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Background: The elimination of malaria in Southeast Asia has become more challenging as a result of rising knowlesi malaria cases. In addition, naturally occurring human infections with other zoonotic simian malaria caused by Plasmodium cynomolgi and Plasmodium inui adds another level of complexity in malaria elimination in this region. Unfortunately, data on vectors which are responsible for transmitting this zoonotic disease is very limited., Methodology/principal Findings: We conducted longitudinal studies to investigate the entomological parameters of the simian malaria vectors and to examine the genetic diversity and evolutionary pattern of their simian Plasmodium. All the captured Anopheles mosquitoes were dissected to examine for the presence of oocysts, sporozoites and to determine the parous rate. Our study revealed that the Anopheles Leucosphyrus Group mosquitoes are highly potential competent vectors, as evidenced by their high rate of parity, survival and sporozoite infections in these mosquitoes. Thus, these mosquitoes represent a risk of human infection with zoonotic simian malaria in this region. Haplotype analysis on P. cynomolgi and P. inui, found in high prevalence in the Anopheles mosquitoes from this study, had shown close relationship between simian Plasmodium from the Anopheles mosquitoes with its vertebrate hosts. This directly signifies the ongoing transmission between the vector, macaques, and humans. Furthermore, population genetic analysis showed significant negative values which suggest that both Plasmodium species are undergoing population expansion., Conclusions/significance: With constant microevolutionary processes, there are potential for both P. inui and P. cynomolgi to emerge and spread as a major public health problem, following the similar trend of P. knowlesi. Therefore, concerted vector studies in other parts of Southeast Asia are warranted to better comprehend the transmission dynamics of this zoonotic simian malaria which eventually would aid in the implementation of effective control measures in a rapidly changing environment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Jeyaprakasam et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

We detected 2 natural, asymptomatic Plasmodium inui monoinfections in humans in Malaysia by using nested PCR on concentrated high-volume blood samples. We found a P. inui-positive Anopheles cracens mosquito in the same site as the human infections. Investigators should use ultrasensitive detection methods to identify simian malaria parasite transmission in humans.

We detected the simian malaria parasites Plasmodium knowlesi, P. cynomolgi, P. inui, P. coatneyi, P. inui-like, and P. simiovale among forest fringe-living indigenous communities from various locations in Malaysia. Our findings underscore the importance of using molecular tools to identify newly emergent malaria parasites in humans.

We explored and constructed haplotype network for simian malaria species: Plasmodium knowlesi, P. cynomolgi and P. inui aiming to understand the transmission dynamics between mosquitoes, humans and macaques. Mosquitoes were collected from villages in an area where zoonotic malaria is prevalent. PCR analysis confirmed Anopheles balabacensis as the main vector for macaque parasites, moreover nearly 60% of the mosquitoes harboured more than one Plasmodium species. Fragments of the A-type small subunit ribosomal RNA (SS rRNA) amplified from salivary gland sporozoites, and equivalent sequences obtained from GenBank were used to construct haplotype networks. The patterns were consistent with the presence of geographically distinct populations for P. inui and P. cynomolgi, and with three discrete P. knowlesi populations. This study provides a preliminary snapshot of the structure of these populations, that was insufficient to answer our aim. Thus, collection of parasites from their various hosts and over time, associated with a systematic analysis of a set of genetical loci is strongly advocated in order to obtain a clear picture of the parasite population and the flow between different hosts. This is important to devise measures that will minimise the risk of transmission to humans, because zoonotic malaria impedes malaria elimination., (Copyright © 2019. Published by Elsevier B.V.)

Background: Malaria is a vector-borne parasitic disease which is prevalent in many developing countries. Recently, it has been found that Plasmodium knowlesi, a simian malaria parasite can be life-threatening to humans. Long-tailed macaques, which are widely distributed in Malaysia, are the natural hosts for simian malaria, including P. knowlesi. The aim of the present study was to determine the prevalence of simian malaria parasites in long-tailed macaques in the district of Hulu Selangor, Selangor, Malaysia., Methods: A total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites., Results: Thirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P. fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites., Conclusion: This study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.

