key: cord-1006608-x1lng449
authors: Flikweert, Antine W.; Grootenboers, Marco J.J.H.; Yick, David C.Y.; du Mée, Arthur W.F.; van der Meer, Nardo J.M.; Rettig, Thijs C.D.; Kant, Merijn K.M.
title: Late histopathologic characteristics of critically ill COVID-19 patients: Different phenotypes without evidence of invasive aspergillosis, a case series
date: 2020-07-08
journal: J Crit Care
DOI: 10.1016/j.jcrc.2020.07.002
sha: 81e6c46225573c5dfbeea1019a918244935e4644
doc_id: 1006608
cord_uid: x1lng449

PURPOSE: Pathological data of critical ill COVID-19 patients is essential in the search for optimal treatment options. MATERIAL AND METHODS: We performed postmortem needle core lung biopsies in seven patients with COVID-19 related ARDS. Clinical, radiological and microbiological characteristics are reported together with histopathological findings. MEASUREMENT AND MAIN RESULTS: Patients age ranged from 58 to 83 years, five males and two females were included. Time from hospital admission to death ranged from 12 to 36 days, with a mean of 20 ventilated days. ICU stay was complicated by pulmonary embolism in five patients and positive galactomannan on bronchoalveolar lavage fluid in six patients, suggesting COVID-19 associated pulmonary aspergillosis. Chest CT in all patients showed ground glass opacities, commonly progressing to nondependent consolidations. We observed four distinct histopathological patterns: acute fibrinous and organizing pneumonia, diffuse alveolar damage, fibrosis and, in four out of seven patients an organizing pneumonia. None of the biopsy specimens showed any signs of invasive aspergillosis. CONCLUSIONS: In this case series common late histopathology in critically ill COVID patients is not classic DAD but heterogeneous with predominant pattern of organizing pneumonia. Postmortem biopsy investigations in critically COVID-19 patients with probable COVID-19 associated pulmonary aspergillosis obtained no evidence for invasive aspergillosis.

Up to 20 percent of hospitalized coronavirus disease 2019 patients are admitted to the intensive care unit (ICU) because of acute hypoxemic respiratory failure. [1] [2] [3] [4] These patients usually present with bilateral patchy ground glass opacities on computed tomography (CT) thorax fulfilling the definition for acute respiratory distress syndrome (ARDS). Often an atypical high compliance phenotype (L-type) is observed during mechanical ventilation in COVID-19 patients in contrast to mechanical ventilation characteristics typically seen in ARDS with low lung compliance phenotype (H -type). [5, 6] During ICU stay the radiologic presentation of bilateral patchy ground glass opacities as present at admission often progress to consolidations with or without fibrotic characteristics. [7] Two earlier observed features may play a critical role in the severity of this disease: thromboembolic complications and early onset aspergillosis. The cumulative incidence of venous thromboembolism reported was 49% in COVID-19 patients admitted to the ICU. [8] Presumed pulmonary aspergillosis may be present in as much as 19% of ICU COVID-19 patients. [9] Since the physiology in COVID-19 related ARDS as well as its complications seems to differ from "typical" ARDS, an insight into the pulmonary tissue pathology of this new infectious disease is of the utmost importance. The scarcely available pathological data in COVID-19 patients show diffuse alveolar damage, closely related to ARDS. [10] [11] [12] The clinical relevance of COVID-19associated pulmonary aspergillosis (CAPA) as well as survival benefit with antifungal treatment and associated mortality are under debate since histopathological evidence of CAPA is not obtained. [13] We examined postmortem obtained lung tissue in seven patients, with COVID related ARDS who needed mechanical ventilation. The histopathologic findings, together with clinical features, radiological

All patients with laboratory confirmed severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) admitted to the ICU due to acute hypoxemic respiratory failure between 22 March 2020 and 30 April 2020 and with available postmortem needle core biopsy of the lung were eligible for inclusion in this case series. SARS-CoV-2 was diagnosed using real-time reverse transcription polymerase chain reaction (RT-PCR) on sputum and/or bronchial aspirates.

