

    Somé, Anyirékun Fabrice; Sorgho, Hermann; Zongo, Issaka; Bazié, Thomas; Nikiéma, Frédéric; Sawadogo, Amadé; Zongo, Moussa; Compaoré, Yves-Daniel; Ouédraogo, Jean-Bosco

    2016-01-01

    Background: The emergence of resistance to artemisinin derivatives in western Cambodia is threatening to revert the recent advances made toward global malaria control and elimination. Known resistance-mediating polymorphisms in the K13, pfcrt, pfmdr1, pfdhfr, and pfdhps genes are of greatest importance for monitoring the spread of antimalarial drug resistance. Methods: Samples for the present study were collected from 244 patients with uncomplicated malaria in health centers of Bobo-Dioulasso, Burkina Faso. Blood sample was collected on filter paper before the subject received any treatment. The parasite DNA was then extracted and amplified by Polymerase Chain Reaction (PCR) to evaluate the prevalence of polymorphism of pfcrtK76T, pfmdr1 (N86Y, Y184F), and pfdhps (A437G, K540E). The K13 gene polymorphism was analyzed by nested PCR followed by sequencing. Results: The overall results showed 2.26% (5/221) of K13 synonymous mutant alleles (two C469C, one Y493Y, one G496G, and one V589V), 24.78%, 19.58%, 68.75%, 60.9%, 53.7%, 63.8%, and 64.28%, respectively, for mutant pfcrt 76T, pfmdr1-86Y, pfmdr1-184F, pfdhfr51I, pfdhfr59R, pfdhfr108N, and pfdhps 437G. We did not report any mutation at codon 540 of pfdhps. Conclusion: These results provide baseline prevalence of known drug resistance polymorphisms and suggest that artemisinin combination therapies may retain good efficacy in the treatment of uncomplicated malaria in Burkina Faso. PMID:28004634

  16. Rapid detection of Pfcrt and Pfmdr1 mutations in Plasmodium falciparum isolates by FRET and in vivo response to chloroquine among children from Osogbo, Nigeria

    PubMed Central

    Ojurongbe, Olusola; Ogungbamigbe, Titus O; Fagbenro-Beyioku, Adetayo F; Fendel, Rolf; Kremsner, Peter G; Kun, Jürgen FJ

    2007-01-01

    Background Chloroquine (CQ) has been in use in Africa for a long time. Because of misuse, this drug has now lost its efficacy due to the emergence of resistance strains in most parts of Africa. Recently, it was shown that after chloroquine has been withdrawn from the market, chloroquine-sensitive Plasmodium falciparum re-emerged and chloroquine could again be used successfully as an antimalarial. Surveillance of parasite populations is, therefore, important to decide whether chloroquine could be re-introduced. Methods To estimate the prevalence of the most pivotal polymorphisms, including Pfcrt K76T, Pfmdr1 N86Y and Pfmdr1 Y184F mutations, and their contributions to the outcome of CQ treatment, isolates from Osogbo Western Nigeria were tested using the Fluorescence Resonance Energy Transfer (FRET) method on a real-time PCR instrument. Results 116 children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of CQ and followed-up for 28 days. Blood samples were collected on filter paper at enrollment and during follow-up for identification of parasite carrying the chloroquine resistant transporter (pfcrt) and P. falciparum-multi drug resistance (pfmdr1) gene mutations. Parasitological assessment of response to treatment showed that 62% of the patients were cured and 38% failed the CQ treatment. The presence of single mutant pfcrt (T76) alleles (P = 0.003) and in combination with mutant pfmdr1 Y86 (P = 0.028) was significantly associated with in vivo CQR. No other mutation on its own or in combinations was significantly associated with treatment outcome. Mutant pfcrt was more prevalent in both pre- and post-treatment isolates. No association was observed between age or initial level of parasitaemia and chloroquine treatment outcome. Conclusion The result established the usefulness and accuracy of real time PCR in pfcrt and pfmdr1 mutation detection and also give further evidence to the reliability of the pfcrt T76 point

  17. Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum

    PubMed Central

    Chahar, Madhvi; Mishra, Neelima; Anvikar, Anup; Dixit, Rajnikant; Valecha, Neena

    2017-01-01

    Chloroquine (CQ) resistance in Plasmodium falciparum is determined by the mutations in the chloroquine resistance transporter (Pfcrt) gene. The point mutation at codon 76 (K76T), which has been observed in more than 91% of P. falciparum isolates in India, is the major determinant of CQ resistance. To overcome the limitations and challenges of traditional methods, in this investigation we developed an easy to use loop mediated isothermal amplification (LAMP) protocol for rapid detection of the K76T mutation associated with CQ resistance in P. falciparum with naked eye visualization. In- house designed primers were synthesized and optimized to specifically distinguish the CQ resistant mutants of P. falciparum. The LAMP reaction was optimal at 61 °C for 60 min and calcein dye was added prior to amplification to enable visual detection. We demonstrate the detection limit of <2 ng/μl respectively, supporting the high sensitivity of this calcein based LAMP method. To the best of our knowledge this is the first report on the establishment of an easy, reliable and cost effective LAMP assay for rapid and specific detection of highly CQ resistance in P. falciparum malaria. PMID:28134241

  18. Molecular epidemiology of malaria in Cameroon. XIII. Analysis of pfcrt mutations and in vitro chloroquine resistance.

    PubMed

    Basco, Leonardo K

    2002-10-01

    The key Lys76Thr amino-acid substitution in Plasmodium falciparum chloroquine-resistance transporter (PfCRT) has been shown to be a reliable marker associated with chloroquine-resistant phenotype in reference clones, but few discordant results have been observed in field isolates. To further examine the relationship between in vitro chloroquine response and pfcrt alleles, the entire exon 2 of the pfcrt gene of 157 Cameroonian isolates was sequenced. All isolates were characterized as having either Cys-72, Met-74, Asn-75, and Lys-76 (wild-type alleles), Cys-72, Ile-74, Glu-75, and Thr-76 (mutant alleles), or mixed alleles. The hypothetical threshold 50% inhibitory concentration (IC50) set at 100 nM distinguished between isolates carrying the wild-type alleles and those with mutant alleles in a large majority of cases (135 of 139 isolates with unmixed pfcrt alleles). Isolates presenting discordant results generally had IC50s within an intermediate range. In vitro chloroquine response of isolates with mixed pfcrt alleles was highly variable. Although discordant results between chloroquine-resistant phenotype and pfcrt alleles were not explained by the immediate adjacent codons, the key Lys76Thr codon may prove to be a highly reliable genetic marker for the epidemiologic monitoring of chloroquine resistance by means of molecular techniques.

  19. Molecular surveillance of Plasmodium falciparum drug resistance markers reveals partial recovery of chloroquine susceptibility but sustained sulfadoxine-pyrimethamine resistance at two sites of different malaria transmission intensities in Rwanda.

    PubMed

    Kateera, Fredrick; Nsobya, Sam L; Tukwasibwe, Steven; Hakizimana, Emmanuel; Mutesa, Leon; Mens, Petra F; Grobusch, Martin P; van Vugt, Michèle; Kumar, Nirbhay

    2016-12-01

    Faced with intense levels of chloroquine (CQ) resistance in Plasmodium falciparum malaria, Rwanda replaced CQ with amodiaquine (AQ)+sulfadoxine-pyrimethamine (SP) in 2001, and subsequently with artemether-lumefantrine (AL) in 2006, as first-line treatments for uncomplicated malaria. Following years of discontinuation of CQ use, re-emergence of CQ-susceptible parasites has been reported in countries including Malawi, Kenya and Tanzania. In contrast, high levels of SP resistant mutant parasites continue to be reported even in countries of presumed reduced SP drug selection pressure. The prevalence and distributions of genetic polymorphisms linked with CQ and SP resistance at two sites of different malaria transmission intensities are described here to better understand drug-related genomic adaptations over time and exposure to varying drug pressures in Rwanda. Using filter paper blood isolates collected from P. falciparum infected patients, DNA was extracted and a nested PCR performed to identify resistance-mediating polymorphisms in the pfcrt, pfmdr1, pfdhps and pfdhfr genes. Amplicons from a total of 399 genotyped samples were analysed by ligase detection reaction fluorescent microsphere assay. CQ susceptible pfcrt 76K and pfmdr1 86N wild-type parasites were found in about 50% and 81% of isolates, respectively. Concurrently, SP susceptible pfdhps double (437G-540E), pfdhfr triple (108N-51I-59R), quintuple pfdhps 437G-540E/pfdhfr 51I-59R-108N and sextuple haplotypes were found in about 84%, 85%, 74% and 18% of isolates, respectively. High-level SP resistance associated pfdhfr 164L and pfdhps 581G mutant prevalences were noted to decline. Mutations pfcrt 76T, pfdhfr 59R and pfdhfr 164L were found differentially distributed between the two study sites with the pfdhfr 164L mutants found only at Ruhuha site, eastern Rwanda. Overall, sustained intense levels of SP resistance mutations and a recovery of CQ susceptible parasites were found in this study following 7 years

  20. Molecular epidemiology of malaria in Cameroon. XIV. Plasmodium falciparum chloroquine resistance transporter (PFCRT) gene sequences of isolates before and after chloroquine treatment.

    PubMed

    Basco, Leonardo K; Ndounga, Mathieu; Ngane, Vincent Foumane; Soula, Georges

    2002-10-01

    Laboratory studies have strongly suggested that the gene coding for Plasmodium falciparum chloroquine resistance transporter (PFCRT) may play a determinant role in chloroquine resistance. A clinical study in Mali also found evidence for selection of the key PFCRT amino acid substitution, Lys76Thr, in patients who fail to respond to chloroquine treatment. To test the hypothesis that in vivo selection of mutant PFCRT alleles occurs after chloroquine treatment, PFCRT and merozoite surface antigen 2 (msa-2) polymorphisms were compared between 61 pretreatment and posttreatment paired samples from children with either clinical or parasitologic failure. There were six wild-type PFCRT alleles, 44 mutant alleles, and 11 mixed alleles among pretreatment isolates. All posttreatment parasites had mutant PFCRT alleles. Recrudescence accounted for 42 of 61 posttreatment infections, while 19 posttreatment infections were due to new infection (including all isolates with Lys-76 before treatment and Thr-76 after treatment). Seven pretreatment isolates with mixed PFCRT alleles had only Thr-76 on recrudescence, providing a direct evidence for in vivo selection for mutant PFCRT. Although the presence of mutant PFCRT alleles in pretreatment isolates is not predictive of chloroquine treatment failure, our data support the hypothesis that in vivo selection for recrudescent parasites carrying mutant PFCRT alleles occurs. These results may have important implications for the future surveillance of chloroquine resistance by the use of molecular markers.

  1. Polymorphisms of the Pfatpase 6 and Pfcrt gene and their relationship with the in vitro susceptibility to dihydroartemisinin and chloroquine of Plasmodium falciparum isolates from Abobo, Côte d'Ivoire.

    PubMed

    Bla, Brice K; Yavo, William; Trébissou, Jonhson; Kipré, Rolland G; Yapi, Félix H; N'guessan, Jean D; Djaman, Joseph A

    2014-01-01

    As a result of widespread resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP), artemisinin-based combination therapy (ACT) has been recommended as a first-line anti-malarial regimen in Côte d'Ivoire since 2005. A thorough understanding of the molecular bases of P. falciparum resistance to existing drugs is therefore needed. The aims of this study were to analyze the in vitro sensitivity of P. falciparum field isolates from Abobo to CQ, pyronaridine (PYR) and dihydroartemisinine (DHA), and to investigate the polymorphisms associated with drug resistance. The standard in vitro drug sensitivity microtechnique recommended by the WHO was used to assess the sensitivity of Plasmodium falciparum isolates collected in December 2006. The Pfcrt haplotype 76 was analysed by PCR-RFLP while Pfatpase 6 amplification products were sequenced. Associations between drug sensitivity and parasite gene polymorphisms were evaluated with Cohen's kappa test. The correlation between the IC50 values for different drugs was assessed by the coefficient of determination (r²). Significance was assumed at p<0.05. Of 128 in vitro tests performed, 112 (87.5%) were successful. Of the isolates, 56.2% were resistant for CQ and 48% for PYR. One isolate (3.6%) demonstrated reduced DHA sensitivity (IC50 higher than 10 nM). The mutant K76T pfcrt codon, present in 90% of DNA fragments analyzed, was associated with CQ-R (ĸ=0.76). The N669Y (16.1%), D734Y (28.6%) and D734H (1.8%) isolates were found to have mutant Pfatpase6, however, these mutations were not associated with diminished DHA sensitivity (k=0.01). These high levels of antimalarial drug resistance in Abobo (Côte d'Ivoire) demand further studies of drug efficacy across the whole country.

  2. Evidence for a Central Role for PfCRT in Conferring Plasmodium falciparum Resistance to Diverse Antimalarial Agents

    PubMed Central

    Johnson, David J.; Fidock, David A.; Mungthin, Mathirut; Lakshmanan, Viswanathan; Sidhu, Amar Bir Singh; Bray, Patrick G.; Ward, Stephen A.

    2010-01-01

    Summary Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of cross-resistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials. PMID:15383277

  3. International comparison CCQM-K76: Sulfur dioxide in nitrogen

    NASA Astrophysics Data System (ADS)

    Guenther, Franklin R.; Kelley, Michael E.; Mitchell, Gerald D.; de Jesús Avila Salas, Manuel; Koelliker Delgado, Jorge; Rangel Murillo, Francisco; Serrano Caballero, Victor M.; Pérez Castorena, Alejandro; Shinji, Uehara; Ciecior, Dariusz; Smarçaro da Cunha, Valnei; Rodrigues Augusto, Cristiane; Cipriano Ribeiro, Claudia; de Lima Fioravante, Andreia; Dias, Florbela; Sang-Hyub, Oh; Macé, Tatiana; Sutour, Christophe; Büki, Tamás; Qiao, Han; Botha, Angelique; Mogale, David M.; Tshilongo, James; Ntsasa, Napo; Mphamo, Tshepiso; Uprichard, Ian; Milton, Martin; Vargha, Gergely; Brookes, Chris; Johri, Prabha; Valkova, Miroslava; Konopelko, Leonid; Kustikov, Yury; Pankratov, V. V.; Rumyantsev, D. V.; Pavlov, M. V.; Gromova, E. V.; van der Veen, Adriaan; van Otterloo, Peter; Wessel, Rob M.

    2011-01-01

    The key comparison CCQM-K76 was designed to test the capabilities of the participants to measure and certify sulfur dioxide in nitrogen, and to provide supporting evidence for the CMCs of these institutes for sulfur dioxide. Also, as sulfur dioxide is designated a core compound, and the 100 µmol/mol concentration is within the designated core compound concentration range, this comparison was also designed to demonstrate core capabilities of institutes which qualify under the rules of the Gas Analysis Working Group. The results of all 16 participants in this key comparison, except for three, are consistent with their key comparisons reference values. The three participants which are outside the KCRV interval are NIM, SMU and NPLI. This comparison may be used to demonstrate core analytical capabilities in accordance with the rules and procedures of the CCQM Gas Analysis Working group. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  4. Different Patterns of pfcrt and pfmdr1 Polymorphisms in P. falciparum Isolates from Nigeria and Brazil: The Potential Role of Antimalarial Drug Selection Pressure

    PubMed Central

    Gbotosho, Grace O.; Folarin, Onikepe A.; Bustamante, Carolina; Pereira da Silva, Luis Hildebrando; Mesquita, Elieth; Sowunmi, Akintunde; Zalis, Mariano G.; Oduola, Ayoade M. J.; Happi, Christian T.

    2012-01-01

    The effect of antimalarial drug selection on pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum isolates from two distinct geographical locations was determined in 70 and 18 P. falciparum isolates from Nigeria and Brazil, respectively, using nested polymerase chain reaction and direct DNA sequencing approaches. All isolates from Brazil and 72% from Nigeria harbored the mutant SVMNT and CVIET pfcrt haplotype, respectively. The pfcrt CVMNT haplotype was also observed in (7%) of the Nigerian samples. One hundred percent (100%) and 54% of the parasites from Brazil and Nigeria, respectively, harbored wild-type pfmdr1Asn86. We provide first evidence of emergence of the CVMNT haplotype in West Africa. The high prevalence of pfcrt CVIET and SVMNT haplotypes in Nigeria and Brazil, respectively, is indicative of different selective pressure by chloroquine and amodiaquine. Continuous monitoring of pfcrt SVMNT haplotype is required in endemic areas of Africa, where artesunate-amodiaquine combination is used for treatment of acute uncomplicated malaria. PMID:22302850

  5. Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

    PubMed Central

    Gabryszewski, Stanislaw J.; Dhingra, Satish K.; Lewis, Ian A.; Callaghan, Paul S.; Hassett, Matthew R.; Siriwardana, Amila; Henrich, Philipp P.; Lee, Andrew H.; Gnädig, Nina F.; Musset, Lise; Llinás, Manuel; Egan, Timothy J.; Roepe, Paul D.

    2016-01-01

    Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize

  6. Survey of chloroquine-resistant mutations in the Plasmodium falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen.

    PubMed

    Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

    2015-09-01

    Malaria is still a major public health problem in Yemen. More than 95% of the malaria cases are due to Plasmodium ‎falciparum‎. Recently in Yemen, the antimalarial treatment policy was changed from chloroquine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed in the Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simultaneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detect chloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falciparum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring and assessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individuals from different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specific nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) methods were used to investigate the mutations in the pfmdr1(codons 86 and 1246) and pfcrt (codons 76, 271, 326, 356 and 371) genes. The overall prevalence of pfcrt mutations at codons 76, 271, 326 and 371 were 50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority of pfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association between pfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons 76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance even after 4 years of shifting to ACTs. These findings warrant complete withdrawal of CQ use from the Yemeni market for P. falciparum and careful usage of CQ for treating Plasmodium vivax.

