Molecular surveillance of anti-malarial resistance Pfdhfr and Pfdhps polymorphisms in African and Southeast Asia Plasmodium falciparum imported parasites to Wuhan, China

Abstract

Background Anti-malarial drug resistance is a severe challenge for eventual control and global elimination of malaria. Resistance to sulfadoxine-pyrimethamine (SP) increases as mutations accumulate in the Pfdhfr and Pfdhps genes. This study aimed to assess the polymorphisms and prevalence of mutation in these genes in the Plasmodium falciparum infecting migrant workers returning to Wuhan, China. Methods Blood samples were collected for 9 years (2011–2019). Parasite genomic DNA was extracted from blood spots on filter paper. The mutations were evaluated by nested PCR and sequencing. The single-nucleotide polymorphisms (SNPs) and haplotypes of the Pfdhfr and Pfdhps genes were analysed. Results Pfdhfr codon 108 showed a 94.7% mutation rate, while for Pfdhps, the rate for codon 437 was 79.0%. In total, five unique haplotypes at the Pfdhfr locus and 11 haplotypes at the Pfdhps locus were found while the Pfdhfr-Pfdhps combined loci revealed 28 unique haplotypes. A triple mutant (IRNI) of Pfdhfr was the most prevalent haplotype (84.4%). For Pfdhps, a single mutant (SGKAA) and a double mutant (SGEAA) were detected at frequencies of 37.8 and 22.3%, respectively. Among the combined haplotypes, a quadruple mutant (IRNI-SGKAA) was the most common, with a 30.0% frequency, followed by a quintuplet mutant (IRNI-SGEAA) with a frequency of 20.4%. Conclusion The high prevalence and saturation of Pfdhfr haplotypes and the medium prevalence of Pfdhps haplotypes demonstrated in the present data will provide support for predicting the status and progression of antifolate resistance in malaria-endemic regions and imported malaria in nonendemic areas. Additional interventions to evaluate and prevent SP resistance should be continuously considered.

Polymorphism of Antifolate Drug Resistance in Plasmodium vivax From Local Residents and Migrant Workers Returned From the China-Myanmar Border

“…Given such high rates of coinfection, treatments meant to target chloroquine-resistant P. falciparum likely impose substantial selective pressure for resistance to sulfadoxine-pyrimethamine (SP) in the local P. vivax population, and may induce high-grade antifolate resistance in P. vivax . SP has been the recommended treatment for falciparum and other non- vivax malaria since the 1980s, replacing chloroquine (CQ) as the front-line anti-malaria treatment when large-scale CQ resistance developed in many countries; but growing drug resistance soon necessitated replacement of SP with artemisinin-based combination therapy (ACT) ( Jiang et al., 2020 ). In addition to typical treatment proscribed by WHO guidelines ( WHO, 2013 ), SP is also used for intermittent preventive treatment in infants (IPTi) and pregnant women (IPTp) in areas endemic for malaria.…”

Single-Nucleotide Polymorphisms of Artemisinin Resistance-Related Pfubp1 and Pfap2mu Genes in Plasmodium Falciparum, Central China

“…In most cases, ACTs are still effective against P. falciparum of African and SEA origin, but the cases presented here suggest that this favorable situation may worsen in the future. The presence of molecular markers associated with drug resistance is important for their role in regulating drug sensitivity and subsequent dynamic prediction of drug resistance [21,22]. ART resistance to the global spread of malaria treatment will be a serious blow.…”

“…Up to now, there is no indigenous malaria cases reported in China [20]. It is worth noting that malaria endemic areas including Africa and SEA are the main source of imported malaria in China including Wuhan [21,22]. With the widespread use of ACTs in malaria-endemic areas such as Africa and SEA, resistance to ART has begun to emerge [6,7,11,12,21].…”

Detection of Antimalarial Resistance-Associated Mutations in Plasmodium falciparum via a Platform of Allele-Specific PCR Combined with a Gold Nanoparticle-Based Lateral Flow Assay

“…Therefore, the identification of SNPs is particularly important in disease diagnosis, biomedical research, food safety, and environmental analysis ( 9 ). Currently, PCR-restriction fragment length polymorphism (PCR-RFLP) ( 10 ), nested PCR ( 11 ), allele-specific PCR (AS-PCR) ( 6 ), real-time fluorescence quantitative PCR (qPCR) ( 12 ), loop-mediated isothermal amplification (LAMP) ( 13 ), Sanger sequencing ( 11 , 14 ), and gene chips ( 15 ) are commonly used to detect known SNPs. For unknown SNPs, single-strand conformation polymorphism (SSCP) ( 16 ), random amplified polymorphic DNA (RAPD) ( 17 ), high-throughput sequencing (next-generation sequencing [NGS]) ( 18 ), single-molecule real-time (SMRT) sequencing technology ( 19 ), and nanopore sequencing technology ( 20 ) are commonly used.…”

“…Another common method is sequencing, which is currently the gold standard for testing gene sequences ( 11 , 12 , 14 ). However, the test cycle is long, and the steps are too complicated, so the results cannot be obtained in time and cannot quickly be used in medical treatment for critical patients.…”