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PubMed2004

rpoB gene sequencing for identification of Corynebacterium species.

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.

Base Sequence↗

PubMed2004

Identification to the species level and differentiation between strains of Aspergillus clinical isolates by automated repetitive-sequence-based PCR.

A commercially available repetitive-sequence-based PCR (rep-PCR) DNA fingerprinting assay adapted to an automated format, the DiversiLab system, enables rapid microbial identification and strain typing. We explored the performance of the DiversiLab system as a molecular typing tool for 69 Aspergillus isolates (38 A. fumigatus, 15 A. flavus, and 16 A. terreus isolates) had been previously characterized by morphological analysis. Initially, 27 Aspergillus isolates (10 A. fumigatus, 9 A. flavus, and 8 A. terreus isolates) were used as controls to create a rep-PCR-based DNA fingerprint library with the DiversiLab software. Then, 42 blinded Aspergillus isolates were typed using the system. The rep-PCR-based profile revealed 98% concordance with morphology-based identification. rep-PCR-based DNA fingerprints were reproducible and were consistent for DNA from both hyphae and conidia. DiversiLab dendrogram reports correctly identified all A. fumigatus (n = 28), A. terreus (n = 8), and A. flavus (n = 6) isolates in the 42 blinded Aspergillus isolates. rep-PCR-based identification of all isolates was 100% in agreement with the contiguous internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) sequence-based identification of the respective isolates. Additionally, the DiversiLab system could demonstrate strain-level differentiation of A. flavus and A. terreus. Automated rep-PCR may be a time-efficient, effective, easy-to-use, novel genotyping tool for identifying and determining the strain relatedness of fungi. This system may be useful for epidemiological studies, molecular typing, and surveillance of Aspergillus species.

Aspergillus↗

PubMed2004

Evaluation of the new VITEK 2 card for identification of clinically relevant gram-negative rods.

The VITEK 2 card for gram-negative bacteria (bioMérieux,Marcy-l'Etoile, France) has been redesigned to improve the identification of fermenting and nonfermenting bacilli. Forty-seven biochemical tests, including 19 enzymatic tests, are present in the new card and interpreted in a kinetic mode. Final identification results are available within 10 h. The database allows the identification of 159 different taxa. Six hundred fifty-five gram-negative rods (GNR; 511 fermenters and 144 nonfermenters), representing 54 taxa, were tested. Strains were taken from fresh routine primary isolation plates (n = 157), from stored routine plates (n = 301), and from stock cultures (n = 197). Six hundred thirty-seven strains (97.3%) were correctly identified to the species level, 14 strains (2.1%) gave low discrimination results requiring additional tests, and 4 strains (0.6%) gave discordant results; not a single strain remained unidentified. Nearly 92% of all isolates were correctly identified within 7 h of incubation. The robustness of the system was demonstrated by the fact that strains were grown on four different agar media before testing. The system may also have the potential to be applied directly to primary isolation plates, since in this instance 96.2% of 157 GNR were correctly identified and 3.8% gave low discrimination results. The new VITEK 2 card for gram-negative bacteria seems to be a promising new tool for routine, rapid identification of GNR.

Automation↗

PubMed2005

Identification of 43 Streptococcus species by pyrosequencing analysis of the rnpB gene.

Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.

Bacterial Typing Techniques↗

PubMed2005

Evaluation of 16S rRNA sequencing and reevaluation of a short biochemical scheme for identification of clinically significant Bacteroides species.

Sequence analysis of the 16S rRNA gene represents a highly accurate and versatile method for bacterial classification and identification, even when the species in question is notoriously difficult to identify by phenotypic means. In this study, we evaluated the utility of 16S rRNA gene sequencing as a means of identifying clinically important Bacteroides species. We sequenced 231 clinical isolates that had been identified by a short biochemical scheme. Based on the sequence analysis, 192 clinical isolates were assigned to an established species, with the other 39 clinical strains revealing five unique sequences that may represent five novel species. This is in contrast to identification obtained from a short biochemical scheme, by which only 73.5% (172 of 231) of isolates were correctly identified to species level. Based on the solid identification obtained from 16S rRNA gene sequencing, the short biochemical scheme was modified and improved to provide clinical laboratories with an inexpensive and simple alternative for the identification of isolates of clinically significant Bacteroides species.

