COVID-associated pernio: Mechanistic insights to an abortive, seronegative SARS-CoV-2 infection

Introduction: SARS-CoV-2 replicates primarily in the airways but generates a systemic immune response mediated by Type I interferons (IFN-I). Pernio is a rare skin manifestation of disorders characterized by excessive IFN-I signalling. Although pernio increased in incidence during the pandemic, the relationship to SARS-CoV-2 remains controversial. Because of the pivotal nature of interferons in COVID-19 outcomes, pernio offers a window to investigate the biology underlying host resiliency to SARS-CoV-2 infection. Method(s): To further assess COVID-associated pernio, we characterized clinical samples from affected patients across 4 waves of the pandemic and investigated mechanistic feasibility in a rodent model. Patients were followed longitudinally with banking of blood and tissue. Golden hamsters were mock-treated or intra-nasally infected with SARS-CoV-2 and harvested at 3-and 30-days post-infection. Result(s): In affected tissue, immunophenotyping utilizing multiplex immunohistochemistry profiled a robust IFN-1 signature characterized by plasmacytoid dendritic cell activation. Viral RNA was detectable in a subset of cases using in situ hybridization for the SARS-CoV-2 S gene transcript. Profiling of the systemic immune response did not reveal a durable type 1 interferon signature. Consistent with previous literature, antibody and T-cell specific responses to SARS-CoV-2 were not detected. Nasopharyngeal SARS-CoV-2 inoculation in hamsters resulted in rapid dissemination of viral RNA and the generation of an IFN-I response that were both detectable in the paws of infected animals. Conclusion(s): Our data support a durable local IFN signature, with direct evidence of viral SARS-CoV-2 RNA in acral skin and suggest that COVID-associated pernio results from an abortive, seronegative SARS-CoV-2 infection.

A Novel Method to Map Small RNAs with High Resolution.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

Viral enteritis in cattle: To well known viruses and beyond

Livestock products supply about 13 percent of energy and 28 percent of protein in diets consumed worldwide. Diarrhea is a leading cause of sickness and death of beef and dairy calves in their first month of life and also affecting adult cattle, resulting in large economic losses and a negative impact on animal welfare. Despite the usual multifactorial origin, viruses are generally involved, being among the most important causes of diarrhea. There are several viruses that have been confirmed as etiological agents (i.e., rotavirus and coronavirus), and some viruses that are not yet confirmed as etiological agents. This review summarizes the viruses that have been detected in the enteric tract of cattle and tries to deepen and gather knowledge about them.Copyright © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Pathology Playground: An Interactive Web-Based System for Pathology Unknowns With Immunohistochemistry and Molecular Studies

(Arch Pathol Lab Med. 2023;147:492-495;doi: 10.5858/ arpa.2021 -0516-EP) Interactive, computer-based learning has been used in numerous areas of medicine, and more often than not is more effective than traditional teaching methods.1,2 Patient simulations are even used in the United States Medical Licensing Examination to assess diagnostic decision-making skills, therapeutic intervention skills, and patient management skills.3 Computer-aided learning in pathology has been used in the forms of virtual slides linked to additional content,4 games in which correctly answering questions results in saving a patient or earning virtual prizes,5 and programs that aid the user in following an algorithmic approach to histologic diagnosis.6 Additionally, in the COVID-19 era, Web-based learning is proving more valuable in replacing standard formats.7 Working through a difficult case in pathology typically involves the use of immunohistochemistry and other special studies, including molecular diagnostics, that either confirm a suspected diagnosis or narrow the differential diagnosis and guide further tests. Ideally, a trainee would be able to see the H&E slide, choose what to order, refine the diagnosis, and perhaps order further studies, which would be closer to clinical practice. Images were obtained from in-house cases as well as online sources, primarily the University of Michigan Virtual Slide Box.8 An interface was written in HTML, PHP (PHP Hypertext Preprocessor), and JavaScript. About 50 cases have been presented in this manner in total, such as carcinoid tumor of the pancreas (with synaptophysin and chromogranin), blastic mantle cell lymphoma (with immunohistochemistry, flow cytometry, and fluorescence in situ hybridization [FISH] results), synovial sarcoma (with immunohistochemistry and cytogenetics results), hemochromatosis (with special stains and molecular genetic results confirming the patient's mutation), and histiocytic sarcoma (with immunostains).

