Molecular surveillance of anti-malarial resistance pfcrt, pfmdr1, and pfk13 polymorphisms in African Plasmodium falciparum imported parasites to Wuhan, China

Abstract

Background Imported malaria parasites with anti-malarial drug resistance (ADR) from Africa is a serious public health challenge in non-malarial regions, including Wuhan, China. It is crucial to assess the ADR status in African Plasmodium falciparum isolates from imported malaria cases, as this will provide valuable information for rational medication and malaria control. Methods During 2017–2019, a cross-sectional study was carried out in Wuhan, China. Peripheral blood 3 ml of returned migrant workers from Africa was collected. The target fragments from pfcrt, pfmdr1, and k13 propeller (pfk13) genes were amplified, sequenced, and analysed. Results In total, 106 samples were collected. Subsequently, 98.11% (104/106), 100% (106/106), and 86.79% (92/106) of these samples were successfully amplified and sequenced for the pfcrt (72–76), pfmdr1, and pfk13 genes, respectively. The prevalence of the pfcrt 76 T, pfmdr1 86Y, and pfmdr1 184F mutations was 9.62, 4.72, and 47.17%, respectively. At codons 72–76, the pfcrt locus displayed three haplotypes, CVMNK (wild-type), CVIET (mutation type), CV M/I N/E K/T (mixed type), with 87.50%, 9.62%, and 2.88% prevalence, respectively. For the pfmdr1 gene, NY (wild type), NF and YF (mutant type), N Y/F, Y Y/F, and N/Y Y/F (mixed type) accounted for 34.91, 43.40, 3.77, 15.09, 0.94, and 1.89% of the haplotypes, respectively. A total of 83 isolates with six unique haplotypes were found in pfcrt and pfmdr1 combined haplotypes, of which NY-CVMNK and NF-CVMNK accounted for 40.96% (34/83) and 43.37% (36/83), respectively. Furthermore, 90 cases were successfully sequenced (84.91%, 90/106) at loci 93, 97, 101, and 145, and 78 cases were successfully sequenced (73.58%, 78/106) at loci 343, 353, and 356 for pfcrt. However, the mutation was observed only in locus 356 with 6.41%. For pfk13, mutations reported in Southeast Asia (at loci 474, 476, 493, 508, 527, 533, 537, 539, 543, 553, 568, 574, 578, and 580) and Africa (at loci 550, 561, 575, 579, and 589) were not observed. Conclusions The present data from pfcrt and pfmdr1 demonstrate that anti-malarial drugs including chloroquine, amodiaquine, and mefloquine, remain effective against malaria treatment in Africa. The new mutations in pfcrt related to piperaquine resistance remain at relatively low levels. Another source of concern is the artemether-lumefantrine resistance-related profiles of N86 and 184F of pfmdr1. Although no mutation in pfk13 is detected, molecular surveillance must continue.

Targeted Amplicon Deep Sequencing for Monitoring Antimalarial Resistance Markers in Western Kenya

“…The majority of parasite genotypes were mixed wild-type (i.e., NYT) and variant NFT, which indicates selection by lumefantrine. A newly described mutation was observed at codon 199 ( Table 1 ) ( 33 ), leading to a new haplotype (NYS) that was rare and observed in mixed infections (9.1%). The Pfcrt data indicated that 100% of the parasites were wild type for both single nucleotide polymorphisms (SNPs) genotyped, and mixed infections could not be accurately determined using the capillary sequencing method and thus were not analyzed ( Table 1 ).…”

A Rapid and Specific Genotyping Platform for Plasmodium falciparum Chloroquine Resistance via Allele-Specific PCR with a Lateral Flow Assay

“…Recently, the antimalarial drug resistance of P. falciparum has remained a priority ( 6 , 9 ) and will hamper the global plan for malaria elimination. Therefore, it is necessary to timely monitor the molecular markers involved in drug-resistant Plasmodium and establish an economical, rapid, and accurate detection method.…”

“…Detection through nested PCR with sequencing is the most used genotyping technique ( 6 , 8 , 9 ). It is characterized primarily by its high specificity, but the sequencing still requires additional equipment, and the long waiting time is still an open problem.…”

“…However, the development of drug resistance reduces the efficacy of CQ ( 3 ). The emergence of the triple mutant haplotype, named CV IET , in the P. falciparum chloroquine transporter gene ( pfcrt ), has been confirmed in several countries to increase the risk of CQ treatment failure ( 4 – 6 ). Therefore, close and efficient detection of this single-nucleotide polymorphism (SNPs) loci and alleles will benefit medication guidance.…”

“…To evaluate the extent of drug resistance with genes, SNPs detection technology is considered one of the main fields conquered by contemporary biotechnology ( 7 ). For the detection of Plasmodium parasites mutation, nested PCR with sequencing considers the ‘gold standard’ method for detecting SNPs and genotyping ( 6 , 8 , 9 ). However, due to the long test period and complicated steps, test results cannot be obtained in real-time.…”