Abstract
ABSTRACTEstimation 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.
Highlights
Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures
In in vivo efficacy drug or vaccine trials in settings where malaria is endemic, parasite genotyping for discrimination of newly incoming versus recrudescent clones after antimalarial treatment is essential for accurate estimation of the efficacy of the intervention [2, 3]
Genotyping is further used in malaria molecular epidemiology to assess multiplicity of infection (MOI) as a proxy of transmission level in molecular monitoring of interventions and for tracking of individual clones over time in cohort studies to measure the molecular force of infection or duration of infection [4, 5]
Summary
Estimation of drug efficacy in antimalarial drug trials requires parasite genotyping to distinguish new infections from treatment failures. Sizing protocols, such as insufficient resolution of minimal size differences, among fragments larger than 500 bp, or unequal loading of variable PCR yields leading to apparent but artificial size differences These limitations are relevant for glurp genotyping, since amplicons of the glurp R2 region often surpass 1,000 bp in size, and CE-based glurp genotyping was not routinely used in most laboratories [8, 9]. How much technical reasons contribute to imperfect detectability of individual clones has not been investigated systematically for the three prime P. falciparum genotyping markers msp, msp, and glurp, but circumstantial observations of clone competition during PCR can be found in the literature [15, 16]
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