Atypical presentation of covid-19 as acute leukemia: A case report

In the absence of respiratory system involvement, COVID-19 patients developing ARDS can clinically mimic other diseases including acute leukemia due to presence of atypical lymphocytes in peripheral blood smear and increased serum lactate dehydrogenase and serum uric acid levels. Herein, we report a case who was initially suspected to have acute leukemia based on his atypical symptoms without any respiratory system involvement and deranged laboratory parameters and finally, diagnosed with COVID-19. Our patient presented with fever, myalgia, gum bleed, and petechiae. On clinical and laboratory evaluation, he was suspected to have acute leukemia based on markedly deranged serum lactate dehydrogenase and serum uric acid and the presence of atypical cells in peripheral blood smear and bone marrow. On day 3 of hospitalization, he developed respiratory symptoms, breathing difficulty which progressed to ARDS, and subsequently, he succumbed to his illness. His real-time reverse transcriptase-polymerase chain reaction test for severe acute respiratory syndrome coronavirus-2 yielded positive results. Also, Flow cytometry and fluorescence in situ hybridization studies for leukemia workup did not show any abnormalities. Although we are reporting the findings of only a single case, we aim to enhance and contribute further to the understanding of this novel infection.Copyright © 2021 Necati Ozpinar. All right reserved.

The rapid diagnosis of Mycoplasma pneumonia using in situ hybridization on clinical samples.

The microbiological etiology of seasonal upper respiratory illnesses in the United States is dominated by viruses, including influenza A, B, respiratory syncytial virus, and SARS-CoV2. Mycoplasma pneumonia, treatable with antibiotics, can also cause upper respiratory symptoms and is typically associated with about 15 % of cases. There is no clinical or radiologic finding diagnostic of Mycoplasma pneumonia infection and PCR-based testing is not routinely used in the clinical setting. Further, the bacteria grows slowly in culture and the diagnostic IgM response will take days after the onset of infection. Thus, a rapid diagnostic test for Mycobacterium pneumonia infection is needed. This study documented two cases of Mycoplasma pneumonia infection of the upper respiratory system using in situ hybridization in a series of over 20 patients who were being tested for SARS-CoV2 infection. The respiratory secretions were placed on a glass slide, fixed in 10 % buffered formalin, and then tested using a Mycoplasma pneumonia probe. The high bacterial number associated with acute infection allowed for straightforward detection by in situ hybridization in a few hours. Antibiotic therapy led to rapid resolution of the symptoms. This highlights the ability of standard in situ hybridization as a rapid diagnostic test for Mycoplasma pneumonia in the clinical setting.

Liver pathology in COVID-19 related death and leading role of autopsy in the pandemic.

BACKGROUND: Information on liver involvement in patients with coronavirus disease 2019 is currently fragmented. AIM: To highlight the pathological changes found during the autopsy of severe acute respiratory syndrome coronavirus 2 positive patients. METHODS: A systematic literature search on PubMed was carried out until June 21, 2022. RESULTS: A literature review reveals that pre-existing liver disease and elevation of liver enzyme in these patients are not common; liver enzyme elevations tend to be seen in those in critical conditions. Despite the poor expression of viral receptors in the liver, it seems that the virus is able to infect this organ and therefore cause liver damage. Unfortunately, to date, the search for the virus inside the liver is not frequent (16% of the cases) and only a small number show the presence of the virus. In most of the autopsy cases, macroscopic assessment is lacking, while microscopic evaluation of livers has revealed the frequent presence of congestion (42.7%) and steatosis (41.6%). Less frequent is the finding of hepatic inflammation or necrosis (19%) and portal inflammation (18%). The presence of microthrombi, frequently found in the lungs, is infrequent in the liver, with only 12% of cases presenting thrombotic formations within the vascular tree. CONCLUSION: To date, the greatest problem in interpreting these modifications remains the association of the damage with the direct action of the virus, rather than with the inflammation or alterations induced by hypoxia and hypovolemia in patients undergoing oxygen therapy and decompensated patients.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Development of a high-sensitivity and short-duration fluorescence in situ hybridization method for viral mRNA detection in HEK 293T cells.

