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Background: The gut microbiome is extensively involved in induction of remission in pediatric Crohn's disease (CD) patients by exclusive enteral nutrition (EEN). In this follow-up study of pediatric CD patients undergoing treatment with EEN, we employ machine learning models trained on baseline gut microbiome data to distinguish patients who achieved and sustained remission (SR) from those who did not achieve remission nor relapse (non-SR) by 24 weeks., Methods: A total of 139 fecal samples were obtained from 22 patients (8-15 years of age) for up to 96 weeks. Gut microbiome taxonomy was assessed by 16S rRNA gene sequencing, and functional capacity was assessed by metagenomic sequencing. We used standard metrics of diversity and taxonomy to quantify differences between SR and non-SR patients and to associate gut microbial shifts with fecal calprotectin (FCP), and disease severity as defined by weighted Pediatric Crohn's Disease Activity Index. We used microbial data sets in addition to clinical metadata in random forests (RFs) models to classify treatment response and predict FCP levels., Results: Microbial diversity did not change after EEN, but species richness was lower in low-FCP samples (<250 µg/g). An RF model using microbial abundances, species richness, and Paris disease classification was the best at classifying treatment response (area under the curve [AUC] = 0.9). KEGG Pathways also significantly classified treatment response with the addition of the same clinical data (AUC = 0.8). Top features of the RF model are consistent with previously identified IBD taxa, such as Ruminococcaceae and Ruminococcus gnavus., Conclusions: Our machine learning approach is able to distinguish SR and non-SR samples using baseline microbiome and clinical data., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

Objective: The aim of this study was to evaluate diagnostic practices for suspected community-acquired central nervous system (CNS) infection in an urban pediatric population., Methods: This is an observational, retrospective single-center review of cerebrospinal fluid (CSF) studies in children, 1 month to 21 years old, evaluated for suspected CNS infection from 2004 to 2014. Cases of suspected nosocomial meningitis were excluded. The frequency of N-methyl-D-aspartate receptor antibody (NMDAR ab) encephalitis was analyzed from 2010 to 2014., Results: A total of 940 unique patient visits with CSF studies were included in the final analysis. There were 940 bacterial cultures sent; 4 (0.42%) grew suspected CSF bacterial pathogens, and 18 (1.9%) grew organisms that were suspected contaminants. Bacterial pathogens included late-onset group B Streptococcus in 3 infants younger than 3 months and Streptococcus pneumoniae in an unvaccinated 9-year-old child. Viral CNS infection was 7.5 times more frequent than bacterial infection. Enterovirus was the only virus isolated. Five cases positive for NMDAR ab were identified since 2010., Conclusions: Bacterial studies were performed more frequently than viral and other studies. Cerebrospinal fluid bacterial culture was nearly 5 times more likely to yield a contaminant than a pathogen. The frequency of viral infection was likely underestimated as only 20% were tested, mainly by culture, which is suboptimal. These data suggest diagnostic practices for the evaluation of suspected community-acquired CNS infections in children need to be modified to reflect current epidemiology and highlight the need for greater accessibility to polymerase chain reaction for viral diagnostics. Furthermore, NMDAR ab-mediated encephalitis should be considered early in children presenting with suggestive symptoms.

Introduction: The aim of this study was to assess the epidemiology of VRE colonization among patients in the intensive care units (ICU) of the National Hospital of Sri Lanka (NHSL)., Method: A cross sectional study was carried out on 218 patients admitted to 12 ICUs of the NHSL from January to March 2012. Rectal swabs were collected on day 0, 4, 8 and every 4th day thereafter till discharge. Enterococci were isolated on selective media and identified up to species level using standard bacteriological procedures. Standardized disc diffusion antibiotic susceptibility testing to ampicillin, teicoplanin and vancomycin was performed using the Clinical and Laboratory Standards Institute (CLSI) method. Minimum inhibitory concentrations to vancomycin were determined, using the E-test in strains showing intermediate or frank resistance to vancomycin by disc diffusion. Genotype determination (van A / van B) was carried out on isolates identified as VRE using the polymerase chain reaction. Patients positive for VRE colonization were followed up to discharge or death., Result: VRE prevalence in the study sample was 5%. Univariate analysis showed that the use of metronidazole (odds ratio [OR] :15.73;95% 95% confidence interval [CI] : 3.94-62.67,P<0.05) or teicoplanin (OR: 12.56; 95% CI:2.65 – 59.52, p< 0.05) and diabetes (OR: 05.13; 95% CI: 1.36 – 18.7, p< 0.05) or hemodialysis during ICU stay (OR: 7.38 ;95% CI : 1.69-32.16, P<0.05) were associated with an increased risk of VRE colonization., Conclusion: The 5% prevalence of VRE colonization detected signals the emergence of VRE in the intensive care setting in Sri Lanka.

Whole cell MALDI is regularly used for the identification of bacteria to species level in clinical Microbiology laboratories. However, there remains a need to rapidly characterize and differentiate isolates below the species level to support outbreak management. We describe the implementation of a modified preparative approach for MALDI-MS combined with a custom analytical computational pipeline as a rapid procedure for subtyping Shigatoxigenic E. coli (STEC) and accurately identifying strain-specifying biomarkers. The technique was able to differentiate E. coli O157:H7 from other STEC. Within O157 serotype O157:H7 isolates were readily distinguishable from Sorbitol Fermenting O157 isolates. Overall, nine homogeneous groups of isolates were distinguished, each exhibiting distinct profiles of defining mass spectra features. This offers a robust analytical tool useable in reference/diagnostic public health scenarios., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

Background: Clostridium difficile is a major pathogen responsible for nosocomial infectious diarrhea. We explored optimal laboratory strategies for diagnosis of C. difficile infection (CDI) in our clinical settings, a 1400-bed tertiary care hospital., Methods: Using 191 fresh stool samples from adult patients, we evaluated the performance of Xpert C. difficile (Xpert CD), C. diff Quik Chek Complete (which simultaneously detects glutamate dehydrogenase [GDH] and C. difficile toxins [CDT]), toxigenic culture, and a two-step algorithm composed of GDH/CDT as a screening test and Xpert CD as a confirmatory test., Results: Clostridium difficile was detected in 35 samples (18.3%), and all isolates were toxigenic strains. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value of each assay for detecting CDI were as follows: Quik Chek Complete CDT (45.7%, 100%, 100%, 89.1%), Quik Chek Complete GDH (97.1%, 99.4%, 97.1%, 99.4%), Xpert CD (94.3%, 100%, 100%, 98.7%), and toxigenic culture (91.4%, 100%, 100%, 98.1%). A two-step algorithm performed identically with Xpert CD assay., Conclusion: Our data showed that most C. difficile isolates from adult patients were toxigenic. We demonstrated that a two-step algorithm based on GDH/CDT assay followed by Xpert CD assay as a confirmatory test was rapid, reliable, and cost effective for diagnosis of CDI in an adult patient setting with high prevalence of toxigenic C. difficile., (© 2017 Wiley Periodicals, Inc.)

