Long-Term Follow-Up of Patients Diagnosed with COVID-19-Associated Pulmonary Aspergillosis (CAPA)

COVID-19-associated pulmonary aspergillosis (CAPA) have been documented during the COVID-19 pandemic. The vast majority of these patients do not meet the classic EORTC/MSGERC criteria for invasive pulmonary aspergillosis. The question arises as to whether there may have been an over-diagnosis of this disease. Here we review our experience and analyze the evolution of 27 patients who were diagnosed with CAPA during hospital admission. Surviving patients were followed-up for a mean time of 15 months (SD 3.78) by a group of experts and clinical records of diseased patients were reviewed. After expert evaluation and follow-up, 10 patients were finally assumed as CAPA according to expert opinion. These cases represent 40% of the initially CAPA assumed cases. Our data suggest the need to reconsider actual diagnosis criteria for CAPA what could drive to better identification of these patients.


Introduction
Since the beginning of the pandemic, there has been concern about the possibility of coronavirus disease 2019 (COVID-19)-related fungal infections in these patients. Based on previous experience of bacterial or fungal infections following influenza infection [1,2] many authors have attempted to anticipate this same scenario in relation to COVID-19.
Among the theories supporting an increased risk of invasive fungal infection (IFI) related to COVID-19 is the direct damage to the airway epithelium caused by the virus, which enables Aspergillus to colonize and invade pulmonary tissue, the same as what has been seen with influenza infection [3]. In addition, the fact that the most frequently used treatments for COVID-19, such as steroids and tocilizumab, play an important role in the impairment of immunity supports the possibility of an increased risk of IFI [4] associated with COVID-19.
Reported prevalence of COVID-19-associated pulmonary aspergillosis (CAPA) rates varies widely among the literature (3-34%) [5][6][7][8]. This variability may be due at least in part to the different definitions used, which could have overinflated the reported prevalence of CAPA early in the pandemic [5].
The aim of this study was to briefly review our experience and analyze the long-term evolution of patients who were diagnosed with CAPA in our center in order to assess whether the clinical and radiological evolution was compatible with the diagnosis of invasive aspergillosis.

Study Population and Setting
We performed a retrospective, observational, single-centre study at the University Hospital "12 de Octubre" (Madrid, Spain), a 1250-bed tertiary-care centre. We included all patients admitted with a definite diagnosis of COVID-19 from January 2020 to January • COVID-19 confirmed case: Positive result in the reverse transcription-polymerase chain reaction (RT-PCR) assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swab. • CAPA in immunosuppressed patients: was classified as possible/probable/proven invasive pulmonary aspergillosis according to the EORTC/MSG criteria [9]. • CAPA in non-immunocompromised patients: For these patients we used the 2020 European Confederation of Medical Mycology and International Society for Human and Animal Mycology (ECMM/ISHAM) consensus criteria for the diagnosis of CAPA [10]. According to these criteria, patients with positive SARS-CoV-2 RT-PCR and who develop respiratory insufficiency requiring intensive care could be classified as: Proven CAPA: Proven by histopathological and/or direct microscopic detection of fungal elements that are morphologically consistent with Aspergillus spp., showing invasive growth into tissues, or by PCR from material that was obtained by a sterile aspiration or biopsy from a pulmonary site, showing an infectious disease. Probable CAPA: Pulmonary infiltrate or nodules, preferably documented by chest CT, or cavitating infiltrate (not attributed to another cause), or both, combined with mycological evidence: microscopic detection of fungal elements in bronchoalveolar lavage (positive bronchoalveolar lavage culture); positive galactomannan in serum or bronchoalveolar lavage galactomannan; positive aspergillus PCR tests in plasma, serum, whole blood; or bronchoalveolar lavage fluid (modified from ECMM/ISHAM proposed criteria) [10]. Possible CAPA: Pulmonary infiltrate or nodules, preferably documented by chest CT, or cavitating infiltrate (which is not attributed to another cause) in combination with mycological evidence (e.g., microscopy, culture, or galactomannan, alone or in combination) obtained via non-bronchoscopic lavage.
• Aspergillus colonization: Isolation of Aspergillus spp. in one or more respiratory samples for which the former criteria was not fulfilled. • CAPA confirmed cases after follow-up: Surviving patients from our cohort were followed up for at least 6 months after hospital discharge. Two clinicians (MRR and JMA) evaluated individually clinical and radiological evolution of these patients and classified them as true or false CAPA. We considered as CAPA those patients with compatible imaging evolution; cavitating infiltrate or nodules, infiltrates with air-crescent sign, or those with subacute evolution documented by lung CT. We considered as previously misidentified CAPA cases those presenting pulmonary infiltrates or nodules with an accelerated resolution, or those that could be attributed during the follow-up period to another cause.

Statistical Analysis
Quantitative data were shown as the mean ± standard deviation (SD) or the median with interquartile range (IQR). Qualitative variables were expressed as absolute and relative frequencies. Categorical variables were compared using the χ 2 test. Student's t-test or Mann-Whitney U test were applied for continuous variables, as appropriate.

