key: cord-0778530-fssuqqlu
authors: Fiel, M. Isabel; El Jamal, Siraj M.; Paniz-Mondolfi, Alberto; Gordon, Ronald E.; Reidy, Jason; Bandovic, Jela; Advani, Rashmi; Kilaru, Saikiran; Pourmand, Kamron; Ward, Stephen; Thung, Swan N.; Schiano, Thomas
title: Findings of Severe Hepatic SARS-CoV-2 Infection
date: 2020-09-28
journal: Cell Mol Gastroenterol Hepatol
DOI: 10.1016/j.jcmgh.2020.09.015
sha: 493c35b8ec59048ef324a1d3c620ca3a92acb22b
doc_id: 778530
cord_uid: fssuqqlu

Background and Aims Liver injury due to COVID-19 is being increasingly recognized. Abnormal liver chemistry tests of varying severities occur in a majority of patients. However, there is a dearth of accompanying liver histologic studies in these patients. Methods: The current report details the clinical courses of two patients having severe COVID-19 hepatitis. Liver biopsies were analyzed under light microscopy, portions of liver tissue were hybridized with a target probe to the SARS-CoV-2 S gene, and small sections from formalin-fixed paraffin embedded liver tissue were processed for electron microscopy. Results The liver histology of both cases showed a mixed inflammatory infiltrate with prominent bile duct damage, endotheliitis and many apoptotic bodies. In-situ hybridization and electron microscopy suggest the intrahepatic presence of the severe acute respiratory syndrome corona virus-2 (SARS-CoV-2), the findings of which may indicate the possibility of direct cell injury. Conclusions Based on the abundant apoptosis and severe cholangiocyte injury, these histopathological changes suggest a direct cytopathic injury. Furthermore, some of the histopathological changes may resemble acute cellular rejection occurring after liver transplantation. These two cases demonstrate that severe COVID-19 hepatitis can occur even in the absence of significant involvement of other organs.

Coronavirus disease 2019 , the disease caused by severe acute respiratory syndrome CoV-2, is a global pandemic of unprecedented proportions. [1] During the months of March and April 2020, New York City became the epicenter of the COVID-19 pandemic, and Mount Sinai Hospital was one of the major hospitals in the region that took care of several thousand COVID-19 patients. In the midst of the pandemic, ongoing liver transplantation (LT) and referrals of patients with severe liver disease continued unabated. [2] https://www1.nyc.gov/site/doh/covid/covid- 19-data.page Initial reports focused on COVID-19 as mainly involving the lungs and being the main cause of morbidity and mortality. [3] However, neurologic, cardiac, renal, hepatobiliary, and gastrointestinal tract involvement are increasingly being recognized. [3] The reported incidence of liver injury in patients with COVID-19 ranges from 14-53%. [4] [5] [6] These studies are mainly based on abnormal liver enzyme elevations in the absence of tissue examination. In one study, a significant number of cases were noted to have elevation of liver enzymes occurring at approximately the tenth day of hospitalization and associated with lopinavir/ritonavir treatment. [7] The authors also found that prolonged hospital stays were associated with abnormal liver enzymes noted at the time of initial admission. [7] Risk factors included underlying chronic liver disease such as viral hepatitis and nonalcoholic fatty liver disease (NAFLD). [4] In a study involving 252 COVID-19 patients, the investigators found that patients having NAFLD had a significantly higher risk of disease progression, a higher likelihood of developing abnormal liver tests during hospitalization, and longer viral shedding times as compared to patients without NAFLD. [6] J o u r n a l P r e -p r o o f

In a previous study of post-mortem examinations, liver histology showed lobular lymphocytic inflammation and centrilobular sinusoidal dilatation with only a few cases showing parenchymal necrosis. [8] Furthermore, in another autopsy study of a 51 year old male, microvesicular steatosis and mild lobular and portal inflammation were noted. [9] At the present time, no detailed histological, immunohistochemical, and ultrastructural findings have been reported in the literature regarding infection of the liver by COVID-19. Herein we report two patients who presented with high aminotransferases and underwent liver biopsy that showed acute hepatitis. A detailed histological analysis along with in-situ hybridization and electron microscopy (EM) were performed on liver tissue to suggest direct hepatic involvement by the novel coronavirus, SARS-CoV-2.

