Non-invasive aspergillosis following COVID-19 exacerbates the severity of SARS-CoV-2 infection

Non-invasive aspergillosis following COVID-19 exacerbates the severity of SARS-CoV-2 infection | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 9 May 2025 V1 Latest version Share on Non-invasive aspergillosis following COVID-19 exacerbates the severity of SARS-CoV-2 infection Authors : Jong Seung Kim 0000-0002-1384-6799 , Jun Hyung Park 0009-0005-4614-0249 , Juhyun Kim , Min Ji Kim 0009-0008-9774-4239 , Wankyu Kim , Yong-Chul Lee , and Jae Seok Jeong 0000-0002-4635-8302 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174677788.84836140/v1 294 views 121 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: A limited number of large-scale population-based studies regarding the causality between COVID-19 and respiratory aspergillosis exist. Herein, using nationwide data, we investigated whether SARS-CoV-2 infection increases incidence of respiratory aspergillosis and impact of COVID-19-associated aspergillosis on severity of COVID-19. Further, to assess the biological impact of SARS-CoV-2 infection on airway structural and immune cells, we analyzed publicly available COVID-19 transcriptomic datasets. Methods: Utilizing a nationwide cohort of 8.5 million clinical registries, we included over 550,000 patients diagnosed with COVID-19 between October 8, 2020, and December 31, 2021, along with control-matched group. The primary outcomes were aspergillosis incidence, including both invasive and non-invasive forms, and its impact on the severity of COVID-19. Results: COVID-19 was closely associated with increased incidence of subsequent respiratory aspergillosis. Comorbidities, including diabetes and COPD, increased the incidence of fungal infections in COVID-19 patients. Regarding severity of COVID-19, both invasive and non-invasive aspergillosis exacerbated the severity of the disease. Particularly, systemic corticosteroids had an overwhelming impact on the increased severity and mortality in both forms of aspergillosis. Notably, antifungal-related genes and pathways, including CCR6, CXCL9, and CX3CR1, were consistently downregulated following SARS-CoV-2 infection and/or corticosteroid treatment. Conclusions: Our findings indicate that COVID-19 increases the incidence of respiratory aspergillosis. Moreover, respiratory aspergillosis, irrespective of its clinical invasiveness, significantly exacerbates the severity of COVID-19. Well-designed studies on the therapeutic potential of antifungal agents to improve the outcomes of COVID-19 are warranted. Non-invasive aspergillosis following COVID-19 exacerbates the severity of SARS-CoV-2 infection Running title: Aspergillosis following COVID-19 Aggravates the disease Jong Seung Kim, M.D., Ph.D. 1,2,3,4* , Jun Hyung Park 1* , Juhyun Kim 5* , Min Ji Kim, M.S. 3 , Wankyu Kim, Ph.D. 5† , Yong Chul Lee, M.D., Ph.D. 1,4† and Jae Seok Jeong, M.D., Ph.D. 1,4,6† 1 Research Center for Pulmonary Disorders, Research Institute of Clinical Medicine of Jeonbuk National University-Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju, Republic of Korea 2 Department of Otorhinolaryngology-Head and Neck Surgery, Jeonbuk National University Medical School, Jeonju, Republic of Korea 3 Department of Medical Informatics, Jeonbuk National University Medical School, Jeonju, Republic of Korea 4 Respiratory Drug Development Research Institute, Jeonbuk National University Medical School, Jeonju, Republic of Korea 5 Department of Life Sciences, Ewha Womans University, Seoul, Republic of Korea 6 Laboratory of Respiratory Immunology and Infectious Diseases, Korea Zoonosis Research Institute¸ Jeonbuk National University, Iksan, Republic of Korea The authors declare no conflict of interest. The authors declare no use of AI tools. *These authors contributed equally to this work † Correspondence: JSJ, YCL, WK Jae Seok Jeong, M.D., Ph.D. Department of Internal Medicine, Jeonbuk National University Hospital, Geonji-ro 20, Geumam-dong, Deokjin-gu, Jeonju, 54907, Republic of Korea Laboratory of Respiratory Immunology and Infectious Diseases, Korea Zoonosis Research Institute¸ Jeonbuk National University, Iksan, Republic of Korea Phone: 82-63-259-3604; Fax: 82-63-250-1468; E-mail: [email protected] Yong Chul Lee, M.D., Ph.D. Department of Internal Medicine, Jeonbuk National University Medical School, Geonji-ro 20, Geumam-dong, Deokjin-gu, Jeonju, 54907, Republic of Korea Phone: 82-63-250-1664; Fax: 82-63-254-1663; E-mail: [email protected] Wankyu Kim, Ph.D. Department of Life Sciences, Ewha Womans University, Ewhayeodae-gil 52, Seodaemun-gu, Seoul, 03760, Republic of Korea Phone: 82-2-3277-4132; Fax: 82-2-3277-4295; E-mail: [email protected] Contributors: JHP, YCL, JK, WK and JSJ interpreted the data and wrote the manuscript. JSK, MJK, and JK conducted statistical planning and data analysis. JSJ initially conceived the study. JSJ, JSK, WK and YCL designed and supervised the project. All the authors critically reviewed and approved the final version of the manuscript. Acknowledgments: This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. RS-2024-00356349; JSJ, No. RS-2022-NR069855; WK) and Special Operating Subsidy of Jeonbuk National University Industrial Cooperation Foundation. