Species identification of clinical Prevotella isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry

The performance of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database.     

Identification of clinical isolates of anaerobic bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry

We evaluated the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) for the rapid identification of anaerobic bacteria that had been isolated from clinical specimens and previously identified by 16s rRNA sequencing. The Bruker Microflex MALDI-TOF instrument with the Biotyper Software was used. We tested 152 isolates of anaerobic bacteria from 24 different genera and 75 different species. A total of 125 isolates (82%) had Biotyper software scores greater than 2.0 and the correct identification to genus and species was made by MALDI-TOF for 120 (79%) of isolates. Of the 12 isolates with a score between 1.8 and 2.0, 2 (17%) organisms were incorrectly identified by MALDI-TOF. Only 15 (10%) isolates had a score less than 1.8 and MALDI-TOF gave the wrong genus and species for four isolates, the correct genus for two isolates, and the correct genus and species for nine isolates. Therefore, we found the Bruker MALDI-TOF MicroFlex LT with an expanded database and the use of bacteria extracts rather than whole organisms correctly identified 130 of 152 (86%) isolates to genus and species when the cut-off for an acceptable identification was a spectrum score ≥1.8.     

Identification of HACEK clinical isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.     

Dermatophyte identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry

The performance of the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer (MS) for the identification of dermatophytes from clinical cultures was compared to that of dermatophyte identification using 28S rRNA gene sequencing. The MALDI Biotyper library (MBL; version 3.0) was used alone and in combination with a supplemented library containing an additional 20 dermatophyte spectra (S-MBL). Acquired spectra were interpreted using both the manufacturer-recommended scores (genus, ≥1.7; species, ≥2.0) and adjusted cutoff values established by this study (genus, ≥1.5; species, ≥1.7); identifications required a minimum 10% difference in scores between the top two different organisms to be considered correct. One hundred well-characterized, archived dermatophyte isolates and 71 fresh dermatophyte cultures were evaluated using both libraries and both sets of cutoff criteria. Collectively, the S-MBL significantly outperformed the MBL at both the genus (93% versus 37.4%; P < 0,0001) and species (59.6% versus 20.5%; P < 0.0001) levels when using the adjusted score criteria. Importantly, application of the lowered cutoff values significantly improved genus (P = 0.005)- and species (P < 0.0001)-level identification for the S-MBL, without leading to an increase in misidentifications. MALDI-TOF MS is a cost-effective and rapid alternative to traditional or molecular methods for dermatophyte identification, provided that the reference library is supplemented to sufficiently encompass clinically relevant, intraspecies strain diversity.     

Successful identification of clinical dermatophyte and Neoscytalidium species by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Dermatophytes are keratinolytic fungi responsible for a wide variety of diseases of glabrous skin, nails, and hair. Their identification, currently based on morphological criteria, is hindered by intraspecies morphological variability and the atypical morphology of some clinical isolates. The aim of this study was to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a routine tool for identifying dermatophyte and Neoscytalidium species, both of which cause dermatomycoses. We first developed a spectral database of 12 different species of common and unusual dermatophytes and two molds responsible for dermatomycoses (Neoscytalidium dimidiatum and N. dimidiatum var. hyalinum). We then prospectively tested the performance of the database on 381 clinical dermatophyte and Neoscytalidium isolates. Correct identification of the species was obtained for 331/360 dermatophytes (91.9%) and 18/21 Neoscytalidium isolates (85.7%). The results of MALDI-TOF MS and standard identification disagreed for only 2 isolates. These results suggest that MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes and Neoscytalidium spp. in clinical mycology laboratories.     

Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.     

Rapid identification of Staphylococci isolated in clinical microbiology laboratories by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yields a reproducible spectrum depending upon growth conditions, strain, or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase-negative staphylococci (CoNS). Our aim was, once a bacterium has been recognized as Micrococcaceae, to identify peaks in the spectrum that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CoNS. Similar results were obtained using four different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification.     

Comparison of two matrix-assisted laser desorption ionisation-time of flight mass spectrometry methods for the identification of clinically relevant anaerobic bacteria

Two commercially available MALDI-TOF MS systems, Bruker MS and Shimadzu MS, were compared for the identification of clinically relevant anaerobic bacteria. A selection of 79 clinical isolates, representing 19 different genera, were tested and compared with identification obtained by 16S rRNA gene sequencing. Correct genus identification was achieved for 71% of isolates by Shimadzu MS and for 61% by Bruker MS. Correct identification at the species level occurred in 61% and 51%, respectively. Shimadzu showed markedly better results for identification of Gram-positive anaerobic cocci. In contrast, the Bruker system performed better than Shimadzu for the Bacteroides fragilis group. When strains not present in the database were excluded from the analyses for each database, both systems performed equally well, with 76.7% and 75.0% correct genus identification for Shimadzu and Bruker, respectively. Similarly, when the most recently updated Bruker database was applied, no difference was observed. We conclude that the composition and quality of the database is crucial for a correct identification. The databases currently available for both systems need to be optimized before MS can be implemented for routine identification of anaerobic bacteria.     

