High rate of reduced susceptibility to ciprofloxacin and ceftriaxone among nontyphoid Salmonella clinical isolates in Asia.

This multinational study from Asia revealed that reduced susceptibility to ciprofloxacin (MIC, 0.125 to 1 microg/ml) in nontyphoid Salmonella isolates was common in Taiwan (48.1%) and Thailand (46.2%) and in S. enterica serotype Choleraesuis (68.8%) and S. Virchow (75.0%) from all countries. Reduced susceptibility to ceftriaxone (MIC, 2 to 8 microg/ml) remained uncommon in Asia, except in Taiwan (38.0%) or in S. Typhimurium (25.0%) from all countries.

Methicillin-resistant Staphylococcus aureus control at the National University Hospital, Singapore: a historical perspective.

INTRODUCTION: The National University Hospital (NUH) was the first restructured public hospital in Singapore. As the most recently established hospital in Singapore, it has a unique record of alert organisms including methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: We performed a critical review of multiple data sources including surveillance reports, task force reports, published abstracts and manuscripts concerning MRSA in NUH. RESULTS: Three themes emerged: 1) the MRSA rates have remained relatively stable through the life of the hospital despite the increased complexity of patients and intermittent intensified control efforts; 2) the major MRSA task forces were driven by surgeons and 3) a scientific approach to epidemiology has a critical role in understanding and planning interventions. CONCLUSION: Although containment of MRSA can be accomplished to a certain degree through mobilisation of existing resources, higher goals such as eradication would require massive infusions of infrastructural, scientific and human resources to have a chance of success.

Antimicrobial susceptibility of Shigella isolates in eight Asian countries, 2001-2004.

BACKGROUND AND PURPOSE: Shigellosis is a major health problem in developing countries, causing 91 million episodes and 414,000 deaths in Asia annually. Because of increasing trends towards drug resistance, this study was undertaken to monitor local resistance patterns of Shigella isolates from 8 Asian countries. METHODS: Ninety eight Shigella isolates collected from 8 centers in 8 Asian countries from July 2001 to July 2004 were analyzed in terms of serogroup distribution and antimicrobial susceptibility. RESULTS: The most common serogroup of Shigella isolates was Shigella flexneri (49/98, 50%), followed by Shigella sonnei (44/98, 45%). The highest resistance rate was found for trimethoprim-sulfamethoxazole (81%), followed by tetracycline (74%) and ampicillin (53%). Overall, 76 Shigella isolates (78%) were multidrug-resistant strains; S. flexneri had a higher multidrug resistance rate than S. sonnei (74% vs 23%). Increasing ciprofloxacin and ceftriaxone resistance was observed; approximately 10% and 5% of isolates were resistant to ciprofloxacin and ceftriaxone, respectively. Five ceftriaxone-non-susceptible strains (from Taiwan [3], Hong Kong [1] and The Philippines [1]) and 10 ciprofloxacin-non-susceptible strains (from Hong Kong [2], The Philippines [1], Korea [2], Vietnam [4] and Sri Lanka [1]) were isolated. CONCLUSIONS: High rates of multidrug resistance and steady increases in ceftriaxone and ciprofloxacin resistance of Shigella are serious pubic health concerns in Asian countries. Continuous monitoring of resistance patterns among Shigella isolates is necessary.

Screening for vancomycin-resistant enterococci using stools sent for Clostridium difficile cytotoxin assay is effective: results of a survey of 300 Patients in a large Singapore Teaching Hospital.

