Displaying publications 21 - 40 of 84 in total

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  1. Vythalingam LM, Hossain MAM, Bhassu S
    Mol Cell Probes, 2021 02;55:101683.
    PMID: 33259896 DOI: 10.1016/j.mcp.2020.101683
    Invasive alien fish species have become a silent treat towards the ecosystem especially the native fish population in Malaysia. There has been a need to develop rapid identification methods that can aid management teams in identifying fish species that are not native to our ecosystem. Current visual identification methods are highly tedious and require time, delaying action towards curbing the invasion. The LAMP assay successfully identified six popular invasive fish species in Malaysia. None of the LAMP assays showed false positives and the Limit of Detection of the LAMP primers were highly sensitive and could detect DNA samples up to 1 × 10-15 ng/μl. The LAMP primers designed were highly specific to the target species and did not amplify non target species. DNA sequencing was done to ensure the accuracy of LAMP assay results. This study demonstrates that LAMP is a suitable tool in species identification efforts of invasive fish species in Malaysia.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  2. Hassan SN, Thirumulu Ponnuraj K, Mohamad S, Hassan R, Wan Ab Rahman WS
    Transfus Med Rev, 2019 04;33(2):118-124.
    PMID: 30910255 DOI: 10.1016/j.tmrv.2019.02.003
    Crossover or conversion between the homologous regions of glycophorin A (GYPA) and glycophorin B (GYPB) gives rise to several different hybrid glycophorin genes encoding a number of different glycophorin variant phenotypes which bear low prevalence antigens in the MNS blood group system. GP.Mur is the main glycophorin variant phenotype which causes hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN) in East and Southeast Asians. The detection of glycophorin variant phenotypes using serological methods is limited to phenotyping reagents that are not commercially available. Moreover, the red blood cells used for antibody identification are usually of the GP.Mur phenotype. The current Polymerase Chain Reaction (PCR)-based methods and loop-mediated isothermal amplification (LAMP) are available alternatives to phenotyping that allow for the specific detection of glycophorin variant phenotypes. This review highlights the molecular detection method for glycophorins A and B variant phenotypes and their clinical relevance.
    Matched MeSH terms: Molecular Diagnostic Techniques*
  3. Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.
    Int J Infect Dis, 2022 Jul;120:132-134.
    PMID: 35472524 DOI: 10.1016/j.ijid.2022.04.036
    OBJECTIVES: Preventing reverse transcription loop-mediated isothermal amplification (RT-LAMP) carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil-DNA glycosylase (UDG) into the reaction master mix.

