Genomic analysis of SARS-CoV-2 in forensic autopsy cases of COVID-19.

Numerous genomic analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been conducted, highlighting its variations and lineage transitions. Despite the importance of forensic autopsy in investigating deaths due to coronavirus disease 2019 (COVID-19), including out-of-hospital deaths, viral genomic analysis has rarely been reported due in part to postmortem changes. In this study, various specimens were collected from 18 forensic autopsy cases with SARS-CoV-2 infection. Reverse-transcription quantitative polymerase chain reaction revealed the distribution of the virus in the body, primarily in the respiratory organs. Next-generation sequencing determined the complete genome sequences in 15 of the 18 cases, although some cases showed severe postmortem changes or degradation of tissue RNA. Intrahost genomic diversity of the virus was identified in one case of death due to COVID-19. The accumulation of single-nucleotide variations in the lung of the case suggested the intrahost evolution of SARS-CoV-2. Lung of the case showed diffuse alveolar damage histologically and positivity for SARS-CoV-2 by immunohistochemical analysis and in situ hybridization, indicating virus-associated pneumonia. This study provides insights into the feasibility of genomic analysis of SARS-CoV-2 in forensic autopsy cases and the potential for uncovering important information in COVID-19 deaths, including out-of-hospital deaths.

Stature estimation by semi-automatic measurements of 3D CT images of the femur.

Stature estimation is one of the most basic and important methods of personal identification. The long bones of the limbs provide the most accurate stature estimation, with the femur being one of the most useful. In all the previously reported methods of stature estimation using computed tomography (CT) images of the femur, laborious manual measurement was necessary. A semi-automatic bone measuring method can simplify this process, so we firstly reported a stature estimation process using semi-automatic bone measurement software equipped with artificial intelligence. Multiple measurements of femurs of adult Japanese cadavers were performed using automatic three-dimensional reconstructed CT images of femurs. After manually setting four points on the femur, an automatic measurement was acquired. The relationships between stature and five femoral measurements, with acceptable intraobserver and interobserver errors, were analyzed with single regression analysis using the standard error of the estimate (SEE) and the coefficient of determination (R2). The maximum length of the femur (MLF) provided the lowest SEE and the highest R2; the SEE and R2 in all cadavers, males and females, respectively, were 3.913 cm (R2 = 0.842), 3.664 cm (R2 = 0.705), and 3.456 cm (R2 = 0.686) for MLF on the right femur, and 3.837 cm (R2 = 0.848), 3.667 cm (R2 = 0.705), and 3.384 cm (R2 = 0.699) for MLF on the left femur. These results were non-inferior to those of previous reports regarding stature estimation using the MLF. Stature estimation with this simple and time-saving method would be useful in forensic medical practice.

Impact of the COVID-19 pandemic on pathological autopsy practices in Japan.

During the coronavirus disease 2019 (COVID-19) pandemic, autopsies have provided valuable insights into the pathogenesis of COVID-19. The precise effect of this pandemic on autopsy procedures in Japan, especially in instances unrelated to COVID-19, has not yet been established. Therefore, we conducted a questionnaire survey from December 2020 to January 2021 regarding the status of pathological autopsy practices in Japan during the first year of the COVID-19 pandemic. The questionnaire was sent to 678 medical facilities with pathologists, of which 227 responded. In cases where a confirmed diagnosis of COVID-19 was not made at the time of autopsy, many facilities counted them as suspected COVID-19 cases if pneumonia was suspected clinically. At around half of the sites, autopsies were prohibited for suspected COVID-19 cases. In addition, the number of autopsies of non-COVID-19 cases during the pandemic period was also investigated, and a significant decrease was observed compared with the incidence in the pre-pandemic period. The COVID-19 pandemic has affected not only the autopsies of COVID-19 cases but also the entire practice of pathological autopsies. It is necessary to establish a system that supports the implementation of pathological autopsy practices during the pandemic of an emerging infectious disease.

Age estimation by evaluation of osteophytes in thoracic and lumbar vertebrae using postmortem CT images in a modern Japanese population.

