Assessment of different genotyping markers and algorithms for distinguishing Plasmodium falciparum recrudescence from reinfection in Uganda

Abstract

<title>Abstract</title> Antimalarial therapeutic efficacy studies are vital for monitoring the efficacy of antimalarial drugs in malaria-endemic regions. The WHO recommends genotyping of polymorphic markers including <italic>msp-1</italic>, <italic>msp-2</italic>, and <italic>glurp</italic> to aid distinguishing recrudescences from reinfections. Recently, WHO proposed replacing <italic>glurp</italic> with microsatellites (Poly-α, PfPK2, TA1). However, suitable combinations with <italic>msp-1</italic> and <italic>msp-2</italic> have not been systematically assessed. Additionally, the performance of different algorithms for classifying recrudescence is unclear. This study investigated various microsatellites alongside <italic>msp-1</italic> and <italic>msp-2</italic> for molecular correction and compared genotyping algorithms across three malaria-endemic areas in Uganda. Microsatellites 313, Poly-α, and 383 exhibited the highest diversity, while PfPK2 and Poly-α revealed elevated multiplicities of infection across all sites. The 3/3 match-counting algorithm classified fewer recrudescences than the ≥ 2/3, and Bayesian algorithms at both ≥ 0.7 and ≥ 0.8 probability cutoffs. The <italic>msp-1</italic>/<italic>msp-2</italic>/2490 combination identified more recrudescences using the ≥ 2/3 and 3/3 algorithms in the artemether-lumefantrine (AL) treatment arm, while <italic>msp-1</italic>/<italic>msp-2</italic>/<italic>glurp</italic> combination identified more cases of recrudescence using the ≥ 2/3 in the dihydroartemisinin-piperaquine (DP) arm. Microsatellites PfPK2 and Poly-α, potentially sensitive to detecting minority clones, are promising replacements for <italic>glurp</italic>. Discrepancies in recrudescence classification between match-counting and Bayesian algorithms highlight the need for standardized PCR correction practices.