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Fulltext A case study of illicit preparation of antirheumatic analgesic with phenylbutazone as active ingredient

Chang ET, Lim BH

Med J Malaysia, 1989 Jun;44(2):160-6.
PMID: 2626126

The abuse of phenylbutazone among rheumatoid arthritis patients has recently become a subject of interest. Unscrupulous manufacturers take advantage of the miraculous analgesic property of phenylbutazone and deliberately add this toxic drug in their preparations without declaring its presence on the label. In a recent survey, many such illicit preparations were seized from Chinese medical halls in Johor and sent to the Department of Chemistry, Johor Bahru for analysis. Here a Gas Chromatograph Mass Selective Detector (GC-MSD) method was developed for the determination of phenylbutazone in illicit traditional preparations.
Pelvic tuberculosis: an uncommon gynaecological problem in Malaysia

Sivanesaratnam V, Lim BH, Sivanesan S, Menon A

J Trop Med Hyg, 1986 Aug;89(4):167-9.
PMID: 3783810

Tuberculosis of the genital tract was diagnosed in only 12 patients during the 17 year period from March 1968 to February 1985 at the University Hospital, Kuala Lumpur, which serves as a major referral centre in Malaysia. The incidence was 0.31 per 1000 gynaecological admissions and the peak age incidence was in the age group 26 to 35 years. The surgical management was mainly conservative as infertility was the most frequent mode of presentation (50%). Evidence of previous pulmonary tuberculosis was present in only five cases. Adnexal adhesions were the commonest pelvic finding; the fallopian tubes and endometrium were affected with equal frequency. Positive cultures for Mycobacterium tuberculosis were obtained in only five of the 12 patients. All patients received combination anti-tuberculosis drugs with satisfactory response.
Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified

Rasineni GK, Loh PC, Lim BH

Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.
METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.
RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.
CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.
GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.
Fulltext Thyrotoxicosis in pregnancy--a six year review

Lim BH, Raman S, Sivanesaratnam V, Ngan A

Singapore Med J, 1989 Dec;30(6):539-41.
PMID: 2635396

Twenty eight patients with hyperthyroidism complicating their pregnancies were seen at the Obstetrics and Gynaecology Department, University Hospital, Kuala Lumpur, Malaysia in a six-year period. All patients were treated with antithyroid drugs, carbimazole being the mainstay of treatment. The incidence of the disease was 0.9 per 1000 births and was similar with other series. No cases of fetal goitre were noted. The mean birth weight was 2952 g; there was no significant difference in the birth weight of term live births in patients treated with carbimazole alone or carbimazole combined with propranolol.
Fulltext Comparison of Brugia-Elisa and thick blood smear examination in a prevalence study of brugian filariasis in Setiu, Terengganu, Malaysia

Lim BH, Rahmah N, Afifi SAB, Ramli A, Mehdi R

Med J Malaysia, 2001 Dec;56(4):491-6.
PMID: 12014770

A total of 1,134 finger-pricked blood samples were collected from residents of Setiu, Terengganu. A drop of blood was used to make thick blood smear and about four drops were used for obtaining serum. The smears were stained and examined by the State Vector Control Unit in Kuala Terengganu, while the serum samples were tested for specific IgG4 antibodies to a novel recombinant antigen using Brugia-Elisa. Prevalence of filariasis in these areas were found to be 0.26% (3/1,134) using thick blood smear examination and 2.47% (28/1,134) using Brugia-Elisa, thus demonstrating the greater sensitivity of the latter test. In addtion, Brugia-Elisa showed a high level of specificity (97.8%, 1,106/1,131) when compared to thick blood smear examination.
Selection of suitable pedicle screw for degenerated cortical and cancellous bone of human lumbar spine: A finite element study

Jain P, Sing Ngie DC, Lim SF, Lim BH

Int J Artif Organs, 2020 Oct 13.
PMID: 33045876 DOI: 10.1177/0391398820964483

Pedicular arthrodesis is the traditional procedure in terms of increase in the biomechanical stability with higher fixation rate. The current work aims to identify the effect of three spinal pedicle screws considering cortical and cancellous degeneracy condition. Lumbar section L2-L3 is utilized and various load and moment conditions were applied to depict the various biomechanical parameters for selection of suitable screw. Three dimensional model is considered in finite element analysis to identify the various responses of pedicle screw at bone screw juncture. Computed tomography (CT) images of a healthy male were considered to generate the finite element vertebral model. Generated intact model was further utilized to develop the other implanted models of degenerated cortical and cancellous bone models. The three fused instrumented models with different cortical and cancellous degeneracy conditions were analyzed in finite element analysis. The results were obtained as stress pattern at bone screw boundary and intervertebral disc stress. FE simulated results represents significant changes in the von Mises stress due to various load and moment conditions on degenerated bones during different body movement conditions. Results have shown that among all pedicle screws, the 6.0 mm diameter screw reflects very less stress values at the juncture. Multiple results on biomechanical aspects obtained during the FE study can be considered to design a new stabilization device and may be helpful to plan surgery of critical sections.
Fulltext Neutralizing FGF4 protein in conditioned medium of IL-21-silenced HCT116 cells restores the migratory activity of the colorectal cancer cells