Bacterial Typing Techniques↗

PubMed2005

Evaluation of tRNA gene PCR for identification of mollicutes.

We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.

Acholeplasma↗

PubMed2006

16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory.

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

Bacterial Typing Techniques↗

PubMed1977

Comparative study of three methods of identification of Enterobacteriaceae.

Three separate hospital clinical microbiology laboratories using three different identification systems participated in the identification of Enterobactericeae from a central pool of 'unknown" clinical isolates. With conventional tubed media, API-20E (Anlytab Products Inc.) and R/B tube (Corning Diagnostics) systems, there was a 91.1% agreement in the species designation. No significant differences at the 95% confidence level were found among the systems. Evaluation of individual tests within the systems used revealed lysine decarboxylase of the conventional and citrate of the API-20E system to be significantly different from the same test within the other two systems. The lysine decarboxylase of the conventional system had species relatedness, whereas the differences in citrate of the API-20E system were not related to a particular species. These individual test variations did not affect final organism identification. Reproducibility, evaluated as the system's ability to designate the same identification on two separate occasions, was 92 to 94% for each system. Exact duplication of selected sets of reactions was 60% for conventional, 45% for API-20E, and 61% for R/B. The variations in sets of reactions differed with the system and with the organism involved. The findings suggest equivalency among the three systems in ability to identify common clinical isolates of Enterobacteriaceae and point out the limited usefulness of these systems for biochemical biotyping.

Bacteriological Techniques↗

PubMed1977

Evaluation of a caffeic acid-ferric citrate test for rapid identification of Cryptococcus neoformans.

An evaluation of a rapid caffeic acid-ferric citrate paper disk test for the identification of Cryptococcus neoformans, using 474 isolates of yeasts and yeastlike organisms, showed that 96.6, 97.7, and 98.3% of 176 isolates of C. neoformans produced brown to dark-brown pigment on disks incubated for 6 h at room temperature, 30 degrees C, and 37 degrees C, respectively. All C. neoformans produced brown to dark-brown pigment within 24 h. However, nonspecific pigmentation was encountered at all temperatures of incubation with one isolate of Trichosporon cutaneum and, at room temperature only, with one isolate of C. luteolus after 6 h. Other genera of yeasts produced similar pigmentation after 24 h at all temperatures. The age of the cultures tested or the types of media used before testing did not significantly affect the ability of C. neoformans to produce pigmentation at 37 degrees C. A positive test may prove useful for presumptive identification of C. neoformans, but a negative reaction should not be used to rule out an identification of this yeast. Since a number of false-negative and false-positive tests occur, it is necessary to confirm, by other biochemical tests, the identification of all organisms suspected of being C. neoformans, to reduct the serious risk of missing or misidentifying this important pathogen.

Caffeic Acids↗

PubMed1978

Comparison of a radiometric procedure with conventional methods for identification of Neisseria.

A radiometric procedure was compared with the conventional cystine tryptic agar (CTA) sugar fermentation method for identification of Neisseria species. Four different ATCC cultures of Neisseria were identified by both procedures with identical results. The only difference noted was that the radiometric procedure required 3 h for completion, whereas the conventional CTA sugar method required overnight incubation. The radiometric procedure was also compared with the fluorescent antibody (FA) and CTA methods for identification of Neisseria gonorrhoeae. The organisms examined were gram-negative, oxidase-positive diplococci isolated from 49 clinical specimens sent to the laboratory for bacteriological analysis. Results obtained by both CTA and FA procedures were comparable. However, the radioisotope method appeared to be superior to the other two methods in that only one isolate identified as positive by both the CTA and FA methods was not identified radiometrically, whereas four isolates positive by the radiometric method were not identified by the other two procedures. Thus, a total of seven more positive identifications were made radiometrically than by either of the two other methods. All positive identifications were confirmed by a reference laboratory. These results indicate that the radiometric procedure is more rapid and reliable as compared with both the CTA and direct FA methods and, thus, may serve as a valuable addition to the methodology available for diagnostic microbiology.

