High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations

Abstract

BackgroundEmergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction–restriction fragment length polymorphism (PCR/RFLP) methodology.MethodsFifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR–RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries.ResultsA high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania.ConclusionHere the results demonstrate that HRM is a rapid, sensitive, and field-deployable alternative technique to PCR–RFLP genotyping that is useful in populations harbouring more than one parasite genome (polygenomic infections). In this study, a high levels of resistance polymorphisms was observed in both dhfr and dhps, among samples from Tanzania and Sénégal. A routine monitoring by molecular markers can be a way to detect emergence of resistance involving a change in the treatment policy.

Increased Frequency of Pfdhps A581G Mutation in Plasmodium falciparum Isolates from Gabonese HIV-Infected Individuals

“…SNPs at Pfdhfr 51, 59, 108, and 164 codons and Pfdhps 437, 540, and 581 codons were detected. HRM method was performed as previously described [27, 28]. High resolution melting (HRM) analysis is an alternative method designed to investigate variance in nucleic acid sequences.…”

“…High resolution melting (HRM) analysis is an alternative method designed to investigate variance in nucleic acid sequences. HRM technique is the cheapest and sensitive compared to PCR-RFLP [27, 28].…”

Strengthening Surveillance Systems for Malaria Elimination by Integrating Molecular and Genomic Data

“…Those tools are available and are being progressively deployed in malaria endemic countries [67]. Currently HRM genotyping using SNPs offers a relatively low-cost, robust, and easy to implement method to study parasite population structure and molecular markers associated with antimalarial drug resistance [68,69]. The technique could easily be implemented in laboratories in malaria endemic settings, even though it would need to be implemented only in reference laboratories with qualified personnel and adequate infrastructure.…”

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique

“…Providing evidence for the continued use of SP as IPTp and SMC in the context of SP resistance in Africa requires constant epidemiological surveillance of parasite resistance levels by monitoring polymorphisms in genes associated with SP resistance. Point mutations such as S 436 A, A 437 G, K 540 E, A 581 G and A 613 T/S in dihydropteroate synthetase ( dhps ) gene and N 51 I, C 59 R, S 108 N and I 164 L in dihydrofolate reductase ( dhfr ) gene are observed to play significant roles in SP resistance 4 – 6 . At a population level, the quintuple mutation in P. falciparum parasites, i.e., triple dhfr mutations of I 51 , R 59 , and N 108 , plus double dhps mutations of G 437 , and E 540 (I 51 R 59 N 108 G 437 E 540 ) has also been strongly linked with reduced SP efficacy as IPTp, reduced parasite clearance ability in asymptomatic pregnant women and shortened post-treatment prophylactic activity 7 – 9 .…”

“…The goal of this study was, therefore, to: (i) examine the current status of circulating Dhfr and Dhps haplotypes by describing polymorphisms in codons 51, 59, 108 and 164 of Dhfr and codons 437, 540, 581 and 613 of Dhps and (ii) estimate the prevalence of single, double, triple, quadruple and quintuple mutation 4 in parasites present in the five (5) Nigerian States.…”