ANALYSIS OF PLASMODIUM FALCIPARUM DRUG RESISTANCE MARKERS IN SCHOOLS IN WESTERN KENYA

Abstract

Malaria is a killer disease despite all the advances made in controlling it. Control measures such as using bed nets that have been treated by insecticides nets and treating malaria with artemisinin-based drugs have reduced malaria incidences and prevalence in the African continent and South East Asia. However, the rise of parasites not responding to artemisinin and its derivatives will create a more significant challenge to controlling and eliminating malaria. This study aimed to analyse Plasmodium falciparum drug resistance markers in young children aged 5-16 years. Specifically: 1) to measure the prevalence of Plasmodium falciparum drug resistance markers in school-going children and 2) to describe the spatial distribution of P. falciparum drug resistance markers in western Kenya. Method. Dried blood spot samples (n=8111) were collected from a survey whose aim was to evaluate the incidences of malaria infection among school children in eight counties of western Kenya (Homa Bay county, Migori county, Siaya county, Bungoma county, Busia county, Kakamega county, Vihiga county and Kisumu county) were analysed. A rapid diagnostic test (RDT) was used to determine malaria positivity. I extracted DNA from the RDT positive samples and used a P. falciparum quantitative polymerase chain reaction (Pf qPCR) assay to quantify the parasitemia in the samples. 2247/8111 samples tested P. falciparum positive using CareStart™ Malaria diagnostic kits while 5864/8111 samples were P. falciparum negative. It is from these 2247 dried blood spots (DBS) samples that DNA extraction was done using the chelex saponin method. 1263/2247 samples tested P. falciparum positive by qPCR while 984/2247 were successfully identified as negative. A total of 500 samples were selected and used to generate amplicons by a nested conventional PCR approach to screen for malaria drug resistance markers (Kelch 13, mdr1, dhps, dhfr genes) using the Illumina MiSeq sequencing platform vi Results. None of the Kelch 13 validated genetic changes pointing to artemisinin resistance were found in the population. However, changes in mutant allele numbers were seen in Sulfadoxine Pyrimethamine (SP), Pfdhfr (N51I, C59R, S108N), including a rise in the Pfdhps (S436H, A437G, A540E) resistance alleles. This data also describes emerging mutant alleles such Pfmdr1 codon T199 and Pfdhfr codons 85I and 164L mutations in the Kenyan population. Conclusion. The findings show that there are no mutations linked to parasite resistance to ACTs currently in use. However, the data presented here suggests that ongoing evaluation of antimalarial drug efficacy, particularly for artemisinin-based combinations, is required for Kenya and other malaria-endemic regions to implement timely evidence-based malaria treatment policies. Schools can serve as essential sampling sites for asymptomatic parasite carriers in regions with a relatively stable endemic malaria transmission pattern. Kenya still uses Sulfadoxine pyrimethamine (SP) as an Intermittent Preventive Treatment of pregnant women (IPTp); hence this study proposes the inclusion of molecular monitoring of Pfdhfr/Pfdhps mutations in SP based drug effectiveness studies. There is no literature on the Pfdhps S436H and Pfmdr1 T199S phenotype changes since SP is not the primary drug used to treat malaria in Kenya. However, it is generally used as part of Intermittent preventive treatment of malaria during pregnancy (IPTp), and some studies have shown that it is still available in the market. I also present a robust molecular surveillance tool that the national malaria control program can adapt.