Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology

Souza, Aracele Maria de; Araújo, Flávia Carolina Faustino de; Fontes, Cor Jésus Fernandes; Carvalho, Luzia H.; Brito, Cristiana Ferreira Alves de; Sousa, Taís Nóbrega de

doi:10.1186/s12936-015-0846-5

Cited by 16 publications

(15 citation statements)

References 51 publications

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“…33,[50][51][52] The complexity of infection was somewhat variable among different regions and geographic clusters for both species, and is comparable to that found in a wide range of higher transmission settings in other countries. 27,28,33,38,[53][54][55][56][57] However, the vast majority of P. vivax infections were polyclonal and MOI was almost 2-fold higher than that of P. falciparum. Furthermore, although complexity of infection was associated with prevalence for P. falciparum, there was no association between these parameters for P. vivax.…”

Section: Discussionmentioning

confidence: 99%

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum Across All Malaria Transmission Zones of Papua New Guinea

Fola

Harrison

Hazairin

et al. 2017

The American Society of Tropical Medicine and Hygiene

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Abstract. Plasmodium falciparum and Plasmodium vivax have varying transmission dynamics that are informed by molecular epidemiology. This study aimed to determine the complexity of infection and genetic diversity of P. vivax and P. falciparum throughout Papua New Guinea (PNG) to evaluate transmission dynamics across the country. In 2008-2009, a nationwide malaria indicator survey collected 8,936 samples from all 16 endemic provinces of PNG. Of these, 892 positive P. vivax samples were genotyped at PvMS16 and PvmspF3, and 758 positive P. falciparum samples were genotyped at Pfmsp2. The data were analyzed for multiplicity of infection (MOI) and genetic diversity. Overall, P. vivax had higher polyclonality (71%) and mean MOI (2.32) than P. falciparum (20%, 1.39). These measures were significantly associated with prevalence for P. falciparum but not for P. vivax. The genetic diversity of P. vivax (PvMS16: expected heterozygosity = 0.95, 0.85-0.98; PvMsp1F3: 0.78, 0.66-0.89) was higher and less variable than that of P. falciparum (Pfmsp2: 0.89, 0.65-0.97). Significant associations of MOI with allelic richness (rho = 0.69, P = 0.009) and expected heterozygosity (rho = 0.87, P < 0.001) were observed for P. falciparum. Conversely, genetic diversity was not correlated with polyclonality nor mean MOI for P. vivax. The results demonstrate higher complexity of infection and genetic diversity of P. vivax across the country. Although P. falciparum shows a strong association of these parameters with prevalence, a lack of association was observed for P. vivax and is consistent with higher potential for outcrossing of this species.

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Section: Discussionmentioning

confidence: 99%

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum Across All Malaria Transmission Zones of Papua New Guinea

Fola

Harrison

Hazairin

et al. 2017

The American Society of Tropical Medicine and Hygiene

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“…For microsatellite genotyping, five neutral microsatellite loci of significant variability in the Peruvian Amazon were typed in a previous study ( 32 ). If there was more than one marker at any given locus, the sample was considered multiclonal, per prior genotyping studies ( 45 – 47 ).…”

Section: Methodsmentioning

confidence: 99%

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

Cowell

Loy

Sundararaman

et al. 2017

mBio

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Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ≥5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens.

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“…How much technical reasons contribute to imperfect detectability of individual clones has not been investigated systematically for the three prime P. falciparum genotyping markers msp1 , msp2 , and glurp , but circumstantial observations of clone competition during PCR can be found in the literature (15, 16). …”

Section: Introductionmentioning

confidence: 99%

Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers

Messerli

Hofmann

Beck

et al. 2017

Antimicrob Agents Chemother

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Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. When using length-polymorphic molecular markers, preferential amplification of short fragments can compromise detection of coinfections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of Plasmodium falciparum strains and investigated the impact of template competition on genotyping outcomes in 44 paired field samples. Substantial amplification bias was detected for all three markers, with shorter fragments outperforming larger fragments. The strongest template competition was observed for the marker glurp. Detection of glurp fragments in multiclonal infections was severely compromised. Eight of 44 sample pairs were identified as new infections by all three markers. Ten pairs were defined as new infections based on one marker alone, seven of which were defined by the questionable marker glurp. The impact of size-dependent template competition on genotyping outcomes therefore calls for necessary amendments to the current WHO recommendations for PCR correction of malaria drug trial endpoints. Accuracy of genotyping outcomes could be improved by separate amplification reactions per allelic family and basing results on markers msp1 and msp2 first, with glurp only used to resolve discordant results.

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Multiple-clone infections of Plasmodium vivax: definition of a panel of markers for molecular epidemiology

Cited by 16 publications

References 51 publications

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum Across All Malaria Transmission Zones of Papua New Guinea

Higher Complexity of Infection and Genetic Diversity of Plasmodium vivax Than Plasmodium falciparum Across All Malaria Transmission Zones of Papua New Guinea

Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

Critical Evaluation of Molecular Monitoring in Malaria Drug Efficacy Trials and Pitfalls of Length-Polymorphic Markers

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