Trends In Sulfadoxine/pyrimethamine And Chloroquine Resistance Markers In Malindi And Busia Districts, Kenya.

Abstract

Plasmodium falciparum is responsible for the high mortality rate of malaria in sub-Sahara Africa. In an attempt to monitor this disease and provide valuable information to the anti-malarial drug program, I have surveyed malaria parasites from two geographically distinct areas in Kenya, namely Malindi and Busia, that represent varying degrees of drug resistance, drug usage patterns, and levels of malaria transmission. From each study site a hundred samples of cultured Plasmodium falciparum parasites were obtained through the U.S Army Medical Research unit -Kenya (USAMRU-K) at Kenya Medical Research Institute (KEMRI). Genomic DNA was isolated from these samples for the determination of specific mutations in the genes that have been correlated with chloroquine and Sulfadoxine Pyrimethamine resistance. These are Pfmdr 1 and Pfcrt for chloroquine resistance and Pfdhfr and Pfdhps for Sulfadoxine IPyrimethamine resistance. The DNA obtained was then used for substitution mutation analysis. The nested Polymerase Chain reactions (PCR) approach as described by Duraisingh (1998) was used to amplify regions of the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr 1, genes whose substitution mutations are known to confer resistance to these two drugs. Point mutation PCR analysis was performed for the detection of point mutations in Pfdhfr Ala613Ser), and Pfcrt (Lys76Thr,). Restriction Fragment Length Polymorphism was performed for detection of point mutations in Pfmdrl (ASP86Tyr, Phe184Tyr,Serl034CyS, Asnl042AsEa;nd ASP1246Tyra)ndPfcrT( Ala220se:): The PCR or restriction digest products, were loaded into a 2% agarose gel containing ethidium bromide and electrophoresed in TBE buffer at 100V for 1.5 to 2 hours Gels were stained with 10ug/ml Ethidium bromide and visualized and photographed on a UV trans-illuminator (313nm) using a gel documentation system, For the analysis of the restriction digest products, each batch of samples were run with an undigested PCR Product (acting as a standard for the digestion) and a negative sample carrying no DNA template. Otherwise for the analysis of the mutation-specific PCR products, each sample was loaded into two consecutive wells, one to detect a wild type PCR product and the other, a mutant PCR product. A 100bp ladder marker (Pharmacia Biotech) was used to estimate the band mobility on the gels. The gel was then treated in 1% Clorox solution before discarding it. Baseline frequencies of mutations were determined form all the samples. The Chisquare test for two-tailed significance P (P=O.05) was used for univariate comparisons. From this study it appears that people still use chloroquine for malaria treatment since the prevalence of mutations that confer resistance to this drug is increasing. It should also be noted that SP resistance is very high even though it is the recommended first line treatment for malaria in Kenya. However many Kenyans continue to use chloroquine in either high doses or in combinations with SP (Personal observation). The information here obtained will facilitate the implementation of rational and prudent strategies for reviewing the current malaria drug policy in Kenya