How Long Can a Dead Body Remain Infectious?

Postmortem Nasopharyngeal Swabs and SARS-CoV-2 Culture in a Corpse Over an 87-Day Period

Abstract

The SARS-CoV-2 pandemic involved several changes and difficulties in the work of forensic pathologists. Postmortem nasopharyngeal swabs for the diagnosis of the SARS-CoV-2 infection are recommended before an autopsy examination by the Centers for Disease Control and Prevention.

Autopsy examinations must not be performed for SARS-CoV-2 infection cases when airborne infection isolation rooms or other suitable spaces are unavailable. However, it has not yet been reported whether the presence of SARS-CoV-2 at a low viral load may be enough to infect and disseminate the contagion.

Here, we report the case of a 67-year-old man found dead at home on November 9, 2020, and transferred immediately after to the Genova District Mortuary. As the first postmortem molecular nasopharyngeal swab resulted positive, a weekly sampling was carried until February 4, 2021. All the molecular tests were positive for SARS-CoV-2, including the last swab performed 87 days after the arrival of the corpse at the morgue. Virus isolation conducted on VERO E6 cells revealed no cytopathic effect indicating no viral replication as early as 18 days after the corpse's arrival at the morgue and until January 2021.

Our findings suggest that the presence of the genome of SARS-CoV-2 at low viral load should not be considered a sign of an active infection but a trace of a remaining viral genome from a previous infection. Then, if the virus shows no replication activity, its molecular detection should not constitute a threat to public health. Further studies are required to establish the infection's potential and its correlation with viral load.

The spread of coronavirus disease 2019 (COVID-19) due to SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has firmly established the development of a worldwide pandemic.^1,2 This novel coronavirus has shaken the global economy, society as a whole, and, in particular, the sustainability of national healthcare systems.³

The COVID-19 can cause mild symptoms or asymptomatic infections in at least 80% of patients but is potentially capable of causing acute respiratory distress in humans leading to death.^4,5 The median incubation period is 3 to 4 days (0–24 days).⁶ The most common symptoms observed at the initial onset of the disease are a cough, fever, fatigue, difficulty in breathing, sore throat, headaches, conjunctivitis, and gastrointestinal/stomach upsets.⁷

Approximately 5.0% of the infected subjects experience a severe form of the disease (admission to the intensive care unit), 2.3% of the subjects underwent invasive mechanical ventilation, and 1.4% died as a result of the infection.⁸ Consequently, the treatment of symptoms related to SARS-CoV-2 has required a great effort by health systems and a radical change in patient management, even at the expense of the treatment for other diseases (eg, cancer or cardiovascular diseases).⁹

For these reasons, accurate diagnostic tests are essential for controlling the COVID-19 pandemic; because the first phase of the health emergency, reverse transcriptase–polymerase chain reaction (RT-PCR) to identify viral RNA in nasopharyngeal swabs was identified as the standard test for a rapid diagnosis of the presence of SARS-CoV-2 in samples.¹⁰ Reverse transcriptase–polymerase chain reaction tests present a high sensibility and specificity; however, false negatives are possible depending on the swab type and period since the onset of symptoms.¹¹

Human-to-human SARS-CoV-2 infection is known to occur through close contact, in particular, through respiratory droplets in the air when an infected person coughs or sneezes.¹² It was reported that the virus can remain infectious for days on surfaces but very little is known about the persistence of SARS-CoV-2 in the body after the death of infected individuals. Particularly, it is not known for how long SARS-CoV-2–positive corpses remain infectious and if the virus can be transmitted through and/or during an autopsy examination.

For this reason, many problems difficult to solve have arisen for forensic pathologists. In autopsy routine practice, we currently know that postmortem nasopharyngeal swabs are a fundamental screening tool before an autopsy examination.^13,14

In particular, the Centers for Disease Control and Prevention recommend collecting nasopharyngeal swabs as a standard for the diagnosis of the active SARS-CoV-2 infection before performing an autopsy.¹⁵ The autopsies of corpses infected with SARS-CoV-2 (category hazard group 3) must be carried out in airborne infection isolation rooms (AIIRs), with negative pressure and a minimum of 6 to 12 air renewals per hour with the expulsion of the air directly outside or through HEPA (high-efficiency particulate air) filters. In the absence of AIIRs, autoptic investigations must be conducted in rooms at negative pressure without air recirculation into adjacent rooms.¹⁶ The main objective of this procedures is to safeguard the forensic pathology staff (ie, pathologists, technicians, and biologists).¹⁷ When autoptic rooms, AIIRs, or other adequate spaces lack, autopsies must not be performed.¹⁸ Indeed, the most relevant question is: how long can a corpse remain infectious?

