BackgroundIn recent years, the primate malaria Plasmodium knowlesi has emerged in human populations throughout South East Asia, with the largest hotspot being in Sabah, Malaysian Borneo. Control efforts are hindered by limited knowledge of where and when people get exposed to mosquito vectors. It is assumed that exposure occurs primarily when people are working in forest areas, but the role of other potential exposure routes (including domestic or peri-domestic transmission) has not been thoroughly investigated.Methodology/Principal FindingsWe integrated entomological surveillance within a comprehensive case-control study occurring within a large hotspot of transmission in Sabah, Malaysia. Mosquitoes were collected at 28 pairs households composed of one where an occupant had a confirmed P. knowlesi infection within the preceding 3 weeks (“case”) and an associated “control” where no infection was reported. Human landing catches were conducted to measure the number and diversity of mosquitoes host seeking inside houses and in the surrounding peri-domestic (outdoors but around the household) areas. The predominant malaria vector species was Anopheles balabacensis, most of which were caught outdoors in the early evening (6pm - 9pm). It was significantly more abundant in the peri-domestic area than inside houses (5.5-fold), and also higher at case than control households (0.28±0.194 vs 0.17±0.127, p<0.001). Ten out of 641 An. balabacensis tested were positive for simian malaria parasites, but none for P. knowlesi.Conclusions/SignificanceThis study shows there is a possibility that humans can be exposed to P. knowlesi infection around their homes. The vector is highly exophagic and few were caught indoors indicating interventions using bednets inside households may have relatively little impact.

Background We investigated the effect of five common habitat types on the diversity and abundance of Anopheles spp. and on the biting rate and time of Anopheles balabacensis (currently the only known vector for Plasmodium knowlesi in Sabah) at Paradason village, Kudat, Sabah. The habitats were forest edge, playground area, longhouse, oil palm plantation and shrub-bushes area. Sampling of Anopheles was done monthly using the human landing catch method in all habitat types for 14 months (October 2013 to December 2014, excluding June 2014). The Anopheles species were morphologically identified and subjected to PCR assay for the detection of Plasmodium parasites. Generalised linear mixed models (GLMM) were applied to test the variation in abundance and biting rates of An. balabacensis in different habitat types. 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. Electronic supplementary material The online version of this article (10.1186/s13071-019-3627-0) contains supplementary material, which is available to authorized users.

Human movement into insect vector and wildlife reservoir habitats determines zoonotic disease risks; however, few data are available to quantify the impact of land use on pathogen transmission. Here, we utilise GPS tracking devices and novel applications of ecological methods to develop fine-scale models of human space use relative to land cover to assess exposure to the zoonotic malaria Plasmodium knowlesi in Malaysian Borneo. Combining data with spatially explicit models of mosquito biting rates, we demonstrate the role of individual heterogeneities in local space use in disease exposure. At a community level, our data indicate that areas close to both secondary forest and houses have the highest probability of human P. knowlesi exposure, providing quantitative evidence for the importance of ecotones. Despite higher biting rates in forests, incorporating human movement and space use into exposure estimates illustrates the importance of intensified interactions between pathogens, insect vectors and people around habitat edges.

In lowland areas of Malaysia, Plasmodium knowlesi infection is associated with land use change and high proportions of the vector Anopheles balabacensis. We conducted a 15-month study in two Malaysian villages to determine the effect of habitat on vector populations in understudied high-altitude, high-incidence districts. Anopheles mosquitoes were sampled in human settlements, plantations and forest edges, and screened for Plasmodium species by PCR. We report the first An. donaldi positive for P. knowlesi. This potential vector was associated with habitat fragmentation measured as disturbed forest edge:area ratio, while An. balabacensis was not, indicating fragmented land use could favour An. donaldi. Anopheline species richness and diversity decreased from forest edge, to plantation, to human settlement. Greater numbers of An. balabacensis and An. donaldi were found in forest edges compared to human settlements, suggesting exposure to vectors and associated zoonoses may be greater for people entering this habitat.

BackgroundAnopheles balabacensis of the Leucospyrus group has been confirmed as the primary knowlesi malaria vector in Sabah, Malaysian Borneo for some time now. Presently, knowlesi malaria is the only zoonotic simian malaria in Malaysia with a high prevalence recorded in the states of Sabah and Sarawak.Methodology/Principal findingsAnopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah.PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%–100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality.Conclusions/SignificanceThis is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.

Background Plasmodium knowlesi is found in macaques and is the only major zoonotic malaria to affect humans. Transmission of P. knowlesi between people and macaques depends on the host species preferences and feeding behavior of mosquito vectors. However, these behaviours are difficult to measure due to the lack of standardized methods for sampling potential vectors attracted to different host species. This study evaluated electrocuting net traps as a safe, standardised method for sampling P. knowlesi vectors attracted to human and macaque hosts.Field experiments were conducted within a major focus on P. knowlesi transmission in Malaysian Borneo to compare the performance of human (HENET) or macaque (MENET) odour-baited electrocuting nets, human landing catches (HLC) and monkey-baited traps (MBT) for sampling mosquitoes. The abundance and diversity of Anopheles sampled by different methods were compared over 40 nights, with a focus on the P. knowlesi vector Anopheles balabancensis.ResultsHLC caught more An. balabacensis than any other method (3.6 per night). In contrast, no An. balabacensis were collected in MBT collections, which generally performed poorly for all mosquito taxa. Anopheles vector species including An. balabacensis were sampled in both HENET and MENET collections, but at a mean abundance of less than 1 per night. There was no difference between HENET and MENET in the overall abundance (P = 0.05) or proportion (P = 0.7) of An. balabacensis. The estimated diversity of Anopheles species was marginally higher in electrocuting net than HLC collections, and similar in collections made with humans or monkey hosts.ConclusionsHost-baited electrocuting nets had moderate success for sampling known zoonotic malaria vectors. The primary vector An. balabacensis was collected with electrocuting nets baited both with humans and macaques, but at a considerably lower density than the HLC standard. However, electrocuting nets were considerably more successful than monkey-baited traps and representatively characterised anopheline species diversity. Consequently, their use allows inferences about relative mosquito attraction to be meaningfully interpreted while eliminating confounding factors due to trapping method. On this basis, electrocuting net traps should be considered as a useful standardised method for investigating vector contact with humans and wildlife reservoirs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2277-3) contains supplementary material, which is available to authorized users.

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