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RATIONALE: There is currently no available test for monitoring the effect of treatment of latent tuberculosis infection (LTBI) to indicate cure or predict risk of subsequent progression to disease. OBJECTIVE: We used the T-SPOT.TB assay, which measures T-cell interferon-gamma responses to the Mycobacterium tuberculosis-specific peptides early secretory antigenic target 6-kD protein (ESAT-6) and culture filtrate protein 10 (CFP-10), to determine the effect of LTBI treatment on these responses. METHODS: A total of 226 tuberculosis contacts with positive T-SPOT.TB results underwent repeat testing on LTBI treatment completion. The majority (96%) received 6 months of isoniazid. The pre- and post-treatment T-SPOT.TB results were analyzed according to the combined and separate responses to ESAT-6 and CFP-10 antigens. RESULTS: The T-SPOT.TB reverted to negative in 85 (37.6%) contacts at treatment completion. Treatment had a significant effect on the response to CFP-10 (p < 0.001; reversion rate, 48.6%), but not on the response to ESAT-6 (p = 0.081; reversion rate, 21.6%). The median number of spot-forming cells (SFCs)/2.5 x 10(5) peripheral blood mononuclear cells (PBMCs) pre- and post-treatment was 6 versus 4.5 for ESAT-6 (p = 0.116) and 11 versus 4 for CFP-10 (p < 0.001). There was a significant difference between the change (fall) in the pre- and post-treatment responses to CFP-10 (6 SFCs/2.5 x 10(5) PBMCs) and ESAT-6 (0 SFCs/2.5 x 10(5) PBMCs; p < 0.001). Significantly different age-related T-cell responses to the two antigens were found. CONCLUSION: LTBI treatment had a differential effect on T-cell responses to ESAT-6 and CFP-10 as measured by the T-SPOT.TB. The quantitative response to CFP-10 may be a useful LTBI treatment-monitoring tool. [ABSTRACT FROM AUTHOR]

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.

