Displaying publications 1 - 20 of 30 in total

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  1. Abdelrahman MZ, Zeehaida M, Rahmah N, Norsyahida A, Madihah B, Azlan H, et al.
    Parasitol Int, 2012 Sep;61(3):508-11.
    PMID: 22575692 DOI: 10.1016/j.parint.2012.04.005
    Strongyloides stercoralis infection can persist in the host for several decades, and patients with cancer and other clinical conditions who are exposed to immunosuppressive therapy are at risk of developing hyperinfection.
  2. Uni S, Bain O, Suzuki K, Agatsuma T, Harada M, Motokawa M, et al.
    Parasitol Int, 2013 Feb;62(1):14-23.
    PMID: 22926421 DOI: 10.1016/j.parint.2012.08.004
    Acanthocheilonema delicata n. sp. (Filarioidea: Onchocercidae: Onchocercinae) is described based on adult filarioids and microfilariae obtained from subcutaneous connective tissues and skin, respectively, of Japanese badgers (Meles anakuma) in Wakayama Prefecture, Japan. No endemic species of the genus had been found in Japan. Recently, some filarioids (e.g., Acanthocheilonema reconditum, Dirofilaria spp., and Onchocerca spp.) have come to light as causative agents of zoonosis worldwide. The new species was readily distinguished from its congeners by morphologic characteristics such as body length, body width, esophagus length, spicule length, and the length of microfilariae. Based on the molecular data of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, A. delicata n. sp. was included in the clade of the genus Acanthocheilonema but differed from two other congeneric species available for study, A. viteae and A. reconditum. Acanthocheilonema delicata n. sp. did not harbor Wolbachia. It is likely that the fauna of filarioids from mammals on the Japanese islands is characterized by a high level of endemicity.
  3. Muhamad N, Walker LR, Pedley KC, Simcock DC, Brown S
    Parasitol Int, 2012 Sep;61(3):487-92.
    PMID: 22562002 DOI: 10.1016/j.parint.2012.04.003
    The initial rate of NH(3)/NH(4)(+) accumulation in a medium containing L(3) Teladorsagia circumcincta was 0.18-0.6 pmol h(-1) larva(-1), which increased linearly with larval density. However it appeared that the larva-generated external concentration of NH(3)/NH(4)(+) did not exceed about 130 μM. The rate of NH(3)/NH(4)(+) accumulation increased with temperature between 4 °C and 37 °C, declined with increasing pH or increasing external NH(3)/NH(4)(+) concentration and was not significantly affected by the concentration of the phosphate buffer or by exsheathing the larvae. We infer from these data that the efflux of NH(3)/NH(4)(+) is a diffusive process and that the secreted or excreted NH(3)/NH(4)(+) is generated enzymatically rather than dissociating from the surface of the nematode. The enzymatic source of the NH(3)/NH(4)(+) is yet to be identified. Since the concentration of NH(3)/NH(4)(+) in the rumen and abomasum is higher than 130 μM, it is unlikely that T. circumcincta contributes to it, but NH(3)/NH(4)(+) may be accumulated from the rumen fluid by the nematode.
  4. Tan LH, Fong MY, Mahmud R, Muslim A, Lau YL, Kamarulzaman A
    Parasitol Int, 2011 Jan;60(1):111-3.
    PMID: 20951228 DOI: 10.1016/j.parint.2010.09.010
    Five local Malaysian patients with clinical manifestations consistent with lymphatic filariasis were referred to our medical centre between 2003 and 2006. Although no microfilariae (mf) were detected in their nocturnal blood samples, all were diagnosed to have lymphatic filariasis on the basis of clinical findings and positive serology results. PCR on their blood samples revealed that two of the patients were infected with Brugia pahangi, an animal filarial worm hitherto not known to cause human disease in the natural environment. All the patients were successfully treated with anti-filarial drugs: four patients were treated with a combination of diethylcarbamazine (DEC) and albendazole, and one with doxycycline. Four of them were residents of Petaling Jaya, a residential suburbia located 10 km southwest of Kuala Lumpur city, Malaysia. The fifth patient was a frequent visitor of the suburbia. This suburbia has no history or record of B. malayi infection. The most likely vector of the worm was Armigeres subalbatus as extensive entomological surveys within the suburbia revealed only adult females of this mosquito species were infected with B. pahangi larvae. Wild monkeys caught in the suburbia were free from B. pahangi mf, but domestic cats were mf positive. This suggests that infected cats might be the source of the zoonotic infection in the suburbia.
