Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin-and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA (Staphylococcus genus specific), nuc (S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin-and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin-and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

Clostridium difficile-associated diarrhea (CDAD) is the most commonly identified cause of nosocomial diarrhea (10,12,17). The pathogenicity of C. difficile is due to the production of two exotoxins: toxin A, an enterotoxin, and toxin B. Both toxins A and B contribute to human disease (19).TechLab Inc. (Blacksburg, Va.) has recently marketed an enzyme immunoassay, C. DIFF CHEK-60, which detects glutamate dehydrogenase (GDH), a common C. difficile antigen (the assay is hereinafter referred to as TL-GDH), and also markets an enzyme immunoassay, C. difficile Tox A/B II (Tox-A/B), for the detection of toxins A and B. The aim of this study was to evaluate and compare these two assays with another immunoassay, the Triage C. difficile Panel assay (Biosite Diagnostics, San Diego, Calif.), which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); an in-house cytotoxin assay (C-Tox); and stool cultures for C. difficile.All nonformed stool specimens from inpatients suspected of having CDAD were included in the study. Except for stool cultures, all tests were performed within 48 h of the arrival of specimens in the laboratory. Specimens were kept at 4°C if not processed immediately. A portion of stool was stored at Ϫ70°C for subsequent culture. Specimens of insufficient quantity were excluded from all tests.The results of TL-GDH and Tox-A/B were read by using a dual-wavelength spectrometer (450 and 620 nm). TR-GDH, TR-Tox-A, TL-GDH, and Tox-A/B were carried out according to the manufacturer's instruction. Details of the Triage panels and Tox-A/B and C-Tox have been described previously (16).After thawing, fecal samples were planted onto prereduced cycloserine-cefoxitin-fructose agar (cefoxitin, 8 mg/liter; cycloserine, 250 mg/liter; fructose agar; Oxoid, Ottawa, Canada) and incubated for 96 h at 35°C in an anaerobic glove box. Suspected colonies were identified based on their growth on selective media, their colonial morphology, and a positive reaction by the MicroScreen C. difficile latex slide agglutination test (Microgen Bioproducts Ltd., Surrey, United Kingdom) (2, 11).All C. difficile isolates were tested by PCR for the presence of toxin A and B genes by use of primers NK9 and NK11, derived from the repeating portion of toxin A, and NK104 and NK105, derived from the nonrepeating portion of toxin B. These primers have been described by Kato and coworkers (13). The composition of our master mix and the amplification conditions were the same as used by those investigators.For the purpose of this study, a true positive sample was defined as a sample that was positive for toxin A and/or B and from which a toxigenic C. difficile strain (positive by PCR for a toxin gene) was isolated. If a positive toxin detection test result was seen for a sample which was negative for C. difficile by culture or yielded a nontoxigenic strain, the reaction was considered to be falsely positive. Sensitivity, specificity, and positive and negative predictive values for each test were calculated on this basis.C. difficile was isolated from 93 of 49...

MRSASelect agar was compared to CHROMagar, mannitol-salt agar with oxacillin, and mannitol-salt agar with cefoxitin (MSA-CFOX) for the isolation of methicillin-resistant Staphylococcus aureus (MRSA). The sensitivities and specificities were 97.3% and 99.8%, 82.9% and 99.1%, 80.2% and 79%, and 99.1% and 84.8%, respectively. MSA-CFOX and MRSASelect had a high sensitivity. MRSASelect, however, was more specific and proved to be a more reliable and rapid medium for the detection of MRSA.