We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were >99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by Ϸ75 to 80% and two-step testing was complete in <3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US$143,000 less than if CCNA alone had been performed on all 5,887 specimens.Clostridium difficile-associated diarrhea is an important illness among patients who are extensively treated with antibacterial or other chemotherapeutic agents (4,7,19). While definitive evidence of toxigenic C. difficile comes from microbiologic testing, laboratories are challenged to provide accurate results rapidly and cost-effectively (23). Cell culture assays for cytotoxin (toxin B) are considered the gold standard but require up to 4 days for results, expensive cells and media, and labor-intensive expertise (4,17,19).Consequently, many laboratories use immunoassays for C. difficile toxins, or "common" glutamate dehydrogenase antigen (2,6,13,16,19,21,23,24). Toxin enzyme immunoassays (EIAs) are frequently used as stand-alone assays but clinical sensitivity may be suboptimal, particularly if only toxin A is detected (4,6,8,9,12,19). Current antigen EIAs (Ag-EIAs) accurately detect an essential and constitutively synthesized enzyme (23), thereby rapidly identifying C. difficile while overcoming the low sensitivity of toxin EIAs and suboptimal performance of older, latex-agglutination antigen assays. Because Ag-EIAs detect nontoxigenic as well as toxigenic C. difficile, however, they must be used in combination with a toxin-detecting assay to provide specific laboratory evidence of C. difficile-associated diarrhea (2,3,10,11,16,19,(21)(22)(23)(24).Both of our institutions' C. difficile-testing laboratories had adopted a stand-alone EIA approach for detecting toxins A and B, using C. DIFFICILE TOX A/B II (ToxAB-EIA; TechLab, Blacksburg, Va.; distributed by Wampole Laboratories, Princeton, N.J.). These laboratories are in acute-care hospitals: one in the 900-bed Johns Hopkins Hospital (JHH), which also serves the 190-bed acute-care Howard County General Hospital in Columbia, Md.; the other is at the 350-bed Johns Hopkins Bayview Medical Center (JHBMC) and serves the 220-bed Johns Hopkins Care Center, a colocated facility for subacute and long-term care. During late 2003, it became apparent that the sensitivity of the ToxAB-EIA was unacceptably low at JHH (see below for analogous JHBMC data from 2004). After determining that the performance of similar assays was inadequate (unpublished data; e.g., 71% sensitivity, 73% specificity, and 25% negative predictive value for Premier C. difficile Toxin AϩB [Meridian Diagnostics, Cincinnati, Ohio] versus cytotoxin testing for 63 specimens), we decided to develop an alternati...

Blood culture bottles with antimicrobial removal systems are recommended for patients who develop fever while on antibiotics. This study compared the ability of Becton Dickinson (Sparks, MD) BACTEC PLUS bottles and bioMerieux (Durham, NC) BacT/Alert FA bottles to effectively remove vancomycin, cefoxitin, ceftriaxone, cefepime, piperacillin-tazobactam, ampicillin, oxacillin, gentamicin, and a combination of gentamicin/penicillin, thus allowing bacterial pathogens to grow. Each bottle was spiked with 10 ml of human blood, antibiotic, and strains of organisms susceptible to the antibiotic evaluated. The organisms used were type strains and clinical isolates of Staphylococcus aureus (methicillin susceptible and resistant), Streptococcus pneumoniae, a viridans streptococcus, Enterococcus faecalis, Enterococcus faecium, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Testing was completed in triplicate, using 10 to 100 CFU/ml of organisms with various concentrations of each antibiotic. Two rounds of testing were completed per antibiotic/organism combination. Bottles were mixed and loaded onto their respective instruments as per the manufacturer's instructions. Antimicrobial removal was evaluated on the basis of time to detection of organism growth, for up to 5 days of incubation. Overall, the BacT/Alert FA system recovered 25.1% of strains from test bottles and 96.9% of strains from growth control bottles (no antibiotic added), and the BACTEC PLUS system recovered 95.1% of strains from test bottles and 100% of strains from growth control bottles. Both systems performed well in the detection of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa in the presence of gentamicin. In the presence of ceftriaxone, neither system was able to recover Streptococcus pneumoniae. The ability to remove vancomycin and cefoxitin was also determined by measuring antibiotic levels remaining in bottles after 1 h of incubation. The results demonstrated remaining levels of 72 to 90% of vancomycin and 71 to 72% of cefoxitin in the BacT/Alert system. For the BACTEC system, remaining levels were 0 to 30% of vancomycin and 0% of cefoxitin. Under these simulated conditions, the BACTEC PLUS system was superior to the BacT/Alert FA system in recovering gram-positive and gram-negative bacterial pathogens in the presence of ␤-lactam antibiotics, gentamicin/penicillin, and vancomycin.

