SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

New Mexico's COVID-19 Experience.

ABSTRACT: The 2019 novel coronavirus disease (COVID-19) has spread worldwide, infiltrating, infecting, and devastating communities in all locations of varying demographics. An overwhelming majority of published literature on the pathologic findings associated with COVID-19 is either from living clinical cohorts or from autopsy findings of those who died in a medical care setting, which can confound pure disease pathology. A relatively low initial infection rate paired with a high biosafety level enabled the New Mexico Office of the Medical Investigator to conduct full autopsy examinations on suspected COVID-19-related deaths. Full autopsy examination on the first 20 severe acute respiratory syndrome coronavirus 2-positive decedents revealed that some extent of diffuse alveolar damage in every death due to COVID-19 played some role. The average decedent was middle-aged, male, American Indian, and overweight with comorbidities that included diabetes, ethanolism, and atherosclerotic and/or hypertensive cardiovascular disease. Macroscopic thrombotic events were seen in 35% of cases consisting of pulmonary thromboemboli and coronary artery thrombi. In 2 cases, severe bacterial coinfections were seen in the lungs. Those determined to die with but not of severe acute respiratory syndrome coronavirus 2 infection had unremarkable lung findings.

Fatal Acute Pancreatitis in an Adolescent: A Case Report.

Acute pancreatitis is uncommon in children and adolescents, and when it does occur, it is rarely fatal. The cause of death may be misdiagnosed as a death due to diabetic ketoacidosis or an infectious disease if an autopsy is not performed. Because these deaths are often unexpected and may occur before medical care, the forensic pathologist may be contacted to determine the cause of death. Even deaths that occur after hospitalization may be referred to the medical examiner because of the concern for drug involvement, the unusual age of the decedent, or the decreased availability for a hospital autopsy. We report a fatal case of acute pancreatitis in a 14-year-old adolescent girl who was brought unresponsive to the emergency room of a university hospital and died after unsuccessful resuscitation efforts.

Can computed tomography replace or supplement autopsy?

Postmortem computed tomography (PMCT) has been integrated into the practice of many forensic pathologists. To evaluate the utility of PMCT in supplementing and/or supplanting medicolegal autopsy, we conducted a prospective double-blind comparison of abnormal findings reported by the autopsy pathologist with those reported by a radiologist reviewing the PMCT. We reviewed 890 cases: 167 with blunt force injury (BFI), 63 with pediatric trauma (under 5 years), 203 firearm injuries, and 457 drug poisoning deaths. Autopsy and radiology reports were coded using the Abbreviated Injury Scale and abnormal findings and cause of death (COD) were compared for congruence in consensus conferences with novel pathologists and radiologists. Overall sensitivity for recognizing abnormal findings was 71% for PMCT and 74.6% for autopsy. Sensitivities for PMCT/autopsy were 74%/73.1% for BFI, 61.5%/71.4% for pediatric trauma, 84.9%/83.7% for firearm injuries, and 56.5%/66.4% for drug poisoning deaths. COD assigned by reviewing PMCT/autopsy was correct in 88%/95.8% of BFI cases, 99%/99.5% of firearm fatalities, 82.5%/98.5% of pediatric trauma deaths, and 84%/100% of drug poisoning deaths of individuals younger than 50. Both autopsy and PMCT were imperfect in recognizing injuries. However, both methods identified the most important findings and are sufficient to establish COD in cases of BFI, pediatric trauma, firearm injuries and drug poisoning in individuals younger than 50. Ideally, all forensic pathologists would have access to a CT scanner and a consulting radiologist. This would allow a flexible approach that meets the diagnostic needs of each case and best serves decedents' families and other stakeholders.

Factor Xa cleaves SARS-CoV-2 spike protein to block viral entry and infection.

