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Biomedical subjects

W C Yam

Publications and source records attributed to W C Yam.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.

Cambodia↗
PubMed2006

Escherichia coli producing CTX-M beta-lactamases in food animals in Hong Kong.

A study was conducted to evaluate the occurrence of extended-spectrum beta-lactamases (ESBLs)-producing Escherichia coli from fecal samples of healthy food animals in Hong Kong. Rectal or cloacal swabs were obtained from cattle, pigs, chicken, ducks, geese, and pigeons in slaughterhouses or wholesale markets over a 5- month period in 2002. Antibiotic-containing medium was used for selective isolation of potentially ESBL-producing E. coli. Of 734 samples analyzed, six (2%) from pigs, three (3.1%) from cattle, and one (3%) from pigeons had E. coli strains with the ESBL phenotype. The ESBL content for the 10 isolates include CTXM- 3 (n = 4), CTX-M-13 (n = 3), CTX-M-14 (n = 2), and CTX-M-24 (n = 1). In five isolates, the bla (CTX-M) gene was encoded on transferable plasmids (60 or 90 kb), and the gene was found to transfer to E. coli (J53 or JP995) with frequencies of 10(7) to 10(3) per donor cells. The ten isolates had five distinct pulsotypes with some clonal spread. However, the isolates from the different kinds of animals were not clonally related. These findings imply that bacteria of animal origins may serve as reservoirs of some ESBL genes.

Animals↗
PubMed2005

Clinical utility of genotyping resistance test on determining the mutation patterns in HIV-1 CRF01_AE and subtype B patients receiving antiretroviral therapy in Hong Kong.

BACKGROUND: HIV-1 genotypic resistance test (GRT) has been widely used to monitor HIV infection but only few reports revealed the mutation patterns of non-B HIV-1 subtypes. OBJECTIVE: To evaluate the concordance of GRT and clinical treatment outcomes on different HIV-1 subtypes and monitor the mutation patterns and frequencies. STUDY DESIGN: Pre- and post-treatment plasma samples from 123 patients (39 treatment naïve and 84 treatment experienced) were tested by ViroSeq HIV-1 Genotyping System followed by analysis using the Stanford HIV database. The mutation patterns and frequencies developed in the pol gene were compared among subtypes. RESULTS: HIV-1 subtypes among patients in Hong Kong were mainly subtype B and CRF01_AE. Primary mutation was not detected among all pre-treatment samples. For post-treatment samples, primary mutations were only detected in the treatment failure group. The mutation patterns and frequencies were similar between CRF01_AE and subtype B viruses. However, the frequencies of L74V/I and K103N in the reverse transcriptase region were different between CRF01_AE and subtype B viruses. VirtualPhenotype was unable to analyze an in-frame insertion of arginine and isoleucine at protease codon 35 of one CRF01_AE isolate. CONCLUSIONS: This is the first report to demonstrate the high degree of concordance of longitudinal genotyping data and clinical treatment outcome in patients harboring different HIV-1 subtypes. Our findings shed light to the emergence of resistance mutations and its testing in CRF01_AE, which is relevant to other prevailing places in Asia.

Algorithms↗
PubMed2005

Kocuria kristinae infection associated with acute cholecystitis.

BACKGROUND: Kocuria, previously classified into the genus of Micrococcus, is commonly found on human skin. Two species, K. rosea and K. kristinae, are etiologically associated with catheter-related bacteremia. CASE PRESENTATION: We describe the first case of K. kristinae infection associated with acute cholecystitis. The microorganism was isolated from the bile of a 56-year old Chinese man who underwent laparoscopic cholecystectomy. He developed post-operative fever that resolved readily after levofloxacin treatment. CONCLUSION: Our report of K. kristinae infection associated with acute cholecystitis expands the clinical spectrum of infections caused by this group of bacteria. With increasing number of recent reports describing the association between Kocuria spp. and infectious diseases, the significance of their isolation from clinical specimens cannot be underestimated. A complete picture of infections related to Kocuria spp. will have to await the documentation of more clinical cases.

Acute Disease↗
PubMed2005

Occurrence and molecular analysis of extended-spectrum {beta}-lactamase-producing Proteus mirabilis in Hong Kong, 1999-2002.