INTRODUCTION: To assess the efficacy of screening stools sent for Clostridium difficile cytotoxin assay (CDTA) for surveillance of vancomycin-resistant enterococci (VRE). MATERIALS AND METHODS: From April to May 2005, all stools submitted for CDTA were also cultured for VRE using vancomycin containing culture media. Isolates were identified to species level and vancomycin resistance confirmed, followed by polymerase chain reaction (PCR) for detection of vancomycin resistance genes and DNA fingerprinting. Over 2 consecutive days during that period, stool specimens or rectal swabs were also obtained from all patients in high-risk units (haematology, oncology, renal and intensive care). Fifty-one patients in each group were compared in terms of VRE risk factors previously identified. RESULTS AND DISCUSSION: The prevalence of VRE in both groups was similar [3/204 (1.5%) in the CDTA arm and 1/97 (1.0%) in the high-risk arm; P = 1.0, Fisher's exact test]. Prevalence of risk factors for VRE colonisation, including age, duration of hospitalisation, exposure to antibiotics, exposure to surgical procedures, presence of malignancy and diabetes mellitus was similar in both groups (P > 0.05). Only renal failure (P < 0.05) was more common in the high-risk group. All 4 isolates of VRE identified were genetically distinct by variable number tandem repeat (VNTR) typing; 3 were Enterococcus faecium (2 with the vanB gene, 1 with vanA) and one E. faecalis. CONCLUSION: Less than 2% of our high-risk patients are VRE carriers. In-hospital VRE screening using stools sent for CDTA is a simple, reasonable surrogate for screening individual high-risk patients as the patient risk profile is similar and the yield comparable in a low-prevalence setting.

Predominance of Candida tropicalis bloodstream infections in a Singapore teaching hospital.

An 18-month epidemiologic investigation of Candida bloodstream infections in a Singapore hospital identified 52 candidemic patients: 36% of whose infections were caused by C. tropicalis, 29% were due to C. albicans, 10% with C. parapsilosis and 21% involved C. glabrata. A predominant clonal C. tropicalis strain was demonstrated. No association with ICU stay, prior exposure to fluconazole/broad-spectrum antibiotics or increased mortality was found in this apparent shift towards non-C. albicans Candida species as the primary agents of candidemia.

Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain.

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 muM and 40 muM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10(4) CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Specific detection of enterovirus 71 directly from clinical specimens using real-time RT-PCR hybridization probe assay.

Enterovirus 71 (EV71) is one of the main causative agents of hand, foot and mouth disease (HFMD) in young children. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. Thus, rapid detection of the virus is required to enable measures to be implemented in preventing widespread transmission. Based on primers and probes targeting at the VP1 region, a real-time reverse-transcriptase polymerase chain reaction (RT-PCR) hybridization probe assay was developed for specific detection of EV71 from clinical specimens. Quantitative analysis showed that the assay was able to detect as low as 5 EV71 viral copies and EV71 was detected from 46 of the 55 clinical specimens obtained from pediatric patients suffering from HFMD during the period from 2000 to 2003 in Singapore. This study showed that the single tube real-time RT-PCR assay developed in this study can be applied as a rapid and sensitive method for specific detection of EV71 directly from clinical specimens.

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis.

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.

Direct identification of Pseudomonas aeruginosa from blood culture bottles by PCR-enzyme linked immunosorbent assay using oprI gene specific primers.

A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for direct identification of Pseudomonas aeruginosa from positive BACTEC blood culture bottles. PCR primers were designed to target a 249 bp sequence of the oprI gene in P. aeruginosa. Biotin-labeled probe (PA3) targeted to the species-specific motif were hybridized to the digoxigenin-labeled amplified products from P. aeruginosa and captured on streptavidin-coated microtiter plates. The specificity of the assay using the PA3 probe was investigated with a range of microorganisms, which are commonly isolated from blood culture bottles serving as negative controls. The PCR-ELISA assay was shown to be highly specific for the identification of P. aeruginosa and was 10-fold more sensitive than an agarose gel-based detection method using the same pair of primers, with a detection limit at 10 fg of template. The PCR-ELISA assay developed in this study is 100% sensitive and 100% specific as it correctly identified all 73 positive and 42 negative controls as well as 25 double blind clinical samples. It significantly reduces the time needed for the identification of P. aeruginosa from positive BACTEC blood cultures bottles from 2-3 days to 6-8h.

Rapid detection of codon 460 mutations in the UL97 gene of ganciclovir-resistant cytomegalovirus clinical isolates by real-time PCR using molecular beacons.

