Multicenter Study Demonstrates Standardization Requirements for Mold Identification by MALDI-TOF MS.

OBJECTIVES: Rapid and accurate mold identification is critical for guiding therapy for mold infections. MALDI-TOF MS has been widely adopted for bacterial and yeast identification; however, few clinical laboratories have applied this technology for routine mold identification due to limited database availability and lack of standardized processes. Here, we evaluated the versatility of the NIH Mold Database in a multicenter evaluation. METHODS: The NIH Mold Database was evaluated by eight US academic centers using a solid media extraction method and a challenge set of 80 clinical mold isolates. Multiple instrument parameters important for spectra optimization were evaluated, leading to the development of two specialized acquisition programs (NIH method and the Alternate-B method). RESULTS: A wide range in performance (33-77%) was initially observed across the eight centers when routine spectral acquisition parameters were applied. Use of the NIH or the Alternate-B specialized acquisition programs, which are different than those used routinely for bacterial and yeast spectral acquisition (MBT_AutoX), in combination with optimized instrument maintenance, improved performance, illustrating that acquisition parameters may be one of the key limiting variable in achieving successful performance. CONCLUSION: Successful mold identification using the NIH Database for MALDI-TOF MS on Biotyper systems was demonstrated across multiple institutions for the first time following identification of critical program parameters combined with instrument optimization. This significantly advances our potential to implement MALDI-TOF MS for mold identification across many institutions. Because instrument variability is inevitable, development of an instrument performance standard specific for mold spectral acquisition is suggested to improve reproducibility across instruments.

A Novel Rapidly Growing Mycobacterium Species Causing an Abdominal Cerebrospinal Fluid Pseudocyst Infection.

Nontuberculous mycobacteria (NTM) are a rare cause of ventriculoperitoneal shunt infections. We describe the isolation and identification of a novel, rapidly growing, nonpigmented NTM from an abdominal cerebrospinal fluid pseudocyst. The patient presented with fevers, nausea, and abdominal pain and clinically improved after shunt removal. NTM identification was performed by amplicon and whole-genome sequencing.

Multicenter Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Study for Identification of Clinically Relevant Nocardia spp.

This multicenter study analyzed Nocardia spp., including extraction, spectral acquisition, Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, and score interpretation, using three Nocardia libraries, the Bruker, National Institutes of Health (NIH), and The Ohio State University (OSU) libraries, and compared the results obtained by each center. A standardized study protocol, 150 Nocardia isolates, and NIH and OSU Nocardia MALDI-TOF MS libraries were distributed to three centers. Following standardized culture, extraction, and MALDI-TOF MS analysis, isolates were identified using score cutoffs of ≥2.0 for species/species complex-level identification and ≥1.8 for genus-level identification. Isolates yielding a score of <2.0 underwent a single repeat extraction and analysis. The overall score range for all centers was 1.3 to 2.7 (average, 2.2 ± 0.3), with common species generally producing higher average scores than less common ones. Score categorization and isolate identification demonstrated 86% agreement between centers; 118 of 150 isolates were correctly identified to the species/species complex level by all centers. Nine strains (6.0%) were not identified by any center, and six (4.0%) of these were uncommon species with limited library representation. A categorical score discrepancy among centers occurred for 21 isolates (14.0%). There was an overall benefit of 21.2% from repeat extraction of low-scoring isolates and a center-dependent benefit for duplicate spotting (range, 2 to 8.7%). Finally, supplementation of the Bruker Nocardia MALDI-TOF MS library with both the OSU and NIH libraries increased the genus-level and species-level identification by 18.2% and 36.9%, respectively. Overall, this study demonstrates the ability of diverse clinical microbiology laboratories to utilize MALDI-TOF MS for the rapid identification of clinically relevant Nocardia spp. and to implement MALDI-TOF MS libraries developed by single laboratories across institutions.

Identification and significance of Weissella species infections.

Weissella spp. are non-spore forming, catalase-negative, gram-positive coccobacilli. They are often misidentified by traditional and commercial phenotypic identification methods as Lactobacillus spp. or Lactobacillus-like organisms. Weissella spp. were previously grouped along with Lactobacillus spp., Leuconostoc spp., and Pediococcus spp. Utilization of more sensitive methods like DNA sequencing or Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) has facilitated identification of Weissella as a unique genus. Nineteen species have been identified to date. W. confusa, W. cibaria, and W. viridescens are the only species isolated from humans. The true prevalence of Weissella spp. continues to be probably underestimated. Weissella spp. strains have been isolated from a wide range of habitats including raw milk, feces, fermented cereals, and vegetables. Weisella is believed to be a rare cause of usually nonfatal infections in humans, and is often considered a contaminant. However, in recent years, Weissella spp. have been implicated in bacteremia, abscesses, prosthetic joint infections, and infective endocarditis. Alterations of the gut flora from surgery or chemotherapy are believed to facilitate translocation of Weissella spp. due to disruption of the mucosal barrier, predisposing the host to infection with this organism. Implications of the isolation of Weissella spp. from blood must be interpreted in context of underlying risk factors. Weissella spp. are inherently resistant to vancomycin. Therefore, early consideraton of the pathogenic role of this bacteria and choice of alternate therapy is important to assure better outcomes.

Severe Sepsis Secondary to Persistent Lysinibacillus sphaericus, Lysinibacillus fusiformis and Paenibacillus amylolyticus Bacteremia.

Lysinibacillus and Paenibacillus are pervasive bacteria rarely associated with human disease. Less sophisticated microbiology techniques may frequently incorrectly identify these genera as Bacillus spp., often regarded as environmental contamination. This report describes a case of severe sepsis due to persistent Lysinibacillus and Paenibacillus bacteremia, identified by matrix-assisted laser desorption and ionization time-of-flight mass spectroscopy and 16S rRNA gene sequencing.

Identification of mycobacteria from solid and liquid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry in the clinical laboratory.

Mycobacteria cause significant morbidity in humans. Rapid and accurate mycobacterial identification is important for improvement of patient outcomes. However, identification may be challenging due to the slow and fastidious growth of mycobacteria. Several diagnostic methods, such as biochemical, sequencing, and probe methods, are used for mycobacterial identification. We compared the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonics) to 16S rRNA/hsp65 sequencing and/or DNA probes (Gen-Probe) for mycobacterial identification. One hundred seventy-eight mycobacterial isolates grown on solid and/or broth medium were included in the study. MALDI-TOF MS identified 93.8% of the mycobacteria isolates accurately to the species level and 98.3% to the genus level, independent of the type of medium used for isolation. The identification of mycobacteria directly from cultures using MALDI-TOF MS allows for precise identification in an hour compared to traditional biochemical and phenotypic methods that can take weeks or probes and sequencing that may take a few hours. Identification by MALDI-TOF MS potentially reduces the turnaround time and cost, thereby saving resources within the health care system.

Detection of yeasts in blood cultures by the Luminex xTAG fungal assay.

A multiplex-PCR Luminex xMAP bead probe fluid array using xTAG analyte-specific reagents (multiplex xTAG fungal ASR assay) was employed for detection of clinically significant Candida species, Cryptococcus neoformans, Histoplasma capsulatum, and Blastomyces dermatitidis from blood cultures. We tested 132 blood cultures negative (n = 10) or positive (n = 97) for yeasts and/or bacteria (n = 25). The assay showed sensitivity and specificity of 100% and 99%, respectively. The xTAG fungal ASR assay is a rapid assay that allows simultaneous identification of multiple yeast species.