COVID-19–Associated Pulmonary Aspergillosis, March–August 2020

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 emerged in China at the end of 2019. Because of the severe immunomodulation and lymphocyte depletion caused by this virus and the subsequent administration of drugs directed at the immune system, we anticipated that patients might experience fungal superinfection. We collected data from 186 patients who had coronavirus disease–associated pulmonary aspergillosis (CAPA) worldwide during March–August 2020. Overall, 182 patients were admitted to the intensive care unit (ICU), including 180 with acute respiratory distress syndrome and 175 who received mechanical ventilation. CAPA was diagnosed a median of 10 days after coronavirus disease diagnosis. Aspergillus fumigatus was identified in 80.3% of patient cultures, 4 of which were azole-resistant. Most (52.7%) patients received voriconazole. In total, 52.2% of patients died; of the deaths, 33.0% were attributed to CAPA. We found that the cumulative incidence of CAPA in the ICU ranged from 1.0% to 39.1%.

Pneumonia caused by severe acute respiratory syndrome coronavirus 2 emerged in China at the end of 2019. Because of the severe immunomodulation and lymphocyte depletion caused by this virus and the subsequent administration of drugs directed at the immune system, we anticipated that patients might experience fungal superinfection. We collected data from 186 patients who had coronavirus disease-associated pulmonary aspergillosis (CAPA) worldwide during March-August 2020. Overall, 182 patients were admitted to the intensive care unit (ICU), including 180 with acute respiratory distress syndrome and 175 who received mechanical ventilation. CAPA was diagnosed a median of 10 days after coronavirus disease diagnosis. Aspergillus fumigatus was identifi ed in 80.3% of patient cultures, 4 of which were azole-resistant. Most (52.7%) patients received voriconazole. In total, 52.2% of patients died; of the deaths, 33.0% were attributed to CAPA. We found that the cumulative incidence of CAPA in the ICU ranged from 1.0% to 39.1%. ventilation (7,17). Many of these patients lack the concurrent conditions usually associated with invasive pulmonary aspergillosis (IPA) such as malignancies, neutropenia, or history of allogeneic stem cell or solid organ transplantation (21). Admission to the ICU or severe influenza are also risk factors for IPA in nonneutropenic patients (22)(23)(24)(25). Reports of CAPA have been mostly limited to a few single-center studies; therefore, a comprehensive analysis of international distribution currently is lacking (4).

Methods
We conducted a retrospective analysis using clinical data of patients worldwide who received a CAPA diagnosis during March 1-August 31, 2020. Our analysis comprised data from the FungiScope registry and academic literature ( Figure 1). FungiScope (https://www.clinicaltrials.gov; National Clinical Trials identifier NCT01731353) is a global registry for emerging invasive fungal infections. FungiScope was approved by the local ethics committee of the University of Cologne, Cologne, Germany (study ID 05-102). The registry includes patients with invasive aspergillosis since 2019. Fun-giScope's methods have been described previously (reference 55 in Appendix).
In addition, we conducted a literature search using the PubMed database (https://pubmed.ncbi. nlm.nih.gov) for suspected CAPA cases occurring in March-August 2020. We used the search string "(Aspergill*) AND (invasive OR putative OR probable OR infection OR case OR patient OR report) AND (COVID* OR corona* OR SARS-CoV-2)," which identified 59 published articles. We reviewed and extracted relevant data from each of the publications. When necessary, we contacted authors for additional details (Appendix). Patients were identified in the FungiScope registry and academic literature using the search string "(Aspergill*) AND (invasive OR putative OR probable OR infection OR case OR patient OR report) AND (COVID* OR corona* OR SARS-CoV-2) (Appendix Table  1

