Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus.

The rates of infection with Fusarium molds are increasing, and a diverse number of Fusarium spp. belonging to different species complexes can cause infection. Conventional species identification in the clinical laboratory is time-consuming and prone to errors. We therefore evaluated whether matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a useful alternative. The 289 Fusarium strains from the Belgian Coordinated Collections of Microorganisms (BCCM)/Institute of Hygiene and Epidemiology Mycology (IHEM) culture collection with validated sequence-based identities and comprising 40 species were used in this study. An identification strategy was developed, applying a standardized MALDI-TOF MS assay and an in-house reference spectrum database. In vitro antifungal testing was performed to assess important differences in susceptibility between clinically relevant species/species complexes. We observed that no incorrect species complex identifications were made by MALDI-TOF MS, and 82.8% of the identifications were correct to the species level. This success rate was increased to 91% by lowering the cutoff for identification. Although the identification of the correct species complex member was not always guaranteed, antifungal susceptibility testing showed that discriminating between Fusarium species complexes can be important for treatment but is not necessarily required between members of a species complex. With this perspective, some Fusarium species complexes with closely related members can be considered as a whole, increasing the success rate of correct identifications to 97%. The application of our user-friendly MALDI-TOF MS identification approach resulted in a dramatic improvement in both time and accuracy compared to identification with the conventional method. A proof of principle of our MALDI-TOF MS approach in the clinical setting using recently isolated Fusarium strains demonstrated its validity.

Banana infecting fungus, Fusarium musae, is also an opportunistic human pathogen: are bananas potential carriers and source of fusariosis?

During re-identification of Fusarium strains in the BCCM™/IHEM fungal collection by multilocus sequence-analysis we observed that five strains, previously identified as Fusarium verticillioides, were Fusarium musae, a species described in 2011 from banana fruits. Four strains were isolated from blood samples or biopsies of immune-suppressed patients and one was isolated from the clinical environment, all originating from different hospitals in Belgium or France, 2001-2008. The F. musae identity of our isolates was confirmed by phylogenetic analysis using reference sequences of type material. Absence of the gene cluster necessary for fumonisin biosynthesis, characteristic to F. musae, was also the case for our isolates. In vitro antifungal susceptibility testing revealed no important differences in their susceptibility compared to clinical F. verticillioides strains and terbinafine was the most effective drug. Additional clinical F. musae strains were searched by performing BLAST queries in GenBank. Eight strains were found, of which six were keratitis cases from the U.S. multistate contact lens-associated outbreak in 2005 and 2006. The two other strains were also from the U.S., causing either a skin infection or sinusitis. This report is the first to describe F. musae as causative agent of superficial and opportunistic, disseminated infections in humans. Imported bananas might act as carriers of F. musae spores and be a potential source of infection with F. musae in humans. An alternative hypothesis is that the natural distribution of F. musae is geographically a lot broader than originally suspected and F. musae is present on different plant hosts.

Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library.

The identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) relies mainly on a robust and extensive database of reference spectra. To this end, a large in-house library containing 760 strains and representing 472 species was built and evaluated on 390 clinical isolates by comparing MALDI-TOF MS with the classical identification method based on morphological observations. The use of MALDI-TOF MS resulted in the correct identification of 95.4% of the isolates at species level, without considering LogScore values. Taking into account the Brukers' cutoff value for reliability (LogScore >1.70), 85.6% of the isolates were correctly identified. For a number of isolates, microscopic identification was limited to the genus, resulting in only 61.5% of the isolates correctly identified at species level while the correctness reached 94.6% at genus level. Using this extended in-house database, MALDI-TOF MS thus appears superior to morphology in order to obtain a robust and accurate identification of filamentous fungi. A continuous extension of the library is however necessary to further improve its reliability. Indeed, 15 isolates were still not represented while an additional three isolates were not recognized, probably because of a lack of intraspecific variability of the corresponding species in the database.

Identification of the Trichophyton mentagrophytes complex species using MALDI-TOF mass spectrometry.