The patient is a 63 year-old man who underwent liver transplantation (LT) in 2017 for hepatitis C (HCV) and alcohol-related liver disease. He received a genotype 1b HCV (+) donor with a pre-perfusion liver biopsy showing no underlying fibrosis and mild portal inflammation. He underwent antiviral therapy with sofosbuvir/ledipasvir starting 3 months post-LT and achieved a sustained virological response. He had developed dialysis-dependent chronic renal failure related to calcineurin inhibitor nephrotoxicity, hypertension and insulin-dependent diabetes mellitus, but always had normal liver chemistry tests and never previously had an episode of acute cellular rejection. The J o u r n a l P r e -p r o o f patient was stable for two years post-LT until he suffered a stroke in January 2020.

Laboratory tests upon discharge from the transplant center after his stroke were normal with AST 12 U/L, ALT 14 U/L, alkaline phosphatase 68 U/L and normal serum bilirubin.

The tacrolimus dose was stable with a level of 4-6 ng/mL. He was discharged to a rehabilitation facility on regular doses of his medications which included ASA 81mg, atorvastatin, calcium acetate, dorzolamide drops, famotidine, insulin, labetalol, nifedipine, ropinirole, sodium bicarbonate, tacrolimus 2mg BID, and tamsulosin. While residing at the rehabilitation facility, the patient developed constitutional symptoms and was found to be COVID-19 (+) via nasopharyngeal swab testing and referred to a local hospital where the liver enzymes were noted to be abnormal. (Table 1) The patient was started on N-acetyl cysteine and acyclovir empirically, however, follow-up laboratory tests showed rising liver enzymes.

He was transferred to Mount Sinai Hospital for further management. RT-PCR on a repeat nasopharyngeal swab specimen confirmed SARS-CoV-2 infection and the tacrolimus dose was increased to 3mg BID in the setting of a trough level of 3.0 ng/mL. Upon presentation, the aminotransferases and alkaline phosphatase were elevated as was the total serum bilirubin. Doppler ultrasonography showed no vascular or biliary abnormalities. The following additional labs were obtained: undetectable PCR for herpes simplex 1 and 2 (HSV), HCV, hepatitis B (HBV), Epstein-Barr virus (EBV), and cytomegalovirus (CMV), undetectable antibody to hepatitis E, and undetectable IgM for hepatitis A (HAV). The triglyceride level was 483 mg/dL (0-150). Inflammatory markers were significantly elevated ( Table 1) Table 1 ) until discharge.

The patient is a 36 year old previously healthy female who presented to a local hospital with six days of progressive nausea, vomiting and scleral icterus. She also had anorexia, myalgias and fevers one week prior to her presentation, taking approximately 1.5 grams of acetaminophen and no other prescription or over-the-counter medications.

Initial laboratory tests at presentation on 4/6/20 showed markedly elevated aminotransferases, total serum bilirubin, and a mildly elevated alkaline phosphatase. (Table 1 Table 1 . She had a normal MRI of the liver; a nasopharyngeal swab was (+) for COVID- 19 . The patient was started on an infusion of N-acetyl cysteine and a liver biopsy was performed on hospital day 7. She was thereafter transferred to Mount Sinai Hospital. projections. Ultrastructurally, these virions appeared to be within sinusoidal endothelial cells. [10] [11] [12] (Figure 3b) The patient was deemed not to require treatment for COVID-19 as she was oxygenating well and had no radiographic evidence of pneumonia. She was also found to have a TSH of .008 and diagnosed with thyroiditis, which has recently been reported in COVID-19 infection [13] ,and she was started on cholestyramine and ursodiol. She had repeat J o u r n a l P r e -p r o o f negative PCR testing for SARS CoV-2 on hospital days 15, 18 and 24 at Mount Sinai.

