Comparison of three multiplex real-time PCR assays for detection of enteric viruses in patients with diarrhea.

In this study, the usability and performance of three commercially available multiplex real-time RT-PCR assays for the detection of major enteric viruses were investigated. In total, 481 fecal specimens were analyzed using the Allplex™ GI Virus Assay, the Rida®Gene Viral Stool Panel I, and the FTD Viral Gastroenteritis. The overall agreement between the assays was 99.9%. Despite convergent analytical performance, differences between the multiplex RT-PCR assays were apparent when considering their suitability for routine diagnostics.

Use of an automated PCR assay, the GenomEra S. pneumoniae, for rapid detection of Streptococcus pneumoniae in blood cultures.

BACKGROUND: Streptococcus pneumoniae is recognized as a major cause of pneumonia, meningitis, and bacteremia. Since the mortality rate for pneumococcal bacteremia remains high, the reliable detection of the bacterium in blood samples is important. In this study, the performance of a new automated PCR assay, the GenomEra(™) S. pneumoniae, for direct detection of S. pneumoniae in blood cultures was investigated. METHODS: In total, 200 samples were analyzed, including 90 previously identified culture collection isolates and 110 blood culture specimens. The species identification was confirmed with routine diagnostic methods including MALDI-TOF or 16S rDNA sequencing. RESULTS: From culture collection, the GenomEra S. pneumoniae assay correctly identified all 37 S. pneumoniae isolates consisting of 18 different serotypes, while all 53 non-S. pneumoniae isolates yielded negative test results. Of 110 blood culture specimens, 46 grew S. pneumoniae and all were positive by the GenomEra assay direct from bottle. The detection sensitivity and specificity of the GenomEra assay for direct analysis of S. pneumoniae in signal positive blood culture bottles was 100%, respectively. With a straightforward sample preparation protocol of blood cultures the results were available within 55 min, thus being significantly quicker than by the routinely used identification methods (18-48 h). The two-step, time-resolved fluorometric measurement mode employed by the GenomEra CDX(™) instrument showed no interference from blood or charcoal. CONCLUSION: The GenomEra S. pneumoniae assay is a tool that performs well for the rapid and reliable detection of S. pneumoniae in blood cultures.

The use of molecular methods for the detection and identification of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen in many hospitals and long-term care facilities as well as in the community. To limit the spread of MRSA, early detection and proper treatment are essential. Because conventional culture as gold standard is time consuming, new techniques such as PCR-based and hybridization assays have emerged for the rapid detection of MRSA. This review will focus on the currently available molecular-based assays and on their utility and performance for detection of S. aureus, of its virulence factors and of the markers for acquired resistance.

Comparison of FecalSwab and ESwab devices for storage and transportation of Diarrheagenic bacteria.

Using a collection (n = 12) of ATCC and known stock isolates, as well as 328 clinical stool specimens, we evaluated the ESwab and the new FecalSwab liquid-based microbiology (LBM) devices for storing and transporting diarrheagenic bacteria. The stock isolates were stored in these swab devices up to 48 h at refrigeration (4°C) or room (∼25°C) temperature and up to 3 months at -20°C or -70°C. With the clinical stool specimens, the performances of the ESwab and FecalSwab were compared to those of routinely used transport systems (Amies gel swabs and dry containers). At a refrigeration temperature, all isolates survived in FecalSwab up to 48 h, while in ESwab, only 10 isolates (83.3%) out of 12 survived. At -70°C, all isolates in FecalSwab were recovered after 3 months of storage, whereas in ESwab, none of the isolates were recovered. At -20°C, neither of the swab devices preserved the viability of stock isolates after 2 weeks of storage, and at room temperature, 7 (58.3%) of the stock isolates were recovered in both transport devices after 48 h. Of the 328 fecal specimens, 44 (13.4%) were positive for one of the common diarrheagenic bacterial species with all transport systems used. Thus, the suitability of the ESwab and FecalSwab devices for culturing fresh stools was at least equal to those of the Amies gel swabs and dry containers. Although the ESwab was shown to be an option for collecting and transporting fecal specimens, the FecalSwab device had clearly better preserving properties under different storage conditions.

Performance of SaSelect, a chromogenic medium for detection of staphylococci in clinical specimens.

In a preliminary study, known staphylococcus (n = 86) and other microbial (n = 12) isolates were plated on three chromogenic media, SaSelect (Bio-Rad, Hercules, CA, USA), CHROMagar Staph. aureus (CHROMagar Microbiology, Paris, France), and S. aureus ID (bioMérieux, Marcy l'Etoile, France). The sensitivities of all the media to detect Staphylococcus aureus after 24 h of incubation were high (100.0%). However, their specificities varied at 93.3% (95% confidence interval [CI], 86.0% to 100.0%) (CHROMagar Staph. aureus), 97.8% (95% CI, 93.5% to 100.0%) (S. aureus ID), and 100.0% (SaSelect). SaSelect also showed the highest sensitivity for recovery and differentiation of other staphylococci. As the best performing chromogenic medium, SaSelect was then prospectively compared to conventional culture and identification tests for the detection of staphylococci from 2,780 clinical specimens. A total of 1,589 staphylococcal isolates were recovered. Of these, 912 were S. aureus and 677 were other staphylococci. The sensitivity and specificity of SaSelect to detect S. aureus in clinical specimens after 24 h of incubation were 99.6% and 99.9% (95% CI, 99.2% to 100.0% and 99.8% to 100.0%), respectively, whereas the sensitivity and specificity using conventional plates combined with laboratory identification methods were 96.8% and 99.5% (95% CI, 95.7 to 97.9% and 99.2% to 99.8%). For the recovery and preliminary identification of other staphylococci, the sensitivity and specificity of SaSelect were 94.4% and 99.9%. SaSelect is a well-performing chromogenic medium that significantly improved the detection of staphylococci, especially S. aureus, compared to conventional culture (P < 0.0001).

