Detection of echinostomatid trematode eggs at the forest-oil palm interface in Sabah, Malaysia.

In this study, we report the occurrence of echinostomatid eggs in feces of wildlife, domestic animals and humans frequenting the forest­oil palm plantation interface in the Kinabatangan (Sabah, Malaysia), and discuss potential implications for public health. Using microscopy, we detected echinostomatid eggs in six host species, including Asian palm civets (Paradoxurus hermaphroditus [13/18]), leopard cats (Prionailurus bengalensis [3/4]), long-tailed macaques (Macaca fascicularis [1/10]), domestic dogs [3/5] and cats [1/1], and humans [7/9]. Molecular analysis revealed a close genetic proximity of civet echinostomatids to Artyfechinostomum malayanum, a zoonotic parasite of public health relevance. The intermediate hosts for A. malayanum have been reported in at least 3 districts in Sabah, suggesting that all the necessary elements required for the completion of the parasite's life cycle are present. Our findings point at the presence of zoonotic trematodes in an area with high human­wildlife interaction and highlight the potential public and animal health concern of zoonotic trematode infection in the context of Southeast Asia's rapidly changing ecosystems.

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

BACKGROUND: The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. METHODS: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. RESULTS: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi. CONCLUSION: The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

Natural Human Infections With Plasmodium cynomolgi and Other Malaria Species in an Elimination Setting in Sabah, Malaysia.

To determine the presence and species composition of malaria infections, we screened a subset of samples collected during a cross-sectional survey in Northern Sabah, Malaysia using highly sensitive molecular techniques. Results identified 54 asymptomatic submicroscopic malaria infections, including a large cluster of Plasmodium falciparum and 3 P. knowlesi infections. We additionally identified 2 monoinfections with the zoonotic malaria Plasmodium cynomolgi, both in individuals reporting no history of forest activities or contact with macaques. Results highlight the need for improved surveillance strategies to detect these infections and determine public health impacts.

Defining the ecological and evolutionary drivers of Plasmodium knowlesi transmission within a multi-scale framework.

Plasmodium knowlesi is a zoonotic malaria parasite normally residing in long-tailed and pig-tailed macaques (Macaca fascicularis and Macaca nemestrina, respectively) found throughout Southeast Asia. Recently, knowlesi malaria has become the predominant malaria affecting humans in Malaysian Borneo, being responsible for approximately 70% of reported cases. Largely as a result of anthropogenic land use changes in Borneo, vectors which transmit the parasite, along with macaque hosts, are both now frequently found in disturbed forest habitats, or at the forest fringes, thus having more frequent contact with humans. Having access to human hosts provides the parasite with the opportunity to further its adaption to the human immune system. The ecological drivers of the transmission and spread of P. knowlesi are operating over many different spatial (and, therefore, temporal) scales, from the molecular to the continental. Strategies to prevent and manage zoonoses, such as P. knowlesi malaria require interdisciplinary research exploring the impact of land use change and biodiversity loss on the evolving relationship between parasite, reservoir hosts, vectors, and humans over multiple spatial scales.

Surveillance of adult Aedes mosquitoes in Selangor, Malaysia.

OBJECTIVE: To determine the effectiveness of using sticky traps and the NS1 dengue antigen kit for the surveillance of Aedes mosquitoes for dengue control. METHODS: Apartments were selected in a dengue-endemic area, and sticky traps were set to capture adult Aedes mosquitoes. NS1 dengue antigen kit was used to detect dengue antigen in mosquitoes, and positive mosquitoes were serotyped using real-time RT-PCR. RESULTS: The sticky traps were effective in capturing Aedes aegypti, and a minimum of three traps per floor was sufficient. Multiple serotypes were found in individual mosquitoes. CONCLUSION: The sticky trap and the NS1 dengue antigen test kit can be used as surveillance tool in dengue control programmes. This proactive method will be better suited for control programmes than current reactive methods.

Forest Restoration and the Zoonotic Vector Anopheles balabacensis in Sabah, Malaysia.