OBJECTIVES: A study was conducted to evaluate the occurrence and characterization of extended-spectrum beta-lactamases (ESBLs) among blood isolates of Proteus mirabilis collected over a 4 year period in Hong Kong. METHODS: Production of ESBLs among 99 consecutive and non-duplicate isolates was evaluated by the double-disc synergy test. The ESBLs were characterized by isoelectric focusing and PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by the Dienes test and PFGE. RESULTS: ESBLs were identified in 13 isolates, from none in 1999-2000 and up to 18.5% (5/27) in 2001 and 25.8% (8/31) in 2002. The ESBL-producing isolates were more resistant to ceftriaxone than to ceftazidime, and were more likely than non-ESBL-producers to have resistance to ciprofloxacin (76.9% versus 14%) and gentamicin (38.5% versus 9.3%). The ESBL content included CTX-M-13 (n=8), CTX-M-14 (n=3), SHV-5 (n=2), TEM-11 (n=1), and an unidentified ESBL with a pI of 7.5. The Dienes test revealed that the genetic background in the 99 isolates was highly heterogeneous, with 54 distinct types among 92 isolates and seven were non-typeable. Among the 13 ESBL-producing isolates, five different backgrounds, including one cluster (Dienes-pulsotype A) with nine isolates, were identified by both Dienes test and PFGE, thus suggesting both clonal and multi-clonal spread of the CTX-M enzymes. CONCLUSIONS: Our findings indicate the emergence of CTX-M enzymes among P. mirabilis in Hong Kong. More ESBL screening of this species is required to improve their recognition.

Drug Resistance, Bacterial↗
PubMed2005

Detection and characterization of extended-spectrum beta-lactamases among bloodstream isolates of Enterobacter spp. in Hong Kong, 2000-2002.

OBJECTIVES: A total of 139 consecutive and non-duplicate bloodstream isolates of Enterobacter spp. collected from inpatients in Hong Kong during 2000-2002 were studied for production of extended-spectrum beta-lactamases (ESBLs). METHODS: All isolates were evaluated by the modified double-disc synergy test (m-DDST), the combined disc method (CDM) and the three-dimensional (3D) test. The m-DDST and CDM were modified by the use of cefepime discs. beta-Lactamases were characterized by isoelectric focusing and PCR sequencing using specific primers. RESULTS: ESBLs were identified in nine isolates (overall 6.5%), including seven of 39 (17.9%) Enterobacter hormaechei, one of 27 (3.7%) Enterobacter aerogenes and the only Enterobacter intermedius strain. The E. intermedius strain was positive only in the 3D test but not in the other two tests. The other eight strains were positive in all three tests. No ESBL was detected in the other species, including non-hormaechei members of the Enterobacter cloacae complex (n=61), Enterobacter agglomerans (n=7), Enterobacter gergoviae (n=4) and Enterobacter sakazakii (n=1). The ESBL content included five different CTX-M enzymes (CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-24 and a novel CTX-M-2-like beta-lactamase), SHV-12 (n=2) and unidentifiable ESBLs with a pI of 7.7 or 7.9 in two strains. The seven ESBL-producing E. hormaechei were genotyped by pulsed-field gel electrophoresis and were found to be unrelated to each other. In three of the CTX-M-producing strains, ISEcp1-like elements, including promoters for the beta-lactamase gene, were found. CONCLUSIONS: Our data underscore the diversity of CTX-M enzymes among Enterobacter spp. in Hong Kong.

Bacteremia↗
PubMed2005

Clinical evaluation of real-time PCR assays for rapid diagnosis of SARS coronavirus during outbreak and post-epidemic periods.

BACKGROUND: The protocols of WHO network laboratories facilitated development of rapid diagnosis for SARS coronavirus (CoV) using reverse transcription (RT)-PCR assays. However, several reports have shown that conventional and real-time PCR assays were very specific for SARS CoV but lack sensitivity depending on the assay, specimen, and time course of disease. OBJECTIVE: To evaluate an automatic nucleic acid extraction system and two standardized real-time PCR assays for rapid diagnosis of SARS CoV during outbreak and post-epidemic periods in Hong Kong. STUDY DESIGN: Specimens from clinically suspected SARS patients collected during outbreak and post-epidemic periods were tested by an automatic nucleic acid extraction system followed by our first generation conventional RT-PCR and two standardized real-time PCR assays (Artus GmbH, Germany and Roche Diagnostics, Germany). Paired serum samples were assayed for increasing titer against SARS CoV. RESULTS: In the SARS epidemic, Artus and Roche PCR assays exhibited sensitivities of 87% and 85% for respiratory specimens (n = 64), 91% and 88% for stool (n = 44), and 82% for urine (n = 29). A specificity of 100% was exhibited by both PCR assays except Artus attained only a 92% specificity for stool. For post-epidemic period, no SARS CoV was identified among 56 respiratory specimens by all PCR assays. Inhibitors to PCR assays were detected at an average rate of 7-8% among 202 clinical specimens. CONCLUSION: This study highlights the high throughput and performance of automatic RNA extraction in coordination with standardized real-time PCR assays suitable for large-scale routine diagnosis in case of future SARS epidemic. As no SARS CoV was detected among specimens collected during post-epidemic period, the positive predictive value of real-time PCR assays for detection of SARS CoV during low epidemic requires further evaluation.

