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Background: Plasmodium knowlesi is the most common cause of human malaria in Malaysia. Acute kidney injury (AKI) is a frequent complication. AKI of any cause can have long-term consequences, including increased risk of chronic kidney disease, adverse cardiovascular events and increased mortality. Additional management strategies are therefore needed to reduce the frequency and severity of AKI in malaria. In falciparum malaria, cell-free haemoglobin (CFHb)-mediated oxidative damage contributes to AKI. The inexpensive and widely available drug paracetamol inhibits CFHb-induced lipid peroxidation via reduction of ferryl haem to the less toxic Fe3+ state, and has been shown to reduce oxidative damage and improve renal function in patients with sepsis complicated by haemolysis as well as in falciparum malaria. This study aims to assess the ability of regularly dosed paracetamol to reduce the incidence and severity of AKI in knowlesi malaria by attenuating haemolysis-induced oxidative damage. Methods: PACKNOW is a two-arm, open-label randomised controlled trial of adjunctive paracetamol versus no paracetamol in patients aged ≥ 5 years with knowlesi malaria, conducted over a 2-year period at four hospital sites in Sabah, Malaysia. The primary endpoint of change in creatinine from enrolment to 72 h will be evaluated by analysis of covariance (ANCOVA) using enrolment creatinine as a covariate. Secondary endpoints include longitudinal changes in markers of oxidative stress (plasma F2-isoprostanes and isofurans) and markers of endothelial activation/Weibel–Palade body release (angiopoietin-2, von Willebrand Factor, P-selectin, osteoprotegerin) over 72 h, as well as blood and urine biomarkers of AKI. This study will be powered to detect a difference between the two treatment arms in a clinically relevant population including adults and children with knowlesi malaria of any severity. Discussion: Paracetamol is widely available and has an excellent safety profile; if a renoprotective effect is demonstrated, this trial will support the administration of regularly dosed paracetamol to all patients with knowlesi malaria. The secondary outcomes in this study will provide further insights into the pathophysiology of haemolysis-induced oxidative damage and acute kidney injury in knowlesi malaria and other haemolytic diseases. Trial registration Clinicaltrials.gov, NCT03056391 . Registered on 12 October 2016.

Background: The emergence of the zoonotic monkey parasite Plasmodium knowlesi as the dominant cause of malaria in Malaysia has disrupted current national WHO elimination goals. Malaysia has free universal access to malaria care; however, out-of-pocket costs are unknown. This study estimated household costs of illness attributable to malaria due to P. knowlesi against other non-zoonotic Plasmodium species infections in Sabah, Malaysia., Methodology/principal Findings: Household costs were estimated from patient-level surveys collected from four hospitals between 2013 and 2016. Direct costs including medical and associated travel costs, and indirect costs due to lost productivity were included. One hundred and fifty-two malaria cases were enrolled: P. knowlesi (n=108), P. vivax (n=22), P. falciparum (n=16), and P. malariae (n=6). Costs were inflated to 2022 Malaysian Ringgits and reported in United States dollars (US$). Across all cases, the mean total costs were US$138 (SD=108), with productivity losses accounting for 58% of costs (US$80; SD=73). P. vivax had the highest mean total household cost at US$210, followed by P. knowlesi (US$127), P. falciparum (US$126), and P. malariae (US$105). Most patients (80%) experienced direct health costs above 10% of monthly income, with 58 (38%) patients experiencing health spending over 25% of monthly income, consistent with catastrophic health expenditure., Conclusions/significance: Despite Malaysia's free health-system care for malaria, patients and families face other related medical, travel, and indirect costs. Household out-of-pocket costs were driven by productivity losses; primarily attributed to infections in working-aged males in rural agricultural-based occupations. Costs for P. knowlesi were comparable to P. falciparum and lower than P. vivax. The higher P. vivax costs related to direct health facility costs for repeat monitoring visits given the liver-stage treatment required., Author Summary: Knowlesi malaria is due to infection with a parasite transmitted by mosquitos from monkeys to humans. Most people who are infected work or live near the forest. It is now the major type of malaria affecting humans in Malaysia. The recent increase of knowlesi malaria cases in humans has impacted individuals, families, and health systems in Southeast Asia. Although the region has made substantial progress towards eliminating human-only malaria species, knowlesi malaria threatens elimination targets as traditional control measures do not address the parasite reservoir in monkeys. The economic burden of illness due to knowlesi malaria has not previously been estimated or subsequently compared with other malaria species. We collected data on the cost of illness to households in Sabah, Malaysia, to estimate their related total economic burden. Medical costs and time off work and usual activities were substantial in patients with the four species of malaria diagnosed during the time of this study. This research highlights the financial burden which households face when seeking care for malaria in Malaysia, despite the free treatment provided by the government.