  7. Low Prevalence of Pfcrt Resistance Alleles among Patients with Uncomplicated Falciparum Malaria in Niger Six Years after Chloroquine Withdrawal

    PubMed Central

    Salissou, Adamou; Zamanka, Halima; Biyghe Binze, Brigitte; Rivière, Taiana; Tichit, Magalie; Ibrahim, Maman Laminou; Fandeur, Thierry

    2014-01-01

    Chloroquine (CQ) resistance is widespread in Africa, but few data are available for Niger. Pfcrt haplotypes (aa 56–118) and ex vivo responses to CQ and amodiaquine were characterized for 26 isolates collected in South Niger from children under 15 years of age suffering from uncomplicated falciparum malaria, six years after the introduction of artemisinin-based combinations and the withdrawal of CQ. The wild-type Pfcrt haplotype CVMNK was found in 22 of the 26 isolates, with CVIET sequences observed in only three of the samples. We also describe for the first time a new CVINT haplotype. The ex vivo responses were better for CVMNK than for CVIET parasites. Pfcrt sequence data were compared with those obtained for 26 additional parasitized blood samples collected in Gabon, from an area of CQ resistance used as a control. Our findings suggest that there has been a significant decline in CQ-resistant genotypes since the previous estimates for Niger were obtained. No such decline in molecular resistance to CQ was observed in the subset of samples collected in similar conditions from Gabon. These results have important implications for public health and support the policy implemented in Niger since 2005, which aims to increase the efficacy and availability of antimalarial drugs whilst controlling the spread of resistance. PMID:25506465

  8. Chloroquine Clinical Failures in P. falciparum Malaria Are Associated with Mutant Pfmdr-1, Not Pfcrt in Madagascar

    PubMed Central

    Andriantsoanirina, Valérie; Ratsimbasoa, Arsène; Bouchier, Christiane; Tichit, Magali; Jahevitra, Martial; Rabearimanana, Stéphane; Raherinjafy, Rogelin; Mercereau-Puijalon, Odile; Durand, Rémy; Ménard, Didier

    2010-01-01

    Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important

  9. Various pfcrt and pfmdr1 Genotypes of Plasmodium falciparum Cocirculate with P. malariae, P. ovale spp., and P. vivax in Northern Angola

    PubMed Central

    Fançony, Cláudia; Gamboa, Dina; Sebastião, Yuri; Hallett, Rachel; Sutherland, Colin; Sousa-Figueiredo, José Carlos

    2012-01-01

    Artemisinin-based combination therapy for malaria has become widely available across Africa. Populations of Plasmodium falciparum that were previously dominated by chloroquine (CQ)-resistant genotypes are now under different drug selection pressures. P. malariae, P. ovale curtisi, and P. ovale wallikeri are sympatric with P. falciparum across the continent and are frequently present as coinfections. The prevalence of human Plasmodium species was determined by PCR using DNA from blood spots collected during a cross-sectional survey in northern Angola. P. falciparum was genotyped at resistance-associated loci in pfcrt and pfmdr1 by real-time PCR or by direct sequencing of amplicons. Of the 3,316 samples collected, 541 (16.3%) contained Plasmodium species infections; 477 (88.2%) of these were P. falciparum alone, 6.5% were P. falciparum and P. malariae together, and 1.1% were P. vivax alone. The majority of the remainder (3.7%) harbored P. ovale curtisi or P. ovale wallikeri alone or in combination with other species. Of 430 P. falciparum isolates genotyped for pfcrt, 61.6% carried the wild-type allele CVMNK at codons 72 to 76, either alone or in combination with the resistant allele CVIET. No other pfcrt allele was found. Wild-type alleles dominated at codons 86, 184, 1034, 1042, and 1246 of the pfmdr1 locus among the sequenced isolates. In contrast to previous studies, P. falciparum in the study area comprises an approximately equal mix of genotypes associated with CQ sensitivity and with CQ resistance, suggesting either lower drug pressure due to poor access to treatment in rural areas or a rapid impact of the policy change away from the use of standard monotherapies. PMID:22850519

  10. Final report on COOMET.QM-K76 (COOMET project no 484/RU/09): Key comparison of primary standard gas mixtures: SO2 in nitrogen (100 µmol/mol)

    NASA Astrophysics Data System (ADS)

    Konopelko, L. A.; Kustikov, Y. A.; Kolobova, A. V.; Shor, N. B.; Efremova, O. V.; Rozhnov, M. S.; Melnyk, D. M.; Kozia, V. G.; Shpilnyi, S. A.; Petryshyn, P. V.; Iakubov, S. E.; Kluchits, A. S.; Ananyin, V. N.; Mironchik, A. M.; Mokhnach, M. V.; Valkova, M.; Stovcik, V.

    2014-01-01

    Sulfur dioxide is one of the main contaminants present in the atmosphere due to burning of coal, oil and natural gas, smelting of base metals and production of sulfuric acid. Sulfur dioxide has been the subject of three previous CCQM key comparisons: CCQM-K1.d in 1997, CCQM-K26.b in 2005 and CCQM-K76 in 2010. VNIIM proposed a new COOMET project (No 484/RU/09) in this field, which was registered in the KCDB as key comparison COOMET.QM-K76. It was found that all results were consistent with the reference (gravimetric) values, with observed differences not exceeding ±0.60% and not exceeding either the appropriate assigned expanded uncertainties. VNIIM is the linking laboratory to CCQM-K76. SMU has improved the results obtained in CCQM-K76. The mixtures prepared for this exercise were found to be stable during about one year within the uncertainty of the measurements. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

  11. Chloroquine-resistant isoforms of the Plasmodium falciparum chloroquine resistance transporter acidify lysosomal pH in HEK293 cells more than chloroquine-sensitive isoforms.

    PubMed

    Reeves, David C; Liebelt, David A; Lakshmanan, Viswanathan; Roepe, Paul D; Fidock, David A; Akabas, Myles H

    2006-12-01

    The emergence of chloroquine-resistant Plasmodium falciparum malaria imperils the lives of millions of people in Africa, Southeast Asia and South America. Chloroquine resistance is associated with mutations in the P. falciparum chloroquine resistance transporter (PfCRT). We expressed chloroquine-sensitive (HB3) and resistant (Dd2) pfcrt alleles in HEK293 human embryonic kidney cells. PfCRT localized to the lysosomal limiting membrane and was not detected in the plasma membrane. We observed significant acidification of lysosomes containing PfCRT HB3 and Dd2, with Dd2 acidifying significantly more than HB3. A mutant HB3 allele expressing the K76T mutation (earlier found to be key for chloroquine resistance) acidified to the same extent as Dd2, whereas the acidification by a Dd2 allele expressing the T76K "back mutation" was significantly less than Dd2. Thus, the amino acid at position 76 is both an important determinant of chloroquine resistance in parasites and of lysosomal acidification following heterologous expression. PfCRT may be capable of modulating the pH of the parasite digestive vacuole, and thus chloroquine availability. Chloroquine accumulation and glycyl-phenylalanine-2-naphthylamide-induced release of lysosomal Ca(2+) stores were unaffected by PfCRT expression. Cytoplasmic domain mutations did not alter PfCRT sorting to the lysosomal membrane. This heterologous expression system will be useful to characterize PfCRT protein structure and function, and elucidate its molecular role in chloroquine resistance.

  12. Genetics of chloroquine-resistant malaria: a haplotypic view

    PubMed Central

    Awasthi, Gauri; Das, Aparup

    2013-01-01

    The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria. PMID:24402147

  13. Short report: polymorphisms in the chloroquine resistance transporter gene in Plasmodium falciparum isolates from Lombok, Indonesia.

    PubMed

    Huaman, Maria Cecilia; Yoshinaga, Kazumi; Suryanatha, Aan; Suarsana, Nyoman; Kanbara, Hiroji

    2004-07-01

    The polymorphisms in the Plasmodium falciparum multidrug resistance 1 (pfmdr1) and P. falciparum chloroquine resistance transporter (pfcrt) genes, which are associated with chloroquine resistance, were examined in 48 P. falciparum isolates from uncomplicated malaria patients from the West Lombok District in Indonesia. The point mutation N86Y in pfmdr1 was present in 35.4% of the isolates and mutation K76T in pfcrt was found in all but one of the samples studied. Identified pfcrt haplotypes were mainly identical to the Papua New Guinea type S(agt)VMNT (42 of 48, 87.5%), and a few isolates had the Southeast Asia type CVIET (5 of 48, 10.4%). Moreover, one P. falciparum isolate harbored the K76N mutation, giving rise to the haplotype CVMNN, which was not previously reported in field isolates. Our findings suggest that chloroquine resistance in this area might have the same origin as in Papua New Guinea.

  14. Temporal and seasonal changes of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine, and quinine in Guinea-Bissau between 2003 and 2012.

    PubMed

    Jovel, Irina Tatiana; Kofoed, Poul-Erik; Rombo, Lars; Rodrigues, Amabelia; Ursing, Johan

    2015-02-01

    In 2008, artemether-lumefantrine was introduced in Guinea-Bissau, West Africa, but quinine has also been commonly prescribed for the treatment of uncomplicated Plasmodium falciparum malaria. An efficacious high-dose chloroquine treatment regimen was used previously. Temporal and seasonal changes of genetic polymorphisms associated with altered drug susceptibility to chloroquine, lumefantrine, and quinine have been described. P. falciparum chloroquine resistance transporter (pfcrt) K76T, pfmdr1 gene copy numbers, pfmdr1 polymorphisms N86Y and Y184F, and pfmdr1 sequences 1034 to 1246 were determined using PCR-based methods. Blood samples came from virtually all (n=1,806) children<15 years of age who had uncomplicated P. falciparum monoinfection and presented at a health center in suburban Bissau (from 2003 to 2012). The pfcrt K76T and pfmdr1 N86Y frequencies were stable, and seasonal changes were not seen from 2003 to 2007. Since 2007, the mean annual frequencies increased (P<0.001) for pfcrt 76T (24% to 57%), pfmdr1 N86 (72% to 83%), and pfcrt 76+pfmdr1 86 TN (10% to 27%), and pfcrt 76T accumulated during the high transmission season (P=0.001). The pfmdr1 86+184 NF frequency increased from 39% to 66% (from 2003 to 2011; P=0.004). One sample had two pfmdr1 gene copies. pfcrt 76T was associated with a lower parasite density (P<0.001). Following the discontinuation of an effective chloroquine regimen, probably highly artemether-lumefantrine-susceptible P. falciparum (with pfcrt 76T) accumulated, possibly due to suboptimal use of quinine and despite a fitness cost linked to pfcrt 76T. (The studies reported here were registered at ClinicalTrials.gov under registration no. NCT00137514 [PSB-2001-chl-amo], NCT00137566 [PSB-2004-paracetamol], NCT00426439 [PSB-2006-coartem], NCT01157689 [AL-eff 2010], and NCT01704508 [Eurartesim 2012].).

  15. Polymorphism in Plasmodium falciparum Drug Transporter Proteins and Reversal of In Vitro Chloroquine Resistance by a 9,10-Dihydroethanoanthracene Derivative

    PubMed Central

    Millet, Julie; Alibert, Sandrine; Torrentino-Madamet, Marylin; Rogier, Christophe; Santelli-Rouvier, Christiane; Bigot, Patricia; Mosnier, Joel; Baret, Eric; Barbe, Jacques; Parzy, Daniel; Pradines, Bruno

    2004-01-01

    BG958 reverses resistance in chloroquine-resistant isolates from different countries. Five mutations in the Plasmodium falciparum crt (pfcrt) gene resulting in the amino acid changes K76T, M74I, N75E, A220S, and R371I are systematically identified in resistance-reversed Asian, African, and Brazilian parasites which possess the pfcrt (CIET) haplotype. In combination with BG958, the activity of chloroquine is increased in parasites with the N86Y mutation in pfmdr1. PMID:15561869

  16. Polymorphism of the Plasmodium falciparum multidrug resistance and chloroquine resistance transporter genes and in vitro susceptibility to aminoquinolines in isolates from the Peruvian Amazon.

    PubMed

    Huaman, Maria Cecilia; Roncal, Norma; Nakazawa, Shusuke; Long, Ton That Ai; Gerena, Lucia; Garcia, Coralith; Solari, Lely; Magill, Alan J; Kanbara, Hiroji

    2004-05-01

    In vitro drug sensitivity to chloroquine (CQ), mefloquine (MQ) and quinine was investigated in 60 culture-adapted Plasmodium falciparum isolates from malaria patients in Padrecocha, a village in the Amazonian Department of Loreto, Peru. All isolates showed resistance to CQ, decreased susceptibility to quinine, and sensitivity to MQ. These isolates were examined for mutations in the P. falciparum multidrug resistance 1 (pfmdr1) and chloroquine resistance transporter (pfcrt) genes previously linked to CQ resistance. The mutations N86Y and D1246Y, two of the five mutations commonly observed in the pfmdr1 gene of CQ-resistant clones, were not found. The pfcrt mutation K76T, associated with CQ resistance, was identified in all the isolates tested. Sequence analysis of codons 72-76 in the pfcrt gene showed the haplotypes SVMNT and CVMNT.

  17. [Mutant alleles associated to chloroquine and sulfadoxine-pyrimethanime resistance in Plasmodium falciparum of the Ecuador-Peru and Ecuador-Colombia borders].

    PubMed

    Arróspide, Nancy; Hijar-Guerra, Gisely; de Mora, Doménica; Diaz-Cortéz, César Eduardo; Veloz-Perez, Raúl; Gutierrez, Sonia; Cabezas-Sánchez, César

    2014-04-01

    The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.

  18. Molecular diagnosis of resistance to antimalarial drugs during epidemics and in war zones.

    PubMed

    Djimdé, Abdoulaye A; Dolo, Amagana; Ouattara, Amed; Diakité, Sira; Plowe, Christopher V; Doumbo, Ogobara K

    2004-08-15

    Plasmodium falciparum mutations pfcrt K76T and the dhfr/dhps "quintuple mutant" are molecular markers of resistance to chloroquine and sulfadoxine-pyrimethamine, respectively. During an epidemic of P. falciparum malaria in an area of political unrest in northern Mali, where standard efficacy studies have been impossible, we measured the prevalence of these markers in a cross-sectional survey. In 80% of cases of infection, pfcrt K76T was detected, but none of the cases carried the dhfr/dhps quintuple mutant. On the basis of these results, chloroquine was replaced by sulfadoxine-pyrimethamine in control efforts. This example illustrates how molecular markers for drug resistance can provide timely data that inform malaria-control policy during epidemics and other emergency situations.

  19. Intermittent Preventive Treatment with Dihydroartemisinin-Piperaquine in Ugandan Schoolchildren Selects for Plasmodium falciparum Transporter Polymorphisms That Modify Drug Sensitivity

    PubMed Central

    Conrad, Melissa D.; Legac, Jennifer; Tukwasibwe, Stephen; Tumwebaze, Patrick; Wandera, Bonnie; Brooker, Simon J.; Staedke, Sarah G.; Kamya, Moses R.; Nsobya, Sam L.; Dorsey, Grant; Rosenthal, Philip J.

