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PubMed2006

Faster identification of pathogens in positive blood cultures by fluorescence in situ hybridization in routine practice.

Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice. After Gram staining, blood culture fluids were simultaneously further identified with FISH and with conventional culture methods. Results and points in time of FISH and culture identification (provisional and final identifications) were collected and compared. For 91% of microorganisms, the genus or family name was identified, and for 79%, the species name could be attributed. The sensitivity and specificity of the individual probes exceeded 95%, except for the Enterobacteriaceae probe (sensitivity, 89%). Cross-hybridization was obtained with the Klebsiella pneumoniae probe for Klebsiella oxytoca. The time gains of FISH and final culture identification were more than 18 h for bacteria and 42 h for yeasts. With FISH, Staphylococcus aureus was differentiated from coagulase-negative staphylococci 1.4 h faster than by provisional identification (P < 0.001). In conclusion, FISH allows rapid and reliable identification of the majority of microorganisms in growth-positive blood cultures. The substantial time gain of identification with FISH may allow same-day adjustment of antimicrobial therapy, and FISH is especially useful if no provisional identification is obtained. With further extension of the number of probes and a reduction in turnaround time, FISH will become a very useful diagnostic tool in the diagnosis of bloodstream infections.

Bacteremia↗
PubMed1987

Rapid identification of pathogenic Neisseria species and Branhamella catarrhalis.

Two systems, the Identicult-Neisseria (IDN; Scott Laboratories, Inc., Fiskeville, R.I.) strip and the Neisseria/Haemophilus Identification Test Kit (NHI; Vitek Systems, Inc., Hazelwood, Mo.) card, were compared with the 4-h Minitek system (BBL Microbiology Systems, Cockeysville, Md.) for their ability to rapidly identify 157 pathogenic Neisseria and Branhamella catarrhalis isolates. IDN, limited in its identification to four species, when incubated at 35 degrees C for 10 min identified 99% of the isolates. However, when IDN was incubated at 22 degrees C for 20 min, it identified only 92% of the isolates. The NHI card, a rapid semiautomated system with the ability to identify 25 organisms to the species level, correctly identified all of the isolates. A test for beta-lactamase production included in the NHI card identified the 12 Neisseria gonorrhoeae and 10 B. catarrhalis beta-lactamase-positive isolates included in the study. The IDN strip (35 degrees C) and the NHI card compared favorably with the Minitek system.

Moraxella catarrhalis↗
PubMed2002

PCR-based identification of pathogenic Candida species using primer mixes specific to Candida DNA topoisomerase II genes.

For rapid identification of Candida to the species level, degenerated primers and specific primers based on the genomic sequences of DNA topoisomerase II of C. albicans, C. dubliniensis, C. tropicalis (genotypes I and II), C. parapsilosis (genotypes I and II), C. krusei, C. kefyr, C. guilliermondii, C. glabrata, C. lusitaniae and Y. lipolytica were designed and their specificities tested in PCR-based identifications. Each of the specific primers selectively and exclusively amplified its own DNA fragment, not only from the corresponding genomic DNA of the Candida sp. but also from DNA mixtures containing other DNAs from several fungal species. For a simpler PCR-based identification, the specific primers were divided into three groups (PsI, PsII and PsIII), each of which contained four specific primer pairs. PCR with the primer mixes yielded four different sizes of PCR product, corresponding to each Candida sp. in the sample DNA. To obtain higher sensitivity of PCR amplification, sample DNAs were preamplified by the degenerated primer pair (CDF28/CDR148), followed by the main amplification using the primer mixes. By including this nested PCR step, 40 fg yeast genomic DNA was detected in the sample. Furthermore, we applied this nested PCR to a clinical diagnosis, using splenic tissues from experimentally infected mice and several clinical materials from patients. In all cases, the nested PCR amplifications detected proper DNA fragments of Candida spp., which were also identified by the standard identification tests. These results suggest that nested PCR, using primer mixes of the Candida DNA topoisomerase II genes, is simple and feasible for the rapid detection/identification of Candida to species level in clinical materials.

Animals↗
PubMed1991

[Multicenter study of the Rosco-Neisseria system for the identification of pathogenic neisserias and Branhamella catarrhalis].