Serine proteases (SP), including furin, trypsin, and TMPRSS2 cleave the SARS-CoV-2 spike (S) protein, enabling the virus to enter cells. Here, we show that factor (F) Xa, an SP involved in blood coagulation, is upregulated in COVID-19 patients. In contrast to other SPs, FXa exerts antiviral activity. Mechanistically, FXa cleaves S protein, preventing its binding to ACE2, and thus blocking viral entry and infection. However, FXa is less effective against variants carrying the D614G mutation common in all pandemic variants. The anticoagulant rivaroxaban, a direct FXa inhibitor, inhibits FXa-mediated S protein cleavage and facilitates viral entry, whereas the indirect FXa inhibitor fondaparinux does not. In the lethal SARS-CoV-2 K18-hACE2 model, FXa prolongs survival yet its combination with rivaroxaban but not fondaparinux abrogates that protection. These results identify both a previously unknown function for FXa and an associated antiviral host defense mechanism against SARS-CoV-2 and suggest caution in considering direct FXa inhibitors for preventing or treating thrombotic complications in COVID-19 patients.

Protease-anti-protease compartmentalization in SARS-CoV-2 ARDS: Therapeutic implications.

BACKGROUND: Interleukin-6 (IL-6) is elevated in SARS-CoV-2 infection. IL-6 regulates acute-phase proteins, such as alpha-1 antitrypsin (AAT), a key lung anti-protease. We investigated the protease-anti-protease balance in the circulation and pulmonary compartments in SARS-CoV-2 acute respiratory distress syndrome (ARDS) compared to non-SARS-CoV-2 ARDS (nsARDS) and the effects of tocilizumab (IL-6 receptor antagonist) on anti-protease defence in SARS-CoV-2 infection. METHODS: Levels and activity of AAT and neutrophil elastase (NE) were measured in plasma, airway tissue and tracheal secretions (TA) of people with SARS-CoV-2 ARDS or nsARDS. AAT and IL-6 levels were evaluated in people with moderate SARS-CoV-2 infection who received standard of care +/- tocilizumab. FINDINGS: AAT plasma levels doubled in SARS-CoV-2 ARDS. In lung parenchyma AAT levels were increased, as was the percentage of neutrophils involved in NET formation. A protease-anti-protease imbalance was detected in TA with active NE and no active AAT. The airway anti-protease, secretory leukoprotease inhibitor was decreased in SARS-CoV-2-infected lungs and cleaved in TA. In nsARDS, plasma AAT levels were elevated but TA samples had less AAT cleavage, with no detectable active NE in most samples. Induction of AAT in ARDS occurred mainly through IL-6. Tocilizumab down-regulated AAT during SARS-CoV-2 infection. INTERPRETATION: There is a protease-anti-protease imbalance in the airways of SARS-CoV-2-ARDS patients. This imbalance is a target for anti-protease therapy. FUNDING: NIH Serological Sciences Network, National Heart, Lung, and Blood Institute and National Institute of Diabetes and Digestive and Kidney Diseases.

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Cause of death conundrum with methadone use: a case report.

Deaths caused by a methadone intoxication or overdose are becoming more frequent. We report a case involving a patient who had extremely high methadone blood concentrations but whose cause of death may have been unrelated to the drug. A 51-year-old woman was found deceased in bed by her daughter. At the scene were numerous bottles of methadone, with the chronic dosage of 240 mg 3 times a day. There was no history of prior suicide attempts, there were no reports of suicidal ideation having been voiced and there was no suicide note. At autopsy, there were no pills found in the stomach. Microscopic tissue examination revealed lobar pneumonia of the right lower lobe. Postmortem lung cultures grew out Streptococcus pneumoniae. Femoral blood contained methadone, 5.7 mg/L; EDDP, 2.1 mg/L; oxycodone, 0.017 mg/L; doxylamine, 0.022 mg/L; and ethanol, 13.0 mg/dL. The postmortem methadone concentration was consistent with her known dose, plausible pharmacokinetics and conditions of discovery. Various causes of death, such as a methadone-related arrhythmia from QTc prolongation or the contribution of methadone to the development of the pneumonia, cannot be ruled out and may well have caused or contributed to death, but the pneumonia was felt to be a competent cause of death. Ultimately, the most likely cause(s) of death, is a decision left to the individual medical examiner. This case is illustrative of the growing number of similar cases facing forensic pathologists. The cause of death cannot be solely based on drug concentrations and it may not be possible to come to a conclusion as to "the" cause of death and the forensic pathologist must be content with "a" cause of death.

A toxicology-based review of fentanyl-related deaths in New Mexico (1986-2007).

