Detection of drug resistant plasmodium Falciparum by polymerase chain reaction Using mutation-specific oligonucleotide Primers

Abstract

Due to increased chloroquine resistance, the antifolate-sulfa combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, resistance to the antifolates exists and it is important to study it because they are still effective in the above combinations. Point mutations in the dihydrofolate reductase (DHFR) gene lead to resistance to the antifolate drugs. It was considered important to establish the prevalence of the six reported point mutations among Kenyan field isolates of P. falciparum, and look for a correlation between these mutations and resistance of these isolates to antimalarials. Mutation specific polymerase chain reaction, (MSPCR), was carried out on 21 Kenyan P. falciparum isolates to detect point mutations. Optimal concentrations of each peR component were established for each MSPCR reaction. Direct sequencing of the DHFR gene was also carried out to firstly confirm the above point mutations and secondly to look for any other base pair changes. IDso values were calculated from drug sensitivity tests carried out by measuring 3H Hypoxanthine uptake in the presence of increasing concentrations of the appropriate drug. Out of the 21 Kenyan isolates five were found to be pyrimethamine sensitive and 16 were pyrimethamine resistant. Drug sensitivity results have shown that compared to reference strains, the Kenyan isolates examined are not resistant to the biguanides with respect to their IDsovalues. At the same time none of the base pair changes associated with biguanide resistance (at positions 16 and 108 in the DHFR gene) were found and neither was the change at 164 which confers cross resistance to both pyrimethamine and biguanides. Of the reported six mutations, we have so far only found three in Kenyan isolates. These are Ser-108 to Asn-108, Asn51 to lIe-51 and Cys-59 to Arg-59; all associated with pyrimethamine resistance. The results obtained were confirmed by direct sequencing. No other differences in the DHFR sequence were detected. Thus the point mutations correlated well with absence or presence of pyrimethamine resistance and absence of cycloguanil resistance. MSPCR has the advantage over drug sensitivity tests of being specific, quicker and more reproducible. It should therefore be feasible to screen large numbers of patients to monitor the appearance, persistence and spread of anti-folate (e.g ., pyrimethamine or biguanides) resistance of P.falciparum in a population.