Anthropogenic changes to forest cover have been linked to an increase in zoonotic diseases. In many areas, natural forests are being replaced with monoculture plantations, such as oil palm, which reduce biodiversity and create a mosaic of landscapes with increased forest edge habitat and an altered micro-climate. These altered conditions may be facilitating the spread of the zoonotic malaria parasite Plasmodium knowlesi in Sabah, on the island of Borneo, through changes to mosquito vector habitat. We conducted a study on mosquito abundance and diversity in four different land uses comprising restored native forest, degraded native forest, an oil palm estate and a eucalyptus plantation, these land uses varying in their vegetation types and structure. The main mosquito vector, Anopheles balabacensis, has adapted its habitat preference from closed canopy rainforest to more open logged forest and plantations. The eucalyptus plantations (Eucalyptus pellita) assessed in this study contained significantly higher abundance of many mosquito species compared with the other land uses, whereas the restored dipterocarp forest had a low abundance of all mosquitos, in particular, An. balabacensis. No P. knowlesi was detected by PCR assay in any of the vectors collected during the study; however, P. inui, P. fieldi and P. vivax were detected in An. balabacensis. These findings indicate that restoring degraded natural forests with native species to closed canopy conditions reduces abundance of this zoonotic malarial mosquito vector and therefore should be incorporated into future restoration research and potentially contribute to the control strategies against simian malaria.

Serological evaluation of risk factors for exposure to malaria in a pre-elimination setting in Malaysian Borneo.

Malaysia has reported no indigenous cases of P. falciparum and P. vivax for over 3 years. When transmission reaches such low levels, it is important to understand the individuals and locations where exposure risks are high, as they may be at greater risk in the case of a resurgence of transmission. Serology is a useful tool in low transmission settings, providing insight into exposure over longer durations than PCR or RDT. We ran blood samples from a 2015 population-based survey in northern Sabah, Malaysian Borneo on a multiplex bead assay. Using supervised machine learning methods, we characterised recent and historic exposure to Plasmodium falciparum and P. vivax and found recent exposure to P. falciparum to be very low, with exposure to both species increasing with age. We performed a risk-factor assessment on environmental, behavioural, demographic and household factors, and identified forest activity and longer travel times to healthcare as common risk-factors for exposure to P. falciparum and P. vivax. In addition, we used remote-sensing derived data and geostatistical models to assess environmental and spatial associations with exposure. We created predictive maps of exposure to recent P. falciparum in the study area and showed 3 clear foci of exposure. This study provides useful insight into the environmental, spatial and demographic risk factors for P. falciparum and P. vivax at a period of low transmission in Malaysian Borneo. The findings would be valuable in the case of resurgence of human malarias in the region.

Life table analysis of Anopheles balabacensis, the primary vector of Plasmodium knowlesi in Sabah, Malaysia.

BACKGROUND: Plasmodium knowlesi has become a major public health concern in Sabah, Malaysian Borneo, where it is now the only cause of indigenous malaria. The importance of P. knowlesi has spurred on a series of studies on this parasite, as well as on the biology and ecology of its principal vector, Anopheles balabacensis. However, there remain critical knowledge gaps on the biology of An. balabacensis, such as life history data and life table parameters. To fill these gaps, we conducted a life table study of An. balabacensis in the laboratory. Characterising vector life cycles and survival rates can inform more accurate estimations of the serial interval, the time between two linked cases, which is crucial to understanding and monitoring potentially changing transmission patterns. METHODS: Individuals of An. balabacensis were collected in the field in Ranau district, Sabah to establish a laboratory colony. Induced mating was used, and the life history parameters of the progeny were recorded. The age-stage, two-sex life table approach was used in the analysis. The culture conditions in the laboratory were 9 h light:15 h dark, mean temperature 25.7 °C ± 0.05 and relative humidity 75.8% ± 0.31. RESULTS: The eggs hatched within 2 days, and the larval stage lasted for 10.5 days in total, with duration of instar stages I, II, III and IV of 2.3, 3.7, 2.3, 2.2 days, respectively. The maximum total fecundity was 729 for one particular female, while the maximum female age-specific mean fecundity (mx) was 142 at age 59 days. The gross reproductive rate or number of offspring per individual was about 102. On average, each female laid 1.81 ± 0.19 (range 1-7) batches of eggs, with 63% of the females producing only one batch; only one female laid six batches, while one other laid seven. Each batch comprised 159 ± 17.1 eggs (range 5-224) and the female ratio of offspring was 0.28 ± 0.06. The intrinsic rate of increase, finite rate of increase, net reproductive rate, mean generation time and doubling time were, respectively, 0.12 ± 0.01 day-1, 1.12 ± 0.01 day-1, 46.2 ± 14.97, 33.02 ± 1.85 and 5.97 days. CONCLUSIONS: Both the net reproductive rate and intrinsic rate of increase of An. balabacensis are lower than those of other species in published studies. Our results can be used to improve models of P. knowlesi transmission and to set a baseline for assessing the impacts of environmental change on malaria dynamics. Furthermore, incorporating these population parameters of An. balabacensis into spatial and temporal models on the transmission of P. knowlesi would provide better insight and increase the accuracy of epidemiological forecasting.