Bacteriological Techniques↗

PubMed1978

Comparison of micro-ID, API 20E, and conventional media systems in identification of Enterobacteriaceae.

The Micro-ID, a new identification kit for Enterobacteriaceae, consists of 15 biochemical tests, with substrates and reagents impregnated in filter paper disks. A 0.2-ml amount of an organism suspension equal to a 0.5 McFarland standard is pipetted into each of the compartments. After 4 h of incubation and addition of potassium-hydroxide (KOH) to the Voges-Proskauer test, the color reactions are read according to the recommendations of the manufacturer. A five-digit octal code number is derived from each set of reactions from which an identification is derived by using a code book. In a single-blind, comparative study of the Micro-ID system with the API 20E system (Analytab Products Inc.) and conventional biochemical tube media, we found that the Micro-ID and the API 20E systems gave a 90% identification correlation when each was compared with the conventional tube media. A comparison of all three systems gave an 82% overall identification correlation. When the common tests of Micro-ID and API 20E were compared with conventional tube media, we found that the tests in the Micro-ID performed as well as or better than those of the API 20E. Certain groups of organisms, i.e., Citrobacter, Enterobacter, Proteus, Salmonella, and Serratia genera, were found to give low correlation on certain common tests. When using primary isolation MacConkey plates from the clinical laboratory, only 74% of the plates with Enterobacteriaceae had sufficient numbers of colonies of each enteric organism to produce the 0.5 McFarland inoculum density required. Problems concerning the misidentification of some organisms are discussed.

Bacteriological Techniques↗

PubMed1978

Improved version of the exoantigen test for identification of Coccidioides immitis and Histoplasma capsulatum cultures.

A rapid and simple method for extracting specific cell-free antigens of Coccidioides immitis and Histoplasma capsulatum from agar slant cultures was developed. The extracts were analyzed in immunodiffusion tests for the presence of C. immitis or H. capsulatum specific antigens. These extracts were compared with culture filtrates of brain heart infusion broth subcultures in tests with 32 isolates of C. immitis and H. capsulatum and 13 other fungi which might be morphologically confused with them. The studies showed that the slant extracts were as useful as the culture filtrates and allowed more rapid identification of C. immitis and H. capsulatum. In every case, when an identification was made by conventional morphological methods, the immunological test yielded correlating results. The immunological tests were positive for two isolates that were converted to their yeast forms only after 4 months of study by conventional tests. The new procedure permits the identification of C. immitis and H. capsulatum 2 days after receipt of a pure mycelial-form culture. The test is recommended for the presumptive identification of C. immitis and H. capsulatum cultures.

Antigens, Fungal↗

PubMed2006

Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus by a novel bacterial expression cloning approach.

Epstein-Barr virus (EBV)-specific T cells have been successfully used to treat or prevent EBV-positive lymphoproliferative disease in hematopoietic stem cell transplant recipients, but the antigens recognized by the infused CD4+ T cells have remained unknown. Here, we describe a simple procedure that permits the identification of viral T-helper (TH)-cell antigens and epitopes. This direct antigen identification method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria, which are subsequently fed to major histocompatibility complex class II+ antigen-presenting cells and probed with antigen-specific T cells. The fusion of antigenic fragments to CAT offers several advantages. First, chloramphenicol treatment allows the selection of bacteria expressing antigen-CAT fusion proteins in frame, which greatly reduces the number of colonies to be screened. Second, antigenic fragments fused to CAT are expressed at high levels, even when derived from proteins that are toxic to bacteria. Third, the uniformly high expression level of antigen-CAT fusion proteins permits the establishment of large and representative pool sizes. Finally, antigen identification does not require knowledge of the restriction element and often leads directly to the identification of the T-cell epitope. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of the EBV-specific T-helper-cell response, demonstrating that lytic cycle antigens are a relevant component of the EBV-specific TH-cell response.

