Comparison of different genotyping techniques to distinguish recrudescence from new infection in studies assessing the efficacy of antimalarial drugs against Plasmodium falciparum

Distinguishing recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against Plasmodium falciparum (P. falciparum). Different genotyping methods are used and may impede the comparison of drug efficacy estimates in space and time, particularly in patients from high transmission settings with polyclonal infections. We compared five different genotyping methods currently used to assess their sensitivity in detecting minority clones in polyclonal infections, their robustness, and the genetic diversity of the markers used. Our study utilized four well-characterized P. falciparum laboratory strains mixed in varying ratios, and 20 paired patient samples collected from a clinical trial. We found that high-resolution capillary electrophoresis (H-CE) using length-polymorphic markers, as well as targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers, revealed the highest sensitivity in detecting minority clones, while also exhibiting robustness, and high genetic diversity in the used markers. Moreover, markers used by TADS gave more consistent results. We observed that microsatellites had a lower genetic diversity compared to markers such as msp1, msp2, glurp and SNP-rich markers, with some genotypes having allelic frequencies of > 30 %. The replacement of glurp by microsatellites did not result in a change in the genotyping outcome, probably due to the lower genetic diversity of microsatellites used in comparison to glurp. More studies with large sample sizes are necessary to identify the most suitable microsatellites that could replace glurp as per the latest recommendations from the World Health Organization (WHO) on genotyping to distinguish recrudescence from new infections in high transmission settings. Our study indicates that TADS should be considered the gold standard for genotyping to differentiate recrudescence from new infection and should be used to validate other techniques.