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Sample records for falciparum freshly isolates

  1. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia

    PubMed Central

    2013-01-01

    Background Performance of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR Green I fluorescence (MSF) drug sensitivity tests were directly compared using Plasmodium falciparum reference strains and fresh ex vivo isolates from Cambodia against a panel of standard anti-malarials. The objective was to determine which of these two common assays is more appropriate for studying drug susceptibility of “immediate ex vivo” (IEV) isolates, analysed without culture adaption, in a region of relatively low malaria transmission. Methods Using the HRP-2 and MSF methods, the 50% inhibitory concentration (IC50) values against a panel of malaria drugs were determined for P. falciparum reference clones (W2, D6, 3D7 and K1) and 41 IEV clinical isolates from an area of multidrug resistance in Cambodia. Comparison of the IC50 values from the two methods was made using Wilcoxon matched pair tests and Pearson’s correlation. The lower limit of parasitaemia detection for both methods was determined for reference clones and IEV isolates. Since human white blood cell (WBC) DNA in clinical samples is known to reduce MSF assay sensitivity, SYBR Green I fluorescence linearity of P. falciparum samples spiked with WBCs was evaluated to assess the relative degree to which MSF sensitivity is reduced in clinical samples. Results IC50 values correlated well between the HRP-2 and MSF methods when testing either P. falciparum reference clones or IEV isolates against 4-aminoquinolines (chloroquine, piperaquine and quinine) and the quinoline methanol mefloquine (Pearson r = 0.85-0.99 for reference clones and 0.56-0.84 for IEV isolates), whereas a weaker IC50 value correlation between methods was noted when testing artemisinins against reference clones and lack of correlation when testing IEV isolates. The HRP-2 ELISA produced a higher overall success rate (90% for producing IC50 best-fit sigmoidal curves), relative to only a 40% success rate for the

  2. Base isolation: Fresh insight

    SciTech Connect

    Shustov, V.

    1993-07-15

    The objective of the research is a further development of the engineering concept of seismic isolation. Neglecting the transient stage of seismic loading results in a widespread misjudgement: The force of resistance associated with velocity is mostly conceived as a source of damping vibrations, though it is an active force at the same time, during an earthquake type excitation. For very pliant systems such as base isolated structures with relatively low bearing stiffness and with artificially added heavy damping mechanism, the so called `damping`` force may occur even the main pushing force at an earthquake. Thus, one of the two basic pillars of the common seismic isolation philosophy, namely, the doctrine of usefulness and necessity of a strong damping mechanism, is turning out to be a self-deception, sometimes even jeopardizing the safety of structures and discrediting the very idea of seismic isolation. There is a way out: breaking with damping dependancy.

  3. Comparative Transcriptional and Genomic Analysis of Plasmodium falciparum Field Isolates

    PubMed Central

    Mackinnon, Margaret J.; Li, Jinguang; Mok, Sachel; Kortok, Moses M.; Marsh, Kevin; Preiser, Peter R.; Bozdech, Zbynek

    2009-01-01

    Mechanisms for differential regulation of gene expression may underlie much of the phenotypic variation and adaptability of malaria parasites. Here we describe transcriptional variation among culture-adapted field isolates of Plasmodium falciparum, the species responsible for most malarial disease. It was found that genes coding for parasite protein export into the red cell cytosol and onto its surface, and genes coding for sexual stage proteins involved in parasite transmission are up-regulated in field isolates compared with long-term laboratory isolates. Much of this variability was associated with the loss of small or large chromosomal segments, or other forms of gene copy number variation that are prevalent in the P. falciparum genome (copy number variants, CNVs). Expression levels of genes inside these segments were correlated to that of genes outside and adjacent to the segment boundaries, and this association declined with distance from the CNV boundary. This observation could not be explained by copy number variation in these adjacent genes. This suggests a local-acting regulatory role for CNVs in transcription of neighboring genes and helps explain the chromosomal clustering that we observed here. Transcriptional co-regulation of physical clusters of adaptive genes may provide a way for the parasite to readily adapt to its highly heterogeneous and strongly selective environment. PMID:19898609

  4. A cross strain Plasmodium falciparum microarray optimized for the transcriptome analysis of Plasmodium falciparum patient derived isolates.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Middha, Sheetal; Acharya, Jyoti; Rao, Sudha Narayana; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2016-09-01

    Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq. PMID:27489776

  5. Association of Plasmodium falciparum isolates encoding the p. Falciparum chloroquine resistance transporter gene K76T polymorphism with anemia and splenomegaly, but not with multiple infections.

    PubMed

    Abdel-Aziz, Inas Z; Oster, Nadja; Stich, August; Coulibaly, Boubacar; Guigemdé, Wendyam A; Wickert, Hannes; Andrews, Kathy T; Kouyaté, Bocar; Lanzer, Michael

    2005-03-01

    The aim of the study was to assess whether infections with Plasmodium falciparum isolates encoding the P. falciparum chloroquine resistance transporter (pfcrt) gene K76T polymorphism, a molecular marker for chloroquine resistance, are associated with multiple infections, age, or clinical signs of malaria in a semi-immune population in a holoendemic area of Burkina Faso. The parameters of interest were investigated in 210 P. falciparum-positive inhabitants. Logistic regression analysis showed that pfcrt K76T-carrying isolates are significantly more likely to cause anemia and splenomegaly. Furthermore, we found that infections with P. falciparum isolates encoding pfcrt K76T are dependent on age rather than multiple infections. Our findings suggest that pfcrt K76T might serve as a valuable marker for assessing the long-term clinical effect of chronic infections with chloroquine-resistant P. falciparum isolates in populations, without the need of drug efficacy trials.

  6. A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent.

    PubMed

    Prugnolle, Franck; Durand, Patrick; Ollomo, Benjamin; Duval, Linda; Ariey, Frédéric; Arnathau, Céline; Gonzalez, Jean-Paul; Leroy, Eric; Renaud, François

    2011-02-01

    From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.

  7. Pfcrt Gene in Plasmodium falciparum Field Isolates from Muzaffargarh, Pakistan

    PubMed Central

    Sahar, Sumrin; Tanveer, Akhtar; Ali, Akbar; Bilal, Hazrat; Muhammad Saleem, Rana

    2015-01-01

    Background: The aim of the study was to identify the prevalence of different species of Plasmodium and haplotypes of pfcrt in Plasmodium falciparum from the selected area. Methods: Overall, 10,372 blood films of suspected malarial patients were examined microscopically from rural health center Sinawan, district Muzaffargarh, Pakistan from November 2008 to November 2010. P. falciparum positive samples (both whole blood and FTA blood spotted cards) were used for DNA extraction. Nested PCR was used to amplify the pfcrt (codon 72–76) gene fragment. Sequencing was carried out to find the haplotypes in the amplified fragment of pfcrt gene. Result: Over all slide positivity rate (SPR), P. vivax and P. falciparum positivity rate was 21.40 %, 19.37 % and 2.03% respectively. FTA blood spotted cards were equally efficient in the blood storage for PCR and sequencing. Analysis of sequencing results of pfcrt showed only one type of haplotype SagtVMNT (AGTGTAATGAATACA) from codon 72–76 in all samples. Conclusion: The results show high prevalence of CQ resistance and AQ resistant genes. AQ is not recommended to be used as a partner drug in ACT in this locality, so as to ward off future catastrophes. PMID:26623432

  8. MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN.

    PubMed

    Sharifi-Sarasiabi, Khojasteh; Haghighi, Ali; Kazemi, Bahram; Taghipour, Niloofar; Mojarad, Ehsan Nazemalhosseini; Gachkar, Latif

    2016-01-01

    In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment.

  9. K13-Propeller Polymorphisms in Plasmodium falciparum Isolates from Patients in Mayotte in 2013 and 2014.

    PubMed

    Torrentino-Madamet, Marylin; Collet, Louis; Lepère, Jean François; Benoit, Nicolas; Amalvict, Rémy; Ménard, Didier; Pradines, Bruno

    2015-12-01

    Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K, and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from Grande Comore showed polymorphisms (N490H, N554K, and E596G).

  10. K13-Propeller Polymorphisms in Plasmodium falciparum Isolates from Patients in Mayotte in 2013 and 2014.

    PubMed

    Torrentino-Madamet, Marylin; Collet, Louis; Lepère, Jean François; Benoit, Nicolas; Amalvict, Rémy; Ménard, Didier; Pradines, Bruno

    2015-12-01

    Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K, and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from Grande Comore showed polymorphisms (N490H, N554K, and E596G). PMID:26416865

  11. Plasmodium falciparum: genetic diversity and complexity of infections in an isolated village in western Thailand.

    PubMed

    Tanabe, Kazuyuki; Zollner, Gabriela; Vaughan, Jefferson A; Sattabongkot, Jetsumon; Khuntirat, Benjawan; Honma, Hajime; Mita, Toshihiro; Tsuboi, Takafumi; Coleman, Russell

    2015-06-01

    Genetic diversity of Plasmodium falciparum is intimately associated with morbidity, mortality and malaria control strategies. It is therefore imperative to study genetic makeup and population structure of this parasite in endemic areas. In Kong Mong Tha, an isolated village in western Thailand, the majority of P. falciparum infections are asymptomatic. In this study we investigated complexity of infections and single nucleotide polymorphisms (SNPs) in the P. falciparum population of Kong Mong Tha, and compared results with those previously obtained from Mae Sod, in northwestern Thailand, where the majority of infections were symptomatic. Using PCR-based determination of the 5' merozoite surface protein 1 gene (msp1) recombinant types, we found that 39% of 59 P. falciparum isolates from Kong Mong Tha had multiple 5' recombinant types with a mean number of 1.54. These values were much lower than those obtained from Mae Sod: 96% for multiple infections and with a mean number of 3.61. Analysis of full-length sequences of two housekeeping genes, the P-type Ca(2+)-transporting ATPase gene (n=33) plus adenylosuccinate lyase gene (n=33), and three vaccine candidate antigen genes, msp1 (n=26), the circumsporozoite protein gene, csp (n=30) and the apical membrane antigen 1 gene, ama 1 (n=32), revealed that in all of these genes within-population SNP diversity was at similar levels between Kong Mong Tha and Mae Sod, suggesting that the extent of MOI and clinical manifestations of malaria are not strongly associated with genetic diversity. Additionally, we did not detect significant genetic differentiation between the two parasite populations, as estimated by the Wright's fixation index of inter-population variance in allele frequencies, suggesting that gene flow prevented the formation of population structuring. Thus, this study highlights unique features of P. falciparum populations in Thailand. The implications of these finding are discussed.

  12. Molecular surveillance of antimalarial drug resistance related genes in Plasmodium falciparum isolates from Eritrea.

    PubMed

    Menegon, Michela; Nurahmed, Abduselam M; Talha, Albadawi A; Nour, Bakri Y M; Severini, Carlo

    2016-05-01

    The introduction of artemisinin-based combination therapy has led to extraordinary results in malaria control, however the recent emergence of partial resistance to artemisinin therapy in Southeast Asia jeopardizes these successes. This study aimed at investigating resistance to the antimalarial drugs by evaluating the polymorphisms in the PfK13, Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolates obtained from patients in Eritrea.

  13. Susceptibility of Anopheles gambiae and Anopheles stephensi to tropical isolates of Plasmodium falciparum

    PubMed Central

    Hume, Jennifer CC; Tunnicliff, Mark; Ranford-Cartwright, Lisa C; Day, Karen P

    2007-01-01

    Background The susceptibility of anopheline mosquito species to Plasmodium infection is known to be variable with some mosquitoes more permissive to infection than others. Little work, however, has been carried out investigating the susceptibility of major malaria vectors to geographically diverse tropical isolates of Plasmodium falciparum aside from examining the possibility of infection extending its range from tropical regions into more temperate zones. Methods This study investigates the susceptibility of two major tropical mosquito hosts (Anopheles gambiae and Anopheles stephensi) to P. falciparum isolates of different tropical geographical origins. Cultured parasite isolates were fed via membrane feeders simultaneously to both mosquito species and the resulting mosquito infections were compared. Results Infection prevalence was variable with African parasites equally successful in both mosquito species, Thai parasites significantly more successful in An. stephensi, and PNG parasites largely unsuccessful in both species. Conclusion Infection success of P. falciparum was variable according to geographical origin of both the parasite and the mosquito. Data presented raise the possibility that local adaptation of tropical parasites and mosquitoes has a role to play in limiting gene flow between allopatric parasite populations. PMID:17958900

  14. Genetic diversity of vaccine candidate antigens in Plasmodium falciparum isolates from the Amazon basin of Peru

    PubMed Central

    Chenet, Stella M; Branch, OraLee H; Escalante, Ananias A; Lucas, Carmen M; Bacon, David J

    2008-01-01

    Background Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. In this study, characterization of genetic variability was performed in major B and T-cell epitopes within vaccine candidate antigens in isolates of P. falciparum from Peru. Methods DNA sequencing analysis was completed on 139 isolates of P. falciparum collected from endemic areas of the Amazon basin in Loreto, Peru from years 1998 to 2006. Genetic diversity was determined in immunological important regions in circumsporozoite protein (CSP), merozoite surface protein-1 (MSP-1), apical membrane antigen-1 (AMA-1), liver stage antigen-1 (LSA-1) and thrombospondin-related anonymous protein (TRAP). Alleles identified by DNA sequencing were aligned with the vaccine strain 3D7 and DNA polymorphism analysis and FST study-year pairwise comparisons were done using the DnaSP software. Multilocus analysis (MLA) was performed and average of expected heterozygosity was calculated for each loci and haplotype over time. Results Three different alleles for CSP, seven for MSP-1 Block 2, one for MSP-1 Block 17, three for AMA-1 and for LSA-1 each and one for TRAP were identified. There were 24 different haplotypes in 125 infections with complete locus typing for each gene. Conclusion Characterization of the genetic diversity in Plasmodium isolates from the Amazon Region of Peru showed that P. falciparum T and B cell epitopes in these antigens have polymorphisms more similar to India than to Africa. These findings are helpful in the formulation of a vaccine considering restricted repertoire populations. PMID:18505558

  15. Full-length sequence analysis of chloroquine resistance transporter gene in Plasmodium falciparum isolates from Sabah, Malaysia.

    PubMed

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979. PMID:25574497

  16. An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

    PubMed Central

    Subudhi, Amit Kumar; Boopathi, P.A.; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B.; Mugasimangalam, Raja C.; Sirohi, Paramendra; Kochar, Sanjay K.; Kochar, Dhanpat K.; Das, Ashis

    2014-01-01

    Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs). Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO) under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014). PMID:26484136

  17. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype.

    PubMed

    Brown, Tyler S; Jacob, Christopher G; Silva, Joana C; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V; Cummings, Michael P

    2015-03-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives.

  18. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype.

    PubMed

    Brown, Tyler S; Jacob, Christopher G; Silva, Joana C; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V; Cummings, Michael P

    2015-03-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  19. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

    PubMed Central

    Brown, Tyler S.; Jacob, Christopher G.; Silva, Joana C.; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M.; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V.; Cummings, Michael P.

    2015-01-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  20. Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen.

    PubMed

    Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Dawaki, Salwa S; Elyana, Fatin N; Al-Areeqi, Mona A; Nasr, Nabil A; Abdulsalam, Awatif M; Subramaniam, Lahvanya R; Azzani, Meram; Ithoi, Init; Lau, Yee Ling; Surin, Johari

    2016-01-01

    Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72-76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72-76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected in 9

  1. Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen

    PubMed Central

    Al-Jasari, Adel; Sady, Hany; Dawaki, Salwa S.; Elyana, Fatin N.; Al-Areeqi, Mona A.; Nasr, Nabil A.; Abdulsalam, Awatif M.; Subramaniam, Lahvanya R.; Azzani, Meram; Ithoi, Init; Lau, Yee Ling; Surin, Johari

    2016-01-01

    Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72–76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72–76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected

  2. No Polymorphism in Plasmodium falciparum K13 Propeller Gene in Clinical Isolates from Kolkata, India

    PubMed Central

    Chatterjee, Moytrey; Ganguly, Swagata; Saha, Pabitra; Bankura, Biswabandhu; Basu, Nandita; Das, Madhusudan; Guha, Subhasish K.; Maji, Ardhendu K.

    2015-01-01

    Molecular markers associated with artemisinin resistance in Plasmodium falciparum are yet to be well defined. Recent studies showed that polymorphisms in K13 gene are associated with artemisinin resistance. The present study was designed to know the pattern of polymorphisms in propeller region of K13 gene among the clinical isolates collected from urban Kolkata after five years of ACT implementation. We collected 59 clinical isolates from urban Kolkata and sequenced propeller region of K13 gene in 51 isolates successfully. We did not find any mutation in any isolate. All patients responded to the ACT, a combination of artesunate + sulphadoxine-pyrimethamine. The drug regimen is still effective in the study area and there is no sign of emergence of resistance against artemisinin as evidenced by wild genotype of K13 gene in all isolates studied. PMID:26688755

  3. In vitro sensitivity of Plasmodium falciparum field isolates to extracts from Cameroonian Annonaceae plants.

    PubMed

    Kemgne, Eugénie Aimée Madiesse; Mbacham, Wilfred Fon; Boyom, Fabrice Fekam; Zollo, Paul Henri Amvam; Tsamo, Etienne; Rosenthal, Philip J

    2012-01-01

    In a search for new plant-derived antimalarial extracts, 19 fractions were obtained from three Annonaceae species, Uvariopsis congolana (leaf, stem), Polyalthia oliveri (stem bark), and Enantia chlorantha (stem, stem bark) with yields ranging from 0.33% to 4.60%. The extracts were prepared from 500 g of each plant part, using organic solvents to afford five methanolic fractions (acetogenin rich), five water fractions, five hexane fractions, and four interface precipitates. Evaluation of the activity of fractions in vitro against field isolates of the malaria parasite Plasmodium falciparum showed that acetogenin-rich fractions and interface precipitates were the most potent, with IC(50) values ranging from 0.05 to 8.09 μg/ml. Sensitivity of parasite isolates to plant extracts varied greatly, with over 100-fold difference from isolate to isolate in some cases. The active acetogenin-rich fractions and interface precipitates were assessed in combination with chloroquine in the same conditions, and showed additive interaction in the huge majority of cases. Synergistic interactions were found in some cases with acetogenin-rich fractions. Acute toxicity of promising fractions was evaluated through oral administration in Swiss albino mice. Tested fractions appeared to be safe, with LD(50) values higher than 2 g/kg. In summary, acetogenin-rich fractions from Annonaceae species showed high potency against P. falciparum field isolates and safety by oral administration in mice, supporting their detailed investigation for antimalarial drug discovery.

  4. NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli

    SciTech Connect

    Ilatovskaya, Daria V.; Levchenko, Vladislav; Ryan, Robert P.; Cowley, Allen W.; Staruschenko, Alexander

    2011-05-06

    Highlights: {yields} We have established a unique approach to search for physiologically relevant mechanisms of TRPC channels in podocytes. {yields} This study describes endogenous TRPC channels in the isolated decapsulated glomeruli preparation. {yields} We report for the first time that NSAIDs inhibit TRPC channels in podocytes. -- Abstract: Using a novel approach for analysis of TRPC channel activity, we report here that NSAIDs are involved into regulation of TRPC channels in the podocytes of the freshly isolated decapsulated glomeruli. Fluorescence and electron microscopy techniques confirmed the integrity of podocytes in the glomeruli. Western blotting showed that TRPC1, 3 and 6 are highly expressed in the glomeruli. Single-channel patch clamp analysis revealed cation currents with distinct TRPC properties. This is the first report describing single TRPC-like currents in glomerular podocytes. Furthermore, our data provide a novel mechanism of NSAIDs regulation of TRPC channels, which might be implicated in maintaining the glomerular filtration barrier.

  5. Plasmodium falciparum isolates from Angola show the StctVMNT haplotype in the pfcrt gene

    PubMed Central

    2010-01-01

    Background Effective treatment remains a mainstay of malaria control, but it is unfortunately strongly compromised by drug resistance, particularly in Plasmodium falciparum, the most important human malaria parasite. Although P. falciparum chemoresistance is well recognized all over the world, limited data are available on the distribution and prevalence of pfcrt and pfmdr1 haplotypes that mediate resistance to commonly used drugs and that show distinct geographic differences. Methods Plasmodium falciparum-infected blood samples collected in 2007 at four municipalities of Luanda, Angola, were genotyped using PCR and direct DNA sequencing. Single nucleotide polymorphisms in the P. falciparum pfcrt and pfmdr1 genes were assessed and haplotype prevalences were determined. Results and Discussion The most prevalent pfcrt haplotype was StctVMNT (representing amino acids at codons 72-76). This result was unexpected, since the StctVMNT haplotype has previously been seen mainly in parasites from South America and India. The CVIET, CVMNT and CVINT drug-resistance haplotypes were also found, and one previously undescribed haplotype (CVMDT) was detected. Regarding pfmdr1, the most prevalent haplotype was YEYSNVD (representing amino acids at codons 86, 130, 184, 1034, 1042, 1109 and 1246). Wild haplotypes for pfcrt and pfmdr1 were uncommon; 3% of field isolates harbored wild type pfcrt (CVMNK), whereas 21% had wild type pfmdr1 (NEYSNVD). The observed predominance of the StctVMNT haplotype in Angola could be a result of frequent travel between Brazil and Angola citizens in the context of selective pressure of heavy CQ use. Conclusions The high prevalence of the pfcrt SVMNT haplotype and the pfmdr1 86Y mutation confirm high-level chloroquine resistance and might suggest reduced efficacy of amodiaquine in Angola. Further studies must be encouraged to examine the in vitro sensitivity of pfcrt SVMNT parasites to artesunate and amodiaquine for better conclusive data. PMID:20565881

  6. Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates.

    PubMed

    Howard, R F; Ardeshir, F; Reese, R T

    1986-01-01

    Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Molecular Analysis of Chloroquine and Sulfadoxine-Pyrimethamine Resistance-Associated Alleles in Plasmodium falciparum Isolates from Nicaragua

    PubMed Central

    Sridaran, Sankar; Rodriguez, Betzabe; Mercedes Soto, Aida; Macedo De Oliveira, Alexandre; Udhayakumar, Venkatachalam

    2014-01-01

    Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles. PMID:24615126

  8. Molecular analysis of chloroquine and sulfadoxine-pyrimethamine resistance-associated alleles in Plasmodium falciparum isolates from Nicaragua.

    PubMed

    Sridaran, Sankar; Rodriguez, Betzabe; Soto, Aida Mercedes; Macedo De Oliveira, Alexandre; Udhayakumar, Venkatachalam

    2014-05-01

    Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles.

  9. Whole-Genome Scans Provide Evidence of Adaptive Evolution in Malawian Plasmodium falciparum Isolates

    PubMed Central

    Ocholla, Harold; Preston, Mark D.; Mipando, Mwapatsa; Jensen, Anja T. R.; Campino, Susana; MacInnis, Bronwyn; Alcock, Daniel; Terlouw, Anja; Zongo, Issaka; Oudraogo, Jean-Bosco; Djimde, Abdoulaye A.; Assefa, Samuel; Doumbo, Ogobara K.; Borrmann, Steffen; Nzila, Alexis; Marsh, Kevin; Fairhurst, Rick M.; Nosten, Francois; Anderson, Tim J. C.; Kwiatkowski, Dominic P.; Craig, Alister; Clark, Taane G.; Montgomery, Jacqui

    2014-01-01

    Background Selection by host immunity and antimalarial drugs has driven extensive adaptive evolution in Plasmodium falciparum and continues to produce ever-changing landscapes of genetic variation. Methods We performed whole-genome sequencing of 69 P. falciparum isolates from Malawi and used population genetics approaches to investigate genetic diversity and population structure and identify loci under selection. Results High genetic diversity (π = 2.4 × 10−4), moderately high multiplicity of infection (2.7), and low linkage disequilibrium (500-bp) were observed in Chikhwawa District, Malawi, an area of high malaria transmission. Allele frequency–based tests provided evidence of recent population growth in Malawi and detected potential targets of host immunity and candidate vaccine antigens. Comparison of the sequence variation between isolates from Malawi and those from 5 geographically dispersed countries (Kenya, Burkina Faso, Mali, Cambodia, and Thailand) detected population genetic differences between Africa and Asia, within Southeast Asia, and within Africa. Haplotype-based tests of selection to sequence data from all 6 populations identified signals of directional selection at known drug-resistance loci, including pfcrt, pfdhps, pfmdr1, and pfgch1. Conclusions The sequence variations observed at drug-resistance loci reflect differences in each country's historical use of antimalarial drugs and may be useful in formulating local malaria treatment guidelines. PMID:24948693

  10. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

  11. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  12. Plasmodium falciparum: genetic polymorphism in apical membrane antigen-1 gene from Indian isolates.

    PubMed

    Rajesh, Vidya; Singamsetti, Vijay Kumar; Vidya, S; Gowrishankar, M; Elamaran, M; Tripathi, Jyotsna; Radhika, N B; Kochar, Dhanpat; Ranjan, Akash; Roy, S K; Das, Ashis

    2008-05-01

    A number of stage-specific antigens have been characterized for vaccine development against Plasmodium falciparum malaria. This study presents a comprehensive analysis of the sequence polymorphism in Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) in population samples from the eastern and western parts of India. This is the first study of its kind for the nearly full length PfAMA-1 gene from these regions in India. Our observations confirmed that sequence diversity of PfAMA-1 confines only to point mutations and shows 4-8% variation as compared to the prototypes. As opposed to the previous studies on PfAMA-1, our study revealed a greater degree of polymorphism in the Domain II region of PfAMA-1 protein, though signature for diversifying selection is seen throughout the gene. Our present investigation also indicates a very high degree of variation in the reported T- and B-cell epitopes of PfAMA-1. Few noteworthy and unique observations made in this study are the substitution of Cysteine residues responsible for the disulfide bond structure of the protein and the presence of premature termination after 595 amino acids in 3 of the 13 isolates under consideration. These crucial findings add new perspectives to the future of AMA-1 research and could have major implications in establishing AMA-1 as a vaccine candidate. PMID:18343371

  13. Large Variation in Detection of Histidine-Rich Protein 2 in Plasmodium falciparum Isolates from Colombia

    PubMed Central

    Pava, Zuleima; Echeverry, Diego F.; Díaz, Gustavo; Murillo, Claribel

    2010-01-01

    Most rapid diagnostic tests (RDTs) available use histidine-rich protein 2 (HRP2) as a target. However, it has been reported that sequence variations of this protein affects its sensitivity. Currently, there is insufficient evidence for HRP2 variability in Plasmodium falciparum isolates from Colombia and its relationship with RDT performance. To determine possible geographic differences and their effects on the performance of RDTs, 22 blood samples from patients with P. falciparum malaria from Tumaco and Buenaventura, Colombia were assessed by measurement of HRP2 concentration by an HRP2 enzyme-linked immunosorbent assay, RDTs, and thick blood smear. Statistical analysis showed an association between RDT performance and HRP2 concentrations. No significant difference was found between locations. A large variation of antigen concentration in samples was found at same parasitemia. In contrast to previously reports, there was no correlation between initial parasitemia and HRP2 concentration. Our results indicate that antigen quantity should be studied more carefully because the sensitivity of the RDT is affected more by antigen concentration than by parasitemia. PMID:20889875

  14. Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

    PubMed

    Urdaneta, L; Plowe, C; Goldman, I; Lal, A A

    1999-09-01

    The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area. PMID:10497990

  15. Artemisinin resistance-associated polymorphisms at the K13-propeller locus are absent in Plasmodium falciparum isolates from Haiti.

    PubMed

    Carter, Tamar E; Boulter, Alexis; Existe, Alexandre; Romain, Jean R; St Victor, Jean Yves; Mulligan, Connie J; Okech, Bernard A

    2015-03-01

    Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations. PMID:25646258

  16. Artemisinin resistance-associated polymorphisms at the K13-propeller locus are absent in Plasmodium falciparum isolates from Haiti.

    PubMed

    Carter, Tamar E; Boulter, Alexis; Existe, Alexandre; Romain, Jean R; St Victor, Jean Yves; Mulligan, Connie J; Okech, Bernard A

    2015-03-01

    Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations.

  17. Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand.

    PubMed

    Gaillard, Tiphaine; Sriprawat, Kanlaya; Briolant, Sébastien; Wangsing, Chirapat; Wurtz, Nathalie; Baragatti, Meïli; Lavina, Morgane; Pascual, Aurélie; Nosten, François; Pradines, Bruno

    2015-08-01

    Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 μM and 31.60 μM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72). PMID:26055380

  18. Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand

    PubMed Central

    Gaillard, Tiphaine; Sriprawat, Kanlaya; Briolant, Sébastien; Wangsing, Chirapat; Wurtz, Nathalie; Baragatti, Meïli; Lavina, Morgane; Pascual, Aurélie; Nosten, François

    2015-01-01

    Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 μM and 31.60 μM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72). PMID:26055380

  19. Pooled deep sequencing of Plasmodium falciparum isolates: an efficient and scalable tool to quantify prevailing malaria drug-resistance genotypes.

    PubMed

    Taylor, Steve M; Parobek, Christian M; Aragam, Nash; Ngasala, Billy E; Mårtensson, Andreas; Meshnick, Steven R; Juliano, Jonathan J

    2013-12-15

    Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies.

  20. Plasmodium falciparum Field Isolates from South America Use an Atypical Red Blood Cell Invasion Pathway Associated with Invasion Ligand Polymorphisms

    PubMed Central

    Lopez-Perez, Mary; Villasis, Elizabeth; Machado, Ricardo L. D.; Póvoa, Marinete M.; Vinetz, Joseph M.; Blair, Silvia; Gamboa, Dionicia; Lustigman, Sara

    2012-01-01

    Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five members of the PfRh (PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5) families were determined. We found that most P. falciparum field isolates from Colombia and Peru invade RBCs through an atypical invasion pathway phenotypically characterized as resistant to all enzyme treatments (NrTrCr). Moreover, the invasion pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant, the PfRh5 variant 1 and EBA-181 RVNKN variant. The ebl and Pfrh expression levels in a field isolate displaying the NrTrCr profile also pointed to PfRh2a, PfRh5 and EBA-181 as being possibly the major players in this invasion pathway. Notably, our studies demonstrate the uniqueness of the Peruvian P. falciparum field isolates in terms of their invasion profiles and ligand polymorphisms, and present a unique opportunity for studying the ability of P. falciparum parasites to expand their invasion repertoire after being reintroduced to human populations. The present study is directly relevant to asexual blood stage vaccine design focused on invasion pathway proteins, suggesting that regional invasion variants and global geographical variation are likely to preclude a simple

  1. Expression of Plasmodium falciparum genes involved in erythrocyte invasion varies among isolates cultured directly from patients.

    PubMed

    Nery, Susana; Deans, Anne-Marie; Mosobo, Moses; Marsh, Kevin; Rowe, J Alexandra; Conway, David J

    2006-10-01

    Plasmodium falciparum merozoites invade erythrocytes using a range of alternative ligands that includes erythrocyte binding antigenic proteins (EBAs) and reticulocyte binding protein homologues (Rh). Variation in the expression of some of these genes among culture-adapted parasite lines correlates with the use of different erythrocyte receptors. Here, expression profiles of four Rh genes and eba175 are analysed in a sample of 42 isolates cultured from malaria patients in Kenya. The profiles cluster into distinct groups, largely because of very strong negative correlations between the levels of expression of particular gene pairs (Rh1 versus Rh2b, eba175 versus Rh2b, and eba175 versus Rh4), previously associated with alternative invasion pathways in culture-adapted parasite lines. High levels of eba175 are seen in isolates in expression profile group I, and may be associated with sialic acid-dependent invasion. Groups II and III are, respectively, characterized by high levels of Rh2b and Rh4, and are more likely to be associated with sialic acid-independent invasion.

  2. A YAC contig map of plasmodium falciparum chromosome 4: Characterization of a DNA amplification between two recently separated isolates

    SciTech Connect

    Rubio, J.P.; Triglia, T.; Cowman, A.F.

    1995-03-20

    We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen. 42 refs., 6 figs., 1 tab.

  3. Postzygotic Isolation Evolves before Prezygotic Isolation between Fresh and Saltwater Populations of the Rainwater Killifish, Lucania parva

    PubMed Central

    Kozak, Genevieve M.; Rudolph, Arthur B.; Colon, Beatrice L.; Fuller, Rebecca C.

    2012-01-01

    Divergent natural selection has the potential to drive the evolution of reproductive isolation. The euryhaline killifish Lucania parva has stable populations in both fresh water and salt water. Lucania parva and its sister species, the freshwater L. goodei, are isolated by both prezygotic and postzygotic barriers. To further test whether adaptation to salinity has led to the evolution of these isolating barriers, we tested for incipient reproductive isolation within L. parva by crossing freshwater and saltwater populations. We found no evidence for prezygotic isolation, but reduced hybrid survival indicated that postzygotic isolation existed between L. parva populations. Therefore, postzygotic isolation evolved before prezygotic isolation in these ecologically divergent populations. Previous work on these species raised eggs with methylene blue, which acts as a fungicide. We found this fungicide distorts the pattern of postzygotic isolation by increasing fresh water survival in L. parva, masking species/population differences, and underestimating hybrid inviability. PMID:22518334

  4. Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

    PubMed

    Wirjanata, Grennady; Sebayang, Boni F; Chalfein, Ferryanto; Prayoga; Handayuni, Irene; Noviyanti, Rintis; Kenangalem, Enny; Poespoprodjo, Jeanne Rini; Burgess, Steven J; Peyton, David H; Price, Ric N; Marfurt, Jutta

    2015-09-01

    Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species.

  5. PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

    PubMed

    Kaewpruk, Napaporn; Tan-ariya, Peerapan; Ward, Stephen A; Sitthichot, Naruemon; Suwandittakul, Nantana; Mungthin, Mathirut

    2016-05-01

    Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC₅₀. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development. PMID:27405118

  6. PFMDR1 POLYMORPHISMS INFLUENCE ON IN VITRO SENSITIVITY OF THAI PLASMODIUM FALCIPARUM ISOLATES TO PRIMAQUINE, SITAMAQUINE AND TAFENOQUINE.

    PubMed

    Kaewpruk, Napaporn; Tan-ariya, Peerapan; Ward, Stephen A; Sitthichot, Naruemon; Suwandittakul, Nantana; Mungthin, Mathirut

    2016-05-01

    Primaquine (PQ), an 8-aminoquinoline, is considered a tissue schizonticide drug for radical cure in vivax and ovale malaria, with minimal impact on asexual erythrocytic stages at therapeutic concentrations. Tafenoquine (TQ), a new 8-aminoquinoline analog of PQ, is active against both malaria parasite tissue and blood stages and is being promoted as a drug candidate for antimalarial chemotherapy and chemoprophylaxis and potential transmission blocking against Plasmodium vivax and P. falciparum. This study compared in vitro sensitivity of Thai P. falciparum isolates against three 8-aminoquinolines, PQ, TQ and sitamaquine (SQ), a related 8-aminoquinoline and assessed the importance of pfmdr1 polymorphism on the in vitro response. Seventy-eight laboratory adapted Thai P. falciparum isolates were evaluated for in vitro sensitivity to the three 8-aminoquinolines using a radioisotopic assay, and pfmdr1 polymorphisms were determined using PCR-based methods. All three drugs have weak antiplasmodial activity against asexual erythrocytic stage with SQ being the most potent by almost 10 folds. Cross susceptibility was observed in all three 8-aminoquinolines. Parasites containing pfmdr1 86Y, 184Y or 1034S allele exhibit significantly higher PQ IC₅₀. TQ sensitivity was reduced in those parasites containing pfmdr1 86Y, 1034S or 1042N allele. However, there was no significant influence of pfmdr1 alleles on SQ sensitivity. The data highlight unique differences among three representative 8-aminoquinoline drugs that may be useful in understanding their potential utility in antimalarial development.

  7. Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

    SciTech Connect

    Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi; Kuo, Fu-Hua; Lee, Chi-Ya; Liu, Der-Zen; Jan, Tong-Rong

    2010-08-01

    It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-{sub L}-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

  8. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Bin Dajem, Saad M; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries.

  9. The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture

    PubMed Central

    Yeda, Redemptah; Ingasia, Luicer A.; Cheruiyot, Agnes C.; Okudo, Charles; Chebon, Lorna J.; Cheruiyot, Jelagat; Akala, Hoseah M.; Kamau, Edwin

    2016-01-01

    The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24–48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long

  10. The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture.

    PubMed

    Yeda, Redemptah; Ingasia, Luicer A; Cheruiyot, Agnes C; Okudo, Charles; Chebon, Lorna J; Cheruiyot, Jelagat; Akala, Hoseah M; Kamau, Edwin

    2016-01-01

    The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long

  11. Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries.

    PubMed

    Munitz, Martin S; Resnik, Silvia L; Pacin, Ana; Salas, Paula M; Gonzalez, Hector H L; Montti, Maria I T; Drunday, Vanesa; Guillin, Eduardo A

    2014-11-01

    Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B1 (FB1); two of them also produced Fumonisin B2 (FB2) and one Fumonisin B3 (FB3). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and aflatoxin G1 (AFG1). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.

  12. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  13. Genetic diversity and natural selection at the domain I of apical membrane antigen-1 (AMA-1) of Plasmodium falciparum in isolates from Iran.

    PubMed

    Mardani, Ahmad; Keshavarz, Hossein; Heidari, Aliehsan; Hajjaran, Homa; Raeisi, Ahmad; Khorramizadeh, Mohammad Reza

    2012-04-01

    The apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is a prime malaria asexual blood-stage vaccine candidate. Antigenic variation is one of the main obstacles in the development of a universal effective malaria vaccine. The extracellular region of P. falciparum AMA-1 (PfAMA-1) consists of three domains (I-III), of which the domain I is the most diverse region of this antigen. The objective of our study was to investigate and analyze the extent of genetic diversity and the effectiveness of natural selection at the AMA-1 domain I of P. falciparum in isolates from Iran. A fragment of ama-1 gene spanning domain I was amplified by nested PCR from 48 P. falciparum isolates collected from two major malaria endemic areas of Iran during 2009 to August 2010 and sequenced. Genetic polymorphism and statistical analyses were performed using DnaSP and MEGA software packages. Analysis of intrapopulation diversity revealed relatively high nucleotide and haplotype diversity at the PfAMA-1 domain I of Iranian isolates. Neutrality tests provided strong evidence of positive natural selection acting on the sequenced gene region. The findings also demonstrated that, in addition to natural selection, intragenic recombination may contribute to the diversity observed at the domain I. The results obtained will have significant implications in the design and the development of an AMA-1-based vaccine against falciparum malaria.

  14. Genetic diversity and multiplicity of infection of Plasmodium falciparum isolates from Kolkata, West Bengal, India.

    PubMed

    Saha, Pabitra; Ganguly, Swagata; Maji, Ardhendu K

    2016-09-01

    The study of genetic diversity of Plasmodium falciparum is necessary to understand the distribution and dynamics of parasite populations. The genetic diversity of P. falciparum merozoite surface protein-1 and 2 has been extensively studied from different parts of world. However, limited data are available from India. This study was aimed to determine the genetic diversity and multiplicity of infection (MOI) of P. falciparum population in Kolkata, West Bengal, India. A total of 80day-zero blood samples from Kolkata were collected during a therapeutic efficacy study in 2008-2009. DNA was extracted; allelic frequency and diversity were investigated by PCR-genotyping method for msp1 and msp2 gene and fragment sizing was done by Bio-Rad Gel-Doc system using Image Lab (version 4.1) software. P. falciparum msp1 and msp2 markers were highly polymorphic with low allele frequencies. In Kolkata, 27 msp1 different genotypes (including 11of K1, 6 of MAD20 and 10 of Ro33 allelic families) and 30 different msp2 genotypes (of which 17 and 13 belonged to the FC27 and 3D7 allelic families, respectively) were recorded. The majority of these genotypes occurred at a frequency below 10%. The mean MOI for msp1 and msp2 gene were 2.05 and 3.72, respectively. The P. falciparum population of Kolkata was genetically diverse. As the frequencies of most of the msp1 and msp2 alleles were low, the probability of new infection with genotype identical to that in pretreatment infection was very rare. This information will serve as baseline data for evaluation of malaria control interventions as well as for monitoring the parasite population structure. PMID:27259367

  15. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

    PubMed

    Rao, Pavitra N; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C; Carlton, Jane M

    2016-06-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.

  16. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

    PubMed Central

    Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.

    2016-01-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  17. Emergence of Mutations in the K13 Propeller Gene of Plasmodium falciparum Isolates from Dakar, Senegal, in 2013-2014.

    PubMed

    Boussaroque, Agathe; Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Benoit, Nicolas; Fall, Mansour; Nakoulima, Aminata; Dionne, Pierre; Fall, Kadidiatou Ba; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar; Pradines, Bruno

    2015-10-26

    The kelch 13 (K13) propeller gene is associated with artemisinin resistance. In a previous work, there were no mutations found in 138 Plasmodium falciparum isolates collected in 2012 and 2013 from patients residing in Dakar, Senegal (M. Torrentino-Madamet et al., Malar J 13:472, 2014, http://dx.doi.org/10.1186/1475-2875-13-472). However, the N554H, Q613H, and V637I mutations were identified in the propeller region of K13 in 92 (5.5%) isolates in 2013 and 2014. There were five polymorphisms identified in the Plasmodium/Apicomplexa-specific domain (K123R, N137S, N142NN/NNN, T149S, and K189T/N).

  18. Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa

    PubMed Central

    St. Laurent, Brandyce; Miller, Becky; Burton, Timothy A.; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P.; Miotto, Olivo; Gwadz, Robert W.; Anderson, Jennifer M.; Fairhurst, Rick M.

    2015-01-01

    Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread. PMID:26485448

  19. Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa.

    PubMed

    St Laurent, Brandyce; Miller, Becky; Burton, Timothy A; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P; Miotto, Olivo; Gwadz, Robert W; Anderson, Jennifer M; Fairhurst, Rick M

    2015-01-01

    Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread. PMID:26485448

  20. Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil.

    PubMed

    Koch, M O; Weiss, R R; Cruz, A A; Soccol, V T; Gonçalves, K A; Bertol, Maf; Beltrame, O C; Dittrich, R L

    2016-08-01

    The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii.

  1. Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil.

    PubMed

    Koch, M O; Weiss, R R; Cruz, A A; Soccol, V T; Gonçalves, K A; Bertol, Maf; Beltrame, O C; Dittrich, R L

    2016-08-01

    The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii. PMID:27287987

  2. Markers of anti-malarial drug resistance in Plasmodium falciparum isolates from Swaziland: identification of pfmdr1-86F in natural parasite isolates

    PubMed Central

    2010-01-01

    Background The development of Plasmodium falciparum resistance to chloroquine (CQ) has limited its use in many malaria endemic areas of the world. However, despite recent drug policy changes to adopt the more effective artemisinin-based combination (ACT) in Africa and in the Southern African region, in 2007 Swaziland still relied on CQ as first-line anti-malarial drug. Methods Parasite DNA was amplified from P. falciparum isolates from Swaziland collected in 1999 (thick smear blood slides) and 2007 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing. Results Retrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2007 carried detectable P. falciparum. The pfcrt haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 70% (n = 64) in 1999 and 83% (n = 6) in 2007. Prevalence of the pfmdr1-86N allele was 24% in 1999 and 67% in 2007. A novel substitution of phenylalanine for asparagine at codon 86 of pfmdr1 (N86F) occurred in two of 51 isolates successfully amplified from 1999. The pfmdr1-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2007. The 86N/184F/1246D pfmdr1 haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2007 isolates. The pfdhfr triple-mutant 16C/51I/59R/108N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n = 34) and 2007 (50%, n = 6), as was the wild-type 431I/436S/437A/540K/581A/613A haplotype of pfdhps (100% and 93% respectively in 1999 and 2007). The quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2007 isolates. Conclusions The prevalence of pfcrt and pfmdr1 alleles reported in this study is

  3. The seasonality of human campylobacter infection and Campylobacter isolates from fresh, retail chicken in Wales.

    PubMed Central

    Meldrum, R. J.; Griffiths, J. K.; Smith, R. M. M.; Evans, M. R.

    2005-01-01

    Seasonal peaks in both human campylobacter infections and poultry isolates have been observed in several European countries but remain unexplained. We compared weekly data on human campylobacter infections with thermophilic Campylobacter isolation rates from fresh, retail chicken samples (n = 514) purchased weekly in Wales between January and December 2002. Human isolates (n = 2631) peaked between weeks 22 and 25 (early June) and chicken isolates (n = 364) between weeks 24 and 26 (late June). In the absence of a temporal association, we postulate that the seasonal rise in humans is not caused by a rise in isolation rates in poultry but that both are more likely to be associated with a common, but as yet unidentified, environmental source. PMID:15724710

  4. Prevalence and Characteristics of Salmonella Serotypes Isolated from Fresh Produce Marketed in the United States.

    PubMed

    Reddy, Shanker P; Wang, Hua; Adams, Jennifer K; Feng, Peter C H

    2016-01-01

    Salmonella continues to rank as one of the most costly foodborne pathogens, and more illnesses are now associated with the consumption of fresh produce. The U.S. Department of Agriculture Microbiological Data Program (MDP) sampled select commodities of fresh fruit and vegetables and tested them for Salmonella, pathogenic Escherichia coli, and Listeria. The Salmonella strains isolated were further characterized by serotype, antimicrobial resistance, and pulsed-field gel electrophoresis profile. This article summarizes the Salmonella data collected by the MDP between 2002 and 2012. The results show that the rates of Salmonella prevalence ranged from absent to 0.34% in cilantro. A total of 152 isolates consisting of over 50 different serotypes were isolated from the various produce types, and the top five were Salmonella enterica serotype Cubana, S. enterica subspecies arizonae (subsp. IIIa) and diarizonae (subsp. IIIb), and S. enterica serotypes Newport, Javiana, and Infantis. Among these, Salmonella serotypes Newport and Javiana are also listed among the top five Salmonella serotypes that caused most foodborne outbreaks. Other serotypes that are frequent causes of infection, such as S. enterica serotypes Typhimurium and Enteritidis, were also found in fresh produce but were not prevalent. About 25% of the MDP samples were imported produce, including 65% of green onions, 44% of tomatoes, 42% of hot peppers, and 41% of cantaloupes. However, imported produce did not show higher numbers of Salmonella-positive samples, and in some products, like cilantro, all of the Salmonella isolates were from domestic samples. About 6.5% of the Salmonella isolates were resistant to the antimicrobial compounds tested, but no single commodity or serotype was found to be the most common carrier of resistant strains or of resistance. The pulsed-field gel electrophoresis profiles of the produce isolates showed similarities with Salmonella isolates from meat samples and from outbreaks, but

  5. Prevalence and Characteristics of Salmonella Serotypes Isolated from Fresh Produce Marketed in the United States.

    PubMed

    Reddy, Shanker P; Wang, Hua; Adams, Jennifer K; Feng, Peter C H

    2016-01-01

    Salmonella continues to rank as one of the most costly foodborne pathogens, and more illnesses are now associated with the consumption of fresh produce. The U.S. Department of Agriculture Microbiological Data Program (MDP) sampled select commodities of fresh fruit and vegetables and tested them for Salmonella, pathogenic Escherichia coli, and Listeria. The Salmonella strains isolated were further characterized by serotype, antimicrobial resistance, and pulsed-field gel electrophoresis profile. This article summarizes the Salmonella data collected by the MDP between 2002 and 2012. The results show that the rates of Salmonella prevalence ranged from absent to 0.34% in cilantro. A total of 152 isolates consisting of over 50 different serotypes were isolated from the various produce types, and the top five were Salmonella enterica serotype Cubana, S. enterica subspecies arizonae (subsp. IIIa) and diarizonae (subsp. IIIb), and S. enterica serotypes Newport, Javiana, and Infantis. Among these, Salmonella serotypes Newport and Javiana are also listed among the top five Salmonella serotypes that caused most foodborne outbreaks. Other serotypes that are frequent causes of infection, such as S. enterica serotypes Typhimurium and Enteritidis, were also found in fresh produce but were not prevalent. About 25% of the MDP samples were imported produce, including 65% of green onions, 44% of tomatoes, 42% of hot peppers, and 41% of cantaloupes. However, imported produce did not show higher numbers of Salmonella-positive samples, and in some products, like cilantro, all of the Salmonella isolates were from domestic samples. About 6.5% of the Salmonella isolates were resistant to the antimicrobial compounds tested, but no single commodity or serotype was found to be the most common carrier of resistant strains or of resistance. The pulsed-field gel electrophoresis profiles of the produce isolates showed similarities with Salmonella isolates from meat samples and from outbreaks, but

  6. In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon.

    PubMed

    Silva, Luiz Francisco Rocha e; Magalhães, Pedro Melillo de; Costa, Mônica Regina Farias; Alecrim, Maria das Graças Costa; Chaves, Francisco Célio Maia; Hidalgo, Ari de Freitas; Pohlit, Adrian Martin; Vieira, Pedro Paulo Ribeiro

    2012-11-01

    Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions. PMID:23147140

  7. Genome Sequence of Lactobacillus brevis Strain D6, Isolated from Smoked Fresh Cheese.

    PubMed

    Kant, Ravi; Uroić, Ksenija; Hynönen, Ulla; Kos, Blaženka; Šušković, Jagoda; Palva, Airi

    2016-01-01

    The autochthonousLactobacillus brevisstrain D6, isolated from smoked fresh cheese, carries a 45-kDa S-layer protein. Strain D6 has shown adhesion to extracellular matrix proteins and to Caco-2 intestinal epithelial cells, as well as immunomodulatory potential and beneficial milk technological properties. Hence, it could be used as a potential probiotic starter culture for cheese production. PMID:27056237

  8. Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data.