Since its approval in the United States, fentanyl has become increasingly popular for the medical management of pain and as a substance of abuse. Fentanyl is unique among the opioids in its widespread use with a transdermal delivery system, which contributes to its unique pharmacokinetics and abuse potential. We examined the demographics of deaths with fentanyl identified on toxicologic analysis and reviewed specific challenges in the laboratory detection of postmortem fentanyl levels. The New Mexico Office of the Medical Investigator database was searched for all cases from January 1986 through December 2007 with fentanyl reported as present or quantified. Those deaths with a cause of death identified as drug overdose were then analyzed separately. From 1986 to 2007, 154 cases were identified with fentanyl present in postmortem samples, with 96 of the cases identified as fentanyl-related drug overdoses. The number of fentanyl-related deaths has increased over the past 20 years, corresponding to both statewide increases in the medical use of fentanyl and the abuse of prescription opioids. The demographics of these fentanyl-related overdoses showed that subjects were more likely to be female, white non-Hispanic, and older than those in previously described overdose deaths. Several cases were identified with central and peripheral blood samples and antemortem and postmortem samples available for fentanyl quantification. Given the uncharacteristic demographics of fentanyl-related deaths and the complexity of the laboratory analysis of fentanyl, forensic scientists must use caution in both the detection and interpretation of fentanyl concentrations.

Depressed right ventricular systolic function in heart failure due to constrictive pericarditis.

AIMS: Heart failure in constrictive pericarditis (CP) is attributed to impaired biventricular diastolic filling. However, diseases that cause CP due to myocardial infiltration and fibrosis can also impair biventricular systolic function (sf) and contribute to heart failure. This study of patients with CP examined biventricular sf and the effect of myocardial infiltration by pericardial diseases and the resulting fibrosis on ventricular sf. METHODS AND RESULTS: Histopathologic examinations of right ventricular (RV) and left ventricular (LV) myocardia and pericardia were performed on three autopsied hearts of patients with pericardial diseases. Additionally, in 40 adults with clinical heart failure and 40 healthy adults (controls), sf of both ventricles was examined by echocardiography, including strain measurements, and biventricular diastolic filling and pulmonary artery pressures were assessed by cardiac catheterization. Cardiac histopathology indicated thickening of the pericardium with fibrosis, disease infiltrating the myocardium, greater infiltration of the RV than the LV, and an association of pericardial thickness with myocardial infiltrations. Functional analysis indicated that RVsf was impaired on all echo indices, including strain measurement, but LVsf was preserved. CONCLUSIONS: Diseases causing CP are not restricted to the pericardium but also infiltrate the biventricular myocardium and affect the thin-walled RV more than the thick-walled LV, resulting in depressed RVsf. The present results help explain clinical heart failure in the presence of restricted diastolic filling in CP. Depression of RVsf due to progression of fibrosis in the RV myocardium may increase the risk of delayed pericardiectomy.

FXa cleaves the SARS-CoV-2 spike protein and blocks cell entry to protect against infection with inferior effects in B.1.1.7 variant.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human natural defense mechanisms against SARS-CoV-2 are largely unknown. Serine proteases (SPs) including furin and TMPRSS2 cleave SARS-CoV-2 spike protein, facilitating viral entry. Here, we show that FXa, a SP for blood coagulation, is upregulated in COVID-19 patients compared to non-COVID-19 donors and exerts anti-viral activity. Mechanistically, FXa cleaves the SARS-CoV-2 spike protein, which prevents its binding to ACE2, and thus blocks viral entry. Furthermore, the variant B.1.1.7 with several mutations is dramatically resistant to the anti-viral effect of FXa compared to wild-type SARA-CoV-2 in vivo and in vitro. The anti-coagulant rivaroxaban directly inhibits FXa and facilitates viral entry, whereas the indirect inhibitor fondaparinux does not. In a lethal humanized hACE2 mouse model of SARS-CoV-2, FXa prolonged survival while combination with rivaroxaban but not fondaparinux abrogated this protection. These preclinical results identify a previously unknown SP function and associated anti-viral host defense mechanism and suggest caution in considering direct inhibitors for prevention or treatment of thrombotic complications in COVID-19 patients.

"Med-X": a medical examiner surveillance model for bioterrorism and infectious disease mortality.