As she was feeling much better she was discharged, with the LDH being 209, and ddimer < 0.27. Laboratory testing one week later showed an ongoing decrease in her liver enzymes although the bilirubin was still elevated to 10.7 mg/dL with an indirect fraction of 7.8 mg/dL.

The cases presented in the current report had markedly elevated liver enzymes at presentation, with both fitting the clinical course timeline as well as the histological features of severe COVID-19 hepatitis. RNA localization of SARS-CoV-2 S protein using in-situ hybridization and EM findings suggest that SARS-CoV-2 may have played a major role in the liver damage incurred by these two patients. Despite the initial presentation of severe liver damage, both patients recovered from COVID-19 hepatitis.

Currently, it is known that the development of abnormal liver chemistry tests and various forms of liver injury may result from COVID-19 [14] , with the majority of cases having mildly elevated liver enzymes. [4] In a series of 99 COVID-19 patients, only one case presented with severe liver injury with markedly elevated liver enzymes, while 43 of 99 showed mild ALT and AST elevations (in 28% and 35%, respectively). [15] In patients with significantly increased liver enzymes, there were longer hospital stays and most patients were taking medications such as lopinavir/ritonavir, the latter raising the possibility of drug-induced liver injury (DILI). in the liver. [26] Widespread endothelial cell dysfunction in heart, kidney, lung, and small intestine resulted in apoptosis and prominent endotheliitis of submucosal vessels. [26] Another mechanism believed to be the underlying cause of liver injury are ischemic J o u r n a l P r e -p r o o f changes in those with severe COVID-19 and DILI. [4, 27, 28] Besides the direct damage, translocation and DILI, immune-mediated liver injury is another mechanism to consider. This is especially important in light of the increased inflammatory markers noted on serological testing. [16] Although SARS-CoV-2 virus is a new virus and our understanding of its tissue and cellular localization and replication is very limited, the sparse distribution of the virus as seen in the liver in the two current cases is not surprising given the comparable sporadic and occasional cellular distribution described with the first SARS-CoV coronavirus in the early and mid-2000s. [29, 30] Despite the fact that we could not prove that the virus is replicating within the liver tissue as we did not see positive signals for the anti-sense strand of the S gene of the virus, we could not rule out such a possibility given that the viral particles are sparsely distributed and there is a limited amount of liver tissue in these core needle biopsies.

The clinical picture of the two patients is that of a spontaneously resolving COVID-19 infection with the markedly elevated liver tests and inflammatory markers decreasing concurrently. Although the patients did not have severe pulmonary, cardiac or neurologic manifestations of COVID-19 infection that warranted treatment, significant liver chemistry abnormalities developed that correlated with the histological liver damage noted. Neither patient had known underlying intrinsic liver disease but if they did in light of the severe histological liver damage noted, it is possible that they might have developed acute-on-chronic liver failure due to COVID-19. [14, 31] J o u r n a l P r e -p r o o f Some of the histological findings seen in Case #1 are also typically seen in ACR, such as mixed inflammatory infiltrate in portal tracts, endotheliitis and severe bile duct damage. Additionally, there was histological evidence of an acute hepatitis, likely to be due to COVID-19, i.e., COVID-19 hepatitis. Lagana et al showed similar findings in their case which occurred 7 days post-transplantation. [18] Although it is possible that this patient had both mild ACR and concurrent COVID-19 hepatitis, it is more likely that the histological features are due to COVID-19 because the magnitude of the aminotransferase elevation is not typical of mild ACR, nor is the substantive decline in liver tests with only a minimal increase in tacrolimus dose typical of ACR.

Although both of the reported post-LT cases may be episodes of ACR that were triggered by COVID-19 as can occur with other viral infections [32] , it is more likely that 

Histopathology Transjugular needle liver biopsy was performed for patient 1 while a percutaneous liver biopsy was performed for patient 2. Core biopsy specimens were immediately placed in formalin and fixed for a minimum of 3 hours. Routine tissue processing through graded alcohols was performed. Thereafter, the liver biopsy tissues were embedded in paraffin 

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