    PubMed

    O'Brien, John D; Iqbal, Zamin; Wendler, Jason; Amenga-Etego, Lucas

    2016-06-01

    We present a rigorous statistical model that infers the structure of P. falciparum mixtures-including the number of strains present, their proportion within the samples, and the amount of unexplained mixture-using whole genome sequence (WGS) data. Applied to simulation data, artificial laboratory mixtures, and field samples, the model provides reasonable inference with as few as 10 reads or 50 SNPs and works efficiently even with much larger data sets. Source code and example data for the model are provided in an open source fashion. We discuss the possible uses of this model as a window into within-host selection for clinical and epidemiological studies. PMID:27362949

  9. Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data

    PubMed Central

    O’Brien, John D.; Amenga-Etego, Lucas

    2016-01-01

    We present a rigorous statistical model that infers the structure of P. falciparum mixtures—including the number of strains present, their proportion within the samples, and the amount of unexplained mixture—using whole genome sequence (WGS) data. Applied to simulation data, artificial laboratory mixtures, and field samples, the model provides reasonable inference with as few as 10 reads or 50 SNPs and works efficiently even with much larger data sets. Source code and example data for the model are provided in an open source fashion. We discuss the possible uses of this model as a window into within-host selection for clinical and epidemiological studies. PMID:27362949

  10. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

    PubMed

    Chaorattanakawee, Suwanna; Saunders, David L; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap; Lanteri, Charlotte A

    2015-08-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure.

  11. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

    PubMed

    Chaorattanakawee, Suwanna; Saunders, David L; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap; Lanteri, Charlotte A

    2015-08-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  12. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

    PubMed Central

    Chaorattanakawee, Suwanna; Saunders, David L.; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T.; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D.; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap

    2015-01-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  13. Antimicrobial Susceptibility of Escherichia coli Isolated from Fresh-Marketed Nile Tilapia (Oreochromis niloticus)

    PubMed Central

    Rocha, Rafael dos Santos; Leite, Lana Oliveira; de Sousa, Oscarina Viana; Vieira, Regine Helena Silva dos Fernandes

    2014-01-01

    The contamination of seafood by bacteria of fecal origin, especially Escherichia coli, is a widely documented sanitary problem. The objective of the present study was to isolate E. coli strains from the gills, muscle, and body surface of farmed Nile tilapias (Oreochromis niloticus) fresh-marketed in supermarkets in Fortaleza (Ceará, Brazil), to determine their susceptibility to antibiotics of different families (amikacin, gentamicin, imipenem, cephalothin, cefotaxime, ciprofloxacin, aztreonam, ampicillin, nalidixic acid, tetracycline, and sulfametoxazol-trimetoprim), and to determine the nature of resistance by plasmid curing. Forty-four strains (body surface = 25, gills = 15, muscle = 4) were isolated, all of which were susceptible to amikacin, aztreonam, cefotaxime, ciprofloxacin, gentamicin, and imipenem. Gill and body surface samples yielded 11 isolates resistant to ampicillin, tetracycline, and sulfametoxazol-trimetoprim, 4 of which of plasmidial nature. The multiple antibiotic resistance index was higher for strains isolated from body surface than from gills. The overall high antibiotic susceptibility of E. coli strains isolated from fresh-marketed tilapia was satisfactory, although the occasional finding of plasmidial resistance points to the need for close microbiological surveillance of the farming, handling, and marketing conditions of aquaculture products. PMID:24808957

  14. Antiplasmodial properties of kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii against chloroquine-resistant Plasmodium falciparum

    PubMed Central

    BARLIANA, MELISA I.; SURADJI, EKA W.; ABDULAH, RIZKY; DIANTINI, AJENG; HATABU, TOSHIMITSU; NAKAJIMA-SHIMADA, JUNKO; SUBARNAS, ANAS; KOYAMA, HIROSHI

    2014-01-01

    Previous intervention studies have shown that the most effective agents used in the treatment of malaria were isolated from natural sources. Plants consumed by non-human primates serve as potential drug sources for human disease management due to the similarities in anatomy, physiology and disease characteristics. The present study investigated the antiplasmodial properties of the primate-consumed plant, Schima wallichii (S. wallichii) Korth. (family Theaceae), which has already been reported to have several biological activities. The ethanol extract of S. wallichii was fractionated based on polarity using n-hexane, ethyl acetate and water. The antiplasmodial activity was tested in vitro against chloroquine-resistant Plasmodium falciparum (P. falciparum) at 100 μg/ml for 72 h. The major compound of the most active ethyl acetate fraction was subsequently isolated using column chromatography and identified by nuclear magnetic resonance. The characterized compound was also tested against chloroquine-resistant P. falciparum in culture to evaluate its antiplasmodial activity. The ethanol extract of S. wallichii at 100 μg/ml exhibited a significant parasite shrinkage after 24 h of treatment. The ethyl acetate fraction at 100 μg/ml was the most active fraction against chloroquine-resistant P. falciparum. Based on the structural characterization, the major compound isolated from the ethyl acetate fraction was kaempferol-3-O-rhamnoside, which showed promising antiplasmodial activity against chloroquine-resistant P. falciparum with an IC50 of 106 μM after 24 h of treatment. The present study has provided a basis for the further investigation of kaempferol-3-O-rhamnoside as an active compound for potential antimalarial therapeutics. PMID:24944812

  15. Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

    PubMed Central

    Pavlov, Tengis S.; Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Pochynyuk, Oleh; Staruschenko, Alexander

    2015-01-01

    Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays. PMID:26381526

  16. Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

    PubMed

    Wang, Li-Hsueh; Chen, Hung-Kai; Jhu, Chu-Sian; Cheng, Jing-O; Fang, Lee-Shing; Chen, Chii-Shiarng

    2015-12-01

    The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly-isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral-associated Symbiodinium had higher protein content than did cultured and sea anemone-associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone-associated Symbiodinium had a distinct pattern compared coral-associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral-associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts. PMID:26987007

  17. Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.

    PubMed

    Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

    2014-08-01

    Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species.

  18. Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.

    PubMed

    Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

    2014-08-01

    Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species. PMID:24953504

  19. Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

    PubMed

    Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

    2016-03-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera.

  20. Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

    PubMed

    Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

    2016-03-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera. PMID:26832643

  1. High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo.

    PubMed

    Koukouikila-Koussounda, Felix; Bakoua, Damien; Fesser, Anna; Nkombo, Michael; Vouvoungui, Christevy; Ntoumi, Francine

    2015-07-01

    Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here

  2. Sequence diversity of the merozoite surface protein 1 of Plasmodium falciparum in clinical isolates from the Kilombero District, Tanzania.

    PubMed

    Jiang, G; Daubenberger, C; Huber, W; Matile, H; Tanner, M; Pluschke, G

    2000-01-01

    Merozoite surface protein 1 of Plasmodium falciparum (PfMSP-1) is regarded as a key candidate antigen for malaria vaccine development. It exhibits significant antigenic polymorphism and has been divided into 17 building blocks based on the analysis of sequence diversity. Differences in the antigenic composition of PfMSP-1 in local P. falciparum populations may result in differences in the efficacy of vaccines, which contain sequences of particular allelic variant(s) of PfMSP-1. To contribute to the required knowledge of genetic diversity of malaria parasites in geographically diverse regions, we have used the polymerase chain reaction (PCR) to analyze the sequence diversity of blocks 1-4 of PfMSP-1 in disease isolates from the Kilombero District in Tanzania. In the semi-conserved block 1, in which dimorphic amino acid variances have been described at three positions, we found three of the five previously described combinations of these three pairs of amino acids. In addition one combination was found, which has not been reported before in parasite isolates from different locations worldwide. Of the two sequence variants, which were dominating, one (S44-Q47-V52) corresponded to the 83.1 sequence incorporated into the SPf66 malaria peptide vaccine, while the other one (G44-H47-I52) differed from the previous in all three dimorphic amino acids. The partial protection observed in a phase III SPf66 trial conducted in the Kilombero District in children aged 1-5, thus does not seem to be associated with a clear dominance of favourable variants of block 1 of PfMSP-1 in this area. All three different principle types of block 2, the major polymorphic region of PfMSP-1, were found in the Tanzanian isolates. Most of the sequences contained K1-type tripeptide repeats, but clones with MAD20-type repeats or no repetitive sequence (RO33-type block 2) were also present. K1- and MAD20-type tripeptide repeat motifs were never mixed within one parasite clone. In one sequence a

  3. Ex vivo drug sensitivity profiles of Plasmodium falciparum field isolates from Cambodia and Thailand, 2005 to 2010, determined by a histidine-rich protein-2 assay

    PubMed Central

    2012-01-01

    Background In vitro drug susceptibility assay of Plasmodium falciparum field isolates processed “immediate ex vivo” (IEV), without culture adaption, and tested using histidine-rich protein-2 (HRP-2) detection as an assay, is an expedient way to track drug resistance. Methods From 2005 to 2010, a HRP-2 in vitro assay assessed 451 P. falciparum field isolates obtained from subjects with malaria in western and northern Cambodia, and eastern Thailand, processed IEV, for 50% inhibitory concentrations (IC50) against seven anti-malarial drugs, including artesunate (AS), dihydroartemisinin (DHA), and piperaquine. Results In western Cambodia, from 2006 to 2010, geometric mean (GM) IC50 values for chloroquine, mefloquine, quinine, AS, DHA, and lumefantrine increased. In northern Cambodia, from 2009–2010, GM IC50 values for most drugs approximated the highest western Cambodia GM IC50 values in 2009 or 2010. Conclusions Western Cambodia is associated with sustained reductions in anti-malarial drug susceptibility, including the artemisinins, with possible emergence, or spread, to northern Cambodia. This potential public health crisis supports continued in vitro drug IC50 monitoring of P. falciparum isolates at key locations in the region. PMID:22694953

  4. Genetic Polymorphism of msp1 and msp2 in Plasmodium falciparum Isolates from Côte d'Ivoire versus Gabon

    PubMed Central

    Yavo, William; Konaté, Abibatou; Mawili-Mboumba, Denise Patricia; Kassi, Fulgence Kondo; Tshibola Mbuyi, Marie L.; Angora, Etienne Kpongbo; Menan, Eby I. Hervé; Bouyou-Akotet, Marielle K.

    2016-01-01

    Introduction. The characterization of genetic profile of Plasmodium isolates from different areas could help in better strategies for malaria elimination. This study aimed to compare P. falciparum diversity in two African countries. Methods. Isolates collected from 100 and 73 falciparum malaria infections in sites of Côte d'Ivoire (West Africa) and Gabon (Central Africa), respectively, were analyzed by a nested PCR amplification of msp1 and msp2 genes. Results. The K1 allelic family was widespread in Côte d'Ivoire (64.6%) and in Gabon (56.6%). For msp2, the 3D7 alleles were more prevalent (>70% in both countries) compared to FC27 alleles. In Côte d'Ivoire, the frequencies of multiple infections with msp1 (45.1%) and msp2 (40.3%) were higher than those found for isolates from Gabon, that is, 30.2% with msp1 and 31.4% with msp2. The overall complexity of infection was 1.66 (SD = 0.79) in Côte d'Ivoire and 1.58 (SD = 0.83) in Gabon. It decreased with age in Côte d'Ivoire in contrast to Gabon. Conclusion. Differences observed in some allelic families and in complexity profile may suggest an impact of epidemiological facies as well as immunological response on genetic variability of P. falciparum. PMID:27110390

  5. Virulence Potential of Activatable Shiga Toxin 2d–Producing Escherichia coli Isolates from Fresh Produce

    PubMed Central

    Melton-Celsa, Angela R.; O'Brien, Alison D.; Feng, Peter C. H.

    2016-01-01

    Shiga toxin (Stx)–producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  6. Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

    PubMed

    Melton-Celsa, Angela R; O'Brien, Alison D; Feng, Peter C H

    2015-11-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

  7. Potent Ex Vivo Activity of Naphthoquine and Methylene Blue against Drug-Resistant Clinical Isolates of Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Wirjanata, Grennady; Sebayang, Boni F; Chalfein, Ferryanto; Prayoga; Handayuni, Irene; Trianty, Leily; Kenangalem, Enny; Noviyanti, Rintis; Campo, Brice; Poespoprodjo, Jeanne Rini; Möhrle, Jörg J; Price, Ric N; Marfurt, Jutta

    2015-10-01

    The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC50]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC50s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic.

  8. In vitro amodiaquine resistance and its association with mutations in pfcrt and pfmdr1 genes of Plasmodium falciparum isolates from Nigeria.

    PubMed

    Folarin, O A; Bustamante, C; Gbotosho, G O; Sowunmi, A; Zalis, M G; Oduola, A M J; Happi, C T

    2011-12-01

    Amodiaquine (AQ) is currently being used as a partner drug in combination with artesunate for treatment of uncomplicated malaria in most endemic countries of Africa. In the absence of molecular markers of artemisinin resistance, molecular markers of resistance to AQ may be useful for monitoring the development and spread of parasites resistance to Artesunate-Amodiaquine combination. This study was designed to assess the potential role of polymorphisms on pfcrt and pfmdr1 genes and parasite in vitro susceptibility for epidemiological surveillance of amodiaquine resistance in Plasmodium falciparum. The modified schizont inhibition assay was used to determine in vitro susceptibility profiles of 98 patients' isolates of P. falciparum to amodiaquine. Polymorphisms on parasites pfcrt and pfmdr1 genes were determined with nested PCR followed by sequencing. The geometric mean (GM) of AQ 50% inhibitory concentration (IC-50) in the 97 P. falciparum isolates was 20.48 nM (95% CI 16.53-25.36 nM). Based on the cut-off value for AQ in vitro susceptibility, 87% (84) of the P. falciparum isolates were sensitive to AQ (GM IC-50=16.32 nM; 95%CI 13.3-20.04 nM) while 13% were resistant to AQ in vitro (GM IC-50=88.73nM; 95%CI 69.67-113.0nM). Molecular analysis showed presence of mutant CVIET pfcrt haplotype, mutant pfmdr1Tyr86 allele and the double mutant CVIET pfcrt haplotype+pfmdr1Tyr86 in 72%, 49% and 35%, respectively. The GM IC-50 of isolates harboring the wild-type pfcrt CVMNK haplotype+pfmdr1Asn86 allele (3.93nM; 95%CI 1.82-8.46 nM) was significantly lower (p=0.001) than those isolates harboring the double mutant pfcrt CVIET haplotype+pfmdr1Tyr86 allele (50.40 nM; 95%CI 40.17-63.24 nM). Results from this study suggest that polymorphisms in pfcrt and pfmdr1 genes are important for AQ resistance and therefore may be useful for epidemiological surveillance of P. falciparum resistance to AQ.

  9. Emergence of sulfadoxine-pyrimethamine resistance in Indian isolates of Plasmodium falciparum in the last two decades.

    PubMed

    Kumar, Amit; Moirangthem, Romilla; Gahlawat, Suresh Kumar; Chandra, Jagdish; Gupta, Purva; Valecha, Neena; Anvikar, Anup; Singh, Vineeta

    2015-12-01

    Genotyping the sulfadoxine-pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country.

  10. Disease specific modules and hub genes for intervention strategies: A co-expression network based approach for Plasmodium falciparum clinical isolates.

    PubMed

    Subudhi, Amit Kumar; Boopathi, Pon Arunachalam; Pandey, Isha; Kaur, Ramandeep; Middha, Sheetal; Acharya, Jyoti; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2015-10-01

    Systems biology approaches that are based on gene expression and bioinformatics analysis have been successful in predicting the functions of many genes in Plasmodium falciparum, a protozoan parasite responsible for most of the deaths due to malaria. However, approaches that can provide information about the biological processes that are active in this parasite in vivo during complicated malaria conditions have been scarcely deployed. Here we report the analysis of a weighted gene co-expression based network for P. falciparum, from non-cerebral clinical complications. Gene expression profiles of 20 P. falciparum clinical isolates were utilized to construct the same. A total of 20 highly interacting modules were identified post network creation. In 12 of these modules, at least 10% of the member genes, were found to be differentially regulated in parasites from patient isolates showing complications, when compared with those from patients with uncomplicated disease. Enrichment analysis helped identify biological processes like oxidation-reduction, electron transport chain, protein synthesis, ubiquitin dependent catabolic processes, RNA binding and purine nucleotide metabolic processes as associated with these modules. Additionally, for each module, highly connected hub genes were identified. Detailed functional analysis of many of these, which have known annotated functions underline their importance in parasite development and survival. This suggests, that other hub genes with unknown functions may also be playing crucial roles in parasite biology, and, are potential candidates for intervention strategies.

  11. Freshly isolated mitochondria from failing human hearts exhibit preserved respiratory function.

    PubMed

    Cordero-Reyes, Andrea M; Gupte, Anisha A; Youker, Keith A; Loebe, Matthias; Hsueh, Willa A; Torre-Amione, Guillermo; Taegtmeyer, Heinrich; Hamilton, Dale J

    2014-03-01

    In heart failure mitochondrial dysfunction is thought to be responsible for energy depletion and contractile dysfunction. The difficulties in procuring fresh left ventricular (LV) myocardium from humans for assessment of mitochondrial function have resulted in the reliance on surrogate markers of mitochondrial function and limited our understanding of cardiac energetics. We isolated mitochondria from fresh LV wall tissue of patients with heart failure and reduced systolic function undergoing heart transplant or left ventricular assist device placement, and compared their function to mitochondria isolated from the non-failing LV (NFLV) wall tissue with normal systolic function from patients with pulmonary hypertension undergoing heart-lung transplant. We performed detailed mitochondrial functional analyses using 4 substrates: glutamate-malate (GM), pyruvate-malate (PM) palmitoyl carnitine-malate (PC) and succinate. NFLV mitochondria showed preserved respiratory control ratios and electron chain integrity with only few differences for the 4 substrates. In contrast, HF mitochondria had greater respiration with GM, PM and PC substrates and higher electron chain capacity for PM than for PC. Surprisingly, HF mitochondria had greater respiratory control ratios and lower ADP-independent state 4 rates than NFLV mitochondria for GM, PM and PC substrates demonstrating that HF mitochondria are capable of coupled respiration ex vivo. Gene expression studies revealed decreased expression of key genes in pathways for oxidation of both fatty acids and glucose. Our results suggest that mitochondria from the failing LV myocardium are capable of tightly coupled respiration when isolated and supplied with ample substrates. Thus energy starvation in the failing heart may be the result of dysregulation of metabolic pathways, impaired substrate supply or reduced mitochondrial number but not the result of reduced mitochondrial electron transport capacity. PMID:24412531

  12. Development of a Potential Probiotic Fresh Cheese Using Two Lactobacillus salivarius Strains Isolated from Human Milk

    PubMed Central

    Cárdenas, Nivia; Peirotén, Ángela; Rodríguez, Juan M.; Fernández, Leónides

    2014-01-01

    Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study. PMID:24971351

  13. Development of a potential probiotic fresh cheese using two Lactobacillus salivarius strains isolated from human milk.

    PubMed

    Cárdenas, Nivia; Calzada, Javier; Peirotén, Angela; Jiménez, Esther; Escudero, Rosa; Rodríguez, Juan M; Medina, Margarita; Fernández, Leónides

    2014-01-01

    Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study. PMID:24971351

  14. Increased pfmdr1 copy number in Plasmodium falciparum isolates from Suriname.

    PubMed

    Labadie-Bracho, Mergiory; Adhin, Malti R

    2013-07-01

    Amplification of the pfmdr1 gene is associated with clinical failures and reduced in vivo and in vitro sensitivity to both mefloquine and artemether-lumefantrine in South-East Asia. Several African countries have reported the absence or very low prevalence of increased copy number, whilst South American reports are limited to Peru without and Venezuela with increased pfmdr1 multiplication. The relative pfmdr1 copy numbers were assessed in 68 isolates from Suriname collected from different endemic villages (2005) and from mining areas (2009). 11% of the isolates harbour multiple copies of the pfmdr1 gene. Isolates originating from mining areas do not yet display a higher tendency for increased copy number and no significant differences could be registered within a time span of 4 years, but the mere presence of increased copy number warrants caution and should be considered as an early warning sign for emerging drug resistance in Suriname and South America. PMID:23621761

  15. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli.

    PubMed

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca(2+) concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  16. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  17. 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

  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. Crystal violet reactions of fresh clinical isolates of Staphylococcus aureus from two British hospitals.

    PubMed

    Freeman, R; Hudson, S J; Burdess, D

    1990-12-01

    When 168 fresh clinical isolates of Staphylococcus aureus were examined for their reactions on a medium containing 1 part in 100,000 crystal violet 50.6% of strains produced a purple appearance, 39.3% produced a white appearance and 10.1% produced a yellow appearance. Purple-reacting isolates were significantly associated with both invasive infections (P less than 0.01) and hospital origin (P less than 0.001). There were no significant associations between the crystal violet reactions and either animal contact or other properties previously reported to be characteristic of white and yellow-reacting strains (beta haemolysin and bovine coagulase production). The results of phage typing showed associations between susceptibility to group III phages and purple-reacting strains and between phage group II susceptibility and white and yellow-reacting strains. There was also a highly significant association between white reactions on crystal violet agar and susceptibility to lysis by a combination of all three groups (that is, I + II + III) and white-reacting strains were significantly more susceptible to lysis by phages 94 and/or 96, whether as a restricted pattern or as part of a broader pattern. The purple reaction on crystal violet medium may be a reliable marker of the 'hospital staphylococcus'.

  20. Enterobacter and Klebsiella species isolated from fresh vegetables marketed in Valencia (Spain) and their clinically relevant resistances to chemotherapeutic agents.

    PubMed

    Falomir, María Pilar; Rico, Hortensia; Gozalbo, Daniel

    2013-12-01

    Occurrence of antibiotic-resistant pathogenic or commensal enterobacteria in marketed agricultural foodstuffs may contribute to their incorporation into the food chain and constitutes an additional food safety concern. In this work, we have determined the clinically relevant resistances to 11 common chemotherapeutic agents in Enterobacter and Klebsiella isolates from fresh vegetables from various sources (supermarkets and greengrocers' shops in Valencia, Spain). A total of 96 isolates were obtained from 160 vegetables analyzed (50% positive samples): 68 Enterobacter isolates (59 E. cloacae, two E. aerogenes, two E. cancerogenus, one E. gergoviae, and four E. sakazakii, currently Cronobacter spp.), and 28 Klebsiella isolates (19 K. oxytoca and 9 K. pneumoniae). Only seven isolates were susceptible to all agents tested, and no resistances to ceftazidime, ciprofloxacin, gentamicin, and chloramphenicol were detected. Most isolates were resistant to amoxicillin/clavulanic acid (74 [58 Enterobacter and 16 Klebsiella]) or to ampicillin (80 [55/25]). Other resistances were less frequent: nitrofurantoin (13 isolates [12/1]), tetracycline (6 [5/1]), co-trimoxazole (3 [3/0]), cefotaxime (1 [1/0]), and streptomycin (2 [1/1]). Multiresistant isolates to two (56 [41/15]), three (10 E. cloacae isolates), four (one E. cloacae and one K. pneumoniae isolate), and five (two E. cloacae isolates) chemotherapeutic agents were also detected. The presence of potential pathogens points to marketed fresh produce, which often is eaten raw, as a risk factor for consumer health. In addition, these results support the usefulness of these bacterial species as indicators of the spreading of antibiotic resistances into the environment, particularly in the food chain, and suggest their role as carriers of resistance determinants from farms to consumers, which may constitute an additional "silent" food safety concern. Therefore, there is a need to improve the hygienic quality of marketed fresh

  1. Reduced in vitro doxycycline susceptibility in plasmodium falciparum field isolates from Kenya is associated with PfTetQ KYNNNN sequence polymorphism.

    PubMed

    Achieng, Angela O; Ingasia, Luiser A; Juma, Dennis W; Cheruiyot, Agnes C; Okudo, Charles A; Yeda, Redemptah A; Cheruiyot, Jelagat; Akala, Hoseah M; Johnson, Jacob; Andangalu, Ben; Eyase, Fredrick; Jura, Walter G Z O; Kamau, Edwin

    2014-10-01

    Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing. PMID:25070109

  2. Natural antisense transcripts in Plasmodium falciparum isolates from patients with complicated malaria.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Orekondy, Harsha B; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2014-06-01

    Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how the parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which are best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. Using a custom designed strand specific whole genome microarray, a total of 797 NATs targeted against annotated loci have been detected. Out of these, 545 NATs are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. However, 96 genes showed a change in sense/antisense ratio on comparison between uncomplicated and complicated disease conditions. The antisense transcripts map to a broad range of biochemical/metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action of NATs, which would further help in

  3. Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

    PubMed Central

    2012-01-01

    Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment. PMID:23077722

  4. Antioxidant properties in some selected cyanobacteria isolated from fresh water bodies of Sri Lanka.

    PubMed

    Hossain, Md Fuad; Ratnayake, R R; Meerajini, Kirisnashamy; Wasantha Kumara, K L

    2016-09-01

    Phytonutrients and pigments present in cyanobacteria act as antioxidants, which facilitate the formation of body's defense mechanism against free radical damage to cells. The aim of this investigation was to study the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, phycobiliproteins (PBPs), and active compounds in four cyanobacterial species, that is, Oscillatoria sp., Lyngbya sp., Microcystis sp., and Spirulina sp. isolated from fresh water bodies of Sri Lanka. In this study, Lyngbya sp., showed highest TPC (5.02 ± 0.20 mg/g), TFC (664.07 ± 19.76 mg/g), and total PBPs (127.01 mg/g) value. The ferric reducing antioxidant power (FRAP) was recorded highest in Oscillatoria sp. (39.63 ± 7.02), whereas the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was also reported the highest in Oscillatoria sp. (465.31 ± 25.76) followed by Lyngbya sp. (248.39 ± 11.97). In FTIR spectroscopy, Lyngbya sp. does not show any N-H stretching band which is ultimately responsible for the inhibition of antioxidant activity. The study revealed that Lyngbya sp. and Oscillatoria sp. can be an excellent source for food, pharmaceutical, and other industrial uses. PMID:27625779

  5. Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts.

    PubMed

    Ravesloot, J H; Ypey, D L; Vrijheid-Lammers, T; Nijweide, P J

    1989-09-01

    Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.

  6. Protective Effects of N-acetylcysteine Against the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes

    PubMed Central

    Abdoli, Narges; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2014-01-01

    Purpose: Hepatotoxicity is one of the most important side effects of the statins therapy as lipid-lowering agents. However, the mechanism(s) of hepatotoxicity induced by these drugs is not clearly understood yet, and no hepatoprotective agent has been developed against this complication. Methods: The protective effect of N-acetylcysteine (NAC) against statins-induced cytotoxicity was evaluated by using freshly isolated rat hepatocytes. Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. This technique is based on liver perfusion with collagenase after removal of calcium ion (Ca2+) with a chelator (ethylene glycol tetra acetic acid (EGTA) 0.5 mM). The level of parameters such as cell death, ROS formation, lipid peroxidation, mitochondrial membrane potential (MMP) in the statins-treated hepatocytes were determined. Additionally, the mentioned markers were assessed in the presence of NAC. Results: Incubation of hepatocytes with the statins resulted in cytotoxicity characterized by an elevation in cell death, increasing ROS generation and consequently lipid peroxidation and impairment of mitochondrial function. Administration of NAC caused reduction in amount of ROS formation, lipid peroxidation and finally, cell viability and mitochondrial membrane potential (MMP) were improved. Conclusion: This study confirms that oxidative stress and consequently mitochondrial dysfunction is one of the mechanisms underlying the statins-induced liver injury and treating hepatocytes by NAC (200 μM) attenuates this cytotoxicity. PMID:24754008

  7. siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts.

    PubMed

    Lokossou, Adjimon Gatien; Toufaily, Chirine; Vargas, Amandine; Barbeau, Benoit

    2016-01-01

    Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols. PMID:27685614

  8. Antibiotic susceptibility and resistance of Staphylococcus aureus isolated from fresh porcine skin xenografts: risk to recipients with thermal injury.

    PubMed

    Busby, Stacey-Ann; Robb, Andrew; Lang, Sue; Takeuchi, Yasu; Vesely, Pavel; Scobie, Linda

    2014-03-01

    The previous use of fresh porcine xenografts at the Prague Burn Centre had raised concerns over the transmission of zoonotic pathogens. This study examines the risk of zoonotic Staphylococcus aureus colonisation of burn patients from fresh porcine skin xenografts. Samples were collected from the nares, skin and perineum of commercial pigs (n=101) and were screened for methicillin sensitive S. aureus (MSSA) and resistant S. aureus (MRSA). The efficacy of the antibiotic wash used in decontamination of the pigskin was tested against planktonic- and biofilm-grown isolates. The spa type of each isolate was also confirmed. All pig swabs were negative for MRSA but 86% positive for MSSA. All planktonic-grown isolates of MSSA were sensitive to chloramphenicol and nitrofurantoin and 44% of isolates were resistant to streptomycin. Isolates grown as biofilm exhibited higher rates of antimicrobial resistance. Sequence analysis revealed three distinct spa types of the MRSA ST398 clonal type. This finding demonstrates the existence of a MSSA reservoir containing spa types resembling those of well-known MRSA strains. These MSSA exhibit resistance to antibiotics used for decontamination of the pigskin prior to xenograft. Amended use of procurement could allow the use of fresh pigskin xenografts to be reinstated.

  9. Isolation, purification and identification of etiolation substrate from fresh-cut Chinese water-chestnut (Eleocharis tuberosa).

    PubMed

    Pan, Yong-Gui; Li, Yi-Xiao; Yuan, Meng-Qi

    2015-11-01

    Fresh cut Chinese water-chestnut is a popular ready-to-eat fresh-cut fruit in China. However, it is prone to etiolation and the chemicals responsible for this process are not known yet. To address this problem, we extracted phytochemicals from etiolated Chinese water-chestnut and separated them using MPLC and column chromatography. Four compounds were obtained and their structures were determined by interpretation of UV, TLC, HPLC and NMR spectral data and by comparison with reported data. We identified these compounds as eriodictyol, naringenin, sucrose and ethyl D-glucoside. Among those, eriodictyol and naringenin were both isolated for the first time in fresh-cut Chinese water-chestnut and are responsible for the yellowing of this fruit cutting.

  10. Characterization of drug resistance associated genetic polymorphisms among Plasmodium falciparum field isolates in Ujjain, Madhya Pradesh, India

    PubMed Central

    2014-01-01

    Background Since 2011, artesunate + sulphadoxine-pyrimethamine (ASP), instead of chloroquine, has been recommended for treatment of uncomplicated malaria in India. In Ujjain, central India, with an annual parasite index <0.1, the prevalence of drug-resistant Plasmodium falciparum is unknown. In other parts of India chloroquine and sulphadoxine-pyrimethamine-resistant P. falciparum is prevalent. The aim of this study was to determine the prevalence of anti-malarial drug resistance-associated genetic polymorphisms in P. falciparum collected in Ujjain in 2009 and 2010, prior to the introduction of ASP. Methods Blood samples from 87 patients with P. falciparum mono-infection verified by microscopy were collected on filter-paper at all nine major pathology laboratories in Ujjain city. Codons Pfcrt 72–76, pfmdr1 1034–1246, pfdhfr 16–185, pfdhps 436–632 and pfnhe1 ms4760 haplotypes were identified by sequencing. Pfcrt K76T and pfmdr1 N86Y were identified by restriction fragment length polymorphism, and pfmdr1 gene copy number by real-time PCR. Results Sulphadoxine-pyrimethamine resistance-associated pfdhfr 108 N and 59R alleles were found in 75/78 (96%) and 70/78 (90%) samples, respectively, and pfdhps 437G was found in 7/77 (9%) samples. Double mutant pfdhfr 59R + 108 N were found in 62/76 (82%) samples. Triple mutant pfdhfr 59R + 108 N and pfdhps 437G were found in 6/76 (8%) samples. Chloroquine-resistance-associated pfcrt 76 T was found in 82/87 (94%). The pfcrt 72–76 haplotypes found were: 80/84 (95%) SVMNT, 3/84 (4%) CVMNK and 1/84 (1%) CVMNT. Pfmdr1 N86 and 86Y were identified in 70/83 (84%) and 13/83 (16%) samples, respectively. Pfmdr1 S1034 + N1042 + D1246 were identified together in 70/72 (97%) of successfully sequenced samples. One pfmdr1 gene copy was found in 74/75 (99%) successfully amplified samples. Conclusion This is the first characterization of key anti-malarial drug resistance-associated genetic markers among P

  11. Formation of Osteogenic Colonies on Well-Defined Adhesion Peptides by Freshly-Isolated Human Marrow Cells

    PubMed Central

    Au, Ada; Boehm, Cynthia A.; Mayes, Anne M.; Muschler, George F.; Griffith, Linda G.

    2007-01-01

    Bone graft performance can be enhanced by addition of connective tissue progenitors (CTPs) from fresh bone marrow in a manner that concentrates the CTP cell population within the graft. Here, we used small peptide adhesion ligands presented against an otherwise adhesion-resistant synthetic polymer background in order to illuminate the molecular basis for the attachment and colony formation by osteogenic CTPs from fresh human marrow, and contrast the behavior of fresh marrow to many commonly-used osteogenic cell sources. The linear GRGDSPY ligand was as effective as tissue culture polystyrene in fostering attachment of culture-expanded porcine CTPs. Although this GRGDSPY peptide was more effective than control peptides in fostering alkaline phosphatase-positive (AP) colony formation from primary human marrow in 5 of the 7 patients tested, GRGDSPY was as effective as the control glass substrate in only one patient of 7. Thus, the peptide appears capable of enabling osteoblastic development from only a subpopulation of CTPs in marrow. The bone sialoprotein-derived peptide FHRRIKA was ineffective in fostering attachment of primary culture-expanded pig CTPs, although it was as effective as GRGDSPY in fostering AP-positive colonies from fresh human marrow. This study provides insights into integrin-mediated behaviors of CTPs and highlights differences between freshly-isolated marrow and culture-expanded cells. PMID:17222453

  12. Expression of nucleoside transporter in freshly isolated neurons and astrocytes from mouse brain.

    PubMed

    Li, B; Gu, L; Hertz, L; Peng, L

    2013-11-01

    Nucleoside transporters comprise equilibrative ENT1-4 and concentrative CNT1-3. CNTs transport against an intracellular/extracellular gradient and are essential for transmitter removal, independently of metabolic need. ENT1-4 mediate transport until intracellular/extracellular equilibrium of the transported compound, but are very efficient, when the accumulated nucleoside or nucleobase is rapidly eliminated by metabolism. Most nucleoside transporters are membrane-bound, but ENT3 is mainly intracellular. This study uses freshly isolated neurons and astrocytes from two adult mouse strains. In one transgenic strain the neuronal marker Thy1 was associated with a compound fluorescing at one wavelength, and in the other the astrocytic marker GFAP was associated with a compound fluorescent at a different wavelength. Highly purified astrocytic and neuronal populations (as determined by presence/absence of cell-specific genes) were obtained from these mice by fluorescence-activated cell sorting. In each population mRNA analysis was performed by reverse-transcription polymerase chain reaction. CNT1 was absent in both cell types; all other nucleoside transporters were expressed to at least a similar degree (in relation to applied amount of RNA and to a house-keeping gene) in astrocytes as in neurons. Astrocytic ENT3 enrichment was dramatic, but it was not up-regulated after fluoxetine-mediated increase in DNA synthesis. A comparison with results obtained in cultured astrocytes shows that the latter are generally compatible with the present findings and suggests that many observations obtained in intact tissue, mainly by in situ hybridization (which also determines mRNA expression) may underestimate astrocytic nucleoside transporter expression.

  13. Effect of olive oil phenols on the production of inflammatory mediators in freshly isolated human monocytes.

    PubMed

    Rosignoli, Patrizia; Fuccelli, Raffaela; Fabiani, Roberto; Servili, Maurizio; Morozzi, Guido

    2013-08-01

    Recent in vitro and in vivo studies suggest that the anti-inflammatory properties of extra virgin olive oil may be involved in the prevention of chronic degenerative diseases. In this study, the ability of olive oil phenols to influence the release of superoxide anions (O2-), prostaglandin E2 (PGE2) and tumor necrosis factor α (TNFα) and the expression of cyclooxygenase2 (COX2) in human monocytes, freshly isolated from healthy donors, was investigated. O2- were measured by superoxide dismutase-inhibitable cytochrome c reduction and PGE2 and TNFα production were determined in culture medium with appropriate enzyme immunoassay kits. COX2 mRNA and protein were evaluated by quantitative reverse transcription-polymerase chain reaction and Western immunoblotting, respectively. Treatment of monocytes for 24 h with 100 μM of hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and their secoiridoid derivatives (3,4-DHPEA and p-HPEA linked to the dialdehydic form of elenolic acid: 3,4-DHPEA-EDA and p-HPEA-EDA, respectively) significantly (P<.05) inhibited the production of O2(-) as follows: 3,4-DHPEA (40%,), p-HPEA (9%), 3,4-DHPEA-EDA (25%) and p-HPEA-EDA (36%). Hydroxytyrosol also considerably reduced the expression of COX2 at both the mRNA and protein level (P<.05) and caused a clear dose-dependent reduction of PGE2 released into the culture medium (45% and 71% at 50 and 100 μM, respectively, P<.05). The COX2 mRNA was also efficiently inhibited by the secoiridoids. Moreover, it was shown that hydroxytyrosol increased the monocytes TNFα production. In addition to other chemopreventive properties, these results suggest that the health effects of olive oil phenols may be related to their ability to modulate the production of pro-inflammatory molecules, a property common to non-steroidal anti-inflammatory drugs. PMID:23477728

  14. Metabolism of cyclosporin A. I. Study in freshly isolated rabbit hepatocytes

    SciTech Connect

    Fabre, G.; Bertault-Peres, P.; Fabre, I.; Maurel, P.; Just, S.; Cano, J.P.

    1987-05-01

    The metabolism of cyclosporin A (CsA), a widely used immunosuppressive agent, was evaluated in freshly isolated rabbit hepatocytes by HPLC which separated CsA from its major group of derivatives, e.g. first generation metabolites (monohydroxylated and N-demethylated) and second generation derivatives (dihydroxylated and dihydroxy-N-demethylated). After exposure of hepatocytes to radiolabeled CsA (0.5 mg/liter), CsA was rapidly accumulated inside the cells and metabolized. The dihydroxylated metabolites represent the major intracellular forms after 1 hr. CsA metabolites synthesized inside the cells are then rapidly detected in the extracellular compartment. Unchanged drug and the various metabolites are concentrated inside the cells with transmembrane chemical gradients ranging between 20:1 and 40:1. Transport and metabolic processes for CsA have been evaluated over the following CsA extracellular concentration range, 0.1-10 mg/liter. Metabolism appears to be the rate-limiting step. The apparent affinity constant of CsA for the enzyme system involved in its metabolism is approximately 15 microM. Besides the lipophilicity of the molecule, which is responsible for the retention of CsA and its metabolites in the intracellular compartment, the presence of a binding component(s) in the hepatocytes was also demonstrated. CsA and its metabolites seem to have similar affinities for this binding site. These studies demonstrate that CsA is rapidly transformed inside the hepatocytes to various metabolites which may play an important role in the pharmacological activity of the drug and/or in its clinical toxicity.

  15. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  16. A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs

    PubMed Central

    2013-01-01

    Background Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. Methods A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. Results Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy

  17. N sub 2 -fixation by freshly isolated Nostoc from coralloid roots of the cycad Macrozamia riedlei (Fisch. ex Gaud. ) Gardn

    SciTech Connect

    Lindblad, P.; Atkins, C.A.; Pate, J.S. Univ. of Western Australia, Nedlands )

    1991-03-01

    Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation ({sup 15}N{sub 2} fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. The data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O{sub 2}, and a direct dependence on the cycad for substrates to support nitrogenase activity.

  18. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from (U-/sup 14/C)glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  19. Genetic polymorphism of merozoite surface protein-1 in Plasmodium falciparum isolates from patients with mild to severe malaria in Libreville, Gabon.

    PubMed

    Bouyou-Akotet, Marielle Karine; M'Bondoukwé, Noé Patrick; Mawili-Mboumba, Denise Patricia

    2015-01-01

    We assessed Plasmodium (P.) falciparum allelic diversity based on clinical severity and age. The study was conducted from 2011 to 2012 in Libreville, Gabon where malaria prevalence was 24.5%. The polymorphism of the merozoite surface protein-1 (msp1) locus was analyzed in isolates from patients with complicated and uncomplicated malaria. Blood was collected on filter paper. After DNA extraction, genotyping of the msp1 gene was performed using nested PCR. The K1, Ro33, and Mad20 allelic families were detected in 71 (63%), 64 (57%), and 38 (34%) of the 112 analyzed samples, respectively. Overall, 17 K1 and 11 Mad20 alleles were detected. There was no association between msp1 allelic families and age. Mad20 allelic diversity increased with the severity of malaria. The number of K1 and Mad20 alleles decreased with age. The multiplicity of infection (MOI) was 1-6 genotypes and the complexity of infection (COI) 1.8 ± 1. The COI differed based on age: it was 1.9 (±1.1) in the isolates from adults, 1.8 (±1.1) in those from 0-5 year-old children, whereas it tended to be lower (1.6 ± 0.8) in those from 6-15 year-old children. Extensive genetic diversity is found in P. falciparum strains circulating in Libreville. The number of specific msp1 alleles increased with clinical severity, suggesting an association between the diversity and the severity of malaria.

  20. N2-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn. 1

    PubMed Central

    Lindblad, Peter; Atkins, Craig A.; Pate, John S.

    1991-01-01

    Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (15N2 fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and 15N2 fixation in light. The relationship between ethylene formation and N2-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C2H4 per nanomole 15N2. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O2 in light, while in darkness there was a broad optimum around 5 to 8% O2. Inhibition of nitrogenase, in light, by pO2 above 0.6% was irreversible. Measurements of light-dependent O2 evolution and 14CO2 fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O2 levels above 6% O2 and the inhibition was reversible. These cells showed rates of light-dependent O2 evolution and 14CO2 fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O2, and a direct dependence on the cycad for substrates to support nitrogenase activity. PMID:16668050

  1. Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

    PubMed

    Markell, Lauren K; Mingoia, Robert T; Peterson, Heather M; Yao, Jianhong; Waters, Stephanie M; Finn, James P; Nabb, Diane L; Han, Xing

    2014-08-18

    Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17β-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together

  2. Limited artemisinin resistance-associated polymorphisms in Plasmodium falciparum K13-propeller and PfATPase6 gene isolated from Bioko Island, Equatorial Guinea

    PubMed Central

    Li, Jian; Chen, Jiangtao; Xie, Dongde; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Ondo Obono, Maximo Miko; Ehapo, Carlos Sala; Yang, Liye; Yang, Huitian; Lin, Min

    2016-01-01

    Objective With emergence and geographically expanding of antimalarial resistance worldwide, molecular markers are essential tool for surveillance of resistant Plasmodium parasites. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain are shown to be associated with artemisinin (ART) resistance in vivo and in vitro. This study aims to investigate the ART resistance-associated polymorphisms of K13-propeller and PfATPase6 genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea (EG). Methods A total of 172 samples were collected from falciparum malaria patients on Bioko Island between 2013 and 2014. The polymorphisms of K13-propeller and PfATPase6 genes were analyzed by Nest-PCR and sequencing. Results Sequences of K13-propeller and PfATPase6 were obtained from 90.74% (98/108) and 91.45% (139/152) samples, respectively. The 2.04% (2/98) cases had non-synonymous K13-propeller A578S mutation but no found the mutations associated with ART resistance in Southeast Asia. For PfATPase6, the mutations were found at positions N569K and A630S with the mutation prevalence of 7.91% (11/139) and 1.44% (2/139), respectively. In addition, a sample with the mixed type at position I723V was discovered (0.72%, 1/139). Conclusions This study initially offers an insight of K13-propeller and PfATPase6 polymorphisms on Bioko Island, EG. It suggests no widespread ART resistance or tolerance in the region, and might be helpful for developing and updating guidance for the use of ART-based combination therapies (ACTs). PMID:27054064

  3. Short communication: Characterization of methicillin-resistant Staphylococcus aureus isolated from raw milk fresh cheese in Colombia.

