MyMedR

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.

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 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 Clustering and genetic differentiation of the normocyte binding protein (nbpxa) of Plasmodium knowlesi clinical isolates from Peninsular Malaysia and Malaysia Borneo

Ahmed MA, Fong MY, Lau YL, Yusof R

Malar J, 2016;15(1):241.

PMID: 27118390 DOI: 10.1186/s12936-016-1294-6

The zoonotic malaria parasite Plasmodium knowlesi has become an emerging threat to South East Asian countries particular in Malaysia. A recent study from Sarawak (Malaysian Borneo) discovered two distinct normocyte binding protein xa (Pknbpxa) types of P. knowlesi. In the present study, the Pknbpxa of clinical isolates from Peninsular Malaysia and Sabah (Malaysian Borneo) were investigated for the presence of Pknbpxa types and natural selection force acting on the gene.

Fulltext Collective behavior quantification on human odor effects against female Aedes aegypti mosquitoes-Open source development

Poh AH, Moghavvemi M, Leong CS, Lau YL, Safdari Ghandari A, Apau A, et al.

PLoS One, 2017;12(2):e0171555.

PMID: 28152031 DOI: 10.1371/journal.pone.0171555

Classifying and quantifying mosquito activity includes a plethora of categories, ranging from measuring flight speeds, repellency, feeding rates, and specific behaviors such as home entry, swooping and resting, among others. Entomologists have been progressing more toward using machine vision for efficiency for this endeavor. Digital methods have been used to study the behavior of insects in labs, for instance via three-dimensional tracking with specialized cameras to observe the reaction of mosquitoes towards human odor, heat and CO2, although virtually none was reported for several important fields, such as repellency studies which have a significant need for a proper response quantification. However, tracking mosquitoes individually is a challenge and only limited number of specimens can be studied. Although tracking large numbers of individual insects is hailed as one of the characteristics of an ideal automated image-based tracking system especially in 3D, it also is a costly method, often requiring specialized hardware and limited access to the algorithms used for mapping the specimens. The method proposed contributes towards (a) unlimited open source use, (b) a low-cost setup, (c) complete guide for any entomologist to adapt in terms of hardware and software, (d) simple to use, and (e) a lightweight data output for collective behavior analysis of mosquitoes. The setup is demonstrated by testing a simple response of mosquitoes in the presence of human odor versus control, one session with continuous human presence as a stimuli and the other with periodic presence. A group of female Aedes aegypti (Linnaeus) mosquitoes are released into a white-background chamber with a transparent acrylic panel on one side. The video feed of the mosquitoes are processed using filtered contours in a threshold-adjustable video. The mosquitoes in the chamber are mapped on the raster where the coordinates of each mosquito are recorded with the corresponding timestamp. The average distance of the blobs within the frames against time forms a spectra where behavioral patterns can be observed directly, whether any collective effect is observed. With this method, 3D tracking will not be required and a more straightforward data output can be obtained.

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 Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification

Lau YL, Lai MY, Teoh BT, Abd-Jamil J, Johari J, Sam SS, et al.

PLoS One, 2015;10(9):e0138694.

PMID: 26384248 DOI: 10.1371/journal.pone.0138694

Dengue is usually diagnosed by isolation of the virus, serology or molecular diagnostic methods. Several commercial kits for the diagnosis of dengue are existing, but concerns have arisen regarding to the affordability and performance characteristics of these kits. Hence, the loop-mediated isothermal amplification (LAMP) is potentially ideal to be used especially in resource limited environments. Serum was collected from healthy donors and patients diagnosed with dengue infection. RNA extracted from the serum samples were tested by reverse-transcription-LAMP assay developed based on 3'-NCR gene sequences for DENV 1-4. Results were interpreted by a turbidity meter in real time or visually at the end of the assay. Sensitivity and specificity of RT-LAMP results were calculated and compared to qRT-PCR and ELISA. RT-LAMP is highly sensitive with the detection limit of 10 RNA copies for all serotypes. Dengue virus RNA was detected in all positive samples using RT-LAMP and none of the negative samples within 30-45 minutes. With continuing efforts in the optimization of this assay, RT-LAMP may provide a simple and reliable test for detecting DENV in areas where dengue is prevalent.

Fulltext Colorimetric Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Rapid Detection of SARS-CoV-2

Lai MY, Tang SN, Lau YL

Am J Trop Med Hyg, 2021 Jun 15;105(2):375-377.

PMID: 34129521 DOI: 10.4269/ajtmh.21-0150

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been spreading rapidly all over the world. In the absence of effective treatments or a vaccine, there is an urgent need to develop a more rapid and simple detection technology of COVID-19. We describe a WarmStart colorimetric reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2. The detection limit for this assay was 1 copy/µL SARS-CoV-2. To test the clinical sensitivity and specificity of the assay, 37 positive and 20 negative samples were used. The WarmStart colorimetric RT-LAMP had 100% sensitivity and specificity. End products were detected by direct observation, thereby eliminating the need for post-amplification processing steps. WarmStart colorimetric RT-LAMP provides an opportunity to facilitate virus detection in resource-limited settings without a sophisticated diagnostic infrastructure.

