Epidemiologic versus genetic relatedness to define an outbreak-associated uropathogenic Escherichia coli group.
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Disease Outbreaks↗
Biomedical subjects
Lee W Riley
Publications and source records attributed to Lee W Riley.
Bacteriophage-based assays for the rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a meta-analysis.
OBJECTIVE: To summarize, using meta-analysis, the accuracy of bacteriophage-based assays for the detection of rifampicin resistance in Mycobacterium tuberculosis. METHODS: By searching multiple databases and sources we identified a total of 21 studies eligible for meta-analysis. Of these, 14 studies used phage amplification assays (including eight studies on the commercial FASTPlaque-TB kits), and seven used luciferase reporter phage (LRP) assays. Sensitivity, specificity, and agreement between phage assay and reference standard (e.g. agar proportion method or BACTEC 460) results were the main outcomes of interest. RESULTS: When performed on culture isolates (N=19 studies), phage assays appear to have relatively high sensitivity and specificity. Eleven of 19 (58%) studies reported sensitivity and specificity estimates > or =95%, and 13 of 19 (68%) studies reported > or =95% agreement with reference standard results. Specificity estimates were slightly lower and more variable than sensitivity; 5 of 19 (26%) studies reported specificity <90%. Only two studies performed phage assays directly on sputum specimens; although one study reported sensitivity and specificity of 100 and 99%, respectively, another reported sensitivity of 86% and specificity of 73%. CONCLUSIONS: Current evidence is largely restricted to the use of phage assays for the detection of rifampicin resistance in culture isolates. When used on culture isolates, these assays appear to have high sensitivity, but variable and slightly lower specificity. In contrast, evidence is lacking on the accuracy of these assays when they are directly applied to sputum specimens. If phage-based assays can be directly used on clinical specimens and if they are shown to have high accuracy, they have the potential to improve the diagnosis of MDR-TB. However, before phage assays can be successfully used in routine practice, several concerns have to be addressed, including unexplained false positives in some studies, potential for contamination and indeterminate results.
Antibiotics, Antitubercular↗
Mycobacterium tuberculosis infection in health care workers in rural India: comparison of a whole-blood interferon gamma assay with tuberculin skin testing.
CONTEXT: Mycobacterium tuberculosis infection in health care workers has not been adequately studied in developing countries using newer diagnostic tests. OBJECTIVES: To estimate latent tuberculosis infection prevalence in health care workers using the tuberculin skin test (TST) and a whole-blood interferon gamma (IFN-gamma) assay; to determine agreement between the tests; and to compare their correlation with risk factors. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional comparison study of 726 health care workers aged 18 to 61 years (median age, 22 years) with no history of active tuberculosis conducted from January to May 2004, at a rural medical school in India. A total of 493 (68%) of the health care workers had direct contact with patients with tuberculosis and 514 (71%) had BCG vaccine scars. INTERVENTIONS: Tuberculin skin testing was performed using 1-TU dose of purified protein derivative RT23, and the IFN-gamma assay was performed by measuring IFN-gamma response to early secreted antigenic target 6, culture filtrate protein 10, and a portion of tuberculosis antigen TB7.7. MAIN OUTCOME MEASURES: Agreement between TST and the IFN-gamma assay, and comparison of the tests with respect to their association with risk factors. RESULTS: A large proportion of the health care workers were latently infected; 360 (50%) were positive by either TST or IFN-gamma assay, and 226 (31%) were positive by both tests. The prevalence estimates of TST and IFN-gamma assay positivity were comparable (41%; 95% confidence interval [CI], 38%-45% and 40%; 95% CI, 37%-43%, respectively). Agreement between the tests was high (81.4%; kappa = 0.61; 95% CI, 0.56-0.67). Increasing age and years in the health profession were significant risk factors for both IFN-gamma assay and TST positivity. BCG vaccination had little impact on TST and IFN-gamma assay results. CONCLUSIONS: Our study showed high latent tuberculosis infection prevalence in Indian health care workers, high agreement between TST and IFN-gamma assay, and similar association between positive test results and risk factors. Although TST and IFN-gamma assay appear comparable in this population, they have different performance and operational characteristics; therefore, the decision to select one test over the other will depend on the population, purpose of testing, and resource availability.