Routine ICU management included, among other things, selective digestive tract decontamination (SDD), chloroquine until the Dutch National Institute for Public Health and Environment advised against its use at the end of March 2020, and high dose anticoagulation with low-molecular-weight heparin (LMWH) (nadroparin 87IE/kg twice daily). Bronchoscopy, with or without lavage, and testing for pulmonary aspergillosis were performed at the discretion of the attending physician. Pulmonary aspergillosis was diagnosed using clinical, radiological and mycological data and included galactomannan (serum and sputum), tracheal or bronchial culture. For galactomannan (GM) testing from bronchoalveolar lavage (BAL) fluid Platelia Aspergillis antigen ELISA (Biorad) was used. Recently, a case definition for influenza associated pulmonary aspergillosis (IAPA) was proposed by an expert panel, which could be used to classify patients with CAPA. [14] Diagnostic criteria include proven infection with clinical symptoms and a GM index of ≥1 on BAL or of ≥ 0ꞏ5 on serum;

or Aspergillus spp. cultured from BAL. J o u r n a l P r e -p r o o f

During the study period, seven patients underwent postmortem needle core biopsy of the lungs. Patients age ranged from 58 to 83 years (median 74 year) and five patients were male. None of the patients had a history of (chronic) pulmonary disease. One patient used immunosuppressive medication before hospital admission, in this case a short course of prednisolone. Median time from hospital to ICU admission was 0 days (interquartile range 0-4). Time from hospital admission to death ranged from 12 to 36 days. Patients deceased at median of 21(range 9-36) ventilated days. In five out of seven patients ICU stay was complicated by pulmonary embolism. Adopting the proposed definition of CAPA by van Arkel et al [9] , six patients were classified as having probable CAPA (table 1) 

A 73-year old female was transferred to our ICU due to shortage of ICU beds in a nearby hospital. Prior to ICU admission, she was healthy but complained of diarrhoea and shortness of breath. Non-contrast chest CT at initial hospital admission showed multiple bilateral areas of ground-glass opacity along the bronchovascular bundles and periphery. There were some small areas of consolidation in the upper lobes.

Subtle bronchiectasis were present in affected areas. Her respiratory condition required mechanical ventilation in prone position due to ARDS. After seven days, prednisolone treatment was started because of lack of improvement. Repeat chest CT-angiography showed segmental pulmonary emboli in the right lung. Ground-glass opacities persisted while the consolidations had disappeared. A reticular pattern combined with GGO was more pronounced with increasing traction bronchiectasis. Biopsy specimens showed a pattern of lung injury, that was partially identical to that of case 1. Intra-alveolar depositions of fibroblastic tissue were found, consistent with organizing pneumonia. However, a predominant, diffuse component of fibrinous exudate in the alveoli was present, which was not the case in the aforementioned case with organizing pneumonia. Other histologic findings were a chronic inflammatory infiltrate, and mild interstitial changes, including widening of alveolar septa. Microthrombi in small septal blood vessels were also observed. Neither J o u r n a l P r e -p r o o f remnants hyaline membranes nor prominent eosinophils were present. Additional PAS-D stain did not show any fungi. The overall histologic pattern of this case was classified as acute fibrinous and organizing pneumonia (AFOP), figure 2D.

We report pathology in deceased critically ill ICU COVID patient in the late phase of disease to be heterogeneous. Histopathologically, we observed four distinct histopathological patterns: AFOP, DAD, fibrosis and, in four out of seven patients an organizing pneumonia (OP). Interestingly, our findings are in contrast to previously reported postmortem studies in COVID-19 patients in which DAD is the most common predominant pattern. [10] [11] [12] [15] [16] [17] [18] [19] In two recent autopsy studies of 21 and 12 deceased COVID-19 patients, prevalence of DAD was 76% and 67% respectively.

None of the patients had the postmortem diagnosis of organizing pneumonia [10, 11] .

In the study of Ackermann and colleagues, pulmonary histology of all seven studied patients showed DAD [19] . Although the organizing stage of DAD may overlap with the histopathological features of OP in lung biopsy, the histologic hallmark of DAD, namely remnants of hyaline membranes were not present in our four OP cases.

