The Pandemic as a Catalyst for Digital Pathology Adoption as an LDT

Whole-slide images (WSI) are the basis for the application of artificial intelligence/machine learning and other informatics methods to histological diagnosis and will further blur the line separating anatomic and clinical pathology. FDA classified WSI systems for primary diagnosis as class III (highest risk) medical devices until 2017. This discouraged anatomic pathology laboratories at risk-averse domestic institutions like mine from investing in these digital pathology (DP) platforms. In 2017, FDA downgraded WSI to class II (moderate risk) when they de-novo approved a system marketed by Philips. We were not interested in that system at my institution, but the downgrade caused us to reset our perception of the risk of validating a RUO system for primary diagnosis. Cost remained a barrier. In April 2020, FDA issued temporary guidance stating they would not enforce premarket approval of WSI systems to facilitate pathologists working remotely during the SARS-CoV-2 pandemic. The guidance included a statement that "laboratories and hospitals consider performing a validation study.” In January 2021, FDA proposed making the temporary non-enforcement guidance permanent. So, in a little more than three years, WSI for primary diagnosis had gone from class III to exempted from pre-market approval! This nicely aligned the approval framework for WSI with the approval framework for our conventional optical microscopes, which are statutorily exempted from approval, and further reset our perception of risk. In April 2021, FDA withdrew the proposal to make non-enforcement permanent, but the temporary non-enforcement guidance is still in effect at the time of writing. Amid all this FDA activity, the College of American Pathologists updated and reissued their consensus guidelines for validating WSI systems for diagnostic purposes in March 2021. The narrative mentions the FDA's recent approval of a few WSI systems and anticipates more, but the expert panel recommendations do not include any related to the approval status of systems. The reissue of this document reminded us that, as clinical laboratorians, we are capable of safely validating WSI as a laboratory-developed test and are supported in doing so by consensus guidelines from one of our leading professional organizations. In early 2021 we committed to funding a DP initiative to make WSI part of our routine histological process for 10% of our anatomic pathology cases. The initial capital investment is $1.5M. When realized, the microscope slides for designated pathology services will be transported directly from the cover slipper to a slide scanner and electronically distributed to pathologists using a clinical-grade image management system that we share with our radiology department. We made the decision to fund this in the context of the regulatory (decreased perception of risk), sociological (demand for remote telepathology), and technological (availability of scalable WSI systems) changes that occurred during the pandemic.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination

Most existing studies characterising SARS-CoV-2-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of ACE-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations. Graphical Yin et al. utilize two informative systems for evaluating overall T cell responses to SARS-CoV-2 and variants, enabling greater understanding of T cell responses to the virus, cross-reactivity to viral variants and the differences between vaccine- and infection-induced immunity to SARS-CoV-2, and other emerging viruses in the future.

Manipulation of innate immune signaling pathways by SARS-CoV-2 non-structural proteins.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic, induces an unbalanced immune response in the host. For instance, the production of type I interferon (IFN) and the response to it, which act as a front-line defense against virus invasion, are inhibited during SARS-CoV-2 infection. In addition, tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, is upregulated in COVID-19 patients with severe symptoms. Studies on the closely related betacoronavirus, SARS-CoV, showed that viral proteins such as Nsp1, Orf6 and nucleocapsid protein inhibit IFN-ß production and responses at multiple steps. Given the conservation of these proteins between SARS-CoV and SARS-CoV-2, it is not surprising that SARS-CoV-2 deploys similar immune evasion strategies. Here, we carried out a screen to examine the role of individual SARS-CoV-2 proteins in regulating innate immune signaling, such as the activation of transcription factors IRF3 and NF-κB and the response to type I and type II IFN. In addition to established roles of SARS-CoV-2 proteins, we report that SARS-CoV-2 proteins Nsp6 and Orf8 inhibit the type I IFN response but at different stages. Orf6 blocks the translocation of STAT1 and STAT2 into the nucleus, whereas ORF8 inhibits the pathway in the nucleus after STAT1/2 translocation. SARS-CoV-2 Orf6 also suppresses IRF3 activation and TNF-α-induced NF-κB activation.

Manipulation of innate immune signaling pathways by SARS-CoV-2 non-structural proteins

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic, induces an unbalanced immune response in the host. For instance, the production of type I interferon (IFN) and the response to it, which act as a front-line defense against virus invasion, are inhibited during SARS-CoV-2 infection. In addition, tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, is upregulated in COVID-19 patients with severe symptoms. Studies on the closely related betacoronavirus, SARS-CoV, showed that viral proteins such as Nsp1, Orf6 and nucleocapsid protein inhibit IFN-β production and responses at multiple steps. Given the conservation of these proteins between SARS-CoV and SARS-CoV-2, it is not surprising that SARS-CoV-2 deploys similar immune evasion strategies. Here, we carried out a screen to examine the role of individual SARS-CoV-2 proteins in regulating innate immune signaling, such as the activation of transcription factors IRF3 and NF-κB and the response to type I and type II IFN. In addition to established roles of SARS-CoV-2 proteins, we report that SARS-CoV-2 proteins Nsp6 and Orf8 inhibit the type I IFN response but at different stages. Orf6 blocks the translocation of STAT1 and STAT2 into the nucleus, whereas ORF8 inhibits the pathway in the nucleus after STAT1/2 translocation. SARS-CoV-2 Orf6 also suppresses IRF3 activation and TNF-α-induced NF-κB activation.

