Paracetamol may improve renal function in patients with severe Plasmodium knowlesi malaria, particularly in those with acute kidney injury and hemolysis, via inhibition of cell-free hemoglobin mediated oxidative kidney damage. We developed a population pharmacokinetic/pharmacodynamic (PK/PD) model to assess effects of paracetamol on creatinine, hepatotoxicity, fever clearance, and parasite clearance among Malaysian patients with predominantly non-severe knowlesi malaria using data from the PACKNOW trial (Clinical Trials Registration: NCT03056391). A total of 372 patients were included in the PK/PD analyses (paracetamol: n = 183, control: n = 189). Paracetamol PK was described using a prior PK model published in patients with falciparum malaria. The PK/PD demonstrated that higher paracetamol exposures were associated with a faster decline in both creatinine and fever clearance time, supporting its renoprotective and antipyretic effects. Increased paracetamol exposure was not associated with hepatotoxicity or serious adverse events, despite a weak positive association with liver transaminases over time. No significant relationship was observed between paracetamol exposure and parasite clearance. Overall, these findings highlight an exposure-response relationship for paracetamol and a decline in creatinine, supporting its use as a renoprotective drug in treating Plasmodium knowlesi malaria.

Development of a chemical precipitated-LAMP assay for the diagnosis of knowlesi malaria.

Zoonotic malaria, particularly Plasmodium knowlesi, has emerged as a growing public health concern across Southeast Asia, with increasing incidence reported in multiple countries. The recent detection of additional zoonotic Plasmodium species further complicates the epidemiological landscape, underscoring the expanding interface among humans, wildlife reservoirs, and mosquito vectors. Despite this trend, progress towards effective control and elimination remains limited, largely due to substantial gaps in entomological knowledge across many endemic regions. Currently, only a narrow range of vector control approaches is available, and these strategies are often ill-suited to the ecological and behavioural characteristics of zoonotic malaria transmission. A major challenge lies in the complexity of vector species assemblages in Southeast Asia. Some of the vectors belong to species complexes with high morphological similarity, making accurate taxonomic identification difficult. Misidentification of mosquito species can distort assessments of vector competence, biting behaviour, and ecological niche, leading to inaccurate inferences about transmission dynamics and potentially resulting in ineffective surveillance and misdirected vector control interventions. Compounding this issue are rapid landscape changes driven by deforestation, agricultural expansion, and habitat fragmentation, which alter mosquito bionomics. Growing evidence suggests that such environmental disturbances may promote outdoor and forest-associated biting, enhance vector adaptability, and shift transmission risk toward human settlements. Thus, this review synthesises current knowledge on the ecological, behavioural, and taxonomic complexities of zoonotic malaria vectors within a rapidly changing landscape. By critically examining challenges in vector identification, surveillance, and control, we highlight key gaps that impede effective intervention. Finally, we discuss pathways forward, emphasising the need for integrated, context-specific, and One Health-oriented strategies to address the unique challenges posed by zoonotic malaria transmission in Southeast Asia.