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

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

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

    Matched MeSH terms: Molecular Diagnostic Techniques/methods
  4. Lai MY, Ponnampalavanar SSS, Omar SFS, Lau YL
    Acta Trop, 2024 Mar;251:107120.
    PMID: 38199452 DOI: 10.1016/j.actatropica.2024.107120
    Combining the advantages of PCR and LAMP, we described a new technique, namely PCR-LAMP, for malaria diagnosis. The whole process of DNA amplification can be completed in 35 min. This hybrid amplification technique markedly improved the sensitivity of detection compared to the classic single PCR or LAMP assay alone. PCR-LAMP assay had a detection limit of 1 copy/µL for P. knowlesi and P. ovale, 0.1 copy/µL for P. vivax, P. falciparum and P. malariae, respectively. To facilitate the endpoint detection, xylenol orange was added. Positive samples were indicated in orange while negative reactions were violet. The inclusion of xylenol orange into the LAMP reaction mix significantly reduces the post-amplification workload. Without relying on the use of specific instruments, the color changes of the amplicons could be visualized directly through the naked eye. In conclusion, PCR-LAMP poses the potential to be developed as a new malaria molecular diagnosis tool.
    Matched MeSH terms: Molecular Diagnostic Techniques*
  5. Lai MY, Ooi CH, Lau YL
    Am J Trop Med Hyg, 2017 Nov;97(5):1597-1599.
    PMID: 28820700 DOI: 10.4269/ajtmh.17-0427
    In this study, we developed a recombinase polymerase amplification (RPA) assay for specific diagnosis of Plasmodium knowlesi. Genomic DNA was extracted from whole blood samples using a commercial kit. With incubation at 37°C, the samples were successfully amplified within 20 minutes. The end product of RPA was further examined by loading onto agarose gel and a specific band was observed with a size of 128 bp. The RPA assay exhibited high sensitivity with limits of detection down to one copy of the plasmid. From the specificity experiments, it was demonstrated that all P. knowlesi samples (N = 45) were positive while other Plasmodium spp. (N = 42) and negative samples (N = 6) were negative. Therefore, the RPA assay is a highly promising approach with the potential to be used in resource-limited settings. This assay can be further optimized for bedside and on field application.
    Matched MeSH terms: Molecular Diagnostic Techniques*
  6. Al-Talib H, Yean CY, Al-Khateeb A, Hasan H, Ravichandran M
    J Microbiol Immunol Infect, 2014 Dec;47(6):484-90.
    PMID: 23927820 DOI: 10.1016/j.jmii.2013.06.004
    Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for significant numbers of nosocomial and community-acquired infections worldwide. Molecular diagnosis for MRSA nasal carriers is increasingly important for rapid detection and screening of MRSA colonization because the conventional methods are time consuming and labor intensive. However, conventional polymerase chain reaction (PCR) tests still require cold-chain storage as well as trained personnel, which makes them unsuitable for rapid high-throughput analysis. The aim of this study was to develop a thermostabilized PCR assay for MRSA in a ready-to-use form that requires no cold chain.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*; Molecular Diagnostic Techniques/standards
  7. Amir A, Cheong FW, De Silva JR, Lau YL
    Parasit Vectors, 2018 01 23;11(1):53.
    PMID: 29361963 DOI: 10.1186/s13071-018-2617-y
    Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.
    Matched MeSH terms: Molecular Diagnostic Techniques/instrumentation*; Molecular Diagnostic Techniques/methods*
  8. Chew CH, Lim YA, Lee PC, Mahmud R, Chua KH
    J Clin Microbiol, 2012 Dec;50(12):4012-9.
    PMID: 23035191 DOI: 10.1128/JCM.06454-11
    Malaria remains one of the major killers of humankind and persists to threaten the lives of more than one-third of the world's population. Given that human malaria can now be caused by five species of Plasmodium, i.e., Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and the recently included Plasmodium knowlesi, there is a critical need not only to augment global health efforts in malaria control but also, more importantly, to develop a rapid, accurate, species-sensitive/species-specific, and economically effective diagnostic method for malaria caused by these five species. Therefore, in the present study, a straightforward single-step hexaplex PCR system targeting five human Plasmodium 18S small-subunit rRNAs (ssu rRNAs) was designed, and the system successfully detected all five human malaria parasites. In addition, this system enables the differentiation of single infection as well as mixed infections up to the two-species level. This assay was validated with 50 randomly blinded test and 184 clinical samples suspected to indicate malaria. This hexaplex PCR system is not only an ideal alternative for routine malaria diagnosis in laboratories with conventional PCR machines but also adds value to diagnoses when there is a lack of an experienced microscopist or/and when the parasite morphology is confusing. Indeed, this system will definitely enhance the accuracy and accelerate the speed in the diagnosis of malaria, as well as improve the efficacy of malaria treatment and control, in addition to providing reliable data from epidemiological surveillance studies.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*; Molecular Diagnostic Techniques/standards
  9. Cools P, van Lieshout L, Koelewijn R, Addiss D, Ajjampur SSR, Ayana M, et al.
    PLoS Negl Trop Dis, 2020 Jun;14(6):e0008231.
    PMID: 32544158 DOI: 10.1371/journal.pntd.0008231
    BACKGROUND: Nucleic acid amplification tests (NAATs) are increasingly being used as diagnostic tools for soil-transmitted helminths (STHs; Ascaris lumbricoides, Trichuris trichiura, Necator americanus, Ancylostoma duodenale and A. ceylanicum), Strongyloides stercoralis and Schistosoma in human stool. Currently, there is a large diversity of NAATs being applied, but an external quality assessment scheme (EQAS) for these diagnostics is lacking. An EQAS involves a blinded process where test results reported by a laboratory are compared to those reported by reference or expert laboratories, allowing for an objective assessment of the diagnostic performance of a laboratory. In the current study, we piloted an international EQAS for these helminths (i) to investigate the feasibility of designing and delivering an EQAS; (ii) to assess the diagnostic performance of laboratories; and (iii) to gain insights into the different NAAT protocols used.