Poxviruses and paramyxoviruses use a conserved mechanism of STAT1 antagonism to inhibit interferon signaling.

The induction of interferon (IFN)-stimulated genes by STATs is a critical host defense mechanism against virus infection. Here, we report that a highly expressed poxvirus protein, 018, inhibits IFN-induced signaling by binding to the SH2 domain of STAT1, thereby preventing the association of STAT1 with an activated IFN receptor. Despite encoding other inhibitors of IFN-induced signaling, a poxvirus mutant lacking 018 was attenuated in mice. The 2.0 Å crystal structure of the 018:STAT1 complex reveals a phosphotyrosine-independent mode of 018 binding to the SH2 domain of STAT1. Moreover, the STAT1-binding motif of 018 shows similarity to the STAT1-binding proteins from Nipah virus, which, similar to 018, block the association of STAT1 with an IFN receptor. Overall, these results uncover a conserved mechanism of STAT1 antagonism that is employed independently by distinct virus families.

Is it COVID-19? The value of medicolegal autopsies during the first year of the COVID-19 pandemic.

OBJECTIVES: We describe the experience of a busy metropolitan medical examiner's office in the United States and share our navigation of the COVID-19 autopsy decision-making process. We describe key gross and microscopic findings that, with appropriate laboratory testing, should direct a pathologist towards a COVID-19-related cause of death. MATERIAL AND METHODS: We performed a retrospective review of 258 suspected and/or confirmed COVID-19 associated deaths that occurred between March 5, 2020, and March 4, 2021. RESULTS: A total of 62 cases due to fatal COVID-19 were identified; autopsy findings included diffuse alveolar damage, acute bronchopneumonia and lobar pneumonia, and pulmonary thromboemboli. Nine additional decedents had a nasopharyngeal swab positive for SARS-CoV-2 and a cause of death unrelated to COVID-19. Forty-seven cases with COVID-19-like symptoms showed no laboratory or histopathologic evidence of SARS-CoV-2 infection; the most common causes of death in this group were hypertensive or atherosclerotic cardiovascular disease, complications of chronic alcoholism, and pulmonary thromboemboli unrelated to infection. CONCLUSIONS: The clinical findings associated with COVID-19 are not specific; a broad differential diagnosis should be embraced when decedents present with cough or shortness of breath. An autopsy may be indicated to identify a cause of death unrelated to COVID-19.

The Impact of Inpatient Telemedicine on Personal Protective Equipment Savings During the COVID-19 Pandemic: Cross-sectional Study.

With the emergence of the COVID-19 pandemic and shortage of adequate personal protective equipment (PPE), hospitals implemented inpatient telemedicine measures to ensure operational readiness and a safe working environment for clinicians. The utility and sustainability of inpatient telemedicine initiatives need to be evaluated as the number of COVID-19 inpatients is expected to continue declining. In this viewpoint, we describe the use of a rapidly deployed inpatient telemedicine workflow at a large academic medical center and discuss the potential impact on PPE savings. In early 2020, videoconferencing software was installed on patient bedside iPads at two academic medical center teaching hospitals. An internal website allowed providers to initiate video calls with patients in any patient room with an activated iPad, including both COVID-19 and non-COVID-19 patients. Patients were encouraged to use telemedicine technology to connect with loved ones via native apps or videoconferencing software. We evaluated the use of telemedicine technology on patients' bedside iPads by monitoring traffic to the internal website. Between May 2020 and March 2021, there were a total of 1240 active users of the Video Visits website (mean 112.7, SD 49.0 connection events per month). Of these, 133 (10.7%) connections were made. Patients initiated 63 (47.4%) video calls with family or friends and sent 37 (27.8%) emails with videoconference connection instructions. Providers initiated a total of 33 (24.8%) video calls with the majority of calls initiated in August (n=22, 67%). There was a low level of adoption of inpatient telemedicine capability by providers and patients. With sufficient availability of PPE, inpatient providers did not find a frequent need to use the bedside telemedicine technology, despite a high census of patients with COVID-19. Compared to providers, patients used videoconferencing capabilities more frequently in September and October 2020. We did not find savings of PPE associated with the use of inpatient telemedicine.

Rapid Generation of Neutralizing Antibody Responses in COVID-19 Patients.

SARS-CoV-2, the virus responsible for COVID-19, is causing a devastating worldwide pandemic, and there is a pressing need to understand the development, specificity, and neutralizing potency of humoral immune responses during acute infection. We report a cross-sectional study of antibody responses to the receptor-binding domain (RBD) of the spike protein and virus neutralization activity in a cohort of 44 hospitalized COVID-19 patients. RBD-specific IgG responses are detectable in all patients 6 days after PCR confirmation. Isotype switching to IgG occurs rapidly, primarily to IgG1 and IgG3. Using a clinical SARS-CoV-2 isolate, neutralizing antibody titers are detectable in all patients by 6 days after PCR confirmation and correlate with RBD-specific binding IgG titers. The RBD-specific binding data were further validated in a clinical setting with 231 PCR-confirmed COVID-19 patient samples. These findings have implications for understanding protective immunity against SARS-CoV-2, therapeutic use of immune plasma, and development of much-needed vaccines.