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PubMed1989

Accuracy and reproducibility of the 4-hour ATB 32A method for anaerobe identification.

The ATB 32A system (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) was evaluated for use in the identification of 214 anaerobes. Organisms included 73 isolates of the Bacteroides fragilis group, 24 Bacteroides spp., 10 fusobacteria, 43 clostridia, 28 cocci, and 36 gram-positive, nonsporeforming rods. With the concomitant use of Gram stain, pigmentation, catalase testing, and aerobic growth, the ATB 32A system correctly identified 97% of the B. fragilis group isolates, 88% of Bacteroides spp., 50% of fusobacteria, 74% of clostridia, 100% of cocci, and 86% of the gram-positive, nonsporeforming rods. Overall, 188 strains (88%) were correctly identified, with 18 (8%) requiring extra tests, other than the four mentioned above, for correct identification. Eight strains were misidentified, including one Bacteroides sp., three fusobacteria, one Clostridium sp., and three gram-positive, nonsporeforming rods. Reproducibility was very good, with 12 of 14 strains (86%) tested in triplicate yielding identical correct results on each of three occasions and 2 strains (14%) yielding identical correct results on two occasions. There was a low-probability identification (including the correct species) on the third testing. The ATB 32A system represents a worthwhile advance in systems used for the identification of clinically significant anaerobic bacteria.

Bacteria, Anaerobic↗

PubMed1990

Use of a novel phosphatase test for simplified identification of species of the tribe Proteeae.

Phosphatase activity of 334 isolates of the tribe Proteeae carefully identified to species level has been evaluated by using both a number of conventional tests and three versions of the novel methyl green-phenolphthalein (MGP) method (G. Satta. R. Pompei, G. Grazi, and G. Cornaglia, J. Clin. Microbiol. 26:2637-2641, 1988). We found that the different species of Proteeae show different and easily distinguishable. behaviors by the MGP method, while all of them behave in a uniform way in the conventional tests. On studying the mechanism underlying these different behaviors, we found that the phosphatase activity of Morganella morganii and Providencia stuartii is higher than that of all other members of the family Enterobacteriaceae. Furthermore, we found that all P. stuartii strains display the property (shared by virtually none of the other species of Enterobacteriaceae) of excreting significant amounts of phosphatase. This extracellular activity is reliably detected by the MGP method but not by conventional tests. Finally, by exploiting the peculiar phosphatase activity of this tribe, as revealed by the MGP method, we devised a simple scheme for routine identification of the species of Proteeae and evaluated its reliability by comparing it with three commonly used commercial kits. The new scheme proved much simpler, but also more reliable, since identifications obtained by this method were in almost complete accord (99%) with those of the reference identification schemes, while in the commercial systems examined the percentage of errors ranged from 13 to 14 in the identification of Providencia species.

Bacteriological Techniques↗

PubMed1991

Evaluation of the MicroScan Rapid Yeast Identification panel.

The MicroScan Rapid Yeast Identification (RYI) panel is a 4-h microdilution system for identification of clinical yeastlike isolates. Its accuracy was evaluated by using 357 isolates encompassing 11 genera and 30 species. The RYI panel identifications were compared with those obtained by the API 20C system assisted with morphological characterization on cornmeal-Tween 80 agar. The panels were read both visually and with the AutoScan-4, a computer-controlled microplate reader. Both the RYI panel and the API 20C system correctly identified 78% of the strains within 4 and 72 h, respectively, with no additional tests. Supplementary tests recommended by the manufacturers made it possible to identify up to 96.6% (AutoScan-4) and 98.9% (API 20C) of the strains. The accuracy of the RYI panel was 99.5% with common strains and 92.1% with less common strains. The RYI panel misidentified 10 or 12 strains and failed to identify 2 or 3 strains, depending on whether it was read with the AutoScan-4 or visually. Errors occurred with one strain of Torulopsis glabrata and the less common yeasts T. candida, Candida lusitaniae, C. lambica, C. rugosa, C. stellatoidea, Cryptococcus albidus, C. laurentii, and C. uniguttulatus. Overall, the RYI panel appears to be a reliable system for identification of the more common clinical yeast isolates.

