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  1. Divis PCS, Singh B, Conway DJ
    Adv Parasitol, 2021;113:191-223.
    PMID: 34620383 DOI: 10.1016/bs.apar.2021.08.003
    Molecular epidemiology has been central to uncovering P. knowlesi as an important cause of human malaria in Southeast Asia, and to understanding the complex nature of this zoonosis. Species-specific parasite detection and characterization of sequences were vital to show that P. knowlesi was distinct from the human parasite species that had been presumed to cause all malaria. With established sensitive and specific molecular detection tools, surveys subsequently indicated the distribution of P. knowlesi infections in humans, wild primate reservoir host species, and mosquito vector species. The importance of studying P. knowlesi genetic polymorphism was indicated initially by analysing a few nuclear gene loci as well as the mitochondrial genome, and subsequently by multi-locus microsatellite analyses and whole-genome sequencing. Different human infections generally have unrelated P. knowlesi genotypes, acquired from the diverse local parasite reservoirs in macaques. However, individual human infections are usually less genetically complex than those of wild macaques which experience more frequent superinfection with different P. knowlesi genotypes. Multi-locus analyses have revealed deep population subdivisions within P. knowlesi, which are structured both geographically and in relation to different macaque reservoir host species. Simplified genotypic discrimination assays now enable efficient large-scale surveillance of the sympatric P. knowlesi subpopulations within Malaysian Borneo. The whole-genome sequence analyses have also identified loci under recent positive natural selection in the P. knowlesi genome, with evidence that different loci are affected in different populations. These provide a foundation to understand recent adaptation of the zoonotic parasite populations, and to track and interpret future changes as they emerge.
  2. Divis PCS, Duffy CW, Kadir KA, Singh B, Conway DJ
    Mol Ecol, 2018 02;27(4):860-870.
    PMID: 29292549 DOI: 10.1111/mec.14477
    Plasmodium knowlesi is a significant cause of human malaria transmitted as a zoonosis from macaque reservoir hosts in South-East Asia. Microsatellite genotyping has indicated that human infections in Malaysian Borneo are an admixture of two highly divergent sympatric parasite subpopulations that are, respectively, associated with long-tailed macaques (Cluster 1) and pig-tailed macaques (Cluster 2). Whole-genome sequences of clinical isolates subsequently confirmed the separate clusters, although fewer of the less common Cluster 2 type were sequenced. Here, to analyse population structure and genomic divergence in subpopulation samples of comparable depth, genome sequences were generated from 21 new clinical infections identified as Cluster 2 by microsatellite analysis, yielding a cumulative sample size for this subpopulation similar to that for Cluster 1. Profound heterogeneity in the level of intercluster divergence was distributed across the genome, with long contiguous chromosomal blocks having high or low divergence. Different mitochondrial genome clades were associated with the two major subpopulations, but limited exchange of haplotypes from one to the other was evident, as was also the case for the maternally inherited apicoplast genome. These findings indicate deep divergence of the two sympatric P. knowlesi subpopulations, with introgression likely to have occurred recently. There is no evidence yet of specific adaptation at any introgressed locus, but the recombinant mosaic types offer enhanced diversity on which selection may operate in a currently changing landscape and human environment. Loci responsible for maintaining genetic isolation of the sympatric subpopulations need to be identified in the chromosomal regions showing fixed differences.
  3. Ang JXD, Kadir KA, Mohamad DSA, Matusop A, Divis PCS, Yaman K, et al.
    Parasit Vectors, 2020 Sep 15;13(1):472.
    PMID: 32933567 DOI: 10.1186/s13071-020-04345-2
    BACKGROUND: Plasmodium knowlesi is a significant cause of human malaria in Sarawak, Malaysian Borneo. Only one study has been previously undertaken in Sarawak to identify vectors of P. knowlesi, where Anopheles latens was incriminated as the vector in Kapit, central Sarawak. A study was therefore undertaken to identify malaria vectors in a different location in Sarawak.

    METHODS: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.

    RESULTS: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.

    CONCLUSIONS: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.

