Validity of the Rapid Nasopharyngeal Antigen Swab for the Detection of SARS-CoV-2 on Cadavers

Abstract

Background: SARS-CoV-2 is classified as a class 3 biological agent; therefore, autopsies on positive subjects must be performed in BSL 3 sectorial rooms. However, many centers lacking such facilities perform molecular nasopharyngeal swabs for SARS-CoV-2 on corpses before autopsy. This approach, though, is marked by prolonged reporting times and extremely high costs. This study aims to compare the results of molecular swabs (RT-PCR) with rapid antigen swabs (RAT) in order to assess if RAT can serve as the sole test for determining corpse positivity or negativity. Methods: Sixty corpses with positive molecular nasopharyngeal swabs for SARS-CoV-2, performed either ante-mortem or post-mortem, were selected. Afterward, they underwent rapid antigen swabs within 0 to 11 days after the last molecular exam. Results: Out of 60 corpses with positive molecular swabs, 52 antigen swabs were positive (86.67%), and 8 were negative (13.33%), indicating a sensitivity of 86.66% and specificity of 100%. Conclusions: Considering the sensitivity and specificity values observed in this study, RAT could be used as the primary investigation on corpses, especially in centers that lack BSL 3 sectorial rooms. Molecular swabs could then serve as a secondary test for subjects negative on RAT.

1. Introduction

From the onset of the first outbreak, the coronavirus disease (COVID-19) has led to over 7 million deaths [1]. The pandemic caused serious health system problems, such as hospital-acquired infections [2].

SARS-CoV-2 is classified as a class 3 biological agent [3], corresponding to agents capable of causing severe diseases in humans and posing a serious risk to workers.

Therefore, autopsies on SARS-CoV-2 positive corpses must be performed in biosafety level 3 (BSL-3) autopsy rooms [4,5]. As defined by the Centers for Disease Control and Prevention in Atlanta, the use of the BSL-3 room is appropriate when there is a risk of transmission of serious infections via aerosols [6]. A growing body of scientific research has demonstrated that SARS-CoV-2, the virus responsible for COVID-19, can persist in the bodies of deceased individuals for extended periods post-mortem. Studies have shown that the virus can remain detectable in cadavers for up to 12–13 days [7,8,9,10,11,12]. This finding has significant implications for public health, particularly in the context of forensic pathology and infection control practices. The persistence of SARS-CoV-2 in cadavers underscores the importance of rigorous infection control measures during autopsies and other post-mortem procedures. Healthcare workers performing these procedures should adhere to strict guidelines to minimize the risk of exposure to the virus. Understanding the persistence of SARS-CoV-2 in post-mortem individuals is crucial for informing public health policies and guidelines. By recognizing the potential risks and implementing appropriate measures, we can effectively mitigate the spread of the virus and protect healthcare workers. Due to this requirement, several centers lacking such safe autopsy rooms, starting from the first wave of the pandemic, began conducting molecular nasopharyngeal swabs on corpses before performing autopsies in lower safety-level containment rooms. The latter is characterized by both a lengthy reporting time (24–72 h depending on the availability of the processing laboratory) and extremely high costs.

During the pandemic, in addition to molecular testing, various rapid antigen tests (RAT) have become available. These tests provide results of infection positivity or negativity within 15–20 min, and most importantly, they do not require processing at a laboratory, significantly reducing reporting times and costs.

While several studies have been conducted on the validity of molecular nasopharyngeal swabs in corpses during the pandemic, little is known about the use of rapid antigen swabs.

The aim of this study is to compare the results of rapid antigen swabs performed on 60 corpses that tested positive for SARS-CoV-2 with molecular swabs. The goal is to evaluate its accuracy with the intention of using it as the sole test for determining corpse positivity or negativity.

2. Materials and Methods

Approximately three hundred autopsies are performed in Genoa (Liguria, Italy) per year; until December 2023, an autopsy room with BSL-3 safety criteria was not available in our center, so it was necessary to perform molecular swabs on each corpse in order to ensure the safety of the operators. However, molecular swabs are burdened with high costs and long reporting times. Therefore, with this study, we sought to evaluate the validity of using rapid antigen swabs for the detection of SARS-CoV-2 in cadavers in order to reduce the cost and time of swabbing but preserving the safety of operators. Sixty bodies with positive molecular nasopharyngeal swabs for SARS-CoV-2, performed ante-mortem or post-mortem, were selected. The study included cases managed by the Department of Forensic Medicine over a one-year period, specifically from July 2022 to July 2023. The bodies underwent RAT within 0 to 11 days after the last molecular test. The timing of RATs versus molecular swabs depended on the availability of materials and personnel.