A rapid real-time polymerase chain reaction (PCR) assay using molecular beacons has been developed for the simultaneous detection of wild-type and mutant strains of cytomegaloviruses (CMV) with respect to codon 460 of the UL97 gene has been developed. The molecular beacons were designed to complement the wild-type codon 460 or the mutant sequence arising from a single base-pair difference (point mutation). Discrimination between wild-type and mutant templates was demonstrated as the beacons did not generate fluorescence with their respective mismatch targets but only with those that they were designed to perfectly anneal with. Samples that harbor mixed populations of CMV could also be readily recognized. Applied to a small number of clinical samples, the retrospective screening by this assay are in general concordance with that obtained by PCR-RFLP. Using molecular beacons strategy, codon 460 mutation was detected in ten out o the total number of 40 samples, whereas the latter method identified nine samples as containing the mutation. The discrepant result arose from the genotyping of one clinical sample as mixed (containing both wild-type and mutant CMV strains) by molecular beacons but as wild-type by PCR-RFLP, suggesting that this real-time strategy is possibly more sensitive for mutation analysis.

Fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae from Asian countries: ANSORP study.

Seventeen clinical isolates of Streptococcus pneumoniae showing reduced susceptibility to ciprofloxacin (MIC >/= 4 micro g/ml) collected from eight different Asian countries were analyzed by antimicrobial susceptibility, serotyping, pulsed-field gel electrophoresis (PFGE), and DNA sequencing of the quinolone resistance-determining regions (QRDRs) in gyrA, gyrB, parC, and parE. All isolates but one showed more than one amino acid alteration in QRDRs of four responsible genes. Ile460 --> Val in parE was the most common mutation. Data suggest that Lys137 --> Asn in parC may be a primary step in the development of high-level and multiple FQ resistance. An additional mutation of Ser81 --> Phe in gyrA resulted in high-level resistance to ciprofloxacin, levofloxacin, and gatifloxacin, whereas Ser79 --> Phe in parC may exert an important role in the development of moxifloxacin resistance. Two novel amino acid changes in gyrB, Ala390 --> Val and Asn423 --> Thr, were found. Data from PFGE suggest an introduction and local spread of multiple resistant Spain(23F)-1 clone in Hong Kong, but isolates from other Asian countries were not related to this clone.

High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia (an ANSORP study).

A total of 685 clinical Streptococcus pneumoniae isolates from patients with pneumococcal diseases were collected from 14 centers in 11 Asian countries from January 2000 to June 2001. The in vitro susceptibilities of the isolates to 14 antimicrobial agents were determined by the broth microdilution test. Among the isolates tested, 483 (52.4%) were not susceptible to penicillin, 23% were intermediate, and 29.4% were penicillin resistant (MICs >/= 2 mg/liter). Isolates from Vietnam showed the highest prevalence of penicillin resistance (71.4%), followed by those from Korea (54.8%), Hong Kong (43.2%), and Taiwan (38.6%). The penicillin MICs at which 90% of isolates are inhibited (MIC(90)s) were 4 mg/liter among isolates from Vietnam, Hong Kong, Korea, and Taiwan. The prevalence of erythromycin resistance was also very high in Vietnam (92.1%), Taiwan (86%), Korea (80.6%), Hong Kong (76.8%), and China (73.9%). The MIC(90)s of erythromycin were >32 mg/liter among isolates from Korea, Vietnam, China, Taiwan, Singapore, Malaysia, and Hong Kong. Isolates from Hong Kong showed the highest rate of ciprofloxacin resistance (11.8%), followed by isolates from Sri Lanka (9.5%), the Philippines (9.1%), and Korea (6.5%). Multilocus sequence typing showed that the spread of the Taiwan(19F) clone and the Spain(23F) clone could be one of the major reasons for the rapid increases in antimicrobial resistance among S. pneumoniae isolates in Asia. Data from the multinational surveillance study clearly documented distinctive increases in the prevalence rates and the levels of antimicrobial resistance among S. pneumoniae isolates in many Asian countries, which are among the highest in the world published to date.

