Prevalence of Plasmodium falciparum kelch 13-propeller region, Pfap2-mu, Pfcrt, and Pfmdr1 mutations associated with resistance to artemisinin-based combination therapies and chloroquine in Nyando, Kisumu County

Abstract

The treatment and eradication of malaria disease is still a major challenge due to the acquisition and spread of resistant parasites to the antimalarial drug in use. Resistance to the mainstay antimalarial drugs is a significant concern in the control of malaria. Delayed Plasmodium falciparum parasite clearance following artemisinin-based combination therapy (ACT) administration has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch 13 propeller region (k13). However, SNPs in the Pf-adaptor protein complex two mu subunit (Pfap2-mu), Pfcrt, and Pfmdr1 are possible markers associated with multi-drug resistance. This study aimed at establishing the prevalence of mutations in the kelch 13 propeller region, Pfap2-mu, Pfcrt, and Pfmdr1 gene(s) associated with resistance to ACT and chloroquine (CQ) antimalarial drugs in Nyando, Kisumu County. Moreover, the study determined the functional implications of mutation present in the k13 gene of P. falciparum based on the structural analysis. A total of 94 dried blood spot field isolates collected from children aged below 12 years infected with P. falciparum during a cross-sectional study were utilized in this study. The samples were collected in 2015 during the peak malaria transmission season in the Nyando region of Western Kenya before treatment with Artemether-lumefantrine (AL), the first-line artemisinin-based combination therapy (ACT) in Kenya. However, 47 of the 94 samples had recurrent parasitemia and were interrogated for the SNPs' presence in k13 and Pfap2-mu. The PCR amplification and sequencing were used to evaluate specific regions of k13 (codons 432–702), Pfap2-mu (codons 1–350), Pfmdr1 (codons 86, 1034–1246), and Pfcrt (codons 72–76) gene(s). The majority of parasites harbored the wild-type k13 sequence. However, one unique non-synonymous W611S change was detected. In silico studies on the impact of the W611S predicted structural changes in the overall topology of the k13 protein. Of the 47 samples analyzed for SNPs in the Pfap2-mu gene, 14 (29%) had S160N mutation. The CVIET haplotype associated with CQ resistance in the Pfcrt yielded a 7.44% (7/94), while the CVMNK haplotype was at 92.56%. Mutations in the Pfmdr1 region were detected only in three samples (3/94; 3.19%) at codon D1246Y. This study suggests that parasites in the western part of Kenya harbor wild-type strains and might be susceptible to AL and CQ antimalarial drugs. However, detecting the unique SNP in k13 and Pfap2-mu linked with ACT delayed parasite clearance may suggest slow filtering of AL resistant parasites. In this view, the study recommends continued surveillance to monitor the possibility of unprecedented ACT resistance in the future.

Keywords: Plasmodium falciparum, ACT, Delayed Clearance, SNPs, Pfap2mu, PfK13