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2024-00440408; JSJ). This research was supported by the Bio&Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. RS-2023-00236157; JSJ). This paper was supported by Fund of Biomedical Research Institute, Jeonbuk National University Hospital. Word count: 3722 Background: A limited number of large-scale population-based studies regarding the causality between COVID-19 and respiratory aspergillosis exist. Herein, using nationwide data, we investigated whether SARS-CoV-2 infection increases incidence of respiratory aspergillosis and impact of COVID-19-associated aspergillosis on severity of COVID-19. Further, to assess the biological impact of SARS-CoV-2 infection on airway structural and immune cells, we analyzed publicly available COVID-19 transcriptomic datasets. Methods: Utilizing a nationwide cohort of 8.5 million clinical registries, we included over 550,000 patients diagnosed with COVID-19 between October 8, 2020, and December 31, 2021, along with control-matched group. The primary outcomes were aspergillosis incidence, including both invasive and non-invasive forms, and its impact on the severity of COVID-19. Results: COVID-19 was closely associated with increased incidence of subsequent respiratory aspergillosis. Comorbidities, including diabetes and COPD, increased the incidence of fungal infections in COVID-19 patients. Regarding severity of COVID-19, both invasive and non-invasive aspergillosis exacerbated the severity of the disease. Particularly, systemic corticosteroids had an overwhelming impact on the increased severity and mortality in both forms of aspergillosis. Notably, antifungal-related genes and pathways, including CCR6, CXCL9, and CX3CR1, were consistently downregulated following SARS-CoV-2 infection and/or corticosteroid treatment. Conclusions: Our findings indicate that COVID-19 increases the incidence of respiratory aspergillosis. Moreover, respiratory aspergillosis, irrespective of its clinical invasiveness, significantly exacerbates the severity of COVID-19. Well-designed studies on the therapeutic potential of antifungal agents to improve the outcomes of COVID-19 are warranted. key words: COVID-19, Respiratory aspergillosis, Transcriptome Word count: 233 Introduction Fungi are ubiquitous in daily life and often infect patients with various immune states. Aspergillus fumigatus , a saprotrophic fungus dominant in indoor and outdoor environments, imposes a considerable impact on the respiratory system. 1 In modern medicine, the widespread use of immunomodulatory agents has increased the incidence and mortality of invasive aspergillosis, especially in immunocompromised patients. 2 Therefore, invasive aspergillosis poses a substantial threat to public health, as exemplified by the marked rise in mortality. 3,4 Moreover, even in immunocompetent hosts, accumulating evidence has demonstrated an increasing incidence of aspergillosis, posing a significant burden on public health. 5,6 In the era of coronavirus disease 2019 (COVID-19) , mounting studies have shown an increasing incidence of mycosis associated with viral infections. 7 In particular, invasive aspergillosis, such as COVID-19-associated pulmonary aspergillosis (CAPA) , has been increasingly reported in patients with severe COVID-19. 4 Although the epidemiological impact of viral infections on the incidence and severity of fungal diseases was repeatedly reported during the 2009 influenza A virus pandemic, the causality between COVID-19 and invasive aspergillosis has yet to be clearly elucidated on a nationwide basis. 5 Furthermore, whereas studies evaluating CAPA are accumulating, the outcomes of COVID-19-associated respiratory non-invasive aspergillosis (CARNIA) remain unclear, and there is a paucity of large-scale, nationwide population-based cohort research on the impact of non-invasive aspergillosis on patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Recently, we performed preclinical mechanistic studies using animal models of non-invasive aspergillosis and demonstrated the impact of this condition on the increased severity and mortality in COVID-19. 