Toxicologic Pathology Forum Opinion: Apoptosis/Single Cell Necrosis as a Possible Procedural Effect in Primate Brain Following Ice-Cold Saline Perfusion.

Nonclinical studies of test articles (TAs) in nonhuman primates are often designed to assess both biodistribution and toxicity. For this purpose, studies commonly use intravenous perfusion of ice-cold (2°C-8°C) saline to facilitate measurements of TA-associated nucleic acids and proteins, after which tissues undergo later fixation by immersion for histological processing and microscopic evaluation. Intriguingly, minimal apoptosis/single cell necrosis (A/SCN) of randomly distributed neural cells is evident in the cerebral cortex and less often the hippocampus in animals from all groups, including vehicle-treated controls. Affected cells exhibit end-stage features such as cytoplasmic hypereosinophilia, nuclear condensation or fragmentation, and shape distortions, so their lineage(s) generally cannot be defined; classical apoptotic bodies are exceedingly rare. In addition, A/SCN is not accompanied by glial reactions, leukocyte infiltration/inflammation, or other parenchymal changes. The severity is minimal in controls but may be slightly exacerbated (to mild) by TA that accumulate in neural cells. One plausible hypothesis explaining this A/SCN exacerbation is that cold shock (perhaps complicated by concurrent tissue acidity and hypoxia) drives still-viable but TA-stressed cells to launch a self-directed death program. Taken together, these observations indicate that A/SCN in brain processed by cold saline perfusion with delayed immersion fixation represents a procedural artifact and not a TA-related lesion.

Incidental Gliosis in the Central Nervous System of Control Nonhuman Primates and Rats.

Gliosis, including microgliosis and astrocytosis, can be challenging to interpret in nonclinical studies. Incidences of glial foci in brains and spinal cords of control rats and nonhuman primates (NHPs) were reviewed in the historical control databases from two contract research organizations, including one specializing in neuropathology. In the brain, minimal to mild (grades 1-2) microgliosis was the most common diagnosis, especially in NHPs, although occasional moderate or marked microgliosis (grades 3 and 4) was encountered in both species. Microgliosis was more common in the cerebral cortex, cerebellum, and medulla oblongata in both species and was frequent in the white matter (brain), thalamus, and basal nuclei of NHPs. Gliosis ("not otherwise specified") of minimal severity was diagnosed in similar brain sub-sites for both species and was more common in NHPs compared with rats. Astrocytosis was most prominent in the cerebellum (molecular layer) of NHPs but was otherwise uncommon. In the spinal cord, microgliosis was most common in the lateral white matter tracts in rats and NHPs, and in the dorsal white matter tracts in NHPs. These data indicate that low-grade spontaneous glial responses occur with some frequency in control animals of two common nonclinical species.

Influenza A virus infection and pathology in nasal and periocular tissues after ocular inoculation in ferrets.

Influenza A viruses (IAV) cause mammalian infections following several transmission routes. Considering the anatomic proximity and connection between the nasopharynx and periocular tissues, there is a need to understand the dynamics of virus spread between these sites following both respiratory and nonrespiratory viral transmission. We examined virus distribution and associated inflammation within nasal and periocular tissues during the acute phase of H1N1 IAV infection in ferrets following intranasal or ocular inoculation. Ocular and intranasal inoculations with IAV caused comparable viral antigen distribution and inflammation in the nasal passages, though infection kinetics and magnitude differed by inoculation route. Ocular inoculation was associated with inflammation in the conjunctiva and lacrimal glands. Although intranasal inoculation was also associated with periocular inflammation, the onset was delayed relative to ocular inoculation. This work underscores the importance of investigating extrapulmonary tissues following mammalian infection with respiratory pathogens, even after intranasal inoculation.