Viral pathogens but also bacteria and fungus can remain viable within a deceased body and maintain their infectious capacity, but there is little literature on this topic¹⁹: for example, viable HIV can be detected in blood up until 2 weeks from death.²⁰ On the contrary, HCV-RNA has been found in the cadaveric blood of 19% of the cases,²¹ but according to the authors, this result does not necessarily entail the presence of viable virus.

In the present article, we report the case of a 67-year-old man found dead in his home on November 9, 2020, and brought to the Genova District Mortuary. At the arrival at the morgue for an autopsy examination, the corpse underwent nasopharyngeal postmortem swab, to determine the presence of SARS-CoV-2. The swab tested positive and the body remained at the morgue until February 4, 2021. This circumstance allowed us to perform weekly swab tests and evaluate the persistence of SARS-CoV-2 in a corpse coupled with the replication capability of the virus on cells cultures.

CASE REPORT

Case History

On November 9, 2020, the corpse of a 67-year-old man, found dead at his home, was brought to the Genoa District Mortuary. Considering the absence of adequate autopsy rooms (AIIRs) in the Liguria region during the COVID-19 pandemic, to ensure a safe environment in which to carry out an autopsy, upon the arrival of the corpse at the morgue our protocol contemplated a preliminary nasopharyngeal swab to identify the presence of the SARS-CoV-2 virus in the body before postmortem examinations.

The man was affected from coronary artery disease, arterial hypertension, and atrial fibrillation treated with β-blockers drugs. Data obtained from the general practitioner of the deceased reported that the man had received the flu vaccination 15 days before death, while no respiratory symptoms, cough, dyspnea, or fever was indicated. No further health or circumstantial data are available.

Upon arrival at the morgue, the body was stored in a cold chamber with a constant temperature of 5°C. The first nasopharyngeal swab performed the day of the arrival at the morgue resulted positive for SARS-CoV-2. The body presented a green discoloration over the entire abdomen and face as a result of the putrefactive phenomena; normal rigor mortis was in the process of settling, and the hypostases were fixed to the posterior regions of the back and limbs. Circumstantial data and putrefactive phenomena suggested that the man's death occurred at least 48 hours before the arrival at the morgue.

After the diagnosis of infection, the autopsy was no longer performed. The cause of death was attributed to natural causes (ie, cardiorespiratory failure due to cardiological condition). The contribution of SARS-CoV-2 infection to the death cannot be excluded.

Because the corpse was never claimed for a significant amount of time, it remained at the morgue for 87 days (until February 4, 2021); during this period, 15 postmortem nasopharyngeal swabs were collected. Regarding putrefactive phenomena, during the aforementioned period, the corpse developed a progressive drying of the tissues and the formation of mold in the perioral, nasal, and periocular regions.

Materials and Methods

Fifteen nasopharyngeal swabs were collected and stored approximately once a week from November 9, 2020, to February 4, 2021.

Multiplex RT-PCR tests were performed for SARS-CoV-2 diagnosis by means of Allplex SARS-CoV-2 Assay kit (Seegene, Inc, South Korea), according to the manufacturer's instructions.²² All nasopharyngeal swabs were stored at room temperature and processed for molecular analysis within 12 hours from their arrival to the laboratory and then stored to −80°C.

To evaluate the infectiousness and the replication capability of the virus, 3 samples were tested for viral isolation on VERO E6 cells cultures. Cultures were observed after 24, 48, 72, and 96 hours for evidence of cytopathic effects. The culture supernatant was collected in each point of observation to perform RT-PCR. RNA was manually extracted from both nasopharyngeal swabs and culture supernatant from 200 μL of samples with QIAamp Viral RNA kit (Qiagen, Venlo, the Netherlands) according to the manufacturer's instructions. RNA was eluted in 50 μL of water and used as the template for RT-PCR. For qPCR (https://international.neb.com/products/e3005-luna-universal-one-step-rt-qpcr-kit, Luna Universal One-Step RT-qPCR; New England BioLabs) and SARS-CoV-2 (2019-nCoV) Centers for Disease Control and Prevention qPCR Probe Assay (Integrated DNA Technologies, Inc) kits were used.