COVID-19-Associated Pulmonary Aspergillosis
We reviewed each patient report using multiple diagnostic definitions. First, we evaluated the patients according to the consensus definition of Koehler et al.
(reference 56 in Appendix); we classified the patients as having proven, probable, or possible CAPA. We used alternative definitions to evaluate patients who were nonclassifiable because of lack of essential information, such as the volume of saline recovered by nondirected bronchial lavage (NBL) fluid applied. We categorized the nonclassifiable patients as proven or probable according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group criteria for invasive fungal infections (21) or as proven, putative, and colonized according to the AspICU algorithm for IPA in critically ill ICU patients by Blot et al. (23). We considered severe COVID-19 with acute respiratory distress syndrome (ARDS) to be a valid host criterion (i.e., acquired immunodeficiency) (8). We considered patients who met >1 definition to have CAPA; we categorized the rest as nonclassifiable.
We collected data on patients' demographic characteristics and baseline conditions. We also collected data on abnormal radiographic images, mycologic evidence, signs and symptoms at CAPA diagnosis, site of infection, antifungal susceptibility testing, antifungal treatment, death at 6 and 12 weeks after CAPA diagnosis, and absolute death. In addition, we calculated the length of time between COVID-19 and CAPA diagnoses, CAPA diagnosis and most recent healthcare contact with the patient, ICU admission and CAPA diagnosis, and installation of mechanical ventilation and CAPA diagnosis. The contribution of CAPA to patient death (i.e., attributable mortality) was assessed by the treating medical team (Appendix Table 2). To determine the cumulative incidence of CAPA in the facilities included in the analysis, we asked each institution for 3 different denominators: the total number of COVID-19 patients, the number of COVID-19 patients admitted to the ICU, and the number of CO-VID-19 patients admitted to the ICU who needed mechanical ventilation during March-August 2020.

Statistical Analysis
We did not calculate an a priori sample size for this exploratory study. To analyze the demographic and clinical characteristics of patients with CAPA, we describe categorical variables using frequencies and percentages; we describe continuous variables using medians and interquartile ranges (IQRs). We used SPSS Statistics 25.0 (IBM, https://www.ibm.com) for statistical analyses.  Table 2).
In total, 19 of 39 institutions provided denominators for cumulative incidence over the duration of the study period. The CAPA incidence among all COVID-19 patients ranged from 0.1%-9.7%. Among COVID-19 patients admitted to ICU, cumulative incidences ranged from 1.0%-39.1%. Among patients admitted to ICU who needed mechanical ventilation, cumulative incidences ranged from 1.1%-47.4% (Table 3).

COVID-19-Associated Pulmonary Aspergillosis
and therefore complications such as CAPA (reference 59 in Appendix).
Most (97.8%) patients were admitted to the ICU, mainly because of ARDS, need for mechanical ventilation, or both. We found that corticosteroid administration, chronic cardiovascular disease, renal failure, diabetes mellitus, and obesity were common characteristics among these patients. Approximately 1 in 5 patients had chronic pulmonary disease. Patients had many similarities to influenza-associated pulmonary aspergillosis (IAPA) patients from Schauwvlieghe et al. (22), including similar rates of mechanical ventilation (IAPA 90.0% vs. CAPA 94.1%), corticosteroid administration (IAPA 56.0% vs. CAPA 52.7%), baseline renal failure (IAPA 42.0% vs. CAPA 39.8%), obesity (IAPA 30.0% vs. CAPA 25.3%), and chronic pulmonary disease (IAPA 16.0% vs. CAPA 21.5%). IAPA patients had a higher proportion of malignancies (30.0% vs. 11.3%) and solid organ transplantation (13.0% vs. 2.7%); however, CAPA patients had a higher prevalence of diabetes mellitus (12.0% vs. 34.4%). In our study, 50.5% of patients had chronic cardiovascular disease. These differences in the distribution of baseline characteristics between IAPA and CAPA patients reflects the epidemiology of COVID-19, which is more common among those with chronic cardiovascular disease, whereas hematologic or oncologic malignancies (22) are more common among those with IAPA (reference 60 in Appendix). Only 2% of COVID-19 patients have cancer (reference 61 in Appendix).
Available guidelines for aspergillosis management recommend diagnostic procedures such as respiratory culture and galactomannan index of BAL samples (references 60,62 in Appendix). However, these procedures have a high risk for aerosolization; safety precautions should be used when handling samples from COVID-19 patients (references 63,64 in Appendix). The elevated risk for SARS-CoV-2 transmission and the initial recommendation against using bronchoscopy for COVID-19 diagnosis (references 63,64 in Appendix) might explain the low number of BAL tests used to diagnose CAPA in our study. Schauwvlieghe (21). **Culture was used to analyze 50 BAL, 47 tracheal aspirate, 34 bronchial aspirate, 17 nondirected bronchial lavage, 3 sputum, 2 nonspecified lower respiratory tract, and 1 BAL and tracheal aspirate sample. † †Microscopy was used to analyze 1 BAL, 1 bronchial aspirate, and 1 tracheal aspirate sample. ‡ ‡Histologic techniques were used to analyze 7 lung tissue samples. § §PCR was used to analyze 16 BAL, 12 tracheal aspirate, 10 nondirected bronchial lavage, 3 bronchial aspirate, 1 lung tissue, and 1 serum sample. ¶ ¶Galactomannan tests were used to analyze 63 BAL, 30 serum or plasma, 22 nondirected bronchial lavage, 9 tracheal aspirate, 3 bronchial aspirate, and 1 sputum sample.