    2016-01-01

    Dihydroartemisinin-piperaquine (DP) offers prolonged protection against malaria, but its impact on Plasmodium falciparum drug sensitivity is uncertain. In a trial of intermittent preventive treatment in schoolchildren in Tororo, Uganda, in 2011 to 2012, monthly DP for 1 year decreased the incidence of malaria by 96% compared to placebo; DP once per school term offered protection primarily during the first month after therapy. To assess the impact of DP on selection of drug resistance, we compared the prevalence of key polymorphisms in isolates that emerged at different intervals after treatment with DP. Blood obtained monthly and at each episode of fever was assessed for P. falciparum parasitemia by microscopy. Samples from 160 symptomatic and 650 asymptomatic episodes of parasitemia were assessed at 4 loci (N86Y, Y184F, and D1246Y in pfmdr1 and K76T in pfcrt) that modulate sensitivity to aminoquinoline antimalarials, utilizing a ligase detection reaction-fluorescent microsphere assay. For pfmdr1 N86Y and pfcrt K76T, but not the other studied polymorphisms, the prevalences of mutant genotypes were significantly greater in children who had received DP within the past 30 days than in those not treated within 60 days (86Y, 18.0% versus 8.3% [P = 0.03]; 76T, 96.0% versus 86.1% [P = 0.05]), suggesting selective pressure of DP. Full sequencing of pfcrt in a subset of samples did not identify additional polymorphisms selected by DP. In summary, parasites that emerged soon after treatment with DP were more likely than parasites not under drug pressure to harbor pfmdr1 and pfcrt polymorphisms associated with decreased sensitivity to aminoquinoline antimalarials. (This study has been registered at ClinicalTrials.gov under no. NCT01231880.) PMID:27401569

  20. Plasmodium falciparum Drug Resistance in Madagascar: Facing the Spread of Unusual pfdhfr and pfmdr-1 Haplotypes and the Decrease of Dihydroartemisinin Susceptibility▿

    PubMed Central

    Andriantsoanirina, Valérie; Ratsimbasoa, Arsène; Bouchier, Christiane; Jahevitra, Martial; Rabearimanana, Stéphane; Radrianjafy, Rogelin; Andrianaranjaka, Voahangy; Randriantsoa, Tantely; Rason, Marie Ange; Tichit, Magali; Rabarijaona, Léon Paul; Mercereau-Puijalon, Odile; Durand, Rémy; Ménard, Didier

    2009-01-01

    The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required. PMID:19704124

  1. Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

    PubMed Central

    Juliano, Jonathan J; Randrianarivelojosia, Milijaona; Ramarosandratana, Benjamin; Ariey, Frédéric; Mwapasa, Victor; Meshnick, Steven R

    2009-01-01

    Background Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings. Methods This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples. Results Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates. Conclusion These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the

  2. Prevalence of Plasmodium falciparum Molecular Markers of Antimalarial Drug Resistance in a Residual Malaria Focus Area in Sabah, Malaysia

    PubMed Central

    Mohd Abd Razak, Mohd Ridzuan; Abdullah, Noor Rain; Sastu, Umi Rubiah; Imwong, Mallika; Muniandy, Prem Kumar; Saat, Muhammad Nor Farhan; Muhammad, Amirrudin; Jelip, Jenarun; Tikuson, Moizin; Yusof, Norsalleh; Rundi, Christina; Mudin, Rose Nani; Syed Mohamed, Ami Fazlin

    2016-01-01

    Chloroquine (CQ) and fansidar (sulphadoxine-pyrimethamine, SP) were widely used for treatment of Plasmodium falciparum for several decades in Malaysia prior to the introduction of Artemisinin-based Combination Therapy (ACT) in 2008. Our previous study in Kalabakan, located in south-east coast of Sabah showed a high prevalence of resistance to CQ and SP, suggesting the use of the treatment may no longer be effective in the area. This study aimed to provide a baseline data of antimalarial drug resistant markers on P. falciparum isolates in Kota Marudu located in the north-east coast of Sabah. Mutations on genes associated with CQ (pfcrt and pfmdr1) and SP (pfdhps and pfdhfr) were assessed by PCR amplification and restriction fragment length polymorphism. Mutations on the kelch13 marker (K13) associated with artemisinin resistance were determined by DNA sequencing technique. The assessment of pfmdr1 copy number variation associated with mefloquine resistant was done by real-time PCR technique. A low prevalence (6.9%) was indicated for both pfcrt K76T and pfmdr1 N86Y mutations. All P. falciparum isolates harboured the pfdhps A437G mutation. Prevalence of pfdhfr gene mutations, S108N and I164L, were 100% and 10.3%, respectively. Combining the different resistant markers, only two isolates were conferred to have CQ and SP treatment failure markers as they contained mutant alleles of pfcrt and pfmdr1 together with quintuple pfdhps/pfdhfr mutation (combination of pfdhps A437G+A581G and pfdhfr C59R+S108N+I164L). All P. falciparum isolates carried single copy number of pfmdr1 and wild type K13 marker. This study has demonstrated a low prevalence of CQ and SP resistance alleles in the study area. Continuous monitoring of antimalarial drug efficacy is warranted and the findings provide information for policy makers in ensuring a proper malaria control. PMID:27788228

  3. Prevalence of mutations associated with antimalarial drugs in Plasmodium falciparum isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran

    PubMed Central

    Zakeri, Sedigheh; Afsharpad, Mandana; Raeisi, Ahmad; Djadid, Navid Dinparast

    2007-01-01

    Background This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran. Methods The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005. Results All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas. Conclusion Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria. PMID:17999755

  4. Molecular surveillance as monitoring tool for drug-resistant Plasmodium falciparum in Suriname.

    PubMed

    Adhin, Malti R; Labadie-Bracho, Mergiory; Bretas, Gustavo

    2013-08-01

    The aim of this translational study was to show the use of molecular surveillance for polymorphisms and copy number as a monitoring tool to track the emergence and dynamics of Plasmodium falciparum drug resistance. A molecular baseline for Suriname was established in 2005, with P. falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance (pfmdr1) markers and copy number in 40 samples. The baseline results revealed the existence of a uniformly distributed mutated genotype corresponding with the fully mefloquine-sensitive 7G8-like genotype (Y184F, S1034C, N1042D, and D1246Y) and a fixed pfmdr1 N86 haplotype. All samples harbored the pivotal pfcrtK76T mutation, showing that chloroquine reintroduction should not yet be contemplated in Suriname. After 5 years, 40 samples were assessed to trace temporal changes in the status of pfmdr1 polymorphisms and copy number and showed minor genetic alterations in the pfmdr1 gene and no significant changes in copy number, thus providing scientific support for prolongation of the current drug policy in Suriname.

  5. Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Rohrbach, Petra; McLean, Jeremy E.; Fidock, David A.; Stein, Wilfred D.; Lanzer, Michael

    2010-01-01

    Summary The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. PMID:17493125

  6. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether–lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

    PubMed Central

    Achieng, Angela O.; Muiruri, Peninah; Ingasia, Luicer A.; Opot, Benjamin H.; Juma, Dennis W.; Yeda, Redemptah; Ngalah, Bidii S.; Ogutu, Bernhards R.; Andagalu, Ben; Akala, Hoseah M.; Kamau, Edwin

    2015-01-01

    Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to

  7. Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether-lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya.

    PubMed

    Achieng, Angela O; Muiruri, Peninah; Ingasia, Luicer A; Opot, Benjamin H; Juma, Dennis W; Yeda, Redemptah; Ngalah, Bidii S; Ogutu, Bernhards R; Andagalu, Ben; Akala, Hoseah M; Kamau, Edwin

    2015-12-01

    Artemether-lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995-2003) and 745 after (post-ACT; 2008-2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995-1996 to 93.2% in 2014 and 0.0% in 2002-2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use

  8. Dihydroethanoanthracene Derivatives as In Vitro Malarial Chloroquine Resistance Reversal Agents

    PubMed Central

    Millet, Julie; Torrentino-Madamet, Marylin; Alibert, Sandrine; Rogier, Christophe; Santelli-Rouvier, Christiane; Mosnier, Joel; Baret, Eric; Barbe, Jacques; Parzy, Daniel; Pradines, Bruno

    2004-01-01

    The ability of four 9,10-dihydroethanoanthracene derivatives (BG920, BG932, BG958, and BG996), as well as verapamil and promethazine, to reverse chloroquine resistance was assessed against 24 chloroquine-resistant and 10 chloroquine-susceptible strains of Plasmodium falciparum from different countries. The 9,10-dihydroethanoanthracene derivatives clearly increase chloroquine susceptibility only in chloroquine-resistant isolates. PMID:15215144

  9. Plasmodium falciparum chloroquine resistance transporter is a H+-coupled polyspecific nutrient and drug exporter

    PubMed Central

    Juge, Narinobu; Moriyama, Sawako; Miyaji, Takaaki; Kawakami, Mamiyo; Iwai, Haruka; Fukui, Tomoya; Nelson, Nathan; Omote, Hiroshi; Moriyama, Yoshinori

    2015-01-01

    Extrusion of chloroquine (CQ) from digestive vacuoles through the Plasmodium falciparum CQ resistance transporter (PfCRT) is essential to establish CQ resistance of the malaria parasite. However, the physiological relevance of PfCRT and how CQ-resistant PfCRT gains the ability to transport CQ remain unknown. We prepared proteoliposomes containing purified CQ-sensitive and CQ-resistant PfCRTs and measured their transport activities. All PfCRTs tested actively took up tetraethylammonium, verapamil, CQ, basic amino acids, polypeptides, and polyamines at the expense of an electrochemical proton gradient. CQ-resistant PfCRT exhibited decreased affinity for CQ, resulting in increased CQ uptake. Furthermore, CQ competitively inhibited amino acid transport. Thus, PfCRT is a H+-coupled polyspecific nutrient and drug exporter. PMID:25733858

  10. High prevalence of asymptomatic malaria in a tribal population in eastern India.

    PubMed

    Ganguly, Swagata; Saha, Pabitra; Guha, Subhasish K; Biswas, Asit; Das, Sonali; Kundu, Pratip K; Maji, Ardhendu K

    2013-05-01

    Asymptomatic infection by Plasmodium falciparum is an important obstacle to eliminating malaria. Asymptomatic carriers do not seek treatment for infection, and therefore they become a reservoir for the parasite. For this reason, these carriers pose a real public health risk. The systematic identification and treatment of asymptomatic infections should reduce the parasite reservoir. A large reduction in this pool will lower the chance of transmission of the disease. In this study, we screened a tribal population of 1,040 individuals in the Purulia district of West Bengal by using a dual-antigen rapid diagnostic kit (RDK), microscopy, and species-specific PCR. All positive individuals were treated with artemisinin-based combination therapy (ACT) (artesunate plus sulfadoxine-pyrimethamine) and followed for 42 days. Polymorphisms in candidate genes were screened by DNA sequencing. A significant proportion (8.4%) of the study population was infected with P. falciparum but showed no clinical manifestations. The PCR method was more sensitive in detecting infection than the RDK or microscopy. The efficacy of the ACT was 97%. In the pfcrt gene, the mutation K76T (the mutated amino acid is indicated by bold type) was found in 100% of the cases. In the pfmdr1 gene, the mutations N86Y and Y184F were noted in 55.5% and 11% of the cases, respectively. Six different haplotypes were identified in the pfdhfr-pfdhps genes. Most importantly, the quintuple mutant A(16)I(51)R(59)N(108)I(164)-S(436)G(437)E(540)A(581)A(613) was found in 10% of the isolates, which is potentially important for the development of sulfadoxine-pyrimethamine resistance. A significant proportion of the study population harboring P. falciparum does not seek treatment and therefore serves as a reservoir for the parasite, maintaining the natural cycle. If the National Vector Borne Disease Control Programme (NVBDCP) of India is to eliminate malaria, then this hidden parasite burden needs to be addressed properly

  11. In vivo and in vitro analysis of chloroquine resistance in Plasmodium falciparum isolates from Senegal.

    PubMed

    Sarr, Ousmane; Myrick, Alissa; Daily, Johanna; Diop, Bernard M; Dieng, Therese; Ndir, Omar; Sow, Pape Salif; Mboup, Souleymane; Wirth, Dyann F

    2005-09-01

    To determine the predictive value of chloroquine (CQ) resistance markers in Senegal, Plasmodium falciparum DNA polymorphisms in pfmdr1and pfcrt were examined in relation to clinical outcome. Despite CQ treatment, 17% of patients had parasitemia after 28 days. Examination of molecular markers of CQ resistance revealed that 64% of all isolates had the T76 resistant allele at the pfcrt locus, while 30% carried the Y86 resistant allele at the pfmdr1 locus. The pfcrt T76 allele was present not only in all in vivo resistant isolates, 89% of in vitro resistant isolates, but also in 35% of in vitro sensitive isolates. The pfmdr1 N86Y polymorphism did not correlate with in vitro or in vivo CQ resistance. Our data suggest that the pfcrt T76 allele alone is required but not a sufficient predictor for in vivo CQ resistance.

  12. Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria.

    PubMed

    Happi, C T; Gbotosho, G O; Folarin, O A; Sowunmi, A; Bolaji, O M; Fateye, B A; Kyle, D E; Milhous, W; Wirth, D F; Oduola, A M J

    2006-12-01

    Chloroquine (CQ) resistance in Plasmodium falciparum is associated with polymorphisms in loci on pfcrt and pfmdr1 genes. In this study, we determined the association and linkage disequilibrium between in vivo CQ resistance and P. falciparum polymorphisms in pfcrt gene at codon 76 and pfmdr1 gene at codon 86 in isolates obtained from 111 children with acute uncomplicated falciparum malaria in Nigeria. Patients were treated with standard dosage of CQ and followed up for 28 days. Filter paper samples were collected at enrollment and during follow-up for parasites genotypes and identification of pfcrt and pfmdr1 mutations. Association and linkage disequilibrium between mutant pfcrtT76 and pfmdr1Y86 alleles in pretreatment isolates of P. falciparum was determined. Fifty-five out of the 111 patients (49.5%) failed treatment. Single mutant pfcrtT76 or pfmdr1Y86 alleles were found in 55 out of 111 P. falciparum isolates screened at enrollment. Of these 55 isolates, the mutant pfcrtT76 and pfmdr1Y86 alleles were found in 84%. Both mutant pfcrtT76 (p=0.0196) and pfmdr1Y86 (p=0.000042) alleles were associated with in vivo CQ resistance. In addition, the mutant pfcrtT76 (p=0.047) and pfmdr1Y86 (p=0.006) alleles were significantly selected by CQ in patients who failed treatment. Association analysis between paired single alleles at pfcrt and pfmdr1 loci showed a significant association (p=0.0349 and chi(2)=4.45) between the pfcrt T76 allele on chromosome 7 and the pfmdr1Y86 allele on chromosome 5 and that these two mutant alleles were in linkage disequilibrium (p=0.000, D'=0.64, and r(2)=0.28). Considering the high level of CQ resistance and drug use in the study area, the observed linkage disequilibrium between the mutant pfcrtT76 and pfmdr1Y86 alleles is maintained epistatically through directional CQ selective pressure.

  13. Molecular epidemiology of malaria in Cameroon. XXIV. Trends of in vitro antimalarial drug responses in Yaounde, Cameroon.

    PubMed

    Basco, Leonardo K; Ringwald, Pascal

    2007-01-01

    In vitro response to chloroquine, monodesethylamodiaquine, mefloquine, lumefantrine, and dihydroartemisinin was assessed by the radioisotopic microtest in Yaoundé, Cameroon, during 2000-2004 and compared with our previous data obtained during 1996-1999. Based on the cut-off value of 100 nmol/L, 36.3% of isolates were chloroquine-susceptible (N = 175; geometric mean IC(50), 40.3 nmol/L) and 63.7% were chloroquine-resistant (N = 307; geometric mean IC(50), 211 nmol/L). There was no significant difference (P > 0.05) in the mean IC(50)s from 1996 to 2004, but a significant linear trend (P < 0.05) toward an increased proportion of chloroquine-resistant isolates was observed from 1996 (49%) to 2004 (69%). All chloroquine-susceptible isolates and most chloroquine-resistant isolates were susceptible to monodesethylamodiaquine (i.e., IC(50) < 60 nmol/L). Despite the positive correlation between chloroquine and monodesethylamodiaquine (r = 0.739, P < 0.05), the IC(50)s for monodesethylamodiaquine remained stable during 1997-2004, with no increase in the proportion of monodesethylamodiaquine-resistant isolates. Mefloquine, lumefantrine, and dihydroartemisinin were equally active against the chloroquine-susceptible and chloroquine-resistant parasites. The responses to these three drugs were positively correlated, and a significant decrease (P < 0.05) in the mean IC(50)s was observed during the study period compared with our earlier data in 1997-1999, probably because of their inverse relationship with chloroquine response. The in vitro results were in general agreement with the in vivo response to chloroquine and amodiaquine. In vitro drug susceptibility assay is a useful, complementary laboratory tool for determining the trend of response to drugs for which there is still no established molecular marker and may serve as an early warning system for emerging drug resistance.

  14. In vitro antimalarial activity of a new organometallic analog, ferrocene-chloroquine.

    PubMed

    Domarle, O; Blampain, G; Agnaniet, H; Nzadiyabi, T; Lebibi, J; Brocard, J; Maciejewski, L; Biot, C; Georges, A J; Millet, P

    1998-03-01

    The in vitro activities of new organometallic chloroquine analogs, based on 4-amino-quinoleine compounds bound to a molecule of ferrocene, were evaluated against chloroquine-susceptible, chloroquine-intermediate, and chloroquine-resistant, culture-adapted Plasmodium falciparum lineages by a proliferation test. One of the ferrocene analogs totally restored the activity of chloroquine against chloroquine-resistant parasites. This compound, associated with tartaric acid for better solubility, was highly effective. The role of the ferrocene in reversing chloroquine resistance is discussed, as is its potential use for human therapy.

  15. In Vitro Antimalarial Activity of a New Organometallic Analog, Ferrocene-Chloroquine

    PubMed Central

    Domarle, O.; Blampain, G.; Agnaniet, H.; Nzadiyabi, T.; Lebibi, J.; Brocard, J.; Maciejewski, L.; Biot, C.; Georges, A. J.; Millet, P.

    1998-01-01

    The in vitro activities of new organometallic chloroquine analogs, based on 4-amino-quinoleine compounds bound to a molecule of ferrocene, were evaluated against chloroquine-susceptible, chloroquine-intermediate, and chloroquine-resistant, culture-adapted Plasmodium falciparum lineages by a proliferation test. One of the ferrocene analogs totally restored the activity of chloroquine against chloroquine-resistant parasites. This compound, associated with tartaric acid for better solubility, was highly effective. The role of the ferrocene in reversing chloroquine resistance is discussed, as is its potential use for human therapy. PMID:9517929

  16. Basis of antimalarial action: non-weak base effects of chloroquine on acid vesicle pH

    SciTech Connect

    Krogstad, D.J.; Schlesinger, P.H.

    1987-03-01

    Biologically active concentrations of chloroquine increase the pH of the parasite's acid vesicles within 3-5 min. This increase in pH results from two mechanisms, one of which is markedly reduced in chloroquine-resistant parasites. Because chloroquine is a weak base, it increases vesicle pH by that mechanism in chloroquine-susceptible and resistant parasites and mammalian cells (based on its two pKs and on the delta pH between the acid vesicle and the extracellular environment). In chloroquine-susceptible parasites, but not resistant parasites or mammalian cells, chloroquine increases the pH of acid vesicles 700- to 800-fold more than can be accounted for by its properties as a weak base. The increase in acid vesicle pH caused by these non-weak base effects of nanomolar chloroquine in susceptible parasites suggests that chloroquine acts by interfering with acid vesicle functions in the parasite such as the endocytosis and proteolysis of hemoglobin, and the intracellular targeting of lysosomal enzymes. The non-weak base effects of nanomolar chloroquine on parasite vesicle pH are also responsible for its safety because these chloroquine concentrations do not affect mammalian cells.

