Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i) conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii) molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii) when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.
b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species-and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications. Recent decades have seen advances in automation of traditional phenotypic and biochemical methods for microbial identification (ID), and advances in sequencing and the proliferation of genomic data hold great promise for further improvements. The development of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has brought microbial diagnostics to another cusp of rapid development. The speed and low cost of bacterial identification by MALDI-TOF MS make it an attractive technology in the clinical microbiology laboratory, and it has shown promise for identification of Gram-positive cocci (2, 6, 8), enteric and nonfermenting Gram-negative rods (11,21,24), HACEK organisms (10), anaerobes (14,17,19,20,31), and broad cohorts of clinically relevant bacteria (3,4,22,27,30).Commercial MALDI-TOF systems identify a broad range of microorganisms based on analysis of unique "fingerprints" of abundant proteins from whole cells or cellular extracts (15,23,26,28). These profiles are searched against databases of reference spectra, and similarity scores for the top database matches are used to determine the identification of unknown isolates. As observed previously, a systematic evaluation of scoring criteria on diverse isolates could improve results (2,10,25,27,29). Identification may be complicated when multiple species-or genus-level matches are among the top 10 results. Most current publications on the MALDI Biotyper system (Bruker Daltonics, Billerica, MA) do not address these complicated situations; however, one example where this problem is addressed is the use of the "10% rule," which stat...
Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.Clinical microbiology laboratories strive to identify infectious organisms from patient samples in the most-accurate yet time-and cost-efficient manner possible. This process may be impeded by rare or difficult-to-identify organisms such as those of the HACEK group (Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae). HACEK organisms are fastidious Gram-negative rods found in the healthy oropharynx or upper respiratory tract and can cause endocarditis, especially in young children and patients with heart defects (3). HACEK organisms are also involved in a wide array of serious infections involving the head and neck, bone, joints, lungs, and other soft tissues (17).Numerous reports have recently been published showing the successful integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in the identification of microorganisms routinely encountered in clinical microbiology labs (1,2,12,15). These reports have focused largely on easy-to-culture bacteria and yeasts, including Staphylococcus aureus and nonfermenting 14). To date, three genera of fastidious Gram-negative rods (Legionella, Bartonella, and Francisella) have been extensively evaluated by the newer MALDI-TOF MS identification systems (4, 7, 10, 11). Several studies have also investigated subsets of organisms, such as difficult-to-identify Nocardia spp., anaerobes, and oral flora, as well as fastidious upper respiratory flora (e.g., Neisseria spp., Moraxella spp., Haemophilus spp.) (5,9,12,13,15,16). Haemophilus spp., including H. influenzae, were previously shown to be distinguishable by MALDI-TOF MS (5), and since H. influenzae and HACEK organisms have similar growth requirements, this suggested that MALDI-TOF MS may be useful in their identification. HACEK organisms are not readily identified on most automated bacterial identification systems, and most require either timeconsuming biochemical profiling or genetic analysis, such as 16S rRNA gene sequencing, for definitive identification (8). This study aimed to determine whether MALDI-TOF MS could accurately identify a large panel of HACEK clinical isolates.HACEK and H. influenzae isolates were grown on chocolate agar plates (Hardy Diagnostics, Santa Maria, CA), and 24-to 48-h-old cult...
The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecTCampylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 μg/ml), amphotericin B (10 μg/ml), and teicoplanin (4 μg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolateCampylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.
Phenotypic identification of AmpC, KPC and extended-spectrum b-lactamases (ESBLs) among members of the Enterobacteriaceae remains challenging. This study compared the Phoenix Automated Microbiology System (BD Diagnostics) with the Clinical and Laboratory Standards Institute confirmatory method to identify ESBL production among 200 Escherichia coli and Klebsiella pneumoniae clinical isolates. The Phoenix system misclassified nearly half of the isolates as ESBL-positive, requiring manual testing for confirmation. Inclusion of aztreonam±clavulanic acid (CA) and cefpodoxime±CA in the testing algorithm increased the ESBL detection rate by 6 %. Boronic acid-based screening identified 24 isolates as AmpC + , but in a subset of genotypically characterized isolates, appeared to have a high false-positivity rate. PCR screening revealed eight KPC + isolates, all of which tested as ESBL + or ESBL + AmpC + by phenotypic methods, but half were reported as carbapenem-susceptible by the Phoenix system. Overall, these results indicate that laboratories should use the Phoenix ESBL results only as an initial screen followed by confirmation with an alternative method.
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