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At least 289 records · Page 16Linked to original sources
PubMed2004

Use of the BD PHOENIX Automated Microbiology System for direct identification and susceptibility testing of gram-negative rods from positive blood cultures in a three-phase trial.

The present study describes the use of the automated BACTEC 9240 blood culture system, the Serum Separator Tube (SST), and the BD PHOENIX Automated Microbiology System in combination for the direct identification and antimicrobial susceptibility testing (AST) of gram-negative rods (GNRs) from positive blood cultures (BCs) without subculture. The study was conducted in three phases: (i) the recovery yield of Escherichia coli ATCC 25922 was determined with the SST between 0 and 8 h after spiked BC bottles turned positive; (ii) the identifications and susceptibility testing results obtained with the PHOENIX system for nine American Type Culture Collection strains of GNRs processed by the SST procedure and for colonies from agar medium were compared; and (iii) the procedure with the BACTEC system, SSTs, and the PHOENIX system was applied to positive cultures of blood from 309 patients during a 3-month period. The SST procedure with E. coli yielded sufficient numbers of cells to perform direct inoculation at any time between 0 and 8 h after a BC bottle turned positive. By using the identities obtained from pure cultures with the PHOENIX system and other biochemical identification systems as reference methods, the agreement between the reference methods and the PHOENIX system tested directly by using cultures of blood from patients was 92.9%. The 7.1% discrepant results were due to 6.5% incorrect identifications with the PHOENIX system with BC samples and 0.6% incorrect identifications with the PHOENIX system with samples from agar cultures. By AST the overall categorical accuracy was 99.0%, with 0.1% very major errors, 0.1% major errors, and 0.8% minor errors. In conclusion, use of the combination of the BACTEC system, SSTs, and the PHOENIX system has the potential to allow the agar isolation step to be skipped and the procedures for rapid direct identification and susceptibility testing of GNRs from positive BCs to be improved both in hospital-based and in central non-hospital-based laboratories.

Aerobiosis↗
PubMed2004

Evaluation of the automated phoenix system for potential routine use in the clinical microbiology laboratory.

A comparative study was designed to evaluate the identification (ID) and antimicrobial susceptibility testing (AST) performances of the BD Phoenix Automated Microbiology System (Becton Dickinson Diagnostic Systems [BD], Pont de Claix, France). A total of 305 single clinical isolates were collected, and comparisons were made with routine manual methods in use in our microbiology laboratories. The percentages of correct IDs were 93.3, 89.4, 91.8, and 85.7% for enterobacteria, nonfermenting gram-negative bacilli, staphylococci, and streptococci-enterococci, respectively. The median ID time was 3 h, and the median time for AST was 10 h 30 min. AST results showed variable percentages of errors for the different antibiotics. None of the enterobacteria and 0.3% of Pseudomonas aeruginosa isolates showed a very major error (VME). Only one strain of Staphylococcus aureus showed a VME with oxacillin. We demonstrate here the efficiency of the Phoenix system, which can be used for the majority of strains encountered in a university-based laboratory, for ID and AST.

Anti-Bacterial Agents↗
PubMed2004

Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory.

Over a period of 18 months we have evaluated the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic catalase-negative gram-positive cocci in the clinical laboratory. A total of 171 clinically relevant strains were studied. The results of molecular analyses were compared with those obtained with a commercially available phenotypic identification system (API 20 Strep system; bioMérieux sa, Marcy l'Etoile, France). Phenotypic characterization identified 67 (39%) isolates to the species level and 32 (19%) to the genus level. Seventy-two (42%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 138 (81%) isolates to the species level and 33 (19%) to the genus level. For 42 of 67 isolates assigned to a species with the API 20 Strep system, molecular analyses yielded discrepant results. Upon further analysis it was concluded that among the 42 isolates with discrepant results, 16S rDNA sequencing was correct for 32 isolates, the phenotypic identification was correct for 2 isolates, and the results for 8 isolates remained unresolved. We conclude that 16S rDNA sequencing is an effective means for the identification of aerobic catalase-negative gram-positive cocci. With the exception of Streptococcus pneumoniae and beta-hemolytic streptococci, we propose the use of 16S rDNA sequence analysis if adequate species identification is of concern.