Wastewater discharged from clinical isolation and general wards at two hospitals in Singapore was examined to determine the emerging trends of antibiotic resistance (AR). We quantified the concentrations of 12 antibiotic compounds by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibiotic-resistant bacteria (ARB), the class 1 integrase gene (intI1), and 16 antibiotic resistance genes (ARGs) that confer resistance to 10 different clinically relevant antibiotics. A subset of 119 antibiotic-resistant isolates were phylogenetically classified and tested for the presence of ARGs encoding resistance to β-lactam antibiotics (bla NDM , bla KPC , bla SHV , bla CTX-M ), amikacin [aac(6')-Ib], co-trimoxazole (sul1, sul2, dfrA), ciprofloxacin (qnrA, qnrB), and the intI1 gene. Among these resistant isolates, 80.7% were detected with intI1 and 66.4% were found to carry at least 1 of the tested ARGs. Among 3 sampled locations, the clinical isolation ward had the highest concentrations of ARB and the highest levels of ARGs linked to resistance to β-lactam (bla KPC ), co-trimoxazole (sul1, sul2, dfrA), amikacin [aac(6')-Ib], ciprofloxacin (qnrA), and intI1 We found strong positive correlations (P < 0.05) between concentrations of bacteria resistant to meropenem, ceftazidime, amikacin, co-trimoxazole, and ciprofloxacin and abundances of bla KPC , aac(6')-Ib, sul1, sul2, dfrA, qnrA, and intI1 genes., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The tuberculin skin test (TST) and interferon gamma (IFN-γ) release assays (IGRAs) are used as adjunctive tests for the evaluation of suspected cases of active tuberculosis (TB). However, a positive test does not differentiate latent from active TB. We investigated whether flow cytometric measurement of novel combinations of intracellular cytokines and surface makers on CD4 T cells could differentiate between active and latent TB after stimulation with Mycobacterium tuberculosis-specific proteins. Blood samples from 60 patients referred to the Singapore Tuberculosis Control Unit for evaluation for active TB or as TB contacts were stimulated with purified protein derivative (PPD), ESAT-6 and CFP-10, or heparin-binding hemagglutinin (HBHA). The CD4 T cell cytokine response (IFN-γ, interleukin-2 [IL-2], interleukin-17A [IL-17A], interleukin-22 [IL-22], granulocyte-macrophage colony-stimulating factor [GM-CSF], and tumor necrosis factor alpha [TNF-α]) and surface marker expression (CD27, CXCR3, and CD154) were then measured. We found that the proportion of PPD-specific CD4 T cells, defined as CD154(+) TNF-α(+) cells that were negative for CD27 and positive for GM-CSF, gave the strongest discrimination between subjects with latent and those with active TB (area under the receiver operator characteristic [ROC] curve of 0.9277; P < 0.0001). Also, the proportions and absolute numbers of HBHA-specific CD4 T cells were significantly higher in those with latent TB infection, particularly CD154(+) TNF-α(+) IFN-γ(+) IL-2(+) and CD154(+) TNF-α(+) CXCR3(+). Finally, we found that the ratio of ESAT-6- and CFP-10-responding to HBHA-responding CD4 T cells was significantly different between the two study populations. In conclusion, we found novel markers of M. tuberculosis-specific CD4 cells which differentiate between active and latent TB., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: Clostridium difficile infection (CDI) is an increasingly recognized nosocomial infection in Singapore. Surveillance methods include laboratory reporting of Clostridium difficile toxin assays (CDTA) or use of International Classification of Diseases, 9(th) Revision (ICD-9) discharge code 008.45. Previous US studies showed good correlation between CDTA and ICD-9 codes. However, the use of ICD-9 codes for CDI surveillance has not been validated in other healthcare settings., Methods: We compared CDI rates based on CDTA to ICD-9 codes for all discharges in 2007 from our hospital to determine sensitivity and specificity of ICD-9 codes. Demographic and hospitalization data were analyzed to determine predictors for missing ICD-9 codes., Results: During 2007, there were 56,352 discharges. Of these, 268 tested CDTA-positive but only 133 were assigned the CDI ICD-9 code. A total of 141 discharges had the ICD-9 code; 8 were CDTA-negative, the rest were CDTA-positive. Community-acquired CDI accounted for only 3.2% of cases. The sensitivity and specificity of ICD-9 codes compared to CDTA were 49.6% and 100% respectively. Concordance between CDTA and ICD-9 codes was 0.649 (p<.001). Comparing concordant patients (CDTA+/ICD9+) to discordant patients (CDTA+/ICD9-), concordant patients were more likely to be over 50 years of age (OR 3.49, 95% CI 1.66-7.34, p = .001) and have shorter time from admission to testing (OR 0.98, 95% CI 0.97-0.99, p = .009)., Discussion: Unlike previous studies in the US, ICD-9 codes substantially underestimate CDI in Singapore compared to microbiological data. Older patients with shorter time to testing were less likely to have missing ICD-9 codes.

The hypothesis that T-cell interferon-gamma responses to Mycobacterium tuberculosis-specific antigens decline as disease activity diminishes with tuberculosis (TB) treatment has generated interest in the interferon-gamma release assays (IGRAs) as treatment-monitoring tools. We studied the effect of TB treatment on these responses as measured by the QuantiFERON-TB Gold In-tube (QFT-IT) and T-SPOT.TB assays. 275 sputum culture-positive, HIV-uninfected pulmonary TB patients were tested with QFT-IT and T-SPOT.TB at baseline, treatment completion and 6 months thereafter. The QFT-IT was also performed at the end of the intensive phase. The time-treatment effect on the qualitative and quantitative IGRA results was determined. There were significant declines in the positivity rates and quantitative results of both IGRAs with treatment. The QFT-IT positivity rate was significantly lower than the T-SPOT.TB. The test reversion rate was significantly different for the two assays (13.9% for T-SPOT.TB versus 39.2% for QFT-IT). 79% and 46% tested positive with T-SPOT.TB and QFT-IT respectively at 6 months post-treatment completion. The kinetics of the quantitative responses was not significantly different between subjects with and without risk factors for disease relapse. That a substantial proportion of patients remained test-positive after TB treatment would suggest a limited role of IGRAs as treatment monitoring tools.

