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.

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.

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.

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.

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.

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.

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.

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.

SmartAmp method can rapidly detect SARS-CoV-2 in dead bodies.

Rapid and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in dead bodies is essential to prevent infection among those working with dead bodies. This study focused on the Smart Amplification (SmartAmp) method, which has a short examination time (approximately an hour), is simple to perform, and demonstrates high specificity and sensitivity. This method has already been used for clinical specimens; however, its effectiveness in dead bodies has not been reported. This study examined the SmartAmp method using 11 autopsies or postmortem needle biopsies performed from January to May, 2021 (of these, five cases tested positive for SARS-CoV-2 by quantitative real-time polymerase chain reaction (qRT-PCR) and six cases tested negative). Swab samples were collected from the nasopharynx, oropharynx, or anus and the SmartAmp and qRT-PCR results were compared. For the nasopharynx and oropharynx samples, the same results were obtained for both methods in all cases; however, for the anal swabs, there was one case that was positive according to qRT-PCR but negative according to the SmartAmp method. The SmartAmp method may therefore be less sensitive than qRT-PCR and results may differ in specimens with a low viral load, such as anal swabs. However, in the nasopharynx and oropharynx specimens, which are normally used for testing, the results were the same using each method, suggesting that the SmartAmp method is useful in dead bodies. In the future, the SmartAmp method may be applied not only during autopsies, but also in various situations where dead bodies are handled.

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.

Generation of disease-specific and CRISPR/Cas9-mediated gene-corrected iPS cells from a patient with adult progeria Werner syndrome.

Adult progeria Werner syndrome (WS), a rare autosomal recessive disorder, is characterized by accelerated aging symptoms after puberty. The causative gene, WRN, is a member of the RecQ DNA helicase family and is predominantly involved in DNA replication, repair, and telomere maintenance. Here, we report the generation of iPS cells from a patient with WS and correction of the WRN gene by the CRISPR/Cas9-mediated method. These iPSC lines would be a valuable resource for deciphering the pathogenesis of WS.

Fatal airway obstruction due to Ludwig's angina from severe odontogenic infection during antipsychotic medication: A case report and a literature review.

Ludwig's angina is characterized by inflammation of the sublingual and submandibular spaces and is mainly caused by odontogenic infection, which leads to cellulitis of the soft tissues of the floor of the mouth and the neck. This causes asphyxia due to elevation and posterior deviation of the tissues of the floor of the mouth. We report a fatal case of airway obstruction due to Ludwig's angina. A woman in her forties who had no physical complications, but had a mental illness, was undergoing outpatient dental treatment for caries in the first premolar of the left mandible. She was admitted to a psychiatric hospital because of insomnia caused by pain, where she developed cardiopulmonary arrest while sleeping and died 14 days after onset of the dental infection. Postmortem computed tomography (PMCT) prior to autopsy showed swelling of the soft tissues-from the floor of the mouth to the oropharyngeal cavity, the supraglottic larynx, and the prevertebral tissue. Autopsy revealed a markedly swollen face and neck, an elevated tongue, and a highly edematous epiglottis and laryngopharyngeal mucosa. There was also cellulitis and abscess of the facial, suprahyoid, and neck musculature, which suggested that the cause of death was asphyxiation due to airway obstruction. This was an alarming case, with mental illness leading to risk of severe odontogenic infection, and in which obesity and use of antipsychotic medication might have acted synergistically leading to airway obstruction. This is also a case of Ludwig's angina captured by PMCT, which has rarely been reported.

The composition of chlorinated or oxidized phosphatidylcholine products changes with hypochlorite concentration: Application to abscess lipid analysis.

Hypochlorous acid, produced by myeloperoxidase upon neutrophil activation, can oxidize various compounds and exert antimicrobial activity in vivo. To elucidate the mechanisms underlying the reactions of the unsaturated phosphatidylcholines, which abound in cell membranes, with hypochlorous acid, we identified and examined phosphatidylcholine chlorination and oxidation products formed under various reaction conditions. We first investigated the products of unsaturated phosphatidylcholine and hypochlorous acid reaction with respect to hypochlorite concentration and reaction time. Next, we examined the lipids extracted postmortem from human abscesses. For all the analyses, we used liquid chromatography-quadrupole time-of-flight mass spectrometry. Various compounds, including phosphatidylcholine chlorohydrin and phosphatidylcholine hydroxide/epoxide, were detected. Oxidized phosphatidylcholines were mainly detectable upon reaction with low concentrations of sodium hypochlorite, whereas chlorinated phosphatidylcholines formed in the presence of higher concentrations. In human abscesses, oxidized phosphatidylcholines were detected in the cases with high procalcitonin concentration, whereas chlorinated phosphatidylcholines were undetected. The detections of oxidized phosphatidylcholines in human tissues might indicate previous exposure to hypochlorous acid in septic cases. Our results provide insight into the mechanisms underlying pathogen survival following inflammation associated with neutrophil activation and topical myeloperoxidase release and show postmortem biomarkers candidates for sepsis.