Histopathology and localization of SARS-CoV-2 and its host cell entry receptor ACE2 in tissues from naturally infected US-farmed mink (Neovison vison).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes respiratory disease in mink similar to human COVID-19. We characterized the pathological findings in 72 mink from US farms with SARS-CoV-2 outbreaks, localized SARS-CoV-2 and its host cellular receptor angiotensin-converting enzyme 2 (ACE2) in mink respiratory tissues, and evaluated the utility of various test methods and specimens for SARS-CoV-2 detection in necropsy tissues. Of SARS-CoV-2-positive animals found dead, 74% had bronchiolitis and diffuse alveolar damage (DAD). Of euthanized SARS-CoV-2-positive animals, 72% had only mild interstitial pneumonia or minimal nonspecific lung changes (congestion, edema, macrophages); similar findings were seen in SARS-CoV-2-negative animals. Suppurative rhinitis, lymphocytic perivascular inflammation in the lungs, and lymphocytic infiltrates in other tissues were common in both SARS-CoV-2-positive and SARS-CoV-2-negative animals. In formalin-fixed paraffin-embedded (FFPE) upper respiratory tract (URT) specimens, conventional reverse transcription-polymerase chain reaction (cRT-PCR) was more sensitive than in situ hybridization (ISH) or immunohistochemistry (IHC) for detection of SARS-CoV-2. FFPE lung specimens yielded less detection of virus than FFPE URT specimens by all test methods. By IHC and ISH, virus localized extensively to epithelial cells in the nasal turbinates, and prominently within intact epithelium; olfactory mucosa was mostly spared. The SARS-CoV-2 receptor ACE2 was extensively detected by IHC within turbinate epithelium, with decreased detection in lower respiratory tract epithelium and alveolar macrophages. This study expands on the knowledge of the pathology and pathogenesis of natural SARS-CoV-2 infection in mink and supports their further investigation as a potential animal model of SARS-CoV-2 infection in humans.

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2 Replication and Tropism in the Lungs, Airways, and Vascular Endothelium of Patients With Fatal Coronavirus Disease 2019: An Autopsy Case Series.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic continues to produce substantial morbidity and mortality. To understand the reasons for the wide-spectrum complications and severe outcomes of COVID-19, we aimed to identify cellular targets of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tropism and replication in various tissues. METHODS: We evaluated RNA extracted from formalin-fixed, paraffin-embedded autopsy tissues from 64 case patients (age range, 1 month to 84 years; 21 COVID-19 confirmed, 43 suspected COVID-19) by SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR). For cellular localization of SARS-CoV-2 RNA and viral characterization, we performed in situ hybridization (ISH), subgenomic RNA RT-PCR, and whole-genome sequencing. RESULTS: SARS-CoV-2 was identified by RT-PCR in 32 case patients (21 COVID-19 confirmed, 11 suspected). ISH was positive in 20 and subgenomic RNA RT-PCR was positive in 17 of 32 RT-PCR-positive case patients. SARS-CoV-2 RNA was localized by ISH in hyaline membranes, pneumocytes, and macrophages of lungs; epithelial cells of airways; and endothelial cells and vessel walls of brain stem, leptomeninges, lung, heart, liver, kidney, and pancreas. The D614G variant was detected in 9 RT-PCR-positive case patients. CONCLUSIONS: We identified cellular targets of SARS-CoV-2 tropism and replication in the lungs and airways and demonstrated its direct infection in vascular endothelium. This work provides important insights into COVID-19 pathogenesis and mechanisms of severe outcomes.

A Guide for the Use of the Ferret Model for Influenza Virus Infection.

As influenza viruses continue to jump species barriers to cause human infection, assessments of disease severity and viral replication kinetics in vivo provide crucial information for public health professionals. The ferret model is a valuable resource for evaluating influenza virus pathogenicity; thus, understanding the most effective techniques for sample collection and usage, as well as the full spectrum of attainable data after experimental inoculation in this species, is paramount. This is especially true for scheduled necropsy of virus-infected ferrets, a standard component in evaluation of influenza virus pathogenicity, as necropsy findings can provide important information regarding disease severity and pathogenicity that is not otherwise available from the live animal. In this review, we describe the range of influenza viruses assessed in ferrets, the measures of experimental disease severity in this model, and optimal sample collection during necropsy of virus-infected ferrets. Collectively, this information is critical for assessing systemic involvement after influenza virus infection in mammals.