Coronavirus disease 2019 (COVID-19) is an extremely contagious illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Early disease recognition of COVID-19 is crucial not only for prompt diagnosis and treatment of the patients, but also for effective public health surveillance and response. The reverse transcription-polymerase chain reaction (RT-PCR) is the most common method for the detection of SARS-CoV-2 viral mRNA and is regarded as the gold standard test for COVID-19. However, this test and those for antibodies (IgM and IgG) and antigens have certain limitations (e.g., by yielding false-negative and false-positive results). We have developed an RNA fluorescence in situ hybridization (FISH) method for high-sensitivity detection of SARS-CoV-2 mRNAs in HEK 293T cell cultures as a model. After transfection of HEK 293T cells with plasmids, Spike (S)/envelope (E) proteins and their mRNAs were clearly detected inside the cells. In addition, hybridization time could be reduced to 2 hours for faster detection when probe concentration was increased. Our approach might thus significantly improve the sensitivity and specificity of SARS-CoV-2 detection and be widely applied for the high-sensitivity single-molecular detection of other RNA viruses (e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Hepatitis A virus, all influenza viruses, and human immunodeficiency virus (HIV)) in various types of samples including tissue, body fluid, blood, and water. RNA FISH can also be utilized for the detection of DNA viruses (e.g., Monkeypox virus, human papillomavirus (HPV), and cytomegalovirus (CMV)) by detection of their mRNAs inside cells or body fluid.

Successful Kidney Transplantation from a Deceased Donor with Severe COVID-19: Undetectable SARS-CoV-2 in Donor Kidney and Aorta by Sensitive Molecular Testing

Purpose: Kidney transplantation (KT) from coronavirus disease 2019 (COVID-19) positive donors has been avoided due to concerns for donor-derived transmission and possibility of the kidney being a viral reservoir. There is no long-term safety data, and sensitive molecular testing for SARS-CoV-2 in donor kidney is not routinely performed. We report a case of successful KT from a deceased donor who died from severe COVID-19 respiratory illness whose donor kidney and aorta were probed for virus using in situ hybridization (ISH) and quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). Method(s): A 30-year-old female was admitted to the hospital with severe COVID-19 pneumonia with a positive RT-PCR test for SARS-CoV-2 on nasopharyngeal swab. With clinical worsening, she was placed on extracorporeal membrane oxygenation, but developed hypoxic brain injury and progressed to brain death. Renal function was stable during her hospital course with serum creatinine concentration of 0.7 mg/dL. SARS-CoV-2 RT-PCR on bronchoalveolar lavage and nasopharyngeal samples tested again three days prior to donation was negative. A 55-year-old male recipient with an end-stage renal disease secondary to hypertension was transplanted with the left kidney from the above donor. The donor kidney was studied using pre-implantation surgical biopsy tissues to investigate the presence of SARS-CoV-2 RNA. Aorta tissue with the kidney was also studied given high expression of angiotensin-converting enzyme 2 receptors in vasculature. Result(s): ISH analyses did not show any positive signal for SARS-CoV-2 RNA in the donor kidney sample compared to a SARS-CoV-2 positive lung control. All samples tested by qRT-PCR were also negative for SARS-CoV-2. We found no evidence of SARS-CoV-2 mRNA in the donor kidney and aorta. The recipient has been free of COVID-19 related signs or symptoms and tested negative for SARSCoV- 2 by nasopharyngeal swab RT-PCR on days 20, 30, and 90 following KT. After an initial period of delayed graft function requiring hemodialysis, the recipient now has excellent renal recovery over 6 months following the transplant, and the most recent creatinine is 1.3 mg/dL. Conclusion(s): Taken together with recent observations of successful KT outcomes from mild or asymptomatic COVID-19 donors, we believe that the transmission risk of SARS-CoV-2 through KT is likely to be very low. Use of deceased donors who died after severe COVID-19 can be considered for KT. Larger scale studies are needed to confirm our findings.