Aerobiosis↗
PubMed2004

PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients.

Pseudomonas aeruginosa is the major opportunistic bacterial pathogen in persons with cystic fibrosis (CF); pulmonary infection occurs in approximately 80% of adult CF patients. Much of CF patient management depends on accurate identification of P. aeruginosa from sputum culture. However, identification of this species may be problematic due to the marked phenotypic variability demonstrated by CF sputum isolates and the presence of other closely related species. To facilitate species identification, we used 16S ribosomal DNA (rDNA) sequence data to design PCR assays intended to provide genus- or species-level identification. Both assays yielded DNA fragments of the predicted size. We tested 42 culture collection strains (including 14 P. aeruginosa strains and 28 strains representing 16 other closely related Pseudomonas species) and 43 strains that had been previously identified as belonging to 28 nonpseudomonal species also recovered from CF patient sputum. Based on these 85 strains, the specificity and sensitivity of both assays were 100%. To further assess the utility of the PCR assays, we tested 66 recent CF sputum isolates. The results indicated that preliminary phenotypic testing had misidentified several isolates. The 16S rDNA sequence was determined for 38 isolates, and in all cases it confirmed the results of the PCR assays. Thus, we have designed two PCR assays: one is specific for the genus Pseudomonas, while the other is specific for P. aeruginosa. Both assays show 100% sensitivity and specificity.

Bacterial Typing Techniques↗
PubMed2005

Recurrent melioidosis in patients in northeast Thailand is frequently due to reinfection rather than relapse.

Human melioidosis is associated with a high rate of recurrent disease, despite adequate antimicrobial treatment. Here, we define the rate of relapse versus the rate of reinfection in 116 patients with 123 episodes of recurrent melioidosis who were treated at Sappasithiprasong Hospital in Northeast Thailand between 1986 and 2005. Pulsed-field gel electrophoresis was performed on all isolates; isolates from primary and recurrent disease for a given patient different by one or more bands were examined by a sequence-based approach based on multilocus sequence typing. Overall, 92 episodes (75%) of recurrent disease were caused by the same strain (relapse) and 31 episodes (25%) were due to infection with a new strain (reinfection). The interval to recurrence differed between patients with relapse and reinfection; those with relapses had a median time to relapse of 228 days (range, 15 to 3,757 days; interquartile range [IQR], 99.5 to 608 days), while those with reinfection had a median time to reinfection of 823 days (range, 17 to 2,931 days; IQR, 453 to 1,211 days) (P = 0.0001). A total of 64 episodes (52%) occurred within 12 months of the primary infection. Relapse was responsible for 57 of 64 (89%) episodes of recurrent infection within the first year after primary disease, whereas relapse was responsible for 35 of 59 (59%) episodes after 1 year (P < 0.0001). Our data indicate that in this setting of endemicity, reinfection is responsible for one-quarter of recurrent cases. This finding has important implications for the clinical management of melioidosis patients and for antibiotic treatment studies that use recurrent disease as a marker for treatment failure.

Bacterial Typing Techniques↗
PubMed2005

Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants.

To identify the most rapid and reliable technique for recovery and identification of Staphylococcus aureus small-colony variants (SCVs), the colonial appearance of 106 isolates representing SCVs and the normal phenotype were evaluated on two newly described chromogenic agar media. Although almost all of the SCVs grew on the chromogenic agar media, they did not exhibit a change of color. In comparison with conventional media, S. aureus ID agar (SAID; bioMerieux, La Balme Les Grottes, France) showed the most reliable results, with 49 of 53 SCVs tested growing either as an SCV colony or with a normal phenotype after only 24 h of incubation. Growth of SCVs was often not detected before 72 h of incubation on some of the media tested. In conclusion, the most accurate and rapid method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens onto both Columbia blood agar and SAID.