For each selected case, the following data were collected: gender, age, date, and the results of the molecular swabs performed during life or post-mortem, cause of death, and the date and results of post-mortem antigenic swabs. All swabs conducted post-mortem were performed by the medical examiner; the operator was wearing the necessary personal protective equipment (FFP-2 mask, gloves, disposable gown, and shoe covers). In subjects with positive molecular swabs in life, these were performed at the hospital ward; RAT, however, was in all cases performed by the medical examiner. Post-mortem molecular and rapid swabs were performed at the same site. A single swab (one molecular and one rapid) was used for both nostrils during specimen collection.

Furthermore, 10 control cases with a negative molecular swab, either during life or post-mortem, were also selected. Molecular nasopharyngeal swabs were performed using the multiplex RT-PCR method with the Allplex SARS-CoV-2 Assay kit (Seegene, Inc., Seoul, Republic of Korea), according to the manufacturer’s instructions [13]. Regarding post-mortem molecular swabs, these were stored at room temperature and processed for molecular analysis within 12 h of their arrival at the laboratory.

Antigenic nasopharyngeal swabs were performed using the SARS-CoV-2 Antigen Rapid Test Cassette (Sejoy) kit according to specific instructions. Specifically, the swab was inserted into both nostrils of the corpse until resistance, indicative of reaching the nasopharynx, was felt. At least 5 rotations were performed in each nostril, and then the swab was slowly removed. The swab was then immersed in an extraction tube containing the extraction reagent, rotated, and pressed for at least 10 s. The samples were immediately processed by adding 2 drops of the treated sample into the small well of the cassette. Finally, the results were read after 10 min and no later than 15 min.

Regarding the interpretation of the results, according to the specific instructions, a single colored line in the control region (C) of the cassette indicates a negative test. On the other hand, the presence of both this line and another colored line in the test region (T) of the cassette indicates a positive result for the rapid swab, signifying the detection of SARS-CoV-2 in the sample.

In eight cases that tested positive on the post-mortem molecular swab for SARS-CoV-2, the autopsy was not performed due to biosecurity reasons as our Forensic Medical Center was not equipped with a BSL-3 autopsy room in accordance with the contemporary national recommendations. In these cadavers, a thorough external examination was performed to exclude traumatic injuries, and death was attributed to undefined natural causes.

It should be noted that these investigations of the cadaver were carried out for diagnostic purposes and, therefore, in accordance with Presidential Decree 285/1990 (Approval of the Mortuary Police Regulations).

3. Results

Of the 60 cases in this series, 35 were male (58.33%) and 25 were female (41.67%). The average age was 83.43 years (minimum 60–maximum 99).

Regarding symptomatology, 36 patients (60%) presented respiratory failure before death, of which 30 cases were associated with instrumentally diagnosed interstitial pneumonia and 2 with pulmonary embolism (PE). Four cases presented with high fever, hypotension, tachycardia and anuria; three cases presented marked asthenia, declivous edema, and severe respiratory fatigue with dyspnea; one case urinary disorders associated with altered consciousness; one case intense abdominal pain, fever and altered consciousness; one case abdominal pain, vomiting, and altered consciousness; one case presented with post-traumatic extradural hemorrhage and associated altered state of consciousness, and one case had a femur fracture with functional impotence and pain in the inferior limb and acute and terminal cardiorespiratory failure. Another three cases were in a comatose state at the time of death: of these, two cases entered the hospital already in a comatose state, including one post-traumatic with subarachnoid hemorrhage and one due to diabetic ketoacidosis, while one case developed a coma during hospitalization following acute heart failure. Finally, for an additional nine cases (15%), there were no known symptoms before death.

In 44 cases (73.33%), the cause of death was attributed to a SARS-CoV-2 infection. As for the other 16 cases the cause of death was caused by other pathologies, and the finding of SARS-CoV-2 positivity was considered incidental. Among these, in eight cases, due to the impossibility of performing an autopsy, death was attributed to undefined natural causes in subjects who tested positive on the post-mortem molecular swab. In four cases death was secondary to unrelated COVID-19 septic shock (one case for urinary tract infection by Escherichia Coli and septicemia by Sthaphylococcus Capitis, one case Staphylococcus Epidermidis septic shock, one case Staphylococcus epidermidis and Candida albicans septic shock and urinary tract infection by Enterecoccus fecium, and in the last case infection Candida Glabrata starting from the urinary tract); in one case to acute heart failure, in one case to post-traumatic cerebral hemorrhage, in one case to intestinal perforation with stercoraceous peritonitis, and in one case to intestinal occlusion.

Regarding the ten control cases, eight were male and two were female. The average age was 65 (minimum 26–maximum 80).

Out of the 60 corpses with a positive molecular nasopharyngeal swab, 52 antigenic swabs tested positive (86.67%), and 8 tested negative (13.33%).