Sixty-six animal workers received primary rabies vaccination with purified Vero cell vaccine (PVRV, Verorab). One year later, 26 (39%) demonstrated antibody titers below the recommended minimum of 0.5 IU/ml, and required a booster. All 15 of a separate group reporting primary vaccination with at least one booster had titers above 0.5 IU/ml 1 year later, demonstrating long-term boostable immunity. Rabies antibody titers should be checked 1 year after primary rabies vaccination in persons at high risk of frequent rabies exposure. If access to serological surveillance is unavailable, such high-risk individuals should receive booster vaccination., (Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Dengue reemerged in Mauritius in 2009 after an absence of >30 years, and >200 cases were confirmed serologically. Molecular studies showed that the outbreak was caused by dengue virus type 2. Phylogenetic analysis of the envelope gene identified 2 clades of the virus. No case of hemorrhagic fever was recorded.

Background: Surveillance for latent tuberculosis in high-risk groups such as healthcare workers is limited by the nonspecificity of the tuberculin skin test (TST) in BCG-vaccinated individuals. The Mycobacterium tuberculosis antigen-specific interferon-gamma release assays (IGRAs) show promise for more accurate latent tuberculosis detection in such groups., Objective: To compare the utility of an IGRA, the T-SPOT.TB assay, with that of the TST in healthcare workers with a high rate of BCG vaccination., Methods: Two hundred seven medical students from 2 consecutive cohorts underwent the T-SPOT.TB test and the TST in their final year of study. Subjects with negative baseline test results underwent repeat testing after working for 1 year as junior physicians in Singapore's public hospitals., Results: The baseline TST result was an induration 10 mm or greater in diameter in 177 of the 205 students who returned to have their TST results evaluated (86.3%), while the baseline T-SPOT.TB assay result was positive in 9 (4.3%) of the students. Repeat T-SPOT.TB testing in 182 baseline-negative subjects showed conversion in 9 (4.9%). A repeat TST in 18 subjects with baseline-negative TST results did not reveal any TST result conversion., Conclusions: The high rate of positive baseline TST results in our BCG-vaccinated healthcare workers renders the TST unsuitable as a surveillance tool in this tuberculosis risk group. Use of an IGRA has enabled the detection and treatment of latent tuberculosis in this group. Our T-SPOT.TB conversion rate highlights the need for greater tuberculosis awareness and improved infection control practices in our healthcare institutions.

The objective was to compare the quantitative T-cell responses measured by the commercial interferon-gamma (IFNgamma) release assays (IGRAs) in active and latent tuberculosis (TB) states. T-cell responses of culture-proven TB cases were compared with those of contacts with positive IGRA results and tuberculin skin tests >or= 15 mm. T-SPOT.TB results in 270 active TB cases and 183 community contacts showed the median spot-forming cells (SFCs) above negative control/2.5 x 10(5) peripheral blood mononuclear cells to be 27 (-1 to 203) vs 10 (-2 to 174) in response to ESAT-6 (p < 0.001); and 37 (0 to 293) vs 13 (0 to 225) to CFP-10 (p < 0.001). The median IFNgamma levels (antigen minus nil control) as measured by QuantiFERON-TB Gold In-tube in 270 cases and 142 contacts in congregate settings was 2.3 IU/ml (-0.58 to 31.44) vs 1.7 IU/ml (0.35 to 26.51, p = 0.98). Quantitative T-cell responses as measured by the T-SPOT.TB may indicate mycobacterial burden and disease activity, but cannot be used to discriminate active from latent TB.