The commercial Rosco-Neisseria system was evaluated in the identification of 228 oxidase-positive Gram-negative diplococci and it was compared with conventional tests. The procedure detects gamma-glutamyl aminopeptidase, ONPG, tributyrin hydrolysis, and sensitivity to the disk of 10 micrograms of colistin. A correct identification was obtained in the 65 strains of Neisseria gonorrhoeae, the 33 of N. meningitidis, the 12 of N. lactamica, and the 56 of B. catarrhalis. The method was also able to discriminate 54 out of the 62 strains of nonpathogenic Neisseria. However, the 7 strains of Neisseria polysaccharea and one strain of N. subflava biovar perflava were erroneously identified as N. gonorrhoeae. None of the latter was superoxol positive in contrast with the 100% of cases of gonococcal strains. The Rosco-Neisseria system is simple and inexpensive but it should be applied on specimens that grow on selective media for gonococci (such as Thayer-Martin and others) and it should be complemented by superoxol test.

Bacterial Infections↗
PubMed1993

[Mobile species of the genus Aeromonas: difficulties of identification and pathogenicity].

Sixty-two Aeromonas strains (39 of clinical and 23 of environmental origin) were identified. The suicide phenomenon and autoagglutination were studied. Identification is based on esculin hydrolysis; fermentation of arabinose salicin, sucrose and mannitol; gas production from glucose, indole and beta hemolysis; Voges-Proskauer and decarboxylation reactions; and finally resistance to cephalothin (30 micrograms) and colistin (4 micrograms/ml). Thirty-four per cent of A hydrophila, 33% A caviae, 28% A veronii subspecies sobria, 3% A jandaei, 2% A veronii subspecies veronii were accurately identified. Also, several new species were identified such as A trota, A enteropelogenes, A schubertii, A ichthiosmia, according to the more recently proposed taxa. This identification scheme could enhance our knowledge concerning virulence factors, pathogenicity and environmental distribution.

Aeromonas↗
PubMed1995

rDNA targeted oligonucleotide primers for the identification of pathogenic yeasts in a polymerase chain reaction.

Species-specific oligonucleotide primers were designed for PCR identification of the basidiomycetous yeasts Cryptococcus neoformans, Trichosporon cutaneum and Rhodotorula mucilaginosa. The procedure uses standard PCR components including DNA from the test species and three primers: two universal external (upstream and downstream) limiting primers and a species-specific internal primer. Species identification requires the formation of a species-specific rDNA nucleotide segment that is significantly smaller (approximately 200 bp) than a non-target segment (approximately 600 bp). The procedure can be used to identify yeasts from single and mixed populations.

Base Sequence↗
PubMed1988

Evaluation of three commercial systems for the identification of pathogenic Neisseria and Branhamella species against the conventional method.

We compared three commercial systems for the identification of Neisseria and Branhamella spp. with the conventional method using cystine-tryptic digest agar (CTA) supplemented with carbohydrates, DNase production, and nitrate reduction. We evaluated the API quadFerm+ [( API], Analytab Products, Inc., Plainview, N.Y.), NEISSERIA [( Pasteur], Diagnostics Pasteur, Marnes-la-Coquette, France), and Neisseria Identification Discs [( Oxoid], Oxoid Ltd., Basingstoke, England) using the conventional method as a reference. One hundred and twenty-nine strains were included in this study. The conventional method identified 125 strains (96.9%). Four strains of N. gonorrhoeae remained glucose-negative in the CTA media but gave positive reactions in the API system. API, Pasteur, and Oxoid identified 126 (97.7%), 124 (96.1%), and 62 strains (48.1%), respectively. API and Pasteur seem to be very useful systems for the differentiation of clinically significant species of Neisseria and Branhamella. API has the additional advantage of requiring only a 3 h incubation period.

Carbohydrate Metabolism↗
PubMed2005

Identification of pathogenic Helicobacter species by chaperonin-60 differentiation on plastic DNA arrays.

A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.

Campylobacter jejuni↗
PubMed1979

Comparison of three methods for identification of pathogenic Neisseria species.