Among the 52 positive antigenic swabs, they included 37 out of 44 decedents whose death was attributed to a SARS-CoV-2 infection (84.10%) and 15 out of 16 subjects where SARS-CoV-2 positivity was considered incidental and not causative of death (93.75%).

Among the 52 corpses with positive post-mortem antigenic swabs, the average time interval between this test and the molecular nasopharyngeal swab was 2.63 days (minimum 0–maximum 11).

On the other hand, in the group of eight corpses with negative post-mortem antigenic swabs, it included seven subjects whose death was related to clinical manifestations of COVID-19 (15.90%) and one subject in whom SARS-CoV-2 positivity was classified as an incidental finding (12.5%).

In this group, the average time elapsed between the last molecular swab performed and the post-mortem antigenic swab was 3 days (minimum 1–maximum 9).

Table 1. Symptoms at the time of death, associated diseases, correlation of death with COVID-19, and postmortem rapid antigen test (RAT) outcome in the 60 cases with positive molecular swab.

Symptoms		Associated Disease	Death COVID-19 Related	RAT Outcome
Respiratory failure	n. 36	n. 30 interstitial pneumonia n. 2 PE n. 4 not known	32 cases	Positive n. 33 Negative n. 3
High fever, hypotension, tachycardia, and anuria	n. 4	Septic shock and multiorgan failure	2 cases	Positive n. 2 Negative n. 2
Marked asthenia, declivous edema, and severe respiratory fatigue with dyspnea	n. 3	Acute heart failure	3 cases	Positive n. 2 Negative n. 1
Urinary disorders associated with altered consciousness	n. 1	Urinary tract infection and acute glycometabolic decompensation	1 case	Positive n. 1 Negative n. 0
Intense abdominal pain, fever, and altered consciousness	n. 1	Intestinal perforation	1 case	Positive n. 1 Negative n. 0
Abdominal pain, vomiting, and altered consciousness	n. 1	Intestinal occlusion	1 case	Positive n. 1 Negative n. 0
Altered state of consciousness	n. 1	Post-traumatic cerebral blood collection	0 case	Positive n. 1 Negative n. 0
Functional impotence and pain in the inferior limb	n. 1	Femur fracture and acute and terminal cardiorespiratory failure	1 case	Positive n. 1 Negative n. 0
Irreversible coma	n. 3	n. 1 trauma n. 1 diabetic ketoacidosis n. 1 acute heart failure	2 cases	Positive n. 1 Negative n. 2
Not known	n. 9		1 case (8 cases in which an autopsy could not be performed)	Positive n. 9 Negative n. 0

summarizes the pre-death symptomatology, associated diseases, cause of death related or unrelated to COVID-19, and the post-mortem rapid antigen test (RAT) outcome of the 60 subjects with a positive molecular swab.

In all 10 control cases, the post-mortem nasopharyngeal molecular swab for the detection of SARS-CoV-2 was negative. The post-mortem antigenic swab was carried out after an average time of 2.1 days (minimum 0–maximum 7) from the molecular swab and was negative in all cases. The causes of death of these ten subjects were all unrelated to SARS-CoV-2 (seven deaths from common pathological causes of cardiovascular, cerebrovascular, neoplastic and septic, one case of trauma secondary to traffic accident, one suicide by firearm, and one case of drug intoxication).

4. Discussion

The SARS-CoV-2 pandemic has had a significant impact on the healthcare system worldwide. In the field of forensic medicine, this infection has brought about profound changes, drawing attention to the need for departments to have an autopsy room with an appropriate level of safety to conduct autopsies on infected corpses.

In the field of forensic pathology, several studies have investigated the diagnostic accuracy of molecular swabs for detecting SARS-CoV-2 in cadavers. Specifically, Skok K. et al. [14] conducted 125 post-mortem molecular swabs, revealing the virus’s survival for up to 128 h after death. Similarly, Heinrich et al. [15] found consistently high SARS-CoV-2 titers persisting for a week (168 h) after death. Plenzing et al. [16] explored the duration of SARS-CoV-2 infection in four subjects who had died while being positive during life. They demonstrated that in two bodies, infectivity lasted for a shorter duration, with no positive results after 2 and 11 days, while in the other two cases, positivity was detected 4 and 17 days after death.

In another study, Plezing et al. [17] indicated the persistence of SARS-CoV-2 positivity in molecular swabs of the oropharynx and lung tissue in two exhumed bodies, conducted four months after death.

Additionally, Ventura et al. [12] demonstrated the survival of the SARS-CoV-2 virus in a deceased individual by assessing its presence through serial molecular swabs. In this case, the positivity of SARS-CoV-2 was not known during the person’s life but was detected in the first post-mortem molecular swab, remaining positive even after 87 days post-mortem.

As for the use of swabs on cadavers, currently, laboratory-based nucleic acid amplification tests (NAATs), including reverse transcription polymerase chain reactions (RT-PCR), remain the gold standard for the diagnostic detection of SARS-CoV-2 [18].