Background: Urinary tract infections (UTI) are among the most common reasons for antibiotic prescriptions. Due to increasing resistance rates, antibiotic therapy should be targeted and based on evidence-based recommendations. Test results and recommendations by medical laboratories have a relevant impact on the choice of antibiotics. The extent to which medical laboratories consider antibiotics recommended by evidence-based treatment guidelines in microbiological testing is unclear., Objectives: The aim of the present study is to assess whether guideline recommendations for antibiotic therapy of UTI are taken into account by medical laboratories in northern Germany., Materials and Methods: A standardized and piloted questionnaire was used in our telephone survey. All microbiological laboratories in northern Germany (Hamburg, Bremen, Lower Saxony, Schleswig-Holstein, and Mecklenburg-Western Pomerania; n = 82) were asked about their standards in diagnosing and microbiological testing of urine cultures., Results: A total of 71 of the 82 contacted laboratories perform microbiological tests, whereby 40 of these participated (56 %). Most of the laboratories (43 %) routinely perform microbiological testing when bacterial counts were ≥ 10(4) colony forming units per ml (CFU/ml), 15 % ≥ 10(5) CFU/ml, 17 % ≥ 10(3) CFU/ml, and 8 % ≥ 10(2) CFU/ml. Antibiotic testing includes ciprofloxacin (95 %), cotrimoxazole (87.5 %), trimethoprim (57 %), fosfomycin (85 %), and nitrofurantoin (72 %)., Conclusions: The diagnostic threshold recommended in evidence-based guidelines (10(3) CFU/ml) is used only by a few laboratories. Antibiotics recommended as a first line therapy are only partly taken into account in microbiological testing. This variance in different diagnostic thresholds and microbiological testing is a barrier to guideline implementation.

Background: Elderly patients represent an increasing population in the emergency department (ED) and physicians often have to deal with multimorbidity and complexity. Infections are one of the major reasons for ED presentations of older patients and the main cause of mortality; however, infections are often difficult to diagnose in older patients., Aim: This article provides a review of important indicators for infections, diagnostic tools and limitations in elderly patients., Material and Methods: A literature search was carried out using PubMed in the period 1990-2015 and in addition own published data are presented., Results and Conclusion: Infections in the elderly are difficult to assess in the emergency department due to atypical symptoms. Even subtle changes need to be recognized. For the diagnosis of infections in older ED patients unspecific symptoms, vital parameters, laboratory parameters, including C-reactive protein (CRP) and procalcitonin levels, cognitive function and functionality of the patient need to be taken into account.

Recent years have seen a world-wide increase in antimicrobial resistance (AMR) in cases of infection with Neisseria gonorrhoeae (NG). NG infection is not notifiable in Germany and there is a lack of information available about the spread and AMR of NG infections. The objective of the study was to provide information on diagnostic methods and AMR testing in cases of NG infections in German laboratories. A cross-sectional survey was undertaken in Germany between June and August 2013 using an online questionnaire. Laboratories performing NG diagnostics were identified and described with regard to the diagnostic methods used, the number of tests performed, the antibiotics tested and the AMR observed, in addition to general laboratory information. In total, 188 of the 521 participating laboratories performed NG diagnostics; these were included in the further statistical analysis. 92.6 % of the 188 laboratories performed culture. A median of 60 (IQR 15-270) samples per quarter (SPQ) were tested, with an overall positivity rate of 4.1 and 6.9 % among men. Most (82.1 %) of the 151 laboratories performing NG culture tested for AMR as well. The most frequently tested antibiotics were ciprofloxacin (94.8 %), penicillin (93.1 %), doxycycline (70.0 %) and ceftriaxone (67.2 %). The most frequently observed AMR ever were those against ciprofloxacin (87.1 %), penicillin (78.3 %), doxycycline (56.6 %) and azithromycin (35.1 %; all percentages refer to laboratories). The laboratories used different standards regarding susceptibility criteria. The emergence and spread of AMR shows that it is crucial to assess and monitor the scope and trends of multidrug-resistant gonorrhea. The data collected on diagnostic methods and AMR testing in cases of NG infections in German laboratories constitute an important basis for future monitoring.

Introduction: Next to official surveillance system in Poland, established by Public Statistic Act in 1995, other surveillance data collection system exists - historically connected with Ministry of Health Decree, which is not in force from 1995 y. Data in this system comes from National Sanitary Inspection laboratories and contains results of clinical samples examinations for Salmonella and Shigella presence., Material and Methods: Analysed data were collected since 2007 to 2014 y. Database has been created, which contains data - results of Salmonella / Shigella (SS) detection in faecal samples, rectal swabs, urine samples, blood samples collected from patients, people exposed in outbreaks, Salmonella carriers, food workers and other people., Results: The number of performed tests for SS in 2014 is 447033. Systematic decrease of this number has been observed since 2008. Most common material tested for SS was stool samples, then rectal swabs. Number of Salmonella identification going down each year during analysed time. The highest decrease in group of sick people has been observed. Most common serotype in 2008-2014 years was S. Enteritidis, then S. Typhimurium. Only in 2010 y. second most common serotype was S. Mbandaka. In 2014 y. highest number of S. enterica 1,4,[5],12:i:- has been observed (37 cases)., Conclusions: The reason of decreased number of performed SS test and positive Salmonella results is unclear. One of theories is decreased level of quality of SS diagnostic. From the other hand, decreased number of false-positive Salmonella identification outside of National Sanitary Inspection laboratories is observed. This demonstrates improving quality of Salmo nella diagnostic in laboratories performing such tests. High disproportion between number of Salmonella cases in described database and official notification system is observed. It comes from, that both systems collect different data. Both systems could exist independently and could supplement each-other.

