Detection of group B Streptococcus bacteria in LIM enrichment broth by peptide nucleic acid fluorescent in situ hybridization (PNA FISH) and rapid cycle PCR.

The sensitivity, specificity, and negative and positive predictive values for the detection of group B Streptococcus (GBS) in 206 LIM enrichment broths by the use of subculture, GBS peptide nucleic acid fluorescent in situ hybridization (PNA FISH), and GBS PCR were 96.9%, 100%, 98.6%, and 100%; 98.4%, 100%, 99.3%, and 100%; and 100%, 100%, 100%, and 100%, respectively.

Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide.

AIMS: The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells by selectively penetrating through the damaged membrane of dead cells and blocking the DNA amplification during the polymerase chain reaction (PCR). We optimized and tested the assay in vitro using Staphylococcus aureus and Staphylococcus epidermidis cultures to distinguish viable from dead bacteria, with the goal of reducing false positive PCR results. METHODS AND RESULTS: Viable and heat-inactivated bacteria were treated with EMA or left untreated before DNA extraction. A real-time PCR assay for the detection of the tuf gene in each DNA extract was used. Our results indicated that EMA influenced viable bacteria as well as dead bacteria, and the effect of EMA depended on the EMA concentration and bacterial number. CONCLUSIONS: EMA is not a suitable indicator of bacterial viability, at least with respect to Staphylococcus species. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the viability of pathogens has a major impact on interpreting the results of molecular tests for bacteria and subsequent clinical management of patients. To this end, several methods are being evaluated. One of these methods--intercalating DNA of dead bacteria by EMA--looked very promising, but our study found it unsatisfactory for S. aureus and coagulase-negative Staphylococci.

Evaluation of radical prostatectomy specimens. A comparative analysis of sampling methods.

We evaluated 104 radical prostatectomies for clinical stage B (n = 93) and stage A (n = 11) prostate cancer. Seven (8%) stage B cases had no gross cancer. By submitting only gross stage B cancer along with standard sections of proximal and distal margins, base of seminal vesicles, and most apical section (next to distal margin), we identified 91% of capsular penetration and 96% of positive margins as compared with identification by complete microscopic examination. Although this method identified 100% capsular penetration and positive margins in stage A cases, 28% of all the cases were grossly normal. Stage A tumor was often difficult to identify because of its heterogeneous location, its gross similarity to nodular hyperplasia, and the confounding presence of post-transurethral resection scarring. In 98% of all stages B and A cases, this method identified to within 1, the Gleason sum of the totally embedded radical prostatectomy. Using this sampling method, key pathologic parameters were identified with an average of 13 blocks (range 7-36) as compared with totally embedding the prostate, using an average of 42 blocks (range 21-81). Based on our study and our understanding of stages A and B disease, we recommend that in grossly normal glands, alternate posterior sections (stage B) and alternate entire sections (stage A) be submitted. Use of this sampling method will achieve a greater uniformity in the processing of specimens and a more accurate pathologic analysis of radical prostatectomy specimens.

Risk factors for an outbreak of multi-drug-resistant Acinetobacter nosocomial pneumonia among intubated patients.

INTRODUCTION: Acinetobacter baumanii is a Gram-negative coccobacillus that is normally a commensal pathogen but can be a nosocomial pathogen. An epidemiologic study was performed to investigate an outbreak of A baumanii that occurred in our medical intensive care unit (MICU) from March to September 1995. METHODS: A case-control study was performed by retrospective chart review, comparing case patients to randomly selected patients who were mechanically ventilated in the MICU for at least 1 week during the outbreak. A case patient was defined as any patient with an Acinetobacter infection in which the epidemic strain was considered to be a pathogen. The epidemic strain was defined by its antibiogram. Case patients and control patients were compared for age, gender, underlying disease, acute physiology and chronic health evaluation III score, length of MICU stay, prior antibiotic use, presence of fever, sepsis, type of pulmonary infiltrate, and outcome. Environmental and hand-washing studies also were performed during the period of the outbreak. Molecular typing was performed on available bloodstream isolates. RESULTS: There were 15 cases of A baumanii nosocomial pneumonia. Fifty percent were bacteremic; one chart was unavailable for review. Twenty-nine patients were identified as control patients. The mean age for case patients was 50 (range, 21 to 84). The mean duration of time from admission to the ICU to infection was 12.8 days (range, 4 to 40). Sepsis developed in 35% of the case patients. Forty-three percent of the case patients died during their hospitalization, with two of those deaths attributed to Acinetobacter infection. Univariate analysis showed that prior use of ceftazidime was associated with infection with Acinetobacter (11/14 case patients compared to 11/29 control patients; p < 0.01). Pulsed-field gel electrophoresis revealed two strains to be responsible for the outbreak. Hand washing was performed before patient contact by only 10% of health-care workers, and only 32% washed their hands after patient contact. CONCLUSION: The use of ceftazidime was associated with an increased risk of nosocomial pneumonia with resistant strains of Acinetobacter. Health-care workers need to improve compliance with hand-washing recommendations.

