Displaying all 11 publications

  1. Michelle Wong Tzeling J, Yean Yean C
    Analyst, 2016 Feb 21;141(4):1246-9.
    PMID: 26783560 DOI: 10.1039/c5an01741f
    A shelf-stable loop-mediated isothermal amplification (LAMP) reagent for Burkholderia pseudomallei detection is described. The coupling of LAMP reagents with the indirect colorimetric indicator and consequently its lyophilization enable the simple evaluation of results without the need for any advance laboratory instruments. The reagents were found to have a stable shelf life of at least 30 days with well-maintained sensitivity and specificity.
  2. Ojha SC, Yean Yean C, Ismail A, Singh KK
    Biomed Res Int, 2013;2013:412370.
    PMID: 23509722 DOI: 10.1155/2013/412370
    The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 10(4) CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.
  3. Azali MA, Yean Yean C, Harun A, Aminuddin Baki NN, Ismail N
    J Trop Med, 2016;2016:2060241.
    PMID: 27127522 DOI: 10.1155/2016/2060241
    The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n = 33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n = 1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n = 1/33, and L. licerasiae, n = 7/33) and nonpathogenic leptospire, L. meyeri (n = 22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors.
  4. 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.
  5. Mohd Ali MR, Mohamad Safiee AW, Yusof NY, Fauzi MH, Yean Yean C, Ismail N
    J Infect Public Health, 2017 12 23;11(4):578-580.
    PMID: 29277333 DOI: 10.1016/j.jiph.2017.12.008
    BACKGROUND: Environmental sampling provides important information that enhances the understanding of the leptospiral human-environment-animal relationship. Several studies have described the distribution of Leptospira in the environment. However, more targeted sites, that is, areas surrounding leptospirosis patients' houses, remain under-explored. Therefore, this study aims to detect the presence of Leptospira spp. in the residential areas of patients with leptospirosis.

    METHODS: Soil and water samples near leptospirosis patients' residences were collected, processed and cultured into EMJH media. Partial 16S rRNA gene sequencing was performed to confirm the identity of Leptospira.

    RESULTS: EMJH culture and partial 16S rRNA gene sequencing revealed predominant growth of pathogenic Leptospira kmetyi (17%, n=7/42). All tested locations had at least one Leptospira sp., mostly from the soil samples.

    CONCLUSION: More than one species of Leptospira may be present in a sampling area. The most common environmental isolates were pathogenic L. kmetyi.

  6. 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.

  7. Ahmad NI, Yean Yean C, Foo PC, Mohamad Safiee AW, Hassan SA
    PMID: 32653480 DOI: 10.1016/j.jiph.2020.06.018
    BACKGROUND: Panton-Valentine Leukocidin (PVL), is one of the virulence gene expressed by Methicillin Resistant Staphylococcus aureus (MRSA) and is known to be associated with severe form of community acquired MRSA infection. The aim of this study is to investigate its prevalence in our setting and patient's clinical outcome.

    METHODS: A cross sectional study involve retrospective record review were done involving 90 MRSA positive isolates between November 2016 and October 2017. Multiplex PCR was performed to detect femA, mecA and PVL genes. Clinical presentation and outcomes of patients were reviewed and presented as descriptive analysis.

    RESULTS: All of the 90 MRSA isolates included in this study were positive for femA and mecA genes following PCR. PVL gene was detected in 20% (n = 18) of the isolates of which 61.1% (n = 11) were community acquired infections and 38.8% (n = 7) were hospital acquired. Case distribution from community acquired infections include patients with skin and soft tissue infections (33.3%, n = 6), infected diabetic foot ulcers (16.7%, n = 3), and one patient each (5.5%, n = 1) for community acquired pneumonia and meningitis. Half of the PVL positive MRSA cases (50%, n = 9) were having sepsis and four of them succumbed to death due to severe infection.

    CONCLUSION: This study shows a high prevalence of PVL positive MRSA infection in our population. Skin and soft tissue infections accounting for the major sources. In addition, the presence of the PVL gene is associated with increased risk for developing sepsis.

  8. Mohd Ali MR, Mohamad Safiee AW, Thangarajah P, Fauzi MH, Muhd Besari A, Ismail N, et al.
    J Infect Public Health, 2017 Nov-Dec;10(6):894-896.
    PMID: 28330585 DOI: 10.1016/j.jiph.2017.02.009
    Leptospirosis and melioidosis are important tropical infections caused by Leptospira and Burkholdheria pseudomallei, respectively. As both infections share similar clinical manifestations yet require different managements, complementary laboratory tests are crucial for the diagnosis. We describe a case of Leptospira and B. pseudomallei co-infection in a diabetic 40-year-old woman with history of visit to a freshwater camping site in northern Malaysia. To our knowledge, this is the first case of such double-infection, simultaneously demonstrated by molecular approach. This case highlights the possibility of leptospirosis and melioidosis co-infections and their underlying challenges in the rapid and accurate detection of the etiologic microorganism.
  9. Mohd Ali MR, Mohd Safee AW, Ismail NH, Abu Sapian R, Mat Hussin H, Ismail N, et al.
    Mol. Cell. Probes, 2018 04;38:1-6.
    PMID: 29524642 DOI: 10.1016/j.mcp.2018.03.001
    BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely.

    METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients.

    RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri.

    CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.

  10. Mohd Ali MR, Lih Huey L, Foo PC, Goay YX, Ismail AS, Mustaffa KMF, et al.
    Biomed Res Int, 2019;2019:9451791.
    PMID: 31355287 DOI: 10.1155/2019/9451791
    Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.
  11. Yu CY, Ang GY, Chua AL, Tan EH, Lee SY, Falero-Diaz G, et al.
    J. Microbiol. Methods, 2011 Sep;86(3):277-82.
    PMID: 21571011 DOI: 10.1016/j.mimet.2011.04.020
    Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.
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