Long-Term Persisting SARS-CoV-2 RNA and Pathological Findings: Lessons Learnt From a Series of 35 COVID-19 Autopsies

Abstract

Background: Long-term sequelae of coronavirus disease 2019 (COVID-19), including the interaction between persisting viral-RNA and specific tissue involvement, pose a challenging issue. In this study, we addressed the chronological correlation (after first clinical diagnosis and postmortem) between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and organ involvement.

Methods: The presence of postmortem SARS-CoV-2 RNA from 35 complete COVID-19 autopsies was correlated with the time interval between the first diagnosis of COVID-19 and death and with its relationship to morphologic findings.

Results: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be evident up to 40 days after the first diagnosis and can persist to 94 hours after death. Postmortem SARS-CoV-2 RNA was mostly positive in lungs (70%) and trachea (69%), but all investigated organs were positive with variable frequency. Late-stage tissue damage was evident up to 65 days after initial diagnosis in several organs. Positivity for SARS-CoV-2 RNA in pulmonary swabs correlated with diffuse alveolar damage (p = 0.0009). No correlation between positive swabs and other morphologic findings was present. Cerebral (p = 0.0003) and systemic hemorrhages (p = 0.009), cardiac thrombi (p = 0.04), and ischemic events (p = 0.03) were more frequent in the first wave, whereas bacterial pneumonia (p = 0.03) was more prevalent in the second wave. No differences in biometric data, clinical comorbidities, and other autopsy findings were found.

Conclusions: Our data provide evidence not only of long-term postmortem persisting SARS-CoV-2 RNA but also of tissue damage several weeks after the first diagnosis of SARS-CoV-2 infection. Additional conditions, such as concomitant bacterial pulmonary superinfection, lung aspergillosis, thromboembolic phenomena, and hemorrhages can further worsen tissue damage.

Keywords: COVID-19; SARS-CoV-2 RNA PCR; autopsy; histopathology; long-COVID; postmortal swabs; pulmonary superinfections.

Figure 1

Representative histopathological findings in autopsies from the first wave. (A) Lung tissue with diffuse alveolar damage in the exudative phase (hematoxylin and eosin (H&E), magnification 13 x, arrow: hyaline membranes). (B) Lung tissue with diffuse alveolar damage in the proliferative phase, which is defined by the presence of organization of the intra-alveolar and interstitial exudate, infiltration with chronic inflammatory cells, and interstitial myofibroblastic reaction. Proliferation and reactive atypias of type II cells are also noted (H&E, magnification 20 x). Inset: reactive pneumocytes type II, magnification 40 x). (C) Lung tissue with diffuse alveolar damage in the proliferative phase, showing an excessive collagen deposition (H&E, magnification 20 x). (D) Lung arterioles with endotheliitis, which is defined by the presence of subendothelial mononuclear inflammatory infiltrates (arrows) and damage of the endothelium (H&E, magnification 25 x) (E) Double immunohistochemistry [red: CD31 (endothelial marker), brown: CD68 (monocytic/macrophage marker)] of another representative case with endotheliitis shows endotheliitis of a venule in the lung with endothelial damage (arrow) and detachment with an associated mononuclear infiltrate (arrowhead) (H&E, magnification 28 x).

Figure 2

Representative histopathological findings in autopsies from the first wave. (A) An overview of multifocal intracerebral microhemorrhages (arrowheads) in a sample from the brain stem (H&E, magnification 1.2 x). (B) The detail of image A with microhemorrhages (magnification 25 x, arrows: microhemorrhages). (C) An overview of acute brain infarction (arrows) in a sample from basal ganglia (H&E, magnification.62 x). (D) Details of image C showing red neurons (arrowheads) and beginning necrosis (arrows) of brain tissue (H&E, magnification 40 x).

Figure 3

Representative histopathological findings in autopsies from the second wave. (A) Acute pulmonary hemorrhage (H&E, magnification 10 x); the inset shows erythrocytes invading alveolar spaces (magnification 40 x). (B) Acute hemorrhagic pulmonary infarct (H&E, magnification 5 x); the inset shows a necrotic alveolar septum and hemorrhagic effusion in alveolar space (magnification 40 x). (C) Large thrombus (arrow) in a lung arteriole (H&E, magnification 1.4 x); the inset shows a fibrin thrombus in a lung capillary (magnification 28 x). (D) Glomerulus (kidney) with fibrin microthrombus (arrow) in a glomerular capillary (H&E, magnification 25 x). (E) Acid fuchsin orange G stain (AFOG-stain) shows several fibrin microthrombi (arrows) in the glomerular capillaries (magnification 25 x).

Figure 4

Representative histopathological findings in autopsies from the second wave, illustrating examples of coinfections in the lung of the patients with coronavirus disease 2019 (COVID-19). (A) Lung tissue with aspergillosis (arrow) and surrounding acute inflammation (H&E, magnification 10 x). (B) Details of image A showing the typical hyphae of Aspergillus spp. (PAS, magnification 30 x). (C) Acute (bacterial) bronchopneumonia showing granulocytic exudate in the alveolar space and destruction of alveolar septa (H&E, magnification 6 x); the inset illustrates granulocytic inflammation with destruction of a septum (magnification 50 x). (D) Herpes simplex pneumonia exhibiting typical herpes-associated nuclear changes (molding, multinucleation, margination of chromatin, see the arrow) (H&E, magnification 25 x). (E) Immunohistochemistry for Herpes simplex virus demonstrates a granular cytoplasmic and nuclear positivity along with the typical nuclear changes (magnification 25 x).

Figure 5

Postmortem findings in the patients of the second wave (green: finding present, red: finding absent. Concerning micro- and macrothrombi, hemorrhages, and infarcts; the following colors to specify the anatomic localization are used: heart and/or major vessels = violet, lung = blue, brain/intracranial = yellow, liver/digestive tract = brown, kidney = orange). The patients are ordered from left to right in a crescent pattern based on the number of days between diagnosis of COVID-19 through nasopharyngeal swab and death. DAD, diffuse alveolar damage; NA, not available.

Figure 6

Persistence of histopathologic findings in relation to the time interval between diagnosis and death (in days). The lines do not show absolute percentages but only the relative variation in prevalence of the findings. The different levels of the line along the y-axis are chosen to avoid their overlapping and to facilitate the visual interpretation but do not reflect an absolute percentage, for which we refer the reader to the text.

Figure 7

Positivity of the postmortem swabs in the different organs (green: positive, red: negative, black: not available). The patients are ordered from left to right in a crescent pattern based on the number of days between diagnosis of COVID-19 through nasopharyngeal swab and death. Abbreviations: NA, not available.

Figure 8

The tendency of positivity of swabs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in relation to the time interval between diagnosis and death (in days). The lines do not show absolute percentages but only the relative variation in prevalence of the positivity for SARS-CoV-2 RNA. The different levels of the line along the y-axis are selected to avoid their overlapping and to facilitate the visual interpretation but do not reflect an absolute percentage, for which we refer the reader to the text.

Figure 9

The tendency of postmortem persistence and anatomical distribution of SARS-CoV-2 RNA in relation to the time interval between death and autopsy (postmortem interval, in hours). The lines do not show absolute percentages but only the relative variation in prevalence of the positivity for SARS-CoV-2 RNA. The different levels of the line along the y-axis are chosen to avoid their overlapping and to facilitate the visual interpretation but do not reflect an absolute percentage, for which we refer the reader to the text.

Figure 10

Comparison of morphologic findings at autopsy between first and second pandemic waves.