key: cord-327601-4uqgwlnx authors: bangash, mansoor n.; patel, jaimin m.; parekh, dhruv; murphy, nicholas; brown, rachel m.; elsharkawy, ahmed m.; mehta, gautam; armstrong, matthew j.; neil, desley title: sars-cov-2: is the liver merely a bystander to severe disease? date: 2020-06-02 journal: j hepatol doi: 10.1016/j.jhep.2020.05.035 sha: doc_id: 327601 cord_uid: 4uqgwlnx nan we read the recent article from wang et al with great interest. 1 their study shows sars-cov-2 positive patients with ≥1 week history of increased aminotransferases have worse acute pulmonary disease (radiological and physiological) than those without. they also report higher ferritin levels, higher proportions of patients with a low albumin and raised direct bilirubin, and histological features (albeit in only two patients) possibly in keeping with a viral-mediated liver injury in this group. considering that interleukin (il)-6 and c-reactive protein (crp) are similar between patients with normal and prolonged abnormal liver aminotransferases, the authors speculate that liver injury is a direct effect of sars-cov-2 viral hepatitis rather than an indirect immune mediated injury. the fact that increases in liver aminotransferases occur and tend to parallel the severity of pulmonary disease remains unquestioned 2 , however, whether the liver injury is a true viral hepatitis rather than a bystander to the multi-organ pathophysiology of critical illness requires further discussion. wang et al provide evidence for direct viral infection based on electron microscopy where they identified multiple intra-hepatocyte microvesicular structures with "crowns" as sars-cov-2 virions. however, normally occurring clathrin-coated vesicles have a similar appearance. additionally, the tissue is undergoing autolysis, as is usual for post-mortem tissue, and autolysed multi-vesicular bodies (mvbs) are seen in the images. it is therefore possible that the observed cytosolic microvesicles are the intraluminal vesicles of autolysed mvbs. in the context of systemic inflammation, hepatocytes are known to produce mvbs which release the contained vesicles as extracellular vesicles by exocytosis during non-apoptotic cell death (e.g. pyroptosis). 3 indeed, the authors demonstrate tunel-positive hepatocytes (not specific for apoptosis, but also positive in non-apoptotic cell death and autolysis 4 ) and elevated ldh levels (a marker of non-apoptotic cell death), supporting pyroptosis and autolysis as alternate explanations for these clinical and tissue findings, respectively. moreover, as the authors acknowledge, hepatocytes express little to no angiotensin converting enzyme-2 (ace2) receptors, the cellular entry point for sars-cov-2. taken together, and in the absence of sar-cov-2 in situ hybridisation, immunohistochemistry/immunoelectron microscopy or demonstration of sars-cov-2 rna or proteins within the liver, we believe the authors, as others, have mislabelled these electron microscopic structures as sars-cov-2 virions. 5 regarding the blood parameters in the study, aminotransferases (in particularly ast) are not specific to liver injury and are also released after acute muscle injury. the authors identify higher levels of creatinine kinase in patients with raised aminotransferases raising the possibility of a predominantly muscle rather than the liver source. acute and chronic infective illnesses drive catabolic processes that involve muscle (protein) breakdown. 6 in keeping with this, patients with severe pulmonary sars-cov-2 infection lose weight and we have found them to have a high incidence of critical illness neuromyopathy on recovery from their respiratory failure. notwithstanding this, the real elephant in the room is the greater degree of respiratory compromise that associates with only modest liver aminotransferase derangement and the complete lack of clinical correlation with clinically significant liver disease. parameters disturbed in severe acute liver failure are lactate, glucose and inr -these were all well preserved in the data presented by the authors. the patterns of direct bilirubin and albumin are therefore unlikely due to poor synthetic liver function. reductions in albumin more likely reflect increased systemic endothelial permeability and albumin loss from the circulation, something which commonly and rapidly occurs in acute systemic illnesses in patients without liver disease. 7 despite il-6 and crp being similar between patient groups, lymphocyte subset depletion, neutrophil counts, ferritin and markers of fibrinolysis are all significantly increased in patients with prolonged abnormal aminotransferases, clearly suggesting increased immune activation, as we have previously highlighted. 2 furthermore recent studies have confirmed increased netosis, a form of non-apoptotic and highly immunogenic cell death causing bystander damage and coagulation changes, accompanies disease severity. 8 immune-mediated bystander damage then remains a credible mechanism for liver enzyme release and has already been shown to be involved in chimeric antigen receptor t cell-mediated cytokine release syndrome. 9 in conclusion, we do not believe that the findings of wang et al conclusively demonstrate a direct cytotoxic effect of sars-cov-2 on the liver. based on the above perspectives, we feel that raised liver aminotransferases associated with sars-cov-2 positivity are more likely attributable to illness severity, in which host response and iatrogenic harm (i.e. drugs, ventilation) drive bystander liver injury, thus explaining its association with mortality and in an analogous fashion to patterns seen in sepsis. 10 we still encourage clinicians to remain vigilant for drug-induced liver injury, and for liver damage in high risk groups (i.e. drug/alcohol abusers, family history etc), but not to get overly distracted by raised liver aminotransferases in this context. sars-cov-2 infection of the liver directly contributes to hepatic impairment in patients with covid-19 covid-19 and the liver: little cause for concern lps induces active hmgb1 release from hepatocytes into exosomes through the coordinated activities of tlr4 and caspase-11/gsdmd signaling in situ detection of fragmented dna (tunel assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note visualization of putative coronavirus in kidney not the usual suspect: type i interferon-responsive t cells drive infectioninduced cachexia capillary leak syndrome: etiologies, pathophysiology, and management neutrophil extracellular traps in covid-19. jci insight gasdermin e-mediated target cell pyroptosis by car t cells triggers cytokine release syndrome sepsis-associated liver injury: incidence, classification and the clinical significance key: cord-303046-unksl7p4 authors: pawlotsky, jean-michel title: covid-19 and the liver-related deaths to come date: 2020-06-11 journal: nat rev gastroenterol hepatol doi: 10.1038/s41575-020-0328-2 sha: doc_id: 303046 cord_uid: unksl7p4 coronavirus disease 2019 (covid-19) itself and/or the use of hepatotoxic drugs might negatively affect the course and management of patients with pre-existing chronic liver diseases. however, the greatest effect of covid-19 on liver diseases will be indirect and delayed, resulting from the impending global economic crisis. coronavirus disease 2019 (covid-19) was first reported in december 2019 in wuhan, china and was considered harmless by many observers. as of may 30th 2020, according to the covid-19 dashboard by the center for systems science and engineering at johns hopkins university, usa, six million people worldwide have been diagnosed with covid-19 and more than 365,000 of them have died from its complications. meanwhile, the world economy is collapsing. covid-19, which is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was initially thought to present essentially as an upper and/or lower respiratory tract infection. we now know that sars-cov-2 induces a systemic disease attacking many organs, potentially causing major damage, including mortality, and long-term sequelae. surprisingly, the liver seems to be relatively spared by the virus. indeed, apart from patients with the most severe forms of the disease resulting from so-called cytokine storm, who die from multivisceral deficiencies including the liver, manifestations of the infection in the liver are generally mild and transient and have no effect on the course of covid-19 disease. studies based on single-cell rna sequencing in healthy liver tissues suggest that the sars-cov-2 cellular receptor, angiotensin-converting enzyme 2 (ace2), is present mainly on cholangiocytes, whereas expression is 20-fold lower in hepatocytes and ace2 is not expressed in kupffer cells or intrahepatic immune cells 1,2 . the low or lack of expression of ace2 in hepatocytes explains that sars-cov-2 does not actually cause viral hepatitis. covid-19 is gene rally associated with mild-to-moderate elevations of aspartate aminotransferase (ast) and, to a lower extent, alanine aminotransferase (alt) levels, which are more frequent in patients with severe disease than in those with a benign outcome 3 . total bilirubin levels can also be slightly elevated, whereas alkaline phosphatase and γ-glutamyltransferase elevations have been rarely reported 3 . nonspecific histological changes in the liver have been described in small autopsy series, ranging from moderate microvesicular steatosis with mild, mixed lobular and portal activity to focal necrosis, with detection of the virus in the liver in some cases 4 . changes in liver biochemistry might result from the systemic inflammatory response, pneumonia-induced hypoxaemia and/or drug-induced hepatic injury, especially in patients with severe forms of covid-19 requiring hospitalization. the frequency of abnormal liver tests was reported to increase during hospitalization, possibly owing to the administration of hepatotoxic drugs, including antibiotics, nonsteroidal anti-inflammatory drugs, herbal products and antiviral agents, such as ribavirin, interferons, or the fixed-dose combination of lopinavir and ritonavir 5 . elevations in aminotransferase levels have also been occasionally reported with other drugs used in clinical trials in patients with covid-19, including chloroquine and hydroxychloroquine, azithromycin, remdesivir, favipiravir, camostat or tocilizumab. however, liver test abnormalities disappear when the infection recovers and/or when hepatotoxic drugs are removed. patients with chronic liver disease do not seem to be at greater risk of acquiring the infection than other indivi duals in the general population, but the potential onset of covid-19 in these patients raises two questions: will patients with chronic liver disease develop a more severe form of covid-19; and will covid-19 aggravate the course of their liver disease and induce liver-related mortality? one study has suggested that 65 patients with non-alcoholic fatty liver disease (nafld) related to the metabolic syndrome were four times more likely to develop severe manifestations of covid-19 than 65 patients without nafld, regardless of the presence of diabetes 5 . however, it is unclear whether nafld itself plays a part, rather than the associated metabolic comorbidities that include obesity, diabetes and hypertension, which are well-known risk factors for developing severe covid-19. patients with cirrhosis seem to be at greater risk than other individuals for severe covid-19 and the occurrence of complications, including mortality, regardless of the aetiology covid-19 and the liver-related deaths to come www.nature.com/nrgastro of liver disease 6, 7 . the risk could also be increased in indivi duals with decomp ensated cirrhosis, but few cases have been reported. data from registries from a large, multicentre international cohort of patients with chronic liver disease suggest that patients with compensated cirrhosis are at increased risk of decompensation and death during the course of covid-19, even in the absence of respiratory symptoms 8 . the contribution of thromboembolic disorders to liver injury in patients with the most severe forms of covid-19 has been suggested 4 . such thromboembolic events might expose patients with pre-existing chronic liver disease to serious hepatic complications. as a result of