Daniel R. Gustafson

Comparison of Real-Time PCR for Detection of the tcdC Gene with Four Toxin Immunoassays and Culture in Diagnosis of Clostridium difficile Infection

ABSTRACT 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.

Reemergence of Anaerobic Bacteremia

BACKGROUND During 1974-1988, the incidence of anaerobic bacteremia at the Mayo Clinic (Rochester, MN) decreased. This trend occurred nationally, prompting calls for discontinuation of routine anaerobic blood cultures. However, recently, the sites of anaerobic infection have been shown not to be as predictable as once thought, and since 1993, the incidence of anaerobic bacteremia has increased significantly in our medical center. METHODS Records from the Mayo Clinic Division of Clinical Microbiology were used to tabulate the number of cases of anaerobic bacteremia in patients at the clinic for the 12-year period from 1993 through 2004. Medical records for patients with anaerobic bacteremia were reviewed from the periods of 1993-1994 and 2004 to identify differences between these 2 patient populations with different rates of bacteremia. RESULTS The mean incidence of anaerobic bacteremias increased from 53 cases per year during 1993-1996 to 75 cases per year during 1997-2000 to 91 cases per year during 2001-2004 (an overall increase of 74%). The total number of cases of anaerobic bacteremia per 100,000 patient-days increased by 74% (P<.001). The number of anaerobic blood cultures per 1000 cultures performed increased by 30% (P=.002). Organisms from the Bacteroides fragilis group, other species of Bacteroides, and Clostridium species were most commonly isolated. CONCLUSIONS 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 by Bruker Biotyper Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry with On-Plate Formic Acid Preparation

ABSTRACT 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.

Evaluation of the Etest for susceptibility testing of anaerobic bacteria

We compared the susceptibility test results of 220 anaerobes against 14 antimicrobials using the Etest (AB Biodisk, Solna, Sweden) with those using the National Committee for Clinical Laboratory Standards (NCCLS) standard agar dilution method (Wadsworth version). The Etest medium was brucella blood (whole) agar and the inoculum size was equivalent to a no. 1 McFarland standard. Thirty-six percent of Etest results were unreadable after 24 h of anaerobic incubation compared to only 5% after 48 h. Also, there were more results with categorical agreement with the NCCLS method after 48 h (97.9%) than at 24 h (89%) and more very major errors (VMEs) at 24 h (22% of resistant organisms) than at 48 h (3.2%). VMEs and major errors occurred most frequently with clindamycin, ceftriaxone, and trospectomycin (which should not be used with the Etest) and involved the Bacteroides fragilis group and/or Clostridium most commonly. The Etest is simple to perform and is a generally reliable method that is optimally read after 48 h of incubation. It should be an acceptable alternative to the agar dilution standard, although results with certain organism-antimicrobial combinations should be read very conservatively because of the frequency of VMEs.

Effects of 4 Hand-Drying Methods for Removing Bacteria From Washed Hands: A Randomized Trial

OBJECTIVE To evaluate the effects of 4 different drying methods to remove bacteria from washed hands. SUBJECTS AND METHODS One hundred adult volunteers participated in this randomized prospective study. All bacterial counts were determined using a modified glove-juice sampling procedure. The difference was determined between the amounts of bacteria on hands artificially contaminated with the bacterium Micrococcus luteus before washing with a nonantibacterial soap and after drying by 4 different methods (cloth towels accessed by a rotary dispenser, paper towels from a stack on the hand-washing sink, warm forced air from a mechanical hand-activated dryer, and spontaneous room air evaporation). The results were analyzed using a nonparametric analysis (the Friedman test). By this method, changes in bacterial colony-forming unit values for each drying method were ranked for each subject. RESULTS The results for 99 subjects were evaluable. No statistically significant differences were noted in the numbers of colony-forming units for each drying method (P = .72). CONCLUSION These data demonstrate no statistically significant differences in the efficiency of 4 different hand-drying methods for removing bacteria from washed hands.

Anaerobic Thioglycolate Broth Culture for Recovery of Propionibacterium acnes from Shoulder Tissue and Fluid Specimens

Propionibacterium acnes is an agent of shoulder infection, especially postsurgically ([1][1], [2][2]). The study by Butler-Wu et al. suggests a minimum culture incubation of 13 days for recovery of P. acnes from periprosthetic tissues and fluids ([3][3]). Methods for recovery of anaerobes vary, and

Actinobaculum Bacteremia: a Report of 12 Cases

ABSTRACT 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.

Isolation of Robinsoniella peoriensis from Four Human Specimens

ABSTRACT Robinsoniella peoriensis is a recently described anaerobic, spore-forming, Gram-positive bacillus originally recovered from swine manure. We report four human cases in which R. peoriensis was isolated from clinical samples.

Desulfovibrio legallii prosthetic shoulder joint infection and review of antimicrobial susceptibility and clinical characteristics of Desulfovibrio infections.

ABSTRACT We describe a case of shoulder hemiarthroplasty infection with Desulfovibrio legallii. Antimicrobial susceptibilities of 36 Desulfovibrio isolates are presented. Metronidazole and carbapenems exhibited reliable activity, although piperacillin-tazobactam did not. Eleven previous cases of Desulfovibrio infection are reviewed; most arose from a gastrointestinal tract-related source.

Shuttleworthia satelles endocarditis: Evidence of non-dental human disease

lease cite this article in press as: Ne ect (2010), doi:10.1016/j.jinf.2010. A 65-year-old man with rheumatic carditis and a mitral valve mechanical prosthesis presented with weakness and expressive aphasia which prompted hospital admission at another institution. For four months prior to admission, he had nightly symptoms of muscle aches and sweats. Six months prior to admission, he recalled a ‘‘boil’’ and bleeding from the mandibular gingiva. Magnetic resonance imaging showed two small acute infarctions in the distribution of the left middle cerebral artery. An echocardiogram showed two distinct mobile thrombi on the prosthetic mitral valve. Three sets of blood cultures were obtained, but no antimicrobial therapy was administered and the patient was transferred to our institution for further management three days later. On arrival, two sets of blood cultures were collected. Therapy with ampicillin/sulbactam was started when three of the five blood culture sets grew an anaerobic Gram-positive bacillus after 72 h of incubation. Gram stain showed short Gram-positive bacilli in diphtheroidal arrangements. Small satellite colonies grew within and from the margins of the primary colony after 10e14 days of incubation. The isolate was resistant to colistin (10 mg disc) and susceptible to vancomycin (5 mg disc), bile and indole positive, and catalase negative. Esculin was hydrolyzed, but arginine and urea were not. Arabinose, glucose, lactose, maltose, melezitose, sucrose, trehalose, fructose and rhamnose were fermented, but cellobiose, melibiose, mannitol, salicin, and sorbitol were not. Gelatin was not liquefied and H2S was not produced. The organism was N-acetyl-glucosidase and a-fucosidase negative, and b-D-galactosidase, a-D-galactosidase, and a-glucosidase positive. Acetic acid, formic acid, butyric acid, and lactic acid were detected by gas liquid chromatography.