As Southeast Asian countries advance toward malaria elimination, challenges such as residual transmission reservoirs and zoonotic spill-over require high-resolution micro-surveillance. While conventional diagnostics often miss hidden transmission foci in low transmission settings, serological assays provide an essential supplement by measuring both historical and recent exposure. This scoping review assesses the implementation of antibody surveillance across Southeast Asian countries and highlights its potential in the concerted effort towards regional malaria elimination. This review adhered to the PRISMA-ScR guidelines to identify peer-reviewed studies on malaria serology in Southeast Asian countries. Systematic searches of PubMed, Web of Science and Scopus were conducted for articles published between January 2000 and May 2025. Multiple independent reviewers screened titles, abstracts and full texts against predefined eligibility criteria, focusing on English-language primary studies involving human samples. Extracted data including study location, population, serological assay type, serological markers and key findings were synthesised to provide a comprehensive overview of malaria antibody surveillance. Of 6,849 records identified, 33 studies across seven Southeast Asian countries met the inclusion criteria. Study designs consisted of 26 cross-sectional, five longitudinal and one each of a prospective cohort and a control trial. Over time, research focuses have shifted from high-transmission assessments to low-transmission and elimination contexts, increasingly utilising multiplex assays and protein microarrays. While Plasmodium falciparum and Plasmodium vivax markers predominated, recent studies have incorporated Plasmodium knowlesi and vector exposure antigens. Furthermore, seroconversion rate estimation and machine learning approaches effectively identified historical transmission declines and residual hotspots missed by conventional microscopy or molecular methods. Serological surveillance offers a transformative supplement to the diagnostic gaps prevalent in Southeast Asia's low-transmission settings. By generating a high-resolution exposure profile, advanced seroepidemiological tools enable precise micro-stratification of transmission foci, the differentiation of recent from historical exposure and the targeted management of zoonotic spill-over. Ultimately, integrating these methods provides a robust, cost-effective framework for identifying high-risk populations, verifying transmission interruption and achieving regional malaria-free certification.

Plasmodium knowlesi has emerged as an important zoonotic malaria in Southeast Asia. While most cases are reported from Malaysia, increasing reports from Indonesia, including Aceh Province, highlight the need for local epidemiological data. A retrospective, hospital-based study was conducted using secondary data from a hospital in Aceh Besar, Indonesia. Medical records of all patients confirmed positive P. knowlesi infection by microscopy and polymerase chain reaction (PCR) between January 2023 and April 2024 were reviewed. Data on socio-demographic variables, clinical presentation, treatment, outcomes, and parasite density were extractedand analysed. A total of 40 confirmed P. knowlesi malaria patients' data were analysed. The median age was 31years (IQR: 25-43). Farmers and plantation workers represented the largest occupational group (67.5%), followed by orangutan (great ape) caretakers (10%). All patients presented with fever and headache and were treated with dihydroartemisinin-piperaquine and primaquine, with complete recovery. Most cases occurred between April and July 2023. Parasite density analysis was performed on 35 patients with completed data, which was classified as high in 42.9%, moderate in 45.7%, and low in 11.4% of patients. Median hospital stay was three days, with longer stays observed among patients with lower parasite density. This study demonstrates that P. knowlesi malaria predominantly affects adult males engaged in forest-related occupations in Aceh Province, Indonesia. Therefore, continue surveillance of infection and context-specific, targeted interventions among high-risk occupational groups are needed to prevent and control zoonotic malaria in Indonesia.

We conducted the noninvasive surveillance of Plasmodium knowlesi in wild macaques using 4752 fecal samples collected across 9 endemic countries. Parasite DNA was detected in 390 samples (8.2%), with positivity rates ranging from 1.4% to 18.4%. This provides the first field-based evidence that P. knowlesi DNA in feces shed by macaques and present under natural conditions can be detected. These findings validate fecal sampling as a practical and scalable tool for tracking zoonotic-malaria. The results support integration into forest-runoff and rural wastewater surveillance systems, offering new opportunities for early detection of pathogens and environmental monitoring at the human-wildlife interface.