Candida↗

PubMed1991

Enterosistem 18-R: description and comparative evaluation with conventional methods for identification of members of the family Enterobacteriaceae.

The efficiency and accuracy of Enterosistem 18-R (Liofilchem s.r.l., Roseto degli Abruzzi, Teramo, Italy) were compared with those of conventional biochemical methods to identify 360 members (38 species) of the family Enterobacteriaceae. Overall, 329 strains (91.3%) were correctly identified (percentage of identification, greater than or equal to 90.0), with 37 (11.2%) requiring additional tests for complete identification. For 11 isolates (3.1%), Enterosistem 18-R gave only genus identifications, and for 14 (3.9%), the strains did not correspond to any key in the codebook and could not be identified by the manufacturer's computer service. Only six isolates (1.7%) were misidentified. The new system accurately identified common and several newly described isolates of the family Enterobacteriaceae, such as Enterobacter gergoviae, Providencia rustigianii, Serratia odorifera, and Serratia rubidaea. The system is highly reproducible, simple to perform, easy to handle, and inexpensive. With adjustments in supplementary code numbers for some strains, Enterosistem 18-R is a suitable alternative for identification of members of the Enterobacteriaceae in clinical laboratories.

Bacteriological Techniques↗

PubMed1991

Clinical evaluation of the RapID-ANA II panel for identification of anaerobic bacteria.

The accuracy of the RapID-ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) was evaluated by comparing the results obtained with that system with results obtained by the methods described by the Virginia Polytechnic Institute and State University. Three hundred anaerobic bacteria were tested, including 259 clinical isolates and 41 stock strains of anaerobic microorganisms representing 16 genera and 48 species. When identifications to the genus level only were included, 96% of the anaerobic gram-negative bacilli, 94% of the Clostridium species, 83% of the anaerobic, nonsporeforming, gram-positive bacilli, and 97% of the anaerobic cocci were correctly identified. When correct identifications to the genus and species levels were compared, 86% of 152 anaerobic gram-negative bacilli, 76% of 34 Clostridium species, 81% of 41 anaerobic, nonsporeforming, gram-positive bacilli, and 97% of 73 anaerobic cocci were correctly identified. Eight isolates (3%) produced inadequate identification in which the correct identification was listed with one or two other possible choices and extra tests were required for separation. A total of 9 isolates (3%) were misidentified by the RapID-ANA II panel. Overall, the system was able to correctly identify 94% of all the isolates to the genus level and 87% of the isolates to the species level in 4 h by using aerobic incubation.

Bacteria, Anaerobic↗

PubMed1991

Enzyme-linked immunosorbent assay using monoclonal antibodies for identification of mycobacteria from early cultures.

A simple enzyme-linked immunosorbent assay (ELISA) for the identification of cultured mycobacteria belonging to the Mycobacterium tuberculosis complex, the Mycobacterium avium complex, and Mycobacterium kansasii has been developed (R. Schöningh, C. P. H. J. Verstijnen, S. Kuijper, and A. H. J. Kolk. J. Clin. Microbiol. 28:708-713, 1990). The test for the routine identification of cultured mycobacteria was introduced in five clinical laboratories located in Tanzania, Thailand, Vietnam, and The Netherlands. The ELISA can be conducted without an ELISA reader since the test can be read visually. The results of identification of 255 strains of the M. tuberculosis complex by microbiological means and by ELISA were compared; the specificity and the sensitivity were 100%. For the M. avium complex, the specificity was 100% and the sensitivity was 64%. All 26 M. kansasii strains tested could be identified as M. kansasii. The ELISA described here proved to be useful in both well- and modestly equipped laboratories and may replace the microbiological method of identification of M. tuberculosis and M. kansasii.

Antibodies, Monoclonal↗

PubMed1976

Evaluation of simplified dichotomous schemata for the identification of anaerobic bacteria from clinical material.

Simplified dichotomous schemata are described for the identification of anaerobic bacteria commonly encountered in clinical material. The procedures used are combinations of routine biochemical tests and techniques that are used to uniformly characterize these organisms. Over 200 anaerobic organisms were used in a three-stage evaluation in which data were compared with those obtained by conventional methods. When there was inconsistency between the biochemical tests described in the presumptive identification schemes and gas-liquid chromatography, additional biochemical tests or reference procedures were used to confirm identification. Strains from the American Type Culture Collection and the Center for Disease Control, as well as recent clinical isolates, were included in this evaluation. The results show the simplified procedures to be useful for the identification of anaerobic isolates from clinical material.

