Displaying all 10 publications

  1. Shaminie J, Peh SC, Tan J
    Pathology, 2005 Feb;37(1):39-44.
    PMID: 15875732
    AIM: Tumour suppressor gene p53 is a common target in carcinogenesis, reported to be altered and functionally inactive in 70% of human cancers. Although p53 mutations are less commonly present in haematological malignancies when compared with other solid tumours, they have been reported in histological transformation of follicular lymphoma. We aimed to investigate the frequency of p53 gene alterations in paraffin-embedded tissue using commercially available PCR-SSCP, and to correlate the results with P53 protein expression by immunohistochemistry.

    METHODS: Surgical samples from seven patients with a total of 17 sequential biopsies were retrieved for the study of p53 gene expression using immunohistochemical stain, and gene status by PCR-SSCP for exons 5-8. The tumours were graded according to the WHO classification criteria. P53 was distinctly over-expressed in five transformed higher grade biopsies, and all except one showed electrophoretic mobility shift in PCR-SSCP analysis. Sequencing analysis revealed single nucleotide substitutions in three of four of these high-grade transformed cases with band shift (75%), whereas some other studies reported a lower frequency of 25-30%, and mobility shift result was found to correlate with P53 expression. Lower grade tumours without P53 over-expression did not demonstrate band shift, and sequencing analysis did not reveal mutations.

    CONCLUSIONS: We demonstrated the feasibility of adopting PCR-SSCP for screening of p53 mutations in archival tissue samples in this study, and there is a strong correlation of p53 gene over-expression and mutation events in high-grade transformed tumours.

  2. George E, Teh LK, Tan J, Lai MI, Wong L
    Pathology, 2013 01;45(1):62-5.
    PMID: 23222244 DOI: 10.1097/PAT.0b013e32835af7c1
    AIMS: Classical carriers of β-thalassaemia are identified by a raised HbA2 level. Earlier studies indicated that the Filipino β-deletion has high raised HbA2 levels. The introduction of automated high performance liquid chromatography (HPLC) for thalassaemia screening is an important advance in technology for haematology laboratories. The BioRad Variant II Hb analyser is a common instrument used to quantify HbA2 levels in thalassaemia screening. This study aimed to determine HbA2 levels in carriers of Filipino β-mutation using the BioRad Variant II Hb analyser.

    METHODS: The Filipino β-deletion was identified using gap-polymerase chain reaction (PCR) in the parents of transfusion dependent β-thalassaemia patients who were homozygous for the Filipino β-deletion in the indigenous population of Sabah, Malaysia. Hb subtypes were quantified on the BioRad Variant II Hb analyser. Concurrent α-thalassaemia was identified by multiplex gap-PCR for deletions and amplification refractory mutation system (ARMS)-PCR for non-deletional mutations.

    RESULTS: The mean HbA2 level for Filipino β-thalassaemia trait was 5.9 ± 0.47 and with coinheritance of α-thalassaemia was 6.3 ± 0.44 (-α heterozygous) and 6.7 ± 0.36 (-α homozygous). The HbA2 levels were all >4% in keeping with the findings of classical β-thalassaemia trait and significantly higher than levels seen in non-deletional forms of β-thalassaemia.

    CONCLUSION: The HbA2 level measured on the BioRad Variant II Hb analyser was lower than the level in the first description of the Filipino β-thalassaemia. β-thalassaemia trait with coinheritance of α-thalassaemia (-α) is associated with significantly higher HbA2 level.

