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Fulltext Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples

Rahumatullah A, Khoo BY, Noordin R

Trop Biomed, 2015 Jun;32(2):376-85.
PMID: 26691266 MyJurnal

Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.
Fulltext Protein expression in sera of patients with amoebic liver abscess (ALA): potential use of haptoglobin as a surrogate disease marker

Othman N, Zainudin NS, Mohamed Z, Yahya MM, Leow VM, Noordin R

Trop Biomed, 2013 Jun;30(2):257-66.
PMID: 23959491 MyJurnal

The protein profile of serum samples from patients with amoebic liver abscess (ALA) was compared to those of normal individuals to determine their expression levels and to identify potential surrogate disease markers. Serum samples were resolved by two dimensional electrophoresis (2-DE) followed by image analysis. The up and down-regulated protein spots were excised from the gels and analysed by MS/MS. The concentration of three clusters of proteins i.e. haptoglobin (HP), α1-antitrypsin (AAT) and transferrin in serum samples of ALA patients and healthy controls were compared using competitive ELISA. In addition, serum concentrations of HP and transferrin in samples of patients with ALA and pyogenic liver abscess (PLA) were also compared. The results of the protein 2-DE expression analysis showed that HP cluster, AAT cluster, one spot each from unknown spots no. 1 and 2 were significantly up-regulated and transferrin cluster was significantly down-regulated in ALA patients' sera (p<0.05). The MS/MS analysis identified the unknown protein spot no.1 as human transcript and haptoglobin and spot no. 2 as albumin. Competitive ELISA which compared concentrations of selected proteins in sera of ALA and healthy controls verified the up-regulated expression (p<0.05) of HP and the down-regulated expression (p<0.01) of transferrin in the former, while there was no significant difference in AAT expression (p> 0.05). However, when ALA and PLA samples were compared, competitive ELISA showed significant increased concentration of HP (p<0.05) while transferrin levels were not different. In conclusion, this study showed that HP is a potential surrogate disease marker for ALA.
Fulltext Efficacies of two in-house indirect ELISAs for diagnosis of amoebic liver abscess

Tan ZN, Wong WK, Noordin R, Zeehaida M, Olivos GA, Lim BH

Trop Biomed, 2013 Jun;30(2):250-6.
PMID: 23959490 MyJurnal

Entamoeba histolytica causes amoebic diarrhoea, colitis and liver abscess (ALA). Diagnosis of ALA is difficult, as most patients do not have simultaneous intestinal amoebic infection. At Hospital Universiti Sains Malaysia (HUSM), diagnosis of ALA relies on a combination of clinical findings, ultrasound examination of the liver and serodiagnosis using a commercial kit. In this study, two in-house indirect ELISAs were developed and evaluated. One of the in-house assays utilises E. histolytica crude soluble antigen (CSA) to detect serum IgG specific to the parasite whereas the other uses E. histolytica ether extract antigen (EEA). Preparation of CSA requires a sonicator to lyse the amoeba whereas EEA was prepared by chemically solubilizing the trophozoites. Based on the cut-off value of mean optical density + 3SD, CSA-ELISA showed 100% (24/24) sensitivity and 93.33% (210/225) specificity; while EEA-ELISA showed 91.67% (22/24) sensitivity and 95.11% (214/225) specificity. In conclusion, both the in-house indirect ELISAs were found to be efficacious for diagnosis of ALA; and the EEA is easier to prepare than the commonly used CSA.
Fulltext Duration of detection of anti-BmR1 IgG4 antibodies after mass-drug administration (MDA) in Sarawak, Malaysia

Noordin R, Muhi J, Md Idris Z, Arifin N, Kiyu A

Trop Biomed, 2012 Mar;29(1):191-6.
PMID: 22543621 MyJurnal

The detection rates of brugian filariasis in three regions of Sarawak namely Central, North and South after three courses of mass drug administration (MDA) from year 2004 to 2006 was investigated. A recombinant BmR1 antigen-based IgG4 detection test, named Brugia Rapid and night blood smear for microfilaria (mf) detection were used. All three regions recorded a sharp fall in mf positive rates after a year post-MDA. Meanwhile Brugia Rapid positive rates declined more gradually to 3.8% and 5.6% of the pre-MDA levels in the Central and North regions, respectively. This study showed that in filariasis endemic areas in Sarawak, anti-filarial IgG4 antibodies to BmR1, as detected by the Brugia Rapid test, were positive for one to two years after mf disappearance.
Fulltext Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR

Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R

Trop Biomed, 2012 Sep;29(3):434-42.
PMID: 23018507

Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.
Fulltext Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE)

Tan AA, Azman SN, Abdul Rani NR, Kua BC, Sasidharan S, Kiew LV, et al.