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid identification of Beta-hemolytic streptococci

This study was undertaken to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid identification of beta-hemolytic streptococci. We compared Bruker Biotyper 2.0 with Vitek2 coupled to the agglutination test. MALDI-TOF MS analysis of 386 beta-hemolytic streptococcal isolates yielded high-confidence identification to the species level for all 386 isolates. The Vitek2 gave high-confidence identification to the species level for 88% of Streptococcus agalactiae isolates (n = 269/306), 92% of Streptococcus pyogenes isolates (n = 48/52), and 39% of isolates of Streptococcus dysgalactiae serogroups C and G (n = 11/28).     

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.     

Differentiation of cfiA-negative and cfiA-positive Bacteroides fragilis isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Carbapenem resistance in Bacteroides fragilis is associated with cfiA-encoded class B metallo-beta-lactamase. cfiA-negative and cfiA-positive isolates belong to genotypically distinct groups. Of a total of 248 B. fragilis isolates included in this study, 214 were susceptible, 10 were intermediate, and 24 were resistant to meropenem. We show that matrix-assisted laser desorption ionization-time of flight mass spectrometry is able to differentiate between cfiA-negative and cfiA-positive isolates and predict carbapenem resistance in a routine laboratory setting.     

Comparison of the Microflex LT and Vitek MS systems for routine identification of bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

This study compared the performance of three matrix-assisted laser desorption ionization-time of flight mass spectrometry systems: Microflex LT (Bruker Daltonics, Bremen, Germany), Vitek MS RUO (Axima Assurance-Saramis database; bioMérieux, Marcy l'Etoile, France), and Vitek MS IVD (bioMérieux). A total of 1,129 isolates, including 1,003 routine isolates, 73 anaerobes, and 53 bacterial enteropathogens, were tested on the Microflex LT and Axima Assurance devices. The spectra were analyzed using three databases: Biotyper (Bruker Daltonics), Saramis, and Vitek MS (bioMérieux). Among the routine isolates requiring identification to the species level (n = 986), 92.7% and 93.2% were correctly identified by the Biotyper and Vitek MS databases, respectively. The Vitek MS database is more specific for the identification of Streptococcus viridans. For the anaerobes, the Biotyper database often identified Fusobacterium isolates to only the genus level, which is of low clinical significance, whereas 20% of the Bacteroides species were not identified or were misidentified by the Vitek MS database. For the enteropathogens, the poor discrimination between Escherichia coli and Shigella explains the high proportion of unidentified organisms. In contrast to the Biotyper database, the Vitek MS database properly discriminated all of the Salmonella entrica serovar Typhi isolates (n = 5). The performance of the Saramis database was globally poorer. In conclusion, for routine procedures, the Microflex LT and Vitek-MS systems are equally good choices in terms of analytical efficiency. Other factors, including price, work flow, and lab activity, will affect the choice of a system.     

Performances of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for rapid identification of bacteria in routine clinical microbiology

Rapid and cost-effective matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based systems will replace conventional phenotypic methods for routine identification of bacteria. We report here the first evaluation of the new MALDI-TOF MS-based Vitek MS system in a large clinical microbiology laboratory. This system uses an original spectrum classifier algorithm and a specific database designed for the identification of clinically relevant species. We have tested 767 routine clinical isolates representative of 50 genera and 124 species. Vitek MS-based identifications were performed by means of a single deposit on a MALDI disposable target without any prior extraction step and compared with reference identifications obtained mainly with the VITEK2 phenotypic system; if the identifications were discordant, molecular techniques provided reference identifications. The Vitek MS system provided 96.2% correct identifications to the species level (86.7%), to the genus level (8.2%), or within a range of species belonging to different genera (1.3%). Conversely, 1.3% of isolates were misidentified and 2.5% were unidentified, partly because the species was not included in the database; a second deposit provided a successful identification for 0.8% of isolates unidentified with the first deposit. The Vitek MS system is a simple, convenient, and accurate method for routine bacterial identification with a single deposit, considering the high bacterial diversity studied and as evidenced by the low prevalence of species without correct identification. In addition to a second deposit in uncommon cases, expanding the spectral database is expected to further enhance performances.     

