Evaluation of a new real-time PCR assay for the direct detection of diarrheagenic Escherichia coli in stool specimens.

Diarrheagenic E. coli (DEC) are one of the most common causes for diarrhea worldwide, especially in children. We evaluated the rapid RIDA ® GENE (RG) real-time multiplex PCR assays (R-Biopharm, Darmstadt, Germany) for the detection of the most important diarrheagenic E. coli. Three hundred fifteen liquid or non-formed stool specimens were examined. The results of the RG multiplex assays were compared to specific PCR methods. The sensitivity and specificity of the RG PCRs were as follows, 100%/100% for the detection of EHEC, 96.3% and 99% for EPEC, 100% and 100% for the detection of EAEC, ETEC and EIEC, respectively. Overall, the RG real-time PCR system for the detection of DEC tested in this study provided reliable and rapid results and shows the ability as a useful addendum for the detection of diarrheagenic E. coli in the medical laboratory.

Evaluation of six PCR assays in combination with patient related data for the diagnosis of Clostridium difficile-associated infections.

BACKGROUND: The aim of this multicenter study was to establish a diagnostic algorithm using molecular methods for the diagnosis of C. difficile-associated infection (CDI). In addition patient specific data were taken into consideration for the interpretation of the results. METHODS: We compared the performance of six different commercially available PCR-tests, two toxin immunoassays, and a glutamat-dehydrogenase test by analysing liquid stool specimens from patients with suspected CDI. Toxigenic culture on CLO-agar was used as reference method. RESULTS: In total 250 stool specimens were collected at two study sites. 77 (30.8%) stool samples were culture-positive for toxigenic C. difficile. 173 (69.2%) specimens showed no growth of C. difficile. As a result, each of the PCR assays tested for C. difficile had a significantly higher sensitivity (94.8% - 100%) and NPV (97.6% - 100%) than the TOX-EIA with a sensitivity of 57.1% and NPV of 83.8%. Specificity of the PCR tests was 94.1% to 96.0% and PPV between 86.5% and 91.6%. The analysis of the patient data revealed a significant difference (p-value 0.0202) between toxin-positive and toxin-negative patients regarding prior antibiotic treatment, especially for cephalosporins. CONCLUSIONS: Our findings support the recommendation to restrict the use of antibiotics as a cornerstone in the prevention of CDI. We conclude that all of the PCR assays evaluated in this study can be applied in a diagnostic algorithm.

Differentiation of Candida dubliniensis from Candida albicans by means of MALDI-TOF mass spectrometry.

BACKGROUND: Species specific differentiation of the two closely related yeasts, Candida albicans and C. dubliniensis, is difficult in routine diagnosis. METHODS AND RESULTS: Here we show that MALDI-TOF MS is a practical and useful tool for the discrimination of C. albicans and C. dubliniensis, demonstrated by the analysis of reference strains from type culture collections and other well characterized isolates. The spectra of C. albicans and C. dubliniensis further revealed that each species consists of several clades. CONCLUSIONS: Reliable, routinely applicable methods for species specific differentiation of C. albicans and C. dubliniensis appear to be of particular importance to better understand the epidemiology and virulence of C. dubliniensis.

Evaluation of a new selective medium, BD BBL CHROMagar MRSA II, for detection of methicillin-resistant Staphylococcus aureus in different specimens.

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.

High interlaboratory reproducibility of matrix-assisted laser desorption ionization-time of flight mass spectrometry-based species identification of nonfermenting bacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).

Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay.

The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) is still increasing worldwide and is associated with significant morbidity, mortality and hospital costs. Screening for MRSA plays a key role in limiting further nosocomial spread of this organism. Control measures require a rapid and sensitive test for direct detection of MRSA carriage. This study evaluated an easy-to-use PCR-hybridisation assay for the direct detection of MRSA in clinical swab specimens. In total, 508 pairs of swabs from 242 patients at risk for MRSA carriage were analysed by the standard culture method and the PCR assay. One swab was used for PCR and culture, while the second was used for culture only. Of the 508 pairs tested, 37 were positive by culture and 35 were positive by PCR. Among the 471 culture-negative specimens, 465 were negative by PCR and six were PCR-positive. The PCR assay had a sensitivity of 94.59%, a specificity of 98.73%, a positive predictive value of 85.37%, and a negative predictive value of 99.57%. The PCR-hybridisation assay enabled reliable detection of MRSA carriage in c. 4 h, thereby allowing its effective use in an MRSA control strategy.

Spread of ampicillin/vancomycin-resistant Enterococcus faecium of the epidemic-virulent clonal complex-17 carrying the genes esp and hyl in German hospitals.

The incidence of vancomycin-resistant Enterococcus faecium isolation was low (

Evaluation of a rapid direct assay for identification of bacteria and the mec A and van genes from positive-testing blood cultures.