SARS-CoV-2 amplicons to assess the full genome were obtained using 2 different primers pool (https://artic.network/ncov-2019), purified with AMPure XP beads (Beckman Coulter) and checked for their size using TapeStation 200 (Agilent Technologies). The amplicon library for Illumina deep sequencing was prepared by using Illumina DNA Prep and IDT ILMN DNA/RNA Index kit set A (Illumina) according to the manufacturer's manual. The library concentration was determined by using the Invitrogen Quant-iT Picogreen dsDNA assay. The resulting libraries were normalized and pooled for subsequent sequencing on an Illumina MiSeq platform using the 2 × 200 cycle paired-end sequencing protocol. SARS-CoV-2 full-length genomes were submitted in GISAID (http://gisaid.org/).

Results were mapped and aligned to the reference genome available on GISAID (https://www.gisaid.org/, accession ID: EPI_ISL_406800). Consensus sequences were generated by Geneious software (v. 9.1.5; Biomatters, Auckland, New Zealand, http://www.geneious.com).²³

SARS-CoV-2 sequences were classified using the Pangolin COVID-19 Lineage Assigner tool v. 2.3.2 and Nextclade v. 0.14.1.^24,25 The consensus sequence has been deposited into GISAID (accession ID: EPI_ISL_2637801).

Results

All nasopharyngeal swabs resulted positive for the presence of SARS-CoV-2 (median cycle threshold, Ct: 30, 31, 29 for E, RdRp/S, and N genes, respectively) including the last one obtained 87 days after the arrival of the corpse at the morgue (Table 1).

TABLE 1 - Series of 15 Postmortem Nasopharyngeal Swabs Collected After the Arrival of the Corpse at the Morgue

Postmortem Nasopharyngeal Swab (Date/Day)	Positive/Negative	Cycle Threshold
09/11/2020 (day 0)	Positive	30.2
15/11/2020 (day 6)	Positive	33.5
21/11/2020 (day 12)	Positive	31.7
27/11/2020 (day 18)	Positive	34.1
03/12/2020 (day 24)	Positive	32.6
09/12/2020 (day 30)	Positive	30.9
15/12/2020 (day 36)	Positive	31.5
21/12/2020 (day 42)	Positive	33.9
27/12/2020 (day 48)	Positive	30
02/01/2021 (day 54)	Positive	33
08/01/2021 (day 60)	Positive	36.5
14/01/2021 (day 66)	Positive	36
20/01/2021 (day 72)	Positive	30.6
26/01/2021 (day 78)	Positive	34.5
04/02/2021 (day 87)	Positive	35.1

In VERO E6 cultures, the swabs collected on November 27, 2020 (18 days), December 27, 2020 (48 days), and January 20, 2021 (72 days) showed no cytopathic effect at the 48/72 hour (Table 2). Despite supernatants were tested positive by using RT-PCR at the 24-, 36-, and 48-hour marks after culture start there was no evidence of viral growth.

TABLE 2 - Trend of Molecular Detection of SARS-CoV-2 on Cell Culture Specimen

Day Cell Culture	Cycle Threshold
1°	30.35
2°	30.62
3°	31.3
4°	30.83
5°	0
6°	0
7°	0
Real-time variant	0
Virus isolation	None

A full genome sequence was obtained from the first original sample. The patient sequence belonged to clade 20E, corresponding to lineage B.1.177 (Fig. 1).

FIGURE 1: Phylogenic analysis of the viral strain (June 22, 2021; https://clades.nextstrain.org ²¹).

DISCUSSION

In this study, 15 postmortem nasopharyngeal swab tests were performed in an individual who had died of natural causes to determine the presence of SARS-CoV-2 viral RNA in the upper respiratory tract as well as the possibility of infectiousness after death.

In a study conducted by Skok et al²⁶ on 125 postmortem swabs, viral RNA was discovered up to 128 hours after death. The cycle threshold values for the throat swabs revealed an average of 28.12, with only a slow decrease of positivity, comparable with those observe in our case. However, in our patient, we did not highlight relevant changes in Ct values considering all samples examined.

