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Background   Plasmodium 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.    Methods  The 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.    Results  The 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.    Conclusions  Depending 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 material  The online version of this article (doi:10.1186/s12936-015-0846-5) contains supplementary material, which is available to authorized users.

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