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

Abstract

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.

Malaria Epidemiology at the Clone Level

“…Importantly, the imperfect detection of minority clones due to day-to-day fluctuations in clone densities or template competition in PCR reactions impact genotyping outcomes more than imperfect resolution of markers [33–35]. …”

“…Protocols for genotyping are straightforward and widely applied. On the other hand, detectability of minority clones is limited due to template competition in the PCR reaction [35]. As a result, substantial day-to-day variation was observed, with up to 40% of all clones not detected in a single sample [33].…”

“…It is also the only method that may allow reliable reconstruction of several multi-locus haplotypes in multiclone infections, as read numbers represent the proportion of each clone in a mixed infection with high accuracy [89, 90]. This is difficult with microsatellite data; the minority clone might not be detected due to template competition in the PCR, and due to preferred amplification of smaller alleles, peak height does not always reflect template density [35]. Given that the proportion of multiple clone infection is high in many populations, the ability to reconstruct minority haplotypes might be crucial.…”

Population Genetics and Molecular Epidemiology of Eukaryotes

“…For most P. falciparum studies, the highly polymorphic loci merozoite surface proteins 1 and 2 (msp1, msp2), and the glutamate-rich protein (glurp) have become standard tools for infection control (75, 108). They are specifically recommended by the WHO (36) to accompany trials of drug efficacy, and there is an extensive literature on their application for this purpose (2, 3, 22, 46, 52, 75, 80, 82, 83, 91, 101, 103, 118). …”

“…To reduce cost, these markers are often analyzed on agarose gels. The joint amplification of these markers in multiplex assays, and the resolution available on agarose gels likely underestimates diversity compared to their detection by capillary electrophoresis (75). There is also inherent variability in agarose gel electrophoresis that needs to be taken into account when establishing criteria for band sizes (83, 101).…”