Agar↗
PubMed2005

Interpretive criteria for use of AccuProbe for identification of Mycobacterium avium complex directly from 7H9 broth cultures.

Rapid identification of Mycobacterium avium complex (MAC) is possible by use of AccuProbe (Gen-Probe, San Diego, Calif.). To evaluate the reliability of the MAC AccuProbe for testing 7H9 cultures inoculated with broth from MGIT cultures positive for acid-fast bacilli or growth on a solid medium, we compared probe results to results obtained by sequencing a portion of the 16S rRNA gene. Isolates were sequenced if the MAC probe result was <100,000 relative light units (RLU) or if it was > or =100,000 RLU and the colony morphology was not classic or there were two colony types. For the 1,389 cultures tested in phase 1, conducted to evaluate cutoff values for the MAC probe in testing of 7H9 cultures inoculated with broth from MGIT cultures, the sensitivity and specificity of the MAC AccuProbe were 97.7% and 88.8%, respectively, according to the manufacturer's interpretive criteria (> or =30,000 RLU is positive). If the cutoff for a positive result were 80,000 RLU, the specificity would be 100% and the sensitivity 92.3%. Of the 344 isolates in phase 2, which was conducted to confirm the 80,000-RLU cutoff for a positive result and therefore included only isolates with a MAC probe result of < or =100,000 RLU, 13 of 16 with results of > or =30,000 but <80,000 RLU were identified as mycobacteria other than MAC, including five Mycobacterium tuberculosis complex isolates. These data support the use of 80,000 RLU as the cutoff for a positive result in testing of 7H9 broth cultures with the MAC AccuProbe.

Bacterial Typing Techniques↗
PubMed2005

Performances of VITEK 2 colorimetric cards for identification of gram-positive and gram-negative bacteria.

The purpose of this study was to evaluate the new VITEK 2 identification cards that use colorimetric reading to identify gram-positive and gram-negative bacteria (GP and GN cards, respectively) in comparison to fluorimetric cards (ID-GPC and ID-GNB, respectively). A total of 580 clinical isolates and stock collection strains belonging to 116 taxa were included in the study. Of the 249 gram-positive strains tested with both the ID-GPC and GP cards, 218 (87.5%) and 235 (94.4%) strains were correctly identified (to the genus and species level), respectively. Of the 331 gram-negative strains tested with the ID-GNB and GN cards, 295 (89.1%) and 321 (97%) strains were correctly identified, respectively. Another focus of the study was to apply the percentages of correct identifications obtained in this study to the list of bacteria isolated in our laboratory (32,739 isolates) in the year 2004. We obtained 97.9% correct identifications with the colorimetric cards and 93.9% with fluorescent cards.

Bacterial Typing Techniques↗
PubMed2005

Anticoagulant carryover may influence clot formation in direct tube coagulase tests from blood cultures.

The tube coagulase test (TCT) performed directly from positive blood culture bottles has been used to reduce the turnaround time for identifying Staphylococcus aureus. Most reports have shown the test to be specific but often lacking sufficient sensitivity to be useful. In a prospective study of blood culture bottles (BCB) signaling positive, with a Gram-stained smear showing gram-positive cocci resembling staphylococci, the sensitivity of the direct TCT was improved by diluting the BCB broth 1:10 in saline before inoculating 0.1 ml into 1.0 ml of 10% pooled human plasma. It was hypothesized that the improved sensitivity might be explained by reduced carryover of the anticoagulant sodium polyanetholesulfonate (SPS) used in blood culture media. By titrating the inoculum size and the concentration of SPS in an in vitro checkerboard assay, it was shown that concentrations of SPS >0.0008% prevented plasma coagulation. The 1:10 dilution of blood culture broth reduced the amount of residual SPS carried over to the TCT to a level (0.0005%) that did not impair plasma coagulation. The direct TCT inoculated with a 1:10 saline dilution of blood culture broth achieved 100% specificity and sensitivity within 4 h of inoculation without reducing the quality or quantity of coagulum.