    PubMed

    Herrera, Fanny C; García-López, María-Luisa; Santos, Jesús A

    2016-10-01

    The aim of this study was the characterization of a collection of 8 methicillin-resistant Staphylococcus aureus (MRSA) isolates, obtained from samples of fresh cheese (Doble Crema) produced from raw cow milk in small dairies in Colombia. All the isolates harbored the mecA and Panton-Valentine leukocidin (PVL) genes, presented with SCCmec type IV, and belonged to multilocus sequence type 8 and spa type 024. Seven isolates presented 3 closely related pulsed-field gel electrophoresis profiles. Three of them carried the staphylococcal enterotoxin B gene. The isolates were resistant to cefoxitin, oxacillin, penicillin, and ampicillin and susceptible to all non-β-lactams antibiotics tested, with minimum inhibitory concentration values for oxacillin of 4 to 8mg/L. The isolates belonged to the community-acquired MRSA group, suggesting a human source of contamination. The risk of human infection by MRSA via contaminated foods is considered low, but contaminated food commodities can contribute to the worldwide dissemination of clones of community-acquired MRSA. PMID:27423957

  4. Short communication: Characterization of methicillin-resistant Staphylococcus aureus isolated from raw milk fresh cheese in Colombia.

    PubMed

    Herrera, Fanny C; García-López, María-Luisa; Santos, Jesús A

    2016-10-01

    The aim of this study was the characterization of a collection of 8 methicillin-resistant Staphylococcus aureus (MRSA) isolates, obtained from samples of fresh cheese (Doble Crema) produced from raw cow milk in small dairies in Colombia. All the isolates harbored the mecA and Panton-Valentine leukocidin (PVL) genes, presented with SCCmec type IV, and belonged to multilocus sequence type 8 and spa type 024. Seven isolates presented 3 closely related pulsed-field gel electrophoresis profiles. Three of them carried the staphylococcal enterotoxin B gene. The isolates were resistant to cefoxitin, oxacillin, penicillin, and ampicillin and susceptible to all non-β-lactams antibiotics tested, with minimum inhibitory concentration values for oxacillin of 4 to 8mg/L. The isolates belonged to the community-acquired MRSA group, suggesting a human source of contamination. The risk of human infection by MRSA via contaminated foods is considered low, but contaminated food commodities can contribute to the worldwide dissemination of clones of community-acquired MRSA.

  5. Characterization of freshly retrieved preantral follicles using a low-invasive, mechanical isolation method extended to different ruminant species.

    PubMed

    Langbeen, A; Jorssen, E P A; Fransen, E; Rodriguez, A P A; García, M Chong; Leroy, J L M R; Bols, P E J

    2015-10-01

    Due to the increased interest in preantral follicular physiology, non-invasive retrieval and morphological classification are crucial. Therefore, this study aimed: (1) to standardize a minimally invasive isolation protocol, applicable to three ruminant species; (2) to morphologically classify preantral follicles upon retrieval; and (3) to describe morphological features of freshly retrieved follicles compared with follicle characteristics using invasive methods. Bovine, caprine and ovine ovarian cortex strips were retrieved from slaughterhouse ovaries and dispersed. This suspension was filtered, centrifuged, re-suspended and transferred to a Petri dish, to which 0.025 mg/ml neutral red (NR) was added to assess the viability of the isolated follicles. Between 59 and 191 follicles per follicle class and per species were collected and classified by light microscopy, based on follicular cell morphology. Subsequently, follicle diameters were measured. The proposed isolation protocol was applicable to all three species and showed a significant, expected increase in diameter with developmental stage. With an average diameter of 37 ± 5 μm for primordial follicles, 47 ± 6.3 μm for primary follicles and 67.1 ± 13.1 μm for secondary follicles, no significant difference in diameter among the three species was observed. Bovine, caprine and ovine follicles (63, 59 and 50% respectively) were graded as viable upon retrieval. Using the same morphological characteristics as determined by invasive techniques [e.g. haematoxylin-eosin (HE) sections], cumulus cell morphology and follicle diameter could be used routinely to classify freshly retrieved follicles. Finally, we applied a mechanical, minimally invasive, follicle isolation protocol and extended it to three ruminant species, yielding viable preantral follicles without compromising further in vitro processing and allowing routine follicle characterization upon retrieval.

  6. Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro.

    PubMed

    Zhang, Chao; He, Xinlong; Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni2+-NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+-NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  7. Localization, biosynthesis, processing and isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium falciparum.

    PubMed

    Delplace, P; Fortier, B; Tronchin, G; Dubremetz, J F; Vernes, A

    1987-04-01

    Monoclonal antibodies prepared against a 50 kDa antigen found in Plasmodium falciparum culture supernatants identify a 126 kDa polypeptide which can be localized by immunofluorescence and immunoelectronmicroscopy at the periphery of the schizonts. This polypeptide is released from the infected erythrocytes by mild saponin lysis and is probably a component of the parasitophorous vacuole. Pulse chase kinetic analysis demonstrated its disappearance from the parasitized red blood cell from 6 to 10 h after being synthesized and the concomitant appearance of the 50 kDa molecule in the culture supernatant. Purification of metabolically labeled, schizont infected cells demonstrated that spontaneous release of merozoites is needed for the processing of the 126 to the 50 kDa whereas reinvasion is not. Polyclonal antibodies were raised in rabbit against affinity purified 126 kDa protein. These antibodies, together with another 126 kDa specific monoclonal antibody have enabled us to characterize two other cleavage products of the 126 kDa antigen in culture supernatants, namely 47 and 18 kDa polypeptides. We believe that the processing of the 126 kDa protein into low molecular weight fragments reflects a proteolytic event which may participate in merozoite release.

  8. Pantoea sp. isolated from tropical fresh water exhibiting N-acyl homoserine lactone production.

    PubMed

    Tan, Wen-Si; Muhamad Yunos, Nina Yusrina; Tan, Pui-Wan; Mohamad, Nur Izzati; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry.

  9. Pantoea sp. Isolated from Tropical Fresh Water Exhibiting N-Acyl Homoserine Lactone Production

    PubMed Central

    Tan, Wen-Si; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry. PMID:25197715

  10. Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

    PubMed Central

    Khor, Wei Ching; Puah, Suat Moi; Tan, Jin Ai Mary Anne; Puthucheary, SD; Chua, Kek Heng

    2015-01-01

    Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism. PMID:26710336

  11. Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia.

    PubMed

    Khor, Wei Ching; Puah, Suat Moi; Tan, Jin Ai Mary Anne; Puthucheary, S D; Chua, Kek Heng

    2015-01-01

    Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.

  12. Insulin resistance in uremia. Characterization of lipid metabolism in freshly isolated and primary cultures of hepatocytes from chronic uremic rats.

    PubMed Central

    Caro, J F; Lanza-Jacoby, S

    1983-01-01

    We have studied the mechanism(s) of hyperlipidemia and liver insulin sensitivity in a rat model of severe chronic uremia (U). Basal lipid synthesis was decreased in freshly isolated hepatocytes from U when compared with sham-operated ad lib.-fed controls (alfC). Basal lipid synthesis in pair-fed controls (pfC) was in between U and alfC. Similarly, the activity of liver acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase was diminished in U. Muscle and adipose tissue lipoprotein lipase was also decreased. Insulin stimulated lipid synthesis in freshly isolated hepatocytes from alfC. Hepatocytes from U and pfC were resistant to this effect of insulin. To ascertain if the insulin resistance in U was due to starvation (chow intake 50% of alfC) or to uremia itself, the U and pfC were intragastrically fed an isocaloric diet via a Holter pump the last week of the experimental period. Hepatocytes from orally fed U and pfC were also cultured for 24 h in serum-free medium. While freshly isolated and cultured U hepatocytes remained insulin resistant, those from pfC normalized, in vivo and in vitro, when they were provided with enough nutrients. Conclusions: (a) Hyperlipidemia in uremia is not due to increased synthesis, but to defect(s) in clearance. (b) Insulin does not stimulate lipid synthesis in uremia. This finding, along with our recent demonstration that insulin binding and internalization are not decreased in the uremic liver, suggests that a post-binding defect(s) in the liver plays an important role in the mechanism(s) of insulin resistance in uremia. (c) Cultured hepatocytes from uremic rats remain insulin resistant. This quality renders these cells useful in studying the postinsulin binding events responsible for the insulin-resistant state in the absence of complicating hormonal and substrate changes that occur in vivo. PMID:6350367

  13. Qualitative toxicity assessment of silver nanoparticles on the fresh water bacterial isolates and consortium at low level of exposure concentration.

    PubMed

    Kumar, Deepak; Kumari, Jyoti; Pakrashi, Sunandan; Dalai, Swayamprava; Raichur, Ashok M; Sastry, T P; Mandal, A B; Chandrasekaran, N; Mukherjee, Amitava

    2014-10-01

    Silver nanoparticles (AgNPs) pose a high risk of exposure to the natural environment owing to their extensive usage in various consumer products. In the present study we attempted to understand the harmful effect of AgNPs at environmentally relevant low concentration levels (≤1ppm) towards two different freshwater bacterial isolates and their consortium. The standard plate count assay suggested that the AgNPs were toxic towards the fresh water bacterial isolates as well as the consortium, though toxicity was significantly reduced for the cells in the consortium. The oxidative stress assessment and membrane permeability studies corroborated with the toxicity data. The detailed electron microscopic studies suggested the cell degrading potential of the AgNPs, and the FT-IR studies confirmed the involvement of the surface groups in the toxic effects. No significant ion leaching from the AgNPs was observed at the applied concentration levels signifying the dominant role of the particle size, and size distribution in bacterial toxicity. The reduced toxicity for the cells in the consortium than the individual isolates has major significance in further studies on the ecotoxicity of the AgNPs.

  14. Helicobacter pullorum isolated from fresh chicken meat: antibiotic resistance and genomic traits of an emerging foodborne pathogen.

    PubMed

    Borges, Vítor; Santos, Andrea; Correia, Cristina Belo; Saraiva, Margarida; Ménard, Armelle; Vieira, Luís; Sampaio, Daniel A; Pinheiro, Miguel; Gomes, João Paulo; Oleastro, Mónica

    2015-12-01

    Meat and meat products are important sources of human intestinal infections. We report the isolation of Helicobacter pullorum strains from chicken meat. Bacteria were isolated from 4 of the 17 analyzed fresh chicken meat samples, using a membrane filter method. MIC determination revealed that the four strains showed acquired resistance to ciprofloxacin; one was also resistant to erythromycin, and another one was resistant to tetracycline. Whole-genome sequencing of the four strains and comparative genomics revealed important genetic traits within the H. pullorum species, such as 18 highly polymorphic genes (including a putative new cytotoxin gene), plasmids, prophages, and a complete type VI secretion system (T6SS). The T6SS was found in three out of the four isolates, suggesting that it may play a role in H. pullorum pathogenicity and diversity. This study suggests that the emerging pathogen H. pullorum can be transmitted to humans by chicken meat consumption/contact and constitutes an important contribution toward a better knowledge of the genetic diversity within the H. pullorum species. In addition, some genetic traits found in the four strains provide relevant clues to how this species may promote adaptation and virulence.

  15. Occurrence of pfatpase6 Single Nucleotide Polymorphisms Associated with Artemisinin Resistance among Field Isolates of Plasmodium falciparum in North-Eastern Tanzania

    PubMed Central

    Chilongola, Jaffu; Ndaro, Arnold; Tarimo, Hipolite; Shedrack, Tamara; Barthazary, Sakurani; Kaaya, Robert; Masokoto, Alutu; Kajeguka, Debora; Kavishe, Reginald A.; Lusingu, John

    2015-01-01

    We aimed to determine the current prevalence of four P. falciparum candidate artemisinin resistance biomarkers L263E, E431K, A623E, and S769N in the pfatpase6 gene in a high transmission area in Tanzania in a retrospective cross sectional study using 154 archived samples collected from three previous malaria studies in 2010, 2011 and 2013. Mutations in pfatpase6 gene were detected in parasite DNA isolated from Dried Blood Spots by using PCR-RFLP. We observed overall allelic frequencies for L263E, E431K, A623E, and S769N to be 5.8% (9/154), 16.2% (25/154), 0.0% (0/154), and 3.9% (6/154). The L263E mutation was not detected in 2010 but occurred at 3.9% and 2.6% in 2011 and 2013 respectively. The L263E mutation showed a significant change of frequency between 2010 and 2011, but not between 2011 and 2013 (P < 0.05). Frequency of E431K was highest of all without any clear trend whereas S769N increased from 2.2% in 2010 to 3.6% in 2011 and 5.1% in 2013. A623E mutation was not detected. The worrisome detection and the increase in the frequency of S769N and other mutations calls for urgent assessment of temporal changes of known artemisinin biomarkers in association with in vivo ACT efficacy. PMID:25685593

  16. Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.

    PubMed

    Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

    2011-07-01

    Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance.

  17. Coupling reverse osmosis with electrodialysis to isolate natural organic matter from fresh waters.

    PubMed

    Koprivnjak, J F; Perdue, E M; Pfromm, P H

    2006-10-01

    Reverse osmosis (RO) has proven to be an effective method for the concentration of natural organic matter (NOM) from fresh waters, but an undesirable consequence of this process is the co-concentration of some inorganic solutes. Accordingly, current practice yields solutions of NOM that, upon desalting and freeze-drying, are converted into dry solids containing finely dispersed sulfuric acid and silicic acid (H(4)SiO(4)). These acids will contribute to the apparent carboxylic and phenolic contents of NOM, leading to an overestimation of both. NOM may also be chemically altered by sulfuric acid, which reacts strongly with many classes of organic compounds. The sulfur content and ash content of NOM will be elevated in the presence of sulfuric acid and H(4)SiO(4). The goal of this study is to develop and test a method in which the removal of water by RO is coupled with the removal of salts by electrodialysis (ED). Like RO, ED is a relatively mild treatment that enables the desalting of NOM solutions without subjecting those samples to conditions of extremely high or low pH. The end product of the coupled process is a desalted, concentrated liquid sample from which low-ash NOM can be obtained as a freeze-dried solid material. In this study, the efficacy of ED for desalting NOM is evaluated using concentrated synthetic river waters and actual concentrated (by RO) river waters. Under optimal operating conditions, both sulfate and silica can be largely removed from RO-concentrated solutions of riverine NOM with only an average loss of 3% of total organic carbon. PMID:16952387

  18. Fumonisin production by Fusarium species isolated from freshly harvested corn in Iran.

    PubMed

    Ghiasian, Seyed Amir; Rezayat, Seyed Mahdi; Kord-Bacheh, Parivash; Maghsood, Amir Hossein; Yazdanpanah, Hassan; Shephard, Gordon S; van der Westhuizen, Liana; Vismer, Hester F; Marasas, Walter F O

    2005-01-01

    Fifty-one strains of Fusarium verticillioides and F. proliferatum isolated from corn collected from four different geographic areas in Iran, namely Fars, Khuzestan, Kermanshah and Mazandaran (an endemic oesophageal cancer (OC) area) were evaluated for their ability to produce fumonisins B1 (FB1), B2 (FB2) and B3 (FB3) in corn culture. Fumonisin levels were determined by high-performance liquid chromatography. All tested strains of F. verticillioides and F. proliferatumproduced fumonisins within a wide range of concentrations, 197-9661 microg/g, 18-1974 microg/g, and 21-1725 microg/g for FB1, FB2, and FB3, respectively. The highest mean concentrations of FB1, FB2, and FB3 were 3897, 806 and 827 microg/g, respectively. Overall, 61% of the F. verticillioides and F. proliferatum strains produced higher levels of FB3 than FB2. The mean ratios of FB1:FB2, FB1:FB3 and FB1:total fumonisins were 8, 7 and 0.7 for F. verticillioides and 5.7, 10.7 and 0.7 for F. proliferatum, respectively. Significant differences in some of the meteorological data (rainfall, relative humidity and minimum temperature) from the four provinces were observed. Fumonisin levels produced by F. verticillioides strains isolated from Khuzestan province (tropical zone) were significantly (P < 0.01) higher than the other three provinces. This is the first report of the fumonisin-producing ability of F.verticillioides and F. proliferatum strains isolated from corn harvested from different geographic areas in Iran.

  19. Plasmodium Falciparum Malaria

    PubMed Central

    Schmidt, John A.; Udeinya, Iroka J.; Leech, James H.; Hay, Robert J.; Aikawa, Masamichi; Barnwell, John; Green, Ira; Miller, Louis H.

    1982-01-01

    Erythrocytes infected with Plasmodium falciparum trophozoites and schizonts are not seen in the peripheral circulation because they attach to venular endothelium via knoblike structures on the infected erythrocyte membrane. We have recently shown that erythrocytes containing P. falciparum trophozoites and schizonts likewise attach to cultured human venous endothelial cells via knobs. In search of a more practical target cell for large scale binding studies designed to characterize and isolate the knob ligand, we tested various normal cells and continuous cell lines for their ability to bind P. falciparum-infected erythrocytes. Of the 18 cell types tested, binding of infected erythrocytes was observed to a human amelanotic melanoma cell line and amnion epithelial cells as well as to human aortic and umbilical vein endothelial cells. 96-100% of amelanotic melanoma cells bound 17±4 (±1 SEM) infected erythrocytes per positive cell, whereas fewer endothelial cells (4-59%) and amnion epithelial cells (8-19%) were capable of binding 12±5 and 4±1 infected erythrocytes per positive cell, respectively. Further studies designed to compare the mechanism of binding to the amelanotic melanoma cell line and endothelial cells showed the following results. First, that adhesion of infected erythrocytes to these two cell types was parasite stage-specific in that only erythrocytes containing late ring forms, trophozoites, and schizonts bound. Erythrocytes containing early ring forms, which do not attach to venular endothelium in vivo, did not bind to either cell type. Second, erythrocytes infected with trophozoites and schizonts of P. vivax or a knobless strain of P. falciparum, both of which continue to circulate in vivo, did not bind to either target cell type. Third, transmission electron microscopy showed that infected erythrocytes attached to the amelanotic melanoma cells via knobs. We conclude that cultured human endothelial cells and an amelanotic melanoma cell line share

  20. 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. PMID:25706423

  1. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    PubMed Central

    2005-01-01

    The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F−) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid. PMID:16192686

  2. In-vitro response of Plasmodium falciparum to the main alkaloids of Cinchona in northwestern Thailand.

    PubMed

    Knauer, Ariane; Sirichaisinthop, Jeeraphat; Reinthaler, Franz F; Wiedermann, Gerhard; Wernsdorfer, Gunther; Wernsdorfer, Walther H

    2003-01-01

    The blood schizontocidal activity of the four main Cinchona alkaloids against Plasmodium falciparum was compared in 46 fresh parasite isolates, using an in-vitro test measuring the drug-specific inhibition of schizont maturation. The studies were conducted in June-August 2001 at Mae Sot, northwestern Thailand, an area where quinine alone is no longer able to eliminate infections with P. falciparum. Quinidine showed the highest blood schizontocidal activity, followed by cinchonine, cinchonidine and finally quinine, which was identified as the least active compound. The isolates showed marked heterogeneity in their response to the Cinchona alkaloids. There was also high correlation of activity among all four alkaloids. The mean EC50 values for quinine, quinidine, cinchonine and cinchonidine were 144 nM, 80 nM, 104 nM and 225 nM, respectively, and the EC99 values 8040 nM, 861 nM, 1176 nM and 6531 nM. The EC99 values for quinine and cinchonidine are beyond the therapeutic concentration range and those for quinidine within it. For cinchonine, values are likely to be within this range, but toxicological and pharmacokinetic studies on this compound are required for clarifying its potential future role in the treatment of falciparum malaria.

  3. Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control.

    PubMed

    Dorado, Erika Jimena; Okoth, Sheila Akinyi; Montenegro, Lidia Madeline; Diaz, Gustavo; Barnwell, John W; Udhayakumar, Venkatachalam; Murillo Solano, Claribel

    2016-01-01

    Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive

  4. Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control.

    PubMed

    Dorado, Erika Jimena; Okoth, Sheila Akinyi; Montenegro, Lidia Madeline; Diaz, Gustavo; Barnwell, John W; Udhayakumar, Venkatachalam; Murillo Solano, Claribel

    2016-01-01

    Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive

  5. Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control

    PubMed Central

    Dorado, Erika Jimena; Okoth, Sheila Akinyi; Montenegro, Lidia Madeline; Diaz, Gustavo; Barnwell, John W.; Udhayakumar, Venkatachalam; Murillo Solano, Claribel

    2016-01-01

    Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive

  6. Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China.

    PubMed

    He, Yu; Jin, Lanlan; Sun, Fengjiao; Hu, Qiongxia; Chen, Lanming

    2016-08-01

    Vibrio parahaemolyticus is a causative agent of human serious seafood-borne gastroenteritis disease and even death. Shrimps, often eaten raw or undercooked, are an important reservoir of the bacterium. In this study, we isolated and characterized a total of 400 V. parahaemolyticus strains from commonly consumed fresh shrimps (Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Exopalaemon carinicauda) in Shanghai fish markets, China in 2013-2014. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying two major toxic genes (tdh and trh, 0.0 and 0.5 %). However, high incidences of antibiotic resistance were observed among the strains against ampicillin (99 %), streptomycin (45.25 %), rifampicin (38.25 %), and spectinomycin (25.50 %). Approximately 24 % of the strains derived from the P. monodon sample displayed multidrug resistant (MDR) phenotypes, followed by 19, 12, and 6 % from the E. carinicauda, L. vannamei, and M. rosenbergii samples, respectively. Moreover, tolerance to heavy metals of Cr(3+) and Zn(2+) was observed in 90 antibiotic resistant strains, the majority of which also displayed resistance to Cu(2+) (93.3 %), Pb(2+) (87.8 %), and Cd(2+)(73.3 %). The pulsed-field gel electrophoresis (PFGE)-based genotyping of these strains revealed a total of 71 distinct pulsotypes, demonstrating a large degree of genomic variation among the isolates. The wide distribution of MDR and heavy-metal resistance isolates in the PFGE clusters suggested the co-existence of a number of resistant determinants in V. parahaemolyticus population in the detected samples. This study provided data in support of aquatic animal health management and food safety risk assessment in aquaculture industry.

  7. Mechanical properties of nerve roots and rami radiculares isolated from fresh pig spinal cords

    PubMed Central

    Nishida, Norihiro; Kanchiku, Tsukasa; Ohgi, Junji; Ichihara, Kazuhiko; Chen, Xian; Taguchi, Toshihiko

    2015-01-01

    No reports have described experiments designed to determine the strength characteristics of spinal nerve roots and rami radiculares for the purpose of explaining the complexity of symptoms of medullary cone lesions and cauda equina syndrome. In this study, to explain the pathogenesis of cauda equina syndrome, monoaxial tensile tests were performed to determine the strength characteristics of spinal nerve roots and rami radiculares, and analysis was conducted to evaluate the stress-strain relationship and strength characteristics. Using the same tensile test device, the nerve root and ramus radiculares isolated from the spinal cords of pigs were subjected to the tensile test and stress relaxation test at load strain rates of 0.1, 1, 10, and 100 s-1 under identical settings. The tensile strength of the nerve root was not rate dependent, while the ramus radiculares tensile strength tended to decrease as the strain rate increased. These findings provide important insights into cauda equina symptoms, radiculopathy, and clinical symptoms of the medullary cone. PMID:26807127

  8. The nature of laboratory domestication changes in freshly isolated Escherichia coli strains.

    PubMed

    Eydallin, Gustavo; Ryall, Ben; Maharjan, Ram; Ferenci, Thomas

    2014-03-01

    Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies.

  9. The nature of laboratory domestication changes in freshly isolated Escherichia coli strains.

    PubMed

    Eydallin, Gustavo; Ryall, Ben; Maharjan, Ram; Ferenci, Thomas

    2014-03-01

    Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies. PMID:23889812

  10. Gram-negative bacterial isolates from fresh-cut processing plants enhance the presence of Escherichia Coli O157:H7 in dual-species biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilms formed by resident microflora may provide a microenvironment for foodborne bacterial pathogens to survive and cause cross-contamination in fresh-cut processing and handling facilities. The objective of this study is to determine the impact of individual bacteria strains isolated from two l...

  11. Genome sequences of Ralstonia insidiosa type strain ATCC 49129 and strain FC1138, a strong biofilm producer isolated from a fresh-cut produce-processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ralstonia insidiosa FC1138 is a strong biofilm producer, isolated from a local fresh-cut produce processing plant. Here, we present the complete genome sequence of Ralstonia insidiosa FC1138 which includes two circular chromosomes and a plasmid. To our knowledge, this is the first reported complete ...

  12. Transmission blocking activity of Azadirachta indica and Guiera senegalensis extracts on the sporogonic development of Plasmodium falciparum field isolates in Anopheles coluzzii mosquitoes

    PubMed Central

    2014-01-01

    Background Targeting the stages of the malaria parasites responsible for transmission from the human host to the mosquito vector is a key pharmacological strategy for malaria control. Research efforts to identify compounds that are active against these stages have significantly increased in recent years. However, at present, only two drugs are available, namely primaquine and artesunate, which reportedly act on late stage gametocytes. Methods In this study, we assessed the antiplasmodial effects of 5 extracts obtained from the neem tree Azadirachta indica and Guiera senegalensis against the early vector stages of Plasmodium falciparum, using field isolates. In an ex vivo assay gametocytaemic blood was supplemented with the plant extracts and offered to Anopheles coluzzii females by membrane feeding. Transmission blocking activity was evaluated by assessing oocyst prevalence and density on the mosquito midguts. Results Initial screening of the 5 plant extracts at 250 ppm revealed transmission blocking activity in two neem preparations. Up to a concentration of 70 ppm the commercial extract NeemAzal® completely blocked transmission and at 60 ppm mosquitoes of 4 out of 5 replicate groups remained uninfected. Mosquitoes fed on the ethyl acetate phase of neem leaves at 250 ppm showed a reduction in oocyst prevalence of 59.0% (CI95 12.0 - 79.0; p < 10-4) and in oocyst density of 90.5% (CI95 86.0 - 93.5; p < 10-4 ), while the ethanol extract from the same plant part did not exhibit any activity. No evidence of transmission blocking activity was found using G. senegalensis ethyl acetate extract from stem galls. Conclusions The results of this study highlight the potential of antimalarial plants for the discovery of novel transmission blocking molecules, and open up the potential of developing standardized transmission blocking herbal formulations as malaria control tools to complement currently used antimalarial drugs and combination treatments. PMID:24735564

  13. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    PubMed Central

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Wu, Jichuan; Hubert, Terrence L.; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  14. Comparative performance of isolation methods using Preston broth, Bolton broth and their modifications for the detection of Campylobacter spp. from naturally contaminated fresh and frozen raw poultry meat.

    PubMed

    Seliwiorstow, T; De Zutter, L; Houf, K; Botteldoorn, N; Baré, J; Van Damme, I

    2016-10-01

    The performance of different isolation methods was evaluated for the detection of Campylobacter from naturally contaminated raw poultry meat. Therefore, fresh and frozen poultry meat samples were analysed using the standard procedure (ISO 10272-1:2006), enrichment in Preston broth, and enrichment in modified Bolton broth (supplemented with (i) potassium clavulanate (C-BB), (ii) triclosan (T-BB), (iii) polymyxin B (P-BB)). The enrichment cultures were streaked onto both modified charcoal cefoperazone deoxycholate agar (mCCDA) and RAPID'Campylobacter agar (RCA). Moreover, direct plating on mCCDA and RCA was performed to quantify Campylobacter. In total, 33 out of 59 fresh retail meat samples (55.9%) were Campylobacter positive. For both fresh and frozen poultry meat samples, enrichment in Bolton broth (ISO 10272-1:2006) resulted in a higher number of positive samples than enrichment in Preston broth. Supplementation of Bolton broth with potassium clavulanate (C-BB) and triclosan (T-BB) enhanced the Campylobacter recovery from fresh poultry meat compared to non-supplemented Bolton broth, although the use of C-BB was less applicable than T-BB for Campylobacter recovery from frozen samples. Additionally, the use of RCA resulted in a higher isolation rate compared to mCCDA. The present study demonstrates the impact of culture medium on the recovery of Campylobacter from fresh and frozen naturally contaminated poultry meat samples and can support laboratories in choosing the most appropriate culturing method to detect Campylobacter. PMID:27391222

  15. Mitigation of statins-induced cytotoxicity and mitochondrial dysfunction by L-carnitine in freshly-isolated rat hepatocytes.

    PubMed

    Abdoli, N; Azarmi, Y; Eghbal, M A

    2015-01-01

    Statins are widely used as anti hyperlipidemic agents. Hepatotoxicity is one of their adverse effects appearing in some patients. No protective agents have yet been developed to treat statins-induced hepatotoxicity. Different investigations have suggested L-carnitine as a hepatoprotective agent against drugs-induced toxicity. This study was designed to evaluate the effect of L-carnitine on the cytotoxic effects of statins on the freshly-isolated rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via portal vein. Cells were treated with the different concentrations of statins (simvastatin, lovastatin and atorvastatin), alone or in combination with L-carnitine. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Furthermore, the effects of statins on cellular reduced and oxidized glutathione reservoirs were evaluated. In accordance with previous studies, an elevation in ROS formation, cellular oxidized glutathione and lipid peroxidation were observed after statins administration. Moreover, a decrease in cellular reduced glutathione level and cellular mitochondrial membrane potential collapse occurred. L-carnitine co-administration decreased the intensity of aforementioned toxicity markers produced by statins treatment. This study suggests the protective role of L-carnitine against statins-induced cellular damage probably through its anti oxidative and reactive radical scavenging properties as well as its effects on sub cellular components such as mitochondria. The mechanism of L-carnitine protection may be related to its capacity to facilitate fatty acid entry into mitochondria; possibly adenosine tri-phosphate or the reducing equivalents are increased, and the toxic effects of statins toward mitochondria are encountered.

  16. Mitigation of statins-induced cytotoxicity and mitochondrial dysfunction by L-carnitine in freshly-isolated rat hepatocytes

    PubMed Central

    Abdoli, N.; Azarmi, Y.; Eghbal, M.A.

    2015-01-01

    Statins are widely used as anti hyperlipidemic agents. Hepatotoxicity is one of their adverse effects appearing in some patients. No protective agents have yet been developed to treat statins-induced hepatotoxicity. Different investigations have suggested L-carnitine as a hepatoprotective agent against drugs-induced toxicity. This study was designed to evaluate the effect of L-carnitine on the cytotoxic effects of statins on the freshly-isolated rat hepatocytes. Hepatocytes were isolated from male Sprague-Dawley rats by collagenase enzyme perfusion via portal vein. Cells were treated with the different concentrations of statins (simvastatin, lovastatin and atorvastatin), alone or in combination with L-carnitine. Cell death, reactive oxygen species (ROS) formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers. Furthermore, the effects of statins on cellular reduced and oxidized glutathione reservoirs were evaluated. In accordance with previous studies, an elevation in ROS formation, cellular oxidized glutathione and lipid peroxidation were observed after statins administration. Moreover, a decrease in cellular reduced glutathione level and cellular mitochondrial membrane potential collapse occurred. L-carnitine co-administration decreased the intensity of aforementioned toxicity markers produced by statins treatment. This study suggests the protective role of L-carnitine against statins-induced cellular damage probably through its anti oxidative and reactive radical scavenging properties as well as its effects on sub cellular components such as mitochondria. The mechanism of L-carnitine protection may be related to its capacity to facilitate fatty acid entry into mitochondria; possibly adenosine tri-phosphate or the reducing equivalents are increased, and the toxic effects of statins toward mitochondria are encountered. PMID:26487891

  17. Characterization of Listeria monocytogenes isolated from a fresh mixed sausage processing line in Pelotas-RS by PFGE

    PubMed Central

    von Laer, Ana Eucares; de Lima, Andréia Saldanha; Trindade, Paula dos Santos; Andriguetto, Cristiano; Destro, Maria Teresa; da Silva, Wladimir Padilha

    2009-01-01

    Listeria monocytogenes is a bacterium capable to adhere to the surfaces of equipment and utensils and subsequently form biofilms. It can to persist in the food processing environmental for extended periods of time being able to contaminate the final product. The aim of this study was to trace the contamination route of L. monocytogenes on a fresh mixed sausage processing line, from raw material to the final product. The isolates obtained were characterized by serotyping and molecular typing by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes ApaI and AscI. L. monocytogenes was detected in 25% of the samples. The samples of raw material were not contaminated, however, the microorganism was detected in 21% of the environmental samples (food contact and non-food contact), 20.8% of the equipments, 20% of the food worker’s hands, 40% of the mass ready to packaging and in all the final products samples, demonstrating that the contamination of final product occurred during the processing and the importance of cross contamination. PFGE yielded 22 pulsotypes wich formed 7 clusters, and serotyping yielded 3 serotypes and 1 serogroup, however, the presence of serotypes 4b and 1/2b in the final product is of great concern for public health. The tracing of contamination showed that some strains are adapted and persisted in the processing environment in this industry. PMID:24031402

  18. GLUCOSE METABOLITE PATTERNS AS MARKERS OF FUNCTIONAL DIFFERENTIATION IN FRESHLY ISOLATED AND CULTURED MOUSE MAMMARY EPITHELIAL CELLS

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissell, M.J.

    1980-06-01

    In the mammary gland of nonruminant animals, glucose is utilized in a characteristic and unique way during lactation. We have measured the incorporation of glucose carbon from [U-{sup 14}C] glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice and demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate were important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells from pregnant mice have a pattern similar to freshly isolated cells from pregnant mice. The pattern of cells from lactating mice is different from that of the cells of origin, and resembles that of the cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  19. Transplantation of freshly isolated adipose tissue-derived regenerative cells enhances angiogenesis in a murine model of hind limb ischemia.

    PubMed

    Harada, Yusuke; Yamamoto, Yasutaka; Tsujimoto, Shunsuke; Matsugami, Hiromi; Yoshida, Akio; Hisatome, Ichiro

    2013-02-01

    Therapeutic angiogenesis has emerged as one of the most promising therapies for severe ischemic cardiovascular diseases with no optional therapy. Several investigators have reported that transplantation of cultured adipose-derived regenerative cells (cADRCs) to ischemic tissues promotes neovascularization and blood perfusion recovery; however, cell therapy using cultured cells has several restrictions. To resolve this problem, the angiogenic capacity of freshly isolated ADRCs (fADRCs) obtained from Lewis rats was compared with cADRCs, both in vivo and in vitro. Flow cytometric analysis showed that fADRCs contained several cell types such as endothelial progenitor cells and endothelial cells; however, these cells were present in a very small proportion in cADRCs. Transplantation of fADRCs in mice significantly improved blood perfusion, capillary density, and production of several angiogenic factors in transplanted ischemic limbs compared with a saline-injected group, whereas these effects were not observed in the cADRCs-injected group. fADRCs also showed significantly higher expression levels of angiogenic factors than cADRCs in the in vitro study. Furthermore, fADRC stimulated tube formation more remarkably than cADRC in an in vitro tube formation assay. These results suggested that fADRCs have an effective angiogenic capacity, and they would be more valuable as a source for cell-based therapeutic angiogenesis than cADRCs or other stem/progenitor cells.

  20. Isolation of Shiga toxin-producing Escherichia coli from fresh produce using STEC heart infusion washed blood agar with mitomycin-C.

    PubMed

    Lin, Andrew; Nguyen, Lam; Clotilde, Laurie M; Kase, Julie A; Son, Insook; Lauzon, Carol R

    2012-11-01

    The ability to detect and isolate Shiga toxin-producing Escherichia coli (STEC) remains a major challenge for food microbiologists. Although methods based on nucleic acids and antibodies have improved detection of STECs in foods, isolation of these bacteria remains arduous. STEC isolation is necessary for matching food, environmental, and clinical isolates during outbreak investigations and for distinguishing between pathogenic and nonpathogenic organisms. STEC heart infusion washed blood agar with mitomycin-C (SHIBAM) is a modification of washed sheep blood agar prepared by adding mitomycin-C and optimizing both the washed blood and base agar to better isolate STECs. Most STEC isolates produce a zone of hemolysis on SHIBAM plates and are easily distinguishable from background microbiota. Here, we present data supporting the use of SHIBAM to isolate STECs from fresh produce. SHIBAM was tested for accuracy in identifying STECs (365 of 410 STEC strains were hemolytic, and 63 of 73 E. coli strains that did not produce Shiga toxin were not hemolytic) and for recovery from artificially inoculated fresh produce (11 of 24 romaine lettuce samples and 6 of 24 tomato samples). STEC recovery with SHIBAM agar was greatly improved when compared with recovery on Levine's eosin-methylene blue agar as a reference method.

  1. Antigen capture and major histocompatibility class II compartments of freshly isolated and cultured human blood dendritic cells

    PubMed Central

    1995-01-01

    Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation. We have now used immunoelectron microscopy and antigen presentation assays to compare freshly isolated DC (f-DC) and cultured DC (c-DC). f-DC display a round appearance, whereas c-DC display characteristic long processes. c-DC express much more cell surface major histocompatibility complex (MHC) class II than f-DC. The uptake of colloidal gold-labeled bovine serum albumin (BSA), however, is greater in f-DC, as is the presentation of 65-kD heat shock protein to T cell clones. The most striking discovery is that the majority of MHC class II molecules in both f-DC and c-DC occur in intracellular vacuoles with a complex shape (multivesicular and multilaminar). These MHC class II enriched compartments (MIIC) represent the site to which BSA is transported within 30 min. Although MIIC appear as more dense structures with less MHC class II molecules in f-DC than c-DC, the marker characteristics are very similar. The MIIC in both types of DC are acidic, contain invariant chain, and express the recently described HLA-DM molecule that can contribute to antigen presentation. CD19+ peripheral blood B cells have fewer MIIC and surface MHC class II expression than DCs, while monocytes had low levels of MIIC and surface MHC class II. These results demonstrate in dendritic cells the elaborate development of MIIC expressing several of the components that are required for efficient antigen presentation. PMID:7790816

  2. Antimalarial drug interactions of compounds isolated from Kigelia africana (Bignoniaceae) and their synergism with artemether, against the multidrug-resistant W2mef Plasmodium falciparum strain.

    PubMed

    Zofou, Denis; Tene, Mathieu; Tane, Pierre; Titanji, Vincent P K

    2012-02-01

    For decades, drug resistance has been the major obstacle in the fight against malaria, and the search for new drugs together with the combination therapy constitutes the major approach in responding to this situation. The present study aims at assessing the in vitro antimalarial activity of four compounds isolated from Kigelia africana stem bark (atranorin - KAE1, specicoside - KAE7, 2β,3β,19α-trihydroxy-urs-12-20-en-28-oic acid - KAE3, and p-hydroxy-cinnamic acid - KAE10) and their drug interactions among themselves and their combination effects with quinine and artemether. The antiplasmodial activity and drug interactions were evaluated against the multidrug-resistant W2mef strain of Plasmodium falciparum using the parasite lactate dehydrogenase assay. Three of the four compounds tested were significantly active against W2mef: specicoside (IC(50) = 1.02 ± 0.17 μM), 2β,3β,19α-trihydroxy-urs-12-en-28-oic acid (IC(50) = 1.86 ± 0.15 μM) and atranorin (IC(50) = 1.78 ± 0.18 μM), whereas p-hydroxy-cinnamic acid showed a weak activity (IC(50) = 12.89 ± 0.87 μM). A slight synergistic effect was observed between atranorin and 2β,3β,19α-trihydroxy-urs-12-en-28-oic acid (Combination index, CI = 0.82) whereas the interaction between specicoside and p-hydroxy-cinnamic acid were instead antagonistic (CI = 2.67). All the three compounds showed synergistic effects with artemether, unlike the slight antagonistic interactions of atranorin and 2β,3β,19α-trihydroxy-urs-12-en-28-oic acid in combination with quinine. K. africana compounds are therefore likely to serve as leads in the development of new partner drugs in artemether-based combination therapy. PMID:21814840

  3. Comparison of Chemical Sensitivity of Fresh and Long-Stored Heat Resistant Neosartorya fischeri Environmental Isolates Using BIOLOG Phenotype MicroArray System.

    PubMed

    Panek, Jacek; Frąc, Magdalena; Bilińska-Wielgus, Nina

    2016-01-01

    Spoilage of heat processed food and beverage by heat resistant fungi (HRF) is a major problem for food industry in many countries. Neosartorya fischeri is the leading source of spoilage in thermally processed products. Its resistance to heat processing and toxigenicity makes studies about Neosartorya fischeri metabolism and chemical sensitivity essential. In this study chemical sensitivity of two environmental Neosartorya fischeri isolates were compared. One was isolated from canned apples in 1923 (DSM3700), the other from thermal processed strawberry product in 2012 (KC179765), used as long-stored and fresh isolate, respectively. The study was conducted using Biolog Phenotype MicroArray platforms of chemical sensitivity panel and traditional hole-plate method. The study allowed for obtaining data about Neosartorya fischeri growth inhibitors. The fresh isolate appeared to be much more resistant to chemical agents than the long-stored isolate. Based on phenotype microarray assay nitrogen compounds, toxic cations and membrane function compounds were the most effective in growth inhibition of N. fischeri isolates. According to the study zaragozic acid A, thallium(I) acetate and sodium selenate were potent and promising N. fischeri oriented fungicides which was confirmed by both chemical sensitivity microplates panel and traditional hole-plate methods. PMID:26815302

  4. Comparison of Chemical Sensitivity of Fresh and Long-Stored Heat Resistant Neosartorya fischeri Environmental Isolates Using BIOLOG Phenotype MicroArray System

    PubMed Central

    Panek, Jacek; Frąc, Magdalena; Bilińska-Wielgus, Nina

    2016-01-01

    Spoilage of heat processed food and beverage by heat resistant fungi (HRF) is a major problem for food industry in many countries. Neosartorya fischeri is the leading source of spoilage in thermally processed products. Its resistance to heat processing and toxigenicity makes studies about Neosartorya fischeri metabolism and chemical sensitivity essential. In this study chemical sensitivity of two environmental Neosartorya fischeri isolates were compared. One was isolated from canned apples in 1923 (DSM3700), the other from thermal processed strawberry product in 2012 (KC179765), used as long-stored and fresh isolate, respectively. The study was conducted using Biolog Phenotype MicroArray platforms of chemical sensitivity panel and traditional hole-plate method. The study allowed for obtaining data about Neosartorya fischeri growth inhibitors. The fresh isolate appeared to be much more resistant to chemical agents than the long-stored isolate. Based on phenotype microarray assay nitrogen compounds, toxic cations and membrane function compounds were the most effective in growth inhibition of N. fischeri isolates. According to the study zaragozic acid A, thallium(I) acetate and sodium selenate were potent and promising N. fischeri oriented fungicides which was confirmed by both chemical sensitivity microplates panel and traditional hole-plate methods. PMID:26815302

  5. Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy

    PubMed Central

    Ndong Ngomo, Jacques-Mari; Mawili-Mboumba, Denise Patricia; M'Bondoukwe, Noé Patrick; Nikiéma Ndong Ella, Rosalie; Bouyou Akotet, Marielle Karine

    2016-01-01

    In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon. PMID:27190671

  6. High prevalence of pfdhfr-pfdhps triple mutations associated with anti-malarial drugs resistance in Plasmodium falciparum isolates seven years after the adoption of sulfadoxine-pyrimethamine in combination with artesunate as first-line treatment in Iran.

    PubMed

    Rouhani, Maryam; Zakeri, Sedigheh; Pirahmadi, Sakineh; Raeisi, Ahmad; Djadid, Navid Dinparast

    2015-04-01

    The spread of anti-malarial drug resistance will challenge any malaria control and elimination strategies, and routine monitoring of resistance-associated molecular markers of commonly used anti-malarial drugs is very important. Therefore, in the present investigation, the extent of mutations/haplotypes in dhfr and dhps genes of Plasmodium falciparum isolates (n=72) was analyzed seven years after the introduction of sulfadoxine-pyrimethamine (SP) plus artesunate (AS) as first-line anti-malarial treatment in Iran using PCR-RFLP methods. The results showed that the majority of the patients (97.2%) carried both 59R and 108N mutations in pure form with wild-type genotype at positions N51 and I164. Additionally, a significant increase (P<0.05) was observed in the frequency of R59N108/G437 haplotype (79.2%) during 2012-2014. This raise was because of the significant increase (P<0.05) in the frequency of 437G mutation (81.9%), which more likely was due to more availability of SP as anti-malarial drug for treatment of falciparum patients in these malaria-endemic areas of Iran. However, no quintuple mutations associated with treatment failure were detected. In conclusion, the present results along with in vivo assays suggest that seven years after the adoption of SP-AS as the first-line treatment in Iran, this drug remains efficacious for treatment of uncomplicated falciparum malaria, as a partner drug with AS in these malaria-endemic areas.

  7. The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya.

    PubMed

    Ochola-Oyier, Lynette Isabella; Okombo, John; Mwai, Leah; Kiara, Steven M; Pole, Lewa; Tetteh, Kevin K A; Nzila, Alexis; Marsh, Kevin

    2015-03-01

    The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). PMID:25534732

  8. The MSPDBL2 Codon 591 Polymorphism Is Associated with Lumefantrine In Vitro Drug Responses in Plasmodium falciparum Isolates from Kilifi, Kenya

    PubMed Central

    Okombo, John; Mwai, Leah; Kiara, Steven M.; Pole, Lewa; Tetteh, Kevin K. A.; Nzila, Alexis; Marsh, Kevin

    2014-01-01

    The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). PMID:25534732

  9. Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes.

    PubMed

    Udomsangpetch, Rachanee; Pipitaporn, Busaba; Silamut, Kamolrat; Pinches, Robert; Kyes, Sue; Looareesuwan, Sornchai; Newbold, Christopher; White, Nicholas J

    2002-09-01

    In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37 degrees C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance. PMID:12177447

  10. Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria

    PubMed Central

    Imwong, Mallika; Woodrow, Charles J.; Hendriksen, Ilse C. E.; Veenemans, Jacobien; Verhoef, Hans; Faiz, M. Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D.; Day, Nicholas P. J.; Dondorp, Arjen M.; White, Nicholas J.

    2015-01-01

    In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples. PMID:25344520

  11. Isolation and characterization by conventional methods and genetic transformation of Psychrobacter and Acinetobacter from fresh and spoiled meat, milk and cheese.

    PubMed

    Gennari, M; Parini, M; Volpon, D; Serio, M

    1992-01-01

    Of 126 samples of fresh and spoiled meat and dairy products, 40% were positive for the presence of Moraxella-like bacteria and 64% of Acinetobacter; 279 and 466 strains, respectively, were isolated and a part of these were tested by biochemical methods and DNA transformation assays. In some cases, the Moraxellaceae in the samples examined reached considerable quantitative levels, but their percentage in the microflora was generally low. Moraxella-like bacteria were predominant in fresh meat, Acinetobacter in spoiled meat and milk. Most acinetobacters belonged to biotype lwoffii (sensu lato) and all 90 strains tested were positive for DNA transformation with an auxotrophic Acinetobacter. Moraxella-like bacteria were identified as Psychrobacter immobilis in 96% of 103 transformation assays. Moraxellaceae show lipolytic activity but they are considered of low incidence in food spoilage. Only 3.7% of acinetobacters from dairy sources was able to produce ropy milk. Unlike strains from clinical isolates, psychrobacters and acinetobacters isolated from food often do not grow at 37 degrees C.