Antigen Presentation↗

PubMed1989

The ordinary light microscope: an appropriate tool for provisional detection and identification of crystals in synovial fluid.

To determine if the ordinary light microscope is a useful tool in the detection and identification of crystals in synovial fluid 63 fluids (13 with monosodium urate (MSU), 14 with calcium pyrophosphate dihydrate (CPPD), one with both types of crystal, and 35 without crystals) were examined blindly by two observers, first with an ordinary light microscope, then with a polarising light microscope. Tentative identification of the crystals by their shape was attempted. The sensitivity of the ordinary light microscope for the detection of crystals was 96.2% and 100%, with specificity of 100% and 97.1% for observers 1 and 2, both with respect to the polarising light microscope. The concordance between observers for the ordinary light microscope was chi = 0.90 (0.897, 0.902) and for the polarising light microscope chi = 0.96 (0.958, 0.961). The specificity for identification of MSU and CPPD crystals by their shape with respect to the polarising light microscope was 92.3% and 85.7% for observers 1 and 2. When a polarising light microscope is not available examination of synovial fluid by an ordinary light microscope may allow a provisional diagnosis; definitive identification of the crystals requires a polarising light microscope.

Crystallization↗

PubMed1997

Efficacy of the alcohol use disorders identification test as a screening tool for hazardous alcohol intake and related disorders in primary care: a validity study.

OBJECTIVE: To determine the properties of the alcohol use disorders identification test in screening primary care attenders for alcohol problems. DESIGN: A validity study among consecutive primary care attenders aged 18-65 years. Every third subject completed the alcohol use disorders identification test (a 10 item self report questionnaire on alcohol intake and related problems) and was interviewed by an investigator with the composite international diagnostic interview alcohol use module (a standardised interview for the independent assessment of alcohol intake and related disorders). SETTING: 10 primary care clinics in Verona, north eastern Italy. PATIENTS: 500 subjects were approached and 482 (96.4%) completed evaluation. RESULTS: When the alcohol use disorders identification test was used to detect subjects with alcohol problems the area under the receiver operating characteristic curve was 0.95. The cut off score of 5 was associated with a sensitivity of 0.84, a specificity of 0.90, and a positive predictive value of 0.60. The screening ability of the total score derived from summing the responses to the five items minimising the probability of misclassification between subjects with and without alcohol problems provided an area under the receiver operating characteristic curve of 0.93. A score of 5 or more on the five items was associated with a sensitivity of 0.79, a specificity of 0.95, and a positive predictive value of 0.73. CONCLUSIONS: The alcohol use disorders identification test performs well in detecting subjects with formal alcohol disorders and those with hazardous alcohol intake. Using five of the 10 items on the questionnaire gives reasonable accuracy, and these are recommended as questions of choice to screen patients for alcohol problems.

Adolescent↗

PubMed2002

Importance of emergency identification schemes.

BACKGROUND: Millions of people worldwide may have a hidden medical condition that could endanger their life in an emergency. These conditions may include cardiac conditions, severe allergies, or diabetes. Emergency identification schemes such as Medic Alert produce emblems that alert health care professionals to potential problems and can ensure appropriate and prompt treatment. This paper uses mechanical failure of the Björk-Shiley convexo-concave (BSCC) heart valve as an example of a hidden medical condition. These patients have been encouraged to carry information to alert staff in an emergency that they have a BSCC patient in their care and to be alert to the signs and symptoms of acute valve malfunction. OBJECTIVE: To establish awareness and credibility of emergency identification schemes among emergency personnel and to assess if information on specific medical conditions would influence ambulance personnel regarding destination hospitals. METHODS: Questionnaires were sent to senior staff (n=380) of accident and emergency (A&E) departments and operational directors of ambulance headquarters (n=39) throughout the United Kingdom. Hospitals were divided into regional divisions to assess differences in responses across regions. RESULTS: The majority of respondents (99%) had heard of emergency identification schemes and felt that it was important for patients with special conditions to carry some form of identification. Nearly all ambulance respondents (97%) indicated it was routine to search for body worn emblems in contrast with only 71% of A & E staff. However, more than half of ambulance respondents (53.9%) stated information on emblems/cards would not influence their choice of destination hospital. CONCLUSIONS: The importance of how information on pre-existing medical conditions can influence care, is highlighted by the BSCC valve issue, where immediate diagnosis is essential for patient survival. It is vital that all staff routinely search patients for this information and if necessary act upon the information provided.