This scoping review synthesizes molecular evidence on Plasmodium genetic diversity and multiplicity of infection (MOI) in Southeast Asia and describes variation by species and epidemiological setting. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews guidelines, we searched PubMed, Scopus, ProQuest, and EBSCO for English language, open-access obersvational studies published between January 1, 2015 until October 11,2025. Included studies involved laboratory-confirmed human malaria and reported MOI and genetic diversity data derived from appropriate molecular genotyping methods. Findings were summarized by species, geography, markers, and clinical categories. Sixteen studies were included, comprising 1,609 genotyped Plasmodium falciparum infections, 1,526 Plasmodium vivax infections, 37 Plasmodium malariae infections, and 188 human Plasmodium knowlesi infections. P. falciparum exhibited marked heterogeneity, with polyclonal infection rates ranging from 3.9% to 73.9% and mean MOI ranging from 1.05 to 4.9. P. vivax consistently showed high diversity, with polyclonal infection rates ranging from 16.9% to 71.4% and mean MOI ranging from 1.1 to 1.91, alongside expected heterozygosity values of 0.66-0.87. P. malariae displayed low MOI, whereas P. knowlesi showed polyclonal infection rates ranging from 7.3% to 21% and mean MOI ranging from 1.04 to 1.06. No data were available for Plasmodium ovale. P. falciparum and P. vivax remain genetically diverse across Southeast Asia, with heterogeneous transmission patterns, particularly in border regions. Limited data on P. malariae, human P. knowlesi, and P. ovale highlight the need for standardized molecular surveillance to support targeted elimination efforts.

Following the elimination of human malaria, Malaysia has experienced an increasing burden of zoonotic Plasmodium knowlesi malaria, particularly in Sabah. Evidence on the cost-effectiveness of vector control interventions is needed to inform policy decisions and optimise resource allocation. This study assessed the cost-effectiveness of Outdoor Residual Spraying (ORS), Long-Lasting Insecticidal Nets (LLINs), and their combined use for controlling P. knowlesi malaria in Sabah, Malaysian Borneo. A cost-effectiveness analysis was conducted from the healthcare provider perspective over a one-year time horizon in three high-endemic districts: Keningau (LLINs), Ranau (ORS), and Kota Marudu (ORS + LLINs). Capital and recurrent components, were estimated using an ingredient-based costing approach. Effectiveness was measured as the number of P. knowlesi cases averted based on routine surveillance data. Incremental cost-effectiveness ratios (ICERs) were calculated relative to a no-intervention scenario, defined as health promotion activities without vector control. One-way sensitivity analyses assessed uncertainty in key parameters. All costs were reported in Malaysian Ringgit (MYR) and adjusted to 2025 values. Annual provider costs ranged from MYR 3912.92 (no intervention) to MYR 39,764.14 (ORS + LLINs). ORS was associated with the highest number of cases averted (n = 13) and the lowest ICER (MYR 2046 per case averted; approximately USD 470). LLINs and the combined strategy were associated with fewer cases averted and higher ICERs. ORS consistently demonstrated the most favourable cost-effectiveness profile across all sensitivity analyses. ORS demonstrated the most favourable cost-effectiveness profile under the observed programme conditions for controlling P. knowlesi malaria in Sabah. A stratified approach prioritising ORS, with targeted use of LLINs among high-risk populations, may represent an economically efficient strategy in resource-limited settings.

The zoonotic parasite Plasmodium knowlesi is an increasing cause of human malaria in Southeast Asia. We aimed to develop a P. knowlesi induced blood-stage malaria (IBSM) model to facilitate study of parasite biology, host responses to infection, and activity of antimalarial treatments. We manufactured and characterised a P. knowlesi parasite bank using the P. knowlesi YH1 strain adapted to grow in human serum. This P. knowlesi bank was evaluated in an IBSM study involving four adults intravenously inoculated with P. knowlesi-infected erythrocytes using a dose escalation strategy (10-fold increases from ∼2,800 parasites). Parasitaemia was monitored by qPCR. All participants received curative treatment with artemether-lumefantrine. Endpoints included safety and infectivity of the P. knowlesi bank. Two of the four participants developed detectable parasitaemia. Participant two (administered ∼28,000 parasites) had detectable parasites (∼6 parasites/mL) immediately prior to protocol-defined treatment on day 21. Participant three (administered ∼280,000 parasites) developed detectable parasites on day 17, peaking at 202 parasites/mL just after protocol-defined treatment on day 24; the parasite multiplication rate over 32 hours (PMR32) was 2.33 (95% CI 1.66-3.25). Participant four (also administered ∼280,000 parasites) did not become infected. Adverse events were mostly mild and unrelated to the challenge agent. A GMP-grade P. knowlesi YH1-HS parasite bank was successfully manufactured and shown to establish patent infection in humans. The finding that only one of two participants became infected at the highest inoculum dose highlights the potential for this IBSM model to evaluate factors influencing inter-individual variability in susceptibility to infection.