lockdowns and suspension of usual clinical care activities for the benefit of patients with covid-19, the sars-cov-2 pandemic is having a major effect on the management of patients with chronic liver diseases, in particular those with cirrhosis, hepatocellular carcinoma and in liver transplantation programmes. the feared risk of acquiring covid-19 at the hospital also prevented many patients from being appropriately managed by hepatology teams. the effect of the pandemic on care of patients with cirrhosis was anti cipated to follow three waves: first, an intense period with prioritized high-acuity care with delayed elective procedures and routine care during physical distancing; then a challenging 'return to normal' following the end of physical distancing, with increased emergent decompensations, morbidity and systems of care overwhelmed by the backlog of deferred care; and finally, a protracted period of suboptimal outcomes characterized by missed diagnoses, progressive disease and loss to follow-up 9 . the covid-19 pandemic will also negatively affect the care and management of patients with hepatocellular carcinoma, generating delayed diagnosis, deferred treatment (including medical and surgical, such as access to liver transplantation), loss to follow-up and, ultimately, increased mortality. liver transplantation has been challenging during the covid-19 pandemic, as many centres had to virtually stop or drastically reduce their transplantation programmes owing to a dramatic reduction in the number of donors and the switch of many care facilities into covid-19 units. sars-cov-2 testing in donors and recipients has been implemented in many places, but the effect of covid-19 on the outcome of liver transplantation is unknown. the role of post-transplantation immune suppression on the course of covid-19 is also largely unknown owing to the scarcity of data. overall, information is still limited, but it is likely that covid-19 will result in a substantial, delayed increase in liver-related mortality that will become visible in the coming months and will substantially contribute to the overall pandemic-related mortality. the covid-19 pandemic will also negatively affect viral hepatitis elimination programmes. the world health organization has set the goal of eliminating viral hepatitis b and c as major public health threats by 2030. this aim includes reducing their incidence, prevalence, morbidity and mortality by means of prevention measures, including hepatitis b vaccination, extensive screening and improved access to care and antiviral treatments. during the covid-19 crisis, attention has been diverted from chronic viral hepa titis, despite the fact that global mortality attributed to viral hepatitis, which has been estimated by the who global hepatitis report 2017 to be approximately 1.5 million per year, currently remains higher than that from covid-19, while resources for public health interventions are already shrinking. lockdown, quarantine and social distancing, closing of harm reduction and treatment facilities, including primary care settings and general practitioners, will probably hamper the major efforts made to achieve the viral hepatitis elimination goals in many areas, further increasing indirect covid-19-linked mortality 10 . the greatest effect of covid-19 on liver-related morbidity and mortality remains to come, as a result of the global economic crisis that has already begun. bankruptcies, job losses, money and food shortage, social isolation and family issues will lead to increases in alcohol and drug use, whereas access to care will suffer from the collapse of health-care structures and organizations, and from government policies diverting resources elsewhere. this situation could translate into a substantial increase in blood-borne virus transmissions (in addition to the current opioid epidemic in north america, which is already rapidly increasing the incidence of new hepatitis c cases), as well as in alcoholic liver disorders and decompensations, resulting in many more patients with cirrhosis, hepatocellular carcinoma, liver transplantation and liver-related death. these unforeseen effects of the covid-19 pandemic will take years to become visible, but they are inevitable. in summary (box 1), sars-cov-2 infection appears to relatively spare the liver, with negligible liver-related mortality during the course of covid-19 (with the exceptions of liver failure in patients with very severe covid-19 infection leading to multivisceral deficiencies and of patients with decompensated cirrhosis). nevertheless, the effect of the covid-19 pandemic on liver-related mortality could be enormous, though hidden, delayed and not ascribed. similar conclusions apply to many other medical specialties and should be considered when drawing up a final assessment of the covid-19 pandemic, the policies put in place to combat it and the lessons to be learnt for the next pandemic. coronavirus disease 2019 (covid-19) has a modest effect on the liver, and liver-related mortality is unusual during the course of the disease, except in patients with severe pulmonary disease who die from multivisceral deficiencies including the liver or in patients with pre-existing advanced liver disease. most covid-19-induced liver-mortality will be delayed, resulting from deferred care for liver diseases, reduced funding for public health interventions and the global economic crisis, which will lead to increases in alcohol and drug use and in blood-borne virus transmissions, while access to care and funding are reduced. prevention of liver-related mortality following the covid-19 epidemic will require rapid resumption of care activities, in particular for patients with advanced cirrhosis or hepatocellular carcinoma and for those requiring liver transplantation, a massive investment in viral hepatitis elimination efforts and courageous economic and social measures to mitigate the health consequences of the coming economic crisis. single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection covid-19: abnormal liver function tests autopsy findings and venous thromboembolism in patients with covid-19 metabolic associated fatty liver disease increases covid-19 disease severity in non-diabetic patients clinical characteristics and outcomes of covid-19 among patients with pre-existing liver disease in united states: a multi-center research network study prognosis of covid-19 in patients with liver and kidney diseases: an early systematic review and meta-analysis high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry the covid-19 pandemic will have a long-lasting impact on the quality of cirrhosis care covid-19 and viral hepatitis elimination programs: are we stepping backward? liver int the author has served as an advisor for abbvie, gilead, glaxosmithkline, merck, regulus and siemens healthcare. covid-19 dashboard by the center for systems science and engineering at johns hopkins university, baltimore, maryland: https://systems.jhu.edu/ who global hepatitis report 2017: https://www.who.int/hepatitis/ publications/global-hepatitis-report2017/en/ key: cord-324509-5c6fzdjm authors: huang, haijun; chen, shanshan; li, hong; zhou, xian‐long; dai, yining; jia, wu; zhang, jun; shao, lina; yan, rong; wang, mingshan; wang, jiafeng; tu, yuexing; ge, minghua title: the association between markers of liver injury and clinical outcomes in patients with covid‐19 in wuhan date: 2020-07-22 journal: aliment pharmacol ther doi: 10.1111/apt.15962 sha: doc_id: 324509 cord_uid: 5c6fzdjm background: the outbreak of coronavirus disease 2019 (covid‐19) is a critical challenge for public health. the effect of covid‐19 on liver injury has not been fully presented. aims: to evaluate the dynamic changes in liver function and the relationship between liver function damage and prognosis in patients with covid‐19. methods: retrospective analysis of clinical data of 675 patients with covid‐19 in zhongnan hospital of wuhan university from january 3 to march 8, 2020. patients were classified as normal, abnormal liver function and liver injury. results: of 675 patients, 253 (37.5%) had abnormal liver function during hospitalisation, and 52 (7.7%) had liver injury. the dynamic changes of alt and ast levels were more significant in patients with liver injury and in those who died. ast >3‐fold uln had the highest risk of death and mechanical ventilation. compared to patients with normal ast levels, mortality and risk of mechanical ventilation significantly increased 19.27‐fold (95% confidence interval [ci], 4.89‐75.97; p < 0.0001) and 116.72‐fold (95% ci, 31.58‐431.46; p < 0.0001), respectively, in patients with ast above 3‐fold uln. increased leucocytes, decreased lymphocytes and female sex were independently associated with liver injury. conclusions: the dynamic changes in liver function may have a significant correlation with the severity and prognosis of covid‐19. increased index of liver injury was closely related to mortality and need for mechanical ventilation. therefore, these indicators should be closely monitored during hospitalisation. in december 2019, unexplained pneumonia cases emerged in wuhan, hubei province, china, 1,2 which spread rapidly throughout the country and became a public health emergency of international concern. on january 7, a novel coronavirus was detected in a swab sample of a patient by the china center for disease control and prevention (cdc). the disease was subsequently named the novel coronavirus disease 2019 (2019-ncov). 3 the pathogen of covid-19 pneumonia is severe acute respiratory syndrome coronavirus 2 (sars-cov-2), 1,4 which mainly causes respiratory, intestinal, liver and nervous system diseases. 5, 6 a study showed that more than 50% of patients with covid-19 have different degrees of liver injury. 7 some studies have reported the clinical characteristics of patients with coronavirus disease 2019 (covid19) , including some factors that may lead to covid-19-related liver damage and the relationship between liver function damage and disease prognosis. 1, 3, [8] [9] [10] [11] in these studies, different degrees of elevated levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were reported. 1, 3, 8, 12 however, the effect of covid-19 on liver injury has not been fully presented. liver injury is related to the severity and mortality of covid-19. 1316 cai et al systematically described the clinical characteristics of covid-19 patients with liver injury and revealed that liver injury was related to disease severity. 14 in addition, a study reported that liver injury was related to death in patients with covid-19, and mortality was related to an increase in liver enzyme levels. 13 however, mechanical ventilation, which is the main auxiliary treatment for critical patients and an important clinical outcome of covid-19, was not involved. on the other hand, dynamic changes in liver functions may indicate a certain relationship between liver injury and mortality. there were few studies on the dynamic changes of liver functions in covid-19-related liver injury. 