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Fulltext Antigenic membrane proteins of virulent variant of Entamoeba histolytica HM-1:IMSS

Kumarasamy G, Abdus Sani AA, Olivos-García A, Noordin R, Othman N

Pathog Glob Health, 2020 09;114(6):333-342.
PMID: 32536281 DOI: 10.1080/20477724.2020.1780402

Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.
Fulltext Entamoeba histolytica: Quantitative Proteomics Analysis Reveals Putative Virulence-Associated Differentially Abundant Membrane Proteins

Ng YL, Olivos-García A, Lim TK, Noordin R, Lin Q, Othman N

Am J Trop Med Hyg, 2018 12;99(6):1518-1529.
PMID: 30298805 DOI: 10.4269/ajtmh.18-0415

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.
Analysis of Entamoeba histolytica Membrane Proteome Using Three Extraction Methods

Ujang J, Sani AAA, Lim BH, Noordin R, Othman N

Proteomics, 2018 12;18(23):e1700397.
PMID: 30284757 DOI: 10.1002/pmic.201700397

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.
Proteomics technology - a powerful tool for the biomedical scientists

Noordin R, Othman N

Malays J Med Sci, 2013 Mar;20(2):1-2.
PMID: 23983570

"Proteomics" refers to the systematic analysis of proteins. It complements other "omics" technologies such as genomics and transcriptomics in elucidating the identity of proteins of an organism, and understanding their functions. Proteomics is used in many areas of research such as discovery of markers for diagnosis and vaccine candidates, understanding pathogenic mechanisms, in the study of expression patterns at different time points and in response to different stimuli, and in elucidating functional protein networks. Proteomics analysis involves sample preparation, protein separation, and protein identification. The 'heart' of current proteomics is mass-spectrometry, with LC-MS/MS and MALDI-TOF/TOF being commonly used equipment. However, the high costs of the equipment, software, databases, and the need for skilled personnel limit the wide utilization of this technology in the less developed countries. Therefore, there need to be sharing of facilities, better networking and collaborations among our scientists and laboratories to take advantage of this powerful technology.
Fulltext Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity

Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R

BMC Res Notes, 2014;7:809.
PMID: 25406411 DOI: 10.1186/1756-0500-7-809

Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
Limited diagnostic value of two commercial rapid tests for acute leptospirosis detection in Malaysia

Chang CH, Riazi M, Yunus MH, Osman S, Noordin R

Diagn Microbiol Infect Dis, 2014 Dec;80(4):278-81.
PMID: 25241641 DOI: 10.1016/j.diagmicrobio.2014.08.012

This study evaluated 2 rapid leptospirosis serological tests, Leptorapide® (Linnodee, Northern Ireland) and VISITECT®-LEPTO (Omega Diagnostics, Scotland, UK), which are commonly used in Malaysia. A total of 183 samples comprised 113 sera from leptospirosis patients, and 70 sera from other infections and healthy controls were used. The leptospirosis sera were grouped into 2 serum panels, i.e., Group I (MAT+, PCR+) and Group II (MAT+). When inconclusive results were interpreted as positives, both tests showed lower diagnostic sensitivities (≤ 34%) with Group I sera, as compared to Group II sera (Leptorapide®, 93%; VISITECT®-LEPTO, 40%). When inconclusive results were interpreted as negatives, the 2 tests showed ~20% sensitivity with both serum panels. The diagnostic specificity of VISITECT®-LEPTO (94%) was superior to Leptorapide® (69%). Since both tests had misdiagnosed a large proportion of Group I patients and showed many inconclusive results among Group II patients, they have limited diagnostic value in detecting acute leptospirosis.
Fulltext Lateral flow test using Echinococcus granulosus native antigen B and comparison of IgG and IgG4 dipsticks for detection of human cystic echinococcosis

Khalilpour A, Sadjjadi SM, Moghadam ZK, Yunus MH, Zakaria ND, Osman S, et al.