A radiometric procedure was compared with the Minitek and Cystine Trypticase Agar sugar degradation methods for identification of 113 Neisseria species (58 Neisseria meningitidis, 51 Neisseria gonorrhoeae, 2 Neisseria lactamica, 2 Neisseria sicca). Identification of meningococci and gonococci was confirmed by agglutination and fluorescent antibody techniques, respectively. The Minitek method identified 97% of meningococci, 92% of gonococci, and 100% of other Neisseria after 4 h of incubation. The radiometric (Bactec) procedure identified 100% of gonococci and 100% of miscellaneous Neisseria after 3 h, but problems were encountered with meningococci: 45% of the later strains yielded index values for fructose between 20 and 28 (recommended negative cut-off point, less than 20), with strongly positive (greater than 100) glucose and maltose and negative o-nitrophenyl-beta-D-galactopyranoside reactions in all 58 strains. The Cystine Trypticase Agar method identified 91% of meningococci, 90% of gonococci, and 100% of other Neisseria after 24 to 48 h. Prolongation of the Cystine Trypticase Agar incubation period led to abnormal lactose/sucrose reactions in some meningococci and gonococci. Radiometric and Minitek systems are more accurate and convenient than Cystine Trypticase Agar techniques, but, on the basis of these results, radiometric fructose sensitivity levels for meningococci need reevaluation.

Bacterial Infections↗
PubMed2004

Identification of pathogenic microbial cells and spores by electrochemical detection on a biochip.

BACKGROUND: Bacillus cereus constitutes a significant cause of acute food poisoning in humans. Despite the recent development of different detection methods, new effective control measures and better diagnostic tools are required for quick and reliable detection of pathogenic micro-organisms. Thus, the objective of this study was to determine a simple method for rapid identification of enterotoxic Bacillus strains. Here, a special attention is given to an electrochemical biosensor since it meets the requirements of minimal size, lower costs and decreased power consumption. RESULTS: A bead-based sandwich hybridization system was employed in conjugation with electric chips for detection of vegetative cells and spores of Bacillus strains based on their toxin-encoding genes. The system consists of a silicon chip based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the DNA probes. The specific signals from 20 amol of bacterial cell or spore DNA were achieved in less than 4 h. The method was also successful when applied directly to unpurified spore and cell extract samples. The assay for the haemolytic enterotoxin genes resulted in reproducible signals from B. cereus and B. thuringiensis while haemolysin-negative B. subtilis strain did not yield any signal. CONCLUSIONS: The sensitivity, convenience and specificity of the system have shown its potential. In this respect an electrochemical detection on a chip enabling a fast characterization and monitoring of pathogens in food is of interest. This system can offer a contribution in the rapid identification of bacteria based on the presence of specific genes without preceding nucleic acid amplification.

Journal Article↗
PubMed1988

Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods.

The combination of radiometric methodology (BACTEC 12B) and probe technology for recovery and identification of mycobacteria was studied in two large hospital laboratories. The sediment from vials with positive growth indices was tested with DNA probes specific for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The sensitivity of the radiometric method and the specificity of the probes resulted in a marked reduction in the time to the final report. Biochemical testing could be eliminated on isolates giving a positive reaction with one of the probes. Some 176 isolates of M. tuberculosis, 110 of M. avium, and 5 of M. intracellulare were recovered. Two-thirds of these isolates were detected and identified within 2 weeks of inoculation and the remainder was detected by 4 weeks, a reduction of 5 to 7 weeks to the final report.

DNA, Bacterial↗
PubMed2006

Rapid identification of pathogens in blood cultures with a modified fluorescence in situ hybridization assay.

We evaluated a modified fluorescence in situ hybridization (FISH) assay for rapid (<1 h) identification of microorganisms in growth-positive blood cultures. The results were compared to those of the standard FISH technique and conventional culturing. The rapid identification of microorganisms with modified FISH can have important effects on clinical management of patients with bloodstream infections.

Aged↗
PubMed1991

Genotypic identification of pathogenic Mycobacterium species by using a nonradioactive oligonucleotide probe.

Commercial DNA hybridization assays (Syngene, Inc., San Diego, Calif.) utilizing alkaline phosphatase-labeled oligonucleotide probes for the identification of Mycobacterium tuberculosis complex and M. avium complex (MAC) were evaluated with 261 isolates of mycobacteria. On the basis of biochemical criteria, the test for MAC was 98% specific and more sensitive (95 of 99, 95%) than Gen-Probe (88 of 99, 89% sensitivity); the major difference in sensitivity noted between the two systems was related to the hybridization of seven MAC strains to the SNAP X probe. The M. tuberculosis complex probe correctly identified all 62 isolates of M. tuberculosis and all 11 isolates of M. bovis, for a sensitivity of 100%. There were two discrepant reactions with mycobacteria other than M. tuberculosis complex isolates.