Abdalkareem EA, Ong CY, Lim BH, Khoo BY

Cytotechnology, 2018 Oct;70(5):1363-1374.
PMID: 29802489 DOI: 10.1007/s10616-018-0228-2

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.
Short communication: use of a recombinant antigen-based ELISA to determine prevalence of brugian filariasis among Malaysian schoolchildren near Pasir Mas, Kelantan-Thailand border

Rahmah N, Lim BH, Azian H, Ramelah TS, Rohana AR

Trop Med Int Health, 2003 Feb;8(2):158-63.
PMID: 12581442

Brugian filariasis infects 13 million people in Asia. The routine prevalence survey method using night thick blood smear is not sensitive enough to reflect the actual infection prevalence. In 1997-2001, only three microfilaraemic cases (of 5601 individuals screened; 0.05%) were reported in Pasir Mas, a district in Kelantan (Malaysia), which shares a border with Thailand. We therefore investigated the infection prevalence in this district by employing a sensitive and specific serological assay (Brugia-Elisa). This test is based on detection of specific IgG4 antibody against a Brugia malayi recombinant antigen. A total of 5138 children, aged 7-12 years, from 16 primary schools, were tested. Eighteen pupils in eight schools, located in five subdistricts, tested positive, giving an overall prevalence rate of 0.35%. Infection in these children is significant as they represent more recent cases. These subdistricts should be included in the national filariasis elimination programme.
Fulltext Comparison and optimization of conventional and ultrasound-assisted solvent extraction for synthetization of lemongrass (Cymbopogon)-infused cooking oil

Lim SF, Hamdan A, David Chua SN, Lim BH

Food Sci Nutr, 2021 May;9(5):2722-2732.
PMID: 34026085 DOI: 10.1002/fsn3.2234

The lemongrass plant, which is widely cultivated in Asia, Australia, and Africa, has been reported to have many significant health benefits such as antimicrobial, insecticide, anticancer, fight fever, and disinfection. Therefore, it is an added benefit to have lemongrass compounds in cooking oil. This study was aimed to compare the conventional (CSE), and ultrasound-assisted solvent extraction (UASE) for citral compounds from lemongrass (Cymbopogon) leaves and to optimize the best extraction method using the response surface methodology (RSM) and ANOVA. RSM design of experiments using three types of cooking oils; palm oil, sunflower oil, and corn oil. The effect of three independent variables, which are temperature (48.2-81.8°C), extraction time (4.8-55.2 min), and solvent to leaves ratio (5.3-18.7), was investigated. The characterization of lemongrass-infused cooking oil was evaluated by Fourier transform infrared spectroscopy (FT-IR), Gas Chromatography-Mass Spectrometry (GC-MS) and Scanning Electron Microscopy (SEM) analysis for confirmation of the citral compound extraction. This extraction process is optimized using Response Surface Methodology (RSM) for producing the lemongrass-infused cooking oil. After optimization, the UASE process gives 1.009 × 106 maximum citral area for palm oil and 1.767 × 106 maximum citral area for sunflower oil. CSE process only can give 2.025 × 105 and 2.179 × 105 citral area in the GC-MS spectrum for palm oil and sunflower oil respectively. For both the UASE and the CSE, the optimum operating conditions are 81.8°C of extraction temperature and 55.2 min of extraction time except for lemongrass-infused palm oil in the CSE process with 45 min extraction time. The optimum solvent to leaves ratio varies from 5.3:1 to 12.9:1. This study found that corn oil cannot be used as a solvent to extract lemongrass-infused cooking oil due to the insignificant changes and no citral peak. The lemongrass (Cymbopogon)-infused palm oil and sunflower oil extracted using the UASE have a higher maximum citral area than the CSE process.
Fulltext Seroprevalence of anti-amoebic antibody among blood donors by indirect hemeagglutination assay

Zeehaida M, Zairi NZ, Tan ZN, Wong WK, Lim BH

Trop Biomed, 2009 Dec;26(3):366-8.
PMID: 20237453

The screening for anti-amoebic antibody among a group of donors was to obtain negative control serum samples for an on-going antigen development assay in diagnosis of amoebic liver abscess. Out of 200 samples, 125 (62.5%) were negative, whereas 44 (21.5%) had IHA titer of less than 1:128 and 31 (16.0%) of the samples had significant IHA titers of 1:128 or more, in which 2 serum samples gave titers of 1:4096.
Evaluation of competency and job satisfaction by positive human psychology among physical education teachers at the university level: A systematic review

