b Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the rapid and highly accurate identification of clinical pathogens but has not been utilized extensively in clinical mycology due to challenges in developing an effective protein extraction method and the limited databases available. Here, we developed an alternate extraction procedure and constructed a highly stringent database comprising 294 individual isolates representing 76 genera and 152 species. To our knowledge, this is the most comprehensive clinically relevant mold database developed to date. When challenged with 421 blinded clinical isolates from our institution, by use of the BioTyper software, accurate species-level (score of >2.0) and genus-level (score of >1.7) identifications were obtained for 370 (88.9%) and 18 (4.3%) isolates, respectively. No isolates were misidentified. Of the 33 isolates (7.8%) for which there was no identification (score of <1.7), 25 were basidiomycetes not associated with clinical disease and 8 were Penicillium species that were not represented in the database. Our library clearly outperformed the manufacturer's database that was obtained with the instrument, which identified only 3 (0.7%) and 26 (6.2%) isolates at species and genus levels, respectively. Identification was not affected by different culture conditions. Implementation into our routine workflow has revolutionized our mycology laboratory efficiency, with improved accuracy and decreased time for mold identification, eliminating reliance on traditional phenotypic features.

e Mycobacterium abscessus (M. abscessus sensu lato, or the M. abscessus group) comprises three closely related taxa whose taxonomic statuses are under revision, i.e., M. abscessus sensu stricto, Mycobacterium bolletii, and Mycobacterium massiliense. We describe here a simple, robust, and cost-effective PCR-based method for distinguishing among M. abscessus, M. massiliense, and M. bolletii. Based on the M. abscessus ATCC 19977 T genome, regions that discriminated between M. abscessus and M. massiliense were identified through array-based comparative genomic hybridization. A typing scheme using PCR primers designed for four of these locations was applied to 46 well-characterized clinical isolates comprising 29 M. abscessus, 15 M. massiliense, and 2 M. bolletii isolates previously identified by multitarget sequencing. Interestingly, 2 isolates unequivocally identified as M. massiliense were shown to have a full-length erm(41) gene instead of the expected gene deletion and showed inducible clarithromycin resistance after 14 days. We propose using this PCR-based typing scheme combined with erm(41) PCR for straightforward identification of M. abscessus, M. massiliense, and M. bolletii and the assessment of inducible clarithromycin resistance. This method can be easily integrated into a routine workflow to provide subspecies-level identification within 24 h after isolation of the M. abscessus group. Rapidly growing mycobacteria (RGM) are ubiquitous environmental microorganisms and a significant cause of human disease (1). The prevalence of lung disease due to RGM is increasing and in many areas of the United States exceeds that of Mycobacterium tuberculosis (2). Within the RGM, the Mycobacterium abscessus group is a prominent cause of lung infections in patients with cystic fibrosis and chronic pulmonary disease (bronchiectasis, nodules, and cavitations) and of posttraumatic and postsurgical infections (1, 3). Infections with organisms in the M. abscessus group are difficult to treat, due to both natural broad-spectrum resistance and acquired resistance, with various antibiotic susceptibility patterns being observed among isolates (4). M. abscessus sensu lato, or the M. abscessus group, was recently divided into three closely related taxa, i.e., M. abscessus sensu stricto (hereinafter referred to as M. abscessus), Mycobacterium massiliense, and Mycobacterium bolletii. M. massiliense has been recognized increasingly as an emerging pathogen causing postsurgical wound infection outbreaks (5), and recently it was identified as a cause of respiratory outbreaks in two cystic fibrosis centers, with evidence of transmission between patients (6, 7).The taxonomic status of the M. abscessus group remains controversial. M. massiliense and M. bolletii were initially proposed as new species mainly on the basis of divergence of their rpoB sequences. However, further studies showed that these organisms could not be separated by biochemical tests and mycolic acid pattern analysis (8) and showed less genomic divergence than would be exp...

