Changes in SARS-CoV-2 viral load and titers over time in SARS-CoV-2-infected human corpses.

High viral titers of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been detected in human corpses long after death. However, little is known about the kinetics of infectious SARS-CoV-2 in corpses. In this case series study, we investigated the postmortem kinetics of infectious SARS-CoV-2 in human corpses by collecting nasopharyngeal swab samples at multiple time points from six SARS-CoV-2-infected patients after their death. SARS-CoV-2 RNA was detected by quantitative reverse transcription-polymerase chain reaction from nasopharyngeal swab samples collected from all six deceased patients. A viral culture showed the presence of infectious virus in one deceased patient up to 12 days after death. Notably, this patient had a shorter time from symptom onset to death than the other patients, and autopsy samples showed pathological findings consistent with viral replication in the upper respiratory tract. Therefore, this patient died during the viral shedding phase, and the amount of infectious virus in the corpse did not decrease over time up to the date of autopsy (12 days after death). The findings of this study indicate that the persistence of SARS-CoV-2 in corpses can vary among individuals and may be associated with the stage of the disease at the time of death. These important results complement many previously reported findings on the infectivity of SARS-CoV-2 at postmortem.

COVID-19 Analysis in Tissue Samples Acquired by Minimally Invasive Autopsy in Out-of-Hospital Deaths with Postmortem Degeneration.

Minimally invasive autopsy (MIA) is an alternative to a full autopsy for the collection of tissue samples from patients' bodies using instruments such as a biopsy needle. MIA has been conducted in many cases of coronavirus disease 2019 (COVID-19) and has contributed to the elucidation of the disease pathogenesis. However, most cases analyzed are hospital deaths, and there are few reports on the application of MIA in out-of-hospital deaths with varying extents of post-mortem changes. In this study, MIA and autopsies were performed in 15 patients with COVID-19 2-30 days after death, including 11 out-of-hospital deaths. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome detection by reverse transcriptase quantitative polymerase chain reaction using MIA samples was mostly consistent with autopsy samples, particularly lung tissue, even in out-of-hospital cases. MIA had high sensitivity and specificity (> 0.80). Histological examination of lung tissue obtained by MIA showed characteristics of COVID-19 pneumonia, with 91% agreement with autopsy samples, whereas localization of SARS-CoV-2 protein in lung tissue was indicated by immunohistochemistry, with 75% agreement. In conclusion, these results suggest that MIA is applicable to out-of-hospital deaths due to COVID-19 with various postmortem changes, especially when autopsies are not available.

Detection of SARS-CoV-2 and Its Related Factors on the Mucosal Epithelium of the Tongue.

SARS-CoV-2 infects a variety of tissues, including the oral cavity. However, there are few reports examining the association of SARS-CoV-2 with tongue mucosal tissues with sticky tongue debris. This study investigated the presence of SARS-CoV-2 and its associated molecules by dissecting tongue tissue from autopsy specimens of 23 patients who died of COVID-19-related illness (pneumonia). Immunohistochemical staining, electron microscopy, and PCR analysis were performed on the tongue tissue specimens. The mucosal epithelium of the tongue formed a very thick keratinized with well-developed filiform papillae in all cases. ACE2 and TMPRSS2 were consistently co-expressed in all samples in the epithelium. The S-protein was strongly expressed in basal cells and the epithelial surface. S-protein-positive viral particles were detected in the tongue's stratified squamous epithelium via an immunoelectron microscope. Based on PCR amplification of the N1 and N2 regions, the SARS-CoV-2 gene was detected on the tongue epithelium, tongue submucosa, and in tongue debris. This suggests that tongue debris, including the squamous epithelial tissue, could be a source of SARS-CoV-2 in saliva. Furthermore, removing tongue debris may decrease the amount of SARS-CoV-2 in the oral cavity.

High titers of infectious SARS-CoV-2 in corpses of patients with COVID-19.

