Khajohnpong Manopwisedjaroen’s research while affiliated with Mahidol University and other places

The evolution of genetic diversity and population structure of Plasmodium vivax as malaria elimination approaches remains unclear. This study analyzed the genetic variation and molecular epidemiology of P. vivax from Yala Province in southern Thailand, an area in the pre-elimination phase. Seventy P. vivax isolates, collected between 2017 and 2020, were genotyped for domain II of pvdbp and the 42-kDa region of pvmsp1 using amplicon deep sequencing. Data from Yala province were compared to published data from Tak province, where transmission was higher. Key analyses included nucleotide diversity (π), haplotype diversity (Hd), natural selection, recombination rates, and complexity of infection (COI). Genetic diversity in Yala was relatively low (π = 0.008dbp and 0.014msp1; Hd = 0.774dbp and 0.407msp1) compared to Tak (π = 0.012dbp and 0.027msp1; Hd = 0.849dbp and 0.962msp1). In Yala, polyclonal infections were found in 53.7% of pvdbpII and 47.8% of pvmsp142 isolates, with average COI of 1.6 and 1.7. Both genes were under balancing selection. Distinct genetic differences were found between Yala and Tak in pvmsp142, providing a local genotypic profile useful for tracing parasite origins.

Background Plasmodium knowlesi, identified as the fifth human malaria parasite, has rapidly spread across various Southeast Asian countries, yet uncertainties persist regarding its human-mosquito-human transmission. Therefore, this study aims to explore the transmission potential of P. knowlesi from human blood to mosquitoes. Methods A direct membrane-feeding assay was conducted by infecting laboratory-reared female Anopheles dirus mosquitoes with P. knowlesi-infected human blood from a single patient presenting with febrile malaria. Mosquitoes were dissected 7 days post-infection under a stereomicroscope to detect oocysts in the midgut, stained with mercurochrome. Salivary glands were examined 14 days post-infection for the presence of sporozoites. Malaria diagnosis employed microscopy by expert microscopists and nested PCR assays. Results Upon dissecting 745 out of 1439 blood-fed An. dirus mosquitoes on day 7 post-infection, two oocysts were identified in the midguts of two mosquitoes (0.27%). An additional 694 mosquitoes were dissected for salivary glands on day 14 post-infection, with three mosquitoes (0.43%) exhibiting sporozoites. Further confirmation by nested-PCR assay verified these sporozoites as belonging to the P. knowlesi species. Conclusions The findings underscore the potential transmission of P. knowlesi from human blood to mosquitoes. The significance of these findings necessitates further investigation, such as repeating similar experiments among natural vectors, to gain deeper insights into the transmission dynamics of P. knowlesi in Southeast Asia. Graphical Abstract

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbpII and pvmsp142kDa, respectively. Nucleotide diversity was higher in pvmsp142kDa than in pvdbpII (π = 0.027 and 0.012), as was haplotype diversity (Hd = 0.962 and 0.849). pvmsp142kDa also showed a higher recombination rate and higher levels of genetic differentiation (Fst) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbpII may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbpII decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp142kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand.

Background Dihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. This study aimed to assess whether the resistance genotypes have arisen the western part of country. Methods Fifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt. Results Among the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance. Conclusions Kelch13 mutation was common in Tak Province in 2013–2019. A new mutation G533S emerged in 2014 and rose to dominance in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.

Background: Dihydroartemisinin-piperaquine (DHA-PPQ) combination therapy is the current first-line treatment for Plasmodium falciparum malaria in Thailand. Since its introduction in 2015, resistance to this drug combination has emerged in the eastern part of the Greater Mekong Subregion including the eastern part of Thailand near Cambodia. Our aim is to assess whether the resistance genotypes have arisen the western part of country. Methods: Fifty-seven P. falciparum-infected blood samples were collected in Tak province of northwestern Thailand between 2013 and 2019. Resistance to DHA was examined through the single nucleotide polymorphisms (SNPs) of kelch13. PPQ resistance was examined through the copy number plasmepsin-2 and the SNPs of Pfcrt. Results: Among the samples whose kelch13 were successfully sequenced, approximately half (31/55; 56%) had mutation associated with artemisinin resistance, including G533S (23/55; 42%), C580Y (6/55; 11%), and G538V (2/55; 4%). During the study period, G533S mutation appeared and increased from 20% (4/20) in 2014 to 100% (9/9) in 2019. No plasmepsin-2 gene amplification was observed, but one sample (1/54) had the Pfcrt F145I mutation previously implicated in PPQ resistance. Conclusions: Kelch13 mutation was common in Tak Province in 2013-2019. A new mutation G533S emerged in 2014 and rose to dominance, possibly fixation, in 2019. PPQ resistance marker Pfcrt F145I was also detected in 2019. Continued surveillance of treatment efficacy and drug resistance markers is warranted.

Objectives Malaria cross-sectional surveys are rarely conducted in very low transmission settings. This study aimed to determine the prevalence and risk factors of Plasmodium infection in a near-elimination setting in southern Thailand. Methods Two cross-sectional surveys were conducted in areas of active transmission in Surat Thani province of Thailand in January and May of 2019. PCR was used to detect Plasmodium infection. Results The prevalence of Plasmodium blood infection was 0.45 and 0.61% in January and May of 2019, respectively. The major parasite species was P. falciparum in January and P. vivax in May. Unexpectedly, P. knowlesi infections were also detected. Most infections, including those of P. knowlesi, were asymptomatic. Being male and staying outdoor at nighttime were the only significant risk factors identified. 28.0% of people infected in January were positive in May for the same parasite species, suggesting persistent asymptomatic infections. Conclusions Despite the very low incidence rate in Surat Thani, most malaria infections were asymptomatic. Outdoor mosquito biting at nighttime is likely an important mode of malaria transmission. Unexpectedly, asymptomatic P. knowlesi infection was found, confirming previous reports of such infection in mainland Southeast Asia.

Although human infections of Plasmodium knowlesi have been found throughout Southeast Asia, most cases originated from Malaysian Borneo. In Thailand, P. knowlesi malaria was considered extremely rare. However, during October 2017-September 2018, there was a surge in the number of reported P. knowlesi cases. Here, a series of six cases of P. knowlesi malaria found during this period in Songkhla and Narathiwat provinces of southern Thailand are presented. All cases were confirmed by polymerase chain reaction. The unprecedented case number in the affected area is a warning sign of an increasing P. knowlesi burden in the south of Thailand.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form—dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.