No evidence of molecular markers of piperaquine resistance in southeastern Nigeria

Abstract

Background: Artemisinin‑based combination therapy (ACT) remains the first‑line treatment for uncomplicated malaria, yet its long‑term efficacy is threatened by Plasmodium falciparum resistance to both artemisinin derivatives and partner drugs. Routine surveillance of clinical efficacy and molecular‑resistance markers is therefore essential. Piperaquine (PPQ), the partner drug in dihydroartemisinin–piperaquine (DHA‑PPQ) is already compromised in Southeast Asia, and key PPQ‑resistance mutations have begun to surface in parts of Africa. No such data are available from Nigeria. Three PPQ‑resistanceassociated singlenucleotide polymorphisms (SNPs)—T93S, H97Y and F145Ilocated in exons 2 and 3 of the P. falciparum chloroquineresistance transporter gene (pfcrt), and copy‑number variation of plasmepsin 2/3 (pfpm2/3), were retrospectively investigated using 299 archived patient isolates collected in Awka (n = 200) and Onitsha (n = 99), South‑East Nigeria (2018–2019). Methods: Allelic states at pfcrt codons 93, 97 and 145 were determined by nested PCR followed by Sanger sequencing. Pfpm2/3 copy number was quantified by real‑time qPCR, using β‑tubulin as the single‑copy reference. All assays incorporated PPQ‑resistant RF7 and wild‑type 3D7 control strains, plus no‑template controls. Results: High‑quality amplification was achieved for 268 of 299 isolates (89.6%). None harboured the pfcrt T93S, H97Y or F145I mutations, and all exhibited single‑copy pfpm2 and pfpm3. Two novel exon‑2 variants (K115I and synonymous L111L) were each detected once (0.4%) and are regarded as neutral polymorphisms without known phenotypic impact. Conclusions: The complete absence of validated PPQresistance markers indicates that piperaquine remains a viable partner drug in DHA‑PPQ for South‑East Nigeria. Nonetheless, because DHA‑PPQ is increasingly available over the counter, ongoing molecular and therapeutic‑efficacy surveillance is imperative to detect any future emergence of resistance.