Molecular surveillance of pfcrt, pfmdr1 and pfk13-propeller mutations in Plasmodium falciparum isolates imported from Africa to China

Background The emergence and spread of multidrug resistance poses a significant risk to malaria control and eradication goals in the world. There has been no indigenous malaria cases reported in China since 2017, and China is approaching national malaria elimination. Therefore, anti-malarial drug resistance surveillance and tracking the emergence and spread of imported drug-resistant malaria cases will be necessary in a post-elimination phase in China. Methods Dried blood spots were obtained from Plasmodium falciparum-infected cases returned from Africa to China between 2012 and 2015, prior to anti-malarial drug treatment. Whole DNA were extracted and known polymorphisms relating to drug resistance of pfcrt, pfmdr1 gene, and the propeller domain of pfk13 were evaluated by nested PCR and sequencing. The haplotypes and prevalence of these three genes were evaluated separately. Chi-squared test and Fisher's exact test were used to evaluate differences among the different sub-regions of Africa. A P value < 0.05 was used to evaluate differences with statistical significance. The maps were created using ArcGIS. Results A total of 731 P. falciparum isolates were sequenced at the pfcrt locus. The wild type CVMNK was the most prevalent haplotype with prevalence of 62.8% and 29.8% of the isolates showed the triple mutant haplotype CVIET. A total of 434 P. falciparum isolates were successfully sequenced and pfmdr1 allelic variants were observed in only codons 86, 184 and 1246. Twelve haplotypes were identified and the prevalence of the wild type pfmdr1 NYD was 44.1%. The single mutant pfmdr1 in codons 86 and 184 was predominant but the haplotype NYY with single mutation in codon 1246 was not observed. The double mutant haplotype YFD was common in Africa. About 1,357 isolates were successfully sequenced of pfk13-propeller domain, the wild type was found in 1,308 samples (96.4%) whereby 49 samples (3.6%) had mutation in pfk13. Of 49 samples with pfk13 mutations, 22 non-synonymous and 4 synonymous polymorphic sites were confirmed. The A578S was the most common mutation in pfk13-propeller domain and three mutations associated with artemisinin resistance (M476I, R539T, P553L) were identified in three isolates. Conclusion This study provides evidence that could give insight into potential issues with anti-malarial drug resistance to inform national drug policy in China in order to treat imported cases.