Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and has now been responsible for millions of hospitalizations (1, 2). Most patients recover from COVID-19 but some—mainly critically ill patients—are vulnerable to dangerous secondary infection by opportunistic pathogens (3–5). Among the relevant microbes, Aspergillus spp. is considered very serious and is potentially deadly, particularly if it induces invasive pulmonary aspergillosis (IPA) (3, 6, 7).

COVID-19-associated IPA (CAPA) is a life-threatening complication in intensive care unit (ICU) patients, and the reported incidence of CAPA varies from 4 to 35% (4, 5, 8–13). The variation in estimates may be partly due to different diagnostic approaches and definitions (3, 4, 6). Regardless of the definition used, IPA is reportedly associated with both viral and host factors (3–6). For example, SARS-CoV-2 can induce severe lung or immune damage that impairs the host's ability to clear the Aspergillus spp. Some critical patients have been immunocompromised due to diabetes and chronic kidney disease (14), which are associated with an increased risk of developing IPA (4, 14). Some drugs used to treat COVID-19 including steroids may also render patients more vulnerable to Aspergillus spp. infection (15). Notably however, few studies have focused on these host and infection variables in an effort to facilitate early stratification of COVID-19 patients at high risk of developing CAPA.

The current study was conducted to determine the incidence of CAPA in COVID-19 patients admitted to ICUs, and investigate risk factors associated with CAPA occurrence and long-term outcomes.

Early identification of CAPA in critically ill COVID-19 patients admitted to ICUs will facilitate the optimal use of medical resources. The current multicenter retrospective study investigated risk factors associated with CAPA, and long-term outcomes in COVID-19 patients admitted to ICUs. Patients with thrombocytopenia, vasopressor use before CAPA diagnosis, and the use of methylprednisolone at a daily dose of ≥40 mg were much more likely to develop CAPA.

The reported incidences of CAPA in patients admitted to ICUs vary widely from 2.5 to 39.1%, and in the current cohort it was 23.3% (8, 9, 20, 21). Differences may be associated with different diagnostic approaches or interpretations of IPA markers (3). BAL culture and galactomannan testing of BAL are a gold standard for IPA diagnosis, but BAL and autopsy are usually lacking in patients with COVID-19 due to fear of viral particles spreading. In the present cohort BAL culture was only performed in 62 (79.5%) CAPA patients, and in 48.7% of patients the BAL cultures were Aspergillus spp.-positive. Given the limited sensitivity of BAL Aspergillus spp. culture (13, 22–24), a serum galactomannan test—which was performed in 316 (94.3%) of 335 patients including 66 (84.6%) of 78 patients with CAPA—was used to classify patients in the present study. Of the patients with CAPA in the current cohort 61.5% yielded positive serum galactomannan tests.

In a study of 630 patients with influenza in the ICU, invasive aspergillosis was detected in 17%. The slightly lower incidence in an influenza cohort could be explained by the different pathogenesis of SARS-CoV-2 infection (18). In most influenza patients respiratory epithelium damage and mucociliary clearance dysfunction may facilitate invasion by Aspergillus spp. Unlike influenza however, in addition to respiratory system damage SARS-CoV-2 sends parts of the immune system into overdrive or depletes certain immune cells, impairing the lung's capacity to clear Aspergillus spp. or leaving the patient less able to fight off other infections (3, 6).

Steroids such as dexamethasone (6 mg daily dose) have been shown to curb overactive immune responses and improve survival rates in severely ill COVID-19 patients (25), but steroids are a double-edged sword because they can render the patient susceptible to other infections, particularly Aspergillus spp. In the current cohort the use of ≥40 mg of methylprednisolone (equivalent to 7.5 mg of dexamethasone) before CAPA diagnosis was independently associated with the development of invasive pulmonary aspergillosis, consistent with corticosteroid treatment increasing the risks of hyperglycemia and infection in severe COVID-19 (15). In a recent observational study COVID-19 patients received higher than recommended steroid doses and were likely have Aspergillus spp. infections (26), therefore IPA infections in critically ill COVID-19 patients should be inversely related to the dosage of immunosuppressive therapy.

