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MALDI-TOF MS-based yeast identification requires a pretreatment step for which four are described in the literature, i.e., direct smear, fast formic acid and two complete formic acid/acetonitrile extractions. In this study we compared the impact of these procedures on the performance of MALDI-TOF MS-based yeast identification of samples from colonies grown on Sabouraud or chromogenic media. A total of 103 yeast isolates recovered from clinical samples were identified in parallel using the four pretreatment procedures. The proportions of both correct identifications (regardless of LogScore values) and of reliable identifications (i.e., correct identifications with a LogScore 2, as recommended by the manufacturer) obtained with the four techniques were compared. Even if the proportion of correct identifications exceeded 85% independent of the pretreatment procedure, results obtained with complete formic acid/acetonitril extractions of colonies grown on Sabouraud media were significantly superior to those with smear and fast formic acid extraction procedures. If one considers only reliable identifications, then both smear and fast formic acid extraction procedures yielded lower (<40%) correct identification rates than the use of the two complete extraction procedures (>77%) of portions of colonies on both Sabouraud and chromogenic media. The data would indicate that the direct smear and fast formic acid procedures cannot be recommended due to the LogScore values which were continually below those recommended by the manufacturer for biological validation. Thus, complete extraction methods are better suited for MALDI-TOF MS-based yeast identification in the clinical laboratory setting although they are more labor-intensive.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014–2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2 , and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 μg/ml, 2/64], itraconazole (MIC > 0.5 μg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 μg/ml, 4/64), and voriconazole (MIC ≥ 1 μg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) is routinely used in mycology laboratories to rapidly identify pathogenic yeasts. Various methods have been proposed to perform routine MS-based identification of clinically relevant species. In this study, we focused on Bruker technology and assessed the identification performance of three protocols: two pretreatment methods (rapid formic acid extraction directly performed on targets and full extraction using formic acid/acetonitrile in tubes) and a direct deposit protocol that omits the extraction step. We also examined identification performance using three target types (ground-steel, polished-steel, and biotargets) and two databases (Bruker and online MSI [biological-mass-spectrometry-identification application]) in a multicenter manner. Ten European centers participated in the study, in which a total of 1511 yeast isolates were analyzed. The 10 centers prospectively performed the three protocols on approximately 150 yeast isolates each, and the corresponding spectra were then assessed against two reference spectra databases (MSI and Bruker), with appropriate thresholds. Three centers evaluated the impact of the targets. Scores were compared between the various combinations, and identification accuracy was assessed. The protocol omitting the extraction step was inappropriate for yeast identification, while the full extraction method yielded far better results. Rapid formic acid extraction yielded variable results depending on the target, database and threshold. Selecting the optimal extraction method in combination with the appropriate target, database and threshold may enable simple and accurate identification of clinically relevant yeast samples. Concerning the widely used polished-steel targets, the full extraction method still ensured better scores and better identification rates. [ABSTRACT FROM AUTHOR]

The non-lipid-dependent yeast Malassezia pachydermatis is predominantly zoophilic but occasionally colonizes the human skin. This yeast caused an outbreak in a neonatal iIntensive care unit (NICU). This study aimed to describe the molecular epidemiology of this M. pachydermatis outbreak. All the M. pachydermatis isolates collected at a French University Hospital from January 2012 to April 2013 were included in the study. M. pachydermatis isolates, sampled from various biological samples sites in 25 patients, were identified via MALDI-TOF mass spectrometry and typed using intergenic-spacer 1 (IGS1) nucleotide sequence polymorphisms analysis. By analyzing 90 IGS1 sequences (including 43 deposited in GenBank), we found that of the 186 M. pachydermatis isolates, 47 were viable for typing and all of them clustered within type 3; 78.7% clustered within the 3D subtype; the remaining clustered within three newly described subtypes: 3E (4.3%), 3F (8.5%) and 3 G (8.5%). No particular subtype was associated with a collection site or a particular time period. This first molecular investigation of a M. pachydermatis outbreak in neonates showed that multiple genotypes can colonize the same neonate patient by. The source of this polyclonal outbreak could not be identified. It stopped after infection control measures, including the prohibition of a lipid-rich moisturizing hand cream used by the health care staff, had been implemented. [ABSTRACT FROM AUTHOR]