There are few head-to-head comparisons of the commercial gamma interferon release assays (GIRAs). We compared the performance of the T-SPOT.TB and QuantiFERON-TB Gold In-Tube (QFT-IT) assays in patients with culture-proven pulmonary tuberculosis. Blood was drawn for both assays within 14 days of starting antituberculosis treatment. The QFT-IT indeterminate rate was 3.5%; the T-SPOT.TB failure rate was 1.4%. There was poor agreement between the GIRAs (kappa = 0.257) among the 270 patients with valid results for both tests. The sensitivities of the T-SPOT.TB and QFT-IT assays were 94.1 and 83.0%, respectively, with a significant difference in the performance of the assays (P = 0.001 [McNemar test]). Factors independently associated with indeterminate QFT-IT results were an age of >or=60 years (odds ratio [OR] 11.18, 95% confidence interval [CI] = 1.841 to 67.823, P = 0.009), female sex (OR = 7.47, 95% CI = 1.517 to 36.733, P = 0.013) and non-Chinese (i.e., Indian or Malay) race (OR = 7.89, 95% CI = 1.585 to 39.267, P = 0.012). The QFT-IT assay was significantly less sensitive in patients >or=60 years old (OR = 0.41, 95% CI = 0.181 to 0.918, P = 0.030) and in Indian compared to Chinese patients (OR = 0.27, 95% CI = 0.073 to 0.990, P = 0.048). The T-SPOT.TB assay was significantly less sensitive in Malay (OR = 0.23, 95% CI = 0.063 to 0.815, P = 0.023) and Indian patients (OR = 0.09, 95% CI = 0.017 to 0.429, P = 0.003) compared to Chinese patients. The performance of both assays was not significantly altered in diabetics. The diminished sensitivity of the GIRAs in persons of Malay and Indian race merits further study.

The laboratory diagnosis of dengue has largely relied on serological assays, although many different RT-PCR protocols have been reported. Owing to its limited use, the value of RT-PCR in the clinical laboratory has not been fully evaluated. During the outbreak of severe acute respiratory syndrome (SARS) in Singapore in 2003, RT-PCR to detect dengue viral RNA was used as a rapid diagnostic tool to differentiate dengue from SARS among patients who presented to a hospital designated to manage and quarantine SARS cases. A total of 343 results for RT-PCR and 439 results for serology were analysed and compared with the final discharge diagnosis. Our experience indicates that RT-PCR for dengue can be set up rapidly in a clinical laboratory, with very sensitive and specific results for the diagnosis of dengue, particularly in the first 5 days from onset of symptoms.

The severe acute respiratory syndrome (SARS)-associated coronavirus causes severe disease, is transmissible to the community and there is no effective prophylaxis or treatment--perhaps fulfilling the criteria for biohazard group 3 or 4. The recommendation to use Biosafety Level (BSL)3 practices within a BSL2 environment appears to have been a practical decision based on available resources; most diagnostic laboratories operate at BSL2. Safety is achieved with controls in administration, engineering and personal protective equipment/behaviour. At the heart of every safety policy is a risk assessment based on the exact manipulations employed. Excessive administrative and engineering controls are less important than the training and personal attitudes, abilities and understanding of the staff. The SARS outbreak focused our attention on the safety aspects of common mundane tasks, such as decapping blood tubes. Laboratories often claim they follow certain practices but casual observation does not always support these claims. Guidelines differed and created uncertainty. This was stressful for laboratory staff held accountable for their implementation. Attempts to categorise risks and their management into neatly wrapped parcels are attractive, but closer inspection reveals a subjective element that allows doubt to creep in with varying interpretations of the literature. Staff most at risk were those handling respiratory samples. Staff receiving samples via pneumatic tubes had least control over their exposure and were potentially exposed to aerosols from leaking samples. Risk assessment remains a balance between cost and benefit.

We document the absence of carriage of Neisseria meningitidis W-135 of the sequence type 11 in returning pilgrims after the Hajj 2002. This finding contrasts with the 15% carriage rate we previously reported in pilgrims returning from the Hajj 2001. The epidemiology of carriage may be changing or may have been controlled by vaccination and a policy of administering antibiotics to pilgrims from countries with a high incidence of meningococcal disease.