Unpredictability of commercially available exoantigen culture confirmation tests in confirming the identity of five Blastomyces dermatitidis isolates.

Colonial and microscopic features of five fungal isolates from three patients suggested Blastomyces dermatitidis. Extracts from the mold forms of all isolates were tested on several occasions with commercially available Exoantigen immunodiffusion culture-confirmation test reagents and Nolan reagents. All three isolates from patient 1 were negative on four separate attempts with Exoantigen reagents using conventional ("slant") and "broth" extraction methods, and were also negative on one attempt with the Nolan reagents. The isolate from patient 3 was negative on three attempts using both reagent kits. The isolate from patient 2 was negative on four of five attempts with Exoantigen test reagents and positive on one attempt with Nolan reagents. Commercially prepared chemiluminescence-labeled DNA probes (Gen-Probe, San Diego, CA) directed at ribosomal RNA from B. dermatitidis and Histoplasma capsulatum confirmed all five isolates as B. dermatitidis. The cost and labor of the exoantigen and DNA Probe culture confirmation tests were evaluated. New methods for confirming the identity of cultural isolates of B. dermatitidis that are sensitive, specific, and commercially available are greatly needed.

Rapid identification of Staphylococcus epidermidis.

A panel of Minitek sugar disks, consisting of trehalose, mannitol, xylose, and sucrose, was evaluated for its ability to identify blood culture isolates of Staphylococcus epidermidis (SE). Using a heavy suspension of organism in Mueller-Hinton broth, 50 microL was pipetted onto each disk in wells of a flat-bottomed microtiter tray. The tray was covered, incubated in a moist chamber in non-CO2 at 35 degrees C, and examined after 5 and 24 hours. A color change of yellow or orange was positive; no color change (red) was negative. Expected reactions for SE were as follows: negative trehalose, mannitol, and xylose; positive, sucrose. On evaluation of 227 coagulase-negative staphylococci (CNS) at 5 and 24 hours, the panel had a sensitivity of 94 and 96%, specificity of 92 and 89%, predictive value of positive tests of 97 and 96%, and predictive value of negative tests of 84 and 87%. This panel offered an inexpensive and convenient method for differentiating SE from the other CNS in five hours.

Clinical comparison of the Baxter MicroScan Yeast Identification Panel and the Vitek Yeast Biochemical Card.

To determine the reliability of the Baxter MicroScan Yeast Identification Panel, processed by the Walkaway-96, and the Vitek Yeast Biochemical Card, 150 clinical yeast isolates (30 Candida albicans, 67 Candida species, not albicans, 26 Torulopsis glabrata, 13 Cryptococcus neoformans, 4 Saccharomyces cerevisiae, 6 Trichosporon beigelii, 3 Rhodotorula species, and 1 Geotrichum species) were tested on both systems. Results were compared with those obtained by the API 20C and the appearance of yeast cells on cornmeal Tween-80 agar. After inoculation of each system, results were available in 4 hours with MicroScan panels, 24-48 hours with Vitek cards, and 72 hours with the API 20C strips. On initial testing, 101 (67%) and 128 (85%) isolates, respectively, were correctly identified by MicroScan and Vitek. After repeat testing, the number of correctly identified isolates increased to 123 (82%) by MicroScan and to 142 (95%) by Vitek. Yeasts most commonly misidentified were Candida tropicalis, T glabrata, and Candida parapsilosis by MicroScan and C tropicalis and T glabrata by Vitek.