Objective: Characterize the clinical and molecular epidemiology of new methicillin-resistant Staphylococcus aureus (MRSA) acquisitions at nasal and extranasal sites among high-risk nursing home (NH) residents., Design: Multicenter prospective observational study., Setting: Six NHs in southeast Michigan., Participants: A total of 120 NH residents with an indwelling device (feeding tube and/or urinary catheter)., Methods: Active surveillance cultures from the nares, oropharynx, groin, perianal area, wounds (if present), and device insertion site(s) were collected upon enrollment, at day 14, and monthly thereafter. Pulsed-field gel electrophoresis and polymerase chain reaction for SCCmec, agr, and Panton-Valentine leukocidin were performed., Results: Of 120 participants observed for 16,290 device-days, 50 acquired MRSA (78% transiently, 22% persistently). New MRSA acquisitions were common in extranasal sites, particularly at device insertion, groin, and perianal areas (27%, 23%, and 17.6% of all acquisitions, respectively). Screening extranasal sites greatly increases the detection of MRSA colonization (100% of persistent carriers and 97.4% of transient carriers detected with nares, groin, perianal, and device site sampling vs 54.5% and 25.6%, respectively, for nares samples alone). Colonization at suprapubic urinary catheter sites generally persisted. Healthcare-associated MRSA (USA100 and USA100 variants) were the dominant strains (79.3% of all new acquisition isolates). Strain diversity was more common in transient carriers, including acquisition of USA500 and USA300 strains., Conclusion: Indwelling device insertion sites as well as the groin and perianal area are important sites of new MRSA acquisitions in NH residents and play a role in the persistency of MRSA carriage. Clonal types differ among persistent and transient colonizers.

Background: Staphylococcus aureus (SA) is a recognized cause of nosocomial infections with 8,767 SA bacteraemia (SAB) cases reported in England only in 2012. Different typing methods have been developed but they are not generally performed as a routine investigation in hospital laboratories., Findings: We collected epidemiological data and spa-typed all SAB isolates over a 12 months period. Spa-typing was useful to detect two potential outbreaks of methicillin-sensitive SA (MSSA). In addition, the analysis of spa-types from individuals with multiple bacteriaemias helped to distinguish between relapse and re-infection., Conclusions: Spa-typing could be used as a rapid tool to understand the epidemiology of SAB, in particular the detection of hospital clusters and to distinguish relapse from re-infection, but clinicians should be aware of its possible limitations.

In a meta-analysis examining respiratory specimen Gram stain for diagnosis of ventilator-associated pneumonia, absence of bacteria on Gram stain had a high negative predictive value, but a positive Gram stain correlated poorly with organisms recovered in culture. Rapid and accurate diagnosis of ventilator-associated pneumonia (VAP) is a major challenge and no generally accepted gold standard exists for VAP diagnosis. We conducted a meta-analysis to examine the role of respiratory specimen Gram stain to diagnose VAP, and the correlation with final culture results. In 21 studies, pooled sensitivity of Gram stain for VAP was 0.79 (95% confidence interval [CI], .77-0.81; P < .0001) and specificity was 0.75 (95% CI, .73-.78; P < .0001). Negative predictive value of Gram stain for a VAP prevalence of 20%-30% was 91%, suggesting that VAP is unlikely with a negative Gram stain but the positive predictive value of Gram stain was only 40%. Pooled kappa was 0.42 for gram-positive organisms and 0.34 for gram-negative organisms, suggesting fair concordance between organisms on Gram stain and recovery by culture. Therefore, a positive Gram stain should not be used to narrow anti-infective therapy until culture results become available.

Background: Biopsy with demonstration of the infectious organism is the gold standard for diagnosing spondylodiscitis. The purpose of this study is to evaluate the positive culture rate of image-guided percutaneous biopsy in cases of radiologically suspected and unsuspected spinal osteomyelitis and to assess the role of pathology in diagnosis., Methods: With IRB approval and in compliance with HIPAA regulations, the charts of patients undergoing 323 consecutive image-guided percutaneous spinal biopsies performed by one musculoskeletal radiology department between January 2001 and March 2007 were reviewed. Image guidance was via fluoroscopy or computed tomography. Radiological and clinical suspicion, cultures, and pathology were assessed and compared to previously published reports., Results: In 92 cases radiographically and clinically consistent with infection (high probability of infection), 28 specimens yielded positive cultures (30.4%). Positive cultures resulted from 5 of 31 cases (16.1%) radiographically indeterminate for infection (intermediate probability of infection versus tumor). When radiographically not suggestive of infection (low probability of infection, i.e. suspicious for tumor), 10 of 200 cultures were positive (5.0%). From 113 cases sent to pathology with an intermediate or high suspicion for infection, 63 were histopathologically diagnosed as such (55.8%). Cultures were positive in 19 of those 63 cases (30.2%). Culture and/or pathology was positive in 73 (64.6%) of the 113 cases. There were no significant differences in rates of positive culture or pathology by vertebral region (p=0.51, p=0.81). The most frequently identified organisms were Staphylococcus aureus (13) and coagulase negative staphylococci (13)., Conclusions: Our results suggest that the positive culture rate of percutaneous spinal biopsy specimens is 30.4% with radiographically high probability for infection, which is lower than previously published. Infection may also be present in cases with imaging characteristics atypical for infection (5.0%). Careful consideration must be given to the interpretation of negative culture results., Level of Evidence: This retrospective review of 323 consecutive percutaneous spine biopsies is level III evidence., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Accurate species-level identification of viridans group streptococci (VGS) is very important for understanding of their pathogenicity and virulence. However, an extremely high level of the similarity between VGS, especially Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis and Streptococcus pseudopneumoniae, often results in misidentification of these organisms, so there is an urgent need of novel approaches to species identification. A set of 50 randomly selected clinical isolates of alpha-hemolytic streptococci from upper respiratory tract were characterized by the routine phenotypic methods (alpha-hemolysis, colony morphology, Gram stain and optochin susceptibility). Modern proteomic and genetic approaches - the direct bacterial profiling (DBP) by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technique and multilocus sequence analysis (MLSA) scheme (http://viridans.emlsa.net/) - were applied for the accurate species identification. After that all isolates were stored at -70°C. Later they were re-inoculated, and a number of additional tests (bile solubility, latex agglutination by commercial "Slidex® pneumo-kit" and repeated optochin test) were performed. A considerable discrepancy was discovered in the results of the different approaches. Looking in the future, one could say that MLSA-like schemes based on the analysis of the nucleotide sequences of seven or more loci of the bacterial genome, appeared to be the most useful instrument in the VGS discrimination, in contrast to the numerous one-target identification schemes, which have been introduced into practice by now., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In order to identify the agent responsible of the infectious process and understanding the pathogenic/pathological implications, clinical course, and to implement an effective antimicrobial therapy, a mainstay in the practice of clinical microbiology is the allocation of species to a microbial isolation. In daily routine practice microbiology laboratory phenotypic techniques are applied to achieve this goal. However, they have some limitations that are seen more clearly for some kinds of microorganism. Molecular methods can circumvent some of these limitations, although its implementation is not universal. This is due to higher costs and the level of expertise required for thei implementation, so molecular methods are often centralized in reference laboratories and centers. Recently, proteomics-based methods made an important breakthrough in the field of diagnostic microbiology and will undoubtedly have a major impact on the future organization of the microbiology services. This paper is a short review of the most noteworthy aspects of the three bacterial identification methods described above used in microbiology laboratories., (Copyright © 2011 Elsevier España, S.L. All rights reserved.)