We created a model surveillance system (Med-X) designed to enable medical examiners and coroners to recognize fatal infections of public health importance and deaths due to bioterrorism. All individuals who died in New Mexico and fell under medical examiner jurisdiction between November 23, 2000, and November 22, 2002, were prospectively evaluated using sets of surveillance symptoms and autopsy-based pathologic syndromes. All infectious disease deaths were evaluated to identify the specific causative agent. Of 6104 jurisdictional cases, 250 (4.1%) met Med-X criteria, of which 141 (56.4%) had a target pathologic syndrome. Ultimately, 127 (51%) of the 250 cases were due to infections. The causative organism was identified for 103 (81%) of the infectious disease deaths, of which 60 (58.3%) were notifiable conditions in New Mexico. Flu-like symptoms, fever and respiratory symptoms, and encephalopathy or new-onset seizures had predictive values positive for fatal infections of 65%, 72%, and 50%, respectively, and are useful as autopsy performance criteria. Before the development of surveillance criteria, 37 (14.8%) of the cases ordinarily would not have been autopsied resulting in a 1% increase in autopsy workload. Med-X is an effective method of detecting infectious disease deaths among medical examiner cases. Uniform criteria for performing medical examiner autopsies and reporting cases to public health authorities enhance surveillance for notifiable infectious diseases and increase the likelihood of recognizing deaths related to bioterrorism.

Repeat Coronary Artery Dissection in Pregnancy: A Case Report and Review of the Literature.

Non-atherosclerotic spontaneous coronary artery dissection (NA-SCAD) is a rare cause of morbidity and mortality with a propensity for young, healthy, and often peripartum women. NA-SCAD etiology is poorly understood, with possible hormonal and hereditary mechanisms. Current treatment strategies range from conservative management (often showing resolution on angiographic follow-up) to invasive angiographic procedures. Rarely, NA-SCAD has recurred in another coronary artery, ranging hours to years later. We report NA-SCAD of the right coronary artery (RCA) in a 30-year old, 3-month postpartum female with an additional autopsy finding of remote myocardial infarction (MI) in the left anterior descending (LAD) coronary artery territory. The remote MI is consistent with prior NA-SCAD of the LAD and, given the medical history, may have occurred in the peripartum period of the decedent first pregnancy 3 years earlier. As such, to the best of our knowledge, this may represent the first reported case of NA-SCAD recurrence in a subsequent pregnancy.

Amended cause and manner of death certification: a six-year review of the New Mexico experience.

Little is known about the amendment of death certificates (DCs) issued by medical examiners and coroners. This retrospective study examined why, how, and with what frequency cause and manner of death were amended on DCs issued by forensic pathologists over a 6-year period at the New Mexico Office of the Medical Investigator. Approximately 1% of DCs had either cause or manner of death amendments, with arteriosclerotic cardiovascular disease and intoxicants the most commonly amended and resulting causes of death, respectively. There was a significant association between manner of death and number of DCs amended (p<0.001). By percent, natural and suicide DCs were the most frequently amended. The way in which manner of death changed was significantly associated with the amount of time elapsed between DCs (p=0.04). Toxicology was the most common reason for DC amendment.

The influence of site of collection on postmortem morphine concentrations in heroin overdose victims.

When assaying for postmortem morphine concentration, significant site sampling variability exists between central and peripheral sampling sites and even within sampling regions of the body. To study the variation, 76 suspected heroin overdoses were identified. Each had femoral artery (FA) and vein (FV), left and right ventricle and pooled heart blood samples obtained at autopsy. Forty-four tested positive for morphine. Morphine concentrations were determined by gas chromatography/mass spectrometry, with sampling site differences reported as log-transformed ratios and compared by signed rank test. The mean FA to FV ratio for total morphine was 1.2 (range 0-4.5). The ratio for left heart to right heart total morphine was 1.1 (range 0.4-3.2). Left ventricular to FV total morphine ratio was 2.0 (range 0.6-6.9). In these opioid overdose deaths, FA and FV morphine concentrations are usually similar, although up to 4.5-fold differences were noted. Centrally obtained morphine concentrations are on average twice as high compared with peripheral morphine concentrations. Left and right ventricular morphine concentrations were usually similar, although up to 3.2-fold differences were noted (left side higher).

Forensic bitemark identification: weak foundations, exaggerated claims.

