MyMedR

Molecular Evidence of Sarcocystis Species Infecting Reptiles in Peninsular Malaysia

Mohd Fadil NF, Tengku-Idris TIN, Shahari S, Fong MY, Lau YL

Iran J Parasitol, 2020 2 27;14(4):623-630.

PMID: 32099565

Background: The genus Sarcocystis consists of intracellular coccidian protozoan parasites with the ability to invade muscle tissue and mature into sarcocysts, causing the zoonotic disease sarcocystosis. These parasites have an obligatory two-host life cycle, which correlates with prey-predator relationship. The distribution and prevalence of Sarcocystis in reptiles remains unclear, despite several previous reports. The aim of this study was to identify the genetic assemblage of the species of Sarcocystis infecting Malaysian snakes and lizards by screening stool samples.

Methods: Overall, 54 fecal samples of various snake species and four fecal samples of several lizard species in Malaysia were taken within the course of August 2015 to January 2016 from Seremban, Melaka, Tioman Island, Pahang, Klang and Langkawi Wildlife Park located in Malaysia. The samples were examined for Sarcocystis through PCR amplification of the 18S rDNA sequence at the Department of Parasitology, University of Malaya.

Results: Fourteen snake fecal samples were positive via PCR; however, only eight samples (14%) were found positive for Sarcocystis species, whereas four were positive for other genera and the identity of another three samples was unable to be determined. Further phylogenetic analysis of the 18S rDNA sequences revealed that the snakes were infected with either S. singaporensis, S. lacertae, or undefined Sarcocystis species closely related to either S. singaporensis or S. zuoi. Sarcocystis nesbitti infection was not identified in any of the infected snakes.

Conclusion: This is the first report of identification of S. lacertae in the black-headed cat snake.

Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization

Jessie K, Fong MY, Devi S, Lam SK, Wong KT

J Infect Dis, 2004 Apr 15;189(8):1411-8.

PMID: 15073678

Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.

Short communication: Genetic variants of Sarcocystis cruzi in infected Malaysian cattle based on 18S rDNA

Ng YH, Fong MY, Subramaniam V, Shahari S, Lau YL

Res Vet Sci, 2015 Dec;103:201-4.

PMID: 26679818 DOI: 10.1016/j.rvsc.2015.10.009

Sarcocystis species are pathogenic parasites that infect a wide range of animals, including cattle. A high prevalence of cattle sarcocystosis has been reported worldwide, but its status is unknown in Malaysia. This study focused on utilizing 18S rDNA to identify Sarcocystis species in Malaysian cattle and to determine their genetic variants. In this study, only Sarcocystis cruzi was detected in Malaysian cattle. The intra-species S. cruzi phylogenetic tree analysis and principal coordinate analysis (PCoA), respectively displayed two minor groups among the parasite isolates. This finding was supported by high Wright FST value (FST=0.647). The definitive hosts (dogs) may play a fundamental role in the development of S. cruzi genetic variants. Additionally, the existence of microheterogeneity within the S. cruzi merozoites and/or distinct genetic variants arisen from independent merozoites in mature sarcocysts, possibly contributed to the existence of intra-species variations within the population.

Experimental Study on Plasmodium knowlesi Normocyte Binding Protein Xa Region II (PkNBPXaII) for Erythrocyte Binding

Wong KC, Lai MY, De Silva JR, Cheong FW, Fong MY, Lau YL

Trop Biomed, 2021 Jun 01;38(2):143-148.

PMID: 34172703 DOI: 10.47665/tb.38.2.051

Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.

Autoantibody profile of patients infected with knowlesi malaria

Liew J, Amir A, Chen Y, Fong MY, Razali R, Lau YL

Clin Chim Acta, 2015 Aug 25;448:33-8.

PMID: 26086445 DOI: 10.1016/j.cca.2015.06.006

Autoantibodies or antibodies against self-antigens are produced either during physiological processes to maintain homeostasis or pathological process such as trauma and infection. Infection with parasites including Plasmodium has been shown to generally induce elevated self-antibody (autoantibody) levels. Plasmodium knowlesi is increasingly recognized as one of the most important emerging human malaria in Southeast Asia that can cause severe infection leading to mortality. Autoimmune-like phenomena have been hypothesized to play a role in the protective immune responses in malaria infection.

Fulltext Perspectives of vector management in the control and elimination of vector-borne zoonoses

Wong ML, Zulzahrin Z, Vythilingam I, Lau YL, Sam IC, Fong MY, et al.