We describe a novel molecular method for the differentiation and identification of 29 mycobacterial species. The target is the secA1 gene that codes for the essential protein SecA1, a key component of the major pathway of protein secretion across the cytoplasmic membrane. A 700-bp region of the secA1 gene was amplified and sequenced from 47 American Type Culture Collection strains of 29 Mycobacterium species as well as from 59 clinical isolates. Sequence variability in the amplified segment of the secA1 gene allowed the differentiation of all species except for the members of the Mycobacterium tuberculosis (MTB) complex, which had identical sequences. A range of 83.3 to 100% interspecies similarity was observed. All species could also be differentiated by their amino acid sequences as deduced from the sequenced region of the secA1 gene, with the exception of the MTB complex. Partial sequences of secA1 from clinical isolates belonging to nine frequently isolated species of mycobacteria revealed a very high intraspecies similarity at the DNA level (typically >99%; range, 96.0 to 100%); all clinical isolates were correctly identified. Comparison of the deduced 233-amino-acid sequences among clinical isolates of the same species showed between 99.6 and 100% similarity. To our knowledge, this is the first time a secretion-related gene has been used for the identification of the species within a bacterial genus.The global increase of infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) has received attention worldwide. Traditionally, the definitive diagnosis of mycobacterial infections depends on the isolation and phenotypic identification of the causative agent, a procedure that is time-consuming and labor intensive.The development of DNA probes for identification of mycobacteria greatly improved the identification speed and accuracy in the mycobacteriology laboratory. The Accuprobe system (Gen-Probe, San Diego, Calif.) is a rapid and sensitive method, but it offers only a limited number of species-or complex-specific probes (39). Recently, a new DNA probe kit, INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), targeting the 16S-23S rRNA spacer region, was developed for the identification of Mycobacterium spp. This assay can presently identify 16 mycobacterial species and was reported to be sensitive (100%) and specific (94.4 to 100%) (37). However, its complex procedure makes it difficult to implement in a clinical laboratory, and it is not available in the United States. The transcription-mediated amplification test (Amplified Mycobacterium Tuberculosis Direct [MTD] test) from Gen-Probe is the first approved commercial method for amplification of mycobacterial nucleic acids directly from respiratory specimens. This method is only useful for detecting members of the M. tuberculosis complex. Compared to culture, the sensitivity of the MTD test is variable, ranging from 65 to 97% (29).PCR has been used to analyze various mycobacterial genes for diagnostic purposes, including the 16...

Two hundred twenty-four isolates of Bacteroides sp. were recovered from recurrently inflamed tonsils, infected peritoneal fluid, abscesses, wounds, and burns of hospitalized children. Isolates were examined for beta-lactamase production by the chromogenic cephalosporin analog 87/312 methodology. Altogether, 119 isolates were beta-lactamase producers. Of these, 53 were in the B. fragilis group, 28 were in the B. melaninogenicus group, 12 were B. oralis, 4 were B. ruminicola subsp. brevis, and 22 were Bacteroides sp. Of 28 beta-lactamaseproducing strains of B. melaninogenicus, 25 were recovered from tonsils. These observations indicate that in pediatric patients there is a significant incidence of beta-lactamase producers among anaerobes other than B. fragilis.Although the pediatric literature suggests that anaerobic bacteria, including various Bacteroides spp., can cause morbidity and mortality in children, these organisms have received only limited attention. This is surprising, for species of Bacteroides are frequently isolated from clinical specimens (3), and significant, although variable fractions of these isolates are resistant to beta-lactam antibiotics via production of betalactamase.The purposes of this study were to determine the incidence of beta-lactamase-producing Bacteroides sp. isolates in children and whether there is correlation between the sources of the isolates and ability to produce this enzyme. MATERIALS AND METHODSThe Bacteroides spp. examined in this study were obtained during 1979 from clinical specimens processed in the Clinical Microbiological Laboratories at Children's Hospital National Medical Center. The methods of isolation and identification were those detailed in the Virginia Polytechnic Institute (5) and Wadsworth (13) manuals. The chromogenic cephalosporin analog 87/312 methodology (7) was used to determine beta-lactamase activity. RESULTSOf the 224 isolates, 95 were derived from core and surface specimens of surgically excised tonsils (removed because of recurrent tonsillitis), 75 were from infected peritoneal fluid, 24 were from abscesses, 20 were from wounds, 5 were from burns, and 5 were from miscellaneous sources (Table 1). Of the total, 119 (53%) produced betalactamase. These included 53 of 65 in the B. fragilis group, 28 of 73 in the B. melaninogenicus group, 12 of 17 B. oralis, 4 of 13 B. ruminicola subsp. brevis, and 22 of 61 other Bacteroides spp.

The preemptive therapy of cytomegalovirus (CMV) reactivation is useful for the prevention of CMV disease in allogeneic hematopoietic stem-cell transplant (HSCT) recipients. We compared results of the pp65 CMV antigenemia test with quantitative touch-down polymerase chain reaction (Q-PCR) on unfractionated whole blood for the detection of CMV reactivation in 51 HSCT recipients. Forty episodes of reactivation in 28 patients were detected by antigenemia and treated by antiviral drugs. Q-PCR detected CMV DNA in 39 (97.5%) of 40 reactivation episodes. False-positive results occurred in 3% of tests, of which 63% were borderline positive. Q-PCR results were positive earlier than antigenemia results in 30 (77%) of 39 episodes detected by antigenemia. Q-PCR remained positive after treatment was discontinued in 14 (36%) of 39 episodes and predicted the return of CMV reactivation in 4 (31%) of 13 episodes. Q-PCR was more sensitive than the antigenemia test and had sufficient specificity for clinical use.