Boon Huat Lim

A rapid DNA biosensor for the molecular diagnosis of infectious disease

Treating patients with infectious diseases relies heavily on rapid and proper diagnosis. Molecular detection such as PCR has become increasingly important and efforts have been made to simplify these detection methods. This study reports the development of a glass fibre-based lateral flow DNA biosensor that uses capture reagents coupled to carrier beads and detector reagent bioconjugated to gold nanoparticles, for the detection of foodborne pathogen, Vibrio cholerae. The DNA biosensor contains a test line which captures target PCR amplicons, an internal amplification control (IC) line which captures IC amplicons and a control line which acts as membrane control to validate the functionality of this device. The test line captures biotin labelled DNA, while the IC line captures digoxigenin labelled DNA. The detector reagent recognizes the fluorescein haptens of the amplified DNA and produces visual red lines. Scanning electron microscopy (SEM) studies performed indicated that the capture reagents remained relatively immobile within the matrix of the membrane even after binding of the detector reagent. The DNA biosensor recorded a limit of detection (LoD) of 5 ng of target DNA. A clinical evaluation was carried out with 174 strains of V. cholerae and non V. cholerae bacteria and the DNA biosensor recorded 100% for both sensitivity and specificity when compared to conventional agarose gel detection of DNA. Thus it is a viable alternative to agarose gel analysis and is easy-to-use, disposable and do not require any specialized equipment and use of carcinogenic chemicals.

Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae

Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.

Analysis of Entamoeba histolytica Excretory-Secretory Antigen and Identification of a New Potential Diagnostic Marker

ABSTRACT Serodiagnosis of amoebiasis remains the preferred method for diagnosis of amoebic liver abscess (ALA). However, the commercially available kits are problematic in areas of endemicity due to the persistently high background antibody titers. Human serum samples (n = 38) from patients with ALA who live in areas of endemicity were collected from Hospital Universiti Sains Malaysia during the period of 2008 to 2010. Western blots using excretory-secretory antigen (ESA) collected from axenically grown Entamoeba histolytica were probed with the above serum samples. Seven antigenic proteins of ESA with various reactivities were identified, i.e., 152 kDa, 131 kDa, 123 kDa, 110 kDa, 100 kDa, 82 kDa, and 76 kDa. However, only the 152-kDa and 110-kDa proteins showed sensitivities above 80% in the Western blot analysis. All the antigenic proteins showed undetectable cross-reactivity when probed with healthy human serum samples (n = 30) and serum samples from other infections (n = 33). From the matrix-assisted laser desorption ionization–two-stage time of flight (MALDI-TOF/TOF) analysis, the proteins were identified as heavy subunits of E. histolytica lectin and E. histolytica pyruvate phosphate dikinase, respectively. Use of the E. histolytica lectin for diagnosis of ALA has been well reported by researchers and is being used in commercialized kits. However, this is the first report on the potential use of pyruvate phosphate dikinase for diagnosis of ALA; thus, this molecule merits further evaluation on its diagnostic value using a larger panel of serum samples.

Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples

Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC–ESI–MS/MS and LC–MALDI–TOF/TOF

BackgroundExcretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa.MethodsE. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC–ESI–MS/MS and LC–MALDI–TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB).ResultsA complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC–ESI–MS/MS and 107 proteins by LC–MALDI–TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC–ESI–MS/MS and LC–MALDI–TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts.ConclusionThis study showed that complementary use of LC–ESI–MS/MS and LC–MALDI–TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

Current progress of immunoinformatics approach harnessed for cellular- and antibody-dependent vaccine design

Abstract Immunoinformatics plays a pivotal role in vaccine design, immunodiagnostic development, and antibody production. In the past, antibody design and vaccine development depended exclusively on immunological experiments which are relatively expensive and time-consuming. However, recent advances in the field of immunological bioinformatics have provided feasible tools which can be used to lessen the time and cost required for vaccine and antibody development. This approach allows the selection of immunogenic regions from the pathogen genomes. The ideal regions could be developed as potential vaccine candidates to trigger protective immune responses in the hosts. At present, epitope-based vaccines are attractive concepts which have been successfully trailed to develop vaccines which target rapidly mutating pathogens. In this article, we provide an overview of the current progress of immunoinformatics and their applications in the vaccine design, immune system modeling and therapeutics.

Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.

Analysis of Entamoeba histolytica Membrane Proteome Using Three Extraction Methods

Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC–ESI–MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.

Neutralizing FGF4 protein in conditioned medium of IL-21-silenced HCT116 cells restores the migratory activity of the colorectal cancer cells

The interleukin-21 (IL-21) protein was found to be expressed at an elevated level in clinical samples of colorectal cancer patients without or with a parasitic infection that were collected from Sudan in our previous study. The IL-21 gene in HT29 and HCT116 cells was then correlated to cell proliferation and cell migration, as well as the cellular mechanisms associated with gene expressions in our present study. Our results demonstrated that silencing the IL-21 gene in HCT116 cells increased the cytotoxic level and fibroblast growth factor-4 (FGF4) mRNA expression in the cancer cells. Moreover, specific gene silencing reduced the migration of cancer cells compared to non-silenced cancer cells. These events were not observed in IL-21-silenced HT29 cells. Neutralizing FGF4 in conditioned medium of IL-21-silenced HCT116 cells further increased the cytotoxic level and restored the migratory activity of HCT116 cells in the culture compared to silencing the IL-21 gene alone in the cancer cells. Our results indicate the importance of both silencing the IL-21 gene and co-expression of the FGF4 protein in HCT116 cells, which pave the way for the discovery of important factors to be used as biomarkers for the design of drugs or cost-effective supplements to effectively treat the patients having infectious disease and HCT116 cells of colorectal cancer simultaneously in the future.

Helminthic Infection and Nutritional Studies among Orang Asli Children in Sekolah Kebangsaan Pos Legap, Perak.

Background. Orang Asli (aborigine) children are susceptible to soil-transmitted helminth (STH) infections due to their lifestyle and substandard sanitation system. Objectives. This study aimed to examine the helminthic and nutritional status of Orang Asli school children in Sekolah Kebangsaan Pos Legap, a remote primary school at Kuala Kangsar District in the state of Perak, Malaysia. In addition, the sensitivities of four STH stool examination techniques were also compared. Methods. Demography and anthropometry data were collected by one-to-one interview session. Collected stools were examined with four microscopy techniques, namely, direct wet mount, formalin ether concentration (FEC), Kato-Katz (KK), and Parasep™. Results. Anthropometry analysis showed that 78% (26/33) of children in SK Pos Legap were malnourished and 33% (11/33) of them were stunted. Stool examinations revealed almost all children (97%) were infected by either one of the three commonest STHs. FEC was the most sensitive method in detection of the three helminth species. Conclusion. This study revealed that STH infections and nutritional status still remain a health concern among the Orang Asli children. These communal problems could be effectively controlled by regular monitoring of STH infection loads, administration of effective antihelminthic drug regimen, and also implementation of effective school nutritional programs.