A Large Postmortem Database of COVID-19 Patients Can Inform Disease Research and Public Policy Decision Making.

CONTEXT.­: Autopsies performed on COVID-19 patients have provided critical information about SARS-CoV-2's tropism, mechanisms of tissue injury, and the spectrum of disease. OBJECTIVE.­: To provide an updated database of postmortem disease in COVID-19 patients, assess relationships among clinical and pathologic variables, evaluate the accuracy of death certification, and correlate disease variables to causes of death. DESIGN.­: The 272 postmortem examinations reported in this paper were submitted by 14 pathologists from 9 medical or forensic institutions across the United States. The study spans the eras of the 3 principal COVID-19 strains and incorporates surveyed demographic, clinical, and postmortem data from decedents infected with SARS-CoV-2, including primary and contributing causes of death. It is the largest database of its kind to date. RESULTS.­: Demographics of the decedents reported here correspond well to national statistics. Primary causes of death as determined by autopsy and official death certificates were significantly correlated. When specifically cited disease conditions found at autopsy were correlated with COVID-19 versus non-COVID-19 death, only lung findings characteristic of SARS-CoV-2 infection or the absence of lung findings were significantly associated. CONCLUSIONS.­: Changes in hospitalization and disease likely stem from longer lifespans after COVID-19 diagnosis and alteration in treatment approaches. Although Omicron variants preferentially replicate in the upper airways, autopsied patients who died of COVID-19 in that time period showed the same lung damage as earlier decedents. Most importantly, findings suggest that there are still unelucidated risk factors for death from COVID-19 including possibly genetic susceptibility.

Diagnostic yield of postmortem brain examination following premortem brain biopsy for neoplastic and nonneoplastic disease.

Medical autopsies have decreased in frequency due in part to advances in radiological techniques and increased availability of molecular and other ancillary testing. However, premortem diagnosis of CNS disease remains challenging; while ∼90% of brain tumor biopsies are diagnostic, only 20%-70% of biopsies for presumed nonneoplastic disease result in a specific diagnosis. The added benefits of performing an autopsy following surgical brain biopsy are not well defined. A retrospective analysis was performed of patients who underwent brain biopsy and autopsy at Brigham and Women's Hospital from 2003 to 2022. A total of 135 cases were identified, including 95 (70%) patients with primary CNS neoplasms, 16 (12%) with metastatic tumors, and 24 (18%) with nonneoplastic neurological disease. Diagnostic concordance between biopsy and autopsy diagnosis was excellent both for primary CNS neoplasms (98%) and metastatic tumors (94%). Conversely, patients with nonneoplastic disease received definitive premortem diagnoses in 7/24 (29%) cases. Five (21%) additional patients received conclusive diagnoses following autopsy; 8 (33%) received a more specific differential diagnosis compared to the biopsy. Overall, autopsy confirmed premortem diagnoses or provided new diagnostic information in 131/135 (97%) cases, highlighting the value in performing postmortem brain examination in patients with both neoplastic and nonneoplastic diseases.

Cognition-associated long noncoding RNAs are dysregulated upon severe COVID-19.

Severe COVID-19 leads to widespread transcriptomic changes in the human brain, mimicking diminished cognitive performance. As long noncoding RNAs (lncRNAs) play crucial roles in the regulation of gene expression, identification of the lncRNAs differentially expressed upon COVID-19 may nominate key regulatory nodes underpinning cognitive changes. Here we identify hundreds of lncRNAs differentially expressed in the brains of COVID-19 patients relative to uninfected age/sex-matched controls, many of which are associated with decreased cognitive performance and inflammatory cytokine response. Our analyses reveal pervasive transcriptomic changes in lncRNA expression upon severe COVID-19, which may serve as key regulators of neurocognitive changes in the brain.

Cerebrospinal fluid metagenomics has greatest added value as a test for Powassan virus among patients in New England with suspected central nervous system infection.

