Evaluation of Plasmodium vivax Genotyping Markers for Molecular Monitoring in Clinical Trials

BackgroundMany antimalarial interventions are accompanied by molecular monitoring of parasite infections, and a number of molecular typing techniques based on different polymorphic marker genes are used. Here, we describe a genotyping technique that provides a fast and precise approach to study Plasmodium vivax infection dynamics during circumstances in which individual clones must be followed over time. The method was tested with samples from an in vivo drug efficacy study MethodsThe sizes of polymerase chain reaction fragments were evaluated by capillary electrophoresis to determine the extent of size polymorphism for 9 potential genetic markers (5 genes of merozoite surface proteins [msp] and 4 microsatellites) in 93-108 P. vivax-positive blood samples from 3 villages in Papua New Guinea ResultsThe microsatellites MS16 and Pv3.27 showed the greatest diversity in the study area, with 66 and 31 different alleles, respectively, followed by 2 fragments of msp1 and 2 other microsatellites. msp3α, msp4 and msp5 revealed limited polymorphism ConclusionsEven for the most diverse markers, the highest allelic frequencies reached 6% (MS16) or 13% (Pv3.27). To reduce the theoretical probability of superinfection with parasites that have the same haplotype as that detected at baseline, we propose to combine at least 2 markers for genotyping individual P. vivax infection

Molecular Markers of In Vivo Plasmodium vivax Resistance to Amodiaquine Plus Sulfadoxine-Pyrimethamine: Mutations in pvdhfr and pvmdr1

Background. Molecular markers for sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax have been reported. However, data on the molecular correlates involved in the development of resistance to 4-aminoquinolines and their association with the in vivo treatment response are scarce. Methods. We assessed pvdhfr (F57L/I, S58R, T61M, S117T/N, and I173F/L) and pvmdr1 (Y976F and F1076L) mutations in 94 patients who received amodiaquine (AQ) plus SP in Papua New Guinea (PNG). We then investigated the association between parasite genotype and treatment response. Results. The treatment failure (TF) rate reached 13%. Polymorphisms in pvdhfr F57L, S58R, T61M, and S117T/N and in pvmdr1 Y976F were detected in 60%, 67%, 20%, 40%, and 39% of the samples, respectively. The single mutant pvdhfr 57 showed the strongest association with TF (odds ratio [OR], 9.04; P=.01). The combined presence of the quadruple mutant pvdhfr 57L+58R+61M+117T and pvmdr1 mutation 976F was the best predictor of TF (OR, 8.56; P=.01). The difference in TF rates between sites was reflected in the genetic drug-resistance profile of the respective parasites. Conclusions. The present study identified a new molecular marker in pvmdr1 that is associated with the in vivo response to AQ+SP. We suggest suitable marker sets with which to monitor P. vivax resistance against AQ+SP in countries where these drugs are use

Heterogeneous distribution of Plasmodium falciparum drug resistance haplotypes in subsets of the host population

BackgroundThe emergence of drug resistance is a major problem in malaria control. For mathematical modelling of the transmission and spread of drug resistance the determinant parameters need to be identified and measured. The underlying hypothesis is that mutations associated with drug resistance incur fitness costs to the parasite in absence of drug pressure. The distribution of drug resistance haplotypes in different subsets of the host population was investigated. In particular newly acquired haplotypes after radical cure were characterized and compared to haplotypes from persistent infections. MethodsMutations associated with antimalarial drug resistance were analysed in parasites from children, adults, and new infections occurring after treatment. Twenty-five known single nucleotide polymorphisms from four Plasmodium falciparum genes associated with drug resistance were genotyped by DNA chip technology. ResultsHaplotypes were found to differ between subsets of the host population. A seven-fold mutated haplotype was significantly reduced in adults compared to children and new infections, whereas parasites harbouring fewer mutations were more frequent in adults. ConclusionThe reduced frequency of highly mutated parasites in chronic infections in adults is likely a result of fitness costs of drug resistance that increases with number of mutations and is responsible for reduced survival of mutant parasites

Heterogeneous distribution of <it>Plasmodium falciparum </it>drug resistance haplotypes in subsets of the host population

Abstract Background The emergence of drug resistance is a major problem in malaria control. For mathematical modelling of the transmission and spread of drug resistance the determinant parameters need to be identified and measured. The underlying hypothesis is that mutations associated with drug resistance incur fitness costs to the parasite in absence of drug pressure. The distribution of drug resistance haplotypes in different subsets of the host population was investigated. In particular newly acquired haplotypes after radical cure were characterized and compared to haplotypes from persistent infections. Methods Mutations associated with antimalarial drug resistance were analysed in parasites from children, adults, and new infections occurring after treatment. Twenty-five known single nucleotide polymorphisms from four Plasmodium falciparum genes associated with drug resistance were genotyped by DNA chip technology. Results Haplotypes were found to differ between subsets of the host population. A seven-fold mutated haplotype was significantly reduced in adults compared to children and new infections, whereas parasites harbouring fewer mutations were more frequent in adults. Conclusion The reduced frequency of highly mutated parasites in chronic infections in adults is likely a result of fitness costs of drug resistance that increases with number of mutations and is responsible for reduced survival of mutant parasites.</p

Comparison of haplotypes between adults (cross section) and new infections of the treatment to reinfection study

* indicates significant difference of haplotype frequency between the two compared groups.<p><b>Copyright information:</b></p><p>Taken from "Heterogeneous distribution of drug resistance haplotypes in subsets of the host population"</p><p>http://www.malariajournal.com/content/7/1/78</p><p>Malaria Journal 2008;7():78-78.</p><p>Published online 6 May 2008</p><p>PMCID:PMC2391149.</p><p></p

Evaluation of Plasmodium vivax Genotyping Markers for Molecular Monitoring in Clinical Trials

BACKGROUND: Many antimalarial interventions are accompanied by molecular monitoring of parasite infections, and a number of molecular typing techniques based on different polymorphic marker genes are used. Here, we describe a genotyping technique that provides a fast and precise approach to study Plasmodium vivax infection dynamics during circumstances in which individual clones must be followed over time. The method was tested with samples from an in vivo drug efficacy study. METHODS: The sizes of polymerase chain reaction fragments were evaluated by capillary electrophoresis to determine the extent of size polymorphism for 9 potential genetic markers (5 genes of merozoite surface proteins [msp] and 4 microsatellites) in 93-108 P. vivax-positive blood samples from 3 villages in Papua New Guinea. RESULTS: The microsatellites MS16 and Pv3.27 showed the greatest diversity in the study area, with 66 and 31 different alleles, respectively, followed by 2 fragments of msp1 and 2 other microsatellites. msp3alpha, msp4, and msp5 revealed limited polymorphism. CONCLUSIONS: Even for the most diverse markers, the highest allelic frequencies reached 6% (MS16) or 13% (Pv3.27). To reduce the theoretical probability of superinfection with parasites that have the same haplotype as that detected at baseline, we propose to combine at least 2 markers for genotyping individual P. vivax infection

Comparison of haplotypes between children of the cross sectional surveys and baseline samples and new infections from the treatment to reinfection study (TRS)

There are no significant differences in haplotype frequencies between groups.<p><b>Copyright information:</b></p><p>Taken from "Heterogeneous distribution of drug resistance haplotypes in subsets of the host population"</p><p>http://www.malariajournal.com/content/7/1/78</p><p>Malaria Journal 2008;7():78-78.</p><p>Published online 6 May 2008</p><p>PMCID:PMC2391149.</p><p></p