Antimicrobial susceptibility testing of a clinical isolate of vancomycin-dependent enterococcus using D-alanine-D-alanine as a growth supplement.

Bacteremia due to a vancomycin-dependent enterococcus (VDE) occurred during long-term vancomycin therapy in a renal transplant recipient with underlying pancreatitis and a vancomycin-resistant enterococcal (VRE) wound infection and bacteremia. The VDE was isolated from blood during vancomycin therapy and grew only in the presence of vancomycin and D-alanine-D-alanine (DADA), a substance required for cell-wall synthesis. Colonies beyond the periphery of growth of the VDE around a vancomycin disk contained vancomycin-independent revertant mutants after 48 hours of incubation. Pulsed-field gel electrophoresis of the VDE, revertant mutant, the initial blood culture isolate of VRE, and an autopsy isolate showed that the four strains were identical. Antimicrobial susceptibility testing was performed using standard macrobroth and microbroth dilution methods. DADA was used as a growth supplement for macrobroth dilution susceptibility testing of the VDE isolate. Minimum inhibitory concentrations (MICs) were similar for the VRE isolate and the VDE revertant, which were both resistant to ampicillin, high-level gentamicin, ciprofloxacin, imipenem, vancomycin, and daptomycin, and were susceptible to fusidic acid, high-level streptomycin, rifampin, and a quinupristin-dalfopristin combination. The MICs of teicoplanin were 2 microg/mL or less and 16 microg/mL for the clinical VRE isolate and the VDE revertant, respectively. The autopsy isolate was resistant to all antimicrobials tested and showed a fourfold increase in MICs for quinupristin-dalfopristin compared with that of the original blood isolate. The VDE was susceptible to all drugs tested except vancomycin.

Isolation of agent associated with cat scratch disease bacillus from pretibial biopsy.

We describe the isolation and cultural characteristics of a Gram-negative bacillus that is very similar to the presumed etiologic agent of cat scratch disease. The organism was isolated from a tibial lesion of a male patient who had been hospitalized for severe necrotizing pancreatitis. The significance of the isolate in this patient remains uncertain.

Screening and confirmatory testing for extended spectrum beta-lactamases (ESBL) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca clinical isolates.

Escherichia coli and Klebsiella spp. were screened for ESBL based on routine susceptibility testing results. Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporins or aztreonam were reported as probable ESBL producers. By using the NCCLS proposed ESBL confirmatory method, we tested 61 screen-positive isolates from 42 patients, 30 randomly selected susceptible isolates, and 12 isolates with previously characterized beta-lactamases. Ceftazidime contributed to 97% of screen-positive isolates, whereas aztreonam added a single patient isolate. An ESBL was confirmed in 86% of K. pneumoniae, 100% of K. oxytoca, and 20% of E. coli screen-positive single patient isolates. None of the susceptible isolates were shown to produce ESBL. Based on these findings a comment regarding the presence of ESBL seems sufficient for Klebsiella spp. but confirmatory testing is indicated for E. coli. 0.25 microg/mL was used to indicate the presence of ESBL, the specificity of the assay increased to 100%. The NCCLS ESBL phenotypic confirmatory method was reproducible and accurate enough to be used in the clinical laboratory.

Aberrant Histoplasma capsulatum. Confirmation of identity by a chemiluminescence-labeled DNA probe.

A cottony, light tan, filamentous fungus with pear-shaped microconidia and lacking tuberculated macroconidia was isolated from a bronchial lavage specimen. Subculture on several media at 37 degrees C failed to convert the fungus to a yeast form after several weeks; attempts at in vivo conversion in mice were also unsuccessful. Sera obtained several months apart showed M bands with Histoplasma capsulatum (HC) antigen by immunodiffusion and an increase in complement fixation titers with mycelial and yeast phase antigens of HC. Parallel identity was obtained on two occasions with exoantigen culture confirmation reagents for HC from Immuno-Mycologics as well as one of identity with Nolan reagents. Extracts from four Chrysosporium spp. strains had no identity reactions with HC with either kit. The fungus was identified as HC by the Accuprobe Histoplasma chemiluminescence-labeled DNA probe directed at ribosomal RNA, whereas all four Chrysosporium spp. isolates tested negative. DNA probes are a fast and accurate method to confirm the identity of aberrant fungal isolates.

