key: cord-0778966-4ulo69yz authors: Lubnow, Matthias; Schmidt, Barbara; Fleck, Martin; Salzberger, Bernd; Müller, Thomas; Peschel, Georg; Schneckenpointner, Roland; Lange, Tobias; Hitzenbichler, Florian; Kieninger, Martin; Lunz, Dirk; Graf, Bernhard; Brochhausen, Christoph; Weber, Florian; Lüke, Florian; Peterhoff, David; Schuster, Philipp; Hiergeist, Andreas; Offner, Robert; Hehr, Ute; Wallner, Stefan; Hanses, Frank; Schmid, Stephan; Weigand, Kilian; Geismann, Florian; Poeck, Hendrik; Pukrop, Tobias; Evert, Matthias; Gessner, Andre; Burkhardt, Ralph; Herr, Wolfgang; Maier, Lars S.; Heudobler, Daniel title: Secondary hemophagocytic lymphohistiocytosis and severe liver injury induced by hepatic SARS-CoV-2 infection unmasking Wilson’s disease: balancing immunosuppression date: 2021-01-04 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2020.12.047 sha: 1f3c4b230bc0fe7e6ea5f450eff1cfe0dfe41ec0 doc_id: 778966 cord_uid: 4ulo69yz A 21 year old woman was hospitalized due to Covid-19 associated respiratory and hepatic impairment concomitant with severe hemolytic anemia. Upon diagnosis of secondary hemophagocytic lymphohistiocytosis immunosuppression with anakinra and steroids was started, leading to hepatic SARS-CoV-2 infection and viremia. Subsequent liver biopsy revealed virus particles in hepatocytes by electron microscopy and SARS-CoV-2 virus could be isolated and cultured. Immunosuppression was stopped and convalescent donor plasma given. As differential diagnosis acute crisis of Wilson’s disease was raised by laboratory and genetic testing. This case highlights the complexity of balancing immunosuppression to control hyperinflammation versus systemic SARS-CoV-2 dissemination.  We present an unusual case of a young COVID-19 patient presenting with hyperinflammation and severe liver injury.  When immunosuppression is applied, (systemic) virus replication has to be closely monitored and any deterioration of organ function under immunosuppression has to raise the suspicion of direct organ infection warranting further diagnostic work-up for instance by biopsy as systemic viral replication and dissemination is a major driver of severe disease. A 21 year old woman was hospitalized due to Covid-19 associated respiratory and hepatic impairment concomitant with severe hemolytic anemia. Upon diagnosis of secondary hemophagocytic lymphohistiocytosis immunosuppression with anakinra and steroids was started, leading to hepatic SARS-CoV-2 infection and viremia. Subsequent liver biopsy revealed virus particles in hepatocytes by electron microscopy and SARS-CoV-2 virus could be isolated and cultured. Immunosuppression was stopped and convalescent donor plasma given. As differential diagnosis acute crisis of Wilson's disease was raised by laboratory and genetic testing. This case highlights the complexity of balancing immunosuppression to control hyperinflammation versus systemic SARS-CoV-2 dissemination. SARS-CoV-2; COVID-19; liver injury; hemophagocytic lymphohistiocytosis; Wilson's disease; On March 27 th , 2020 a 21 year old female patient without known comorbidities complained about nausea, loss of appetite and frontal headache (illness day 1). The following day she noticed limb and back pain, a dry cough, scleral icterus as well as discoloration of the feces and dark urine. On day 3, she was hospitalized with fever (39°C) and painless icterus. Abdominal imaging revealed hepatosplenomegaly and sludge in the gallbladder without cholestasis or signs of cholecystitis. She was started on on ceftriaxon and metronidazol. The next day she developed diarrhea, emesis and acute renal failure (inactive urine sediment). Examination of a stool sample showed no evidence of pathogenic bacteria. Laboratory work-up revealed a progressive hemolytic , macrocytic anemia without vitamin B12 or folate deficiency. Peripheral blood smear showed no sphaerocytes, or schistocytes. Direct Coombs-test as well as Hanta virus and leptospirosis testing were negative. SARS-CoV-2 RNA testing was positive from an oropharyngeal sample. Since her condition deteriorated, she was transferred to our University hospital on day 5. Our diagnostic work up showed COVID-19 typical infiltrates in a thoracic CT scan and respiratory insufficiency (SpO2 92% with 6l/min of oxygen). Peripheral blood smear showed thrombocytopenia and activated lymphocytes likely associated with COVID-19. (Lüke et al., 2020) Ferritin was massively elevated while ceruloplasmin was severely depressed hinting possible Wilson's disease, thus 24h urine collection and copper analysis was initiated. Liver function tests were elevated combined with a severe impairment of liver synthesis parameters (supplementary Table 1 ). Autoimmune hepatitis was ruled out by serological tests. Additional clinical examination revealed apart from peripheral edema no abnormalities (especially no Kayser-Fleischer rings). A bone marrow aspirate showed hemophagocytosis (supplementary Figure 1A+B ). The HScore, yielded 273 points representing a > 99% probability for secondary hemophagocytic lymphohistiocytosis (sHLH). (Fardet et al., 2014; Mehta et al., 2020) Genetic testing revealed no clinically relevant sequence variants or copy number variations in the coding regions and flanking splice sites of the 4 genes currently assciated with familial hemophagocytic lymphohistiocytos is (PRF1, UNC13D, STX11, STXBP2). As elevated ferritin was found to be a predictor of fatality in a retrospective study (Ruan et al., 2020) suggesting mortality in COVID-19 is partly caused by virus-induced hyperinflammation and these patients likely benefit from immunosuppression (Mehta et al., 2020) , anakinra (interleukin-1ß antagonist) 100 mg s.c./d was initiated on day 6 resulting in mild clinical and laboratory improvement (Figure 1 ). We decided to apply anakinra, which has a short half live and therefore is easy to adjust to the patient's clinical course. On day 9, leukopenia was detected and anakinra was stopped. On day 10 she deteriorated clinically, developed orthostatic hypotension and central venous oxygen saturation