Molecular monitoring of PfATPase6 single nucleotide polymorphism (SNPs) in relation to artemisinin resistance in Western Kenya

Abstract

P. falciparum resistance to drugs is a major public health problem. Early last decade, a concern was raised regarding resistance to Sulphadoxine-Pyrimethamine by falciparum malaria in the country. Following recognition that S-P therapy was failing, in April 2004, replacement options resulted in a decision to adopt artemether-lumefantrine (AL), as the first line therapy in Kenya. Subsequent to the policy change by the Kenyan government, it was reported elsewhere that artemisinin resistance is associated with the S769N mutation in the PfATPase6 gene. Furthermore, N569K and L89E mutations have been investigated in West Africa. The aim of this study was therefore to investigate whether mutations have occurred in the PfATPase6 genes of parasites circulating in Western Kenya and if so, whether the mutations have affected malaria treatment by artemisinin drugs. One hundred and fifty P. falciparum field isolates were collected in 2009 from blood obtained from consenting patients at Kisumu, Kericho and Kisii district Hospitals. A nested PCR and sequencing assay was developed to analyze the genotype polymorphisms of the PfATPase6 involving S769N, N569K and L89E changes. Bioinformatics tools were applied for these analyses. The tools included DNA BASER for contig assembly of nucleotide sequences, NCBI ORF finder to translate the nucleotide sequences to protein code, MUSCLE 3.6 to align the genetic sequences and GENEDOC software to view multiple sequence files. In-vitro culture of the parasites was followed by artemisinin sensitivity testing of the parasites using Malaria SYBR Green I-based fluorescence assay. xv Results were analyzed by Kruskol-wallis one-way ANOVA test using graph pad prism software. Western Kenya PfATPase6 genes presented polymorphisms at five codons, viz.: 569, 679, 630, 639 and 657. Kisumu District hospital had the highest prevalence of 73.07% at codon S679F followed by Kericho with 70% and Kisii with 12.5%. At codon N569K, Kericho District Hospital had the highest prevalence of 37.5% followed by Kisumu District Hospital at 23% and Kisii District Hospital with 20%. Codons A630S and T657P had equal prevalence of 7.69% in Kisumu District Hospital and 12.5% at the Kisii District Hospital. G639D codon mutation was at 2.7% occurring only in Kericho District Hospital. All mutations observed were rare, except the PfATPase6 S679F in 61.36% and PfATPase6 N569K found in 25% of parasites. No polymorphisms were observed at codons 89 and 769. In general, the highest rate of polymorphism was observed in Kisumu District Hospital, suggesting sustained drug pressure at this site compared to the other sites studied. Amongst 92 samples tested and sensitive to artemisinin, 42.39% had S679F mutations while 17.39% had N569K. 15.21% had both S679F and N569K mutations. This might suggests a synergistic relationship between the two mutations. Only 2 (2.89%) parasite isolates had all five mutations present. Analysis of the IC50 values to determine association between the mutations and resistance to artemisinin revealed no such corelation. It can be concluded that in 2009, P. falciparum parasites circulating in Western Kenya were mainly polymorphic at codons 569 and 679 in the PfATPase6 genes. Continual xvi usage of ACT in the region may influence this polymorphism hence suggesting need for close and sustained monitoring of these polymorphisms in this gene as well as artemisinin sensitivity in western Kenya.