No association between thePlasmodium vivax crt-oMS334 or In9pvcrtpolymorphisms 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 In9 pvcrt . Longer TGAAGH motifs at MS334 were associated with CQ resistance, as were shorter motifs at the In9 pvcrt locus. In this study, high-grade CQR clinical isolates of P. vivax from Malaysia were used to investigate the association between the MS334 and In9 pvcrt variants and treatment efficacy. Amongst a total of 49 independent monoclonal P. vivax isolates assessed, high-quality MS334 and In9 pvcrt sequences could be derived from 30 (61%) and 23 (47%), respectively. Five MS334 and six In9 pvcrt 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 In9 pvcrt 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 .