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

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 msp-1 , msp-2 , and glurp to aid distinguishing recrudescences from reinfections. Recently, WHO proposed replacing glurp with microsatellites (Poly-α, PfPK2, TA1). However, suitable combinations with msp-1 and msp-2 have not been systematically assessed. Additionally, the performance of different algorithms for classifying recrudescence is unclear. This study investigated various microsatellites alongside msp-1 and msp-2 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 msp-1 / msp-2 /2490 combination identified more recrudescences using the ≥ 2/3 and 3/3 algorithms in the artemether-lumefantrine (AL) treatment arm, while msp-1 / msp-2 / glurp 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 glurp . Discrepancies in recrudescence classification between match-counting and Bayesian algorithms highlight the need for standardized PCR correction practices.