OBJECTIVES: The prolonged presence of infectious SARS-CoV-2 in deceased patients with COVID-19 has been reported. However, infectious virus titers have not been determined. Such information is important for public health, death investigation, and handling corpses. The aim of this study was to assess the level of SARS-CoV-2 infectivity in the corpses of patients with COVID-19. METHODS: We collected 11 nasopharyngeal swabs and 19 lung tissue specimens from 11 autopsy cases with COVID-19 in 2021. We then investigated the viral genomic copy number by real-time reverse transcription-polymerase chain reaction and infectious titers by cell culture and virus isolation. RESULTS: Infectious virus was present in six of 11 (55%) cases, four of 11 (36%) nasopharyngeal swabs, and nine of 19 (47%) lung specimens. The virus titers ranged from 6.00E + 01 plaque-forming units/ml to 2.09E + 06 plaque-forming units/g. In all cases in which an infectious virus was found, the time from death to discovery was within 1 day and the longest postmortem interval was 13 days. CONCLUSION: The corpses of patients with COVID-19 may have high titers of infectious virus after a long postmortem interval (up to 13 days). Therefore, appropriate infection control measures must be taken when handling corpses.

Mutations in CHCHD2 cause α-synuclein aggregation.

Mutations in CHCHD2 are linked to a familial, autosomal dominant form of Parkinson's disease (PD). The gene product may regulate mitochondrial respiratory function. However, whether mitochondrial dysfunction induced by CHCHD2 mutations further yields α-synuclein pathology is unclear. Here, we provide compelling genetic evidence that mitochondrial dysfunction induced by PD-linked CHCHD2 T61I mutation promotes α-synuclein aggregation using brain autopsy, induced pluripotent stem cells (iPSCs) and Drosophila genetics. An autopsy of an individual with CHCHD2 T61I revealed widespread Lewy pathology with both amyloid plaques and neurofibrillary tangles that appeared in the brain stem, limbic regions and neocortex. A prominent accumulation of sarkosyl-insoluble α-synuclein aggregates, the extent of which was comparable to that of a case with α-synuclein (SNCA) duplication, was observed in CHCHD2 T61I brain tissue. The prion-like activity and morphology of α-synuclein fibrils from the CHCHD2 T61I brain tissue were similar to those of fibrils from SNCA duplication and sporadic PD brain tissues. α-Synuclein insolubilization was reproduced in dopaminergic neuron cultures from CHCHD2 T61I iPSCs and Drosophila lacking the CHCHD2 ortholog or expressing the human CHCHD2 T61I. Moreover, the combination of ectopic α-synuclein expression and CHCHD2 null or T61I enhanced the toxicity in Drosophila dopaminergic neurons, altering the proteolysis pathways. Furthermore, CHCHD2 T61I lost its mitochondrial localization by α-synuclein in Drosophila. The mislocalization of CHCHD2 T61I was also observed in the patient brain. Our study suggests that CHCHD2 is a significant mitochondrial factor that determines α-synuclein stability in the etiology of PD.

High-throughput determination of valproate in human samples by modified QuEChERS extraction and GC-MS/MS.

A new high-throughput method was developed for analysis of valproate in human plasma samples by QuEChERS extraction and gas chromatography-tandem mass spectrometry (GC-MS/MS). Plasma samples (0.2 ml) spiked with valproate and secobarbital-d5 (internal standard) were diluted with 1.3 ml of distilled water. Acetonitrile (1 ml) was added followed by 0.4 g MgSO4 and 0.1 g NaOAC. After a centrifugation step (2000 g for 10 min), 1 ml of the supernatant was transferred to a dispersive-solid phase extraction (dSPE) tube containing 150 mg MgSO4 and 50 mg C18. This mixture was vortexed and centrifuged at 3000 g for 5 min, and then the upper layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 40 µl ethyl acetate, and a 1-µl aliquot was injected into the GC-MS/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rxi-5Sil MS (30 m × 0.25 mm i.d.; 0.25-µm film thickness) and detected by MS/MS operating in electron ionization ion source mode. The regression equations showed excellent linearity (r > 0.9997) from 50 to 5000 ng/ml for plasma, with limit of detection of 10 ng/ml. The extraction efficiency of valproate for plasma ranged between 71.2%-103.5%. The coefficient of variation was <18.5%. The method was successfully applied to actual analyses of an autopsy case. This method can be useful for simple and reliable measurements of valproate in clinical and toxicological analyses; it can be integrated in screening and simultaneous determination methods for multiple drugs and poisons in the further studies.