Bacteria↗

PubMed1993

Collaborative evaluation of the Radiometer Sensititre AP80 for identification of gram-negative bacilli.

A multicenter trial of the Sensititre AP80 panel read on the Sensititre AutoReader (Radiometer America, Westlake, Ohio) for the automated identification of gram-negative bacilli was conducted with 1,023 clinical isolates (879 members of the family Enterobacteriaceae plus 144 nonenteric organisms). Assignment of taxa was based on the computer-assisted interpretation of the results of a series of reactions with fluorogenic enzyme substrates after 5 h of incubation, with an incubation interval of approximately 18 h used when indicated. Accuracy was determined initially by comparison with the results obtained with the API 20E or Rapid NFT system (Analytab Products, Plainview, N.Y.). Isolates showing discrepancies were identified by using conventional biochemical profiles. Identifications were available after 5 h of incubation for 918 isolates (90%). Agreements with reference results for members of the family Enterobacteriaceae were 95.3 and 92.5% at the genus and species levels, respectively, and for the nonmembers of the family Enterobacteriaceae, the agreements with reference results were 95.1 and 84.7%, respectively. The Sensititre AP80 panel was found to be simple and convenient to use, allowed for the testing of three isolates per panel, required minimal supplementary testing for completion of identification, performed in a reproducible fashion, and demonstrated an accuracy of same-day identification comparable to that reported for other automated systems. The AP80 panel appears well suited for routine use in the clinical microbiology laboratory as an automated means of identifying both members of the family Enterobacteriaceae and nonenteric gram-negative bacilli.

Bacterial Typing Techniques↗

PubMed1994

Enzyme capture assay for rapid identification of Escherichia coli in blood cultures.

An enzyme capture assay (ECA) for rapid identification of Escherichia coli in blood cultures by using beta-D-glucuronidase as a marker was developed. Microdilution plates coated with antiglucuronidase were used to capture this enzyme from the cell lysates of blood cultures which showed growth of gram-negative bacteria. The assay, using 4-methylumbelliferyl-beta-D-glucuronide as a fluorogenic substrate, had a detection limit of 0.1 ng/ml (3 x 10(-13) M) for the enzyme; this was approximately equal to a cell concentration of 10(6) CFU of E. coli per ml. Among 212 blood cultures showing growth of gram-negative bacteria, 77 specimens were found to contain E. coli by conventional culture procedures and 73 samples were positive by ECA. Among the 135 blood cultures from which E. coli was not isolated, ECA gave one false-positive (Salmonella enteritidis) reaction. Thus, the sensitivity and specificity for the identification of E. coli in blood cultures by ECA were 94.8% (73/77) and 99.3% (134/135), respectively. From the finding of positive growth in the culture bottle, the assay can be completed within 4 h. In view of the high rate of isolation of E. coli from bacteremic patients, the test can be performed in parallel with conventional culture protocols; this may shorten the identification time for E. coli, and proper antimicrobial treatments may be started 24 h earlier than when results of conventional identification systems are used.

Antibodies, Bacterial↗

PubMed1995

Four-year prospective study of STAPH-IDENT system and conventional method for reference identification of Staphylococcus, Stomatococcus, and Micrococcus spp..