Background: Malaria parasites such as Plasmodium knowlesi, P. inui, and P. cynomolgi are spread from macaques to humans through the Leucosphyrus Group of Anopheles mosquitoes. It is crucial to know the distribution of these vectors to implement effective control measures for malaria elimination. Plasmodium knowlesi is the most predominant zoonotic malaria parasite infecting humans in Malaysia. Methods: Vector data from various sources were used to create distribution maps from 1957 to 2021. A predictive statistical model utilizing logistic regression was developed using significant environmental factors. Interpolation maps were created using the inverse distance weighted (IDW) method and overlaid with the corresponding environmental variables. Results: Based on the IDW analysis, high vector abundances were found in the southwestern part of Sarawak, the northern region of Pahang and the northwestern part of Sabah. However, most parts of Johor, Sabah, Perlis, Penang, Kelantan and Terengganu had low vector abundance. The accuracy test indicated that the model predicted sampling and non-sampling areas with 75.3% overall accuracy. The selected environmental variables were entered into the regression model based on their significant values. In addition to the presence of water bodies, elevation, temperature, forest loss and forest cover were included in the final model since these were significantly correlated. Anopheles mosquitoes were mainly distributed in Peninsular Malaysia (Titiwangsa range, central and northern parts), Sabah (Kudat, West Coast, Interior and Tawau division) and Sarawak (Kapit, Miri, and Limbang). The predicted Anopheles mosquito density was lower in the southern part of Peninsular Malaysia, the Sandakan Division of Sabah and the western region of Sarawak. Conclusion: The study offers insight into the distribution of the Leucosphyrus Group of Anopheles mosquitoes in Malaysia. Additionally, the accompanying predictive vector map correlates well with cases of P. knowlesi malaria. This research is crucial in informing and supporting future efforts by healthcare professionals to develop effective malaria control interventions. [ABSTRACT FROM AUTHOR]

Although Malaysia is considered free of human malaria, there has been a growing number of Plasmodium knowlesi cases. This alarming trend highlighted the need for our understanding of this parasite and its associated vectors, especially considering the role of genetic diversity in the adaptation and evolution among vectors in endemic areas, which is currently a significant knowledge gap in their fundamental biology. Thus, this study aimed to investigate the genetic diversity of Anopheles balabacensis, Anopheles cracens, Anopheles introlatus, and Anopheles latens—the vectors for P. knowlesi malaria in Malaysia. Based on cytochrome c oxidase 1 (CO1) and internal transcribed spacer 2 (ITS2) markers, the genealogic networks of An. latens showed a separation of the haplotypes between Peninsular Malaysia and Malaysia Borneo, forming two distinct clusters. Additionally, the genetic distances between these clusters were high (2.3–5.2% for CO1) and (2.3–4.7% for ITS2), indicating the likely presence of two distinct species or cryptic species within An. latens. In contrast, no distinct clusters were observed in An. cracens, An. balabacensis, or An. introlatus, implying a lack of pronounced genetic differentiation among their populations. It is worth noting that there were varying levels of polymorphism observed across the different subpopulations, highlighting some levels of genetic variation within these mosquito species. Nevertheless, further analyses revealed that all four species have undergone demographic expansion, suggesting population growth and potential range expansion for these vectors in this region. [ABSTRACT FROM AUTHOR]