  17. Antimalarial activity of WR 243251, a Dihydroacridinedione.

    PubMed Central

    Berman, J; Brown, L; Miller, R; Andersen, S L; McGreevy, P; Schuster, B G; Ellis, W; Ager, A; Rossan, R

    1994-01-01

    WR 243251 is a dihydroacridinedione that was evaluated for antimalarial blood schizonticidal activity in vitro and in vivo. The in vitro doses calculated to kill 50% of organisms were 11 nM for a chloroquine-susceptible, mefloquine-resistant standard strain and 25 nM for a chloroquine- and pyrimethamine-resistant standard strain. The total dose needed to cure 100% of mice infected with a drug-susceptible strain of Plasmodium berghei was 12 to 20 mg/kg of body weight for both oral and subcutaneous administration. The regimen needed to cure 100% of Aotus monkeys infected with Plasmodium falciparum was 8 mg/kg/day for 3 days (chloroquine-susceptible strain) and 16 mg/kg/day for 3 days (chloroquine-resistant strain). The 100% curative doses for Aotus monkeys did not increase for parasites previously exposed to subcurative doses. The absolute value of the curative doses of WR 243251 was comparable to or lower than the values for clinical antimalarial agents. The high absolute activity, comparability of activities against susceptible and resistant parasites, and inability to induce resistance by exposure to subcurative doses suggest that WR 243251 has strong potential as a blood schizonticidal agent. PMID:7986005

  18. Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter.

    PubMed

    Petersen, Ines; Gabryszewski, Stanislaw J; Johnston, Geoffrey L; Dhingra, Satish K; Ecker, Andrea; Lewis, Rebecca E; de Almeida, Mariana Justino; Straimer, Judith; Henrich, Philipp P; Palatulan, Eugene; Johnson, David J; Coburn-Flynn, Olivia; Sanchez, Cecilia; Lehane, Adele M; Lanzer, Michael; Fidock, David A

    2015-07-01

    The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within-host competition with wild-type drug-sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC50 values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild-type pfcrt in co-culture competition assays. These three alleles mediated cross-resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first-line artemisinin-based combination therapy. These data reveal ongoing region-specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine-resistant malaria.

  19. A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras.

    PubMed

    Fontecha, Gustavo A; Sanchez, Ana L; Mendoza, Meisy; Banegas, Engels; Mejía-Torres, Rosa E

    2014-07-01

    Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt "CVMNK" genotype in codons 72-76.

  20. Synthesis and preliminary biological evaluation of a small library of hybrid compounds based on Ugi isocyanide multicomponent reactions with a marine natural product scaffold.

    PubMed

    Avilés, Edward; Prudhomme, Jacques; Le Roch, Karine G; Franzblau, Scott G; Chandrasena, Kevin; Mayer, Alejandro M S; Rodríguez, Abimael D

    2015-11-15

    A mixture-based combinatorial library of five Ugi adducts (4-8) incorporating known antitubercular and antimalarial pharmacophores was successfully synthesized, starting from the naturally occurring diisocyanide 3, via parallel Ugi four-center three-component reactions (U-4C-3CR). The novel α-acylamino amides obtained were evaluated for their antiinfective potential against laboratory strains of Mycobacterium tuberculosis H37Rv and chloroquine-susceptible 3D7 Plasmodium falciparum. Interestingly, compounds 4-8 displayed potent in vitro antiparasitic activity with higher cytotoxicity in comparison to their diisocyanide precursor 3, with the best compound exhibiting an IC50 value of 3.6 nM. Additionally, these natural product inspired hybrids potently inhibited in vitro thromboxane B2 (TXB2) and superoxide anion (O2(-)) generation from Escherichia coli lipopolysaccharide (LPS)-activated rat neonatal microglia, with concomitant low short-term toxicity.

  1. In vivo and in vitro antiplasmodial activities of some plants traditionally used in Guatemala against malaria.

    PubMed Central

    Franssen, F F; Smeijsters, L J; Berger, I; Medinilla Aldana, B E

    1997-01-01

    We present an evaluation of the antiplasmodial and cytotoxic effects of four plants commonly used in Guatemalan folk medicine against malaria. Methanol extracts of Simarouba glauca D. C., Sansevieria guineensis Willd, Croton guatemalensis Lotsy, and Neurolaena lobata (L.)R.Br. significantly reduced parasitemias in Plasmodium berghei-infected mice. Dichloromethane fractions were screened for their cytotoxicities on Artemia salina (brine shrimp) larvae, and 50% inhibitory concentrations were determined for Plasmodium falciparum in in vitro cultures. Both chloroquine-susceptible and -resistant strains of P. falciparum were significantly inhibited by these extracts. Of all dichloromethane extracts, only the S. glauca cortex extract was considered to be toxic to nauplii of A. salina in the brine shrimp test. PMID:9210673

  2. Chloroquine-resistant malaria in travelers returning from Haiti after 2010 earthquake.

    PubMed

    Gharbi, Myriam; Pillai, Dylan R; Lau, Rachel; Hubert, Véronique; Khairnar, Krishna; Existe, Alexandre; Kendjo, Eric; Dahlström, Sabina; Guérin, Philippe J; Le Bras, Jacques

    2012-08-01

    We investigated chloroquine sensitivity to Plasmodium falciparum in travelers returning to France and Canada from Haiti during a 23-year period. Two of 19 isolates obtained after the 2010 earthquake showed mixed pfcrt 76K+T genotype and high 50% inhibitory concentration. Physicians treating malaria acquired in Haiti should be aware of possible chloroquine resistance.

  3. Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

    PubMed Central

    Warring, Sally D.; Dou, Zhicheng; Carruthers, Vern B.; McFadden, Geoffrey I.

    2014-01-01

    Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites. PMID:24859994

  4. A Redox-Active Fluorescent pH Indicator for Detecting Plasmodium falciparum Strains with Reduced Responsiveness to Quinoline Antimalarial Drugs.

    PubMed

    Jida, Mouhamad; Sanchez, Cecilia P; Urgin, Karène; Ehrhardt, Katharina; Mounien, Saravanan; Geyer, Aurelia; Elhabiri, Mourad; Lanzer, Michael; Davioud-Charvet, Elisabeth

    2017-02-10

    Mutational changes in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) have been associated with differential responses to a wide spectrum of biologically active compounds including current and former quinoline and quinoline-like antimalarial drugs. PfCRT confers altered drug responsiveness by acting as a transport system, expelling drugs from the parasite's digestive vacuole where these drugs exert, at least part of, their antiplasmodial activity. To preserve the efficacy of these invaluable drugs, novel functional tools are required for epidemiological surveys of parasite strains carrying mutant PfCRT variants and for drug development programs aimed at inhibiting or circumventing the action of PfCRT. Here we report the synthesis and characterization of a pH-sensitive fluorescent chloroquine analogue consisting of 7-chloro-N-{2-[(propan-2-yl)amino]ethyl}quinolin-4-amine functionalized with the fluorochrome 7-nitrobenzofurazan (NBD) (henceforth termed Fluo-CQ). In the parasite, Fluo-CQ accumulates in the digestive vacuole, giving rise to a strong fluorescence signal but only in parasites carrying the wild type PfCRT. In parasites carrying the mutant PfCRT, Fluo-CQ does not accumulate. The differential handling of the fluorescent probe, combined with live cell imaging, provides a diagnostic tool for quick detection of those P. falciparum strains that carry a PfCRT variant associated with altered responsiveness to quinoline and quinoline-like antimalarial drugs. In contrast to the accumulation studies, chloroquine (CQ)-resistant parasites were observed cross-resistant to Fluo-CQ when the chemical probe was tested in various CQ-sensitive and -resistant parasite strains. NBD derivatives were found to act as redox cyclers of two essential targets, using a coupled assay based on methemoglobin and the NADPH-dependent glutathione reductase (GRs) from P. falciparum. This redox activity is proposed to contribute to the dual action of Fluo-CQ on redox

  5. Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria.

    PubMed

    Happi, C T; Gbotosho, G O; Folarin, O A; Bolaji, O M; Sowunmi, A; Kyle, D E; Milhous, W; Wirth, D F; Oduola, A M J

    2006-07-01

    This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

  6. Chloroquine and sulphadoxine-pyrimethamine sensitivity of Plasmodium falciparum parasites in a Brazilian endemic area

    PubMed Central

    Gama, Bianca Ervatti; de Oliveira, Natália K Almeida; Zalis, Mariano G; de Souza, José Maria; Santos, Fátima; Daniel-Ribeiro, Cláudio Tadeu; Ferreira-da-Cruz, Maria de Fátima

    2009-01-01

    Background The goal of the present study was the characterization of Plasmodium falciparum genes associated to malaria drug resistance (pfcrt, pfdhfr and pfdhps), in samples from two Brazilian localities. Methods Parasites from 65 P. falciparum samples were genotyped using nested-PCR and direct DNA sequencing. Results Six resistant sulphadoxine-pyrimethamine (SP) pfdhfr genotypes and one haplotype associated to SP sensitivity were detected. For pfcrt gene, SVMNT chloroquine (CQ)-resistant genotype was detected as well as the CVMNK CQ-sensitive haplotype in the same sample from Paragominas, that showed a SP-sensitive genotype. Conclusion This study is the first to document the sensitivity of P. falciparum parasites to CQ and SP in Brazilian field samples. The importance of these findings is discussed. PMID:19602248

  7. Quinine dimers are potent inhibitors of the Plasmodium falciparum chloroquine resistance transporter and are active against quinoline-resistant P. falciparum.

    PubMed

    Hrycyna, Christine A; Summers, Robert L; Lehane, Adele M; Pires, Marcos M; Namanja, Hilda; Bohn, Kelsey; Kuriakose, Jerrin; Ferdig, Michael; Henrich, Philipp P; Fidock, David A; Kirk, Kiaran; Chmielewski, Jean; Martin, Rowena E

    2014-03-21

    Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

  8. 1H-NMR metabolite profiles of different strains of Plasmodium falciparum

    PubMed Central

    Teng, Rongwei; Lehane, Adele M.; Winterberg, Markus; Shafik, Sarah H.; Summers, Robert L.; Martin, Rowena E.; van Schalkwyk, Donelly A.; Junankar, Pauline R.; Kirk, Kiaran

    2014-01-01

    Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite. PMID:25405893

  9. Molecular epidemiology of malaria in Cameroon. XII. In vitro drug assays and molecular surveillance of chloroquine and proguanil resistance.

    PubMed

    Basco, Leonardo K

    2002-10-01

    Chloroquine-proguanil combination is one of the options for chemoprophylaxis. The rapid evolution of drug resistance status requires a constant upgrade of epidemiologic data. Due to various difficulties in conducting prospective clinical studies on the prophylactic efficacy of the drug combination, especially in highly chloroquine-resistant zones, in vitro drug sensitivity assays and specific molecular markers for chloroquine (Plasmodium falciparum chloroquine-resistance transporter, pfcrt) and cycloguanil (a biologically active metabolite of proguanil; dihydrofolate reductase, dhfr) resistance were evaluated as an alternative approach in this study. Of 116 isolates, 62 (53.4%) were doubly resistant in vitro to chloroquine (IC50 > or = 100 nM) and cycloguanil (IC50 > or = 15 nM). Likewise, 62 of 118 isolates (52.5%) carried both the mutant Thr-76 pfcrt allele and at least one dhfr mutant allele (1 with a single Asn-108 allele, 8 with double Arg-59 and Asn-108 mutations, and 53 with triple Ile-51, Arg-59, and Asn-108 mutations). The in vitro drug response corresponded with the presence or absence of key mutation(s) in the pfcrt and dhfr genes. These results suggest the high proportion of P. falciparum isolates in southern Cameroon that may not respond to chloroquine-proguanil combination.

  10. Prospects of intermittent preventive treatment of adults against malaria in areas of seasonal and unstable malaria transmission, and a possible role for chloroquine.

    PubMed

    Giha, Hayder A

    2010-04-01

    Chloroquine (CQ) is outmoded as an antimalarial drug in most of the malarial world because of the high resistance rate of parasites. The parasite resistance to CQ is attributed to pfcrt/pfmdr1 gene mutations. Recent studies showed that parasites with mutations of pfcrt/pfmdr1 genes are less virulent, and that those with dhfr/dhps mutations are more susceptible to host immune clearance; the former and latter mutations are linked. In the era of artemisinin-based combination therapy, the frequency of pfcrt/pfmdr1 wild variants is expected to rise. In areas of unstable malaria transmission, the unpredictable severe epidemics of malaria and epidemics of severe malaria could result in high mortality rate among the semi-immune population. With this in mind, the use of CQ for intermittent preventive treatment of adults (IPTa) is suggested as a feasible control measure to reduce malaria mortality in adults and older children without reducing uncomplicated malaria morbidity. The above is discussed in a multidisciplinary approach validating the deployment of molecular techniques in malaria control and showing a possible role for CQ as a rescue drug after being abandoned.

  11. Molecular targets of 5-fluoroorotate in the human malaria parasite, Plasmodium falciparum.

    PubMed Central

    Rathod, P K; Leffers, N P; Young, R D

    1992-01-01

    5-Fluoroorotate is known to have potent antimalarial activity against chloroquine-susceptible as well as chloroquine-resistant clones of Plasmodium falciparum. It was hypothesized that this activity was mediated through synthesis of 5-fluoro-2'-deoxyuridylate, an inactivator of thymidylate synthase, or through incorporation of 5-fluoropyrimidine residues into nucleic acids. Treatment of P. falciparum in culture with 100 nM 5-fluoroorotate resulted in rapid inactivation of malarial thymidylate synthase activity. A 50% loss of thymidylate synthase activity as well as a 50% decrease in parasite proliferation were seen with 5 nM 5-fluoroorotate. Dihydrofolate reductase activity, which resides on the same bifunctional protein as thymidylate synthase, was not affected by 5-fluoroorotate treatment. Incubation of malarial parasites with 3 to 10 microM radioactive 5-fluoroorotic acid for 48 h resulted in significant incorporation of radioactivity into the RNA fraction of P. falciparum; approximately 9% of the uridine residues were substituted with 5-fluorouridine. However, compared with the 50% inhibitory concentrations of 5-fluoroorotate, a 1,000-fold higher concentration of the pyrimidine analog was required to see significant modification of RNA molecules. Results of these studies are consistent with the hypothesis that thymidylate synthase is the primary target of 5-fluoroorotate in malarial parasites. PMID:1503432

  12. Medicinal chemistry discoveries among 1,3,5-triazines: recent advances (2000-2013) as antimicrobial, anti-TB, anti-HIV and antimalarials.

    PubMed

    Patel, Rahul V; Keum, Young-Soo; Park, Se Won

    2014-01-01

    The chemistry and an extensive spectrum of biological activities of s-triazines have been examined since several decades and this heterocyclic core has received emerging consensus. This article aims to summarize recent advances (2000-2013) made towards the discovery of antimicrobial, antituberculosis, anti-HIV and antimalarial agents holding 1,3,5-triazine ring as a nucleus with the substitution of several types of nucleophiles. Molecular patterns associated with particular potency have been identified targeting several Gram-positive and Gram-negative bacteria and some fungal species, mycobacterium tuberculosis H37Rv, HIV type I and HIV type II, particularly, HIV-1I IIB and HIV- 1ROD strains as well as a variety of P. falciparum malarial strains as chloroquine-resistant K1, chloroquine-susceptible NF54, chloroquine-sensitive 3D7, P. falciparum (D6 clone), P. falciparum (W2 clone), cycloguanil-resistant FCR-3, chloroquine sensitive RKL2. The report will be of considerable interest to gain useful information for the furtherance of drug discovery with extended 1,3,5-triazine designs.