8 Using nationwide data derived from the largest population cohort available to date, we focused on whether SARS-CoV-2 infection increases the incidence of respiratory aspergillosis. Moreover, we aimed to provide insights into the impact of COVID-19-associated aspergillosis on the severity of COVID-19 with a particular focus on CARNIA . Importantly, to further validate our epidemiological findings, we investigated whether SARS-CoV-2 infection and associated viral-targeted corticosteroid therapy could influence the antifungal defense of the host using a publicly available COVID-19 dataset. Materials and Methods Study Design and Data Source In the primary analysis, the cohort was divided into the COVID-19 and matched control groups to assess the presence (primary outcome) and severity (the other primary outcome) of aspergillosis. This study used data from the National Health Insurance Service-National Health Information Database (NHIS-NHID) (NHIS-2022-1-623). Nearly every person in the Republic of Korea is enrolled in the National Health Insurance System, thus providing access to comprehensive nationwide COVID-19 medical data. The NHIS database includes demographic data, such as age, sex, economic status, and residential area, as well as clinical data including medical records, diagnoses, prescriptions, and medication information. The NHIS-NHID database encompasses detailed records of approximately 580,000 confirmed SARS-CoV-2 cases and about 7.9 million individuals in the control group, covering the period from 2020 to 2021. This dataset integrates demographic information, comorbidity profiles, and medical histories collected between 2015 and 2019. All data were anonymized to ensure privacy protection. Study Population and Operational Definitions The study period was from October 8, 2020, to December 31, 2021 (Figure 1). The COVID-19 group included individuals with laboratory-confirmed COVID-19 and those diagnosed with the International Classification of Diseases, 10th Revision (ICD-10) code U07.1 during this period. The control group comprised individuals matched in a 1:1 ratio with the COVID-19 group, using propensity score (PS) matching to ensure similar demographics and medical histories. The primary outcome was the occurrence of aspergillosis, and the other primary outcome included the following severity indicators: 1) admission to the intensive care unit (ICU), 2) extracorporeal membrane oxygenation treatment, 3) intubation or mechanical ventilation, 4) death, and 5) oxygen supply. Follow-up was conducted until the occurrence of the aforementioned outcomes, death, or the study end date (December 31, 2021), whichever came first . 9 Systemic steroid therapy within 2 weeks after SARS-CoV-2 infection included intravenous dexamethasone or oral prednisolone administration. Comorbidities were defined using data from the previous 5 years. The comorbidities identified using the ICD-10 codes included angina (I20), cancer (any C code), congestive heart failure (CHF; I43 or I50), chronic kidney disease (CKD; N18 or N19), myocardial infarction (MI; I21 or I22), diabetes mellitus (DM; E10–14), hypertension (HTN; I10–13, I15), asthma (J43 or J44), chronic obstructive pulmonary disease (COPD; J45–46), and chronic liver disease (K74, K703, or B18). Patients with at least one diagnosis within the last 5 years were included in the study. Aspergillosis was identified using ICD-10 codes B44 and B49, with invasive fungal aspergillosis classified under B440 and non-invasive aspergillosis under B441–B449 or B49. Statistical Analysis Descriptive statistics are expressed as frequencies (percentages) for categorical data and means ± standard deviations for continuous data. The standard mean difference (SMD) was used to quantify the differences between the groups, with an SMD < 0.1 indicating a balanced distribution. The COVID-19 and matched control groups were analyzed after 1:1 PS matching to ensure balanced baseline characteristics. Hazard ratios (HRs) were calculated using the Cox regression model. Unadjusted HRs were based on the Kaplan–Meier formula without considering other variables, whereas adjusted HRs accounted for all covariates. Statistical significance was determined using two-sided p-values < 0.05 and 95% confidence intervals (CIs). Analyses were performed using SAS 9.4 and R 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria). Transcriptome Analysis We collected a transcriptome dataset of SARS-Cov-2 infected cell lines from the NCBI Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). The datasets included the transcriptome profiles of human lung adenocarcinoma (Calu-3), normal human bronchial epithelial (NHBE) (GSE147507), and human airway epithelial (HAE) cells (GSE153970). The transcriptomes of blood samples from patients with COVID-19 were obtained from EBI ArrayExpress (https://www.ebi.ac.uk/biostudies/arrayexpress) (E-MTAB-10926). We calculated the expression profiles of approximately 20,000 protein-coding genes for downstream analysis. Differentially expressed genes (DEGs) were identified using DESeq2 10 (FC > 1.5 for cell lines, FC > 2 for blood samples, and FDR < 0.05 [Benjamini-Hochberg