Summary
Matrix‐assisted laser desorption/ionisation mass spectrometry (MALDI‐TOF MS) is increasingly used by clinical microbiology laboratories to cope with the need for rapid, cost‐effective and ...accurate identification of microorganisms. Several research teams have recently succeed in identifying moulds using MALDI‐TOF MS, which was first adapted to bacteria, then to yeast identification. Since 2004, different commercial firms have released several ready‐to‐use MALDI‐TOF MS platforms. This review describes the similarities and differences between the commercially available systems. In two parts, we first describe and compare the preprocessing and identification steps between the platforms and then compare the identification efficacy of yeast, moulds and dermatophytes species.
Several Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry protocols, which differ in identification criteria, have been developed for mold and dermatophyte identification. ...Currently, the most widely used approach is Bruker technology, although no consensus concerning the log(score) threshold has been established. Furthermore, it remains unknown how far increasing the number of spots to compare results might improve identification performance. In this study, we used in-house and Bruker reference databases as well as a panel of 422 isolates belonging to 126 species to test various thresholds. Ten distinct identification algorithms requiring one to four spots were tested.
Our findings indicate that optimal results were obtained by applying a decisional algorithm in which only the highest score of four spots was taken into account with a 1.7 log(score) threshold. Testing the entire panel enabled identification of 87.41% (in-house database) and 35.15% (Bruker database) of isolates, with a positive predictive value (PPV) of 1 at the genus level for both databases as well as 0.89 PPV (in-house database) and 0.72 PPV (Bruker database) at the species level. Applying the same rules to the isolates for which the species were represented by at least three strains in the database enabled identification of 92.1% (in-house database) and 46.6% (Bruker database) of isolates, with 1 PPV at the genus level for both databases as well as 0.95 PPV (in-house database) and 0.93 PPV (Bruker database) at the species level.
Depositing four spots per extract and lowering the threshold to 1.7, a threshold which is notably lower than that recommended for bacterial identification, decreased the number of unidentified specimens without altering the reliability of the accepted results. Nevertheless, regardless of the criteria used for mold and dermatophyte identification, commercial databases require optimization.
Summary
Matrix‐assisted laser desorption ionisation‐time of flight mass spectrometry (MALDI‐TOF MS) is commonly used by clinical microbiology laboratories to identify bacterial pathogens and yeasts, ...but not for the identification of moulds. Recent progress in extraction protocols and the composition of comparative libraries support potential application of MALDI‐TOF MS for mould identification in clinical microbiology laboratories. We evaluated the performance of the Bruker Microflex™ MALDI‐TOF MS instrument (Billerica, MA, USA) to identify clinical isolates and reference strains of moulds using 3 libraries, the Bruker mould library, the National Institutes of Health (NIH) library and the Mass Spectrometry Identification (MSI) online library, and compared those results to conventional (morphological) and molecular (18S/ITS; gold standard) identification methods. All 3 libraries demonstrated greater accuracy in genus identification (≥94.9%) than conventional methods (86.4%). MALDI‐TOF MS identified 73.3% of isolates to species level compared to only 31.7% by conventional methods. The MSI library demonstrated the highest rate of species‐level identification (72.0%) compared to NIH (19.5%) and Bruker (13.6%) libraries. Greater than 20% of moulds remained unidentified to species level by all 3 MALDI‐TOF MS libraries primarily because of library limitations or imperfect spectra. The overall identification rate of each MALDI‐TOF MS library depended on the number of species and the number of spectra representing each species in the library.
MALDI-TOF MS recently emerged as a valuable identification tool for bacteria and yeasts and revolutionized the daily clinical laboratory routine. But it has not been established for routine mould ...identification. This study aimed to validate a standardized procedure for MALDI-TOF MS-based mould identification in clinical laboratory.
First, pre-extraction and extraction procedures were optimized. With this standardized procedure, a 143 mould strains reference spectra library was built. Then, the mould isolates cultured from sequential clinical samples were prospectively subjected to this MALDI-TOF MS based-identification assay. MALDI-TOF MS-based identification was considered correct if it was concordant with the phenotypic identification; otherwise, the gold standard was DNA sequence comparison-based identification.