GenomEra MRSA/SA, a fully automated homogeneous PCR assay for rapid detection of Staphylococcus aureus and the marker of methicillin resistance in various sample matrixes.

The GenomEra MRSA/SA assay (Abacus Diagnostica, Turku, Finland) is the first commercial homogeneous PCR assay using thermally stable, intrinsically fluorescent time-resolved fluorometric (TRF) labels resistant to autofluorescence and other background effects. This fully automated closed tube PCR assay simultaneously detects Staphylococcus aureus specific DNA and the mecA gene within 50 min. It can be used for both screening and confirmation of methicillin-resistant and -sensitive S. aureus (MRSA and MSSA) directly in different specimen types or from preceding cultures. The assay has shown excellent performance in comparisons with other diagnostic methods in all the sample types tested. The GenomEra MRSA/SA assay provides rapid assistance for the detection of MRSA as well as invasive staphylococcal infections and helps the early targeting of antimicrobial therapy to patients with potential MRSA infection.

Evaluation of a new automated homogeneous PCR assay, GenomEra C. difficile, for rapid detection of Toxigenic Clostridium difficile in fecal specimens.

We evaluated a new automated homogeneous PCR assay to detect toxigenic Clostridium difficile, the GenomEra C. difficile assay (Abacus Diagnostica, Finland), with 310 diarrheal stool specimens and with a collection of 33 known clostridial and nonclostridial isolates. Results were compared with toxigenic culture results, with discrepancies being resolved by the GeneXpert C. difficile PCR assay (Cepheid). Among the 80 toxigenic culture-positive or GeneXpert C. difficile assay-positive fecal specimens, 79 were also positive with the GenomEra C. difficile assay. Additionally, one specimen was positive with the GenomEra assay but negative with the confirmatory methods. Thus, the sensitivity and specificity were 98.8% and 99.6%, respectively. With the culture collection, no false-positive or -negative results were observed. The analytical sensitivity of the GenomEra C. difficile assay was approximately 5 CFU per PCR test. The short hands-on (<5 min for 1 to 4 samples) and total turnaround (<1 h) times, together with the high positive and negative predictive values (98.8% and 99.6%, respectively), make the GenomEra C. difficile assay an excellent option for toxigenic C. difficile detection in fecal specimens.

First isolation of Dietzia cinnamea from a dog bite wound in an adult patient.

We report the first case of deep-wound colonization by Dietzia cinnamea in a patient who had been bitten by a dog.

Usability and performance of CHROMagar STEC medium in detection of Shiga toxin-producing Escherichia coli strains.

The performance and usability of CHROMagar STEC medium (CHROMagar Microbiology, Paris, France) for routine detection of Shiga toxin-producing Escherichia coli (STEC) strains were examined. The ability of the medium to selectively propagate STEC strains differing by their serotypes and virulence genes was studied with a collection of diarrheagenic E. coli isolates (n = 365) consisting of 49 different serotypes and with non-STEC and other bacterial isolates (n = 264). A total of 272 diarrheagenic E. coli (75.0%) isolates covering 24 different serotypes grew on CHROMagar STEC. The highest detection sensitivities were observed within the STEC serogroups O26 (90.0%), O111 (100.0%), O121 (100.0%), O145 (100.0%), and O157 (84.9%), and growth on CHROMagar STEC was highly associated with the presence of the tellurite resistance gene (terD). The specificity of the medium was 98.9%. In addition, CHROMagar STEC was used in parallel with a Shiga toxin-detecting immunoassay (Ridaquick Verotoxin/O157 Combi; R-biopharm, Darmstadt, Germany) to screen fecal specimens (n = 47) collected from patients suffering from hemorrhagic diarrhea. Positive growth on CHROMagar STEC was confirmed by the Premier EHEC enzyme immunoassay (Meridian Bioscience, Inc., Cincinnati, OH), and discrepant results between the two screening methods were confirmed by stx gene-detecting PCR. All 16 of the 47 stool samples that showed positive growth on CHROMagar STEC were also positive in the confirmatory tests. CHROMagar STEC proved to be an interesting option for STEC screening, allowing good detection sensitivity and specificity and permitting strain isolation for further outbreak investigations when required.

Shiga toxin-producing Escherichia coli serotype O78:H(-) in family, Finland, 2009.

Shiga toxin-producing Escherichia coli (STEC) is a pathogen that causes gastroenteritis and bloody diarrhea but can lead to severe disease, such as hemolytic uremic syndrome (HUS). STEC serotype O78:H(-) is rare among humans, and infections are often asymptomatic. We detected a sorbitol-fermenting STEC O78:H(-):stx(1c):hlyA in blood and fecal samples of a 2-week-old boy who had bacteremia and HUS and in fecal samples of his asymptomatic family members. The phenotypic and genotypic characteristics and the virulence properties of this invasive STEC were investigated. Our findings demonstrate that contrary to earlier suggestions, STEC under certain conditions can invade the human bloodstream. Moreover, this study highlights the need to implement appropriate diagnostic methods for identifying the whole spectrum of STEC strains associated with HUS.

Bacteriophage ϕ6 nucleocapsid surface protein 8 interacts with virus-specific membrane vesicles containing major envelope protein 9.

Enveloped double-stranded RNA (dsRNA) bacterial virus Pseudomonas phage ϕ6 has been developed into an advanced assembly system where purified virion proteins and genome segments self-assemble into infectious viral particles, inferring the assembly pathway. The most intriguing step is the membrane assembly occurring inside the bacterial cell. Here, we demonstrate that the middle virion shell, made of protein 8, associates with the expanded viral core particle and the virus-specific membrane vesicle.