• Plasmodium knowlesi is a serious public health problem in Southeast Asia. • Other Non-human primate malarias are also being reported in humans. • Leucosphyrus group of anopheles mosquitoes have been incriminated as vectors. • Deforestation and changes in land use has led to this serious problem. • NHP will remain as a problem for countries to obtain malaria elimination status in the absence of control measures. Malaria continues to be a global public health problem although it has been eliminated from many countries. Sri Lanka and China are two countries that recently achieved malaria elimination status, and many countries in Southeast Asia are currently in the pipeline for achieving the same goal by 2030. However, Plasmodium knowlesi , a non-human primate malaria parasite continues to pose a threat to public health in this region, infecting many humans in all countries in Southeast Asia except for Timor-Leste. Besides, other non-human primate malaria parasite such as Plasmodium cynomolgi and Plasmodium inui are infecting humans in the region. The non-human primates, the long-tailed and pig-tailed macaques which harbour these parasites are now increasingly prevalent in farms and forest fringes close by to the villages. Additionally, the Anopheles mosquitoes belonging to the Lecuosphyrus Group are also present in these areas which makes them ideal for transmitting the non-human primate malaria parasites. With changing landscape and deforestation, non-human primate malaria parasites will affect more humans in the coming years with the elimination of human malaria. Perhaps due to loss of immunity, more humans will be infected as currently being demonstrated in Malaysia. Thus, control measures need to be instituted rapidly to achieve the malaria elimination status by 2030. However, the zoonotic origin of the parasite and the changes of the vectors behaviour to early biting seems to be the stumbling block to the malaria elimination efforts in this region. In this review, we discuss the challenges faced in malaria elimination due to deforestation and the serious threat posed by non-human primate malaria parasites. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The zoonotic malaria parasite Plasmodium knowlesi has emerged across Southeast Asia and is now the main cause of malaria in humans in Malaysia. A critical priority for P. knowlesi surveillance and control is understanding whether transmission is entirely zoonotic or is also occurring through human-mosquito-human transmission. Methods: A systematic literature review was performed to evaluate existing evidence which refutes or supports the occurrence of sustained human-mosquito-human transmission of P. knowlesi. Possible evidence categories and study types which would support or refute non-zoonotic transmission were identified and ranked. A literature search was conducted on Medline, EMBASE and Web of Science using a broad search strategy to identify any possible published literature. Results were synthesized using the Synthesis Without Meta-analysis (SWiM) framework, using vote counting to combine the evidence within specific categories. Results: Of an initial 7,299 studies screened, 131 studies were included within this review: 87 studies of P. knowlesi prevalence in humans, 14 studies in non-human primates, 13 studies in mosquitoes, and 29 studies with direct evidence refuting or supporting non-zoonotic transmission. Overall, the evidence showed that human-mosquito-human transmission is biologically possible, but there is limited evidence of widespread occurrence in endemic areas. Specific areas of research were identified that require further attention, notably quantitative analyses of potential transmission dynamics, epidemiological and entomological surveys, and ecological studies into the sylvatic cycle of the disease. Conclusion: There are key questions about P. knowlesi that remain within the areas of research that require more attention. These questions have significant implications for malaria elimination and eradication programs. This paper considers limited but varied research and provides a methodological framework for assessing the likelihood of different transmission patterns for emerging zoonotic diseases. [ABSTRACT FROM AUTHOR]

