Carole Cassagne

Mould Routine Identification in the Clinical Laboratory by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry

Background 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. Materials and Methods 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. Results 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. Conclusions This works 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.

Cultivable microbial communities in raw cow milk and potential transfers from stables of sixteen French farms

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.

Evaluation of four pretreatment procedures for MALDI-TOF MS yeast identification in the routine clinical laboratory

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.

Pseudallescheria/Scedosporium complex species identification by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry.

Because timely and accurate identification of members of the Pseudallescheria/ Scedosporium species complex (PSC) is clinically relevant, the objective of this investigation was to study the stability and influence of the main variable factors in the routine clinical laboratory to the potential use the Matrix-Assisted Laser Desorption Ionization-Time-Of-Flight (MALDI-TOF MS) in the identification of these fungi. Twenty-two PSC reference strains, three clinical isolates, an αHCCA matrix, and an Autoflex I spectrometer with BioTyper software (Bruker) were employed in this study. Intra-and inter-specimen composite correlation indices for each MS spectrum as compared to a reference spectrum were computed. MS identification was stable after the fungi were subcultured over a 1-month period. While neither culture medium (Sabouraud vs. Malt extract) nor protein extraction methods (formic acid vs. trifluoroacetic acid) significantly influenced the quality of the MS identifications, they were considerably increased from day 3 to day 6 of incubation. MALDI-TOF MS can be used in the routine clinical laboratory in the identification of members of the complex provided that valid spectra libraries are developed. Although preliminary results are encouraging, further studies are warranted to demonstrate whether MS can distinguish the species that have recently been described using multilocus sequence analysis within P. boydii sl. and to validate its use in the routine clinical laboratory for identifying clinically relevant moulds.

Assessment of various parameters to improve MALDI-TOF MS reference spectra libraries constructed for the routine identification of filamentous fungi

BackgroundThe poor reproducibility of matrix-assisted desorption/ionization time-of-flight (MALDI-TOF) spectra limits the effectiveness of the MALDI-TOF MS-based identification of filamentous fungi with highly heterogeneous phenotypes in routine clinical laboratories. This study aimed to enhance the MALDI-TOF MS-based identification of filamentous fungi by assessing several architectures of reference spectrum libraries.ResultsWe established reference spectrum libraries that included 30 filamentous fungus species with various architectures characterized by distinct combinations of the following: i) technical replicates, i.e., the number of analyzed deposits for each culture used to build a reference meta-spectrum (RMS); ii) biological replicates, i.e., the number of RMS derived from the distinct subculture of each strain; and iii) the number of distinct strains of a given species. We then compared the effectiveness of each library in the identification of 200 prospectively collected clinical isolates, including 38 species in 28 genera.Identification effectiveness was improved by increasing the number of both RMS per strain (p<10-4) and strains for a given species (p<10-4) in a multivariate analysis.ConclusionAddressing the heterogeneity of MALDI-TOF spectra derived from filamentous fungi by increasing the number of RMS obtained from distinct subcultures of strains included in the reference spectra library markedly improved the effectiveness of the MALDI-TOF MS-based identification of clinical filamentous fungi.

Performance of MALDI-TOF MS platforms for fungal identification.

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.

Outbreak of Corynebacterium pseudodiphtheriticum infection in cystic fibrosis patients, France.

Respiratory tract colonization with these bacteria may be common in this population.

Identification of Leishmania at the species level with matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption ionization time-of-flightMALDI-TOF mass spectrometry (MS) is now widely recognized as a powerful tool with which to identify bacteria and fungi at the species level, and sometimes in a rapid and accurate manner. We report herein an approach to identify, at the species level, Leishmania promastigotes from in vitro culture. We first constructed a reference database of spectra including the main Leishmania species known to cause human leishmaniasis. Then, the performance of the reference database in identifying Leishmania promastigotes was tested on a panel of 69 isolates obtained from patients. Our approach correctly identified 66 of the 69 isolates tested at the species level with log (score) values superior to 2. Two Leishmania isolates yielded non-interpretable MALDI-TOF MS patterns, owing to low log (score) values. Only one Leishmania isolate of Leishmania peruviana was misidentified as the closely related species Leishmania braziliensis, with a log (score) of 2.399. MALDI-TOF MS is a promising approach, providing rapid and accurate identification of Leishmania from in vitro culture at the species level.

Routine identification and mixed species detection in 6,192 clinical yeast isolates

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 sequence-based 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.

Decision criteria for MALDI-TOF MS-based identification of filamentous fungi using commercial and in-house reference databases

BackgroundSeveral 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.ResultsOur 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.ConclusionsDepositing 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.