BackgroundThe emergence of Plasmodium knowlesi as the predominant cause of malaria in Malaysian Borneo raises important questions about protective genetic factors in endemic populations. This study investigates two common erythrocyte polymorphisms, glucose-6-phosphate dehydrogenase (G6PD) deficiency and Filipino β-thalassemia, for association with P. knowlesi infection risk.MethodsA retrospective case–control study compared 106 PCR-confirmed P. knowlesi cases with 89 controls from Sabah, Malaysia. Genotyping used multiplex PCR (8 major Southeast Asian G6PD variants) and gap-PCR (Filipino β-thalassemia deletions). Statistical analysis employed Fisher’s exact tests and logistic regression models to evaluate associations with infection risk, including controlling for age, sex, ethnicity and district site.ResultsG6PD deficiency genetic variants were present in 4/106 (3.8%, 95%CI 1.0–9.4%) P. knowlesi cases compared to 11/89 (12.4%, 95%CI 6.3–21.0%) controls (OR = 0.28, 95%CI 0.09–0.91; p = 0.034). The Viangchan variant (871G>A) was predominant (71%), with Coimbra and Mediterranean variants also present. No relationship with G6PD variants and severe malaria or sex was detected, although analysis was constrained by small numbers. Filipino β-thalassemia demonstrated no association.ConclusionsG6PD deficiency related variants confer substantial protection against P. knowlesi susceptibility, while β-thalassemia appears neutral. Findings have implications for zoonotic malaria transmission risk in Southeast Asia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12936-026-05908-x.

Zoonotic malaria caused by infection with the monkey parasite Plasmodium knowlesi has emerged across Southeast Asia, particularly in areas previously close to elimination of non-zoonotic malaria. In Indonesia, some rural and remote areas must now consider strategies which target various Plasmodium species in hard-to-reach populations. Indonesia has mostly subsidised care at local health clinics for patients with malaria and other febrile illnesses; however, patients still face out-of-pocket costs. This study estimated household cost of illness due to malaria and non-malarial febrile illness in North Kalimantan and North Sumatra, Indonesia.Household costs were estimated from individual patients as part of health facility-based cross-sectional surveys in eight health clinics across North Sumatra and North Kalimantan between January 2022 and October 2023. Direct costs due to medical and travel expenses, and indirect costs resulting from productivity losses were included. Overall, 2244 patients were recruited, including 153 (6.8%) malaria-confirmed cases. Five Plasmodium species were identified using validated PCR conducted on all participants: P. vivax (n=97), P. knowlesi (n=35), P. malariae (n=12), P. falciparum (n=3) and P. ovale (n=1), in addition to five mixed infections. Costs were inflated to 2023 Indonesian Rupiah and reported in US dollars (US$). A mean total cost of US$33 (SD=57) was reported for malaria episodes and US$17 for non-malarial fever episodes (SD=38), primarily composed of indirect productivity losses from time away from usual activities (70% and 61% of total cost for patients with malaria and other febrile illnesses, respectively). Overall, 16% of patients with malaria and 11% of patients with other febrile illnesses experienced catastrophic health expenditure from their illness episode.Despite a largely subsidised health system, patients and families face other medical, travel and indirect costs when seeking care for febrile illnesses. These costs need consideration when designing malaria control policies, particularly in near-elimination settings, with few malaria cases among broader febrile illness.

