The protein “mycoarray”: a novel immunoassay for the serological diagnosis of primitive invasive mycoses

Objectives. Invasive fungal infections are an important cause of morbidity and mortality in an increasingly higher number of patients, also because of difficulties in providing a rapid and appropriate diagnosis. In some cases, detection of a specific antibody response is a crucial diagnostic tool; however, the available serological assays often provide qualitative results only, their sensitivity and specificity are poor and long time procedures are required. In addition, patients who suffer from an invasive mycosis may have multiple infections likely underestimated by conventional diagnostic approaches. In order to couple the serology of primitive invasive mycoses to the protein microarray technology, a “mycoarray” assay has been designed and set up.Methods. Four antigen extracts (histoplasmin, coccidioidin, Coccidioides “TP” antigen and aspergillin) and the appropriate controls were spotted in various conditions onto a restricted area of a microscope slide. The printed slides were then incubated with immune sera produced in goat against each single antigen or, subsequently, with human sera (6 from patients affected by primitive invasive mycoses and 7 from healthy individuals). The occurring immunocomplexes were detected by indirect immunofluorescence.Results. The pilot experiments, conducted using the goat immune sera, allowed to establish the optimal spotting conditions for each antigen in terms of both spotting buffer and extracts’ dilution. The “mycoarrays”, obtained by spotting all the fungal antigens with the best condition, were then processed with sera either from patients or control subjects. The reactivity observed in the arrays processed with the patients’ samples was in agreement with the clinical and microbiological diagnosis; no reactivity was ever observed in the arrays processed with the negative control sera.Conclusions. The “protein mycoarray” is sensitive enough to discriminate between healthy individuals and patients affected by histoplasmosis or coccidioidomycosis. This novel diagnostic tool, because of its intrinsic features, miniaturization and multiparametricity, can contribute to cut out costs and to shorten times-to-results, with the potentiality to be included in the daily clinical practice in the near future.