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Background: Plasmodium knowlesi, a simian malaria parasite infection, increases as Plasmodium falciparum and Plasmodium vivax infections decrease in Johor, Malaysia. Therefore, this study aimed to identify the distribution of vectors involved in knowlesi malaria transmission in Johor. This finding is vital in estimating hotspot areas for targeted control strategies. Methods: Anopheles mosquitoes were collected from the location where P. knowlesi cases were reported. Cases of knowlesi malaria from 2011 to 2019 in Johor were analyzed. Internal transcribed spacers 2 (ITS2) and cytochrome c oxidase subunit I (COI) genes were used to identify the Leucosphyrus Group of Anopheles mosquitoes. In addition, spatial analysis was carried out on the knowlesi cases and vectors in Johor. Results: One hundred and eighty-nine cases of P. knowlesi were reported in Johor over 10 years. Young adults between the ages of 20–39 years comprised 65% of the cases. Most infected individuals were involved in agriculture and army-related occupations (22% and 32%, respectively). Four hundred and eighteen Leucosphyrus Group Anopheles mosquitoes were captured during the study. Anopheles introlatus was the predominant species, followed by Anopheles latens. Spatial analysis by Kriging interpolation found that hotspot regions of P. knowlesi overlapped or were close to the areas where An. introlatus and An. latens were found. A significantly high number of vectors and P. knowlesi cases were found near the road within 0–5 km. Conclusions: This study describes the distribution of P. knowlesi cases and Anopheles species in malaria-endemic transmission areas in Johor. Geospatial analysis is a valuable tool for studying the relationship between vectors and P. knowlesi cases. This study further supports that the Leucosphyrus Group of mosquitoes might be involved in transmitting knowlesi malaria cases in Johor. These findings may provide initial evidence to prioritize diseases and vector surveillance. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi, a simian malaria parasite of great public health concern has been reported from most countries in Southeast Asia and exported to various countries around the world. Currently P. knowlesi is the predominant species infecting humans in Malaysia. Besides this species, other simian malaria parasites such as P. cynomolgi and P. inui are also infecting humans in the region. The vectors of P. knowlesi and other Asian simian malarias belong to the Leucosphyrus Group of Anopheles mosquitoes which are generally forest dwelling species. Continual deforestation has resulted in these species moving into forest fringes, farms, plantations and human settlements along with their macaque hosts. Limited studies have shown that mosquito vectors are attracted to both humans and macaque hosts, preferring to bite outdoors and in the early part of the night. We here review the current status of simian malaria vectors and their parasites, knowledge of vector competence from experimental infections and discuss possible vector control measures. The challenges encountered in simian malaria elimination are also discussed. We highlight key knowledge gaps on vector distribution and ecology that may impede effective control strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites (P. knowlesi, P. coatneyi, P. fieldi, P. cynomolgi, and P. inui) present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore's wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore. Author summary: Malaria is one of the most important vector-borne diseases in the world. In Southeast Asia, the incidence of human malaria infections caused by simian malaria parasites that originate from macaques has been increasing in the last decade, possibly due to closer proximity between the macaque and human population as a result of deforestation. In Singapore, urbanization and change in land use have resulted in closer proximity and interactions between macaques and the general human population. In order to assess the risk of potential simian malaria zoonosis, 1039 macaques' blood samples were screened in this study for simian malaria parasites. Our results showed that simian malaria parasites were only detected in macaques living deep in the forest within the military protected zone, while macaques living close to human habitations are malaria-free. This study illustrated a low risk of zoonotic transmission of simian malaria parasites to the general population in Singapore. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi, a simian malaria parasite, has been in the limelight since a large focus of human P. knowlesi infection was reported from Sarawak (Malaysian Borneo) in 2004. Although this infection is transmitted across Southeast Asia, the largest number of cases has been reported from Malaysia. The increasing number of knowlesi malaria cases has been attributed to the use of molecular tools for detection, but environmental changes including deforestation likely play a major role by increasing human exposure to vector mosquitoes, which coexist with the macaque host. In addition, with the reduction in human malaria transmission in Southeast Asia, it is possible that human populations are at a greater risk of P. knowlesi infection due to diminishing cross-species immunity. Furthermore, the possibility of increasing exposure of humans to other simian Plasmodium parasites such as Plasmodium cynomolgi and Plasmodium inui should not be ignored. We here review the current status of these parasites in humans, macaques, and mosquitoes to support necessary reorientation of malaria control and elimination in the affected areas. [ABSTRACT FROM AUTHOR]

Background Anopheles 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 findings Anopheles 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/Significance This 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. [ABSTRACT FROM AUTHOR]

Plasmodium knowlesi , a malaria parasite of macaques, has emerged as an important parasite of humans. Despite the significance of P. knowlesi malaria in parts of Southeast Asia, very little is known about the genetic variation in this parasite. Our aim here was to explore sequence variation in a molecule called the 42 kDa merozoite surface protein-1 (MSP-1), which is found on the surface of blood stages of Plasmodium spp. and plays a key role in erythrocyte invasion. Several studies of P. falciparum have reported that the C-terminus (a 42 kDa fragment) of merozoite surface protein-1 (MSP-1 42 ; consisting of MSP-1 19 and MSP-1 33 ) is a potential candidate for a malaria vaccine. However, to date, no study has yet investigated the sequence diversity of the gene encoding P. knowlesi MSP-1 42 (comprising Pk-msp - 1 19 and Pk - msp - 1 33 ) among isolates in Malaysia. The present study explored this aspect. Twelve P. knowlesi isolates were collected from patients from hospitals in Selangor and Sabah Borneo, Malaysia, between 2012 and 2014. The Pk - msp - 1 42 gene was amplified by PCR and directly sequenced. Haplotype diversity (Hd) and nucleotide diversity (л) were studied among the isolates. There was relatively high genetic variation among P. knowlesi isolates; overall Hd and л were 1 ± 0.034 and 0.01132 ± 0.00124, respectively. A total of nine different haplotypes related to amino acid alterations at 13 positions, and the Pk -MSP-1 19 sequence was found to be more conserved than Pk-msp-1 33 . We have found evidence for negative selection in Pk-msp- 42 as well as the 33 kDa and 19 kDa fragments by comparing the rate of non-synonymous versus synonymous substitutions. Future investigations should study large numbers of samples from disparate geographical locations to critically assess whether this molecule might be a potential vaccine target for P. knowlesi . [ABSTRACT FROM AUTHOR]

