RESULTS: Over 31 days, 2243 mosquitoes were collected in 5748 discrete collections. Nine mosquito genera were sampled with Aedes and Culex species being present in all habitats and most abundant. RB and CDC backpack aspiration were most efficient for sampling Culex whereas CDC backpack aspiration and SRB were most efficient for Aedes. Most Aedes identified to species level were Ae. albopictus (91%), with their abundance being highest in forest edge habitats. In contrast, Culex were most abundant under houses. Most blood-fed mosquitoes (76%) were found in human settlements; with humans and chickens being the only blood source.
CONCLUSIONS: RB and SRB traps proved capable of sampling mosquitoes resting in all sampled habitats. However, sampling efficiency was generally low (c.0.1 per trap per day), necessitating traps to be deployed in high numbers for mosquito detection. None of the traps were effective for sampling zoonotic malaria vectors; however, SRB collected relatively higher numbers of the dengue vector Ae. albopictus. The higher abundance of mosquitoes in forest edge habitats indicates the potential value of these traps for investigating sylvatic dengue transmission. This study has demonstrated the merits in application of simple resting traps for characterising mosquito vector resting behaviour outside of the home.
RESULTS: A total of 1599 Anopheles specimens were collected in the village, of which about 90% were An. balabacensis. Anopheles balabacensis was present throughout the year and was the dominant Anopheles species in all habitat types. The shrub bushes habitat had the highest Anopheles species diversity while forest edge had the greatest number of Anopheles individuals caught. GLMM analysis indicated that An. balabacensis abundance was not affected by the type of habitats, and it was more active during the early and late night compared to predawn and dawn. PCR assay showed that 1.61% of the tested An. balabacensis were positive for malaria parasites, most of which were caught in oil palm estates and infected with one to two Plasmodium species.
CONCLUSIONS: The identification of infected vectors in a range of habitats, including agricultural and farming areas, illustrates the potential for humans to be exposed to P. knowlesi outside forested areas. This finding contributes to a growing body of evidence implicating environmental changes due to deforestation, expansion of agricultural and farming areas, and development of human settlements near to forest fringes in the emergence of P. knowlesi in Sabah.
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.
METHODOLOGY/PRINCIPAL FINDINGS: We conducted a one year longitudinal study of P. knowlesi vectors in three sites within an endemic area of Sabah, Malaysia. All mosquitoes were captured using human landing catch. Anopheles mosquitoes were dissected to determine, oocyst, sporozoites and parous rate. Anopheles balabacensis is confirmed as the primary vector of. P. knowlesi (using nested PCR) in Sabah for the first time. Vector densities were significantly higher and more seasonally variable in the village than forest or small scale farming site. However An. balabacensis survival and P. knowlesi infection rates were highest in forest and small scale farm sites. Anopheles balabacensis mostly bites humans outdoors in the early evening between 1800 to 2000 hrs.
CONCLUSIONS/SIGNIFICANCE: This study indicates transmission is unlikely to be prevented by bednets. This combined with its high vectorial capacity poses a threat to malaria elimination programmes within the region.
METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.
RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).
CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.
METHODOLOGY/PRINCIPAL FINDINGS: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.
CONCLUSIONS/SIGNIFICANCE: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.