RDTs as a source of DNA to study Plasmodium falciparum drug resistance in isolates from Senegal and the Comoros Islands

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.

Proof of concept: Malaria rapid diagnostic tests and massively parallel sequencing for surveillance of molecular markers of antimalarial resistance in Bissau, Guinea-Bissau during 2014-2017

“…SP was never first-line treatment in Guinea-Bissau, but was recommended as second-line treatment from 1996-2007. Selection may also be caused by use of SP for self-treatment of malaria, the use of sulfamethoxazole-trimethoprim for bacterial infections, and finally it is also possible that quadruple mutants are imported from neighbouring countries where SP has been used as first-line treatment and where mutant haplotypes have historically been more prevalent than in Guinea-Bissau (44-46).…”

Molecular assays for antimalarial drug resistance surveillance: A target product profile

“…However, good quality filter paper should be used to ensure optimal yield and quality of DNA, especially after long term storage [ 57 ]. Optimally, the assay should be able to use DNA extracted from a positive rapid diagnostic test (RDT), as RDTs are currently widely used in malaria endemic countries, especially in Africa, offering at times the best access to samples [ 58 ]. Importantly, assays should routinely include negative and positive controls.…”

Molecular Epidemiology of Plasmodium Falciparum by Multiplexed Amplicon Deep Sequencing in Senegal

“…All PCR reactions were conducted in a 20 µl reaction mixture containing 2 µl of template DNA, 4 µl Phusion high-delity (HF) PCR master mix, 0.2 µl HF Phusion Taq Polymerase, 10 mM deoxyribonucleotide triphosphate (dNTPs), 10 µM of each forward and reverse primer of each gene. Cycling conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 1 min and extension at 68 °C for 1 min 40 s; a nal extension was done at 68 °C for 5 min [23,35]. The PCR products were revealed by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized under ultraviolet (UV) trans-illumination (VersaDoc®, BIORAD, Hercules, USA).…”

“…Microsatellite typing is an alternative tool to assess genetic diversity but might fail to detect many minor clones as it requires a cut-off of 33% of the predominant peak for a minimal peak height [20][21][22]. Similarly, high resolution melting (HRM) has been used for genotyping of single nucleotide polymorphisms (SNP), but shows limitations in the detection of indels [23]. Advances in next generation sequencing (NGS) technologies and bioinformatic analysis has enabled the accurate detection of clones and even minor clones, overcoming the limitations of these other methods [24].…”