Effects of solid-medium type on routine identification of bacterial isolates by use of 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 rapid method for the identification of bacteria. Factors that may alter protein profiles, including growth conditions and presence of exogenous substances, could hinder identification. Bacterial isolates identified by conventional methods were grown on various media and identified using the MALDI Biotyper (Bruker Daltonics, Billerica, MA) using a direct smear method and an acid extraction method. Specimens included 23 Pseudomonas isolates grown on blood agar, Pseudocel (CET), and MacConkey agar (MAC); 20 Staphylococcus isolates grown on blood agar, colistin-nalidixic acid agar (CNA), and mannitol salt agar (MSA); and 25 enteric isolates grown on blood agar, xylose lysine deoxycholate agar (XLD), Hektoen enteric agar (HE), salmonella-shigella agar (SS), and MAC. For Pseudomonas spp., the identification rate to genus using the direct method was 83% from blood, 78% from MAC, and 94% from CET. For Staphylococcus isolates, the identification rate to genus using the direct method was 95% from blood, 75% from CNA, and 95% from MSA. For enteric isolates, the identification rate to genus using the direct method was 100% from blood, 100% from MAC, 100% from XLD, 92% from HE, and 87% from SS. Extraction enhanced identification rates. The direct method of MALDI-TOF analysis of bacteria from selective and differential media yields identifications of varied confidence. Notably, Staphylococci spp. from CNA exhibit low identification rates. Extraction enhances identification rates and is recommended for colonies from this medium.     

Utility of matrix-assisted laser desorption ionization-time of flight mass spectrometry following introduction for routine laboratory bacterial identification

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated prospectively in a diagnostic laboratory. Nine hundred twenty-seven organisms were tested in triplicate; 2,351/2,781 (85%) species and 2,681/2,781 (96%) genus identifications were correct. Known issues such as the misidentification of alpha-hemolytic streptococci as Streptococcus pneumoniae were easily corrected. Identifications cost AUD$0.45 per isolate and were available in minutes. MALDI-TOF MS is rapid, accurate, and inexpensive.     

Rapid identification of Cryptococcus neoformans and Cryptococcus gattii by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Compared to DNA sequence analysis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) correctly identified 100% of Cryptococcus species, distinguishing the notable pathogens Cryptococcus neoformans and C. gattii. Identification was greatly enhanced by supplementing a commercial spectral library with additional entries to account for subspecies variability.     

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for the identification of bacterial and fungal isolates

Identification of bacterial and fungal isolates from clinical specimens has traditionally been performed by examination of colony morphology and by biochemical characterization using classic tube sets. While these methods remain the gold standard for identification, they can be laborious to perform and subjective to interpret. These drawbacks have largely been alleviated with the advent of automated biochemical testing platforms and with the development of DNA sequencing and polymerase chain reaction techniques. Though reliable and rapid, molecular-based platforms are associated with significantly higher cost and require advanced user expertise for assay development and performance. These factors limit the routine implementation of molecular diagnostic methods to large hospitals and reference laboratories. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is an automated, molecular platform recently adopted by many clinical laboratories worldwide, which offers a rapid, straightforward, and inexpensive method for the identification of bacterial and fungal organisms.     

Rapid identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry

Purpose: The purpose of this study was to evaluate the identification of Mycobacterium tuberculosis which is often plagued with ambiguity. It is a time consuming process requiring 4-8 weeks after culture positivity, thereby delaying therapeutic intervention. For a successful treatment and disease management, timely diagnosis is imperative. We evaluated a rapid, proteomic based technique for identification of clinical mycobacterial isolates by protein profiling using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Materials and Methods: Freshly grown mycobacterial isolates were used. Acetonitrile/trifluoroacetic acid extraction procedure was carried out, following which cinnamic acid charged plates were subjected to identification by MALDI-TOF MS. Results: A comparative analysis of 42 clinical mycobacterial isolates using the MALDI-TOF MS and conventional techniques was carried out. Among these, 97.61% were found to corroborate with the standard methods at genus level and 85.36% were accurate till the species level. One out of 42 was not in accord with the conventional assays because MALDI-TOF MS established it as Mycobacterium tuberculosis (log (score)>2.0) and conventional methods established it to be non-tuberculous Mycobacterium. Conclusions: MALDI-TOF MS was found to be an accurate, rapid, cost effective and robust system for identification of mycobacterial species. This innovative approach holds promise for early therapeutic intervention leading to better patient care.