method] for blood samples). Pathway enrichment was calculated as an enrichment factor (EF). \begin{equation} EF=\frac{n\left(A\cap B\right)+1}{\frac{n\left(A\right)*n\left(B\right)}{N}+1}\nonumber \\ \end{equation} where A is the number of DEGs, B is the number of pathway genes, and N is the total number of genes. Pathway information and protein-protein interaction (PPI) networks were obtained from MSigDB (v2023.2 11 ) and STRING (v12.0 12 ), respectively. Ethics Approval The study protocol was approved by the Institutional Review Board of Jeonbuk National University Hospital (No. 2024-02-014). The requirement for informed consent was waived because all patient records were anonymized before use. Results Baseline Characteristics The baseline characteristics of the study population are shown in Supplementary Table 1. The COVID-19 (n = 557,680) and control (n = 557,680) groups were well matched in terms of demographics such as sex, age, economic status, and residential area, as well as pre-existing conditions such as HTN, DM, CKD, CHF, cardiovascular disease (CVD), cancer, MI, asthma, and COPD (all SMDs < 0.1, Figure 1). Incidence and Risk of Aspergillosis in Patients with COVID-19 The incidence and risk of new-onset aspergillosis were compared between COVID-19 and PS-matched control cohorts. New-onset aspergillosis occurred in 0.06% of the COVID-19 group (353 of 557,680) and 0·02% of the control group (94 of 557,680), with incidences of 1.99 and 0.52 per 1000 person-years, respectively (Supplementary Table 2). A significant 3.81-fold difference was observed in the incidence of aspergillosis between the COVID-19 and the matched control groups (HR, 3.81; 95% CI: 3·03–4·78; p < 0.001; Figure 2A). Subgroup analysis showed that the risk of COVID-19-associated respiratory invasive aspergillosis (CARIA) was 27.58 times higher (95% CI: 12.21–62.30) and that for CARNIA was 2.18 times higher (95% CI: 1.69–2.80) in the COVID-19 group compared to the control group (Figures 2B and 2C, Supplementary Table 2). Notably, the risk of aspergillosis was 1.95 times higher (95% CI: 1.51–2.51) in patients with COVID-19 not receiving systemic steroids and 15·68 times higher (95% CI: 12.21–20.14) in patients receiving systemic steroids (Figure 2A, Supplementary Figure 1, Supplementary Table 2). Furthermore, the risk of CARIA was 9.27 times higher (95% CI: 3.96–21.70) in patients with COVID-19 not receiving systemic steroids and 124.89 times higher (95% CI: 54.86–284.34) in patients receiving systemic steroids (Figure 2B, Supplementary Figure 2, Supplementary Table 2). The risk of CARNIA was 1.44 times higher (95% CI: 1.09–1.91) in patients not receiving systemic steroids and 7.35 times higher (95% CI: 5·39–10·02) in patients receiving systemic steroids (Figure 2C, Supplementary Figure 3, Supplementary Table 2). The risk of aspergillosis was higher in males than in females (HR: 1.34, 95% CI: 1.09–1.66), primarily for invasive aspergillosis (HR: 1.80, 95% CI: 1.30–2.48). Younger age was associated with a lower risk of aspergillosis (HR: 0.58, 95% CI: 0.43–0.80), particularly for invasive aspergillosis (HR: 0.18, 95% CI: 0.07–0.44). Individuals aged ≥ 65 years were associated with a higher risk of aspergillosis (HR: 1.81, 95% CI: 1.42–2.31) for both the invasive (HR: 2.17, 95% CI: 1.47–3.20) and non-invasive aspergillosis (HR: 1.59, 95% CI: 1.16–2.18). The presence of comorbidities such as DM, CKD, CHF, cancer, and COPD increased the risk of aspergillosis by 2.69 (95% CI: 2.14–3.38), 1.94 (95% CI: 1.38–2.72), 1.68 (95% CI: 1.32–2.14), 1·34 (95% CI: 1.09–1.66), and 2.36 (95% CI: 1.82–3.07) times, respectively. This trend was consistent for both the CARIA and CARNIA cohorts, except for cancer in CARIA (p = 0.711) and CHF in CARNIA (p = 0.191) (Figures 2A-C, Supplementary Table 2). Economic status and residential area were not significantly associated with aspergillosis risk (HR: 0.99, 95% CI: 0.81–1.20 and HR: 0.96, 95% CI: 0.80–1.16, respectively). Similarly, other comorbidities such as HTN, CVD, MI, and asthma were not significantly associated with the risk of aspergillosis (HR: 1.26, 95% CI: 0.98–1.61; HR: 1.20, 95% CI: 0.94–1.54; HR: 1.17, 95% CI: 0.85–1.62; and HR: 0.69, 95% CI: 0.48–1.01, respectively) (Figure 2A-C, Supplementary Table 2). Incidence and Risk of Severe COVID-19 in Patients with Aspergillosis The incidence and risk of severe COVID-19 in patients with aspergillosis were compared between COVID-19 and PS-matched control cohorts. Severe clinical outcomes occurred in 52.9% of patients with aspergillosis in the COVID-19 group (108 204) and 3.96% of those in the matched control group (22,055 of 557,476), with an incidence of 3254.89 and 165.03 per 1000 person-years, respectively (Figures 3A and 3D, Supplementary Table 3). Patients with aspergillosis had a 1.67-fold higher risk (95% CI: 1.38–2.02) of severe