MyMedR

Fulltext Specific, sensitive and rapid detection of human plasmodium knowlesi infection by loop-mediated isothermal amplification (LAMP) in blood samples

Lau YL, Fong MY, Mahmud R, Chang PY, Palaeya V, Cheong FW, et al.

Malar J, 2011;10:197.

PMID: 21774805 DOI: 10.1186/1475-2875-10-197

The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.

Fulltext Detection of Naegleria species in environmental samples from Peninsular Malaysia

Ithoi I, Ahmad AF, Nissapatorn V, Lau YL, Mahmud R, Mak JW

PLoS One, 2011;6(9):e24327.

PMID: 21915311 DOI: 10.1371/journal.pone.0024327

BACKGROUND: In Malaysia, researchers and medical practitioners are unfamiliar with Naegleria infections. Thus little is known about the existence of pathogenic Naegleria fowleri, and the resultant primary amoebic meningoencephalitis (PAM) is seldom included in the differential diagnosis of central nervous system infections. This study was conducted to detect the presence of Naegleria species in various environmental samples.

METHODS/FINDINGS: A total of 41 Naegleria-like isolates were isolated from water and dust samples. All these isolates were subjected to PCR using two primer sets designed from the ITS1-ITS2 regions. The N. fowleri species-specific primer set failed to produce the expected amplicon. The Naegleria genus-specific primers produced amplicons of 408 bp (35), 450 bp (2), 457 bp (2) or 381 bp (2) from all 41 isolates isolated from aquatic (33) and dust (8) samples. Analysis of the sequences from 10 representative isolates revealed that amplicons with fragments 408, 450 and 457 bp showed homology with non-pathogenic Naegleria species, and 381 bp showed homology with Vahlkampfia species. These results concurred with the morphological observation that all 39 isolates which exhibited flagella were Naegleria, while 2 isolates (AC7, JN034055 and AC8, JN034056) that did not exhibit flagella were Vahlkampfia species.

CONCLUSION: To date, pathogenic species of N. fowleri have not been isolated from Malaysia. All 39 isolates that produced amplicons (408, 450 and 457 bp) from the genus-specific primers were identified as being similar to nonpathogenic Naegleria. Amplicon 408 bp from 5 representative isolates showed 100% and 99.7% identity to Naegleria philippinensis isolate RJTM (AM167890) and is thus believed to be the most common species in our environment. Amplicons 450 bp and 457 bp were respectively believed to be from 2 new species of Naegleria, since representative isolates showed lower homology and had a longer base pair length when compared to the reference species in the Genbank, Naegleria schusteri (AJ566626) and Naegleria laresi (AJ566630), respectively.

Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris

Lau YL, Fong MY, Idris MM, Ching XT

Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):10-6.

PMID: 23082548

Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.

Fulltext Prevalence of intestinal and blood parasites among wild rats in Kuala Lumpur, Malaysia

Siti Shafiyyah CO, Jamaiah I, Rohela M, Lau YL, Siti Aminah F

Trop Biomed, 2012 Dec;29(4):544-50.

PMID: 23202599 MyJurnal

A survey was undertaken to investigate the prevalence of intestinal and blood parasites among wild rats in urban area of Kuala Lumpur, Malaysia. A total of 137 stool and blood samples were collected from wild rats from Sentul and Chow Kit areas. Five species of rats were captured and supplied by Kuala Lumpur City Hall. The most common was Rattus rattus diardii (Malayan Black rat), 67%, followed by Rattus norvegicus (Norway rat), 10%, Rattus argentiventer (rice-field rat), 10%, Rattus tiomanicus (Malaysian field rat), 9% and Rattus exulans (Polynesian rat), 4%. Rattus rattus diardii is commonly known to live in human environment and they are normally identified as pests to human community. More male rats were captured (61%) compared to female (39%). Out of 137 samples, 81.8% samples were positive with intestinal parasites, with 86.2% from Sentul area and 78.5% from Chow Kit area. Six different parasites were detected. The most common intestinal helminth parasite detected was Nippostrongylus brasiliensis (80.3%), followed by Hymenolepis nana (23.4%), Capillaria hepatica (13.9%) and Hymenolepis diminuta (2.9%). Intestinal protozoan detected was Entamoeba histolytica/E. dispar (8.8%). Trypanosoma lewisi (1.5%) was the only blood parasite detected.

Comparative analysis of ITS1 nucleotide sequence reveals distinct genetic difference between Brugia malayi from Northeast Borneo and Thailand

Fong MY, Noordin R, Lau YL, Cheong FW, Yunus MH, Idris ZM

Parasitology, 2013 Jan;140(1):39-45.

PMID: 22917270 DOI: 10.1017/S0031182012001242

Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based molecular techniques.