13, 17 nevertheless, the dynamic changes in liver function based on fatal and nonfatal individuals have never been reported. moreover, there is little research on what abnormalities occur at what time and how those may relate to clinical outcomes. therefore, we retrospectively analysed the clinical characteristics and dynamic changes in liver function based on different liver function levels at admission and different prognosis, in the purpose of finding out risk factors related to liver injury, and associations between markers of liver injury and clinical outcomes in covid-19, including mortality and mechanical ventilation. from january 3 to march 8, 2020, the medical records of in-patients diagnosed with covid-19 were analysed retrospectively at zhongnan hospital of wuhan university. information on epidemiological, demographic, clinical symptoms or physical signs and comorbidities was extracted from the electronic medical records. according to the diagnosis and treatment standard of covid-19 18 issued by the national health committee, the disease severity was divided into three groups: mild, severe and critical. patients with mild type might have fever and respiratory symptoms, and pneumonia was revealed by imaging. severe covid-19 was defined when the patients met any of the following criteria: (a) respiratory distress (≥30 breaths/min); (b) resting oxygen saturation ≤93%; and (c) arterial blood oxygen partial pressure (pao2)/fio2 ≤300 mm hg. in the critical group, at least one of the following three diagnostic criteria fisher's exact test. dynamic changes in liver function based on different liver function levels at admission and different prognosis were presented using locally weighted scatterplot smoothing (loess). the mixed-effect cox proportional risk regression model was used to study the relationship between liver enzyme level and mortality and mechanical ventilation. the mixed-effect cox model was adjusted for gender, age, smoking, chronic liver disease and comorbidities (including hypertension, diabetes mellitus, coronary heart disease and chronic obstructive pulmonary disease). to explore the factors associated with covid-19-related liver injury, logistic regression analysis was performed. the variables with p < 0.1 in a univariate analysis were then included in a forward stepwise regression model. a twosided p of less than 0.05 was considered statistically significant. table 1 . among the 675 patients, 370 (54.8%) patients had normal liver function, 253 (37.5%) patients had abnormal liver function and 52 (7.7%) patients had liver injury. in patients with liver injury, the median age was 51.50 (35.75-60.25), and the ratio of males to females was 4:1. the body mass index (bmi) of patients with liver injury was 24.66 (23.14-26.37), which was higher than that of patients with normal and abnormal liver function. the incidences of hypertension, diabetes mellitus (dm), coronary heart disease (chd) and chronic obstructive pulmonary disease (copd) were 12 (23.08%), 6 (11.54%), 6 (11.54%) and 1 (1.92%), respectively, in these patients with liver injury. among 52 patients with liver injury, 42 (80.77%) patients had fever, and 20 (38.46%) had dyspnoea, which were significantly higher than those of patients without liver injury (p < 0.001). twenty-eight (53.85%) patients with liver injury were in the mild group, 8 (15.38%) patients were in the severe group and 16 (30.77%) patients were in the critical group. the median values of alt, ast and tbil were 105.00 (49.25-159.50), 58.50 (45.00-90.50) and 12.55 (9.50-17.67), respectively, which were much higher than those of patients with normal and abnormal liver function (p < 0.001 for all). the number of lymphocytes in patients with liver injury was 1.08 (0.58-1.63), which was significantly lower than that in patients without liver injury. figure 3c depicted that the fluctuation in tbil levels was mild and normal in the nonfatal group, and the tbil levels increased much more slowly than the alt and ast in the fatal group. nevertheless, tbil continued to rise slowly until it surpassed the uln at the third week. kaplan-meier survival curves were used to evaluate the survival probability and mechanical ventilation-free survival probability during hospitalisation in patients of covid-19 with different levels of alt, ast and tbil. among these indexes of liver function, ast over 3-fold uln had the highest risks of death and mechanical ventilation. in addition, abnormal levels of alt and tbil were also significantly associated with the risk of death and mechanical ventilation (figures 4 and 5) . the relationship between impaired liver function, mortality and mechanical ventilation was evaluated by a mixed-effect cox model adjusted for age, gender, smoking, chronic liver disease and comorbidities, with the hazard ratios of alt, ast and tbil to mortality and the risk of mechanical ventilation showed in logistic regression analysis of the influencing factors of liver injury, such as epidemiological and clinical characteristics, and laboratory variables was performed to select the predictor parameters of covid-19 patients. factors significantly associated with liver injury were increased leucocytes, decreased lymphocytes and female (table 3 ). in this study, we retrospectively and systematically analysed the there were few studies on the dynamic changes of liver functions in covid-19-related liver injury. 13, 17 one study had suggested that the dynamic changes in liver enzyme levels in severe patients were more significant, and ast was the parameter most correlated with mortality. 13 another study indicated that the pattern of liver biochemical was consistent with the damage of hepatocytes, especially ast. the correlation between ast and alt was very strong on admission and throughout the hospitalisation. this suggested that liver injury was the predominant source of aminotransferase elevation. 17 it is in agreement with our findings. in our study, the dynamic changes of alt and ast levels were more significant in patients with liver injury and in the fatal group. moreover, ast over 3-fold uln had the highest risks of death and mechanical ventilation. in the group of patients with liver injury, alt and ast levels disease. the sight increase of ast levels in the early stage of disease may be related to immune-mediated inflammation in the liver. 13 on the other hand, a multicentre study has reported that the incidence of increased tbil in covid-19 was 10%. 12 in our study, level of tbil increased only in the later stage of increased leucocytes and decreased lymphocytes were proved to be risk factors for liver injury. [21] [22] [23] this occurred because of inflammatory response having some effect on the occurrence of covid-19-related liver injury. 23 a study has showed that lymphopenia may be a key factor related to disease severity and mortality, 24 and this is consistent with the conclusion of our research. the study has several limitations. firstly, this is a retrospective study. the data are not able to assess the causality of covid-19related liver injury and poor clinical outcomes. secondly, some cases did not have enough clinical data on past liver injury. thirdly, the sample size of this study is small. a large cohort study is needed to clarify the association of dynamic changes in liver function and clinical outcomes. in conclusion, the dynamic changes in the markers of liver injury have a significant correlation with severity and prognosis of covid-19. elevated liver function was closely related to mortality and risk of the study was supported by the national natural science foundation a 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transmission: a study of a family cluster the association between markers of liver injury and clinical outcomes in patients with covid-19 in wuhan shanshan chen: department of infectious disease, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college zhang: department of orthopaedic surgery, zhejiang provincial people's hospital & people's hospital affiliated of department of nephrology, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college yuexing tu: department of intensive care unit, zhejiang provincial people's hospital & people's hospital affiliated of hangzhou medical college key: cord-339786-elrzlbsg authors: gurala, dhineshreddy; al moussawi, hassan; philipose, jobin; abergel, jeffrey r title: acute liver failure in a covid-19 patient without any preexisting liver disease date: 2020-08-26 journal: cureus doi: 10.7759/cureus.10045 sha: doc_id: 339786 cord_uid: elrzlbsg in december 2019, an outbreak of novel coronavirus started in wuhan, china, which gradually spread to the entire world. the world health organization (who) on february 11, 2020, officially announced the name for the disease as coronavirus disease 2019, abbreviated as covid-19. it is caused by severe respiratory distress syndrome coronavirus 2 (sars-cov-2). the who declared sars-cov-2 as a pandemic on march 11, 2020. sars-cov-2 mainly causes fever as well as respiratory symptoms such as cough and shortness of breath. gastrointestinal/hepatic sequelae such as diarrhea, nausea, vomiting, and elevated liver enzymes have been reported as well. studies and data so far on coronavirus infections from china, singapore, and other countries showed that liver enzymes elevation could be seen in 20-50% of cases. more severe disease can correlate with the worsening of liver enzymes. however, acute liver failure in patients with covid-19 has not been described. herein we report a case of acute liver failure in an elderly patient with covid-19 infection who did not have a history of preexisting liver disease. until recently, seven types of coronaviruses had been reported to cause infections in humans [1] . coronaviruses can use animal hosts and then can evolve to infect humans. this process is thought to explain the emergence of sars-cov (severe respiratory distress syndrome coronavirus) in 2003, mers-cov (middle eastern respiratory syndrome) in 2012, and sars-cov-2 in 2019. sars-cov-2 has 82% genome sequence similarity to sars-cov and 50% genome sequence homology to mers-cov. covid-19 symptoms range from mild (fever, cough, or dyspnea) to moderate (respiratory failure requiring oxygen support) and can progress to ards (acute respiratory distress syndrome) and multiorgan failure. in one of the earlier studies, 80% of cases were mild, but the mortality rate ranged from 1.86% to 9.86% [2] . higher mortality rates were reported in countries like italy, possibly secondary to resource depletion in an overwhelmed health care system. gastrointestinal symptoms such as diarrhea have been reported in approximately 2-10% of patients [3] , with a lower rate in china (3.7%) as compared to singapore (17%) [4] . liver injury has been reported in 60% of patients with sars-cov [5] and has also been reported in patients infected with mers-cov [6] . studies also suggest that sars-cov-2 can affect the liver [7] [8] [9] . in a recent study published in shanghai, 75 (50.7%) out of 148 patients were found to have elevated liver enzymes with sars-cov-2 [7] . in another study published in the lancet in february 2020 by huang et al., an increase in aspartate aminotransferase (ast) was observed in 62% in intensive care unit (icu) patients compared to 25% in non-icu patients, indicating that more severe disease correlates with worsening of liver enzymes [10] . several other studies showed that liver injury in the form of an increase in ast/alanine aminotransferase (alt) levels with a mild increase in bilirubin ranging from 14.8% to 53% [11] . in patients who died of sars-cov-2, liver injury was reported as high as 58.06% [12] . the highest levels recorded included an alt of 7,590 u/l and an ast of 1,445 u/l [13] . generally speaking, transaminase elevations are mild in patients with covid-19. here, we report a case of acute liver failure in an elderly patient with covid-19 infection who did not have a history of preexisting liver disease. an 80-year-old male with a medical history of diabetes, hypertension, dyslipidemia, asthma, coronary artery disease with bypass graft, atrial fibrillation on warfarin, and heart failure with preserved ejection fraction with an automatic implantable cardiac defibrillator and pacemaker presented to the emergency department (ed) in march 2020 with intermittent fever, productive cough, and shortness of breath (sob) for four to five days. he initially started noticing fever that was partially relieved by acetaminophen five days prior to presentation (maximum temperature of 102°f at home). this was associated with sob on exertion, which progressed to sob at rest and a productive cough. he denied any recent travel, contact with sick person, herbal medications use, or a recent change in home medications. his home medications included oral warfarin daily, oral metoprolol tartrate two times daily, oral metformin er daily, oral aspirin daily, oral atorvastatin, and budesonide-formoterol inhaler twice daily. the review of systems was otherwise negative. the patient did not have a history of smoking, alcohol consumption, illicit drugs, or high-risk sexual behavior. vitals at the time of admission showed a temperature of 101.6°f, heart rate of 80 beats/minute, blood pressure 140/70 of mm hg, respiratory rate of 20 breaths/minute, and oxygen saturation of 98% on room air. physical examination was positive for bilateral wheeze and rhonchi in all lung fields, 1+ pedal edema bilateral. his chest was without spider angiomas and abdomen with no hepatosplenomegaly, and he had no shifting dullness, with normoactive bowel sounds and no palmar erythema. on neurological examination, he was alert, oriented to time, place/person, followed commands, and had no focal deficits. laboratory examination results are shown in table 1 the patient had normal liver enzymes at presentation but had elevated transaminases on day 4. the examination at that time was negative for asterixis or encephalopathy. atorvastatin was stopped, and the recommendation was made to start n acetylcysteine (nac), and workup for acute and chronic liver disease was ordered. his respiratory status continued to deteriorate, requiring increased oxygen support. his radiologic findings worsened with enlarging infiltrates on a chest x-ray on day 4, as shown in figures 1-3 . the patient then developed cytokine release syndrome (crs) (elevated interleukin [il]-6 and il-10 as mentioned in table 5 ), and he expired on day 9. covid-19 is a pandemic illness that primarily affects the respiratory system with a wide spectrum of disease presentation that ranges from mild disease (fever, cough) to severe (ards, multiorgan failure). the gastrointestinal/hepatic systems are the next most commonly affected, with symptoms such as nausea, vomiting, diarrhea, and an increase in liver enzymes. currently, studies on the exact pathophysiology of liver injury in these patients are limited, but it is believed either to be a direct effect of the virus or immune-mediated inflammatory response, such as crs, hypoxemia, and failure of innate immune regulation, or to be drug-induced. 