Front Microbiol, 2023;14:1135977.

PMID: 37025644 DOI: 10.3389/fmicb.2023.1135977

The complex transmission profiles of vector-borne zoonoses (VZB) and vector-borne infections with animal reservoirs (VBIAR) complicate efforts to break the transmission circuit of these infections. To control and eliminate VZB and VBIAR, insecticide application may not be conducted easily in all circumstances, particularly for infections with sylvatic transmission cycle. As a result, alternative approaches have been considered in the vector management against these infections. In this review, we highlighted differences among the environmental, chemical, and biological control approaches in vector management, from the perspectives of VZB and VBIAR. Concerns and knowledge gaps pertaining to the available control approaches were discussed to better understand the prospects of integrating these vector control approaches to synergistically break the transmission of VZB and VBIAR in humans, in line with the integrated vector management (IVM) developed by the World Health Organization (WHO) since 2004.

Fulltext Human malaria in the highlands of Yemen

Al-Mekhlafi AM, Al-Mekhlafi HM, Mahdy MA, Azazy AA, Fong MY

Ann Trop Med Parasitol, 2011 Apr;105(3):187-95.

PMID: 21801497 DOI: 10.1179/136485911X12987676649421

Between June 2008 and March 2009, a cross-sectional study of human malaria was carried out in four governorates of Yemen, two (Taiz and Hodiedah) representing the country's highlands and the others (Dhamar and Raymah) the country's coastal plains/foothills. The main aims were to determine the prevalences of Plasmodium infection among 455 febrile patients presenting for care at participating health facilities and to investigate the potential risk factors for such infection. Malarial infection was detected in 78 (17·1%) of the investigated patients and was more likely to be detected among the febrile patients from the highlands than among those presenting in the coastal plains/foothills (22·6% v.13·9%; χ(2)=10·102; P=0·018). Binary logistic-regression models identified low household income [odds ratio (OR)=13·52; 95% confidence interval (CI)=2·62-69·67; P=0·002], living in a household with access to a water pump (OR=4·18; CI=1·60-10·96; P=0·004) and living in a household near a stream (OR=4·43; CI=1·35-14·56; P=0·014) as significant risk factors for malarial infection in the highlands. Low household income was the only significant risk factor identified for such infection in the coastal plains and foothills (OR = 8·20; CI=1·80-37·45; P=0·007). It is unclear why febrile patients in the highlands of Yemen are much more likely to be found to have malarial infection than their counterparts from the coastal plains and foothills. Although it is possible that malarial transmission is relatively intense in the highlands, it seems more likely that, compared with those who live at lower altitudes, those who live in the highlands are less immune to malaria, and therefore more likely to develop febrile illness following malarial infection. Whatever the cause of the symptomatic malarial infection commonly found in the highlands of Yemen, it is a matter of serious concern that should be addressed in the national strategy to control malaria.

Recombinant expression of Toxocara canis excretory-secretory antigen TES-120 in Escherichia coli

Fong MY, Lau YL, Init I, Jamaiah I, Anuar AK, Rahmah N

Southeast Asian J Trop Med Public Health, 2003 Dec;34(4):723-6.

PMID: 15115078

The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

Restriction enzyme digestion analysis of PCR-amplified DNA of Blastocystis hominis isolates

Init I, Foead AL, Fong MY, Yamazaki H, Rohela M, Yong HS, et al.

Southeast Asian J Trop Med Public Health, 2007 Nov;38(6):991-7.

PMID: 18613539

Genomic DNA of Blastocystis isolates released into 0.1% Triton X-100 was suitable for amplification and yielded similar results as the genomic DNA extracted with standard kit. The specific B. hominis primers (BH1: GCT TAT CTG GTT GAT CCT GCC AGT and BH2: TGA TCC TTC CGC AGG TTC ACC TAC A) successfully produced the PCR product of about 1,770 bp with all the 7 Blastocystis isolates tested. The restriction fragment length polymorphism (RFLP) patterns yielded by 13 out of 25 restriction endonucleases showed that the 7 isolates could be grouped into 4 subgroups: subgroup-1 consisted of isolate C; subgroup-2 of isolates H4 and H7; subgroup-3 of isolates KP1, Y51 and M12; and subgroup-4 of isolate 27805. The differences between subgroups manifested as clear-cut RFLP patterns. A common band of 230 bp was revealed by Eco R1 in all the Blastocystis isolates tested. The band of about 180 bp was revealed by Alu I, differentiated symptomatic from asymptomatic isolates of this parasite, and might indicate the pathogenicity of this parasite.