Early Clinical Experience with ALVR109, a Partially HLA-Matched SARS-CoV-2 Specific T-Cell Therapy, in Immunocompromised Patients with COVID-19

Purpose: Therapies for COVID-19 in immunocompromised (IC) patients (pts), including transplant (tx) pts, are limited. We describe our experience with ALVR109, an allogeneic, partially HLA-matched T-cell product, given through emergency investigational new drug (eIND) application to 4 consecutive IC pts with protracted COVID-19. Method(s): To measure SARS-CoV-2 viral loads, SARS-2 RNA was quantified by RT-PCR (N gene) in plasma and saliva. ALVR109 was manufactured for Allovir at Baylor College of Medicine. Result(s): Between May and October 2021, ALVR109 was given to 4 IC pts with COVID-19 (details in Table 1). 2 pts had lymphoma (1 post auto-tx) and 2 had lung tx. All pts had SARS-CoV-2 RNA detected in plasma (viremia) in the weeks leading up to ALVR109 administration. Infusions (20-40 million cells (MC) per dose) were well-tolerated with no adverse events. Prior to ALVR109, pts 1 and 3 had progressive COVID-19 and ongoing SARS-CoV-2 viremia despite monoclonal antibodies (mABs) and remdesivir. Following ALVR109 administration both patients had a decrease in viremia with marked clinical improvement in pt 1, but both eventually died from their underlying disease. Viral loads (plasma/saliva) and functional scores for pt 1 are shown in the figure. Autopsy of pt 3 showed no evidence of SARS-CoV-2 infection by lung in-situ hybridization (ISH). Pts 2 and 4 received ALVR109 as adjunctive therapy to mABs and remdesivir;viremia continued to decline following ALVR109 and both pts survived and were discharged home. Conclusion(s): This initial experience suggests a potential role of ALVR109 in the treatment of IC and tx pts with COVID-19. SARS-CoV-2-specific T-cells appear to be safe and may control viremia in IC pts. Larger studies are needed to confirm this observation, define the best candidates for ALVR109, and determine optimal timing of administration. (Table Presented).

SARS-CoV-2 infection causes cardiomyocyte swelling, cardiac pericyte loss, increased permeability, and diastolic dysfunction in a hamster model

Background/Introduction: Recovered COVID-19 patients often display cardiac dysfunction, even after a relatively mild infection. Purpose: We present an in-depth physiological and histological timeline of the cardiac consequences of SARS-CoV-2 infection using a hamster model. Methods: We used several methods, including transthoracic echocardiography, RNA sequencing on in vitro cultures, and in-situ hybridization techniques, complemented with histological analysis. Results: We analysed cardiac function by echocardiography over a period of 35 dpi. Already by 14 dpi and continuing at 35 dpi, infected hamsters presented with an increased E/E', decreased MV deceleration time, and an increased isovolumetric contraction time as compared to control, indicating the presence of diastolic dysfunction. Histologically, cardiomyocytes were enlarged already by 4 dpi and remained enlarged over 5 weeks. We observed the presence of fibrin-rich microthrombi at 4 dpi, which were resolved by 14 dpi. SARS-CoV-2 RNA was present in cardiac pericytes, accompanied by reduced pericyte coverage of capillaries at 4 dpi and 14 dpi, which mostly recovered by 35 dpi. At 14 dpi, the reduced pericyte coverage coincided with increased vascular permeability, suggesting that SARS-CoV-2 infection of pericytes affects microvascular integrity. SARS-CoV-2 infection of pericytes in vitro induced the expression of genes involved in viral defence, and affected genes involved in pericyte contractility and extracellular matrix proteins. Loss of cardiac pericytes was observed in cardiac biopsies from patients recovered from SARSCoV- 2 infection. Conclusion(s): Overall, our results demonstrate that SARS-CoV-2 infection causes a phenotype similar to ischemia-reperfusion, without overt ischemia. We propose that partial occlusion by microthrombi and microvascular dilation caused by pericyte loss induces regional variations in blood flow, and results in a stiffer ;swollen' heart that shows diastolic dysfunction.