  13. Management of relapsing Plasmodium vivax malaria

    PubMed Central

    Chu, Cindy S; White, Nicholas J

    2016-01-01

    ABSTRACT Introduction: Relapses are important contributors to illness and morbidity in Plasmodium vivax and P. ovale infections. Relapse prevention (radical cure) with primaquine is required for optimal management, control and ultimately elimination of Plasmodium vivax malaria. A review was conducted with publications in English, French, Portuguese and Spanish using the search terms ‘P. vivax’ and ‘relapse’. Areas covered: Hypnozoites causing relapses may be activated weeks or months after initial infection. Incidence and temporal patterns of relapse varies geographically. Relapses derive from parasites either genetically similar or different from the primary infection indicating that some derive from previous infections. Malaria illness itself may activate relapse. Primaquine is the only widely available treatment for radical cure. However, it is often not given because of uncertainty over the risks of primaquine induced haemolysis when G6PD deficiency testing is unavailable. Recommended dosing of primaquine for radical cure in East Asia and Oceania is 0.5 mg base/kg/day and elsewhere is 0.25 mg base/kg/day. Alternative treatments are under investigation. Expert commentary: Geographic heterogeneity in relapse patterns and chloroquine susceptibility of P. vivax, and G6PD deficiency epidemiology mean that radical treatment should be given much more than it is today. G6PD testing should be made widely available so primaquine can be given more safely. PMID:27530139

  14. In vitro activities of novel catecholate siderophores against Plasmodium falciparum.

    PubMed Central

    Pradines, B; Ramiandrasoa, F; Basco, L K; Bricard, L; Kunesch, G; Le Bras, J

    1996-01-01

    The activities of novel iron chelators, alone and in combination with chloroquine, quinine, or artemether, were evaluated in vitro against susceptible and resistant clones of Plasmodium falciparum with a semimicroassay system. N4-nonyl,N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound 7) demonstrated the highest level of activity: 170 nM against a chloroquine-susceptible clone and 1 microM against a chloroquine-resistant clone (50% inhibitory concentrations). Compounds 6, 8, and 10 showed antimalarial activity with 50% inhibitory concentrations of about 1 microM. Compound 7 had no effect on the activities of chloroquine, quinine, and artemether against either clone, and compound 8 did not enhance the schizontocidal action of either chloroquine or quinine against the chloroquine-resistant clone. The incubation of compound 7 with FeCI3 suppressed or decreased the in vitro antimalarial activity of compound 7, while no effect was observed with incubation of compound 7 with CuSO4 and ZnSO4. These results suggest that iron deprivation may be the main mechanism of action of compound 7 against the malarial parasites. Chelator compounds 7 and 8 primarily affected trophozoite stages, probably by influencing the activity of ribonucleotide reductase, and thus inhibiting DNA synthesis. PMID:8878587

  15. Update on the in vivo tolerance and in vitro reduced susceptibility to the antimalarial lumefantrine.

    PubMed

    Nzila, Alexis; Okombo, John; Ohuma, Eric; Al-Thukair, Assad

    2012-10-01

    Coartem(®), the combination of artemether (an artemisinin derivative) and lumefantrine, has been adopted as the first-line treatment for uncomplicated malaria in many countries. The emergence of resistance to artemisinin derivatives has now been proven in South-East Asia, and there is concern that this may spread to other endemic areas. Strategies to contain and control the spread of artemisinin resistance have been proposed. On the other hand, not much attention has been given to lumefantrine. Indeed, for more than 7 years, reports have been emerging that the use of Coartem(®) is associated with rapid selection of lumefantrine-tolerant parasites. These parasites can survive in the presence of sub-therapeutic lumefantrine concentrations, and, interestingly, this in vivo phenotype is translated in vitro into reduced susceptibility to lumefantrine. As a result, such parasites could form the setting in which lumefantrine resistance would emerge. Thus, identifying genetic markers that reflect this phenotype (both in vitro and in vivo) could yield information on the mechanisms of lumefantrine resistance. More interestingly, lumefantrine tolerance is associated with an increase in chloroquine susceptibility, raising the possibility of re-introducing chloroquine. In this work, we have reviewed the current knowledge, and we present existing challenges and gaps with regard to the mechanisms of in vivo tolerance and in vitro reduced susceptibility to lumefantrine. The re-introduction of chloroquine in areas of high lumefantrine resistance is also discussed.

  16. Drug resistance associated genetic polymorphisms in Plasmodium falciparum and Plasmodium vivax collected in Honduras, Central America

    PubMed Central

    2011-01-01

    Background In Honduras, chloroquine and primaquine are recommended and still appear to be effective for treatment of Plasmodium falciparum and Plasmodium vivax malaria. The aim of this study was to determine the proportion of resistance associated genetic polymorphisms in P. falciparum and P. vivax collected in Honduras. Methods Blood samples were collected from patients seeking medical attention at the Hospital Escuela in Tegucigalpa from 2004 to 2006 as well as three regional hospitals, two health centres and one regional laboratory during 2009. Single nucleotide polymorphisms in P. falciparum chloroquine resistance transporter (pfcrt), multidrug resistance 1 (pfmdr1), dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes and in P. vivax multidrug resistance 1 (pvmdr1) and dihydrofolate reductase (pvdhfr) genes were detected using PCR based methods. Results Thirty seven P. falciparum and 64 P. vivax samples were collected. All P. falciparum infections acquired in Honduras carried pfcrt, pfmdr1, pfdhps and pfdhfr alleles associated with chloroquine, amodiaquine and sulphadoxine-pyrimethamine sensitivity only. One patient with parasites acquired on a Pacific Island had pfcrt 76 T and pfmdr1 86Y alleles. That patient and a patient infected in West Africa had pfdhfr 51I, 59 R and 108 N alleles. Pvmdr1 976 F was found in 7/37 and two copies of pvmdr1 were found in 1/37 samples. Pvdhfr 57 L + 58 R was observed in 2/57 samples. Conclusion The results indicate that P. falciparum from Honduras remain sensitive to chloroquine and sulphadoxine-pyrimethamine. This suggests that chloroquine and sulphadoxine-pyrimethamine should be efficacious for treatment of uncomplicated P. falciparum malaria, supporting current national treatment guidelines. However, genetic polymorphisms associated with chloroquine and sulphadoxine-pyrimethamine tolerance were detected in local P. vivax and imported P. falciparum infections. Continuous monitoring of the prevalence

  17. Mutant Plasmodium falciparum chloroquine resistance transporter in Hodeidah, Yemen: association with parasitologic indices and treatment-seeking behaviors.

    PubMed

    Abdul-Ghani, Rashad; Farag, Hoda F; Allam, Amal F; Shawky, Sherine M; Al-Mekhlafi, Abdulsalam M

    2013-12-01

    Malaria still represents a major health problem in Yemen, particularly in Hodeidah, despite continuing efforts to eliminate it. With the absence of clinically proven vaccines, chemotherapy with antimalarials is still greatly needed. Chloroquine (CQ) has been popular as the drug of choice for malaria control. However, Plasmodium falciparum resistance to CQ has been one of the main obstacles in malaria control and elimination. Although CQ is no longer the recommended antimalarial chemotherapy, it has remained the number one over-the-counter antimalarial drug in many endemic areas, including Yemen, and is still used for self-medication. In addition, promising reports on CQ efficacy reversal in many African countries brought it again into the scene. This has led to a growing interest in the possibility of its re-introduction, particularly with the concerns raised about the parasite resistance to artemisinin-based combination therapies. Therefore, the present study aimed at analyzing the CQ-associated pfcrt 76T mutation in P. falciparum isolates from patients with uncomplicated falciparum malaria in Hodeidah, west of Yemen. The association of treatment-seeking behaviors and antimalarial drug use with the pfcrt 76T mutant allele was also studied. It was revealed that there is still a sustained high frequency of this molecular marker among parasite isolates associated with younger age, decreased parasite density and the presence of gametocytes in blood. Delay in seeking treatment and frequent use of antimalarials were the behaviors significantly associated with the presence of the pfcrt 76T mutant allele among patients reporting a history of malaria treatment.

  18. Molecular markers associated with resistance to commonly used antimalarial drugs among Plasmodium falciparum isolates from a malaria-endemic area in Taiz governorate-Yemen during the transmission season.

    PubMed

    Alareqi, Lina M Q; Mahdy, Mohammed A K; Lau, Yee-Ling; Fong, Mun-Yik; Abdul-Ghani, Rashad; Mahmud, Rohela

    2016-10-01

    Since 2005, artesunate (AS) plus sulfadoxine/pyrimethamine (SP) combination has been adopted as the first-line treatment for uncomplicated malaria in Yemen in response to the high level of Plasmodium falciparum resistance to chloroquine (CQ). Therefore, the aim of the present study was to determine the frequency distribution of molecular markers associated with resistance to CQ and AS plus SP combination among P. falciparum isolates from a malaria-endemic area in Taiz governorate, Yemen. Fifty P. falciparum isolates were collected during a cross-sectional study in Mawza district, Taiz, in the period from October 2013 to April 2014. The isolates were investigated for drug resistance-associated molecular markers in five genes, including P. falciparum CQ resistance transporter (pfcrt) 76T and P. falciparum multidrug resistance 1 (pfmdr1) 86Y as markers of resistance to CQ, mutations in the Kelch 13 (K13) propeller domain for resistance to AS, and P. falciparum dihydrofolate reductase (pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) genes for resistance to SP. Nested polymerase chain reaction was used to amplify target genes in DNA extracts of the isolates followed by restriction fragment length polymorphism for detecting 76T and 86Y mutations in pfcrt and pfmdr1, respectively, and by DNA sequencing for detecting mutations in K13, pfdhfr and pfdhps. All the investigated isolates from Mawza district were harboring the pfcrt 76T mutant and the pfmdr1 N86 wild-type alleles. The pfdhfr 51I/108N double mutant allele was found in 2.2% (1/45) of the isolates; however, no mutations were detected at codons 436, 437, 540, 581 and 613 of pfdhps. All P. falciparum isolates that were successfully sequenced (n=47) showed the K13 Y493, R539, I543 and C580 wild-type alleles. In conclusion, the pfcrt 76T mutant allele is fixed in the study area about six years after the official withdrawal of CQ, possibly indicating its over-the-counter availability and continued use as a

  19. A Purine Analog Synergizes with Chloroquine (CQ) by Targeting Plasmodium falciparum Hsp90 (PfHsp90)

    PubMed Central

    Shahinas, Dea; Folefoc, Asongna; Taldone, Tony; Chiosis, Gabriela; Crandall, Ian; Pillai, Dylan R.

    2013-01-01

    Background Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling Plasmodium falciparum malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of P. falciparum Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants. Methods The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. In vivo studies were conducted using the Plasmodium berghei infection mouse model. Results PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine in vitro. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the P. falciparum chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the P.berghei mouse model of malaria to reduce parasitemia and improve survival. Conclusions We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy. PMID:24098696

  20. Drug resistance and genetic diversity of Plasmodium falciparum parasites from suriname.

    PubMed

    Peek, Ron; VAN Gool, Tom; Panchoe, Daynand; Greve, Sophie; Bus, Ellen; Resida, Lesley

    2005-11-01

    Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the chloroquine resistance marker of the P. falciparum chloroquine resistance transporter (pfcrt) gene (codon 76) and the pyrimethamine-sulfadoxine resistance markers in the dihydrofolate reductase (dhfr) gene (codons 16, 51, 59, 108, and 164) and dihydropteroate synthase (dhps) gene (codons 436, 437, 540, 581, and 613). Genetic variability was determined by sequence analysis of the polymorphic segments of the merozoite surface protein 2 (msp-2) and glutamate-rich protein (glurp) genes. Mutations in the pfcrt, dhps, and dhfr genes were found in all samples tested, suggesting that resistance to chloroquine and antifolate drugs is present at a high frequency. A low number of alleles was found for the msp-2 and glurp genes. This indicates limited genetic diversity and, based on geographic data, a genetically homogeneous P. falciparum population in Suriname.

  1. New Antimalarial Hits from Dacryodes edulis (Burseraceae) - Part I: Isolation, In Vitro Activity, In Silico “drug-likeness” and Pharmacokinetic Profiles

    PubMed Central

    Zofou, Denis; Tematio, Esther Laure; Ntie-Kang, Fidele; Tene, Mathieu; Ngemenya, Moses N.; Tane, Pierre; Titanji, Vincent P. K.

    2013-01-01

    The aims of the present study were to identify the compounds responsible for the anti-malarial activity of Dacryoedes edulis (Burseraceae) and to investigate their suitability as leads for the treatment of drug resistant malaria. Five compounds were isolated from ethyl acetate and hexane extracts of D. edulis stem bark and tested against 3D7 (chloroquine-susceptible) and Dd2 (multidrug-resistant) strains of Plasmodium falciparum, using the parasite lactate dehydrogenase method. Cytotoxicity studies were carried out on LLC-MK2 monkey kidney epithelial cell-line. In silico analysis was conducted by calculating molecular descriptors using the MOE software running on a Linux workstation. The “drug-likeness” of the isolated compounds was assessed using Lipinski criteria, from computed molecular properties of the geometry optimized structures. Computed descriptors often used to predict absorption, distribution, metabolism, elimination and toxicity (ADMET) were used to assess the pharmacokinetic profiles of the isolated compounds. Antiplasmodial activity was demonstrated for the first time in five major natural products previously identified in D. edulis, but not tested against malaria parasites. The most active compound identified was termed DES4. It had IC50 values of 0.37 and 0.55 µg/mL, against 3D7 and Dd2 respectively. In addition, this compound was shown to act in synergy with quinine, satisfied all criteria of “Drug-likeness” and showed considerable probability of providing an antimalarial lead. The remaining four compounds also showed antiplasmodial activity, but were less effective than DES4. None of the tested compounds was cytotoxicity against LLC-MK2 cells, suggesting their selective activities on malaria parasites. Based on the high in vitro activity, low toxicity and predicted “Drug-likeness” DES4 merits further investigation as a possible drug lead for the treatment of malaria. PMID:24282507

  2. In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase

    PubMed Central

    Kusch, Peter; Deininger, Susanne; Specht, Sabine; Maniako, Rudeka; Haubrich, Stefanie; Pommerening, Tanja; Lin, Paul Kong Thoo; Hoerauf, Achim; Kaiser, Annette

    2011-01-01

    Balanites aegyptiaca (Balanitaceae) is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 μg/μL ± 3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H)-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a Ki of 2.35 μg/μL and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a Ki of 4.8 μg/μL. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 μM ± 3 and 47.82 μM ± 2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed. PMID:21687598

  3. Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite’s Chloroquine Resistance Transporter

    PubMed Central

    Baker, Eileen S.; Webster, Michael W.; Lehane, Adele M.; Shafik, Sarah H.; Martin, Rowena E.

    2016-01-01

    Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite’s digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite’s hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite’s survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite’s hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite

  4. Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010–2012

    PubMed Central

    Okoth, Sheila Akinyi; Arrospide, Nancy; Gonzalez, Rommell V.; Sánchez, Juan F.; Macedo, Silvia; Conde, Silvia; Tapia, L. Lorena; Salas, Carola; Gamboa, Dionicia; Herrera, Yeni; Edgel, Kimberly A.; Udhayakumar, Venkatachalam; Lescano, Andrés G.

    2015-01-01

    During 2010–2012, an outbreak of 210 cases of malaria occurred in Tumbes, in the northern coast of Peru, where no Plasmodium falciparum malaria case had been reported since 2006. To identify the source of the parasite causing this outbreak, we conducted a molecular epidemiology investigation. Microsatellite typing showed an identical genotype in all 54 available isolates. This genotype was also identical to that of parasites isolated in 2010 in the Loreto region of the Peruvian Amazon and closely related to clonet B, a parasite lineage previously reported in the Amazon during 1998–2000. These findings are consistent with travel history of index case-patients. DNA sequencing revealed mutations in the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr1 loci, which are strongly associated with resistance to chloroquine and sulfadoxine/pyrimethamine, and deletion of the Pfhrp2 gene. These results highlight the need for timely molecular epidemiology investigations to trace the parasite source during malaria reintroduction events. PMID:25897626

  5. Efficacy of chloroquine for the treatment of uncomplicated Plasmodium falciparum malaria in Honduras.

    PubMed

    Mejia Torres, Rosa Elena; Banegas, Engels Ilich; Mendoza, Meisy; Diaz, Cesar; Bucheli, Sandra Tamara Mancero; Fontecha, Gustavo A; Alam, Md Tauqeer; Goldman, Ira; Udhayakumar, Venkatachalam; Zambrano, Jose Orlinder Nicolas

    2013-05-01

    Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated using the Pan American Health Organization-World Health Organization protocol with a follow-up of 28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations (pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras.

  6. Enhancement of the antimalarial efficacy of amodiaquine by chlorpheniramine in vivo.

    PubMed

    Sowunmi, Akintunde; Gbotosho, Grace O; Happi, Christian T; Adedeji, Ahmed A; Bolaji, Olayinka M; Fehintola, Fatai A; Fateye, Babasola A; Oduola, Ayoade M J

    2007-06-01

    Resistance in Plasmodium falciparum to amodiaquine (AQ) can be reversed in vitro with with antihistaminic and tricyclic antidepressant compounds, but its significance in vivo is unclear. The present report presents the enhancement of the antimalarial efficacy of AQ by chlorpheniramine, an H1 receptor antagonist that reverses chloroquine (CQ) resistance in vitro and enhances its efficacy in vivo, in five children who failed CQ and/or AQ treatment, and who were subsequently retreated and cured with a combination of AQ plus CP, despite the fact that parasites infecting the children harboured mutant pfcrtT76 and pfmdr1Y86 alleles associated with AQ resistance. This suggests a potential clinical application of the reversal phenomenon.

  7. Evaluation of antimalarial resistance marker polymorphism in returned migrant workers in China.

    PubMed

    Feng, Jun; Li, Jun; Yan, He; Feng, Xinyu; Xia, Zhigui

    2015-01-01

    Imported malaria has been a great challenge for public health in China due to decreased locally transmitted cases and frequent exchange worldwide. Plasmodium falciparum has been mainly responsible for the increasing impact. Currently, artesunate plus amodiaquine, one of the artemisinin combination therapies recommended by the World Health Organization, has been mainly used against uncomplicated P. falciparum malaria in China. However, drug resistance marker polymorphism in returning migrant workers has not been demonstrated. Here, we have evaluated the prevalence of pfmdr1 and pfcrt polymorphisms, as well as the K13 propeller gene, a molecular marker of artemisinin resistance, in migrant workers returned from Ghana to Shanglin County, Guangxi Province, China, in 2013. A total of 118 blood samples were randomly selected and used for the assay. Mutations of the pfmdr1 gene that covered codons 86, 184, 1034, and 1246 were found in 11 isolates. Mutations at codon N86Y (9.7%) were more frequent than at others, and Y(86)Y(184)S(1034)D(1246) was the most prevalent (63.6%) of the four haplotypes. Mutations of the pfcrt gene that covered codons 74, 75, and 76 were observed in 17 isolates, and M(74)N(75)T(76) was common (70.6%) in three haplotypes. Eight different genotypes of the K13 propeller were first observed in 10 samples in China, 2 synonymous mutations (V487V and A627A) and 6 nonsynonymous mutations. C580Y was the most prevalent (2.7%) in all the samples. The data presented might be helpful for enrichment of molecular surveillance of antimalarial resistance and will be useful for developing and updating antimalarial guidance in China.