Results
From January 2020 to January 2021, Aspergillus spp. were recovered from respiratory samples of 279 patients. Of these, 200 were isolated in sputum samples and 79 in lower respiratory tract samples. Twenty-seven patients with Aspergillus spp. isolated in a lower respiratory tract sample had a concomitant diagnosis of COVID-19. Aspergillus was isolated from tracheal aspirate (BAS) in 25 patients (92.6%), and from bronchoalveolar lavage (BAL) in the remaining two cases.
Demographics and clinical characteristics of these 27 patients are detailed in Table 1. Mean age at diagnosis was 65 ± 9.17 years, with 77.8% (21/27) of the episodes occurring in males. Prior immunosuppression was present in 18.5% (5/27) of the patients, two lung transplant recipients, one allogenic stem cell transplant recipient, one patient with melanoma, and one with an autoimmune disease. Most of the patients (25/27) had previously received broad-spectrum antibiotics. We analyzed how many patients from our cohort met the ECMM/ISHAM proposed definition criteria for CAPA [10], 25/27 (92,5%) patients met criteria for probable CAPA. Only 5 out of these 25 patients (18.5%) presented prior immunosuppression and thoracic CT patterns compatible with invasive pulmonary aspergillosis, and therefore could be classified as probable cases according to the EORTC/MSG criteria [9]. Thirteen patients with probable CAPA died and therefore no follow-up was possible. Clinical records of these 13 diseased patients were reviewed by experts (MRR and JMA), 10 of them died in the first 10 days of admission: these cases were considered as not CAPA related deaths after expert review. For the remaining three patients death related to CAPA could not be exclude (Figure 1). We analyzed how many patients from our cohort met the ECMM/ISHAM proposed definition criteria for CAPA [10], 25/27 (92,5%) patients met criteria for probable CAPA. Only 5 out of these 25 patients (18.5%) presented prior immunosuppression and thoracic CT patterns compatible with invasive pulmonary aspergillosis, and therefore could be classified as probable cases according to the EORTC/MSG criteria [9]. Thirteen patients with probable CAPA died and therefore no follow-up was possible. Clinical records of these 13 diseased patients were reviewed by experts (MRR and JMA), 10 of them died in the first 10 days of admission: these cases were considered as not CAPA related deaths after expert review. For the remaining three patients death related to CAPA could not be exclude (Figure 1).  In order to analyze the evolution of the surviving patients, they were followed-up in a dedicated COVID outpatient clinic by a group of experts with experience in pulmonary fungal pathology. Twelve surviving patients were followed-up for a mean time of 15 months (SD 3.78); patients clinical, microbiological, and radiological characteristics are represented in Table 2. We observed that 5 of the 12 survivors (41.6%) were eventually considered as colonization and antifungal treatment was not initiated or was withdrawn within less than 48 h; however, they were followed-up to ensure that they did not subsequently develop symptoms compatible with IFI. Of these five patients, only one had a control CT scan in which no lesions compatible with IFI were observed despite not having received antifungal treatment. The other four patients were followed-up by experts in infectious diseases, none presented respiratory complications during follow-up.
The remaining seven patients were finally assumed as CAPA confirmed cases after follow-up according to expert opinion ( Figure 1). Two of these patients had a CT scan in which resolution of pulmonary infiltrates was confirmed. The remaining five patients had at least one chest X-ray performed during the follow-up without IFI compatible images. All seven patients were considered as cured and did not present clinical relapse during follow-up.

Discussion
Most reported cases of CAPA occurred in patients without classic risk factors of invasive aspergillus infection such as neutropenia or hematological malignancy [11][12][13]; this fact has complicated the diagnosis as patients without histologic evidence of invasive fungal disease cannot meet the classic research definitions of the EORTC/MSG for probable aspergillosis, because these require the presence of immunocompromising host factors [9].
As the pandemic evolves, the question arises as to whether there may have been missed or misidentified individuals. The difficulty of establishing a diagnosis of certainty has been a constant in all series, especially because limited use of diagnostic bronchoscopy due to the need to protect health-care workers from aerosol exposure, and due to the low sensitivity of detection of circulating galactomannan in serum in non-severely immunocompromised patients. Alternative research definitions for CAPA have been proposed [10,14,15], their major limitation may be the characterization of CAPA by nonspecific clinical and radiographic findings that are difficult to distinguish from COVID-19 alone. All these circumstances bring up the concern of the impossibility of distinguishing between colonization and invasive disease.
In the present study we investigated how many patients from the first wave of the pandemic had been initially considered as CAPA and how many of these actually could be considered as CAPA after follow-up. As shown in the flowchart, only 10 patients could be considered as probable CAPA (including 7 survivors and 3 deceased): these cases represent 40% of the initially CAPA assumed cases.
Due to high COVID-related mortality in critical ill patients specially in the first part of the pandemic, we understand the prompt initiation of antifungal treatment in ICU patients for whom respiratory Aspergillus spp. has been isolated and fulfil CAPA criteria. Today, with a better knowledge and management of the disease, an increase in the proportion of immunized population and new and promising therapeutic alternatives, it seems necessary to rethink the diagnostic criteria for CAPA.
Our research has some limitations. It is a single-centre study with a small sample size, and there was an absence of tissue-proven diagnosis, which compromised our ability to detect the actual prevalence of CAPA. The strength of our study was the long-term follow-up of the survivors by a group of experts in order to determine whether the clinical picture was compatible with invasive aspergillosis. This long-term follow-up has allowed us to analyze with greater perspective the reality of this problem, concluding that in a considerable proportion of assumed CAPA cases the diagnosis has been overestimated.

Conclusions
Based on our data and the growing body of published evidence, it seems reasonable to consider the need for stricter criteria that allow a better diagnosis of COVID-19-associated pulmonary aspergillosis.