  12. Recent developments in production and purification of malaria antigens: Evidence for environmental modulation of gametocytogenesis in Plasmodium falciparum in continuous culture*

    PubMed Central

    Carter, Richard; Miller, Louis H.

    1979-01-01

    With the introduction of continuous culture of Plasmodium falciparum it has become possible to study the factors involved in gametocyte production in vitro and thus eliminate the uncontrollable in vivo variables of the host. The authors have developed a method for measuring quantitatively the rate of production of gametocytes at any time in such cultures. The method is based on an estimation of the percentage of ring forms that develop into stage II gametocytes. Using this approach, it was found that dilution of cultures with fresh red blood cells so as to lower the parasitaemia led to rapid fall in the rate of conversion to gametocytes. The conversion rates subsequently rose again to levels typically in the order of 10% after several days of growth in the new culture. In the parental cultures from which the dilutions were made, conversion rates remained high at all times. This pattern was consistently observed in three different isolates of P. falciparum from Africa and the results indicate that the reduction of parasitaemia by addition of fresh cells was responsible for reducing production of gametocytes and that conditions associated with a period of growth in culture induced renewed gametocytogenesis. The authors conclude, therefore, that environmental conditions directly modulate the rate of gametocyte production by P. falciparum in culture. After 1½ years in culture, parasites have retained their ability to produce gametocytes and the gametocytes to undergo exflagellation. ImagesFig. 1 PMID:397008

  13. Antiplasmodial activity-aided isolation and identification of quercetin-4'-methyl ether in Chromolaena odorata leaf fraction with high activity against chloroquine-resistant Plasmodium falciparum.

    PubMed

    Ezenyi, I C; Salawu, O A; Kulkarni, R; Emeje, M

    2014-12-01

    The present study was undertaken to evaluate the antiplasmodial activity of Chromolaena odorata leaf extract and gradient fractions through in vivo and in vitro tests, aimed at identifying its antiplasmodial constituents. Sub-fractions obtained from the most active gradient fraction were further tested for cytotoxicity against THP-1 cells, chloroquine-sensitive (HB3) and chloroquine-resistant (FCM29) Plasmodium falciparum. Our results showed the dichloromethane gradient fraction was most effective, significantly (P < 0.05) suppressing infection by 99.46% at 100 mg/kg body weight. Amongst its 13 sub-fractions (DF1-DF13), DF11 was highly active, with IC50 of 4.8 and 6.74 μg/ml against P. falciparum HB3 and FCM29, respectively. Cytotoxicity of DF11 was estimated to be above 50 μg/ml, and its separation by column chromatography yielded a flavonoid which was characterized as 3, 5, 7, 3' tetrahydroxy-4'-methoxyflavone from its spectroscopic data. It significantly suppressed infection (65.43-81.48%) in mice at 2.5-5 mg/kg doses and compared favourably with the effects of chloroquine and artemisinin. It may therefore serve as a useful phytochemical and antiplasmodial activity marker of C. odorata leaves, which exhibit potential for development as medicine against malaria.

  14. Mechanisms of trazodone-induced cytotoxicity and the protective effects of melatonin and/or taurine toward freshly isolated rat hepatocytes.

    PubMed

    Taziki, Shohreh; Sattari, Mohammad Reza; Eghbal, Mohammad Ali

    2013-10-01

    It has been reported that the bioactive intermediate metabolites of trazodone might cause hepatotoxicity. This study was designed to investigate the exact mechanism of hepatocellular injury induced by trazodone as well as the protective effects of taurine and/or melatonin against this toxicity. Freshly isolated rat hepatocytes were used. Trazodone was cytotoxic and caused cell death with LC50 of 300 µm within 2 h. Trazodone caused an increase in reactive oxygen species (ROS) formation, malondialdehyde accumulation, depletion of intracellular reduced glutathione (GSH), rise of oxidized glutathione disulfide (GSSG), and a decrease in mitochondrial membrane potential, which confirms the role of oxidative stress in trazodone-induced cytotoxicity. Preincubation of hepatocytes with taurine prevented ROS formation, lipid peroxidation, depletion of intracellular reduced GSH, and increase of oxidized GSSG. Taurine could also protect mitochondria against trazodone-induced toxicity. Administration of melatonin reduced the toxic effects of trazodone in isolated rat hepatocytes. PMID:24023050

  15. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    PubMed

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting.

  16. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    PubMed

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting. PMID:27581019

  17. Dual-species biofilm formation by Escherichia coli O157:H7 and environmental bacteria isolated from fresh-cut processing facilities.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2014-02-01

    Biofilm formation is a mechanism adapted by many microorganisms that enhances the survival in stressful environments. In food processing facilities, foodborne bacterial pathogens, which many are poor biofilm formers, could potentially take advantage of this protective mechanism by interacting with other strong biofilm producers. The objective of this study was to determine the influence of bacteria native to fresh produce processing environments on the incorporation of Escherichia coli O157:H7 in biofilms. Bacteria strains representing 13 Gram-negative species isolated from two fresh produce processing facilities in a previous study were tested for forming dual-species biofilms with E. coli O157:H7. Strong biofilm producing strains of Burkholderia caryophylli and Ralstonia insidiosa exhibited 180% and 63% increase in biofilm biomass, and significant thickening of the biofilms (B. caryophylli not tested), when co-cultured with E. coli O157:H7. E. coli O157:H7 populations increased by approximately 1 log in dual-species biofilms formed with B. caryophylli or R. insidiosa. While only a subset of environmental isolates with strong biofilm formation abilities increased the presence of E. coli O157:H7 in biofilms, all tested E. coli O157:H7 exhibited higher incorporation in dual-species biofilms with R. insidiosa. These observations support the notion that E. coli O157:H7 and specific strong biofilm producing bacteria interact synergistically in biofilm formation, and suggest a route for increased survival potential of E. coli O157:H7 in fresh produce processing environments.

  18. Transfer and Detection of Freshly Isolated or Cultured Chicken (Gallus gallus) and Exotic Species’ Embryonic Gonadal Germ Stem Cells in Host Embryos

    PubMed Central

    Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

    2015-01-01

    The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ova sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds. PMID:24882096

  19. Sebaceous gland differentiation: III. The uses and limitations of freshly isolated mouse preputial gland cells for the in vitro study of hormone and drug action.

    PubMed

    Wheatley, V R; Brind, J L

    1981-04-01

    The effects of selected hormones and drugs on freshly isolated mouse preputial gland cells have been studied. The steroid hormones, testosterone, DHT, androstanediol, androsterone and androstanedione all failed to stimulate, and estradiol failed to inhibit, either DNA or lipid synthesis under the conditions studied. Thyroxine and insulin had no effect on lipogenesis but epinephrine and PGE2 caused significant stimulation as did Bt2cAMP. The antilipemic drugs clofibrate, nicotinic acid and hydroxycitrate were all able to inhibit lipogenesis. Of the anti-acne drugs only L-DOPA was able to inhibit lipogenesis, neither tetracycline nor trans-retinoic acid showed any effect. Pyridoxine was unable to inhibit lipogenesis but DMSO caused dramatic stimulation though it was without effect on DNA synthesis. Evidence is presented which suggests that the lack of response to steroid hormones is not due to the inability of the cells to take up and metabolize the steroids but is due to the fact that the time-span of exposure is not long enough to elicit a cellular response. It is concluded that these freshly isolated cells are suitable for the study of those effects of hormones and drugs which occur within the first 3 hr after exposure to the compound. PMID:6162901

  20. Combining Freshly Isolated Chondroprogenitor Cells from the Infrapatellar Fat Pad with a Growth Factor Delivery Hydrogel as a Putative Single Stage Therapy for Articular Cartilage Repair

    PubMed Central

    Ahearne, Mark; Liu, Yurong

    2014-01-01

    Growth factor delivery systems incorporating chondroprogenitor cells are an attractive potential treatment option for damaged cartilage. The rapid isolation, processing, and implantation of therapeutically relevant numbers of autologous chondroprogenitor cells, all performed “in-theatre” during a single surgical procedure, would significantly accelerate the clinical translation of such tissue engineered implants by avoiding the time, financial and regulatory challenges associated with in vitro cell expansion, and differentiation. The first objective of this study was to explore if rapid adherence to a specific substrate could be used as a simple means to quickly identify a subpopulation of chondroprogenitor cells from freshly digested infrapatellar fat pad (IFP) tissue. Adhesion of cells to tissue culture plastic within 30 min was examined as a mechanism of isolating subpopulations of cells from the freshly digested IFP. CD90, a cell surface marker associated with cell adhesion, was found to be more highly expressed in rapidly adhering cells (termed “RA” cells) compared to those that did not adhere (termed “NA” cells) in this timeframe. The NA subpopulation contained a lower number of colony forming cells, but overall had a greater chondrogenic potential but a diminished osteogenic potential compared to the RA subpopulation and unmanipulated freshly isolated (FI) control cells. When cultured in agarose hydrogels, NA cells proliferated faster than RA cells, accumulating significantly higher amounts of total sGAG and collagen. Finally, we sought to determine if cartilage tissue could be engineered by seeding such FI cells into a transforming growth factor-β3 delivery hydrogel. In such a system, both RA and NA cell populations demonstrated an ability to proliferate and produced a matrix rich in sGAG (∼2% w/w) that stained positively for type II collagen; however, the tissues were comparable to that generated using FI cells. Therefore, while the

  1. Whole-Genome Sequences of Mycobacterium bovis Strain MbURU-001, Isolated from Fresh Bovine Infected Samples

    PubMed Central

    Lasserre, Moira; Berná, Luisa; Greif, Gonzalo; Díaz-Viraqué, Florencia; Naya, Hugo; Castro-Ramos, Miguel; Juambeltz, Arturo

    2015-01-01

    Bovine tuberculosis in cattle has a high incidence in Uruguay, where it is considered a disease of national importance. We present the genome sequence of Mycobacterium bovis strain MbURU-001, isolated from pectoral lymph nodes of a bovine host from a cattle farm. PMID:26543108

  2. Pectic polysaccharides of the fresh plum Prunus domestica L. isolated with a simulated gastric fluid and their anti-inflammatory and antioxidant activities.

    PubMed

    Popov, Sergey V; Ovodova, Raisa G; Golovchenko, Victoria V; Khramova, Daria S; Markov, Pavel A; Smirnov, Vasily V; Shashkov, Alexandre S; Ovodov, Yury S

    2014-01-15

    A pectic polysaccharide, designated as PD, was extracted from fresh plums (Prunus domestica L.) with a simulated gastric fluid. Galacturonan, which was partially substituted with methyl and O-acetyl ester groups, and rhamnogalacturonan were the main constituents of the linear regions of the sugar chains of PD. The ramified region contained mainly 1,4-linked β-d-galactopyranose residues and, to a lesser extent, 1,5-linked α-l-arabinofuranose residues. The separation of PD, by DEAE-cellulose column chromatography, yielded two pectic fractions: PD-1 and PD-2, eluted with 0.1 and 0.2 M NaCl, respectively. Enzymatic digestion of PD with 1,4-α-d-polygalacturonase yielded the fraction PD-E. The parent pectin PD and the PD-1 fraction were found to diminish the adhesion of peritoneal leukocytes at the concentrations of 0.05-1.0mg/ml. However, the PD-E fraction failed to have an effect on cell adhesion at the concentrations of 0.05-0.1mg/ml. PD, PD-1 and PD-E were found to inhibit the production of superoxide anion radicals by reducing xanthine oxidase activity by 38%, 97% and 47%, respectively. Therefore, the PD-1 fraction appeared to be an active fragment of pectic macromolecule isolated from fresh plum with a simulated gastric fluid.

  3. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2015-01-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  4. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2015-03-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  5. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2015-03-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products.

  6. Small, clonally variant antigens expressed on the surface of the Plasmodium falciparum-infected erythrocyte are encoded by the rif gene family and are the target of human immune responses.

    PubMed

    Fernandez, V; Hommel, M; Chen, Q; Hagblom, P; Wahlgren, M

    1999-11-15

    Disease severity in Plasmodium falciparum infections is a direct consequence of the parasite's efficient evasion of the defense mechanisms of the human host. To date, one parasite-derived molecule, the antigenically variant adhesin P. falciparum erythrocyte membrane protein 1 (PfEMP1), is known to be transported to the infected erythrocyte (pRBC) surface, where it mediates binding to different host receptors. Here we report that multiple additional proteins are expressed by the parasite at the pRBC surface, including a large cluster of clonally variant antigens of 30-45 kD. We have found these antigens to be identical to the rifins, predicted polypeptides encoded by the rif multigene family. These parasite products, formerly called rosettins after their identification in rosetting parasites, are prominently expressed by fresh isolates of P. falciparum. Rifins are immunogenic in natural infections and strain-specifically recognized by human immune sera in immunoprecipitation of surface-labeled pRBC extracts. Furthermore, human immune sera agglutinate pRBCs digested with trypsin at conditions such that radioiodinated PfEMP1 polypeptides are not detected but rifins are detected, suggesting the presence of epitopes in rifins targeted by agglutinating antibodies. When analyzed by two-dimensional electrophoresis, the rifins resolved into several isoforms in the pI range of 5.5-6.5, indicating molecular microheterogeneity, an additional potential novel source of antigenic diversity in P. falciparum. Prominent polypeptides of 20, 22, 76-80, 140, and 170 kD were also detected on the surfaces of pRBCs bearing in vitro-propagated or field-isolated parasites. In this report, we describe the rifins, the second family of clonally variant antigens known to be displayed by P. falciparum on the surface of the infected erythrocyte.

  7. The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil.

    PubMed

    Lagos, N; Onodera, H; Zagatto, P A; Andrinolo, D; Azevedo, S M; Oshima, Y

    1999-10-01

    The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries. PMID:10414862

  8. Iodinated contrast media inhibit oxygen consumption in freshly isolated proximal tubular cells from elderly humans and diabetic rats: Influence of nitric oxide

    PubMed Central

    Liss, Per; Hansell, Peter; Fasching, Angelica; Palm, Fredrik

    2016-01-01

    Objectives Mechanisms underlying contrast medium (CM)-induced nephropathy remain elusive, but recent attention has been directed to oxygen availability. The purpose of this study was to evaluate the effect of the low-osmolar CM iopromide and the iso-osmolar CM iodixanol on oxygen consumption (QO2) in freshly isolated proximal tubular cells (PTC) from kidneys ablated from elderly humans undergoing nephrectomy for renal carcinomas and from normoglycemic or streptozotocin-diabetic rats. Materials PTC were isolated from human kidneys, or kidneys of normoglycemic or streptozotocin-diabetic rats. QO2 was measured with Clark-type microelectrodes in a gas-tight chamber with and without each CM (10 mg I/mL medium). L-NAME was used to inhibit nitric oxide (NO) production caused by nitric oxide synthase. Results Both CM reduced QO2 in human PTC (about –35%) which was prevented by L-NAME. PTC from normoglycemic rats were unaffected by iopromide, whereas iodixanol decreased QO2 (–34%). Both CM decreased QO2 in PTC from diabetic rats (–38% and –36%, respectively). L-NAME only prevented the effect of iopromide in the diabetic rat PTC. Conclusions These observations demonstrate that CM can induce NO release from isolated PTC in vitro, which affects QO2. Our results suggest that the induction of NO release and subsequent effect on the cellular oxygen metabolism are dependent on several factors, including CM type and pre-existing risk factors for the development of CM-induced nephropathy. PMID:26933994

  9. Metabolism of benzo[a]pyrene and (-)-trans-benzo[a]pyrene-7,8-dihydrodiol by freshly isolated hepatocytes of brown bullheads.

    PubMed

    Steward, A R; Zaleski, J; Sikka, H C

    1990-01-01

    The metabolism of [3H]benzo[a]pyrene (BP) and (-)-trans-[14C]7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP-7,8-diol) was studied in freshly isolated hepatocytes of the wild benthic fish, brown bullhead (Ictalurus nebulosus). Bullhead hepatocytes incubated with 40 microM [3H]BP for 1 h metabolized BP to water soluble metabolites which were separated on silica gel t.l.c. plates to reveal conjugates with glucuronic acid, glutathione, and sulfate (51%, 14% and 4% of total metabolites, respectively). Additional metabolites that were extractable with ethyl acetate were separated by reversed phase HPLC to reveal only two major metabolites: BP-9,10-dihydrodiol and BP-7,8-diol (13% and 2.6% of total metabolites, respectively). Hepatocytes isolated from individual fish displayed an 11-fold variability in the rates at which they metabolized BP (756 +/- 167 pmol x mg dry wt-1 x h-1), which correlated negatively (r = -0.7, P less than 0.01) with an 18-fold variability in the glycogen content of the cells. Hepatocytes isolated from the same fish, in parallel incubations under the same optimum conditions, metabolized BP-7,8-diol 4.5-fold faster than they metabolized BP. The variability in the rate of BP-7,8-diol metabolism was about 7-fold. Major metabolites included glutathione conjugates, glucuronides and sulfates (35%, 25% and 30% of total metabolites, respectively). These conjugates, like those formed from BP, were degradable with gamma-glutamyltransferase, beta-glucuronidase and arylsulfatase, respectively. Ethyl acetate extractable metabolites were predominantly isomeric benzo-ring tetrahydrotetrols (9% of total metabolites). In summary, this study indicates that during short-term incubations bull-head hepatocytes metabolize BP and BP-7,8-diol primarily to conjugated derivatives. The usefulness of thin-layer chromatography for the convenient determination of the rate of BP-7,8-diol metabolism is demonstrated.

  10. Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model

    PubMed Central

    2013-01-01

    Introduction The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. Methods A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6. Results Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1β and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. Conclusions We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic

  11. Differential expression and upregulation of interleukin-1alpha, interleukin-1beta and interleukin-6 by freshly isolated human small intestinal epithelial cells.

    PubMed Central

    Madrigal-Estebas, Laura; Doherty, Derek G; O'Donoghue, Diarmuid P; Feighery, Conleth; O'Farrelly, Cliona

    2002-01-01

    BACKGROUND: Small intestinal epithelial cells (SIEC) may contribute to local immune regulation. AIM: To examine production of interleukin (IL)-1alpha, IL-1beta and IL-6 by freshly isolated human SIEC. METHODS: IL-1alpha and IL-1beta mRNA in epithelial layers (EL) prepared from small intestine and in intestinal epithelial cell (EC) lines were examined by reverse transcription-polymerase chain reaction. IL-1alpha, IL-1beta and IL-6 protein expression by SIEC was examined by flow cytometry before and after activation with lipopolysaccharide and epithelial growth factor. RESULTS: IL-1alpha and IL-1beta mRNA was detected in EL and EC lines. Background expression of IL-1alpha and IL-1beta protein by SIEC was observed, which did not increase even following activation. IL-6 protein was expressed by SIEC, in a proportion that increased in two out of three samples following activation. CONCLUSIONS: IL-6 expression and the presence of IL-1alpha and IL-1beta mRNA suggest a role for SIEC in the regulation of local inflammation. PMID:12467524

  12. Effect of whey protein isolate-pullulan edible coatings on the quality and shelf life of freshly roasted and freeze-dried Chinese chestnut.

    PubMed

    Gounga, M E; Xu, S-Y; Wang, Z; Yang, W G

    2008-05-01

    Harvested chestnut is characterized by a short shelf life, exposing many Chinese producers to a storage problem as product losses are very high. The objective of this study was to develop a suitable technology to extend the shelf life of harvested chestnut fruits for commercial use. The effect of whey protein isolate-pullulan (WPI-Pul) coating on fresh-roasted chestnuts (FRC) and roasted freeze-dried chestnut (RFDC) quality and shelf life was studied under 2 different storage temperature (4 and 20 degrees C) conditions. Coatings were formed directly onto the surface of the fruits by dipping them into a film solution. SEM micrographs showed homogeneous WPI-Pul to cover the whole surface of chestnut with good adherence and perfect integrity. Moisture loss or gain, fruit quality, and shelf life were evaluated by weight loss or gain, surface color development, and visible decay during the storage period of 15 to 120 d at 4 and 20 degrees C, respectively. WPI-Pul coating had a low, yet significant effect on reducing moisture loss and decay incidence of FRC, hence delaying changes in their external color. The results were satisfactory when the coating was done with freeze-drying at low temperature storage, thus improving the quality and increasing the shelf life. This provides an alternative strategy to minimize the significant losses in harvested chestnut.

  13. Physico-chemical properties of whey protein isolate films containing oregano oil and their antimicrobial action against spoilage flora of fresh beef.

    PubMed

    Zinoviadou, Kyriaki G; Koutsoumanis, Konstantinos P; Biliaderis, Costas G

    2009-07-01

    Antimicrobial films were prepared by incorporating different levels of oregano oil (0.5%, 1.0%, and 1.5% w/w in the film forming solution) into sorbitol-plasticized whey protein isolate (WPI) films. The moisture uptake behavior and the water vapor permeability (WVP) were not affected by the addition of oregano oil at any of the concentrations used. A reduction of the glass transition temperature (∼10-20°C), as determined by dynamic mechanical thermal analysis (DMTA), was caused by addition of oil into the protein matrix. A decrease of Young modulus (E) and maximum tensile strength (σ(max)) accompanied with an increase in elongation at break (%EB) was observed with increasing oil concentration up to a level of 1.0% (w/w). Wrapping of beef cuts with the antimicrobial films resulted in smaller changes in total color difference (ΔΕ) and saturation difference (Δ(chroma)) during refrigeration (5°C, 12days). The maximum specific growth rate (μ(max)) of total flora (total viable count, TVC) and pseudomonads were significantly reduced (P<0.05) by a factor of two with the use of antimicrobial films (1.5% w/w oil in the film forming solution), while the growth of lactic acid bacteria was completely inhibited. These results pointed to the effectiveness of oregano oil containing whey protein films to increase the shelf life of fresh beef.

  14. Multiple independent introductions of Plasmodium falciparum in South America

    PubMed Central

    Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J.; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N.; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J.; Renaud, François; Prugnolle, Franck

    2012-01-01

    The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

  15. Functional link between muscarinic receptors and large-conductance Ca2+ -activated K+ channels in freshly isolated human detrusor smooth muscle cells.

    PubMed

    Parajuli, Shankar P; Hristov, Kiril L; Cheng, Qiuping; Malysz, John; Rovner, Eric S; Petkov, Georgi V

    2015-04-01

    Activation of muscarinic acetylcholine receptors (mAChRs) constitutes the primary mechanism for enhancing excitability and contractility of human detrusor smooth muscle (DSM). Since the large-conductance Ca(2+)-activated K(+) (KCa1.1) channels are key regulators of human DSM function, we investigated whether mAChR activation increases human DSM excitability by inhibiting KCa1.1 channels. We used the mAChR agonist, carbachol, to determine the changes in KCa1.1 channel activity upon mAChR activation in freshly isolated human DSM cells obtained from open bladder surgeries using the perforated whole cell and single KCa1.1 channel patch-clamp recordings. Human DSM cells were collected from 29 patients (23 males and 6 females, average age of 65.9 ± 1.5 years). Carbachol inhibited the amplitude and frequency of KCa1.1 channel-mediated spontaneous transient outward currents and spontaneous transient hyperpolarizations, which are triggered by the release of Ca(2+) from ryanodine receptors. Carbachol also caused membrane potential depolarization, which was not observed in the presence of iberiotoxin, a KCa1.1 channel inhibitor, indicating the critical role of the KCa1.1 channels. The potential direct carbachol effects on KCa1.1 channels were examined under conditions of removing the major cellular Ca(2+) sources for KCa1.1 channel activation with pharmacological inhibitors (thapsigargin, ryanodine, and nifedipine). In the presence of these inhibitors, carbachol did not affect the single KCa1.1 channel open probability and mean KCa1.1 channel conductance (cell-attached configuration) or depolarization-induced whole cell steady-state KCa1.1 currents. The data support the concept that mAChR activation triggers indirect functional KCa1.1 channel inhibition mediated by intracellular Ca(2+), thus increasing the excitability in human DSM cells.

  16. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  17. Cloning of Plasmodium falciparum by single-cell sorting.

    PubMed

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples.

  18. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014.

    PubMed

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar; Pradines, Bruno

    2016-05-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August-December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  19. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014

    PubMed Central

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar

    2016-01-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August–December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  20. In vitro response of chloroquine-resistant Plasmodium falciparum to mefloquine.

    PubMed

    López Antuñano, F J; Wernsdorfer, W H

    1979-01-01

    The present study was conducted to evaluate the application of the in vitro microtechnique system in determining the response of chloroquine-resistant Plasmodium falciparum to mefloquine.Using isolates of P. falciparum from Boa Vista, Brazil, and Villavicencio, Colombia, mefloquine was more than 7.7, 7.1, 7.1, and 6.4 times more effective than chloroquine in vitro at the ED(90), ED(95), ED(99), and ED(99.9) levels, respectively.

  1. Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 genes are associated with reduced in vitro activities of quinine in Plasmodium falciparum isolates from western Kenya.

    PubMed

    Cheruiyot, Jelagat; Ingasia, Luicer A; Omondi, Angela A; Juma, Dennis W; Opot, Benjamin H; Ndegwa, Joseph M; Mativo, Joan; Cheruiyot, Agnes C; Yeda, Redemptah; Okudo, Charles; Muiruri, Peninah; Bidii, Ngalah S; Chebon, Lorna J; Angienda, Paul O; Eyase, Fredrick L; Johnson, Jacob D; Bulimo, Wallace D; Andagalu, Ben; Akala, Hoseah M; Kamau, Edwin

    2014-07-01

    In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pfmdr1codons 86 and 184, Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC50), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and quinine susceptibility (P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pfmdr1-N86, Pfmdr1-Y184, and Pfcrt-K76, respectively). In addition, quinine IC50 values for parasites with Pfnhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats (P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

  2. Population genetic study of Plasmodium falciparum parasites pertaining to dhps gene sequence in malaria endemic areas of Assam.

    PubMed

    Sharma, J; Dutta, P; Khan, S A

    2015-01-01

    Plasmodium falciparum malaria parasite had developed resistance to almost all the currently used antimalarial drugs. The purpose of the study was to come across the genetic distances in P. falciparum dhps gene sequences circulating in Assam. A partial fragment of P. falciparum dhps gene containing major single nucleotide polymorphisms associated with sulphadoxine resistance were amplified and sequenced. Thereafter specific bioinformatics tools like BioEdit v7.0.9, ClustalW in Mega 5, DnaSP version v.5.10.01 etc were used for the analysis. A total of 100 P. falciparum positive cases in different malaria endemic areas of Assam were included for the study. Based upon the mutation analysis, a total of seven different P. falciparum dhps genotypes were observed with five variable sites. Maximum five haplotypes were found in the P. falciparum isolates from Jorhat district of Assam. Four polymorphic sites were observed in the P. falciparum dhps gene sequences in Karbi Anglong, NC Hills, Chirang and Jorhat whereas the isolates from other study areas had three polymorphic sites. A statistically significant positive value of Tajima's D were observed among the P. falciparum field isolates in Assam indicating that there is an excess of intermediate frequency alleles and can result from population bottlenecks, structure and/or balancing selection. Extensive gene flow took place among the P. falciparum population of Jorhat with Sivasagar, Chirang with Sivasagar and Chirang with Karbi Anglong. However, large genetic differentiation was observed among the P. falciparum isolates of NC Hills with Lakhimpur, Tinsukia, Dibrugarh and Golaghat and also the parasite population of Karbi Anglong with Lakhimpur and Tinsukia signifying little gene flow among the population. This finding has shown that mutant Pfdhps gene associated with sulphadoxine resistance is circulating in Assam. It is believed that, the parasite population may have undergone high level of breeding.

  3. Induction of gene amplification in Plasmodium falciparum

    SciTech Connect

    Rogers, P.L.

    1985-01-01

    Human erythrocytic in vitro cultures of Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with /sup 35/S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.

  4. Polysome profiling of the malaria parasite Plasmodium falciparum.

    PubMed

    Lacsina, Joshua R; LaMonte, Gregory; Nicchitta, Christopher V; Chi, Jen-Tsan

    2011-09-01

    In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.

  5. Plasmodium falciparum genetic crosses in a humanized mouse model

    PubMed Central

    Vaughan, Ashley M.; Pinapati, Richard S.; Cheeseman, Ian H.; Camargo, Nelly; Fishbaugher, Matthew; Checkley, Lisa A.; Nair, Shalini; Hutyra, Carolyn A.; Nosten, François H.; Anderson, Timothy J. C.; Ferdig, Michael T.; Kappe, Stefan H. I.

    2015-01-01

    Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here, we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations. PMID:26030447

  6. Evolution of genetic polymorphisms of Plasmodium falciparum merozoite surface protein (PfMSP) in Thailand.

    PubMed

    Kuesap, Jiraporn; Chaijaroenkul, Wanna; Ketprathum, Kanchanok; Tattiyapong, Puntanat; Na-Bangchang, Kesara

    2014-02-01

    Plasmodium falciparum malaria is a major public health problem in Thailand due to the emergence of multidrug resistance. The understanding of genetic diversity of malaria parasites is essential for developing effective drugs and vaccines. The genetic diversity of the merozoite surface protein-1 (PfMSP-1) and merozoite surface protein-2 (PfMSP-2) genes was investigated in a total of 145 P. falciparum isolates collected from Mae Sot District, Tak Province, Thailand during 3 different periods (1997-1999, 2005-2007, and 2009-2010). Analysis of genetic polymorphisms was performed to track the evolution of genetic change of P. falciparum using PCR. Both individual genes and their combination patterns showed marked genetic diversity during the 3 study periods. The results strongly support that P. falciparum isolates in Thailand are markedly diverse and patterns changed with time. These 2 polymorphic genes could be used as molecular markers to detect multiple clone infections and differentiate recrudescence from reinfection in P. falciparum isolates in Thailand.

  7. First evidence of pfcrt mutant Plasmodium falciparum in Madagascar.

    PubMed

    Randrianarivelojosia, Milijaona; Fidock, David A; Belmonte, Olivier; Valderramos, Stephanie G; Mercereau-Puijalon, Odile; Ariey, Frédéric

    2006-09-01

    The island of Madagascar, lying in the Indian Ocean approximately 250 miles from the African coast, has so far remained one of the few areas in the world without noticeable Plasmodium falciparum high-grade chloroquine (CQ) resistance. Here we report genotyping data on pfcrt in Madagascar. The pfcrt K76T mutation, which is critical for resistance to CQ, was detected in six (3.3%) of 183 P. falciparum isolates screened, within the mutant haplotypes CVIET and CVIDT. This is the first observation of pfcrt mutant parasites on the island. The current massive distribution of CQ for in-home management of fever in children will promote the dissemination of these mutant CQ-resistant parasites. In this context, genotyping of pfcrt remains a useful tool for CQ resistance surveillance as the prevalence of pfcrt mutations is far from saturation in Madagascar.

  8. A simple protocol for platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes in a resource poor setting.

    PubMed

    Tembo, Dumizulu L; Montgomery, Jacqui; Craig, Alister G; Wassmer, Samuel C

    2013-01-01

    most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps (10). Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM (11). In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian (12) and Mozambican patients (13), (although not in Malian (14)). With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay (15). Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation. PMID:23711755

  9. A simple protocol for platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes in a resource poor setting.

    PubMed

    Tembo, Dumizulu L; Montgomery, Jacqui; Craig, Alister G; Wassmer, Samuel C

    2013-01-01

    most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps (10). Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM (11). In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian (12) and Mozambican patients (13), (although not in Malian (14)). With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay (15). Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

  10. Antigenic Variation in Plasmodium falciparum.

    PubMed

    Petter, Michaela; Duffy, Michael F

    2015-01-01

    Plasmodium falciparum is the protozoan parasite that causes most malaria-associated morbidity and mortality in humans with over 500,000 deaths annually. The disease symptoms are associated with repeated cycles of invasion and asexual multiplication inside red blood cells of the parasite. Partial, non-sterile immunity to P. falciparum malaria develops only after repeated infections and continuous exposure. The successful evasion of the human immune system relies on the large repertoire of antigenically diverse parasite proteins displayed on the red blood cell surface and on the merozoite membrane where they are exposed to the human immune system. Expression switching of these polymorphic proteins between asexual parasite generations provides an efficient mechanism to adapt to the changing environment in the host and to maintain chronic infection. This chapter discusses antigenic diversity and variation in the malaria parasite and our current understanding of the molecular mechanisms that direct the expression of these proteins. PMID:26537377

  11. Factors associated with regional bias of pfcrt (plasmodium falciparum chloroquine resistance transporter) haplotypes in Nepal.

    PubMed

    Banjara, Megha Raj; Imwong, Mallika; Petmitr, Songsak; Sirawaraporn, Worachart; Joshi, Anand B; Chavalitshewinkoon-Petmitr, Porntip

    2011-01-01

    Evidences of reappearance of chloroquine sensitive Plasmodium falciparum haplotypes after cessation of chloroquine in many countries provide a rationale for the search of chloroquine sensitive haplotypes in P. falciparum isolates in Nepal where the use of chloroquine for falciparum malaria treatment has been ceased since 1988. P. falciparum chloroquine resistant transporter gene (pfcrt) haplotypes were determined and the factors associated with pfcrt haplotypes in the Eastern and Central regions of Nepal were identified. Blood samples from 106 microscopy-positive falciparum malaria patients (62 from the Eastern and 44 from the Central region) were collected on filter paper. Pfcrt region covering codons 72-76 was amplified by PCR and sequenced. SVMNT haplotype was predominant in the Central region, whereas CVIET haplotype significantly more common in the Eastern region. In multivariable analysis of factors associated with CVIET haplotype, the Eastern region and parasite isolates from patients visiting India within one month are significant at 5% level of significance. These findings suggest that antimalarial pressure is different between Eastern and Central regions of Nepal and there is a need of an effective malaria control program in the border areas between India and Nepal.

  12. Bacterial count comparisons on examination gloves from freshly opened boxes versus nearly empty boxes and from examination gloves before treatment versus after dental dam isolation.

    PubMed

    Luckey, Jeffrey B; Barfield, Robert D; Eleazer, Paul D

    2006-07-01

    Use of gloves in dentistry is primarily for protection of the healthcare worker, yet little information is available regarding potential patient issues such as microbial contamination of gloves before and during use. The purposes of this study were to compare gloves from newly opened boxes with those from boxes that had been in dental operatories until they were nearly empty and to determine if gloves are contaminated though diagnostic procedures and rubber dam placement. Eight endodontic residents provided samples by streaking gloved fingers on nutrient agar plates. Results showed no statistically significant difference between counts from new boxes versus nearly empty boxes (n = 32, p < 0.6216). Gloves after rubber dam placement yielded a mean colony count of 158 versus a 1.5 mean of fresh gloves (n = 64, p < 0.0001). These results suggest that the use of new gloves before opening a tooth for endodontic therapy may be warranted.

  13. In vitro sensitivity of Plasmodium falciparum to artesunate in Thailand.

    PubMed Central

    Wongsrichanalai, C.; Wimonwattrawatee, T.; Sookto, P.; Laoboonchai, A.; Heppner, D. G.; Kyle, D. E.; Wernsdorfer, W. H.

    1999-01-01

    Reported are the in vitro susceptibilities of Plasmodium falciparum to artesunate, mefloquine, quinine and chloroquine of 86 isolates and to dihydroartemisinin of 45 isolates collected from areas of high resistance to mefloquine within Thailand near the borders with Myanmar and Cambodia, and from southern Thailand where P. falciparum is generally still sensitive to mefloquine. All the isolates were highly sensitive to artesunate, but the geometric mean IC50S were higher in isolates from the Thai-Myanmar and Thai-Cambodian borders than in those from southern Thailand. The IC50S for mefloquine and artesunate were strongly correlated (Pearson r = 0.605; n = 86; P < 0.00001). As expected, the in vitro sensitivities to dihydroartemisinin and artesunate were similar and strongly correlated (at IC50, Pearson r = 0.695; n = 45; P < 0.00002). The correlation between the activity of mefloquine and artesunate requires further investigation in order to determine the potential for development of cross-resistance in nature. Our results suggest that combination with mefloquine is not the ideal way of protecting the usefulness of artemisinin and its derivatives. A search for more suitable partner drugs to these compounds and careful regulation of their use are necessary in the interest of ensuring their long therapeutic life span. PMID:10361756

  14. Chromogenic agar medium for detection and isolation of Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from fresh beef and cattle feces.

    PubMed

    Kalchayanand, Norasak; Arthur, Terrance M; Bosilevac, Joseph M; Wells, James E; Wheeler, Tommy L

    2013-02-01

    Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically important foodborne pathogens. Unlike E. coli O157:H7, these foodborne pathogens have no unique biochemical characteristics to readily distinguish them from other E. coli strains growing on plating media. In this study, a chromogenic agar medium was developed in order to differentiate among non-O157 STEC strains of serogroups O26, O45, O103, O111, O121, and O145 on a single agar medium. The ability of this chromogenic agar medium to select and distinguish among these pathogens is based on a combination of utilization of carbohydrates, b -galactosidase activity, and resistance to selective agents. The agar medium in combination with immunomagnetic separation was evaluated and successfully allowed for the detection and isolation of these six serogroups from artificially contaminated fresh beef. The agar medium in combination with immunomagnetic separation also allowed successful detection and isolation of naturally occurring non-O157 STEC strains present in cattle feces. Thirty-five strains of the top six non-O157 STEC serogroups were isolated from 1,897 fecal samples collected from 271 feedlot cattle. This chromogenic agar medium could help significantly in routine screening for the top six non-O157 STEC serogroups from beef cattle and other food.

  15. Chloroquine resistance of Plasmodium falciparum is associated with severity of disease in Nigerian children.

    PubMed

    Olumese, P E; Amodu, O K; Björkman, A; Adeyemo, A A; Gbadegesin, R A; Walker, O

    2002-01-01

    Chloroquine resistance of Plasmodium falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease. This association may be due to either progression of uncomplicated to severe disease following chloroquine failure or increased virulence of chloroquine-resistant parasites. The implication of this for antimalarial treatment policy is discussed. PMID:12497979

  16. Isolation and characterization of starch from industrial fresh pasta by-product and its potential use in sugar-snap cookie making.

    PubMed

    Ellouzi, Soumaya Zouari; Driss, Dorra; Maktouf, Sameh; Neifar, Mohamed; Kobbi, Ameni; Kamoun, Hounaida; Chaabouni, Semia Ellouze; Ghorbel, Raoudha Ellouze

    2015-09-01

    In this paper, starch was extracted from fresh pasta by-product (PS) and its chemical composition and physical and microscopic characteristics were determined. Commercial wheat starch (CS) was used as reference. In general, purity was similar between starches studied. However, others compounds such as protein, lipid and ash were significantly different. PS starch granules had large lenticular-shape (25-33 μm) and small spherical-shape (5-8 μm). The pH and color of PS starch were similar to those reported for CS starch. On the other hand, PS had higher water absorption capacity, viscosity and cooking stability than CS. The gelatinization temperature of PS was similar to that of CS (60 and 61 °C). At high temperature (90 °C) both starches had similar rheological behavior. The results achieved suggest that PS starch has potential for application in food systems requiring high processing temperatures such the manufacture of sugar snap cookie. The effects of PS starch addition on the dough making stage and the final cookie quality were analyzed. Improvements in dough cohesiveness (24 %) and springiness (10 %) were significant relative to those of CS dough. Texture profile analysis confirmed the rheological changes.

  17. Isolation and characterization of starch from industrial fresh pasta by-product and its potential use in sugar-snap cookie making.

    PubMed

    Ellouzi, Soumaya Zouari; Driss, Dorra; Maktouf, Sameh; Neifar, Mohamed; Kobbi, Ameni; Kamoun, Hounaida; Chaabouni, Semia Ellouze; Ghorbel, Raoudha Ellouze

    2015-09-01

    In this paper, starch was extracted from fresh pasta by-product (PS) and its chemical composition and physical and microscopic characteristics were determined. Commercial wheat starch (CS) was used as reference. In general, purity was similar between starches studied. However, others compounds such as protein, lipid and ash were significantly different. PS starch granules had large lenticular-shape (25-33 μm) and small spherical-shape (5-8 μm). The pH and color of PS starch were similar to those reported for CS starch. On the other hand, PS had higher water absorption capacity, viscosity and cooking stability than CS. The gelatinization temperature of PS was similar to that of CS (60 and 61 °C). At high temperature (90 °C) both starches had similar rheological behavior. The results achieved suggest that PS starch has potential for application in food systems requiring high processing temperatures such the manufacture of sugar snap cookie. The effects of PS starch addition on the dough making stage and the final cookie quality were analyzed. Improvements in dough cohesiveness (24 %) and springiness (10 %) were significant relative to those of CS dough. Texture profile analysis confirmed the rheological changes. PMID:26344989

  18. Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan

    PubMed Central

    2010-01-01

    Background Plasmodium vivax and Plasmodium falciparum are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of P. vivax and P. falciparum in this understudied region. Methods The genetic diversities of P. vivax and P. falciparum populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (msp) genes by PCR-RFLP. Specifically, the Pvmsp-3α and Pvmsp-3β genes of P. vivax and the Pfmsp-1 and Pfmsp-2 genes of P. falciparum were analysed. Results In P. vivax, genotyping of Pvmsp-3α and Pvmsp-3β genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for Pvmsp-3α, type A being the most prevalent (82%). Conversely, amplification of the P. vivax msp-3β locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of Pvmsp-3α and Pvmsp-3β yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In P. falciparum, all three known genotypes of Pfmsp-1 and two of Pfmsp-2 were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% P. falciparum samples exhibited mixed-strain infections. Conclusion These results indicate that both P. vivax and P. falciparum populations in Pakistan are highly diverse. PMID:20416089

  19. Molecular Investigation into a Malaria Outbreak in Cusco, Peru: Plasmodium falciparum BV1 Lineage is Linked to a Second Outbreak in Recent Times.

    PubMed

    Okoth, Sheila Akinyi; Chenet, Stella M; Arrospide, Nancy; Gutierrez, Sonia; Cabezas, Cesar; Matta, Jose Antonio; Udhayakumar, Venkatachalam

    2016-01-01

    In November 2013, a Plasmodium falciparum malaria outbreak of 11 cases occurred in Cusco, southern Peru, where falciparum malaria had not been reported since 1946. Although initial microscopic diagnosis reported only Plasmodium vivax infection in each of the specimens, subsequent examination by the national reference laboratory confirmed P. falciparum infection in all samples. Molecular typing of four available isolates revealed identity as the B-variant (BV1) strain that was responsible for a malaria outbreak in Tumbes, northern Peru, between 2010 and 2012. The P. falciparum BV1 strain is multidrug resistant, can escape detection by PfHRP2-based rapid diagnostic tests, and has contributed to two malaria outbreaks in Peru. This investigation highlights the importance of accurate species diagnosis given the potential for P. falciparum to be reintroduced to regions where it may have been absent. Similar molecular epidemiological investigations can track the probable source(s) of outbreak parasite strains for malaria surveillance and control purposes.

  20. Molecular Investigation into a Malaria Outbreak in Cusco, Peru: Plasmodium falciparum BV1 Lineage is Linked to a Second Outbreak in Recent Times.

    PubMed

    Okoth, Sheila Akinyi; Chenet, Stella M; Arrospide, Nancy; Gutierrez, Sonia; Cabezas, Cesar; Matta, Jose Antonio; Udhayakumar, Venkatachalam

    2016-01-01

    In November 2013, a Plasmodium falciparum malaria outbreak of 11 cases occurred in Cusco, southern Peru, where falciparum malaria had not been reported since 1946. Although initial microscopic diagnosis reported only Plasmodium vivax infection in each of the specimens, subsequent examination by the national reference laboratory confirmed P. falciparum infection in all samples. Molecular typing of four available isolates revealed identity as the B-variant (BV1) strain that was responsible for a malaria outbreak in Tumbes, northern Peru, between 2010 and 2012. The P. falciparum BV1 strain is multidrug resistant, can escape detection by PfHRP2-based rapid diagnostic tests, and has contributed to two malaria outbreaks in Peru. This investigation highlights the importance of accurate species diagnosis given the potential for P. falciparum to be reintroduced to regions where it may have been absent. Similar molecular epidemiological investigations can track the probable source(s) of outbreak parasite strains for malaria surveillance and control purposes. PMID:26483121

  1. Investigating the activity of quinine analogues vs. chloroquine resistant Plasmodium falciparum

    PubMed Central

    Dinio, Theresa; Gorka, Alexander P.; McGinniss, Andrew; Roepe, Paul D.; Morgan, Jeremy B.

    2012-01-01

    Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide well-tolerated therapy with improved activity vs. CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC50 values relative to QN. PMID:22512909

  2. Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration.

    PubMed

    Curiel, J A; Ruiz-Capillas, C; de Las Rivas, B; Carrascosa, A V; Jiménez-Colmenero, F; Muñoz, R

    2011-07-01

    The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.

  3. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion **

    PubMed Central

    Egan, Elizabeth S.; Jiang, Rays H.Y.; Moechtar, Mischka A.; Barteneva, Natasha S.; Weekes, Michael P.; Nobre, Luis V.; Gygi, Steven P.; Paulo, Joao A.; Frantzreb, Charles; Tani, Yoshihiko; Takahashi, Junko; Watanabe, Seishi; Goldberg, Jonathan; Paul, Aditya S.; Brugnara, Carlo; Root, David E.; Wiegand, Roger C.; Doench, John G.; Duraisingh, Manoj T.

    2015-01-01

    Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, precluding genetic manipulation in the cell where the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis. PMID:25954012

  4. Branch Point Identification and Sequence Requirements for Intron Splicing in Plasmodium falciparum ▿ †

    PubMed Central

    Zhang, Xiaohong; Tolzmann, Caitlin A.; Melcher, Martin; Haas, Brian J.; Gardner, Malcolm J.; Smith, Joseph D.; Feagin, Jean E.

    2011-01-01

    Splicing of mRNA is an ancient and evolutionarily conserved process in eukaryotic organisms, but intron-exon structures vary. Plasmodium falciparum has an extreme AT nucleotide bias (>80%), providing a unique opportunity to investigate how evolutionary forces have acted on intron structures. In this study, we developed an in vivo luciferase reporter splicing assay and employed it in combination with lariat isolation and sequencing to characterize 5′ and 3′ splicing requirements and experimentally determine the intron branch point in P. falciparum. This analysis indicates that P. falciparum mRNAs have canonical 5′ and 3′ splice sites. However, the 5′ consensus motif is weakly conserved and tolerates nucleotide substitution, including the fifth nucleotide in the intron, which is more typically a G nucleotide in most eukaryotes. In comparison, the 3′ splice site has a strong eukaryotic consensus sequence and adjacent polypyrimidine tract. In four different P. falciparum pre-mRNAs, multiple branch points per intron were detected, with some at U instead of the typical A residue. A weak branch point consensus was detected among 18 identified branch points. This analysis indicates that P. falciparum retains many consensus eukaryotic splice site features, despite having an extreme codon bias, and possesses flexibility in branch point nucleophilic attack. PMID:21926333

  5. Aspergillus clavatus Y2H0002 as a New Endophytic Fungal Strain Producing Gibberellins Isolated from Nymphoides pe ltata in Fresh Water.

