Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been described as a fast and inexpensive method for the identification of mycobacteria. Although mycobacteria require extraction prior to MALDI-TOF MS analysis, previously published protocols have been relatively complex, involving significant hands-on time and materials not often found in the clinical laboratory. In this study, we tested two simplified protein extraction protocols developed at the University of Washington (UW) and by bioMérieux (BMX) for use with two different mass spectrometry platforms (the Bruker MALDI Biotyper and the bioMérieux Vitek MS, respectively). Both extraction protocols included vortexing with silica beads in the presence of ethanol. The commercial Bruker database was also augmented with an in-house database composed of 123 clinical Mycobacterium strains. A total of 198 clinical strains, representing 18 Mycobacterium species, were correctly identified to the species level 94.9% of the time when extracted using the UW protocol and compared to the augmented database. The BMX protocol and Vitek MS system resulted in correct species-level identifications for 94.4% of these strains. In contrast, only 79.3% of the strains were identified to the species level by the nonaugmented Bruker database, although the use of a lower identification score threshold (>1.7) increased the identification rate to 93.9%, with two misidentifications that were unlikely to be clinically relevant. The two simplified protein extraction protocols described in this study are easy to use for identifying commonly encountered Mycobacterium species.

Vancomycin is the standard of care for the treatment of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections. Infections with vancomycin-nonsusceptible MRSA, including vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA), are clinically challenging and are associated with poor patient outcomes. The identification of VISA in the clinical laboratory depends on standard susceptibility testing, which takes at least 24 h to complete after isolate subculture, whereas hVISA is not routinely detected in clinical labs. We therefore sought to determine whether VISA and hVISA can be differentiated from vancomycin-susceptible S. aureus (VSSA) using the spectra produced by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Strains of MRSA were characterized for vancomycin susceptibility phenotype by broth microdilution and modified population analysis. We tested 21 VISA, 21 hVISA, and 38 VSSA isolates by MALDI-TOF MS. Susceptibility phenotypes were separated by using a support vector machine (SVM) machine learning algorithm. The resulting model was validated by leave-one-out cross validation. Models were developed and validated by using spectral profiles generated under various subculture conditions, as well as with and without hVISA strains. Using SVM, we correctly identified 100% of the VISA and 97% of the VSSA isolates with an overall classification accuracy of 98%. Addition of hVISA to the model resulted in 76% hVISA identification, 100% VISA identification, and 89% VSSA identification, for an overall classification accuracy of 89%. We conclude that VISA/hVISA and VSSA isolates are separable by MALDI-TOF MS with SVM analysis. Delayed initiation of appropriate antibiotic therapy results in increased mortality rates for patients with sepsis (1-6). Staphylococcus aureus is a pathogen frequently isolated from patients with sepsis, with worse clinical outcomes noted for patients with methicillin-resistant S. aureus (MRSA) (7). Vancomycin remains the standard of care for the treatment of invasive infections with MRSA (8, 9). Infections with vancomycin-nonsusceptible isolates are associated with prolonged bacteremia, longer hospital stays, and greater rates of clinical treatment failure than infections with vancomycin-susceptible S. aureus (VSSA) (10, 11).Vancomycin nonsusceptibility falls into two related phenotypes: vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA). The VISA phenotype is reliably detected by broth microdilution, and these strains are characterized by a vancomycin MIC of 4 or 8 g/ml. Reliance on susceptibility testing to identify the VISA phenotype therefore delays the identification of these strains and thus the initiation of appropriate antimicrobial therapy. Because only a subpopulation of cells of hVISA strains (Յ10 Ϫ5 to 10 Ϫ6 ) have vancomycin MICs in the intermediate range, hVISA isolates frequently test susceptible to vancomycin (i.e., MICs of Յ2 g/ml) by broth microdilution methods, which typically tes...

Summary Characteristic but rare vascular neoplasms in the adult liver composed of small vessels with an infiltrative border were collected from an international group of collaborators over a 5-year period (N = 17). These tumors were termed hepatic small vessel neoplasm (HSVN), and the histologic differential diagnosis was angiosarcoma (AS). The average age of patients was 54 years (range, 24–83 years). HSVN was more common in men. The average size was 2.1 cm (range, 0.2–5.5 cm). Diagnosis was aided by immunohistochemical stains for vascular lineage (CD31, CD34, FLI-1), which were uniformly positive in HSVN. Immunohistochemical stains (p53, c-Myc, GLUT-1, and Ki-67) for possible malignant potential are suggestive of a benign/low-grade tumor. Capture-based next-generation sequencing (using an assay that targets the coding regions of more than 500 cancer genes) identified an activating hotspot GNAQ mutation in 2 of 3 (67%) tested samples, and one of these cases also had a hotspot mutation in PIK3CA. When compared with hepatic AS (n = 10) and cavernous hemangioma (n = 6), the Ki-67 proliferative index is the most helpful tool in excluding AS, which demonstrated a tumor cell proliferative index greater than 10% in all cases. Strong p53 and diffuse c-Myc staining was also significantly associated with AS but not with HSVN or cavernous hemangioma. There have been no cases with rupture/hemorrhage, disseminated intravascular coagulation, or Kasabach-Merritt syndrome. Thus far, there has been no metastasis or recurrence of HSVN, but complete resection and close clinical follow-up are recommended because the outcome remains unknown.

PURPOSESeveral in silico tools have been shown to have reasonable research sensitivity and specificity for classifying sequence variants in coding regions. The recently-developed Combined Annotation Dependent Depletion (CADD) method generates predictive scores for single nucleotide variants (SNVs) in all areas of the genome, including non-coding regions. We sought to determine the clinical validity of non-coding variant CADD scores.METHODSWe evaluated 12,391 unique SNVs in 624 patient samples submitted for germline mutation testing in a cancer-related gene panel. We compared the distributions of CADD scores of rare SNVs, common SNVs in our patient population, and the null distribution of all possible SNVs stratifying by genomic region.RESULTSThe median CADD scores of intronic and nonsynonymous variants were significantly different between rare and common SNVs (p<0.0001). Despite these different distributions, no individual variants could be identified as plausibly causative among rare intronic variants with the highest scores. The ROC AUC for non-coding variants is modest, and the positive predictive value of CADD for intronic variants in panel testing was found to be 0.088.CONCLUSIONFocused in-silico scoring systems with much higher predictive value will be necessary for clinical genomic applications.

Rectus muscle trauma is proposed as a complication of sub-Tenon's local anaesthesia and caution is advised to operators to clearly identify the sub-Tenon's space for injection of local anaesthetic.