Dermatophytes are fungi capable of invading keratinized tissues and are responsible for the most common fungal infection worldwide: dermatophytosis. Identification of these organisms to the species level is often necessary for the correct treatment of these infections, and is always recommended from an epidemiological point of view. Since the identification of dermatophytes is sometimes problematic, we assessed whether Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) could provide a useful tool to identify dermatophytes of the Trichophyton mentagrophytes complex. A reference database was constructed with 17 strains of six different species belonging to this complex. A total of 54 dermatophyte strains of the Belgian co-ordinated collections of micro-organisms, Scientific Institute of Public Health, Brussels, Belgium (BCCM™/IHEM) collection were used to challenge this database; 89% of the tested strains (not used as reference strains in the database) could readily be identified. When incorrect identifications were encountered, the confusion was always between phylogenetically closely related taxa which indicates that observations made by MALDI-TOF MS correlate with phylogenetic data. To assess this observation, a dendrogram outlining the similarities between the obtained spectra was constructed. Strikingly, the relationships found in this dendrogram were highly similar to the ones observed in the phylogenetic tree recently reported by Beguin and co-workers. In conclusion, MALDI-TOF MS is a fast and reliable tool for the identification of dermatophytes, since it can even discriminate between the closely related species of the T. mentagrophytes complex. Moreover, our data indicate that the data obtained by MALDI-TOF MS correlate with phylogenetic data.

Is Trichophyton simii endemic to the Indian subcontinent?

Trichophyton simii is considered to be prevalent only in the Indian subcontinent where it was isolated from soil, as well as from infections of humans and animals. We have investigated a case of onychomycosis caused by this exotic dermatophyte, not traceable to endemic areas. This case, as in others due to this fungus in man or animals, that have been previously and sporadically reported worldwide, suggests infections caused by T. simii might be underestimated, especially outside its primary geographic areas. Indeed, there are isolates that do not show species-specific morphology, as in our case isolate, and as a result may be misidentified by classical methods. By checking the identity of some strains preserved in the collection BCCM/IHEM, we found several that proved to be T. simii, originating from non-endemic areas (Belgium, France and Ivory Coast). Therefore, the natural distribution of T. simii is probably not as restricted as has previously been proposed.

The taxonomic status of Trichophyton quinckeanum and T. interdigitale revisited: a multigene phylogenetic approach.

Trichophyton quinckeanum, known as the causative agent of mouse favus, has been a subject of controversy since its discovery, 125 years ago. The purpose of this study was to examine the phylogenetic relationships between this fungus and related taxa. To achieve this objective, the ITS rDNA region, as well as actin and ß-tubulin gene regions of various isolates were sequenced. Bayesian inference and maximum likelihood analyses were conducted with T. rubrum as outgroup. Our study showed that strains identified as T. quinckeanum and others identified as T. schoenleinii are part of the complex T. mentagrophytes, and that their genotype cannot be confused with any other dermatophytes. Furthermore, this study demonstrates that the choice of the neotype of T. mentagrophytes was inappropriate. The beta-tubulin topology also revealed that isolates of T. interdigitale form a genetically distinct population from the type strains of Arthroderma vanbreuseghemii. Therefore, contrary to what is generally accepted, the anthropophilic species T. interdigitale cannot be considered as the anamorph associated with the latter.

Changes to airborne pollen counts across Europe.

A progressive global increase in the burden of allergic diseases has affected the industrialized world over the last half century and has been reported in the literature. The clinical evidence reveals a general increase in both incidence and prevalence of respiratory diseases, such as allergic rhinitis (common hay fever) and asthma. Such phenomena may be related not only to air pollution and changes in lifestyle, but also to an actual increase in airborne quantities of allergenic pollen. Experimental enhancements of carbon dioxide (CO[Formula: see text]) have demonstrated changes in pollen amount and allergenicity, but this has rarely been shown in the wider environment. The present analysis of a continental-scale pollen data set reveals an increasing trend in the yearly amount of airborne pollen for many taxa in Europe, which is more pronounced in urban than semi-rural/rural areas. Climate change may contribute to these changes, however increased temperatures do not appear to be a major influencing factor. Instead, we suggest the anthropogenic rise of atmospheric CO[Formula: see text] levels may be influential.

Screening of strains of the Candida parapsilosis group of the BCCM/IHEM collection by MALDI-TOF MS.

One hundred sixty-three strains stored as Candida parapsilosis in the BCCM/IHEM collection were reidentified based on internal transcribed spacer sequencing: 92% were identified as true C. parapsilosis, while 4.3% and 3% belonged to the closely related species C. metapsilosis and C. orthopsilosis, respectively, providing important epidemiologic information. Furthermore, we showed that matrix-assisted laser desorption ionisation time-of-flight mass spectrometry is a fast method that can discriminate between these species.