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Abstract
BackgroundPlasmodium vivax infections commonly contain multiple genetically distinct parasite clones. The detection of multiple-clone infections depends on several factors, such as the accuracy of the genotyping method, and the type and number of the molecular markers analysed. Characterizing the multiplicity of infection has broad implications that range from population genetic studies of the parasite to malaria treatment and control. This study compared and evaluated the efficiency of neutral and non-neutral markers that are widely used in studies of molecular epidemiology to detect the multiplicity of P. vivax infection.MethodsThe performance of six markers was evaluated using 11 mixtures of DNA with well-defined proportions of two different parasite genotypes for each marker. These mixtures were generated by mixing cloned PCR products or patient-derived genomic DNA. In addition, 51 samples of natural infections from the Brazil were genotyped for all markers. The PCR-capillary electrophoresis-based method was used to permit direct comparisons among the markers. The criteria for differentiating minor peaks from artifacts were also evaluated.ResultsThe analysis of DNA mixtures showed that the tandem repeat MN21 and the polymorphic blocks 2 (msp1B2) and 10 (msp1B10) of merozoite surface protein-1 allowed for the estimation of the expected ratio of both alleles in the majority of preparations. Nevertheless, msp1B2 was not able to detect the majority of multiple-clone infections in field samples; it identified only 6 % of these infections. The merozoite surface protein-3 alpha and microsatellites (PvMS6 and PvMS7) did not accurately estimate the relative clonal proportions in artificial mixtures, but the microsatellites performed well in detecting natural multiple-clone infections. Notably, the use of a less stringent criterion to score rare alleles significantly increased the sensitivity of the detection of multi-clonal infections.ConclusionsDepending on the type of marker used, a considerable amplification bias was observed, which may have serious implications for the characterization of the complexity of a P. vivax infection. Based on the performance of markers in artificial mixtures of DNA and natural infections, a minimum panel of four genetic markers (PvMS6, PvMS7, MN21, and msp1B10) was defined, and these markers are highly informative regarding the genetic variability of P. vivax populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0846-5) contains supplementary material, which is available to authorized users.
Highlights
Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones
Characterizing the within-host diversity is essential to address several issues, such as differentiation between new infection and recrudescence, in order to better estimate the true risk of treatment failure and explore the dynamics of clones influenced by host immunity during anti-malarial treatment or challenge with vaccine [12, 20, 21]
All six molecular markers accurately identified the correct alleles in samples containing a single clone
Summary
Plasmodium vivax infections commonly contain multiple genetically distinct parasite clones. Dormant forms of the parasite in the liver, i.e., hypnozoites, act as a reservoir for the disease and have hindered the control of malaria caused by P. vivax [7]. Plasmodium vivax infections are often characterized by the presence of two or more genetically distinct parasites in the same individual [9,10,11] These infections are very common in malaria-endemic areas worldwide [12,13,14,15] and can arise from a single mosquito bite carrying a mixture of parasites or from inoculation by different mosquitoes carrying single clones. Characterizing the multiplicity of infection has broad implications ranging from population genetic studies of the parasite to malaria treatment and control. A broad understanding of the genetic diversity of parasite populations can contribute to the definition of control measures, including an appropriate anti-malarial treatment
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