Background: Zoonotic P. knowlesi and P. cynomolgi symptomatic and asymptomatic infections occur across endemic areas of Southeast Asia. Most infections are low-parasitemia, with an unknown proportion below routine microscopy detection thresholds. Molecular surveillance tools optimizing the limit of detection (LOD) would allow more accurate estimates of zoonotic malaria prevalence., Methods: An established ultra-sensitive Plasmodium genus quantitative-PCR (qPCR) assay targeting the 18S rRNA gene underwent LOD evaluation with and without reverse transcription (RT) for P. knowlesi , P. cynomolgi and P. vivax using total nucleic acid preserved (DNA/RNA Shield ™ ) isolates and archived dried blood spots (DBS). LODs for selected P. knowlesi- specific assays, and reference P. vivax- and P. cynomolgi -specific assays were determined with RT. Assay specificities were assessed using clinical malaria samples and malaria-negative controls., Results: The use of reverse transcription improved Plasmodium species detection by up to 10,000-fold ( Plasmodium genus), 2759-fold ( P. knowlesi ), 1000-fold ( P. vivax ) and 10-fold ( P. cynomolgi ). The median LOD with RT for the Kamau et al. Plasmodium genus RT-qPCR assay was ≤0.0002 parasites/μL for P. knowlesi and 0.002 parasites/μL for both P. cynomolgi and P. vivax . The LODs with RT for P. knowlesi -specific PCRs were: Imwong et al. 18S rRNA (0.0007 parasites/μL); Divis et al. real-time 18S rRNA (0.0002 parasites/μL); Lubis et al. hemi-nested SICAvar (1.1 parasites/μL) and Lee et al. nested 18S rRNA (11 parasites/μL). The LOD for P. vivax- and P. cynomolgi- specific assays with RT were 0.02 and 0.20 parasites/μL respectively. For DBS P. knowlesi samples the median LOD for the Plasmodium genus qPCR with RT was 0.08, and without RT was 19.89 parasites/uL (249-fold change); no LOD improvement was demonstrated in DBS archived beyond 6 years. The Plasmodium genus and P. knowlesi -assays were 100% specific for Plasmodium species and P. knowlesi detection, respectively, from 190 clinical infections and 48 healthy controls. Reference P. vivax- specific primers demonstrated known cross-reactivity with P. cynomolgi ., Conclusion: Our findings support the use of an 18S rRNA Plasmodium genus qPCR and species-specific nested PCR protocol with RT for highly-sensitive surveillance of zoonotic and human Plasmodium species infections.

Background: The interaction between iron deficiency and malaria is incompletely understood. We evaluated longitudinal changes in iron homeostasis in volunteers enrolled in malaria volunteer infection studies (VIS) and in Malaysian patients with falciparum and vivax malaria., Methods: We retrieved samples and associated data from 55 participants enrolled in malaria VIS, and 171 malaria patients and 30 healthy controls enrolled in clinical studies in Malaysia. Ferritin, hepcidin, erythropoietin, and soluble transferrin receptor (sTfR) were measured by ELISA., Results: In the VIS, participants' parasitaemia was correlated with baseline mean corpuscular volume (MCV), but not iron status (ferritin, hepcidin or sTfR). Ferritin, hepcidin and sTfR all increased during the VIS. Ferritin and hepcidin normalised by day 28, while sTfR remained elevated. In VIS participants, baseline iron status (ferritin) was associated with post-treatment increases in liver transaminase levels. In Malaysian malaria patients, hepcidin and ferritin were elevated on admission compared to healthy controls, while sTfR increased following admission. Hepcidin normalised by day 28; however, ferritin and sTfR both remained elevated 4 weeks following admission., Conclusion: Our findings demonstrate that parasitaemia is associated with an individual's MCV rather than iron status. The persistent elevation in sTfR 4 weeks post-infection in both malaria VIS and clinical malaria may reflect a causal link between malaria and iron deficiency., Competing Interests: Declaration of interests None of the authors have conflicts of interests to declare.

Background: Plasmodium knowlesi is a zoonotic parasite that causes malaria in humans. The pathogen has a natural host reservoir in certain macaque species and is transmitted to humans via mosquitoes of the Anopheles Leucosphyrus Group. The risk of human P. knowlesi infection varies across Southeast Asia and is dependent upon environmental factors. Understanding this geographic variation in risk is important both for enabling appropriate diagnosis and treatment of the disease and for improving the planning and evaluation of malaria elimination. However, the data available on P. knowlesi occurrence are biased towards regions with greater surveillance and sampling effort. Predicting the spatial variation in risk of P. knowlesi malaria requires methods that can both incorporate environmental risk factors and account for spatial bias in detection., Methods & Results: We extend and apply an environmental niche modelling framework as implemented by a previous mapping study of P. knowlesi transmission risk which included data up to 2015. We reviewed the literature from October 2015 through to March 2020 and identified 264 new records of P. knowlesi , with a total of 524 occurrences included in the current study following consolidation with the 2015 study. The modelling framework used in the 2015 study was extended, with changes including the addition of new covariates to capture the effect of deforestation and urbanisation on P. knowlesi transmission., Discussion: Our map of P. knowlesi relative transmission suitability estimates that the risk posed by the pathogen is highest in Malaysia and Indonesia, with localised areas of high risk also predicted in the Greater Mekong Subregion, The Philippines and Northeast India. These results highlight areas of priority for P. knowlesi surveillance and prospective sampling to address the challenge the disease poses to malaria elimination planning.