A 4-year prospective study compared the accuracy of the STAPH-IDENT system (bioMérieux Vitek, Inc., Hazelwood, Mo.) with that of the reference procedure of the Centers for Disease Control and Prevention for the identification of Staphylococcus species, Stomatococcus mucilaginosus, and Micrococcus species. The study compared the results from 1,106 cultures (500 eye cultures, 217 strains submitted for reference identification, and 389 known stock strains) representing 21 species of the family Micrococcaceae. The overall agreement of genus and species identifications was 81.1%. The percent agreement for the five most common clinical isolates was as follows: Staphylococcus epidermidis, 97.1% (517 isolates); Staphylococcus hominis, 82.5% (57 isolates); Staphylococcus aureus, 77.2% (162 isolates); Staphylococcus haemolyticus, 75.8% (61 isolates); and Staphylococcus warneri, 64.1% (39 isolates). The lowest percent agreement was with Staphylococcus cohnii (11.1%; (9 isolates). Of the 217 isolates sent to the Centers for Disease Control and Prevention for identification, 60.4% (131) were correctly identified by the STAPH-IDENT system. Of these, S. epidermidis accounted for 23.9%, S. aureus accounted for 15.6%, S. warneri accounted for 6.9%, Staphylococcus lugdunensis accounted for 6.5%, S. haemolyticus accounted for 5.5%, and S. hominis accounted for 4.1%. The STAPH-IDENT system did not perform adequately when dealing with commonly encountered organisms and is unsuitable for identifying uncommon isolates.

Bacterial Typing Techniques↗

PubMed1995

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.

Bacteria↗

PubMed1995

Identification of Acinetobacter isolates in the A. calcoaceticus-A. baumannii complex by restriction analysis of the 16S-23S rRNA intergenic-spacer sequences.

Members of the genus Acinetobacter are reported to be involved in hospital-acquired infections with an increasing frequency. However, clinical laboratories still lack simple methods that allow complete identification of some pathogenic species, i.e., those corresponding to A. baumannii (DNA group or genospecies 2), unnamed genospecies 3 and 13, and two new genospecies that have recently been described. In fact, a complete discrimination between these species is possible only by DNA-DNA hybridization or ribotyping. Both of these techniques are complex and time-consuming and cannot be performed in most clinical laboratories. As a consequence, isolates belonging to these genospecies are often not differentiated and included, together with the environmental genospecies 1, in the A. calcoaceticus-A. baumannii complex. In this report, a simple and rapid method for the identification of the genospecies belonging to the A. calcoaceticus-A. baumannii complex is proposed. It is based on the combined digestion by the restriction endonuclease AluI and NdeII of the DNA fragments resulting from the amplification of the 16S-23S rRNA intergenic spacer sequences. The analysis of 36 strains characterized by DNA-DNA hybridization in previous studies showed that the restriction profiles obtained are highly reproducible and characteristic for each genospecies. Moreover, extending this study to 68 clinical strains, which were assigned to the A. calcoaceticus-A. baumannii complex by phenotypic tests, confirmed the existence of a panel of limited and well-conserved restriction patterns and allowed the identification of the strains tested. This study thus proposes the detection of restriction length polymorphism in the spacer sequences between the 16S and 23S rRNA genes as a method for the identification of isolates in the A. calcoaceticus-A. baumannii complex.

Acinetobacter↗

PubMed1995

Comparison of phenotypic characteristics, DNA-DNA hybridization results, and results with a commercial rapid biochemical and enzymatic reaction system for identification of viridans group streptococci.

The rapid ID 32 Strep system (bioMérieux, Marcy l'Etoile, France) was evaluated for its ability to identify 21 species of viridans group streptococci; results were compared with DNA-DNA hybridization results and results of conventional physiological tests. A total of 171 strains of the 21 species including 147 clinical strains was analyzed. Of the 156 strains of species included in the database of this system, 136 strains (87%) were correctly identified. Incorrect identification occurred for 13 strains (8%), and no identification was given for 7 strains (5%). It was difficult to differentiate S. mitis and S. oralis accurately with this system. Of the 17 strains identified as S. mitis by the rapid ID 32 Strep system, the results of DNA-DNA hybridization were in agreement for only 3 strains. S. crista and S. parasanguis, which are not included in the database, were identified as S. mitis or S. sanguis or were not identified, but S. parasanguis could probably be identified by using the rapid ID 32 Strep system because the biochemical profile is well characterized for this species. The rapid ID 32 Strep system can be used to differentiate most species for which phenotypic characteristics have been described if the database is revised according to recently reported amended criteria for the identification of viridans group streptococci. However, identification of a few species such as S. mitis and S. oralis is problematic with this system.

Bacterial Typing Techniques↗

PubMed1995

Inter- and intraspecies identification of Bartonella (Rochalimaea) species.

