key: cord-0694071-l9p7cmjj
authors: Fortarezza, Francesco; Boscolo, Annalisa; Pezzuto, Federica; Lunardi, Francesca; Jesús Acosta, Manuel; Giraudo, Chiara; Del Vecchio, Claudia; Sella, Nicolò; Tiberio, Ivo; Godi, Ilaria; Cattelan, Annamaria; Vedovelli, Luca; Gregori, Dario; Vettor, Roberto; Viale, Pierluigi; Navalesi, Paolo; Calabrese, Fiorella
title: Proven COVID‐19—associated pulmonary aspergillosis in patients with severe respiratory failure
date: 2021-07-04
journal: Mycoses
DOI: 10.1111/myc.13342
sha: 435c3eb3d3bfbf12d4959c67caa1d8634bce004f
doc_id: 694071
cord_uid: l9p7cmjj

BACKGROUND: An increasing number of reports have described the COVID‐19–associated pulmonary aspergillosis (CAPA) as being a further contributing factor to mortality. Based on a recent consensus statement supported by international medical mycology societies, it has been proposed to define CAPA as possible, probable, or proven on the basis of sample validity and thus diagnostic certainty. Considering current challenges associated with proven diagnoses, there is pressing need to study the epidemiology of proven CAPA. METHODS: We report the incidence of histologically diagnosed CAPA in a series of 45 consecutive COVID‐19 laboratory‐confirmed autopsies, performed at Padova University Hospital during the first and second wave of the pandemic. Clinical data, laboratory data and radiological features were also collected for each case. RESULTS: Proven CAPA was detected in 9 (20%) cases, mainly in the second wave of the pandemic (7/17 vs. 2/28 of the first wave). The population of CAPA patients consisted of seven males and two females, with a median age of 74 years. Seven patients were admitted to the intensive care unit. All patients had at least two comorbidities, and concomitant lung diseases were detected in three cases. CONCLUSION: We found a high frequency of proven CAPA among patients with severe COVID‐19 thus confirming at least in part the alarming epidemiological data of this important complication recently reported as probable CAPA.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still represents a worldwide sanitary challenge, particularly regarding the severe forms. Severe COVID-19 is characterised by acute respiratory distress syndrome, often requiring mechanical ventilation or extracorporeal membrane oxygenation. In this case, the occurrence of respiratory bacterial and fungal superinfections, including invasive pulmonary aspergillosis, represents an additional risk factor for worse outcome. Similarly to influenza-associated pulmonary aspergillosis, an increasing number of reports 1-3 have described the COVID-19-associated pulmonary aspergillosis (CAPA) as being a further contributing factor to mortality. A recent consensus statement on defining and managing CAPA has been prepared by experts and supported by medical mycology societies. 4 It has been proposed to define CAPA as possible, probable or proven on the basis of sample validity and thus diagnostic certainty. 4 Table 1 ).

All radiographs were evaluated using a previously published and validated composed COVID-19 chest X-rAy scoRE (i.e., CARE). 6 The score was based on the subdivision of each lung in three areas (i.e., WBC (10 9 /L) a N (10 9 /L) a L (10 9 /L) a upper, middle and lower areas) and a three-grade score describing, separately, the extension of ground glass and consolidations.

Additional findings like pleural effusion, pneumothorax, pneumomediastinum and subcutaneous emphysema were also recorded. Each available computed tomography (CT) scan was assessed using a modified version of the semi-quantitative score of Francone et al. 7 which was based on the separate evaluation of ground glass, consolidations and crazy paving in each of the six lobes, separately. The extension of each finding at CT was rated from 0 to 5 (i.e., 0 = no involvement; 1 = <5% involvement; 2 = 5%-25% involvement; 3 = 26%-50% involvement; 4 = 51%-75% involvement; and 5 = >75% involvement). 7

The global score at CT ranged from 0 to 30. The occurrence of fibrosis, subpleural lines, reversed 'halo sign', pleural effusion, tracheal and bronchial wall thickening, and enlarged lymph nodes. In the case of contrast medium injection, the presence of pulmonary embolism was also documented. Moreover, pulmonary nodules, which can occur in case of fungal infections, were separately assessed.