Adult↗
Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor.
Gene expression systems that allow the regulation of bacterial genes during an infection are valuable molecular tools but are lacking for mycobacterial pathogens. We report the development of mycobacterial gene regulation systems that allow controlling gene expression in fast and slow-growing mycobacteria, including Mycobacterium tuberculosis, using anhydrotetracycline (ATc) as inducer. The systems are based on the Escherichia coli Tn10-derived tet regulatory system and consist of a strong tet operator (tetO)-containing mycobacterial promoter, expression cassettes for the repressor TetR and the chemical inducer ATc. These systems allow gene regulation over two orders of magnitude in Mycobacterium smegmatis and M.tuberculosis. TetR-controlled gene expression was inducer concentration-dependent and maximal with ATc concentrations at least 10- and 20-fold below the minimal inhibitory concentration for M.smegmatis and M.tuberculosis, respectively. Using the essential mycobacterial gene ftsZ, we showed that these expression systems can be used to construct conditional knockouts and to analyze the function of essential mycobacterial genes. Finally, we demonstrated that these systems allow gene regulation in M.tuberculosis within the macrophage phagosome.
Animals↗
Rapid detection of Neisseria meningitidis in cerebrospinal fluid by one-step polymerase chain reaction of the nspA gene.
A polymerase chain reaction (PCR) protocol for the rapid detection of meningococcal DNA in cerebrospinal fluid (CSF) was developed and optimized. A set of primers based on Neisseria surface protein A (nspA) gene sequence was designed to amplify a 481-bp product specific for N. meningitidis. We tested 85 N. meningitidis strains obtained from patients with meningococcal meningitis and 112 CSF samples from patients with suspected meningococcal meningitis. No amplification of the nspA gene was observed from other Neisseriaceae species (except from N. gonorrhoeae) and from other bacteria frequently associated with meningitis. N. meningitidis belonging to different serogroups yielded the same product after PCR amplification. The sensitivity and specificity of our protocol was determined by comparing the results of specific amplification of nspA gene by PCR reaction (nspA-PCR) with those obtained by conventional methods. All positive samples by conventional methods were confirmed by nspA-PCR, whereas 48% of negative samples after culture and latex agglutination tested positive by nspA-PCR. The use of nspA-PCR proved to be a rapid diagnostic method, in which sensitivity and specificity may not be affected by prior antibiotic treatment.
Base Sequence↗
Hepatitis C virus (HCV) 5'NC sequence variants and their association with hepatitis C risk groups.
BACKGROUND: Genotype distribution of hepatitis C virus (HCV) is relatively uniform in Prague, Czech Republic. Unlike the other developed countries where HCV genotype 3 is increasingly associated with injection drug users (IDU), subtype 1b remains the most prevalent HCV subtype in Prague, regardless of risk factors. OBJECTIVE: We wished to determine if subtype 1b strains could be further differentiated by comparing the conserved 5'NC sequences of the strains infecting IDU and non-IDU populations. STUDY DESIGN: All prospectively collected serum samples that were HCV RNA positive were genotyped according to the 5'NC and NS5b regions. All 5'NC sequences were further analyzed for new mutations and these data were compared to patient epidemiologic information. RESULTS: We found eight 5'NC sequence variants among 96 specimens tested. Further analysis of subtype 1b strains showed that a variant with a nucleotide insertion at -138 positions was found only among non-IDU subjects, while the variant with T-->C substitution at -94 was found only among the IDUs. CONCLUSIONS: These observations suggest that the current HCV transmission between the IDU and non-IDU populations is uncommon, and may reflect the beginning of divergence of HCV genotypes in the IDU population in Prague.
5' Untranslated Regions↗
Exogenous reinfection in tuberculosis.
