Objectives: Aspergillus spp. and other molds have been shown to induce elevated levels of several cytokines. It remains unknown whether these cytokines hold value for clinical routine and enhance diagnostic performances of established and novel biomarkers/molecular tests for invasive aspergillosis (IA) and other invasive mold infections (IMI). The objective of this prospective cohort study was to determine the diagnostic potential of a bundle of cytokines as well as established and emerging tests for IA and IMI, in patients with underlying hematological malignancies in a setting that frequently uses mold-active prophylaxis. 

Methods: This cohort study included prospectively enrolled cases (n=106, adults; 04/2014-08/2017; Medical University of Graz, Austria) with underlying hematological malignancies and suspected pulmonary infections who underwent bronchoscopy. Serum samples were collected within 24 hours of bronchoalveolar lavage fluid (BALF) sampling. Both, serum and BALF samples, were used to evaluate diagnostic performances of the Aspergillus-specific lateral-flow device test (LFD; OLM Diagnostics), an Aspergillus-specific PCR (conducted at the University Hospital of Mannheim, Heidelberg University, Germany), galactomannan (GM), β-D-glucan (BDG), and a bundle of cytokines previously associated with IA. Cytokines included interleukin (IL)-4/-6/-8/-10/-15/-17A/-22, soluble IL-2 receptor, tumor necrosis factor α, interferon-γ and C-C motif chemokine ligand 5 (CCL5); concentrations were retrospectively tested using a custom made ProcartaPlex 11plex immunoassay (eBioscience, Vienna, Austria). IA and IMI were classified according to the 2008 revised EORTC/MSG criteria.

Results: Among the 106 cases, 11 had probable IA, 32 possible IA (of which 7 had probable IMI), and 63 no evidence for IMI. Underlying diseases in probable IMI cases (including probable IA) were acute myeloid leukemia/myelodysplastic syndrome (n=11), acute lymphoblastic leukemia (n=3), Non-Hodgkin lymphoma (n=3) and multiple myeloma (n=1). Neutropenia <500/µL for >10 days before BALF sampling were seen in 28/106 cases (including 5/11 of those with probable IA). Overall 85/106 cases (including 10/11 of those with probable IA) had received ≥2 defined daily doses of mold-active antifungal prophylaxis or treatment at the time of sample collection. Combinations of serum IL-8 with either BALF Aspergillus LFD (sensitivity 100%, specificity 94%) or BALF Aspergillus PCR (sensitivity 91%, specificity 97%) were highly sensitive and specific for differentiating probable IA from no IA. In Receiver Operating Characteristics (ROC) curve analysis, serum IL-8 (p=0.004) and serum IL-6 (p=0.008) were significantly associated with probable IA, while other cytokines and also serum BDG (p=0.271) were not associated. ROC curve analysis also showed potential for serum IL-8 (p=0.014) for differentiating possible and probable IMI from no IMI, followed by serum CCL5 (p=0.038), and marginally also serum IL-10 (p=0.045 when extrapolated low levels were included; p=0.089 after extrapolated levels were excluded). In contrast, the other serum and BALF cytokines, serum GM, serum BDG and BALF GM were not discriminative.

Conclusion: Our study indicates that serum IL-8 testing may be a valuable addition to clinical routine for diagnosing IA and IMI in high risk patients who have a suspected pulmonary infection with an unknown microbiologic entity while receiving mold-active antifungals.

3 / Invasive Aspergillosis: Performance of New and Established Diagnostic Approaches with Same-Day Blood and Bronchoalveolar Lavage -- a Prospective Cohort Study