Objectives:

Species of the genus Scedosporium are emergent pathogens ranking the second, only behind Aspergillus spp., among filamentous fungi causing a chronic colonization of the airways of cystic fibrosis (CF) patients. This may lead to chronic inflammation or even to life-threatening invasive disease in cases of severe immunosuppression. 
The lack of standardization and the low sensitivity and specificity of the detection methods commonly used result in a delayed diagnosis and inadequate monitoring and treatment of the patients. To contribute to the finding of new diagnostic targets, our aim was to identify the most immunoreactive antigens of Scedosporium boydii recognized specifically by serum IgGs from CF patients colonized by Scedosporium spp.

Methods:

Sera from CF patients were classified into three groups: CF without fungal colonization, CF colonized by Scedosporium spp. and CF colonized by Aspergillus spp. Five sera from each group were pooled to carry out the experiments. To compare the serological response against S. boydii and A. fumigatus, an ELISA assay was performed with serial dilutions of the pooled sera to calculate the titer of IgG and IgE antibodies against both fungal species. 
Furthermore, a total protein extract from S. boydii cultured during 24 h at 37 ºC into potato dextrose broth was obtained and separated by 2-D electrophoresis. Then, proteins were transferred to PVDF membranes to perform immunoblotting with the pooled sera. Comparative image analysis of the results obtained with the sera from the three groups of CF patients allowed characterizing the antigens of S. boydii cross-reacting with sera from CF patients colonized by Aspergillus spp, as well as those specifically detected by sera from CF patients colonized by Scedosporium spp. Among the latter, the most immunoreactive ones were identified by LC-MS/MS.

Results:

The ELISA test revealed that IgG antibody titer against both fungi, S. boydii and A. fumigatus, in CF patients sera with Scedosporium was higher (1/2000) than in those with Aspergillus (1/500). Conversely, the measure of IgE levels showed very low titers in both sera types, similar to the control sera values.
Regarding the immunoproteomics study, different serological profiles were obtained. On the one hand, sera from CF patients colonized by Scedosporium reacted with a great number antigens of high molecular weight (Mr 75-250 kDa) located in the acid region of the gel (pI 3-5), while they were not detected by control sera. On the other hand, sera from patients colonized by Aspergillus showed cross-reactivity with some antigens of S. boydii, but the immunomic pattern was very different. 

Conclusion:

This study characterizes the most immunoreactive Scedosporium antigens recognized by pooled sera from CF patients colonized by these fungi but not by sera from CF patients with an Aspergillus. These proteins should be studied further in order to develop a sensitive and specific serological test for improved detection of Scedosporium species in CF patients

Characterization of Scedosporium boydii antigens recognized by serum IgGs from cystic fibrosis patients