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

Antimalarial therapeutic efficacy studies are vital for monitoring drug efficacy in malaria-endemic regions. The WHO recommends genotyping polymorphic markers including msp-1 , msp-2 , and glurp for distinguishing recrudescences from reinfections. Recently, WHO proposed replacing glurp with microsatellites (Poly-α, PfPK2, TA1). However, suitable combinations with msp-1 and msp-2 , as well as the performance of different algorithms for classifying recrudescence, have not been systematically assessed. This study investigated various microsatellites alongside msp-1 and msp-2 for molecular correction and compared different genotyping algorithms across three sites in Uganda. Microsatellites 313, Poly-α, and 383 exhibited the highest diversity, while PfPK2 and Poly-α revealed elevated multiplicity of infection (MOI) across all sites. The 3/3 match-counting algorithm classified significantly fewer recrudescences than both the ≥ 2/3 and Bayesian algorithms at probability cutoffs of ≥ 0.7 and ≥ 0.8 ( P  < 0.05). 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 classified 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.