dropped. In suspicion of worsening sHLH anakinra was restarted together with 250 mg of prednisolone. Over the next two days, she developed fever and plasma ferritin levels, transaminases and lactate levels rose. Blood cultures and urine as well as peripheral blood PCR-screening for systemic viral infection with cytomegalovirus, adenovirus, Epstein-Barr virus, parvovirus B19, human herpesvirus 6, and human immunodeficiency virus/hepatitis A-E serology were negative. Both the antibiotic regimen and the immunosuppressive therapy were intensified (Figure 1 ). Oral zinc supplementation was started as therapeutic option with minimal side effects for suspected Wilson's disease. Over the next two days the fever disappeared but liver function (supplementary Table 1 , Figure 1 ) and clinical condition worsened. A transjugular liver biopsy and further microbiological workup were performed. Coronavirus viremia (10^3 SARS-CoV-2 RNA copies/ml) and an increase in respiratory tract virus load accompanied by worsening respiratory failure were detected. The liver biopsy showed 10^7 SARS-CoV-2 RNA copies per 10^6 cells of liver tissue ( Figure 1C ). Histology revealed signs of hepatitis, cholestasis and periportal fibrosis (supplementary Figure 1C+D ) and virus-like particles in hepatocytes were detected by electron microscopy (supplementary Figure 1E+F ). The absence of typical findings of autoimmune inflammation or copper in the liver biopsy made immune-mediated inflammation or toxic drug effects less likely. (Zhang et al., 2020 ) Furthermore, we isolated and sequenced SARS-CoV-2 directly from liver tissue. A recent report by Wang et al. showed infection of hepatocytes by EM only in two postmortem cases. Because of systemic virus infection immunosuppressive therapy was stopped on day 15. Since only borderline SARS-CoV-2 IgG-antibodies were present ( Figure 1C ), three doses of convalescent plasma (CP) were administered. Consecutively clinical status, hematologic and liver function improved, ferritin levels dropped and the SARS-CoV-2 IgG-antibody levels rose ( Figure 1C ). Virus levels in serum and respiratory tract dropped also. There were no signs of recurrence of sHLH. Results of two repeated 24 hour urine collections showed a markedly increased copper excretion (supplementary Table 1 Interestingly, the RECOVERY trial as well as a meta-analysis reported that the administration of systemic corticosteroids in moderate doses, compared with usual care or placebo, was associated with lower 28-day mortality in critically ill patients. (Horby et al., 2020; Sterne et al., 2020) Concerning SARS-CoV-2 clearance in patients treated with corticosteriods, several studies report conflicting data maybe due to different doses of steriods used. (Schoot et al., 2020) While higher doses of steriods seem to be associated with prolonged viral shedding (Li et al., 2020b) With barely detectable SARS-CoV-2 antibody titers, we initiated the intravenous application of CP (compassionate use). Afterwards a rapid increase in SARS-CoV-2 IgG antibodies combined with an improvement of the patient's condition was detected. Noteworthy, after discontinuation of prednisolone a slight rise in the SARS-CoV-2 IgG titer was already observed before CP application ( Figure 1C ). The pronounced increase after CP treatment might have been facilitated by virus inactivation/opsonisation. While initial reports described effectiveness of CP leading to an increase in the amount of neutralizing antibodies like in our patient, a recently published randomized trial showed no statistically significant improvement in time to clinical improvement (Li et al., 2020a) . In summary, this case highlights also the complex differential diagnostic process dissecting the underlying causative events: sHLH, hepatic and systemic SARS-CoV-2 infection likely induced by immunosuppression and Wilson's disease, having two rare diseases in one patient. To our knowledge, this is the first report showing detection of SARS-CoV-2 in hepatocytes by EM as well as virus isolation from a diagnostic liver biopsy in a convalescent patient. Another key aspect from our patient is that, when immunosuppression is applied, (systemic) virus replication has to be monitored closely and any deterioration of organ function under immunosuppression has to raise the suspicion of direct organ infection warranting further diagnostic work-up for instance by biopsy. We acknowledge financial support through the pandemic responsiveness of The Bavarian Ministry of Science and Art. The study was approved by the Ethics Committee of the University Regensburg (ethics statement No.: 20-1785-101) and written informed consent was obtained from the patient. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. J o u r n a l P r e -p r o o f COVID-19) Treatment Guidelines. National Institutes of Health Development and validation of the HScore, a score for the diagnosis of reactive hemophagocytic syndrome. Arthritis & rheumatology (Hoboken Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial High-dose but not low-dose corticosteroids potentially delay viral shedding of patients with COVID-19. Clinical infectious diseases an official publication of the Infectious Diseases Society of America Coronavirus disease 2019 induces multi-lineage, morphologic changes in peripheral blood cells COVID-19: consider cytokine storm syndromes and immunosuppression Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan Immunosuppressive Drugs and COVID-19: A Review Association Between Administration of Systemic Corticosteroids and Mortality Among Critically Ill Patients With COVID-19: A Meta-analysis SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19 Liver injury in COVID-19: management and challenges The authors thank the patient and her family; Anette Rohrhofer, Rawia Kserawi, and Leonid Tydykov for excellent technical assistance; the nursing staff and physicians of the wards 91, 93 and 11 for their excellent patient care, the entire COVUR study team at the University Hospital of Regensburg.J o u r n a l P r e -p r o o f