Autoptic examinations and systematic histological analyses were performed as previously described. 8 Briefly, autoptic examinations were carried out with a post-mortem interval ranging from 24 h to 6 days. Lungs were removed in block and fixed in 10% buffered formalin. Eighteen tissue blocks from airways and lungs for each case were sampled ( in the supernatant was further confirmed by RT-PCR using primers described previously. 9

The principal clinical, radiological and pathological data are summarised in Table 1 (3) are shown in Figure 1 .

Upper airway inflammation was evident in all cases with four acute and five chronic inflammatory infiltrates, but no tracheal fungal infections were detected. The population consisted of seven males and two females, with a median age of 74 years (Table 1) . All patients had at least two comorbidities, and concomitant lung diseases were detected in three cases (interstitial lung disease and lung cancer). Seven patients were admitted to the intensive care unit.

Five patients showed lymphopenia without any case of neutropenia.

Mycological tests (i.e., Aspergillus bronchial aspirate culture, galactomannan (GM) index on serum or respiratory specimens, or serum The principal differences between the CAPA and non-CAPA groups are listed in Table 2 .

In this study, we evaluated the incidence of proven pulmonary as- The diagnosis of CAPA is also challenging from a radiological point of view because invasive aspergillosis and COVID-19 pneumonia can show overlapping radiological features as seen in the index case ( Figure 1 ) included in this paper. For example, the so-called halo sign, typical of invasive aspergillosis in neutropenic patients, can also be related to a pulmonary infarction, which may be an expression of the vascular injury and the microthrombosis, peculiar feature of COVID-19. 10, 13 It has been suggested that the presence of multiple pulmonary nodules or cavitation should lead to suspicion of pulmonary aspergillosis, as these changes are not typical of SARS-CoV-2 pneumonia. 14 In our case series, the chest X-ray did not highlight any signs of CAPA with similar CARE-scores between the two groups of F I G U R E 1 An index case of CAPA (patient 3, Table 1 ). Axial chest CT demonstrating typical COVID-19 signs such as bilateral patchy ground glass opacities with interstitial thickening (A). Gross sample of the left lung. The cut surface shows several roundish haemorrhagic areas (B). At the panoramic view, the lung parenchyma appears edematous with multiples aggregates of fungal hyphae (C, haematoxylin and eosin stain, original magnification 20×). The fungal aggregate fill the alveolar space (arrow) and invade the arteriolar vessel with necrosis of the vascular wall (asterisks). Small aggregates of hyphae are also visible in the lumen (D, haematoxylin and eosin stain, original magnification 200×). At higher magnification, the hyphae show the typical morphological features (septation, diameter from 2.5 to 4.5 μm, dichotomous branching at 45 degrees angles) of the Aspergillus spp (E, haematoxylin and eosin stain, original magnification 400×)

patients. Only in one case (case 8) did the CT scan show pulmonary nodules corresponding to foci of invasive aspergillosis at histological examination. Further studies using CT-scans are recommended to obtain more accurate characterisations of this superinfection.

Interestingly, most cases were detected in the second wave of the pandemic (7/17 vs. 2/28 of the first wave). This may be due to a larger use of immunosuppression, at least in our experience (16/17 patients were treated with steroids in the second wave vs. 12/28 of the first wave). Indeed, steroid therapies have been more widely used following the results of the RECOVERY trial 11 which showed lower mortality in ventilated patients treated with dexamethasone.

Although of undoubted usefulness, steroid treatments can make patients more susceptible to superinfections, as observed in the majority of our patients most of whom were treated with steroids. The majority of our patients with CAPA recovered in ICU (7/9, 78%) and

were mechanically ventilated (6/9, 67%) in a larger percentage compared to non-CAPA cases ( Table 2 ). In a recent study by Ichai et al. 15 some Another food for thought is the occurrence of concomitant lung diseases and other pathological lesions in five cases, including lung cancer, interstitial lung diseases, severe emphysema and bacterial pneumonia. These lesions were more frequent in the CAPA group compared to patients without invasive aspergillosis. Such lung alterations could make patients more susceptible to developing CAPA and could contribute to defining an alarming clinical phenotype.

Our study is limited due to small sample size and data from a single centre. The limited number of patients, the extreme imbalance in the two groups (9 vs. 36 cases) and the lack of a precise timing of fungal superinfection did not allow reliable statistical analyses. It should be noted, however, that this is one of the largest single-centre case series where all the cases received protocolised treatment 16 and standardised lung sampling methodology 5 which allowed us to identify even single foci of pulmonary aspergillosis.