Cerebrospinal fluid (CSF) metagenomic next generation sequencing (mNGS) can detect diverse pathogens in patients with central nervous system infection. Due to its high cost and unclear clinical utility, it is typically reserved for patients with unrevealing routine workups. A multi-center retrospective analysis of real-world CSF mNGS was performed involving orders between 2017 and 2022 at a large New England healthcare system. CSF mNGS was performed 64 times with 17 positive results (27 %). In 11/17 positive samples (65 %), the infectious agent had not been previously detected using routine methods. Arboviruses (n = 8) were the most frequently detected agents, particularly Powassan virus (n = 6). Results changed therapy in 3/64 cases (5 %). Positive results were associated with immunodeficiency (p = 0.06), especially anti-B-cell therapy (p = 0.02), and earlier sample collection (p = 0.06). The association with compromised humoral immunity was stronger in the arbovirus and Powassan virus subgroups (p = 0.001), whose constituents were older than the overall cohort and had higher mortality rates.

Persistence of intact HIV-1 proviruses in the brain during antiretroviral therapy.

HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the CNS. Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies.

Approximately 39 million people in the world live with HIV infection. Currently available treatments can reduce the amount of virus to near undetectable levels. But they do not eliminate the virus. A reservoir of HIV-infected cells persists during treatment. If treatment stops, these cells can cause rebounding virus levels and a return of symptoms. As a result, patients living with HIV must remain on treatment their entire lives. HIV reservoir cells often do not express viral proteins, making them hard for the immune system to find and destroy. Many of these reservoir cells occur in lymph nodes, which makes them difficult for researchers to access for study. Learning more about where these cells hide in the body may enable scientists to develop new treatments to help eliminate them. Sun et al. show that HIV reservoir cells exist in many body tissues, including the brain. In the experiments, Sun et al. used single HIV genome sequencing to identify HIV genetic sequences in the brain and other body tissues from three recently deceased individuals with HIV. The individuals agreed to donate their tissues for postmortem studies before their deaths. All received antiretroviral therapy until death. The experiments identified functional HIV genetic sequences in lymph nodes and gastrointestinal tissues, known hotspots for HIV-infected cells. Sun et al. also found genetically intact HIV in brain tissue from two of the individuals. The HIV genetic sequences were identical to sequences found in other body tissues. This discovery suggests HIV-infected cells had divided into more HIV-infected cells and spread. The results suggest that cells harboring intact HIV invade the brain and persist there for extended periods during antiretroviral therapy. To eradicate the virus, interventions targeting HIV reservoir cells must be able to reach the brain. This new information may help researchers developing HIV-reservoir targeting drugs decide which candidates will likely be the most effective. Future studies may also shed light on how HIV reaches the brain and how the infected cells escape destruction by immune cells, which may suggest more treatment strategies.

Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

A 28-Year-Old Woman With Left-Sided Weakness and Atypical MRI Lesions: From the National Multiple Sclerosis Society Case Conference Proceedings.

A 28-year-old woman presented with subacute relapsing left-sided weakness. MRI demonstrated both enhancing C3-C6 and nonenhancing T2-T4 lesions. Initial provisional diagnosis was inflammatory/autoimmune. Her left-sided weakness progressed despite immunosuppressive therapies. We reassessed our original suspected diagnosis because of an atypical clinicoradiologic course, leading to biopsy and a definitive diagnosis.

Persistence of intact HIV-1 proviruses in the brain during antiretroviral therapy.

HIV-1 reservoir cells that circulate in peripheral blood during suppressive antiretroviral therapy (ART) have been well characterized, but little is known about the dissemination of HIV-1-infected cells across multiple anatomical tissues, especially the central nervous system (CNS). Here, we performed single-genome, near full-length HIV-1 next-generation sequencing to evaluate the proviral landscape in distinct anatomical compartments, including multiple CNS tissues, from 3 ART-treated participants at autopsy. While lymph nodes and, to a lesser extent, gastrointestinal and genitourinary tissues represented tissue hotspots for the persistence of intact proviruses, we also observed intact proviruses in CNS tissue sections, particularly in the basal ganglia. Multi-compartment dissemination of clonal intact and defective proviral sequences occurred across multiple anatomical tissues, including the CNS, and evidence for the clonal proliferation of HIV-1-infected cells was found in the basal ganglia, in the frontal lobe, in the thalamus and in periventricular white matter. Deep analysis of HIV-1 reservoirs in distinct tissues will be informative for advancing HIV-1 cure strategies.

Eastern Equine Encephalitis Virus Diversity in Massachusetts Patients, 1938-2020.