Unlabelled: TARGET OF THE STUDY: Strain typing of pathogens is essential to pinpoint the sources and routes of transmission and to forecast future trends. In a general hospital, we studied possible changes in the MRSA population., Patients and Methods: MRSA isolates received from a Belgian general hospital, during 2002 (n=150) and the second half of 2007 (n=105), were compared by phage and spa typing., Results: In 2002, [J]* phage types characterized 45% of the MRSA isolates, 13% belonged to the [O]* phage types, 12% to a local phage type 29/42E/54/D11* and 28% were not assigned to a defined group. Thirteen different spa types were found among the isolates: 39% belonged to t038, 27% to t121, 14% to t041, 5% to t740, and 4% to t002 and t024 each. Two spa types were found respectively in two and three isolates, five were unique. In 2007, 35% belonged to [J]*, 23% to [O]* and 39% could not be put in a defined group. Eighteen different spa types were found: 30% belonged to t740, 29% to t121, 13% to t038 and 10% to t002. Three spa types were represented in two isolates, eleven were unique. The t041 spa type was specific for the 29/42E/54/D11* and the majority of the t121 isolates were related to [J]*., Conclusion: [J]* remained the dominant phage types group but decreased whereas [O]*, the second phage types group, increased. As to the spa types, t740 became dominant while t121 remained second. Phage and spa typing point to some quantitative changes among the Belgian MRSA population., (Copyright (c) 2009. Published by Elsevier SAS.)

The Micro Imaging Technology (MIT) 1000 Rapid Microbial Identification (RMID) System is a device that uses the principles of light scattering coupled with proprietary algorithms to identify bacteria after being cultured and placed in a vial of filtered water. This specific method is for pure culture identification of Listeria spp. A total of 81 microorganisms (55 isolates) were tested by the MIT 1000 System, of which 25 were Listeria spp. and 30 a variety of other bacterial species. In addition, a total of 406 tests over seven different ruggedness parameters were tested by the MIT 1000 System to determine its flexibility to the specifications stated in the MIT 1000 System User Guide in areas where they might be deviated by a user to shorten the test cycle. Overall, MIT concluded that the MIT 1000 System had an accuracy performance that should certify this Performance Test Method for the identification of Listeria spp. This report discusses the tests performed, results achieved, and conclusions, along with several reference documents to enable a higher understanding of the technology used by the MIT 1000 System.

The classification of bacteria by using genomic methods or expensive biochemical-based commercial kits is sometimes beyond the reach of many laboratories that need to perform numerous classifications of unknown bacterial strains in a fast, cheap, and reliable way. A new computer program, Identax, for the computer-assisted identification of microorganisms by using only results obtained from conventional biochemical tests is presented. Identax improves current microbial identification software and provides a multiplatform and user-friendly program. It can be executed from any operating system and can be downloaded without any cost from the Identax website (www.identax.org).

Objectives: Information on clinical practice regarding the diagnosis of pneumonia in European intensive care units is limited. The aim of this study was to describe the spectrum of actual diagnostic practices in a large sample of European intensive care units., Design: Prospective, observational, multicenter study., Setting: Twenty-seven intensive care units of nine European countries., Patients: Consecutive patients requiring invasive mechanical ventilation for an admission diagnosis of pneumonia or receiving mechanical ventilation for >48 hrs irrespective of admission diagnosis., Interventions: None., Measurements and Main Results: A total of 2,436 patients were evaluated; 827 were admitted with or developed nosocomial pneumonia (hospital-acquired pneumonia [HAP], 27.1%; ventilator-associated pneumonia [VAP], 56.2%; very early onset VAP, 16.7%). Mean age was 59.4 +/- 18.1 yrs, 65.0% were men, and mean admission Simplified Acute Physiology Score II was 46.7 +/- 17.1. Worsening oxygenation (76.8%), purulent/changing respiratory secretions (72.1%), and new temperature elevation (69.2%) were the most frequent clinical signs of nosocomial pneumonia. Etiological diagnosis was based on noninvasive respiratory specimens in 74.8% of episodes. Bronchoscopy was performed in 23.3% of episodes. Bronchoscopy performance, after adjustment by severity of illness, age, and type of hospital, were predicted by worsening oxygenation (odds ratio 2.03; 95% confidence interval, 1.27-3.24) and male sex (odds ratio 1.77; 95% confidence interval, 1.19-2.65). Definite cause was documented in 69.5% of nosocomial pneumonia cases. The most common isolates were Staphylococcus aureus (16.3% methicillin-sensitive S. aureus and 16.0% methicillin-resistant S. aureus), Pseudomonas aeruginosa (23.1%), and Acinetobacter baumannii (19.1%). Presence of nosocomial pneumonia significantly prolonged mean length of mechanical ventilation (10.3 days, p < .05) and mean intensive care unit length of stay (12.2 days, p < .05) in intensive care unit survivors. Mortality rate was 37.7% for nosocomial pneumonia vs. 31.6% for patients without pneumonia (p < .05)., Conclusions: Etiological diagnosis of nosocomial pneumonia in a large sample of European intensive care units was based mainly on noninvasive techniques. However, there was high variability in bronchoscopy use between the participating intensive care units.