Social contribution of forensic odontology in Japan.

Half a century has passed since the department for education and research on forensic odontology was established at dentistry-related universities in Japan in 1964. In order to meet the demands of society, the number of universities with a department of forensic odontology increased up until around 2005. In 2007, the Japanese Society of Forensic Dental Science was established, and then a series of reforms such as establishment of the Study Council on Death Cause Investigation in both the National Police Agency and the Cabinet Office of the Japanese government, cabinet decision of enactment and enforcement of new laws on death cause investigation, publication of an article on the Model Core Curriculum of Dental Education, publication of the results of a fact-finding survey on education and research on forensic odontology conducted by the Ministry of Education, Culture, Sports, Science and Technology, inclusion of questions about forensic odontology in the National Board Dental Examination, and compilation of a database on dental findings by the Ministry of Health, Labor and Welfare, proceeded in succession. We introduced the half century of forensic odontology in Japan in chronological order.

Preparation of dentin standard samples for age estimation based on increased aspartic acid racemization rate by heating.

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p < 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p < 0.01) and a maxillary third molar (20 years, r = 0.944; p < 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.

Estimation of birth year by radiocarbon dating of tooth enamel: Approach to obtaining enamel powder.

Atmospheric radiocarbon (14C) levels increased from 1955 to 1963 due to atmospheric nuclear weapon tests, and then decreased. As 14C accumulates in human tooth enamel while the enamel is being formed, 14C can be used to estimate the birth year of unidentified bodies. Measurement results of 14C content in tooth enamel using accelerator mass spectrometry vary depending on the enamel's sample site. To address this problem, a method for equalizing samples using a pulverizer was considered in this study. Regarding the tube and cone used as the pulverizer, (1) a polycarbonate tube and stainless steel cone, (2) a stainless steel tube and cone, and (3) a tungsten carbide tube and cone, were compared. In (1), the modern carbon ratio was approximately half that of the normal ratio of 100 pMC, with which accurate dating was impossible, and in (2), a high background value was obtained for IAEA-C1, which was pulverized using a reusable tube and cone. In (3), the 14C content for IAEA-C1 pulverized using reusable tube and cone, which was washed with quartz sand, was 0.31 ±â€¯0.01 pMC. This result did not show any problems regarding background value. Therefore, the use of tungsten carbide products and washing with quartz sand is recommended for 14C measurement of pulverized teeth.

Age estimation based on maturation of the medial clavicular epiphysis in a Japanese population using multidetector computed tomography.

To estimate forensic age, the Study Group of Forensic Age Diagnostics of the German Association of Forensic Medicine (AGFAD) has recommended the radiological investigation of the medial clavicular epiphysis (MCE). This study aimed to investigate the relation between the maturation of the MCE and age in a contemporary Japanese population using a standard five-stage system by Schmeling et al. and Kellinghaus substage system. A total of 207 cadavers (128 males, 79 females) of known age and sex that underwent postmortem computed tomography (CT) and subsequent forensic autopsy were obtained. The earliest appearance for stage 3c was 18.1 years in the male subjects and 18.4 years in the female subjects. Our results were consistent with those in previous studies and indicate that stage 3c is crucial for medico-legal processes in evaluating age in various countries. Similar studies on the staging of the MCE using CT images in other populations, particularly in Asians, are required.

Relationship between KCNQ1 (LQT1) and KCNH2 (LQT2) gene mutations and sudden death during illegal drug use.

Long QT syndrome (LQTS), a congenital genetic disorder, can cause torsades de pointes (TdP), and lethal cardiac arrhythmia may result from ingestion of cardiotoxic drugs. Methamphetamine (MP) and new psychoactive substances (NPSs) can trigger TdP due to QT prolongation, leading to sudden death. We therefore analysed variations in the LQTS-associated genes KCNQ1 (LQT1) and KCNH2 (LQT2) using cardiac blood and myocardial tissue from subjects having died suddenly during MP or NPS use to investigate the relationship between congenital genetic abnormalities and sudden death during illegal drug use. We amplified and sequenced all exons of these genes using samples from 20 subjects, half of whom had died taking MP and half after using NPSs. G643S, a KCNQ1 missense polymorphism, was significantly more common among sudden deaths involving NPSs (6 subjects) than those involving MP (1 subject) and healthy Japanese subjects (P = 0.001). Notably, synthetic cathinones were detected in 2 of 3 cases involving G643S carriers. Previous functional analyses have indicated that the G643S polymorphism in the KCNQ1 potassium channel gene causes mild IKs channel dysfunction. Our data suggest that use of NPSs, particularly synthetic cathinones, is associated with elevated risk of serious cardiac arrhythmia and sudden death for subjects carrying KCNQ1 G643S.