Rapid detection of Klebsiella pneumoniae from blood culture bottles by real-time PCR.

A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.

Role of clinical judgment in the application of a nucleic acid amplification test for the rapid diagnosis of pulmonary tuberculosis.

BACKGROUND: Several nucleic acid amplification (NAA) tests for Mycobacterium tuberculosis (MTB) have been licensed for the rapid diagnosis of active pulmonary tuberculosis (PTB) in respiratory secretions. There is uncertainty however regarding the practical application of these tests in clinical decision making. OBJECTIVE: To evaluate the utility of the COBAS AMPLICOR assay (Roche Diagnostics; Singapore) for MTB as applied by specialists for the rapid diagnosis of PTB in the routine clinical setting. DESIGN: A prospective study of consecutive patients suspected of PTB and tested with the AMPLICOR assay under the care of respiratory physicians. The final diagnosis was based on all relevant clinical information after at least 3 months of follow-up. Accuracy of the NAA test was compared with that of the initial expectant treatment. Expectant treatment was based on an integrated approach that incorporated clinical evaluation with results of direct smear and NAA tests. RESULTS: The incidence of PTB in 168 patients was 32%. The basis for expectant treatment of PTB was positive smear result in 47%, clinical suspicion in 26%, and positive AMPLICOR result in 23%. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the AMPLICOR test were 77%, 100%, 99%, 90%, and 93%, respectively. In comparison, they were 96%, 97%, 94%, 98%, and 97%, respectively, for the integrated clinical approach. CONCLUSIONS: In the rapid diagnosis of PTB, the clinical judgment of specialists augmented the utility of the NAA test: (1) specialists selected patients with high-to-moderate pretest probabilities, (2) they commenced treatment promptly on a positive NAA test result, and (3) they were willing to start treatment in some patients on the basis of high clinical suspicion despite negative smear and negative NAA test results.

Community-acquired methicillin-resistant Staphylococcus aureus infection in Singapore is usually "healthcare associated".

OBJECTIVE: To assess the frequency of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infections. SETTING: A teaching hospital in Singapore. METHODS: Prospectively collected surveillance data were reviewed during a 1-year period to determine the extent and origin of community-acquired MRSA infections. RESULTS: Whereas 32% of 383 MRSA infections were detected less than 48 hours after hospital admission and would, by convention, be classified as "community acquired," all but one of these were among patients who had been exposed to outpatient centers including dialysis or chemotherapy clinics, visiting nurses, community hospitals, or all three. CONCLUSIONS: With health care increasingly being delivered in an outpatient setting, community-acquired MRSA infections are often acquired in hospital-related sites and most may be more accurately described as "healthcare acquired." Infection control measures need to move beyond the traditional paradigm of acute care hospitals to effectively control the spread of resistant pathogens.

What is the optimal approach for using a direct amplification test in the routine diagnosis of pulmonary tuberculosis? A preliminary assessment.

OBJECTIVE: The aim of this study was to determine the most appropriate strategy for the rapid diagnosis of pulmonary tuberculosis (PTB) using a nucleic acid amplification (NAA) test. METHODOLOGY: This was a prospective study of 128 adult patients in whom respiratory secretions were tested for Mycobacterium tuberculosis by the AMPLICOR assay. The basis for starting PTB treatment was noted for each patient. The optimal approach was determined by using Bayes' theorem to compare different combinations of pretest probability, smear results with the AMPLICOR test. RESULTS: The incidence of PTB was 15.6%. In only one patient was treatment for PTB commenced because of a positive AMPLICOR result. The rest were managed according to the conventional approach which relied upon clinical judgment and direct smear. The optimal approach was to treat patients with high or intermediate pretest risk for PTB who returned positive AMPLICOR tests. The overall accuracies of the conventional approach, AMPLICOR test and optimal approach were 89.8, 95.3 and 96.1%, respectively. CONCLUSION: This small study suggests that NAA testing be limited to patients with high or intermediate pretest risk of PTB. In this group, positive results demand treatment while the management of those with negative results still relies on clinical judgment.