    METHODS AND PRINCIPAL FINDINGS: A panel of twelve stool samples and eight DNA samples was validated by six expert laboratories for the presence of six helminths (Ascaris, Trichuris, N. americanus, Ancylostoma, Strongyloides and Schistosoma). Subsequently this panel was sent to 15 globally dispersed laboratories. We found a high degree of diversity among the different DNA extraction and NAAT protocols. Although most laboratories performed well, we could clearly identify the laboratories that were poorly performing.

    CONCLUSIONS/SIGNIFICANCE: We showed the technical feasibility of an international EQAS for the NAAT of STHs, Strongyloides and Schistosoma. In addition, we documented that there are clear benefits for participating laboratories, as they can confirm and/or improve the diagnostic performance of their NAATs. Further research should aim to identify factors that explain poor performance of NAATs.

    Matched MeSH terms: Molecular Diagnostic Techniques/methods*; Molecular Diagnostic Techniques/standards
  10. Khan AH, Noordin R
    Eur J Clin Microbiol Infect Dis, 2020 Jan;39(1):19-30.
    PMID: 31428897 DOI: 10.1007/s10096-019-03680-2
    Infection by Toxoplasma gondii is prevalent worldwide. The parasite can infect a broad spectrum of vertebrate hosts, but infection of fetuses and immunocompromised patients is of particular concern. Easy-to-perform, robust, and highly sensitive and specific methods to detect Toxoplasma infection are important for the treatment and management of patients. Rapid diagnostic methods that do not sacrifice the accuracy of the assay and give reproducible results in a short time are highly desirable. In this context, rapid diagnostic tests (RDTs), especially with point-of-care (POC) features, are promising diagnostic methods in clinical microbiology laboratories, especially in areas with minimal laboratory facilities. More advanced methods using microfluidics and sensor technology will be the future trend. In this review, we discuss serological and molecular-based rapid diagnostic tests for detecting Toxoplasma infection in humans as well as animals.
    Matched MeSH terms: Molecular Diagnostic Techniques
  11. Hamzan NI, Ab Rahman N, Suraiya S, Mohamad I, George Kalarakkal T, Mohamad S
    Arch Oral Biol, 2021 Apr;124:105051.
    PMID: 33581498 DOI: 10.1016/j.archoralbio.2021.105051
    OBJECTIVE: The present study established a real-time loop-mediated isothermal amplification (qLAMP) for rapid detection of human papillomavirus subtype 16 (HPV-16) in oral squamous cell carcinoma (OSCC).

    METHODS: The qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays.

    RESULTS: The detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/μl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/μl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR.

    CONCLUSION: This study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.