  3. Cheok YY, Puah SM, Chua KH, Tan JAMA
    Acta Vet Hung, 2020 10 13;68(2):130-139.
    PMID: 33055305 DOI: 10.1556/004.2020.00029
    Aeromonads are recognised as important pathogens of fishes. In this study, ten water samples were randomly collected from pet shops' fish tanks and home aquaria inhabited by several fish species (silver arowana, koi, goldfish, catfish, pictus fish, silver shark and silver dollar fish). Altogether 298 colonies were isolated using Aeromonas selective agar. A total of 154 isolates were then confirmed as belonging to the genus Aeromonas using the GCAT gene. Using ERIC-PCR, a total of 40 duplicate isolates were excluded from the study and 114 isolates were subjected to PCR-RFLP targeting the RNA polymerase sigma factor (rpoD) gene using lab-on-chip. A total of 13 different Aeromonas species were identified. The most prevalent species were A. veronii (27%, 31/114), followed by A. dhakensis (17%, 19/114), A. finlandiensis (9%, 10/114), A. caviae (8%, 9/114), A. hydrophila (4%, 4/114), A. jandaei (4%, 4/114), A. rivuli (3%, 3/114), A. enteropelogens (2%, 2/114), A. tecta (2%, 2/114), A. allosaccharophila (1%, 1/114), A. eucrenophila (1%, 1/114), A. media (1%, 1/114) and A. diversa (1%, 1/114). Twenty-six isolates (23%) were unidentifiable at species level. The present study demonstrates that Aeromonas species are highly diverse in freshwater fish tanks, and suggests the potential risks posed by the isolated bacteria to the health of ornamental fish species.
  4. Puah SM, Tan JAMA, Chew CH, Chua KH
    J Food Sci, 2018 Sep;83(9):2337-2342.
    PMID: 30101982 DOI: 10.1111/1750-3841.14300
    Staphylococcus aureus is able to form multilayer biofilms embedded within a glycocalyx or slime layer. Biofilm formation poses food contamination risks and can subsequently increase the risk of food poisoning. Identification of food-related S. aureus strains will provide additional data on staphylococcal food poisoning involved in biofilm formation. A total of 52 S. aureus strains isolated from sushi and sashimi was investigated to study their ability for biofilm formation using crystal violet staining. The presence of accessory gene regulator (agr) groups and 15 adhesion genes was screened and their associations in biofilm formation were studied. All 52 S. aureus strains showed biofilm production on the tested hydrophobic surface with 44% (23/52) strains classified as strong, 33% (17/52) as moderate, and 23% (12/52) as weak biofilm producers. The frequency of agr-positive strains was 71% (agr group 1 = 21 strains; agr group 2 = 2 strains; agr group 3 = 12 strains; agr group 4 = 2 strains) whereas agr-negative strains were 29% (15/52). Twelve adhesion genes were detected and 98% of the S. aureus strains carried at least one adhesion gene. The ebps was significantly (p < .05) associated with strong biofilm producing strains. In addition, eno, clfA, icaAD, sasG, fnbB, cna, and sasC were significantly higher in the agr-positive group compared to the agr-negative group. The results of this study suggest that the presence of ebps, eno, clfA, icaAD, sasG, fnbB, cna, and sasC may play an important role in enhancing the stage of biofilm-related infections and warrants further investigation.

    PRACTICAL APPLICATION: This work contributes to the knowledge on the biofilm formation and the distribution of agr groups in S. aureus strains as well as microbial surface components in recognizing adherence matrix molecules of organisms isolated from ready-to-eat sushi and sashimi. The findings provide valuable information to further study the roles of specific genes in causing biofilm-related infections.