The optimized procedure comprised a culture on sabouraud-gentamicin-chloramphenicol agar followed by a chemical extraction of the fungal colonies with formic acid and acetonitril. The identification was done using a reference database built with references from at least four culture replicates. For five months, 197 clinical isolates were analyzed; 20 were excluded because they were not identified at the species level. MALDI-TOF MS-based approach correctly identified 87% (154/177) of the isolates analyzed in a routine clinical laboratory activity. It failed in 12% (21/177), whose species were not represented in the reference library. MALDI-TOF MS-based identification was correct in 154 out of the remaining 156 isolates. One Beauveria bassiana was not identified and one Rhizopus oryzae was misidentified as Mucor circinelloides.
This work's seminal finding is that a standardized procedure can also be used for MALDI-TOF MS-based identification of a wide array of clinically relevant mould species. It thus makes it possible to identify moulds in the routine clinical laboratory setting and opens new avenues for the development of an integrated MALDI-TOF MS-based solution for the identification of any clinically relevant microorganism.
Children who grow up in environments that afford them a wide range of microbial exposures, such as traditional farms, are protected from childhood asthma and atopy. In previous studies, markers of ...microbial exposure have been inversely related to these conditions.
In two cross-sectional studies, we compared children living on farms with those in a reference group with respect to the prevalence of asthma and atopy and to the diversity of microbial exposure. In one study--PARSIFAL (Prevention of Allergy-Risk Factors for Sensitization in Children Related to Farming and Anthroposophic Lifestyle)--samples of mattress dust were screened for bacterial DNA with the use of single-strand conformation polymorphism (SSCP) analyses to detect environmental bacteria that cannot be measured by means of culture techniques. In the other study--GABRIELA (Multidisciplinary Study to Identify the Genetic and Environmental Causes of Asthma in the European Community GABRIEL Advanced Study)--samples of settled dust from children's rooms were evaluated for bacterial and fungal taxa with the use of culture techniques.
In both studies, children who lived on farms had lower prevalences of asthma and atopy and were exposed to a greater variety of environmental microorganisms than the children in the reference group. In turn, diversity of microbial exposure was inversely related to the risk of asthma (odds ratio for PARSIFAL, 0.62; 95% confidence interval CI, 0.44 to 0.89; odds ratio for GABRIELA, 0.86; 95% CI, 0.75 to 0.99). In addition, the presence of certain more circumscribed exposures was also inversely related to the risk of asthma; this included exposure to species in the fungal taxon eurotium (adjusted odds ratio, 0.37; 95% CI, 0.18 to 0.76) and to a variety of bacterial species, including Listeria monocytogenes, bacillus species, corynebacterium species, and others (adjusted odds ratio, 0.57; 95% CI, 0.38 to 0.86).
Children living on farms were exposed to a wider range of microbes than were children in the reference group, and this exposure explains a substantial fraction of the inverse relation between asthma and growing up on a farm. (Funded by the Deutsche Forschungsgemeinschaft and the European Commission.).
The worldwide emergence of multidrug-resistant pathogenic fungi is a threat to human health. At this very moment, an emergence of
isolates harbouring a resistance to fluconazole, one of the most ...popular antifungal drugs, is being described in several countries. We seek to better understanding the epidemiology, pathogenicity and transmission of resistant
Faced with an outbreak of invasive infections due to resistant isolates of
, we performed a 7-year retrospective and prospective analysis of 283
isolates collected in 240 patients, among who 111 had invasive candidiasis. Study included review of hospital records, genotyping analysis and susceptibility testing that allow determining the type and outcome of infections, as well as the spatial and temporal spread of clusters. Overall the incidence of azole resistance was 7.5%. Genotyping analysis unveiled several previously undetected outbreaks and clonal spread of susceptible and resistant isolates over a long period of time. In comparison with susceptible isolates, resistant ones have a more restricted genetic diversity and seem to be more likely to spread and more frequently associated with invasive infections. In intensive care units, patients with invasive infections due to resistant isolates had poorer outcome (overall mortality at day 30 of 40%; 4/10) than susceptible ones (overall mortality at day 30 of 26.5%; 9/34). Our results suggest that the propensity of
to spread on an epidemic fashion is underestimated, which warrants reinforced control and epidemiological survey of this species.
The spread of fungal clones is hard to detect in the daily routines in clinical laboratories, and there is a need for new tools that can facilitate clone detection within a set of strains. Currently, ...Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry is extensively used to identify microbial isolates at the species level. Since most of clinical laboratories are equipped with this technology, there is a question of whether this equipment can sort a particular clone from a population of various isolates of the same species. We performed an experiment in which 19 clonal isolates of Aspergillus flavus initially collected on contaminated surgical masks were included in a set of 55 A. flavus isolates of various origins. A simple convolutional neural network (CNN) was trained to detect the isolates belonging to the clone. In this experiment, the training and testing sets were totally independent, and different MALDI-TOF devices (Microflex) were used for the training and testing phases. The CNN was used to correctly sort a large portion of the isolates, with excellent (> 93%) accuracy for two of the three devices used and with less accuracy for the third device (69%), which was older and needed to have the laser replaced.