  8. New molecular detection methods of malaria parasites with multiple genes from genomes.

    PubMed

    Gupta, Himanshu; Srivastava, Shikha; Chaudhari, Sima; Vasudevan, Thanvanthri G; Hande, Manjunath H; D'souza, Sydney C; Umakanth, Shashikiran; Satyamoorthy, Kapaettu

    2016-08-01

    For the effective control of malaria, development of sensitive, accurate and rapid tool to diagnose and manage the disease is essential. In humans subjects, the severe form of malaria is caused by Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) and there is need to identify these parasites in acute, chronic and latent (during and post-infection) stages of the disease. In this study, we report a species specific and sensitive diagnostic method for the detection of Pf and Pv in humans. First, we identified intra and intergenic multiloci short stretch of 152 (PfMLS152) and 110 (PvMLS110) nucleotides which is present up to 44 and 34 times in the genomes of Pf and Pv respectively. We developed the single-step amplification-based method using isolated DNA or from lysed red blood cells for the detection of the two malaria parasites. The limit of detection of real-time polymerase chain reaction based assays were 0.1copyof parasite/μl for PfMLS152 and PvMLS110 target sequences. Next, we have tested 250 clinically suspected cases of malaria to validate the method. Sensitivity and specificity for both targets were 100% compared to the quantitative buffy coat microscopy analysis and real-time PCR (Pf-chloroquine resistance transporter (PfCRT) and Pv-lactate dehydrogenase (PvLDH)) based assays. The sensitivity of microscopy and real-time PCR (PfCRT and PvLDH primers) assays were 80.63%; 95%CI 75.22%-85.31%; p<0.05 and 97.61%; 95%CI 94.50%-99.21%; p<0.05 in detecting malaria infection respectively when compared to PfMLS152 and PvMLS110 targets to identify malaria infection in patients. These improved assays may have potential applications in evaluating malaria in asymptomatic patients, treatment, blood donors and in vaccine studies.

  9. Limited genetic variation in the Plasmodium falciparum heme detoxification protein (HDP).

    PubMed

    Vinayak, Sumiti; Rathore, Dharmendar; Kariuki, Simon; Slutsker, Laurence; Shi, Ya Ping; Villegas, Leopoldo; Escalante, Ananias A; Udhayakumar, Venkatachalam

    2009-03-01

    Malaria parasites infecting host red blood cells degrade hemoglobin by detoxifying heme into hemozoin. This conversion of heme to hemozoin is performed by a potent protein called heme detoxification protein (HDP), making HDP an attractive target for antimalarial drug development. We studied the genetic variation in Plasmodium falciparum HDP and also investigated if HDP due to its involvement in the heme detoxification pathway is under any potential chloroquine (CQ) selection pressure. We sequenced the complete HDP gene encompassing three exons and two introns (AT and ATTT repeats in intron 1; AT repeats in intron 2) from five P. falciparum laboratory strains with known CQ sensitivity and 50 field isolates from Venezuela (n=26) and Kenya (n=24), with high levels of CQ resistance. Sequencing revealed two mutations, C41F and F91L in exon 1 and exon 2, respectively. The F41 mutation was present only in the CQ sensitive (CQS) HB3 strain. However, all the isolates harbored the 91L mutation, except for the CQS 3D7 strain. The sequencing of the intron 2 region revealed no variation in the number of AT repeats. In contrast, there was a wide variation in the AT and ATTT repeats in intron 1. Overall with respect to the intron 1 repeats, the Venezuelan isolates (Expected heterozygosity, He=0.685) showed less genetic variation as compared to the Kenyan isolates (He=0.986). Furthermore, we also genotyped the 72-76 codons of the pfcrt gene but did not observe any correlation of the pfcrt CQ resistant genotypes (SVMNT or CVIET) with variation in the HDP, thus indicating HDP not to be under any CQ selection pressure. In conclusion, HDP is a conserved target for future antimalarial development.

  10. Akt Phosphorylation and PI (3, 4, 5) P3 Binding Coordinately Inhibit the Tumor Suppressive Activity of Merlin

    DTIC Science & Technology

    2010-02-01

    K76R K364R K455R K543R FITC DAPI Merge EQLNE LKTE IEALK R KEADQ LKQD LQEARR RRLLQ MKEE ATMAN R DTAVW LKMD KKVLD R Figure 4 0 In te rc el lu la... Xiaoling Tang, 1 Luca M. Neri, 2 and Keqiang Ye 1 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia...l e t t e r s Akt phosphorylation regulates the tumour-suppressor merlin through ubiquitination and degradation Xiaoling Tang1, Sung-Wuk Jang1

  11. Reference Guide for Federal Government Acquisition.

    DTIC Science & Technology

    1983-09-01

    ELEMENT, PROJECT. TAWK ARA A &WORK UNIT NUNGERS Naval Postgraduate School Monterey, California 93943 ’I.I CONTROLLING OPIPICE RARE AUG ADDRESS 12...78 7 -. -- - -NOW I. uh!.gkglJ..I2! Purchase requests, which must be controlled through a sometimes long and ccmplex acquisition process...Q12eria 2Z 2.A" JZ21J1lSS iiA sI=Ag = S. 2rUnb gle, 3 March 1966 2. Cffice cf Managesent and Budget Circular No. k-76 (Revised), Subject: 1 s1jlgi I iurig

  12. Malaria prevalence in Nias District, North Sumatra Province, Indonesia

    PubMed Central

    Syafruddin, Din; Asih, Puji BS; Wahid, Isra; Dewi, Rita M; Tuti, Sekar; Laowo, Idaman; Hulu, Waozidohu; Zendrato, Pardamean; Laihad, Ferdinand; Shankar, Anuraj H

    2007-01-01

    Background The Nias district of the North Sumatra Province of Indonesia has long been known to be endemic for malaria. Following the economic crisis at the end of 1998 and the subsequent tsunami and earthquake, in December 2004 and March 2005, respectively, the malaria control programme in the area deteriorated. The present study aims to provide baseline data for the establishment of a suitable malaria control programme in the area and to analyse the frequency distribution of drug resistance alleles associated with resistance to chloroquine and sulphadoxine-pyrimethamine. Methods Malariometric and entomology surveys were performed in three subdistricts. Thin and thick blood smears were stained with Giemsa and examined under binocular light microscopy. Blood blots on filter paper were also prepared for isolation of parasite and host DNA to be used for molecular analysis of band 3 (SAO), pfcrt, pfmdr1, dhfr, and dhps. In addition, haemoglobin measurement was performed in the second and third surveys for the subjects less than 10 years old. Results Results of the three surveys revealed an average slide positivity rate of 8.13%, with a relatively higher rate in certain foci. Host genetic analysis, to identify the Band 3 deletion associated with Southeast Asian Ovalocytosis (SAO), revealed an overall frequency of 1.0% among the 1,484 samples examined. One hundred six Plasmodium falciparum isolates from three sub-districts were successfully analysed. Alleles of the dhfr and dhps genes associated with resistance to sulphadoxine-pyrimethamine, dhfr C59R and S108N, and dhps A437G and K540E, were present at frequencies of 52.2%, 82.5%, 1.18% and 1.18%, respectively. The pfmdr1 alleles N86Y and N1042D, putatively associated with mefloquine resistance, were present at 31.4% and 2%, respectively. All but one sample carried the pfcrt 76T allele associated with chloroquine resistance. Entomologic surveys identified three potential anopheline vectors in the area, Anopheles

  13. A HECT Ubiquitin-Protein Ligase as a Novel Candidate Gene for Altered Quinine and Quinidine Responses in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Cyrklaff, Marek; Mu, Jianbing; Ferdig, Michael T.; Stein, Wilfred D.; Lanzer, Michael

    2014-01-01

    The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors. PMID:24830312

  14. Genome-wide association analysis identifies genetic loci associated with resistance to multiple antimalarials in Plasmodium falciparum from China-Myanmar border

    PubMed Central

    Wang, Zenglei; Cabrera, Mynthia; Yang, Jingyun; Yuan, Lili; Gupta, Bhavna; Liang, Xiaoying; Kemirembe, Karen; Shrestha, Sony; Brashear, Awtum; Li, Xiaolian; Porcella, Stephen F.; Miao, Jun; Yang, Zhaoqing; Su, Xin-zhuan; Cui, Liwang

    2016-01-01

    Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr. Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine – an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy. PMID:27694982

  15. Clonal Outbreak of Plasmodium falciparum Infection in Eastern Panama

    PubMed Central

    Obaldia, Nicanor; Baro, Nicholas K.; Calzada, Jose E.; Santamaria, Ana M.; Daniels, Rachel; Wong, Wesley; Chang, Hsiao-Han; Hamilton, Elizabeth J.; Arevalo-Herrera, Myriam; Herrera, Socrates; Wirth, Dyann F.; Hartl, Daniel L.; Marti, Matthias; Volkman, Sarah K.

    2015-01-01

    Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated. PMID:25336725

  16. Combination therapy counteracts the enhanced transmission of drug-resistant malaria parasites to mosquitoes.

    PubMed

    Hallett, Rachel L; Sutherland, Colin J; Alexander, Neal; Ord, Rosalynn; Jawara, Musa; Drakeley, Chris J; Pinder, Margaret; Walraven, Gijs; Targett, Geoffrey A T; Alloueche, Ali

    2004-10-01

    Malaria parasites carrying genes conferring resistance to antimalarials are thought to have a selective advantage which leads to higher rates of transmissibility from the drug-treated host. This is a likely mechanism for the increasing prevalence of parasites with resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine in sub-Saharan Africa. Combination therapy is the key strategy being implemented to reduce the impact of resistance, but its effect on the transmission of genetically resistant parasites from treated patients to mosquito vectors has not been measured directly. In a trial comparing CQ monotherapy to the combination CQ plus artesunate (AS) in Gambian children with uncomplicated falciparum malaria, we measured transmissibility by feeding Anopheles gambiae mosquitoes with blood from 43 gametocyte-positive patients through a membrane. In the CQ-treated group, gametocytes from patients carrying parasites with the CQ resistance-associated allele pfcrt-76T prior to treatment produced infected mosquitoes with 38 times higher Plasmodium falciparum oocyst burdens than mosquitoes fed on gametocytes from patients infected with sensitive parasites (P < 0.001). Gametocytes from parasites carrying the resistance-associated allele pfmdr1-86Y produced 14-fold higher oocyst burdens than gametocytes from patients infected with sensitive parasites (P = 0.011). However, parasites carrying either of these resistance-associated alleles pretreatment were not associated with higher mosquito oocyst burdens in the CQ-AS-treated group. Thus, combination therapy overcomes the transmission advantage enjoyed by drug-resistant parasites.

  17. A Whole Cell Pathway Screen Reveals Seven Novel Chemosensitizers to Combat Chloroquine Resistant Malaria

    PubMed Central

    Ch'ng, Jun-Hong; Mok, Sachel; Bozdech, Zbynek; Lear, Martin James; Boudhar, Aicha; Russell, Bruce; Nosten, Francois; Tan, Kevin Shyong-Wei

    2013-01-01

    Due to the widespread prevalence of resistant parasites, chloroquine (CQ) was removed from front-line antimalarial chemotherapy in the 1990s despite its initial promise of disease eradication. Since then, resistance-conferring mutations have been identified in transporters such as the PfCRT, that allow for the efflux of CQ from its primary site of action, the parasite digestive vacuole. Chemosensitizing/chemoreversing compounds interfere with the function of these transporters thereby sensitizing parasites to CQ once again. However, compounds identified thus far have disappointing in vivo efficacy and screening for alternative candidates is required to revive this strategy. In this study, we propose a simple and direct means to rapidly screen for such compounds using a fluorescent-tagged CQ molecule. When this screen was applied to a small library, seven novel chemosensitizers (octoclothepin, methiothepin, metergoline, loperamide, chlorprothixene, L-703,606 and mibefradil) were quickly elucidated, including two which showed greater potency than the classical chemosensitizers verapamil and desipramine. PMID:23615863

  18. A broad analysis of resistance development in the malaria parasite.

    PubMed

    Corey, Victoria C; Lukens, Amanda K; Istvan, Eva S; Lee, Marcus C S; Franco, Virginia; Magistrado, Pamela; Coburn-Flynn, Olivia; Sakata-Kato, Tomoyo; Fuchs, Olivia; Gnädig, Nina F; Goldgof, Greg; Linares, Maria; Gomez-Lorenzo, Maria G; De Cózar, Cristina; Lafuente-Monasterio, Maria Jose; Prats, Sara; Meister, Stephan; Tanaseichuk, Olga; Wree, Melanie; Zhou, Yingyao; Willis, Paul A; Gamo, Francisco-Javier; Goldberg, Daniel E; Fidock, David A; Wirth, Dyann F; Winzeler, Elizabeth A

    2016-06-15

    Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.

  19. A broad analysis of resistance development in the malaria parasite

    PubMed Central

    Corey, Victoria C.; Lukens, Amanda K.; Istvan, Eva S.; Lee, Marcus C. S.; Franco, Virginia; Magistrado, Pamela; Coburn-Flynn, Olivia; Sakata-Kato, Tomoyo; Fuchs, Olivia; Gnädig, Nina F.; Goldgof, Greg; Linares, Maria; Gomez-Lorenzo, Maria G.; De Cózar, Cristina; Lafuente-Monasterio, Maria Jose; Prats, Sara; Meister, Stephan; Tanaseichuk, Olga; Wree, Melanie; Zhou, Yingyao; Willis, Paul A.; Gamo, Francisco-Javier; Goldberg, Daniel E.; Fidock, David A.; Wirth, Dyann F.; Winzeler, Elizabeth A.

    2016-01-01

    Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance. PMID:27301419

  20. Mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents.

    PubMed

    Gemma, Sandra; Camodeca, Caterina; Brindisi, Margherita; Brogi, Simone; Kukreja, Gagan; Kunjir, Sanil; Gabellieri, Emanuele; Lucantoni, Leonardo; Habluetzel, Annette; Taramelli, Donatella; Basilico, Nicoletta; Gualdani, Roberta; Tadini-Buoninsegni, Francesco; Bartolommei, Gianluca; Moncelli, Maria Rosa; Martin, Rowena E; Summers, Robert L; Lamponi, Stefania; Savini, Luisa; Fiorini, Isabella; Valoti, Massimo; Novellino, Ettore; Campiani, Giuseppe; Butini, Stefania

    2012-12-13

    The intramolecular hydrogen bond formed between a protonated amine and a neighboring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum ) and in vivo (against Plasmodium berghei ). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasite's "chloroquine resistance transporter" (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising analogue of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei -infected mice.

  1. Baseline in vitro efficacy of ACT component drugs on Plasmodium falciparum clinical isolates from Mali.

    PubMed

    Kaddouri, Halima; Djimdé, Abdoulaye; Dama, Souleymane; Kodio, Aly; Tekete, Mamadou; Hubert, Véronique; Koné, Aminatou; Maiga, Hamma; Yattara, Oumar; Fofana, Bakary; Sidibe, Bakary; Sangaré, Cheick P O; Doumbo, Ogobara; Le Bras, Jacques

    2008-06-01

    In vitro susceptibility to antimalarial drugs of Malian Plasmodium falciparum isolates collected between 2004 and 2006 was studied. Susceptibility to chloroquine and to three artemisinin-based combination therapy (ACT) component drugs was assessed as a first, to our knowledge, in vitro susceptibility study in Mali. Overall 96 Malian isolates (51 from around Bamako and 45 collected from French travellers returning from Mali) were cultivated in a CO(2) incubator. Fifty percent inhibitory concentrations (IC(50)s) were measured by either hypoxanthine incorporation or Plasmodium lactate dehydrogenase (pLDH) ELISA. Although the two sets of data were generated with different methods, the global IC(50) distributions showed parallel trends. A good concordance of resistance phenotype with pfcrt 76T mutant genotype was found within the sets of clinical isolates tested. We confirm a high prevalence of P. falciparum in vitro resistance to chloroquine in Mali (60-69%). While some isolates showed IC(50)s close to the cut-off for resistance to monodesethylamodiaquine, no decreased susceptibility to dihydroartemisinin or lumefantrine was detected. This study provides baseline data for P. falciparum in vitro susceptibility to ACT component drugs in Mali.

  2. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

    PubMed Central

    Veiga, M. Isabel; Dhingra, Satish K.; Henrich, Philipp P.; Straimer, Judith; Gnädig, Nina; Uhlemann, Anne-Catrin; Martin, Rowena E.; Lehane, Adele M.; Fidock, David A.

    2016-01-01

    Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes. PMID:27189525

  3. Genomic sequencing of Plasmodium falciparum malaria parasites from Senegal reveals the demographic history of the population.