  5. Khadri MS, Depaquit J, Bargues MD, Ferté H, Mas-coma S, Lee HL, et al.
    Parasitol Int, 2008 Sep;57(3):295-9.
    PMID: 18378490 DOI: 10.1016/j.parint.2008.01.003
    The male of Phlebotomus (Larroussius) betisi is described from Malayan caves. Several males have been caught in association with P. betisi females. Males and females have been associated by ecology, biogeography, morphology and molecular biology (homology of the ND4 mtDNA sequences).
  6. Waikagul J
    Parasitol Int, 2006;55 Suppl:S297-300.
    PMID: 16326132
    The SEAMEO TROPMED Network is a regional cooperative network established in 1967 for education, training and research in tropical medicine and public health under the Southeast Asia Ministers of Education Organization. The Network operates through four Regional Centers with respective areas of specialization and host institutions: Community Nutrition/Tropmed Indonesia; Microbiology, Parasitology and Entomology/Tropmed Malaysia; Public Health/Tropmed Philippines; and Tropical Medicine/Tropmed Thailand. To train health workers, to support research on endemic and newly emerging diseases, and to advocate relevant health policies are the main functions of these centers. SEAMEO TROPMED Network in collaboration with the Faculty of Tropical Medicine, Mahidol University and other institutions has regularly organized the Seminar on Food-borne Parasitic Zoonoses every 3-5 years over the past 15 years. The Faculty of Tropical Medicine has organized the annual Joint International Tropical Medicine Meeting since 1996. Full papers of the presentations at these two meetings have been published as supplementary issues to the Southeast Asian Journal of Tropical Medicine and Public Health, an in-house journal of SEAMEO TROPMED Network. Recently, the Parasitology Association of ASEAN Countries has rotated the hosting of the ASEAN Congress of Parasitology and Tropical Medicine. These institutional and conference networks will enable closer links, to promote the health of people in the Southeast Asian region.
  7. Boo YL, Lim HT, Chin PW, Lim SY, Hoo FK
    Parasitol Int, 2016 Feb;65(1):55-57.
    PMID: 26454133 DOI: 10.1016/j.parint.2015.10.003
    Plasmodium knowlesi, a zoonotic malaria, is now considered the fifth species of Plasmodium causing malaria in humans. With its 24-hour erythrocytic stage of development, it has raised concern regarding its high potential in replicating and leading to severe illness. Spleen is an important site for removal of parasitized red blood cells and generating immunity. We reported a case of knowlesi malaria in a non-immune, splenectomized patient. We observed the delay in parasite clearance, high parasitic counts, and severe illness at presentation. A thorough search through literature revealed several case reports on falciparum and vivax malaria in splenectomized patients. However, literature available for knowlesi malaria in splenectomized patient is limited. Further studies need to be carried out to clarify the role of spleen in host defense against human malaria especially P. knowlesi.
  8. Jaganathan A, Murugan K, Panneerselvam C, Madhiyazhagan P, Dinesh D, Vadivalagan C, et al.
    Parasitol Int, 2016 Jun;65(3):276-84.
    PMID: 26873539 DOI: 10.1016/j.parint.2016.02.003
    The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW-AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW-AgNP showed plasmon resonance reduction in UV-vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW-AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW-AgNP were toxic to Anopheles stephensi larvae and pupae, LC(50) were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW-AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW-AgNP IC(50) were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC(50) were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW-AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW-AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW-AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW-AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.
  9. Uni S, Fukuda M, Agatsuma T, Bain O, Otsuka Y, Nakatani J, et al.
    Parasitol Int, 2015 Dec;64(6):493-502.
    PMID: 26165205 DOI: 10.1016/j.parint.2015.07.001
    Human zoonotic onchocercosis is caused by Onchocerca dewittei japonica, parasitic in wild boars (Sus scrofa leucomystax) in Japan. Previously, microfilariae longer than those of Onchocerca dewittei japonica were observed in skin snips from wild boars during the study of O. dewittei japonica. Moreover, the third-stage larvae (L3) of these longer microfilariae were obtained from the blackfly Simulium bidentatum after experimental injections. Based on morphometric and molecular studies, similar L3 were found in blackflies during fieldwork in Oita, Japan. However, except for O. dewittei japonica, adult worms of Onchocerca have not been found in wild boars. In this study, we discovered adult females of a novel Onchocerca species in the skin of a wild boar in Oita, and named it Onchocerca takaokai n. sp. Females of this new species had longer microfilariae and differed from O. dewittei japonica in terms of their morphological characteristics and parasitic location. The molecular characteristics of the cytochrome c oxidase subunit 1 and 12S rRNA genes of the new species were identical to those of the longer microfilariae and L3 previously detected, but they differed from those of O. dewittei japonica at the species level. However, both species indicated a close affinity among their congeners and Onchocerca ramachandrini, parasitic in the warthog in Africa, was basal in the Suidae cluster of the 12S rRNA tree.