  5. Wee YC, Tan KL, Tan PC, Yap SF, Tan JAMA
    Med J Malaysia, 2005 Oct;60(4):447-53.
    PMID: 16570706
    Haemoglobin Bart's hydrops foetalis syndrome (--SEA/--SEA) is not compatible with life and contributes to a majority of the hydropic foetuses in the Malaysian Chinese alpha-thalassaemia carriers who possess the 2-alpha-gene deletion in cis (--SEA/alphaalpha). A duplex-PCR which simultaneously amplifies a normal 136 bp sequence between the psialpha-alpha2-globin genes and a 730 bp Southeast Asian deletion-specific sequence (--SEA) between the psialpha2-theta1-globin genes was established. The duplex-PCR which detects the --SEA deletion in both chromosomes serves as a rapid and cost-effective confirmatory test in the antenatal diagnosis of Haemoglobin Bart's hydrops foetalis syndrome in Malaysia. In addition, the duplex-PCR is simple to perform as both the normal and deletion-specific alpha-globin gene sequences are amplified in the same PCR reaction.
  6. Chan YF, Tan KL, Wong YC, Wee YC, Yap SF, Tan JAMA
    PMID: 12041567
    Molecular characterization and prenatal diagnosis for beta-thalassemia can be carried out using the Amplification Refractory Mutation System (ARMS). The ARMS is a rapid and direct molecular technique in which beta-thalassemia mutations are visualized immediately after DNA amplification by gel electrophoresis. In the University of Malaya Medical Center, molecular characterization and prenatal diagnosis for beta-thalassemia is carried out using ARMS for about 96% of the Chinese and 84.6% of the Malay patients. The remaining 4% and 15.4% of the uncharacterized mutations in the Chinese and Malay patients respectively are detected using DNA sequencing. DNA sequencing is an accurate technique but it is more time-consuming and expensive compared with the ARMS. The ARMS for the rare Chinese beta-mutations at position -29 (A-->G) and the ATG-->AGG base substitution at the initiator codon for translation in the beta-gene was developed. In the Malays, ARMS was optimized for the beta-mutations at codon 8/9 (+G), Cap (+1) (A-->C) and the AATAAA-->AATAGA base substitution in the polyadenylation region of the beta-gene. The ARMS protocols were developed by optimization of the parameters for DNA amplification to ensure sensitivity, specificity and reproducibility. ARMS primers (sequences and concentration), magnesium chloride concentration, Taq DNA polymerase and PCR cycling parameters were optimized for the specific amplification of each rare beta-thalassemia mutation. The newly-developed ARMS for the 5 rare beta-thalassemia mutations in the Chinese and Malays in Malaysia will allow for more rapid and cost-effective molecular characterization and prenatal diagnosis for beta-thalassemia in Malaysia.
  7. Tan JAMA, Yap SF, Tan KL, Wong YC, Wee YC, Kok JL
    Acta Haematol., 2003;109(4):169-75.
    PMID: 12853688 DOI: 10.1159/000070965
    Molecular characterization of the compound heterozygous condition - (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia - in four families showing mild beta-thalassemia intermedia was carried out using DNA amplification techniques. Using the Amplification Refractory Mutation System (ARMS) to confirm the beta-mutations and DNA amplification to detect the 100-kb Chinese-specific (G)gamma((A)gammadeltabeta)(o)-deletion, ()two families were confirmed to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia with the IVSII No. 654 beta(+)-allele. In the third family, the (G)gamma((A)gammadeltabeta)(o)-deletion was confirmed in the father and the mother was a beta-thalassemia carrier with the cd 41-42 beta(o)-allele. Their affected child with (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia was found to be transfusion dependent. The same (G)gamma((A)gammadeltabeta)(o)-deletion and beta-thalassemia (cd 41-42) was also confirmed in a fourth family. In addition, the mother was also diagnosed with Hb H disease (genotype -alpha(3.7)/-(SEA)). Both the children were found to possess (G)gamma((A)gammadeltabeta)(o)/beta-thalassemia but they were not transfusion dependent and this could be due to co-inheritance of alpha-thalassemia-2 (genotype-alpha(3.7)/alphaalpha) in the children together with their compound heterozygous condition.
  8. Puah SM, Khor WC, Kee BP, Tan JAMA, Puthucheary SD, Chua KH
    J Med Microbiol, 2018 Sep;67(9):1271-1278.
    PMID: 30024365 DOI: 10.1099/jmm.0.000796
    PURPOSE: The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.

    METHODOLOGY: A pair of degenerate primers (Aero F: 5'-YGARATCGAYATCGCCAARCGB-3' and Aero R: 5'-GRCCDATGCTCATRCGRCGGTT-3') was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.

    CONCLUSION: This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.

  9. Wong FN, Chua KH, Tan JAMA, Wong CM, Kuppusamy UR
    PeerJ, 2018;6:e4421.
    PMID: 29610703 DOI: 10.7717/peerj.4421
    Background: Chronic kidney disease (CKD) is characterised by long-term kidney damage and renal function decline. Diabetic CKD is the principal subtype of kidney disease in Malaysia and is associated with oxidative stress which plays an important role in development and progression of the disease. Glycaemic control slows down the progression of diabetic complications, including diabetic CKD. However, the implication of glycaemic control on enzymatic antioxidants and soluble RAGE (sRAGE) in CKD patients remains elusive. The aim of this study was to investigate the effect of glycaemic control on the levels or activities of glutathione peroxidase (GPx), superoxide dismutase (SOD) and sRAGE in CKD patients.