Quantifying human-animal contact rates in Malaysian Borneo: Influence of agricultural landscapes on contact with potential zoonotic disease reservoirs.

Changing landscapes across the globe, but particularly in Southeast Asia, are pushing humans and animals closer together and may increase the likelihood of zoonotic spillover events. Malaysian Borneo is hypothesized to be at high risk of spillover events due to proximity between reservoir species and humans caused by recent deforestation in the region. However, the relationship between landscape and human-animal contact rates has yet to be quantified. An environmentally stratified cross-sectional survey was conducted in Sabah, Malaysia in 2015, collecting geolocated questionnaire data on potential risk factors for contact with animals for 10,100 individuals. 51% of individuals reported contact with poultry, 46% with NHPs, 30% with bats, and 2% with swine. Generalised linear mixed models identified occupational and demographic factors associated with increased contact with these species, which varied when comparing wildlife to domesticated animals. Reported contact rates with each animal group were integrated with remote sensing-derived environmental data within a Bayesian framework to identify regions with high probabilities of contact with animal reservoirs. We have identified high spatial heterogeneity of contact with animals and clear associations between agricultural practices and high animal rates. This approach will help inform public health campaigns in at-risk populations and can improve pathogen surveillance efforts on Malaysian Borneo. This method can additionally serve as a framework for researchers looking to identify targets for future pathogen detection in a chosen region of study.

Dengue surveillance using gravid oviposition sticky (GOS) trap and dengue non-structural 1 (NS1) antigen test in Malaysia: randomized controlled trial.

Dengue remains a major public threat and existing dengue control/surveillance programs lack sensitivity and proactivity. More efficient methods are needed. A cluster randomized controlled trial was conducted for 18 months to determine the efficacy of using a combination of gravid oviposition sticky (GOS) traps and dengue non-structural 1 (NS1) antigen for early surveillance of dengue among Aedes mosquito. Eight residential apartments were randomly assigned into intervention and control groups. GOS traps were placed at the intervention apartments weekly to trap Aedes mosquitoes and these tested for dengue NS1 antigen. When dengue-positive pool was detected, the community were notified and advised to execute protective measures. Fewer dengue cases were recorded in the intervention group than the control. Detection of NS1-positive mosquitoes was significantly associated with GOS Aedes index (rs = 0.68, P < 0.01) and occurrence of dengue cases (rs = 0.31, P < 0.01). Participants' knowledge, attitude, and practice (KAP) toward dengue control indicated significant improvement for knowledge (P < 0.01), practice (P < 0.01) and total scores (P < 0.01). Most respondents thought this surveillance method is good (81.2%) and supported its use nationwide. Thus, GOS trap and dengue NS1 antigen test can supplement the current dengue surveillance/control, in alignment with the advocated integrated vector management for reducing Aedes-borne diseases.

Assessing seroprevalence and associated risk factors for multiple infectious diseases in Sabah, Malaysia using serological multiplex bead assays.

