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

Abstract

Background Thailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand. Methods Under Thailand’s integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed. Results The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance. Conclusions This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.

A novel sensitive hexaplex high-resolution melt assay for identification of five human Plasmodium species plus internal control

“…2 and Supplementary file 2). The hexaplex PCR-HRM assay provides 91.76 % sensitivity and 98.04 % specificity to detect and identify the five Plasmodium species-related malaria infections using the real-time PCR assay described previously as the reference method for P. falciparum, P. vivax, P. ovale , and P. malariae identification ( Perandin et al., 2004 ; Rougemont et al., 2004 ), and DNA sequencing reference methods from other sources ( Snounou et al., 1993b ; Sugaram et al., 2021 ) for P. knowlesi identification (Supplementary file 3). To demonstrate the effect of the PCR competition of the hexaplex PCR-HRM assay, we prepared a plasmid DNA mix of the five Plasmodium species and the hexaplex PCR-HRM assay could detect all species-specific peaks of the mix, which limited the PCR competition effect (Supplementary file 1b).…”

“…To validate the sensitivity and specificity, achieved clinical samples confirmed Plasmodium species ( N = 85) and the samples collected from healthy participants ( N = 102). The achieved samples with confirmed Plasmodium species included P. falciparum ( N = 20), P. vivax ( N = 30), P. ovale ( N = 10) including P. ovale wallikeri ( N = 5) and P. ovale curtisi ( N = 5), P. malariae ( N = 10), and P. knowlesi ( N = 15) obtained from a previous study, describing a malaria infection outbreak at Ubon Ratchathani, Northeastern Thailand, in 2014 ( Imwong et al., 2014 ) and at Chumphon, Southern Thailand in 2018 and 2019 ( Sugaram et al., 2021 ). These samples have been obtained from the ethical review committees of the Faculty of Tropical Medicine, Mahidol University (approval no.…”

“…We interpreted the result as positive when the Ct-value of the real-time PCR was lower than 40. For P. knowlesi , the species identification was analyzed using the sequencing of previous protocols ( Snounou et al., 1993b ; Sugaram et al., 2021 ).…”

Multidisciplinary Investigations of Sustained Malaria Transmission in the Greater Mekong Subregion

“…In recent years, we and others have identified an increasing trend of clinical P. knowlesi cases in Thailand. 63 , 64 Increased incidences of P. knowlesi are likely due to environmental changes such as deforestation, increased forest-related human activities, and potentially peridomestic transmission. 74 Plasmodium knowlesi diagnosis is challenging 75 —it is often misdiagnosed by microscopy due to its resemblance to P. malariae and P. falciparum, current RDTs are not sufficiently sensitive to detect P. knowlesi, and confirmation requires the use of molecular methods.…”

“…Zoonotic Plasmodium knowlesi malaria. Since the first cluster of Plasmodium knowlesi malaria cases in humans was reported in 2004 in Malaysian Borneo, 59 reports of P. knowlesi incidence have increased strikingly, including in all countries of the GMS-Thailand, [60][61][62][63][64] Laos, 65,66 Cambodia, 67 Myanmar, 68,69 and Vietnam. 65,70,71 This wide range of P. knowlesi in Southeast Asia largely reflects the distribution of the zoonotic hosts (the long-tailed and pig-tailed macaques) and vectors of the Leucosphyrus group of anopheline mosquitoes.…”