COVID-19 outcomes than those without. More importantly, this was consistent for both CARIA (HR: 1.85, 95% CI: 1.44–2.38) and CARNIA (HR: 1.46, 95% CI: 1.09–1.96) (Figure 3B-D, Supplementary Table 3). This trend was more pronounced in males than in females (HR: 1.15, 95% CI: 1.12–1.18) for both CARIA and CARNIA. Younger age was associated with a lower risk of severe COVID-19 (HR: 0.50, 95% CI: 0.48–0.52), while older age was associated with a higher risk (HR: 1.25, 95% CI: 1.21–1.29); these findings were consistent for both CARIA and CARNIA. Urban residents had a 1.11-fold higher risk of severe COVID-19 outcomes compared to other residents (95% CI: 1.08–1.14) (Figure 3D, Supplementary Table 3). Comorbidities such as HTN, DM, CKD, CHF, CVD, MI, and COPD were associated with an increased risk of severe COVID-19 outcomes by 1.10 (95% CI: 1.06–1.14), 1.22 (95% CI: 1.18–1.26), 1.25 (95% CI: 1.18–1.33), 1.27 (95% CI: 1.22–1.31), 1.23 (95% CI: 1.19–1.28), 1.09 (95% CI: 1.03–1.15), and 1.17 (95% CI: 1.11–1.22) times, respectively. These associations were consistent in both CARIA and CARNIA (Figure 3D, Supplementary Table 3). Notably, the risk of severe COVID-19 outcomes was 23.54 times higher (95% CI: 22.88–24.22) in patients receiving systemic steroids than in those that did not (Figure 3D, Supplementary Table 3). Transcriptomic Changes after SARS-CoV-2 Infection and Steroid Treatment To explore the molecular pathobiological context of our epidemiological findings, we analyzed the transcriptome profiles of SARS-CoV-2-infected epithelial cell lines and COVID-19 patient blood samples (Figure 4A). We focused specifically on the immune and inflammatory pathways associated with fungal infections. First, we compared the DEGs of the three infected epithelial cell lines and identified 121 down-DEGs common to at least two cell lines. For patient blood samples, we observed a distinct grouping of expression patterns according to disease severity and steroid treatment/non-treatment (Figure 4B). Consistent with our nationwide epidemiological findings, the antifungal and immune-related pathways were consistently downregulated by SARS-CoV-2 infection, disease progression, and steroid treatment (Figure 4C). In blood samples from patients with COVID-19, these results suggest an attenuation of early T helper 2 (Th2)-type response involving interleukin-5 (IL-5), which induces eosinophil-mediated immune clearance in the immune cells of these patients. 13 Additionally, this attenuation leads to the downregulation of subsequent IL-17 signaling, which reduces A. fumigatus fungal burden 14 in both epithelial and immune cells. Moreover, we observed a decrease in the function of natural killer (NK) cells in blood samples. This dysfunction of NK cell responses may contribute to reduced cytotoxic activity by perforin, compromising antifungal defense. 15 Furthermore, in SARS-CoV-2-infected epithelial cells, reductions in lipid metabolism involving cholesterol and steroid hormones were noted. Interestingly, lipid metabolic pathways such as lipid export from cell, cholesterol metabolism, and surfactant metabolism are known to play a crucial role in antifungal immunity within the host bronchial epithelial cell. 16 In addition, one of the most important lipids in the airway defense, surfactants, are necessary for phagocytosis ( e.g. SP-A and SP-D) 17 and enhance antifungal drug activity. 18 Lastly, phosphatidylinositol 3-kinase (PI3K) signaling pathways, which have been known to be involved in the recognition of A. fumigatus conidia by neutrophils via integrin CD11b/CD18, 19 were also significantly downregulated in both SARS-CoV-2-infected epithelial cells and COVID-19 immune cells in our data. We then investigated the interactions between the downregulated pathway genes using the STRING protein-protein network (Figure 4D). 12 These genes are densely connected to each other in the networks of both disease and treatment cases (p < 0.000175 and p < 0.000167, respectively, estimated by sampling 1,000,000 random subnetworks of the same number of nodes). Interferon-gamma (IFN-γ) signaling genes are enriched with the highly connected nodes. CD4, IFNG, and CXCL9, related to IFN-γ signaling, emerged as significant nodes in our network. CD4 is the top connected node in both networks, which activates T cells by toll-like receptor (TLR)-dependent IFN-γ production. 20 IFN-γ shows a synergistic effect with antifungal drugs such as amphotericin B to treat invasive aspergillosis. 21 IFN-γ also induces CXCL9, a CXC chemokine 22 that enhances NADPH oxidase activity through plasmacytoid dendritic cells (DCs). 23 Specifically, CD4 is consistently downregulated and is the most connected node across all networks, while IFNG and CXCL9 are prominently present only in the disease and treatment networks, respectively. Additionally, CD3E and CCR6 are notably connected within both networks and CX3CR1, stimulated by CD3, is observed specifically in the treatment network. Among the downregulated DEGs, CD3E has anti-inflammatory and repair effects in mouse A. fumigatus keratitis and regulates IL-10. 