Tissue-specific evidence for SARS-CoV-2 within intraocular tissues

Purpose : To systematically investigate ocular changes in autopsied eyes from fatal cases of Coronavirus disease 2019 (COVID-19) and to investigate the localization of severe acute respiratory syndrome coronavirus (SARS-CoV-2) within ocular structures. Methods : Macroscopic and microscopic histopathological evaluation was performed and the localization of SARS-CoV-2 RNA within ocular tissues investigated using an in situ hybridization (ISH) technique in 13 eyes. Contralateral eyes were freshly dissected, and droplet digital polymerase chain reaction (ddPCR) assay was performed on ocular fluids and tissues to quantify SARS-CoV-2 RNA. Results : A total of 21 fatal COVID-19 cases were included (mean age, 60.2 years [range, 27- 91 years];23.8% female). Histopathological abnormalities include vascular changes (61.9%), cytoid bodies (52.4%), and retinal edema (23.8%) with minimal inflammation (0.09%) were observed. Non-CMV viral inclusions were identified in one eye. No CMV positivity was detected. Of the 21 contralateral eyes tested by ddPCR, 14 tested positive for SARS-CoV-2. Using ddPCR and ISH, SARS-CoV-2 localization was observed in the following ocular tissues and fluid: cornea (27.3%), aqueous (26.3%), lens (54.5%), vitreous (15.0%), retina (22.2%), choroid/sclera (47.4%), and optic nerve (50.0%). The choroid/sclera, optic nerve and lens were the most frequent ocular structures found to be ddPCR positive. Evidence of replication was detected in four cases. Conclusions : Our results suggest that SARS-CoV-2 localizes to intraocular tissues. However, histological changes observed are likely a secondary hemodynamic change rather than primary effect of the virus.

Haematological Malignancy Presenting in an Unusual Manner

Chronic lymphocytic leukaemia is a haematological malignancy that occurs due to an increased proliferation of mature B lymphocytes. It is considered to be the most common leukaemia in adults. Hyponatremia is commonly seen in such patients. This case report is about a 75-year-old male, who presented with giddiness, followed by altered sensorium. However, the patient had no motor weakness or sensory loss. Initially, a diagnosis of posterior circulation stroke was made but Magnetic Resonance Imaging (MRI) brain did not show associated signs. The routine investigations showed highly elevated total leukocyte count and hyponatremia. The patient was worked up for malignancy and diagnosed with Chronic lymphocytic leukaemia. Oncology reference was taken and treated with tablet Ibrutinib. On discharge, the patient's mentation improved, and he is on regular follow-up.

Relationship between Uveal Inflammation and Viral Detection in 30 Cats with Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) virus is the most common infectious cause of uveitis in cats. Confirmatory diagnosis is usually only reached at postmortem examination. The relationship between the histologic inflammatory pattern, which depends on the stage of the disease, and the likelihood of detection of the viral antigen and/or RNA has not been investigated. We hypothesized that viral detection rate by either immunohistochemistry, in situ hybridization or RT-qPCR is dependent upon the predominant type of uveal inflammatory response (i.e., pyogranulomatous vs. plasmacytic). Thus, the aims of this study were to evaluate cases of FIP-induced uveitis, localize the viral antigen and RNA, and assess the relationship between the inflammatory pattern (macrophage- vs. plasma cell-rich) and the likelihood of detecting the FIP antigen and/or RNA. We evaluated 30 cats with FIP-induced uveitis. The viral antigen and/or RNA were detected within uveal macrophages in 11/30 cases, of which 8 tested positive by RT-qPCR. Correlation analysis determined a weak to moderate but significant negative correlation between the degree of plasmacytic uveal inflammation and the likelihood of detecting the FIP antigen and RNA. This study suggests that predominance of plasmacytic inflammation in cases of FIP uveitis reduces the odds of a confirmatory diagnosis through the viral detection methods available.

