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

Abstract

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I₅₁R₅₉N₁₀₈, dhps double mutant G₄₃₇G₅₈₁ and quadruple dhfr I₅₁R₅₉N₁₀₈ + dhps G₄₃₇ mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I₅₁R₅₉N₁₀₈ + dhps G₄₃₇E₅₄₀ and sextuple dhfr I₅₁R₅₉N₁₀₈ + dhps G₄₃₇E₅₄₀G₅₈₁ mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.