ARCHIVED GIEMSA-STAINED THICK-SMEAR BLOOD SAMPLES AS A SOURCE OF DNA FOR PLASMODIUM FALCIPARUM DETECTION BY THE POLYMERASE CHAIN REACTION (PCR): AN IDEA FOR LOW TRANSMISSION SETTINGS

Abstract

The absence of a reasonable number of cases of malaria in low transmission settings often presents problems for epidemiologic studies of antimalarial drug resistance. Whole blood from filter paper spots is normally used as a source of deoxyribonucleic acid (DNA). The number of samples available to derive statistical power is usually limited due to low transmission rates. This study suggests an alternative source of DNA from Giemsa-stained thick smears (GSTS) for epidemiological studies of Plasmodium falciparum drug resistance. A total of 73 archived GSTS and 6 whole blood filter paper samples were available for this analysis. DNA obtained from GSTS was successfully extracted, genotyped and sequenced for 64 (88%) samples for the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene, 51 (70%) samples for the Plasmodium falciparum multi-drug resistance 1 (pfmdr1) gene, 34 (47%) samples for the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene and 27 (37%) samples for the Plasmodium falciparum dihydropteroate synthase (pfdhps) gene. Whole blood from the 6 filter paper samples that were also run for comparison were all (100%) successfully genotyped and sequenced in a single attempt for all the four genes. The ease of the analysis of the filter paper DNA samples and the quality of the gel electrophoresis pictures suggests that filter paper DNA sources are much more sensitive than archived GSTS samples. Nonetheless, the results of this study suggest that safely stored and clearly labelled GSTS can provide a cheap and somewhat reliable alternative source of DNA for retrospective epidemiologic studies using the polymerase chain reaction (PCR) analyses where filter paper sources are either not available or insufficient. It is, therefore, strongly recommended that laboratories in malaria low transmission settings develop guidelines for safe storage of GSTS for future use in genotyping and other experiments. Protocols to refine extraction and PCR methods as well as the design of appropriate primer pairs may improve the sensitivity of current PCR methods to improve the results of GSTS.