Yan T, Teo EW, Lim BH, Lin B

Front Psychol, 2022;13:1084961.
PMID: 36605263 DOI: 10.3389/fpsyg.2022.1084961

The study aimed to determine how a teacher's competency at the university level related to professional commitment and job satisfaction. This systematic review intends to determine physical education teachers' competency and job satisfaction. One of the main objectives of physical education programs is to increase various activities of the student, especially physical activities and participation in it. Students who participate in physical activities feel a variety of emotions, and these may enhance their mental and physical well-being. The study concerns the teachers' satisfaction in effectively influencing and teaching the students. Job satisfaction refers to teachers' feelings and positive attitudes toward their work. The five components of job satisfaction are pay, promotion, supervision, co-workers, and work itself. Job satisfaction is broken down into these five components. Work as a teacher is one of the most important components of a teacher's or teacher's success at work and one of the key criteria for the school's achievement. Electronic libraries were explored, including Google Scholar, PubMed, Embase, Bing Academic, and Cochrane. Appropriate keywords were used for searching the literature. By applying certain inclusion and exclusion criteria, the final studies were selected for evaluation. The study's findings showed that physical education instructors had high levels of teaching competence and moderate levels of job satisfaction. The findings demonstrated that job satisfaction and competency rise when degree levels rise, and relatedness, skill, and autonomy self-motivation ensue. Students' motivation for physical education was greatly influenced by the teachers' support, motivation, and mastery climate. The self-determination theory positively enhances physical education from the teacher's perspective on PE, increasing instruction effectiveness.
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.
Fulltext Analysis of Entamoeba histolytica excretory-secretory antigen and identification of a new potential diagnostic marker

Wong WK, Tan ZN, Othman N, Lim BH, Mohamed Z, Olivos Garcia A, et al.

Clin. Vaccine Immunol., 2011 Nov;18(11):1913-7.
PMID: 21918120 DOI: 10.1128/CVI.05356-11

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.
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.
Brugia malayi infection in Meriones unguiculatus: antibody response to recombinant BmR1

Lim BH, Noordin R, Nor ZM, Rahman RA, Abdullah KA, Sinnadurai S

Exp Parasitol, 2004 Sep-Oct;108(1-2):1-6.
PMID: 15491542

BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.
Fulltext Intrinsic and extrinsic motivation among adolescent ten-pin bowlers in kuala lumpur, malaysia

Teo EW, Khoo S, Wong R, Wee EH, Lim BH, Rengasamy SS

Journal of human kinetics, 2015 Mar 29;45:241-51.
PMID: 25964827 DOI: 10.1515/hukin-2015-0025

Motivation has long been associated with sports engagement. However, to date no research has been performed to understand the domain of motivation among ten-pin bowlers. The purpose of this study was to investigate different types of motivation (i.e., intrinsic vs. extrinsic) based on self-determination theory from the perspective of gender and the bowler type (competitive vs. casual). A total of 240 bowlers (104 male, 136 female; 152 competitive, 88 casual) with a mean age of 16.61 ± 0.78 years were recruited in Kuala Lumpur. The Sport Motivation Scale, a 28-item self-report questionnaire measuring seven subscales (i.e., intrinsic motivation to know, intrinsic motivation to accomplish, intrinsic motivation to experience stimulation, extrinsic motivation to identify regulation, extrinsic motivation for introjection regulation, extrinsic motivation to external regulation, and amotivation) was administered. Results showed significant differences (t=10.43, df=239, p=0.01) between total scores of intrinsic and extrinsic motivation among ten-pin bowlers. There were significant gender differences with respect to intrinsic motivation to know, intrinsic motivation to accomplish, intrinsic motivation to experience stimulation, and extrinsic motivation to identify regulation. However, no significant bowler type differences were found for either the intrinsic (t=-1.15, df=238, p=0.25) or extrinsic (t=-0.51, df=238, p=0.61) motivation dimensions. In conclusion, our study demonstrated substantial intrinsic motivation for gender effects, but no bowler type effects among adolescent ten-pin bowlers.
A recombinant antigen-based IgG4 ELISA for the specific and sensitive detection of Brugia malayi infection

Rahmah N, Lim BH, Khairul Anuar A, Shenoy RK, Kumaraswami V, Lokman Hakim S, et al.