Rapid and highly sensitive analysis of benzodiazepines and tandospirone in human plasma by automated on-line column-switching UFLC-MS/MS.

A high-throughput method was developed for the detection of 31 benzodiazepine drugs and tandospirone in human plasma by on-line column-switching ultra-fast liquid chromatography-tandem mass spectrometry. Plasma samples (100µl) spiked with the 32 drugs and oxazepam-d5 (internal standard) were diluted with 300µl of 13.3mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced into an analytical column (SUMIPAX ODS D-Swifter column, 30mm×3.0mm i.d.; particle size 2µm) by column switching. Quantification was performed by multiple reaction monitoring with positive-ion electrospray ionization. Distinct peaks appeared for each drug and the internal standard on each channel within 7min, including the extraction time. All drugs spiked into plasma showed recoveries of 83-95%. The regression equations for the 32 drugs showed excellent linearities in the range of 50-2000pg/ml of plasma and the limits of detection ranged from 20 to 50pg/ml. The lower and upper limits of quantitation were 50-100ng/ml and 2000pg/ml, respectively. Intra- and interday coefficients of variation for none of the drugs were greater than 13.6%. The accuracies of quantitation were 87-112%. The multiple reaction monitoring information-dependent acquisition of enhanced product ions method enabled the quantification and confirmation of diazepam, triazolam, and lorazepam obtained from actual plasma.

A forensic autopsy case of death from laryngeal stenosis due to a late complication of radiotherapy.

Irradiation is one of the standard modalities of treatment for neck cancer; however, it occasionally causes severe late complications. Here, we report an autopsy case of a death from laryngeal stenosis due to a late complication of radiotherapy. A man in his 70s who underwent radiotherapy for laryngeal cancer 6months before death was found dead in his home. Complete regression of the cancer was obtained after treatment, and signs suggestive of cancer recurrence were not evident before his death. Postmortem computed tomography showed severe narrowing of the upper airway due to glottic tissue swelling. The autopsy revealed an edematous epiglottis and supraglottic-glottic tissue with an ulcerative lesion, which severely narrowed the upper airway. Histopathological findings showed edema, hyalinization, and a proliferation of granulation tissue at the affected site. Neither a gross recurrence of cancer or finding suggestive of infection was observed in the specimen. This case is instructive to forensic pathologists regarding noting a radiotherapy complication as one of the possible causes of sudden death when the decedent previously underwent radiotherapy for neck cancer, and stresses the importance of detailed history taking and careful examination of the neck organs.

Evaluation of post-mortem lateral cerebral ventricle changes using sequential scans during post-mortem computed tomography.

In the present study, we evaluated post-mortem lateral cerebral ventricle (LCV) changes using computed tomography (CT). Subsequent periodical CT scans termed "sequential scans" were obtained for three cadavers. The first scan was performed immediately after the body was transferred from the emergency room to the institute of legal medicine. Sequential scans were obtained and evaluated for 24 h at maximum. The time of death had been determined in the emergency room. The sequential scans enabled us to observe periodical post-mortem changes in CT images. The series of continuous LCV images obtained up to 24 h (two cases)/16 h (1 case) after death was evaluated. The average Hounsfield units (HU) within the LCVs progressively increased, and LCV volume progressively decreased over time. The HU in the cerebrospinal fluid (CSF) increased at an individual rate proportional to the post-mortem interval (PMI). Thus, an early longitudinal radiodensity change in the CSF could be potential indicator of post-mortem interval (PMI). Sequential imaging scans reveal post-mortem changes in the CSF space which may reflect post-mortem brain alterations. Further studies are needed to evaluate the proposed CSF change markers in correlation with other validated PMI indicators.

Evaluation of thymic volume by postmortem computed tomography.

The thymus is exceedingly sensitive to stress and undergoes abrupt involution as a result of exposure to strong stress in early childhood. Therefore, thymic involution is often utilized to assess the presence of a stressful environment, such as an environment involving child abuse, in forensic medicine. In recent years, computed tomography (CT) has been commonly used in the daily practice of forensic medicine. We have focused on the thymic volume in postmortem CT images to evaluate the presence of a stressful antemortem environment. We calculated the thymus volume from postmortem CT images of children under six years old and demonstrated that the volume showed a positive correlation with the real weight obtained from an autopsy. The evaluation of thymic volume by CT may make it possible for us to identify child maltreatment. The most useful feature of this application of CT is to be able to demonstrate thymic involution less invasively in a surviving victim.