Several forensic sciences, especially of the pattern-matching kind, are increasingly seen to lack the scientific foundation needed to justify continuing admission as trial evidence. Indeed, several have been abolished in the recent past. A likely next candidate for elimination is bitemark identification. A number of DNA exonerations have occurred in recent years for individuals convicted based on erroneous bitemark identifications. Intense scientific and legal scrutiny has resulted. An important National Academies review found little scientific support for the field. The Texas Forensic Science Commission recently recommended a moratorium on the admission of bitemark expert testimony. The California Supreme Court has a case before it that could start a national dismantling of forensic odontology. This article describes the (legal) basis for the rise of bitemark identification and the (scientific) basis for its impending fall. The article explains the general logic of forensic identification, the claims of bitemark identification, and reviews relevant empirical research on bitemark identification-highlighting both the lack of research and the lack of support provided by what research does exist. The rise and possible fall of bitemark identification evidence has broader implications-highlighting the weak scientific culture of forensic science and the law's difficulty in evaluating and responding to unreliable and unscientific evidence.

Epstein-Barr virus-associated intravascular large T-cell lymphoma presenting as acute renal failure in a patient with acquired immune deficiency syndrome.

Intravascular lymphoma (IVL) is a rare neoplasm, recently included as a specific entity in the World Health Organization classification of lymphoid tumors. Most cases are of B-cell lineage; however, rare cases of T-cell phenotype have been reported. We report a human immunodeficiency virus (HIV)-positive patient who died of acute renal failure in whom IVL was identified at autopsy, predominantly involving the renal interstitial vessels. Immunohistochemical stains revealed a T-cell phenotype, which was confirmed by T-cell receptor gamma gene rearrangement studies. The lymphoma cells showed nuclear Epstein-Barr virus (EBV)-encoded RNA transcripts by in situ hybridization, suggesting that EBV might be of etiologic importance in this tumor. The predominant involvement of kidney is unusual. With effective therapy, morbidity and mortality of HIV-1 infection has been substantially reduced, and survival times have been prolonged. However, the relative risk of secondary neoplasms, especially non-Hodgkin's lymphoma (NHL), has increased. Consequently, we conclude that unique types of NHL, such as this case of IVL, may be encountered more frequently in this patient population, and that NHL should be added to the list of differential diagnostic considerations in HIV-1-positive patients who develop acute renal failure.

Fatal bupivacaine intoxication following unusual erotic practices.

A fatal drug overdose is described which involved unusual erotic practices. A 54-year-old male was discovered supine on the floor surrounded by sexual paraphernalia, syringes, and medications including three empty bottles of bupivacaine. Acute and chronic injection sites of the external genitalia with contusions, scarring, focal necrosis, and calcification were present at autopsy. Toxicology revealed femoral blood, heart blood, and vitreous bupivacaine concentrations of 3.8, 2.8 and 1.3 mg/L, respectively. The urine bupivacaine concentration was 11.4 mg/L. The cause of death was attributed to bupivacaine intoxication and the manner of death was accidental.

An examination of the postmortem redistribution of fentanyl and interlaboratory variability.

Fentanyl is a synthetic opioid agonist used for pain control. Often administered as a transdermal patch, it is an interesting drug for study of postmortem redistribution. We hypothesized that fentanyl concentrations would increase over time after death, as measured in blood drawn on the day prior to autopsy and in blood drawn at the time of autopsy in ten cases where fentanyl patches were identified at the scene. Concentrations were compared, and heart blood to femoral blood ratios were calculated as markers of postmortem redistribution. Fentanyl concentrations measured in peripheral blood drawn the day of autopsy (peripheral blood 2 [PB2]) were higher than those drawn the day prior to autopsy (peripheral blood 1 [PB1]) with a mean ratio (PB2/PB1) of 1.80. The ratio of heart blood concentrations (HB) to femoral blood concentrations drawn at autopsy (PB2) had a mean ratio (HB/PB2) of 1.08. Some cases had blood from the same source analyzed at two different laboratories, and concentrations of fentanyl in those samples showed inter- and intralaboratory differences up to 25 ng/mL. Postmortem fentanyl concentrations may be affected by antemortem factors, postmortem redistribution, and laboratory variability. Forensic pathologists must use caution in interpreting fentanyl levels as part of death investigation.