  4. Lubis IND, Wijaya H, Lubis M, Lubis CP, Divis PCS, Beshir KB, et al.
    J Infect Dis, 2017 Apr 01;215(7):1148-1155.
    PMID: 28201638 DOI: 10.1093/infdis/jix091
    Background: As Indonesia works toward the goal of malaria elimination, information is lacking on malaria epidemiology from some western provinces. As a basis for studies of antimalarial efficacy, we set out to survey parasite carriage in 3 communities in North Sumatera Province.

    Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi.

    Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%).

    Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.

  5. Sukumarran D, Hasikin K, Mohd Khairuddin AS, Ngui R, Wan Sulaiman WY, Vythilingam I, et al.
    Trop Biomed, 2023 Jun 01;40(2):208-219.
    PMID: 37650409 DOI: 10.47665/tb.40.2.013
    Timely and rapid diagnosis is crucial for faster and proper malaria treatment planning. Microscopic examination is the gold standard for malaria diagnosis, where hundreds of millions of blood films are examined annually. However, this method's effectiveness depends on the trained microscopist's skills. With the increasing interest in applying deep learning in malaria diagnosis, this study aims to determine the most suitable deep-learning object detection architecture and their applicability to detect and distinguish red blood cells as either malaria-infected or non-infected cells. The object detectors Yolov4, Faster R-CNN, and SSD 300 are trained with images infected by all five malaria parasites and from four stages of infection with 80/20 train and test data partition. The performance of object detectors is evaluated, and hyperparameters are optimized to select the best-performing model. The best-performing model was also assessed with an independent dataset to verify the models' ability to generalize in different domains. The results show that upon training, the Yolov4 model achieves a precision of 83%, recall of 95%, F1-score of 89%, and mean average precision of 93.87% at a threshold of 0.5. Conclusively, Yolov4 can act as an alternative in detecting the infected cells from whole thin blood smear images. Object detectors can complement a deep learning classification model in detecting infected cells since they eliminate the need to train on single-cell images and have been demonstrated to be more feasible for a different target domain.
  6. Sukumarran D, Hasikin K, Khairuddin ASM, Ngui R, Sulaiman WYW, Vythilingam I, et al.
    Parasit Vectors, 2024 Apr 16;17(1):188.
    PMID: 38627870 DOI: 10.1186/s13071-024-06215-7
    BACKGROUND: Malaria is a serious public health concern worldwide. Early and accurate diagnosis is essential for controlling the disease's spread and avoiding severe health complications. Manual examination of blood smear samples by skilled technicians is a time-consuming aspect of the conventional malaria diagnosis toolbox. Malaria persists in many parts of the world, emphasising the urgent need for sophisticated and automated diagnostic instruments to expedite the identification of infected cells, thereby facilitating timely treatment and reducing the risk of disease transmission. This study aims to introduce a more lightweight and quicker model-but with improved accuracy-for diagnosing malaria using a YOLOv4 (You Only Look Once v. 4) deep learning object detector.

    METHODS: The YOLOv4 model is modified using direct layer pruning and backbone replacement. The primary objective of layer pruning is the removal and individual analysis of residual blocks within the C3, C4 and C5 (C3-C5) Res-block bodies of the backbone architecture's C3-C5 Res-block bodies. The CSP-DarkNet53 backbone is simultaneously replaced for enhanced feature extraction with a shallower ResNet50 network. The performance metrics of the models are compared and analysed.

    RESULTS: The modified models outperform the original YOLOv4 model. The YOLOv4-RC3_4 model with residual blocks pruned from the C3 and C4 Res-block body achieves the highest mean accuracy precision (mAP) of 90.70%. This mAP is > 9% higher than that of the original model, saving approximately 22% of the billion floating point operations (B-FLOPS) and 23 MB in size. The findings indicate that the YOLOv4-RC3_4 model also performs better, with an increase of 9.27% in detecting the infected cells upon pruning the redundant layers from the C3 Res-block bodies of the CSP-DarkeNet53 backbone.

    CONCLUSIONS: The results of this study highlight the use of the YOLOv4 model for detecting infected red blood cells. Pruning the residual blocks from the Res-block bodies helps to determine which Res-block bodies contribute the most and least, respectively, to the model's performance. Our method has the potential to revolutionise malaria diagnosis and pave the way for novel deep learning-based bioinformatics solutions. Developing an effective and automated process for diagnosing malaria will considerably contribute to global efforts to combat this debilitating disease. We have shown that removing undesirable residual blocks can reduce the size of the model and its computational complexity without compromising its precision.

