Abstract:

Objective To analyze the polymorphism of Plasmodium vivax multidrug resistance protein 1 gene (Pvmdr1) from imported vivax malaria patients in Yunnan Province. Methods The whole Pvmdr1 gene was amplified and sequenced in the blood samples of patients who diagnosed with vivax malaria in 2020 and 2021 in Yunnan Province. The sequence of P. vivax SalⅠ isolate (GenBank accession number: NC_009915.1) was taken as the reference sequence for designing primers. MEGA 5.04 software was used to align the spliced sequences with the coding region reference sequence (GenBank accession number: XM_001613678.1). Haplotype and single nucleotide polymorphism (SNP) sites and their mutation types, nucleotide diversity π, expected heterozygosity (He), etc were analyzed by DnaSP 6.11.01. Haplotype medium network diagram was constructed using Network 10.0 Software. Results A total of 276 blood samples were collected from vivax malaria patients and were imported from overseas. The full gene sequences with 4 392 bp of Pvmdr1 were obtained from 259 blood samples, and the accession numbers were OP559204-OP559462 assigned by GenBank. The π of 259 DNA coding sequences was 0.000 76. A total of 22 SNPs were detected from 259 coding sequences, including 13 non-synonymous mutant and 9 synonymous mutant loci. Only the detection rate difference of c.4074C>T mutation between 2020 and 2021 [15.0% (21/140), 5.9% (7/119)] was statistically significant (χ² = 5.546, P < 0.05). The minor allele frequency was c.1587A>G (97.9%, 253/259), and the newly discovered SNPs included c.2499G>T (2.3%, 6/259), c.3358C>T (0.4%, 1/259), and c.3832C>T (0.4%, 1/259). All of c.3064C>T, c.4065A>G, c.3358C>T and c.3832C>T mutant sites were detected from the blood samples in 2021. The 259 complete coding region sequences of Pvmdr1 were defined as 26 haplotypes (Hap_2-Hap_27) by comparison with the reference sequence (haplotype Hap_1, all of which were mutant type haplotypes, and He was 0.890 7. Among them, the detection rate of Hap_8 was the highest (18.5%, 48/259). There were 15 and 22 haplotypes found from blood samples in 2020 and 2021, respectively. Among them, 4 haplotypes were only detected in blood samples from 2020, and 10 haplotype were only detected in blood samples from 2021. The difference of Hap_10 detection rate between 2020 and 2021 [15.0% (21/140), 3.4% (4/119)] was statistically significant (χ² = 22.264, P < 0.05). The proportions of quadruple, quintuple, six-fold, seven-fold, nine-fold and ten-fold multiple mutations in 26 haplotypes were 0.4% (1/26), 19.2% (5/26), 38.5% (10/26), 30.8% (8/26), 0.4% (1/26), and 0.4% (1/26), respectively. The haplotype medium network diagram displayed that the gradual evolution of 26 haplotypes started with Hap_1, and then passed through quadruple mutation (Hap_24), moved towards quintuple, six-fold, seven-fold, nine-fold, and ten-fold multiple mutations away from. Conclusion There is high polymorphism in the Pvmdr1 gene in the P. vivax from the imported vivax malaria cases in Yunnan Province.

Key words: Plasmodium vivax, Multidrug resistance protein 1, Mutation, Yunnan Province