Objectives To determine the distribution of Plasmodium ( P ) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis. Methods The study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR. Results Among the participants, 57 (63.3%) were positive and 33 (36.7%) were negative by microscopy. Of positive samples, 39 (68.4%) were Plasmodium falciparum , 17 (29.8%) Plasmodium vivax and 1 (1.8%) Plasmodium malariae , whereas 59-amplified by PCR were 40 (67.8%), 18 (30.5%) and 1 (1.7%) respectively. PCR amplified 2 microscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale , P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (κ = 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diagnosis of P. falciparum were 92.5% (95% CI: 79.6–98.4) and 96.0% (95% CI: 86.3–99.5) respectively, whereas for P. vivax were 83.3% (95% CI: 58.6–96.4) and 97.2% (95% CI: 90.3–99.7). Conclusions P. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists. [ABSTRACT FROM AUTHOR]

Background: In 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 Findings: We 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/Significance: This 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. [ABSTRACT FROM AUTHOR]

Background: Infection by the simian malaria parasite, Plasmodium knowlesi, can lead to severe and fatal disease in humans, and is the most common cause of malaria in parts of Malaysia. Despite being a serious public health concern, the geographical distribution of P. knowlesi malaria risk is poorly understood because the parasite is often misidentified as one of the human malarias. Human cases have been confirmed in at least nine Southeast Asian countries, many of which are making progress towards eliminating the human malarias. Understanding the geographical distribution of P. knowlesi is important for identifying areas where malaria transmission will continue after the human malarias have been eliminated. Methodology/Principal Findings: A total of 439 records of P. knowlesi infections in humans, macaque reservoir and vector species were collated. To predict spatial variation in disease risk, a model was fitted using records from countries where the infection data coverage is high. Predictions were then made throughout Southeast Asia, including regions where infection data are sparse. The resulting map predicts areas of high risk for P. knowlesi infection in a number of countries that are forecast to be malaria-free by 2025 (Malaysia, Cambodia, Thailand and Vietnam) as well as countries projected to be eliminating malaria (Myanmar, Laos, Indonesia and the Philippines). Conclusions/Significance: We have produced the first map of P. knowlesi malaria risk, at a fine-scale resolution, to identify priority areas for surveillance based on regions with sparse data and high estimated risk. Our map provides an initial evidence base to better understand the spatial distribution of this disease and its potential wider contribution to malaria incidence. Considering malaria elimination goals, areas for prioritised surveillance are identified. [ABSTRACT FROM AUTHOR]

Background: Malaria cases caused by Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques, are increasing rapidly in Sabah, Malaysia. One hypothesis is that this increase is associated with changes in land use. A study was carried out to identify the anopheline vectors present in different forest types and to observe the human landing behaviour of mosquitoes. 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 < 0.0001). A greater abundance of mosquitoes were landing in the logged forest compared to the primary forest (p < 0.0001). There was no difference between mosquito abundance in the logged forest and lightly logged forest (p = 0.554). A higher evening temperature (p < 0.0001) and rainfall (p < 0.0001) significantly decreased mosquito abundance during collection nights. Conclusions: This study demonstrates the potential ability of An. balabacensis to transmit 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. [ABSTRACT FROM AUTHOR]

Background: Plasmodium knowlesi is a zoonotic pathogen, transmitted among macaques and to humans by anopheline mosquitoes. Information on P. knowlesi malaria is lacking in most regions so the first step to understand the geographical distribution of disease risk is to define the distributions of the reservoir and vector species. 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. [ABSTRACT FROM AUTHOR]

Background: The simian malaria parasite Plasmodium knowlesi is emerging as a public health problem in Southeast Asia, particularly in Malaysian Borneo where it now accounts for the greatest burden of malaria cases and deaths. Control is hindered by limited understanding of the ecology of potential vector species. 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 2000hrs. 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. [ABSTRACT FROM AUTHOR]