Species of the genus Rochalimaea, recently renamed Bartonella, are of a growing medical interest. Bartonella quintana was reported as the cause of trench fever, endocarditis, and bacillary angiomatosis. B. henselae has been implicated in symptoms and infections of human immunodeficiency virus-infected patients, such as fever, endocarditis, and bacillary angiomatosis, and is involved in the etiology of cat scratch disease. Such a wide spectrum of infections makes it necessary to obtain an intraspecies identification tool in order to perform epidemiological studies. B. vinsonii, B. elizabethae, seven isolates of B. quintana, and four isolates of B. henselae were studied by pulsed-field gel electrophoresis (PFGE) after restriction with the infrequently cutting endonucleases NotI, EagI, and SmaI. Specific profiles were obtained for each of the four Bartonella species. Comparison of genomic fingerprints of isolates of the same species showed polymorphism in DNA restriction patterns, and a specific profile was obtained for each isolate. A phylogenetic analysis of the B. quintana isolates was obtained by using the Dice coefficient, UPGMA (unweighted pair-group method of arithmetic averages), and Package Philip programming. Amplification by PCR and subsequent sequencing using an automated laser fluorescent DNA sequencer (Pharmacia) was performed on the intergenic spacer region (ITS) between the 16 and 23S rRNA genes. It was found that each B. henselae isolate had a specific sequence, while the B. quintana isolates fell into only two groups. When endonuclease restriction analysis of the ITS PCR product was done, three enzymes, TaqI, HindIII, and HaeIII, allowed species identification of Bartonella spp. Restriction fragment length polymorphism after PCR amplification of the 16S-23S rRNA gene ITS may be useful for rapid species identification, and PFGE could be an efficient method for isolate identification.

Animals↗

PubMed1995

Species identification of members of the Streptococcus milleri group isolated from the vagina by ID 32 Strep system and differential phenotypic characteristics.

The importance of bacterial vaginosis as a risk factor in obstetric and gynecological infections has recently been recognized. The bacterial vaginosis group of organisms includes members of the Streptococcus milleri group, the identification of which has caused much confusion. We prospectively surveyed the rates of carriage of S. milleri group organisms in 397 high vaginal swabs received in our laboratory. For the identification of 99 clinical isolates and 23 control strains, we compared the results obtained by the rapid ID 32 Strep system (Analytab Products) and by a scheme utilizing six differential phenotypic characteristics (presence of beta-N-acetylglucosaminidase, alpha-glucosidase, beta-D-fucosidase, beta-galactosidase, beta-N-acetylgalactosaminidase, and beta-glucosidase) as described by Whiley et al. (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). We identified Streptococcus anginosus in 18% and Streptococcus constellatus in 0.05% of the specimens examined. Of the isolates of S. anginosus that reacted with grouping antisera, 20 of 25 belonged to Lancefield group F. The incubation conditions for bacterial cultures and for reaction mixtures affected the results of phenotypic characterization in the production of alpha-glucosidase, beta-galactosidase, and beta-glucosidase. However, by using bacterial cultures grown under hypercapnic conditions and incubating the reaction mixtures aerobically, consistent phenotypic characteristics were obtained, allowing identification similar to that obtained by the ID 32 Strep system. We therefore recommend the phenotypic scheme as an inexpensive, reliable, and convenient method for the initial identification of species of the S. milleri group.

Antibodies, Bacterial↗

PubMed1996

Accuracy of four commercial systems for identification of Burkholderia cepacia and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis.

Burkholderia cepacia has recently been recognized as an important pathogen in chronic lung disease in patients with cystic fibrosis (CF). Because of the social, psychological, and medical implications of the isolation of B. cepacia from CF patients, accurate identification of this organism is essential. We compared the accuracies of four commercial systems developed for the identification of nonfermenting, gram-negative bacilli with that of conventional biochemical testing for 150 nonfermenters including 58 isolates of B. cepacia recovered from respiratory secretions from CF patients. The accuracies of the four systems for identifying all nonfermenters ranged from 57 to 80%, with the RapID NF Plus system being most accurate. The accuracies of these systems for identifying B. cepacia ranged from 43 to 86%, with the Remel system being most accurate. Depending on the commercial system, from two to seven isolates were misidentified as B. cepacia. The relatively poor performance of the commercial systems requires that identification of certain nonfermenters be confirmed by conventional biochemical testing. These organisms include B. cepacia, Burkholderia sp. other than B. cepacia, and infrequently encountered environmental species (Pseudomonas and Flavobacterium species). In addition, conventional biochemical testing should be done if a commercial system fails to assign an identification to an organism. Confirmatory testing should preferably be performed by a reference laboratory with experience in working organisms isolated from CF patients.