We evaluated the accuracy of the BD Phoenix system for the identification (ID) and antimicrobial susceptibility testing (AST) of 251 isolates of the family Enterobacteriaceae representing 31 species. Organisms were inoculated onto the Phoenix panel according to the manufacturer's instructions. The results from conventional biochemical tests were used for the reference method for ID. Agar dilution, performed according to the CLSI guidelines, was the reference AST method. Essential and categorical agreements were determined. The overall levels of agreement for the genus-and species-level identifications were 95.6% and 94.4%, respectively. Fourteen isolates were incorrectly identified by the Phoenix system; 10 of these were incorrectly identified to the species level. Three of these were Enterobacter (Pantoea) species and four of these were Shigella spp. misidentified as Escherichia coli. For AST results, the essential and categorical agreements were 98.7% and 97.9%, respectively. The very major error, major error, and minor error rates were 0.38%, 0.33%, and 1.8%, respectively. Six isolates (three E. coli isolates and three Klebsiella isolates) were extended-spectrum ␤-lactamase producers. All six were flagged by the Phoenix system expert rules. The Phoenix system compares favorably to traditional methods for ID and AST of Enterobacteriaceae.As hospitals face the continuing challenge of treating sicker patients, the burden falls to clinical microbiology laboratories to provide accurate and rapid identification (ID) of the pathogens recovered and, more importantly, to detect antimicrobial resistance. To accomplish this goal, many laboratories rely upon automated microbial identification and antimicrobial susceptibility testing (AST) systems. The newer-generation instruments have more extensive databases; data management tools, including expert systems; and other features unique to each manufacturer. The BD Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) is the newest system to obtain clearance from the Food and Drug Administration.We evaluated the performance of the Phoenix instrument for the identification and susceptibility testing of challenge and clinical isolates of the family Enterobacteriaceae isolated from a variety of specimen sources in a busy tertiary-care medical center. MATERIALS AND METHODSBacterial strains. A total of 251 bacterial isolates were used for this evaluation. A total of 76 archived "challenge" gram-negative bacilli, including 19 isolates of Shigella spp. from Bangladesh, and 175 clinical isolates recovered from routine cultures in the Clinical Microbiology Laboratory of the Johns Hopkins Hospital (JHH) were included in this comparison.Reference identification. The laboratory's routine method for the identification of gram-negative organisms includes testing with the following conventional biochemicals. For Enterobacteriaceae, the following biochemicals incorporated into an in-house agar system were used: colistin, cefazolin, oxidase, phenylalanine deaminase, urea...

