Genotyping methods to distinguish Plasmodium falciparum recrudescence from new infection for the assessment of antimalarial drug efficacy: an observational, single-centre, comparison study

Background

Distinguishing Plasmodium falciparum recrudescence from new infections is crucial for the assessment of antimalarial drug efficacy against P falciparum. We aimed to compare the efficacy of different genotyping methods to assess their effect on drug efficacy estimates, particularly in patients from high-transmission settings with polyclonal infections.

Methods

In this head-to-head comparison study, we compared five different genotyping methods currently used: fast capillary electrophoresis (F-CE) using msp1, msp2, and glurp; high-resolution capillary electrophoresis (H-CE) using msp1, msp2, and glurp; H-CE using microsatellites; targeted amplicon deep sequencing (TADS) using single nucleotide polymorphism (SNP)-rich markers; and high-resolution melting (HRM) analysis using msp1 and msp2. We assessed their sensitivity in detecting minority clones in polyclonal infections, their reproducibility, and the genetic diversity of the markers used. Our study used four well characterised P falciparum laboratory strains mixed in varying ratios, and 20 paired samples collected from an in-vivo clinical trial. The experiments were performed at the Swiss Tropical and Public Health Institute in Basel, Switzerland between May 5, 2020, and Aug 23, 2021.

Findings

H-CE using msp1 and msp2 and TADS revealed the highest sensitivity in detecting minority clones (up to ratios of 1:100 for H-CE and 50:1:1:1 for TADS in the FCB1:HB3 and 3D7:K1:HB3:FCB1 laboratory strain mixtures, respectively), highest reproducibility (intra-assay: 99% and 91% for H-CE and TADS, respectively; inter-assay: 98% and 92% for H-CE and TADS, respectively), and highest genetic diversity in the used markers (up to 36 and 32 unique genotypes in 20 paired samples for H-CE using msp2 and TADS using cpmp, respectively). Microsatellites assessed by H-CE had a lower genetic diversity compared with msp1, msp2, and glurp assessed by H-CE and the SNP-rich markers assessed by TADS, with a maximum of 13 unique genotypes, and some genotypes having allelic frequencies larger than 30%. Markers used by TADS gave the most consistent results in distinguishing recrudescence from new infection across all methods (in 18 of 20 pairs of samples vs 15 of 20 pairs for H-CE).

Interpretation

WHO currently recommends replacing glurp with microsatellites. However, in this study, the replacement of glurp with microsatellites did not change the genotyping outcome, probably due to the lower genetic diversity of microsatellites. More studies with large sample sizes are required to identify the most suitable microsatellites that could replace glurp. Our study indicates that TADS should be considered the gold standard for genotyping to distinguish recrudescence from new infection, and that it should be used to validate other methods.

Funding

Swiss Tropical and Public Health Institute.