Fulltext Plasmodium knowlesi reinfection in human

Lau YL, Tan LH, Chin LC, Fong MY, Noraishah MA, Rohela M

Emerg Infect Dis, 2011 Jul;17(7):1314-5.

PMID: 21762601 DOI: 10.3201/eid1707.101295

Fulltext Comparison of three molecular methods for the detection and speciation of five human Plasmodium species

Lau YL, Lai MY, Anthony CN, Chang PY, Palaeya V, Fong MY, et al.

Am J Trop Med Hyg, 2015 Jan;92(1):28-33.

PMID: 25385862 DOI: 10.4269/ajtmh.14-0309

In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar's test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar's test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar's test: P = 0.0012).

Fulltext Sarcocystis nesbitti infection in human skeletal muscle: possible transmission from snakes

Lau YL, Chang PY, Tan CT, Fong MY, Mahmud R, Wong KT

Am J Trop Med Hyg, 2014 Feb;90(2):361-4.

PMID: 24420776 DOI: 10.4269/ajtmh.12-0678

Sarcocystis nesbitti is an intracellular protozoan parasite found as sarcocysts within muscle fibers of intermediate hosts (monkey and baboon). The definitive host is suspected to be the snake. We report two cases from a larger cohort of 89 patients who had fever, headache, and generalized myalgia after a trip to Pangkor Island, Malaysia. Sarcocysts were detected in skeletal muscle biopsy specimens by light and electron microscopy from these two patients. DNA sequencing based on the 18S ribosomal DNA region identified the Sarcocystis species as S. nesbitti. We also identified S. nesbitti sequences in the stools of a snake (Naja naja). Phylogenetic analysis showed that these sequences form a cluster with most of the other known Sarcocystis species for which the snake is a definitive host. We believe these two patients were likely to have symptomatic acute muscular sarcocystosis after S. nesbitti infection that may have originated from snakes.

Fulltext Phylogenetic study of six species of Anopheles mosquitoes in Peninsular Malaysia based on inter-transcribed spacer region 2 (ITS2) of ribosomal DNA

Sum JS, Lee WC, Amir A, Braima KA, Jeffery J, Abdul-Aziz NM, et al.

Parasit Vectors, 2014;7:309.

PMID: 24993022 DOI: 10.1186/1756-3305-7-309

Molecular techniques are invaluable for investigation on the biodiversity of Anopheles mosquitoes. This study aimed at investigating the spatial-genetic variations among Anopheles mosquitoes from different areas of Peninsular Malaysia, as well as deciphering evolutionary relationships of the local Anopheles mosquitoes with the mosquitoes from neighbouring countries using the anopheline ITS2 rDNA gene.

Fulltext High proportion of knowlesi malaria in recent malaria cases in Malaysia

Yusof R, Lau YL, Mahmud R, Fong MY, Jelip J, Ngian HU, et al.

Malar J, 2014;13:168.

PMID: 24886266 DOI: 10.1186/1475-2875-13-168

Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.

Fulltext Malaysian child infected with Plasmodium vivax via blood transfusion: a case report

Anthony CN, Lau YL, Sum JS, Fong MY, Ariffin H, Zaw WL, et al.

Malar J, 2013;12:308.

PMID: 24007496 DOI: 10.1186/1475-2875-12-308

Malaria may be a serious complication of blood transfusion due to the asymptomatic persistence of parasites in some donors. This case report highlights the transfusion-transmitted malaria of Plasmodium vivax in a child diagnosed with germ cell tumour. This child had received blood transfusion from three donors and a week later started developing malaria like symptoms. Nested PCR and sequencing confirmed that one of the three donors was infected with P. vivax and this was transmitted to the 12-year-old child. To the best of the authors' knowledge, this is the first reported transfusion-transmitted malaria case in Malaysia.

Fulltext The suitability of P. falciparum merozoite surface proteins 1 and 2 as genetic markers for in vivo drug trials in Yemen

Al-abd NM, Mahdy MA, Al-Mekhlafi AM, Snounou G, Abdul-Majid NB, Al-Mekhlafi HM, et al.

PLoS One, 2013;8(7):e67853.

