Polymorphisms of pfcrt, pfmdr1, and K13-propeller genes in imported falciparum malaria isolates from Africa in Guizhou province, China.

BACKGROUND: Imported falciparum malaria from Africa has become a key public health challenge in Guizhou Province since 2012. Understanding the polymorphisms of molecular markers of drug resistance can guide selection of antimalarial drugs for the treatment of malaria. This study was aimed to analyze the polymorphisms of pfcrt, pfmdr1, and K13-propeller among imported falciparum malaria cases in Guizhou Province, China. METHOD: Fifty-five imported falciparum malaria cases in Guizhou Province during 2012-2016 were included in this study. Their demographic information and filter paper blood samples were collected. Genomic DNA of Plasmodium falciparum was extracted from the blood samples, and polymorphisms of pfcrt, pfmdr1, and K13-propeller were analyzed with nested PCR amplification followed by sequencing. Data were analyzed with the SPSS17.0 software. RESULTS: The prevalence of pfcrt K76T, pfmdr1 N86Y, and pfmdr1 Y184F mutation was 56.6, 22.2, and 72.2%, respectively, in imported falciparum malaria cases in Guizhou Province. We detected two mutant haplotypes of pfcrt, IET and MNT, with IET being more commonly found (54.7%), and five mutant haplotypes of pfmdr1, of which NFD was the most frequent (53.7%). There were totally 10 combined haplotypes of pfcrt and pfmdr1, of which the haplotype IETNFD possessed a predominance of 28.8%. In addition, three nonsynonymous mutations (S459T, C469F, and V692L) and two synonymous mutations (R471R and V589V) were detected in K13-propeller, all having prevalence less than 6.0%. In particular, a candidate K13 resistance mutation, C469F, was identified for the first time from Democratic Republic of the Congo with the prevalence of 2.0%. CONCLUSIONS: The high prevalence of IET haplotype of pfcrt and NFD haplotype of pfmdr1 suggests the presence of chloroquine, artemether/lumefantrine, and dihydroartemisinin/piperaquine resistance in these cases. Therefore cautions should be made to artemisinin therapy for P. falciparum in Africa. Continuous monitoring of anti-malarial drug efficacy in imported malaria cases is helpful for optimizing antimalarial drug therapy in Guizhou Province, China.

[Comparison of Malaria Diagnosis between County and Provincial Laboratories in Guizhou Province].

Malaria cases reported by county laboratories were further tested in the provincial laboratory in Guizhou province by using PCR test and microscopy. The consistency between PCR and microscopic results in the provincial laboratory was set as the basis for evaluation of microscopic results in county laboratories. In 89 samples, 24 were identified by PCR to be positive for malaria, among which 15 were infected with P. falciparum, 7 with P. vivax, and 2 with P. ovale; all were imported cases. And 21 samples had consistent identifications by PCR test and microscopic examination in the provincial laboratory. The total coincidence rate between county and provincial laboratories was 79.8%(67/84), and the undetected and error rates in county laboratories were 9.5%(2/21) and 23.8% (15/63), respectively. The Kappa value between county and provicial diagnosis was 0.6, being at the medium-to-high level of consistency.

[Single-tube Single-run Multiplex PCR Detects Mixed Samples with Four Species of Plasmodium].

OBJECTIVE: To establish a single-tube single-run multiplex PCR technique that can detect single or mixed samples with four species of Plasmodium. METHODS: Folding primers were designed based on the fast nested PCR. The reaction component concentrations were optimized and the primers were selected based on the annealing temperature. The established single-tube single-run folding-primer multiplex PCR (FP-PCR) was tested for its sensitivity and specificity to detect single-species and mixed samples with P. vivax, P. falciparum, P. ovale (including P. ovale wallikeri) and/or P. malariae. RESULTS: In all the seven experimental repeats, FP-PCR successfully detected single-species infection for all the four species, with the detection limit reaching or close to 1 parasite/µl blood. For mixed infections with 2-4 species at different densities with the highest being 100 times of the lowest, FP-PCR identified all the species in each combination in 57 out of 84 tests. Further, in 10 dried blood samples on filter paper from healthy subjects, no FP-PCR amplification was found, except weak formation of dimers. CONCLUSION: FP-PCR is a simple and sensitive method for detecting both single-species and mixed infections with human Plasmodium, and can be applied for malaria diagnosis, screening and monitoring.