Antonietta Vella

Species identification of Aspergillus, Fusarium and Mucorales with direct surface analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Accurate species discrimination of filamentous fungi is essential, because some species have specific antifungal susceptibility patterns, and misidentification may result in inappropriate therapy. We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for species identification through direct surface analysis of the fungal culture. By use of culture collection strains representing 55 species of Aspergillus, Fusarium and Mucorales, a reference database was established for MALDI-TOF MS-based species identification according to the manufacturers recommendations for microflex measurements and MALDI BioTyper 2.0 software. The profiles of young and mature colonies were analysed for each of the reference strains, and species-specific spectral fingerprints were obtained. To evaluate the database, 103 blind-coded fungal isolates collected in the routine clinical microbiology laboratory were tested. As a reference method for species designation, multilocus sequencing was used. Eighty-five isolates were unequivocally identified to the species level (≥99% sequence similarity); 18 isolates producing ambiguous results at this threshold were initially rated as identified to the genus level only. Further molecular analysis definitively assigned these isolates to the species Aspergillus oryzae (17 isolates) and Aspergillus flavus (one isolate), concordant with the MALDI-TOF MS results. Excluding nine isolates that belong to the fungal species not included in our reference database, 91 (96.8%) of 94 isolates were identified by MALDI-TOF MS to the species level, in agreement with the results of the reference method; three isolates were identified to the genus level. In conclusion, MALDI-TOF MS is suitable for the routine identification of filamentous fungi in a medical microbiology laboratory.

MALDI-TOF mass spectrometry in the clinical mycology laboratory: identification of fungi and beyond

MALDI-TOF mass spectrometry (MS) is becoming essential in most clinical microbiology laboratories throughout the world. Its successful use is mainly attributable to the low operational costs, the universality and flexibility of detection, as well as the specificity and speed of analysis. Based on characteristic protein spectra obtained from intact cells – by means of simple, rapid and reproducible preanalytical and analytical protocols – MALDI-TOF MS allows a highly discriminatory identification of yeasts and filamentous fungi starting from colonies. Whenever used early, direct identification of yeasts from positive blood cultures has the potential to greatly shorten turnaround times and to improve laboratory diagnosis of fungemia. More recently, but still at an infancy stage, MALDI-TOF MS is used to perform strain typing and to determine antifungal drug susceptibility. In this article, the authors discuss how the MALDI-TOF MS technology is destined to become a powerful tool for routine mycological diagnostics.

Comparative Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time Of Flight (MALDI-TOF) Mass Spectrometry Systems for Identification of Yeasts of Medical Importance

ABSTRACT We report the first comparative evaluation between the Bruker Biotyper MS (BMS) and the Vitek MS (VMS) for the identification of yeasts. The rate of correct identifications at the species level was comparable using the commercial databases (89.8% versus 84.3%; P = 0.712), but higher for BMS using an in-house-extended database (100% versus 84.3%; P = 0.245). Importantly, the rate of misidentification was significantly higher for VMS (1% versus 12.1%; P < 0.0001), including the rate of major errors (0% versus 4.5%; P = 0.0036).

Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Caspofungin Susceptibility Testing of Candida and Aspergillus Species

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was evaluated for testing susceptibility to caspofungin of wild-type and fks mutant isolates of Candida and Aspergillus. Complete essential agreement was observed with the CLSI reference method, with categorical agreement for 94.1% of the Candida isolates tested. Thus, MALDI-TOF MS is a reliable and accurate method to detect fungal isolates with reduced caspofungin susceptibility.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Method for Discrimination between Molecular Types of Cryptococcus neoformans and Cryptococcus gattii

ABSTRACT We evaluated the usefulness of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for Cryptococcus identification at the species and subspecies levels by using an in-house database of 25 reference cryptococcal spectra. Eighty-one out of the 82 Cryptococcus isolates (72 Cryptococcus neoformans and 10 Cryptococcus gattii) tested were correctly identified with respect to their molecular type designations. We showed that MALDI-TOF MS is a practicable alternative to conventional mycology or DNA-based methods.

Rapid Antifungal Susceptibility Testing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

ABSTRACT The widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicans after a 3-h incubation in the presence of “breakpoint” level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) μg/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 μg/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 μg/ml. In this way, the drug resistance of C. albicans isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1 genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.

Typing of Nosocomial Outbreaks of Acinetobacter baumannii by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been evaluated for the identification of multidrug-resistant Acinetobacter baumannii nosocomial outbreaks in comparison with the repetitive sequence-based PCR DiversiLab system. The results suggest that MALDI-TOF MS can be used for real-time detection of Acinetobacter outbreaks before results from DNA-based systems are available.

Application of MALDI-TOF mass spectrometry in clinical diagnostic microbiology

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently emerged as a powerful technique for identification of microorganisms, changing the workflow of well-established laboratories so that its impact on microbiological diagnostics has been unparalleled. In comparison with conventional identification methods that rely on biochemical tests and require long incubation procedures, MALDI-TOF MS has the advantage of identifying bacteria and fungi directly from colonies grown on culture plates in a few minutes and with simple procedures. Numerous studies on different systems available demonstrate the reliability and accuracy of the method, and new frontiers have been explored besides microbial species level identification, such as direct identification of pathogens from positive blood cultures, subtyping, and drug susceptibility detection.

Antifungal Susceptibility Profiles of Bloodstream Yeast Isolates by Sensititre YeastOne over Nine Years at a Large Italian Teaching Hospital

ABSTRACT Sensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, the MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or the wild-type [WT] phenotype) to systemic antifungal agents. Excluding Candida albicans (57.4% of all isolates [n = 1,250]), the most predominant species were Candida parapsilosis complex (20.9%), Candida tropicalis (8.2%), Candida glabrata (6.4%), Candida guilliermondii (1.6%), and Candida krusei (1.3%). Among the non-Candida species (1.9%), 7 were Cryptococcus neoformans and 17 were other species, mainly Rhodotorula species. Over 97% of Candida isolates were susceptible (WT phenotype) to amphotericin B and flucytosine. Rates of susceptibility (WT phenotype) to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in the C. parapsilosis complex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates consisted of 6 C. parapsilosis complex isolates, 3 C. glabrata isolates, 2 C. albicans isolates, 2 C. tropicalis isolates, and 1 Candida lusitaniae isolate. Of the non-Candida isolates, 2 C. neoformans isolates had the non-WT phenotype for susceptibility to fluconazole, whereas Rhodotorula isolates had elevated azole MICs. Overall, 99.7% to 99.8% of Candida isolates were susceptible (WT phenotype) to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicans and C. guilliermondii), and micafungin (C. albicans). However, when the intrinsically resistant non-Candida isolates were included, the rate of echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.

Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure

ABSTRACT In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of ≥2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies.In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization‒time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis, the preanalytical sample processing was emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to the discrepancy between methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), that were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts, routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) isolates were successfully identified to the species level (scores of ≥2.0), with 1,676 (39.6%) having scores higher than 2.3. For the remaining 23 (0.5%) isolates, no reliable identification (score of