Performance evaluation of fungal DNA PCR amplification from formalin-fixed paraffin-embedded tissue for diagnosis: Experience of a tertiary reference laboratory.

BACKGROUND: Diagnosis of invasive fungal infections from formalin-fixed paraffin-embedded (FFPE) tissues by PCR amplification is a developing technology. One of the difficulties of establishing a validated protocol for this testing is that the gold standard, culture, is much less sensitive than the test being validated. OBJECTIVES: To validate FFPE PCR as a refence laboratory identification methodology in the absence of abundant gold standard specimens. METHODS: In this validation, PCR from FFPE tissue was compared to other diagnostic methods for genus/species identification. Four different groups of correlative data from FFPE tissues were used to validate this procedure. Thirteen specimens had culture or serology results and FFPE PCR results, 49 specimens had both immunohistochemistry (IHC) identification and FFPE PCR results, 118 specimens had histological evidence of fungal elements, 64 of which also had FFPE PCR results, and 36 fungal mock tissues or fungal negative tissues were used. RESULTS: The sensitivity determined from the tissues with positive fungal histopathology was 54%. The specificity of the cases for which there were both culture and FFPE PCR results was 100%. For the correlation with IHC, the specificity was 98%. For the mock tissues and fungal negative tissues, the calculated analytical sensitivity was 94%, specificity was 95%, and accuracy was 94%. CONCLUSIONS: By uniquely combining various data sources, this study provides a comprehensive framework for how validation can be achieved in the absence of a gold standard and outlines the excellent performance of PCR from FFPE tissue, despite relatively the low sensitivity when compared to histopathology.

Pathology and Pathogenesis of SARS-CoV-2 Associated with Fatal Coronavirus Disease, United States.

An ongoing pandemic of coronavirus disease (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Characterization of the histopathology and cellular localization of SARS-CoV-2 in the tissues of patients with fatal COVID-19 is critical to further understand its pathogenesis and transmission and for public health prevention measures. We report clinicopathologic, immunohistochemical, and electron microscopic findings in tissues from 8 fatal laboratory-confirmed cases of SARS-CoV-2 infection in the United States. All cases except 1 were in residents of long-term care facilities. In these patients, SARS-CoV-2 infected epithelium of the upper and lower airways with diffuse alveolar damage as the predominant pulmonary pathology. SARS-CoV-2 was detectable by immunohistochemistry and electron microscopy in conducting airways, pneumocytes, alveolar macrophages, and a hilar lymph node but was not identified in other extrapulmonary tissues. Respiratory viral co-infections were identified in 3 cases; 3 cases had evidence of bacterial co-infection.

Outbreak of Anthrax Associated with Handling and Eating Meat from a Cow, Uganda, 2018.

On April 20, 2018, the Kween District Health Office in Kween District, Uganda reported 7 suspected cases of human anthrax. A team from the Uganda Ministry of Health and partners investigated and identified 49 cases, 3 confirmed and 46 suspected; no deaths were reported. Multiple exposures from handling the carcass of a cow that had died suddenly were significantly associated with cutaneous anthrax, whereas eating meat from that cow was associated with gastrointestinal anthrax. Eating undercooked meat was significantly associated with gastrointestinal anthrax, but boiling the meat for >60 minutes was protective. We recommended providing postexposure antimicrobial prophylaxis for all exposed persons, vaccinating healthy livestock in the area, educating farmers to safely dispose of animal carcasses, and avoiding handling or eating meat from livestock that died of unknown causes.

Pathology and Telepathology Methods in the Child Health and Mortality Prevention Surveillance Network.