Two forensic autopsy cases of death due to upper gastrointestinal hemorrhage: a comparison of postmortem computed tomography and autopsy findings.

In this report, we describe two autopsy cases of death due to upper gastrointestinal hemorrhage (Case 1: gastric ulcer, Case 2: aortoduodenal fistula). Postmortem computed tomography (CT) images from both cases revealed pooling of gastric fluid, which contained high attenuation areas, although these images also mirrored the different sources of the gastrointestinal hemorrhage. Fluid collection was observed in the small intestine for both cases, although the high attenuation areas were only remarkable in Case 2. The autopsy in Case 1 revealed a peptic ulcer, with small vessels exposed on the surface of the ulcer. Melena was also observed throughout the intestine, although clotting was only observed inside the stomach. The autopsy in Case 2 revealed diffuse massive clotting from the stomach to the upper portion of the ileum, which was due to a primary aortoduodenal fistula. Given our autopsy findings, the extent of the high attenuation areas in the digestive tract during postmortem CT scanning may be correlated with the speed of the gastrointestinal hemorrhage before death. Carefully evaluating the radiodensity of the gastrointestinal contents during postmortem CT scanning may indicate the primary site of the hemorrhage before the autopsy, thereby facilitating the accurate identification of the cause of death during forensic autopsy.

Criminal gunshot wound and iatrogenic tension pneumothorax detected by post-mortem computed tomography.

Post-mortem imaging at autopsy is gradually increasing in popularity among forensic practitioners. The objective of the present paper was to demonstrate that it is essential to survey the cadaver using computed tomography (CT) before autopsy. This case report presents an iatrogenic tension pneumothorax caused by left subclavian vein puncture undertaken during treatment for a gunshot-related wound. The victim, a 64-year-old woman, was shot by her husband at home, and transferred to the hospital emergency unit. Before surgical procedures were carried out, left subclavian vein puncture was performed; however, during the operation, the victim experienced sudden cardiac arrest. Subsequent intensive resuscitation was unsuccessful. The clinical cause of death was recorded as traumatic shock caused by the gunshot injury. However, before the legal autopsy took place, CT clarified the existence of tension pneumothorax not on the same side as the gunshot wound, but on the side of the iatrogenic subclavian vein puncture. Because of this information gained prior to legal dissection, a typical dissection procedure for tension pneumothorax could be performed. Post-mortem imaging prior to regular dissection is essential as an adjunct diagnostic tool.

[Corpse disposal by concealment in transport containers or packaging materials: Examination by post-mortem computed tomography (PMCT) before forensic autopsy]. / Leichenbeseitigung durch Verstecken in Transport- behältnissen oder Verpackungsmaterialien: präautoptische Untersuchung mittels postmortaler Computertomographie (PMCT).

The presented case reports illustrate the value of post-mortem computed tomography in cases of homicide in which the body was hidden in a container or packaging material and could not be inspected directly from outside. In Case 1, the body was forced into a suitcase, which was then thrown into a flood basin. Post-mortem computed tomography (PMCT) visualized the compressed and extremely flexed body inside the suitcase as well as fractures of the left temporal bone, the left ulna and the left side of the mandible. After recovery of the body, the consequences of long-term immersion in fresh water became visible. At autopsy, a laceration in the left temporal region and a temporal bone fracture were observed. The fractures of the left ulna and the left jaw were associated with moderate hematoma. In Case 2, the body of an 11-year-old girl was retrieved from a plastic bag concealed in a garden shed. The cause of death was ligature strangulation. In the presented cases, PMCT was performed as part of the police investigations for reliable primary documentation of the contents of the containers without further manipulation. Thus the human body, its position inside the container and the presence of bone injuries could be demonstrated before opening the container. In such cases, post-mortem imaging provides reliable and rapid information to the investigating authorities before autopsy already.

"Dead in hot bathtub" phenomenon: accidental drowning or natural disease?

