Changing genetic profiles of <i>Plasmodium falciparum</i> piperaquine resistance in Southeast Asia over 25 years

Piperaquine resistance in <i>Plasmodium falciparum</i> has emerged in Southeast Asia and is strongly associated with mutations in the <i>pfcrt</i> gene and amplification of <i>pfplasmepsin2/3</i>. This study assessed the frequency of <i>pfcrt</i> mutations and <i>pfplasmepsin2/3</i> gene amplifications across Southeast Asia over 25 years, which is critical for tracking resistant parasites. A total of 898 <i>P</i>. <i>falciparum</i> isolates collected from Thailand, Myanmar, Cambodia, Laos, and Vietnam between 1995 and 2023 were analyzed for <i>pfcrt</i> mutations, <i>pfplasmepsin2</i> copy number, and microsatellite variation around the <i>pfcrt</i> locus. During the study period, <i>pfcrt</i> mutations in Cambodia across the study period showed that H97Y had the highest prevalence at 15%, followed by T93S at 8%, while I218F was common in 24% of specimens in Srisaket and Ubon in Thailand at 24.44%, followed by G353V at 20%, T93S at 17%, F145I at 13%, and H97Y at 4%. In Tak, Thailand, a mutation was found only in 1995 with 40% at I218F and the remaining wild-type. Yala in Thailand and Kayin State in Myanmar remained wild-type. Vietnam showed frequent T93S at 21%. The prevalence of <i>pfcrt</i> mutations observed in this study changed throughout the study period. Over 65% of parasites with <i>pfcrt</i> mutations at positions 93, 97, 145, 218, 343, and 353 also had <i>pfplasmepsin2</i> amplification. Temporal analysis revealed that in Cambodia, <i>pfplasmepsin2</i> CNV emerged first, peaking at 88% prevalence in 2015 after the introduction of DHA-piperaquine, with the subsequent increase of <i>pfcrt</i> mutations H97Y and G353V. In Eastern Thailand, both <i>pfcrt</i> mutations and <i>pfplasmepsin2</i> CNV were highly prevalent, exceeding 70% during 2015-2018. In Vietnam, rapid increases in both markers were observed after 2012, reaching peak by 2017. Microsatellite analysis revealed reduced genetic diversity around mutant <i>pfcrt</i> alleles, indicating selective sweeps. This study demonstrates that frequencies of <i>pfcrt</i> mutations and <i>pfplasmepsin2</i> amplification linked to piperaquine resistance have changed over time, highlighting the importance of ongoing genetic monitoring to inform strategies for preserving artemisinin-based combination therapy efficacy.