Am J Trop Med Hyg, 2014 Nov;91(5):994-9.
PMID: 25200268 DOI: 10.4269/ajtmh.14-0170

Cystic echinococcosis (CE) caused by infection with Echinococcus granulosus is of major concern for humans in many parts of the world. Antigen B was prepared from E. granulosus hydatid fluid, and Western blots confirmed eight batches showing a band corresponding to the 8-/12-kDa subunit with positive serum and no low-molecular mass band (< 15 kDa) with negative serum. The batches were pooled and used to prepare lateral flow immunoglobulin G4 (IgG4) and IgG dipsticks. Diagnostic sensitivity was determined using serum samples from 21 hydatidosis patients, and diagnostic specificity was established using sera from 17 individuals infected with other parasites and 15 healthy people. IgG4 dipstick had a diagnostic sensitivity of 95% (20 of 21) and a specificity of 100% (32 of 32). The IgG dipstick had a sensitivity of 100% (21 of 21) and a specificity of 87.5% (28 of 32). Thus, both IgG and IgG4 dipsticks had high sensitivities, but IgG4 had greater specificity for the diagnosis of human CE.
Fulltext Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess

Saidin S, Yunus MH, Zakaria ND, Razak KA, Huat LB, Othman N, et al.

BMC Infect Dis, 2014 Apr 04;14:182.
PMID: 24708664 DOI: 10.1186/1471-2334-14-182

BACKGROUND: Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.
METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.
RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.
CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
Fulltext Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.

BMC Infect Dis, 2013;13:287.
PMID: 23800344 DOI: 10.1186/1471-2334-13-287

Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
Antigenic proteins of Helicobacter pylori of potential diagnostic value

Khalilpour A, Santhanam A, Wei LC, Saadatnia G, Velusamy N, Osman S, et al.

Asian Pac J Cancer Prev, 2013;14(3):1635-42.
PMID: 23679248

Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (≥ 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by mass spectrometry. The relative molecular weights and the identities of the proteins, based on MALDI TOF/ TOF, were as follows: CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline- 5-carboxylate dehydrogenase (118 KDa). These identified proteins, singly and/or in combinations, may be useful for diagnosis of H. pylori infection in patients.
Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess

Othman N, Mohamed Z, Yahya MM, Leow VM, Lim BH, Noordin R

Exp Parasitol, 2013 Aug;134(4):504-10.
PMID: 23680184 DOI: 10.1016/j.exppara.2013.05.001

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.
Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera

Amerizadeh A, Idris ZM, Khoo BY, Kotresha D, Yunus MH, Karim IZ, et al.

Microb Pathog, 2013 Jan;54:60-6.
PMID: 23044055 DOI: 10.1016/j.micpath.2012.09.006

Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.
Fulltext Triplex PCR using new primers for the detection of Toxoplasma gondii

Rahumatullah A, Khoo BY, Noordin R

Exp Parasitol, 2012 Jun;131(2):231-8.
PMID: 22561042 DOI: 10.1016/j.exppara.2012.04.009

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.
Fulltext A pilot study on cytotoxic T lymphocyte-4 gene polymorphisms in urinary schistosomiasis

Idris ZM, Yazdanbakhsh M, Adegnika AA, Lell B, Issifou S, Noordin R

Genet Test Mol Biomarkers, 2012 Jun;16(6):488-92.
PMID: 22288822 DOI: 10.1089/gtmb.2011.0209