CLIPPERS and lymphoma: a unified syndrome

Background and aims: Chronic Lymphocytic Inflammation with Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS) is a defined inflammatory central nervous system disorder characterized by established lesions, predominantly on the cerebellum and pons, with punctate gadolinium enhancement on the MRI and responsiveness to glucocorticosteroid-based immunosuppression. Its pathogenesis is unknown. Methods: A 54-year-old man, with history of obesity and alcoholism, was admitted to our emergency ward due to fever, disorientation, and behavior disorder for 3 days. The neurological examination manifested left eye ptosis and unceasing left gaze torsional nystagmus. He tested positive for SARS-CoV2. A lumbar puncture revealed 41 leucocytes/ mm3, with negative antibody determinations. MRI depicted hyperintense lesions on cerebellum, brain stem, left temporal lobe and right parieto-occipital cortex, with punctate gadolinium enhancement. There was a radiological and clinical response to glucocorticosteroid treatment. He was diagnosed of possible CLIPPERS. Results: Several months following the discharge, after the patient voluntarily abandoned the treatment, the symptoms reappeared. A new MRI showed extensive lesions suitable with primary lymphoma. A biopsy confirmed the diagnosis. In situ hybridization detected Epstein-Barr virus (EBV). Conclusion: Reports describing the development of lymphoma on patients previously diagnosed with CLIPPERS, and the fact that both conditions share anatomopathological findings such as infiltrating CD4+ lymphocytes, support the theory of a syndrome with various prestages encompassing both entities. Furthermore, a nexus between B-cell lymphoma and EBV has already been established, but recent studies dwell on the possibility of this same infection being one of the main triggers of CLIPPERS and its progression into lymphoma.

Primary analysis of MOUNTAINEER: A phase 2 study of tucatinib and trastuzumab for HER2-positive mCRC

