Role of stop codons in development and loss of vancomycin non-susceptibility in methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Problems of vancomycin non-susceptible Staphylococcus aureus (VISA) and subsequent treatment failure are increasing. This study aimed to observe development and loss of vancomycin non-susceptibility, determine exposure time needed for resistance development, and follow mutations in the VraSR and GraSR two-component systems during these processes. METHODS: Sequences of vraS, graR and rpoB, proposed as critical sites of mutation associated with non-susceptibility development, were compared in susceptible clinical methicillin-resistant S. aureus isolates both initially and following vancomycin induction and its withdrawal, to identify mutations. Mutations were correlated with exposure time, increase in vancomycin MIC and phenotypic changes. RESULTS: Both time required for heterogeneous VISA and VISA development, and maximum MIC attained (6-20 mg/L) varied between strains. Sequence analysis revealed the presence of stop codons in an initial strain with delayed non-susceptibility development. Other changes in vraS and graR occurred during VISA development in all isolates. After removal of vancomycin pressure, most strains reverted to susceptibility accompanied by emergence of stop codons in both vraS and graR. One strain not displaying stop codons remained resistant in the absence of vancomycin pressure. A substitution in GraR (D148Q) appeared to be associated with an elevated MIC (20 mg/L). No rpoB mutations were observed throughout VISA development. CONCLUSIONS: Vancomycin non-susceptibility developed in all strains tested. Mutations in vraS and graR appeared to be essential for VISA development, with stop codons playing an important role in delaying non-susceptibility development and reversion. Absence of mutations in rpoB suggests that these are not essential for vancomycin resistance. Further work is required to confirm consistent changes involved in non-susceptibility development.

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.

Evaluation of an in-house genotyping resistance test for HIV-1 drug resistance interpretation and genotyping.

INTRODUCTION: The human immunodeficiency virus type 1 (HIV-1) genotyping resistance test (GRT) has been considered essential for HIV-1 drug resistance monitoring. However, it is not commonly used in some developing countries in Asia and Africa due to its high running cost. OBJECTIVE: This study aims to evaluate a new low-cost in-house GRT for both subtype B and non-B HIV-1. STUDY DESIGN: The in-house GRT sequenced the entire protease and 410 codons of reverse transcriptase (RT) in the pol gene. Its performance on drug resistance interpretation was evaluated against the FDA-approved ViroSeq HIV-1 Genotyping System. Particularly, a panel of 235 plasma samples from 205 HIV-1-infected patients in Hong Kong was investigated. The HIV-1 drug resistance-related mutations detected by the two systems were compared. The HIV-1 subtypes were analyzed through the REGA HIV-1 Genotyping Tool and env phylogenetic analysis. RESULTS: Among the 235 samples, 229 (97.4%) were successfully amplified by both in-house and ViroSeq systems. All PCR-negative samples harbored viral RNA at <400 copies/mL. The in-house and ViroSeq system showed identical drug resistance-related mutation patterns in 216 out of 229 samples (94.3%). The REGA pol genotyping results showed 93.9% (215/229) concordance with the env phylogenetic results including HIV-1 subtype A1, B, C, D, G, CRF01_AE, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC, CRF15_01B and other recombinant strains. The cost of running the in-house GRT is only 25% of that for the commercial system, thus making it suitable for the developing countries in Asia and Africa. CONCLUSIONS: Overall, our in-house GRT provided comparable results to those of the commercial ViroSeq genotyping system on diversified HIV-1 subtypes at a more affordable price which make it suitable for HIV-1 monitoring in developing countries.

Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens.

