We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific.

Anaerobic bacteremia has reemerged as a significant clinical problem. Although there are probably multiple reasons for this change, the increasing number of patients with complex underlying diseases makes the clinical context for anaerobic infections less predictable than it once was. Anaerobic blood cultures should be routinely performed in medical centers with a patient population similar to ours.

Identification of anaerobic bacteria using phenotypic methods is often time-consuming; methods such as 16S rRNA gene sequencing are costly and may not be readily available. We evaluated 253 clinical isolates of anaerobic bacteria using the Bruker MALDI Biotyper (Bruker Daltonics, Billerica, MA) matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) system with a user-supplemented database and an on-plate formic acid-based preparation method and compared results to those of conventional identification using biochemical testing or 16S rRNA gene sequencing. A total of 179 (70.8%) and 232 (91.7%) isolates were correctly identified to the species and genus levels, respectively, using manufacturer-recommended score cutoffs. MALDI-TOF MS offers a rapid, inexpensive method for identification of anaerobic bacteria. Identification of anaerobic bacteria using phenotypic methods is time-consuming and may produce inconclusive results, leading to application of costly methods, such as 16S rRNA gene sequencing. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been used predominantly for aerobic bacterial identification and is now in use in many clinical laboratories (1-5). A small number of studies have preliminarily evaluated this technology for identification of anaerobic bacteria (4, 6-12), but this application is not in widespread clinical use.We showed that off-plate protein extraction yields increased scores and, consequently, a higher percentage of isolates identified compared to direct on-plate testing of colonies for Corynebacterium species and Gram-positive cocci using the Bruker Biotyper system (Bruker Daltonics, Billerica, MA) (1, 13). Others have applied this strategy to anaerobic bacteria (6,14,15). In our experience, off-plate extraction is cumbersome for laboratory technologists and results in waste production and high cost. We recently showed that an easier-to-use onplate testing method using 70% formic acid demonstrated results comparable to those of off-plate extraction for Corynebacterium species and yeast (16). Using the Bruker Biotyper system, Justesen and coworkers performed a similar on-plate preparation technique using 1 l of 70% formic acid for anaerobic bacteria which were not identified using direct on-plate testing without an extraction step (7). Unpublished studies conducted by our group have shown that on-plate formic acid testing yields results equivalent to those of off-plate extraction for staphylococci, streptococci, and aerobic and facultatively anaerobic Gram-negative bacilli. Universal application of this method for identification of bacterial colonies is potentially fast and cost-effective and allows application of MALDI-TOF MS without antecedent Gram staining.We evaluated the Bruker MALDI Biotyper for identification of clinically isolated anaerobic bacteria using on-plate formic acid preparation. MATERIALS AND METHODS Bacterial isolates.A total of 253 clinical isolates of anaerobic bacteria were obtaine...

Actinobaculum species are anaerobic Gram-positive rods that have previously been associated with urinary tract infection (UTI) in the elderly. We report 12 patients with Actinobaculum bacteremia. Only 40% of blood cultures were clinically considered significant by the treating physicians, but most patients were treated for UTI, suggesting a possible urinary source of bacteremia. Clinicians should be aware of the pathogenic potential of Actinobaculum spp.

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