As the use of nucleotide sequence-based typing has become more widespread in the investigation of microbial epidemiology, there has been a natural requirement for curated Internet-based databases that can act as central authorities for nomenclature and type definitions. These facilitate the sharing and comparison of data between laboratories without the need for reference samples. Here, the use of the most common multilocus sequence typing (MLST) and antigen sequence databases are described. In particular, for MLST, the steps required for allele sequence and profile identification are explained along with a detailed overview of searching and matching isolate records. BLAST searching of antigen sequence databases is also described.

The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of Staphylococcus aureus. The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions. This single-locus sequence-based typing method combines a number of technical advantages, such as rapidity, reproducibility, and portability. Moreover, due to its repeat structure, the spa locus simultaneously indexes micro- and macrovariations, enabling the use of spa typing in both local and global epidemiological studies. These studies are facilitated by the establishment of standardized spa type nomenclature and Internet shared databases.

New methodologies for surveillance and identification of Mycobacterium tuberculosis are required to stem the spread of disease worldwide. In addition, the ability to discriminate mycobacteria at the strain level may be important to contact or source case investigations. To this end, we are developing MALDI-TOF MS methods for the identification of M. tuberculosis in culture. In this report, we describe the application of MALDI-TOF MS, as well as statistical analysis including linear discriminant and random forest analysis, to 16 medically relevant strains from four species of mycobacteria, M. tuberculosis, M. avium, M. intracellulare, and M. kansasii. Although species discrimination can be accomplished on the basis of unique m/z values observed in the MS fingerprint spectrum, discrimination at the strain level is predicted on the relative abundance of shared m/z values among strains within a species. For the 16 mycobacterial strains investigated in the present study, it is possible to unambiguously identify strains within a species on the basis of MALDI-TOF MS data. The error rate for classification of individual strains using linear discriminant analysis was 0.053 using 37 m/z variables, whereas the error rate for classification of individual strains using random forest analysis was 0.023 using only 18 m/z variables. In addition, using random forest analysis of MALDI-TOF MS data, it was possible to correctly classify bacterial strains as either M. tuberculosis or non-tuberculous with 100% accuracy.

The studies that correlate the results obtained by different typing methodologies rely solely on qualitative comparisons of the groups defined by each methodology. We propose a framework of measures for the quantitative assessment of correspondences between different typing methods as a first step to the global mapping of type equivalences. A collection of 325 macrolide-resistant Streptococcus pyogenes isolates associated with pharyngitis cases in Portugal was used to benchmark the proposed measures. All isolates were characterized by macrolide resistance phenotyping, T serotyping, emm sequence typing, and pulsed-field gel electrophoresis (PFGE), using SmaI or Cfr9I and SfiI. A subset of 41 isolates, representing each PFGE cluster, was also characterized by multilocus sequence typing (MLST). The application of Adjusted Rand and Wallace indices allowed the evaluation of the strength and the directionality of the correspondences between the various typing methods and showed that if PFGE or MLST data are available one can confidently predict the emm type (Wallace coefficients of 0.952 for both methods). In contrast, emm typing was a poor predictor of PFGE cluster or MLST sequence type (Wallace coefficients of 0.803 and 0.655, respectively). This was confirmed by the analysis of the larger data set available from http://spyogenes.mlst.net and underscores the necessity of performing PFGE or MLST to unambiguously define clones in S. pyogenes.

The Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.) is an automated system for the identification and antimicrobial susceptibility testing of bacterial isolates. This system with its negative identification (NID) panel was evaluated for its accuracy in the identification of 507 isolates of the family Enterobacteriaceae, 57 other nonenteric gram-negative isolates that are commonly isolated in clinical microbiology laboratories, and 138 isolates of the family Vibrionaceae. All of the isolates had been characterized by using approximately 48 conventional tube biochemicals. Of the 507 isolates of the Enterobacteriaceae, 456 (89.9%) were correctly identified to the genus and species levels. The five isolates of Proteus penneri required an off-line indole test, as suggested by the system to differentiate them from Proteus vulgaris. The identifications of 20 (3.9%) isolates were correct to the genus level but incorrect at the species level. Two (0.4%) isolates were reported as "no identification." Misidentifications to the genus and species levels occurred for 29 (5.7%) isolates of the Enterobacteriaceae. These incorrect identifications were spread over 14 different genera. The most common error was the misidentification of Salmonella species. The shortest time for a correct identification was 2 h 8 min. The longest time was 12 h 27 min, for the identification of a Serratia marcescens isolate. Of the 57 isolates of nonenteric gram-negative bacilli (Acinetobacter, Aeromonas, Burkholderia, Plesiomonas, Pseudomonas, and Stenotrophomonas spp.), 48 (84.2%) were correctly identified to the genus and species levels and 7 (12.3%) were correctly identified to the genus level but not to the species level. The average time for a correct identification was 5 h 11 min. Of the Vibrionaceae spp., 123 (89.1%) were correctly identified at the end of the initial incubation period, which averaged 4 h. Based on the findings of this study, the Phoenix 100 ID/AST system NID panel falls short of being an acceptable new method for the identification of the Enterobacteriaceae, Vibrionaceae, and gram-negative nonenteric isolates that are commonly encountered in many hospital microbiology laboratories.

Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Microorganisms, such as bacteria, which might be present as contamination inside an industrial food or pharmaceutical clean room process need to be identified on short time scales in order to minimize possible health hazards as well as production downtimes causing financial deficits. Here we describe the first results of single-particle micro-Raman measurements in combination with a classification method, the so-called support vector machine technique, allowing for a fast, reliable, and nondestructive online identification method for single bacteria.