Sudden death in a hot bathtub occurs frequently in Japan, particularly among elderly people. This retrospective report describes the epidemiologic circumstances and physical findings at autopsy. In total, 268 victims were found unconscious or dead during tub bathing. After postmortem examination, the manner of death was judged as natural cause in 191 (71.2%) and accidental drowning in 63 (23.5%) cases. Mean age (SD) was 72.1 (15.2) years with no significant difference between males and females. A seasonal difference was evident: the winter displayed the highest frequency. Drowning water inhalation, which was confirmed in 72% of victims, was absent in the others. The most common observations on postmortem examination were cardiac ischemic changes and cardiomegaly. Water inhalation signs were evident in a significantly fewer victims exhibiting these factors. In contrast, inhalational findings were observed more frequently in victims with other backgrounds such as alcohol intake, mobility disturbance, and history of epilepsy. Annual mortality in Japan from accidental drowning in persons aged older than 75 years is 33 deaths per 100,000 population. However, this number may be considerably underestimated as pathologists tend to regard lack of water inhalation as indicating a natural cause of death. Confusion in diagnosis remains consequent to the accidental and natural aspects of "dead in hot bathtub" phenomenon.

Post-mortem computed tomography (PMCT) and PMCT-angiography after transvascular cardiac interventions.

BACKGROUND AND PURPOSE: During the last years, Post Mortem Computed Tomography (PMCT) has become an integral part of the autopsy. PMCT-angiography may augment PMCT. Both exams have proven their value in visualizing complications after heart surgery. Therefore, they should also show complications after transvascular interventions. This assumption initiated our project: to evaluate the possibilities of PMCT and PMCT-angiography after transvascular cardiac interventions. MATERIAL AND METHODS: In our archives of characteristic and typical PMCT findings, we searched for observations on preceding transvascular cardiac interventions. Additionally, we reviewed our PMCT-angiographies (N = 140). RESULTS: After transvascular cardiac interventions, PMCT and PMCT-angiography visualized bleeding, its amount and its origin, cardiac tamponade, free and covered perforations, transvascular implanted valves and their position, catheters and pacemakers with fractures, abnormal loops and bending. Bubbles in the coronary vessels (indicating air embolism) become visible. CONCLUSION: After transvascular cardiac interventions, PMCT and PMCT-angiography show complications and causes of death. They prove a correct interventional approach and also guide autopsy. In isolated cases, they may even replace autopsy.

Fatal Staphylococcus aureus bacteremia in the Felty syndrome: a maltreatment-suspected case.

Familial neglect was suspected when an older deceased female was found to have large decubitus ulcers and weight loss. Postmortem examinations including histopathology and bacterial culture revealed systemic Staphylococcus aureus infection as the cause of death. The victim might have exhibited Felty syndrome, which is characterized by complications of splenomegaly and neutropenia in the underlying rheumatoid arthritis. As a result of neutropenia, the affected individual was susceptible to skin ulcer formation and sepsis. The manifestation of pressure ulcers as abuse biomarkers should also be explored from interaction with intrinsic disease factors.

Sudden death caused by chronic Chagas disease in a non-endemic country: Autopsy report.

Chagas disease is a tropical disease that is prevalent in Latin America. Described herein is an autopsy case of the sudden death of a 48-year-old Brazilian man who had stayed in Japan for 7 years. The man, who had a history of Chagas disease, collapsed unexpectedly at work. Because the cause of death was unknown, forensic autopsy examination was performed. As gross findings, the heart was dilated and rounded with an increase in size and weight. The esophagus and large intestine were dilated moderately, with extensive interstitial inflammatory infiltration in the cardiac muscle, but no apparent parasite nest was observed in various tissues. On post-mortem laboratory examinations, indirect immunofluorescence antibody test indicated the presence of IgG antibody specific to Trypanosoma cruzi in the serum. Subsequent polymerase chain reaction amplification using DNA extracted from blood yielded the specific product derived from T. cruzi genomic DNA. These examinations indicate that the infection had resulted from the Tripanosoma parasite. The cause of death was judged to be chronic cardiomyopathy caused by Chagas disease. It is important for pathologists to know the possible involvement of chronic Chagas disease in sudden unexpected deaths in the current globalized society of Japan.