1) it is postulated that both sars-cov-2 and sars-cov bind to angiotensin-converting enzyme 2 (ace2) receptors to enter the target cell [14] where the virus replication begins and starts to infect cells of the upper respiratory tract. based on the scrna-seq data, chai et al. [15] found that ace 2 receptors also found in the hepatobiliary system (high in bile duct cells, cholangiocytes, when compared to liver cells). cholangiocytes play a critical role in liver regeneration and immune responses [16] . thus, the authors concluded that potential damage of cholangiocytes by 2019-ncov might lead to profound consequences in the liver rather than the direct effect of the virus on hepatocytes. 2) crs is a group of disorders caused by a wide variety of inflammatory etiologies, resulting in a profound increase in inflammatory markers such as il-2, il-7, il-6 granulocyte colonystimulating factor, interferon-γ inducible protein 10, monocyte chemo-attractant protein 1, macrophage, inflammatory protein 1-α, and tumor necrosis factor-α. this can ultimately lead to hemodynamic instability, multiorgan dysfunction, and death [17] . elevations in il-6, il-10, procalcitonin, and ferritin, as well as thrombocytopenia have been associated with severe covid and potentially severe liver injury as seen in our patient [18] . 3) ischemic hepatitis, also known as shock liver, is characterized by a significant increase in serum aminotransferases due to reduced oxygen delivery to the liver, usually seen in shock and thromboembolic disease [19] . 4) clinicians should also consider drug-induced liver injury due to hepatotoxicity associated with drugs used in treating covid such as lopinavir, ritonavir, and hydroxychloroquine that are recently approved by the fda for the treatment of covid. our patient, who had no previous history of liver disease and normal liver enzymes at presentation, developed elevated liver enzyme levels on day 4. initial differential diagnosis was broad, including ischemic hepatitis, drug-induced liver injury, viral hepatitis, cholestasis of sepsis, and autoimmune diseases. on laboratory workup, viral infections such as hepatitis (a, b, c), epstein-barr virus, cytomegalovirus, herpes simplex virus, hiv, autoimmune, and metabolic causes were ruled out. since his blood pressure was stable until day 8 of his hospitalization without any pressor support, ischemic hepatitis was unlikely [20] . tylenol toxicity was excluded (levels were less than 5 ug/ml). the remainder of the patient's medications were reviewed, and none of the patient's medications was likely to be the culprit. for example, the patient's home medication coumadin is a rare cause of acute liver injury and usually results in a cholestatic pattern rather than a hepatocellular one, which is what our patient demonstrated. aspirin has been associated with an increase in liver enzymes but usually only with dosages of more than 1,800 to 3,200 mg daily. metoprolol and metformin have been associated with only mild elevations in liver enzymes. hydroxychloroquine used to treat sars-cov-2 has been rarely associated with clinically apparent liver injury. a single case series (two cases) of acute liver failure attributed to hydroxychloroquine was published, but these patients took the medication for more than two weeks. in summary, we describe the first case of acute liver failure caused by the covid-19 infection. acute liver failure was diagnosed clinically by rising liver function tests and inr, as well as progressive encephalopathy. we could not conclusively prove that the covid-19 was the etiologic agent as the patient declined a liver biopsy. however, alternative causes of acute liver failure were effectively ruled out. bloodwork did not identify another etiology, and the patient's hypotension was too late in his course and too mild to cause ischemic hepatopathy. additionally, none of his medications was among the usual suspect for acute liver failure. as we learn more about this new infection, we expect to better understand the spectrum, pathophysiology, and treatment of the resultant liver injury. human subjects: consent was obtained by all participants in this study. in compliance with the icmje uniform disclosure form, all authors declare the following: payment/services info: all authors have declared that no financial support was received from any organization for the submitted work. financial relationships: all authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. other relationships: all authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. sars-cov-2: an emerging coronavirus that causes a global threat clinical characteristics of 2019 novel coronavirus infection in china enteric involvement of coronaviruses: is faecal-oral transmission of epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore sars-associated viral hepatitis caused by a novel coronavirus: report of three cases histopathology of middle east respiratory syndrome coronovirus (mers-cov) infection -clinicopathological and ultrastructural study clinical features of covid-19-related liver damage epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study liver injury in covid-19: management and challenges clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020 liver injury during highly pathogenic human coronavirus infections clinical characteristics of 36 non-survivors with covid-19 in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the novel coronavirus 2019 (2019-ncov) uses the sars-1 coronavirus receptor2 ace2 and the cellular protease tmprss2 for entry into target cells specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection cholangiocyte pathobiology the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a meta-analysis hypoxic hepatitis: a review and clinical update athlete's hepatitis in a young healthy marathon runner key: cord-328147-61gtx2h2 authors: lopez-mendez, ivan; aquino-matus, jorge; gall, sofia murua-beltrán; prieto-nava, jose d.; juarez-hernandez, eva; uribe, misael; castro-narro, graciela title: association of liver steatosis and fibrosis with clinical outcomes in patients with sars-cov-2 infection (covid-19) date: 2020-10-21 journal: ann hepatol doi: 10.1016/j.aohep.2020.09.015 sha: doc_id: 328147 cord_uid: 61gtx2h2 introduction and objectives: liver function tests (lft) abnormalities are reported in up to 50% of covid-19 patients, metabolic comorbidities are associated with poorer outcomes. the aim of the study is to determine prevalence of liver steatosis and fibrosis in patients with covid-19 and their association with clinical outcomes. material and methods: retrospective study in hospitalized covid-19 patients. risk for liver steatosis was estimated by hsi > 36, and risk for advanced liver fibrosis with apri > 1.0, nafld fs > 0.675 and/or fib-4 > 3.25. clinical outcomes were admission to intensive care unit (icu) and mortality. results: of 155 patients, 71.6% were male (n = 111), and 28.4% (n = 44) were obese. abnormal lft were present in 96.8% (n = 150), prevalence of steatosis was 42.6% (n = 66) and of significative liver fibrosis was 44.5% (n = 69). liver fibrosis by fib-4 was associated with risk of icu admission (or 1.74 [95%ci 1.74-2.68; p = 0.023]) and mortality (or 6.45 [95%ci 2.01-20.83, p = 0.002]), no independent associations were found. conclusions: the prevalence of steatosis and significant liver fibrosis was high in covid-19 patients but was not associated with clinical outcomes. the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus emerged in wuhan, china, in december 2019; it is a highly transmittable pathogen that causes the coronavirus disease 19 . the two previous betacoronavirus epidemics of global concern where the severe acute respiratory syndrome coronavirus (sars-cov) the centers for disease control and prevention (cdc) revised and updated a summary of medical conditions associated with increased risk of severe illness derived from covid-19, which included among others, obesity (body mass index (bmi) >30 kg/m 2 ) and type 2 diabetes mellitus (t2dm). in mexico, 72.5% of the adult population is overweight and 9.4% have t2dm(4) additionally, the prevalence of hepatic steatosis in mexico ranges from 14 .4% to 62.9%, (5) and he prevalence of liver fibrosis has been reported in 8.1% (noninvasive assessment).(6) currently, mexico city is one of the most affected regions in the world with rising numbers of cases and deaths caused by covid-19, and we have very few data regarding gi symptoms and lft abnormalities and their prognostic value in mexican patients. j o u r n a l p r e -p r o o f we conducted a retrospective, cross sectional, descriptive study using electronic medical records of adult patients (>18 years old) with a positive rt-pcr sars-cov-2 test (genefinder tm covid-19 plus realamp kit, osang healthcare co., ltd. korea) in nasopharyngeal swab, admitted to medica sur clinic & foundation between march 14 th through june 5 th , 2020. patients with covid-19 diagnosis but without lft determination in the first 48 hours after admission were excluded. clinical and biochemical data were included in the analysis. the study was approved by the medica sur ethics committee (2020-ext-487). the presence of steatosis was determined by hepatic steatosis index (hsi) by the formula 8 the distribution of data was assessed by the kolmogorov-smirnov test. continuous data were presented as median an interquartile range; categorical data were presented as percentage and frequencies. the mann-whitney u test was used to evaluate differences between patients with and without steatosis, as well as liver fibrosis. the association of categorical data and clinical outcomes was analyzed by chi square. univariate and multivariate analyses were performed for associations between steatosis and liver fibrosis with intensive care unit j o u r n a l p r e -p r o o f (icu) admission and mortality. the p-values <0.05 were considered statistically significant. spss v.21 was used for statistical analysis. a total of 155 patients were included in the final analysis, from which 71.6% (n=111) were male; median of age and bmi were 51 [42-62] years and 27.9 [25.8-30.5 ] kg/m 2 , respectively. obesity (28.4%, n=44), hypertension (23.2%, n=36), and t2dm (15.5%, n=24) were the most frequent comorbidities. only 2 patients (1.3%) reported a preexisting liver disease. among biochemical parameters, the median of hemoglobin was 14.8 [13.7-15.9] g/dl, platelets 198 [162-254] x 10 9 /l, and leukocytes 7.1 [5.3-9.9] x 10 9 /l. demographic data, clinical characteristics, drug history, and biochemical parameters are shown in table 1 . patients had a median of 7 [5] [6] [7] [8] [9] [10] days with symptoms before admission, which included fever in 69.7% (n=108), cough in 65.2% (n=101), and dyspnea in 47.7% (n=74). median of hospital stay was 9 [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] days and 34.2% (n=53) of the patients required admission to the icu, with a median stay of 13 [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] days. at least one gi symptom was reported in 36.1% (n=56) of the patients, which included diarrhea in 25.8% (n=40), nausea in 9% (n=14), and anorexia in 9% (n=14). regarding lft (table 2 ), a total of 96.8% (n=150) of patients presented at least one abnormality, and lactate dehydrogenase was the most frequent in 89.7% (n=139) of the cases. the prevalence of steatosis by hsi score >36 was 42.6% (n=66) and it was not associated with clinical outcomes. on the other hand, the prevalence of advanced liver fibrosis by any model was 44.5% (n=69), which is shown in figure 1 . the prevalence of t2dm was higher among patients with fibrosis (66.7% (n=16) vs. 33.3% (n=8), p=0.025), and mortality was also higher in these patients (81.8% (n=9) vs. 18.2% (n=2), p=0.012); however, no independent associations were found in multivariate analyses. differences in basal biochemical levels were found in patients with advanced liver fibrosis. mortality was 7.1% (n=11) and the associated factors were hypertension, basal total bilirubin, basal direct bilirubin, and liver fibrosis by any method, as well as by nafld fs and fib-4; ( as expected, due to the epidemiological context of mexico, in our study we found a high prevalence of metabolic comorbidities which included obesity, hypertension, and t2dm, which were associated with clinical outcomes in univariate analyses. obese patients face an increased risk for severe complications and mortality. they also represent a challenge for therapeutic maneuvers such as imaging diagnosis, intubation, mechanical ventilation, and pronation, among others.(11) a meta-analysis including 3,207 patients with covid-19 described that underlying chronic conditions such as hypertension, diabetes, and cardiovascular and respiratory diseases were higher in critical/non-surviving patients; clinical manifestations such as fever and dyspnea were also associated with the progression of the disease.(12) we found similar results in our study, with dyspnea as the most important associated symptom for icu admission with or 4.07 (ci95% 1.6-9.86). the pathogenesis of gi symptoms and liver injury in covid-19 is still a subject of research. although ace2 is not expressed in kupffer cells, hepatocytes, and endothelium of liver sinusoids,(15) expression of ace2 is induced by hypoxia in cultured hepatocytes (17) and it has been shown that covid-19 infection is associated with liver disease, ranging from a mild to severe damage, but it is usually transient.(2) currently, there is no consensus on the exact mechanism of liver injury, but it may be related to: 1) direct cytopathic damage, 2) systemic inflammatory response, 3) liver hypoxia and ischemia, 4) acute-on-chronic liver injury, or 5) drug-induced liver injury. (18) it has been recently demonstrated the presence of sars-cov-2 virions inside hepatocytes (19) and that death of cholangiocytes induced by sars-cov-2.(20) even though it has not been demonstrated in humans, hepatic steatosis in mice models increases hepatic ace2 mrna (21) , which could explain, at least in theory, the relationship between sars-cov-2 and liver injury in mafld patients. the liver harvests a great pool of macrophages and immune cells, that in addition to a particular susceptibility of hepatocytes to proinflammatory cytokines, the systemic inflammatory response can easily produce liver injury as a collateral damage. (19) the global prevalence of gi symptoms in covid-19 patients has been reported between 11.3% and 79.1%. (22) in our study, gi symptoms were reported in 36.1% (n=56), which is similar to another recent mexican study which reported a prevalence of 20.8%. (23) additionally, a meta-analysis which included 6,686 patients estimated that 15% of them had at least one gi symptom and that 10% only had gi symptoms upon presentation.(24) another systematic review and meta-analysis which included 12,797 patients reported that mortality in the group of patients with gi symptoms (0.4% [ic95%, 0-1.1%; i 2 =74%]) was similar (p=0.15) to overall mortality (2.1% [ic95%, 0.2-4.7%; i 2 =94%]). (25) abnormal lft were found in 96.8% (n=150) of our patients, which is higher that recent studies reporting a prevalence ranging from 16.1% to 76.3%, (3, 26) including 21.5% of liver injury prevalence during hospitalization.(3) our study did not find an association between abnormal lft and mortality, as a multicentric study in china that included 5,771 patients which found that abnormal ast was associated with increased mortality risk by any cause with an or 4.81 (ic95%, 3.38-6.86; p<0.001), which increased to or 14.87 (ic95%, 9.64-22.93; p<0.001) in the group with ast above 120 iu/l (27) . we described a prevalence of 42.6% (n=66) of steatosis, which was not associated with clinical outcomes. in contrast, a retrospective study (28) j o u r n a l p r e -p r o o f found a prevalence of mafld of 37.6% and higher risk of disease progression. since mafld is currently considered the liver manifestation of metabolic syndrome (29) these patients are considered chronically inflamed and this fact could contribute to the interplay in the cytokine storm described in covid-19, resulting in the progression of the disease, its complications, and fatal outcomes. (30) this explanation is supported by zheng et al's study (31) in china, in which 30% of the patients presented mafld, according to tomographic criteria, and found an increased 6-fold risk of severe covid-19 and obesity in mafld patients, in comparison to non-obese patients (or 6.32, 95%ci 1.16-34.54, p=0.033). the addition of metabolic comorbidities may increase the risk of serious complications in this group of patients, but further research is required. in our study, we found a pooled prevalence of advanced liver fibrosis of 44.5% (n=69) through any non-invasive liver assessment model, which was associated with hospital outcomes. this result is in concordance to the multicenter study by ibáñez-samaniego et al. (32) in spain, which included 160 patients and estimated a risk for advanced fibrosis in 28.1%. a fib-4 ≥2.67 score increased the risk of icu admission as an independent risk factor (or 3.41, 95%ci 1. 30-8.92 ). interestingly, in the multivariate analysis with exclusion of patients with mafld, fib-4≥2.67 remained as a risk factor for mechanical ventilation (or 3.25; 95% ci, 1.24-8.53). in our study, no independent associations were found for liver fibrosis and clinical outcomes. these associations are interesting but must be interpreted with caution, since most of the noninvasive predictive models include variables that may be affected by the covid-19 infection as well, resulting in overdiagnosis of liver fibrosis upon presentation. nonetheless, we consider that screening for liver fibrosis in patients with covid-19 would contribute to further risk stratification and to design follow-up strategies in surviving patients who were unaware of their liver diseases, especially in regions where obesity and other metabolic comorbidities are highly prevalent. our study only included two patients with preexisting chronic liver disease, but a previous report by an international registry (covid-hep.net and covidcirrhosis.org) that included 152 patients showed that 23.3% were admitted to the icu and 17.5% required mechanical ventilation, with a reported mortality of 39.8%. (33) a meta-analysis with 1,558 patients from reported important associations between mafld and covid-19 severity. in a retrospective study of 202 patients in china, mafld was associated with covid-19 progression had (or 6.4; 95% ic, 1.5-31.2). (36) further research, especially in the americas, is required to elucidate the relationship between liver disease and covid-19 regarding clinical outcomes. limitations of our study include the retrospective design, the assessment of liver fibrosis through non-invasive models, and the conduction of the study in a private hospital; additionally, as this is not a prospective design, decisions in management and timing of laboratory tests are based on each attending physician during hospital course. in covid-19 patients, the prevalence of liver steatosis and advanced liver fibrosis by noninvasive assessment prediction models was high and was not associated with clinical outcomes; 96.8% of covid-19 patients had at least one abnormal lft. the presence of metabolic comorbidities was associated with mortality and icu admission. the timely diagnosis of covid-19 patients, including those presenting gi symptoms and abnormal lft, is of utmost importance in the fight towards reducing mortality. j o u r n a l p r e -p r o o f clinical features of patients infected with 2019 novel coronavirus in wuhan gastrointestinal, hepatobiliary, and pancreatic manifestations of covid-19 covid-19: abnormal liver function tests obesity and public health in mexico: transforming the hegemonic food supply and demand pattern the mexican consensus on nonalcoholic fatty liver disease prevalence of liver fibrosis in an unselected general population with high prevalence of obesity and diabetes mellitus. time for screening? hepatic steatosis index: a simple screening tool reflecting nonalcoholic fatty liver disease. dig liver dis performance of the aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis c-related fibrosis: an updated meta-analysis the nafld fibrosis score: a noninvasive system that identifies liver fibrosis in patients with nafld development of a simple noninvasive index to predict significant fibrosis in patients with hiv/hcv coinfection covid-19 infection: origin, transmission, and characteristics of human coronaviruses a new coronavirus associated with human respiratory disease in china tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis review article: gastrointestinal features in covid-19 and the possibility of faecal transmission chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2 what has the covid-19 pandemic taught us so far? addressing the problem from a hepatologist's perspective direct or collateral liver damage in sars-cov-2-infected patients recapitulation of sars-cov-2 infection and cholangiocyte damage with human liver ductal organoids pioglitazone upregulates hepatic angiotensin converting enzyme 2 expression in rats with steatohepatitis beware: gastrointestinal symptoms can be a manifestation of covid-19 solís-gonzález a. initial gastrointestinal manifestations in patients with sars-cov-2 in 112 patients from veracruz manifestations and prognosis of gastrointestinal and liver involvement in patients with covid-19: a systematic review and meta-analysis prevalence and mortality of covid-19 patients with gastrointestinal symptoms: a systematic review and metaanalysis liver injury in covid-19: management and challenges longitudinal association between markers of liver injury and mortality in covid-19 in china non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study a new definition for metabolic dysfunction-associated fatty liver disease: an international expert consensus statement covid-19 and nonalcoholic fatty liver disease: two intersecting pandemics letter to the editor: obesity as a risk factor for greater severity of covid-19 in patients with metabolic associated fatty liver disease elevation of liver fibrosis index fib-4 is associated with poor clinical outcomes in patients with covid-19 high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry does comorbidity increase the risk of patients with covid-19: evidence from meta-analysis clinical characteristics and outcomes of coronavirus disease among patients with preexisting liver disease in the united states: a multicenter research network study non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study key: cord-283120-hyzk59qv authors: sharma, ashish; jaiswal, pragya; kerakhan, yasameen; saravanan, lakshmi; murtaza, zeba; zergham, azka; honganur, nagaraj-sanchitha; akbar, aelia; deol, aran; francis, benedict; patel, shakumar; mehta, deep; jaiswal, richa; singh, jagmeet; patel, urvish; malik, preeti