Abstract
BackgroundThe World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. In addition to providing quick and accurate detection of Plasmodium parasite proteins in the blood, these tests can be used as sources of DNA for further genetic studies. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs.MethodsThe proximal portion of the nitrocellulose membrane of discarded RDTs was used for DNA extraction. This genomic DNA was amplified using HRM to genotype the molecular markers involved in resistance to sulfadoxine-pyrimethamine: dhfr (51, 59, 108, and 164) and dhps (436, 437, 540, 581, and 613). Additionally, the msp1 and msp2 genes were amplified to determine the average clonality between Grande-Comore (Comoros) and Thiès (Senegal).ResultsA total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR. A high prevalence of resistance mutations were observed in the dhfr gene at codons 51, 59, and 108 as well as in the dhps gene at codons 437 and 436. A novel mutant in dhps at codon positions 436Y/437A was observed. The dhfr I164L codon and dhps K540 and dhps A581G codons had 100 % wild type alleles in all samples.ConclusionThe utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting). RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0861-6) contains supplementary material, which is available to authorized users.
Highlights
The World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases
To validate the utility of field-collected RDTs for typing of drug resistance markers, two markers involved in sulfadoxine-pyrimethamine (SP, Fansidar®) resistance were selected: dhfr and dhps genes, as these both drugs are routinely used in the populations of interest (Senegal and Comoros)
Point mutations in the dhfr and dhps genes confer resistance to pyrimethamine and sulfadoxine, respectively, with a decrease of in vitro susceptibility of P. falciparum in relation to the number of mutations of each gene [9,10,11,12,13]. This molecule has seen a relative decline of its therapeutic efficacy, it is used in Senegal for the Intermittent Preventive Treatment (IPT) of pregnant women and children against malaria, and in Comoros, for IPT of pregnant women, a policy change that was implemented in 2003 and 2004, respectively
Summary
The World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs. Malaria remains a major public health problem as it is responsible for 207 million cases and 627,000 deaths. Point mutations in the dhfr and dhps genes confer resistance to pyrimethamine and sulfadoxine, respectively, with a decrease of in vitro susceptibility of P. falciparum in relation to the number of mutations of each gene [9,10,11,12,13] This molecule has seen a relative decline of its therapeutic efficacy, it is used in Senegal for the Intermittent Preventive Treatment (IPT) of pregnant women and children against malaria, and in Comoros, for IPT of pregnant women, a policy change that was implemented in 2003 and 2004, respectively
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