AIMS: To develop a quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) for severe acute respiratory syndrome coronavirus (SARS-CoV) detection and explore the potential of using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control to exclude false negative results. METHODS: SARS-CoV and GAPDH mRNA were both measured in 26 specimens from 16 patients with SARS, 40 follow up specimens from the same batch of patients, and appropriate control subjects. The relation between SARS positivity and GAPDH mRNA concentration was investigated using the chi2 test. Increasing the sensitivity for SARS-CoV and GAPDH mRNA detection was investigated in follow up specimens in which SARS-CoV and GAPDH mRNA were not detected initially. RESULTS: Varying amounts of SARS-CoV were found in the 26 SARS-CoV positive specimens and SARS-CoV was not detected in the 40 follow up specimens and controls. In addition, concentrations of GAPDH mRNA were significantly different between the patients with SARS, follow up specimens, and healthy controls (Kruskal-Wallis test, p<0.05). Moreover, GAPDH mRNA concentrations were highly correlated with SARS-CoV positivity (chi2 = 5.43; p<0.05). Finally, SARS-CoV and GAPDH mRNA were both detected in three follow up urine specimens that were initially negative when the amount of cDNA used was increased from 5 microl to 10 and 15 microl. CONCLUSIONS: This Q-RT-PCR assay can be used to detect SARS-CoV. Moreover, GAPDH mRNA may be useful to rule out false negative results in SARS-CoV detection, and the current extraction method for urine may not be sensitive enough to detect low titres of SARS-CoV.

An active surveillance study of vancomycin-resistant Enterococcus in Queen Elizabeth Hospital, Hong Kong.

OBJECTIVE: To assess the rate of faecal vancomycin-resistant Enterococcus colonisation in high-risk patients in a regional hospital. DESIGN: Prospective observational surveillance study. SETTING: Queen Elizabeth Hospital, Hong Kong. PATIENTS: From September 2001 to December 2002, stool samples from patients in the intensive care unit and patients in whom Clostridium difficile testing was requested were used for study using a broth enrichment method. MAIN OUTCOME MEASURES: Number of faecal vancomycin-resistant Enterococcus colonisation. RESULTS: A total of 2414 cultures from 1792 patients were tested for vancomycin-resistant Enterococcus using a broth enrichment method. Only one (0.06%) patient was found to harbour a vancomycin-resistant Enterococcus faecalis in the gastro-intestinal tract. Surveillance cultures from contacts of the case revealed another six with vancomycin-resistant Enterococcus faecalis. Vancomycin-resistant Enterococcus faecalis was also later reported from a clinical specimen (catheterized urine) of another patient. They were all epidemiologically linked to the index case. Mean inhibitory concentrations of vancomycin and teicoplanin were determined to be higher than 256 and 0.5 microgram/mL, respectively by E-test for all the vancomycin-resistant Enterococcus isolates. Polymerase chain reaction analysis confirmed the presence of vanB genes and the result was in line with the phenotype. Pulsed-field gel electrophoresis confirmed a monoclonal vancomycin-resistant Enterococcus outbreak. Strict infection control measures recommended by the Centers for Disease Control and Prevention were followed and the outbreak was successfully controlled. CONCLUSION: Vancomycin-resistant Enterococcus colonisation is rare, but present among high-risk patients in our hospital. A routine surveillance programme should be implemented that will enable early case detection and prompt initiation of infection control measures to prevent the emergence of an endemic situation.

Importance of early detection of vancomycin-resistant subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus clinical isolates.

This study aimed to investigate the presence of vancomycin-non-susceptible subpopulations in apparently susceptible meticillin-resistant Staphylococcus aureus (MRSA) and the ability of these isolates to develop into homogeneously resistant strains. Vancomycin MICs of 200 clinical MRSA isolates were determined using agar dilution (AD) and spiral gradient endpoint (SGE) technique. Isolates with an MIC≤2mg/L but displaying subpopulations with an MIC>2mg/L by SGE were re-tested by Etest and PAP-AUC and were incubated with 2mg/L vancomycin for 2 weeks. MIC testing was repeated weekly by AD, Etest and SGE to observe progression to non-susceptibility. A total of 17.5% and 16.0% of isolates were non-susceptible to vancomycin (MIC>2mg/L) by SGE and AD, respectively. Eight isolates (4%) displayed a resistant subpopulation; five met the definition of hVISA by PAP-AUC. The initial Etest MIC for these isolates was 2mg/L, but resistant subpopulations were observed in only three isolates on prolonged incubation. MICs of all eight isolates increased rapidly in the presence of vancomycin, reaching ≥3.0mg/L by Day 7 and ≥4mg/L after 14 days by all three methods. The prevalence of vancomycin-non-susceptible MRSA was high, and non-susceptibility developed rapidly in seemingly susceptible isolates with covert subpopulations. These were effectively detected by SGE. With increasing reports of vancomycin clinical failure, early detection of potentially non-susceptible isolates before or early in vancomycin therapy is essential to avoid further resistance development and poor clinical outcomes. SGE offers a novel and cost-effective technique for detection of potentially non-susceptible strains.