    Matched MeSH terms: Molecular Diagnostic Techniques
  12. Khazani NA, Noor NZ, Yean Yean C, Hasan H, Suraiya S, Mohamad S
    J Trop Med, 2017;2017:7210849.
    PMID: 28386286 DOI: 10.1155/2017/7210849
    Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q(10) method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.
    Matched MeSH terms: Molecular Diagnostic Techniques
  13. Ilham NE, Joseph NSM, Bahtiar Affendy N, Mohd Taib N, Vasantha KN, Masri SN
    Trop Biomed, 2020 Jun 01;37(2):357-362.
    PMID: 33612804
    Leptospirosis is a common febrile illness in Malaysia. The disease is caused by pathogenic bacteria called leptospires that are transmitted directly or indirectly from animals to humans via contaminated water or soil. It is a potentially serious but treatable disease. Its symptoms may mimic those of other unrelated febrile illnesses such as dengue, influenza, meningitis, hepatitis or viral haemorrhagic fevers. The spectrum of the disease is extremely wide, ranging from subclinical infection to a severe syndrome of multiorgan infection with high mortality. The diagnosis requires high suspicion with history of exposure to water or environment possibly contaminated with infected animal urine. This is a case of a 13 year-oldgirl with no known medical illness, and a history of exposure to outdoor activities. However, paired sera for leptospirosis serology was not diagnostic. She then developed septic shock on day 14 of illness. But due to high suspicion of leptospirosis, antibiotic therapy was upgraded to ceftriaxone and samples were sent for further testing which revealed that leptospires were detected in the urine, using molecular technique. She improved after treated as leptospirosis.
    Matched MeSH terms: Molecular Diagnostic Techniques
  14. Mohamed Zahidi J, Bee Yong T, Hashim R, Mohd Noor A, Hamzah SH, Ahmad N
    Diagn Microbiol Infect Dis, 2015 Apr;81(4):227-33.
    PMID: 25641125 DOI: 10.1016/j.diagmicrobio.2014.12.012
    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  15. Liam CK, Leow HR, Pang YK
    J Thorac Oncol, 2013 Dec;8(12):e114.
    PMID: 24389448 DOI: 10.1097/JTO.0b013e3182a4e111
    Matched MeSH terms: Molecular Diagnostic Techniques*
  16. Abdullah J, Saffie N, Sjasri FA, Husin A, Abdul-Rahman Z, Ismail A, et al.
    Braz J Microbiol, 2014;45(4):1385-91.
    PMID: 25763045
    An in-house loop-mediated isothermal amplification (LAMP) reaction was established and evaluated for sensitivity and specificity in detecting the presence of Salmonella Typhi (S. Typhi) isolates from Kelantan, Malaysia. Three sets of primers consisting of two outer and 4 inner were designed based on locus STBHUCCB_38510 of chaperone PapD of S. Typhi genes. The reaction was optimised using genomic DNA of S. Typhi ATCC7251 as the template. The products were visualised directly by colour changes of the reaction. Positive results were indicated by green fluorescence and negative by orange colour. The test was further evaluated for specificity, sensitivity and application on field samples. The results were compared with those obtained by gold standard culture method and Polymerase Chain Reaction (PCR). This method was highly specific and -10 times more sensitive in detecting S. Typhi compared to the optimised conventional polymerase chain reaction (PCR) method.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  17. Lau YL, Anthony C, Fakhrurrazi SA, Ibrahim J, Ithoi I, Mahmud R
    Parasit Vectors, 2013;6(1):250.
    PMID: 23985047 DOI: 10.1186/1756-3305-6-250
    Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  18. Issa R, Mohd Hassan NA, Abdul H, Hashim SH, Seradja VH, Abdul Sani A
    Diagn Microbiol Infect Dis, 2012 Jan;72(1):62-7.
    PMID: 22078904 DOI: 10.1016/j.diagmicrobio.2011.09.021
    A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  19. Lau YL, Fong MY, Mahmud R, Chang PY, Palaeya V, Cheong FW, et al.
    Malar J, 2011;10:197.
    PMID: 21774805 DOI: 10.1186/1475-2875-10-197
    The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR.
    Matched MeSH terms: Molecular Diagnostic Techniques/methods*
  20. Rajadurai P, Cheah PL, How SH, Liam CK, Annuar MAA, Omar N, et al.
    Lung Cancer, 2019 10;136:65-73.
    PMID: 31446227 DOI: 10.1016/j.lungcan.2019.08.005
    In the recent years, increased understanding of the molecular profiles of non-small cell lung cancer (NSCLC) has allowed for targeted treatment of actionable genetic mutations. The management of NSCLC now requires multiple molecular tests to guide the treatment strategy. In the light of this, there is a need to establish a molecular testing consensus statement for advanced NSCLC patients in Malaysia. This Malaysian consensus statement was developed by a panel of experts, chaired by a pathologist and composed of three other pathologists, four respiratory physicians and three oncologists. It reflects currently available scientific data and adaptations of recommendations from international guidelines to the local landscape. Expert recommendations on different aspects of molecular testing agreed upon by the panel are provided as structured discussions. These recommendations address the appropriate patients and samples to be tested, as well as when and how these tests should be performed. The algorithms for molecular testing in metastatic NSCLC, in the first line setting and upon disease progression beyond first line therapy, were developed.
    Matched MeSH terms: Molecular Diagnostic Techniques*
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