Disease Outbreaks↗
PubMed2004

Direct detection of Mycobacterium tuberculosis in clinical specimens using single-tube biotinylated nested polymerase chain reaction-enzyme linked immunoassay (PCR-ELISA).

A biotinylated single-tube nested polymerase chain reaction (PCR) assay with microwell hybridization assay (bPCR-ELISA) was developed for detection of Mycobacterium tuberculosis in clinical specimens. A total of 659 specimens (601 respiratory specimens and 58 nonrespiratory specimens) were collected for evaluation using three DNA amplification techniques: newly designed bPCR-ELISA, in-house single-tube nested PCR for IS6110 gene sequence (nPCR), and commercial automated assays, the Cobas Amplicor System from Roche Diagnostic Systems (aPCR). Sixty-four (9.7%) specimens were culture-positive for M. tuberculosis. Eleven (1.7%) specimens culture-positive for nontuberculosis mycobacteria were negative by all three PCR assays. The resolved performance of bPCR-ELISA, nPCR, and aPCR was found at sensitivities of 97%, 94%, and 97%, respectively. All three PCR assays exhibited a 100% specificity. In evaluation of bPCR-ELISA, a clear distinction between PCR-positive and PCR-negative specimens when an OD405 value of 0.6 was chosen as cut-off. With serial dilutions of M. tuberculosis H37Rv DNA, the detection limit of bPCR-ELISA was found to be 0.75 cfu per reaction at OD405 value of 0.6. Our developed bPCR-ELISA provides a highly sensitive and low-costing molecular diagnosis suitable for developing countries with high prevalence of tuberculosis.

Bacteriological Techniques↗
PubMed2004

Direct detection of rifampin-resistant mycobacterium tuberculosis in respiratory specimens by PCR-DNA sequencing.

This study evaluated the feasibility of a molecular strategy based on identification of Mycobacterium tuberculosis by IS6110 PCR or Cobas Amplicor PCR, and rpoB PCR-DNA sequencing of the 81-bp rifampin resistance determining region (RRDR) for direct detection of rifampin resistance in respiratory specimens. A collection of 2,138 respiratory specimens and 352 nonduplicate M. tuberculosis isolates (including 233 isolates from the evaluated respiratory specimens and an additional collection of 119 stored isolates) from Southern China was investigated. Using culture as the reference test, the overall diagnostic sensitivities of an acid-fast bacillus (AFB) smear, Cobas Amplicor PCR, IS6110 PCR were 54.5% (156 of 286), 86.7% (248 of 286), and 89.2% (255 of 286), respectively. The sensitivities of the rpoB PCR for the specimens with positive AFB smears and with positive PCR results in the IS6110 PCR and/or Cobas Amplicor PCR were 100% (156 of 156) and 92.3% (239 of 259), respectively. Of the 352 nonduplicate M. tuberculosis isolates, the agar proportion method for rifampin reported 39 resistant strains. Full agreement (352 of 352) was found with the agar proportion method and the genotype inferred from the rpoB DNA sequencing data for rifampin. Thirty-nine mutations of nine distinct kinds, eight point mutations, and one deletion within the RRDR were found in the 39 resistant strains. For the direct DNA sequencing performed on rpoB PCR-positive respiratory specimens, the concordance with the agar proportion method and the subsequent PCR-sequencing for the culture isolate was 100%. This strategy has potential application for direct and rapid diagnosis of rifampin-resistant M. tuberculosis in IS6110 PCR or Cobas Amplicor PCR-positive respiratory specimens.

Antibiotics, Antitubercular↗
PubMed2004

Clinical evaluation of the polymerase chain reaction for the rapid diagnosis of tuberculosis.

AIMS: Use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis (TB PCR) as a basis for making clinical decisions on the initiation of antituberculosis treatment was studied. METHODS: A retrospective study involving a cohort of 155 patients being investigated for tuberculosis in an infectious disease consultation service was undertaken. TB PCR was performed on pulmonary and extrapulmonary specimens from these patients. The sensitivity of TB PCR was analysed. RESULTS: Of the 155 patients, 144 fitted the clinical diagnosis of tuberculosis, and 112 of them were culture positive for M tuberculosis. Sixty (58.3%) patients with clinical features suggestive of tuberculosis received antituberculosis treatment based on positive TB PCR alone. Of 224 clinical specimens (138 pulmonary and 86 extrapulmonary) sent for TB PCR, 148 (99 pulmonary and 49 extrapulmonary) were positive in 117 patients. Of the 690 clinical specimens sent for culture, 279 were positive for M tuberculosis in 112 patients. The diagnostic sensitivity of TB PCR was 75.9% (85 of 112) and 81.3% (117 of 144) in patients with culture confirmed and clinically diagnosed tuberculosis, respectively. Using culture as the gold standard, the overall sensitivity of TB PCR was 78.3%, and for pulmonary and extrapulmonary specimens it was 82.3% and 72.0%, respectively. CONCLUSIONS: TB PCR is a rapid and reliable test in the diagnosis and management of tuberculosis.