Trop Biomed, 2011 Dec;28(3):620-9.
PMID: 22433892 MyJurnal

There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample.
Enhanced production of short-peptide tagged Ss3a recombinant protein as a novel potential biomarker for strongyloidiasis

Mohd-Hassan NH, Noordin R, Arifin N

Trop Biomed, 2020 Sep 01;37(3):578-586.
PMID: 33612773 DOI: 10.47665/tb.37.3.578

Strongyloidiasis is a mysterious yet important parasitic disease that is hard to diagnose. While microscopic examination remains a "controversial" gold standard method, improved diagnosis is achieved through confirmatory assays with serological and/or molecular diagnostic approaches. In the current serodiagnosis of strongyloidiasis, recombinant proteins have been adopted in place of the use of native parasite antigens, although the availability of diagnostically potential proteins are still limited. Here, we introduce a novel Strongyloides recombinant protein that is uniquely attached to two different short peptide tags as a potential diagnostic biomarker for serodiagnosis of strongyloidiasis, namely lysine (7K) and aspartic acid (7D). The work presented focus on improving the yield and purity of the previously unexpressed recombinant protein. Preliminary diagnostic evaluation of the recombinant favors Ss3a7K protein owing to its higher antigenicity performance with 80% sensitivity and 100% specificity, respectively.
Entamoeba infections and associated risk factors among migrant workers in Peninsular Malaysia

Sahimin N, Yunus MH, Douadi B, Yvonne Lim AL, Noordin R, Behnke JM, et al.

Trop Biomed, 2019 Dec 01;36(4):1014-1026.
PMID: 33597471

The influx of low skilled migrant workers to Malaysia from low socio-economic countries where gastrointestinal parasitic infections are prevalent has raised concerns about transmission to the local population. Three methods for detection (serology, microscopy and molecular techniques) were utilized to identify Entamoeba infections amongst the targeted cohort and determine risk factors associated with infection. Serological screening of 484 migrant workers from five working sectors in Peninsular Malaysia using IgG4 ELISA based on the rPPDK antigen showed an overall seroprevalence of 7.4% (n = 36; CL95 = 5.3-10.1%) with only one factor statistically associated with seropositivity of anti-amoebic antibodies, i.e. years of residence in Malaysia (χ2 1 = 4.007, p = 0.045). Microscopic examination of 388 faecal samples for protozoan cysts and trophozoites showed a slightly higher prevalence (11.6%; n=45; CL95: 8.4-14.8%). Meanwhile, amplification of the 16S rDNA gene detected two species i.e. Entamoeba dispar (23/388; 5.9%; CL95: 3.6-8.3%) and E. histolytica (11/388; 2.8%; CL95: 1.2-4.5%) and mixed infections with both parasites in only three samples (3/388; 0.8%; CL95: 0.2-2.2%). Entamoeba dispar infection was significantly associated with those employed in food and domestic services (χ2 4 = 12.879, p = 0.012). However, none of the factors affected the prevalence of E. histolytica infection. Despite the low prevalence of E. histolytica in faecal samples of the study cohort, the presence of this pathogenic parasite still poses potential public health risks and calls for tighter control strategies based on better availability of chemotherapeutic treatment and accessibility to appropriate health education.
Fulltext Diagnostics and the neglected tropical diseases roadmap: setting the agenda for 2030

Souza AA, Ducker C, Argaw D, King JD, Solomon AW, Biamonte MA, et al.

Trans R Soc Trop Med Hyg, 2021 01 28;115(2):129-135.
PMID: 33169166 DOI: 10.1093/trstmh/traa118

Accurate and reliable diagnostic tools are an essential requirement for neglected tropical diseases (NTDs) programmes. However, the NTD community has historically underinvested in the development and improvement of diagnostic tools, potentially undermining the successes achieved over the last 2 decades. Recognizing this, the WHO, in its newly released draft roadmap for NTD 2021-2030, has identified diagnostics as one of four priority areas requiring concerted action to reach the 2030 targets. As a result, WHO established a Diagnostics Technical Advisory Group (DTAG) to serve as the collaborative mechanism to drive progress in this area. Here, the purpose and role of the DTAG are described in the context of the challenges facing NTD programmes.
Seroprevalence of lymphatic filariasis among migrant workers in Peninsular Malaysia

Noordin R, Mohd Zain SN, Yunus MH, Sahimin N

Trans R Soc Trop Med Hyg, 2017 08 01;111(8):370-372.
PMID: 29206992 DOI: 10.1093/trstmh/trx062