  7. Hocking SE, Divis PCS, Kadir KA, Singh B, Conway DJ
    Emerg Infect Dis, 2020 08;26(8):1749-1758.
    PMID: 32687018 DOI: 10.3201/eid2608.190864
    Most malaria in Malaysia is caused by Plasmodium knowlesi parasites through zoonotic infection from macaque reservoir hosts. We obtained genome sequences from 28 clinical infections in Peninsular Malaysia to clarify the emerging parasite population structure and test for evidence of recent adaptation. The parasites all belonged to a major genetic population of P. knowlesi (cluster 3) with high genomewide divergence from populations occurring in Borneo (clusters 1 and 2). We also observed unexpected local genetic subdivision; most parasites belonged to 2 subpopulations sharing a high level of diversity except at particular genomic regions, the largest being a region of chromosome 12, which showed evidence of recent directional selection. Surprisingly, we observed a third subpopulation comprising P. knowlesi infections that were almost identical to each other throughout much of the genome, indicating separately maintained transmission and recent genetic isolation. Each subpopulation could evolve and present a broader health challenge in Asia.
  8. Sutton PL, Luo Z, Divis PCS, Friedrich VK, Conway DJ, Singh B, et al.
    Infect Genet Evol, 2016 06;40:243-252.
    PMID: 26980604 DOI: 10.1016/j.meegid.2016.03.009
    Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes.
  9. Yunos NE, Sharkawi HM, Hii KC, Hu TH, Mohamad DSA, Rosli N, et al.
    Sci Rep, 2022 Oct 14;12(1):17284.
    PMID: 36241678 DOI: 10.1038/s41598-022-21439-2
    Plasmodium knowlesi infections in Malaysia are a new threat to public health and to the national efforts on malaria elimination. In the Kapit division of Sarawak, Malaysian Borneo, two divergent P. knowlesi subpopulations (termed Cluster 1 and Cluster 2) infect humans and are associated with long-tailed macaque and pig-tailed macaque hosts, respectively. It has been suggested that forest-associated activities and environmental modifications trigger the increasing number of knowlesi malaria cases. Since there is a steady increase of P. knowlesi infections over the past decades in Sarawak, particularly in the Kapit division, we aimed to identify hotspots of knowlesi malaria cases and their association with forest activities at a geographical scale using the Geographic Information System (GIS) tool. A total of 1064 P. knowlesi infections from 2014 to 2019 in the Kapit and Song districts of the Kapit division were studied. Overall demographic data showed that males and those aged between 18 and 64 years old were the most frequently infected (64%), and 35% of infections involved farming activities. Thirty-nine percent of Cluster 1 infections were mainly related to farming surrounding residential areas while 40% of Cluster 2 infections were associated with activities in the deep forest. Average Nearest Neighbour (ANN) analysis showed that humans infected with both P. knowlesi subpopulations exhibited a clustering distribution pattern of infection. The Kernel Density Analysis (KDA) indicated that the hotspot of infections surrounding Kapit and Song towns were classified as high-risk areas for zoonotic malaria transmission. This study provides useful information for staff of the Sarawak State Vector-Borne Disease Control Programme in their efforts to control and prevent zoonotic malaria.
  10. Divis PCS, Hu TH, Kadir KA, Mohammad DSA, Hii KC, Daneshvar C, et al.
    Emerg Infect Dis, 2020 07;26(7):1392-1398.
    PMID: 32568035 DOI: 10.3201/eid2607.190924
    Population genetic analysis revealed that Plasmodium knowlesi infections in Malaysian Borneo are caused by 2 divergent parasites associated with long-tailed (cluster 1) and pig-tailed (cluster 2) macaques. Because the transmission ecology is likely to differ for each macaque species, we developed a simple genotyping PCR to efficiently distinguish between and survey the 2 parasite subpopulations. This assay confirmed differences in the relative proportions in areas of Kapit division of Sarawak state, consistent with multilocus microsatellite analyses. Analyses of 1,204 human infections at Kapit Hospital showed that cluster 1 caused approximately two thirds of cases with no significant temporal changes from 2000 to 2018. We observed an apparent increase in overall numbers in the most recent 2 years studied, driven mainly by increased cluster 1 parasite infections. Continued monitoring of the frequency of different parasite subpopulations and correlation with environmental alterations are necessary to determine whether the epidemiology will change substantially.
  11. Munajat MB, Rahim MAFA, Wahid W, Seri Rakna MIM, Divis PCS, Chuangchaiya S, et al.
    Malar J, 2021 Apr 27;20(1):202.
    PMID: 33906645 DOI: 10.1186/s12936-021-03741-y
    BACKGROUND: Malaysia is on track towards malaria elimination. However, several cases of malaria still occur in the country. Contributing factors and communal aspects have noteworthy effects on any malaria elimination activities. Thus, assessing the community's knowledge, attitudes and practices (KAP) towards malaria is essential. This study was performed to evaluate KAP regarding malaria among the indigenous people (i.e. Orang Asli) in Peninsular Malaysia.