    Methods: A total of 150 CKD patients and 64 non-CKD patients were enrolled. The type 2 diabetic patients in the recruited study participants were categorised based on their glycaemic control; poor glycaemic control (GC) with haemoglobin A1c (HbA1c) > 7% and good GC with HbA1c ≤ 7%. The levels or activities of GPx, SOD and sRAGE in plasma were measured. These biochemical parameters were analysed using Mann-WhitneyUtest and two-way analysis of variance (ANOVA).

    Results: The activities of GPx and SOD as well as plasma level of sRAGE were not significantly different among the CKD patients with varying glycaemic control status. Irrespective of diabetes status and glycaemic control status, CKD patients also exhibited lower plasma SOD activities compared with non-CKD patients. Among the non-CKD patients, SOD activities were significantly higher in diabetic patients with good GC than diabetic patients with poor GC. Two-way ANOVA revealed that both CKD status and glycaemic control had an interaction effect on SOD activities in diabetic subjects with and without CKD. Follow-up analysis showed that SOD activities were significantly higher in non-CKD patients with good GC. There were no overall significant differences in GPx activities among the study participants. Furthermore, plasma sRAGE levels were higher in diabetic patients with CKD than those without CKD, regardless of glycaemic control status. There were no interaction effects between CKD status and glycaemic control status on GPx and sRAGE. Instead, CKD status showed significant main effects on these parameters, indicating significant differences between diabetic subjects with CKD and diabetic subjects without CKD.

    Conclusion: Glycaemic control did not quantitatively alter GPx, SOD and sRAGE in diabetic CKD patients. Despite the advantages of good glycaemic control, a well-controlled diabetes in CKD did not modulate the activities of enzymatic antioxidants and sRAGE levels, therefore may not be the primary mechanism to handle oxidative stress.

  10. Khor WC, Puah SM, Koh TH, Tan JAMA, Puthucheary SD, Chua KH
    Microb Drug Resist, 2018 May;24(4):469-478.
    PMID: 29461928 DOI: 10.1089/mdr.2017.0083
    OBJECTIVE: The objective of this study was to examine the species distribution, genetic relatedness, virulence gene profiles, antimicrobial sensitivities, and resistance gene distribution of clinical Aeromonas strains from Singapore and Malaysia.

    METHODS: A total of 210 Aeromonas clinical isolates were investigated: 116 from Singapore General Hospital and 94 archived clinical isolates from University of Malaya Medical Center, Malaysia. The isolates were genetically identified based on the gcat gene screening and the partial sequences of the rpoD housekeeping gene. Genetic relatedness, distribution of 15 virulence genes and 4 beta-lactamase resistance genes, and susceptibility patterns to 11 antimicrobial agents were compared.

    RESULTS: Of the 210 Aeromonas isolates, A. dhakensis-94 (45%) was the dominant species in Singapore and Malaysia. Species composition was similar and enterobacterial repetitive intergenic consensus-PCR did not show genetic relatedness between strains from the two countries. Of the 15 virulence genes, A. dhakensis and A. hydrophila harbored the most compared with other species. Different combinations of 9 virulence genes (exu, fla, lip, eno, alt, dam, hlyA, aexU, and ascV) were present in A. dhakensis, A. hydrophila, and A. veronii from both the countries. Distribution of virulence genes was species and anatomic site related. Majority (>80%) of the strains were susceptible to all antimicrobial agents tested, except amoxicillin and cephalothin. A. dhakensis strains from Malaysia significantly harbored the cphA gene compared with A. dhakensis from Singapore. Multidrug resistance was mostly detected in strains from peritoneal fluids of dialysis patients.

    CONCLUSION: This study revealed A. dhakensis as the dominant species isolated in both geographic regions, and that it carried a high number of virulence genes. It also highlights the geographic-related differences of virulence gene distribution and antimicrobial resistance profiles of clinical Aeromonas strains from Singapore and Malaysia.

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