Emergency Medical Services↗

PubMed1993

Multipoint identification of Enterobacteriaceae: report of the British Society for Microbial Technology collaborative study.

AIMS: To evaluate the accuracy and reproducibility of multipoint identification schemes in a multicentre trial. METHODS: Forty two strains of Enterobacteriaceae were distributed to 22 laboratories for identification by routine multipoint methods. Analysis of results enabled inter- and intralaboratory reproducibility of a variety of tests, and the ability of laboratories to identify individual organisms to be determined. RESULTS: Interlaboratory reproducibility of most of the biochemical tests was acceptable. The least reproducible tests, both within and between laboratories, were citrate utilisation, production of urease and beta galactosidase, detection of motility, and decarboxylation of lysine and ornithine. Inconsistent results for these tests were often associated with misidentified strains. Most laboratories performed identifications satisfactorily. Most isolates (72.1%) were identified correctly to species level; 9.6% were incorrectly identified, and 6.4% could not be identified at all. The most difficult organisms to identify were Citrobacter freundii, Enterobacter cloacae, Hafnia alvei and Aeromonas hydrophila. Strains of Enterobacter, Serratia sp, and Providencia sp were difficult to speciate. Several laboratories could not identify organisms exhibiting at least one atypical biochemical reaction. CONCLUSION: This study emphasises the need for quality control of media and reagents for multipoint identification of Gram negative enteric bacilli.

Bacteriological Techniques↗

PubMed1994

Comparison of Vitek and Cobas Micro systems with a semiautomated conventional microsystem for identification and susceptibility testing of gram negative bacilli.

AIMS: To compare the sensitivity and specificity of two semiautomated systems against a conventional (MIC 2000) test system for the identification and antibiotic susceptibility of Gram negative bacteria. METHODS: Clinical isolates of Gram negative bacilli (188 urinary and 229 non-urinary strains) were identified and tested for antibiotic susceptibility in the Cobas Micro and MIC 2000 systems. Of these, 359 strains were then tested in the Vitek and MIC 2000 systems. Two hundred and forty three strains were tested in all three systems immediately after isolation. Forty three were also tested only in the Vitek and MIC 2000 systems immediately after isolation. The remaining 174 strains were tested after storage at -20 degrees C for several months. RESULTS: The Cobas Micro and MIC 2000 systems agreed on the identification of 310 of the 417 (74.3%) strains; the Vitek and MIC 2000 systems agreed on 338 of the 359 (94.2%) strains. The Cobas Micro system correctly identified 86.8% of strains tested after storage and 65.4% of those immediately after isolation. Organism-antibiotic combinations (non-urinary isolates) were tested in the Cobas Micro and MIC 2000 systems (n = 2335), in the Vitek and MIC 2000 systems (n = 999). Essential correlation (complete agreement plus minor errors) was observed in 98% (with 90% complete agreement) in the former and in 97% (with 86% complete agreement) in the latter. For the urinary isolates, 1949 organism-antibiotic combinations were analysed in the Cobas Micro and MIC 2000 systems where complete agreement was observed in 92% (with 3% very major discrepancies), for 1382 urinary organism-antibiotic combinations tested in the Vitek and MIC 2000 systems, the figures were 95% and 2%, respectively. CONCLUSIONS: The Vitek system is highly accurate in the identification and antibiotic susceptibility testing of Gram negative bacteria. The Cobas Micro system has many shortcomings in its identification of Gram negative rods, especially freshly isolated strains, but it is comparable with the Vitek system in antibiotic susceptibility testing.

Anti-Bacterial Agents↗