This report analyzes 36 cases of bacteremia or catheter-related infection caused by Roseomonas species, a group of pink, slimy, waterborne, gram-negative coccobacilli. The causative species included the newly described Roseomonas mucosa (22 cases [61%]) and Roseomonas gilardii subspecies rosea (8 cases [22%]) and known species R. gilardii subspecies gilardii (5 cases [14%]) and Roseomonas genomospecies 4 (1 case [3%]). Twenty-nine (81%) of the cases were symptomatic, with fever being the most common symptom (in 27 [75%] of the cases). Twenty (56%) of the infections were monomicrobic. Six cases (17%) involved persistent catheter colonization, and 5 of these cases required removal of the catheter to clear the infection. All infections resolved, most with empirical antibiotic treatment. A summary of the antibiotic susceptibility pattern of these strains and other reported series show that Roseomonas species are consistently susceptible to amikacin and imipenem and frequently susceptible to ciprofloxacin and ticarcillin, but essentially nonsusceptible to ceftazidime and cefepime. This result may guide future therapy for infections due to Roseomonas species.

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of Streptococcus pneumoniae and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.

We evaluated the Vitek 2 ID-GNB identification card (bioMérieux, Inc., Durham, N.C.) for its ability to identify members of the family Enterobacteriaceae and other gram-negative bacilli that are isolated in clinical microbiology laboratories. Using 482 enteric stock cultures and 103 strains of oxidase-positive, gram-negative glucose-fermenting and nonfermenting bacilli that were maintained at -70 degrees C and passaged three times before use, we inoculated cards according to the manufacturer's directions and processed them in a Vitek 2 instrument using version VT2-R02.03 software. All panel identifications were compared to reference identifications previously confirmed by conventional tube biochemical assays. At the end of the initial 3-h incubation period, the Vitek 2 instrument demonstrated an accuracy of 93.0% for the identification of enteric strains; 414 (85.9%) were correctly identified at probability levels ranging from excellent to good, and an additional 34 (7.1%) strains were correctly identified but at a low level of discrimination. Nineteen (3.9%) strains were unidentified, and 15 (3.1%) were misidentified. The 19 unidentified strains were scattered among 10 genera. Three of the 15 misidentified strains were lactose-positive Salmonella spp. and were identified as Escherichia coli; another was a lactose-positive, malonate-negative Salmonella enterica subsp. arizonae strain that was identified as E. coli. Of the 103 glucose-fermenting and nonfermenting nonenteric strains, 88 (85.4%) were correctly identified at probability levels ranging from excellent to good, and 10 (9.7%) were correctly identified, but at a low level of discrimination, for a total of 95.1% accuracy with this group. Two strains were unidentified and three were misidentified. The errors occurred for strains in three different genera. With the increased hands-off approach of the Vitek 2 instrument and accuracies of 93% for the identification of enteric organisms and 95.1% for the identification of nonenteric organisms with the ID-GNB card, use of this product presents an acceptable method for the identification of most gram-negative organisms commonly isolated in the clinical laboratory. A comparison of these results to those obtained by testing 454 of the same strains with the Vitek GNI+ card revealed no significant difference in the abilities of the two cards to identify these organisms accurately.

The detection and molecular typing of Helicobacter pylori virulence genes in human stool specimens by polymerase chain reaction (PCR) require an adequate amount of bacterial DNA and an appropriately adjusted PCR protocol. DNA was isolated from stool samples of 39 H. pylori-infected and nine uninfected Colombian children using the QIAamp Kit following the manufacturer's instructions but with modifications. DNA templates were amplified for the vacA s and m regions and for the cagA gene by PCR using radioactively labeled (32P) primers. The modifications in the standard Qiagen protocol of stool DNA extraction increased the final concentration of eluted total stool DNA 4.7 times (117 +/- 17 versus 22 +/- 3 ng/microl; P < 0.0001). Nevertheless, its amplification by regular PCR programs (30-40 cycles) did not generate visible signals because of the very low ratio of H. pylori DNA to other DNA. PCR for 80 cycles successfully amplified vacA in 36/39 samples (sensitivity, 92.3%) and cagA fragments in 21/39 (53.8%) fecal DNA samples. Both s and m vacA regions were amplified in 33/36 (91.7%) DNA samples. The s1m1 genotype was the most commonly isolated variant, accounting for 17/36 or 47.2% of positive samples. The s2m2 genotype was ascertained to be frequent also (14/36 or 38.9%). Almost all (94.1%) s1m1 genotypes were cagA positive. The majority of s2m2 genotypes (78.6%) were not associated with the cagA gene. Neither cagA nor vacA fragments were amplified from DNA isolates of H. pylori-uninfected children nor from DNA isolated from six gastrointestinal bacterial strains (specificity, 100%). The data suggest that the proposed modified technique of DNA extraction and PCR assay of stool samples may be an effective and reliable noninvasive tool for the detection and typing of H. pylori cagA/vacA virulence genes in infected individuals., (Copyright 2003 Elsevier Science B.V.)

Massachusetts was one of seven sentinel surveillance sites in the National Tuberculosis Genotyping and Surveillance Network. From 1996 through 2000, isolates from new patients with tuberculosis (TB) underwent genotyping. We describe the impact that genotyping had on public health practice in Massachusetts and some limitations of the technique. Through genotyping, we explored the dynamics of TB outbreaks, investigated laboratory cross-contamination, and identified Mycobacterium tuberculosis strains, transmission sites, and accurate epidemiologic links. Genotyping should be used with epidemiologic follow-up to identify how resources can best be allocated to investigate genotypic findings.

Motivation: The Direct Repeat (DR) locus of Mycobacterium tuberculosis is a suitable model to study (i) molecular epidemiology and (ii) the evolutionary genetics of tuberculosis. This is achieved by a DNA analysis technique (genotyping), called sp acer oligo nucleotide typing (spoligotyping ). In this paper, we investigated data analysis methods to discover intelligible knowledge rules from spoligotyping, that has not yet been applied on such representation. This processing was achieved by applying the C4.5 induction algorithm and knowledge rules were produced. Finally, a Prototype Selection (PS) procedure was applied to eliminate noisy data. This both simplified decision rules, as well as the number of spacers to be tested to solve classification tasks. In the second part of this paper, the contribution of 25 new additional spacers and the knowledge rules inferred were studied from a machine learning point of view. From a statistical point of view, the correlations between spacers were analyzed and suggested that both negative and positive ones may be related to potential structural constraints within the DR locus that may shape its evolution directly or indirectly., Results: By generating knowledge rules induced from decision trees, it was shown that not only the expert knowledge may be modeled but also improved and simplified to solve automatic classification tasks on unknown patterns. A practical consequence of this study may be a simplification of the spoligotyping technique, resulting in a reduction of the experimental constraints and an increase in the number of samples processed.