24 CX3CR1 is stimulated by CD3 25 and is negatively correlated with the risk of invasive aspergillosis. 26 CCR6 mediates the migration of myeloid dendritic cells (DCs) in the host challenged with A. fumigatus , 27 and high central in both networks. Discussion This is the first report to conduct a comprehensive analysis of a substantial nationwide cohort of 8.5 million clinical registries with CARIA and CARNIA in the respiratory system. The incidence of both forms of aspergillosis has increased in patients with a SARS-CoV-2 infection. Notably, it was corroborated that the severity including mortality of COVID-19 increased remarkably not only in patients with CARIA, but also in those with CARNIA. Recent studies have reported an increased incidence of invasive aspergillosis in patients with COVID-19 in the ICU, and various underlying pathophysiological mechanisms have been suggested. 3,4,28 Our data validated the increased incidence of CARIA. SARS-CoV-2 infection itself is a cardinal risk factor for developing CARIA, and as previously known, comorbidities also contribute to the increased incidence. 28 Interestingly, the incidence of CARNIA significantly increased in patients with COVID-19. Of particular importance is the substantially increased incidence of CARNIA in individuals with comorbidities, such as DM and COPD. Regarding mechanisms explaining the rise in the incidence of invasive aspergillosis, alterations in both innate and adaptive host immunity or the airway epithelial damage triggered by COVID-19 itself, are attributable to subsequent CARIA. 3,28 Moreover, although little is known about CARNIA, we have recently demonstrated through preclinical models of A. fumigatus-induced severe allergic lung inflammation, one of the clinical spectrums of non-invasive aspergillosis, that a COVID-19-induced dysregulation of innate immune response is closely involved in the clinical course of the disease . 8 Thus, immunological changes owing to viral infections in the micromilieu surrounding the respiratory epithelium could prime the host respiratory tract for the development and aggravation of subsequent aspergillosis, including both invasive and non-invasive forms. Furthermore, CAPA, a term tending towards invasive aspergillosis, has been reported to increase the mortality of patients with COVID-19, and in accordance with previous studies, the severity was 1.85 times higher in invasive aspergillosis. 3,4 Notably, the severity was also 1.46 times higher even in non-invasive aspergillosis, which was corroborated for the first time. The increased severity of COVID-19 caused by CARNIA was also substantiated for the first time. Although myriad mechanisms may play a role, various factors, including hyperinflammation, comorbidities, and medical treatment, may be implicated. With respect to CARIA, invasive aspergillosis is reported to increase the severity of COVID-19 in patients through a cytokine storm. 28,29,30 More importantly, despite the limited knowledge on non-invasive aspergillosis, we have previously demonstrated with animal models and large clinical data that dysregulated immune response associated with underlying non-invasive pulmonary aspergillosis contributes to the increased mortality of patients with COVID-19. CARNIA may activate several crucial innate immune components in the cells, such as NLRP3 inflammasome, thereby inducing pro-inflammatory cytokines (interleukin [IL]-1, IL-6, IL-17, and tumor necrosis factor) that increase the severity of COVID-19. 8 This exuberant immune response, namely hyper-inflammation, results in the accumulation of neutrophils that mediate destruction of the hyphae via reactive oxygen species (ROS) and neutrophil extracellular traps (NETs). 31 Uncontrolled ROS and NETs propagate pulmonary inflammation and contribute to pneumonia or acute respiratory distress syndrome with thrombotic effects. Aberrant release of pro-inflammatory cytokines leads to increased vascular permeability and oedema, culminating in the cytokine storm and multiple organ failure. 32 This profound impact of dysregulated innate immune response on the severity of COVID-19 could explain the results observed in this cohort study. In the era of COVID-19, comorbidities, such as DM, COPD, HTN and CHF, have been highlighted as key drivers of increased COVID-19 severity. 33 Furthermore, our group recently reported the impact of prior respiratory syncytial virus infection within 3 years on the severity of COVID-19. 