This manuscript describes the Child Health and Mortality Prevention Surveillance (CHAMPS) network approach to pathologic evaluation of minimally invasive tissue sampling (MITS) specimens, including guidelines for histopathologic examination and further diagnostics with special stains, immunohistochemistry, and molecular testing, as performed at the CHAMPS Central Pathology Laboratory (CPL) at the Centers for Disease Control and Prevention, as well as techniques for virtual discussion of these cases (telepathology) with CHAMPS surveillance locations. Based on review of MITS from the early phase of CHAMPS, the CPL has developed standardized histopathology-based algorithms for achieving diagnoses from MITS and telepathology procedures in conjunction with the CHAMPS sites, with the use of whole slide scanners and digital image archives, for maximizing concurrence and knowledge sharing between site and CPL pathologists. These algorithms and procedures, along with lessons learned from initial implementation of these approaches, guide pathologists at the CPL and CHAMPS sites through standardized diagnostics of MITS cases, and allow for productive, real-time case discussions and consultations.

Sneathia amnii and Maternal Chorioamnionitis and Stillbirth, Mozambique.

We report a case of Sneathia amnii as the causative agent of maternal chorioamnionitis and congenital pneumonia resulting in a late fetal death in Mozambique, with strong supportive postmortem molecular and histopathologic confirmation. This rare, fastidious gram-negative coccobacillus has been reported to infrequently cause abortions, stillbirths, and neonatal infections.

Lassa Virus Targeting of Anterior Uvea and Endothelium of Cornea and Conjunctiva in Eye of Guinea Pig Model.

Lassa virus (LASV), a hemorrhagic fever virus endemic to West Africa, causes conjunctivitis in patients with acute disease. To examine ocular manifestations of LASV, we histologically examined eyes from infected guinea pigs. In fatal disease, LASV immunostaining was most prominent in the anterior uvea, especially in the filtration angle, ciliary body, and iris and in and around vessels in the bulbar conjunctiva and peripheral cornea, where it co-localized with an endothelial marker (platelet endothelial cell adhesion molecule). Antigen was primarily associated with infiltration of T-lymphocytes around vessels in the anterior uvea and with new vessel formation at the peripheral cornea. In animals that exhibited clinical signs but survived infection, eyes had little to no inflammation and no LASV immunostaining 6 weeks after infection. Overall, in this model, LASV antigen was restricted to the anterior uvea and was associated with mild chronic inflammation in animals with severe disease but was not detected in survivors.

Anthrax Epizootic in Wildlife, Bwabwata National Park, Namibia, 2017.

In late September 2017, Bwabwata National Park in Namibia experienced a sudden die-off of hippopotamuses and Cape buffalo. A multiorganizational response was initiated, involving several ministries within Namibia and the US Centers for Disease Control and Prevention. Rapid interventions resulted in zero human or livestock cases associated with this epizootic.

Zika Virus RNA Replication and Persistence in Brain and Placental Tissue.

Zika virus is causally linked with congenital microcephaly and may be associated with pregnancy loss. However, the mechanisms of Zika virus intrauterine transmission and replication and its tropism and persistence in tissues are poorly understood. We tested tissues from 52 case-patients: 8 infants with microcephaly who died and 44 women suspected of being infected with Zika virus during pregnancy. By reverse transcription PCR, tissues from 32 (62%) case-patients (brains from 8 infants with microcephaly and placental/fetal tissues from 24 women) were positive for Zika virus. In situ hybridization localized replicative Zika virus RNA in brains of 7 infants and in placentas of 9 women who had pregnancy losses during the first or second trimester. These findings demonstrate that Zika virus replicates and persists in fetal brains and placentas, providing direct evidence of its association with microcephaly. Tissue-based reverse transcription PCR extends the time frame of Zika virus detection in congenital and pregnancy-associated infections.

Pathology of congenital Zika syndrome in Brazil: a case series.