Whether the development of active tuberculosis in people with previous tuberculous infection represents an episode of endogenous reactivation or exogenous reinfection has been debated for decades. Articles proposing the unitary concept of pathogenesis of tuberculosis in the 1960s initiated a period in which reinfection was considered to be an uncommon cause of tuberculosis. To review evidence demonstrating the occurrence of tuberculosis due to exogenous reinfection, we did a literature search covering publications from 1966 until the present, and found that there was substantial evidence--both experimental and epidemiological--supporting the role of exogenous reinfection in tuberculosis. However, only models based on estimates of the annual risk of infection and the incidence of tuberculosis provided a quantitative estimate of the relative contribution of exogenous reinfection to the burden of tuberculosis. Better estimates of the contribution of exogenous reinfection to new cases of tuberculosis may need to be considered in tuberculosis control strategies.
Humans↗
Mupirocin for controlling methicillin-resistant Staphylococcus aureus: lessons from a decade of use at a university hospital.
BACKGROUND: From 1990 to 1995 at Hospital Universitário Clementino Fraga Filho, patients colonized or infected with methicillin-resistant Staphylococcus aureus (MRSA) were treated with mupirocin to eliminate MRSA carriage. In 1995, 65% of MRSA patients at this hospital had mupirocin-resistant isolates. Starting in 1996, mupirocin use was restricted to patients colonized, but not infected, with MRSA. OBJECTIVES: To describe the use of mupirocin for controlling MRSA over a decade and to analyze the molecular epidemiology of mupirocin-resistant MRSA infections at this hospital. SETTING: A 490-bed, tertiary-care university hospital. METHODS: The incidence densities of patients with MRSA and acquisition of mupirocin by the hospital were calculated for the period 1992-2001. S. aureus isolates from 1999-2000 were analyzed by pulsed-field gel electrophoresis. Mupirocin-resistant MRSA isolates from 1994-1995 and 1999-2000 were analyzed for ileS-2 gene background polymorphisms. RESULTS: The incidence density of MRSA patients increased slightly over time, whereas the purchase of mupirocin decreased dramatically. Mupirocin-resistant MRSA infections decreased from 65% in 1994-1995 to 15% in 1999-2000. The MRSA Brazilian clone, detected in 1992, was still highly prevalent. The same ileS-2 encoding plasmid found in 1994-1995 persisted in three identical MRSA isolates from 1999-2000 belonging to the Brazilian clone. CONCLUSIONS: After mupirocin use decreased, the ileS-2 encoding plasmid persisted in only a few Brazilian clone isolates. Our data on mupirocin-resistant MRSA incidence and mupirocin use strongly suggested that restricted use was related to decreased rates of mupirocin resistance at our hospital.
Anti-Bacterial Agents↗
Mycobacterium kansasii: antibiotic susceptibility and PCR-restriction analysis of clinical isolates.
Mycobacterium kansasii is the second most common cause of non-tuberculosis mycobacterial diseases in Sao Paulo, Brazil. An important component of the management of infections caused by this organism is antibiotic susceptibility testing. The objective of this study was to determine the drug susceptibility profiles and genotypes of clinical isolates of M. kansasii obtained from patients with or without an infection that met the American Thoracic Society's case definition criteria of M. kansasii disease. One hundred and sixty-nine clinical isolates of M. kansasii collected between 1993 and 1998 in Sao Paulo, Brazil, were tested consecutively. The isolates were genotyped by PCR restriction-enzyme pattern analysis (PRA). Most of the M. kansasii strains were susceptible to isoniazid, streptomycin, rifabutin, rifampicin, clarithromycin, ethionamide, amikacin, clofazimine and cycloserine, and resistant to ethambutol, ciprofloxacin and doxycycline. Of 169 isolates, 167 belonged to the type I PRA genotype and one each belonged to type II and III genotypes. There was no correlation between PRA subtype and M. kansasii disease according to the American Thoracic Society case definition. Clinical trials may be needed to better correlate MIC values with treatment outcomes to identify appropriate parameters for drug-resistance testing of M. kansasii.