Comparison of vidas Clostridium difficile toxin-A assay and premier C. difficile toxin-A assay to cytotoxin-B tissue culture assay for the detection of toxins of C. difficile.

Damage to the intestinal mucosa by Clostridium difficile (CD) is toxin mediated. Two enzyme immunoassays (EIAs) for toxin-A detection, the automated Vitek immunodiagnostic assay system CDA (Vidas CDA), and the Premier toxin A (Premier) were tested for their ability to detect toxin A in 301 stool samples and compared with an in-house tissue culture assay for toxin B (TCA). Of these 301 samples, 49 were TCA positive and 252 were TCA negative. Agreement between Vidas CDA and TCA on the initial run was 85% (255 of 301) and increased to 94% (278 of 296) when discordant samples were retested from available frozen specimens. Corresponding levels of agreement for Premier were 91% (272 of 301) and 98% (284 of 288), respectively. If tissue culture positivity at any titer was used as the sole criterion for positivity of the specimen, agreement with positive TCA before and after repeat testing was 57% (26 of 49) and 74% (34 of 46) for Vidas CDA and 65% (32 of 49) and 95% (36 of 38) for Premier. Agreement with negative TCA titers was good: 90% for Vidas CDA and 95% for Premier, and 98% for Vidas CDA and 99% for Premier after repeat testing. Predictive values positive and negative after repeat testing were, respectively, 88% and 96% for Vidas CDA, and 95% and 99% for Premier. Results for the automated and manual EIA methods for detection of C. difficile toxin A were obtained in 2.5 h as compared with 36-48 h for tissue culture.

Minimum bactericidal concentrations of Propionibacterium acnes isolates from cases of chronic endophthalmitis.

Six isolates of Propionibacterium acnes recovered from cases of chronic infectious endophthalmitis following extracapsular cataract extraction were tested for antibiotic susceptibility. All isolates were susceptible to penicillin, cefazolin, and vancomycin with a macrobroth dilution method. Minimum bactericidal concentrations testing at 72 h revealed that six of six isolates were killed by < or = 1.0 micrograms of vancomycin/ml, one of six isolates by < or = 1.0 micrograms of penicillin/ml, and zero of six isolates by < or = 1.0 micrograms cefazolin/ml.

Absence of skin alkaloids in captive-raised Madagascan mantelline frogs (Mantella) and sequestration of dietary alkaloids.

Mantelline frogs of the genus Mantella contain a variety of pumiliotoxin, allopumiliotoxin and homopumiliotoxin alkaloids in their skin. Pyrrolizidines, indolizidines and quinolizidines are also present. In contrast, captive-raised frogs (Mantella aurantiaca) have no alkaloids detectable in skin extracts. Frogs fed alkaloid-dusted fruit flies accumulate alkaloids into their skin. Thus, these mantelline frogs, like the neotropical dendrobatid frogs, appear dependent on dietary sources for their skin alkaloids and have the requisite alkaloid-sequestering system(s).

Actinomycosis of the cholecystic duct: case report and review.

Actinomycosis of the gall bladder, cholecystic duct or common bile duct is rare, with only 16 cases reported to our knowledge. We report a case of actinomycosis in the cholecystic duct remnant of an 80-year-old woman with a history of cholecystitis, choledocholithiasis and cholecystoduodenal fistula requiring cholecystectomy and fistula closure three years prior. Histologic sections of the cystic duct remnant showed several dense basophilic masses containing numerous, dark blue, Gram-positive branching bacilli compatible with actinomycotic granules. Fluorescent antibody staining was positive for Actinomyces naeslundii. Staining for acid-fast bacilli was negative. The pathogenesis, presentation, diagnosis and management of abdominal actinomycosis with specific reference to disease involving the gall bladder are discussed.

Primary processing of specimens and isolation and cultivation of mycobacteria.