Estimation of age at death is important in forensic investigations of unknown remains. There have been several reports on applying the degree of osteophyte formation-an age-related change in the vertebral body-for age estimation; however, this method is not yet established. This study investigated a method for age estimation of modern Japanese individuals using osteophytes measured on CT images. The sample included 250 cadavers (125 males) aged 20-95 years. The degree of osteophyte formation was evaluated as score O (0-5 points), and the degree of fusion of the osteophytes between the upper and lower vertebrae was evaluated as score B (0-2 points). Age estimation equations were developed using regression analyses with seven variables, determined by scores O and B, and the equation with the smallest standard error of estimate (SEE) was obtained when the number of vertebrae with score O ≥ 2 was used as the explanatory variable. Age estimation with SEE of about 10 years was possible even when partial vertebrae with a high degree of osteophyte formation were used, showing its potential for practical application. The cutoff value for age estimation was established using the receiver operating characteristic curve analysis, wherein good results were obtained for all variables (area under the curve ≥ 0.8). The combination of the estimation equation and the cutoff value can narrow the range of age estimates.

Determination of sex on the basis of hyoid bone measurements in a Japanese population using multidetector computed tomography.

Sex determination for unidentified human remains is important because the determined sex is usually used as a foundation for the estimation of other biological profiles. This study investigated the sexual dimorphism of the adult hyoid bone and developed discriminant equations to accurately estimate sex in a contemporary Japanese population using measurements on two-dimensional computed tomography (CT) images. The study sample comprised 280 cadavers (140 males, 140 females) of known age and sex that underwent postmortem CT and subsequent forensic autopsy. In accordance with previous studies, seven measurements were performed using two-dimensional CT reconstructed images. Most measurements of males were significantly greater than those of females. The perpendicular length from the most anterior edge of the hyoid body to the line that connects the most distal points of the greater horns of the hyoid and the linear distance between the most lateral edges of the hyoid body most significantly contributed to sex determination. This study demonstrated that discriminant functions on the basis of three measurements provided higher rates of accurate sex classification (93.3-94.6%) than univariate functions. Therefore, the hyoid bone is highly sexually dimorphic in the contemporary Japanese population and may be useful in forensic contexts for sex determination with a high level of accuracy.

Uptake of Shiga-toxigenic Escherichia coli†SubAB by HeLa cells requires an actin- and lipid raft-dependent pathway.

The novel cytotoxic factor subtilase cytotoxin (SubAB) is produced mainly by non-O157 Shiga-toxigenic Escherichia coli (STEC). SubAB cleaves the molecular chaperone BiP/GRP78 in the endoplasmic reticulum (ER), leading to activation of RNA-dependent protein kinase (PKR)-like ER kinase (PERK), followed by caspase-dependent cell death. However, the SubAB uptake mechanism in HeLa cells is unknown. In this study, a variety of inhibitors and siRNAs were employed to characterize the SubAB uptake process. SubAB-induced BiP cleavage was inhibited by high concentrations of Dynasore, and methyl-ß-cyclodextrin (mßCD) and Filipin III, but not suppressed in clathrin-, dynamin I/II-, caveolin1- and caveolin2-knockdown cells. We observed that SubAB treatment led to dramatic actin rearrangements, e.g. formation of plasma membrane blebs, with a significant increase in fluid uptake. Confocal microscopy analysis showed that SubAB uptake required actin cytoskeleton remodelling and lipid raft cholesterol. Furthermore, internalized SubAB in cells was found in the detergent-resistant domain (DRM) structure. Interestingly, IPA-3, an inhibitor of serine/threonine kinase p21-activated kinase (PAK1), an important protein of macropinocytosis, directly inhibited SubAB-mediated BiP cleavage and SubAB internalization. Thus, our findings suggest that SubAB uses lipid raft- and actin-dependent, but not clathrin-, caveolin- and dynamin-dependent pathways as its major endocytic translocation route.

Stature estimation in Japanese cadavers based on scapular measurements using multidetector computed tomography.

This study assessed the correlation between stature and scapular measurements in a Japanese population, using three-dimensional (3D) computed tomographic (CT) images, and derived regression equations for predicting stature. A total of 194 cadavers (100 males 94 females) underwent postmortem CT (PMCT) and subsequent forensic autopsy in our department between May 2011 and April 2014. Left and right longitudinal scapular lengths (LLSL and RLSL, respectively) and left and right transverse scapular lengths (LTSL and RTSL, respectively) were measured on 3D CT reconstructed images that extracted only scapular data. The correlation between stature and each of the scapular measurements were analyzed using Pearson product-moment correlation coefficients. The four variables correlated significantly with stature, regardless of sex. The LLSL measurement had the lowest standard error of estimation value among all subjects (4.22 cm) and among all females (4.37 cm), whereas the RLSL measurement had the lowest standard error of estimation value among all males (3.75 cm). The results of this study indicate that scapular measurements may be useful for the forensic estimation of the stature of Japanese individuals, particularly in cases where better predictors, such as long bone lengths, are unavailable.