Amikacin↗
Analysis of a uropathogenic Escherichia coli clonal group by multilocus sequence typing.
Although many strain typing methods exist for pathogenic Escherichia coli, most have drawbacks in terms of resolving power, interpretability, or scalability. For this reason, multilocus sequence typing (MLST) is an appealing alternative. However, its applicability to different pathogens in specific epidemiologic contexts is not well understood. Here, we applied a previously established MLST method based on housekeeping genes to a well-characterized collection of uropathogenic E. coli isolates to compare the discriminatory ability of this procedure with that of enterobacterial repeat intergenic consensus (ERIC2) PCR, serogrouping, and pulsed-field gel electrophoresis (PFGE). Among 45 E. coli isolates studied, 17 different multilocus sequence types (ST) were identified. One MLST group (designated ST69 complex) was comprised of 22 isolates, all belonging to uropathogenic and bacteremic E. coli strains previously defined as clonal group A (CgA) by ERIC2 PCR. The ST69 strains contained five different serogroups and 14 PFGE types. ERIC2 PCR CgA strains belonging to different MLST groups were also identified. Interestingly, one cow E. coli isolate, previously shown by PFGE to be closely related to a human uropathogenic CgA strain, was found to cluster with the ST69 strains. All of the other animal and environmental CgA isolates had different MLST profiles. The discriminatory power of this MLST method based on housekeeping genes appears to be higher than that of ERIC2 PCR but lower than that of PFGE for epidemiologic study of uropathogenic E. coli.
Animals↗
Possible animal origin of human-associated, multidrug-resistant, uropathogenic Escherichia coli.
BACKGROUND: The multistate occurrence of cases of urinary tract infection (UTI) caused by trimethoprim-sulfamethoxazole (TMP-SMZ)-resistant Escherichia coli strains belonging to a single clonal group (designated as clonal group A [CgA]) in the United States has raised an intriguing hypothesis that these infections may have been spread by contaminated food products. The present study attempted to determine if CgA strains could be traced to food animals. METHODS: A total of 495 animal and environmental E. coli isolates, which belonged to serogroups O11, O17, O73, and O77 and were collected between 1965 and 2002 by the Gastroenteric Disease Center at Pennsylvania State University (University Park, PA), were further subtyped by antimicrobial drug susceptibility, enterobacterial repetitive intergenic consensus (ERIC2) PCR, random amplified polymorphic DNA analysis, pulsed-field gel electrophoresis (PFGE), and virulence profile pattern. RESULTS: Of 495 isolates, 128 (26%) had an ERIC2 PCR electrophoretic pattern indistinguishable from that of the human prototype CgA strain, and 14 CgA isolates were resistant to TMP-SMZ. Cluster analysis of PFGE patterns showed that 1 of these 14 isolates, obtained from a cow in 1988, was 94% similar to a CgA uropathogenic human-associated E. coli strain. The pattern for this isolate was included among a cluster of PFGE patterns for 5 human-associated UTI isolates that were >80% similar to each other. CONCLUSIONS: These observations suggest that drug-resistant, uropathogenic human-associated E. coli strains potentially have an animal origin. The possibility that human drug-resistant UTI could be a foodborne illness has serious public health implications.
Animals↗
Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis.
Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.
Binding Sites↗
Trehalose is required for growth of Mycobacterium smegmatis.