Cefepime versus vancomycin plus netilmicin therapy for continuous ambulatory peritoneal dialysis-associated peritonitis.

Cefepime is a cephalosporin with a broad spectrum of activity against most gram-positive and gram-negative pathogens. In this study, we attempted to compare the safety and efficacy of cefepime monotherapy against the potentially more toxic combination of vancomycin and netilmicin in the treatment of continuous ambulatory peritoneal dialysis (CAPD)-associated bacterial peritonitis. Eighty-one consecutive CAPD patients who presented with peritonitis from January 1, 1998, to June 30, 2000, were recruited for study. Patients were randomized to be administered either intraperitoneal (IP) cefepime, 1 g once daily (group A), or intravenous vancomycin and netilmicin at conventional doses (group B) for 10 days. Bacterial growth was obtained in 52 episodes (66%), and pathogens identified included gram-positive organisms (30 episodes; 38%), gram-negative organisms (14 episodes; 18%), mixed organisms (2 episodes; 2.5%), and fungus (6 episodes; 8%). Eight patients were excluded after randomization for various reasons (6 patients, fungal peritonitis; 2 patients, wrong diagnoses). Because of the relatively low peritonitis rate after the use of a disconnect system, the sample size of this study was relatively small, giving a power of 0.45. There were no significant differences in primary response rates and cure rates (no relapse >28 days after completion of antibiotic therapy) between both groups of patients (group A versus group B, 82% [32 of 39 patients] versus 85% [29 of 34 patients] and 72% [28 of 39 patients] versus 76% [26 of 34 patients], respectively; P = not significant). No significant side effect was encountered in either group. Total peritonitis-related hospitalizations were 84 patient-days (1, 7, 8, 11, 20, and 37 patient-days) and 115 patient-days (3, 6, 9, 14, 21, 21, and 41 patient-days), whereas total costs per patient cure were estimated to be US $1,039 and US $1,371 in groups A and B, respectively. We conclude that once-daily 1-g IP cefepime monotherapy is a simple, safe, and cost-effective alternative to vancomycin and netilmicin therapy in the treatment of CAPD-associated bacterial peritonitis.

Comparison of screening methods for detection of extended-spectrum beta-lactamases and their prevalence among Escherichia coli and Klebsiella species in Hong Kong.

Three tests, the disk diffusion test, the double-disc synergy test and the inhibitor-potentiated disc diffusion test, were compared for their abilities to detect production of extended-spectrum beta-lactamases (ESBL) in 702 Escherichia coli and 472 Klebsiella spp. strains from four hospitals. Eleven percent E. coli and 13% Klebsiella spp. were found to produce ESBL. As an indicator of ESBL activity, the sensitivities of the five extended-spectrum beta-lactams were as follows: cefotaxime (100%), cefpodoxime (99.3%), ceftriaxone (98.6%), aztreonam (93%) and ceftazidime (57.7%) when interpreted using the National Committee for Clinical Laboratory Standards criteria. Their positive predictive values ranged from 67.8-83.8%. Both the inhibitor-potentiated disc diffusion test and the double-disc synergy test (at three inter-disc widths of 20, 25 and 30 mm) were capable of identifying all the ESBL-producers. However, at a single inter-disc width of 30 mm, the double-disc synergy test has limited sensitivity (83.8%). As a second test for confirming ESBL activity in strains with reduced susceptibility to beta-lactams, the inhibitor-potentiated disc diffusion test is therefore a simple and reliable option.

Bacteremia due to Staphylococcus aureus with reduced susceptibility to vancomycin.

Four cases of bacteremia caused by Staphylococcus aureus with heteroresistance to vancomycin (hetero-VRSA) were described. In at least two of these four mortalities, the cause of death was temporally related to the hetero-VRSA bacteremia. The vancomycin and teicoplanin MICs of the resistant subpopulations of these four hetero-VRSA were 8 and 24 microg/ml, respectively. All isolates were producers of beta-lactamase, produced penicillin-binding protein PBP2a, and possessed the mecA gene accounting for methicillin resistance. Thickening of the peptidoglycan cell wall was observed by electron microscopy. When ampicillin was combined with vancomycin, in vitro synergism was detected using the checkerboard titration method (epsilonFIC = 0.13). The use of vancomycin plus ampicillin-sulbactam could be a viable option in treating severe hetero-VRSA infection in view of the higher affinity of ampicillin toward PBP2a.