Bacteriological Techniques↗

PubMed1997

Rapid identification of Candida species in blood cultures by a clinically useful PCR method.

Widespread use of fluconazole for the prophylaxis and treatment of candidiasis has led to a reduction in the number of cases of candidemia caused by Candida albicans but has also resulted in the emergence of candidemias caused by innately fluconazole-resistant, non-C. albicans Candida species. Given the fulminant and rapidly fatal outcome of acute disseminated candidiasis, rapid identification of newly emerging Candida species in blood culture is critical for the implementation of appropriately targeted antifungal drug therapy. Therefore, we used a PCR-based assay to rapidly identify Candida species from positive blood culture bottles. This assay used fungus-specific, universal primers for DNA amplification and species-specific probes to identify C. albicans, C. krusei, C. parapsilosis, C. tropicalis, or C. glabrata amplicons. It also used a simpler and more rapid (1.5-h) sample preparation technique than those described previously and used detergent, heat, and mechanical breakage to recover Candida species DNA from blood cultures. A simple and rapid (3.5-h) enzyme immunosorbent assay (EIA)-based format was then used for amplicon detection. One hundred fifty blood culture bottles, including 73 positive blood culture bottle sets (aerobic and anaerobic) from 31 patients with candidemia, were tested. The combined PCR and EIA methods (PCR-EIA) correctly identified all Candida species in 73 blood culture bottle sets, including bottles containing bacteria coisolated with yeasts and 3 cultures of samples from patients with mixed candidemias originally identified as single-species infections by routine phenotypic identification methods. Species identification time was reduced from a mean of 3.5 days by routine phenotypic methods to 7 h by the PCR-EIA method. No false-positive results were obtained for patients with bacteremias (n = 18), artificially produced non-Candida fungemias (n = 3), or bottles with no growth (n = 20). Analytical sensitivity was 1 cell per 2-microl sample. This method is simpler and more rapid than previously described molecular identification methods, can identify all five of the most medically important Candida species, and has the potential to be automated for use in the clinical microbiology laboratory.

Candida↗

PubMed1998

Clinical application of PCR-restriction enzyme pattern analysis for rapid identification of aerobic actinomycete isolates.

The accuracy and practicality of PCR-restriction enzyme pattern analysis (PRA) for routine identification of aerobic actinomycete clinical isolates were evaluated for 299 cultures submitted to the Mycobacteria/Nocardia Laboratory at the University of Texas Health Center at Tyler. PRA identification using an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was compared to identification by traditional methods, including growth characteristics, susceptibility patterns, biochemical testing, and high-performance liquid chromatography analysis. Microbiological examination of six cultures ruled out aerobic actinomycetes, and they were omitted from the study. Amplicons were analyzed with BstEII, HaeIII, MspI, HinfI, and BsaHI. When necessary, AciI, HhaI, and NarI were also used. From March 1995 through May 1997 (27 months), 274 of the remaining 293 (93.5%) isolates were accurately identified by PRA. Major diagnostic groups included 170 mycobacteria, 93 nocardiae, and 30 other aerobic actinomycetes. Mixed cultures were readily recognized by PRA, including a wound culture that contained two Nocardia taxa that were indistinguishable morphologically. Mycobacterium mucogenicum was identified in three cultures heavily contaminated with gram-positive cocci. The 19 isolates that produced PRA patterns that did not match those in the current PRA database were differentiated into 8 Mycobacterium species and 11 other aerobic actinomycetes by the presence or absence of BstEII recognition sites. Identification of 15 of these 19 isolates was also equivocal by traditional methods. PRA results were reportable within 2 to 5 working days and were as accurate as and faster and less expensive to obtain than those of traditional methods.

Actinomycetales↗