Mycobacteria contain high levels of the disaccharide trehalose in free form as well as within various immunologically relevant glycolipids such as cord factor and sulfolipid-1. By contrast, most bacteria use trehalose solely as a general osmoprotectant or thermoprotectant. Mycobacterium tuberculosis and Mycobacterium smegmatis possess three pathways for the synthesis of trehalose. Most bacteria possess only one trehalose biosynthesis pathway and do not elaborate the disaccharide into more complex metabolites, suggesting a distinct role for trehalose in mycobacteria. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. The resulting trehalose biosynthesis mutant was unable to proliferate and enter stationary phase unless supplemented with trehalose. At elevated temperatures, however, the mutant was unable to proliferate even in the presence of trehalose. Genetic complementation experiments showed that each of the three pathways was able to recover the mutant in the absence of trehalose, even at elevated temperatures. From a panel of trehalose analogs, only those with the native alpha,alpha-(1,1) anomeric stereochemistry rescued the mutant, whereas alternate stereoisomers and general osmo- and thermoprotectants were inactive. These findings suggest a dual role for trehalose as both a thermoprotectant and a precursor of critical cell wall metabolites.
Animals↗
Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis.
BACKGROUND: Conventional tests for tuberculous pleuritis have several limitations. A variety of new, rapid tests such as nucleic acid amplification tests--including polymerase chain reaction--have been evaluated in recent times. We conducted a systematic review to determine the accuracy of nucleic acid amplification (NAA) tests in the diagnosis of tuberculous pleuritis. METHODS: A systematic review and meta-analysis of 38 English and Spanish articles (with 40 studies), identified via searches of six electronic databases, hand searching of selected journals, and contact with authors, experts, and test manufacturers. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic curves were used to summarize overall test performance. Heterogeneity in study results was formally explored using subgroup analyses. RESULTS: Of the 40 studies included, 26 used in-house ("home-brew") tests, and 14 used commercial tests. Commercial tests had a low overall sensitivity (0.62; 95% confidence interval [CI] 0.43, 0.77), and high specificity (0.98; 95% CI 0.96, 0.98). The positive and negative likelihood ratios for commercial tests were 25.4 (95% CI 16.2, 40.0) and 0.40 (95% CI 0.24, 0.67), respectively. All commercial tests had consistently high specificity estimates; the sensitivity estimates, however, were heterogeneous across studies. With the in-house tests, both sensitivity and specificity estimates were significantly heterogeneous. Clinically meaningful summary estimates could not be determined for in-house tests. CONCLUSIONS: Our results suggest that commercial NAA tests may have a potential role in confirming (ruling in) tuberculous pleuritis. However, these tests have low and variable sensitivity and, therefore, may not be useful in excluding (ruling out) the disease. NAA test results, therefore, cannot replace conventional tests; they need to be interpreted in parallel with clinical findings and results of conventional tests. The accuracy of in-house nucleic acid amplification tests is poorly defined because of heterogeneity in study results. The clinical applicability of in-house NAA tests remains unclear.
Bacteriological Techniques↗
Multidrug-resistant Escherichia coli clonal groups causing community-acquired pyelonephritis.
From October 1999 through January 2000, an Escherichia coli clonal group (designated "CgA") was isolated from the urine of nearly one-half of all women with urinary tract infections (UTIs) caused by trimethoprim-sulfamethoxazole (TMP-SMZ)-resistant E. coli in a California community. This study describes the prevalence of pyelonephritis caused by CgA in the same community. E. coli isolates were characterized by enterobacterial repetitive intergenic consensus (ERIC2) polymerase chain reaction (PCR), serogrouping, and pulsed-field gel electrophoresis. Fourteen (11%) of 130 women with UTIs received a diagnosis of pyelonephritis. CgA was associated with 4 (57%) of the 7 pyelonephritis cases caused by TMP-SMZ-resistant E. coli and was associated with none of the cases caused by TMP-SMZ-susceptible E. coli (P<.02). Six (86%) of these TMP-SMZ-resistant E. coli isolates belonged to 2 distinct ERIC2 PCR-defined clonal groups, whereas all of the TMP-SMZ-susceptible E. coli strains had unique fingerprints (P<.001). The prevalence of antimicrobial-resistant pyelonephritis in a community may be affected by a limited number of E. coli clonal groups.
Adolescent↗
TRAIL-R as a negative regulator of innate immune cell responses.
