Characteristics of molecular markers associated with chloroquine resistance in Plasmodium vivax strains from vivax malaria cases in Yunnan Province, China

Abstract Background Chloroquine (CQ) has been the preferred clinical treatment for vivax malaria in Yunnan Province since 1958, with over 300,000 patients. This study aimed to help make trend predictions regarding variations the in anti-malarial drug susceptibility of Plasmodium vivax distributed in Yunnan Province and effectively implement monitoring measures on the efficacy of anti-malarial drugs for vivax malaria. Methods Blood samples collected from patients with mono-P. vivax infections were employed in this study based on the principle of cluster sampling. The whole gene of P. vivax multidrug resistance 1 protein gene (pvmdr1) was amplified by nested-PCR techniques and the PCR amplification produce were sequenced by Sanger bidirectional sequencing. The mutant loci and haplotypes of coding DNA sequence (CDS) were identified by comparison with the reference sequence (NC_009915.1) of the P. vivax Sal I isolate. Parameters such as Ka/Ks ratio were calculated using MEGA 5.04 software. Results A total of 753 blood samples from patients infected with mono-P. vivax were collected, of which 624 blood samples yielded the full gene sequence (4392 bp) of the pvmdr1 gene, with 283, 140, 119, and 82 sequences from 2014, 2020, 2021 and 2022, respectively. A total of 52 single nucleotide polymorphic (SNP) loci were detected for the 624 CDSs, of which 92.3% (48/52), 34.6% (18/52), 42.3% (22/52), and 36.5% (19/52) SNPs were detected in 2014, 2020, 2021 and 2022, respectively. All of 624 CDSs were defined for a total of 105 mutant haplotypes, with CDSs of 2014, 2020, 2021, and 2022 containing 88, 15, 21, and 13 haplotypes, respectively. Of the 105 haplotypes, the threefold mutant haplotype (Hap_87) was the starting point for stepwise evolution, and the most drastic tenfold mutations were Hap_14 and Hap_78, and the fivefold, sixfold, sevenfold, and eightfold mutations. Conclusions In the majority of vivax malaria cases in Yunnan Province, most of them were infected with strains carrying demonstrating highly mutated in pvmdr1 genes. However, the dominant mutation strains types varied from year to year, which warrants further exploration in order to confirm the correlation between with phenotypic changes in P. vivax strains and their susceptibility to anti-malarial drugs such as chloroquine