We performed the first evaluation of a DNA strip assay (GenoType blood culture; Hain Lifescience, Nehren, Germany) for the detection of the most relevant bacterial sepsis pathogens directly from positive BACTEC blood culture bottles (Becton Dickinson, Heidelberg, Germany). The test comprises two panels, one for the direct species identification of important gram-positive cocci and the other for gram-negative rods. Additionally, detection of the mec A and the van genes are implemented. The GenoType assay was validated regarding its analytical sensitivity with blood cultures spiked with reference strains. Approximately 10(4) CFU per ml were detected. Analytical specificity was calculated with a test panel of 212 reference strains. Of the strains tested, 99% were correctly identified. Additionally, 279 consecutive blood cultures signaled positive by BACTEC were processed directly, in comparison to conventional methods. The GenoType assays were performed according to Gram stain morphology. A total of 243 (87.1%) of the 279 organisms isolated were covered by specific probes. A total of 152 organisms were gram-positive cocci, of which 148 (97.4%) were correctly identified by the GenoType assay. Ninety-one organisms were gram-negative rods, of which 89 (97.8%) were correctly identified. Concerning mec A gene detection, GenoType assay correctly detected 12 of 13 methicillin-resistant Staphylococcus aureus isolates. One Enterococcus faecium isolate with a positive van A gene isolated was correctly differentiated by the assay. All results were available 4 h after the results of microscopic analysis. The evaluated GenoType blood culture assay showed fast and reliable results in detecting the most important sepsis pathogens and the mec A and van genes directly from positive blood culture bottles.

Analysis of the comparative workflow and performance characteristics of the VITEK 2 and Phoenix systems.

The VITEK 2 (bioMérieux, Marcy L'Etoile, France) and the Phoenix systems (BD Diagnostic Systems, Sparks, Md.) are automated instruments for rapid organism identification and susceptibility testing. We evaluated the workflow, the time to result, and the performance of identification and susceptibility testing of both instruments. A total of 307 fresh clinical isolates were tested: 141 Enterobacteriaceae, 22 nonfermenters, 93 Staphylococcus spp., and 51 Enterococcus spp. Manipulation time was measured in batches, each with seven isolates, for a total of 39 batches. The mean (+/- standard deviation [SD]) manipulation time per batch was 20.9 +/- 1.8 min for Phoenix and 10.6 +/- 1.0 min for VITEK 2 (P < 0.001). Mean (+/-SD) time to result for all bacterial groups was 727 +/- 162 min for Phoenix and 506 +/- 120 min for VITEK 2 (P < 0.001). Concerning identification, Phoenix and VITEK 2 yielded the same results for nonfermenters (100%), staphylococci (97%), and enterococci (100%). For 140 Enterobacteriaceae strains evaluated, 135 (96%) were correctly identified by Phoenix and 137 (98%) by VITEK 2 (P = 0.72). The overall category agreement for all isolates was 97.0% for both instruments. The minor error rate, major error rate, and very major error rate for all bacterial isolates tested were 3.0, 0.3, and 0.6 and 2.8, 0.2, and 1.7 for Phoenix and VITEK 2, respectively (P values of 0.76, 0.75, and 0.09). The VITEK 2 system required less manual manipulation time and less time than the Phoenix system to yield results.

Evaluation of a new molecular system for simultaneous identification of four Enterococcus species and their glycopeptide resistance genotypes.

We compared the performances of a new DNA-based strip assay and the VITEK 2 system for identification of 105 enterococcal strains and differentiation of their van gene-associated resistance levels. Both methods provided excellent results. The molecular assay showed advantages in time to result for identification of van-associated genes.

Evaluation of a new chromogenic medium for the isolation and presumptive identification of Salmonella species from stool specimens.

The performance of BBL CHROMagar Salmonella (Becton Dickinson, France), a new selective chromogenic medium for the isolation and presumptive identification of Salmonella spp., was evaluated. On this medium, which is a modification of CHROMagar Salmonella (CHROMagar Microbiology, France) with enhanced selectivity, the colonies of Salmonella are stained in mauve (rose-violet), while those of other organisms appear in blue-green or are not stained by any of the chromogens of the medium. The medium was evaluated with a total of 176 strains of Salmonella and other organisms, consisting of 18 reference strains and 158 clinical isolates. All Salmonella strains except subspecies IIIa and IIIb strains and Salmonella Gallinarum yielded typical mauve colonies. During the evaluation with 107 known positive and 332 unknown stool specimens in a clinical laboratory, a total of 115 and 105 Salmonella isolates were obtained on BBL CHROMagar Salmonella and Hektoen enteric agar, respectively. From the known positive stool specimens, 92 true positive cultures were obtained on BBL CHROMagar Salmonella and 89 on Hektoen enteric agar, yielding sensitivities of 86 and 83%, respectively. From the unknown stool specimens, a total of 27 Salmonella isolates were obtained, with 23 isolated from BBL CHROMagar Salmonella and 16 from Hektoen enteric agar by direct plating (sensitivity 85 and 59%, specificity 99 and 97%, respectively). Seroagglutination tests could be performed directly from BBL CHROMagar Salmonella. Compared to conventional isolation media, the time needed for confirmatory biochemical and serological tests was shortened by about 1 day when BBL CHROMagar Salmonella was used. On the basis of these results, the medium can be recommended for the primary isolation and presumptive identification of Salmonella spp. from clinical stool specimens.

[The analgesic efficacy of sulfur mud baths in treating rheumatic diseases of the soft tissues. A study using the double-blind control method]. / Ob anal'geticheskoi éffektivnosti sernistykh griazevykh vann pri lechenii revmaticheskikh zabolevanii miagkikh tkanei. Issledovanie s primeneniem metoda slepogo dvoinogo kontrolia.

Changes in pain thresholds assessed in 16 trigger points were traced in two randomly selected groups of patients subjected to diluted mud baths in the presence or absence of sulfur compounds. Sulfur baths produced a significant lowering of pain sensitivity. The effect was augmenting with the number of procedures, the rise slowing down to the end of the course.