Invasive pulmonary aspergillosis (IPA) is a life-threatening fungal infection predominantly affecting neutropenic patients. These patients generally have severe immune suppression, such as malignant hematological diseases, solid organ or hematopoietic stem cell transplantation, etc (Heylen et al., 2024). In non-neutropenic patients, IPA is relatively uncommon but can occur in individuals with other underlying risk factors, such as chronic obstructive pulmonary disease (COPD) or bronchiectasis (Zhu et al., 2023). The diagnosis of IPA in non-neutropenic patients is challenging due to the lack of specific clinical and radiological manifestations. Traditional diagnostic methods, such as culture and histopathology, have limited sensitivity and specificity, leading to delayed diagnosis and treatment. Therefore, in recent years, there has been a significant increase in research related to the diagnosis of pulmonary aspergillosis in non-neutropenic patients.

GM test is also called Aspergillus galactomannan test, which is used to qualitatively detect Aspergillus galactomannan in vitro, which is released from the tips of Aspergillus filaments into blood, bronchoalveolar lavage fluid (BALF) or cerebrospinal fluid during the growth of Aspergillus mycelia in tissues (Cai et al., 2023). According to the Infectious Diseases Society of America guidelines (IDSA), GM testing is considered a mycologic criterion for Aspergillus infections in neutropenic patients (Patterson et al., 2016). However, GM testing has limited diagnostic value in non-neutropenic IPA patients (Dai et al., 2021). Interleukin-17 (IL-17), which is produced primarily by Th17 lymphocytes, is an early trigger for the induction of an inflammatory response, and it plays a crucial role in host defense against fungal infections by recruiting and activating immune cells, such as neutrophils and macrophages (Puerta-Arias et al., 2020; Launder et al., 2024). Previous studies have found that IL-17 levels in bronchoalveolar lavage fluid from Aspergillus-infected mice increased progressively with increasing GM levels (Kimura et al., 2017). In our previous study, plasma IL17 levels have been explored to be significantly higher in patients with IPA and bronchiectasis than in controls (He et al., 2022). However, in patients with fungal lung infections, BALF is a more direct indicator of lung inflammation than blood. In this study, we therefore evaluate the diagnostic value of BALF and plasma IL-17 levels in non-neutropenic patients with IPA.

IL-17 is recognized for its crucial role in the innate immune response to invasive aspergillosis infection. Numerous significant studies have emphasized the importance of IL-17 in this context (Khosravi et al., 2018; Malacco et al., 2018). In the present study, IL-17 was more sensitive than GM for the diagnosis of IPA in non-neutropenic patients, both in plasma and BALF, Combined diagnostic testing of GM and IL-17 may improve the diagnostic efficiency of IPA in non-neutropenic patients.

According to the EORTC/MSG recommendations (Donnelly et al., 2020), IPA can be diagnosed by testing plasma and lavage fluid for GM. However, several studies have found that GM testing has limited diagnostic value in in non-neutropenic patients with IPA. In previous studies in non-neutropenic patients, the sensitivity of plasma GM ranged from 11.6% to 90.9% and the specificity from 66.3% to 100% (Zhang et al., 2013; Zhou et al., 2017; He et al., 2021). In this study, the optimal threshold value for analyzing plasma GM based on the ROC curve was 0.6, and the sensitivity and specificity were 56.5% and 87.7%, respectively. In our previous studies, the sensitivity of plasma GM in patients with bronchiectasis was 39.4% and specificity 89.2% (He et al., 2022). The reason for the difference in sensitivity between the two studies is that this study included not only patients with bronchiectasis, but also patients with COPD, previous tuberculosis, etc.

Current research suggests that indicators in BALF are a better surrogate of lung inflammation. In this study, the optimal threshold value of BALF GM for the diagnosis of IPA was 1.01, at which the sensitivity of the test was 68.7% and the specificity was 91.9%. Previous studies have shown that ROC curve analyses of non-neutropenic patients with pulmonary aspergillosis have reported optimal BALF GM cutoff values between 0.7 and 1.25 (He et al., 2012; Zhou et al., 2017; Liu et al., 2021). Our previous study for patients with bronchiectasis combined with IPA reported the same optimal cutoff value of 1.01. In a study in a Chinese population, the optimal cutoff value for diagnosing IPA in non-neutropenic patients with GM in BALF was 0.7 with a sensitivity of 72.97% and a specificity of 89.16% (Zhou et al., 2017). However, in He’s study, the optimal cutoff value for the diagnosis of IPA by BALF GM could be as high as 1.25, which may be due to the fact that the majority of patients included in this study were ICU patients or received mechanical ventilation (He et al., 2012).