Motivation: Multi Locus Sequence Typing (MLST) is a newly developed typing method for bacteria based on the sequence determination of internal fragments of seven house-keeping genes. It has proved useful in characterizing and monitoring disease-causing and antibiotic resistant lineages of bacteria. The strength of this approach is that unlike data obtained using most other typing methods, sequence data are unambiguous, can be held on a central database and be queried through a web server., Results: A database-driven software system (mlstdb) has been developed, which is used by public health laboratories and researchers globally to query their nucleotide sequence data against centrally held databases over the internet. The mlstdb system consists of a set of perl scripts for defining the database tables and generating the database management interface and dynamic web pages for querying the databases., Availability: http://www.mlst.net.

Aims: Listeria monocytogenes strains isolated in the same geographical area from sewage sludge and from patients presenting with listeriosis were compared., Methods and Results: All isolates were typed by serotyping, phage typing and SmaI/ApaI pulsed-field gel electrophoresis (PFGE). Among the sludge isolates (n=32), 22 subtypes could be distinguished by the combination of all typing methods. The human isolates (n=11) were distributed into 10 subtypes which clearly differed from those observed among sludge isolates, except for one cluster formed by two related human isolates which showed high similarity in PFGE patterns (SmaI: 92%; ApaI: 89.5%) with one sludge isolate., Conclusion: These results suggest the existence of an epidemiological link between sludge and human isolates, but they may also be reflecting the distribution of L. monocytogenes types within the environment., Significance and Impact of the Study: Sludge and human L. monocytogenes may be related but further epidemiological studies are necessary to elucidate this point.

Introduction: The diagnostic test of swabbing the surface of the tonsil as a culture specimen for the determination of the organism responsible for the tonsil infection is still in practice, despite controversy. To date there has been no reports of establishing the reliability and validity of this common diagnostic test by appropriate statistical test of Likelihood ratios taking into consideration the specificity and sensitivity., Aims: To assess the reliability and validity of throat swab in the diagnosis of bacterial microflora in chronic tonsillitis., Patients and Methods: A prospective study of 40 patients clinically diagnosed to have chronic tonsillitis undergoing tonsillectomy was undertaken. The reliability of the throat surface swab was then assessed and validated with the reference (gold) standard of tonsil core culture., Results: The likelihood ratio of this diagnostic test being positive (LR +ve) was 0.84 to similar and 1.3 to general organism pathogen., Conclusion: Routine culture of the throat by surface swab in the accurate diagnosis of bacterial flora in chronic tonsillitis is neither reliable nor valid. The clinical implications of this investigation which is still very popular is discussed.

Methods: A simple and reproducible proposed for Benito et al. scheme for identification biotypes of Gardnerella vaginalis has been developed, based on reactions for lipase, hippurate, hydrolysis, and beta-galactosidase., Results: 11 biotypes were found among 130 strains from women with and without bacterial vaginosis (non-specific vaginitis) 1A, 5B; 8B, 5C, 8C, 1E, 6G and 7G in women with vaginosis and the biotypes 1A, 5B, 8B, 1C, 8F and 6H in women without vaginosis. These biotypes 1A; 5B and 8B were found in two groups., Conclusions: These data suggest that some biotypes of G. vaginalis are associated with bacterial vaginosis.

Objective: To assess state-based surveillance for isolation from a sterile site of vancomycin-resistant enterococci (VRE) in Connecticut., Design: Clinical laboratory reporting (passive surveillance) of VRE isolates to the Connecticut Department of Public Health (CDPH) was followed by state-initiated validation, laboratory proficiency testing, and review of hospital demographic characteristics., Settings: All 45 clinical laboratories and all 37 (36 for 1995 and 1996) acute-care hospitals in Connecticut were included in the study., Main Outcome Measures: The outcome measures included determination of the statewide incidence of VRE and the accuracy of passive reporting, determination of clinical laboratory proficiency in detecting VRE, and analysis of hospital characteristics that might be associated with an increased incidence of VRE., Results: During 1994 through 1996, 29 (78%) of 37 hospital-affiliated clinical laboratories and 1 (11%) of 9 commercial or other laboratories in Connecticut reported to the CDPH the isolation of VRE from sterile sites; 158 isolates were reported for these 3 years. Based on verification, we discovered that these laboratories actually detected 58 VRE isolates in 1994, 104 in 1995, and 104 in 1996 (total, 266). The age-standardized incidence rate of VRE was 14.1 cases per million population in 1994 and 26.8 cases per million population for both 1995 and 1996. Laboratory proficiency testing revealed that high-level vancomycin resistance was identified accurately and that low- and moderate-level resistance was not detected. The incidence of VRE isolates was three times greater in hospitals with over 300 beds compared with categories of hospitals with fewer beds. Increases in the number of VRE isolates were at least twice as likely in hospitals located in areas with a higher population density, or with a residency program or trauma center in the hospital., Conclusions: Passive reporting of VRE isolates from sterile sites markedly underestimated the actual number of iso lates, as determined in a statewide reporting system. Statewide passive surveillance systems for routine or emerging pathogens must be validated and laboratory proficiency ensured if results are to be accurate and substantial underreporting is to be corrected.