Background: Plasmodium knowlesi is a simian malaria parasite that is widespread in humans in Malaysian Borneo. However, little is known about the incidence and distribution of this parasite in the Sandakan division, Malaysian Borneo. Therefore, the aim of the present epidemiological study was to investigate the incidence and distribution of P. knowlesi as well as other Plasmodium species in this division based on a most recent developed hexaplex PCR system (PlasmoNex™). Methods: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene. Results: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/ P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study. Conclusions: The discovery of P. knowlesi in Sandakan division showed that prospective studies on the epidemiological risk factors and transmission dynamics of P. knowlesi in these areas are crucial in order to develop strategies for effective malaria control. The availability of advanced diagnostic tool PlasmoNex™ enhanced the accuracy and accelerated the speed in the diagnosis of malaria. [ABSTRACT FROM AUTHOR]

Background: Anopheles cracens has been incriminated as a vector for the simian malaria parasite, Plasmodium knowlesi, that is the fifth Plasmodium species infecting humans. Little experimental data exists on this mosquito species due to the lack of its availability in laboratories. Findings: The population of An. cracens, collected from Kuala Lipis, Pahang was maintained at the insectary of the Department of Parasitology, Faculty of Medicine, University Malaya at 24-26°C and 60-80% relative humidity. The mosquitoes were maintained with artificial mating and blood-fed on humans and hamsters. The colony has been established since November 2011 and to date has reached its sixth generation. Conclusion: This is the first description of maintaining the Malaysian strain An. cracens colony by artificial mating. Colonization of An. cracens will provide fundamental information for genetic studies and will be useful in assessing comparative susceptibility to Plasmodium parasites. [ABSTRACT FROM AUTHOR]

Background: The first natural infection of Plasmodium knowlesi in humans was recorded in 1965 in peninsular Malaysia. Extensive research was then conducted and it was postulated that it was a rare incident and that simian malaria will not be easily transmitted to humans. However, at the turn of the 21st century, knowlesi malaria was prevalent throughout Southeast Asia and is life threatening. Thus, a longitudinal study was initiated to determine the vectors, their seasonal variation and preference to humans and macaques. Methods: Monthly mosquito collections were carried out in Kuala Lipis, Pahang, peninsular Malaysia, using human-landing collection and monkey-baited traps at ground and canopy levels. All mosquitoes were identified and all anopheline mosquitoes were dissected and the gut and gland examined for oocysts and sporozoites. Nested polymerase chain reaction (PCR) was conducted on positive samples, followed by sequencing of the csp gene. Results and discussion: Anopheles cracens was the predominant mosquito biting humans as well as the macaques. It comprised 63.2% of the total collection and was the only species positive for sporozoites of P. knowlesi. It was exophagic and did not enter houses. Besides An. cracens, Anopheles kochi was also found in the monkey-bait trap. Both species preferred to bite monkeys at ground level compared to canopy. Conclusion: Anopheles cracens, which belongs to the Dirus complex, Leucosphyrus subgroup, Leucosphyrus group of mosquitoes, has been confirmed to be the only vector for this site from Pahang during this study. It was the predominant mosquito at the study sites and with deforestation humans and villages are entering deeper in the forests, and nearer to the mosquitoes and macacques. The close association of humans with macaques and mosquitoes has led to zoonotic transmission of malaria. [ABSTRACT FROM AUTHOR]