IL-17 is primarily produced by a subset of T cells called Th17 cells. Other cell types such as natural killer cells, and neutrophils can also produce IL-17 (Mills, 2023). IL-17 induces the production of chemokines that attract neutrophils to the site of inflammation and enhance the immune response to pathogens (Akhter et al., 2023). It also stimulates the release of pro-inflammatory cytokines such as IL-6 and IL-8, further promoting the inflammatory process (Ritzmann et al., 2022). Current research suggests that IL-17 has emerged as a key factor in host defense against fungal infections (Puerta-Arias et al., 2020). IL-17 promotes the production of antimicrobial peptides such as defensins and cathelicidins, which have direct fungicidal activity against invading pathogens. It can also stimulate the production of chemokines that attract neutrophils and other immune cells to the site of infection, enhancing phagocytosis and clearance of fungal pathogens (Soleimanifar et al., 2023). In an experiment in mice with COPD combined with Aspergillus infection, serum IL-17 levels were significantly elevated in Aspergillus-infected mice compared with controls, and the lung Aspergillus load was almost twice as high in IL-17 knockout mice as compared with controls (Geng et al., 2020). In a study of chronic sinusitis, plasma IL17 levels were found to be significantly higher in patients with combined Aspergillus infections compared to controls (Rai et al., 2018). In the study conducted by Hassan et al., elevated serum IL-17 levels were observed in patients with end-stage liver disease who had invasive fungal infections, particularly Aspergillus infections (present in 41.67% of cases), compared to those with non-invasive fungal infections. In our previous study, we found that plasma IL17 was significantly higher in IPA patients with bronchiectasis than in controls (Hassan et al., 2014). Our current study is consistent with the above findings. In a study of bronchiectasis combined with Aspergillus infection, the sensitivity (78.8%) and specificity (71.9%) at the optimal cut-off value were higher than in this study (He et al., 2022). We believe that differences in the study populations may account for the differences in results. In animal experiments, Kimura et al. observed that levels of IL-17 were markedly increased in BALF from mice infected with Aspergillus fumigatus, which correlated with GM levels. Administration of posaconazole resulted in a notable decrease in IL-17 levels in BALF (Kimura et al., 2017). In our study, we found a significant elevation of IL-17 in the BALF compared to the control group. At the optimal cutoff value, the sensitivity of IL-17 in plasma and BALF was significantly higher than that of GM, but the specificity was lower than that of GM.

For patients who cannot undergo bronchoscopy or have difficulty obtaining bronchoalveolar lavage fluid, the sensitivity of diagnosing invasive IPA can be significantly enhanced through combined plasma diagnostics. this study found that the sensitivity (67.7%) of plasma GM combined with IL-17 in diagnosing IPA in patients with non-neutropenic was significantly higher than that of plasma GM, while there was no significant difference in specificity (83.1%). However, the sensitivity of GM combined with IL-17 in diagnosing IPA in patients with bronchiectasis in He’s study (He et al., 2022) (81.8%) was higher than that in this study. The observed differences may be attributed to the following reasons. Firstly, our study included patients with autoimmune conditions who were receiving immunosuppressive therapy, which can affect IL-17 levels. Secondly, there was a significant increase in the number of patients using glucocorticoids in our study compared to previous research(46.77% vs 30.3%), leading to a poorer IL-17 response. Additionally, previous studies exclusively enrolled bronchiectasis patients, who may be more prone to fungal colonization, thus contributing to IL-17 response. In the combined diagnostic assay of BALF GM and IL17, the sensitivity and specificity could reach more than 80%, and there was a significant increase in sensitivity compared with BALF GM. It was also more sensitive than the combined diagnosis of plasma GM and IL17.

Current research suggests that specific antigens of Aspergillus can stimulate immune cells in peripheral blood, such as generating antigen-specific T cells (Dellière and Aimanianda, 2023). These specific T cells are capable of producing IL-10, IFN-γ, IL-4, and IL-17A. A study found successful generation of Aspergillus-specific T cells in all 8 cases of IPA patients examined, which exhibited cytotoxic subsets capable of lysing Aspergillus hyphae. These antigen-specific T cells can be expanded through short-term culture. They primarily target Aspergillus cell wall antigens, tend to increase during IPA, and are associated with better clinical outcomes (Potenza et al., 2013). Detecting Aspergillus-specific T cells may potentially enhance the diagnosis of IPA. However, in clinical practice, most T cell immune analyses require mononuclear cell isolation, which limits their robustness and practicality, hindering their broader applicability in clinical settings. Therefore, in recent years, researchers have explored a method using dual ɑ-CD28 and ɑ-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus antigens. This approach strongly enhances the release of T cell signature cytokines induced by Aspergillus, detected through enzyme-linked immunosorbent assay (ELISA). Compared to controls, IPA patients tested using this method exhibited a median concentration of key T helper cell cytokines at least 7 times higher, including IL-17 and Th2 cytokines IL-4 and IL-5, demonstrating the method’s high discriminative capability (Lauruschkat et al., 2021).Therefore, future research will continue to explore the use of antigen-stimulated functional immune assays to enhance the ability to diagnose IPA from peripheral blood samples.

In conclusion, the sensitivity of IL-17 for the diagnosis of IPA in patients with non-neutropenic was better than that of GM. The combined detection of GM and IL17 in lavage fluid significantly improved the diagnosis of IPA in patients with non-neutrophilic deficiency; in patients who cannot undergo invasive procedures, the combined detection of GM and IL-17 in plasma was also helpful in the diagnosis of IPA.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The study was approved by the Institutional Review Board of Changzhou first people’s Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

QH: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing – original draft, Writing – review & editing. JC: Conceptualization, Methodology, Project administration, Writing – original draft. ZM: Supervision, Validation, Visualization, Writing – review & editing. CF: Conceptualization, Investigation, Methodology, Writing – review & editing.