BACKGROUND: Zika virus is an arthropod-borne virus that is a member of the family Flaviviridae transmitted mainly by mosquitoes of the genus Aedes. Although usually asymptomatic, infection can result in a mild and self-limiting illness characterised by fever, rash, arthralgia, and conjunctivitis. An increase in the number of children born with microcephaly was noted in 2015 in regions of Brazil with high transmission of Zika virus. More recently, evidence has been accumulating supporting a link between Zika virus and microcephaly. Here, we describe findings from three fatal cases and two spontaneous abortions associated with Zika virus infection. METHODS: In this case series, formalin-fixed paraffin-embedded tissue samples from five cases, including two newborn babies with microcephaly and severe arthrogryposis who died shortly after birth, one 2-month-old baby, and two placentas from spontaneous abortions, from Brazil were submitted to the Infectious Diseases Pathology Branch at the US Centers for Disease Control and Prevention (Atlanta, GA, USA) between December, 2015, and March, 2016. Specimens were assessed by histopathological examination, immunohistochemical assays using a mouse anti-Zika virus antibody, and RT-PCR assays targeting the NS5 and envelope genes. Amplicons of RT-PCR positive cases were sequenced for characterisation of strains. FINDINGS: Viral antigens were localised to glial cells and neurons and associated with microcalcifications in all three fatal cases with microcephaly. Antigens were also seen in chorionic villi of one of the first trimester placentas. Tissues from all five cases were positive for Zika virus RNA by RT-PCR, and sequence analyses showed highest identities with Zika virus strains isolated from Brazil during 2015. INTERPRETATION: These findings provide strong evidence of a link between Zika virus infection and different congenital central nervous system malformations, including microcephaly as well as arthrogryposis and spontaneous abortions. FUNDING: None.

Evaluation of Placental and Fetal Tissue Specimens for Zika Virus Infection - 50 States and District of Columbia, January-December, 2016.

Zika virus infection during pregnancy can cause congenital microcephaly and brain abnormalities (1), and detection of Zika virus RNA in clinical and tissue specimens can provide definitive laboratory evidence of recent Zika virus infection. Whereas duration of viremia is typically short, prolonged detection of Zika virus RNA in placental, fetal, and neonatal brain tissue has been reported and can provide key diagnostic information by confirming recent Zika virus infection (2). In accordance with recent guidance (3,4), CDC provides Zika virus testing of placental and fetal tissues in clinical situations where this information could add diagnostic value. This report describes the evaluation of formalin-fixed paraffin-embedded (FFPE) tissue specimens tested for Zika virus infection in 2016 and the contribution of this testing to the public health response. Among 546 live births with possible maternal Zika virus exposure, for which placental tissues were submitted by the 50 states and District of Columbia (DC), 60 (11%) were positive by Zika virus reverse transcription-polymerase chain reaction (RT-PCR). Among 81 pregnancy losses for which placental and/or fetal tissues were submitted, 18 (22%) were positive by Zika virus RT-PCR. Zika virus RT-PCR was positive on placental tissues from 38/363 (10%) live births with maternal serologic evidence of recent unspecified flavivirus infection and from 9/86 (10%) with negative maternal Zika virus immunoglobulin M (IgM) where possible maternal exposure occurred >12 weeks before serum collection. These results demonstrate that Zika virus RT-PCR testing of tissue specimens can provide a confirmed diagnosis of recent maternal Zika virus infection.

Notes from the Field: Evidence of Zika Virus Infection in Brain and Placental Tissues from Two Congenitally Infected Newborns and Two Fetal Losses--Brazil, 2015.