The indigenous microflora in raw milk plays an important role in the diversity of cheese flavours and may protect against the growth of pathogens, but the sources of contamination and the factors ...that might affect the microbial communities in milk are not well known. The objectives of this study were to broaden knowledge of the microbial composition of milk and to assess microbial transfers from the stable to the milk.
Air (collected in milking parlour and stable), dust (passively collected using plastic box), cow teat surface, and hay and milk samples were collected in 16 French farms with either stanchion barn or freestall barn configurations and plated on various culture media. Bacterial and fungal colonies were identified using phenotypic and DNA sequencing methods.
Results showed that most of the fungal species and environmental bacteria found in the milk were also found in the stable and the milking parlour environments, indicating large microbial transfer from stable to milking parlour then to milk. However, milk from the stanchion barns were more contaminated than milk from freestall barns. Contrasting with other bacterial and fungal species, useful cheese-making bacteria – lactobacilli and PAB – were frequently identified in the milk and on the teat surface but were rarely found in other environments.
In conclusion, milk contamination by the stable environment is considerable, even if it is lower in farms with a milking parlour. Besides this environmental contamination, the teat surface remains the main source of useful cheese-making bacteria.
► Here, we characterised the whole indigenous milk microflora. ► We traced the environmental sources of milk contamination. ► Microorganisms were phenotypically and genotypically identified. ► Milk contamination routes were mapped for the different microbial taxa. ► Our findings are useful for agro-alimentary industries and for farming communities.
Aspergillus section Nigri is a taxonomically difficult but medically and economically important group. In this study, an update of the taxonomy of A. section Nigri strains within the BCCM/IHEM ...collection has been conducted. The identification accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was tested and the antifungal susceptibilities of clinical isolates were evaluated. A total of 175 strains were molecularly analyzed. Three regions were amplified (ITS, benA, and caM) and a multi-locus phylogeny of the combined loci was created by using maximum likelihood analysis. The in-house MALDI-TOF MS reference database was extended and an identification data set of 135 strains was run against a reference data set. Antifungal susceptibility was tested for voriconazole, itraconazole, and amphotericin B, using the EUCAST method. Phylogenetic analysis revealed 18 species in our data set. MALDI-TOF MS was able to distinguish between A. brasiliensis, A. brunneoviolaceus, A. neoniger, A. niger, A. tubingensis, and A. welwitschiae of A. sect. Nigri. In the routine clinical lab, isolates of A. sect. Nigri are often identified as A. niger. However, in the clinical isolates of our data set, A. tubingensis (n = 35) and A. welwitschiae (n = 34) are more common than A. niger (n = 9). Decreased antifungal susceptibility to azoles was observed in clinical isolates of the /tubingensis clade. This emphasizes the importance of identification up to species level or at least up to clade level in the clinical lab. Our results indicate that MALDI-TOF MS can be a powerful tool to replace classical morphology.
Summary
This study aimed to validate the effectiveness of a standardised procedure for the MALDI‐TOF mass spectrometry (MS)‐based identification on a large sample of filamentous fungi routinely ...identified in university hospitals' laboratories. Non‐dermatophyte filamentous fungi prospectively isolated in the routine activity of five teaching hospitals in France were first identified by conventional methods in each laboratory and then by MS in one centre. DNA sequence‐based identification resolved discrepancies between both methods. In this study, of the 625 analysed filamentous fungi of 58 species, 501 (80%) and 556 (89%) were correctly identified by conventional methods and MS respectively. Compared with the conventional method, MS dramatically enhanced the performance of the identification of the non‐Aspergillus filamentous fungi with a 31–61% increase in correct identification rate. In conclusion, this study on a large sample of clinical filamentous fungi taxa demonstrates that species identification is significantly improved by MS compared with the conventional method. The main limitation is that MS identification is possible only if the species is included in the reference spectra library. Nevertheless, for the routine clinical laboratory, MS provides the means to attain markedly accurate results in filamentous fungi identification, which was previously restricted to only a few reference laboratories.
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