    PubMed

    You, Young-Hyun; Kwak, Tae Won; Kang, Sang-Mo; Lee, Myung-Chul; Kim, Jong-Guk

    2015-03-01

    Eighteen endophytic fungi with different colony morphologies were isolated from the roots of Nymphoides peltata growing in the Dalsung wetland. The fungal culture filtrates of the endophytic fungi were treated to Waito-c rice seedling to evaluate their plant growth-promoting activities. Culture filtrate of Y2H0002 fungal strain promoted the growth of the Waito-c rice seedlings. This strain was identified on the basis of sequences of the partial internal transcribed spacer region and the partial beta-tubulin gene. Upon chromatographic analysis of the culture filtrate of Y2H0002 strain, the gibberellins (GAs: GA1, GA3, and GA4) were detected and quantified. Molecular and morphological studies identified the Y2H0002 strain as belonging to Aspergillus clavatus. These results indicated that A. clavatus improves the growth of plants and produces various GAs, and may participate in the growth of plants under diverse environmental conditions.

  6. Aspergillus clavatus Y2H0002 as a New Endophytic Fungal Strain Producing Gibberellins Isolated from Nymphoides pe ltata in Fresh Water

    PubMed Central

    You, Young-Hyun; Kwak, Tae Won; Kang, Sang-Mo; Lee, Myung-Chul

    2015-01-01

    Eighteen endophytic fungi with different colony morphologies were isolated from the roots of Nymphoides peltata growing in the Dalsung wetland. The fungal culture filtrates of the endophytic fungi were treated to Waito-c rice seedling to evaluate their plant growth-promoting activities. Culture filtrate of Y2H0002 fungal strain promoted the growth of the Waito-c rice seedlings. This strain was identified on the basis of sequences of the partial internal transcribed spacer region and the partial beta-tubulin gene. Upon chromatographic analysis of the culture filtrate of Y2H0002 strain, the gibberellins (GAs: GA1, GA3, and GA4) were detected and quantified. Molecular and morphological studies identified the Y2H0002 strain as belonging to Aspergillus clavatus. These results indicated that A. clavatus improves the growth of plants and produces various GAs, and may participate in the growth of plants under diverse environmental conditions. PMID:25892921

  7. Detection of SUMOylation in Plasmodium falciparum.

    PubMed

    Reiter, Katherine H; Matunis, Michael J

    2016-01-01

    Reversible protein modification by small ubiquitin-related modifiers (SUMOs) regulates many cellular processes, including transcription, protein quality control, cell division, and oxidative stress. SUMOylation is therefore essential for normal cell function and represents a potentially valuable target for the development of inhibitors of pathogenic eukaryotic organisms, including the malaria parasite, Plasmodium falciparum (Pf). The specific and essential functions of SUMOylation in Pf, however, remain largely uncharacterized. The further development of antimalarial drugs targeting SUMOylation would benefit significantly from a more detailed understanding of its functions and regulation during the parasite life cycle. The recent development of antibodies specific for Pf SUMO provides a valuable tool to study the functions and regulation of SUMOylation. In preliminary studies, we have used immunoblot analysis to demonstrate that SUMOylation levels vary significantly in parasites during different stages of the red blood cell cycle and also in response to oxidative stress. Owing to the dynamic nature of SUMOylation and to the robust activity of SUMO isopeptidases, analysis of SUMOylation in cultured Pf parasites requires a number of precautions during parasite purification and lysis. Here, we outline methods for preserving SUMO conjugates during isolation of Pf parasites from human red blood cell cultures, and for their detection by immunoblot analysis using PfSUMO-specific antibodies. PMID:27631812

  8. Exchange Transfusion in Severe Falciparum Malaria

    PubMed Central

    Khatib, Khalid Ismail

    2016-01-01

    Malaria is endemic in India with the incidence of P. falciparum Malaria increasing gradually over the last decade. Severe malaria is an acute disease, caused by P. falciparum, but increasingly also by P. vivax with major signs of organ dysfunction and/or high levels of parasitaemia (>10%) in blood smear. Use of exchange transfusion with antimalarial drug therapy as an additional modality of treatment in severe Falciparum malaria is controversial and is unclear. We report a case of severe malaria complicated by multiorgan failure and ARDS. Patient responded well to manual exchange transfusion with standard artesunate-based chemotherapy. PMID:27042503

  9. Frozen cord blood hematopoietic stem cells differentiate into higher numbers of functional natural killer cells in vitro than mobilized hematopoietic stem cells or freshly isolated cord blood hematopoietic stem cells.

    PubMed

    Luevano, Martha; Domogala, Anna; Blundell, Michael; Jackson, Nicola; Pedroza-Pacheco, Isabela; Derniame, Sophie; Escobedo-Cousin, Michelle; Querol, Sergio; Thrasher, Adrian; Madrigal, Alejandro; Saudemont, Aurore

    2014-01-01

    Adoptive natural killer (NK) cell therapy relies on the acquisition of large numbers of NK cells that are cytotoxic but not exhausted. NK cell differentiation from hematopoietic stem cells (HSC) has become an alluring option for NK cell therapy, with umbilical cord blood (UCB) and mobilized peripheral blood (PBCD34(+)) being the most accessible HSC sources as collection procedures are less invasive. In this study we compared the capacity of frozen or freshly isolated UCB hematopoietic stem cells (CBCD34(+)) and frozen PBCD34(+) to generate NK cells in vitro. By modifying a previously published protocol, we showed that frozen CBCD34(+) cultures generated higher NK cell numbers without loss of function compared to fresh CBCD34(+) cultures. NK cells generated from CBCD34(+) and PBCD34(+) expressed low levels of killer-cell immunoglobulin-like receptors but high levels of activating receptors and of the myeloid marker CD33. However, blocking studies showed that CD33 expression did not impact on the functions of the generated cells. CBCD34(+)-NK cells exhibited increased capacity to secrete IFN-γ and kill K562 in vitro and in vivo as compared to PBCD34(+)-NK cells. Moreover, K562 killing by the generated NK cells could be further enhanced by IL-12 stimulation. Our data indicate that the use of frozen CBCD34(+) for the production of NK cells in vitro results in higher cell numbers than PBCD34(+), without jeopardizing their functionality, rendering them suitable for NK cell immunotherapy. The results presented here provide an optimal strategy to generate NK cells in vitro for immunotherapy that exhibit enhanced effector function when compared to alternate sources of HSC.

  10. Contrasting Transmission Dynamics of Co-endemic Plasmodium vivax and P. falciparum: Implications for Malaria Control and Elimination

    PubMed Central

    Noviyanti, Rintis; Coutrier, Farah; Utami, Retno A. S.; Trimarsanto, Hidayat; Tirta, Yusrifar K.; Trianty, Leily; Kusuma, Andreas; Sutanto, Inge; Kosasih, Ayleen; Kusriastuti, Rita; Hawley, William A.; Laihad, Ferdinand; Lobo, Neil; Marfurt, Jutta; Clark, Taane G.; Price, Ric N.; Auburn, Sarah

    2015-01-01

    Background Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions. Methodology/ Principal Findings Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor). Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species. Conclusions/ Significance Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given

  11. Spontaneous rupture of spleen in falciparum malaria.

    PubMed

    Vidyashankar, C; Basu, Arup; Kulkarni, A R; Choudhury, R K

    2003-01-01

    Spontaneous rupture of spleen is an extremely rare complication of falciparum malaria. We report a 3 1/2-year-old girl with splenic rupture who was managed successfully with splenectomy and antimalarials.

  12. Use of a colorimetric (DELI) test for the evaluation of chemoresistance of Plasmodium falciparum and Plasmodium vivax to commonly used anti-plasmodial drugs in the Brazilian Amazon

    PubMed Central

    2013-01-01

    Background The emergence and spread of Plasmodium falciparum and Plasmodium vivax resistance to available anti-malarial drugs represents a major drawback in the control of malaria and its associated morbidity and mortality. The aim of this study was to evaluate the chemoresistance profile of P. falciparum and P. vivax to commonly used anti-plasmodial drugs in a malaria-endemic area in the Brazilian Amazon. Methods The study was carried out in Manaus (Amazonas state), in the Brazilian Amazon. A total of 88 P. falciparum and 178 P. vivax isolates was collected from 2004 to 2007. The sensitivity of P. falciparum isolates was determined to chloroquine, quinine, mefloquine and artesunate and the sensitivity of P. vivax isolates was determined to chloroquine and mefloquine, by using the colorimetric DELI test. Results As expected, a high prevalence of P. falciparum isolates resistant to chloroquine (78.1%) was observed. The prevalence of isolates with profile of resistance or decreased sensitivity for quinine, mefloquine and artesunate was 12.7, 21.2 and 11.7%, respectively. In the case of P. vivax, the prevalence of isolates with profile of resistance for chloroquine and mefloquine was 9.8 and 28%, respectively. No differences in the frequencies of isolates with profile of resistance or geometric mean IC50s were seen when comparing the data obtained in 2004, 2005, 2006 and 2007, for all tested anti-malarials. Conclusions The great majority of P. falciparum isolates in the Brazilian malaria-endemic area remain resistant to chloroquine, and the decreased sensitivity to quinine, mefloquine and artesunate observed in 10–20% of the isolates must be taken with concern, especially for artesunate. Plasmodium vivax isolates also showed a significant proportion of isolates with decreased sensitivity to chloroquine (first-line drug) and mainly to mefloquine. The data presented here also confirm the usefulness of the DELI test to generate results able to impact on public health

  13. Detectability of Plasmodium falciparum clones

    PubMed Central

    2010-01-01

    Background In areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed. Methods A longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion. Results The distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted. Conclusions A simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week

  14. Prevalence of mutation and phenotypic expression associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Zakai, Haytham A; Khan, Wajihullah; Asma, Umme

    2013-09-01

    Therapeutic efficacy of sulfadoxine-pyrimethamine (SP), which is commonly used to treat falciparum malaria, was assessed in isolates of Plasmodium falciparum (Welch, 1897) and Plasmodium vivax (Grassi et Feletti, 1890) ofAligarh, Uttar Pradesh, North India and Taif, Saudi Arabia during 2011-2012. Both the species showed mutations in dihydrofolate reductase (DHFR) enzyme as they have common biochemical drug targets. Mutation rate for pfdhfr was higher compared to pvdhfr because the drug was mainly given to treat falciparum malaria. Since both the species coexist, P. vivax was also exposed to SP due to faulty species diagnosis or medication without specific diagnosis. Low level of mutations against SP in P. falciparum of Saudi isolates indicates that the SP combination is still effective for the treatment of falciparum malaria. Since SP is used as first-line of treatment because of high level of resistance against chloroquine (CQ), it may result in spread of higher level of mutations resulting in drug resistance and treatment failure in near future. Therefore, to avoid further higher mutations in the parasite, use of better treatment regimens such as artesunate combination therapy must be introduced against SP combination.

  15. Comparative ex vivo activity of novel endoperoxides in multidrug-resistant plasmodium falciparum and P. vivax.

    PubMed

    Marfurt, Jutta; Chalfein, Ferryanto; Prayoga, Pak; Wabiser, Frans; Wirjanata, Grennady; Sebayang, Boni; Piera, Kim A; Wittlin, Sergio; Haynes, Richard K; Möhrle, Jörg J; Anstey, Nicholas M; Kenangalem, Enny; Price, Ric N

    2012-10-01

    The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC(50)s) in both species (median IC(50)s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity.

  16. Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants.

    PubMed

    Voepel, Nadja; Boes, Alexander; Edgue, Güven; Beiss, Veronique; Kapelski, Stephanie; Reimann, Andreas; Schillberg, Stefan; Pradel, Gabriele; Fendel, Rolf; Scheuermayer, Matthias; Spiegel, Holger; Fischer, Rainer

    2014-11-01

    Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80°C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP_TSR, PfTRAP_TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails. PMID:25200253

  17. Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal.

    PubMed

    Gendrot, Mathieu; Fall, Bécaye; Madamet, Marylin; Fall, Mansour; Wade, Khalifa Ababacar; Amalvict, Rémy; Nakoulima, Aminata; Benoit, Nicolas; Diawara, Silman; Diémé, Yaya; Diatta, Bakary; Wade, Boubacar; Pradines, Bruno

    2016-08-01

    The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC50) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary. PMID:27185795

  18. 3-Halo Chloroquine Derivatives Overcome Plasmodium falciparum Chloroquine Resistance Transporter-Mediated Drug Resistance in P. falciparum.

    PubMed

    Edaye, Sonia; Tazoo, Dagobert; Bohle, D Scott; Georges, Elias

    2015-12-01

    Polymorphism in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) was shown to cause chloroquine resistance. In this report, we examined the antimalarial potential of novel 3-halo chloroquine derivatives (3-chloro, 3-bromo, and 3-iodo) against chloroquine-susceptible and -resistant P. falciparum. All three derivatives inhibited the proliferation of P. falciparum; with 3-iodo chloroquine being most effective. Moreover, 3-iodo chloroquine was highly effective at potentiating and reversing chloroquine toxicity of drug-susceptible and -resistant P. falciparum.

  19. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

  20. Neutralizing Antibodies against Plasmodium falciparum Associated with Successful Cure after Drug Therapy

    PubMed Central

    Goh, Yun Shan; Peng, Kaitian; Chia, Wan Ni; Siau, Anthony; Chotivanich, Kesinee; Gruner, Anne-Charlotte; Preiser, Peter; Mayxay, Mayfong; Pukrittayakamee, Sasithon; Sriprawat, Kanlaya; Nosten, Francois; White, Nicholas J.

    2016-01-01

    An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection. PMID:27427762

  1. Neutralizing Antibodies against Plasmodium falciparum Associated with Successful Cure after Drug Therapy.

    PubMed

    Goh, Yun Shan; Peng, Kaitian; Chia, Wan Ni; Siau, Anthony; Chotivanich, Kesinee; Gruner, Anne-Charlotte; Preiser, Peter; Mayxay, Mayfong; Pukrittayakamee, Sasithon; Sriprawat, Kanlaya; Nosten, Francois; White, Nicholas J; Renia, Laurent

    2016-01-01

    An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection. PMID:27427762

  2. Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays.

    PubMed

    Cooper, Roland A; Conrad, Melissa D; Watson, Quentin D; Huezo, Stephanie J; Ninsiima, Harriet; Tumwebaze, Patrick; Nsobya, Samuel L; Rosenthal, Philip J

    2015-08-01

    We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda.

  3. Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays.

    PubMed

    Cooper, Roland A; Conrad, Melissa D; Watson, Quentin D; Huezo, Stephanie J; Ninsiima, Harriet; Tumwebaze, Patrick; Nsobya, Samuel L; Rosenthal, Philip J

    2015-08-01

    We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda. PMID:26033725

  4. Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

    PubMed Central

    Conrad, Melissa D.; Watson, Quentin D.; Huezo, Stephanie J.; Ninsiima, Harriet; Tumwebaze, Patrick; Nsobya, Samuel L.; Rosenthal, Philip J.

    2015-01-01

    We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda. PMID:26033725

  5. Toward a high-resolution Plasmodium falciparum linkage map: Polymorphic markers from hundreds of simple sequence repeats

    SciTech Connect

    Su, Xin-Zhuan; Wellems, T.E.

    1996-05-01

    A total of 5.7 simple sequence repeats (SSRs or {open_quotes}microsatellites{close_quotes}) were identified from Plasmodium falciparum sequences in GenBank and from inserts in a genomic DNA library. Oligonucleotide primers from sequences that flank 224 of these SSRs were synthesized and used in PCR assays to test for simple sequence length polymorphisms (SSLPs). Of the 224 SSRs, 188 showed SSLPs were assigned to chromosome linkage groups by physical mapping and by comparing their inheritance patterns against those of restriction fragment length polymorphism markers in a genetic cross (HB3XDd2). The predominant SSLPs in P. falciparum were found to contain [TA]{sub n}, and [TAA]{sub n}, a feature that is reminiscent of plant genomes and is consistent with the proposed algal-like origin of malaria parasites. Since such SSLPs are abundant and readily isolated, they are a powerful resource for genetic analysis of P. falciparum. 38 refs., 2 figs., 2 tabs.

  6. Identification of a cDNA clone encoding a mature blood stage antigen of Plasmodium falciparum by immunization of mice with bacterial lysates.

    PubMed Central

    Coppel, R L; Brown, G V; Mitchell, G F; Anders, R F; Kemp, D J

    1984-01-01

    A cDNA library was constructed in pBR322 using mRNA from blood stages of a Papua New Guinean isolate of Plasmodium falciparum. Expression of parasite antigens was not directly detectable by conventional immunological assays. To circumvent this, mice were immunized with lysates of cDNA clones, and the antisera raised were assayed for anti-parasite reactivity. One cDNA clone was identified which reliably elicited antibodies to P. falciparum. The mouse antisera were used to characterize the native P. falciparum protein as a 120-kd protein, which is antigenic during natural infection. The protein occurs in late trophozoite and schizont stages and is found in isolates of the parasite from widely separated geographical areas. The genomic context of the antigen gene is conserved in the different isolates. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6370681

  7. In vitro activity of pyronaridine against multidrug-resistant Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Price, R N; Marfurt, J; Chalfein, F; Kenangalem, E; Piera, K A; Tjitra, E; Anstey, N M; Russell, B

    2010-12-01

    Pyronaridine, a Mannich base antimalarial, has demonstrated high in vivo and in vitro efficacy against chloroquine-resistant Plasmodium falciparum. Although this drug has the potential to become a prominent artemisinin combination therapy, little is known about its efficacy against drug-resistant Plasmodium vivax. The in vitro antimalarial susceptibility of pyronaridine was assessed in multidrug-resistant P. vivax (n = 99) and P. falciparum (n = 90) isolates from Papua, Indonesia, using a schizont maturation assay. The median 50% inhibitory concentration (IC(50)) of pyronaridine was 1.92 nM (range, 0.24 to 13.8 nM) against P. falciparum and 2.58 nM (range, 0.13 to 43.6 nM) against P. vivax, with in vitro susceptibility correlating significantly with chloroquine, amodiaquine, and piperaquine (r(s) [Spearman's rank correlation coefficient] = 0.45 to 0.62; P < 0.001). P. falciparum parasites initially at trophozoite stage had higher IC(50)s of pyronaridine than those exposed at the ring stage (8.9 nM [range, 0.6 to 8.9 nM] versus 1.6 nM [range, 0.6 to 8.9 nM], respectively; P = 0.015), although this did not reach significance for P. vivax (4.7 nM [range, 1.4 to 18.7 nM] versus 2.5 nM [range, 1.4 to 15.6 nM], respectively; P = 0.085). The excellent in vitro efficacy of pyronaridine against both chloroquine-resistant P. vivax and P. falciparum highlights the suitability of the drug as a novel partner for artemisinin-based combination therapy in regions where the two species are coendemic.

  8. Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

    PubMed Central

    Hoonchaiyapoom, Thirasak; Inorn, Kornnarin

    2014-01-01

    Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy. PMID:25548414

  9. Fucosylated chondroitin sulfate inhibits Plasmodium falciparum cytoadhesion and merozoite invasion.

    PubMed

    Bastos, Marcele F; Albrecht, Letusa; Kozlowski, Eliene O; Lopes, Stefanie C P; Blanco, Yara C; Carlos, Bianca C; Castiñeiras, Catarina; Vicente, Cristina P; Werneck, Claudio C; Wunderlich, Gerhard; Ferreira, Marcelo U; Marinho, Claudio R F; Mourão, Paulo A S; Pavão, Mauro S G; Costa, Fabio T M

    2014-01-01

    Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria.

  10. Fucosylated Chondroitin Sulfate Inhibits Plasmodium falciparum Cytoadhesion and Merozoite Invasion

    PubMed Central

    Bastos, Marcele F.; Albrecht, Letusa; Kozlowski, Eliene O.; Lopes, Stefanie C. P.; Blanco, Yara C.; Carlos, Bianca C.; Castiñeiras, Catarina; Vicente, Cristina P.; Werneck, Claudio C.; Wunderlich, Gerhard; Ferreira, Marcelo U.; Marinho, Claudio R. F.; Mourão, Paulo A. S.; Pavão, Mauro S. G.

    2014-01-01

    Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria. PMID:24395239

  11. Comparison between Flow Cytometry, Microscopy, and Lactate Dehydrogenase-Based Enzyme-Linked Immunosorbent Assay for Plasmodium falciparum Drug Susceptibility Testing under Field Conditions

    PubMed Central

    Woodrow, Charles J.; Wangsing, Chirapat; Sriprawat, Kanlaya; Christensen, Peter R.; Nosten, Francois; Rénia, Laurent; Russell, Bruce

    2015-01-01

    Flow cytometry is an objective method for conducting in vitro antimalarial sensitivity assays with increasing potential for application in field sites. We examined in vitro susceptibility to seven anti-malarial drugs for 40 fresh P. falciparum field isolates via a flow cytometry method (FCM), a colorimetric LDH-based ELISA (DELI), and standard microscopic slide analysis of growth. For FCM, 184/280 (66%) assays met analytical acceptance criteria, compared to 166/280 (59%) for DELI. There was good agreement between FCM and microscopy, while DELI tended to produce higher half-maximal inhibition constants (IC50s) than FCM, with an overall bias of 2.2-fold (Bland-Altman comparison). Values for artesunate and dihydroartemisinin were most affected. Paradoxical increases in signal at very high concentrations of mefloquine and related compounds were more marked with the DELI assay, suggesting that off-target effects on LDH production may be responsible. Loss of FCM signal due to reinvasion or slow growth was observed in a small number of samples. These results extend previous work on use of flow cytometry to determine antimalarial susceptibility in terms of the number of samples, range of drugs, and comparison with other methods. PMID:26269616

  12. Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth.

    PubMed

    Divo, A A; Geary, T G; Davis, N L; Jensen, J B

    1985-02-01

    Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640. PMID:3886898

  13. Evidence of promiscuous endothelial binding by Plasmodium falciparum-infected erythrocytes.

    PubMed

    Esser, Claudia; Bachmann, Anna; Kuhn, Daniela; Schuldt, Kathrin; Förster, Birgit; Thiel, Meike; May, Jürgen; Koch-Nolte, Friedrich; Yáñez-Mó, María; Sánchez-Madrid, Francisco; Schinkel, Alfred H; Jalkanen, Sirpa; Craig, Alister G; Bruchhaus, Iris; Horstmann, Rolf D

    2014-05-01

    The adhesion of infected red blood cells (iRBCs) to human endothelium is considered a key event in the pathogenesis of cerebral malaria and other life-threatening complications caused by the most prevalent malaria parasite Plasmodium falciparum. In the past 30 years, 14 endothelial receptors for iRBCs have been identified. Exposing 10 additional surface proteins of endothelial cells to a mixture of P.  falciparum isolates from three Ghanaian malaria patients, we identified seven new iRBC receptors, all expressed in brain vessels. This finding strongly suggests that endothelial binding of P.  falciparum iRBCs is promiscuous and may use a combination of endothelial surface moieties.

  14. A Unique Plasmodium falciparum K13 Gene Mutation in Northwest Ethiopia.

    PubMed

    Bayih, Abebe Genetu; Getnet, Gebeyaw; Alemu, Abebe; Getie, Sisay; Mohon, Abu Naser; Pillai, Dylan R

    2016-01-01

    Artemisinin combination therapy (ACT) is the first line to treat uncomplicated Plasmodium falciparum malaria worldwide. Artemisinin treatment failures are on the rise in southeast Asia. Delayed parasite clearance after ACT is associated with mutations of the P. falciparum kelch 13 gene. Patients (N = 148) in five districts of northwest Ethiopia were enrolled in a 28-day ACT trial. We identified a unique kelch 13 mutation (R622I) in 3/125 (2.4%) samples. The three isolates with R622I were from Negade-Bahir and Aykel districts close to the Ethiopia-Sudan border. One of three patients with the mutant strain was parasitemic at day 3; however, all patients cleared parasites by day 28. Correlation between kelch 13 mutations and parasite clearance was not possible due to the low frequency of mutations in this study. PMID:26483118

  15. Plasmodium falciparum Polymorphisms associated with ex vivo drug susceptibility and clinical effectiveness of artemisinin-based combination therapies in Benin.

    PubMed

    Dahlström, Sabina; Aubouy, Agnès; Maïga-Ascofaré, Oumou; Faucher, Jean-François; Wakpo, Abel; Ezinmègnon, Sèm; Massougbodji, Achille; Houzé, Pascal; Kendjo, Eric; Deloron, Philippe; Le Bras, Jacques; Houzé, Sandrine

    2014-01-01

    Artemisinin-based combination therapies (ACTs) are the main option to treat malaria, and their efficacy and susceptibility must be closely monitored to avoid resistance. We assessed the association of Plasmodium falciparum polymorphisms and ex vivo drug susceptibility with clinical effectiveness. Patients enrolled in an effectiveness trial comparing artemether-lumefantrine (n = 96), fixed-dose artesunate-amodiaquine (n = 96), and sulfadoxine-pyrimethamine (n = 48) for the treatment of uncomplicated malaria 2007 in Benin were assessed. pfcrt, pfmdr1, pfmrp1, pfdhfr, and pfdhps polymorphisms were analyzed pretreatment and in recurrent infections. Drug susceptibility was determined in fresh baseline isolates by Plasmodium lactate dehydrogenase enzyme-linked immunosorbent assay (ELISA). A majority had 50% inhibitory concentration (IC50) estimates (the concentration required for 50% growth inhibition) lower than those of the 3D7 reference clone for desethylamodiaquine, lumefantrine, mefloquine, and quinine and was considered to be susceptible, while dihydroartemisinin and pyrimethamine IC50s were higher. No association was found between susceptibility to the ACT compounds and treatment outcome. Selection was observed for the pfmdr1 N86 allele in artemether-lumefantrine recrudescences (recurring infections) (4/7 [57.1%] versus 36/195 [18.5%]), and of the opposite allele, 86Y, in artesunate-amodiaquine reinfections (new infections) (20/22 [90.9%] versus 137/195 [70.3%]) compared to baseline infections. The importance of pfmdr1 N86 in lumefantrine tolerance was emphasized by its association with elevated lumefantrine IC50s. Genetic linkage between N86 and Y184 was observed, which together with the low frequency of 1246Y may explain regional differences in selection of pfmdr1 loci. Selection of opposite alleles in artemether-lumefantrine and artesunate-amodiaquine recurrent infections supports the strategy of multiple first-line treatment. Surveillance based on clinical, ex

  16. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes

    PubMed Central

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A.; Sauerwein, Robert W.

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  17. Allelic Diversity of MSP1 Gene in Plasmodium falciparum from Rural and Urban Areas of Gabon.

    PubMed

    Mawili-Mboumba, Denise Patricia; Mbondoukwe, Noé; Adande, Elvire; Bouyou-Akotet, Marielle Karine

    2015-08-01

    The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families' frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (±0.95) higher compared to that found at Owendo (1.55±0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.

  18. Analyses of genetic variations at microsatellite loci present in-and-around the Pfcrt gene in Indian Plasmodium falciparum.

    PubMed

    Chauhan, Kshipra; Pande, Veena; Das, Aparup

    2013-12-01

    Evolution and spread of chloroquine resistant (CQR) malaria parasite Plasmodium falciparum have posed great threat in malaria intervention across the globe. The occurrence of K76T mutation in the P. falciparum chloroquine resistance transporter (pfcrt) gene has been widely attributed to CQR with four neighboring mutations providing compensatory fitness benefit to the parasite survival. Understanding evolutionary patterns of the pfcrt gene is of great relevance not only for devising new malaria control measures but also could serve as a model to understand evolution and spread of other human drug-resistant pathogens. Several studies, mainly based on differential patterns of diversities of the microsatellite loci placed in-and-around the pfcrt gene have indicated the role of positive natural selection under the 'hitchhiking' model of molecular evolution. However, the studies were restricted to limited number of microsatellite loci present inside the pfcrt gene. Moreover, comparatively higher level of diversities in microsatellite loci present inside the pfcrt gene than the loci flanking the pfcrt gene are hallmarks of Indian P. falciparum, presenting contrasting evolutionary models to global isolates. With a view to infer evolutionary patterns of the pfcrt gene in Indian P. falciparum, we have adopted a unique sampling scheme of two types of populations (cultured and field collected) and utilized 20 polymorphic microsatellite loci (16 located inside the pfcrt gene and four in the two flanking regions) to disentangle between genetic drift (inbred cultured isolates) and natural selection (field isolates). Data analyses employing different population genetic tests could not straightforwardly explain either the model invoking 'genetic hitchhiking' or 'genetic drift'. However, complex evolutionary models influenced by both demography and natural selection or an alternative model of natural selection (e.g. diversifying/balancing selection) might better explain the observed

  19. Regular production of infective sporozoites of Plasmodium falciparum and P. vivax in laboratory-bred Anopheles albimanus.

    PubMed

    Hurtado, S; Salas, M L; Romero, J F; Zapata, J C; Ortiz, H; Arevalo-Herrera, M; Herrera, S

    1997-01-01

    One of the major constraints for studies on the sporogonic cycle of the parasites causing human malaria, and on the protective efficacy of pre-erythrocytic vaccines, is the scarcity of laboratory-reared Anopheles mosquitoes as a source of infective sporozoites. The aim of the present study was to reproduce the life-cycles of Plasmodium falciparum and P. vivax in the laboratory and so develop the ability to produce infective sporozoites of these two species regularly under laboratory conditions. Colonized Anopheles albimanus, of Buenaventura and Tecojate strains, were infected by feeding either on Plasmodium-infected blood, from human patients or experimentally inoculated Aotus monkeys, or on gametocytes of the P. falciparum NF-54 isolate grown in vitro. The monkeys were infected with the blood stages of a Colombian P. vivax isolate and then, after recovery, with the Santa Lucia strain of P. falciparum from El Salvador. Although both of the mosquito strains used were successfully infected with both parasite species, the Buenaventura strain of mosquito was generally more susceptible to infection than the Tecojate strain, and particularly to infection with the parasites from the patients, who lived where this strain of mosquitoes was originally isolated. Monkeys injected intravenously with the P. vivax sporozoites produced in the mosquitoes developed patent sexual and asexual parasitaemias; the gametocytes that developed could then be used to infect mosquitoes, allowing the development of more sporozoites. However, experimental infections failed to establish after the P. falciparum sporozoites were used to inoculate monkeys. The ability to reproduce the complete life cycle of P. vivax in the laboratory, from human to mosquito and then to monkey, should greatly facilitate many studies on vivax malaria and on the efficacy of candidate malaria vaccines. The availability of the sporogonic cycles of P. falciparum from three different sources should also permit a variety of

  20. On the diversity of malaria parasites in African apes and the origin of Plasmodium falciparum from Bonobos.

    PubMed

    Krief, Sabrina; Escalante, Ananias A; Pacheco, M Andreina; Mugisha, Lawrence; André, Claudine; Halbwax, Michel; Fischer, Anne; Krief, Jean-Michel; Kasenene, John M; Crandfield, Mike; Cornejo, Omar E; Chavatte, Jean-Marc; Lin, Clara; Letourneur, Franck; Grüner, Anne Charlotte; McCutchan, Thomas F; Rénia, Laurent; Snounou, Georges

    2010-02-12

    The origin of Plasmodium falciparum, the etiological agent of the most dangerous forms of human malaria, remains controversial. Although investigations of homologous parasites in African Apes are crucial to resolve this issue, studies have been restricted to a chimpanzee parasite related to P. falciparum, P. reichenowi, for which a single isolate was available until very recently. Using PCR amplification, we detected Plasmodium parasites in blood samples from 18 of 91 individuals of the genus Pan, including six chimpanzees (three Pan troglodytes troglodytes, three Pan t. schweinfurthii) and twelve bonobos (Pan paniscus). We obtained sequences of the parasites' mitochondrial genomes and/or from two nuclear genes from 14 samples. In addition to P. reichenowi, three other hitherto unknown lineages were found in the chimpanzees. One is related to P. vivax and two to P. falciparum that are likely to belong to distinct species. In the bonobos we found P. falciparum parasites whose mitochondrial genomes indicated that they were distinct from those present in humans, and another parasite lineage related to P. malariae. Phylogenetic analyses based on this diverse set of Plasmodium parasites in African Apes shed new light on the evolutionary history of P. falciparum. The data suggested that P. falciparum did not originate from P. reichenowi of chimpanzees (Pan troglodytes), but rather evolved in bonobos (Pan paniscus), from which it subsequently colonized humans by a host-switch. Finally, our data and that of others indicated that chimpanzees and bonobos maintain malaria parasites, to which humans are susceptible, a factor of some relevance to the renewed efforts to eradicate malaria.

  1. Various pfcrt and pfmdr1 genotypes of Plasmodium falciparum cocirculate with P. malariae, P. ovale spp., and P. vivax in northern Angola.

    PubMed

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

    2012-10-01

    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.

  2. The paradoxical population genetics of Plasmodium falciparum.

    PubMed

    Hartl, Daniel L; Volkman, Sarah K; Nielsen, Kaare M; Barry, Alyssa E; Day, Karen P; Wirth, Dyann F; Winzeler, Elizabeth A

    2002-06-01

    Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies. PMID:12036741

  3. Plasma glutamine levels and falciparum malaria.

    PubMed

    Cowan, G; Planche, T; Agbenyega, T; Bedu-Addo, G; Owusu-Ofori, A; Adebe-Appiah, J; Agranoff, D; Woodrow, C; Castell, L; Elford, B; Krishna, S

    1999-01-01

    Glutamine deficiency is associated with increased rates of sepsis and mortality, which can be prevented by glutamine supplementation. Changes in glutamine concentration were examined in Ghanaian children with acute falciparum malaria and control cases. The mean (SD) plasma glutamine concentration was lower in patients with acute malaria (401 (82) mumol/L, n = 50) than in control patients (623 (67) mumol/L, n = 7; P < 0.001). Plasma glutamine concentrations all rose in convalescence. The mean (SD) increase in plasma glutamine was 202 (123) mumol/L (n = 18; P < 0.001) compared with acute infection. We conclude that acute falciparum malaria is associated with large decreases in plasma glutamine and these falls may increase susceptibility to sepsis and dyserythropoeisis.

  4. Acute renal failure due to falciparum malaria.

    PubMed

    Habte, B

    1990-01-01

    Seventy-two patients with severe falciparum malaria are described. Twenty-four (33.3%) were complicated by acute renal failure. Comparing patients with renal failure and those without, statistically significant differences occurred regarding presence of cerebral malaria (83% vs 46%), jaundice (92% vs 33%), and death (54% vs 17%). A significantly higher number of patients with renal failure were nonimmune visitors to malaria endemic regions. Renal failure was oliguric in 45% of cases. Dialysis was indicated in 38%, 29% died in early renal failure, and 33% recovered spontaneously. It is concluded that falciparum malaria is frequently complicated by cerebral malaria and renal failure. As nonimmune individuals are prone to develop serious complications, malaria prophylaxis and vigorous treatment of cases is mandatory. PMID:2236718

  5. The paradoxical population genetics of Plasmodium falciparum.

    PubMed

    Hartl, Daniel L; Volkman, Sarah K; Nielsen, Kaare M; Barry, Alyssa E; Day, Karen P; Wirth, Dyann F; Winzeler, Elizabeth A

    2002-06-01

    Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies.

  6. Plasmodium falciparum Na+/H+ Exchanger 1 Transporter Is Involved in Reduced Susceptibility to Quinine ▿

    PubMed Central

    Henry, Maud; Briolant, Sébastien; Zettor, Agnès; Pelleau, Stéphane; Baragatti, Meili; Baret, Eric; Mosnier, Joel; Amalvict, Rémy; Fusai, Thierry; Rogier, Christophe; Pradines, Bruno

    2009-01-01

    Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC50s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC50 of QN (P = 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC50 of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC50 of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC50 of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC50 of QN (P = 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC50 of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates. PMID:19273668

  7. Activity of a combination of three cinchona bark alkaloids against Plasmodium falciparum in vitro.

    PubMed

    Druilhe, P; Brandicourt, O; Chongsuphajaisiddhi, T; Berthe, J

    1988-02-01

    In vitro studies with quinine, quinidine, cinchonine, and cinchonidine showed that despite a similarity of chemical structure, the effectiveness of these cinchona bark alkaloids against several culture lines of Plasmodium falciparum varied widely. Depending on the strain tested, quinidine and cinchonine were 1 to 10 and 1 to 5 times, respectively, more active than quinine. A combination made of equal parts of quinine, quinidine, and cinchonine was found to have several interesting features; it had activity similar to that of quinine against quinine-susceptible strains but was found to be 2 to 10 times more effective against strains resistant to quinine and had a more consistent effect than any of the alkaloids used singly. The potentiation was found to depend mainly on the presence of cinchonine in the mixtures studied. Synergism was also confirmed in a study of 25 P. falciparum strains isolated from Thai patients. Combinations of cinchona bark alkaloids could thus be of interest in areas where P. falciparum is becoming less susceptible to quinine.

  8. Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in West Africa

    PubMed Central

    2012-01-01

    Background Malaria parasite population genetic structure varies among areas of differing endemicity, but this has not been systematically studied across Plasmodium falciparum populations in Africa where most infections occur. Methods Ten polymorphic P. falciparum microsatellite loci were genotyped in 268 infections from eight locations in four West African countries (Republic of Guinea, Guinea Bissau, The Gambia and Senegal), spanning a highly endemic forested region in the south to a low endemic Sahelian region in the north. Analysis was performed on proportions of mixed genotype infections, genotypic diversity among isolates, multilocus standardized index of association, and inter-population differentiation. Results Each location had similar levels of pairwise genotypic diversity among isolates, although there were many more mixed parasite genotype infections in the south. Apart from a few isolates that were virtually identical, the multilocus index of association was not significant in any population. Genetic differentiation between populations was low (most pairwise FST values < 0.03), and an overall test for isolation by distance was not significant. Conclusions Although proportions of mixed genotype infections varied with endemicity as expected, population genetic structure was similar across the diverse sites. Very substantial reduction in transmission would be needed to cause fragmented or epidemic sub-structure in this region. PMID:22759447

  9. Imputation-Based Population Genetics Analysis of Plasmodium falciparum Malaria Parasites

    PubMed Central

    Samad, Hanif; Coll, Francesc; Preston, Mark D.; Ocholla, Harold; Fairhurst, Rick M.; Clark, Taane G.

    2015-01-01

    Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima’s D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and

  10. Imputation-based population genetics analysis of Plasmodium falciparum malaria parasites.

    PubMed

    Samad, Hanif; Coll, Francesc; Preston, Mark D; Ocholla, Harold; Fairhurst, Rick M; Clark, Taane G

    2015-04-01

    Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86 k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima's D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and

  11. Imputation-based population genetics analysis of Plasmodium falciparum malaria parasites.

    PubMed

    Samad, Hanif; Coll, Francesc; Preston, Mark D; Ocholla, Harold; Fairhurst, Rick M; Clark, Taane G

    2015-04-01

    Whole-genome sequencing technologies are being increasingly applied to Plasmodium falciparum clinical isolates to identify genetic determinants of malaria pathogenesis. However, genome-wide discovery methods, such as haplotype scans for signatures of natural selection, are hindered by missing genotypes in sequence data. Poor correlation between single nucleotide polymorphisms (SNPs) in the P. falciparum genome complicates efforts to apply established missing-genotype imputation methods that leverage off patterns of linkage disequilibrium (LD). The accuracy of state-of-the-art, LD-based imputation methods (IMPUTE, Beagle) was assessed by measuring allelic r2 for 459 P. falciparum samples from malaria patients in 4 countries: Thailand, Cambodia, Gambia, and Malawi. In restricting our analysis to 86 k high-quality SNPs across the populations, we found that the complete-case analysis was restricted to 21k SNPs (24.5%), despite no single SNP having more than 10% missing genotypes. The accuracy of Beagle in filling in missing genotypes was consistently high across all populations (allelic r2, 0.87-0.96), but the performance of IMPUTE was mixed (allelic r2, 0.34-0.99) depending on reference haplotypes and population. Positive selection analysis using Beagle-imputed haplotypes identified loci involved in resistance to chloroquine (crt) in Thailand, Cambodia, and Gambia, sulfadoxine-pyrimethamine (dhfr, dhps) in Cambodia, and artemisinin (kelch13) in Cambodia. Tajima's D-based analysis identified genes under balancing selection that encode well-characterized vaccine candidates: apical merozoite antigen 1 (ama1) and merozoite surface protein 1 (msp1). In contrast, the complete-case analysis failed to identify any well-validated drug resistance or candidate vaccine loci, except kelch13. In a setting of low LD and modest levels of missing genotypes, using Beagle to impute P. falciparum genotypes is a viable strategy for conducting accurate large-scale population genetics and

  12. Sterile Protective Immunity to Malaria is Associated with a Panel of Novel P. falciparum Antigens*

    PubMed Central

    Trieu, Angela; Kayala, Matthew A.; Burk, Chad; Molina, Douglas M.; Freilich, Daniel A.; Richie, Thomas L.; Baldi, Pierre; Felgner, Philip L.; Doolan, Denise L.

    2011-01-01

    The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized. PMID:21628511

  13. Tracing the origins and signatures of selection of antifolate resistance in island populations of Plasmodium falciparum

    PubMed Central

    2010-01-01

    Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes. Methods Blood samples were collected in July and December 2004 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions. Results An increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci. Conclusions This study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal. PMID:20534146

  14. Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

    PubMed Central

    Coronado, Lorena Michelle; Tayler, Nicole Michelle; Correa, Ricardo; Giovani, Rita Marissa; Spadafora, Carmenza

    2013-01-01

    Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ≈40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte. Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite. Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing. PMID:23486405

  15. Lack of artemisinin resistance in Plasmodium falciparum in northwest Benin after 10 years of use of artemisinin-based combination therapy

    PubMed Central

    Ogouyèmi-Hounto, Aurore; Damien, Georgia; Deme, Awa Bineta; Ndam, Nicaise T.; Assohou, Constance; Tchonlin, Didier; Mama, Atika; Hounkpe, Virgile Olivier; Moutouama, Jules Doumitou; Remoué, Franck; Ndiaye, Daouda; Gazard, Dorothée Kinde

    2016-01-01

    Aim: In Benin, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment for uncomplicated Plasmodium falciparum malaria since 2004. The emergence in Southeast Asia of parasites that are resistant to artemisinins poses a serious threat to global control of this disease. The presence of artemisinin resistance genotypes in parasite populations in Benin is currently unknown. The present study investigated the prevalence of relevant K13-propeller gene polymorphisms in parasite isolates from the north-western region of Benin. Method: Plasmodium falciparum isolates were collected from children with a confirmed diagnosis of malaria aged 6 months to 5 years in two towns, Cobly and Djougou, in the north-western part of Benin. The study was conducted during the rainy season from July to November 2014 in local health facilities. The K13-propeller gene was amplified in parasite isolates using nested PCR and subsequently sequenced. Results: A total of 108 children were recruited into the study. The efficiency of amplification reactions was 72% (78/108). The propeller domain of the K13 gene was successfully sequenced in 78 P. falciparum isolates; all of them were wild type with no polymorphisms detectable. Conclusion: The absence of mutations in the K13 gene indicates that P. falciparum parasite populations in the study area are still fully susceptible to artemisinins. PMID:27443837

  16. Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

    PubMed Central

    Perrin, L H; Ramirez, E; Er-Hsiang, L; Lambert, P H

    1980-01-01

    Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis. Images Fig. 2 PMID:6160002

  17. Plasmodium vivax and Plasmodium falciparum at the Crossroads of Exchange among Islands in Vanuatu: Implications for Malaria Elimination Strategies

    PubMed Central

    Chan, Chim W.; Sakihama, Naoko; Tachibana, Shin-Ichiro; Idris, Zulkarnain Md; Lum, J. Koji; Kaneko, Akira

    2015-01-01

    Understanding the transmission and movement of Plasmodium parasites is crucial for malaria elimination and prevention of resurgence. Located at the limit of malaria transmission in the Pacific, Vanuatu is an ideal candidate for elimination programs due to low endemicity and the isolated nature of its island setting. We analyzed the variation in the merozoite surface protein 1 (msp1) and the circumsporozoite protein (csp) of P. falciparum and P. vivax populations to examine the patterns of gene flow and population structures among seven sites on five islands in Vanuatu. Genetic diversity was in general higher in P. vivax than P. falciparum from the same site. In P. vivax, high genetic diversity was likely maintained by greater extent of gene flow among sites and among islands. Consistent with the different patterns of gene flow, the proportion of genetic variance found among islands was substantially higher in P. falciparum (28.81–31.23%) than in P. vivax (-0.53–3.99%). Our data suggest that the current island-by-island malaria elimination strategy in Vanuatu, while adequate for P. falciparum elimination, might need to be complemented with more centrally integrated measures to control P. vivax movement across islands. PMID:25793260

  18. A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

    PubMed Central

    Ardeshir, F; Flint, J E; Richman, S J; Reese, R T

    1987-01-01

    Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:3556166

  19. Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT).

    PubMed

    Callaghan, Paul S; Hassett, Matthew R; Roepe, Paul D

    2015-08-18

    At least 53 distinct isoforms of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein are expressed in strains or isolates of P. falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQR(CS)) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT-mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities, we recently optimized a convenient method for measuring PfCRT-mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 of 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.

  20. Enhancing Blockade of Plasmodium falciparum Erythrocyte Invasion: Assessing Combinations of Antibodies against PfRH5 and Other Merozoite Antigens

    PubMed Central

    Miura, Kazutoyo; Illingworth, Joseph J.; Choudhary, Prateek; Murungi, Linda M.; Furze, Julie M.; Diouf, Ababacar; Miotto, Olivo; Crosnier, Cécile; Wright, Gavin J.; Kwiatkowski, Dominic P.; Fairhurst, Rick M.; Long, Carole A.; Draper, Simon J.

    2012-01-01

    No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines. PMID:23144611

  1. Fresh, Rayed Impact Crater

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-416, 9 July 2003

    This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a fresh, young meteor impact crater on the martian surface. It is less than 400 meters (less than 400 yards) across. While there is no way to know the exact age of this or any other martian surface feature, the rays are very well preserved. On a planet where wind can modify surface features at the present time, a crater with rayed ejecta patterns must be very young indeed. Despite its apparent youth, the crater could still be many hundreds of thousands, if not several million, of years old. This impact scar is located within the much larger Crommelin Crater, near 5.6oN, 10.0oW. Sunlight illuminates the scene from the left.

  2. Sickle cell trait carriage: imbalanced distribution of IgG subclass antibodies reactive to Plasmodium falciparum family-specific MSP2 peptides in serum samples from Gabonese children.

