Critical evaluation of molecular monitoring in malaria drug efficacy trials: pitfalls of length polymorphic markers [PublishAheadOfPrint]

Estimating 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 co-infections, potentially leading to misclassification of treatment outcome. We quantified minority clone detectability and competition among msp1, msp2, and glurp amplicons using mixtures of P. falciparum strains and investigated the impact of template competition on genotyping outcomes in forty-four paired field samples. Substantial amplification bias was detected for all three markers with shorter fragments outperforming larger fragments. Strongest template competition was observed for marker glurp. Detection of glurp fragments in multi-clonal infections was severely compromised. Eight of forty-four sample pairs were identified as new infection by all three markers. Ten pairs were defined as new infection based on one marker alone, seven of which were defined by 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 to resolve discordant results.

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