Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia

Abstract

BackgroundMalaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission.MethodsThe P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.ResultsFourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.ConclusionAlthough moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

Asymptomatic Natural Human Infections With the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi

“…In early PCR protocols, there was cross-reactivity with P. vivax , its closest phylogenetic relative among the human malaria parasites, but more-recent methods such as those used here are highly specific. As with P. cynomolgi, P. knowlesi is not under antimalarial drug selection pressure and so remains fully susceptible to all of the antimalarial drugs [33]. Although the number of subjects with asymptomatic nonhuman-primate malaria parasite infections recorded in this large survey was small, there was still a significant difference in the calculated parasite densities, with P. cynomolgi carried at lower parasitemia levels.…”

Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand

“…The three-dimensional structural models of the two mutant proteins in complex with pyrimethamine showed that both Arg34 and Thr105 are not part of the binding pocket and are located far from the inhibitor-binding site, suggesting that the mutation at residue 34 and deletion of residue 105 are not associated with pyrimethamine resistance. Other studies have found a number of pkdhfr mutations, including Arg34Leu from Sabah, Malaysia, with no signs of positive selection [ 38 ]. Moreover, ex vivo enzyme activity has also been studied, but there was no association with antifolate resistance [ 39 ].…”

<em>Plasmodium knowlesi</em> malaria: current research perspectives

“…Nevertheless, there was no evidence of selective drug pressure in humans. 70 In addition to crt and dhfr, other orthologs of P. falciparum drug resistance genes, including multidrug resistance-1 (mdr1), dihydropteroate synthase (dhps), and kelch K13, were also looked at, with no signs of positive selection. 71 Since only human hosts would be expected to have antimalarial drug exposure and as long as the transmission of P. knowlesi remains zoonotic, the absence of drug selection pressure will make it unlikely to develop antimalarial drug resistance.…”