    PubMed

    Ntoumi, Francine; Ekala, Marie-Thérése; Makuwa, Maria; Lekoulou, Faustin; Mercereau-Puijalon, Odile; Deloron, Philippe

    2002-10-21

    Several mechanisms have been proposed for explaining the protection of young children with hemoglobin AS from severe Plasmodium falciparum malaria. In a previous study carried out in Gabon, we have shown an association between hemoglobin AS carriage and a greater P. falciparum infection complexity. In the present study, we have investigated the presence and fine specificity of merozoite surface protein 2 (MSP2) reactive antibodies using different peptides covering conserved and polymorphic regions (Blocks 1-3) of P. falciparum MSP2 molecules. A cross-sectional study was conducted in the city of Bakoumba (Gabon), where malaria is hyperendemic with perennial P. falciparum transmission. Among the 641 children included, 135 were heterozygous for the sickle cell trait (HbAS). There was no significant difference in age distribution (mean age: 5 years, 0.5-11 years) and sex ratio in both hemoglobin groups (HbAA vs. HbAS). Blood group O was, however, associated with the sickle cell trait (P=0.02). P. falciparum isolates obtained from children with HbAS had a trend to higher infection complexity before the age of 5 years. Plasma samples were tested for the presence of antibodies to the different MSP2 peptides. Total IgG antibodies with a predominant reactivity against the FC27 type (the predominant P. falciparum MSP2 genotype) were found in serum samples from both groups. The profile of the IgG subclasses varied according to the hemoglobin phenotype. IgG3 and IgG2 were predominantly detected in plasma samples from HbAS children, whereas mainly IgG3 was found in children with HbAA. The role of the high multiclonal carriage associated with high family-specific antibodies reactive to MSP2 in HbAS children with asymptomatic P. falciparum parasitism is discussed.

  3. 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

  4. 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.

  5. Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

    PubMed

    Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra

    2012-01-01

    We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards.

  6. Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria

    PubMed Central

    Pain, Arnab; Ferguson, David J. P.; Kai, Oscar; Urban, Britta C.; Lowe, Brett; Marsh, Kevin; Roberts, David J.

    2001-01-01

    Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria. PMID:11172032

  7. Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax

    PubMed Central

    Talundzic, Eldin; Chenet, Stella M.; Goldman, Ira F.; Patel, Dhruviben S.; Nelson, Julia A.; Plucinski, Mateusz M.; Barnwell, John W.; Udhayakumar, Venkatachalam

    2015-01-01

    Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species. PMID:26292024

  8. Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax.

    PubMed

    Talundzic, Eldin; Chenet, Stella M; Goldman, Ira F; Patel, Dhruviben S; Nelson, Julia A; Plucinski, Mateusz M; Barnwell, John W; Udhayakumar, Venkatachalam

    2015-01-01

    Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species. PMID:26292024

  9. Fresh Veggies from Space

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Professor Marc Anderson of the University of Wisconsin-Madison developed a technology for use in plant-growth experiments aboard the Space Shuttle. Anderson's research and WCSAR's technology were funded by NASA and resulted in a joint technology licensed to KES Science and Technology, Inc. This transfer of space-age technology resulted in the creation of a new plant-saving product, an ethylene scrubber for plant growth chambers. This innovation presents commercial benefits for the food industry in the form of a new device, named Bio-KES. Bio-KES removes ethylene and helps to prevent spoilage. Ethylene accounts for up to 10 percent of produce losses and 5 percent of flower losses. Using Bio-KES in storage rooms and displays will increase the shelf life of perishable foods by more than one week, drastically reducing the costs associated with discarded rotten foods and flowers. The savings could potentially be passed on to consumers. For NASA, the device means that astronauts can conduct commercial agricultural research in space. Eventually, it may also help to grow food in space and keep it fresh longer. This could lead to less packaged food being taken aboard missions since it could be cultivated in an ethylene-free environment.

  10. Microbial safety of fresh produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The book entitled “Microbial Safety of Fresh Produce” with 23 chapters is divided into following six sections: Microbial contamination of fresh produce, Pre-harvest strategies, post-harvest interventions, Produce safety during processing and handling, Public, legal, and economic Perspectives, and Re...

  11. Campylobacter and Salmonella contaminating fresh chicken meat.

    PubMed

    Geilhausen, B; Schütt-Gerowitt, H; Aleksic, S; Koenen, R; Mauff, G; Pulverer, G

    1996-07-01

    1853 packages of fresh chicken breast meat of German, Dutch and French origin were investigated for their contamination with Campylobacter and/or Salmonella. Swabs were taken and cultured from dripwater, meat surface, meat interior and packet bowl. Campylobacter was isolated from 619 meat samples (= 33%), Salmonella from 377 meat packages (= 20%). In 111 of these contaminated chicken samples, both Salmonella and Campylobacter were present. The contamination rate and the species spectrum observed differed depending on the origin of the packages and the time of control.

  12. A bedside dipstick method to detect Plasmodium falciparum.

    PubMed

    Shah, Ira; Deshmukh, C T

    2004-11-01

    We conducted this study to determine efficacy of Parasight-F (an HRP-II antigen dipstick method to detect P. Falciparum) in children. A total of 30 children were enrolled in the age group of 2 months to 12 years whose peripheral smear showed asexual forms of Plasmodium falciparum. All patients were tested for presence of HRP-II antigen of Plasmodium falciparum in their blood by the Parasight-F dipstick test by either an EDTA sample or a finger prick blood sample. The sensitivity of Parasight-F was 83.3 % However, the sensitivity of Parasight-F to detect Plasmodium Falciparum in case of mixed Plasmodium (Vivax + Falciparum) infection was only 25 %. Also, all patients less than 6 months of age had a negative Parasight-F test. Parasitic index, prior treatment with antimalarials or severity of Falciparum malaria have no effect on the sensitivity of Parasight-F test. We conclude that Parasight-F is an effective tool for diagnosis of Plasmoduim falciparum malaria in children. PMID:15591666

  13. Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah, East Malaysia

    PubMed Central

    Mohd Abd Razak, Mohd Ridzuan; Sastu, Umi Rubiah; Norahmad, Nor Azrina; Abdul-Karim, Abass; Muhammad, Amirrudin; Muniandy, Prem Kumar; Jelip, Jenarun; Rundi, Christina; Imwong, Mallika; Mudin, Rose Nani; Abdullah, Noor Rain

    2016-01-01

    Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751–800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651–700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0

  14. Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah, East Malaysia.

    PubMed

    Mohd Abd Razak, Mohd Ridzuan; Sastu, Umi Rubiah; Norahmad, Nor Azrina; Abdul-Karim, Abass; Muhammad, Amirrudin; Muniandy, Prem Kumar; Jelip, Jenarun; Rundi, Christina; Imwong, Mallika; Mudin, Rose Nani; Abdullah, Noor Rain

    2016-01-01

    Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751-800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651-700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0

  15. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    NASA Astrophysics Data System (ADS)

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2014-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (`K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

  16. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2016-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. PMID:24352242

  17. Polycyclic amines as chloroquine resistance modulating agents in Plasmodium falciparum.

    PubMed

    Joubert, Jacques; Kapp, Erika; Taylor, Dale; Smith, Peter J; Malan, Sarel F

    2016-02-15

    Pentacycloundecylamines (PCUs) and adamantane amines, such as NGP1-01 (1) and amantadine, have shown significant channel blocking activities. They are postulated to act as chemosensitizers and circumvent the resistance of the plasmodia parasite against chloroquine (CQ) by inhibiting the p-glycoprotein efflux pump and enabling the accumulation of CQ inside the parasite digestive vacuole. Twelve polycyclic amines containing either a PCU or adamantane amine moiety conjugated to different aromatic functionalities through various tethered linkers were selected based on their channel blocking abilities and evaluated as potential chemosensitizers. Compounds 2, 4, 5 and 10 showed significant voltage-gated calcium channel (VGCC) blocking ability (IC50=0.27-35 μM) and were able to alter the CQ IC50 in differing degrees (45-81%) in the multidrug resistant Plasmodium falciparum Dd2 isolate. Among them, the PCU-dansyl amine compound (4) displayed the best potential to act as a chemosensitizer against the Dd2 strain at a 1 μM concentration (RMI=0.19) while displaying moderate antiplasmodial activity (Dd2 IC50=6.25 μM) and low in vitro cytotoxicity against a mammalian cell line (CHO, IC50=119 μM). Compounds 2 and 10 also showed some promising chemosensitizing abilities (RMI=0.36 and 0.35 respectively). A direct correlation was found between the VGCC blocking ability of these polycyclic amines and their capacity to act as CQ resistance modulating agents.

  18. Virulence and transmission success of the malarial parasite Plasmodium falciparum

    PubMed Central

    Hayward, Rhian E.; Tiwari, Bela; Piper, Karen P.; Baruch, Dror I.; Day, Karen P.

    1999-01-01

    Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host–parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages. PMID:10200302

  19. Falciparum malaria-induced hypoglycaemia in a diabetic patient.

    PubMed Central

    Shalev, O.; Tsur, A.; Rahav, G.

    1992-01-01

    We report a patient with diabetes mellitus who suffered severe falciparum malaria complicated by profound and persistent hypoglycaemia. The hypoglycaemia evolved before therapy with quinine was begun and resolved with eradication of the parasitaemia. The patient reverted to her baseline hyperglycaemia despite continuation of quinine. This case illustrates the critical role of falciparum malaria in the pathogenesis of malaria-associated hypoglycaemia, rather than quinine-mediated mechanisms. Anticipation of hypoglycaemia in falciparum malaria and its vigorous treatment may improve the poor prognosis associated with this complication. PMID:1409194

  20. Immune activation during cerebellar dysfunction following Plasmodium falciparum malaria.

    PubMed

    de Silva, H J; Hoang, P; Dalton, H; de Silva, N R; Jewell, D P; Peiris, J B

    1992-01-01

    Evidence for immune activation was investigated in 12 patients with a rare syndrome of self-limiting, delayed onset cerebellar dysfunction following an attack of falciparum malaria which occurred 18-26 d previously. Concentrations of tumour necrosis factor, interleukin 6 and interleukin 2 were all significantly higher in serum samples of patients during cerebellar ataxia than in recovery sera and in the sera of 8 patients who did not develop delayed cerebellar dysfunction following an attack of falciparum malaria. Cytokine concentrations in the cerebrospinal fluid were also significantly higher in ataxic patients than in controls. These findings suggest that immunological mechanisms may play a role in delayed cerebellar dysfunction following falciparum malaria.

  1. Type of in vitro cultivation influences cytoadhesion, knob structure, protein localization and transcriptome profile of Plasmodium falciparum

    PubMed Central

    Tilly, Ann-Kathrin; Thiede, Jenny; Metwally, Nahla; Lubiana, Pedro; Bachmann, Anna; Roeder, Thomas; Rockliffe, Nichola; Lorenzen, Stephan; Tannich, Egbert; Gutsmann, Thomas; Bruchhaus, Iris

    2015-01-01

    In vitro cultivation of Plasmodium falciparum is critical for studying the biology of this parasite. However, it is likely that different in vitro cultivation conditions influence various aspects of the parasite’s life cycle. In the present study two P. falciparum isolates were cultivated using the two most common methods, in which AlbuMAX or human serum as additives are used, and the results were compared. The type of cultivation influenced the knob structure of P. falciparum-infected erythrocytes (IEs). IEs cultivated with AlbuMAX had fewer knobs than those cultivated with human serum. Furthermore, knob size varied between isolates and is also depended on the culture medium. In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum. Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs. Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX. Moreover, the type of cultivation had a marked influence on the transcriptome profile. Compared with cultivation with human serum, cultivation with AlbuMAX increased the expression of approximately 500–870 genes. PMID:26568166

  2. Severe Plasmodium falciparum infection mimicking acute myocardial infarction.

    PubMed

    Sulaiman, Helmi; Ismail, Muhammad Dzafir; Jalalonmuhali, Maisarah; Atiya, Nadia; Ponnampalavanar, Sasheela

    2014-08-30

    This case report describes a case of presumed acute myocardial infarction in a returned traveler who was later diagnosed to have severe malaria. Emergency coronary angiography was normal and subsequent peripheral blood film was positive for Plasmodium falciparum.

  3. Characterization of a 225 kilodalton rhoptry protein of Plasmodium falciparum.

    PubMed

    Roger, N; Dubremetz, J F; Delplace, P; Fortier, B; Tronchin, G; Vernes, A

    1988-01-15

    A monoclonal antibody (24C6 4F12) raised against Plasmodium falciparum culture supernatant antigens gave a multiple dot picture on schizonts when assayed by immunofluorescence on P. falciparum erythrocytic stages. The corresponding antigen was localized in the peduncle of rhoptries by immunoelectronmicroscopy. On Western blots of P. falciparum schizonts, a major antigen of 225 kDa and a minor one of 240 kDa were recognized by this McAb. Pulse chase analysis of [35S]methionine biosynthetic labeling of P. falciparum culture demonstrated that the 240 kDa molecule was the precursor of the 225 kDa and that its processing occurred between 0 and 4 h after synthesis. Biosynthesis of the 240-225 kDa antigen occurred only during schizogony. PMID:3278223

  4. Steroidal saponins from fresh stems of Dracaena angustifolia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six new steroidal saponins (1-6), angudracanosides A-F, were isolated from fresh stems of Dracaena angustifolia, together with eight known compounds. The structures of compounds 1-6 were determined by detailed spectroscopic analyses and chemical methods. Antifungal testing of all compounds showed th...

  5. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been ...

  6. Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

    PubMed Central

    Day, K P; Karamalis, F; Thompson, J; Barnes, D A; Peterson, C; Brown, H; Brown, G V; Kemp, D J

    1993-01-01

    Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria. Images Fig. 1 Fig. 2 Fig. 3 PMID:8367496

  7. Hemoglobinopathies: Slicing the Gordian Knot of Plasmodium falciparum Malaria Pathogenesis

    PubMed Central

    Taylor, Steve M.; Cerami, Carla; Fairhurst, Rick M.

    2013-01-01

    Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits—including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia—are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a “natural experiment” to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the “Gordian knot” of host and parasite

  8. Foods - fresh vs. frozen or canned

    MedlinePlus

    Frozen foods vs. fresh or canned; Fresh foods vs. frozen or canned; Frozen vegetables versus fresh ... a well-balanced diet. Many people wonder if frozen and canned vegetables are as healthy for you ...

  9. The Dynamics of Natural Plasmodium falciparum Infections

    PubMed Central

    Felger, Ingrid; Maire, Martin; Bretscher, Michael T.; Falk, Nicole; Tiaden, André; Sama, Wilson; Beck, Hans-Peter; Owusu-Agyei, Seth; Smith, Thomas A.

    2012-01-01

    Background Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. Methods An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. Results Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5–9 year old children (average duration 319 days, 95% confidence interval 318;320). Conclusions The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections. PMID:23029082

  10. Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010-2012.

    PubMed

    Baldeviano, G Christian; 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

    2015-05-01

    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.

  11. Microbiological status of fresh beef cuts.

    PubMed

    Stopforth, J D; Lopes, M; Shultz, J E; Miksch, R R; Samadpour, M

    2006-06-01

    Fresh beef samples (n = 1,022) obtained from two processing plants in the Midwest (July to December 2003) were analyzed for levels of microbial populations (total aerobic plate count, total coliform count, and Escherichia coli count) and for the presence or absence of E. coli O157:H7 and Salmonella. A fresh beef cut sample was a 360-g composite of 6-g portions excised from the surface of 60 individual representative cuts in a production lot. Samples of fresh beef cuts yielded levels of 4.0 to 6.2, 1.1 to 1.8, and 0.8 to 1.0 log CFU/g for total aerobic plate count, total coliform count, and E. coli count, respectively. There did not appear to be substantial differences or obvious trends in bacterial populations on different cuts. These data may be useful in establishing a baseline or a benchmark of microbiological levels of contamination of beef cuts. Mean incidence rates of E. coli O157:H7 and Salmonella on raw beef cuts were 0.3 and 2.2%, respectively. Of the 1,022 samples analyzed, cuts testing positive for E. coli O157:H7 included top sirloin butt (0.9%) and butt, ball tip (2.1%) and for Salmonella included short loins (3.4%), strip loins (9.6%), rib eye roll (0.8%), shoulder clod (3.4%), and clod, top blade (1.8%). These data provide evidence of noticeable incidence of pathogens on whole muscle beef and raise the importance of such contamination on product that may be mechanically tenderized. Levels of total aerobic plate count, total coliform count, and E. coli count did not (P > or = 0.05) appear to be associated with the presence of E. coli O157:H7 and Salmonella on fresh beef cuts. E. O157:H7 was exclusively isolated from cuts derived from the sirloin area of the carcass. Salmonella was exclusively isolated from cuts derived from the chuck, rib, and loin areas of the carcass. Results of this study suggest that contamination of beef cuts may be influenced by the region of the carcass from which they are derived.

  12. The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum.

    PubMed

    Cassera, María B; Gozzo, Fabio C; D'Alexandri, Fabio L; Merino, Emilio F; del Portillo, Hernando A; Peres, Valnice J; Almeida, Igor C; Eberlin, Marcos N; Wunderlich, Gerhard; Wiesner, Jochen; Jomaa, Hassan; Kimura, Emilia A; Katzin, Alejandro M

    2004-12-10

    Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.

  13. Postharvest treatments of fresh produce.

    PubMed

    Mahajan, P V; Caleb, O J; Singh, Z; Watkins, C B; Geyer, M

    2014-06-13

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  14. Postharvest treatments of fresh produce

    PubMed Central

    Mahajan, P. V.; Caleb, O. J.; Singh, Z.; Watkins, C. B.; Geyer, M.

    2014-01-01

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  15. Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains.

    PubMed

    Chugh, Monika; Scheurer, Christian; Sax, Sibylle; Bilsland, Elizabeth; van Schalkwyk, Donelly A; Wicht, Kathryn J; Hofmann, Natalie; Sharma, Anil; Bashyam, Sridevi; Singh, Shivendra; Oliver, Stephen G; Egan, Timothy J; Malhotra, Pawan; Sutherland, Colin J; Beck, Hans-Peter; Wittlin, Sergio; Spangenberg, Thomas; Ding, Xavier C

    2015-02-01

    Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs.

  16. Complex polymorphisms in the Plasmodium falciparum multidrug resistance protein 2 gene and its contribution to antimalarial response.

    PubMed

    Veiga, Maria Isabel; Osório, Nuno S; Ferreira, Pedro Eduardo; Franzén, Oscar; Dahlstrom, Sabina; Lum, J Koji; Nosten, Francois; Gil, José Pedro

    2014-12-01

    Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.

  17. Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains.

    PubMed

    Chugh, Monika; Scheurer, Christian; Sax, Sibylle; Bilsland, Elizabeth; van Schalkwyk, Donelly A; Wicht, Kathryn J; Hofmann, Natalie; Sharma, Anil; Bashyam, Sridevi; Singh, Shivendra; Oliver, Stephen G; Egan, Timothy J; Malhotra, Pawan; Sutherland, Colin J; Beck, Hans-Peter; Wittlin, Sergio; Spangenberg, Thomas; Ding, Xavier C

    2015-02-01

    Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs. PMID:25487796

  18. Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model

    PubMed Central

    Obaldía III, Nicanor; Dow, Geoffrey S.; Gerena, Lucia; Kyle, Dennis; Otero, William; Mantel, Pierre-Yves; Baro, Nicholas; Daniels, Rachel; Mukherjee, Angana; Childs, Lauren M.; Buckee, Caroline; Duraisingh, Manoj T.; Volkman, Sarah K.; Wirth, Dyann F.; Marti, Matthias

    2016-01-01

    Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model. PMID:26880111

  19. Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

    PubMed Central

    Chugh, Monika; Scheurer, Christian; Sax, Sibylle; Bilsland, Elizabeth; van Schalkwyk, Donelly A.; Wicht, Kathryn J.; Hofmann, Natalie; Sharma, Anil; Bashyam, Sridevi; Singh, Shivendra; Oliver, Stephen G.; Egan, Timothy J.; Malhotra, Pawan; Sutherland, Colin J.; Beck, Hans-Peter; Wittlin, Sergio; Spangenberg, Thomas

    2014-01-01

    Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs. PMID:25487796

  20. Calmidazolium evokes high calcium fluctuations in Plasmodium falciparum.

    PubMed

    Budu, Alexandre; Gomes, Mayrim M; Melo, Pollyana M; El Chamy Maluf, Sarah; Bagnaresi, Piero; Azevedo, Mauro F; Carmona, Adriana K; Gazarini, Marcos L

    2016-03-01

    Calcium and calmodulin (CaM) are important players in eukaryote cell signaling. In the present study, by using a knockin approach, we demonstrated the expression and localization of CaM in all erythrocytic stages of Plasmodium falciparum. Under extracellular Ca(2+)-free conditions, calmidazolium (CZ), a potent CaM inhibitor, promoted a transient cytosolic calcium ([Ca(2+)]cyt) increase in isolated trophozoites, indicating that CZ mobilizes intracellular sources of calcium. In the same extracellular Ca(2+)-free conditions, the [Ca(2+)]cyt rise elicited by CZ treatment was ~3.5 fold higher when the endoplasmic reticulum (ER) calcium store was previously depleted ruling out the mobilization of calcium from the ER by CZ. The effects of the Ca(2+)/H(+) ionophore ionomycin (ION) and the Na(+)/H(+) ionophore monensin (MON) suggest that the [Ca(2+)]cyt-increasing effect of CZ is driven by the removal of Ca(2+) from at least one Ca(2+)-CaM-related (CaMR) protein as well as by the mobilization of Ca(2+) from intracellular acidic calcium stores. Moreover, we showed that the mitochondrion participates in the sequestration of the cytosolic Ca(2+) elicited by CZ. Finally, the modulation of membrane Ca(2+) channels by CZ and thapsigargin (THG) was demonstrated. The opened channels were blocked by the unspecific calcium channel blocker Co(2+) but not by 2-APB (capacitative calcium entry inhibitor) or nifedipine (L-type Ca(2+) channel inhibitor). Taken together, the results suggested that one CaMR protein is an important modulator of calcium signaling and homeostasis during the Plasmodium intraerythrocytic cell cycle, working as a relevant intracellular Ca(2+) reservoir in the parasite.

  1. Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5.

    PubMed

    Tanabe, Kazuyuki; Arisue, Nobuko; Palacpac, Nirianne M Q; Yagi, Masanori; Tougan, Takahiro; Honma, Hajime; Ferreira, Marcelo U; Färnert, Anna; Björkman, Anders; Kaneko, Akira; Nakamura, Masatoshi; Hirayama, Kenji; Mita, Toshihiro; Horii, Toshihiro

    2012-02-21

    SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS=0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wright's fixation index (Fst) of inter-population variance in allele frequencies

  2. Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

    PubMed Central

    2013-01-01

    Background Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic

  3. A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells.

    PubMed

    Herricks, Thurston; Seydel, Karl B; Turner, George; Molyneux, Malcolm; Heyderman, Robert; Taylor, Terrie; Rathod, Pradipsinh K

    2011-09-01

    The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria.

  4. Genetic diversity and population structure of Plasmodium falciparum over space and time in an African archipelago.

    PubMed

    Salgueiro, Patrícia; Vicente, José Luís; Figueiredo, Rita Carrilho; Pinto, João

    2016-09-01

    The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of malaria since the 16th century. Until the last decade, when after a successful control program STP has become a low transmission country and one of the few nations with decreases of more than 90% in malaria admission and death rates. We carried out a longitudinal study to determine the genetic structure of STP parasite populations over time and space. Twelve microsatellite loci were genotyped in Plasmodium falciparum samples from two islands collected in 1997, 2000 and 2004. Analysis was performed on proportions of mixed genotype infections, allelic diversity, population differentiation, effective population size and bottleneck effects. We have found high levels of genetic diversity and minimal inter-population genetic differentiation typical of African continental regions with intense and stable malaria transmission. We detected significant differences between the years, with special emphasis for 1997 that showed the highest proportion of samples infected with P. falciparum and the highest mean number of haplotypes per isolate. This study establishes a comprehensive genetic data baseline of a pre-intervention scenario for future studies; taking into account the most recent and successful control intervention on the territory.

  5. Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

    PubMed Central

    Baker, Joanne; Gatton, Michelle L.; Peters, Jennifer; Ho, Mei-Fong; McCarthy, James S.; Cheng, Qin

    2011-01-01

    Background Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs. PMID:21799910

  6. Selection of drug resistance-mediating Plasmodium falciparum genetic polymorphisms by seasonal malaria chemoprevention in Burkina Faso.

    PubMed

    Somé, Anyirékun Fabrice; Zongo, Issaka; Compaoré, Yves-Daniel; Sakandé, Souleymane; Nosten, François; Ouédraogo, Jean-Bosco; Rosenthal, Philip J

    2014-07-01

    Seasonal malaria chemoprevention (SMC), with regular use of amodiaquine plus sulfadoxine-pyrimethamine (AQ/SP) during the transmission season, is now a standard malaria control measure in the Sahel subregion of Africa. Another strategy under study is SMC with dihydroartemisinin plus piperaquine (DP). Plasmodium falciparum single nucleotide polymorphisms (SNPs) in P. falciparum crt (pfcrt), pfmdr1, pfdhfr, and pfdhps are associated with decreased response to aminoquinoline and antifolate antimalarials and are selected by use of these drugs. To characterize selection by SMC of key polymorphisms, we assessed 13 SNPs in P. falciparum isolated from children aged 3 to 59 months living in southwestern Burkina Faso and randomized to receive monthly DP or AQ/SP for 3 months in 2009. We compared SNP prevalence before the onset of SMC and 1 month after the third treatment in P. falciparum PCR-positive samples from 120 randomly selected children from each treatment arm and an additional 120 randomly selected children from a control group that did not receive SMC. The prevalence of relevant mutations was increased after SMC with AQ/SP. Significant selection was seen for pfcrt 76T (68.5% to 83.0%, P = 0.04), pfdhfr 59R (54.8% to 83.3%, P = 0.0002), and pfdhfr 108N (55.0% to 87.2%, P = 0.0001), with trends toward selection of pfmdr1 86Y, pfdhfr 51I, and pfdhps 437G. After SMC with DP, only borderline selection of wild-type pfmdr1 D1246 (mutant; 7.7% to 0%, P = 0.05) was seen. In contrast to AQ/SP, SMC with DP did not clearly select for known resistance-mediating polymorphisms. SMC with AQ/SP, but not DP, may hasten the development of resistance to components of this regimen. (This study has been registered at ClinicalTrials.gov under registration no. NCT00941785.).

  7. 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.

  8. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... freezing will not preclude use of the term “fresh frozen” to describe the food. “Quickly frozen” means... 21 Food and Drugs 2 2010-04-01 2010-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH...

  9. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  10. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

    PubMed Central

    Malik, A; Egan, J E; Houghten, R A; Sadoff, J C; Hoffman, S L

    1991-01-01

    Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID:1707538

  11. Eleganolone, a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

    PubMed Central

    Gallé, Jean-Baptiste; Attioua, Barthélémy; Kaiser, Marcel; Rusig, Anne-Marie; Lobstein, Annelise; Vonthron-Sénécheau, Catherine

    2013-01-01

    Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6). PMID:23442789

  12. Identification of a chloroquine importer in Plasmodium falciparum. Differences in import kinetics are genetically linked with the chloroquine-resistant phenotype.

    PubMed

    Sanchez, C P; Wünsch, S; Lanzer, M

    1997-01-31

    We demonstrate that uptake of the antimalarial drug chloroquine is temperature-dependent, saturable, and inhibitable in Plasmodium falciparum. These features are indicative of carrier-mediated transport and suggest that a P. falciparum-encoded protein facilitates chloroquine import. Although both chloroquine-resistant and susceptible parasite isolates exhibit facilitated chloroquine uptake, the kinetics differ. Chloroquine-resistant parasite isolates consistently have an import mechanism with a lower transport activity and a reduced affinity for chloroquine. These differences in uptake kinetics are linked with chloroquine resistance in a genetic cross. These data suggest that changes in chloroquine import kinetics constitute a minimal and necessary event in the generation of the resistant phenotype. Competitive inhibition of chloroquine uptake by amiloride derivatives further suggests that chloroquine import is mediated by a plasmodial Na+/H+ exchanger.

  13. Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

    PubMed

    Warimwe, George M; Abdi, Abdirahman I; Muthui, Michelle; Fegan, Gregory; Musyoki, Jennifer N; Marsh, Kevin; Bull, Peter C

    2016-05-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria.

  14. Antibody responses to a novel Plasmodium falciparum merozoite surface protein vaccine correlate with protection against experimental malaria infection in Aotus monkeys.

    PubMed

    Cavanagh, David R; Kocken, Clemens H M; White, John H; Cowan, Graeme J M; Samuel, Kay; Dubbeld, Martin A; Voorberg-van der Wel, Annemarie; Thomas, Alan W; McBride, Jana S; Arnot, David E

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  15. Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

    PubMed Central

    Cavanagh, David R.; Kocken, Clemens H. M.; White, John H.; Cowan, Graeme J. M.; Samuel, Kay; Dubbeld, Martin A.; der Wel, Annemarie Voorberg-van; Thomas, Alan W.; McBride, Jana S.; Arnot, David E.

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  16. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites.

    PubMed

    Lee, Andrew H; Fidock, David A

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or "Accelerated Resistance to Multiple Drugs" (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  17. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites.

    PubMed

    Lee, Andrew H; Fidock, David A

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or "Accelerated Resistance to Multiple Drugs" (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  18. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites

    PubMed Central

    Lee, Andrew H.; Fidock, David A.

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or “Accelerated Resistance to Multiple Drugs” (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  19. Multiple origins of Plasmodium falciparum dihydropteroate synthetase mutant alleles associated with sulfadoxine resistance in India.

    PubMed

    Lumb, Vanshika; Das, Manoj K; Singh, Neeru; Dev, Vas; Khan, Wajihullah; Sharma, Yagya D

    2011-06-01

    With the spread of chloroquine (CQ)-resistant malaria in India, sulfadoxine-pyrimethamine (SP) alone or in combination with artesunate is used as an alternative antimalarial drug. Due to continuous drug pressure, the Plasmodium falciparum parasite is exhibiting resistance to antifolates because of mutations in candidate genes dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps). Our earlier study on flanking microsatellite markers of dhfr mutant alleles from India had shown a single origin of the pyrimethamine resistance and some minor haplotypes which shared haplotypes with Southeast Asian (Thailand) strains. In the present study, we have analyzed 193 of these Indian P. falciparum isolates for 15 microsatellite loci around dhps to investigate the genetic lineages of the mutant dhps alleles in different parts of the country. Eighty-one of these samples had mutant dhps alleles, of which 62 were from Andaman and Nicobar Islands and the remaining 19 were from mainland India. Of 112 isolates with a wild-type dhps allele, 109 were from mainland India and only 3 were from Andaman and Nicobar Islands. Consistent with the model of selection, the mean expected heterozygosity (H(e)) around mutant dhps alleles (H(e) = 0.55; n = 81) associated with sulfadoxine resistance was lower (P ≤ 0.05) than the mean H(e) around the wild-type dhps allele (H(e) = 0.80; n = 112). There was more genetic diversity in flanking microsatellites of dhps than dhfr among these isolates, which confirms the assertion that dhps mutations are at a very early stage of fixation in the parasite population. Microsatellite haplotypes around various mutant dhps alleles suggest that the resistant dhps alleles have multiple independent origins in India, especially in Andaman and Nicobar Islands. Determining the genetic lineages of the resistant dhps alleles on Andaman and Nicobar Islands and mainland India is significant, given the role of Asia in the intercontinental spread of chloroquine

  20. Serum protein concentrations in Plasmodium falciparum malaria.

    PubMed

    Graninger, W; Thalhammer, F; Hollenstein, U; Zotter, G M; Kremsner, P G

    1992-12-01

    In patients with uncomplicated Plasmodium falciparum infection cytokine-mediated serum protein levels of C-reactive protein (CRP), coeruloplasmin (COE), beta 2-microglobulin (B2M), alpha 1-acid glycoprotein (AAG), alpha 1-antitrypsin (AAT), haptoglobin (HPT), prealbumin (PRE), retinol binding protein (RBP), albumin (ALB) and transferrin (TRF) were measured in an endemic area of the Amazonian rain forest. Semi-immune (SI) and nonimmune (NI) patients were investigated. In both patient groups the serum concentrations of CRP, COE and B2M were elevated on admission. In addition AAG and AAT concentrations were increased in NI patients compared to control subjects. Significantly lower serum concentrations of HPT, PRE, RBP, ALB and TRF were seen in both patient groups during the acute phase of the disease, and were more pronounced in NI patients. After a 28-day follow-up, AAT and B2M were normal in SI patients but HPT, AAT and B2M were still significantly altered in NI patients.

  1. Risk factors for UK Plasmodium falciparum cases

    PubMed Central

    2014-01-01

    Background An increasing proportion of malaria cases diagnosed in UK residents with a history of travel to malaria endemic areas are due to Plasmodium falciparum. Methods In order to identify travellers at most risk of acquiring malaria a proportional hazards model was used to estimate the risk of acquiring malaria stratified by purpose of travel and age whilst adjusting for entomological inoculation rate (EIR) and duration of stay in endemic countries. Results Travellers visiting friends and relatives and business travellers were found to have significantly higher hazard of acquiring malaria (adjusted hazard ratio (HR) relative to that of holiday makers 7.4, 95% CI 6.4–8.5, p < 0. 0001 and HR 3.4, 95% CI 2.9-3.8, p < 0. 0001, respectively). All age-groups were at lower risk than children aged 0–15 years. Conclusions These estimates of the increased risk for business travellers and those visiting friends and relatives should be used to inform programmes to improve awareness of the risks of malaria when travelling. PMID:25091803

  2. Plasmodium falciparum Secretome in Erythrocyte and Beyond

    PubMed Central

    Soni, Rani; Sharma, Drista; Bhatt, Tarun K.

    2016-01-01

    Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies. PMID:26925057

  3. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms.

  4. [Research Progress on Artemisinin Resistance in Plasmodium falciparum].

    PubMed

    Zhang, Yi-long; Pan, Wei-qing

    2015-12-01

    Artemisinin (ART) is a novel and effective antimalarial drug discovered in China. As recommended by the World Health Organization, the ART-based combination therapies (ACTs) have become the first-line drugs for the treatment of falciparum malaria. ART and its derivatives have contributed greatly to the effective control of malaria globally, leading to yearly decrease of malaria morbidity and mortality. However, there have recently been several reports on the resistance of Plasmodium falciparum to ART in Southeast Asia. This is deemed a serious threat to the global malaria control programs. In this paper, we reviewed recent research progress on ART resistance to P. falciparum, including new tools for resistance measurement, resistance-associated molecular markers, and the origin and spread of the ART-resistant parasite strains.

  5. [Research Progress on Artemisinin Resistance in Plasmodium falciparum].

    PubMed

    Zhang, Yi-long; Pan, Wei-qing

    2015-12-01

    Artemisinin (ART) is a novel and effective antimalarial drug discovered in China. As recommended by the World Health Organization, the ART-based combination therapies (ACTs) have become the first-line drugs for the treatment of falciparum malaria. ART and its derivatives have contributed greatly to the effective control of malaria globally, leading to yearly decrease of malaria morbidity and mortality. However, there have recently been several reports on the resistance of Plasmodium falciparum to ART in Southeast Asia. This is deemed a serious threat to the global malaria control programs. In this paper, we reviewed recent research progress on ART resistance to P. falciparum, including new tools for resistance measurement, resistance-associated molecular markers, and the origin and spread of the ART-resistant parasite strains. PMID:27089770

  6. Localization of apical sushi protein in Plasmodium falciparum merozoites.

    PubMed

    Srivastava, Anand; Singh, Shailja; Dhawan, Shikha; Mahmood Alam, M; Mohmmed, Asif; Chitnis, Chetan E

    2010-11-01

    Plasmodium falciparum belongs to the Apicomplexan group of parasites and is characterised by presence of specialized secretory organelles at the apical end. These apical organelles, referred to as microneme and rhoptries, contain proteins that play important roles during host cell invasion by mediating specific functions such as initial attachment, apical reorientation and junction formation. Recently, a protein referred to as P. falciparum apical sushi protein (PfASP), which is expressed at late schizont stage, was localized to micronemes of P. falciparum merozoites. In the present study, we have used indirect immunofluorescence assays and immunoelectron microscopy to demonstrate that PfASP is localized in the neck of rhoptries and not in micronemes as previously described.

  7. Plasmodium falciparum: growth response to potassium channel blocking compounds.

    PubMed

    Waller, Karena L; Kim, Kami; McDonald, Thomas V

    2008-11-01

    Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds. PMID:18703053

  8. Active case detection, treatment of falciparum malaria with combined chloroquine and sulphadoxine/pyrimethamine and vivax malaria with chloroquine and molecular markers of anti-malarial resistance in the Republic of Vanuatu

    PubMed Central

    2010-01-01

    Background Chloroquine-resistant Plasmodium falciparum was first described in the Republic of Vanuatu in the early 1980s. In 1991, the Vanuatu Ministry of Health instituted new treatment guidelines for uncomplicated P. falciparum infection consisting of chloroquine/sulphadoxine-pyrimethamine combination therapy. Chloroquine remains the recommended treatment for Plasmodium vivax. Methods In 2005, cross-sectional blood surveys at 45 sites on Malo Island were conducted and 4,060 adults and children screened for malaria. Of those screened, 203 volunteer study subjects without malaria at the time of screening were followed for 13 weeks to observe peak seasonal incidence of infection. Another 54 subjects with malaria were followed over a 28-day period to determine efficacy of anti-malarial therapy; chloroquine alone for P. vivax and chloroquine/sulphadoxine-pyrimethamine for P. falciparum infections. Results The overall prevalence of parasitaemia by mass blood screening was 6%, equally divided between P. falciparum and P. vivax. Twenty percent and 23% of participants with patent P. vivax and P. falciparum parasitaemia, respectively, were febrile at the time of screening. In the incidence study cohort, after 2,303 person-weeks of follow-up, the incidence density of malaria was 1.3 cases per person-year with P. vivax predominating. Among individuals participating in the clinical trial, the 28-day chloroquine P. vivax cure rate was 100%. The 28-day chloroquine/sulphadoxine-pyrimethamine P. falciparum cure rate was 97%. The single treatment failure, confirmed by merozoite surface protein-2 genotyping, was classified as a day 28 late parasitological treatment failure. All P. falciparum isolates carried the Thr-76 pfcrt mutant allele and the double Asn-108 + Arg-59 dhfr mutant alleles. Dhps mutant alleles were not detected in the study sample. Conclusion Peak seasonal malaria prevalence on Malo Island reached hypoendemic levels during the study observation period. The only in

  9. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    PubMed

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

  10. Further Improvements of the P. falciparum Humanized Mouse Model

    PubMed Central

    Meija, Pedro; Swetman, Claire; Gleeson, James; Pérignon, Jean-Louis; Druilhe, Pierre

    2011-01-01

    Background It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines. Materials/Methods We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2Rγ deficiency (NSG mice). Results We demonstrate here the role of aging, of inosine and of the IL-2 receptor γ mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent. Conclusion The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research. PMID:21483851

  11. Intraerythrocytic stages of Plasmodium falciparum biosynthesize vitamin E.

    PubMed

    Sussmann, Rodrigo A C; Angeli, Cláudia B; Peres, Valnice J; Kimura, Emilia A; Katzin, Alejandro M

    2011-12-15

    The 2-C-methyl-D-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.

  12. Analysis of expressed sequence tags from Plasmodium falciparum.

    PubMed

    Chakrabarti, D; Reddy, G R; Dame, J B; Almira, E C; Laipis, P J; Ferl, R J; Yang, T P; Rowe, T C; Schuster, S M

    1994-07-01

    An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

  13. Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

    PubMed

    Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra

    2012-01-01

    We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards. PMID:22326740

  14. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains

    PubMed Central

    Preston, Mark D.; Campino, Susana; Assefa, Samuel A.; Echeverry, Diego F.; Ocholla, Harold; Amambua-Ngwa, Alfred; Stewart, Lindsay B.; Conway, David J.; Borrmann, Steffen; Michon, Pascal; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A.; Doumbo, Ogobara K.; Nosten, Francois; Pain, Arnab; Bousema, Teun; Drakeley, Chris J.; Fairhurst, Rick M.; Sutherland, Colin J.; Roper, Cally; Clark, Taane G.

    2014-01-01

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. PMID:24923250

  15. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains.

    PubMed

    Preston, Mark D; Campino, Susana; Assefa, Samuel A; Echeverry, Diego F; Ocholla, Harold; Amambua-Ngwa, Alfred; Stewart, Lindsay B; Conway, David J; Borrmann, Steffen; Michon, Pascal; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A; Doumbo, Ogobara K; Nosten, Francois; Pain, Arnab; Bousema, Teun; Drakeley, Chris J; Fairhurst, Rick M; Sutherland, Colin J; Roper, Cally; Clark, Taane G

    2014-01-01

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. PMID:24923250

  16. Potential antimalarial candidates from African plants: and in vitro approach using Plasmodium falciparum.

    PubMed

    Khalid, S A; Farouk, A; Geary, T G; Jensen, J B

    1986-02-01

    Twenty-one compounds isolated from nine medicinal plants used in traditional medicine in the Sudan and other African countries were examined in vitro for antimalarial activity against Plasmodium falciparum, the major human malaria parasite. Compounds tested include alkaloids, lignans, triterpenes, coumarins, limonoids and flavonoids. Most were relatively inactive; one limonoid, gedunin, had an IC50 value of about 1 microM after 48 h exposure (0.3 microM after 96 h), roughly equivalent to quinine. In this protocol, the flavonoid quercetin purified from Diosma pilosa was found to have the same activity as a commercially obtained preparation. Simple radiometric assays for antimalarial activity can thus be used to rapidly screen purified plant material or secondary plant metabolites. The high potency and efficacy of quinine and the Chinese herbal antimalarial quinghaosu (artemisinine) illustrate the merit of this approach. PMID:3520157

  17. Branched Tricarboxylic Acid Metabolism in Plasmodium falciparum

    PubMed Central

    Olszewski, Kellen L.; Mather, Michael W.; Morrisey, Joanne M.; Garcia, Benjamin A.; Vaidya, Akhil B.; Rabinowitz, Joshua D.; Llinás, Manuel

    2010-01-01

    A central hub of carbon metabolism is the tricarboxylic acid (TCA) cycle1, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which TCA metabolism plays a minor role2. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen3, yet the parasite genome encodes all of the enzymes necessary for a complete TCA cycle4. By tracing 13C-labeled compounds using mass spectrometry5 we show that TCA metabolism in the human malaria parasite P. falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture than the canonical textbook pathway. We find that this pathway is not cyclic but rather a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction thereby generating two-carbon units in the form of acetyl-coenzyme A (acetyl-CoA). We further show that glutamine-derived acetyl-CoA is used for histone acetylation while glucose-derived acetyl-CoA is used to acetylate aminosugars. Thus the parasite has evolved two independent acetyl-CoA-production mechanisms with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments. PMID:20686576

  18. Branched tricarboxylic acid metabolism in Plasmodium falciparum.

    PubMed

    Olszewski, Kellen L; Mather, Michael W; Morrisey, Joanne M; Garcia, Benjamin A; Vaidya, Akhil B; Rabinowitz, Joshua D; Llinás, Manuel

    2010-08-01

    A central hub of carbon metabolism is the tricarboxylic acid cycle, which serves to connect the processes of glycolysis, gluconeogenesis, respiration, amino acid synthesis and other biosynthetic pathways. The protozoan intracellular malaria parasites (Plasmodium spp.), however, have long been suspected of possessing a significantly streamlined carbon metabolic network in which tricarboxylic acid metabolism plays a minor role. Blood-stage Plasmodium parasites rely almost entirely on glucose fermentation for energy and consume minimal amounts of oxygen, yet the parasite genome encodes all of the enzymes necessary for a complete tricarboxylic acid cycle. Here, by tracing (13)C-labelled compounds using mass spectrometry we show that tricarboxylic acid metabolism in the human malaria parasite Plasmodium falciparum is largely disconnected from glycolysis and is organized along a fundamentally different architecture from the canonical textbook pathway. We find that this pathway is not cyclic, but rather is a branched structure in which the major carbon sources are the amino acids glutamate and glutamine. As a consequence of this branched architecture, several reactions must run in the reverse of the standard direction, thereby generating two-carbon units in the form of acetyl-coenzyme A. We further show that glutamine-derived acetyl-coenzyme A is used for histone acetylation, whereas glucose-derived acetyl-coenzyme A is used to acetylate amino sugars. Thus, the parasite has evolved two independent production mechanisms for acetyl-coenzyme A with different biological functions. These results significantly clarify our understanding of the Plasmodium metabolic network and highlight the ability of altered variants of central carbon metabolism to arise in response to unique environments. PMID:20686576

  19. Artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Fairhurst, Rick M.; Dondorp, Arjen M.

    2016-01-01

    For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins – the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs) – the first-line treatments for malaria – are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in-vitro, genomics, and transcriptomics studies in SEA have defined in-vivo and in-vitro phenotypes of artemisinin resistance; identified its causal genetic determinant; explored its molecular mechanism; and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's ‘K13’ gene; is associated with an upregulated “unfolded protein response” pathway that may antagonize the pro-oxidant activity of artemisinins; and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent; test whether new combinations of currently-available drugs cure ACT failures; and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to Sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest. PMID:27337450

  20. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border

    PubMed Central

    Ye, Run; Hu, Dongwei; Zhang, Yilong; Huang, Yufu; Sun, Xiaodong; Wang, Jian; Chen, Xuedi; Zhou, Hongning; Zhang, Dongmei; Mungthin, Mathirut; Pan, Weiqing

    2016-01-01

    The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0−3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced. PMID:26831371

  1. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border.

    PubMed

    Ye, Run; Hu, Dongwei; Zhang, Yilong; Huang, Yufu; Sun, Xiaodong; Wang, Jian; Chen, Xuedi; Zhou, Hongning; Zhang, Dongmei; Mungthin, Mathirut; Pan, Weiqing

    2016-01-01

    The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0-3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced. PMID:26831371

  2. Origin of the human malaria parasite Plasmodium falciparum in gorillas.

    PubMed

    Liu, Weimin; Li, Yingying; Learn, Gerald H; Rudicell, Rebecca S; Robertson, Joel D; Keele, Brandon F; Ndjango, Jean-Bosco N; Sanz, Crickette M; Morgan, David B; Locatelli, Sabrina; Gonder, Mary K; Kranzusch, Philip J; Walsh, Peter D; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V; Muller, Martin N; Shaw, George M; Peeters, Martine; Sharp, Paul M; Rayner, Julian C; Hahn, Beatrice H

    2010-09-23

    Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here we develop a single-genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in faecal samples from wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed and almost always made up of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas comprised parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla origin and not of chimpanzee, bonobo or ancient human origin.