Fulltext Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.

Int J Infect Dis, 2022 Jul;120:132-134.

PMID: 35472524 DOI: 10.1016/j.ijid.2022.04.036

OBJECTIVES: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix.

METHODS: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method.

RESULTS: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP.

CONCLUSION: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.

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.

Fulltext Comparative study on Toxoplasma infection between Malaysian and Myanmar pregnant women

Andiappan H, Nissapatorn V, Sawangjaroen N, Nyunt MH, Lau YL, Khaing SL, et al.

Parasit Vectors, 2014;7:564.

PMID: 25498432 DOI: 10.1186/s13071-014-0564-9

Toxoplasma gondii, an obligate intracellular protozoan parasite, causes a disease called toxoplasmosis which can sometimes be acquired congenitally by a newborn from an infected mother. This study aimed to determine the seroprevalence of Toxoplasma infection and its associated risks among 219 and 215 pregnant women from Malaysia and Myanmar, respectively.

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 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.

Compound heterozygous mutations in TTC7A cause familial multiple intestinal atresias and severe combined immunodeficiency

Yang W, Lee PP, Thong MK, Ramanujam TM, Shanmugam A, Koh MT, et al.

Clin Genet, 2015 Dec;88(6):542-9.

PMID: 25534311 DOI: 10.1111/cge.12553

Familial multiple intestinal atresias is an autosomal recessive disease with or without combined immunodeficiency. In the last year, several reports have described mutations in the gene TTC7A as causal to the disease in different populations. However, exact correlation between different genotypes and various phenotypes are not clear. In this study, we report identification of novel compound heterozygous mutations in TTC7A gene in a Malay girl with familial multiple intestinal atresias and severe combined immunodeficiency (MIA-SCID) by whole exome sequencing. We found two mutations in TTC7A: one that destroyed a putative splicing acceptor at the junction of intron 17/exon 18 and one that introduced a stop codon that would truncate the last two amino acids of the encoded protein. Reviewing the recent reports on TTC7A mutations reveals correlation between the position and nature of the mutations with patient survival and clinical manifestations. Examination of public databases also suggests carrier status for healthy individuals, making a case for population screening on this gene, especially in populations with suspected frequent founder mutations.

Fulltext Confirmation of five novel susceptibility loci for systemic lupus erythematosus (SLE) and integrated network analysis of 82 SLE susceptibility loci

Molineros JE, Yang W, Zhou XJ, Sun C, Okada Y, Zhang H, et al.

Hum Mol Genet, 2017 03 15;26(6):1205-1216.

PMID: 28108556 DOI: 10.1093/hmg/ddx026

We recently identified ten novel SLE susceptibility loci in Asians and uncovered several additional suggestive loci requiring further validation. This study aimed to replicate five of these suggestive loci in a Han Chinese cohort from Hong Kong, followed by meta-analysis (11,656 cases and 23,968 controls) on previously reported Asian and European populations, and to perform bioinformatic analyses on all 82 reported SLE loci to identify shared regulatory signatures. We performed a battery of analyses for these five loci, as well as joint analyses on all 82 SLE loci. All five loci passed genome-wide significance: MYNN (rs10936599, Pmeta = 1.92 × 10-13, OR = 1.14), ATG16L2 (rs11235604, Pmeta = 8.87 × 10 -12, OR = 0.78), CCL22 (rs223881, Pmeta = 5.87 × 10-16, OR = 0.87), ANKS1A (rs2762340, Pmeta = 4.93 × 10-15, OR = 0.87) and RNASEH2C (rs1308020, Pmeta = 2.96 × 10-19, OR = 0.84) and co-located with annotated gene regulatory elements. The novel loci share genetic signatures with other reported SLE loci, including effects on gene expression, transcription factor binding, and epigenetic characteristics. Most (56%) of the correlated (r2 > 0.8) SNPs from the 82 SLE loci were implicated in differential expression (9.81 × 10-198

Correction to: Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) & Two extraction-free reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2

Lau YL

BMC Infect Dis, 2023 Nov 16;23(1):755.

PMID: 37968620 DOI: 10.1186/s12879-023-08738-3

Fulltext Correction: Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax

Mahendran P, Liew JWK, Amir A, Ching XT, Lau YL

Malar J, 2023 Oct 25;22(1):324.

PMID: 37880730 DOI: 10.1186/s12936-023-04733-w

Correction: Plasmodium knowlesi: the game changer for malaria eradication

Lee WC, Cheong FW, Amir A, Lai MY, Tan JH, Phang WK, et al.

Malar J, 2023 Oct 19;22(1):316.

PMID: 37858164 DOI: 10.1186/s12936-023-04732-x