TRAIL receptor (TRAIL-R) signaling has been implicated in inducing apoptosis in tumor cells, but little is understood about its physiological function. Here, we report the generation and characterization of TRAIL-R(-/-) mice, which develop normal lymphocyte populations but possess enhanced innate immune responses. TRAIL-R(-/-) mice exhibited increased clearance of murine cytomegalovirus that correlated with increased levels of IL-12, IFN-alpha, and IFN-gamma. Stimulation of macrophages with Mycobacterium and Toll-like receptor (TLR)-2, -3, and -4, but not TLR9, ligands resulted in high levels of TRAIL upregulation and enhanced cytokine production in TRAIL-R(-/-) cells. The immediate-early TLR signaling events in TRAIL-R(-/-) macrophages and dendritic cells are normal, but I kappa B-alpha homeostatic regulation and NF-kappa B activity at later time points is perturbed. These data suggest that TRAIL-R negatively regulates innate immune responses.
Amino Acid Sequence↗
Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review.
A major challenge in tuberculosis control is the diagnosis and treatment of latent tuberculosis infection. Until recently, there were no alternatives to the tuberculin skin test (TST) for diagnosing latent tuberculosis. However, an alternative has now emerged in the form of a new in-vitro test: the interferon-gamma assay. We did a systematic review to assess the performance of interferon-gamma assays in the immunodiagnosis of tuberculosis. By searching databases, contacting experts and test manufacturers, we identified 75 relevant studies. The results suggest that interferon-gamma assays that use Mycobacterium tuberculosis-specific region of difference 1 (RD1) antigens (such as early secretory antigenic target 6 and culture filtrate protein 10) may have advantages over the TST, in terms of higher specificity, better correlation with exposure to M tuberculosis, and less cross-reactivity due to BCG vaccination and non-tuberculous mycobacterial infection. However, interferon-gamma assays that use RD1 antigens in isolation may maximise specificity at the cost of sensitivity. Assays that use cocktails of RD1 antigens seem to overcome this problem, and such assays have the highest accuracy. RD1-based interferon-gamma assays can potentially identify those with latent tuberculosis who are at high risk for developing active disease, but this requires confirmation. There is inadequate evidence on the value of interferon-gamma assays in the management of immunocompromised individuals, children, patients with extrapulmonary or non-tuberculous mycobacterial disease, and populations in countries where tuberculosis is endemic. Current evidence suggests that interferon-gamma assays based on cocktails of RD1 antigens have the potential to become useful diagnostic tools. Whether this potential can be realised in practice remains to be confirmed in well designed, long-term studies.
Antigens, Bacterial↗
Risk factors and costs associated with methicillin-resistant Staphylococcus aureus bloodstream infections.
OBJECTIVES: To compare the cost of hospitalization of patients with methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) versus patients with methicillin-sensitive S. aureus (MSSA) BSI, controlling for severity of underlying illness; and to identify risk factors associated with MRSA BSI. DESIGN: Retrospective case-control study based on medical chart review. SETTING: A 640-bed, tertiary-care hospital in Seattle, Washington. PATIENTS: All patients admitted to the hospital between January 1, 1997, and December 31, 1999, with S. aureus BSI confirmed by culture. RESULTS: Twenty patients with MRSA BSI were compared with 40 patients with MSSA BSI. Univariate analysis identified 5 risk factors associated with MRSA BSI. Recent hospital admission (P = .006) and assisted living (P = .004) remained significant in a multivariate model. Costs were significantly higher per patient-day of hospitalization for MRSA BSI than for MSSA BSI (dollar 5,878 vs dollar 2,073; P = .003). When patients were stratified according to severity of illness as measured by the case mix index, a difference of dollar 5,302 per patient-day was found between the two groups for all patients with a case mix index greater than 2 (P < .001). CONCLUSION: These observations suggest that MRSA BSI significantly increases hospitalization costs compared with MSSA BSI, even when controlling for the severity of the patient's underlying illness. As MRSA BSI was also found to be significantly associated with a group of patients who have repeated hospitalizations, such infections contribute substantially to the increasing cost of medical care.
Aged↗