Trans R Soc Trop Med Hyg, 2001 8 9;95(3):280-4.
PMID: 11490997

An IgG4 ELISA based on a novel recombinant antigen was evaluated for detection of Brugia malayi infection, using 2487 sera from various institutions: 2031 samples from Universiti Sains Malaysia, 276 blinded sera from 2 other institutions in Malaysia, 140 blinded sera from India and 40 blinded sera from Thailand. These sera were from various groups of individuals, i.e., microfilaraemics, chronic patients, endemic normals, non-endemic normals and individuals with other parasitic and bacterial infections. Based on a cut-off optical density reading of 0.300, the IgG4 ELISA demonstrated specificity rates of 95.6-100%, sensitivity rates of 96-100%, positive predictive values of 75-100% and negative predictive values of 98.9-100%. These evaluation studies demonstrated the high specificity and sensitivity of this test for the detection of active B. malayi infection. Thus, the IgG4 ELISA would be very useful as a tool in diagnosis and in elimination programmes for brugian filariasis.
A Brugia malayi antigen specifically recognized by infected individuals

Rahmah N, Anuar AK, A'shikin AN, Lim BH, Mehdi R, Abdullah B, et al.

Biochem Biophys Res Commun, 1998 Sep 29;250(3):586-8.
PMID: 9784388

Western blot analyses were performed on 444 serum specimens: 40 sera from microfilaraemic individuals, 10 sera from elephantiasis patients, 24 treated individuals, 50 sera from residents of endemic areas without anti-filarial IgG4 antibodies (endemic normals), 20 sera from amicrofilaraemic individuals with high anti-filarial IgG4 antibodies, 200 sera from healthy city-dwellers (non-endemic samples), and 100 sera from soil-transmitted helminth-infected individuals. Phast electrophoresis system was used to electrophorese Brugia malayi soluble adult worm antigen on 10-15% SDS-PAGE gradient gels followed by electrophoretic transfer onto PVDF membranes. Membrane strips were then successively incubated with blocking solution, human sera, and monoclonal anti-human IgG4 antibody-HRP, with adequate washings done in between each incubation step. Luminol chemiluminescence detection was then used to develop the blots. An antigenic band with the MW of approximately 37 kDa was found to be consistently present in the Western blots of all microfilaraemic sera, all amicrofilaraemic sera with high titres of anti-filarial IgG4 antibodies, some treated patients, and some elephantiasis patients. The antigen did not occur in immunoblots of individuals with other helminthic infections, normal endemic individuals, and city dwellers. Therefore the B. malayi antigen of with the MW of approximately 37 kDa demonstrated specific reactions with sera of B. malayi-infected individuals and thus may be useful for diagnostic application.
Fulltext Loop-mediated isothermal amplification (LAMP) reaction as viable PCR substitute for diagnostic applications: a comparative analysis study of LAMP, conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR) based on Entamoeba histolytica DNA derived from faecal sample

Foo PC, Nurul Najian AB, Muhamad NA, Ahamad M, Mohamed M, Yean Yean C, et al.

BMC Biotechnol, 2020 Jun 22;20(1):34.
PMID: 32571286 DOI: 10.1186/s12896-020-00629-8

BACKGROUND: This study reports the analytical sensitivity and specificity of a Loop-mediated isothermal amplification (LAMP) and compares its amplification performance with conventional PCR, nested PCR (nPCR) and real-time PCR (qPCR). All the assays demonstrated in this study were developed based on Serine-rich Entamoeba histolytica protein (SREHP) gene as study model.
RESULTS: A set of SREHP gene specific LAMP primers were designed for the specific detection of Entamoeba histolytica. This set of primers recorded 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. These primers were later modified for conventional PCR, nPCR and qPCR applications. Besides, 3 different post-LAMP analyses including agarose gel electrophoresis, nucleic acid lateral flow immunoassay and calcein-manganese dye techniques were used to compare their limit of detection (LoD). One E. histolytica trophozoite was recorded as the LoD for all the 3 post-LAMP analysis methods when tested with E. histolytica DNA extracted from spiked stool samples. In contrast, none of the PCR method outperformed LAMP as both qPCR and nPCR recorded LoD of 100 trophozoites while the LoD of conventional PCR was 1000 trophozoites.
CONCLUSIONS: The analytical sensitivity comparison among the conventional PCR, nPCR, qPCR and LAMP reveals that the LAMP outperformed the others in terms of LoD and amplification time. Hence, LAMP is a relevant alternative DNA-based amplification platform for sensitive and specific detection of pathogens.
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

Foo PC, Chan YY, See Too WC, Tan ZN, Wong WK, Lalitha P, et al.

J Med Microbiol, 2012 Sep;61(Pt 9):1219-1225.
PMID: 22556327 DOI: 10.1099/jmm.0.044552-0

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.

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