Urinary schistosomiasis is caused by the digenetic trematode Schistosoma haematobium, characterized by accumulation of eggs in the genitourinary tract. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) can play an important role in parasitic infection due to its major role as a negative regulator of T-cell activation and proliferation. This study was performed in patients with schistosomiasis and healthy controls to analyze the allele and genotype frequencies of four CTLA-4 gene polymorphisms. The CTLA-4 gene was amplified using Taqman real-time polymerase chain reaction, and allele and genotypes of 49 patients with schistosomiasis were analyzed using allelic discrimination analysis followed by subsequent direct sequencing. The results were compared with healthy control subjects. The frequencies of CTLA-4 rs733618 A allele at position -1722 (p=0.001), rs11571316 C allele at position -1577 (p<0.001), and rs231775 A allele at position +49 (p=0.002) in the patient group were significantly higher than the control group. The rs733618 AA genotype (p=0.001), rs11571316 CC genotype (p<0.001), and rs231775 AA genotype (p=0.007) were also significantly overrepresented. Meanwhile, rs733618 AG genotype (p=0.001), rs11571316 CT genotype (p=0.02), and rs231775 GG genotype (p=0.029) were significantly decreased in the patients with schistosomiasis, as compared with the controls. No significant difference was observed in both allele and genotype of rs16841252. The results of this study suggest that the rs733618, rs11571316, and rs231775 polymorphisms in the CTLA-4 gene may influence susceptibility to schistosomiasis infection in the Gabonese children.
Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population

Idris ZM, Miswan N, Muhi J, Mohd TA, Kun JF, Noordin R

Hum Immunol, 2011 Jul;72(7):607-12.
PMID: 21513760 DOI: 10.1016/j.humimm.2011.03.017

Lymphatic filariasis (LF) is a parasitic disease caused by threadlike worms of the Brugia and Wuchereria species that live in the human lymphatic system. Regulatory T cells (Tregs) may play a key role in the pathogenesis of LF, and cytotoxic T-lymphocyte antigen-4 (CTLA4) expressed by Tregs is a potential candidate gene because it modulates T-cell activation. A case-control study was performed to establish a potential association of 5 CTLA4 gene promoter single nucleotide polymorphisms (SNPs; rs733618, rs11571316, rs5742909, rs231775, and rs16840252) with the occurrence of LF in an East Malaysian population (320 LF-infected individuals and 150 healthy controls). Polymorphisms were evaluated using TaqMan real-time polymerase chain reaction followed by direct sequencing. LF carriers of the rs733618 AG genotypes (p = 0.02) and those with combined minor allele G carriers (AG + GG; p = 0.01) exhibited a significantly decreased risk for LF. Among the asymptomatic amicrofilaremic cases, positive associations were reported for all genotypes and variants of rs733618 with odds ratios (ORs) ranging from 0.27 to 0.45. In the asymptomatic microfilaremic cases, marker rs231775 exhibited a significant decreased risk, with ORs ranging from 0.50 to 0.57. The study has identified SNPs in the CTLA4 promoter gene that may be functionally linked with susceptibility to LF.
Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential

Saadatnia G, Mohamed Z, Ghaffarifar F, Osman E, Moghadam ZK, Noordin R

APMIS, 2012 Jan;120(1):47-55.
PMID: 22151308 DOI: 10.1111/j.1600-0463.2011.02810.x

Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase.
Detection of immunogenic parasite and host-specific proteins in the sera of active and chronic individuals infected with Toxoplasma gondii

Yeng C, Osman E, Mohamed Z, Noordin R

Electrophoresis, 2010 Dec;31(23-24):3843-9.
PMID: 21080484 DOI: 10.1002/elps.201000038

Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
Recombinant proteins in the diagnosis of toxoplasmosis

Kotresha D, Noordin R

APMIS, 2010 Aug;118(8):529-42.
PMID: 20666734 DOI: 10.1111/j.1600-0463.2010.02629.x

Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient's serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis.
Comparison of protein-free defined media, and effect of L-cysteine and ascorbic acid supplementation on viability of axenic Entamoeba histolytica

Wong WK, Tan ZN, Lim BH, Mohamed Z, Olivos-Garcia A, Noordin R

Parasitol Res, 2011 Feb;108(2):425-30.
PMID: 20922423 DOI: 10.1007/s00436-010-2083-8

Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
Application of real-time polymerase chain reaction in detection of Entamoeba histolytica in pus aspirates of liver abscess patients

Othman N, Mohamed Z, Verweij JJ, Huat LB, Olivos-García A, Yeng C, et al.

Foodborne Pathog Dis, 2010 Jun;7(6):637-41.
PMID: 20132028 DOI: 10.1089/fpd.2009.0427

Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.

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