Stature estimation in Japanese cadavers based on pelvic measurements in three-dimensional multidetector computed tomographic images.

The aim of this study was to examine the feasibility of stature estimation by measuring the pelvic bones of Japanese cadavers using three-dimensional (3D) computed tomography (CT). We assessed 3D reconstructed images of 210 Japanese subjects (108 males, 102 females) who had undergone postmortem CT between May 2011 and November 2013. We defined the linear distance from the anterosuperior margin of the left and right anterior superior iliac spines (ASIS) to the posterior margin of the left and right ischial spines as the LSS and RSS, respectively. We also defined the linear distance from the anterosuperior margin of the left and right ASIS to the anteroinferior margin of the left and right ischial tuberosities as the LST and RST, respectively. The correlation between the cadaver stature (CS) and each parameter (LSS, LST, RSS, and RST) was evaluated using Pearson product-moment correlation coefficients and regression analysis was performed for stature estimation. All four parameters correlated significantly with stature independent of sex, suggesting that they can be used as a tool for stature estimation. The LST had the closest correlation with stature in both sexes.

Age estimation by quantitative features of pubic symphysis using multidetector computed tomography.

Macroscopic assessment of the pubic symphysis is commonly used for age estimation because its surface changes over time. However, postmortem computed tomography (PMCT), a method several forensic medical departments and institutes have begun to adopt, has the potential to simplify the information gathering process from the pelvic bone without requiring soft tissue removal. Some studies have previously evaluated the use of three-dimensional images of the pubic symphysis, but because of variance in the graphics processing among image analysis software packages, certain differences have been observed between these studies. Therefore, in this study, the PMCT findings of 199 subjects of known age and sex were retrospectively reviewed to examine the feasibility of age estimation using planar images of the pubic bones and soft tissue. The coronal and axial sectional images were observed at the center of the symphyseal surface, and the pubic bone length and thickness of the connective tissue of the pubic symphysis were measured at each slice. Our results revealed a significant positive correlation between the length of the pubic bone of the coronal section and age, suggesting that the use of a cutoff value for pubic bone length might be feasible for age estimations. In addition, the thickness of the connective tissue tended to narrow over time. Although the prediction interval range of planar images obtained by PMCT was major and is not usable in practice at this moment, it may still be a useful tool if used in conjunction with other findings obtained by PMCT.

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.

Viable herpes simplex virus type 1 and varicella-zoster virus in the trigeminal ganglia of human cadavers.

Human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) were isolated in the bilateral trigeminal ganglia of 12 human cadavers with no history of herpes-related symptoms within 1-5 days of death. Sixteen trigeminal ganglia were subjected to explant culture by using Vero cells, but no cytopathogenic effects (CPE) were observed. However, when another eight trigeminal ganglia were placed in a cell strainer and kept from direct contact with Vero cells during culture, CPE were clearly apparent in all cultures. The amount of DNA in the culture supernatants of 16 trigeminal ganglia decreased over time; 12 and 9 of these samples were PCR-positive for HSV-1 and VZV, respectively. In new Vero cells inoculated with supernatants collected 2 days after culture initiation, immunofluorescence staining revealed HSV-1 and VZV in 6 and 5 of 8 trigeminal ganglia, respectively. HSV-1 and VZV DNA was detected in supernatants collected 3 and 7 days after culture initiation and in Vero cells collected after culture completion, but real-time PCR revealed the DNA amounts decreased over time. There was less VZV DNA than HSV-1 DNA. These results demonstrate that infective HSV-1 and VZV can be isolated in culture, and confirm that viable HSV-1 and VZV persist in human trigeminal ganglia for some time after death.

Age estimation by multidetector CT images of the sagittal suture.