Introduction/Objective Agricultural workers in Malaysia are consistently exposed to biological hazards originating from soil, water sources, livestock, and vector populations. Environmental conditions, such as tropical climate, monsoon-related flooding, and deforestation, further exacerbate the risk of zoonotic and vector-borne diseases. Despite these risks, biological hazards remain less extensively studied compared to physical and chemical exposures. This scoping review aimed to synthesize the current evidence on biological hazards affecting Malaysian agricultural workers, with an emphasis on occupational and environmental determinants, surveillance gaps, and preventive challenges. Methods A scoping review was conducted following Arksey and O’Malley’s methodological framework and the PRISMA-ScR guidelines. Peer-reviewed studies published between 2016 and September 2025 were retrieved from major databases. The eligible studies included biological hazards associated with paddy farming, plantation work, livestock production, and aquaculture. Data were charted according to hazard type, agricultural setting, study methodology, and the identified determinants. Results A total of 47 studies met the inclusion criteria. The findings identified a diverse range of pathogens linked to occupational exposure, with Leptospira spp. being the most frequently reported, followed by Plasmodium knowlesi , Burkholderia pseudomallei , and various tick-borne agents. Environmental determinants, including contaminated soil and water, inadequate sanitation, climate variability, and high vector density, were consistently associated with increased disease occurrence. However, surveillance systems remain fragmented, with limited longitudinal monitoring and inconsistent reporting of biological hazards. Discussion This review highlights substantial knowledge gaps in understanding emerging biological hazards, climate–disease interactions, and the effectiveness of existing preventive strategies. Weak integration of occupational health, environmental monitoring, and vector surveillance limits timely detection and response. These gaps reduce the accuracy of disease burden estimates and hinder the development of targeted risk-reduction measures. Conclusion Biological hazards in Malaysian agriculture constitute a critical yet under-monitored occupational health threat, influenced by environmental and climatic dynamics. Strengthening One Health-based surveillance systems, updating national policies, and integrating environmental microbiology into occupational health programs are essential to address these gaps. Enhanced monitoring, adaptive prevention strategies, and cross-sector collaboration are necessary to safeguard worker safety and ecosystem health in changing climates.

Despite Indonesia reporting high numbers of Plasmodium knowlesi malaria cases in North Sumatra Province, the Anopheles mosquito vectors remain unknown. This study identified the Leucosphyrus Group species present in Langkat Regency, North Sumatra, and the Plasmodium species DNA in their heads and thoraces. Mosquitoes collected by human landing catch were morphologically identified and their species identification subsequently confirmed using ITS2 sequencing as well as Dirus Complex (DiCSIP) and Anopheles scanloni-specific PCR. Reverse-transcription real-time and nested PCR assays targeting the 18 S rRNA gene were applied for Plasmodium species detection and identification. Of 597 morphologically identified Leucosphyrus Group mosquitoes, two species of the Dirus Complex were confirmed for the first time in Indonesia: 97.8% of specimens were Anopheles dirus with 2.2% being Anopheles scanloni. Seven An. dirus specimens were Plasmodium-positive, including mixed infections with P. inui, P. knowlesi, and/or P. vivax and one equivocal sample positive for P. coatneyi and P. knowlesi. BLAST analysis indicated possible cross-reactivity of P. fieldi primers with P. inui. This study provides the first molecular confirmation of An. dirus and confirms the presence of An. scanloni, two species of the Dirus Complex in North Sumatra. In addition, it demonstrates the presence of both macaque and human Plasmodium species DNA in An. dirus, suggesting the potential role of this species in zoonotic and human malaria transmission in this Indonesian region.

In silico profiling of the plasmodium knowlesi 32kDa antigen: Diversity, epitope prediction, and structural modeling.

A cold-chain free loop-mediated isothermal amplification test for detection of human malaria in point-of-care settings.