  10. Fukuda M, Uni S, Otsuka Y, Eshita Y, Nakatani J, Ihara K, et al.
    Parasitol Int, 2015 Dec;64(6):519-21.
    PMID: 26209456 DOI: 10.1016/j.parint.2015.07.006
    A case of zoonotic onchocercosis has been found in a resident who lived in Iizuka City, Fukuoka Prefecture, Japan for some time. A 24-year-old male developed a painful nodule on the middle finger of his right hand. The nodule was surgically removed from the vagina fibrosa tendinis of the finger at Beppu Medical Center, Beppu City, Oita Prefecture in 2012. The causative agent was identified as a female Onchocerca dewittei japonica based on its histopathological characteristics. The identity of the filarioid has been confirmed by sequencing the cox1 gene. The present study indicates that the zoonotic onchocercosis caused by O. dewittei japonica has been concentrated in northeast Kyushu.
  11. Claveria FG, Cruz MJ
    Parasitol Int, 2000 Jan;48(3):243-7.
    PMID: 11227764
    Ultrastructural studies of sarcocysts obtained from Philippine water buffaloes revealed the presence of the commonly reported macroscopic species, Sarcocystis fusiformis, and the microscopic species Sarcocystis levinei (Dissanaike A, Kan S. Studies on Sarcocystis in Malaysia. I: Sarcocystis levinei n.sp. from the water buffalo Bubalus bubalis. Z Parasitenkd 1978;55:127-38), (Huong L, Dubey J, Uggla A. Redescription of Sarcocystis levinei Dissanaike and Kan, 1978 (Protozoa: Sarcocystidae) of the water buffalo (Bubalus bubalis). J Parasitol 1997;83:1148-52). The globular to oval microscopic cysts commonly observed in the muscles of the diaphragm and neck exhibit compartmentalized arrangement of zoites with septal partitions and measure 13-48 microns in diameter. The parasitophorous vacuolar membrane of sarcocyst bears minute and hair-like villar protrusions measuring 2.3-2.75 microns long emanating at certain distances from the primary cyst wall and lack microfilaments. Villar protrusions have expanded to dome-shaped base measuring 0.33-1.6 microns long by 0.22-1.0 micron wide, and intermediate and tapering distal segments bent approximately 90 degrees and run parallel to the cyst surface. The distal segments at some areas join to form conical tufts. The primary cyst wall bears numerous prominent undulations that are arranged in small clusters. The ground substance is 0.42-0.57 micron thick. This paper documents the first report of S. levinei in Philippine water buffaloes possessing the type 7 cyst wall.
  12. Tan TK, Low VL, Ng WH, Ibrahim J, Wang D, Tan CH, et al.
    Parasitol Int, 2019 Apr;69:110-113.
    PMID: 30590124 DOI: 10.1016/j.parint.2018.12.007
    This report describes the detection of zoonotic Cryptosporidium muris, C. parvum subgenotype IIa and Giardia duodenalis genotype B in urban rodents in Malaysia. A rare occurrence of C. meleagridis was also reported suggesting a role of rodents in mechanical transmission of this pathogen. Utilization of DNA sequencing and subtyping analysis confirmed the presence of zoonotic C. parvum subtypes IIaA17G2R1 and IIaA16G3R1 for the first time in rodents.
  13. Hata M, Sato S, Kita K
    Parasitol Int, 2019 Apr;69:99-102.
    PMID: 30543864 DOI: 10.1016/j.parint.2018.12.003
    The growth and the survival of the human malaria parasite Plasmodium falciparum are critically dependent on the functions of the two organelles - the mitochondrion and the apicoplast. However, these two organelles have been known to be difficult to separate from each other when they are released from Plasmodium cell. We have been searching for the conditions with which separation of the mitochondrion and the apicoplast is achieved. In this study, we investigated how the two organelle's separation is affected when the pressure of the nitrogen gas to disrupt the Plasmodium cells by nitrogen cavitation method is lowered from the pressure regularly applied (1200 psi). The parasite cell was sufficiently disrupted even when nitrogen cavitation was carried out at 300 psi. The obtained mitochondrial sample was much less contaminated by DNA compared with the sample prepared using the gas at the regular pressure. After the fractionation by Percoll density gradient, the mitochondrion and the apicoplast from the 300 psi cell lysate exhibited different separation profiles. This is the first experimental evidence that indicates the mitochondrion and the apicoplast of P. falciparum are separable from each other.