title: liver disease and outcomes among covid-19 hospitalized patientsa systematic review and meta-analysis date: 2020-10-16 journal: ann hepatol doi: 10.1016/j.aohep.2020.10.001 sha: doc_id: 283120 cord_uid: hyzk59qv introduction and objectives: the coronavirus disease 2019 (covid-19) pandemic has been a challenge globally. in severe acute respiratory syndrome (sars) epidemic 60% of patients had hepatic injury, due to phylogenetic similarities of the viruses it is assumed that covid-19 is associated with acute liver injury. in this meta-analysis, we aim to study the occurrence and association of liver injury, comorbid liver disease and elevated liver enzymes in covid-19 confirmed hospitalizations with outcomes. materials and methods: data from observational studies describing comorbid chronic liver disease, acute liver injury, elevated aspartate aminotransferase (ast), alanine aminotransferase (alt) levels and outcomes of covid-19 hospitalized patients from december 1, 2019, to june 30, 2020 was extracted following prisma guidelines. adverse outcomes were defined as admission to intensive care unit (icu), oxygen saturation <90%, invasive mechanical ventilation (imv), severe disease and in-hospital mortality. odds ratio (or) and 95% confidence interval (95%ci) were obtained. results: 24 studies with 12882 confirmed covid-19 patients were included. overall prevalence of cm-cld was 2.6%, covid-19-ali was 26.5%, elevated ast was 41.1% and elevated alt was 29.1%. cm-cld had no significant association with poor outcomes (pooledor:0.96;95%ci:0.71–1.29; p = 0.78). covid-19-ali (1.68;1.04–2.70; p = 0.03), elevated ast (2.98;2.35–3.77; p < 0.00001) and elevated alt (1.85;1.49–2.29; p < 0.00001) were significantly associated with higher odds of poor outcomes. conclusion: our meta-analysis suggests that acute liver injury and elevated liver enzymes were significantly associated with covid-19 severity. future studies should evaluate changing levels of biomarkers amongst liver disease patients to predict poor outcomes of covid-19 and causes of liver injury during covid-19 infection. the who declared coronavirus disease 2019 (covid-19) as a global pandemic on march 11, 2020 (1) . with approximately 5 million cases and over 160,220 deaths, the usa remains the worst affected country as of august 8, 2020 (2) . following this, india (4.3m), brazil (4.1m), and russia (1m), are among the other countries that are greatly affected. total cases worldwide are around 27 million, and thus it remains an emergency of international concern (3). severe acute respiratory syndrome coronavirus 2 (sars-cov2) mainly affects the respiratory system but as we are gaining more insight about this novel disease, many published studies have also provided evidence of its organotropism and multisystem organ inflammation nature (4, 5) . multiple studies have observed association of elevated liver enzymes in patients with covid-19 infection assuming that it can cause liver damage either via direct hepatotoxic injury with viral infection, or drug toxicity, or immune mediated response. in the past, it has been reported that 60% of patients developed liver damage due to the sars epidemic (6) . since sars-cov-2 belongs to the same coronavirus family, we assume it may cause liver injury. the biliary epithelium has expression of angiotensinconverting enzyme (ace-2) receptor, which is also the binding site of sars-cov-2 (7) . the ace2 receptor expression in hepatocytes has been shown to be upregulated in animals' models of liver injury but hepatocytes have lower expression in humans (8, 9) . (10) . hence, we aim to systematically study the occurrence of liver injury, comorbid liver disease and elevated liver enzymes in covid-19 confirmed hospitalizations and also identify their association with outcomes. j o u r n a l p r e -p r o o f the aim of the study is to evaluate the role of the comorbid chronic liver disease (cm-cld), elevated liver enzymes and covid-19 associated acute liver injury (covid-19 ali) in predicting the outcomes in confirmed covid-19 hospitalized patients. covid-19 confirmation was evaluated by combined findings of rt-pcr, serology, symptoms, and mri chest in majority of those studies. poor outcomes were defined by intensive care unit (icu) admission, oxygen saturation<90%, invasive mechanical ventilation (imv) utilization, severe disease, and in-hospital mortality. study-specific poor outcomes and definitions of cm-cld, covid-19 ali and cut-off levels of liver enzymes in each individual study are mentioned in table 1 . a systematic search was conducted on published studies using prisma guidelines (11) abstracts were reviewed, and articles were retrieved and reviewed for availability of data on comorbid liver disease, elevated liver enzymes, acute liver injury and outcomes of covid-19 patients. studies which gave details on outcomes were selected for quantitative analysis. pm and dm independently screened all identified studies and assessed full-texts to decide eligibility. any disagreement was resolved through discussion with another reviewer up. from the included studies, we extracted the following variables including comorbid liver disease, elevated ast and alt levels, acute liver injury and outcomes. details on binary outcomes like icu vs. non-icu admission, severe vs non-severe disease, imv vs no-imv use, oxygen saturation <90% vs >90%, in-hospital mortality vs discharged alive and survivors were collected using prespecified data collection forms by two authors (pm and dm) with a common consensus of author (up) upon disagreement. we have presented the study characteristics like the first author's last name, publication month and year, country of origin, sample size, mean or median age, males, outcomes and definitions of comorbid liver disease and acute liver injury and cut offs for elevated ast and alt levels assessed in that individual study in table 1 (references for the studies are in supplemental file 1.1). data analysis was performed using review manager version 5.3 (the nordic cochrane centre, the cochrane collaboration, copenhagen, denmark). if the study has more than one outcome comparison j o u r n a l p r e -p r o o f then we have used data from the most severe outcome in the analysis to minimize the overall selection bias of our study. the maentel-haenszel formula was used to calculate dichotomous variables to obtain odds ratios (ors) along with its 95% confidence intervals to describe the association of comorbid liver disease, elevated liver enzymes, acute liver injury and outcomes of covid-19 patients in each study. random-effect models were used regardless of heterogeneity to estimate the combined effect and its precision, to give a more conservative estimate of the ors and 95%ci. the i² statistic was used to assess statistical heterogeneity and i² >50% was considered significant heterogeneity. the p<0.05 was considered significant. publication bias was assessed visually using funnel plots and the newcastle-ottawa scale (nos). newcastle ottawa scale (nos) (13) was used to assess the quality and bias in the included studies, which rates selection, comparability and outcome (supplemental file1.2) . all studies were assessed to be of high quality. the pooled or and 95% ci are represented in the form of forest plots. each square on the chart area represents individual study and the area of each square is equivalent to the weight of the study, which is the inverse of the study variance. the diamond represents the pooled or and the width corresponds to the 95% ci. j o u r n a l p r e -p r o o f discussion in our meta-analysis, we found that acute liver injury and elevated alt and ast levels were associated with poor outcomes. however, our study could not provide significant evidence of the effect of preexisting chronic liver disease on covid-19 patient outcomes. in support of our findings, a recent metaanalysis found that the frequency of underlying chronic liver disease was not statistically different between severe and non-severe diseases (14) . however, another study by singh et al. reported that patients with preexisting liver disease, particularly cirrhosis, are at higher risk for hospitalizations and mortality (15) . few studies have reported that the frequency of patients with liver injury was higher in severe cases compared to mild cases, consistent with our findings of liver injury associated with poor outcomes (16) (17) (18) . there are several theories proposed for liver damage in covid-19 such as direct effect of the virus on hepatocytes or biliary epithelium via angiotensin-converting enzyme 2 (ace 2) receptors expression, liver injury related to increased immune response (cytokine storm) and immune mediated damage, drug toxicity (because of drugs like acetaminophen, antivirals and hydroxychloroquine), and liver failure occurring in patients having multiorgan dysfunction (19) (20) (21) . a study by chai et al., has reported that expression of ace 2 receptors was 57.9 % in bile duct cells (cholangiocytes) and 2.6% in hepatocytes (7). cholangiocytes play a vital role in liver regeneration and immune response (22) . hence one possible theory for liver injury in covid-19 patients is destruction of cholangiocytes by sars-cov-2 virus via ace 2 receptors. additionally, it was also observed that expression of ace 2 in hepatocytes increases in cases of liver injury (9) . recently, post mortem liver biopsies of covid-19 patients have shown moderate microvesicular steatosis and mild lobular and portal activity, indicating the injury could have been caused by either sars-cov-2 infection or druginduced liver injury (23) . whether this liver injury is caused by the virus itself or is due to a severe j o u r n a l p r e -p r o o f inflammatory response with liver damage or sepsis or multisystem organ failure or drug toxicity is not well understood (24) . therefore, temporal relationship of covid-19 induced liver damage cannot be established. furthermore, increased liver enzymes (ast and alt) occur in the setting of hepatocyte damage (abnormal liver function). according to initial studies, more than a third of patients had elevated ast and alt (transaminitis) which was associated with longer hospital stay(17, 25, 26) . in a study done by cai et al., 76 .3% of covid-19 patients had abnormal liver tests while 21.5% developed liver injury during hospitalization, which was defined by alt, ast, total bilirubin and gamma-glutamyl transferase levels elevated to more than 3x the upper limit of normal (27). the study also found that patients with abnormal liver tests had significantly higher odds of developing severe pneumonia (27). these findings are also consistent with our study that shows a significant elevation in ast and alt among covid-19 patients may be helpful in predicting poor outcomes among these patients. sars-cov-2 is phylogenetically similar to sars-cov and mers and, studies have given evidence of association of sars-cov and mers with liver injury. and elevated transaminases associated with severe disease (20, (28) (29) (30) (31) (32) .additionally, autopsies of sars patients found not only virus particles in the hepatocytes and hepatic vascular endothelial cells but also significant increase in mitotic cells, with eosinophilic bodies and ballooning hepatocytes, suggesting that sars-cov may induce liver cells apoptosis and thus cause liver injury (33, 34) . hence, liver injury due to sars-cov, mers and sars the data used in this study is deidentified and collected from the studies published online thus informed consent or irb approval was not needed for this study. the data is collected from the studies published online, publicly available, and specific details related to data and/or analysis will be made available upon request. j o u r n a l p r e -p r o o f who director-general's opening remarks at the media briefing on covid-19 -11 covid-19 coronavirus pandemic. worldometer multiorgan and renal tropism of sars-cov-2 age-adjusted risk factors associated with mortality and mechanical ventilation utilization amongst systematic review and meta-analysis. sn comprehensive clinical medicine covid-19 and liver specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection. biorxiv chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2 upregulation of hepatic angiotensin-converting enzyme 2 (ace2) and angiotensin-(1-7) levels in experimental biliary fibrosis comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis preferred reporting items for systematic reviews and meta-analyses: the prisma statement meta-analysis of observational studies in epidemiology: a proposal for reporting. meta-analysis of observational studies in epidemiology (moose) group scale (nos) for assessing the quality of nonrandomised studies in meta-analyses coronavirus disease (covid-19) and the liver: a comprehensive systematic review and meta-analysis clinical characteristics and outcomes of coronavirus disease 2019 among patients with preexisting liver disease in the united states: a multicenter research network study clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet covid-19 in a designated infectious diseases hospital outside hubei province tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis clinical characteristics and mechanism of liver injury in patients with severe acute respiratory syndrome risk factors related to hepatic injury in patients with corona virus disease 2019. medrxiv cholangiocyte pathobiology associated with acute respiratory distress syndrome. the lancet respiratory medicine covid-19 and liver dysfunction: current insights and emergent therapeutic strategies clinical features of covid-19-related liver functional abnormality clinical features of patients infected with 2019 novel coronavirus in wuhan covid-19: abnormal liver function tests organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways clinical significance of hepatic derangement in severe acute respiratory syndrome clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus sars-associated viral hepatitis caused by a novel coronavirus: report of three cases , and pneumonia can be seen in imaging. severe cases: meeting any of the followingrespiratory distress, respiratory rate ≥ 30 breaths/min; spo2 ≤ 93% at rest; and pao2/fio2 ≤ 300. patients with >50% lesion progression within 24 to 48 hours. critical/extremely severe cases: if they have one of the following: respiratory failure requiring mechanical ventilation, shock, and other organ failure requiring icu treatment. ***patients were included in the mild disease group if they did not need high-flow oxygen support and in the severe disease group if they were provided with high-flow oxygen support. #not mentioned; ##general covid-19 included following criteria:(i) obvious alleviation of respiratory symptoms (e.g. cough, chest distress and breath shortness) after treatment; (ii) maintenance of normal body temperature for ≥3 days without the use of corticosteroid or antipyretics; (iii) improvement in radiological abnormalities on chest ct or x-ray after treatment; (iv) a hospital stays of ≤10 days.otherwise, it was classified as refractory covid-19; $ all the studies mentioned chronic liver disease as a comorbidity. ^liver enzyme abnormalities, ^^acute liver injury defined as an increase in alanine aminotransferase (alt) over two times the upper limit of the normal range (uln) or an increase in conjugated bilirubin or a combined increase in aspartate aminotransferase (ast), alkaline phosphatase and total bilirubin provided that one of them was above two times uln. ^^^ acute liver injury was defined as jaundice with a total bilirubin level of 3mg/dl or higher and an acute increase in alt of at least 5 times the upper limit of the normal range and/or an increase alkaline phosphatase of at least twice the upper limit of the normal range. ^*liver injury was judged alt and ast levels @ studies considered ast (aspartate aminotransferase) and alt (alanine aminotransferase) levels >40iu/l as elevated levels.references of the included studies in meta-analysis is in esupplemental file 1 key: cord-355395-rckzi8vz authors: tian, dandan; ye, qing title: hepatic complications of covid‐19 and its treatment date: 2020-05-21 journal: j med virol doi: 10.1002/jmv.26036 sha: doc_id: 355395 cord_uid: rckzi8vz covid‐19 is highly contagious and has a variety of clinical manifestations, it can affect a number of other organs in addition to the lungs, and liver injury may occur. sars‐cov‐2 can cause liver injury through systemic inflammatory response syndrome (sirs), cytokine storms, ischemia‐reperfusion injury, side effects of treatment drugs, and underlying liver disease and can attack liver cells directly via ace2. clinical studies have found that liver injury in covid‐19 patients mainly manifests as abnormal liver biochemical indicators, but there have been no reports of liver failure caused by this disease. the number of covid‐19 patients with liver injury is increasing, and the incidence of liver injury in covid‐19 patients with severe disease are higher than in patients with mild disease. liver injury may be a risk factor for progresses and worsens in patients with covid‐19, and it is necessary to pay attention to the occurrence of liver injury in the diagnosis and treatment of covid‐19. this article is protected by copyright. all rights reserved. coronavirus disease 2019 (covid19) is an acute respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) 1-3 . because it is highly contagious, people are generally susceptible, and it is mainly transmitted via respiratory droplets, covid-19 is spreading all over the world. it has been reported in more than 200 countries, and the number of deaths worldwide is also increasing. sars-cov-2 infection mainly affects the respiratory tract, and the main clinical manifestations are fever, dry cough, rhinorrhea and fatigue. many cases of covid-19 are acute and resolve quickly, but the disease can also be fatal, with a mortality rate of approximately 3% 4 . the fatality rate of critically ill patients admitted to the intensive care unit (icu) is relatively high, the results from a study of 52 critically ill patients with covid-19 who were admitted to the intensive care unit (icu) of wuhan jin yin-tan hospital (wuhan, china) between late december, 2019, and jan 26, 2020 showed that 32 (61.5%) patients had died within 28 days, and the median duration from icu admission to death was 7 (iqr 3-11) days in the non-survivors 5 . many clinical studies have also suggested that patients with covid-19 have multiple organ damage, including the circulatory system, urinary system, nervous system, and digestive system, among others. the presence and number of these complications are high-risk factors that aggravate a patient's condition and correlate with a poor prognosis. the whole genome sequencing results showed that sars-cov-2 shares 82% genome sequence similarity to sars-cov, and 50% genome sequence homology to middle east respiratory syndrome coronavirus (mers-cov) 22 sars-cov, middle east respiratory syndrome coronavirus (mers-cov) and sars-cov-2 are all coronaviruses and known to cause severe respiratory symptoms 11 . as many as 60% of patients infected with sars-cov have liver damage 23 , and some patients infected with mers-cov also have liver damage 24 19 . the above indicate that the incidence of liver injury in severe or critically ill cases is significantly higher than that in mild cases of covid-19, and the probability of patients with liver function impairment requiring icu treatment is increased. 19 . another single-center study of 138 patients with covid-19 showed that the levels of alt and ast in patients who required icu treatment were significantly higher than those in patients who did not require icu treatment 10 . moreover, in a large cohort of 1099 patients from 552 hospitals in 31 provinces or provincial municipalities, the total incidence of elevated total bilirubin was approximately 10%, of which the incidence in severe and nonsevere patients was approximately 13.3% and 9.9%, respectively; the incidence of increased total bilirubin in patients who needed to be moved to the icu, needed mechanical ventilation, or died was higher than in other patients (20.8% vs 9.8%) 5 . the above studies suggest that liver biochemical indicators may be used as predictors of severity and prognosis of covid-19 patients. thus, clinicians should pay more attention to changes in liver biochemical indicators and also identify patients with liver damage in a timely manner. the results from biopsies in a death covid-19 patient showed moderate microvascular steatosis and mild portal and lobular activity in liver tissue 25 . in another four autopsy in a death covid-19 patient showed mild zone 3 sinusoidal dilatation, patchy hepatic necrosis, mild increase in sinusoidal lymphocytes are the main pathologic changes of liver in case 3 and case 4, and rt-pcr showed direct evidence of the sars-cov-2 rna sequence in the liver tissues in case 1 26 . through learning of tian et al report 26 , li et al observed foci of hepatic necrosis in adjacent to terminal hepatic veins and peri-portal area, and found no significant surrounding inflammatory cellular infiltration, which consistent with the pattern of acute liver injury 27 . the above studies may indicate that hepatic injuries may be related to direct viral attack. it has been confirmed that sars-cov-2 enters host cells through binding of its s protein to angiotensin-converting enzyme 2 (ace2) on the surface of the host cell. ace2 is the most important cell receptor that mediates the entry of sars-cov-2 into target cells [28] [29] . however, based on single-cell sequencing and animal model analysis of liver tissue, chaix's team found that the expression level of ace2 in liver tissue was only approximately 0.31% and that specific expression of ace2 in bile duct epithelial cells was 20 this article is protected by copyright. all rights reserved. times higher than that in hepatocyte 30 . considering limited number of autopsy cases in patients with covid-19 studied and the relatively low expression of ace2 in liver, liver damage directly caused by sars-cov-2 infection of hepatocytes deserves further investigation. moreover, biomarkers for cholangiocyte injury, such as gamma-glutamyl transferase (ggt) and alkaline phosphatase (alp) have also been seen in some patients 31 , and consistent with injury to biliary epithelial cells, and about 10% of covid-19 patients have elevated total bilirubin level.these might suggest that sars-cov-2 might directly bind to cholangiocytes expressing ace2 result in cholangiocyte injury. huang c 18 and colleagues found that liver injury in covid-19 patients differed from that in sars patients. liver injury as the first manifestation in covid-19 patients was very rare, with most being secondary liver injury. it was speculated that in addition to the virus itself causing liver injury, immune injury, systemic inflammatory response syndrome (sirs), cytokine storms, ischemia and hypoxia reperfusion injury, and drug-induced injury may be the main mechanisms that cause secondary liver injury in patients with covid-19 [11] [12] 14, 27 . in addition to receptor-mediated viral infection, antibody-dependent enhancement of infection (ade) may occur in patients with sars 32 . ade refers to the interaction of a virus-specific antibody with fc receptor (fcr) and/or complement receptor (cr) to enhance the ability of the virus to enter granulocytes, monocytes, and macrophages. the virus constantly replicates in the above cells, resulting in increased virus production and aggravating infection. previous studies have reported that antibodies against the sars-cov spike protein trigger ade, causing sars-cov to enter immune cells that do not express ace2 and immune damage 33 . the liver contains a large number of cells related to the immune response. whether ade can also mediate sars-cov-2 to this article is protected by copyright. all rights reserved. infect immune cells by a non-ace2-dependent pathway and participate in liver injury caused by sars-cov-2 is a concern. systemic inflammatory response syndrome (sirs) and cytokine storms( as the liver, myocardium and kidney. these results suggest that the sirs and cytokine storms caused by sars-cov-2 infection may be one of the important mechanisms of liver injury. patients with covid-19 have varying degrees of hypoxemia, with more than 40% requiring oxygen therapy 5 drug hepatotoxicity( figure 