Development of an analytical method based on protein profiling for the rapid identification of Aspergillus spp. from clinical samples by MALDI-TOF-MS Biotyper

Putigani L., Mancinelli L., Dimiziani L., Russo C., Coltella L., Maier R., Menichella D.

Author address: 

NULL

Abstract: 

Objectives: Invasive mould infections are becoming more frequent, resulting in significant morbidity and mortality in children. Paediatric populations are currently at high risk for fungal opportunistic infections due to the high impact of changes in medical practice, intensive care and organ transplantation practises. In our study, we enlarged the library of the MALDI-ToF MS (Matrix-assisted laser description ionization- time of flight-mass spectrometry) Biotyper with the aim to exploit proteome profiling of Aspergillus spp. to provide an advanced and reliable method for the identification of Aspergillus species from clinical specimens. Methods: Reference and clinical strains of ten Aspergillus species were both purchased from the culture collection Centraalbureau voor Schimmelcultures (CBS) and collected in our diagnostic unit to select the most representative species isolated from clinical specimens. Medium growth conditions and protein extraction protocols were optimised to produce suitable template for MALDI-ToF MS analysis. Replicates for each spectrum were collected for each species and analysed for reproducibility. Finally eight overlapping spectra were selected for each species and evaluated for variance by principal component clustering and dendrogram analysis (PCA). Selected spectra were uploaded into the library of the MALDI TOF-MS and added to the pre-existents to perform identification of fungal clinical specimens. Results: The reference spectra of the Aspergillus species showed typical MALDI-TOF-MS spectra with peaks between m/z 2000 and up to about m/z 16,000. On visual inspection, the similarity of spectra produced by different Aspergillus species could be recognized. Obvious differences between spectra produced by the diverse species were also easily noticed. The reproducibility of the method was proved by the high similarity and PCA outcome of spectra belonging to the same species. Enlarged dataset provided high matching scores (2.3-3.0 range) for Aspergillus spp. identification from clinical testing samples. Conclusion: New proteome profiling-based assays for detection of mould fungi may be an optimal diagnostic approach to overcome current culture-based methods, encompass multiple fungal genera, and for being applied to a variety of specimen types. In our experience, MALDI-ToF is currently under setting and may represent a new frontier for the fast and reliable management of fungal infections in paediatric high risk patients.
2009

abstract No: 

P 885

Full conference title: 

19th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 19th (2009)