  3. Blood coagulation in falciparum malaria--a review.

    PubMed

    Ghosh, Kanjaksha; Shetty, Shrimati

    2008-03-01

    Falciparum malaria infection influences blood coagulation by various interacting pathobiological mechanisms, the most important being the overwhelming response of the host to sepsis resulting in a cytokine storm. In addition, the parasite infects the red cells leading to changes in the red cell phospholipid composition which supports blood coagulation. Red cells infected with Plasmodium falciparum also adhere to deeper tissue capillary endothelium leading to profound damage to endothelial cells leading to further activation. This results in widespread consumption of platelets and activation of blood coagulation which at times culminates in a clinically and pathologically detectable disseminated intravascular coagulation (DIC). Monocyte-macrophage system also gets activated in this infection compounding the hypercoagulable state. Heavy parasitaemia leading to occlusion of hepatic microcirculation leads to abnormalities in synthesis and secretion of coagulation factors and their inhibitors. Drugs used in the treatment for falciparum malaria can cause thrombocytopaenia, bone marrow suppression and haemolytic anaemia, all of which can interfere indirectly with blood coagulation. Microparticle formation from platelets, red cells and macrophages also causes widespread activation of blood coagulation, and this recently observed mechanism is the focus of intense research in many other inflammatory and neoplastic conditions where there is activation of blood coagulation system. Thus, in severe falciparum malaria, there is activation of blood coagulation system along with thrombocytopaenia, even before widespread DIC and coagulation failure occur.

  4. Origin of the human malaria parasite Plasmodium falciparum in gorillas

    PubMed Central

    Liu, Weimin; Li, Yingying; Learn, Gerald H.; Rudicell, Rebecca S.; Robertson, Joel D.; Keele, Brandon F.; Ndjango, Jean-Bosco N.; Sanz, Crickette M.; Morgan, David B.; Locatelli, Sabrina; Gonder, Mary K.; Kranzusch, Philip J.; Walsh, Peter D.; Delaporte, Eric; Mpoudi-Ngole, Eitel; Georgiev, Alexander V.; Muller, Martin N.; Shaw, George M.; Peeters, Martine; Sharp, Paul M.; Rayner, Julian C.; Hahn, Beatrice H.

    2010-01-01

    Plasmodium falciparum is the most prevalent and lethal of the malaria parasites infecting humans, yet the origin and evolutionary history of this important pathogen remain controversial. Here, we developed a novel polymerase chain reaction based single genome amplification strategy to identify and characterize Plasmodium spp. DNA sequences in fecal samples of wild-living apes. Among nearly 3,000 specimens collected from field sites throughout central Africa, we found Plasmodium infection in chimpanzees (Pan troglodytes) and western gorillas (Gorilla gorilla), but not in eastern gorillas (Gorilla beringei) or bonobos (Pan paniscus). Ape plasmodial infections were highly prevalent, widely distributed, and almost always comprised of mixed parasite species. Analysis of more than 1,100 mitochondrial, apicoplast and nuclear gene sequences from chimpanzees and gorillas revealed that 99% grouped within one of six host-specific lineages representing distinct Plasmodium species within the subgenus Laverania. One of these from western gorillas was comprised of parasites that were nearly identical to P. falciparum. In phylogenetic analyses of full-length mitochondrial sequences, human P. falciparum formed a monophyletic lineage within the gorilla parasite radiation. These findings indicate that P. falciparum is of gorilla and not of chimpanzee, bonobo or ancient human origin. PMID:20864995

  5. 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.

  6. Antimicrobial packaging for fresh-cut fruits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fresh-cut fruits are minimally processed produce which are consumed directly at their fresh stage without any further kill step. Microbiological quality and safety are major challenges to fresh-cut fruits. Antimicrobial packaging is one of the innovative food packaging systems that is able to kill o...

  7. Plasmodium falciparum Malaria Endemicity in Indonesia in 2010

    PubMed Central

    Elyazar, Iqbal R. F.; Gething, Peter W.; Patil, Anand P.; Rogayah, Hanifah; Kusriastuti, Rita; Wismarini, Desak M.; Tarmizi, Siti N.; Baird, J. Kevin; Hay, Simon I.

    2011-01-01

    Background Malaria control programs require a detailed understanding of the contemporary spatial distribution of infection risk to efficiently allocate resources. We used model based geostatistics (MBG) techniques to generate a contemporary map of Plasmodium falciparum malaria risk in Indonesia in 2010. Methods Plasmodium falciparum Annual Parasite Incidence (PfAPI) data (2006–2008) were used to map limits of P. falciparum transmission. A total of 2,581 community blood surveys of P. falciparum parasite rate (PfPR) were identified (1985–2009). After quality control, 2,516 were included into a national database of age-standardized 2–10 year old PfPR data (PfPR2–10) for endemicity mapping. A Bayesian MBG procedure was used to create a predicted surface of PfPR2–10 endemicity with uncertainty estimates. Population at risk estimates were derived with reference to a 2010 human population count surface. Results We estimate 132.8 million people in Indonesia, lived at risk of P. falciparum transmission in 2010. Of these, 70.3% inhabited areas of unstable transmission and 29.7% in stable transmission. Among those exposed to stable risk, the vast majority were at low risk (93.39%) with the reminder at intermediate (6.6%) and high risk (0.01%). More people in western Indonesia lived in unstable rather than stable transmission zones. In contrast, fewer people in eastern Indonesia lived in unstable versus stable transmission areas. Conclusion While further feasibility assessments will be required, the immediate prospects for sustained control are good across much of the archipelago and medium term plans to transition to the pre-elimination phase are not unrealistic for P. falciparum. Endemicity in areas of Papua will clearly present the greatest challenge. This P. falciparum endemicity map allows malaria control agencies and their partners to comprehensively assess the region-specific prospects for reaching pre-elimination, monitor and evaluate the effectiveness of

  8. Hiding in Fresh Fruits and Vegetables: Opportunistic Pathogens May Cross Geographical Barriers

    PubMed Central

    Al-Kharousi, Zahra S.; Al-Sadi, Abdullah M.; Al-Bulushi, Ismail M.; Shaharoona, Baby

    2016-01-01

    Different microbial groups of the microbiome of fresh produce can have diverse effects on human health. This study was aimed at identifying some microbial communities of fresh produce by analyzing 105 samples of imported fresh fruits and vegetables originated from different countries in the world including local samples (Oman) for aerobic plate count and the counts of Enterobacteriaceae, Enterococcus, and Staphylococcus aureus. The isolated bacteria were identified by molecular (PCR) and biochemical methods (VITEK 2). Enterobacteriaceae occurred in 60% of fruits and 91% of vegetables. Enterococcus was isolated from 20% of fruits and 42% of vegetables. E. coli and S. aureus were isolated from 22% and 7% of vegetables, respectively. Ninety-seven bacteria comprising 21 species were similarly identified by VITEK 2 and PCR to species level. E. coli, Klebsiella pneumoniae, Enterococcus casseliflavus, and Enterobacter cloacae were the most abundant species; many are known as opportunistic pathogens which may raise concern to improve the microbial quality of fresh produce. Phylogenetic trees showed no relationship between clustering of the isolates based on the 16S rRNA gene and the original countries of fresh produce. Intercountry passage of opportunistic pathogens in fresh produce cannot be ruled out, which requires better management. PMID:26989419

  9. [From malaria parasite point of view--Plasmodium falciparum evolution].

    PubMed

    Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa

    2015-12-31

    Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago) than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies.

  10. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

    PubMed

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M R K; Freund, Yvonne R; DeRisi, Joseph; Cusack, Stephen; Rosenthal, Philip J

    2016-08-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS.

  11. Endemicity response timelines for Plasmodium falciparum elimination

    PubMed Central

    Smith, David L; Hay, Simon I

    2009-01-01

    Background The scaling up of malaria control and renewed calls for malaria eradication have raised interest in defining timelines for changes in malaria endemicity. Methods The epidemiological theory for the decline in the Plasmodium falciparum parasite rate (PfPR, the prevalence of infection) following intervention was critically reviewed and where necessary extended to consider superinfection, heterogeneous biting, and aging infections. Timelines for malaria control and elimination under different levels of intervention were then established using a wide range of candidate mathematical models. Analysis focused on the timelines from baseline to 1% and from 1% through the final stages of elimination. Results The Ross-Macdonald model, which ignores superinfection, was used for planning during the Global Malaria Eradication Programme (GMEP). In models that consider superinfection, PfPR takes two to three years longer to reach 1% starting from a hyperendemic baseline, consistent with one of the few large-scale malaria control trials conducted in an African population with hyperendemic malaria. The time to elimination depends fundamentally upon the extent to which malaria transmission is interrupted and the size of the human population modelled. When the PfPR drops below 1%, almost all models predict similar and proportional declines in PfPR in consecutive years from 1% through to elimination and that the waiting time to reduce PfPR from 10% to 1% and from 1% to 0.1% are approximately equal, but the decay rate can increase over time if infections senesce. Conclusion The theory described herein provides simple "rules of thumb" and likely time horizons for the impact of interventions for control and elimination. Starting from a hyperendemic baseline, the GMEP planning timelines, which were based on the Ross-Macdonald model with completely interrupted transmission, were inappropriate for setting endemicity timelines and they represent the most optimistic scenario for

  12. A sequence in subdomain 2 of DBL1α of Plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies.

    PubMed

    Blomqvist, Karin; Albrecht, Letusa; Quintana, Maria del Pilar; Angeletti, Davide; Joannin, Nicolas; Chêne, Arnaud; Moll, Kirsten; Wahlgren, Mats

    2013-01-01

    Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1α-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. PMID:23335956

  13. Multiple dimensions of epigenetic gene regulation in the malaria parasite Plasmodium falciparum: gene regulation via histone modifications, nucleosome positioning and nuclear architecture in P. falciparum.

    PubMed

    Ay, Ferhat; Bunnik, Evelien M; Varoquaux, Nelle; Vert, Jean-Philippe; Noble, William Stafford; Le Roch, Karine G

    2015-02-01

    Plasmodium falciparum is the most deadly human malarial parasite, responsible for an estimated 207 million cases of disease and 627,000 deaths in 2012. Recent studies reveal that the parasite actively regulates a large fraction of its genes throughout its replicative cycle inside human red blood cells and that epigenetics plays an important role in this precise gene regulation. Here, we discuss recent advances in our understanding of three aspects of epigenetic regulation in P. falciparum: changes in histone modifications, nucleosome occupancy and the three-dimensional genome structure. We compare these three aspects of the P. falciparum epigenome to those of other eukaryotes, and show that large-scale compartmentalization is particularly important in determining histone decomposition and gene regulation in P. falciparum. We conclude by presenting a gene regulation model for P. falciparum that combines the described epigenetic factors, and by discussing the implications of this model for the future of malaria research.

  14. A rapid and simple DNA extraction procedure to detect Salmonella spp. and Listeria monocytogenes from fresh produce using real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples, DNA isolation from food samples such as fresh and fresh-cut produce has remained a formidable task and has hampered the sensitivity and accuracy of molecular methods. We...

  15. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area

    PubMed Central

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-01-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  16. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs.

  17. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    PubMed

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  18. Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia

    PubMed Central

    Khim, Nimol; Chim, Pheaktra; Kim, Saorin; Ke, Sopheakvatey; Kloeung, Nimol; Chy, Sophy; Duong, Socheat; Leang, Rithea; Ringwald, Pascal; Dondorp, Arjen M.; Tripura, Rupam; Benoit-Vical, Françoise; Berry, Antoine; Gorgette, Olivier; Ariey, Frédéric; Barale, Jean-Christophe; Mercereau-Puijalon, Odile

    2013-01-01

    The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA. PMID:23208708

  19. Salmonella surveillance on fresh produce in retail in Turkey.

    PubMed

    Gunel, Elif; Polat Kilic, Gozde; Bulut, Ece; Durul, Bora; Acar, Sinem; Alpas, Hami; Soyer, Yeşim

    2015-04-16

    Although Turkey is one of the major producers of fruits and vegetables in the world, there has been no information available on the prevalence of pathogens in fresh produce. To fill this gap, we collected 503 fresh produce samples including tomato, parsley, iceberg lettuce, green-leaf lettuce and five different fresh pepper varieties (i.e., green, kapya, bell, mazamort and Charleston) from 3 major districts within 9 supermarkets and 3 bazaars in Ankara, Turkey to investigate the presence of Salmonella. Salmonella was detected in 0.8% (4/503) of samples by conventional culturing method with molecular confirmation conducted through polymerase chain reaction (PCR). For further characterization of isolates, serotyping, antimicrobial susceptibility testing, multi-locus sequence typing (MLST; aroC, thrA, purE, sucA, hisD, hemD and dnaN) and pulsed-field gel electrophoresis (PFGE) were performed. Salmonella enterica subsp. enterica serotypes Anatum, Charity, Enteritidis and Mikawasima were isolated from two parsley, one pepper and one lettuce samples, respectively. MLST resulted in 4 sequence types (STs) for each serotype, including one novel ST for serotype Mikawasima. Similarly, PFGE revealed four different XbaI PFGE patterns. The results of this survey, obtained by the most common subtyping methods (i.e. serotyping, MLST and PFGE) worldwide, contributes to the development of a national database in Turkey, which is essential for investigating the evolutionary pathways, geographical distribution and genetic diversity of Salmonella strains.

  20. Salmonella surveillance on fresh produce in retail in Turkey.

    PubMed

    Gunel, Elif; Polat Kilic, Gozde; Bulut, Ece; Durul, Bora; Acar, Sinem; Alpas, Hami; Soyer, Yeşim

    2015-04-16

    Although Turkey is one of the major producers of fruits and vegetables in the world, there has been no information available on the prevalence of pathogens in fresh produce. To fill this gap, we collected 503 fresh produce samples including tomato, parsley, iceberg lettuce, green-leaf lettuce and five different fresh pepper varieties (i.e., green, kapya, bell, mazamort and Charleston) from 3 major districts within 9 supermarkets and 3 bazaars in Ankara, Turkey to investigate the presence of Salmonella. Salmonella was detected in 0.8% (4/503) of samples by conventional culturing method with molecular confirmation conducted through polymerase chain reaction (PCR). For further characterization of isolates, serotyping, antimicrobial susceptibility testing, multi-locus sequence typing (MLST; aroC, thrA, purE, sucA, hisD, hemD and dnaN) and pulsed-field gel electrophoresis (PFGE) were performed. Salmonella enterica subsp. enterica serotypes Anatum, Charity, Enteritidis and Mikawasima were isolated from two parsley, one pepper and one lettuce samples, respectively. MLST resulted in 4 sequence types (STs) for each serotype, including one novel ST for serotype Mikawasima. Similarly, PFGE revealed four different XbaI PFGE patterns. The results of this survey, obtained by the most common subtyping methods (i.e. serotyping, MLST and PFGE) worldwide, contributes to the development of a national database in Turkey, which is essential for investigating the evolutionary pathways, geographical distribution and genetic diversity of Salmonella strains. PMID:25643853

  1. Incidence and identification of phospholipase C-producing bacteria in fresh and spoiled homogenized milk.

    PubMed

    Fox, C W; Chrisope, G L; Marshall, R T

    1976-11-01

    Bacteria which produced phospholipase C were isolated from 13 of 34 fresh and 15 of 35 spoiled samples of homogenized milk. No single off flavor was assigned consistently to samples with phospholipase producers, but 75% of them were bitter. Pseudomonads constituted 62% of the isolates. Other phospholipase C-producing genera and their numbers were Acinetobacter, two; Alcaligenes, three; Bacillus, two; Citrobacter, one; Enterobacter, three; and Flavobacterium, two. Two unidentified yeasts also were isolated.

  2. Antimalarial Activity of KAF156 in Falciparum and Vivax Malaria.

    PubMed

    White, Nicholas J; Duong, Tran T; Uthaisin, Chirapong; Nosten, François; Phyo, Aung P; Hanboonkunupakarn, Borimas; Pukrittayakamee, Sasithon; Jittamala, Podjanee; Chuthasmit, Kittiphum; Cheung, Ming S; Feng, Yiyan; Li, Ruobing; Magnusson, Baldur; Sultan, Marc; Wieser, Daniela; Xun, Xiaolei; Zhao, Rong; Diagana, Thierry T; Pertel, Peter; Leong, F Joel

    2016-09-22

    Background KAF156 belongs to a new class of antimalarial agents (imidazolopiperazines), with activity against asexual and sexual blood stages and the preerythrocytic liver stages of malarial parasites. Methods We conducted a phase 2, open-label, two-part study at five centers in Thailand and Vietnam to assess the antimalarial efficacy, safety, and pharmacokinetic profile of KAF156 in adults with acute Plasmodium vivax or P. falciparum malaria. Assessment of parasite clearance rates in cohorts of patients with vivax or falciparum malaria who were treated with multiple doses (400 mg once daily for 3 days) was followed by assessment of the cure rate at 28 days in a separate cohort of patients with falciparum malaria who received a single dose (800 mg). Results Median parasite clearance times were 45 hours (interquartile range, 42 to 48) in 10 patients with falciparum malaria and 24 hours (interquartile range, 20 to 30) in 10 patients with vivax malaria after treatment with the multiple-dose regimen and 49 hours (interquartile range, 42 to 54) in 21 patients with falciparum malaria after treatment with the single dose. Among the 21 patients who received the single dose and were followed for 28 days, 1 had reinfection and 7 had recrudescent infections (cure rate, 67%; 95% credible interval, 46 to 84). The mean (±SD) KAF156 terminal elimination half-life was 44.1±8.9 hours. There were no serious adverse events in this small study. The most common adverse events included sinus bradycardia, thrombocytopenia, hypokalemia, anemia, and hyperbilirubinemia. Vomiting of grade 2 or higher occurred in 2 patients, 1 of whom discontinued treatment because of repeated vomiting after receiving the single 800-mg dose. More adverse events were reported in the single-dose cohort, which had longer follow-up, than in the multiple-dose cohorts. Conclusions KAF156 showed antimalarial activity without evident safety concerns in a small number of adults with uncomplicated P. vivax or P

  3. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance.

    PubMed

    Dogovski, Con; Xie, Stanley C; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A; Simpson, Julie A; Dondorp, Arjen M; Foote, Simon; Klonis, Nectarios; Tilley, Leann

    2015-04-01

    Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART

  4. A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking

    PubMed Central

    Daniels, Rachel; Volkman, Sarah K; Milner, Danny A; Mahesh, Nira; Neafsey, Daniel E; Park, Daniel J; Rosen, David; Angelino, Elaine; Sabeti, Pardis C; Wirth, Dyann F; Wiegand, Roger C

    2008-01-01

    Background Single nucleotide polymorphism (SNP) genotyping provides the means to develop a practical, rapid, inexpensive assay that will uniquely identify any Plasmodium falciparum parasite using a small amount of DNA. Such an assay could be used to distinguish recrudescence from re-infection in drug trials, to monitor the frequency and distribution of specific parasites in a patient population undergoing drug treatment or vaccine challenge, or for tracking samples and determining purity of isolates in the laboratory during culture adaptation and sub-cloning, as well as routine passage. Methods A panel of twenty-four SNP markers has been identified that exhibit a high minor allele frequency (average MAF > 35%), for which robust TaqMan genotyping assays were constructed. All SNPs were identified through whole genome sequencing and MAF was estimated through Affymetrix array-based genotyping of a worldwide collection of parasites. These assays create a "molecular barcode" to uniquely identify a parasite genome. Results Using 24 such markers no two parasites known to be of independent origin have yet been found to have the same allele signature. The TaqMan genotyping assays can be performed on a variety of samples including cultured parasites, frozen whole blood, or whole blood spotted onto filter paper with a success rate > 99%. Less than 5 ng of parasite DNA is needed to complete a panel of 24 markers. The ability of this SNP panel to detect and identify parasites was compared to the standard molecular methods, MSP-1 and MSP-2 typing. Conclusion This work provides a facile field-deployable genotyping tool that can be used without special skills with standard lab equipment, and at reasonable cost that will unambiguously identify and track P. falciparum parasites both from patient samples and in the laboratory. PMID:18959790

  5. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance.

    PubMed

    Dogovski, Con; Xie, Stanley C; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A; Simpson, Julie A; Dondorp, Arjen M; Foote, Simon; Klonis, Nectarios; Tilley, Leann

    2015-04-01

    Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART

  6. Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

    PubMed Central

    Dogovski, Con; Xie, Stanley C.; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M.; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A.; Simpson, Julie A.; Dondorp, Arjen M.; Foote, Simon; Klonis, Nectarios; Tilley, Leann

    2015-01-01

    Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART

  7. Importation of chloroquine-resistant Plasmodium falciparum by Guatemalan peacekeepers returning from the Democratic Republic of the Congo

    PubMed Central

    2013-01-01

    Background Malaria elimination is being pursued in five of seven Central American countries. Military personnel returning from peacekeeping missions in sub-Saharan Africa could import chloroquine-resistant Plasmodium falciparum, posing a threat to elimination and to the continued efficacy of first-line chloroquine (CQ) treatment in these countries. This report describes the importation of P. falciparum from among 150 Guatemalan army special forces and support staff who spent ten months on a United Nations’ peacekeeping mission in the Democratic Republic of the Congo (DRC) in 2010. Methods Investigators reviewed patients’ medical charts and interviewed members of the contingent to identify malaria cases and risk factors for malaria acquisition. Clinical specimens were tested for malaria; isolated parasites were characterized molecularly for CQ resistance. Results Investigators identified 12 cases (8%) of laboratory-confirmed P. falciparum infection within the contingent; one case was from a soldier infected with a CQ-resistant pfcrt genotype resulting in his death. None of the contingent used an insecticide-treated bed net (ITN) or completely adhered to malaria chemoprophylaxis while in the DRC. Conclusion This report highlights the need to promote use of malaria prevention measures, in particular ITNs and chemoprophylaxis, among peacekeepers stationed in malaria-endemic areas. Countries attempting to eliminate malaria should consider appropriate methods to screen peacekeepers returning from endemic areas for malaria infections. Cases of malaria in travellers, immigrants and soldiers returning to Central America from countries with CQ-resistant malaria should be assumed to be carry resistant parasites and receive appropriate anti-malarial therapy to prevent severe disease and death. PMID:24060234

  8. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya.

    PubMed

    Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin

    2016-04-01

    Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p<0.0001) whereas Kisii had the least significant index of association values (0.03; p<0.0001). Our data suggest high genetic diversity in Kenyan parasite population with the exception of parasite from Malindi where malaria has been on the decline. The presence of significant LD suggests that there is occurrence of inbreeding in the parasite population. Parasite populations from Kisii showed the strongest evidence for epidemic population structure whereas the rest of the regions showed panmixia. Defining the genetic diversity of the parasites in different ecological regions of Kenya after

  9. The mechanism of resistance to sulfa drugs in Plasmodium falciparum.

    PubMed

    Triglia, Tony; Cowman, Alan F.

    1999-02-01

    The sulfonamide and sulfone (sulfa) group of antimalarials has been used extensively throughout malaria endemic regions of the world to control this important infectious disease of humans. Sulfadoxine is the most extensively used drug of this group of drugs and is usually combined with pyrimethamine (Fansidar), particularly for the control of Plasmodium falciparum, the causative agent of the most lethal form of malaria. Resistance to the sulfadoxine/pyrimethamine combination is widespread. Analysis using molecular, genetic and biochemical approaches has shown that the mechanism of resistance to sulfadoxine involves mutation of dihydropteroate synthase, the enzyme target of this group of drugs. Understanding the mechanism of resistance of P. falciparum to sulfa drugs has allowed detailed analysis of the epidemiology of the spread of drug resistance alleles in the field(1)and, in the future, opens the way to the development of novel antimalarials to this target enzyme. Copyright 1999 Harcourt Publishers Ltd.

  10. Squalestatin Is an Inhibitor of Carotenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Gabriel, Heloisa B.; Silva, Marcia F.; Kimura, Emília A.; Wunderlich, Gerhard

    2015-01-01

    The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite. PMID:25779575

  11. [Plasmodium falciparum malaria: epidemiology and clinical features at Tarapoto Hospital].

    PubMed

    Calderon, J; Rodriguez, J; Romero, D

    1997-01-01

    A retrospective study was conducted of the clinical records of 41 patients discharged from a hospital in Tarapoto, Peru, between August 1992 and June 1996 following treatment for Plasmodium falciparum malaria. Patients ranged in age from 18 to 65 years; 25 were male. The cases were uniformly distributed throughout the year. The duration of illness averaged 11 days. At admission, 40 patients had fever, 36 had shaking chills, 29 had headache, 21 had nausea and vomiting, 21 had hyporexia, 15 had pallor, and 13 had splenomegaly. 3 of the 16 women were pregnant. 7 patients reported a history of malaria. The admission diagnosis was malaria in 33 cases. 31 patients were treated with chloroquine; 18 were subsequently treated with pyrimethamine-sulfadoxin and 1 received doxycycline. No cases of grave illness or death occurred. The increasing presence of Plasmodium falciparum malaria in the Peruvian lowlands should promote review of the adequacy of control programs.

  12. Microscopic Plasmodium falciparum Gametocytemia and Infectivity to Mosquitoes in Cambodia.

    PubMed

    Lin, Jessica T; Ubalee, Ratawan; Lon, Chanthap; Balasubramanian, Sujata; Kuntawunginn, Worachet; Rahman, Rifat; Saingam, Piyaporn; Heng, Thay Kheang; Vy, Dav; San, Savoeun; Nuom, Sarath; Burkly, Hana; Chanarat, Nitima; Ponsa, Chanudom; Levitz, Lauren; Parobek, Christian; Chuor, Char Meng; Somethy, Sok; Spring, Michele; Lanteri, Charlotte; Gosi, Panita; Meshnick, Steven R; Saunders, David L

    2016-05-01

    Although gametocytes are essential for malaria transmission, in Africa many falciparum-infected persons without smear-detectable gametocytes still infect mosquitoes. To see whether the same is true in Southeast Asia, we determined the infectiousness of 119 falciparum-infected Cambodian adults to Anopheles dirus mosquitoes by membrane feeding. Just 5.9% of subjects infected mosquitoes. The 8.4% of patients with smear-detectable gametocytes were >20 times more likely to infect mosquitoes than those without and were the source of 96% of all mosquito infections. In low-transmission settings, targeting transmission-blocking interventions to those with microscopic gametocytemia may have an outsized effect on malaria control and elimination.

  13. Artemisinin resistance in Plasmodium falciparum: A process linked to dormancy?

    PubMed

    Cheng, Qin; Kyle, Dennis E; Gatton, Michelle L

    2012-12-01

    Artemisinin (ART) based combination therapy (ACT) is used as the first line treatment of uncomplicated falciparum malaria in over 100 countries and is the cornerstone of malaria control and elimination programs in these areas. However, despite the high potency and rapid parasite killing action of ART derivatives there is a high rate of recrudescence associated with ART monotherapy and recrudescence is not uncommon even when ACT is used. Compounding this problem are reports that some parasites in Cambodia, a known foci of drug resistance, have decreased in vivo sensitivity to ART. This raises serious concerns for the development of ART resistance in the field even though no major phenotypic and genotypic changes have yet been identified in these parasites. In this article we review available data on the characteristics of ART, its effects on Plasmodium falciparum parasites and present a hypothesis to explain the high rate of recrudescence associated with this potent class of drugs and the current enigma surrounding ART resistance.

  14. Genotype comparison of Plasmodium vivax and Plasmodium falciparum clones from pregnant and non-pregnant populations in North-west Colombia

    PubMed Central

    2012-01-01

    Background Placental malaria is the predominant pathology secondary to malaria in pregnancy, causing substantial maternal and infant morbidity and mortality in tropical areas. While it is clear that placental parasites are phenotypically different from those in the peripheral circulation, it is not known whether unique genotypes are associated specifically with placental infection or perhaps more generally with pregnancy. In this study, genetic analysis was performed on Plasmodium vivax and Plasmodium falciparum parasites isolated from peripheral and placental blood in pregnant women living in North-west Colombia, and compared with parasites causing acute malaria in non-pregnant populations. Methods A total of 57 pregnant women at delivery with malaria infection confirmed by real-time PCR in peripheral or placental blood were included, as well as 50 pregnant women in antenatal care and 80 men or non-pregnant women with acute malaria confirmed by a positive thick smear for P. vivax or P. falciparum. Five molecular markers per species were genotyped by nested PCR and capillary electrophoresis. Genetic diversity and the fixation index FST per species and study group were calculated and compared. Results Almost all infections at delivery were asymptomatic with significantly lower levels of infection compared with the groups with acute malaria. Expected heterozygosity for P. vivax molecular markers ranged from 0.765 to 0.928 and for P. falciparum markers ranged from 0.331 to 0.604. For P. vivax infections, the genetic diversity was similar amongst the four study groups and the fixation index from each pairwise comparison failed to show significant genetic differentiation. For P. falciparum, no genetic differentiation was observed between placental and peripheral parasites from the same woman at delivery, but the parasites isolated at delivery showed significant genetic differentiation compared with parasites isolated from subjects with acute malaria. Conclusions In

  15. Correlation between 'H' blood group antigen and Plasmodium falciparum invasion.

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2016-06-01

    The ABO blood group system is the most important blood group system in clinical practice. The relationship between Plasmodium falciparum and ABO blood groups has been studied for many years. This study was undertaken to investigate the abilities of different blood group erythrocytes to support in vitro growth of P. falciparum parasites. P. falciparum parasites of four different strains (3D7, 7G8, Dd2 and RKL9) were co-cultured with erythrocytes of blood group 'A', 'B', 'O' (n = 10 for each) and 'O(h)' (Bombay group) (n = 7) for 5 days. Statistically significant differences were observed on the fourth day among the mean percent parasitemias of 'O', non-'O' ('A' and 'B') and 'O(h)' group cultures. The parasitemias of four strains ranged from 12.23 to 14.66, 11.68 to 13.24, 16.89 to 22.3, and 7.37 to 11.27 % in 'A', 'B', 'O' and Bombay group cultures, respectively. As the expression of H antigen decreased from 'O' blood group to 'A' and 'B' and then to Bombay blood group, parasite invasion (percent parasitemia) also decreased significantly (p < 0.01) and concomitantly, indicating the association of parasite invasion with the amount of H antigen present on the surface of erythrocyte. Thus, the question arises, could H antigen be involved in P. falciparum invasion? To evaluate erythrocyte invasion inhibition, 'O' group erythrocytes were virtually converted to Bombay group-like erythrocytes by the treatment of anti-H lectins extracted from Ulex europaeus seeds. Mean percent parasitemia of lectin-treated cultures on the fourth day was significantly lower (p < 0.05) than that of non-treated cultures and was found to be similar with the mean percent parasitemia demonstrated by the Bombay group erythrocyte cultures, thus further strengthening the hypothesis.

  16. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    SciTech Connect

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  17. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  18. Protein farnesyltransferase and protein prenylation in Plasmodium falciparum.

    PubMed

    Chakrabarti, Debopam; Da Silva, Thiago; Barger, Jennifer; Paquette, Steve; Patel, Hetal; Patterson, Shelley; Allen, Charles M

    2002-11-01

    Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.

  19. CRISPR-mediated genome editing of Plasmodium falciparum malaria parasites.

    PubMed

    Lee, Marcus Cs; Fidock, David A

    2014-01-01

    The development of the CRISPR-Cas system is revolutionizing genome editing in a variety of organisms. The system has now been used to manipulate the genome of Plasmodium falciparum, the most lethal malaria-causing species. The ability to generate gene deletions or nucleotide substitutions rapidly and economically promises to accelerate the analysis of novel drug targets and to help elucidate the function of specific genes or gene families, while complementing genome-wide association studies.

  20. Parasite-induced permeation of nucleosides in Plasmodium falciparum malaria.

    PubMed

    Upston, J M; Gero, A M

    1995-06-14

    A mechanism which mediates the transport of the nonphysiological nucleoside, L-adenosine, was demonstrated in Plasmodium falciparum infected erythrocytes and naturally released merozoites. L-Adenosine was not a substrate for influx in freed intraerythrocytic parasites or in normal human erythrocytes nor was L-adenosine transported in a variety of cell types including other parasitic protozoa such as Crithidia luciliae, Trichomonas vaginalis, Giardia intestinalis, or the mammalian cells, Buffalo Green Monkey and HeLa cells. L-Adenosine transport in P. falciparum infected cells was nonsaturable, with a rate of 0.13 +/- 0.01 pmol/microliter cell water per s per microM L-adenosine, yet the transport was inhibited by furosemide, phloridzin and piperine with IC50 values between 1-13 microM, distinguishing the transport pathway from simple diffusion. The channel-like permeation was selective as disaccharides were not permeable to parasitised cells. In addition, an unusual metabolic property of parasitic adenosine deaminase was found in that L-adenosine was metabolised to L-inosine by both P. falciparum infected erythrocytes and merozoites, an activity which was inhibited by 50 nM deoxycoformycin. No other cell type examined displayed this enzymic activity. The results further substantiate that nucleoside transport in P. falciparum infected cells was significantly altered compared to uninfected erythrocytes and that L-adenosine transport and metabolism was a biochemical property of Plasmodium infected cells and merozoites and not found in normal erythrocytes nor any of the other cell types investigated.

  1. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  2. An outbreak of Plasmodium falciparum malaria in the Torres Strait.

    PubMed

    Preston-Thomas, Annie; Gair, Richard W; Hosking, Kelly A; Devine, Gregor J; Donohue, Steven D

    2012-06-01

    This report describes the largest outbreak of Plasmodium falciparum malaria in the Torres Strait for more than 25 years. It details factors that may have contributed to the outbreak, the public health response and implications for the broader region. Eight cases of locally-acquired falciparum malaria occurred on Saibai and Dauan islands during March and April 2011. Including imports, there were 17 P. falciparum notifications between February and May 2011. Three cases of pure P. vivax malaria that might have been locally acquired have been omitted from this report. Malaria is endemic on the nearby coast of Papua New Guinea (PNG), and regularly imported to the Torres Strait where a competent vector exists in sufficient numbers to transmit the disease to the local population. The most common malaria vectors in northern Australia and Torres Strait are the Anopheles farauti complex. Factors contributing to the outbreak may include an increase in travel between the outer islands and PNG, inadequate local vector control and late or missed diagnoses of malaria. Outbreak management involved intensive case finding and treatment, vector control and health promotion. Reducing the risk of future outbreaks requires studies of vector behaviour, ecology and management, health promotion, improvements to protective infrastructure, and clinical guideline revision. Further malaria outbreaks are likely in the Torres Strait and elsewhere in northern Australia. It is important to maintain awareness and be prepared to respond rapidly. PMID:23186217

  3. Systematic Study of the Content of Phytochemicals in Fresh and Fresh-Cut Vegetables.

    PubMed

    Alarcón-Flores, María Isabel; Romero-González, Roberto; Vidal, José Luis Martínez; Frenich, Antonia Garrido

    2015-01-01

    Vegetables and fruits have beneficial properties for human health, because of the presence of phytochemicals, but their concentration can fluctuate throughout the year. A systematic study of the phytochemical content in tomato, eggplant, carrot, broccoli and grape (fresh and fresh-cut) has been performed at different seasons, using liquid chromatography coupled to triple quadrupole mass spectrometry. It was observed that phenolic acids (the predominant group in carrot, eggplant and tomato) were found at higher concentrations in fresh carrot than in fresh-cut carrot. However, in the case of eggplant, they were detected at a higher content in fresh-cut than in fresh samples. Regarding tomato, the differences in the content of phenolic acids between fresh and fresh-cut were lower than in other matrices, except in winter sampling, where this family was detected at the highest concentration in fresh tomato. In grape, the flavonols content (predominant group) was higher in fresh grape than in fresh-cut during all samplings. The content of glucosinolates was lower in fresh-cut broccoli than in fresh samples in winter and spring sampling, although this trend changes in summer and autumn. In summary, phytochemical concentration did show significant differences during one-year monitoring, and the families of phytochemicals presented different behaviors depending on the matrix studied. PMID:26783709

  4. Extreme geographical fixation of variation in the Plasmodium falciparum gamete surface protein gene Pfs48/45 compared with microsatellite loci.

    PubMed

    Conway, D J; Machado, R L; Singh, B; Dessert, P; Mikes, Z S; Povoa, M M; Oduola, A M; Roper, C

    2001-07-01

    Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility. PMID:11420101

  5. Development of a selective agar plate for the detection of Campylobacter spp. in fresh produce.

    PubMed

    Yoo, Jin-Hee; Choi, Na-Young; Bae, Young-Min; Lee, Jung-Su; Lee, Sun-Young

    2014-10-17

    This study was conducted to develop a selective medium for the detection of Campylobacter spp. in fresh produce. Campylobacter spp. (n=4), non-Campylobacter (showing positive results on Campylobacter selective agar) strains (n=49) isolated from fresh produce, indicator bacteria (n=13), and spoilage bacteria isolated from fresh produce (n=15) were plated on four Campylobacter selective media. Bolton agar and modified charcoal cefoperazone deoxycholate agar (mCCDA) exhibited higher sensitivity for Campylobacter spp. than did Preston agar and Hunt agar, although certain non-Campylobacter strains isolated from fresh produce by using a selective agar isolation method, were still able to grow on Bolton agar and mCCDA. To inhibit the growth of non-Campylobacter strains, Bolton agar and mCCDA were supplemented with 5 antibiotics (rifampicin, polymyxin B, sodium metabisulfite, sodium pyruvate, ferrous sulfate) and the growth of Campylobacter spp. (n=7) and non-Campylobacter strains (n=44) was evaluated. Although Bolton agar supplemented with rifampicin (BR agar) exhibited a higher selectivity for Campylobacter spp. than did mCCDA supplemented with antibiotics, certain non-Campylobacter strains were still able to grow on BR agar (18.8%). When BR agar with various concentrations of sulfamethoxazole-trimethoprim were tested with Campylobacter spp. (n=8) and non-Campylobacter (n=7), sulfamethoxazole-trimethoprim was inhibitory against 3 of 7 non-Campylobacter strains. Finally, we validated the use of BR agar containing 50mg/L sulfamethoxazole (BRS agar) or 0.5mg/L ciprofloxacin (BRCS agar) and other selective agars for the detection of Campylobacter spp. in chicken and fresh produce. All chicken samples were positive for Campylobacter spp. when tested on mCCDA, BR agar, and BRS agar. In fresh produce samples, BRS agar exhibited the highest selectivity for Campylobacter spp., demonstrating its suitability for the detection of Campylobacter spp. in fresh produce.

  6. A new world malaria map: Plasmodium falciparum endemicity in 2010

    PubMed Central

    2011-01-01

    Background Transmission intensity affects almost all aspects of malaria epidemiology and the impact of malaria on human populations. Maps of transmission intensity are necessary to identify populations at different levels of risk and to evaluate objectively options for disease control. To remain relevant operationally, such maps must be updated frequently. Following the first global effort to map Plasmodium falciparum malaria endemicity in 2007, this paper describes the generation of a new world map for the year 2010. This analysis is extended to provide the first global estimates of two other metrics of transmission intensity for P. falciparum that underpin contemporary questions in malaria control: the entomological inoculation rate (PfEIR) and the basic reproductive number (PfR). Methods Annual parasite incidence data for 13,449 administrative units in 43 endemic countries were sourced to define the spatial limits of P. falciparum transmission in 2010 and 22,212 P. falciparum parasite rate (PfPR) surveys were used in a model-based geostatistical (MBG) prediction to create a continuous contemporary surface of malaria endemicity within these limits. A suite of transmission models were developed that link PfPR to PfEIR and PfR and these were fitted to field data. These models were combined with the PfPR map to create new global predictions of PfEIR and PfR. All output maps included measured uncertainty. Results An estimated 1.13 and 1.44 billion people worldwide were at risk of unstable and stable P. falciparum malaria, respectively. The majority of the endemic world was predicted with a median PfEIR of less than one and a median PfRc of less than two. Values of either metric exceeding 10 were almost exclusive to Africa. The uncertainty described in both PfEIR and PfR was substantial in regions of intense transmission. Conclusions The year 2010 has a particular significance as an evaluation milestone for malaria global health policy. The maps presented here

  7. Whole transcriptome expression analysis and comparison of two different strains of Plasmodium falciparum using RNA-Seq.

    PubMed

    Antony, Hiasindh Ashmi; Pathak, Vrushali; Parija, Subhash Chandra; Ghosh, Kanjaksha; Bhattacherjee, Amrita

    2016-06-01

    The emergence and distribution of drug resistance in malaria are serious public health concerns in tropical and subtropical regions of the world. However, the molecular mechanism of drug resistance remains unclear. In the present study, we performed a high-throughput RNA-Seq to identify and characterize the differentially expressed genes between the chloroquine (CQ) sensitive (3D7) and resistant (Dd2) strains of Plasmodium falciparum. The parasite cells were cultured in the presence and absence of CQ by in vitro method. Total RNA was isolated from the harvested parasite cells using TRIzol, and RNA-Seq was conducted using an Illumina HiSeq 2500 sequencing platform with paired-end reads and annotated using Tophat. The transcriptome analysis of P. falciparum revealed the expression of ~ 5000 genes, in which ~ 60% of the genes have unknown function. Cuffdiff program was used to identify the differentially expressed genes between the CQ-sensitive and resistant strains. Here, we furnish a detailed description of the experimental design, procedure, and analysis of the transcriptome sequencing data, that have been deposited in the National Center for Biotechnology Information (accession nos. PRJNA308455 and GSE77499). PMID:27222812

  8. Whole transcriptome expression analysis and comparison of two different strains of Plasmodium falciparum using RNA-Seq.

    PubMed

    Antony, Hiasindh Ashmi; Pathak, Vrushali; Parija, Subhash Chandra; Ghosh, Kanjaksha; Bhattacherjee, Amrita

    2016-06-01

    The emergence and distribution of drug resistance in malaria are serious public health concerns in tropical and subtropical regions of the world. However, the molecular mechanism of drug resistance remains unclear. In the present study, we performed a high-throughput RNA-Seq to identify and characterize the differentially expressed genes between the chloroquine (CQ) sensitive (3D7) and resistant (Dd2) strains of Plasmodium falciparum. The parasite cells were cultured in the presence and absence of CQ by in vitro method. Total RNA was isolated from the harvested parasite cells using TRIzol, and RNA-Seq was conducted using an Illumina HiSeq 2500 sequencing platform with paired-end reads and annotated using Tophat. The transcriptome analysis of P. falciparum revealed the expression of ~ 5000 genes, in which ~ 60% of the genes have unknown function. Cuffdiff program was used to identify the differentially expressed genes between the CQ-sensitive and resistant strains. Here, we furnish a detailed description of the experimental design, procedure, and analysis of the transcriptome sequencing data, that have been deposited in the National Center for Biotechnology Information (accession nos. PRJNA308455 and GSE77499).

  9. Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

    PubMed Central

    Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F.; Griffing, Sean M.; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J.; Bacon, David J.; Barnwell, John W.; Udhayakumar, Venkatachalam

    2013-01-01

    The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times. PMID:24077522

  10. Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru.

    PubMed

    Akinyi, Sheila; Hayden, Tonya; Gamboa, Dionicia; Torres, Katherine; Bendezu, Jorge; Abdallah, Joseph F; Griffing, Sean M; Quezada, Wilmer Marquiño; Arrospide, Nancy; De Oliveira, Alexandre Macedo; Lucas, Carmen; Magill, Alan J; Bacon, David J; Barnwell, John W; Udhayakumar, Venkatachalam

    2013-09-30

    The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.

  11. Within-population genetic diversity of Plasmodium falciparum vaccine candidate antigens reveals geographic distance from a Central sub-Saharan African origin.

    PubMed

    Tanabe, Kazuyuki; Mita, Toshihiro; Palacpac, Nirianne M Q; Arisue, Nobuko; Tougan, Takahiro; Kawai, Satoru; Jombart, Thibaut; Kobayashi, Fumie; Horii, Toshihiro

    2013-02-18

    Populations of Plasmodium falciparum, the most virulent human malaria parasite, are diverse owing to wide levels of transmission and endemicity of infection. Genetic diversity of P. falciparum antigens, within and between parasite populations, remains a confounding factor in malaria pathogenesis as well as clinical trials of vaccine candidates. Variation of target antigens in parasite populations may arise from immune pressure depending on the levels of acquired immunity. Alternatively, similar to our study in housekeeping genes [Tanabe et al. Curr Biol 2010;70:1-7], within-population genetic diversity of vaccine candidate antigens may also be determined by geographical distance from a postulated origin in Central sub-Saharan Africa. To address this question, we obtained full-length sequences of P. falciparum genes, apical membrane antigen 1 (ama1) (n=459), circumsporozoite protein (csp) (n=472) and merozoite surface protein 1 (msp1) (n=389) from seven geographically diverse parasite populations in Africa, Southeast Asia and Oceania; and, together with previously determined sequences (n=13 and 15 for csp and msp1, respectively) analyzed within-population single nucleotide polymorphism (SNP) diversity. The three antigen genes showed SNP diversity that supports a model of isolation-by-distance. The standardized number of polymorphic sites per site, expressed as θ(S), indicates that 77-83% can be attributed by geographic distance from the African origin, suggesting that geographic distance plays a significant role in variation in target vaccine candidate antigens. Furthermore, we observed that a large proportion of SNPs in the antigen genes were shared between African and non-African parasite populations, demonstrating long term persistence of those SNPs. Our results provide important implications for developing effective malaria vaccines and better understanding of acquired immunity against falciparum malaria. PMID:23295064

  12. What determines fresh fish consumption in Croatia?

    PubMed

    Tomić, Marina; Matulić, Daniel; Jelić, Margareta

    2016-11-01

    Although fresh fish is widely available, consumption still remains below the recommended intake levels among the majority of European consumers. The economic crisis affects consumer food behaviour, therefore fresh fish is perceived as healthy but expensive food product. The aim of this study was to determine the factors influencing fresh fish consumption using an expanded Theory of Planned Behaviour (Ajzen, 1991) as a theoretical framework. The survey was conducted on a heterogeneous sample of 1151 Croatian fresh fish consumers. The study investigated the relationship between attitudes, perceived behavioural control, subjective norm, moral obligation, involvement in health, availability, intention and consumption of fresh fish. Structural Equation Modeling by Partial Least Squares was used to analyse the collected data. The results indicated that attitudes are the strongest positive predictor of the intention to consume fresh fish. Other significant predictors of the intention to consume fresh fish were perceived behavioural control, subjective norm, health involvement and moral obligation. The intention to consume fresh fish showed a strong positive correlation with behaviour. This survey provides valuable information for food marketing professionals and for the food industry in general. PMID:26721719

  13. DISPERSIBILITY OF CRUDE OIL IN FRESH WATER

    EPA Science Inventory

    The effects of surfactant composition on the ability of chemical dispersants to disperse crude oil in fresh water were investigated. The objective of this research was to determine whether effective fresh water dispersants can be designed in case this technology is ever consider...

  14. Fresh fruit: microstructure, texture and quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fresh-cut produce has a huge following in today’s supermarkets. The trend follows the need to decrease preparation time as well as the desire to follow the current health guidelines for consumption of more whole “heart-healthy” foods. Additionally, consumers are able to enjoy a variety of fresh prod...

  15. Microbial Safety of Fresh Produce - Preface

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fresh produce has been the source of recent outbreaks of foodborne illness which have caused sickness, hospitalizations and deaths of consumers, as well as serious adverse economic impact on growers and processors. The preface for the book entitled “Microbial Safety of Fresh Produce” discusses possi...