Bacteriological Techniques↗
PubMed2002

Evaluation of the BactiCard Neisseria for identification of pathogenic Neisseria species and Moraxella catarrhalis.

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), gamma-glutamyl aminopeptidase (GLUT), and ss-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory.

Bacterial Typing Techniques↗
PubMed2002

Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR.

Bacillus anthracis spores have been shown to be an efficient biological weapon and their recent use in bioterrorist attacks has demonstrated the need for rapid and specific diagnostics. A TaqMan real-time PCR for identification of B. anthracis was developed, based on the two plasmids, pX01 and pX02, both of which are necessary for pathogenicity, as well as on the chromosomally encoded rpoB gene. Bacteria picked from colonies or pelleted from liquid cultures were directly inoculated into the PCR mix, thus avoiding time-consuming DNA preparation and minimizing handling risks. B. anthracis spores were cultivated for a few hours in enrichment broth before PCR analysis, or used directly for real-time PCR, thus allowing to confirm or exclude potential attacks approximately 2-3 h after the material has arrived in the laboratory.

Anthrax↗
PubMed2005

Isolation and identification of pathogenic Acanthamoeba strains in Tenerife, Canary Islands, Spain from water sources.

A comprehensive survey to document the presence of free-living amoebae of the genus Acanthamoeba was conducted in tap water and sea water sources related to human environments in Tenerife, Canary Islands, Spain. Acanthamoeba identification was based on the morphology of cyst and trophozoite forms and PCR amplification with a genus-specific primer pair. The pathogenic potential of Acanthamoeba isolates was characterized by temperature and osmotolerance assays and PCR reactions with two primer pairs related to Acanthamoeba pathogenesis. The results demonstrate the presence of potentially pathogenic strains in both sources. Thus, some of the amoebae in these aquatic habitats can act as opportunistic pathogens, could play a role in the diseases of aquatic organisms, and may present a risk to human health.

Acanthamoeba↗
PubMed1985

Evaluation of the RIM-N, Gonochek II, and Phadebact systems for the identification of pathogenic Neisseria spp. and Branhamella catarrhalis.

Methods for identifying Neisseria spp. include conventional and modified carbohydrate degradation procedures, chromogenic enzyme substrate tests, and immunologic coagglutination tests for Neisseria gonorrhoeae. In this study, we evaluated the abilities of the RIM-N carbohydrate degradation system (American MicroScan, Campbell, Calif.), the Gonochek II enzymatic identification system (Du Pont Co., Wilmington, Del.), and the Phadebact Gonococcus coagglutination test (Pharmacia Diagnostics, Piscataway, N.J.) to identify pathogenic Neisseria spp. and Branhamella catarrhalis. Both stock strains and clinical isolates, including 176 N. gonorrhoeae, 173 Neisseria meningitidis, 48 Neisseria lactamica, and 12 B. catarrhalis strains, were tested. The RIM-N identified 98% of the gonococci, 99% of the meningococci, 94% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 1 h. The Gonochek II system identified 99% of the gonococci, 97% of the meningococci, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 30 min. Phadebact coagglutination provided clearly positive results for only 77% of the N. gonorrhoeae strains, producing negative or equivocal results with 23% of the strains. The RIM-N and Gonocheck II tests generally produced clear-cut reactions. An additional advantage of the Gonocheck II system was the small inoculum required for the performance of the test compared with the other systems, thus allowing the identification of N. gonorrhoeae directly from the primary isolation medium.

Agglutination Tests↗
PubMed1982

Enumeration and identification of pathogenic bacteria from sewage in Kuwait.

Mycobacterium spp. was isolated and enumerated in all sewage samples at different stages of the sewage treatment. Aeromonas hydrophila, Salmonella spp., Shigella dysenteriae and others were isolated from both treated and untreated sewage. Moreover, the serological identification of Salmonella spp. was also done, using antisera specific to their antigenic structures. Counts of bacteria in various sewage sludges and effluents tested showed the efficiency of the activated sludge process in reducing the bacterial number. Seasonal variation in either the number or kind of pathogenic bacteria revealed inconsiderable changes. Almost all species of bacteria tested were found to be resistant to ampicillin.

Aeromonas↗