  14. Mathenge PG, Low SK, Vuong NL, Mohamed MYF, Faraj HA, Alieldin GI, et al.
    Parasitol Int, 2020 Feb;74:101919.
    PMID: 31015034 DOI: 10.1016/j.parint.2019.04.016
    BACKGROUND: Malaria parasites have developed resistance to most of the known antimalarial drugs in clinical practice, with reports of artemisinin resistance emerging in South East Asia (SEA). We sort to find the status of artemisinin resistance and efficacy of different modalities of the current artemisinin-based combination therapies (ACTs).

    METHODS: We carried out a systematic search in 11 electronic databases to identify in vivo studies published between 2001 and 2017 that reported artemisinin resistance. This was then followed by A network meta-analysis to compare the efficacy of different ACTs. Quality assessment was performed using the Cochrane Risk of Bias (ROB) tool for randomized controlled trials and National Institute of Health (NIH) tool for cross-sectional studies. The study protocol was registered in PROSPERO under number CRD42018087574.

    RESULTS: With 8400 studies initially identified, 82 were eligible for qualitative and quantitative analysis. Artemisinin resistance was only reported in South East Asia. K13 mutation C580Y was the most abundant mutation associated with resistance having an abundance of 63.1% among all K13 mutations reported. Although the overall network meta-analysis had shown good performance of dihydroartemisinin piperaquine in the early years, a subgroup analysis of the recent years revealed a poor performance of the drug in relation to recrudescence, clinical failure and parasitological failure especially in the artemisinin resistant regions.

    CONCLUSION: With report of high resistance and treatment failure against the leading artemisinin combination therapy in South East Asia, it is imperative that a new drug or a formulation is developed before further spread of resistance.

  15. Khoo JJ, Husin NA, Lim FS, Oslan SNH, Mohd Azami SNI, To SW, et al.
    Parasitol Int, 2021 Feb;80:102202.
    PMID: 33038482 DOI: 10.1016/j.parint.2020.102202
    Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans. Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing. Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested. The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.
  16. Hassan NA, Thodsapol A, Lim YAL, Wan Sulaiman WY, Sidi Omar SFN, Umsakul K, et al.
    Parasitol Int, 2021 Feb;80:102237.
    PMID: 33144195 DOI: 10.1016/j.parint.2020.102237
    Soil-transmitted helminth (STH) infections are the most common intestinal parasitic infections of medical importance in humans. The infections are widely distributed throughout tropical and subtropical countries, particularly among disadvantaged and underprivileged communities. In Malaysia, STH infections is highly prevalent, especially among the indigenous groups. However, species identification through molecular studies is still lacking. Using molecular techniques, this study was conducted to identify STH species that infect humans. Faecal samples were collected from three Iban tribal longhouses in the rural area of Sarikei Division, Sarawak. Positive faecal samples by microscopy were subjected to PCR and sequenced for species identification. Based on the microscopy examination, the most prevalent STH infection was Ascaris, followed by hookworm and Trichuris. All microscopy-positive samples were then PCR-amplified. The amplicons were sequenced, aligned, and compared with other sequences in the GenBank database. The results showed that Ascaris lumbricoides was the predominant STH species, followed by Trichuris trichiura, Ancylostoma duodenale, and Necator americanus. Our findings were contradictory to past studies, as we showed that A. duodenale was more abundant than N. americanus. Socioeconomic improvement and health education programs should be included in the management and prevention of public health strategies against STH.
  17. Soo OYM, Tan WB
    Parasitol Int, 2021 Apr;81:102282.