COVID-19-Associated Pulmonary Aspergillosis
Almost all (97.8%) patients had imaging abnormalities; however, many had only marginally typical features of aspergillosis, hampering the differential diagnosis of CAPA according to radiologic criteria. Positive isolates were recovered from 81.7% of CAPA patients. Similar to IAPA patients, the most common (80.3%) pathogen was A. fumigatus (22). In total, 5 patients had azole-resistant infections: 4 A. fumigatus and 1 A. lentulus infection. We noted 2 patients who had a possible previous exposure to triazoles. The professions of these 2 patients involved exposure to fungicides and manipulated organic matter containing triazole-resistant A. fumigatus. Therefore, the treating teams hypothesized that workplace exposure might have contributed to these patients' illness. We found a similar proportion of patients with previous azole exposure as Verweij  Triazoles, especially voriconazole, were the most frequently administered antifungal drugs: 52.7% of the study cohort and 71.5% of the patients on antifungal treatment received voriconazole. We found that voriconazole use was associated with decreased death. The first-line use of voriconazole in 79 (80.6%) of 98 patients aligns with current recommendations (references 56,60,62 in Appendix).
We found a 50% mortality rate at 12 weeks after CAPA diagnosis. This finding is similar to the 51.0% mortality rate of IAPA patients in the same timeframe; however, these rates are almost 20 points higher than in other cohorts, such as aspergillosis patients with acute leukemia (33.8%) (reference 66 in Appendix). Nonetheless, in our study CAPA was attributed as the main reason for death in only 17.2% of the patients, whereas in Koehler et al. (reference 66 in Appendix), it was the main cause of death for 26.9% of patients with hematologic conditions.
We found an overall 6.9% cumulative incidence for CAPA among patients during the study period, although incidences varied by institution (1.0%-39.1% of CAPA patients admitted to ICU). In most facilities, the rates of CAPA were lower than those of IAPA (14%-19%) (reference 67 in Appendix). However, these ranges might vary according to diagnostic protocols in the different countries and healthcare facilities. Differences in screening practices for CAPA in COVID-19 patients might have affected detection rates and therefore our calculations of cumulative incidence (8). Further analyses are necessary to establish the geographic variance of this rate.
The first limitation of this study is that, because of the cross-sectional design of this study, we could not control for disease severity. Second, samples from the lower respiratory tract are the best way to differentiate between colonization and infection, but a low percentage of patients in this study had mycologic evidence from BAL culture or galactomannan tests. Third, we analyzed many cases from literature and could not contact certain authors for further details. In addition, institutions might not have documented all CAPA cases in the literature or FungiScope registry. Given the regional variability of the patient distribution, longitudinal studies might be a more appropriate tool to determine rates. Finally, because of the retrospective nature of the study, we could not retrieve the necessary clinical and diagnostic details of all patients. As a result, many patients were not classifiable according to the definitions used in this article, possibly contributing to an