Yellow-legged gulls have been reported to carry antibiotic-resistant Enterobacteriaceae; however, the gut mycobiota of these birds has not yet been described. In this study, we analyzed the gut yeast communities in five yellow-legged gull breeding colonies along the Mediterranean littoral in southern France. Gull fecal samples were inoculated onto four types of culture media, including one supplemented with itraconazole. Yeast species richness, abundance, and diversity were estimated, and factorial analysis was used to highlight correspondences between breeding colonies. Yeast grew in 113 of 177 cultures, and 17 distinct yeast species were identified. The most frequent species were Candida krusei (53.5%), Galactomyces geotrichum (44.1%), C. glabrata (40.9%), C. albicans (20.5%), and Saccharomyces cerevisiae (18.1%). Gut yeast community structure in the gulls at both Pierre-Blanche Lagoon (PB) and Frioul Archipelago (F) were characterized by greater species richness and diversity than in those at the two cities of La Grande-Motte (GM) and Palavas-les-Flots (PF) as well as Riou Archipelago (R). Gulls in these latter three sites probably share a similar type of anthropogenic diet. Notably, the proportion of anthropic yeast species, including C. albicans and C. glabrata, in the gull mycobiota increased with gull colony synanthropy. Antifungal resistance was found in each of the five most frequent yeast species. We found that the gut yeast communities of these yellow-legged gulls include antifungal-resistant human pathogens. Further studies should assess the public health impact of these common synanthropic seabirds, which represent a reservoir and disseminator of drug-resistant human pathogenic yeast into the environment. [ABSTRACT FROM AUTHOR]

The black Aspergillus group comprises A. niger and 18 other species, which are morphologically indistinguishable. Among this species subset, A. tubingensis, described in less than 30 human cases before 2014, is primarily isolated from ear, nose, and throat samples. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has emerged as a powerful technique to identify microbes in diagnostic settings. We applied this method to identify 1,720 filamentous fungi routinely isolated from clinical samples our laboratory over a two-year study period. Accordingly, we found 85 isolates of A. niger, 58 of A. tubingensis, and six other black Aspergillus (4 A. carbonarius and 2 A. japonicus). A. tubingensis was the fifth most frequent mold isolated in our mycology laboratory, primarily isolated from respiratory samples (40/58 isolates). In this study, we mainly aimed to describe the clinical pattern of Aspergillus tubingensis. We analyzed the clinical features of the patients in whom A. tubingensis had been isolated from 40 respiratory samples. Thirty patients suffered from cystic fibrosis, chronic obstructive pulmonary disease or other types of chronic respiratory failure. Strikingly, 20 patients were experiencing respiratory acute exacerbation at the time the sample was collected. Antifungal susceptibility testing of 36 A. tubingensis isolates showed lower amphotericin B MICs (P < 10-4) and higher itraconazole and voriconazole MICs (P < 10-4 and P = .0331, respectively) compared with 36 A. niger isolates. Further studies are required to better establish the role that this fungus plays in human diseases, especially in the context of cystic fibrosis and chronic pulmonary diseases. [ABSTRACT FROM AUTHOR]