Eastern equine encephalitis virus (EEEV) is a relatively little-studied alphavirus that can cause devastating viral encephalitis, potentially leading to severe neurological sequelae or death. Although case numbers have historically been low, outbreaks have been increasing in frequency and scale since the 2000 s. It is critical to investigate EEEV evolutionary patterns, especially within human hosts, to understand patterns of emergence, host adaptation, and within-host evolution. To this end, we obtained formalin-fixed paraffin-embedded tissue blocks from discrete brain regions from five contemporary (2004-2020) patients from Massachusetts, confirmed the presence of EEEV RNA by in situ hybridization (ISH) staining, and sequenced viral genomes. We additionally sequenced RNA from scrapings of historical slides made from brain sections of a patient in the first documented EEE outbreak in humans in 1938. ISH staining revealed the presence of RNA in all contemporary samples, and quantification loosely correlated with the proportion of EEEV reads in samples. Consensus EEEV sequences were generated for all six patients, including the sample from 1938; phylogenetic analysis using additional publicly available sequences revealed clustering of each study sample with like sequences from a similar region, whereas an intrahost comparison of consensus sequences between discrete brain regions revealed minimal changes. Intrahost single nucleotide variant (iSNV) analysis of four samples from two patients revealed the presence of tightly compartmentalized, mostly nonsynonymous iSNVs. This study contributes critical primary human EEEV sequences, including a historic sequence as well as novel intrahost evolution findings, contributing substantially to our understanding of the natural history of EEEV infection in humans.

Double-stranded RNA immunohistochemistry as a screening tool for viral encephalitis.

OBJECTIVES: Viral infections of the central nervous system can be challenging to diagnose because of the wide range of causative agents and nonspecific histologic features. We sought to determine whether detection of double-stranded RNA (dsRNA), produced during active RNA and DNA viral infections, could be used to select cases for metagenomic next-generation sequencing (mNGS) from formalin-fixed, paraffin-embedded brain tissue. METHODS: Eight commercially available anti-dsRNA antibodies were optimized for immunohistochemistry (IHC) and the top antibody tested in a series of cases with confirmed viral infections (n = 34) and cases with inflammatory brain lesions of unclear etiology (n = 62). RESULTS: Among known positives, anti-dsRNA IHC produced a strong cytoplasmic or nuclear staining pattern for Powassan virus, West Nile virus, rabies virus, JC polyoma virus, and adenovirus while failing to detect Eastern equine encephalitis virus, Jamestown Canyon virus, or any herpesvirus. All the unknown cases were negative by anti-dsRNA IHC, while mNGS detected rare viral reads (0.3-1.3 reads per million total reads) in 2 cases (3%), with only 1 having potential clinical significance. CONCLUSIONS: Anti-dsRNA IHC can effectively identify a subset of clinically relevant viral infections but not all. The absence of staining should not exclude cases from mNGS if sufficient clinical and histologic suspicion exists.

What Can We Still Learn from Brain Autopsies in COVID-19?

Neuropathological findings have been published from ∼900 patients who died with or from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, representing less than 0.01% of the close to 6.4 million deaths reported to the World Health Organization 2 years into the coronavirus disease 2019 (COVID-19) pandemic. In this review, we extend our prior work summarizing COVID-19 neuropathology by including information on published autopsies up to June 2022, and neuropathological studies in children, COVID-19 variants, secondary brain infections, ex vivo brain imaging, and autopsies performed in countries outside of the United States or Europe. We also summarize research studies that investigate mechanisms of neuropathogenesis in nonhuman primates and other models. While a pattern of cerebrovascular pathology and microglial-predominant inflammation remains the primary COVID-19-associated neuropathological finding, there is no singular understanding of the mechanisms that underlie neurological symptoms in acute COVID-19 or the post-acute COVID-19 condition. Thus, it is paramount that we incorporate microscopic and molecular findings from brain tissue into what we know about the clinical disease so that we attain best practice guidance and direct research priorities for the study of the neurological morbidity of COVID-19.

Teaching Infectious Disease Pathology and Taking it To Africa.

With the advent of increasing emerging infectious diseases, rising antibiotic resistance, and the growing number of immunocompromised patients, there is increasing demand for infectious disease (ID) pathology expertise and microbiology testing. Currently, ID pathology training and emerging molecular microbiology techniques (eg, metagenomic next-generation sequencing and whole genome sequencing) are not included in the most American Council of Graduate Medical Education medical microbiology fellowship curricula, and not surprisingly, many institutions lack anatomical pathologists with expertise in ID pathology and advanced molecular diagnostics. In this article, we describe the curriculum and structure of the Franz von Lichtenberg Fellowship in Infectious Disease and Molecular Microbiology at Brigham and Women's Hospital in Boston, MA. We emphasize the value of a training model that strives to integrate anatomical pathology, clinical pathology, and molecular pathology by providing examples in a case-based format and presenting selected metrics of the potential effect of such integrative ID pathology service and briefly describing opportunities and challenges of our global health efforts in Rwanda.