Closure of cranial sutures progresses with age; therefore, macroscopic assessment of cranial sutures has been used as one method of age estimation. Postmortem computed tomography (PMCT), which many forensic medical departments and institutes have begun to adopt, has the potential to simplify the gathering of information from cranial sutures for both surface and cross-sectional evaluation. To examine the feasibility of age estimation by cross-sectional multidetector computed tomography images of the sagittal suture, PMCT findings of 125 subjects of known age and sex were retrospectively reviewed. The sagittal suture was divided into four segments, and 20 cross-sectional slices from each segment were analyzed. These slices were each categorized by visual evaluation into one of the seven stages defined by Harth et al. according to the degree of closure. The mean stage value of 20 slices was calculated for each segment. We were able to evaluate cross-sectional images of the sagittal suture by PMCT, and a positive correlation between age and closure degree was observed. Despite the prediction interval achieved with this method not being superior to traditional macroscopic or flat-panel CT assessment, multidetector CT is a potentially useful tool, in conjunction with other methods, for age estimation, particularly in adult females and in cases where only a skull is the sole remain.

Superimposition-based personal identification using skull computed tomographic images: application to skull with mouth fixed open by residual soft tissue.

We previously reported that superimposition of 3-dimensional (3D) images reconstructed from computed tomographic images of skeletonized skulls on photographs of the actual skulls afforded a match of skull contours, thereby demonstrating that superimposition of 3D-reconstructed images provides results identical to those obtained with actual skulls. The current superimposition procedure requires a skeletonized skull with mouth closed and thus is not applicable to personal identification using a skull with residual soft tissue or the mouth fixed open, such as those found in mummified or burned bodies. In this study, we scanned using computed tomography the skulls of mummified and immersed body with mandibles fixed open by residual soft tissue, created 3D-reconstructed skull images, which were digitally processed by computer software to close the mandible, and superimposed the images on antemortem facial photographs. The results demonstrated morphological consistency between the 3D-reconstructed skull images and facial photographs, indicating the applicability of the method to personal identification.

Comparison of racemization rates between vital and endodontically treated teeth for age estimation.

Amino acid racemization of dentin (AAR) is among the most precise methods for age estimation in unidentified adult cadavers. Although vital teeth are generally used for this technique, cases often have endodontically treated (Endo) teeth only. Therefore, the aim of this preliminary pilot study was to determine the applicability of Endo teeth by comparing AAR rates between vital and Endo teeth. Thirty-six dentin samples from vital teeth and 18 dentin samples from Endo teeth were analyzed, including the maxillary first premolar, maxillary second molar, and mandibular second premolar. Standard calibration curves for the mandibular second premolar and maxillary second molar were compared among vital teeth, Endo teeth, and mixed vital/Endo teeth. Discrepancies between estimated and actual ages were assessed by analyzing the AAR rates for the maxillary first premolar between vital and Endo teeth. The AAR rates for Endo teeth were higher than those for vital teeth in both the mandibular second premolar and maxillary second molar, and the correlation of the standard calibration curve for vital teeth only was highest (r = 0.982836; r = 0.92011467), followed by mixed (r = 0.949579; r = 0.76158) and Endo teeth only (r = 0.896082; r = 0.744991). In the maxillary first premolar, discrepancies were more than 10 years for all 5 Endo teeth, based on the standard calibration curve of vital teeth. The present study suggests that the AAR rates for Endo teeth might be affected by variation in dentin structure between vital and Endo teeth.

SARS-CoV-2 Transmission from Virus-Infected Dead Hamsters.

Although it has been 2.5 years since the coronavirus disease 2019 (COVID-19) pandemic began, the transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a dead infected body remains unclear, and in Japan, bereaved family members are often not allowed to view in person a loved one who has died from COVID-19. In this study, we analyzed the possibility of SARS-CoV-2 transmission from a dead body using a hamster model. We also analyzed the effect of "angel care"--in which the pharynx, nostrils, and rectum are plugged--and embalming on reducing transmissibility from dead bodies. We found that SARS-CoV-2 could be transmitted from the bodies of animals that had died within a few days of infection; however, angel care and embalming were effective in preventing transmission from the dead bodies. These results suggest that protection from infection is essential when in contact with a SARS-CoV-2-infected dead body and that sealing the cavities of a dead body is an important infection control step if embalming is not performed. IMPORTANCE We found that SARS-CoV-2 could be transmitted from a dead body, presumably via postmortem gases. However, we also found that postmortem care, such as plugging the pharynx, nostrils, and rectum or embalming the corpse, could prevent transmission from the dead body. These results indicate that protection from infection is essential when handling infected corpses and that appropriate care of SARS-CoV-2-infected corpses is important.