Background: Despite the occurrence of HER2 amplification/overexpression (HER2+) in ~3% to 5% of all patients with metastatic colorectal cancer (mCRC) and up to ~10% of patients with RAS/BRAF wild-type mCRC, there are currently no FDA- or EMA-approved HER2-directed therapies for HER2+ mCRC. Patients with mCRC who progress on early lines of chemotherapy regimens receive limited clinical benefit from current standard-of-care treatments. Tucatinib is a highly selective, HER2-directed, tyrosine kinase inhibitor. The MOUNTAINEER trial (NCT03043313) was initiated to evaluate the efficacy and safety of the investigational combination of tucatinib with trastuzumab in patients with HER2+ mCRC. Here we present results from the primary analysis of MOUNTAINEER. Methods: MOUNTAINEER is a multi-center, open-label, randomised, phase 2 trial conducted in the US and Europe. Eligible patients had HER2+ (one or more local tests: 3+ immunohistochemistry, 2+ immunohistochemistry with amplification by in situ hybridization, or amplification by next‑generation sequencing of tumor tissue) and RAS wild-type mCRC with progression on or intolerance to fluoropyrimidine, oxaliplatin, irinotecan, and an anti-VEGF antibody. Measurable disease and an ECOG performance status of 0–2 were required. Previous HER2-directed therapies were not permitted. The trial initially consisted of a single cohort (Cohort A) to be treated with tucatinib (300 mg PO BID) and trastuzumab (8 mg/kg IV then 6 mg/kg IV every 3 weeks). The trial was expanded to include patients randomised 4:3 to receive tucatinib + trastuzumab (Cohort B) or tucatinib monotherapy (Cohort C). The primary endpoint is confirmed objective response rate (ORR) per RECIST 1.1 by blinded independent central review (BICR) in Cohorts A+B. Secondary endpoints include duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety and tolerability. Results: MOUNTAINEER enrolled 117 patients between 08Aug2017 and 22Sept2021. Data cutoff was 28Mar2022. The median age was 56.0 years (range, 24, 77), and baseline characteristics were balanced across cohorts. Eighty-six patients received at least 1 dose of study treatment in Cohorts A+B, and 30 patients received tucatinib monotherapy in Cohort C (total, 116). The overall median duration of follow-up was 16.3 months (IQR, 10.8, 28.2). In Cohorts A+B, the confirmed ORR by BICR was 38.1% (95% CI, 27.7, 49.3). The median DOR was 12.4 months (95% CI, 8.5, 20.5). The median PFS was 8.2 months (95% CI, 4.2, 10.3), and the median OS was 24.1 months (95% CI, 20.3, 36.7). The most common adverse events (AEs) in Cohorts A+B were diarrhoea (64.0%), fatigue (44.2%), nausea (34.9%), and infusion-related reaction (20.9%);the most common AE of grade ≥3 was hypertension (7.0%). Adverse events leading to tucatinib discontinuation in Cohorts A+B occurred in 5.8% of patients and included alanine amino transferase increase (2.3%), COVID-19 pneumonia (1.2%), cholangitis (1.2%), and fatigue (1.2%). No deaths resulted from AEs. Conclusions: In patients with chemotherapy-refractory HER2+ mCRC, tucatinib in combination with trastuzumab was well tolerated with clinically meaningful antitumor activity including durable responses and a median overall survival of 2 years. Tucatinib in combination with trastuzumab has the potential to become a new standard of care for patients with HER2+ mCRC. Clinical trial identification: NCT03043313. Editorial acknowledgement: The authors thank Joseph Giaconia of MMS Holdings, Michigan, USA for providing medical writing support/editorial support, which was funded by Seagen Inc., Bothell, WA, USA in accordance with Good Publication Practice (GPP3) guidelines. Legal entity responsible for the study: Seagen Inc. Funding: Seagen Inc. Disclosures: J. Strickler: Advisory / Consultancy: Seagen, Bayer, Pfizer;Research grant / Funding (institution): Amgen, Roche/Genentech, Seagen. A. Cercek: Advisory / Consultancy: Bayer, Merck, Seagen;Research grant / Funding (institution): Seagen, GSK, Rgenix. T. André: Honoraria (self : Amgen, Astra-Zeneca, Bristol-Myers Squibb, Gritstone Oncology, GlaxoSmithKline, Haliodx, Kaleido Biosciences, Merck & Co., Inc., Pierre Fabre, Sanofi, Servier, Merck & Co., Inc, Servier;Advisory / Consultancy: Astellas Pharma, BMS, Gritstone Oncology, Transgène, Roche/Ventana, Seagen, Merck & Co., Inc, Servier;Research grant / Funding (institution): BMS, Seagen, GSK;Travel / Accommodation / Expenses: BMS, Merck & Co., Inc. K. Ng: Advisory / Consultancy: Seattle Genetics, Bicara Therapeutics, GlaxoSmithKline;Research grant / Funding (institution): Pharmavite, Evergrande Group, Janssen. E. Van Cutsem: Advisory / Consultancy: AbbVie, Array, Astellas, AstraZeneca, Bayer, Beigene, Biocartis, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Daiichi, Halozyme, GSK, Helsinn, Incyte, Ipsen, Janssen Research, Lilly, Merck Sharp & Dohme, Merck KGaA, Mirati, Novartis, Pierre Fabre, Roche, Seattle Genetics, Servier, Sirtex, Terumo, Taiho, TRIGR, Zymeworks;Research grant / Funding (institution): Amgen, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Ipsen, Lilly, Merck Sharp & Dohme, Merck KGaA, Novartis, Roche, Servier. C. Wu: Research grant / Funding (institution): Seagen. A. Paulson: Research grant / Funding (institution): Seattle Genetics. J. Hubbard: Research grant / Funding (institution): Seattle Genetics. H. Lenz: Honoraria (self): BMS, Bayer, Roche;Advisory / Consultancy: Bayer, Merck, Roche;Travel / Accommodation / Expenses: BMS, Bayer, Merck KG;Shareholder / Stockholder / Stock options: Fulgent. M. Stecher: Full / Part-time employment: SeaGen. W. Feng: Full / Part-time employment: Seagen. T. Bekaii-Saab: Honoraria (self): Royalties: Uptodate;Advisory / Consultancy: Consulting (to institution): Ipsen, Arcus, Pfizer, Seattle Genetics, Bayer, Genentech, Incyte, Eisai and Merck., Consulting (to self): Stemline, AbbVie, Boehringer Ingelheim, Janssen, Daichii Sankyo, Natera, TreosBio, Celularity, Exact Science, Sobi, Beigene, Kanaph, Astra Zeneca, Deciphera, MJH Life Sciences, Aptitude Health, Illumina and Foundation Medicine, IDMC/DSMB: Fibrogen, Suzhou Kintor, Astra Zeneca, Exelixis, Merck/Eisai, PanCan and 1Globe;Research grant / Funding (institution): Agios, Arys, Arcus, Atreca, Boston Biomedical, Bayer, Eisai, Celgene, Lilly, Ipsen, Clovis, Seattle Genetics, Genentech, Novartis, Mirati, Merus, Abgenomics, Incyte, Pfizer, BMS.;Licensing / Royalties: WO/2018/183488: HUMAN PD1 PEPTIDE VACCINES AND USES THEREOF – Licensed to Imugene, WO/2019/055687: METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER CACHEXIA – Licensed to Recursion. All other authors have declared no conflicts of interest.

