High Interlaboratory Reproducibility of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry-Based Species Identification of Nonfermenting Bacteria

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a fast and cost-effective alternative for bacterial species identification in microbiology. On the basis of mass analysis of the protein composition of a bacterial cell, which is assumed to be characteristic for each bacterial species, it is possible to determine the species within few minutes, starting from whole cells, cell lysates, or crude bacterial extracts (2, 3, 5, 6). The proof of principle of MALDI-TOF MS for bacterial species identification was shown a decade ago (2, 5, 6); however, due to low reproducibility, it has not been widely adopted in clinical microbiology. We have recently shown that use of a larger mass range for detection (2,000 to 20,000 Da), dedicated analysis software for spectral pattern matching, and a high-quality reference database of spectra generated from quality-controlled culture collection strains resulted in accurate species identifications, with high intralaboratory reproducibility (7). For interlaboratory reproducibility, there are only very limited data available (8, 10). We therefore evaluated the interlaboratory reproducibility for MALDI-TOF MS-based species identification in a multicenter study, applying the above-described MALDI-TOF MS improvements.(This study was presented in part at the 19th European Congress of Clinical Microbiology and Infectious Diseases [ECCMID] in Helsinki, Finland, 16 to 19 May 2009.)Sixty blind-coded samples were shipped worldwide by mail to eight laboratories with access to Bruker MALDI-TOF MS platforms and personnel trained in MALDI-TOF MS-based species identification (Centre de Ressources Biologiques de l′Institut Pasteur [CRBIP], Department of Microbiology, Institut Pasteur, Paris, France; Department of Microbiology and Molecular Cell Biology, Center for Biomedical Proteomics, Eastern Virginia Medical School, Norfolk, VA; Labor Limbach, Heidelberg, Germany; Research Institute of Physical-Chemical Medicine, Moscow, Russia; Bruker Daltonik, GmbH, Bremen, Germany; Institut de Bactériologie, Strasbourg, France; Microbiology Unit, Bambino Gesù Children''s Hospital, Health Care and Research Institute, Rome, Italy; and Molecular Infectious Diseases Laboratory, Vanderbilt University Hospital, Nashville, TN). Samples 001 to 030 of the 60 samples included pure cultures of different nonfermenting bacteria, either culture collection strains from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and Laboratorium voor Microbiologie, Universiteit Gent (LMG, Gent, Belgium), or strains isolated at the Institute of Hygiene, Münster, Germany, during routine diagnostic efforts. They contained single strains of Alcaligenes faecalis subsp. faecalis (DSMZ 30030), Brevundimonas andropogonis (DSMZ 9511), Brevundimonas aurantiaca (DSMZ 4731), Burkholderia caribensis (DSMZ 13236), Brevundimonas diminuta (DSMZ 7234), Brevundimonas intermedia (DSMZ 4732), Brevundimonas vesicularis (DSMZ 7226), Comamonas nitrativorans (DSMZ 13191), Comamonas testosteroni (DSMZ 50244), Flavobacterium johnsoniae (DSMZ 2064), Inquilinus limosus (DSMZ 16000), Sphingobacterium mizutaii (DSMZ 11724), Pseudomonas beteli (LMGZ 978), Pseudomonas boreopolis (LMGZ 979), Pseudomonas extremorientalis (DSMZ 15824), 13 Pseudomonas aeruginosa strains (DSMZ 50071 and 12 clinical isolates), and 2 Stenotrophomonas maltophilia strains (clinical isolates). The 16 culture collection strains listed above were among the 248 strains used for constructing a nonfermenter reference database (7). All species designations were unambiguously confirmed using partial 16S rRNA gene sequencing as described elsewhere (7). Samples 031 to 060 contained preprocessed cell extracts from the first 30 strains as described recently (7). Accompanying the samples, each participating laboratory received a “sample cultivation and preparation guide” and a “result reporting guide” to facilitate and standardize data generation and interpretation. Briefly, the laboratories were asked to streak out samples 001 to 030 onto blood agar plates (irrespective of the vender) and to incubate them for 48 h at 30°C in an ambient atmosphere. Using a single colony, extraction for MALDI-TOF MS analysis was initiated. For preprocessed samples 031 to 060, the guide included instructions for matrix preparation and MALDI-TOF MS analysis (7). For spectral calibration, the Bruker bacterial test standard (Escherichia coli lysate) was used during the measuring step. All laboratories used the MALDI Biotyper 2.0 software package (Bruker Daltonik, GmbH, Bremen, Germany) and the MALDI Biotyper database, containing spectra of more than 2,800 microorganisms (including the 248 nonfermenter species) as reference data. The software generates a list of probable species identifications ranked by the log(score) value, which reflects the peak matches as well as intensities and results in values between 0 and 3 (0 to 100% pattern match). After comparison of an unknown spectrum with all reference spectra of the database, the log(score)s are ranked. Values of ≥2.0 were required for secure identification at the species level and values between <2 and ≥1.7 for secure identification at the genus level. Results based on log(score) values of <1.7 were rated as not identifiable. These thresholds were empirically determined based on the whole MALDI Biotyper database contents.Each of the eight participating laboratories received 60 blind-coded samples for MALDI-TOF MS-based species identification. The aggregated results for each laboratory and the machines used are shown in Table ​Table1.1. Of the total 480 samples, 474 (98.75%) were correctly identified at the species level by using the highest log(score) value for the identified species after MALDI-TOF MS spectral comparisons. Five of the remaining six samples were misidentified, and one sample did not result in any valid species designation, due to low signal intensity. Overall, six of the eight laboratories identified all 60 samples correctly (Table ​(Table1).1). Four hundred sixty-seven of the 480 samples (97.29%) with log(score) values of ≥2 (mean, 2.353; standard deviation, 0.146) were identified, indicating a probable secure species identification level (Table ​(Table1).1). Twelve of the remaining 13 samples showed log(score) values between 1.7 and 2, which correlated with at least a secure identification at the genus level; only sample 044, which was not identified, due to low signal intensity, had a log(score) value of 0. The 12 samples were distributed among four laboratories; no pattern of an especially problematic sample was discerned. The five misidentified samples showed log(score) values of ≥2. Figure ​Figure11 displays the mean log(score) value for each sample and its standard deviation. Of the 60 samples investigated, only sample 044 showed a significantly higher standard deviation and lower mean value due to the failure in laboratory B. There was no significant difference between the mean log(score) values of cultured samples versus those of preprocessed samples as determined by t test statistics (P = 0.20).Open in a separate windowFIG. 1.Mean log(score) values and standard deviations for all 60 blind-coded samples identified using MALDI-TOF MS, calculated from the results for all eight participating laboratories.

