Abstract
BackgroundResistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine–pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples.MethodsPlasmodium falciparum DNA was collected from the Thailand–Myanmar, Thailand–Malaysia and Thailand–Cambodia borders during 2008–2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated.ResultsMutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes. In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016. Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification.ConclusionsDespite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.
Highlights
Resistance to anti-malarials is a major threat to the control and elimination of malaria
A more recent survey conducted in Ubonratchathani province close the Thailand–Cambodia borders, which had a lot of reports in many anti-malarial drug resistances [2, 3], showed high levels of pfdhfr (N51I, C59R, and S108N, ≥ 76%) and pfdhps (A437G, K540E, A581G or A437G, K540N, A581G or S436A, A437G, K540E, ≥ 90%) triple mutations [10]
Mutations in pfdhfr and pfdhps genes in isolates from the Thailand–Myanmar border A total of 146 P. falciparum samples collected from malaria clinics in five provinces between 2008 and 2016 were analysed (Table 1)
Summary
Resistance to anti-malarials is a major threat to the control and elimination of malaria. A more recent survey conducted in Ubonratchathani province close the Thailand–Cambodia borders, which had a lot of reports in many anti-malarial drug resistances [2, 3], showed high levels of pfdhfr (N51I, C59R, and S108N, ≥ 76%) and pfdhps (A437G, K540E, A581G or A437G, K540N, A581G or S436A, A437G, K540E, ≥ 90%) triple mutations [10]. These border areas are malaria endemic regions. Several factors including drug target, nature of genes and host/parasite genetic background may differently affect the persistence of SP resistance after removal of SP use
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