Fine needle aspiration diagnosis of Penicillium marneffei infection.

A disseminated infection with Penicillium marneffei, a rare human pathogen that may infect both healthy and immunocompromised patients, was diagnosed by fine needle aspiration cytology in a patient infected with the human immunodeficiency virus. The presence of yeast-form organisms with an eccentric or central dot and occasional septate and elongated forms highly suggested the diagnosis, which was confirmed on culture. Establishment of the diagnosis is important because this infection is potentially curable.

Penicillium marneffei in bronchoalveolar lavage fluid.

Penicillium marneffei is a rare human pathogen that may infect either healthy or immunocompromised hosts. In methenamine silver-stained preparations of bronchoalveolar lavage fluid from a patient with dermatomyositis on steroid treatment, round-to-oval intracellular and extracellular microorganisms were found. The finding of occasional septate and elongated forms established the microorganism as probably P marneffei, which was confirmed on culture. Distinguishing this rare fungus from Pneumocystis carinii is important because these two diseases require different forms of treatment.

Stability of urea and creatinine in spent hemodialysate.

Urea and creatinine levels in spent hemodialysates showed only small declines in spite of incubation at 37 degrees C for 36 hours. In the determination of dialysate-side solute removal, it would seem prudent to keep spent dialysate cold during collection to retard bacterial breakdown of these waste products.

Antimicrobial susceptibility in hospitals in Hong Kong: The current status 2009-2011.

Hospitals in Hong Kong, like many hospitals in the world, are constantly challenged by the increasing rate of non-susceptible and multidrug-resistant organisms (MDROs). Accurate and timely surveillance is essential for effective control. The Hospital Authority of Hong Kong has developed a comprehensive antimicrobial susceptibility monitoring system that utilises data obtained from all of its 38 hospitals. In this review, the susceptibility pattern of more than 320000 isolates covering the period 2009-2011 will be discussed. Special attention will be paid to MDROs.

Tracking changes in the vraSR and graSR two component regulatory systems during the development and loss of vancomycin non-susceptibility in a clinical isolate.

We investigated changes in regulatory genes, vraS and graR, during development of vancomycin non-susceptibility in a patient with methicillin-resistant Staphylococcus aureus who failed therapy and following in-vitro vancomycin exposure and a subsequent drug-free growth period. Minimum Inhibitory Concentration (MICs) were determined and genes sequenced at each stage. After 30 days of vancomycin exposure, the strain attained maximum MIC (20 mg/L) and was resistant to all antibiotics. Reversion to vancomycin susceptibility occurred 21 days after removal. We observed mutations in vraS and graR during non-susceptibly development and novel stop codons in the reverted strain. Mutations in graR appear important for development of intermediate susceptibility to vancomycin. The results suggest that monitoring of vancomycin therapy could allow earlier change to appropriate agents.

Molecular epidemiology demonstrated three emerging clusters of human immunodeficiency virus type 1 subtype B infection in Hong Kong.

We conducted a molecular epidemiological study on newly diagnosed human immunodeficiency virus type 1 (HIV-1)-infected patients in Hong Kong to identify the epidemiological linkage of HIV-1 infection in the locality. Reverse transcription polymerase chain reaction (RT-PCR) for HIV-1 was performed on newly diagnosed HIV-1-positive sera collected from January 2002 to December 2006. PCR products correspond to the env C2V3V4 region and gag p17/p24 junction of the HIV-1 genome were nucleotide sequenced. Phylogenetic analyses performed on the acquired nucleotide sequences revealed that CRF01_AE and subtype B were the two dominant HIV-1 subtypes. Analyses also demonstrated the presence of three emerging HIV-1 clusters among the subtype B sequences in Hong Kong. Individual cluster possesses a unique cluster-specific amino acid signature for identification. Data show that one of the clusters (Cluster I) is rapidly expanding. In addition to the unique cluster-specific amino acid signature, the majority of sequences in Cluster I harbor a 6-amino acid insertion at the gag p17/p24 junction in a region that is thought to be closely associated with HIV-1 infectivity.