Assessment of risk factors associated with Plasmodium knowlesi infection in Sarawak, Malaysia

Plasmodium knowlesi can adapt to infect Duffy-negative erythrocytes\u2217

Plasmodium knowlesi poses an emerging challenge for malaria control in Southeast Asia due to its zoonotic nature, diagnostic complexity and lack of species-specific control strategies. These factors complicate both prevention and case management efforts. Understanding both community awareness and healthcare provider perspectives is critical for informing targeted responses. This study aimed to assess awareness of P. knowlesi malaria among at-risk community members in southern Thailand and to explore contextual challenges through qualitative insights from healthcare providers, including provincial officers, district-based malaria control staff, and village health volunteers. An explanatory sequential mixed-methods design was employed between April and May 2025. A cross-sectional survey of 300 adults residing in three P. knowlesi-endemic districts was conducted using a structured questionnaire that included eight items assessing overall awareness. Descriptive statistics, violin plots, and multivariable generalized linear modeling were used to identify factors associated with awareness scores. Subsequently, semi-structured interviews were conducted with 28 healthcare providers across various administrative levels. Qualitative data were thematically analyzed. Overall awareness of P. knowlesi malaria was moderate (mean score: 10.7/23; SD ± 2.9; range: 3.0-20.0). Female individuals, living farther from health facilities, receiving malaria-related health education, and having higher attitude scores were significantly associated with increased awareness (p < 0.05). Healthcare providers reported a decline in P. knowlesi incidence, alongside persistent transmission risks in forested areas. Health education efforts were largely reactive, delivered following case detection, but often included information about monkey-to-human transmission. Awareness of P. knowlesi malaria remains suboptimal among high-risk populations, particularly among men, those who had received limited health education, and individuals with poor attitudes toward malaria. Integrated, proactive, and male-focused health promotion strategies are essential to enhance community knowledge and support disease control efforts.

The relocation of Indonesia's capital to Ibu Kota Nusantara (IKN) in East Kalimantan, a malaria and dengue hotspot, presents new risks of infectious disease transmission due to land-use changes and population movements. Current knowledge on the impact of these changes on vector-borne diseases, especially Plasmodium knowlesi malaria and other arboviruses, is limited. Serological surveillance offers a robust method for assessing population exposure. A community-based cross-sectional study will be conducted in IKN and its surrounding area, in East Kalimantan. Approximately 2,000 individuals aged >1 year will be enrolled. Finger-prick blood samples will be collected for serological analysis (multiplex bead-based assays for malaria species, and dengue virus serotypes) and malaria RDTs. Demographic, clinical, environmental, and geolocation data will also be collected. Statistical and geostatistical models will be used to assess seroprevalence, spatial patterns, and risk factors of exposure to malaria and dengue.

Duffy antigen receptor for chemokines (DARC) is the primary red blood cell (RBC) receptor for invasion of human RBCs by Plasmodium vivax and Plasmodium knowlesi parasites. By contrast, Plasmodium falciparum parasites use multiple RBC receptors for invasion. Whether DARC is one of these receptors has never been systematically explored. We used flow cytometry and microscopy-based approaches to investigate whether P. falciparum parasites preferentially invade specific Duffy RBC phenotypes and explored 2 potential explanations for invasion preference-differences in RBC biophysical properties and surface protein composition. P. falciparum parasites showed a consistent preference for Duffy-positive RBCs, and some biophysical properties and surface protein expression varied between Duffy-positive and Duffy-negative RBCs. We then used our in vitro invasion data to parametrize an evolutionary-epidemiological model of the relationship between P. falciparum and the FYBES allele. Our model accounts for immunity against P. falciparum virulence, gained through exposure, and thus mutations that impede infection are not always advantageous. The inhibition of P. falciparum invasion that we observed in vitro leads to FYBES frequencies increasing at low levels of P. falciparum transmission but decreasing at high levels of transmission. The impact of P. falciparum on the prevalence of Duffy negativity may therefore be most apparent in lower transmission settings. Our findings show a link between Duffy negativity and P. falciparum and suggest that DARC may directly or indirectly be involved in P. falciparum invasion of human RBCs which could, together with P. vivax, explain the distribution of Duffy negativity in sub-Saharan Africa.