The performances of five automated microbial identification systems, relative to that of a reference identification system, for their ability to accurately and repeatedly identify six common food-borne pathogens were assessed. The systems assessed were the MicroLog system (Biolog Inc., Hayward, Calif.), the Microbial Identification System (MIS; MIDI Inc., Newark, Del.), the VITEK system (bioMérieux Vitek, Hazelwood, Mo.), the MicroScan WalkAway 40 system (Dade-MicroScan International, West Sacramento, Calif.), and the Replianalyzer system (Oxoid Inc., Nepean, Ontario, Canada). The sensitivities and specificities of these systems for the identification of food-borne isolates of Bacillus cereus, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and verotoxigenic Escherichia coli were determined with 40 reference positive isolates and 40 reference negative isolates for each pathogen. The sensitivities of these systems for the identification of these pathogens ranged from 42.5 to 100%, and the specificities of these systems for the identification of these pathogens ranged from 32.5 to 100%. Some of the systems had difficulty correctly identifying the reference isolates when the results were compared to those from the reference identification tests. The sensitivity of MIS for the identification of S. aureus, B. cereus, E. coli, and C. jejuni, for example, ranged from 47.5 to 72. 5%. The sensitivity of the Microlog system for the identification of E. coli was 72.5%, and the sensitivity of the VITEK system for the identification of B. cereus was 42.5%. The specificities of four of the five systems for the identification of all of the species tested with the available databases were greater than or equal to 97.5%; the exception was MIS for the identification of C. jejuni, which displayed a specificity of 32.5% when it was tested with reference negative isolates including Campylobacter coli and other Campylobacter species. All systems had >80% sensitivities for the identification of Salmonella species and Listeria species at the genus level. The repeatability of these systems for the identification of test isolates ranged from 30 to 100%. Not all systems included all six pathogens in their databases; thus, some species could not be tested with all systems. The choice of automated microbial identification system for the identification of a food-borne pathogen would depend on the availability of identification libraries within the systems and the performance of the systems for the identification of the pathogen.

Of four tested identification systems (API 20E, API Rapid 32 IDE, Micronaut E, and the PCR-based Yersinia enterocolitica Amplification Set), API 20E is still the system of choice for identifying pathogenic Yersinia isolates. It provides the highest sensitivity both at the genus and at the species level and has the best cost-effectiveness correlation.

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and SphI, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PT11 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

A ligation-mediated PCR (LMPCR) method for the amplification of sequences flanking the IS6110 of the Mycobacterium tuberculosis complex has been developed. The method uses one primer specific for IS6110 and a second specific for a linker ligated to SalI-restricted genomic DNA. LMPCR is a rapid screening method, valuable for the fingerprinting of M. tuberculosis complex strains.

We evaluated the Vitek GNI+ and Becton Dickinson Crystal E/NF identification systems for their ability to accurately identify 619 and 626 strains, respectively, of members of the family Enterobacteriaceae and other glucose-fermenting and non-glucose-fermenting gram-negative rods. All strains tested were taken from a stock collection and passed three times on 5% sheep blood agar prior to testing. These strains represented a more rigorous challenge to both systems than one resulting from the testing of consecutive clinical isolates. Testing with both systems was done according to the manufacturers' instructions, and tests were repeated in duplicate when errors occurred. Vitek version 5.01 and Crystal version 3.0 softwares were used for identifications. The identification results from each system were compared with identifications previously determined with reference biochemicals. At the completion of the appropriate incubation period, the GNI+ and Crystal systems correctly identified 80.1 and 71.1% of the total isolates, respectively. After additional tests suggested by the software programs were completed, the GNI+ had an accuracy of 87.6% and the Crystal system's accuracy had improved to 87.9%. The error rates for the GNI+ and Crystal systems were 6.5 and 5.3%, respectively. A report of "no identification" was given for 6.0 and 6.9% of the isolates, respectively, and was associated with no particular organism group. One isolate each of Acinetobacter lwoffii and Vibrio alginolyticus would not grow in the Vitek card. The average times to detection for correct enteric identifications in the GNI+ system were 4.1 and 6.8 h for nonenteric identifications, while the Crystal results were routinely read at 18 h. We conclude that there was no significant difference (P > 0.05) between the results of the GNI+ card and those of the Crystal E/NF system after additional testing was performed with the group of organisms tested, but the overall accuracy for both systems in this study was below 90%.

Large-restriction-fragment (LRF) polymorphisms in Mycobacterium kansasii isolates from 84 patients with bronchopulmonary infections in Japan between the 1960s and 1995 were studied by pulsed-field gel electrophoresis (PFGE). Chromosomal fragments digested with VspI were most suitable for PFGE separation of 16 to 21 fragments of between 40 and 550 kbp. All 84 isolates and the type strain M. kansasii ATCC 12478 were successfully typed by LRF analysis with VspI digestion. Twenty-one distinctive LRF types were identified, and the LRF patterns tested over time were reproducible and stable. A computer-assisted dendrogram of the percent similarity demonstrated that isolates of 18 LRF types had relatively close genetic relatedness, while isolates of the remaining 3 types showed divergence. Sequence analysis of the 16S rRNA gene in the isolates showing divergent genetic relatedness revealed a sequence identical to that of a previously reported subspecies of M. kansasii. In the Chugoku district of Japan, 11 cases of M. kansasii infection which occurred in workers in a coastal industrial zone between 1982 and 1993 were caused by one particular strain tentatively named LRF type M. When both detailed demographic data for the patients and ecologic data for the M. kansasii isolates are obtained, LRF typing may be of potential use for investigating the source and transmission of M. kansasii infection.

Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms.

We examined the utility of a PCR-based method termed cleaved amplified polymorphic sequences (CAPS) to type 35 well-characterized isolates of Staphylococcus epidermidis. The results were compared with detailed epidemiologic information and typing obtained by using pulsed-field gel electrophoresis (PFGE). To identify CAPS markers for this study, eight pairs of oligonucleotide primers corresponding to five previously sequenced S. epidermidis genes were synthesized and then used to amplify DNA sequences from the S. epidermidis strains by using PCR. Amplified products were reproducibly obtained for seven of eight primer pairs from chromosomal DNA of 33 of the 35 isolates. Seven restriction site polymorphisms were found in five of the amplified products when they were subjected to digestion with a panel of restriction endonucleases. Each fragment-enzyme combination that was polymorphic demonstrated only two alleles in the 33 S. epidermidis isolates analyzed, corresponding to the presence or absence of a single restriction site. Overall, five distinct combinations of alleles were detected and were designated CAPS types A through E. There was a close correlation between the CAPS grouping, the epidemiologic information for the strains, and grouping by PFGE following SmaI digestion of chromosomal DNA. Although PFGE analysis was more discriminatory than typing based on the limited number of CAPS markers used in this study (isolates from the same CAPS group were sometimes distributed into more than one PFGE group), no isolates from the same PFGE group were found in more than one CAPS group. The CAPS procedure was highly reproducible, in contrast to published experience with arbitrarily primed PCR. These preliminary data suggest that CAPS represents a PCR-based technique for strain typing that is highly reproducible, rapid, utilizes widely available technologies, and provides results that are relatively easy to interpret and express.

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.