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SARS‐CoV‐2 NSP12, the viral RNA‐dependent RNA polymerase (RdRp), is required for viral replication and is a therapeutic target to treat COVID‐19. To facilitate research on SARS‐CoV‐2 NSP12 protein, we developed a rat monoclonal antibody (CM12.1) against the NSP12 N‐terminus that can facilitate functional studies. Immunoblotting and immunofluorescence assay (IFA) confirmed the specific detection of NSP12 protein by this antibody for cells overexpressing the protein. Although NSP12 is generated from the ORF1ab polyprotein, IFA of human autopsy COVID‐19 lung samples revealed NSP12 expression in only a small fraction of lung cells including goblet, club‐like, vascular endothelial cells, and a range of immune cells, despite wide‐spread tissue expression of spike protein antigen. Similar studies using in vitro infection also generated scant protein detection in cells with established virus replication. These results suggest that NSP12 may have diminished steady‐state expression or extensive posttranslation modifications that limit antibody reactivity during SARS‐CoV‐2 replication. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease 2019 (COVID-19) patients manifest with pulmonary symptoms reflected by diffuse alveolar damage (DAD), excessive inflammation, and thromboembolism. The mechanisms mediating these processes remain unclear.Methods: We performed multicolor staining for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and lineage markers to define viral tropism and lung pathobiology in 5 autopsy cases.Results: Lung parenchyma showed severe DAD with thromboemboli. Viral infection was found in an extensive range of cells including pneumocyte type II, ciliated, goblet, club-like, and endothelial cells. More than 90% of infiltrating immune cells were positive for viral proteins including macrophages, monocytes, neutrophils, natural killer (NK) cells, B cells, and T cells. Most but not all infected cells were angiotensin-converting enzyme 2 (ACE2) positive. The numbers of infected and ACE2-positive cells are associated with extensive tissue damage. Infected tissues exhibited high levels of inflammatory cells including macrophages, monocytes, neutrophils, and NK cells, and low levels of B cells but abundant T cells consisting of mainly T helper cells, few cytotoxic T cells, and no regulatory T cells. Robust interleukin-6 expression was present in most cells, with or without infection.Conclusions: In fatal COVID-19 lungs, there are broad SARS-CoV-2 cell tropisms, extensive infiltrated innate immune cells, and activation and depletion of adaptive immune cells, contributing to severe tissue damage, thromboemboli, excess inflammation, and compromised immune responses. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM) is an aggressive tumour that can lead to lymphopaenia. Its standard treatment involves temozolomide (TMZ) chemotherapy with radiation, often with addition of corticosteroids for symptomatic management. Although TMZ is also immunosuppressive, patients receiving TMZ rarely develop disseminated opportunistic infections. Here, we report the case of a patient with GBM receiving TMZ, radiotherapy and corticosteroids, who develops an incidental new brain lesion that is found to be disseminated Aspergillus within a new GBM tumour site. The patient received successful early treatment of her central nervous system aspergillosis. This case illustrates the profound immunosuppressive potential of GBM in conjunction with TMZ and corticosteroids, which can lead to high-morbidity opportunistic infections concurrently with tumour progression. Future research is needed to elucidate GBM, TMZ and corticosteroids’ compound immune effects and guide management that strikes a balance between treating high-morbidity infections and continuing with immunosuppressive chemotherapy. [ABSTRACT FROM AUTHOR]

Context.—: The diagnosis of some infectious diseases requires their identification in tissue specimens. As institutions adopt digital pathology for primary diagnosis, the limits of microorganism detection from digital images must be delineated., Objective.—: To assess the reliability of microorganism detection from digitized images of histochemical and immunohistochemical stains commonly used in pathology., Design.—: Original glass slides from 620 surgical pathology cases evaluated for the presence of infectious microorganisms were digitized. Immunohistochemical stains included those for herpes simplex virus (n = 100), cytomegalovirus (n = 100), Helicobacter pylori (n = 100), and spirochetes (n = 80). Histochemical stains included mucicarmine for Cryptococcus spp (n = 20), Grocott methenamine silver for fungi (n = 100), Giemsa for H pylori (n = 100), and Ziehl-Neelsen for acid-fast bacilli (n = 20). The original diagnosis based on the glass slides was regarded as the reference standard. Six pathologists reviewed the digital images., Results.—: Digital review was generally associated with high (ie, ≥90%) specificity and positive predictive value owing to a low percentage of false positive reads, whereas a high percentage of false negatives contributed to low sensitivity and negative predictive value for many stains. Fleiss κ showed substantial interobserver agreement in the interpretation of Grocott methenamine silver and immunostains for herpes simplex virus, H pylori, and cytomegalovirus; moderate agreement for spirochete, Ziehl-Neelsen, and mucicarmine; and poor agreement for Giemsa., Conclusions.—: Digital immunohistochemistry generally outperforms histochemical stains for microorganism detection. Digital interpretation of Ziehl-Neelsen and mucicarmine stains is associated with low scores for interrater reliability, accuracy, sensitivity, and negative predictive value such that it should not substitute for conventional review of glass slides., Competing Interests: The authors have no relevant financial interest in the products or companies described in this article., (© 2024 College of American Pathologists.)