9 In light of this, host-related medical factors, such as comorbidities and previous medical history, may play pivotal roles in increasing the severity of COVID-19 not only in patients with invasive aspergillosis but also in those with non-invasive aspergillosis. As expected, comorbidities including CKD, CHF, and CVD worsened COVID-19 in patients with subsequent aspergillosis. Additionally, our study confirmed that the increased severity of CARIA or CARNIA could be attributed to COVID-19-targeted treatment. Notably, systemic steroids have a profound effect on the severity of COVID-19 in patients with subsequent aspergillosis. This is consistent with the accumulating evidence that a prolonged treatment course with systemic steroid therapy in hospitalized patients with COVID-19 is correlated with higher mortality and poor outcomes. 34 Corticosteroids hamper phagocytosis and inflammatory mechanisms, thereby predisposing patients to secondary infections. 3,35,36 In fungal infections, corticosteroids hinder macrophage anti-conidial activity by repressing reactive oxidant intermediate (ROI) production, neutrophils, and Th cell type 1 cytokine production and increasing Th2 cytokines, leading to the inhibition of the response against aspergillus. 37 Additionally, the hyperglycemia frequently encountered in patients undergoing steroid therapy may compound the antifungal response and lead to increased severity. 3 Furthermore, long-term corticosteroid use may induce various fungal infections, such as fungal rhinosinusitis and chronic necrotizing pulmonary aspergillosis. 2,38 Although not included in our study, side-effects from various medical treatments other than steroids, including excessive use of broad-spectrum antibiotics, may play previously unrecognized roles in the increased incidence and severity of fungal infections. Disturbance of the microbiological composition and diversity of the normal flora in the respiratory tract due to antibiotics may predispose patients to subsequent fungal infections, including allergic bronchopulmonary aspergillosis. 2 Moreover, in patients with COVID-19 receiving excessive oxygen treatment, increased oxidative stress and excessive ROS generation may have detrimental effects on the outcome. 39 However, further studies are required to delineate clear mechanisms. Importantly, the findings from our epidemiological data were validated at the molecular level using a public dataset, particularly for human-derived systems, including human cell lines and patient blood samples. Notably, SARS-CoV-2 infection weakened the antifungal defense pathway. Marked downregulation of T-cell responses, recognized as critical components of antifungal immunity, was observed in both structural and immune cells. In addition, IL-5 signaling and NK cell-mediated immunity were significantly diminished in the blood samples. Furthermore, the suppression of lipid metabolism and PI3K/Akt signaling pathways, both of which are known to play pivotal roles in fungal defense, was evident. Through supplementary analysis using the STRING protein–protein interaction network, we further identified a cluster of highly interconnected nodes associated with the observed impairment in antifungal immunity, particularly involving genes related to IFN-γ signaling. Notably, the CD4, IFNG, CXCL9, CD3E, CCR6, and CX3CR1 genes emerged as central components within this network. These genes have previously been recognized as key regulators of antifungal defense, thereby reinforcing the validity of our epidemiological findings. 20-27 These nodes represent promising targets for preclinical research that focuses on patients vulnerable to secondary fungal infections following viral illnesses. Targeting these molecules may pave the way for translational strategies aimed at developing novel pathogen-specific therapeutics. Conclusion In this study, utilising hitherto the largest cohort of 8.5 million clinical registries, we demonstrated that SARS-CoV-2 increases the incidence of both invasive and non-invasive forms of aspergillosis in the respiratory system. Notably, in addition to the well-known impact of CAPA on the severity of COVID-19, our data demonstrate, for the first time, that CARNIA significantly increases the severity of COVID-19. Moreover, systemic steroids have deleterious effects on the severity of COVID-19 in patients with subsequent aspergillosis. Our results highlight the therapeutic potential of antifungal agents for both invasive and non-invasive aspergillosis to improve the outcomes of patients infected with respiratory viruses, although results from well-designed clinical trials are warranted in the near future. 