Background: Malaysia aims to eliminate lymphatic filariasis (LF) by the year 2020, thus the potential threat of LF from migrant workers needs to be investigated.
Methods: Brugian and bancroftian filariasis among 484 migrant workers from six countries were investigated using rapid tests based on detection of specific IgG4 antibodies against BmR1 (Brugia Rapid) and BmSXP recombinant antigens.
Results: The seroprevalence of brugian filariasis was very low; however, bancroftian filariasis was notable among workers from India, Nepal and Myanmar.
Conclusion: Malaysia is not endemic for Wuchereria bancrofti, but harbors the vectors for the parasite, thus the results showed that migrant workers should be monitored for this infection.
Purification of BmR1 recombinant protein

Arifin N, Basuni M, Lan CA, Yahya AR, Noordin R

Protein J, 2010 Oct;29(7):509-15.
PMID: 20845068 DOI: 10.1007/s10930-010-9281-1

This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
Fulltext Antibodies to somatic L3 antigen not protective against Brugia malayi infection

Noordin R, Abdullah KA, Azahri NA, Ramachandran CP

Southeast Asian J. Trop. Med. Public Health, 1999 Dec;30(4):678-81.
PMID: 10928359

Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.
Comparison of two IgG4 assay formats (ELISA and rapid dipstick test) for detection of brugian filariasis

Noordin R, Shenoy RK, Rahman RA

Southeast Asian J. Trop. Med. Public Health, 2003 Dec;34(4):768-70.
PMID: 15115085

Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
Food beliefs of rural Malay women of Trengganu

Chen PC, Noordin RA, Ngor LY

Med J Malaysia, 1979 Dec;34(2):100-7.
PMID: 548710
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 Lymphatic filariasis and the global elimination program

Noordin R

Malays J Med Sci, 2007 Jan;14(1):1-3.
PMID: 22593644 MyJurnal
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.
Mature Erythrocyte Surface Antigen Protein Identified in the Serum of Plasmodium falciparum-Infected Patients

Zainudin NS, Othman N, Muhi J, Abdu Sani AA, Noordin R

Am J Trop Med Hyg, 2015 Dec;93(6):1268-73.
PMID: 26392156 DOI: 10.4269/ajtmh.15-0333

This study was performed to identify circulating Plasmodium falciparum proteins in patient serum, which may be useful as diagnostic markers. Depletion of highly abundant proteins from each pooled serum sample obtained from P. falciparum-infected patients and healthy individuals was performed using the Proteoseek Antibody-Based Albumin/IgG Removal Kit (Thermo Scientific, Rockford, IL). In analysis 1, the depleted serum was analyzed directly by NanoLC-MS/MS. In analysis 2, the depleted serum was separated by two-dimensional electrophoresis followed by western blot analysis. Subsequently, the selected band was analyzed by NanoLC-MS/MS. The result of analysis 1 revealed the presence of two mature erythrocyte surface antigen (MESA) proteins and chloroquine resistance transporter protein (PfCRT). In addition, analysis 2 revealed an antigenic 75-kDa band when the membrane was probed with purified IgG from the pooled serum obtained from P. falciparum-infected patients. MS/MS analysis of this protein band revealed fragments of P. falciparum MESA proteins. Thus, in this study, two different analyses revealed the presence of Plasmodium MESA protein in pooled serum from malaria patients; thus, this protein should be further investigated to determine its usefulness as a diagnostic marker.
Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

Zahabiun F, Sadjjadi SM, Yunus MH, Rahumatullah A, Moghaddam MH, Saidin S, et al.

Am J Trop Med Hyg, 2015 Aug;93(2):319-25.
PMID: 26033026 DOI: 10.4269/ajtmh.15-0190

Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis.
Development of an Antigen Detection ELISA for Bancroftian Filariasis Using BmSXP-Specific Recombinant Monoclonal Antibody

Rahumatullah A, Lim TS, Yunus MH, Noordin R

Am J Trop Med Hyg, 2019 08;101(2):436-440.
PMID: 31162018 DOI: 10.4269/ajtmh.19-0034

Lymphatic filariasis is a mosquito-borne parasitic disease responsible for morbidity and disability that affects 1.2 billion people worldwide, mainly the poor communities. Currently, filarial antigen testing is the method of choice for the detection of bancroftian filariasis, and to date, there are two commonly used tests. In the present study, a recently reported recombinant monoclonal antibody (5B) specific to BmSXP filarial antigen was used in developing an ELISA for the detection of circulating filarial antigen in sera of patients with bancroftian filariasis. The performance of the ELISA was evaluated using 124 serum samples. The ELISA was positive with all sera from microfilaremic bancroftian filariasis patients (n = 34). It also showed 100% diagnostic specificity when tested with sera from 50 healthy individuals and 40 patients with other parasitic diseases. The developed assay using the novel 5B recombinant monoclonal antibody could potentially be a promising alternative antigen detection test for bancroftian filariasis.

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