METHODS: Mosquito collections were carried out using human landing catches at ground and canopy levels in the Tawau Division of Sabah. Collections were conducted along an anthropogenic disturbance gradient (primary forest, lightly logged virgin jungle reserve and salvage logged forest) between 18:00 and 22:00 h.
RESULTS: Anopheles balabacensis, a vector of P. knowlesi, was the predominant species in all collection areas, accounting for 70 % of the total catch, with a peak landing time of 18:30-20:00 h. Anopheles balabacensis had a preference for landing on humans at ground level compared to the canopy (p knowlesi between canopy-dwelling simian hosts and ground-dwelling humans, and that forest disturbance increases the abundance of this disease vector. These results, in combination with regional patterns of land use change, may partly explain the rapid rise in P. knowlesi cases in Sabah. This study provides essential data on anthropophily for the principal vector of P. knowlesi which is important for the planning of vector control strategies.
METHODOLOGY/PRINCIPAL FINDINGS: After reviewing the published literature we identified potential host and vector species and ranked these based on how informative they are for the presence of an infectious parasite reservoir, based on current evidence. We collated spatial data on parasite occurrence and the ranges of the identified host and vector species. The ranked spatial data allowed us to assign an evidence score to 475 subnational areas in 19 countries and we present the results on a map of the Southeast and South Asia region.
CONCLUSIONS/SIGNIFICANCE: We have ranked subnational areas within the potential disease range according to evidence for presence of a disease risk to humans, providing geographical evidence to support decisions on prevention, management and prophylaxis. This work also highlights the unknown risk status of large parts of the region. Within this unknown category, our map identifies which areas have most evidence for the potential to support an infectious reservoir and are therefore a priority for further investigation. Furthermore we identify geographical areas where further investigation of putative host and vector species would be highly informative for the region-wide assessment.
METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.
RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.
CONCLUSIONS: This test demonstrated excellent sensitivity and specificity, and adds P. knowlesi to the repertoire of Plasmodium targets for the clinical diagnosis of malaria by real-time PCR assays. Furthermore, quantitation of DNA copy number provides a useful advantage over other molecular assays to investigate the correlation between levels of infection and the spectrum of disease.
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: We used macaque and mosquito species presence data, background data that captured sampling bias in the presence data, a boosted regression tree model and environmental datasets, including annual data for land classes, to predict the distributions of each vector and host species. We then compared the predicted distribution of each species with cover of each land class.
RESULTS: Fine-scale distribution maps were generated for three macaque host species (Macaca fascicularis, M. nemestrina and M. leonina) and two mosquito vector complexes (the Dirus Complex and the Leucosphyrus Complex). The Leucosphyrus Complex was predicted to occur in areas with disturbed, but not intact, forest cover (> 60% tree cover) whereas the Dirus Complex was predicted to occur in areas with 10-100% tree cover as well as vegetation mosaics and cropland. Of the macaque species, M. nemestrina was mainly predicted to occur in forested areas whereas M. fascicularis was predicted to occur in vegetation mosaics, cropland, wetland and urban areas in addition to forested areas.
CONCLUSIONS: The predicted M. fascicularis distribution encompassed a wide range of habitats where humans are found. This is of most significance in the northern part of its range where members of the Dirus Complex are the main P. knowlesi vectors because these mosquitoes were also predicted to occur in a wider range of habitats. Our results support the hypothesis that conversion of intact forest into disturbed forest (for example plantations or timber concessions), or the creation of vegetation mosaics, will increase the probability that members of the Leucosphyrus Complex occur at these locations, as well as bringing humans into these areas. An explicit analysis of disease risk itself using infection data is required to explore this further. The species distributions generated here can now be included in future analyses of P. knowlesi infection risk.
MAIN TEXT: Plasmodium knowlesi is the most common cause of malaria in Malaysia, and cases have also been reported in nearly all countries of Southeast Asia. Outside of Malaysia, P. knowlesi is frequently misdiagnosed by microscopy as Plasmodium falciparum or Plasmodium vivax. Thus, P. knowlesi may be underdiagnosed in affected regions and its true incidence underestimated. Acknowledgement in the World Malaria Report of the regional importance of P. knowlesi will facilitate efforts to improve surveillance of this emerging parasite. Furthermore, increased recognition will likely lead to improved delivery of effective treatment for this potentially fatal infection, as has occurred in Malaysia where P. knowlesi case-fatality rates have fallen despite rising incidence. In a number of knowlesi-endemic countries, substantial progress has been made towards the elimination of P. vivax and P. falciparum. However, efforts to eliminate these human-only species should not preclude efforts to reduce human malaria from P. knowlesi. The regional importance of knowlesi malaria was recognized by the WHO with its recent Evidence Review Group meeting on knowlesi malaria to address strategies for prevention and mitigation.
CONCLUSION: The WHO World Malaria Report has an appropriate focus on falciparum and vivax malaria, the major causes of global mortality and morbidity. However, the authors hope that in future years this important publication will also incorporate data on the progress and challenges in reducing knowlesi malaria in regions where transmission occurs.
METHODS: Blood samples were collected from P. knowlesi malaria patients within a period of 4 years (2008-2012). The pkmsp3 gene of the isolates was amplified via PCR, and subsequently cloned and sequenced. The full length pkmsp3 sequence was divided into Domain A and Domain B. Natural selection, genetic diversity, and haplotypes of pkmsp3 were analysed using MEGA6 and DnaSP ver. 5.10.00 programmes.
RESULTS: From 23 samples, 48 pkmsp3 sequences were successfully obtained. At the nucleotide level, 101 synonymous and 238 non-synonymous mutations were observed. Tests of neutrality were not significant for the full length, Domain A or Domain B sequences. However, the dN/dS ratio of Domain B indicates purifying selection for this domain. Analysis of the deduced amino acid sequences revealed 42 different haplotypes. Neighbour Joining phylogenetic tree and haplotype network analyses revealed that the haplotypes clustered into two distinct groups.
CONCLUSIONS: A moderate level of genetic diversity was observed in the pkmsp3 and only the C-terminal region (Domain B) appeared to be under purifying selection. The separation of the pkmsp3 into two haplotype groups provides further evidence of the existence of two distinct P. knowlesi types or lineages. Future studies should investigate the diversity of pkmsp3 among P. knowlesi isolates in North Borneo, where large numbers of human knowlesi malaria infection still occur.