Antitubercular Agents↗
PubMed2003

Risk factors for development of paradoxical response during antituberculosis therapy in HIV-negative patients.

The risk factors for development of paradoxical response were studied in a cohort of 104 patients with culture-documented Mycobacterium tuberculosis infection. Paradoxical deterioration occurred in 16 (15.4%) patients (case group) during antituberculosis therapy, involving lungs and pleura (n=4), spine and paraspinal tissue (n=5), intracranium (n=3), peritoneum (n=2), bone and joint (n=1), and lymph node (n=1). The median time from commencement of treatment to paradoxical deterioration was 56 days (range, 20-109 days). Compared with 53 patients without clinical deterioration after antituberculosis therapy (control group), patients with paradoxical response were more likely to have extrapulmonary involvement (62.5% vs. 17.0%; P<0.05) at initial diagnosis, to have lower baseline lymphocyte counts (672+/-315 cells/microl vs. 1,328+/-467 cells/microl; P<0.001), and to exhibit a greater surge in lymphocyte counts (627+/-465 cells/microl vs. 225+/-216 cells/microl; P<0.05) during paradoxical response. Further studies on lymphocyte subsets and cytokine levels would be useful in understanding the exact immunological mechanisms involved in immunorestitution.

Adult↗
PubMed2003

A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli.

A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.

Amino Acid Sequence↗
PubMed2003

Evaluation of reverse transcription-PCR assays for rapid diagnosis of severe acute respiratory syndrome associated with a novel coronavirus.

The reverse transcription (RT)-PCR protocols of two World Health Organization (WHO) severe acute respiratory syndrome (SARS) network laboratories (WHO SARS network laboratories at The University of Hong Kong [WHO-HKU] and at the Bernhard-Nocht Institute in Hamburg, Germany [WHO-Hamburg]) were evaluated for rapid diagnosis of a novel coronavirus (CoV) associated with SARS in Hong Kong. A total of 303 clinical specimens were collected from 163 patients suspected to have SARS. The end point of both WHO-HKU and WHO-Hamburg RT-PCR assays was determined to be 0.1 50% tissue culture infective dose. Using seroconversion to CoV as the "gold standard" for SARS CoV diagnosis, WHO-HKU and WHO-Hamburg RT-PCR assays exhibited diagnostic sensitivities of 61 and 68% (nasopharyngeal aspirate specimens), 65 and 72% (throat swab specimens), 50 and 54% (urine specimens), and 58 and 63% (stool specimens), respectively, with an overall specificity of 100%. For patients confirmed to have SARS CoV and from whom two or more respiratory specimens were collected, testing the second specimen increased the sensitivity from 64 and 71% to 75 and 79% for the WHO-HKU and WHO-Hamburg RT-PCR assays, respectively. Testing more than one respiratory specimen will maximize the sensitivity of PCR assays for SARS CoV.

Humans↗
PubMed2002

Characterization of a laboratory-generated variant of BPS beta-lactamase from Burkholderia pseudomallei that hydrolyses ceftazidime.

Burkholderia pseudomallei produces an Ambler class A beta-lactamase, known as BPS-1. The beta-lactamase gene from a laboratory-derived, ceftazidime-resistant strain of B. pseudomallei (LH-1-2) was cloned and expressed in Escherichia coli. The beta-lactamase, named BPS-1m, had an identical isoelectric focusing point (pI 7.7) to that of BPS-1, but differed in having a stronger hydrolytic activity against ceftazidime. Susceptibility testing showed that BPS-1m when expressed in E. coli conferred resistance to ceftazidime (MIC >or= 32 mg/L). The amino acid sequence of BPS-1m differed from that of BPS-1 by a Pro-to-Ser change at position 167 in the omega loop.

Amino Acid Substitution↗
PubMed2002

Polyclonal antibodies to glutathione S-transferase--verotoxin subunit a fusion proteins neutralize verotoxins.

The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% +/- 7.9% and 3.6% +/- 2.3%, respectively, and those of VT2 were 1.7% +/- 2.3% and 82.5% +/- 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.

Animals↗
PubMed2001

The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong.

The aim of the study was to define the prevalence of verotoxin-producing Escherichia coli (VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin (VT) by PCR and cytotoxicity assays. VTEC was isolated from 415 and 1-8% of cattle faecal and carcass samples and from 2.1 and 0.2% of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157:H7 and eaeA+. The most prevalent vt genotype among bovine VTEC was vtl+vt2 (73.8%) and in porcine VTEC was vt2e+ (30%). None of the porcine VTEC isolates and 9.3% of the bovine VTEC isolates was eaeA+. The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef), the low VTEC carriage in pigs, the rarity of additional virulence factors (caeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eacA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.

Abattoirs↗