According to La Scola et al,²⁷ the median Ct of 30 for E gene indicates a low viral RNA load as well as a low probability of contagion. The authors suggested that patients with Ct values of 33 to 34 (solely for E gene) were no longer infectious and could be discharged from hospital.

Similarly, Heinrich et al²⁸ revealed a lack of a time-dependent effect on SARS-CoV-2 viral loads in a series of 9 sequential pharyngeal swab samples. They also determined the SARS-CoV-2 RNA persistence at constantly high titers up to 1 week (168 hours) after death.²⁸

Finally, according to Edler et al,²⁹ postmortem evidence of COVID-19 through a nasopharyngeal or oropharyngeal swab was discovered up to a maximum postmortem interval of 12 days, whereas Plenzig et al¹⁸ showed that positivity still persists in a corpse with visible signs of decomposition (internal and external putrefaction) after a postmortem interval of 17 days. In particular, the reported Ct values were 25.08 and 20.88 in the right lung and left, respectively.

As the corpse, in this case, was stored in a cold chamber with a constant temperature of 4°C, it can be suggested that at low temperatures, the viral load does not show a rapid decrease but remains constant over time.

Similarly, by using respiratory tract swabs, Beltempo et al³⁰ documented the persistence of SARS-CoV-2 RNA in the upper respiratory tract 35 days after death.

In all cited studies, positivity to SARS-CoV-2 was already diagnosed before death. However, in our study, the diagnosis was attained only by postmortem swabs.

Moreover, we cannot exclude that putrefaction can alter the results of the test. In addition, environmental conditions and temperature could also interfere with such results. It is also postulated that virus can be detected at higher concentrations in decomposition because the viral genome is released with cell lysis; on the other hand, no cases are reported in the literature of a putrefied corpse with positive swab results.

Regarding microbiological culture analysis, according to a study conducted by Bullard et al,³¹ SARS-CoV-2 VERO cell infectivity (growth of the virus in culture) is Ct dependent and was solely observed for an RT-PCR Ct value less than 24, indicating lower infectivity in patients with a Ct value greater than 24. In addition, these results were obtained from live human samples and not proven in cadaveric cases. In particular, and to the best of our knowledge, there are no serial culture studies of SARS-CoV-2 on corpses.

In the presented case, the positivity of the nasopharyngeal swab test was observed in all performed tests. The corpse remained at the morgue for 87 days, and during this period, all the samples obtained from nasopharyngeal swabs tested positive for the presence of SARS-CoV-2 with a Ct value greater than 29. This represents the longest time frame (ie, time lapse between the arrival of the corpse at the morgue and the nasopharyngeal swab test) described in the literature. Therefore, RT-PCR of the nasopharyngeal swab is confirmed to be the most reliable test to evaluate the possible presence of the virus in the corpse.³²

As reported by Plenzig et al,¹⁸ it is possible that the stationing of the corpse at a constant temperature of approximately 5°C had favored the persistence of the virus and prevented the degradation of the viral genome.

Viral growth in culture was not identified in our study. Therefore, we can hypothesize that although virus has always been detectable in the upper respiratory tract (through a nasopharyngeal swab test), with the identical number of cycles, the virus was no longer able to replicate and infect at least after approximately 20 days of time of death. Comparable results were observed in live patients showing same Ct values.

The major limitation of the present study is the lack of culture analysis conducted on samples from the first swab obtained after the corpse's arrival at the morgue. Moreover, the impossibility of performing an autopsy due to the absence of AIIRs rooms or other adequate spaces did not allow us to determine whether the man died with or because of SARS-CoV-2. At the same way, our Institute of Legal Medicine does not have a whole-body computed tomography scan dedicated to corpse. Moreover, the hospital's computed tomography scans were not available because of the presence of too many hospitalized COVID-19 patients.

Another limitation of the study has been the use of only Allplex SARS-CoV-2 assay (RT-PCR; Seegene, Inc, South Korea). In fact, we had not the possibility to use another test kit (eg, rapid PCR) to assess the diagnostic performance of the assay against RT-PCR through the calculation of accuracy.

Our study represents a preliminary assessment; further studies are needed testing different viral load and larger cases to validate our results.