    PMID: 33444771 DOI: 10.1016/j.parint.2021.102282
    Hamatopeduncularia longiangusticirrata sp. nov. and H. petalumvaginata sp. nov. were collected from Arius maculatus and Nemapteryx caelata, respectively from Tanjung Karang, Peninsular Malaysia. Morphological and molecular investigations were carried out to ascertain the identity of the new species. The two new species differ from previously described Hamatopeduncularia species in the morphology of the male and female reproductive organs. Hamatopeduncularia longiangusticirrata sp. nov. possesses a long penis similar to H. elongata, H. longicopulatrix, H. brisbanensis, H. major and H. petalumvaginata sp. nov., but differs in having a thread-like tapering distal end and can be distinguished from H. brisbanensis and H. major in not having an accessory piece. Hamatopeduncularia longiangusticirrata sp. nov. is also unique in having an ornamented penis initial and a vaginal tube surrounded by fine hair-like structures. Hamatopeduncularia petalumvaginata sp. nov. possesses a simple penis without an accessory piece and a petaloid vaginal opening that resembles the arrangement of petals on a flower. Maximum likelihood trees were constructed from partial 28S and 18S rDNA sequences of the two new species and other ancylodiscoidids to reveal a strongly supported monophyletic branch consisting of the two new species for both markers. According to Lim's classification in 1996 of Hamatopeduncularia species penis type, H. petalumvaginata sp. nov. has been classified within the elegans-type and H. longiangusticirrata sp. nov. is proposed as the longiangusticirrata-type.
  18. Uni S, Fukuda M, Uga S, Agatsuma T, Nakatani J, Suzuki K, et al.
    Parasitol Int, 2021 Aug;83:102313.
    PMID: 33662527 DOI: 10.1016/j.parint.2021.102313
    Reports of zoonotic infections with Onchocerca japonica (Nematoda: Filarioidea), which parasitizes the Japanese wild boar, Sus scrofa leucomystax, have recently increased in Japan. To predict the occurrence of infection in humans, it is necessary to determine the prevalence of O. japonica infection in the natural host animals. We investigated the presence of adult worms in the footpads, and of microfilariae in skin snips, taken from the host animals, between 2000 and 2018. Onchocerca japonica was found in 165 of 223 (74%) Japanese wild boars in Honshu and Kyushu. Among the nine regions studied, the highest prevalence of O. japonica infection was found in Oita, Kyushu, where 47 of 52 (90.4%) animals were infected. The ears were the predilection sites for O. japonica microfilariae. Adult worms of O. japonica were found more frequently in the hindlimbs than in the forelimbs of the host animals. Onchocerca takaokai was found in 14 of 52 (26.9%) Japanese wild boars in Oita. In Kakeroma Island among the Nansei Islands, both O. japonica and O. takaokai were isolated from the Ryukyu wild boar, S. s. riukiuanus. These observations could help predict future occurrences of human zoonotic onchocercosis in Japan.
  19. Uni S, Fukuda M, Ogawa K, Lim YA, Agatsuma T, Bunchom N, et al.
    Parasitol Int, 2017 Oct;66(5):593-595.
    PMID: 28648713 DOI: 10.1016/j.parint.2017.06.006
    An 11-year-old boy living in Otsu City, Shiga Prefecture, Kansai Region, Western Honshu, Japan had zoonotic onchocercosis. The patient developed a painful swelling on the little finger of his left hand. The worm detected in the excised mass had external transverse ridges but did not have inner striae in the cuticle. On the basis of the parasite's histopathological characteristics, the causative agent was identified as a female Onchocerca dewittei japonica (Spirurida: Onchocercidae). The species of the filarial parasite was confirmed by sequencing the cox1 gene of the parasite. The Japanese wild boar Sus scrofa leucomystax is a definitive host for O. dewittei japonica, which is then transmitted by blackflies as the vector to humans. The current case described occurred in the Kansai Region, Western Honshu, where such infections were previously not reported.
  20. Ola-Fadunsin SD, Gimba FI, Abdullah DA, Sharma RSK, Abdullah FJF, Sani RA
    Parasitol Int, 2018 Dec;67(6):659-665.
    PMID: 29960083 DOI: 10.1016/j.parint.2018.06.013
    Bovine anaplasmosis is a major concern to cattle farming in most parts of the world. Anaplasmosis negatively impacts the profitability of cattle farming by reducing the production, reproduction, and draft ability of cattle. Here, we report results from a one-year cross sectional study to determine the epidemiology and the risk factors for Anaplasma marginale infection of cattle in Peninsular Malaysia. Examination of one thousand and forty five blood samples of apparently healthy cattle from forty-three farms in all the states of Peninsular Malaysia by polymerase chain reaction (PCR) assay revealed an overall prevalence of A. marginale infection of cattle of 72.6%, showing high endemicity of this heamoprotozoan among cattle in the country. Cattle breeds, production type, herd owner, herd size, management system, farm size, farm age, prophylactic treatment against blood parasites, presence of ticks, frequency of deticking, zones, closeness to forest, closeness to waste area, closeness to human settlement and closeness to body of water were the risk factors significantly associated (P 
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