Diagnostic Practices for Suspected Community-Acquired Central Nervous System Infection in the Post-Conjugate Vaccine Era.

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.

Vancomycin resistant enterococcal (VRE) colonization among patients treated in intensive care units at the National Hospital of Sri Lanka, and determination of genotype/s responsible for resistance

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.

Laboratory diagnosis of Clostridium difficile infection: Comparison of Techlab C. diff Quik Chek Complete, Xpert C. difficile, and multistep algorithmic approach.

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.

[Laboratory practices: diagnostics and antibiotics resistance testing of Neisseria gonorrhoeae in Germany]. / Die deutsche Laborlandschaft zu Diagnostik und Antibiotikaresistenztestung bei Neisseria gonorrhoeae.

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.

[Recognition of infections in elderly emergency patients]. / Erkennen von Infektionen beim älteren Notfallpatienten.

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.

[Analysis of laboratory tests results for Salmonella infections performed since 2008 to 2014 in Poland]. / Analiza wyników badan laboratoryjnych w kierunku zakazen wywolanych przez paleczki Salmonella w latach 2008-2014 w Polsce.

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.

Is the gram stain useful in the microbiologic diagnosis of VAP? A meta-analysis.

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.

[Bacterial identification methods in the microbiology laboratory]. / Métodos de identificación bacteriana en el laboratorio de microbiología.

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.

The use of spa and phage typing for characterization of a MRSA population in a Belgian hospital: comparison between 2002 and 2007.

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.

Evaluation of the MIT RMID 1000 system for the identification of Listeria species.

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.

Bacterial Taxa and Functions Are Predictive of Sustained Remission Following Exclusive Enteral Nutrition in Pediatric Crohn's Disease.

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.

New multiplatform computer program for numerical identification of microorganisms.

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).

Spectrum of practice in the diagnosis of nosocomial pneumonia in patients requiring mechanical ventilation in European intensive care units.

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.

Internet-based sequence-typing databases for bacterial molecular epidemiology.

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.

spa typing for epidemiological surveillance of Staphylococcus aureus.

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.

Rapid and robust analytical protocol for E. coli STEC bacteria subspecies differentiation using whole cell MALDI mass spectrometry.

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.

[Resistance testing for urinary tract infections. A barrier to guideline implementation]. / Resistenzprüfung bei Harnwegsinfektionen. Eine Barriere für die Leitlinienimplementierung.

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.

Clinical significance of Roseomonas species isolated from catheter and blood samples: analysis of 36 cases in patients with cancer.

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.

Reporting of vancomycin-resistant enterococci in Connecticut: implementation and validation of a state-based surveillance system.

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.

Evaluation of PCR-based methods and ribotyping performed with a mixture of PstI and SphI to differentiate strains of Salmonella serotype Enteritidis.

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.