Background: Infectious diseases continue to burden populations in Malaysia, especially among rural communities where resources are limited and access to health care is difficult. Current epidemiological trends of several neglected tropical diseases in these populations are at present absent due to the lack of habitual and efficient surveillance. To date, various studies have explored the utility of serological multiplex beads to monitor numerous diseases simultaneously. We therefore applied this platform to assess population level exposure to six infectious diseases in Sabah, Malaysia. Furthermore, we concurrently investigated demographic and spatial risk factors that may be associated with exposure for each disease. Methods: This study was conducted in four districts of Northern Sabah in Malaysian Borneo, using an environmentally stratified, population-based cross-sectional serological survey targeted to determine risk factors for malaria. Samples were collected between September to December 2015, from 919 villages totaling 10,100 persons. IgG responses to twelve antigens of six diseases (lymphatic filariasis- Bm33, Bm14, BmR1, Wb123; strongyloides- NIE; toxoplasmosis-SAG2A; yaws- Rp17 and TmpA; trachoma- Pgp3, Ct694; and giardiasis- VSP3, VSP5) were measured using serological multiplex bead assays. Eight demographic risk factors and twelve environmental covariates were included in this study to better understand transmission in this community. Results: Seroprevalence of LF antigens included Bm33 (10.9%), Bm14+ BmR1 (3.5%), and Wb123 (1.7%). Seroprevalence of Strongyloides antigen NIE was 16.8%, for Toxoplasma antigen SAG2A was 29.9%, and Giardia antigens GVSP3 + GVSP5 was 23.2%. Seroprevalence estimates for yaws Rp17 was 4.91%, for TmpA was 4.81%, and for combined seropositivity to both antigens was 1.2%. Seroprevalence estimates for trachoma Pgp3 + Ct694 were 4.5%. Age was a significant risk factors consistent among all antigens assessed, while other risk factors varied among the different antigens. Spatial heterogeneity of seroprevalence was observed more prominently in lymphatic filariasis and toxoplasmosis. Conclusions: Multiplex bead assays can be used to assess serological responses to numerous pathogens simultaneously to support infectious disease surveillance in rural communities, especially where prevalences estimates are lacking for neglected tropical diseases. Demographic and spatial data collected alongside serosurveys can prove useful in identifying risk factors associated with exposure and geographic distribution of transmission.

Environmental and spatial risk factors for the larval habitats of Plasmodium knowlesi vectors in Sabah, Malaysian Borneo.

Land-use changes, such as deforestation and agriculture, can influence mosquito vector populations and malaria transmission. These land-use changes have been linked to increased incidence in human cases of the zoonotic malaria Plasmodium knowlesi in Sabah, Malaysian Borneo. This study investigates whether these associations are partially driven by fine-scale land-use changes creating more favourable aquatic breeding habitats for P. knowlesi anopheline vectors. Using aerial remote sensing data, we developed a sampling frame representative of all land use types within a major focus of P. knowlesi transmission. From 2015 to 2016 monthly longitudinal surveys of larval habitats were collected in randomly selected areas stratified by land use type. Additional remote sensing data on environmental variables, land cover and landscape configuration were assembled for the study site. Risk factor analyses were performed over multiple spatial scales to determine associations between environmental and spatial variables and anopheline larval presence. Habitat fragmentation (300 m), aspect (350 m), distance to rubber plantations (100 m) and Culex larval presence were identified as risk factors for Anopheles breeding. Additionally, models were fit to determine the presence of potential larval habitats within the areas surveyed and used to generate a time-series of monthly predictive maps. These results indicate that land-use change and topography influence the suitability of larval habitats, and may partially explain the link between P. knowlesi incidence and deforestation. The predictive maps, and identification of the spatial scales at which risk factors are most influential may aid spatio-temporally targeted vector control interventions.

Parasite community structure in sympatric Bornean primates.

