Nonfermenting bacteria are ubiquitous environmental opportunists that cause infections in humans, especially compromised patients. Due to their limited biochemical reactivity and different morphotypes, misidentification by classical phenotypic means occurs frequently. Therefore, we evaluated the use of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) for species identification. By using 248 nonfermenting culture collection strains composed of 37 genera most relevant to human infections, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturer's recommendations for microflex measurement and MALDI BioTyper software (Bruker Daltonik GmbH, Leipzig, Germany), i.e., by using a mass range of 2,000 to 20,000 Da and a new pattern-matching algorithm. To evaluate the database, 80 blind-coded clinical nonfermenting bacterial strains were analyzed. As a reference method for species designation, partial 16S rRNA gene sequencing was applied. By 16S rRNA gene sequencing, 57 of the 80 isolates produced a unique species identification (>99% sequence similarity); 11 further isolates gave ambiguous results at this threshold and were rated as identified to the genus level only. Ten isolates were identified to the genus level (>97% similarity); and two isolates had similarity values below this threshold, were counted as not identified, and were excluded from further analysis. MALDI-TOF MS identified 67 of the 78 isolates (85.9%) included, in agreement with the results of the reference method; 9 were misidentified and 2 were unidentified. The identities of 10 randomly selected strains were 100% correct when three different mass spectrometers and four different cultivation media were used. Thus, MALDI-TOF MS-based species identification of nonfermenting bacteria provided accurate and reproducible results within 10 min without any substantial costs for consumables.

A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluoresceinlabeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ‫؍‬ 72), C. dubliniensis (n ‫؍‬ 58), C. glabrata (n ‫؍‬ 5), C. krusei (n ‫؍‬ 2), C. parapsilosis (n ‫؍‬ 4), and C. tropicalis (n ‫؍‬ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.

(2) and is associated with prolonged hospitalization, with higher health care costs (3). The clinical microbiology laboratory plays a vital role in the treatment of patients with bloodstream infections. While current methods for the detection and characterization of bloodstream pathogens can take days, the risk of death from sepsis increases by 6 to 10% per hour from the onset of shock to the start of effective antimicrobial treatment (4).Over 50% of organisms identified in positive blood cultures are Gram-positive bacteria (5). Gram-positive organisms also are common contaminants of blood cultures. Therefore, rapid differentiation of pathogens from contaminants would be clinically useful.The Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (IUO) (Nanosphere, Northbrook, IL) is a random-access, automated test that performs nucleic acid extraction directly from positive blood culture media, hybridization onto a microarray, and analysis in 2.5 h. Targets in the assay include Staphylococcus spp., Streptococcus spp., Micrococcus spp., Listeria spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus anginosus group, Enterococcus faecalis, and Enterococcus faecium. In addition, if S. aureus or S. epidermidis is detected, then the presence or absence of the mecA gene is reported. Similarly, if E. faecium or E. faecalis is detected, then the presence or absence of the vanA and vanB genes is reported. The BC-GP test was approved by the U.S. Food and Drug Administration (FDA) on 27 June 2012 for the reporting of all IUO targets except Micrococcus spp. (6).We evaluated the performance of the Verigene BC-GP IUO assay in comparison with routine laboratory methods used in the clinical laboratory. A recent evaluation of the BC-GP assay (7) tested blood cultures from a blood culture system (VersaTREK; Trek Diagnostic Systems, Cleveland, OH) that is used by Ͻ10% of U.S. laboratories, while our study utilized the BacT/Alert instrument (bioMérieux, Durham, NC), which is found in ϳ50% of laboratories (N. Safwat, personal communication). Another novel aspect of our study is the assessment of the time from Gram stain result reporting to identification and susceptibility result reporting for current laboratory methods, in comparison to the time for BC-GP results. This research was conducted at the Cleveland Clinic, after approval by the institutional internal review board.(This study was presented in part at ID Week, San Diego, CA, 20 October 2012.) MATERIALS AND METHODSBlood culture samples. The BC-GP IUO test was evaluated with blood cultures submitted for testing at the Cleveland Clinic microbiology laboratory in February to August 2012. Positive aerobic blood culture samples (in BacT/Alert FA bottles [bioMérieux, Durham, NC]) that contained Gram-positive cocci from unique patients were included in the study. Specimens were excluded if multiple morphologies were observed ...

We evaluated the performance of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method, a rapid two-color assay for detection of C. albicans and C. glabrata, in a multicenter study. The assay is designed for use directly from positive blood culture bottles in a FISH format. Intact, fixed cells are labeled fluorescent green (C. albicans) or fluorescent red (C. glabrata) by rRNA hybridization of fluorophore-labeled PNA probes. Results are available <3 h after cultures signal positive. An evaluation of 197 routine blood culture bottles newly positive for yeast by Gram staining was performed at five hospitals. The sensitivities of detection for C. albicans, and C. glabrata were 98.7% (78/79) and 100% (37/37), respectively, and the specificity for both components of the assay was 100% (82/82). The assay was also evaluated with 70 fungal reference strains and was challenged in the BacT/ALERT microbiological detection system with spiked blood culture bottles. These results support the use of the assay for rapid, simultaneous identification of C. albicans and C. glabrata in positive blood culture bottles. This rapid assay may aid in the selection of initial antifungal drugs, leading to improved patient outcomes.