The clinical laboratory methods used to diagnose yeast infections should be rapid, reliable, and capable of detecting mixed infections with species exhibiting a distinct antifungal susceptibility profile. In this study, we report the performance of a procedure combining the detection of mixed yeast cultures with a chromogenic medium and MALDI-TOF identification of the colonies. We then evaluated the impact that (i) the isolation medium and (ii) lowering the identification log score (LS) threshold value have on yeast identification performance in the routine laboratory. Among 15,661 clinical samples analyzed, 5,671 tested positive and 6,192 yeasts of 42 distinct species were identified. Overall, 6,117 isolates (98.79%) were identified on the first or second MALDI-TOF Mass Spectrometry (MS) attempt, yielding an average yeast species identification turnaround time of 0.346 days (95% CI [0.326 to 0.364]). The 75 remaining isolates were identified via nucleotide sequencing. Mixed infections accounted for 498 (8.78%) of the positive samples. The MALDI-TOF MS identification procedure performed well, regardless of the culture media tested. Lowering the recommended 2.0 LS threshold value to 1.8 would reduce the number of required (i) second MALDI-TOF MS identification attempts (178 vs. 490) and (ii) ITS2 and D1-D2 sequencebased identifications (17 vs. 75), while achieving an adequate identification rate (6,183/6,192, 99.85%). In conclusion, we propose applying a 1.8 LS threshold combined with chromogenic medium subculture to optimize the yeast identification workflow and detect mixed infection in the clinical laboratory. [ABSTRACT FROM AUTHOR]

Rapid and accurate identification of yeast isolates from clinical samples is essential, given their innately variable antifungal susceptibility profiles, and the proposal of species-specific antifungal susceptibility interpretive breakpoints. Here we have evaluated the utility of MALDI-ToF MS analysis for the identification of clinical isolates of pathogenic yeasts. A simplified, rapid extraction method, developed in our laboratory, was applied to 6343 isolates encompassing 71 different yeast species, which were then subjected to MALDI-ToF MS analysis using a Bruker Microflex and the resulting spectra were assessed using the supplied Bruker database. In total, 6328/6343 (99.8%) of isolates were correctly identified by MALDI-ToF MS. Our simplified extraction protocol allowed the correct identification of 93.6% of isolates, without the need for laborious full extraction, and a further 394 (6.2%) of isolates could be identified after full extraction. Clinically relevant identifications with both extraction methods were achieved using the supplied Bruker database and did not require the generation of bespoke, in-house databases created using profiles obtained with the adapted extraction method. In fact, the mean LogScores obtained using our method were as robust as those obtained using the recommended, published full extraction procedures. However, an in-house database can provide a useful additional identification tool for unusual or rarely encountered organisms. Finally, the proposed methodology allowed the correct identification of over 75% of isolates directly from the initial cultures referred to our laboratory, without the requirement for additional sub-culture on standardised mycological media. [ABSTRACT FROM AUTHOR]

Utilizing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra for Candida glabrata typing would be a cost-effective and easy-to-use alternative to classical DNA-based typing methods. This study aimed to use MALDI-TOF for the typing of C. glabrata clinical isolates from various geographical origins and test its capacity to differentiate between fluconazole-sensitive and -resistant strains. Both microsatellite length polymorphism (MLP) and MALDI-TOF mass spectra of 58 C. glabrata isolates originating from Marseilles (France) and Tunis (Tunisia) as well as collection strains from diverse geographic origins were analyzed. The same analysis was conducted on a subset of C. glabrata isolates that were either susceptible (MIC ≤ 8 mg/l) or resistant (MIC ≥ 64 mg/l) to fluconazole. According to the seminal results, both MALDI-TOF and MLP classifications could highlight C. glabrata population structures associated with either geographical dispersal barriers (p < 10-5) or the selection of antifungal drug resistance traits (<10-5). In conclusion, MALDI-TOF geographical clustering was congruent with MPL genotyping and highlighted a significant population genetic structure according to fluconazole susceptibility in C. glabrata. Furthermore, although MALDI-TOF and MLP resulted in distinct classifications, MALDI-TOF also classified the isolates with respect to their fluconazole susceptibility profile. Further prospective studies are required to evaluate the capacity of MALDI-TOF typing to investigate C. glabrata infection outbreaks and predict the antifungal susceptibility profile of clinical laboratory isolates. [ABSTRACT FROM AUTHOR]