Usefulness of powdered crown of mandibular first premolars in birth year estimation by radiocarbon dating.

BACKGROUND: Radiocarbon (14C), whose levels increased in the atmosphere between 1955 and 1963, accumulates in the enamel of human teeth only during the process of tooth formation and has been applied to estimate the birth year of unidentified corpses. However, enamel isolation from teeth is time-consuming and labor-intensive. Therefore, this study aimed to estimate the birth year using 14C in the crown of a single mandibular first premolar tooth. MATERIALS AND METHODS: Fourteen mandibular first premolars collected from forensic autopsies were analyzed. For nine teeth, each tooth was cut longitudinally: half of the tooth was enamel, and the other half was crown (1). For the other five teeth, the entire crown was used (2). Thereafter, the 14C concentration in each tooth was measured using accelerator mass spectrometry. RESULTS: The absolute error between the actual date of birth (DOB) and the estimated DOB was within the range of 1.0-8.8 years in the enamel of five teeth and 0.1-4.0 years in the crown halves of seven teeth (1). For the five teeth with entire crowns, the absolute errors ranged between 0.3 and 3.0 years (2). CONCLUSION: The absolute error of 14C-based year of birth estimation using the powdered crown of the mandibular first premolar teeth ranged between 0.1 and 4.0 years. Our method, which involves pulverizing an entire crown, eliminates the need for the equipment, time, and labor associated with enamel isolation. Therefore, 14C dating using powdered crowns of mandibular premolars can be useful for birth year estimation.

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.

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.

DNA identification of formalin-fixed organs is affected by fixation time and type of fixatives: using the AmpF l STR(R) Identifiler(R) PCR Amplification Kit.

Personal identification using DNA typing of formalin-fixed tissue is very important in the forensic sciences. However, few studies have been conducted to determine the detection limit of DNA typing of formalin fixation time in samples using the AmpFℓSTR(®) Identifiler(®) PCR Amplification Kit (Identifiler Kit). We collected samples from five cadavers submitted for forensic autopsies, and fixed them either in a 10% formalin solution, or in a 10% neutral-buffered formalin solution. The amount of template DNA for polymerase chain reaction (PCR) amplification and the detection limit of DNA typing for the Identifiler Kit were determined. When tissues were fixed in 10% formalin, 10 ng of DNA template was required for successful genotyping even after three-hour fixation and 100 ng was required after one-week fixation for PCR amplification. However, when tissues were fixed in 10% neutral-buffered formalin, the required amount of DNA template was 1 ng for a fixation time of three hours to three days and 125 ng for three months. Fixation time in neutral-buffered formalin was longer for successful PCR than that in formalin solution. Dropout was more common with increasing formalin fixation time. These results suggest that neutral-buffered formalin is preferred to formalin for fixation of tissues if they are to be subjected to DNA typing and that tissues fixed with neutral-buffered formalin can be used for DNA typing using the Identifiler Kit unless the fixation time exceeds one month.

Sixty-eight cases of postmortem pink teeth observed in dental autopsies of unidentified cadavers.

The pink teeth phenomenon was first reported in people who died by hanging or drowning in 1829. In recent years, there have been many reports that the occurrence of postmortem pink teeth is closely related to the condition of surroundings and time after death rather than to the cause of death, as had previously been thought. We observed pink teeth in 68 of 324 unidentified cadavers autopsied during 2015-2021, and we investigated the relationship of pink teeth with age, sex, cadaveric condition, time after death, tooth type, and cause of death. The results showed no significant differences in the occurrence of pink teeth according to sex or place of discovery (indoors, outdoors, or in water). Pink teeth tended to be observed below the age of 60 years. In the 68 cases with pink teeth, 29.1% of all vital teeth were affected, with the anterior teeth being most common. No evidence was found that supported an association between pink teeth and cause of death. These results are largely consistent with recent reports on postmortem pink teeth. As it stands, the pink teeth phenomenon may be nonspecific and must not be misinterpreted in medico-legal investigations. This case series provides further evidence supporting recent reports and contributes to clarifying in greater detail the mechanisms of the coloration and fading of pink teeth.