We evaluated the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) for the identification (ID) and antimicrobial susceptibility testing (AST) of challenge and clinical staphylococci and enterococci recovered from patients in a tertiary-care medical center. In total, 424 isolates were tested: 90 enterococci; 232 Staphylococcus aureus isolates, including 14 vancomycin-intermediate S. aureus isolates; and 102 staphylococci other than S. aureus (non-S. aureus). The Phoenix panels were inoculated according to the manufacturer's instructions. The reference methods for ID comparisons were conventional biochemicals and cell wall fatty acid analysis with the Sherlock microbial identification system (v 3.1; MIDI, Inc. Newark, DE). Agar dilution was the reference AST method. The overall rates of agreement for identification to the genus and the species levels were 99.7% and 99.3%, respectively. All S. aureus isolates and enterococci were correctly identified by the Phoenix panels. For the non-S. aureus staphylococci, there was 98.0% agreement for the ID of 16 different species. The AST results were stratified by organism group. For S. aureus, the categorical agreement (CA) and essential agreement (EA) were 98.2% and 98.8%, respectively. Three of three very major errors (VMEs; 1.7%) were with oxacillin. For non-S. aureus staphylococci, the CA, EA, VME, major errors, and minor error rates were 95.7%, 96.8%, 0.7%, 1.7%, and 2.9%, respectively. The two VMEs were with oxacillin. For the enterococci, there was 100% CA and 99.3% EA. All 36 vancomycin-resistant enterococci were detected by the Phoenix system. The Phoenix system compares favorably to traditional methods for the ID and AST of staphylococci and enterococci.The burden of the rapid and accurate detection of antimicrobial resistance among gram-positive bacteria remains a continuous challenge for clinical microbiology laboratories. The last decade has seen increases in the numbers of vancomycinresistant enterococcal (VRE) infections (11, 18), the appearance of glycopeptide resistance among staphylococci (15, 18), a continued rise in nosocomial methicillin-resistant Staphylococcus aureus (MRSA) isolates (9, 11), and a surge in communityassociated MRSA isolates (5,12,19). In addition, other staphylococci continue to cause serious nosocomial infections, such as endocarditis caused by S. lugdunensis and infections of intravascular and prosthetic devices caused primarily by S. epidermidis (1, 7, 13). This fact, combined with the change in breakpoints for oxacillin/methicillin testing of coagulase-negative staphylococci (10), has presented additional challenges for laboratories, which must adopt systems with improved abilities for the identification (ID) and antimicrobial susceptibility testing (AST) of non-S. aureus staphylococci.The newer automated instruments have more extensive databases, data management tools (including expert systems), and other features unique to the instrument of each manufacturer. In addition, the accuracy of susceptibility tes...

The performance characteristics of Xpect RSV (XP) and Binax Now RSV (BN) were compared to those of direct fluorescent-antibody staining and/or tissue culture for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirate and wash samples from children (n ‫؍‬ 110) and adults (n ‫؍‬ 66). The sensitivity, specificity, positive predictive value, and negative predictive value of XP were 75%, 98%, 95%, and 90%, respectively; and those of BN were 74%, 100%, 100%, and 90%, respectively. The performances of the assays were similar within a given age group and specimen type (nasopharyngeal aspirate or wash specimen). XP and BN are useful for screening for RSV in respiratory specimens when large volumes are tested or low levels of staffing occur.Respiratory syncytial virus (RSV) is a negative-strand RNA virus and is a member of the pneumovirus subfamily of the family Paramyxoviridae. It is known as a major cause of respiratory tract illness in children, and it is increasingly recognized as a significant respiratory pathogen in adults (3). Nosocomial outbreaks are well recognized (2, 4). Effective approaches to decreasing the rates of nosocomial transmission rely on rapid laboratory diagnosis (5, 6). Rapid direct RSV detection methods vary in their complexities. Lateral flow, membrane-based immunochromatographic assays have certain advantages compared to direct fluorescent-antibody staining (DFA), including an ultrarapid turnaround time and low levels of complexity. The goal of this study was to determine the performance characteristics of two immunochromatographic methods (Xpect RSV [XP] and Binax Now RSV [BN]) to those of a combined standard of DFA and/or culture (shell vial and conventional tube assays) with nasopharyngeal swab specimens collected in an urban tertiary-care setting.Nasopharyngeal samples (n ϭ 176 total samples; nasopharyngeal aspirates [NPAs], n ϭ 130; nasopharyngeal washes [NPW], n ϭ 46) were collected from children (n ϭ 110; age range, 6 months to 18 years) and adults (n ϭ 66) for routine medical care. Specimens were received in M4RT viral transport medium (approximate volume, 3 ml; Remel Inc., Lenexa, KS). Specimens were not further diluted prior to testing. Each specimen was tested by XP, BN, DFA (IMAGEN, Dakocytomation, Carpinteria, CA), and culture, according to the manufacturers' instructions. Uncentrifuged specimens were used for testing by XP and BN. NPAs have been approved for use with XP but not BN. Both tests have been approved for use with NPWs. After migration control failure in the XP device, samples were extracted and retested according to the instructions in the package insert. For extractions, 2 drops of proprietary extraction buffer were added to 250 l of specimen. Following mixing of the contents, XP was repeated by applying 3 to 4 drops of extracted specimen to an XP device. For DFA, specimens were considered adequate if three or more cells were present per ϫ200 field. Cell culture was performed with R-Mix shell vials (Diagnostic Hybrids, Inc.[DHI], Athens, OH) and tub...