Most plausible explanation for the more common pattern of OP in our study population when compared to previous mentioned studies is the difference in length of hospital and ICU stay correlating with more advanced disease and longer treatment with mechanical ventilation. In the studies mentioned above the mean hospital stay was six days or less and most of the patients did not receive mechanical ventilatory support. In our present series mean hospital stay of 22 days is significant longer and all patients died in the ICU with a mean of 20 days on mechanical ventilation. Secondary OP can be seen in association with many types of non-specific J o u r n a l P r e -p r o o f lung injury, including viral infections and drug reactions. OP is reported following severe influenza infections [20] [21] [22] [23] [24] and Middle East Respiratory Syndrome [25] . Most recently, Copin et al. reported postmortem biopsies on six patients with COVID and reported in five patients with phenotype H and AFOP histology in contrast to their patient with DAD. [26] Estimated elapsed disease time in the AFOP group was 20 days versus 6 days in the DAD patients. In our opinion this supports the theory that pathology changes over time.

Although the exact pathogenesis of OP remains unknown, it is thought that OP is a consequence of alveolar epithelial injury. This initial epithelial injury is followed by leakage of plasma proteins, leading to a cascade of host responses with hyperinflammation [27, 28] . Subsequent fibroblast recruitment and connective tissue and fibroblast organisation is seen within the alveolar space. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play a central role in organizing pneumonia and are highly expressed in intraluminal fibromyxoid lesion in organizing pneumonia. [29] Interestingly, binding to ACE2 receptor is recognized as a critical initial step for SARS-CoV-2 to entry alveolar type II cells, resulting in loss of ACE2 at the membrane. ACE 2 is a negative regulator of the renin-angiotensin system (RAS) and this depletion of ACE2 upregulates the RAS. [30] An activated RAS can induce Fibroblastic Growth Factor. ACE2 also plays a role in regulating the effect of VEGF. [31] Therefore, depletion of ACE2 due to the high affinity of SARS-CO-V2 to ACE2 might play a role in the pathogenesis of COVID19 related organizing pneumonia.

Being aware that medication can be the cause of OP it is notable is that in our study six out of seven patients received chloroquine, at that time the advised treatment by the Dutch National Institute for Public Health and Environment. Although chloroquine J o u r n a l P r e -p r o o f use is associated with cardiovascular disorders, pulmonary side effects, i.e. drug induced interstitial lung disease, are not described before, and therefore an unlikely cause of the observed histologic OP. We found no studies reporting possible relations between antifungal therapy and OP.

In retrospect, chest CT scans of each of the patients showed a different development during hospital admission, concordant with the histopathological diagnosis (table 2).

in the presence of an organizing pneumonia. Excluding nosocomial infections in such cases is essential. The diversity in histopathological findings correlating with radiological findings is interesting considering possible therapeutic implications and should be subject for further research. Although steroids are not routinely recommended to be used in de early phase of SARS-CoV-2 pneumonia, they might have a role in the late phase of COVID19 when an organizing pneumonia is suspected. [32] Recently there is increasing awareness and concern for development of secondary infection in COVID patients e.g. invasive aspergillus co-infection, a contra-indication for (long-term) systemic steroid therapy. Criteria and risk factors for invasive pulmonary aspergillosis are well defined in immunocompromised populations.

Furthermore it is a well known complication of severe influenza pneumonia with reported incidences of 19% in ICU patients admitted for influenza related acute respiratory failure with high mortality rates. [33] . In CAPA case definition is absent although recently an expert panel proposed a classification. [14] Pathophysiology of COVID associated pulmonary aspergillosis (CAPA) consists of lung damage with bilateral alveolar-interstitial damage due to viral replication and cytokine storm in combination with marked low T-lymphocytes CD4+T and CD8+T cells. [27] Secondary J o u r n a l P r e -p r o o f infection due to lung tissue damage develop within a median of 17 days. [34] COVID associated pulmonary aspergillosis (CAPA) data are scarce but increasingly reported, although histological confirmation is still absent [9, 13, 35, 36] . Of the first 31 COVID-19 patients admitted to our ICU, six where highly suspected for COVID-19 associated pulmonary aspergillosis (CAPA). [9] In this case series, BAL fluid galactomannan was positive in six out of the seven cases, concluding in the clinical diagnosis of probable CAPA.

To our surprise, none of the lung biopsies showed any presence of invasive aspergillosis. Lack of evidence for IPA in our patients with probable CAPA raises the question whether patients with suspected CAPA truly develop invasive aspergillosis and require antifungal therapy. For instance, the three deaths in the CAPA report from France were attributed to bacterial septic shock, and not to aspergillosis. 

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