In conclusion, we found a high frequency of invasive pulmonary aspergillosis histologically documented among patients with severe COVID-19, in which the viral infection was further confirmed by molecular analyses on the lung tissues. These observations confirm at least in part the alarming epidemiological data of this important complication recently reported as probable CAPA. This important evidence underscores the need of tools and updated methodologies to more precisely diagnose proven CAPA.

We wish to thank Judith Wilson for English revision. The work was partially supported by a fellowship from the University of Padova (Intesa San Paolo Vita bank) for a young pathologist (FF) involved in the autopsy study (2020A08).

The main clinical, radiological and pathological differences between CAPA and non-CAPA patients IMV length (days) 6 (6, 6) 6 (3, 20) Hospital stay (days) 12 (7, 15) 6 (4, 17)

Steroid treatments 8 (88%) 19 (54%) CARE score 21 (15, 30) 21 (12, 29) Other lung lesions c 2.00 (1.00, 2.00) 0.50 (0.00, 1.00) Acute tracheitis 4 (44%) 8 (22%)

Abbreviations: CAPA, COVID-19-associated pulmonary aspergillosis; CARE,COVID-19 chest X-rAy scoRE; ICU, intensive care unit; IMV, invasive mechanical ventilation. a n (%); Median (IQR).

b At hospital admission.

c Diffuse alveolar damage and invasive pulmonary aspergillosis not included.

Additional supporting information may be found online in the Supporting Information section. 

Francesco Fortarezza: Data curation (equal); Formal analysis (equal)

Investigation (equal)

Annalisa Boscolo: Data curation (equal); Formal analysis (equal)

Investigation (equal)

Federica Pezzuto: Data curation (equal); Formal analysis (equal)

Investigation (equal)

Writing-original draft (supporting). Francesca Lunardi: Data curation (equal); Formal analysis (equal)

Investigation (equal)

Manuel Jesùs Acosta: Data curation (equal)

Chiara Giraudo: Data curation (equal)

Formal analysis (equal)

Investigation (equal)

Claudia Del Vecchio: Data curation (equal); Formal analysis (equal)

Nicolò Sella: Data curation (equal)

Formal analysis (equal)

Ivo Tiberio: Data curation (equal); Formal analysis (equal)

Ilaria Godi: Data curation (equal); Formal analysis (equal)

Investigation (equal)

Data curation (equal); Formal analysis (equal)

Luca Vedovelli: Data curation (equal); Formal analysis (equal)

Dario Gregori: Data curation (equal); Formal analysis (equal)

Roberto Vettor: Conceptualization (equal)

Writing-review & editing (equal)

Pierluigi Viale: Conceptualization (equal)

Paolo Navalesi: Conceptualization (equal); Writing-review & editing (equal). Fiorella Calabrese: Conceptualization (equal)

Writing-review & editing (equal)

COVID-19 Associated pulmonary aspergillosis

Prevalence of putative invasive pulmonary aspergillosis in critically ill patients with COVID-19

COVID-19-associated pulmonary aspergillosis (CAPA) in patients admitted with severe COVID-19 pneumonia: an observational study from Pakistan

Defining and managing COVID-19-associated pulmonary aspergillosis: the 2020 ECMM/ ISHAM consensus criteria for research and clinical guidance

Pulmonary pathology and COVID-19: lessons from autopsy. The experience of european pulmonary pathologists

Validation of a composed COVID-19 chest radiography score: the CARE project

Chest CT score in COVID-19 patients: correlation with disease severity and short-term prognosis

Machine learning-based analysis of alveolar and vascular injury in SARS-CoV-2 acute respiratory failure

The diagnostic yield of the multidisciplinary discussion in patients With COVID-19 pneumonia

COVID-19 pulmonary pathology: a multi-institutional autopsy cohort from Italy and New York City

Dexamethasone in Hospitalized Patients with Covid-19

Epidemiology of invasive pulmonary aspergillosis among COVID-19 intubated patients: a prospective study

Two sorts of microthrombi in a patient with coronavirus disease 2019 and lung cancer

A national strategy to diagnose COVID-19 associated invasive fungal disease in the ICU

Impact of negative air pressure in ICU rooms on the risk of pulmonary aspergillosis in COVID-19 patients

Regional COVID-19 network for coordination of SARS-CoV-2 outbreak in Veneto, Italy