Characterization of antimalarial drugs resistant I plasmodium falciparum isolates, using dot blot hybridization molecular technique
Kimani, Francis T.
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One of the major obstacles to the control of malaria is the emergence and spread of parasites resistant to anti-malarial drugs. Resistance by Plasmodium fulciparum to antifolate chemotherapy is a significant problem where drugs such as Fansidar® (sulfadoxine - pyrimethamine; S/P) have been in use for the treatment of chloroquine-resistant malaria. With the current trend in malaria treatment policies, this study aimed at assessing the level of resistance to one of the artemesinin combination therapy candidate sulfadoxine/ pyrimethamine in Kisii, an epidemic area of Kenya. Standard clinical efficacy testing in vivo, as recommended is time consuming and expensive method for establishing the levels of drug resistance, especially for large scale epidemiological surveys. The study also aimed at evaluating the dot-blot hybridization, a radionuclide based technique, for use in the laboratory settings and for drug efficacy and resistance monitoring at an epidemiological scale. This was taken up alongside the conventional PCR-RFLP technique. Pre-treatment peripheral blood was collected by lancet prick from a fingertip for direct microscopic diagnosis. Some amount of blood obtained was spotted directly onto Glass Fiber Membrane (GFM) or on 3MM Whatman filter paper, Parasitic DNA was extracted using GFM and Chelex-100®methods respectively. PCIZ amplifications were done using specific primers for RFLP and blotting. RFLP was by the use of specific restriction enzymes by overnight digests. Single stranded fragments were blotted on nitrocellulose membranes for probing using [Gama-32P] dATP 5' end labeled allele specific probes. The nature and distribution of mutations in the dhfr gene (chromosome 4) in codons 51, 59 and 108 were profiled in Kisii, a I'. falciparum malaria epidemic area in Kenya. Mutation profiles after dot blotting dhfr 108 showed that there were mutations in 69 (58.9%) of the study sample with no wild type, dhfr 59 showed 14 (11.97%) wild and 67 (59.82%) being mutant type while dhfr 51 had 2 (1.7%) wilds and 70 (59.82%) being mutants. Fifty six (47.8%) of the study population carried mutations in the three codons that were studied and these represented 80% of the PCR positive samples. Sixty one (52.1%) of these samples had double mutations in dhfr codons 108 and 59 and this was equivalent to 87.1% of the PCR positive isolates. Fifteen (12.82%) isolates which had been characterized as wild type by RFLP turned out to be mixed infections by radio probing. Moreover 29 (24.79%) of the isolates which had been characterized as mutant type actually came out as mixed type by radio probing. These findings would imply a high failure rate on the S/P drugs making them not to be good combination candidates in this epidemic area. Continued use of the drugs would lead to increased resistance due to drug pressure selection. The dot blot technique was found to have similar levels of specificity but more PCR/RFLP. Moreover it was found to be suited for large-scale epidemiological surveys of genes associated with antimalarial drug resistance. From the study it is recommended that this technique be adopted even in the monitoring of the efficacy of the new antimalarials which would help in detecting any possible resistance early enough for appropriate policy change.