To date, only a few studies have focused on evaluating the use of antigen swabs on bodies undergoing autopsy examination. Zacharis et al. [19] compared the results of rapid antigen swabs with molecular swabs in a total of 30 patients, concluding that rapid antigen swabs should not be considered a potential replacement for molecular testing but rather an addition to current post-mortem analysis strategies. Matsumoto et al. [20] highlighted a sensitivity of 91.67% and a specificity of 100% of postmortem RATs, concluding that the combined execution of rapid antigen testing with molecular testing can be useful in the post-mortem diagnosis of SARS-CoV-2 infection.

In live subjects, the diagnostic accuracy of rapid antigen swabs has been more extensively studied. For example, Albert et al. [21] investigated its efficiency in 54 symptomatic patients (Supplementary Materials) referred to a point of care (POC) and testing positive with a molecular swab. They detected concordance between the two swabs in 43 cases, while in 11 cases, the molecular swab was positive, and the rapid antigen swab was negative. This study demonstrated a specificity of the RAT of 100% (95%CI 98.7–100%) and a sensitivity of 79.6% (95%CI 67.0–88.8%). Furthermore, it was indicated that the viral load was significantly higher in subjects with both positive swabs compared to the 11 cases with negative rapid antigen swabs.

Iglòi et al. [22] evaluated the accuracy of the rapid antigen swab compared to the molecular swab in a population of non-hospitalized symptomatic subjects, revealing a sensitivity of 84.9% (95%CI 79.1–89.4), which increased to 95.8% (95%CI 90.5–98.2) in subjects who sought medical assistance within the first 7 days of the symptom onset. The specificity was 99.5% (95%CI 98.7–99.8).

In our study, out of 60 subjects positive (either in life or post-mortem) for the nasopharyngeal molecular swab (RT-PCR), 52 also tested positive for the rapid antigen swab, while all 10 cadavers that tested negative on the molecular swab were also negative on the RAT. Therefore, the execution of the rapid antigen swab revealed a sensitivity of 86.66% and a specificity of 100%.

From the literature analysis, it emerges that negativity in the rapid antigen swab is correlated with the viral load and infectivity of the patient, as individuals with a positive RT-PCR but negative rapid swab are unlikely to be infectious [21,23]. Recent studies have reported that there are no cases of COVID-19 transmission with viral RNA loads < 4 log10 copies/mL on respiratory tract swabs [24]. Viral research methodologies based on RT-PCR are able to detect SARS-CoV-2 RNA even after the infectious virus is no longer detectable: in most positive patients, it is not possible to isolate the infectious virus from upper respiratory tract samples after 10 days from the onset of symptoms, and it is unlikely these individuals pose a transmission risk to others. Furthermore, there is no evidence that persistent or recurrent detection of viral RNA after clinical recovery represents a risk of disease transmission [22].

Based on these data, it is possible to hypothesize that in the eight cases of discordance between the molecular swab and the antigen swab in this study, the individuals had a low viral load, and therefore, were not detectable in the rapid antigen swab. In support of this, in six of the eight cases, the last positive molecular swab performed during life was weakly positive, while in another case, the time interval between the last positive molecular swab and the negative post-mortem antigen swab was nine days. From this perspective, the main limitation of this study is related to the inability, due to lack of available data, to correlate the positivity/negativity of the rapid antigen test (RAT) with the onset and possible disappearance of the clinical symptoms of COVID-19. Another limitation of the present study is represented by the relatively small cohort; nevertheless, considering the few studies currently available regarding the comparison of the effectiveness of molecular and antigenic swabs in cadavers, this work still provides useful insights, adding further information to what is already known.

Therefore, the main limitations of this study are the limited sample number and the inability to correlate the RAT outcome with the clinical presentation over the subject’s lifetime; thus, it will be necessary to perform further in-depth studies on larger case series.

In conclusion, currently the gold standard for detecting SARS-CoV-2 in cadavers is RT-PCR, while the use of rapid antigen tests (RAT) as the sole diagnostic test is widely adopted with living subjects. The RAT result is closely related to the subject’s infectivity, making it valuable in preventing the transmission of the disease from person to person. On the other hand, RT-PCR, with its higher sensitivity and specificity, can detect the pathogen even at low viral loads in symptomatic, pre-symptomatic, or asymptomatic individuals [25].

According to the findings of this study and considering the observed sensitivity and specificity values, the RAT could be used as a frontline investigation for cadavers and, especially for centers without BSL-3 containment facilities, molecular testing could be reserved as a second-line test for individuals who test negative with RAT. Based on the data from this study, the use of RAT as a first-line investigation, as it has proven to be a reliable test in cases of positivity, could lead to a substantial reduction in reporting time and the cost of the test.