Processing of specimens for mycobacteria need to proceed as rapidly as possible to provide 24 hour turnaround for smears and 21-day turnaround for the identification of Mycobacterium tuberculosis. If these goals are to be accomplished, a broth culture or microcolony plate method must be part of the media and in the planting process. Use of conventional solid media alone does not provide adequate turnaround time.

Case report: acromegaly and Cushing's disease in a patient with synchronous pituitary adenomas.

A 40-year-old white woman presented with hirsutism, amenorrhea, generalized fatigue, diffuse weight gain, acral changes, and coarsened facial features. Physical examination revealed mild diastolic hypertension, acromegalic features, hirsutism, and seborrhea. The growth hormone concentration was elevated and did not suppress after glucose administration. Urinary free cortisol excretion was increased and was not suppressed during a 2 mg low-dose dexamethasone suppression test. Magnetic resonance imaging of the sella demonstrated a 1.3 x 1.2 x 0.8 cm pituitary adenoma. Trans-sphenoidal resection was performed, and portions of the resected tumor were analyzed by routine pathologic methods. Histopathologic and immunohistochemical findings indicated discrete growth hormone- and adrenocorticotropic hormone-producing pituitary adenomas. Coexisting acromegaly and Cushing's syndrome due to pituitary neoplasia was previously reported in two patients. However, to the authors' knowledge, this represents the first description of a patient with acromegaly and Cushing's disease resulting from discrete synchronous adenomas of the pituitary gland as defined by modern histopathologic techniques.

Growth curve for Propionibacterium acnes.

We established growth curves for Propionibacterium acnes isolates recovered from eyes with chronic postoperative endophthalmitis. The growth curve plotted the average of the duplicate bacterial concentration against time. The generation time for P. acnes calculated from the growth curves was approximately 5.1 hours. The growth of P. acnes is slower than other anaerobic bacteria. This may account for its delayed appearance in culture of ocular specimens. It may also explain treatment failure if the concentration of an antibiotic injected into the vitreous does not remain at an effective level during the critical replicative phase of the organism.

Bacterial contamination of anaerobic vitreous cultures: using techniques employed for endophthalmitis.

PURPOSE: To investigate the occurrence of contaminated cultures of vitreous specimens from non-infected eyes obtained using anaerobic techniques employed for endophthalmitis. METHODS: Vitreous specimens were obtained using meticulous sterile techniques employed for endophthalmitis from seventeen patients undergoing pars plana vitrectomy for non-infective indications: vitreous hemorrhage (12 eyes), retinal detachment (3), Coat's disease (1), and congenital dislocated lens(1). Vitreous specimens were inoculated in the operating room onto an anaerobic blood agar plate and into thioglycolate broth. Bacterial growth occurring before 10 days was considered positive. RESULTS: Three organisms were isolated from three separate eyes. One colony of Staphylococcus species was isolated on an anaerobic blood agar plate on day 3. A single colony of Propionibacterium acnes grew on an anaerobic blood agar plate on day 6. Alpha-hemolytic streptococci grew from thioglycolate broth on day 10. CONCLUSIONS: Growth as detected in this study might represent contaminating rather than an infecting organism in an eye suspected of having endophthalmitis.

Probe technology for the clinical microbiology laboratory.

Molecular-based techniques, such as use of genetic probes, can greatly reduce turnaround time of the identification of some isolates in the clinical microbiology laboratory. For example, culture confirmation of an acid-fast bacillus such as Mycobacterium tuberculosis or Mycobacterium avium-intracellulare by a gene probe can be performed within hours of the isolation of the organism. Conventional methods might require more than 2 to 4 weeks for this identification. Identification of a mold, such as Histoplasma capsulatum, with a gene probe, can be done in less than 24 hours, with minimal growth available; conventional fungal identification takes 1 to 2 weeks or more. Whether a probe is used in the clinical microbiology laboratory depends on a number of factors including accuracy of the probe, cost, commercial availability, and ease of performance. In addition, the relevance of a more rapid result needs to be considered. A gene probe may be more specific than conventional methodologies, and this may increase its advantage. Direct specimen testing with probes is presently hampered by inadequate sensitivities. Prior amplification may decrease this limitation. It is hoped that methods will be available in the near future that provide amplification and probing of clinical specimens for laboratory diagnosis to be made within a short time following specimen collection.