Background
Between 2010 and 2018, the incidence of malaria declined globally from 71 to 57 cases per 1000 head of at-risk populations. However, malaria still kills over 400,000 individuals every year [
1]. In the Greater Mekong subregion (GMS), including Cambodia, China (Yunnan Province), Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam, the reported number of malaria cases fell by 76% between 2010 and 2018, and malaria deaths fell by 95% over the same period [
1]. Thailand is committed to eliminating malaria by 2024. Between 2013 and 2020, the overall malaria incidence decreased from 37,741 to 4474 cases (88.1% reduction). The incidence of both
Plasmodium falciparum and
Plasmodium vivax malaria is declining, but the proportion of the two species has changed, with
P. falciparum accounting for 5.7% (257/4474) of cases and
P. vivax for 91.6% (4099/4474) in 2020 [
2].
One of the
Plasmodium species that infects humans,
Plasmodium knowlesi [
3], is a natural parasite of the long-tailed macaque,
Macaca fascicularis and the pig-tailed macaque,
Macaca nemestrina. Human malarial infection with this parasite was first reported in 1965 [
4], and a second case presented in 1971 in Malaysia [
5].
Plasmodium knowlesi infections have also shown distribution across the Greater Mekong Subregion (GMS), and previous
P. knowlesi infections in the GMS have been recorded in Malaysia [
6‐
9], Thailand [
10‐
12], Myanmar [
13,
14], Laos [
15,
16], Vietnam [
17,
18], and Cambodia [
19]. The distribution of
P. knowlesi may obstruct the malaria elimination agendas of countries of the GMS of Southeast Asia, especially due to asymptomatic cases, which have been previously reported [
20].
In Thailand, the malaria information system set up by the National Malaria Control Programme (NMCP) of Thailand does not include information on
P. knowlesi infections that occurred during the early stages of the system’s development; this is because the programme used Giemsa staining of thick and thin blood films and the pfHRPII-pLDH antigen rapid diagnostic test (pf-pan RDT) for diagnosis [
21], which do not clearly distinguish
P. knowlesi from other
Plasmodium species. The NMCP of Thailand began using molecular techniques for confirmation as the most effective tool for malaria verification in quality control and quality assurance.
Plasmodium knowlesi cases were subsequently detected in malaria patients who had visited the forest habitats of
M. fascicularis and
M. nemestrina macaques.
The present study aimed to analyse the genetic population of
P. knowlesi parasites in Thailand and compare them with previous published findings of parasites isolated from Thailand [
22,
23], Cambodia [
20] and Malaysia [
24‐
26]. Network analyses based on microsatellite markers were performed and constructed a phylogenetic tree based on the nucleotide sequences of the
P. knowlesi merozoite surface protein 1 gene (
pkmsp1). Furthermore, the
P. knowlesi dihydrofolate reductase gene was isolated and analysed for mutations, and homology modelling of PkDHFR mutants was conducted.
Discussion
The six GMS countries have endorsed a malaria elimination plan with the goal of eliminating
P. falciparum malaria by 2024 and all malaria by 2030 [
31]. Although the number of
P. falciparum and
P. vivax infections has decreased substantially, the incidence of zoonotic malaria from
P. knowlesi continues to increase in the GMS subregion [
32]. The ongoing increase in
P. knowlesi incidence presents a major challenge to regional malaria control and prevention activities.
P. knowlesi infections have been reported in almost all countries in Southeast Asia, and cases have occurred in travelers returning from these countries. However, most infections were reported in Malaysian Borneo [
32,
33]. The
P. knowlesi infections (3.1%) included in this study were found during the Thailand iDES scheme between 2018 and 2020. Furthermore, asymptomatic
P. knowlesi infections have previously been found at the Thai-Cambodia border [
20].
Plasmodium knowlesi infection can result in high parasitaemia and death, and the diagnosis should be confirmed by PCR [
32]. Therefore, highly specific and sensitive molecular tools and identification are required for malaria detection.
To understand the source of
P. knowlesi infections in Thailand, microsatellite markers and nucleotide sequences of
pkmsp1 were analysed for comparison with those of
P. knowlesi isolated from prior reported findings of Thailand [
22,
23], Cambodia [
20] and Malaysia [
24‐
26], which share borders with Thailand and may be the sources of the
P. knowlesi. The microsatellite marker and nucleotide sequencing results of
pkmsp1 obtained in this study showed that
P. knowlesi isolated from southern Thailand were similar to parasites isolated from Malaysia [
24‐
26], suggesting that
P. knowlesi in southern Thailand may be transmitted from Malaysia. Contrastingly,
P. knowlesi isolated from eastern Thailand were highly similar to those isolated from Cambodia [
20], suggesting this country may be the source of the parasites in that area. The clustering of the parasite lineages is likely to be a result of the migration of macaques, as human-to-human transmission has not been identified and the
Anopheles vector can only fly a few kilometres. These findings provide information on the source of infection and how
P. knowlesi malaria may be transmitted.
Molecular clinical and epidemiological studies have clearly shown that specific point mutations in the parasite dihydrofolate reductase gene (
dhfr) lead to resistance to pyrimethamine. The mutations cause alterations in crucial residues in the active sites of these enzymes, resulting in reduced drug affinity [
34‐
37].
Plasmodium knowlesi dihydrofolate reductase (
pkdhfr) mutations, found in field isolates from many countries, and ex vivo enzyme activity has been the focus of a number of studies. In this study, Arg34Leu and Thr105 deletions were observed in isolates from 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]. Anti-malarial drug exposure only occurs in human hosts, and if the transmission of
P. knowlesi remains zoonotic and there is no selection pressure, the malaria would be unlikely to develop anti-malarial resistance. Although there has yet been no anti-malarial resistance reported in
P. knowlesi, new anti-malarials should be adopted to counteract emerging anti-malarial resistance in the GMS [
40]. These new anti-malarials could aid in resolving anti-malarial resistance issues with other
Plasmodium species or used in combination to increase anti-malarial efficiency. Furthermore, as monkeys are not treated for malaria, the elimination of
P. knowlesi is impossible as long as macaques continue to act as zoonotic hosts. This is particularly evident from the experience in Sarawak in Malaysia, where
P. knowlesi is now almost the only remaining malaria infecting humans [
41].
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