    METHODS: A household-based cross-sectional study was conducted in five remote villages (clusters) of Orang Asli located in the State of Kelantan, a central region of the country. Community members aged six years and above were interviewed. Demographic, socio-economic and KAP data on malaria were collected using a structured questionnaire and analysed using descriptive statistics.

    RESULTS: Overall, 536 individuals from 208 households were interviewed. Household indoor residual spraying (IRS) coverage and bed net ownership were 100% and 89.2%, respectively. A majority of respondents used mosquito bed nets every night (95.1%), but only 50.2% were aware that bed nets were used to prevent malaria. Nevertheless, almost all of the respondents (97.9%) were aware that malaria is transmitted by mosquitoes. Regarding practice for managing malaria, the most common practice adopted by the respondents was seeking treatment at the health facilities (70.9%), followed by self-purchase of medication from a local shop (12.7%), seeking treatment from a traditional healer (10.5%) and self-healing (5.9%). Concerning potential zoonotic malaria, about half of the respondents (47.2%) reported seeing monkeys from their houses and 20.1% reported entering nearby forests within the last 6 months.

    CONCLUSION: This study found that most populations living in the villages have an acceptable level of knowledge and awareness about malaria. However, positive attitudes and practices concerning managing malaria require marked improvement.

  12. Nainggolan IRA, Syafutri RD, Sinambela MN, Devina C, Handayani, Hasibuan BS, et al.
    Malar J, 2022 Nov 05;21(1):316.
    PMID: 36333701 DOI: 10.1186/s12936-022-04335-y
    BACKGROUND: Indonesia is progressing towards malaria elimination. To achieve this goal, intervention measures must be addressed to cover all Plasmodium species. Comprehensive control measures and surveillance programmes must be intensified. This study aims to determine the prevalence of microscopic and submicroscopic malaria in Langkat district, North Sumatera Province, Indonesia.

    METHODS: A cross-sectional survey was conducted in six villages in Langkat district, North Sumatera Province in June 2019. Data were recorded using a standardized questionnaire. Finger pricked blood samples were obtained for malaria examination using rapid diagnostic test, thick and thin blood smears, and polymerase chain reaction.

    RESULTS: A total of 342 individuals were included in the study. Of them, one (0.3%) had a microscopic Plasmodium malariae infection, no positive RDT examination, and three (0.9%) were positive for P. malariae (n = 1) and Plasmodium knowlesi (n = 2). The distribution of bed net ownership was owned by 40% of the study participants. The participants had a house within a radius of 100-500 m from the forest (86.3%) and had the housing material of cement floor (56.1%), a tin roof (82.2%), wooden wall (35.7%), bamboo wall (28.1%), and brick wall (21.6%).

    CONCLUSION: Malaria incidence has substantially decreased in Langkat, North Sumatera, Indonesia. However, submicroscopic infection remains in the population and may contribute to further transmission. Surveillance should include the detection of microscopic undetected parasites, to enable the achievement of malaria elimination.