Evaluation of Toxoplasma gondii-recombinant dense granular protein (GRA2) for serodiagnosis by western blot

Ching XT, Lau YL, Fong MY, Nissapatorn V

Parasitol Res, 2013 Mar;112(3):1229-36.

PMID: 23274488 DOI: 10.1007/s00436-012-3255-5

Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.

Fulltext Protective immune response in BALB/c mice induced by DNA vaccine of the ROP8 gene of Toxoplasma gondii

Parthasarathy S, Fong MY, Ramaswamy K, Lau YL

Am J Trop Med Hyg, 2013 May;88(5):883-7.

PMID: 23509124 DOI: 10.4269/ajtmh.12-0727

Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.

Fulltext Evaluation of recombinant Plasmodium knowlesi merozoite surface protein-1(33) for detection of human malaria

Cheong FW, Lau YL, Fong MY, Mahmud R

Am J Trop Med Hyg, 2013 May;88(5):835-40.

PMID: 23509118 DOI: 10.4269/ajtmh.12-0250

Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-1(33) has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-133. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-1(33) (pkMSP-1(33)) was expressed by using an Escherichia coli system. The purified pkMSP-1(33) reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-1(33) also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-1(33).

Fulltext Comparison of two nested PCR methods for the detection of human malaria

Anthony C, Mahmud R, Lau YL, Syedomar SF, Sri La Sri Ponnampalavanar S

Trop Biomed, 2013 Sep;30(3):459-66.

PMID: 24189676 MyJurnal

Battling malaria will be a persistent struggle without the proper means to diagnose the parasitic infection. However, the inherent limitations of microscopy, the conventional method of diagnosing malaria, affect the accuracy of diagnosis. The present study aimed to compare the accuracy of two different set of primers targeting the small subunit ribosomal RNA (ssRNA) and the dihydrofolate reductase-thymidylate synthase linker region (dhfr-ts) in detecting species specific malaria infections by nested PCR. The sensitivity and specificity of nested PCR assay using the two primers were calculated with reference to microscopy as the 'gold standard'. The results show that 18S rRNA primers had 91.9% sensitivity and 100% specificity in detecting human Plasmodium species as opposed to dhfr-ts primers which had 51.4% sensitivity and 100% specificity. The higher sensitivity of 18S rRNA primers suggests that it may be a better diagnostic tool for detecting human malaria.

Fulltext Genetic assemblage of Sarcocystis spp. in Malaysian snakes

Lau YL, Chang PY, Subramaniam V, Ng YH, Mahmud R, Ahmad AF, et al.

Parasit Vectors, 2013 Sep 09;6(1):257.

PMID: 24010903 DOI: 10.1186/1756-3305-6-257

BACKGROUND: Sarcocystis species are protozoan parasites with a wide host range including snakes. Although there were several reports of Sarcocytis species in snakes, their distribution and prevalence are still not fully explored.

METHODS: In this study, fecal specimens of several snake species in Malaysia were examined for the presence of Sarcocystis by PCR of 18S rDNA sequence. Microscopy examination of the fecal specimens for sporocysts was not carried as it was difficult to determine the species of the infecting Sarcocystis.

RESULTS: Of the 28 snake fecal specimens, 7 were positive by PCR. BLASTn and phylogenetic analyses of the amplified 18S rDNA sequences revealed the snakes were infected with either S. nesbitti, S. singaporensis, S. zuoi or undefined Sarcocystis species.

CONCLUSION: This study is the first to report Sarcocystis infection in a cobra, and S. nesbitti in a reticulated python.

Fulltext Immunogenicity of bacterial-expressed recombinant Plasmodium knowlesi merozoite surface protein-142 (MSP-142)

Cheong FW, Fong MY, Lau YL, Mahmud R

Malar J, 2013;12:454.

PMID: 24354660 DOI: 10.1186/1475-2875-12-454

Plasmodium knowlesi is the fifth Plasmodium species that can infect humans. The Plasmodium merozoite surface protein-1(42) (MSP-1(42)) is a potential candidate for malaria vaccine. However, limited studies have focused on P. knowlesi MSP-1(42).

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 Armigeres subalbatus incriminated as a vector of zoonotic Brugia pahangi filariasis in suburban Kuala Lumpur, Peninsular Malaysia

Muslim A, Fong MY, Mahmud R, Lau YL, Sivanandam S

Parasit Vectors, 2013;6:219.

PMID: 23898840 DOI: 10.1186/1756-3305-6-219

In 2011, we reported occurrence of natural human infections with Brugia pahangi, a filarial worm of dogs and cats, in a surburb of Kuala Lumpur, the capital city of Malaysia. Our preliminary entomological survey at that time suggested the mosquito species Armigeres subalbatus as the vector of the zoonotic infections. In this present report, we provide biological evidence to confirm our preliminary finding.

