Infectivity of deceased COVID-19 patients

Corpses were stored at 6–8 °C within about 12–24 h after death. Intervals between death and autopsy were 1, 4, 9 and 17 days, respectively. A full autopsy was carried out for all corpses. Tissue samples of the brain, heart, lungs, liver, spleen, pancreas, kidneys, adrenal glands, thyroid gland and paratracheal lymph nodes, as well as small intestine and colon, were taken for histopathological examination. The samples were fixed in buffered 4.5% formaldehyde solution, processed according to standard procedures and stained with haematoxylin-eosin. For toxicological analysis, cardiac blood, femoral venous blood and urine were obtained. Samples were analysed using gas chromatography and high-performance liquid chromatography-mass spectrometry.

For the acquisition of virological examination specimen, care was taken to avoid contamination during the process. Instruments were disinfected appropriately (Incidin^TM Plus 0,5 %; Ecolab Healthcare; Monheim am Rhein, Germany) before and after removal of the specimen and specimen were taken as soon as possible during the course of the autopsy. UTM^TM swabs (Hain Lifescience; Nehren, Germany) were taken from perioral, both palms of the hands and inner parts of the elbow, reflecting the areas with highest probability of virus particle deposition during coughing, as well as from the oropharynx, trachea and both lungs. Swabs from the lungs were obtained after disinfection of the organ surface (Octenisept®; Schülke & Mayr GmbH; Norderstedt, Germany) and subsequent incision with disinfected instruments (Incidin^TM Plus 0,5 %; Ecolab Healthcare; Monheim am Rhein, Germany). In addition, small tissue samples of the heart, lungs, brain, liver, spleen, pancreas, left kidney, thyroid gland, small intestine and colon were obtained and stored in physiological sodium chloride solution.

Tissue samples and swabs were stored at −80 °C and thawed directly before preparation. Approximately 0.5 cm³ tissue specimen was dissected from original sample material and homogenised with 300 μL PBS (phosphate buffered saline). Both tissue homogenate and swabs were mixed with MEM (minimum essential medium) containing 1% FCS (foetal calf serum; Sigma-Aldrich; St. Louis, Missouri, USA), 3% Amphotericin B and 0.2% Primocin (InvivoGen; San Diego, California, USA). Swabs-inoculums were immediately transferred to Caco-2 cells (human colon adenocarcinoma cell line) seeded in 12.5 cm² culture tubes, whilst tissue-inoculum was transferred to Caco-2 cells after removing cell debris by centrifugation and filtration with 0.45 μm filter. Cytopathogenic effect (CPE) was assessed daily for up to 7 days. After 7 days or when cell lysis occurred, supernatants were inspected for the presence of viral RNA by real-time reverse transcription-polymerase chain reaction (RT-qPCR). Total RNA from cell-culture supernatants was isolated using AVL buffer (viral lysis buffer) and the QIAamp Viral RNA Kit (Qiagen; Hilden, Germany) according to the manufacturer’s instructions. SARS-CoV-2 RNA was analysed by RT-qPCR using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs; Ipswich, Massachusetts, USA) and primers targeting RNA-dependent RNA polymerase (RdRp):

If there were remnants of original tissue/swab, specimen RNA was extracted out of the remaining original sample prepared for virus isolation using the QIAsymphony instrument (DSP Virus/Pathogen Midi Kit; Qiagen; Hilden, Germany). SARS-CoV-2 RNA was detected by using the Allplex^TM 2019-nCoV (Seegene; Seoul, Korea) according to the manufacturer’s recommendations.

The 65-years-old male patient was admitted to the hospital with severe respiratory failure and suspected SARS-CoV-2 infection. A positive test result was gained 2 days after admission. During the course of his hospitalization he was intubated and ventilated, finally receiving extracorporeal membrane oxygenation (ECMO) treatment. He died in the hospital one month after admission. There were no known pre-existing medical conditions. Upon autopsy, a highly solidified dense lung tissue (weight left: 845 g, right: 1018 g) with fibrinous overlays of the visceral pleura, cardiac hypertrophy, coronary heart disease (CHD) and moderate atherosclerosis were noted. Histologically, a high level of diffuse irreversible alveolar damage with fibrosis, fibrin deposits of the visceral pleura and coronary atherosclerosis was identified. Close to small vessels, interstitial lymphocytic infiltrates were noted. The alveolar walls showed fibrotic reorganisation. Nuclei of alveolar macrophages appeared to be enlarged with prominent nucleoli. Toxicological tests of autopsy samples showed morphine, amantadine, midazolam, propofol, amiodarone and metabolic products of the aforementioned in non-toxic concentrations. Acute respiratory distress syndrome (ARDS) in the fibroproliferative phase of the disease due to COVID-19 was diagnosed as the cause of death.

