Identification and validation of diagnostic cut-offs of the ELISpot assay for the diagnosis of invasive aspergillosis in high-risk patients

by Francesca Bettelli, Daniela Vallerini, Ivana Lagreca, Patrizia Barozzi, Giovanni Riva, Vincenzo Nasillo, Ambra Paolini, Roberto D’Amico, Fabio Forghieri, Monica Morselli, Valeria Pioli, Andrea Gilioli, Davide Giusti, Andrea Messerotti, Paola Bresciani, Angela Cuoghi, Elisabetta Colaci, Roberto Marasca, Livio Pagano, Anna Candoni, Johan Maertens, Pierluigi Viale, Cristina Mussini, Rossella Manfredini, Enrico Tagliafico, Mario Sarti, Tommaso Trenti, Russell Lewis, Patrizia Comoli, Albino Eccher, Mario Luppi, Leonardo Potenza

Objective

We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies.

Methods

We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10.

Results

In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/10⁶ PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases.

Conclusions

ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients’ different pre-test probability of infection can widen its use in patients at risk.