    PubMed

    Chang, Hsiao-Han; Park, Daniel J; Galinsky, Kevin J; Schaffner, Stephen F; Ndiaye, Daouda; Ndir, Omar; Mboup, Souleymane; Wiegand, Roger C; Volkman, Sarah K; Sabeti, Pardis C; Wirth, Dyann F; Neafsey, Daniel E; Hartl, Daniel L

    2012-11-01

    Malaria is a deadly disease that causes nearly one million deaths each year. To develop methods to control and eradicate malaria, it is important to understand the genetic basis of Plasmodium falciparum adaptations to antimalarial treatments and the human immune system while taking into account its demographic history. To study the demographic history and identify genes under selection more efficiently, we sequenced the complete genomes of 25 culture-adapted P. falciparum isolates from three sites in Senegal. We show that there is no significant population structure among these Senegal sampling sites. By fitting demographic models to the synonymous allele-frequency spectrum, we also estimated a major 60-fold population expansion of this parasite population ∼20,000-40,000 years ago. Using inferred demographic history as a null model for coalescent simulation, we identified candidate genes under selection, including genes identified before, such as pfcrt and PfAMA1, as well as new candidate genes. Interestingly, we also found selection against G/C to A/T changes that offsets the large mutational bias toward A/T, and two unusual patterns: similar synonymous and nonsynonymous allele-frequency spectra, and 18% of genes having a nonsynonymous-to-synonymous polymorphism ratio >1.

  4. Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum.

    PubMed

    Garg, Aprajita; Wesolowski, Donna; Alonso, Dulce; Deitsch, Kirk W; Ben Mamoun, Choukri; Altman, Sidney

    2015-09-22

    Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome.

  5. Analyzing Thiol-Dependent Redox Networks in the Presence of Methylene Blue and Other Antimalarial Agents with RT-PCR-Supported in silico Modeling

    PubMed Central

    Zirkel, J.; Cecil, A.; Schäfer, F.; Rahlfs, S.; Ouedraogo, A.; Xiao, K.; Sawadogo, S.; Coulibaly, B.; Becker, K.; Dandekar, T.

    2012-01-01

    Background In the face of growing resistance in malaria parasites to drugs, pharmacological combination therapies are important. There is accumulating evidence that methylene blue (MB) is an effective drug against malaria. Here we explore the biological effects of both MB alone and in combination therapy using modeling and experimental data. Results We built a model of the central metabolic pathways in P. falciparum. Metabolic flux modes and their changes under MB were calculated by integrating experimental data (RT-PCR data on mRNAs for redox enzymes) as constraints and results from the YANA software package for metabolic pathway calculations. Several different lines of MB attack on Plasmodium redox defense were identified by analysis of the network effects. Next, chloroquine resistance based on pfmdr/and pfcrt transporters, as well as pyrimethamine/sulfadoxine resistance (by mutations in DHF/DHPS), were modeled in silico. Further modeling shows that MB has a favorable synergism on antimalarial network effects with these commonly used antimalarial drugs. Conclusions Theoretical and experimental results support that methylene blue should, because of its resistance-breaking potential, be further tested as a key component in drug combination therapy efforts in holoendemic areas. PMID:23236254

  6. Thermodynamic Temperature Measurement to the Indium Point Based on Radiance Comparison

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Y.; Yamada, Y.

    2017-04-01

    A multi-national project (the EMRP InK project) was completed recently, which successfully determined the thermodynamic temperatures of several of the high-temperature fixed points above the copper point. The National Metrology Institute of Japan contributed to this project with its newly established absolute spectral radiance calibration capability. In the current study, we have extended the range of thermodynamic temperature measurement to below the copper point and measured the thermodynamic temperatures of the indium point (T_{90} = 429.748 5 K), tin point (505.078 K), zinc point (692.677 K), aluminum point (933.473 K) and the silver point (1 234.93 K) by radiance comparison against the copper point, with a set of radiation thermometers having center wavelengths ranging from 0.65 μm to 1.6 μm. The copper-point temperature was measured by the absolute radiation thermometer which was calibrated by radiance method traceable to the electrical substitution cryogenic radiometer. The radiance of the fixed-point blackbodies was measured by standard radiation thermometers whose spectral responsivity and nonlinearity are precisely evaluated, and then the thermodynamic temperatures were determined from radiance ratios to the copper point. The values of T-T_{90} for the silver-, aluminum-, zinc-, tin- and indium-point cells were determined as -4 mK (U = 104 mK, k=2), -99 mK (88 mK), -76 mK (76 mK), -68 mK (163 mK) and -42 mK (279 mK), respectively.

  7. Polymorphism of Plasmodium falciparum Na+/H+ exchanger is indicative of a low in vitro quinine susceptibility in isolates from Viet Nam

    PubMed Central

    2011-01-01

    Background The Plasmodium falciparum NA+/H+ exchanger (pfnhe1, gene PF13_0019) has recently been proposed to influence quinine (QN) susceptibility. However, its contribution to QN resistance seems to vary geographically depending on the genetic background of the parasites. Here, the role of this gene was investigated in in vitro QN susceptibility of isolates from Viet Nam. Method Ninety-eight isolates were obtained from three different regions of the Binh Phuoc and Dak Nong bordering Cambodia provinces during 2006-2008. Among these, 79 were identified as monoclonal infection and were genotyped at the microsatellite pfnhe1 ms4760 locus and in vitro QN sensitivity data were obtained for 51 isolates. Parasite growth was assessed in the field using the HRP2 immunodetection assay. Results Significant associations were found between polymorphisms at pfnhe1 microsatellite ms4760 and susceptibility to QN. Isolates with two or more DNNND exhibited much lower susceptibility to QN than those harbouring zero or one DNNND repeats (median IC50 of 682 nM versus median IC50 of 300 nM; p = 0.0146) while isolates with one NHNDNHNNDDD repeat presented significantly reduced QN susceptibility than those who had two (median IC50 of 704 nM versus median IC50 of 375 nM; p < 0.01). These QNR associated genotype features were mainly due to the over representation of profile 7 among isolates (76.5%). The majority of parasites had pfcrt76T and wild-type pfmdr1 (> 95%) thus preventing analysis of associations with these mutations. Interestingly, area with the highest median QN IC50 showed also the highest percentage of isolates carrying the pfnhe1 haplotype 7. Conclusions The haplotype 7 which is the typical Asian profile is likely well-adapted to high drug pressure in this area and may constitute a good genetic marker to evaluate the dissemination of QNR in this part of the world. PMID:21669011

  8. Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

    PubMed

    Moers, A P H A; Hallett, R L; Burrow, R; Schallig, H D F H; Sutherland, C J; van Amerongen, A

    2015-01-01

    The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries.

  9. Parasite Polymorphism and Severe Malaria in Dakar (Senegal): A West African Urban Area

    PubMed Central

    Bob, Ndeye Sakha; Diop, Bernard Marcel; Renaud, Francois; Marrama, Laurence; Durand, Patrick; Tall, Adama; Ka, Boubacar; Ekala, Marie Therese; Bouchier, Christiane; Mercereau-Puijalon, Odile; Jambou, Ronan

    2010-01-01

    Background Transmission of malaria in West African urban areas is low and healthcare facilities are well organized. However, malaria mortality remains high. We conducted a survey in Dakar with the general objective to establish who died from severe malaria (SM) in urban areas (particularly looking at the age-groups) and to compare parasite isolates associated with mild or severe malaria. Methodology/Principal Findings The current study included mild- (MM) and severe malaria (SM) cases, treated in dispensaries (n = 2977) and hospitals (n = 104), We analysed Pfdhfr/Pfcrt-exon2 and nine microsatellite loci in 102 matched cases of SM and MM. Half of the malaria cases recorded at the dispensaries and 87% of SM cases referred to hospitals, occurred in adults, although adults only accounted for 26% of all dispensary consultations. This suggests that, in urban settings, whatever the reason for this adult over-representation, health-workers are forced to take care of increasing numbers of malaria cases among adults. Inappropriate self treatment and mutations in genes associated with drug resistance were found associated with SM in adults. SM was also associated with a specific pool of isolates highly polymorphic and different from those associated with MM. Conclusion In this urban setting, adults currently represent one of the major groups of patients attending dispensaries for malaria treatment. For these patients, despite the low level of transmission, SM was associated with a specific and highly polymorphic pool of parasites which may have been selected by inappropriate treatment. PMID:20352101

  10. Detection of Single-Nucleotide Polymorphisms in Plasmodium falciparum by PCR Primer Extension and Lateral Flow Immunoassay

    PubMed Central

    Moers, A. P. H. A.; Hallett, R. L.; Burrow, R.; Schallig, H. D. F. H.; Sutherland, C. J.

    2014-01-01

    The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries. PMID:25367901

  11. Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers

    PubMed Central

    2013-01-01

    Background The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. Methods Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC50 estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. Results Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC50 estimates, and carried the CVIET haplotype at codons 72–76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC50 estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. Conclusions This study describes the

  12. Molecular Evidence of Increased Resistance to Anti-Folate Drugs in Plasmodium falciparum in North-East India: A Signal for Potential Failure of Artemisinin Plus Sulphadoxine-Pyrimethamine Combination Therapy

    PubMed Central

    Mohapatra, Pradyumna Kishore; Sarma, Devojit Kumar; Prakash, Anil; Bora, Khukumoni; Ahmed, Md. Atique; Sarma, Bibhas; Goswami, Basanta Kumar; Bhattacharyya, Dibya Ranjan; Mahanta, Jagadish

    2014-01-01

    North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in

  13. Molecular evidence of increased resistance to anti-folate drugs in Plasmodium falciparum in North-East India: a signal for potential failure of artemisinin plus sulphadoxine-pyrimethamine combination therapy.

    PubMed

    Mohapatra, Pradyumna Kishore; Sarma, Devojit Kumar; Prakash, Anil; Bora, Khukumoni; Ahmed, Md Atique; Sarma, Bibhas; Goswami, Basanta Kumar; Bhattacharyya, Dibya Ranjan; Mahanta, Jagadish

    2014-01-01

    North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76 T), while 68% had mutant pfmdr-1 genotype (86 Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51 I/59 R/108 N/164 L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine

  14. Measurements of CP-Violating Asymmetries and Branching Fractions in B Meson Decays to η'K

    NASA Astrophysics Data System (ADS)

    Aubert, B.; Barate, R.; Boutigny, D.; Gaillard, J.-M.; Hicheur, A.; Karyotakis, Y.; Lees, J. P.; Robbe, P.; Tisserand, V.; Zghiche, A.; Palano, A.; Pompili, A.; Chen, J. C.; Qi, N. D.; Rong, G.; Wang, P.; Zhu, Y. S.; Eigen, G.; Ofte, I.; Stugu, B.; Abrams, G. S.; Borgland, A. W.; Breon, A. B.; Brown, D. N.; Button-Shafer, J.; Cahn, R. N.; Charles, E.; Day, C. T.; Gill, M. S.; Gritsan, A. V.; Groysman, Y.; Jacobsen, R. G.; Kadel, R. W.; Kadyk, J.; Kerth, L. T.; Kolomensky, Yu. G.; Kral, J. F.; Kukartsev, G.; Leclerc, C.; Levi, M. E.; Lynch, G.; Mir, L. M.; Oddone, P. J.; Orimoto, T. J.; Pripstein, M.; Roe, N. A.; Romosan, A.; Ronan, M. T.; Shelkov, V. G.; Telnov, A. V.; Wenzel, W. A.; Harrison, T. J.; Hawkes, C. M.; Knowles, D. J.; Penny, R. C.; Watson, A. T.; Watson, N. K.; Deppermann, T.; Goetzen, K.; Koch, H.; Lewandowski, B.; Pelizaeus, M.; Peters, K.; Schmuecker, H.; Steinke, M.; Barlow, N. R.; Bhimji, W.; Boyd, J. T.; Chevalier, N.; Cottingham, W. N.; Mackay, C.; Wilson, F. F.; Hearty, C.; Mattison, T. S.; McKenna, J. A.; Thiessen, D.; Kyberd, P.; McKemey, A. K.; Blinov, V. E.; Bukin, A. D.; Golubev, V. B.; Ivanchenko, V. N.; Kravchenko, E. A.; Onuchin, A. P.; Serednyakov, S. I.; Skovpen, Yu. I.; Solodov, E. P.; Yushkov, A. N.; Best, D.; Chao, M.; Kirkby, D.; Lankford, A. J.; Mandelkern, M.; McMahon, S.; Mommsen, R. K.; Roethel, W.; Stoker, D. P.; Buchanan, C.; Hadavand, H. K.; Hill, E. J.; Macfarlane, D. B.; Paar, H. P.; Rahatlou, Sh.; Schwanke, U.; Sharma, V.; Berryhill, J. W.; Campagnari, C.; Dahmes, B.; Kuznetsova, N.; Levy, S. L.; Long, O.; Lu, A.; Mazur, M. A.; Richman, J. D.; Verkerke, W.; Beringer, J.; Eisner, A. M.; Heusch, C. A.; Lockman, W. S.; Schalk, T.; Schmitz, R. E.; Schumm, B. A.; Seiden, A.; Turri, M.; Walkowiak, W.; Williams, D. C.; Wilson, M. G.; Albert, J.; Chen, E.; Dorsten, M. P.; Dubois-Felsmann, G. P.; Dvoretskii, A.; Hitlin, D. G.; Narsky, I.; Porter, F. C.; Ryd, A.; Samuel, A.; Yang, S.; Jayatilleke, S.; Mancinelli, G.; Meadows, B. T.; Sokoloff, M. D.; Barillari, T.; Blanc, F.; Bloom, P.; Clark, P. J.; Ford, W. T.; Nauenberg, U.; Olivas, A.; Rankin, P.; Roy, J.; Smith, J. G.; van Hoek, W. C.; Zhang, L.; Harton, J. L.; Hu, T.; Soffer, A.; Toki, W. H.; Wilson, R. J.; Zhang, J.; Altenburg, D.; Brandt, T.; Brose, J.; Colberg, T.; Dickopp, M.; Dubitzky, R. S.; Hauke, A.; Lacker, H. M.; Maly, E.; Müller-Pfefferkorn, R.; Nogowski, R.; Otto, S.; Schubert, K. R.; Schwierz, R.; Spaan, B.; Wilden, L.; Bernard, D.; Bonneaud, G. R.; Brochard, F.; Cohen-Tanugi, J.; Thiebaux, Ch.; Vasileiadis, G.; Verderi, M.; Khan, A.; Lavin, D.; Muheim, F.; Playfer, S.; Swain, J. E.; Tinslay, J.; Bozzi, C.; Piemontese, L.; Sarti, A.; Treadwell, E.; Anulli, F.; Baldini-Ferroli, R.; Calcaterra, A.; de Sangro, R.; Falciai, D.; Finocchiaro, G.; Patteri, P.; Peruzzi, I. M.; Piccolo, M.; Zallo, A.; Buzzo, A.; Contri, R.; Crosetti, G.; Lo Vetere, M.; Macri, M.; Monge, M. R.; Passaggio, S.; Pastore, F. C.; Patrignani, C.; Robutti, E.; Santroni, A.; Tosi, S.; Bailey, S.; Morii, M.; Aspinwall, M. L.; Bowerman, D. A.; Dauncey, P. D.; Egede, U.; Eschrich, I.; Morton, G. W.; Nash, J. A.; Sanders, P.; Taylor, G. P.; Grenier, G. J.; Lee, S.-J.; Mallik, U.; Cochran, J.; Crawley, H. B.; Lamsa, J.; Meyer, W. T.; Prell, S.; Rosenberg, E. I.; Yi, J.; Davier, M.; Grosdidier, G.; Höcker, A.; Laplace, S.; Le Diberder, F.; Lepeltier, V.; Lutz, A. M.; Petersen, T. C.; Plaszczynski, S.; Schune, M. H.; Tantot, L.; Wormser, G.; Bionta, R. M.; Brigljević, V.; Cheng, C. H.; Lange, D. J.; Wright, D. M.; Bevan, A. J.; Fry, J. R.; Gabathuler, E.; Gamet, R.; Kay, M.; Payne, D. J.; Sloane, R. J.; Touramanis, C.; Back, J. J.; Bellodi, G.; Harrison, P. F.; Shorthouse, H. W.; Strother, P.; Vidal, P. B.; Cowan, G.; Flaecher, H. U.; George, S.; Green, M. G.; Kurup, A.; Marker, C. E.; McMahon, T. R.; Ricciardi, S.; Salvatore, F.; Vaitsas, G.; Winter, M. A.; Brown, D.; Davis, C. L.; Allison, J.; Barlow, R. J.; Forti, A. C.; Hart, P. A.; Jackson, F.; Lafferty, G. D.; Lyon, A. J.; Weatherall, J. H.; Williams, J. C.; Farbin, A.; Jawahery, A.; Kovalskyi, D.; Lae, C. K.; Lillard, V.; Roberts, D. A.; Blaylock, G.; Dallapiccola, C.; Flood, K. T.; Hertzbach, S. S.; Kofler, R.; Koptchev, V. B.; Moore, T. B.; Staengle, H.; Willocq, S.; Cowan, R.; Sciolla, G.; Taylor, F.; Yamamoto, R. K.; Mangeol, D. J.; Milek, M.; Patel, P. M.; Lazzaro, A.; Palombo, F.; Bauer, J. M.; Cremaldi, L.; Eschenburg, V.; Godang, R.; Kroeger, R.; Reidy, J.; Sanders, D. A.; Summers, D. J.; Zhao, H. W.; Hast, C.; Taras, P.; Nicholson, H.; Cartaro, C.; Cavallo, N.; de Nardo, G.; Fabozzi, F.; Gatto, C.; Lista, L.; Paolucci, P.; Piccolo, D.; Sciacca, C.; Baak, M. A.; Raven, G.; Losecco, J. M.; Gabriel, T. A.; Brau, B.; Pulliam, T.; Brau, J.; Frey, R.; Iwasaki, M.; Potter, C. T.; Sinev, N. B.; Strom, D.; Torrence, E.; Colecchia, F.; Dorigo, A.; Galeazzi, F.; Margoni, M.; Morandin, M.; Posocco, M.; Rotondo, M.; Simonetto, F.; Stroili, R.; Tiozzo, G.; Voci, C.; Benayoun, M.; Briand, H.; Chauveau, J.; David, P.; de La Vaissière, Ch.; del Buono, L.; Hamon, O.; Leruste, Ph.; Ocariz, J.; Pivk, M.; Roos, L.; Stark, J.; T'jampens, S.; Manfredi, P. F.; Re, V.; Gladney, L.; Guo, Q. H.; Panetta, J.; Angelini, C.; Batignani, G.; Bettarini, S.; Bondioli, M.; Bucci, F.; Calderini, G.; Carpinelli, M.; Forti, F.; Giorgi, M. A.; Lusiani, A.; Marchiori, G.; Martinez-Vidal, F.; Morganti, M.; Neri, N.; Paoloni, E.; Rama, M.; Rizzo, G.; Sandrelli, F.; Walsh, J.; Haire, M.; Judd, D.; Paick, K.; Wagoner, D. E.; Danielson, N.; Elmer, P.; Lu, C.; Miftakov, V.; Olsen, J.; Smith, A. J.; Varnes, E. W.; Bellini, F.; Cavoto, G.; del Re, D.; Faccini, R.; Ferrarotto, F.; Ferroni, F.; Gaspero, M.; Leonardi, E.; Mazzoni, M. A.; Morganti, S.; Pierini, M.; Piredda, G.; Safai Tehrani, F.; Serra, M.; Voena, C.; Christ, S.; Wagner, G.; Waldi, R.; Adye, T.; de Groot, N.; Franek, B.; Geddes, N. I.; Gopal, G. P.; Olaiya, E. O.; Xella, S. M.; Aleksan, R.; Emery, S.; Gaidot, A.; Ganzhur, S. F.; Giraud, P.-F.; Hamel de Monchenault, G.; Kozanecki, W.; Langer, M.; London, G. W.; Mayer, B.; Schott, G.; Vasseur, G.; Yeche, Ch.; Zito, M.; Purohit, M. V.; Weidemann, A. W.; Yumiceva, F. X.; Aston, D.; Bartoldus, R.; Berger, N.; Boyarski, A. M.; Buchmueller, O. L.; Convery, M. R.; Coupal, D. P.; Dong, D.; Dorfan, J.; Dujmic, D.; Dunwoodie, W.; Field, R. C.; Glanzman, T.; Gowdy, S. J.; Grauges-Pous, E.; Hadig, T.; Halyo, V.; Hryn'ova, T.; Innes, W. R.; Jessop, C. P.; Kelsey, M. H.; Kim, P.; Kocian, M. L.; Langenegger, U.; Leith, D. W.; Luitz, S.; Luth, V.; Lynch, H. L.; Marsiske, H.; Menke, S.; Messner, R.; Muller, D. R.; O'Grady, C. P.; Ozcan, V. E.; Perazzo, A.; Perl, M.; Petrak, S.; Ratcliff, B. N.; Robertson, S. H.; Roodman, A.; Salnikov, A. A.; Schindler, R. H.; Schwiening, J.; Simi, G.; Snyder, A.; Soha, A.; Stelzer, J.; Su, D.; Sullivan, M. K.; Tanaka, H. A.; Va'Vra, J.; Wagner, S. R.; Weaver, M.; Weinstein, A. J.; Wisniewski, W. J.; Wright, D. H.; Young, C. C.; Burchat, P. R.; Meyer, T. I.; Roat, C.; Ahmed, S.; Ernst, J. A.; Bugg, W.; Krishnamurthy, M.; Spanier, S. M.; Eckmann, R.; Kim, H.; Ritchie, J. L.; Schwitters, R. F.; Izen, J. M.; Kitayama, I.; Lou, X. C.; Ye, S.; Bianchi, F.; Bona, M.; Gallo, F.; Gamba, D.; Borean, C.; Bosisio, L.; della Ricca, G.; Dittongo, S.; Grancagnolo, S.; Lanceri, L.; Poropat, P.; Vitale, L.; Vuagnin, G.; Panvini, R. S.; Banerjee, Sw.; Brown, C. M.; Fortin, D.; Jackson, P. D.; Kowalewski, R.; Roney, J. M.; Band, H. R.; Dasu, S.; Datta, M.; Eichenbaum, A. M.; Hu, H.; Johnson, J. R.; Liu, R.; di Lodovico, F.; Mohapatra, A. K.; Pan, Y.; Prepost, R.; Sekula, S. J.; von Wimmersperg-Toeller, J. H.; Wu, J.; Wu, S. L.; Yu, Z.; Neal, H.