Parasites are important components of ecosystems, influencing trophic networks, competitive interactions and biodiversity patterns. Nonetheless, we are not nearly close to disentangling their complex roles in natural systems. Southeast Asia falls within global areas targeted as most likely to source parasites with zoonotic potential, where high rates of land conversion and fragmentation have altered the circulation of wildlife species and their parasites, potentially resulting in altered host-parasite systems. Although the overall biodiversity in the region predicts equally high, or even higher, parasite diversity, we know surprisingly little about wild primate parasites, even though this constitutes the first step towards a more comprehensive understanding of parasite transmission processes. Here, we characterise the gastrointestinal helminth parasite assemblages of a community of Bornean primates living along the Kinabatangan floodplain in Sabah (Malaysian Borneo), including two species endemic to the island. Through parasitological analyses, and by using several measures of parasite infection as proxies for parasite diversity and distribution, we show that (i) most parasite taxonomic groups are not limited to a single host, suggesting a greater flexibility for habitat disturbance, (ii) parasite infracommunities of nocturnal primates differ from their diurnal counterparts, reflecting both phylogenetic and ecological constraints, and (iii) soil-transmitted helminths such as whipworm, threadworm and nodule worm are widespread across the primate community. This study also provides new parasite records for southern pig-tailed macaques (Macaca nemestrina), silvered langurs (Trachypithecus cristatus) and Western tarsiers (Cephalopachus bancanus) in the wild, while adding to the limited records for the other primate species in the community. Given the information gap regarding primate-parasite associations in the region, the information presented here should prove relevant for future studies of parasite biodiversity and infectious disease ecology in Asia and elsewhere.

Development of allele-specific single-nucleotide polymorphism-based polymerase chain reaction markers in cytochrome oxidase I for the differentiation of Bactrocera papayae and Bactrocera carambolae (Diptera: Tephritidae).

Differentiation of Bactrocera papayae Drew & Hancock and Bactrocera carambolae Drew & Hancock (Diptera: Tephritidae) based on morphological characters has often been problematical. We describe here a single-nucleotide polymorphism (SNP)-based polymerase chain reaction (PCR) assay to differentiate between these two species. For detection of SNPs, fragments derived from each species were amplified using two primer pairs, COIF/COIR and UEA7/UEA10, sequenced, and aligned to obtain a contiguous 1,517-bp segment. Two new sets of primers were designed based on the 11 SNPs identified in the region. Results of the SNP-PCR test using any one of these species-specific primer sets indicate that these two species could be differentiated on basis of presence or absence of a band in the gel profile. We also tested the SNP-PCR primers on Bactrocera umbrosa F., Bactrocera cucurbitae Coquillett, Bactrocera latifrons Hendel, and Bactrocera tau (Walker) but did not detect any band in the gel, indicating the likelihood of a false positive for B. papayae is nil. This SNP-PCR method is efficient and useful, especially for immature life stages or when only adult body parts of the two species are available for identification, as encountered often in quarantine work.

Prevalence and risk factors of geohelminthiasis among the rural village children in Kota Marudu, Sabah, Malaysia.

Geohelminthiasis is a worldwide problem, especially in low-income countries. Children from rural areas and those living in poverty, lacking basic health amenities and having poor environmental sanitation are likely to be affected. Adverse effects such as anemia, protein malnutrition, colitis are common which can affect both the children's physical and mental growing development. A cross-sectional study on geohelminthiasis was conducted among children from 238 households in 13 villages in Kota Marudu of northern Sabah, East Malaysia. The study involved interviewing villagers using questionnaires to collect demographic and socio-economic data, getting faecal samples from the children, collecting soil samples and identifying parasite eggs with microscopy and molecular methods. A total of 407 children (6 months-17 years old) enrolled in the study. Geohelminthiasis was detected in the faecal samples of children from 54% (7/13) of the villages with mean prevalence of infection per village of 9.0% (0%-34.9%). On a household basis, 18% (43/238) of the households sampled had infected children, with mean prevalence rate per household of 11% (0%-43%). The prevalence was for Ascaris lumbricoides: 9.6% (39/407), Trichuris trichiura: 2.7% (11/407) and hookworms (Necator americanus and Ancylostoma sp.): 2.7% (11/407). The overall mean infection rate of the children examined was 14.3%. Significantly higher prevalence was recorded for the children of mothers who did not have any formal education (p = 0.003); household income of less than USD119 (RM500) (p<0.001); children from homes without proper sanitation facilities (p<0.001); children who usually go about barefoot (p<0.001) and not washing feet before entering the house (p = 0.017). Soil samples were found to have geohelminth eggs or larvae which could be due to unhygienic sanitation practices. This study shows the geohelminthiasis is prevalent in the villages, and the risk factors are lack of maternal education, low income, poor sanitation facilities and irregular deworming practice. Expanding deworming coverage in the study region may help reduce the worm infections in these communities, so that the mental and physical development of the children would not be affected by geohelminthiasis. The data on the prevalence of geohelminthiasis in this study would contribute to better public health monitoring and operation to reduce the infection in rural areas.