The 88-year-old male patient was living in a geriatric institution affected by COVID-19. SARS-CoV-2 testing was carried out 11 days prior to death and showed a positive result. He was admitted to a hospital, where he died in the isolation ward. Known pre-existing medical conditions included chronic obstructive pulmonary disease (COPD), giant cell arteritis, mitral regurgitation and chronic kidney failure. Upon autoptical examination pulmonary oedema (weight left: 500 g, right: 780 g), COPD, cardiac hypertrophy, CHD, mitral insufficiency and granular renal atrophy were noted. Histologically, pronounced pulmonary oedema, patch-like lymphocytic infiltrates in alveolar septa, occasional microthrombi, signs of severe COPD and moderate coronary atherosclerosis were seen. Toxicological analysis of autopsy samples showed the presence of melperon in a non-toxic concentration. A combination of exacerbated pre-existing medical conditions and COVID-19 was the presumed cause of death.

The 88-year-old male patient was transferred to a geriatric ward after hip surgery, where he was then tested positive for SARS-CoV-2. His condition deteriorated over the next days with death occurring in the isolation unit of a hospital 19 days after the initial diagnosis. Pre-existing medical conditions included atrial fibrillation with pacemaker implantation, CHD, arterial hypertension and chronic cardiac and renal insufficiency. Upon autopsy solidified and partially oedematous lung tissue (weight left: 850 g, right: 875 g), COPD, cardiac hypertrophy, CHD, mitral insufficiency, signs of chronic right heart failure and severe atherosclerosis were seen. Histologically, severe pulmonary oedema, beginning diffuse alveolar damage with occasional hyaline membranes (exudative phase of the disease), desquamated alveolar macrophages partially with dysplastic nuclei, interstitial lymphocytic pneumonia and coronary atherosclerosis were seen. Toxicological tests yielded morphine in a non-toxic concentration. A combination of pre-existing diseases and COVID-19 was diagnosed as the cause of death.

The 82-year-old female patient had been tended by relatives after showing flu-like symptoms. Two days prior to death she began to suffer from fever, dyspnoea and cough. She was found on the floor in the living room inanimate by her husband. The death certificate stated 12:55 pm as the time of death and that no resuscitation efforts had been started after the initial contact due to existent signs of irreversible death. The woman had last been seen alive after 9 pm on the previous day. Pre-existing medical conditions included chronic heart failure, arterial hypertension, defibrillator implantation and schizophrenia. Not having been tested prior to death, a tracheal swab was taken 2 days after death, respectively 15 days before autopsy, which showed SARS-CoV-2 infection (cycle threshold: 21.6). Upon external examination, there were beginning signs of putrefaction with marbling of vessels, bloating of the body and mould infestation in the perioral and neck regions. Upon autoptical examination congested brittle lung tissue (weight left 570 g, right 585 g), CHD, cardiac hypertrophy, signs of chronic right heart overload, mitral insufficiency, defibrillator implantation and moderate general atherosclerosis were noted. Histologically, intraalveolar lung oedema with few lymphoid infiltrates in lung tissue, coronary atherosclerosis, beginning signs of putrefaction with post mortem bacterial overgrowth, starting cell fragmentation and weak staining of the nuclei in all organs were seen. Histological assessment was severely limited due to putrefaction. Toxicological tests revealed tramadol, melperon, quetiapine and olanzapine in non-toxic concentrations. Severe pre-existing medical conditions combined with COVID-19 were assumed as the cause of death.

Tissue samples of the lungs and swabs of some parts of the respiratory tract of case 2 und 4 showed infectivity in cell culture. In case 1 and 3, no cytopathogenic effect was observed in any sample. RNA of SARS-CoV-2 was detected in original tissue samples of the lungs in all four cases and in tissue specimen of the brain (case 3), small intestine and kidney (case 2). All results of post mortem virological examinations (cell culture and RT-qPCR from original tissue samples and swabs) are listed in Table 2.