Anticoagulants↗
PubMed2005

Rapid identification of Staphylococcus aureus in blood cultures by a combination of fluorescence in situ hybridization using peptide nucleic acid probes and flow cytometry.

Fluorescence in situ hybridization (FISH) using peptide nucleic acid probes (PNAs) allows the identification of Staphylococcus aureus from human blood culture samples. We present data revealing that the combination of PNA FISH and flow cytometry is a possible approach for the noncultural identification of staphylococci in blood cultures.

Bacteremia↗
PubMed2006

Evaluation of the Phoenix 100 ID/AST system and NID panel for identification of Enterobacteriaceae, Vibrionaceae, and commonly isolated nonenteric gram-negative bacilli.

The Phoenix 100 ID/AST system (Becton Dickinson Co., Sparks, Md.) is an automated system for the identification and antimicrobial susceptibility testing of bacterial isolates. This system with its negative identification (NID) panel was evaluated for its accuracy in the identification of 507 isolates of the family Enterobacteriaceae, 57 other nonenteric gram-negative isolates that are commonly isolated in clinical microbiology laboratories, and 138 isolates of the family Vibrionaceae. All of the isolates had been characterized by using approximately 48 conventional tube biochemicals. Of the 507 isolates of the Enterobacteriaceae, 456 (89.9%) were correctly identified to the genus and species levels. The five isolates of Proteus penneri required an off-line indole test, as suggested by the system to differentiate them from Proteus vulgaris. The identifications of 20 (3.9%) isolates were correct to the genus level but incorrect at the species level. Two (0.4%) isolates were reported as "no identification." Misidentifications to the genus and species levels occurred for 29 (5.7%) isolates of the Enterobacteriaceae. These incorrect identifications were spread over 14 different genera. The most common error was the misidentification of Salmonella species. The shortest time for a correct identification was 2 h 8 min. The longest time was 12 h 27 min, for the identification of a Serratia marcescens isolate. Of the 57 isolates of nonenteric gram-negative bacilli (Acinetobacter, Aeromonas, Burkholderia, Plesiomonas, Pseudomonas, and Stenotrophomonas spp.), 48 (84.2%) were correctly identified to the genus and species levels and 7 (12.3%) were correctly identified to the genus level but not to the species level. The average time for a correct identification was 5 h 11 min. Of the Vibrionaceae spp., 123 (89.1%) were correctly identified at the end of the initial incubation period, which averaged 4 h. Based on the findings of this study, the Phoenix 100 ID/AST system NID panel falls short of being an acceptable new method for the identification of the Enterobacteriaceae, Vibrionaceae, and gram-negative nonenteric isolates that are commonly encountered in many hospital microbiology laboratories.

Automation↗
PubMed1978

Mucosal-associated bacterial flora of the intestine in patients with Crohn's disease and in a control group.

The bacterial flora intimately associated with the intestinal mucosa of patients with Crohn's disease has been examined and compared with the mucosal flora of a control group. Specimens were obtained at operation from patients undergoing intestinal surgery. Whole thickness intestinal sections were taken from the diseased segment and from a portion of uninvolved intestine from patients with Crohn's disease as well as from a control group. A section of each specimen was examined histologically. Twenty-two specimens of Crohn's tissue (12 ileum, 10 colon) and 46 control samples from small and large bowel were examined using strictly anaerobic bacteriological techniques in an anaerobic chamber. Organisms were grown under both aerobic and anaerobic conditions. A mucosal flora was found to exist in all the large bowel samples and in three-quarters of the small bowel samples. It was qualitatively similar in all the samples, consisting mainly of Gram positive bacteria, aerobic Gram negative rods and bacteroides. Greater numbers of bacteria were associated with colonic tissue (10(7) - 10(8) per g) than with tissue from the jejunum (10(3) - 10(4) per g). This difference was statistically significant (p<0.03). Samples from the terminal ileum were quantitatively intermediate between jejunum and colon. There was no statistical difference in the numbers of bacteria associated with Crohn's tissue compared with histologically normal tissue from the same patients and from the control group of patients. Among the bacterial isolates, however, Enterobacteria were more commonly associated with Crohn's tissue.