  16. High Performance of Histidine-Rich Protein 2 Based Rapid Diagnostic Tests in French Guiana are Explained by the Absence of pfhrp2 Gene Deletion in P. falciparum

    PubMed Central

    Berger, Franck; Volney, Béatrice; Blanchet, Denis; Faway, Emilie; Donato, Damien; Legrand, Eric; Carme, Bernard; Musset, Lise

    2013-01-01

    Background Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs). Most RDTs target the histidine-rich protein-2 antigen (PfHRP2) to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. Methods The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. Results Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9–99.3), and 86.0% (95% CI: 78.9–91.5) for the detection of P. vivax. No isolates (95% CI: 0–4.5) lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8–15.4) lacked the exon 2 part of the pfhrp3 gene. Conclusions Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment. PMID:24086328

  17. Possible Clinical Failure of Artemether-Lumefantrine in an Italian Traveler with Uncomplicated Falciparum Malaria.

    PubMed Central

    Repetto, Ernestina C.; Traverso, Antonio; Giacomazzi, Claudio G.

    2011-01-01

    Artemisinin-combination therapies (ACTs) are recommended for the treatment of uncomplicated malaria in endemic areas with multidrug resistant Plasmodium falciparum. We report a case of possible artemether-lumefantrine clinical failure in an Italian traveler with uncomplicated P. falciparum malaria imported from Democratic Republic of Congo. PMID:22084655

  18. Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

    1989-06-01

    The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

  19. Falciparum malaria and climate change in the northwest frontier province of Pakistan.

    PubMed

    Bouma, M J; Dye, C; van der Kaay, H J

    1996-08-01

    Following a striking increase in the severity of autumnal outbreaks of Plasmodium falciparum during the last decade in the Northwest Frontier Province (NWFP) of Pakistan, the role of climatologic variables was investigated. A multivariate analysis showed that during the transmission season of P. falciparum, the amount of rainfall in September and October, the temperature in November and December, and the humidity in December were all correlated (r2 = 0.82) with two measures of P. falciparum, the falciparum rate (percent of slides examined positive for P. falciparum) since 1981 and the annual P. falciparum proportion (percent of all malaria infections diagnosed as P. falciparum) since 1978. Climatologic records since 1876 show an increase in mean November and December temperatures by 2 degrees C and 1.5 degrees C, respectively, and in October rainfall. Mean humidity in December has also been increasing since 1950. These climatologic changes in the area appear to have made conditions for transmission of P. falciparum more favorable, and may account for the increase in incidence observed in the NWFP in recent years.

  20. 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.

  1. Immunogenicity and protective efficacy of affinity-purified Plasmodium falciparum exoantigens in Aotus nancymai monkeys.

    PubMed

    James, M A; Fajfar-Whetstone, C J; Kakoma, I; Buese, M M; Clabaugh, G W; Hansen, R; Ristic, M

    1991-03-01

    Soluble Plasmodium falciparum polypeptides, affinity-purified from culture supernatant fluids using sequential immunoadsorptions employing both monoclonal and polyclonal antibodies, induced protective immunity against experimental falciparum malaria in Peruvian Aotus nancymai monkeys. Susceptible monkeys were vaccinated with polypeptides affinity-purified from supernatant fluids of P. falciparum Indochina I/CDC cultures. Eighteen animals (6 immunized with purified antigens plus adjuvants, 6 injected with only the adjuvant preparation, and 6 untreated) were challenged with whole blood containing monkey-adapted virulent organisms of the Indochina I/CDC strain. Selected hematologic, serologic and parasitologic profiles served as potential indicators of protection. This immunogen, when fortified with an aluminum hydroxide/Quil-A saponin adjuvant combination, elicited good antibody responses to major P. falciparum antigens. Protection in vaccinated animals was evidenced by a significantly limited reduction in hematocrit and hemoglobin levels and a relatively moderate course of infection after homologous needle-challenge with Aotus monkey-adapted P. falciparum parasites.

  2. Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

    PubMed Central

    2010-01-01

    Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation. PMID:20470441

  3. 9 CFR 319.141 - Fresh pork sausage.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Fresh pork sausage. 319.141 Section... INSPECTION AND CERTIFICATION DEFINITIONS AND STANDARDS OF IDENTITY OR COMPOSITION Sausage Generally: Fresh Sausage § 319.141 Fresh pork sausage. “Fresh Pork Sausage” is sausage prepared with fresh pork or...

  4. 9 CFR 319.142 - Fresh beef sausage.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Fresh beef sausage. 319.142 Section... INSPECTION AND CERTIFICATION DEFINITIONS AND STANDARDS OF IDENTITY OR COMPOSITION Sausage Generally: Fresh Sausage § 319.142 Fresh beef sausage. “Fresh Beef Sausage” is sausage prepared with fresh beef or...

  5. [Plasmodium falciparum malaria: evaluation of three imported cases].

    PubMed

    İnkaya, Ahmet Çağkan; Kaya, Filiz; Yıldız, İrem; Uzun, Ömrüm; Ergüven, Sibel

    2016-04-01

    Among Plasmodium species the causative agent of malaria in Turkey is P.vivax, however the incidence of imported falciparum malaria cases is steadily increasing. P.falciparum may cause severe malaria with the involvement of central nervous system, acute renal failure, severe anemia or acute respiratory distress syndrome. Furhermore most of the casualties due to malaria are related with P.falciparum. There is recently, a considerable increase in malaria infections especially in tropical areas. In this report, three cases, who have admitted to our hospital with three different clinical presentations of falciparum malaria, and all shared common history of travelling to Africa were presented. First case was a 27 years old, male patient who returned from Malawi seven days ago where he stayed for two weeks. He admitted to our hospital with the complaints of sensation of cold, shivering and fever. In physical examination his body temperature was 37.9°C, C-reactive protein level was high, and the other systemic results were normal. The second case was a 25 years old, male patient who returned from Gambia two weeks ago. He was suffering from fever, headache, shivering and unable to maintain his balance. The patient's body temperature was 38°C. Laboratory tests revealed hyperbilirubinemia and thrombocytopenia. Parasitological examination of the Giemsa-stained peripheral blood smear of these two patients demonstrated ring forms compatible with P.falciparum. Treatment was commenced with arthemeter plus lumefantrine, resulting with complete cure. Third case was a 46 years old, male patient who had been working in Uganda, and returned to Turkey two weeks ago. He had sudden onset of fever, headache, nausea and vomiting and impaired consciousness. His peripheral blood smear revealed ring-formed trophozoites and banana-shaped gametocytes of P.falciparum. Arthemeter plus lumefantrine therapy was started, however, he developed severe thrombocytopenia and jaundice under treatment

  6. A World Malaria Map: Plasmodium falciparum Endemicity in 2007

    PubMed Central

    Hay, Simon I; Guerra, Carlos A; Gething, Peter W; Patil, Anand P; Tatem, Andrew J; Noor, Abdisalan M; Kabaria, Caroline W; Manh, Bui H; Elyazar, Iqbal R. F; Brooker, Simon; Smith, David L; Moyeed, Rana A; Snow, Robert W

    2009-01-01

    Background Efficient allocation of resources to intervene against malaria requires a detailed understanding of the contemporary spatial distribution of malaria risk. It is exactly 40 y since the last global map of malaria endemicity was published. This paper describes the generation of a new world map of Plasmodium falciparum malaria endemicity for the year 2007. Methods and Findings A total of 8,938 P. falciparum parasite rate (PfPR) surveys were identified using a variety of exhaustive search strategies. Of these, 7,953 passed strict data fidelity tests for inclusion into a global database of PfPR data, age-standardized to 2–10 y for endemicity mapping. A model-based geostatistical procedure was used to create a continuous surface of malaria endemicity within previously defined stable spatial limits of P. falciparum transmission. These procedures were implemented within a Bayesian statistical framework so that the uncertainty of these predictions could be evaluated robustly. The uncertainty was expressed as the probability of predicting correctly one of three endemicity classes; previously stratified to be an informative guide for malaria control. Population at risk estimates, adjusted for the transmission modifying effects of urbanization in Africa, were then derived with reference to human population surfaces in 2007. Of the 1.38 billion people at risk of stable P. falciparum malaria, 0.69 billion were found in Central and South East Asia (CSE Asia), 0.66 billion in Africa, Yemen, and Saudi Arabia (Africa+), and 0.04 billion in the Americas. All those exposed to stable risk in the Americas were in the lowest endemicity class (PfPR2−10 ≤ 5%). The vast majority (88%) of those living under stable risk in CSE Asia were also in this low endemicity class; a small remainder (11%) were in the intermediate endemicity class (PfPR2−10 > 5 to < 40%); and the remaining fraction (1%) in high endemicity (PfPR2−10 ≥ 40%) areas. High endemicity was widespread in the

  7. Numbers of fecal streptococci and Escherichia coli in fresh and dry cattle, horse, and sheep manure.

    PubMed

    Weaver, R W; Entry, J A; Graves, Alexandria

    2005-10-01

    Livestock are known contributors to stream pollution. Numbers of fecal streptococci and Escherichia coli in manure naturally deposited by livestock in the field are needed for activities related to bacterial source tracking and determining maximum daily bacterial loading of streams. We measured populations of fecal streptococci and E. coli in fresh and dry manure from cattle (Bos taurus L.), horses (Equus caballus L.), and sheep (Ovis aires L.) on farms in southern Idaho. Populations of indicator bacteria in dry manure were often as high as that in fresh manure from horse and sheep. There was a 2 log10 drop in the population of fecal coliform numbers in dry cattle manure from cattle in pastures but not from cattle in pens. Bacterial isolates used in source tracking should include isolates from both fresh and dry manure to better represent the bacterial source loading of streams.

  8. Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

    PubMed

    Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

    2016-10-01

    Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. PMID:27458230

  9. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  10. Transition State Analogues of Plasmodium falciparum and Human Orotate Phosphoribosyltransferases*

    PubMed Central

    Zhang, Yong; Evans, Gary B.; Clinch, Keith; Crump, Douglas R.; Harris, Lawrence D.; Fröhlich, Richard F. G.; Tyler, Peter C.; Hazleton, Keith Z.; Cassera, María B.; Schramm, Vern L.

    2013-01-01

    The survival and proliferation of Plasmodium falciparum parasites and human cancer cells require de novo pyrimidine synthesis to supply RNA and DNA precursors. Orotate phosphoribosyltransferase (OPRT) is an indispensible component in this metabolic pathway and is a target for antimalarials and antitumor drugs. P. falciparum (Pf) and Homo sapiens (Hs) OPRTs are characterized by highly dissociative transition states with ribocation character. On the basis of the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states, analogues were designed, synthesized, and tested as inhibitors. Iminoribitol mimics of the ribocation transition state in linkage to pyrimidine mimics using methylene or ethylene linkers gave dissociation constants (Kd) as low as 80 nm. Inhibitors with pyrrolidine groups as ribocation mimics displayed slightly weaker binding affinities for OPRTs. Interestingly, p-nitrophenyl riboside 5′-phosphate bound to OPRTs with Kd values near 40 nm. Analogues designed with a C5-pyrimidine carbon–carbon bond to ribocation mimics gave Kd values in the range of 80–500 nm. Acyclic inhibitors with achiral serinol groups as the ribocation mimics also displayed nanomolar inhibition against OPRTs. In comparison with the nucleoside derivatives, inhibition constants of their corresponding 5′-phosphorylated transition state analogues are largely unchanged, an unusual property for a nucleotide-binding site. In silico docking of the best inhibitor into the HsOPRT active site supported an extensive hydrogen bond network associated with the tight binding affinity. These OPRT transition state analogues identify crucial components of potent inhibitors targeting OPRT enzymes. Despite their tight binding to the targets, the inhibitors did not kill cultured P. falciparum. PMID:24158442

  11. [Artemisinin resistance in Plasmodium falciparum: global status and basic research].

    PubMed

    Zhao, Shao-min; Wang, Man-yuan

    2014-10-01

    Artemisinin-resistant Plasmodium falciparum has been identified by WHO in the Greater Mekong subregion. While there is no report on artemisinin resistance in Africa and South America by now, related surveillance measures have been taken place. The genes related artemisinin-resistance has been identified and the molecular markers will be used for large-scale surveillance efforts to contain artemisinin resistance. The emergence and spread of artemisinin resistance worldwide is a present danger and needs more attention. This article reviews the progress of artemisininresistance malaria parasites and artemisinin-based combination therapies. PMID:25726605

  12. The epidemiology of Plasmodium falciparum gametocytes: weapons of mass dispersion.

    PubMed

    Drakeley, Chris; Sutherland, Colin; Bousema, J Teun; Sauerwein, Robert W; Targett, Geoffrey A T

    2006-09-01

    Much of the epidemiology of Plasmodium falciparum in Sub-Saharan Africa focuses on the prevalence patterns of asexual parasites in people of different ages, whereas the gametocytes that propagate the disease are often neglected. One expected benefit of the widespread introduction of artemisinin-based combination therapy for malaria is a reduction in gametocyte carriage. However, the factors that affect the transmission of parasites from humans to mosquitoes show complex dynamics in relation to the intensity and seasonality of malaria transmission, and thus such benefits might not be automatic. Here, we review data on gametocyte carriage in the context of the development of naturally acquired immunity and population infectivity. PMID:16846756

  13. Replication and maintenance of the Plasmodium falciparum apicoplast genome.

    PubMed

    Milton, Morgan E; Nelson, Scott W

    2016-08-01

    Members of the phylum Apicomplexa are responsible for many devastating diseases including malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), babesiosis (Babesia bovis), and cyclosporiasis (Cyclospora cayetanensis). Most Apicomplexans contain a unique and essential organelle called the apicoplast. Derived from an ancient chloroplast, the apicoplast replicates and maintains a 35 kilobase (kb) circular genome. Due to its essential nature within the parasite, drugs targeted to proteins involved in DNA replication and repair of the apicoplast should be potent and specific. This review summarizes the current knowledge surrounding the replication and repair of the Plasmodium falciparum apicoplast genome and identifies several putative proteins involved in replication and repair pathways. PMID:27338018

  14. Antimicrobial activity of fresh garlic juice: An in vitro study

    PubMed Central

    Yadav, Seema; Trivedi, Niyati A.; Bhatt, Jagat D.

    2015-01-01

    Introduction: Antimicrobial resistance has been a global concern. Currently, interest has been focused on exploring antimicrobial properties of plants and herbs. One such botanical is Allium sativum (garlic). Aim: To evaluate the antimicrobial activity of fresh juice of garlic. Materials and Methods: Varying concentrations of fresh garlic juice (FGJ) were tested for their antimicrobial activity against common pathogenic organisms isolated at SSG Hospital, Vadodara, using well diffusion method. Moreover, minimum inhibitory concentration (MIC) and minimum lethal concentration (MLC) of FGJ were tested using broth dilution method. Sensitivity pattern of the conventional antimicrobials against common pathogenic bacteria was tested using disc diffusion method. Results: FGJ produced dose-dependent increase in the zone of inhibition at a concentration of 10% and higher. MIC of FGJ against the pathogens ranged from 4% to 16% v/v whereas MLC value ranged from 4% to 32% v/v with Escherichia coli and Staphylococcus aureus spp. showed highest sensitivity. Conclusion: FGJ has definite antimicrobial activity against common pathogenic organisms isolated at SSG Hospital, Vadodara. Further studies are needed to find out the efficacy, safety, and kinetic data of its active ingredients. PMID:27011724

  15. Two novel aromatic glucosides, marylaurencinosides D and E, from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'.

    PubMed

    Yoshikawa, Kazuko; Okahuji, Mariko; Iseki, Kanako; Ito, Takuya; Asakawa, Yoshinori; Kawano, Sachiko; Hashimoto, Toshihiro

    2014-04-01

    Two novel aromatic glucosides, named marylaurencinosides D (1) and E (2), were isolated from the fresh flowers of Cymbidium Great Flower 'Marylaurencin'. In addition, eight known aromatic compounds (3-10) were isolated. These structures were determined on the basis of NMR experiments as well as chemical evidence.

  16. Polyamine quinoline rhodium complexes: synthesis and pharmacological evaluation as antiparasitic agents against Plasmodium falciparum and Trichomonas vaginalis.

    PubMed

    Stringer, Tameryn; Taylor, Dale; Guzgay, Hajira; Shokar, Ajit; Au, Aaron; Smith, Peter J; Hendricks, Denver T; Land, Kirkwood M; Egan, Timothy J; Smith, Gregory S

    2015-09-01

    A series of mono- and bis-salicylaldimine ligands and their corresponding Rh(i) complexes were prepared. The compounds were characterised using standard spectroscopic techniques including NMR, IR spectroscopy and mass spectrometry. The salicylaldimine ligands and complexes were screened for antiparasitic activity against two strains of Plasmodium falciparum i.e. the NF54 CQ-sensitive and K1 CQ-resistant strain as well as against the G3 isolate of Trichomonas vaginalis. The monomeric salicylaldimine quinolines exhibited good activity against the NF54 strain and the dimeric salicylaldimine quinolines exhibited no cross resistance across the two strains. The binuclear 5-chloro Rh(i) complex displayed the best activity against the Trichomonas vaginalis parasite, possibly a consequence of its enhanced lipophilicity. The compounds were also screened for cytotoxicity in vitro against WHCO1 oesophageal cancer cells. The monomeric salicylaldimine quinolines exhibited high selectivity towards malaria parasites compared to cancer cells, while the dimeric compounds were less selective.

  17. Dispersibility of crude oil in fresh water.

    PubMed

    Wrenn, B A; Virkus, A; Mukherjee, B; Venosa, A D

    2009-06-01

    The effects of surfactant composition on the ability of chemical dispersants to disperse crude oil in fresh water were investigated. The objective of this research was to determine whether effective fresh water dispersants can be designed in case this technology is ever considered for use in fresh water environments. Previous studies on the chemical dispersion of crude oil in fresh water neither identified the dispersants that were investigated nor described the chemistry of the surfactants used. This information is necessary for developing a more fundamental understanding of chemical dispersion of crude oil at low salinity. Therefore, we evaluated the relationship between surfactant chemistry and dispersion effectiveness. We found that dispersants can be designed to drive an oil slick into the freshwater column with the same efficiency as in salt water as long as the hydrophilic-lipophilic balance is optimum.

  18. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... freezing will not preclude use of the term “fresh frozen” to describe the food. “Quickly frozen” means frozen by a freezing system such as blast-freezing (sub-zero Fahrenheit temperature with fast moving...

  19. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... freezing will not preclude use of the term “fresh frozen” to describe the food. “Quickly frozen” means frozen by a freezing system such as blast-freezing (sub-zero Fahrenheit temperature with fast moving...

  20. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... freezing will not preclude use of the term “fresh frozen” to describe the food. “Quickly frozen” means frozen by a freezing system such as blast-freezing (sub-zero Fahrenheit temperature with fast moving...

  1. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... freezing will not preclude use of the term “fresh frozen” to describe the food. “Quickly frozen” means frozen by a freezing system such as blast-freezing (sub-zero Fahrenheit temperature with fast moving...

  2. The redox systems of Plasmodium falciparum and Plasmodium vivax: comparison, in silico analyses and inhibitor studies.

    PubMed

    Mohring, F; Pretzel, J; Jortzik, E; Becker, K

    2014-01-01

    Plasmodium falciparum is responsible for the most severe form of human malaria. P. vivax, in contrast, is the most widespread malaria parasite with an enormous impact on health and economy, since the infection is characterized by high rates of relapses. Due to the mild course of malaria tertiana and complicated in vitro culturing conditions of P. vivax, most of the research on malaria parasites has focused on P. falciparum so far. The redox metabolism of P. falciparum is a promising target for novel antimalarial drugs, since maintaining a redox equilibrium is of fundamental importance for the parasite. P. falciparum contains a cytosolic glutathione and thioredoxin system, as well as redox systems in the apicoplast and the mitochondrion. In contrast to P. falciparum, little is known about the redox processes in P. vivax so far. This review summarizes the current knowledge of the redox metabolism in malaria parasites and provides a detailed in silico comparison of the known and mostly well characterized redox enzymes from P. falciparum and the largely unknown redox proteins from P. vivax. Known antimalarials at least partially mediating their antiparasitic activity by influencing the redox balance of Plasmodium, including dehydroepiandrosterone, Mannich bases, methylene blue, and naphthoquinones, are discussed. Furthermore, we present novel inhibitors identified via screening of a compound library from the Leibniz Institute for Natural Product Research and Infection Biology, Jena that are active against the redox-related enzymes thioredoxin reductase, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase 6- phosphoglucono- lactonase from P. falciparum.

  3. Potentiation of antimalarial drug action by chlorpheniramine against multidrug-resistant Plasmodium falciparum in vitro.

    PubMed

    Nakornchai, Sunan; Konthiang, Phattanapong

    2006-09-01

    Chlorpheniramine, a histamine H1 receptor antagonist, was assayed for in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum K1 strain and chloroquine-resistant P. falciparum T9/94 clone, by measuring the 3H-hypoxanthine incorporation. Chlorphenirame inhibited P. falciparum K1 and T9/94 growth with IC50 values of 136.0+/-40.2 microM and 102.0+/-22.6 microM respectively. A combination of antimalarial drug and chlorpheniramine was tested against resistant P. falciparum in vitro. Isobologram analysis showed that chlorpheniramine exerts marked synergistic action on chloroquine against P. falciparum K1 and T9/94. Chlorpheniramine also potentiated antimalarial action of mefloquine, quinine or pyronaridine against both of the resistant strains of P. falciparum. However, chlorpheniramine antagonism with artesunate was obtained in both P. falciparum K1 and T9/94. The results in this study indicate that antihistaminic drugs may be promising candidates for potentiating antimalarial drug action against drug resistant malarial parasites.

  4. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

    PubMed Central

    Crabb, B S; Cowman, A F

    1996-01-01

    Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest. Images Fig. 4 Fig. 5 PMID:8692985

  5. Global distribution of polymorphisms associated with delayed Plasmodium falciparum parasite clearance following artemisinin treatment: genotyping of archive blood samples.

    PubMed

    Murai, Kenji; Culleton, Richard; Hisaoka, Teruhiko; Endo, Hiroyoshi; Mita, Toshihiro

    2015-06-01

    The recent emergence and spread of artemisinin-resistant Plasmodium falciparum isolates is a growing concern for global malaria-control efforts. A recent genome-wide analysis study identified two SNPs at genomic positions MAL10-688956 and MAL13-1718319, which are linked to delayed clearance of parasites following artemisinin combination therapy (ACT). It is expected that continuous artemisinin pressure will affect the distribution of these SNPs. Here, we investigate the worldwide distribution of these SNPs using a large number of archived samples in order to generate baseline data from the period before the emergence of ACT resistance. The presence of SNPs in MAL10-688956 and MAL13-1718319 was assessed by nested PCR RFLP and direct DNA sequencing using 653 global P. falciparum samples obtained before the reported emergence of ACT resistance. SNPs at MAL10-688956 and MAL13-1718319 associated with delayed parasite clearance following ACT administration were observed in 8% and 3% of parasites, respectively, mostly in Cambodia and Thailand. Parasites harbouring both SNPs were found in only eight (1%) isolates, all of which were from Cambodia and Thailand. Linkage disequilibrium was detected between MAL10-688956 and MAL13-1718319, suggesting that this SNP combination may have been selected by ACT drug pressure. Neither of the SNPs associated with delayed parasite clearance were observed in samples from Africa or South America. Baseline information of the geographical difference of MAL10-688956 and MAL13-1718319 SNPs provides a solid basis for assessing whether these SNPs are selected by artemisinin-based combination therapies.

  6. Immune characterization of Plasmodium falciparum parasites with a shared genetic signature in a region of decreasing transmission.

    PubMed

    Bei, Amy K; Diouf, Ababacar; Miura, Kazutoyo; Larremore, Daniel B; Ribacke, Ulf; Tullo, Gregory; Moss, Eli L; Neafsey, Daniel E; Daniels, Rachel F; Zeituni, Amir E; Nosamiefan, Iguosadolo; Volkman, Sarah K; Ahouidi, Ambroise D; Ndiaye, Daouda; Dieye, Tandakha; Mboup, Souleymane; Buckee, Caroline O; Long, Carole A; Wirth, Dyann F

    2015-01-01

    As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population. PMID:25368109

  7. Global distribution of polymorphisms associated with delayed Plasmodium falciparum parasite clearance following artemisinin treatment: genotyping of archive blood samples.

    PubMed

    Murai, Kenji; Culleton, Richard; Hisaoka, Teruhiko; Endo, Hiroyoshi; Mita, Toshihiro

    2015-06-01

    The recent emergence and spread of artemisinin-resistant Plasmodium falciparum isolates is a growing concern for global malaria-control efforts. A recent genome-wide analysis study identified two SNPs at genomic positions MAL10-688956 and MAL13-1718319, which are linked to delayed clearance of parasites following artemisinin combination therapy (ACT). It is expected that continuous artemisinin pressure will affect the distribution of these SNPs. Here, we investigate the worldwide distribution of these SNPs using a large number of archived samples in order to generate baseline data from the period before the emergence of ACT resistance. The presence of SNPs in MAL10-688956 and MAL13-1718319 was assessed by nested PCR RFLP and direct DNA sequencing using 653 global P. falciparum samples obtained before the reported emergence of ACT resistance. SNPs at MAL10-688956 and MAL13-1718319 associated with delayed parasite clearance following ACT administration were observed in 8% and 3% of parasites, respectively, mostly in Cambodia and Thailand. Parasites harbouring both SNPs were found in only eight (1%) isolates, all of which were from Cambodia and Thailand. Linkage disequilibrium was detected between MAL10-688956 and MAL13-1718319, suggesting that this SNP combination may have been selected by ACT drug pressure. Neither of the SNPs associated with delayed parasite clearance were observed in samples from Africa or South America. Baseline information of the geographical difference of MAL10-688956 and MAL13-1718319 SNPs provides a solid basis for assessing whether these SNPs are selected by artemisinin-based combination therapies. PMID:25449286

  8. Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

    PubMed Central

    Wurtz, Nathalie; Fall, Bécaye; Pascual, Aurélie; Fall, Mansour; Baret, Eric; Camara, Cheikhou; Nakoulima, Aminata; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Wade, Boubacar

    2014-01-01

    The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites. PMID:25199781

  9. Hazardous materials in Fresh Kills landfill

    SciTech Connect

    Hirschhorn, J.S.

    1997-12-31

    No environmental monitoring and corrective action programs can pinpoint multiple locations of hazardous materials the total amount of them in a large landfill. Yet the consequences of hazardous materials in MSW landfills are considerable, in terms of public health concerns, environmental damage, and cleanup costs. In this paper a rough estimation is made of how much hazardous material may have been disposed in Fresh Kills landfill in Staten Island, New York. The logic and methods could be used for other MSW landfills. Fresh Kills has frequently been described as the world`s largest MSW landfill. While records of hazardous waste disposal at Fresh Kills over nearly 50 years of operation certainly do not exist, no reasonable person would argue with the conclusion that large quantities of hazardous waste surely have been disposed at Fresh Kills, both legally and illegally. This study found that at least 2 million tons of hazardous wastes and substances have been disposed at Fresh Kills since 1948. Major sources are: household hazardous waste, commercial RCRA hazardous waste, incinerator ash, and commercial non-RCRA hazardous waste, governmental RCRA hazardous waste. Illegal disposal of hazardous waste surely has contributed even more. This is a sufficient amount to cause serious environmental contamination and releases, especially from such a landfill without an engineered liner system, for example. This figure is roughly 1% of the total amount of waste disposed in Fresh Kills since 1948, probably at least 200 million tons.

  10. Distribution of Drug Resistance Genotypes in Plasmodium falciparum in an Area of Limited Parasite Diversity in Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Farsi, Hissa M.; Al-Hashami, Zainab S.; Al-Sheikh, Adel Ali H.; Al-Qahtani, Ahmed; Babiker, Hamza A.

    2012-01-01

    Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent. PMID:22556074

  11. Plasmodial surface anion channel-independent phloridzin resistance in Plasmodium falciparum.

    PubMed

    Desai, Sanjay A; Alkhalil, Abdulnaser; Kang, Myungsa; Ashfaq, Umar; Nguyen, My-Le

    2005-04-29

    The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasite- or host-encoded remains to be determined. PMID:15701633

  12. Reduced Parasite Burden in Children with Falciparum Malaria and Bacteremia Coinfections: Role of Mediators of Inflammation.

    PubMed

    Davenport, Gregory C; Hittner, James B; Otieno, Vincent; Karim, Zachary; Mukundan, Harshini; Fenimore, Paul W; Hengartner, Nicolas W; McMahon, Benjamin H; Kempaiah, Prakasha; Ong'echa, John M; Perkins, Douglas J

    2016-01-01

    Bacteremia and malaria coinfection is a common and life-threatening condition in children residing in sub-Saharan Africa. We previously showed that coinfection with Gram negative (G[-]) enteric Bacilli and Plasmodium falciparum (Pf[+]) was associated with reduced high-density parasitemia (HDP, >10,000 parasites/μL), enhanced respiratory distress, and severe anemia. Since inflammatory mediators are largely unexplored in such coinfections, circulating cytokines were determined in four groups of children (n = 206, aged <3 yrs): healthy; Pf[+] alone; G[-] coinfected; and G[+] coinfected. Staphylococcus aureus and non-Typhi Salmonella were the most frequently isolated G[+] and G[-] organisms, respectively. Coinfected children, particularly those with G[-] pathogens, had lower parasite burden (peripheral and geometric mean parasitemia and HDP). In addition, both coinfected groups had increased IL-4, IL-5, IL-7, IL-12, IL-15, IL-17, IFN-γ, and IFN-α and decreased TNF-α relative to malaria alone. Children with G[-] coinfection had higher IL-1β and IL-1Ra and lower IL-10 than the Pf[+] group and higher IFN-γ than the G[+] group. To determine how the immune response to malaria regulates parasitemia, cytokine production was investigated with a multiple mediation model. Cytokines with the greatest mediational impact on parasitemia were IL-4, IL-10, IL-12, and IFN-γ. Results here suggest that enhanced immune activation, especially in G[-] coinfected children, acts to reduce malaria parasite burden. PMID:27418744

  13. High levels of Plasmodium falciparum rosetting in all clinical forms of severe malaria in African children.

    PubMed

    Doumbo, Ogobara K; Thera, Mahamadou A; Koné, Abdoulaye K; Raza, Ahmed; Tempest, Louisa J; Lyke, Kirsten E; Plowe, Christopher V; Rowe, J Alexandra

    2009-12-01

    Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific syndromes such as cerebral malaria. We investigated the relationship between rosetting and clinical malaria in 209 Malian children enrolled in a case-control study of severe malaria. Rosetting was significantly higher in parasite isolates from severe malaria cases compared with non-severe hyperparasitemia and uncomplicated malaria controls (F(2,117) = 8.15, P < 0.001). Analysis of sub-categories of severe malaria (unrousable coma, severe anemia, non-comatose neurological impairment, repeated seizures or a small heterogeneous group with signs of renal failure or jaundice) showed high levels of rosetting in all sub-categories, and no statistically significant differences in rosetting between sub-categories (F(4,67) = 1.28, P = 0.28). Thus rosetting may contribute to the pathogenesis of all severe malaria syndromes in African children, and interventions to disrupt rosetting could be potential adjunctive therapies for all forms of severe malaria in Africa. PMID:19996426

  14. PlasmoView: a web-based resource to visualise global Plasmodium falciparum genomic variation.

    PubMed

    Preston, Mark D; Assefa, Samuel A; Ocholla, Harold; Sutherland, Colin J; Borrmann, Steffen; Nzila, Alexis; Michon, Pascal; Hien, Tran Tinh; Bousema, Teun; Drakeley, Christopher J; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A; Doumbo, Ogobara K; Nosten, Francois; Fairhurst, Rick M; Conway, David J; Roper, Cally; Clark, Taane G

    2014-06-01

    Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (eg, biological function) in a geographic setting. The first release contains over 600,000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania). PMID:24338354

  15. PlasmoView: A Web-based Resource to Visualise Global Plasmodium falciparum Genomic Variation

    PubMed Central

    Preston, Mark D.; Assefa, Samuel A.; Ocholla, Harold; Sutherland, Colin J.; Borrmann, Steffen; Nzila, Alexis; Michon, Pascal; Hien, Tran Tinh; Bousema, Teun; Drakeley, Christopher J.; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A.; Doumbo, Ogobara K.; Nosten, Francois; Fairhurst, Rick M.; Conway, David J.; Roper, Cally; Clark, Taane G.

    2014-01-01

    Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (eg, biological function) in a geographic setting. The first release contains over 600 000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania). PMID:24338354

  16. Human immune responses that reduce the transmission of Plasmodium falciparum in African populations

    PubMed Central

    Bousema, Teun; Sutherland, Colin J.; Churcher, Thomas S.; Mulder, Bert; Gouagna, Louis C.; Riley, Eleanor M.; Targett, Geoffrey A.T.; Drakeley, Chris J.

    2011-01-01

    Malaria-infected individuals can develop antibodies which reduce the infectiousness of Plasmodium gametocytes to biting Anopheles mosquitoes. When ingested in a bloodmeal together with gametocytes, these antibodies reduce or prevent subsequent parasite maturation in the insect host. This transmission-blocking immunity is usually measured in human sera by testing its effect on the infectivity of gametocytes grown in vitro. Here we evaluate evidence of transmission-blocking immunity in eight studies conducted in three African countries. Plasmodium falciparum gametocytes isolated from each individual were fed to mosquitoes in both autologous plasma collected with the parasites, and permissive serum from non-exposed donors. Evidence of transmission reducing effects of autologous plasma was found in all countries. Experiments involving 116 Gambian children (aged 0.5–15 years) were combined to determine which factors were associated with transmission reducing immune responses. The chances of infecting at least one mosquito and the average proportion of infected mosquitoes were negatively associated with recent exposure to gametocytes and sampling late in the season. These results suggest that effective malaria transmission-reducing antibodies do not commonly circulate in African children, and that recent gametocyte carriage is required to initiate and/or boost such responses. PMID:20974145

  17. Response of falciparum malaria to different antimalarials in Myanmar.

    PubMed Central

    Ejov, M. N.; Tun, T.; Aung, S.; Sein, K.

    1999-01-01

    The purpose of the study was to ascertain the therapeutic efficacy of different treatments for uncomplicated falciparum malaria in the hospitals in Sagaing, northern and eastern Shan, to facilitate updating the existing national antimalarial drug policy. The proposed 14-day trial for monitoring the efficacy of treatments of uncomplicated falciparum malaria is an efficient method for identifying treatment failure patterns at the intermediate level (township hospital) in the Union of Myanmar. Minimal clinical and parasitological data for days 0-14 were required to classify treatment failure and success. Clinical and parasitiological responses on day 3 and days 4-14 were used as clear examples of early and late treatment failure, respectively. Mefloquine is five times more likely to be effective than chloroquine and sulfadoxine pyrimethamine (S-P), whereas chloroquine and S-P treatments have nearly identical failure patterns. The alarming frequency of clinical and parasitological failure (failure rate > 50%) following chloroquine treatment was reported in Sagaing and following S-P treatment in Sagaing and eastern Shan. PMID:10212515

  18. Dynamic alteration in splenic function during acute falciparum malaria

    SciTech Connect

    Looareesuwan, S.; Ho, M.; Wattanagoon, Y.; White, N.J.; Warrell, D.A.; Bunnag, D.; Harinasuta, T.; Wyler, D.J.

    1987-09-10

    Plasmodium-infected erythrocytes lose their normal deformability and become susceptible to splenic filtration. In animal models, this is one mechanism of antimalarial defense. To assess the effect of acute falciparum malaria on splenic filtration, we measured the clearance of heated /sup 51/Cr-labeled autologous erythrocytes in 25 patients with acute falciparum malaria and in 10 uninfected controls. Two groups of patients could be distinguished. Sixteen patients had splenomegaly, markedly accelerated clearance of the labeled erythrocytes (clearance half-time, 8.4 +/- 4.4 minutes (mean +/- SD) vs. 62.5 +/- 36.5 minutes in controls; P less than 0.001), and a lower mean hematocrit than did the patients without splenomegaly (P less than 0.001). In the nine patients without splenomegaly, clearance was normal. After institution of antimalarial chemotherapy, however, the clearance in this group accelerated to supernormal rates similar to those in the patients with splenomegaly, but without the development of detectable splenomegaly. Clearance was not significantly altered by treatment in the group with splenomegaly. Six weeks later, normal clearance rates were reestablished in most patients in both groups. We conclude that splenic clearance of labeled erythrocytes is enhanced in patients with malaria if splenomegaly is present and is enhanced only after treatment if splenomegaly is absent. Whether this enhanced splenic function applies to parasite-infected erythrocytes in patients with malaria and has any clinical benefit will require further studies.

  19. Glycerol inhibits water permeation through Plasmodium falciparum aquaglyceroporin.

    PubMed

    Chen, Liao Y

    2013-01-01

    Plasmodium falciparum aquaglyceroporin (PfAQP) is a multifunctional membrane protein in the plasma membrane of P. falciparum, the parasite that causes the most severe form of malaria. The current literature has established the science of PfAQP's structure, functions, and hydrogen-bonding interactions but left unanswered the following fundamental question: does glycerol modulate water permeation through aquaglyceroporin that conducts both glycerol and water? This paper provides an affirmative answer to this question of essential importance to the protein's functions. On the basis of the chemical-potential profile of glycerol from the extracellular bulk region, throughout PfAQP's conducting channel, to the cytoplasmic bulk region, this study shows the existence of a bound state of glycerol inside aquaglyceroporin's permeation pore, from which the dissociation constant is approximately 14μM. A glycerol molecule occupying the bound state occludes the conducting pore through which permeating molecules line up in single file by hydrogen-bonding with one another and with the luminal residues of aquaglyceroporin. In this way, glycerol inhibits permeation of water and other permeants through aquaglyceroporin. The biological implications of this theory are discussed and shown to agree with the existent in vitro data. It turns out that the structure of aquaglyceroporin is perfect for the van der Waals interactions between the protein and glycerol to cause the existence of the bound state deep inside the conducting pore and, thus to play an unexpected but significant role in aquaglyceroporin's functions.

  20. Delayed Diagnosis of Falciparum Malaria with Acute Kidney Injury.

    PubMed

    Choi, Iee Ho; Hwang, Pyoung Han; Choi, Sam Im; Lee, Dae Yeol; Kim, Min Sun

    2016-09-01

    Prompt malaria diagnosis is crucial so antimalarial drugs and supportive care can then be rapidly initiated. A 15-year-old boy who had traveled to Africa (South Africa, Kenya, and Nigeria between January 3 and 25, 2011) presented with fever persisting over 5 days, headache, diarrhea, and dysuria, approximately 17 days after his return from the journey. Urinalysis showed pyuria and hematuria. Blood examination showed hemolytic anemia, thrombocytopenia, disseminated intravascular coagulation, and hyperbilirubinemia. Plasmapheresis and hemodialysis were performed for 19 hospital days. Falciparum malaria was then confirmed by peripheral blood smear, and antimalarial medications were initiated. The patient's condition and laboratory results were quickly normalized. We report a case of severe acute renal failure associated with delayed diagnosis of falciparum malaria, and primary use of supportive treatment rather than antimalarial medicine. The present case suggests that early diagnosis and treatment is important because untreated tropical malaria can be associated with severe acute renal failure and fatality. Physicians must be alert for correct diagnosis and proper management of imported tropical malaria when patients have travel history of endemic areas. PMID:27510397

  1. The Molecular Basis of Folate Salvage in Plasmodium falciparum

    PubMed Central

    Salcedo-Sora, J. Enrique; Ochong, Edwin; Beveridge, Susan; Johnson, David; Nzila, Alexis; Biagini, Giancarlo A.; Stocks, Paul A.; O'Neill, Paul M.; Krishna, Sanjeev; Bray, Patrick G.; Ward, Stephen A.

    2011-01-01

    Tetrahydrofolates are essential cofactors for DNA synthesis and methionine metabolism. Malaria parasites are capable both of synthesizing tetrahydrofolates and precursors de novo and of salvaging them from the environment. The biosynthetic route has been studied in some detail over decades, whereas the molecular mechanisms that underpin the salvage pathway lag behind. Here we identify two functional folate transporters (named PfFT1 and PfFT2) and delineate unexpected substrate preferences of the folate salvage pathway in Plasmodium falciparum. Both proteins are localized in the plasma membrane and internal membranes of the parasite intra-erythrocytic stages. Transport substrates include folic acid, folinic acid, the folate precursor p-amino benzoic acid (pABA), and the human folate catabolite pABAGn. Intriguingly, the major circulating plasma folate, 5-methyltetrahydrofolate, was a poor substrate for transport via PfFT2 and was not transported by PfFT1. Transport of all folates studied was inhibited by probenecid and methotrexate. Growth rescue in Escherichia coli and antifolate antagonism experiments in P. falciparum indicate that functional salvage of 5-methyltetrahydrofolate is detectable but trivial. In fact pABA was the only effective salvage substrate at normal physiological levels. Because pABA is neither synthesized nor required by the human host, pABA metabolism may offer opportunities for chemotherapeutic intervention. PMID:21998306

  2. Identification of a Plasmodium falciparum Phospholipid Transfer Protein*

    PubMed Central

    van Ooij, Christiaan; Withers-Martinez, Chrislaine; Ringel, Alessa; Cockcroft, Shamshad; Haldar, Kasturi; Blackman, Michael J.

    2013-01-01

    Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte. PMID:24043620

  3. Molecular Aspects of Plasmodium falciparum Infection during Pregnancy

    PubMed Central

    Ndam, Nicaise Tuikue; Deloron, Philippe

    2007-01-01

    Cytoadherence of Plasmodium-falciparum-parasitized red blood cells (PRBCs) to host receptors is the key phenomenon in the pathological process of the malaria disease. Some of these interactions can originate poor outcomes responsible for 1 to 3 million annual deaths mostly occurring among children in sub-Saharan Africa. Pregnancy-associated malaria (PAM) represents an important exception of the disease occurring at adulthood in malaria endemic settings. Consequences of this are shared between the mother (maternal anemia) and the baby (low birth weight and infant mortality). Demonstrating that parasites causing PAM express specific variant surface antigens (VSAPAM), including the P. falciparum erythrocyte membrane protein 1 (P f EMP1) variant VAR2CSA, that are targets for protective immunity has strengthened the possibility for the development of PAM-specific vaccine. In this paper, we review the molecular basis of malaria pathogenesis attributable to the erythrocyte stages of the parasites, and findings supporting potential anti-PAM vaccine components evidenced in PAM. PMID:17641725

  4. Efficacy of oral and intravenous artesunate in male Tanzanian adults with Plasmodium falciparum malaria and in vitro susceptibility to artemisinin, chloroquine, and mefloquine.

    PubMed

    Alin, M H; Kihamia, C M; Bjorkman, A; Bwijo, B A; Premji, Z; Mtey, G J; Ashton, M

    1995-12-01

    The clinical efficacy of oral and intravenous (iv) artesunate was compared in an open randomized trial in 50 male adult patients with uncomplicated Plasmodium falciparum malaria in Kibaha, Tanzania. Oral artesunate treatment was started with 2 x 50 mg initially followed by 50 mg 12 hr later and then 50 mg twice a day for four days (total dose = 550 mg or 9.6 mg/kg). Intravenous artesunate administration began with 2 x 0.8 mg/kg initially followed by 0.8 mg/kg 12 hr later and then 0.8 mg/kg twice a day for four days (total dose = 8.8 mg/kg). The mean +/- SD parasite clearance times (PCTs) were nearly identical at 23.4 +/- 5.9 hr and 24.2 +/- 7.2 hr after oral and iv administration, respectively. Mean +/- SD fever subsidence times (FSTs) were also similar at 18.7 +/- 8.3 hr and 21.0 +/- 4.8 hr, respectively. All patients remained negative for P. falciparum for at least 14 days. Recrudescence/reinfection occurred between days 21 and 28 in five of 25 patients (20%) after oral treatment and in four of 25 patients (16%) after iv treatment. The mean erythrocyte count and hemoglobin concentration were slightly reduced after iv treatment but remained in the normal range. Otherwise, there was no change in blood biochemistry, hematology, and electrocardiograms monitored prior to and during the last dose. It is concluded that treatment with oral and iv artesunate was equally efficacious and well tolerated. A 24-hr in vitro susceptibility test of P. falciparum to artemisinin, chloroquine, and mefloquine was performed in samples from all patients. The three compounds exhibited 100% inhibition with the exception of three isolates, which showed chloroquine resistance. Parameter estimates of a sigmoid Emax model (drug concentration at which 50% of the growth inhibition occurs [EC50]), the sigmoidicity factor s and EC95 fitted to the growth inhibition data differed between compounds and isolates, indicating different sensitivity of P. falciparum isolates. There was no correlation

  5. The use of activated protein C in severe Plasmodium falciparum malaria.

    PubMed

    Rankin, L G; Austin, D L H

    2007-06-01

    A 56-year-old man presented to a peripheral hospital in New Zealand with severe Plasmodium falciparum malaria with cerebral involvement and subsequently developed multi-system organ failure. Activated protein C was used in an attempt to stop the cascade of events into multi-organ failure. Severe infection with P. falciparum is life-threatening and appears to activate a hypercoagulable state similar to that of severe sepsis. Activated protein C is currently used in the treatment of severe sepsis and may provide a new adjuvant therapy for severe P. falciparum malaria.

  6. [Is Plasmodium falciparum, the parasite responsible for tropical malaria, resistant to fansidar?].

    PubMed

    Holzer, B; Keller, H; Frossard, E; Stürchler, D

    1980-03-01

    A world-wide increase of malaria infections is observed. Malaria is imported into Switzerland mainly by tourists and recently by refugees from South East Asia. The strains of P. falciparum resistant to treatment are of increasing importance. A patient with P. falciparum infection from Cambodia is reported, who suffered from three episodes of malaria recrudescence within ten weeks, in spite of adequate therapy with quinine and Fansidar. The definition, the significance and the geographical distribution of resistances and the possible cause for a P. falciparum recrudescence are discussed. For the treatment of repeating recrudescence quinine and Fansidar are recommended, followed by a suppressive Fansidar prophylaxy for 4--8 weeks.

  7. Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment.

    PubMed

    Leang, Rithea; Taylor, Walter R J; Bouth, Denis Mey; Song, Lijiang; Tarning, Joel; Char, Meng Chuor; Kim, Saorin; Witkowski, Benoit; Duru, Valentine; Domergue, Anais; Khim, Nimol; Ringwald, Pascal; Menard, Didier

    2015-08-01

    Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia (P <10(-3)). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC50) of PP were independent of treatment outcomes, in contrast to median mefloquine IC50, which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.). PMID:26014949

  8. Evidence of Plasmodium falciparum Malaria Multidrug Resistance to Artemisinin and Piperaquine in Western Cambodia: Dihydroartemisinin-Piperaquine Open-Label Multicenter Clinical Assessment