Molecular Epidemiology of Anti-malarial Drugs Resistance Markers in Clinical Plasmodium falciparum Isolates and Infectiousness of Symptomatic Patients to Mosquitoes in East Shewa Zone, Oromia Regional State, Ethiopia

Abstract

Surveillance of antimalarial drug resistance markers and membrane feeding experiments are among the efforts of controlling and/or eliminating malaria. Plasmodium falciparum resistance to every class of anti-malarial drugs is a major challenge in efforts to control and/or eliminate malaria worldwide. Due to widespread chloroquine (CQ) resistant P. falciparum Ethiopia changed its treatment policy from CQ to sulfadoxine-pyrimethamine (SP) in 1998. However, resistance to SP developed shortly and in 2004 artemether–lumefantrine (AL, Coartem®) was recommended as a first-line treatment for the uncomplicated falciparum malaria. Data on the prevalence of CQ resistance markers after more than two decades of its removal is important to map the selection pressure behind the targets codons of interest. Understanding human malaria infectious reservoir is important for malaria elimination initiatives. It is poorly studied in low malaria transmission settings. As a consequence, data of onward transmission potential of malaria infected patients to mosquitoes are scant in Ethiopia. The present study was conducted to determine the prevalence of mutations in Pfcrt K76T and Pfmdr1 N86Y codons and to assess infectiousness of symptomatic Plasmodium infected patients to colonies of Anopheles arabiensis mosquitoes in East Shewa Zone, Oromia Regional State, Ethiopia. Finger-prick blood samples were collected on 3MM Whatman ® filter papers from a total of 121 microscopically confirmed P. falciparum infected patients. Parasite DNA was extracted by Chelex-100 method from dried blood spot (DBS). Genomic DNA was used to amplify both codons by nested PCR. Nested PCR products were subjected to APoI endonuclease digestion to determine mutations at codons 76 and 86 of Pfcrt and Pfmdr1 genes, respectively. Direct membrane feeding assays (DMFAs) were conducted on 63 patients infected with P. falciparum, P. vivax and mixed infections. Mosquitoes’ mid-guts and salivary glands were dissected to evaluate oocyst and sprozoite infection rates, respectively. Out of 83 P. falciparum isolates successfully genotyped for Pfcrt K76T, 91.6% harbored 76T mutant genotypes. The prevalence of Pfcrt 76T was 95.7%, 92.5% and 84.5% in Adama, Metehara and Olenchiti study areas, respectively. However, the prevalence of this mutation in the three study areas showed no statistical significance difference (χ2 = 1.895; P = 0.388). On the other hand, among 80 P. falciparum samples successfully amplified for Pfmdr1 N86Y all carried Pfmdr1 N86 wild-type genotypes. Out of 63 DMFAs, 68.3% resulted in at least one infected mosquito with a total infection rate of 21.6% of mosquitoes infected with 1-285 oocysts per mid-gut. Among 28 randomly selected assays with oocyst infected mosquitoes, 60.7% had sprozoites in the salivary glands of mosquitoes. Both mosquitoes (oocysts) and sporozoite infection rates demonstrated positive correlation with parasitaemia and gametocytaemia. Demonstrating oocyst infection in the mosquitoes might predict infectiousness of mosquitoes, although not all infected mosquitoes became infectious mosquitoes. The greater proportions of P. falciparum clinical isolates carrying mutant 76T genotypes may be due to indirect CQ pressure following partial withdrawal of CQ in the country. The current fixation of Pfmdr1 N86 allele may be favored by the use of AL for the treatment of uncomplicated falciparum malaria. Study by DMFA showed high infectiousness of symptomatic patients. Patients with symptomatic P. vivax infections were found to be more infectious to mosquitoes than patients with P. falciparum symptomatic infections. The current high prevalence of 76T mutant allele (CQ-resistant) and N86 wild-type allele (Al less susceptible) showed that continues monitoring implementation of CQ and AL should take place in order to contain the spread of antimalarial drug resistance P. falciparum. Further DMFA studies on human and mosquito determinant factors including both symptomatic and asymptomatic malaria infections with larger sample size should be carried out using microscopy and molecular techniques in order to guide malaria control and elimination efforts.