    2003-10-01

    We present measurements of the branching fractions of the decays B+→η'K+ and B0→η'K0. For B0→η'K0S we also measure the time-dependent CP-violation parameters Sη'K0S and Cη'K0S, and for B+→η'K+ the time-integrated charge asymmetry Ach. The data sample corresponds to 88.9×106 BB¯ pairs produced by e+e- annihilation at the ϒ(4S). The results are B(B+→η'K+)=(76.9±3.5±4.4)×10-6, B(B0→η'K0)=(60.6±5.6±4.6)×10-6, Sη'K0S=0.02±0.34±0.03, Cη'K0S=0.10±0.22±0.04, and Ach=0.037±0.045±0.011.

  15. Spectroscopic, thermal analyses, structural and antibacterial studies on the interaction of some metals with ofloxacin

    NASA Astrophysics Data System (ADS)

    Zordok, W. A.; El-Shwiniy, W. H.; El-Attar, M. S.; Sadeek, S. A.

    2013-09-01

    Reaction between the fluoroquinolone antibacterial agent ofloxacin and V(IV), Zr(IV) and U(VI) in methanol and acetone was studied. The ability of ofloxacin to form metal complexes is high. The isolated solid complexes were characterized by elemental analysis, magnetic moment, conductance measurements, infrared, electronic, 1H NMR spectra and thermal investigation. In all complexes the ofloxacin ligand is coordinated through the pyridone and carboxylate oxygen forming 1:2 M:HOfl complexes. The calculated bond length and force constant, F(Udbnd O), in the uranyl complex are 1.73 Å and 640.83 N m-1, respectively. The metal-ligand binding of the V(IV) and Zr(IV) complexes was predicted by using the density functional theory (DFT) at the B3LYP-CEP-31G level of theory and total energy, dipole moment estimation of different V(IV) and Zr(IV) ofloxacin structures. All the synthesized complexes exhibited higher biocidal activity against S. aureus K1, Bacillus subtilis K22, Br. otitidis K76, Escherichia coli K32, Pseudomonas aeruginosa SW1 and Klebsiella oxytoca K42. compared to parent compounds and standard drugs.

  16. Identification of novel resistance gene sources to cowpea aphid (Aphis craccivora Koch) in cowpea (Vigna unguiculata L.).

    PubMed

    Aliyu, H; Ishiyaku, M F

    2013-08-01

    The development of cowpea aphid larvae was monitored on seven cowpea genotypes (IAR-48, TVu-15866, IT84S-2246-4, SAKA BABBA SATA, IT90K-76, KANANNADO and TVX 3236). The aim of the study was to determine the developmental response of the larvae as an indication of antibiotic resistance of the genotypes. Highly significant differences (p < 0.01) were observed with respect to fertility, larval development, adult longevity, life span, multiplication rate and intrinsic rate of increase. KANANNADO and TVX 3236 show minimum antibiotic effects while a landrace SAKA BABBA SATA shows relatively high antibiotic effects. This result further reveals the potential of SAKA BABBA SATA as a resistance source to aphid. The reaction of IT84S-2246-4, a hitherto aphid resistant genotype, which supported higher levels of survival of the larvae relative to other known susceptible genotype IAR-48, may be an indication of the presence of a new biotype of Aphis craccivora endemic to Zaria environs, or that of the ability of insects to overcome hindrances to their survival including various forms of resistance.

  17. Continuing Measurements of CO2 Crystals with a Hand-Held 35 GHz Radiometer

    NASA Technical Reports Server (NTRS)

    Foster, J.; Chang, A.; Hall, D.; Tait, A.; Wergin, W.; Erbe, E.

    2000-01-01

    In order to increase our knowledge of the Martian polar caps, an improved understanding of the behavior of both frozen H2O and CO2 in different parts of the electromagnetic spectrum is needed. The thermal microwave part of the spectrum has received relatively little attention compared to the visible and infrared wavelengths. A simple experiment to measure the brightness temperature of frozen CO2 was first performed in the winter of 1998 using a 35 GHz radiometer. in experiments performed during the winter of 1999 and 2000, passive microwave radiation emanating from within layers of manufactured CO2 (dry ice) crystals was again measured with a 35 GHz handheld radiometer. Both large (0.8 cm) and small (0.3 cm) cylindrical-shaped dry ice pellets, at a temperature of 197 K (-76 C), were measured. A 1 sq m plate of aluminum sheet metal was positioned beneath the dry ice so that microwave emissions from the underlying soil layers would be minimized. Non-absorbing foam was positioned around the sides of the plate in order to keep the dry ice in place and to assure that the incremental deposits were level. Thirty-five GHz measurements of this plate were made through the dry ice deposits in the following way. Layers of dry ice were built up and measurements were repeated for the increasing CO2 pack. First, 7 cm of large CO2 pellets were poured onto the sheet metal plate, then an additional 7 cm were added, and finally, 12 cm were added on top of the 14 cm base. Hand-held 35 GHz measurements were made each time the thickness of the deposit was increased. The same process was repeated for the smaller grain pellets. Furthermore, during the past winter, 35 GHz measurements were taken of a 25 kg (27 cm x 27 cm x 27 cm) solid cube Of CO2, which was cut in half and then re-measured. Additional information is contained in the original extended abstract.

  18. Management and characteristics of recycled manure solids used for bedding in Midwest freestall dairy herds.

    PubMed

    Husfeldt, A W; Endres, M I; Salfer, J A; Janni, K A

    2012-04-01

    Interest in using recycled manure solids (RMS) as a bedding material for dairy cows has grown in the US Midwest. Cost of common bedding materials has increased in recent years and availability has decreased. Information regarding the composition of RMS and its use as a bedding material for dairy cows in the Midwest is very limited. The objectives of this study were to characterize RMS as a bedding material, observe bedding management practices, document methods of obtaining RMS, and describe housing facilities. We visited 38 Midwest dairy operations bedding freestalls with RMS to collect data. Methods of obtaining RMS for bedding included separation of anaerobic digested manure, separation of raw manure, and separation of raw manure followed by mechanical drum-composting for 18 to 24 h. Average bedding moisture of unused RMS was 72.4% with a pH of 9.16. Unused samples contained (on a dry basis) 1.4% N, 44.9% C, 32.7C:N ratio, 0.44% P, 0.70% K, 76.5% neutral detergent fiber, 9.4% ash, 4.4% nonfiber carbohydrates, and 1.1% fat. Moisture was lowest for drum-composted solids before and after use as freestall bedding. After use in the stalls, digested solids had lower neutral detergent fiber content (70.5%) than drum-composted (75.0%) and separated raw (73.1%) solids. Total N content was greater in digested solids (2.0%) than in separated raw (1.7%) solids. Total bacterial populations in unused bedding were greatest in separated raw manure solids but were similar between digested and drum-composted manure solids. Drum-composted manure solids had no coliform bacteria before use as freestall bedding. After use as bedding, digested manure solids had lower total bacteria counts compared with drum-composted and separated raw manure solids, which had similar counts. Used bedding samples of digested solids contained fewer environmental streptococci than drum-composted and separated raw solids and had reduced Bacillus counts compared with separated raw solids. Coliform counts

  19. Spectroscopic studies, thermal analyses and biological evaluation of new V(IV), Zr(IV) and U(VI) moxifloxacin complexes

    NASA Astrophysics Data System (ADS)

    Sadeek, Sadeek A.; El-Shwiniy, Walaa H.; Zordok, Wael A.; Kotb, Essam

    2011-12-01

    The synthesis and characterization of the new solid complexes [VO(MOX) 2H 2O]SO 4·11H 2O, [ZrO(MOX) 2Cl]Cl·15H 2O and [UO 2(MOX) 3](NO 3) 2·3H 2O formed in the interaction of moxifloxacin (MOX) with VOSO 4·H 2O, ZrOCl 2·8H 2O and UO 2(NO 3) 2·6H 2O in methanol and acetone as a solvents at room temperature were reported. The isolated solid complexes have been characterized with melting points, elemental analysis, molar conductance, magnetic moments studies, spectral (UV-Visible, IR and 1HNMR) as well as thermal analyses (TGA and DTG). The results support the formation of the complexes and indicate that moxifloxacin reacts as a bidentate ligand chelate to the metal ion through the pyridone oxygen and one carboxylato oxygen. The kinetic parameters of thermogravimetric (TGA) and its differential (DTG), such as activation energies, E*, enthalpies, Δ H*, entropies, Δ S* and Gibbs free energies, Δ G*, have been evaluated by using Coats-Redfern (CR) and Horowitz-Metzeger (HM) methods. The proposed structure of the ligand and their complexes were detected by using the density functional theory (DFT) at the B3LYP/CEP-31G level of theory. The bond stretching force constant and length of the U dbnd O for the [UO 2(MOX) 3](NO 3) 2·3H 2O complex were calculated. The antibacterial activity of the free moxifloxacin ligand and their metal complexes have been tested against some selected bacterial strains such as: Streptococcus aureus K1, Bacillus subtilis K22, Brevibacterium otitidis K76, Escherichia coli K32, Pseudomonas aeruginosa SW1 and Klebsiella oxytoca K42. The complexes showed good antibacterial effect to the selected bacterial strains as compared to the free ligand and Zr(IV) complex is very highly significant compared with the other two complexes.

  20. Immunological and Chemical Identification of Intracellular Forms of Adenovirus Type 2 Terminal Protein

    PubMed Central

    Green, Maurice; Symington, Janey; Brackmann, Karl H.; Cartas, Maria A.; Thornton, Helen; Young, Leann

    1981-01-01

    Highly purified adenovirus type 2 terminal protein (TP) with an apparent Mr of 55,000 (55K) was prepared in quantities of 10 to 30 μg from guanidine hydrochloride- or sodium dodecyl sulfate-disrupted virions (60 to 120 mg). Guinea pigs were immunized with 14 to 20 injections of TP in amounts of 1 to 2 μg. Antiserum to TP was used to study the intracellular polypeptides related to adenovirus type 2 TP. By immunoprecipitation with anti-TP serum, we identified 80K and 76K polypeptides in the nucleoplasmic and cytoplasmic S100 fractions of [35S]methionine-labeled cells early and late after infection with Ad2. By immunoautoradiographic analysis which eliminates coprecipitation of unrelated proteins, we identified an 80K polypeptide (probably an 80K-76K doublet) in unlabeled, late infected cells, using anti-TP serum and 125I-labeled staphylococcal protein A. About two- to threefold-higher levels of the 80K and 76K polypeptides were present in the nucleoplasm than in the S100 fraction, and two- to threefold-higher levels were found in late infected cells than in early infected cells (cycloheximide enhanced, arabinofuranosylcytosine treated). We did not detect the 80K or 76K polypeptide in uninfected cells, indicating that these polypeptides are virus coded. Tryptic peptide map analysis showed that the 80K and 76K polypeptides are very closely related and that they share peptides with the DNA-bound 55K TP. Our data provide the first direct demonstration of intracellular 80K and 76K forms of TP. The intracellular 80K and 76K polypeptides are closely related or identical to the 80K polypeptide that Challberg and co-workers (Proc. Natl. Acad. Sci. U.S.A. 77:5105-5109, 1980) detected at the termini of adenovirus DNA synthesized in vitro and to the 87K polypeptide that Stillman and co-workers (Cell 23:497-508, 1981) translated in vitro. We did not detect the 55K TP in early or late infected cells, consistent with the proposal by Challberg and co-workers that the 80K

  1. Active and passive properties of rabbit descending colon: a microelectrode and nystatin study.

    PubMed

    Wills, N K; Lewis, S A; Eaton, D C

    1979-03-28

    The electrical properties of the basolateral membrane of rabbit descending colon were studied with microelectrode methods in conjunction with the polyene antibiotic nystatin. Two problems were examined: (i) the relative distribution of tight junctional, apical membrane and basolateral membrane resistances, and (ii) the ionic basis of the basolateral membrane potential. Intracellular K+ activity (K+) was measured using liquid ion exchanger microelectrodes ((K+) = 76 +/- 2 MM) and was found not to be in equilibrium with the basolateral membrane potential. In order to measure membrane resistances and to estimate the selective permeability of the basolateral membrane, the apical membrane was treated with nystatin and bathed with a K2SO4 Ringer's solution which was designed to mimic intracellular K+ composition. This procedure virtually eliminated the resistance and electromotive force of the apical membrane. Shunt resistance was calculated by two independent methods based on microelectrode and transepithelial measurements. Both methods produced similar results (Rs = 691 +/- 63 omega cm2 and 770 +/- 247 omega cm2, respectively). These findings indicate that the shunt has no significant selectivity, contrary to previous reports. Native apical membrane resistance was estimated as 705 +/- 123 V cm2 and basolateral membrane resistance was 95 +/- 14 V cm2. To estimate basolateral membrane selectivity, the serosa was bathed in a NaCl Ringer's solution followed by a series of changes in which all or part of the Na+ was replaced by equimolar amounts of K+. From measures of bi-ionic potentials and conductance during these replacements, we calculated potassium permeability and selectivity ratios for the nystatin-treated colon by fitting these results to the constant field equations. By correcting for shunt conductance, it was then possible to estimate the selective permeability of the basolateral membrane alone. Selectivity estimates were as follows: PNa/PK = .08 and PCl/ PK