Zika virus is a mosquito-borne flavivirus that is related to dengue virus and transmitted primarily by Aedes aegypti mosquitoes, with humans acting as the principal amplifying host during outbreaks. Zika virus was first reported in Brazil in May 2015 (1). By February 9, 2016, local transmission of infection had been reported in 26 countries or territories in the Americas.* Infection is usually asymptomatic, and, when symptoms are present, typically results in mild and self-limited illness with symptoms including fever, rash, arthralgia, and conjunctivitis. However, a surge in the number of children born with microcephaly was noted in regions of Brazil with a high prevalence of suspected Zika virus disease cases. More than 4,700 suspected cases of microcephaly were reported from mid-2015 through January 2016, although additional investigations might eventually result in a revised lower number (2). In response, the Brazil Ministry of Health established a task force to further investigate possible connections between the virus and brain anomalies in infants (3).

B cell-specific deficiencies in mTOR limit humoral immune responses.

Generation of high-affinity Abs in response to Ags/infectious agents is essential for developing long-lasting immune responses. B cell maturation and Ab responses to Ag stimulation require Ig somatic hypermutation (SHM) and class-switch recombination (CSR) for high-affinity responses. Upon immunization with either the model Ag 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to chicken γ globulin lysine (NP-CGG) or heat-killed Streptococcus pneumoniae capsular type 14 protein (Pn14), knock-in (KI) mice hypomorphic for mTOR function had a decreased ability to form germinal centers, develop high-affinity anti-NP-specific or anti-Pn14-specific Abs, and perform SHM/CSR. Hypomorphic mTOR mice also had a high mortality (40%) compared with wild-type (WT) (0%) littermates and had lower pneumococcal surface protein A-specific Ab titers when immunized and challenged with live S. pneumoniae infection. Mice with mTOR deleted in their B cell lineage (knockout [KO]) also produced fewer splenic germinal centers and decreased high-affinity Ab responses to NP-CGG than did their WT littermates. CSR rates were lower in mTOR KI and KO mice, and pharmacologic inhibition of mTOR in WT B cells resulted in decreased rates of ex vivo CSR. RNA and protein levels of activation-induced cytidine deaminase (AID), a protein essential for SHM and CSR, were lower in B cells from both KI and B cell-specific KO mice, concomitant with increases in phosphorylated AKT and FOXO1. Rescue experiments increasing AID expression in KI B cells restored CSR levels to those in WT B cells. Thus, mTOR plays an important immunoregulatory role in the germinal center, at least partially through AID signaling, in generating high-affinity Abs.

Pharmacokinetic Study of Islatravir and Etonogestrel Implants in Macaques.

The prevention of HIV and unintended pregnancies is a public health priority. Multi-purpose prevention technologies capable of long-acting HIV and pregnancy prevention are desirable for women. Here, we utilized a preclinical macaque model to evaluate the pharmacokinetics of biodegradable ε-polycaprolactone implants delivering the antiretroviral islatravir (ISL) and the contraceptive etonogestrel (ENG). Three implants were tested: ISL-62 mg, ISL-98 mg, and ENG-33 mg. Animals received one or two ISL-eluting implants, with doses of 42, 66, or 108 µg of ISL/day with or without an additional ENG-33 mg implant (31 µg/day). Drug release increased linearly with dose with median [range] plasma ISL levels of 1.3 [1.0-2.5], 1.9 [1.2-6.3] and 2.8 [2.3-11.6], respectively. The ISL-62 and 98 mg implants demonstrated stable drug release over three months with ISL-triphosphate (ISL-TP) concentr54ations in PBMCs above levels predicted to be efficacious for PrEP. Similarly, ENG implants demonstrated sustained drug release with median [range] plasma ENG levels of 495 [229-1110] pg/mL, which suppressed progesterone within two weeks and showed no evidence of altering ISL pharmacokinetics. Two of the six ISL-98 mg implants broke during the study and induced implant-site reactions, whereas no reactions were observed with intact implants. We show that ISL and ENG biodegradable implants are safe and yield sufficient drug levels to achieve prevention targets. The evaluation of optimized implants with increased mechanical robustness is underway for improved durability and vaginal efficacy in a SHIV challenge model.