METHODS AND ANALYSIS: A population-based case-control study will be conducted over a 2-year period at two adjacent districts in north-west Sabah, Malaysia. Confirmed malaria cases presenting to the district hospital sites meeting relevant inclusion criteria will be requested to enrol. Three community controls matched to the same village as the case will be selected randomly. Study procedures will include blood sampling and administration of household and individual questionnaires to evaluate potential exposure risks associated with acquisition of P. knowlesi malaria. Secondary outcomes will include differences in exposure variables between P. knowlesi and other Plasmodium spp, risk of severe P. knowlesi malaria, and evaluation of P. knowlesi case clustering. Primary analysis will be per protocol, with adjusted ORs for exposure risks between cases and controls calculated using conditional multiple logistic regression models.
ETHICS: This study has been approved by the human research ethics committees of Malaysia, the Menzies School of Health Research, Australia, and the London School of Hygiene and Tropical Medicine, UK.
METHODS: We did this population-based case-control study over a 2 year period in the state of Sabah in Malaysia. We enrolled cases with microscopy-positive, PCR-confirmed malaria who presented to two primary referral hospitals serving the adjacent districts of Kudat and Kota Marudu. We randomly selected three malaria-negative community controls per case, who were matched by village within 2 weeks of case detection. We obtained questionnaire data on demographics, behaviour, and residential malaria risk factors, and we also assessed glucose-6-phosphate dehydrogenase (G6PD) enzyme activity. We used conditional logistic regression models to evaluate exposure risk between P knowlesi cases and controls, and between P knowlesi and human-only Plasmodium spp malaria cases.
FINDINGS: From Dec 5, 2012, to Jan 30, 2015, we screened 414 patients and subsequently enrolled 229 cases with P knowlesi malaria mono-infection and 91 cases with other Plasmodium spp infection. We enrolled 953 matched controls, including 683 matched to P knowlesi cases and 270 matched to non-P knowlesi cases. Age 15 years or older (adjusted odds ratio [aOR] 4·16, 95% CI 2·09-8·29, p<0·0001), male gender (4·20, 2·54-6·97, p<0·0001), plantation work (3·50, CI, 1·34-9·15, p=0·011), sleeping outside (3·61, 1·48-8·85, p=0·0049), travel (2·48, 1·45-4·23, p=0·0010), being aware of the presence of monkeys in the past 4 weeks (3·35, 1·91-5·88, p<0·0001), and having open eaves or gaps in walls (2·18, 1·33-3·59, p=0·0021) were independently associated with increased risk of symptomatic P knowlesi infection. Farming occupation (aOR 1·89, 95% CI 1·07-3·35, p=0·028), clearing vegetation (1·89, 1·11-3·22, p=0·020), and having long grass around the house (2·08, 1·25-3·46, p=0·0048) increased risk for P knowlesi infection but not other Plasmodium spp infection. G6PD deficiency seemed to be protective against P knowlesi (aOR 0·20, 95% CI 0·04-0·96, p=0·045), as did residual insecticide spraying of household walls (0·52, 0·31-0·87, p=0·014), with the presence of young sparse forest (0·35, 0·20-0·63, p=00040) and rice paddy around the house (0·16, 0·03-0·78, 0·023) also associated with decreased risk.
INTERPRETATION: Adult men working in agricultural areas were at highest risk of knowlesi malaria, although peri-domestic transmission also occurrs. Human behavioural factors associated with P knowlesi transmission could be targeted in future public health interventions.
FUNDING: United Kingdom Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.
METHODS: The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket.
RESULTS: Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates.
CONCLUSION: Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.
METHODS: We did an environmentally stratified, population-based, cross-sectional survey across households in the Kudat, Kota Marudu, Pitas, and Ranau districts in northern Sabah, Malaysia, encompassing a range of ecologies. Using blood samples, the transmission intensity of P knowlesi and other malaria species was measured by specific antibody prevalence and infection detected using molecular methods. Proportions and configurations of land types were extracted from maps derived from satellite images; a data-mining approach was used to select variables. A Bayesian hierarchical model for P knowlesi seropositivity was developed, incorporating questionnaire data about individual and household-level risk factors with selected landscape factors.
FINDINGS: Between Sept 17, 2015, and Dec 12, 2015, 10 100 individuals with a median age of 25 years (range 3 months to 105 years) were sampled from 2849 households in 180 villages. 5·1% (95% CI 4·8-5·4) were seropositive for P knowlesi, and marked historical decreases were observed in the transmission of Plasmodium falciparum and Plasmodium vivax. Nine Plasmodium spp infections were detected. Age, male sex, contact with macaques, forest use, and raised house construction were positively associated with P knowlesi exposure, whereas residing at higher geographical elevations and use of insecticide were protective. Agricultural and forest variables, such as proportions and fragmentation of land cover types, predicted exposure at different spatial scales from households.
INTERPRETATION: Although few infections were detected, P knowlesi exposure was observed in all demographic groups and was associated with occupational factors. Results suggest that agricultural expansion and forest fragmentation affect P knowlesi exposure, supporting linkages between land use change and P knowlesi transmission.
FUNDING: UK Medical Research Council, Natural Environment Research Council, Economic and Social Research Council, and Biotechnology and Biosciences Research Council.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted comprehensive surveys in three areas where P. knowlesi transmission is reported: Limbuak, Pulau Banggi and Matunggung, Kudat, Sabah, Malaysia and Bacungan, Palawan, the Philippines. Infection prevalence was low with parasites detected by PCR in only 0.2% (4/2503) of the population. P. knowlesi PkSERA3 ag1 antibody responses were detected in 7.1% (95% CI: 6.2-8.2%) of the population, compared with 16.1% (14.6-17.7%) and 12.6% (11.2-14.1%) for P. falciparum and P. vivax. Sero-prevalence was low in individuals <10 years old for P. falciparum and P. vivax consistent with decreased transmission of non-zoonotic malaria species. Results indicated marked heterogeneity in transmission intensity between sites and P. knowlesi exposure was associated with agricultural work (OR 1.63; 95% CI 1.07-2.48) and higher levels of forest cover (OR 2.40; 95% CI 1.29-4.46) and clearing (OR 2.14; 95% CI 1.35-3.40) around houses. Spatial patterns of P. knowlesi exposure differed from exposure to non-zoonotic malaria and P. knowlesi exposed individuals were younger on average than individuals exposed to non-zoonotic malaria.
CONCLUSIONS/SIGNIFICANCE: This is the first study to describe serological exposure to P. knowlesi and associated risk factors within endemic communities. Results indicate community-level patterns of infection and exposure differ markedly from demographics of reported cases, with higher levels of exposure among women and children. Further work is needed to understand these variations in risk across a wider population and spatial scale.