No association between the Plasmodium vivax crt-o MS334 or In9pvcrt polymorphisms and chloroquine failure in a clinical cohort from Malaysia

Increasing reports of resistance to a frontline malaria blood-stage treatment, chloroquine (CQ), raise concerns for the elimination of Plasmodium vivax. The absence of an effective molecular marker of CQ resistance in P. vivax greatly constrains surveillance of this emerging threat. A recent genetic cross between CQ sensitive (CQS) and CQ resistant (CQR) NIH-1993 strains of P. vivax linked a moderate CQR phenotype with two candidate markers in P. vivax CQ resistance transporter gene (pvcrt-o): MS334 and In9pvcrt. Longer TGAAGH motifs at MS334 were associated with CQ resistance, as were shorter motifs at the In9pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from Malaysia were used to investigate the association between the MS334 and In9pvcrt variants and treatment efficacy. Amongst a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9pvcrt alleles were observed, with allele frequencies ranging from 2 to 76% and 3 to 71%, respectively. None of the clinical isolates had the same variant as the NIH-1993 CQR strain, and none were associated with CQ treatment failure (all p>0.05). Our findings suggest that the pvcrt-o MS334 and In9pvcrt markers cannot be used universally as markers of CQ treatment efficacy in an area of high-grade CQ resistance. Further studies applying hypothesis-free genome-wide approaches are warranted to identify more effective CQR markers for P. vivax.