PMID: 23861823 DOI: 10.1371/journal.pone.0067853

The accuracy of the conclusions from in vivo efficacy anti-malarial drug trials depends on distinguishing between recrudescences and re-infections which is accomplished by genotyping genes coding P. falciparum merozoite surface 1 (MSP1) and MSP2. However, the reliability of the PCR analysis depends on the genetic markers' allelic diversity and variant frequency. In this study the genetic diversity of the genes coding for MSP1 and MSP2 was obtained for P. falciparum parasites circulating in Yemen.

Fulltext Hyperparasitaemic human Plasmodium knowlesi infection with atypical morphology in peninsular Malaysia

Lee WC, Chin PW, Lau YL, Chin LC, Fong MY, Yap CJ, et al.

Malar J, 2013;12:88.

PMID: 23496970 DOI: 10.1186/1475-2875-12-88

Plasmodium knowlesi is a potentially life-threatening zoonotic malaria parasite due to its relatively short erythrocytic cycle. Microscopic identification of P. knowlesi is difficult, with "compacted parasite cytoplasm" being one of the important identifying keys. This report is about a case of hyperparasitaemic human P. knowlesi infection (27% parasitaemia) with atypical amoeboid morphology. A peninsular Malaysian was admitted to the hospital with malaria. He suffered anaemia and acute kidney function impairment. Microscopic examination, assisted by nested PCR and sequencing confirmed as P. knowlesi infection. With anti-malarial treatment and several medical interventions, patient survived and recovered. One-month medical follow-up was performed after recovery and no recrudescence was noted. This case report highlights the extreme hyperparasitaemic setting, the atypical morphology of P. knowlesi in the patient's erythrocytes, as well as the medical interventions involved in this successfully treated case.

Fulltext Invasion characteristics of a Plasmodium knowlesi line newly isolated from a human

Amir A, Russell B, Liew JW, Moon RW, Fong MY, Vythilingam I, et al.

Sci Rep, 2016 Apr 21;6:24623.

PMID: 27097521 DOI: 10.1038/srep24623

Plasmodium knowlesi is extensively used as an important malaria model and is now recognized as an important cause of human malaria in Malaysia. The strains of P. knowlesi currently used for research were isolated many decades ago, raising concerns that they might no longer be representative of contemporary parasite populations. We derived a new P. knowlesi line (University Malaya line, UM01), from a patient admitted in Kuala Lumpur, Malaysia, and compared it with a human-adapted laboratory line (A1-H.1) derived from the P. knowlesi H strain. The UM01 and A1-H.1 lines readily invade human and macaque (Macaca fascicularis) normocytes with a preference for reticulocytes. Whereas invasion of human red blood cells was dependent on the presence of the Duffy antigen/receptor for chemokines (DARC) for both parasite lines, this was not the case for macaque red blood cells. Nonetheless, differences in invasion efficiency, gametocyte production and the length of the asexual cycle were noted between the two lines. It would be judicious to isolate and characterise numerous P. knowlesi lines for use in future experimental investigations of this zoonotic species.

Fulltext Field evaluation of a PfHRP-2/pLDH rapid diagnostic test and light microscopy for diagnosis and screening of falciparum malaria during the peak seasonal transmission in an endemic area in Yemen

Alareqi LM, Mahdy MA, Lau YL, Fong MY, Abdul-Ghani R, Ali AA, et al.

Malar J, 2016 Jan 28;15:49.

PMID: 26821911 DOI: 10.1186/s12936-016-1103-2

Malaria is a public health threat in Yemen, with 149,451 cases being reported in 2013. Of these, Plasmodium falciparum represents 99%. Prompt diagnosis by light microscopy (LM) and rapid diagnostic tests (RTDs) is a key element in the national strategy of malaria control. The heterogeneous epidemiology of malaria in the country necessitates the field evaluation of the current diagnostic strategies, especially RDTs. Thus, the present study aimed to evaluate LM and an RDT, combining both P. falciparum histidine-rich protein-2 (PfHRP-2) and Plasmodium lactate dehydrogenase (pLDH), for falciparum malaria diagnosis and survey in a malaria-endemic area during the transmission season against nested polymerase chain reaction (PCR) as the reference method.

Multicentre evaluation of dengue IgM dot enzyme immunoassay

Lam SK, Fong MY, Chungue E, Doraisingham S, Igarashi A, Khin MA, et al.

Clin Diagn Virol, 1996 Nov;7(2):93-8.

PMID: 9137865 DOI: 10.1016/S0928-0197(96)00257-7

The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings.

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