underdiagnosis of CAPA. In conclusion, we described a large cohort of CAPA patients using cases from the literature and the FungiScope registry. CAPA occurs mostly in ICU patients on mechanical ventilation. We found that CAPA patients had high rates of chronic cardiovascular disease, renal failure, diabetes mellitus, and corticosteroid use. We also found that CAPA substantially contributed to a high death rate in COVID-19 patients, although cumulative incidence varied by treatment site. We believe that improved screening can identify and enable early treatment of CAPA. and a nonfinancial scientific grant from IMMY, outside the context of the submitted work. D.R.G. has received honoraria from Stepstone Pharma GmbH and unconditional grants from MSD Italia Srl and Correvio Pharma Corp. J.F.M. reports grants from F2G Ltd. and Pulmocide, consultancy fees from Scynexis, Inc., and speaker fees from Gilead Sciences Inc., United Medical, and Teva Pharmaceutical Industries Ltd., outside the context of the submitted work. J.P.G. has participated in advisory boards and received speaker honoraria from Pfizer Inc. and Gilead Sciences Inc., outside the context of the submitted work. E.S. has received grants from the Philipp Schwartz Initiative of the Alexander von Humboldt Foundation. O.A.C. is financially supported by the German Federal Ministry of Research and Education; is funded by the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy (CECAD, EXC 2030 -390661388); has received research grants from Actelion Pharmaceuticals Global, Amplyx Pharmaceuticals, Inc., Astellas Pharma Inc., Basilea Pharmaceutica International Ltd., Cidara Therapeutics, Inc., Da Volterra, F2G Ltd., Gilead Sciences Inc., Janssen Pharmaceuticals, The Medicines Company, Melinta Therapeutics, Merck Sharp & Dohme Corp., Octapharma AG, Pfizer Inc., and Scynexis, Inc.; is a consultant to Actelion Pharmaceuticals Global, Allecra Therapeutics GmbH, Amplyx Pharmaceuticals, Inc., Astellas Pharma Inc., Basilea Pharmaceutica International Ltd., BIOSYS USA LLC, Cidara Therapeutics, Inc., Da Volterra, Entasis Therapeutics, F2G Ltd., Gilead Sciences Inc., Matinas BioPharma Holdings, Inc., MedPace, Inc., The Menarini Group, Merck Sharp & Dohme Corp., Mylan Inc., Nabriva Therapeutics plc, NOXXON Pharma, Octapharma AG, Paratek Pharmaceuticals, Inc., Pfizer Inc., Pharmaceutical Solutions Industry, Roche Diagnostics, Scynexis, Inc., and Shionogi Inc.; and received lecture honoraria from Al-Jazeera Pharmaceutical Industries, Astellas Pharma Inc., Basilea Pharmaceutica International Ltd., Gilead Sciences Inc., Grupo Biotoscana, Merck Sharp & Dohme Corp., and Pfizer Inc., outside the context of the submitted work. P.K. has received nonfinancial scientific grants from Miltenyi Biotec GmbH and the Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, and lecture honoraria from or is advisor to Akademie für Infektionsmedizin e.V., Ambu GmbH, Astellas Pharma Inc., European Confederation of Medical Mycology, Gilead Sciences Inc., Gesundheits und Pflegezentrum Rüsselsheim gemeinnützige GmbH, Merck Sharp & Dohme Corp., and University Hospital, Ludwig Maximilian University of Munich, and is advisor to Gilead Sciences Inc. and NOXXON N.V. outside the context of the submitted work.

About the Author
Dr. Salmanton-García is an epidemiologist at University Hospital Cologne, Cologne. His primary research interests are invasive fungal infections, infectious diseases, epidemiology, and database management.