  13. Sugiarto SR, Natalia D, Mohamad DSA, Rosli N, Davis WA, Baird JK, et al.
    Sci Rep, 2022 Nov 03;12(1):18546.
    PMID: 36329096 DOI: 10.1038/s41598-022-21570-0
    The simian parasite Plasmodium knowlesi is the predominant species causing human malaria infection, including hospitalisations for severe disease and death, in Malaysian Borneo. By contrast, there have been only a few case reports of knowlesi malaria from Indonesian Borneo. This situation seems paradoxical since both regions share the same natural macaque hosts and Anopheles mosquito vectors, and therefore have a similar epidemiologically estimated risk of infection. To determine whether there is a true cross-border disparity in P. knowlesi prevalence, we conducted a community-based malaria screening study using PCR in Kapuas Hulu District, West Kalimantan. Blood samples were taken between April and September 2019 from 1000 people aged 6 months to 85 years attending health care facilities at 27 study sites within or close to jungle areas. There were 16 Plasmodium positive samples by PCR, five human malarias (two Plasmodium vivax, two Plasmodium ovale and one Plasmodium malariae) and 11 in which no species could be definitively identified. These data suggest that, if present, simian malarias including P. knowlesi are rare in the Kapuas Hulu District of West Kalimantan, Indonesian Borneo compared to geographically adjacent areas of Malaysian Borneo. The reason for this discrepancy, if confirmed in other epidemiologically similar regions of Indonesian Borneo, warrants further studies targeting possible cross-border differences in human activities in forested areas, together with more detailed surveys to complement the limited data relating to monkey hosts and Anopheles mosquito vectors in Indonesian Borneo.
  14. Hu TH, Rosli N, Mohamad DSA, Kadir KA, Ching ZH, Chai YH, et al.
    Sci Rep, 2021 10 11;11(1):20117.
    PMID: 34635723 DOI: 10.1038/s41598-021-99644-8
    Plasmodium knowlesi, a simian malaria parasite responsible for all recent indigenous cases of malaria in Malaysia, infects humans throughout Southeast Asia. There are two genetically distinct subpopulations of Plasmodium knowlesi in Malaysian Borneo, one associated with long-tailed macaques (termed cluster 1) and the other with pig-tailed macaques (cluster 2). A prospective study was conducted to determine whether there were any between-subpopulation differences in clinical and laboratory features, as well as in epidemiological characteristics. Over 2 years, 420 adults admitted to Kapit Hospital, Malaysian Borneo with knowlesi malaria were studied. Infections with each subpopulation resulted in mostly uncomplicated malaria. Severe disease was observed in 35/298 (11.7%) of single cluster 1 and 8/115 (7.0%) of single cluster 2 infections (p = 0.208). There was no clinically significant difference in outcome between the two subpopulations. Cluster 1 infections were more likely to be associated with peri-domestic activities while cluster 2 were associated with interior forest activities consistent with the preferred habitats of the respective macaque hosts. Infections with both P. knowlesi subpopulations cause a wide spectrum of disease including potentially life-threatening complications, with no implications for differential patient management.
  15. Dian ND, Muhammad AB, Azman EN, Eddie NA, Azmi NI, Yee VCT, et al.
    Am J Trop Med Hyg, 2023 Nov 01;109(5):1081-1085.
    PMID: 37748768 DOI: 10.4269/ajtmh.23-0184
    Malaysia has maintained zero cases of indigenous human malaria since 2018. However, zoonotic malaria is still prevalent in underdeveloped areas and hard-to-reach populations. This study aimed to determine the prevalence of malaria among remote indigenous communities in Peninsular Malaysia. A cross-sectional survey was conducted in six settlements in Kelantan state, from June to October 2019. Blood samples were tested for malaria using microscopy and nested polymerase chain reaction (nPCR) targeting the Plasmodium cytochrome c oxidase subunit III (cox3) gene. Of the 1,954 individuals who appeared healthy, no malaria parasites were found using microscopy. However, nPCR revealed seven cases of Plasmodium knowlesi mono-infection (0.4%), and six out of seven infections were in the group of 19 to 40 years old (P = 0.026). No human malaria species were detected by nPCR. Analysis of the DNA sequences also showed high similarity that reflects common ancestry to other P. knowlesi isolates. These findings indicate low submicroscopic P. knowlesi infections among indigenous communities in Malaysia, requiring PCR-based surveillance to support malaria control activities in the country.
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