Platelets have versatile antifungal immune functions, and thrombocytopenic hosts are highly susceptible to IPA and can rapidly succumb to infection (27–29). To the best of our knowledge no study has investigated the correlation between thrombocytopenia in COVID-19 patients and IPA susceptibility. In the present study thrombocytopenia was an independent risk factor for CAPA, but the underlying mechanisms are not clear. Mechanisms of IPA-related thrombocytopenia have been suggested (27–29). One is that when infected with Aspergillus spp. the galactosaminogalactan secreted by the pathogen is deposited on the surfaces of platelets, inducing their activation and resulting in fungal damage (27, 29, 30). Another is that the altered platelet surface triggers a complement cascade reaction that attracts and activates phagocytes to eliminate the invading Aspergillus spp. (27, 28). In the current cohort platelets were extremely low in critically ill patients with COVID-19, thus their susceptibility to IPA was increased.

In the present study mortality in patients with CAPA was 65.4% at 180 days after CAPA diagnosis, and the study is the first report on the long-term outcomes of CAPA. The rate is higher than that previously reported in patients admitted to the ICU with influenza-associated IPA at 90 days (51%) (18), and in other cohorts such as aspergillosis patients with phlebovirus (~40%) (31). The different incidence may be because the pathophysiology of COVID-19 differs substantially from that of influenza and phlebovirus infections. On 04 April 2021 the European Centre for Disease Prevention and Control (CDC) stated that in 186 patients with CAPA the mortality rate was 52.2%, which was similar to the ICU mortality in the current study (52.6%). The study was somewhat cross-sectional in that it included data at 6 and 12 weeks after CAPA diagnosis. The number of patients with CAPA had decreased by the 90-day timepoint because a lot of them had died by that time. In the present cohort, a number of patients with CAPA decreased after 90 days. Mortality was also considerably high in case series from France, Germany, Belgium, and the Netherlands, ranging from 44.5–66.7% (10, 11, 13, 32). Of particular importance was the 100% fatality rate of patients with underlying diseases reported in the Netherlands (10).

The current study had several limitations. Due to its retrospective design confounding cannot be ruled out and a standardized diagnostic approach toward IPA was not used. Considerations of mycological criteria and antigen testing were included in case classifications however, and the galactomannan index was beneficial for the early identification of CAPA patients at risk of a poor outcome. Thus, we believe that the diagnosis of CAPA in the study was more accurate and clinically pertinent. Another potential limitation was that 13 patients with only positive serum galactomannan data were categorized as CAPA patients, and this was likely to have included some false-positive cases. A higher serum galactomannan index represents an increased Aspergillus spp. burden however, because severe SARS-CoV-2 infection is likely to contribute to Aspergillus spp. micro-angioinvasion at the interface of the alveoli and blood vessels. Indeed, there was no difference in mortality between the galactomannan-positive and the galactomannan-negative groups among the CAPA patients in the study. Whether serum galactomannan should be considered as a standardized diagnostic approach should be a focus of future prospective studies.

Thrombocytopenia, vasopressor use before CAPA diagnosis, and the use of methylprednisolone at a daily dose ≥ 40 mg are independent predictors of the development of CAPA. The occurrence of CAPA increases the long-term mortality of critically ill COVID-19 patients. The results of the current study may help physicians to optimize the management of critically ill COVID-19 patients, and improve antifungal management.

All datasets generated for this study are included in the article/Supplementary Material. Deidentified participant data will be provided after approval from the corresponding author and study center.

Research approval (2020-0041-1) was granted by the institutional review board of Wuhan Union Hospital as the central coordinating center. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.

JX, ZL, XZ, LZ, WC, and BL collected the epidemiological and clinical data. JX, XY, and TZ summarized all data. JX, XY, HL, YY, and YS contributed to literature search and writing of the manuscript. YS, CH, SY, and MH designed the study, had full access to all data in the study, and take full responsibility for accuracy of the analyses and their interpretation. All authors contributed to the article and approved the submitted version.

The research was designed, conducted, analyzed, and interpreted by the authors entirely independently of the funding sources.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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