Neuropathological features of SARS-CoV-2 delta and omicron variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continually evolving resulting in variants with increased transmissibility, more severe disease, reduced effectiveness of treatments or vaccines, or diagnostic detection failure. The SARS-CoV-2 Delta variant (B.1.617.2 and AY lineages) was the dominant circulating strain in the United States from July to mid-December 2021, followed by the Omicron variant (B.1.1.529 and BA lineages). Coronavirus disease 2019 (COVID-19) has been associated with neurological sequelae including loss of taste/smell, headache, encephalopathy, and stroke, yet little is known about the impact of viral strain on neuropathogenesis. Detailed postmortem brain evaluations were performed for 22 patients from Massachusetts, including 12 who died following infection with Delta variant and 5 with Omicron variant, compared to 5 patients who died earlier in the pandemic. Diffuse hypoxic injury, occasional microinfarcts and hemorrhage, perivascular fibrinogen, and rare lymphocytes were observed across the 3 groups. SARS-CoV-2 protein and RNA were not detected in any brain samples by immunohistochemistry, in situ hybridization, or real-time quantitative PCR. These results, although preliminary, demonstrate that, among a subset of severely ill patients, similar neuropathological features are present in Delta, Omicron, and non-Delta/non-Omicron variant patients, suggesting that SARS-CoV-2 variants are likely to affect the brain by common neuropathogenic mechanisms.

High-depth sequencing characterization of viral dynamics across tissues in fatal COVID-19 reveals compartmentalized infection.

SARS-CoV-2 distribution and circulation dynamics are not well understood due to challenges in assessing genomic data from tissue samples. We develop experimental and computational workflows for high-depth viral sequencing and high-resolution genomic analyses from formalin-fixed, paraffin-embedded tissues and apply them to 120 specimens from six subjects with fatal COVID-19. To varying degrees, viral RNA is present in extrapulmonary tissues from all subjects. The majority of the 180 viral variants identified within subjects are unique to individual tissue samples. We find more high-frequency (>10%) minor variants in subjects with a longer disease course, with one subject harboring ten such variants, exclusively in extrapulmonary tissues. One tissue-specific high-frequency variant was a nonsynonymous mutation in the furin-cleavage site of the spike protein. Our findings suggest adaptation and/or compartmentalized infection, illuminating the basis of extrapulmonary COVID-19 symptoms and potential for viral reservoirs, and have broad utility for investigating human pathogens.

Mimics of Pediatric Small Vessel Primary Angiitis of the Central Nervous System.

OBJECTIVE: Small vessel primary angiitis of the central nervous system is a rare and often severe disease characterized by central nervous system-restricted inflammatory vasculitis on histopathology. Diagnosis requires brain biopsy for confirmation and is suggested prior to starting immunotherapy when feasible. However, emerging evidence suggests that other neuroinflammatory conditions may have a clinical and radiographic phenotype that mimics small vessel primary angiitis, at times with overlapping pathologic features as well. Such diagnoses, including myelin oligodendrocyte glycoprotein antibody-associated disease and central nervous system-restricted hemophagocytic lymphohistiocytosis, can be non-invasively diagnosed with serum antibody or genetic testing that would prompt different monitoring and treatment paradigms. To determine the ultimate diagnosis of patients who were suspected prior to biopsy to have small vessel primary angiitis, we reviewed the clinical, radiographic, and pathological features of a cohort of patients at a single center undergoing brain biopsy for non-oncologic indications. METHODS: Clinical data were retrospectively extracted from the medical record. Pathology and neuroimaging review was conducted. RESULTS: We identified 21 patients over a 19-year time-period, of whom 14 (66.7%) were ultimately diagnosed with entities other than small vessel primary angiitis that would have obviated the need for brain biopsy. Diagnoses included anti-myelin oligodendrocyte glycoprotein antibody associated disease (n = 9), central nervous system-restricted hemophagocytic lymphohistiocytosis (n = 3), anti-GABAA receptor encephalitis (n = 1), and Aicardi-Goutières syndrome (n = 1). INTERPRETATION: This study highlights the importance of pursuing now readily available non-invasive testing for mimicking diagnoses before performing a brain biopsy for suspected small vessel primary angiitis of the central nervous system. ANN NEUROL 2023;93:109-119.