TABLE 1.

Aggregated log(score) values and final species identification results for each of the eight participating laboratories (not specified) for 60 blind-coded samples (n = 480 in total) containing nonfermenting bacteria either as pure culture or as preprocessed cell extract

Solitary fibrous tumor of the scalp: a case report

The solitary fibrous tumor is a rare tumor, ubiquitous, mesenchymatous essentially affecting serosal surfaces. The location in the scalp is exceptional. We report a case of tumor in left occipital scalp discovered after recurrence of a tumor initially taken for a lipoma. The motive for consultation was mainly aesthetic. The pathological examination is the main element of diagnosis. The assumption is based on a large lumpectomy and an extended and regular clinical monitoring. The forecast is favourable with a decline of one year. However, relapses may occur in the long-term and there are malignant forms.     

Ultrastrukturelle Veränderungen am Nierenparenchym durch ESWL unter Acetylsalicylsäure (ASS)

BACKGROUND: The risk of hemorrhagic complications after extracorporeal shock-wave lithotripsy (ESWL) increases in patients with aspirin intake, but the hematoma-inducing mechanism has not been understood completely at the ultrastructural level. METHODS: The effect off shock-waves on the kidneys of male Wistar-rats (n=24) was investigated in an experimental setting using a special ESWL device. Ultrastructural examination was performed by light-, transmission electron- and scanning electron microscopy. RESULTS: Shock-wave induced tissue damage appeared in all kidneys independently of aspirin intake. Endothelial detachment, lethal cell injury, gaps and mechanical disruption of the glomerular basement membrane were regularly found. After 1 week, repair processes were completed with evidence of permanent fibrosis in some cases. CONCLUSIONS: ESWL can induce modest as well as fatal damage to renal tissue cells. Therefore, after an ESWL-induced hematoma a second ESWL should not be performed within 1 week of the first treatment.     

Lymphangiomyomatosis: CT, chest radiographic, and functional correlations

Eight patients with the diagnosis of lymphangiomyomatosis were evaluated with computed tomography (CT), chest radiography, and pulmonary function tests to determine the relationship between the extent of disease seen on imaging studies and functional status. Chest radiographic assessment included the subjective determination of disease extent and measurements of lung length and the arc of the right hemidiaphragm. Disease extent on CT scans was scored as a percentage of lung that was abnormal on the basis of visual assessment of the degree of cystic replacement of the lung parenchyma. Significant correlations were observed between CT scores and percentages of predicted forced expiratory volume in 1 second/forced vital capacity (r = -.92, P less than .002) and diffusing capacity of the lungs for carbon monoxide (r = -.80, P less than .017). No significant correlations were observed between subjective chest radiographic scores and pulmonary function tests, although measurements of lung length and percentage of predicted total lung capacity were correlated (r = .76, P less than .045). CT was more accurate than chest radiography in defining the presence and extent of parenchymal cysts and provided for greater morphologic-physiologic correlation. CT, particularly high-resolution CT, may be useful in the diagnosis and longitudinal evaluation of patients with this disease and may be more sensitive than pulmonary function tests in the early stages of lung damage.     

Miliary tuberculosis: A complication of intravesical BCG treatment

A case of miliary tuberculosis following intravesical bacillus Calmette-Guerin (BCG) treatment is described.     

Gene Probe Analysis in an Informative Family with Multiple Endocrine Neoplasia Syndrome Type 2A (MEN 2A). Improvement in Carrier Risk Estimation

Gene probe analysis of the MEN 2A locus on chromosome 10 hasbeen undertaken using the markers TB10.163, RBP 3 and TB14.34in a large kindred with familial medullary thyroid carcinomas,with or without phaeochromocytomas or primary hyperparathyroidism.A maximum LOD score of 2.97 gave strong evidence of close linkagewith zero recombination. For 12 members of the family so far not known to be affectedby any form of the disease the estimated risk of carrying thegene has been considerably decreased in all but one, whose riskhas been greatly increased.