Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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.

New rapid scheme for distinguishing the subspecies of the Mycobacterium abscessus group and identifying Mycobacterium massiliense isolates with inducible clarithromycin resistance.

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.

Legionella feeleii serotype 2 pneumonia in a man with chronic lymphocytic leukemia: a challenging diagnosis.

Legionella feeleii has rarely been reported as causing pneumonia in patients with hematologic malignancies. We present a case of Legionella feeleii serotype 2 pneumonia with empyema in a man with chronic lymphocytic leukemia and describe the methods of identifying this organism using both standard methods and newer diagnostic techniques.

Identification of Mycobacterium species by secA1 sequences.

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.

Clinical trial of quantitative real-time polymerase chain reaction for detection of cytomegalovirus in peripheral blood of allogeneic hematopoietic stem-cell transplant recipients.

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.