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The primary analysis divided the cohort into COVID-19 and matched control groups to assess the presence (primary outcome) and severity (the other primary outcome) of aspergillosis. This study used data from the National Health Insurance Service-National Health Information Database (NHIS-NHID) (NHIS-2022-1-623). The NHIS-NHID database encompasses detailed records of approximately 580,000 confirmed SARS-CoV-2 cases and about 7.9 million individuals in the control group, covering the period from 2020 to 2021. This dataset integrates demographic information, comorbidity profiles, and medical histories collected between 2015 and 2019. The COVID-19 (n = 557,680) and control (n = 557,680) groups were well-matched in terms of demographics, such as sex, age, economic status, and residential area, as well as pre-existing conditions, such as HTN, DM, CKD, CHF, cardiovascular disease (CVD), cancer, MI, asthma, and COPD (all SMDs < 0.1). Figure.2 The impact of COVID-19 on the incidence of aspergillosis. (A) Cumulative incidence rate for the aspergillosis susceptibility in the COVID and control (non-COVID) groups; forest plot of the adjusted hazard ratio for each factor: prior COVID-19 infection, use of systemic steroids, demography (male, old age ≥ 65 years), socioeconomic status, comorbidities. (B) Cumulative incidence rate for CARIA susceptibility in the COVID and control (non-COVID) groups; forest plot of the adjusted hazard ratio for each factor: prior COVID-19 infection, use of systemic steroids, demography (male, old age ≥ 65 years), socioeconomic status, comorbidities. (C) Cumulative incidence rate for CARNIA susceptibility in the COVID and control (non-COVID) groups; forest plot of the adjusted hazard ratio for each factor: prior COVID-19 infection, use of systemic steroids, demography (male, old age ≥ 65 years), socioeconomic status, comorbidities. Figure.3 The impact of aspergillosis on COVID-19 severity. (A) Cumulative incidence rate for the severity after COVID-19 in the aspergillosis and control (no aspergillosis) groups. (B) Cumulative incidence rate for the severity after COVID-19 in the CARIA and control (no CARIA) groups. (C) Cumulative incidence rate for the severity after COVID-19 in the CARNIA and control (no CARNIA) groups. (D) Forest plot of the adjusted hazard ratio for each factor: aspergillosis, CARIA, CARNIA group at risk for severe COVID-19, demography (male, old age ≥ 65 years), socioeconomic status, comorbidities. Figure.4 The antifungal responses are downregulated at the transcriptional level. (A) A schematic representation of the public transcriptome dataset, which was obtained from two different sources (cell lines and blood) and categorized based on study design: SARS-CoV-2 infection and steroid treatment after infection. (B) Principal component analysis (PCA) plot of the study design. Genes differentially expressed in cell lines overlapped with each other. (C) Significantly enriched pathways with downregulated DEG sets were categorized into five groups: antifungal response, immune response, lipid metabolism, PI3K/Akt signaling, and TGF-β signaling. (D) Protein-protein interaction (PPI) involved in five key groups of pathways. The interactions were obtained from STRING and filtered using a confidence score cutoff (> 0.7). Inner nodes are colored by the fold change of gene expression (log2FC) in immunological aspects, and the outer color represents mean log2FC in structural aspects. The weight of edge indicates the interaction strength between proteins. Overlapped proteins between disease and treatment effect are denoted by diamond. Figure.4 Supplementary Material File (figures_merged.pdf) Download 2.18 MB File (supplementary figures and tables_2.pdf) Download 131.01 KB Information & Authors Information Version history V1 Version 1 09 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords bioinformatics covid epidemiology infections prevention sars-cov Authors Affiliations Jong Seung Kim 0000-0002-1384-6799 Jeonbuk National University Hospital Biomedical Research Institute View all articles by this author Jun Hyung Park 0009-0005-4614-0249 Jeonbuk National University Hospital Biomedical Research Institute View all articles by this author Juhyun Kim Ewha Womans University View all articles by this author Min Ji Kim 0009-0008-9774-4239 Jeonbuk National University Medical School View all articles by this author Wankyu Kim Ewha Womans University View all articles by this author Yong-Chul Lee Jeonbuk National University Hospital Biomedical Research Institute View all articles by this author Jae Seok Jeong 0000-0002-4635-8302 [email protected] Jeonbuk National University Hospital Biomedical Research Institute View all articles by this author Metrics & Citations Metrics Article Usage 294 views 121 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jong Seung Kim, Jun Hyung Park, Juhyun Kim, et al. Non-invasive aspergillosis following COVID-19 exacerbates the severity of SARS-CoV-2 infection. Authorea . 09 May 2025. DOI: https://doi.org/10.22541/au.174677788.84836140/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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