SPECIFIC DETECTION and REPLICATION KINETICS of SARS-CoV-2 USING smRNA-FISH

Background: SARS-CoV-2 is a positive-sense single-stranded RNA virus and its replication begins after the synthesis of virally encoded polymerase complex that is required for replication and transcription of genomic RNA (gRNA) within the infected cells. Despite the global interest in the study of SARS-CoV-2, the kinetics of SARS-CoV2 RNA replication and transcription during the early phase of viral infection is poorly understood. Here, we used the single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) for sensitive detection of SARS-CoV-2 at single molecule level and to determine the replication of genomic RNA (gRNA) and sub-genomic RNA (sgRNA) in the infected cells, at very early stages of infection. Methods: We designed highly specific smRNA-FISH probes targeted to gRNA and Spike gene sgRNA of SARS-CoV-2 virus, using stellaris method and optimized the method to simultaneously visualize these two RNAs at single cell and single molecule level. Because of the high sensitivity of our probes, we applied smRNA-FISH technology to detect SARS-CoV-2 positive cells from autopsy samples obtained from diseased COVID-19 patients. Furthermore, we used high-resolution and high-speed scanning microscopy to detect extent of infection in cell models of SARS-CoV-2 and in COVID-19 patient samples. Results: A time course analysis SARS-CoV-2 replication indicated that single molecules of gRNA could be detected as little as 30 min to 2 hr. post-infection. Distinct "Replication Centers" (RC) began to appear one to two hours post-infection and the sgRNAs began to migrate out of these RCs. Replication after the initial delay appeared to be rapid and gRNA and sgRNAs dispersed throughout the cell within 4-5 hours post infection forming multiple RCs. We found that our RNA-FISH correctly detected the SARS-CoV-2 positive samples from patient autopsy samples that were characterized by qRT-PCR or immunological detection methods. The signals of spike gRNA and sgRNA along with the spike proteins co-localized within the same cells of the SARS-CoV-2 infected patients within the cells of lung, kidney, and heart autopsy samples. Conclusion: We propose that the specific probes and the methodology that we have developed will be highly applicable to the study of SARS-CoV-2 replication in depth and to characterize SARS-CoV-2 infection in COVID-19 patient samples. This study may open a novel direction towards COVID-19 pathophysiology, drug screening and diagnostics.

RESTRICTED INFECTION of MACROPHAGES by SARS-CoV-2 INDUCES PROINFLAMMATORY RESPONSES

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.

Fluorescence In Situ Hybridization (FISH) Tests for Identifying Protozoan and Bacterial Pathogens in Infectious Diseases.

Diagnosing and treating many infectious diseases depends on correctly identifying the causative pathogen. Characterization of pathogen-specific nucleic acid sequences by PCR is the most sensitive and specific method available for this purpose, although it is restricted to laboratories that have the necessary infrastructure and finance. Microscopy, rapid immunochromatographic tests for antigens, and immunoassays for detecting pathogen-specific antibodies are alternative and useful diagnostic methods with different advantages and disadvantages. Detection of ribosomal RNA molecules in the cytoplasm of bacterial and protozoan pathogens by fluorescence in-situ hybridization (FISH) using sequence-specific fluorescently labelled DNA probes, is cheaper than PCR and requires minimal equipment and infrastructure. A LED light source attached to most laboratory light microscopes can be used in place of a fluorescence microscope with a UV lamp for FISH. A FISH test hybridization can be completed in 30 min at 37 °C and the whole test in less than two hours. FISH tests can therefore be rapidly performed in both well-equipped and poorly-resourced laboratories. Highly sensitive and specific FISH tests for identifying many bacterial and protozoan pathogens that cause disease in humans, livestock and pets are reviewed, with particular reference to parasites causing malaria and babesiosis, and mycobacteria responsible for tuberculosis.