Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.

Rapid detection of Group B streptococcus directly from vaginal-rectal specimens using liquid swabs and the BD Max GBS assay.

OBJECTIVE: We adapted the BD Max GBS assay, an automated platform for the detection of Group B streptococcus (GBS) DNA in vaginal-rectal swab specimens after LIM broth enrichment, to directly detect GBS in specimens collected using cellular foam swabs in Amies liquid medium. We compared the BD Max GBS assay performance to that of enriched culture and the BD GeneOhm StrepB assay. METHODS: Seventy-two reference vaginal-rectal specimens were employed to determine the limit of GBS detection and the preferred test volume for direct detection of GBS. A total of 304 clinical specimens were then tested by the optimized BD Max GBS assay, both by direct testing and following broth enrichment. RESULTS: The limit of GBS detection was 75 CFU/mL and the preferred test volume was 100 µL. Of 304 clinical specimens tested, GBS was detected in 62 specimens by enriched culture (20.4%); 61 of these yielded GBS by the BD Max GBS assay when performed directly from the liquid swab (sensitivity 98.4%). All 242 culture-negative specimens also yielded negative results by the BD Max GBS assay (specificity 100%). When this assay was performed following broth enrichment, GBS was detected in all 62 culture-positive specimens (100% sensitivity). The sensitivity and specificity of the BD GeneOhm StrepB assay was 90.3% and 99%, respectively. CONCLUSIONS: The BD Max GBS assay is highly sensitive, requires minimal technical skill with <2 min required to set-up, and results are available in under 80 min (versus 24-48 h for culture). It is configured for 'on demand' testing and vaginal-rectal specimens can be rapidly screened for GBS without the need for enrichment. The results obtained in this study demonstrate that rapid GBS screening using the BD Max GBS assay at the time of delivery is a viable alternative to the current recommended screening at 35-37 weeks of gestation with pre-enrichment testing methods.

Difficulties in detection and identification of Enterococcus faecium with low-level inducible resistance to vancomycin, during a hospital outbreak.

Between June and November 2004, a vancomycin-resistant Enterococcus faecium (VRE) strain was isolated from 13 patients in the haematology/bone marrow transplant unit. There were difficulties in identifying the organism, which had low-level, inducible vancomycin resistance, and standard screening methods did not reveal carriage in patients or their contacts. These technical failures led to spread of VRE and delays in providing appropriate management, which might otherwise have been avoided. Therefore, we reviewed our laboratory methods and compared three identification systems to determine which would best identify this VRE strain. The VITEK 2 (BioMerieux) correctly identified, as E. faecium, only two of 16 isolates, whereas API Rapid ID 32 Strep (BioMerieux) and Phoenix 100 (Becton Dickinson and Co.) correctly identified 13 of 15 and 12 of 13 isolates tested, respectively. Isolates from urine, tested by the CLSI disk diffusion method, were apparently susceptible or of intermediate susceptibility to vancomycin, upon primary testing. VITEK 2 and Phoenix 100 identified all isolates as vancomycin-resistant, although the MICs, measured by Etest, were in the susceptible range for three of 16 isolates. Reducing the vancomycin concentration in screening media substantially increased the sensitivity for detection of VRE. Isolates were characterized as genotype vanB2/3 by PCR and were indistinguishable from each other by pulsed-field gel electrophoresis. VRE with low-level inducible resistance can be missed by routine screening methods. Better identification and screening methods for detection of low-level vancomycin resistance are needed to improve surveillance and prevent transmission of VRE.

Evaluation of four methods for rapid identification of Staphylococcus aureus from blood cultures.

The identification of Staphylococcus aureus directly from blood cultures is clinically relevant, but it requires a test that is both rapid and reliable. Previously, biochemical, immunological, tube coagulase, and thermostable-endonuclease methods have shown variable sensitivity and specificity. Testing directly from blood culture broth has not been described for the latex kit Staphaurex Plus (Murex Diagnostics Ltd.), and the modified conventional tests have not been used with the newer, continuously monitored blood culture systems. In addition, the commercial RAPIDEC staph kit (bioMerieux Vitek, Inc.) has been used to detect S. aureus directly from the Vital blood culture system (bioMerieux, Marcy l'Etoile, France), but its performance has not been evaluated with other continuously monitored systems. A total of 201 clinical blood cultures (BACTEC 9240 culture system; Johnston Laboratories, Inc.) in which a Gram stain showed gram-positive cocci resembling staphylococci were evaluated prospectively. The Staphaurex Plus kit, the tube coagulase test, the thermostable-endonuclease test, and the RAPIDEC staph kit were compared. The sensitivities were 23, 92, 85, and 98% and the specificities were 99, 100, 93, and 100%, respectively. The RAPIDEC staph kit was the most reliable test, with a diagnostic accuracy comparable to that of the best published results for any of the rapid tests. However, it was the most expensive of the tests and relatively labor-intensive. The tube coagulase test was also sensitive, the simplest to perform, and inexpensive.