Simple Summary: Plasmodium parasites cause malaria. The bites of infected female Anopheles mosquitoes, known as "malaria vectors," transmit the parasites to people. To prevent the spread of malaria, precise mosquito species identification is essential. This study aims to develop a quick and accurate method for identifying the Anopheles species (An. introlatus, An. latens, An. cracens, and An. balabacensis), which have been incriminated as vectors for simian malaria in Malaysia. Overall, six primers targeting the internal transcribed spacer 2 (ITS2) region of each species were designed for this assay. This study is helpful for the researchers or vector-related field workers to correctly identify the mosquitoes for control activities. The Leucosphyrus Group of mosquitoes are the major simian malaria vectors in Malaysia. Accurate species identification is required to help in curbing the spread of simian malaria. The aim of the study is to provide an accurate molecular method for identifying the four important Anopheles vector species found in Malaysia. Mosquito specimens were collected from various localities in Malaysia, where simian malaria cases were reported. DNA from 122 mosquito specimens was tested to develop a multiplex polymerase chain reaction (PCR) assay. The specificity of this assay was tested against other mosquito species. Molecular identification of the species was further confirmed by analysing the internal transcribed spacer 2 (ITS2) DNA region of the specimens. Anopheles balabacensis and An. latens showed two distinct clades in the phylogenetic tree. The multiplex PCR assay was developed based on the ITS2 region for the identification of Anopheles introlatus (298–299 bp), Anopheles latens (197–198 bp), Anopheles cracens (421–426 bp), and Anopheles balabacensis (224–228 bp). This method will be useful to accurately identify the major Anopheles Leucosphyrus Group species in Malaysia, which are difficult to identify morphologically, to determine the correct vector as well as its geographical distribution. [ABSTRACT FROM AUTHOR]

Background: A large focus of human infections with Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques was discovered in the Kapit Division of Sarawak, Malaysian Borneo. A study was initiated to identify the vectors of malaria, to elucidate where transmission is taking place and to understand the bionomics of the vectors in Kapit. Methods: Three different ecological sites in the forest, farm and longhouse in the Kapit district were selected for the study. Mosquitoes were collected by human landing collection at all sites and at the forest also by monkey-baited-traps situated on three different levels. All mosquitoes were identified and salivary glands and midguts of anopheline mosquitoes were dissected to determine the presence of malaria parasites. Results and Discussions: Over an 11-month period, a total of 2,504 Anopheles mosquitoes comprising 12 species were caught; 1,035 at the farm, 774 at the forest and 425 at the longhouse. Anopheles latens (62.3%) and Anopheles watsonii (30.6%) were the predominant species caught in the forested ecotypes, while in the farm Anopheles donaldi (49.9%) and An. latens (35.6%) predominated. In the long house, An. latens (29.6%) and An. donaldi (22.8%) were the major Anopheline species. However, An. latens was the only mosquito positive for sporozoites and it was found to be attracted to both human and monkey hosts. In monkey-baited net traps, it preferred to bite monkeys at the canopy level than at ground level. An. latens was found biting early as 18.00 hours. Conclusion: Anopheles latens is the main vector for P. knowlesi malaria parasites in the Kapit District of Sarawak, Malaysian Borneo. The study underscores the relationship between ecology, abundance and bionomics of anopheline fauna. The simio-anthropophagic and acrodendrophilic behaviour of An. latens makes it an efficient vector for the transmission of P. knowlesi parasites to both human and monkey hosts. [ABSTRACT FROM AUTHOR]

A recently published comment on a report of Plasmodium knowlesi infections in Vietnam states that this may not accurately represent the situation in the study area because the PCR primers used may cross-hybridize with Plasmodium vivax. Nevertheless, P. knowlesi infections have been confirmed by sequencing. In addition, a neighbour-joining tree based on the 18S S-Type SSUrRNA gene shows that the Vietnamese samples clearly cluster with the P. knowlesi isolates identified in Malaysia and are distinct from the corresponding P. vivax sequences. All samples came from asymptomatic individuals who did not consult for fever during the months preceding or following the survey, indicating that asymptomatic P. knowlesi infections occur in this population, although this does not exclude the occurrence of symptomatic cases. Large-scale studies to determine the extent and the epidemiology of P. knowlesi malaria in Vietnam are further needed. [ABSTRACT FROM AUTHOR]

Background: The C-terminal 42 kDa domain of Plasmodium knowlesi merozoite surface protein 1 (PkMSP1) is a potential asexual blood-stage vaccine candidate, however, only a limited number of clinical isolates have been analysed from Malaysia and no inter-country comparative diversity study has been conducted. In the present study, nucleotide diversity, haplotypes and natural selection levels of pkmsp1 in clinical samples from geographically distinct regions of Malaysia and Thailand were investigated. The overall population structure of the parasite from the region was determined. Methods: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5. Results: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain. Conclusions: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries. [ABSTRACT FROM AUTHOR]