Human exposure to zoonotic malaria vectors in village, farm and forest habitats in Sabah, Malaysian Borneo.

The zoonotic malaria parasite, Plasmodium knowlesi, is now a substantial public health problem in Malaysian Borneo. Current understanding of P. knowlesi vector bionomics and ecology in Sabah comes from a few studies near the epicentre of human cases in one district, Kudat. These have incriminated Anopheles balabacensis as the primary vector, and suggest that human exposure to vector biting is peri-domestic as well as in forest environments. To address the limited understanding of vector ecology and human exposure risk outside of Kudat, we performed wider scale surveillance across four districts in Sabah with confirmed transmission to investigate spatial heterogeneity in vector abundance, diversity and infection rate. Entomological surveillance was carried out six months after a cross-sectional survey of P. knowlesi prevalence in humans throughout the study area; providing an opportunity to investigate associations between entomological indicators and infection. Human-landing catches were performed in peri-domestic, farm and forest sites in 11 villages (3-4 per district) and paired with estimates of human P. knowlesi exposure based on sero-prevalence. Anopheles balabacensis was present in all districts but only 6/11 villages. The mean density of An. balabacensis was relatively low, but significantly higher in farm (0.094/night) and forest (0.082/night) than peri-domestic areas (0.007/night). Only one An. balabacensis (n = 32) was infected with P. knowlesi. Plasmodium knowlesi sero-positivity in people was not associated with An. balabacensis density at the village-level however post hoc analyses indicated the study had limited power to detect a statistical association due low vector density. Wider scale sampling revealed substantial heterogeneity in vector density and distribution between villages and districts. Vector-habitat associations predicted from this larger-scale surveillance differed from those inferred from smaller-scale studies in Kudat; highlighting the importance of local ecological context. Findings highlight potential trade-offs between maximizing temporal versus spatial breadth when designing entomological surveillance; and provide baseline entomological and epidemiological data to inform future studies of entomological risk factors for human P. knowlesi infection.

Effect of different habitat types on abundance and biting times of Anopheles balabacensis Baisas (Diptera: Culicidae) in Kudat district of Sabah, Malaysia.

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.

The Role of Ecological Linkage Mechanisms in Plasmodium knowlesi Transmission and Spread.

Defining the linkages between landscape change, disease ecology and human health is essential to explain and predict the emergence of Plasmodium knowlesi malaria, a zoonotic parasite residing in Southeast Asian macaques, and transmitted by species of Anopheles mosquitos. Changing patterns of land use throughout Southeast Asia, particularly deforestation, are suggested to be the primary drivers behind the recent spread of this zoonotic parasite in humans. Local ecological changes at the landscape scale appear to be increasing the risk of disease in humans by altering the dynamics of transmission between the parasite and its primary hosts. This paper will focus on the emergence of P. knowlesi in humans in Malaysian Borneo and the ecological linkage mechanisms suggested to be playing an important role.

Author Correction: Vector compositions change across forested to deforested ecotones in emerging areas of zoonotic malaria transmission in Malaysia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vector compositions change across forested to deforested ecotones in emerging areas of zoonotic malaria transmission in Malaysia.

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.