New insights into the immunodiagnosis of Scedosporium/Lomentospora in Cystic Fibrosis patients

Resumen

Scedosporium and Lomentospora genera are emergent fungal pathogens ranking the second, only behind Aspergillus spp., among filamentous fungi causing a chronic colonization of the airways of CF patients. This may lead to chronic inflammation or even to life-threatening invasive disease in cases of severe immunosuppression. The detection of these microorganisms is currently performed by non-standardized and low-sensitivity culture-based methods. With this concern, the aim of this thesis project was to gain insights into the immunodiagnosis of Scedosporium/Lomentospora fungi in CF patients. In this thesis project different protein extracts ¿ including a total whole cell protein extract from conidia and hyphae (Total WCP), secreted proteins (Secretome), conidial and hyphal cell surface associated proteins (Total CSP) and conidial surface associated proteins (Conidial CSP) ¿ were studied by ELISA to select the most useful for anti-Scedosporium/Lomentospora-IgG detection in sera from CF patients. Furthermore, a rapid Dot Immunobinding Assay (DIA) that employed S. boydii Total WCP was developed. This rapid system allowed a naked-eye detection of anti-Scedosporium/Lomentospora-IgG in 15 minutes in sera from CF patients with a sensitivity and specificity of 90.48% and 79.30%, respectively. Finally, an immunoproteomics-based study was performed to identify the S. boydii antigens specifically recognized by serum IgGs from CF patients and infected mice. In this sense, 22 proteins were identified as specific antigens. After evaluating their diagnostic capacity by in-gel purification and western blotting with individual sera, 4 proteins in particular stood out as novel promising diagnostic targets which might be included in future diagnostic methods. // Scedosporium and Lomentospora genera are emergent fungal pathogens ranking the second, only behind Aspergillus spp., among filamentous fungi causing a chronic colonization of the airways of CF patients. This may lead to chronic inflammation or even to life-threatening invasive disease in cases of severe immunosuppression. The detection of these microorganisms is currently performed by non-standardized and low-sensitivity culture-based methods. With this concern, the aim of this thesis project was to gain insights into the immunodiagnosis of Scedosporium/Lomentospora fungi in CF patients. In this thesis project different protein extracts ¿ including a total whole cell protein extract from conidia and hyphae (Total WCP), secreted proteins (Secretome), conidial and hyphal cell surface associated proteins (Total CSP) and conidial surface associated proteins (Conidial CSP) ¿ were studied by ELISA to select the most useful for anti-Scedosporium/Lomentospora-IgG detection in sera from CF patients. Furthermore, a rapid Dot Immunobinding Assay (DIA) that employed S. boydii Total WCP was developed. This rapid system allowed a naked-eye detection of anti-Scedosporium/Lomentospora-IgG in 15 minutes in sera from CF patients with a sensitivity and specificity of 90.48% and 79.30%, respectively. Finally, an immunoproteomics-based study was performed to identify the S. boydii antigens specifically recognized by serum IgGs from CF patients and infected mice. In this sense, 22 proteins were identified as specific antigens. After evaluating their diagnostic capacity by in-gel purification and western blotting with individual sera, 4 proteins in particular stood out as novel promising diagnostic targets which might be included in future diagnostic methods.