Fulltext Is there a risk of suburban transmission of malaria in Selangor, Malaysia?

Braima KA, Sum JS, Ghazali AR, Muslimin M, Jeffery J, Lee WC, et al.

PLoS One, 2013;8(10):e77924.

PMID: 24194901 DOI: 10.1371/journal.pone.0077924

BACKGROUND: The suburban transmission of malaria in Selangor, Malaysia's most developed and populous state still remains a concern for public health in this region. Despite much successful control efforts directed at its reduction, sporadic cases, mostly brought in by foreigners have continued to occur. In addition, cases of simian malaria caused by Plasmodium knowlesi, some with fatal outcome have caused grave concern to health workers. The aim of this study was to investigate the possibility of local malaria transmission in suburban regions of Selangor, which are adjacent to secondary rainforests.

FINDINGS: A malaria survey spanning 7 years (2006 - 2012) was conducted in Selangor. A total of 1623 laboratory confirmed malaria cases were reported from Selangor's nine districts. While 72.6% of these cases (1178/1623) were attributed to imported malaria (cases originating from other countries), 25.5% (414/1623) were local cases and 1.9% (31/1623) were considered as relapse and unclassified cases combined. In this study, the most prevalent infection was P. vivax (1239 cases, prevalence 76.3%) followed by P. falciparum (211, 13.0%), P. knowlesi (75, 4.6%), P. malariae (71, 4.4%) and P. ovale (1, 0.06%). Mixed infections comprising of P. vivax and P. falciparum were confirmed (26, 1.6%). Entomological surveys targeting the residences of malaria patients' showed that the most commonly trapped Anopheles species was An. maculatus. No oocysts or sporozoites were found in the An. maculatus collected. Nevertheless, the possibility of An. maculatus being the malaria vector in the investigated locations was high due to its persistent occurrence in these areas.

CONCLUSIONS: Malaria cases reported in this study were mostly imported cases. However the co-existence of local cases and potential Plasmodium spp. vectors should be cause for concern. The results of this survey reflect the need of maintaining closely monitored malaria control programs and continuous extensive malaria surveillance in Peninsula Malaysia.

Fulltext Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia

Lau YL, Anthony C, Fakhrurrazi SA, Ibrahim J, Ithoi I, Mahmud R

Parasit Vectors, 2013;6(1):250.

PMID: 23985047 DOI: 10.1186/1756-3305-6-250

Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.

Fulltext Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome

Lau YL, Lee WC, Tan LH, Kamarulzaman A, Syed Omar SF, Fong MY, et al.

Malar J, 2013;12:389.

PMID: 24180319 DOI: 10.1186/1475-2875-12-389

Plasmodium ovale is one of the causative agents of human malaria. Plasmodium ovale infection has long been thought to be non-fatal. Due to its lower morbidity, P. ovale receives little attention in malaria research.

Fulltext Colonization of Anopheles cracens: a malaria vector of emerging importance

Amir A, Sum JS, Lau YL, Vythilingam I, Fong MY

Parasit Vectors, 2013;6:81.

PMID: 23537404 DOI: 10.1186/1756-3305-6-81

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.

Fulltext Cloning, expression, and immunocharacterization of surface protein containing an altered thrombospondin repeat domain (SPATR) from Plasmodium knowlesi

Palaeya V, Lau YL, Mahmud R, Chen Y, Fong MY

Malar J, 2013;12:182.

PMID: 23734702 DOI: 10.1186/1475-2875-12-182

Plasmodium knowlesi is the fifth species identified to cause malaria in humans and is often misdiagnosed as Plasmodium malariae due to morphological similarities. The development of an inexpensive, serological detection method utilizing antibodies specific to P. knowlesi would be a valuable tool for diagnosis. However, the identification of specific antigens for these parasites remains a major challenge for generating such assays. In this study, surface protein containing an altered thrombospondin repeat domain (SPATR) was selected as a potentially specific antigen from P. knowlesi. Its multistage expression by sporozoites, asexual erythrocytic forms and gametocytes, along with its possible role in liver cell invasion, suggests that SPATR could be used as a biomarker for diagnosis of P. knowlesi.

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 Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings

Lee WC, Russell B, Lau YL, Fong MY, Chu C, Sriprawat K, et al.

PLoS One, 2013;8(4):e60303.

PMID: 23565221 DOI: 10.1371/journal.pone.0060303

The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on 'subvital' staining. The specialist 'subvital' stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa's stain (commonly used microbiology and parasitology) in a 'subvital' manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.

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