Aerobiosis↗
PubMed1992

Evaluation of enzyme linked immunosorbent assay for screening urinary tract infection in elderly people.

AIMS: To evaluate the Uristat test, an indirect enzyme linked immunosorbent assay for the qualitative detection of antibodies in urine, as a screening, and in the diagnosis of urinary tract infection in the elderly. METHODS: Semiquantitative culture was compared with conventional microscopy, dipstick analysis and the ELISA. In the ELISA, 371 urine samples were examined for antibodies to an antigen mixture of six common urinary pathogens. RESULTS: The sensitivity was 91% and the specificity 25% for the ELISA. The negative predictive value was 81% and the positive predictive value was 43%. CONCLUSIONS: In its present form the Uristat test has no clear advantages over conventional bacteriological techniques for screening urine samples for infection in an elderly population.

Aged↗
PubMed1992

New rapid identification test for Clostridium difficile.

AIMS: A set of five tests were developed and tested for their ability to confirm the identity of C difficile colonies within 30 minutes. METHODS: The relevant substrates were incorporated into four filter paper squares attached to a plastic carrier (Diffstrip), five enzymes/products (prolyl aminopeptidase, galactosidase, leucine aminopeptidase, acid phosphatase and indole). The strips were inoculated, incubated for 20 minutes, and reagents added. RESULTS: 96.4% (212 of 220) strains of C difficile were immediately differentiated from 51 other Clostridium spp tested. The remaining 3.6% (eight of 220) of C difficile isolates produced a reaction pattern similar to some of the Clostridium sporogenes tested and required additional tests. None of the other Clostridium spp tested produced reaction patterns similar to C difficile. CONCLUSION: The Diffstrip allowed colonies of C difficile to be confirmed within 30 minutes for 96.4% of isolates, with less than 4% requiring any additional tests. No strains of C difficile were misidentified and no strains of other Clostridium spp tested were misidentified as C difficile.

Bacterial Typing Techniques↗
PubMed1993

Development of an internal quality assessment scheme in a clinical bacteriology laboratory.

AIM: To develop an internal quality assessment (IQA) scheme in a clinical bacteriology laboratory. METHODS: Over 24 months, 1230 diagnostic specimens, representing 0.42% of laboratory workload, were anonymised and resubmitted for analysis. Six hundred and twenty one (48.7%) of these gave positive culture results; 44 fecal and upper respiratory specimens were "spiked" (artificially inoculated) to increase the proportion of positive samples. RESULTS: Discrepancies between IQA and clinical sample results occurred in 188 cases (14.8%): 76.6% of these were in culture results, 13.3% in microscopy performance, and 10.1% in clerical recording. The culture discrepancy rate for each positive sample was lowest for wound (17.5%) and urine (18.1%) specimens, and highest for faeces (34.9%) and upper respiratory (37.7%) samples. Discrepancies in several areas responded to staff training and improvement in technical methods. CONCLUSIONS: An IQA programme of this type assesses the reproducibility of tests within a diagnostic laboratory when analysing common specimen types and organisms. It permits blind assessment of many areas of diagnostic work that are not readily amenable to other quality assurance methods, and it raises the awareness of all staff to the importance of quality in every aspect of specimen and data processing.

Bacteriological Techniques↗