Systemic administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque CNS.

BACKGROUND: Adeno-associated virus (AAV) vectors are a promising vehicle for noninvasive gene delivery to the central nervous system via intravenous infusion. However, naturally occurring serotypes have a limited ability to transduce the brain, and translating engineered capsids from mice to nonhuman primates has proved challenging. METHODS: In this study, we use an mRNA-based directed-evolution strategy in multiple strains of mice as well as a de novo selection in cynomolgus macaques to identify families of engineered vectors with increased potency in the brain and decreased tropism for the liver. FINDINGS: We compare the transgene expression capabilities of several engineered vectors and show that while some of our novel macaque-derived variants significantly outperform AAV9 in transducing the macaque brain following systemic administration, mouse-derived variants-both those identified in this study and those reported by other groups-universally do not. CONCLUSIONS: Together, the results of this work introduce a class of primate-derived engineered AAV capsids with increased therapeutic potential and highlight the critical need for using appropriate animal models to both identify and evaluate novel AAVs intended for delivery to the human central nervous system. FUNDING: This work was funded primarily through an anonymous philanthropic gift to the P.C.S. lab at the Broad Institute of MIT and Harvard and by a grant from the Howard Hughes Medical Institute to P.C.S.

A single-nucleus and spatial transcriptomic atlas of the COVID-19 liver reveals topological, functional, and regenerative organ disruption in patients.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

Powassan Virus Encephalitis.

Powassan virus is an increasingly recognized cause of severe encephalitis that is transmitted by Ixodes ticks. Given the nonspecific clinical, laboratory, and imaging features of Powassan virus disease, providers should consider it in patients with compatible exposures and request appropriate testing.

Absence of SARS-CoV-2 Spike glycoprotein expression in placentas from individuals after mRNA SARS-CoV-2 vaccination.

Current public health initiatives to contain the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) global pandemic focus on expanding vaccination efforts to include vulnerable populations such as pregnant people. Vaccines using messenger ribonucleic acid (mRNA) technology rely on translation by immune cells, primarily at the injection site. Hesitancy remains among the general population regarding the safety of mRNA vaccines during gestation, and it remains unknown whether the SARS-CoV-2 Spike protein (the product of mRNA vaccines available) accumulates in the placenta after vaccination. Objective: To determine whether Spike protein translation and accumulation occurs in placental tissue in the context of recent mRNA SARC-CoV-2 vaccination during pregnancy. We identified 48 patients receiving one or two doses of mRNA SARS-CoV-2 vaccine during gestation and used immunohistochemistry against SARS-CoV-2 Spike protein in formalin-fixed, paraffin-embedded placental tissue. One placenta, positive for SARS-CoV-2 RNA by in situ hybridization (ISH) was used as positive control. Seven term placentas collected prior to the emergence of SARS-CoV-2 served as negative controls. Eighty one percent of patients in the study group underwent third-trimester delivery; remaining had a first-trimester spontaneous abortion or elective second-trimester termination. Patients received two (52%) or one (48%) vaccine doses during pregnancy, with a median interval between latest dose and delivery of 13 days (range 2-79 days). Most (63%) cases had their latest dose within 15 days prior to delivery. All the placentas in the study and negative control groups were negative for SARS-CoV-2 immunohistochemistry. Six study cases with short vaccine-delivery intervals (2-7 days) were subjected to SARS-CoV-2 ISH and were negative. Our findings suggest that mRNA vaccines do not reach significant concentrations in the placenta given the absence of definitive SARS-CoV-2 Spike protein accumulation in placental tissue. This observation provides evidence supporting the safety of mRNA vaccines to the placental-fetal unit.

Rigidoporus corticola Colonization and Invasive Fungal Disease in Immunocompromised Patients, United States.

We report 2 cases of Rigidoporus corticola (Oxyporus corticola) infection in humans in the United States. Clinical manifestations consisted of angioinvasive fungal sinusitis in 1 patient and pulmonary intracavitary fungus ball in the other patient. These cases illustrate previously undescribed clinicopathologic manifestations of infection by this filamentous basidiomycete in humans.