Background: Moslems from all over the world go to Mecca and Medina in Saudi Arabia for two types of pilgrimage: the major pilgrimage (hajj) and the minor (umra). An international outbreak of meningococcal disease with serogroup W-135 occurred in association with hajj pilgrimage in the years 2000 and 2001, and it has been shown that pharyngeal carriage of a single W-135 strain was high in returning hajj pilgrims. We investigated the meningococcal carriage in umra pilgrims to determine the extent of circulation of this strain, during the minor pilgrimage., Method: Tonsillopharyngeal swabs were taken from umra returnees. Serogrouping and pulsed field gel electrophoresis were performed on all meningococcal isolates. Subjects were questioned about the occurrence of symptoms of upper respiratory tract infection and use of antibiotics during the pilgrimage., Results: were compared with those previously reported in hajj pilgrims. Results: We enrolled 160 pilgrims returning from the umra pilgrimage in 2001. The meningococcal carriage rate was 1.3%, which is significantly lower compared with the hajj pilgrimage (17%; p<0.001). None of the umra pilgrims carried serogroup W-135, whereas 90% of the isolates in returning hajj pilgrims were Neisseria meningitidis W-135., Conclusions: Meningococcal carriage during the umra pilgrimage was significantly lower compared with the hajj pilgrimage in the year 2001. No carriage of N. meningitidis W-135 was documented in umra pilgrims, whereas this was the predominant serogroup in hajj pilgrims. Public health measures to reduce the potential introduction of N. meningitidis W-135 into the countries of origin of returning pilgrims need to be prioritized for the hajj pilgrimage.

After an outbreak of meningococcal disease caused by Neisseria meningitidis W135, associated with the Hajj pilgrimage in 2001, 15% of returning vaccinated pilgrims carried a single W135 clone, and 55% were still carriers 6 months later. Transmission to 8% of their unvaccinated household contacts occurred within the first few weeks, but no late transmission took place. Public health interventions are needed to protect household contacts.

We present data on the prevalence of Candida species isolated from inpatients in three Singapore hospitals and from vaginal samples collected at community clinics. Strikingly, approximately 20% of the isolates from blood and vaginal samples were fluconazole-resistant species. By analyzing species-specific gene sequence signatures, we identified Candida dubliniensis from both groups of patients.

Collaboration between clinicians and microbiologists revealed that many patients with subsequently proven urinary tract infection (UTI) present with symptoms suggestive of chest infection. A retrospective analysis was performed on patients over 50 years old with community acquired bacteraemic UTI proven by blood cultures. The main presenting features were confusion (30%), cough (27%), dyspnoea (28%) and new urinary symptoms (20%). The initial clinical diagnosis was UTI in 43% and chest infection in 24%. Chest infection was diagnosed more often in those over 70 years old than those aged 50-70 years old (chi 2 = 7.2, p = 0.007). The majority had pyuria but less than half of the urine samples arrived in the laboratory on the day of admission, fewer from the older patients than the younger (chi 2 = 2.57, p = 0.10). These results demonstrate that UTI frequently presents with respiratory features and that the diagnosis of UTI is often delayed. Sampling the urine with a catheter may be justified to enable diagnosis on the day of admission.

We report a marked increase in the rate of notifications of tuberculosis in young adults in the London Borough of Lambeth. Analysis of notifications made to the Proper Officer over a 10-year period showed that the age specific notification rate in the cohort aged 20-44 years increased from 30/100,000 in 1983 to 51/100,000 in 1992. Analysis of St. Thomas' Hospital laboratory records of patients seen between 1984 and 1991 from whom Mycobacterium tuberculosis was isolated showed an increase in the number of patients of African origin from five in the first half of the study period (1984-7) to 25 in the second half (1988-91): 21 of these 25 had immigrated into England within 4 years of their illness. This finding is being further investigated in a prospective study of ethnicity, travel history and date of immigration of Lambeth residents notified with tuberculosis.