Evaluations of candidate markers of dihydroartemisinin-piperaquine resistance in Plasmodium falciparum isolates from the China-Myanmar, Thailand-Myanmar, and Thailand-Cambodia borders.

backgroundThe fast-declining clinical efficacy of dihydroartemisinin-piperaquine (DHA-PPQ) in Cambodia is a warning of the underlying westward dissemination of piperaquine resistance in the Greater Mekong Subregion (GMS). Mutations in the Plasmodium falciparum Kelch 13-propeller (PfK13) and the P. falciparum chloroquine resistance transporter (PfCRT), as well as plasmepsin 2/3 gene amplification, have been discovered as molecular markers for predicting DHA-PPQ treatment failure. Determining whether these genetic variations of P. falciparum are linked to DHA-PPQ resistance is critical, especially along the China-Myanmar (CM) border, where PPQ has been utilized for decades.

methodsA total of 173 P. falciparum samples of dried blood spots (DBS) were collected along the CM border between 2007 and 2010, the Thailand-Cambodia (TC) border between 2009 and 2013, and the Thailand-Myanmar (TM) border between 2012 and 2014. PCR and sequencing were used to identified PfCRT mutations, while qPCR was used to determine the copy number of plasmepsin 2/3. The prevalence of DHA-PPQ resistance in three locations was investigated using data paired with K13 mutations.

resultsThree fragments of the pfcrt gene were amplified for all 173 samples, and seven SNPs were identified (M74I, N75E/D, K76T, H97L, I218F, A220S, I356L). No new PfCRT mutations conferring resistance to PPQ (T93S, H97Y, F145I, M343L, and G353V) were discovered, except for one mutant I218F identified in the TM border (2.27%, 1/44). Additionally, mutant H97L was found in the TC, TM, and CM borders at 3.57% (1/28), 6.82% (3/44), and 1% (1/101), respectively. A substantial K13 C580Y variant prevalence was found in the TC and TM border, accounting for 64.29% (18/28) and 43.18% (19/44), respectively, while only 1% (1/101) was found in the CM border. The K13 F446I variant was only identified and found to reach a high level (28.71%, 29/101) in the CM border. Furthermore, 10.71% (3/28) of TC isolates and 2.27% (1/44) of TM isolates carried more than one copy of plasmepsin 2/3 and K13 C580Y variant, while no plasmepsin 2/3 amplification was identified in the CM isolates.

conclusionsCompared with the P. falciparum samples collected from the TC and TM borders, fewer parasites carried plasmepsin 2/3 amplification and novel PfCRT variants, while more parasites carried predominant K13 mutations at position F446I, in the CM border. Clear evidence of DHA-PPQ resistance associated with candidate markers was not found in this border region suggesting a further evaluation of these markers and continuous surveillance is warranted.