2) in china, the incidence of drug-induced liver injury is second only to viral hepatitis and fatty liver disease (including alcoholic and non-alcoholic). drugs commonly causing liver injury include traditional chinese patent medicines containing components of saikosaponins [38] [39] , antitumor drugs, antituberculosis drugs, antimalarial drugs and antibiotics [40] [41] . most patients with covid-19 have fever, and many of them use antipyretic and analgesic drugs, which contain this article is protected by copyright. all rights reserved. acetaminophen and are known to cause liver damage, overdose of these drugs can cause liver damage. currently, there is no clearly effective antiviral drug, and many patients in the outbreak were given lopinavir, abidor, ritonavir and other antiviral drugs. the latest study published in jci 42 reported that cyp3a4 plays an important role in ritonavir's mediating of hepatotoxicity, and the cyp3a metabolic pathways can produce electrophilic content, oxygen free radical, etc, they can be covalent binding with macromolecular substances within the liver cells, cause system membrane lipid peroxidation, destruction of membrane integrity and membrane ca 2 + -atpase, disrupt cell internal and external ca 2 + homeostasis, influence function of critical organelles such as mitochondria, endoplasmic reticulum, and eventually lead to liver cell damage and even death. moreover, the combination of overdose of lopinavir and ritonavir can activate the endoplasmic reticulum stress pathway in the liver, inhibit the proliferation of hepatocytes, induce apoptosis of hepatocytes through the caspase cascade system, induce inflammatory reactions and accelerate liver injury by aggravating oxidative stress [43] [44] . some scholars believe that hiv protease inhibitors can effectively suppress sars-cov-2 replication, nevertheless, shen's team confirmed that the risk of liver damage was increased in patients taking both hormones and hiv protease inhibitors 45 . the incidence of liver injury caused by different drugs is varies, but the incidence increases with the increase in drug types. the diagnosis of drug-induced liver injury requires the combination of medical history and relevant tests to exclude other liver diseases and assess the association between liver injury and suspected drugs by causality. chronic liver disease is a major disease burden globally. liver diseases, including chronic viral hepatitis, alcohol-related liver disease, non-alcoholic fatty liver disease, and autoimmune hepatitis, affect approximately 300 million people in china. a retrospective analysis of 333 covid-19 inpatients 19 showed that 12 accepted article had a history of hepatitis b and that 2 had a history of hepatitis c. in another study of baseline liver biochemical parameters in 324 cases in the shanghai area, the hbsag-positivity rate in covid-19 patients reached 6.5% 46 for covid-19 patients with mild liver biochemical abnormalities, the primary disease should be treated actively when antiviral and supportive treatments are given to inhibit viral replication, reduce inflammation and improve immunity, and preventive application of liver-protecting and enzyme-lowering drugs is not recommended. with acute liver injury, clinicians should analyze and judge the causes of liver injury and take appropriate measures, while closely monitoring alt, ast, total bilirubin, direct bilirubin, albumin, and pta (inr). the occurrence of acute liver failure should also be identified, and liver-protecting and enzyme-lowering drugs, for which the composition and mechanism of action are relatively clear, should be chosen. nonetheless, too many drug types should not be administered (generally no more than 2), and the dosage should not be too large. for patients with severe and critical covid-19 disease with liver injury, which should be considered to be caused by cytokine storms and microcirculation ischemia and hypoxia, respiratory and circulatory support should be strengthened. if necessary, extracorporeal membrane oxygenation should be performed to improve the blood oxygen saturation of patients. patients with acute liver failure should be given intensive monitoring and symptomatic and supportive treatment, and hypoproteinemia should be corrected. in cases of liver injury caused by drugs, in addition to conventional anti-inflammatory liver protection treatment, stopping or reducing the amount of the suspected drugs should be considered, and the degree of liver damage should be assessed, followed by adjustment of the treatment plan. anti-hbv or anti-hcv treatment should not be discontinued, but large doses of hormones should not be used simultaneously. overall, the prevention and treatment experience of liver injury in sars patients can provide some reference for covid-19 patients with liver injury. for example, active prevention and control of the inflammatory response in the early stage of the disease is not only conducive to reducing the nonspecific inflammation of the liver this article is protected by copyright. all rights reserved. but also to preventing the occurrence of systemic inflammatory response syndrome to reduce the probability of mild disease developing into severe or critical disease. covid-19 combined with liver injury is very common, and the incidence of liver injury in patients with severe or critical covid-19 disease is higher than that in patients with mild disease. liver injury in patients with covid-19 may be caused by a variety of mechanisms, such as direct virus infection, immune injury, drug-induced liver injury, systemic inflammatory response, ischemia and hypoxia, and recurrence or exacerbation of the underlying liver disease, among others. since the liver is an important processing organ of the body, impaired liver function seriously affects the body's anabolism and prognosis of the disease. therefore, clinicians should pay more attention to the occurrence of liver damage in the diagnosis and 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nicolls, r.; mcgonigle, j.; kelly, m.; banerjee, r. title: high liver fat associates with higher risk of developing symptomatic covid-19 infection initial uk biobank observations date: 2020-06-05 journal: nan doi: 10.1101/2020.06.04.20122457 sha: doc_id: 275637 cord_uid: ea6w2kqv background a high proportion of covid-19 patients develop acute liver dysfunction. early research has suggested that pre-existing fatty liver disease may be a significant risk factor for hospitalisation. liver fat, in particular, is a modifiable parameter and can be a target for public health policy and individual patient plans. in this study we aimed to assess pre-existing liver disease as a risk factor for developing symptomatic covid-19. methods from 502,506 participants from the uk biobank, 42,146 underwent mri (aged 45-82), and had measures of liver fat, liver fibroinflammatory disease and liver iron. patients were censored on may 28th to determine how many had tested for covid-19 with symptomatic disease. uk testing was restricted to those with symptoms in hospital. covid-19 symptoms included fever, dry cough, sore throat, diarrhoea and fatigue. univariate analysis was performed on liver phenotypic biomarkers to determine if these variables increased risk of symptomatic covid-19, and compared to previously described risk factors associated with severe covid-19, including to age, ethnicity, gender and obesity, findings increased liver fat was associated with a higher risk for symptomatic confirmed covid-19 in this population in univariate analysis(or:1.85, p=0.03). in obese participants, only those with concomitant fatty liver([≥]10%) were at increased risk(or:2.96, p=0.02), with those having normal liver fat (<5%) showing no increased risk(or:0.36, p=0.09). conclusions uk biobank data demonstrated an association between pre-existing liver disease and obesity with severe covid-19, with higher proportions of liver fat in obese individuals a likely risk factor for symptomatic disease and severity. public policy measures to protect patients with liver disease who may have almost double the risk of the general population should be considered, especially as dietary and pharmacological strategies to reduce body weight and liver fat already exist. funding university of oxford, innovate uk, uk biobank. authors are employees of perspectum ltd. coronavirus disease is an infectious disease caused by the recently discovered sars-cov-2 coronavirus. risk factors leading to severe infection have been identified in previous studies around the world (1-3) including increased age, male sex, non-caucasian ethnicity and the presence of pre-existing co-morbidities, in particular cardiovascular and metabolic conditions, particularly those conditions involving liver function. the majority of patients experiencing covid-19 developed abnormal liver function(4), with studies reporting abnormal alanine transaminase (alt), aspartate aminotransferase (ast), albumin levels, and increased gamma-glutamyl transferase (ggt) serum in severe patients (6) . the development of liver injury was more common in severe covid-19, with liver injury in mild disease often transient and resolving without treatment. the mechanistic link between covid-19 and the liver may relate to the expression of the ace2 protein, through which sars-cov-2 is believed to enter cells (7) in cholangiocytes (8) . however, recent studies have also shown a hepatocellular profile of damage rather than cholestasis (9) . alternative hypotheses suggest that liver injury in severe cases is being caused by an immune response triggered by the infection (6) or due to a drug-induced liver injury from the antivirals, antibiotics and steroids widely being used for treating covid-19 (10) . traditionally, characterisation of chronic liver disease has relied on invasive liver biopsy to evaluate the pathological hallmarks of disease: fibrosis, inflammation, and steatosis (11) . more recently, scalable, non-invasive alternatives with precise and accurate quantitative measurements have been developed using mri biomarkers of liver fat (also known as proton density fat fraction, pdff, and fibroinflammation, ct1). multiparametric mri measuring liver fat and fibroinflammation has demonstrated value in characterising liver disease (12, 13) and predicting clinical outcomes (14) . this has allowed for determination of the current prevalence . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . of fatty liver disease in the uk adult population, along with genetic associations with liver fat and fibroinflammation (15) . the uk biobank has recently released covid-19 data on testing status for a subset of participants, thus offering a unique resource for investigating the associations between demographic, phenotypic and genotypic factors with covid-19 disease severity and related healthcare outcomes. the aim of this study was to test the hypothesis that liver disease, and specifically liver fat accumulation, is a risk factor for developing symptomatic covid-19. 42,146 participants were extracted from the uk biobank (ukb) imaging study with complete liver imaging to measure fat and fibroinflammatory disease. of these, 287 had a covid-19 test result available with 79 testing positive and 208 testing negatives (figure 1 ). patient meta-data including information on covid-19 testing status, demographics, diabetes status, cardiometabolic risk factors and genetic variants associated with liver disease were available. no biochemistry data was available for alt, ast, albumin or ggt levels. mri measurement of liver fat (proton density fat fraction, pdff, measured in %) and fibroinflammation (iron corrected t1 mapping, ct1, measured in ms) biomarkers were obtained. both liver fat and ct1 have been included in the large cohort uk biobank study (ukb) (16, 17) due to the repeatability and reproducibility of livermultiscan (18) , and suitability for large-scale population imaging. liver fat and ct1 are both continuous variables, with population reference ranges and cut-offs for disease previously described. liver fat >5% is diagnostic of fatty liver disease (19) ; liver fat >10% is considered to qualify as severe fatty liver disease and is a commonly used inclusion criteria in clinical trials of patients with non-alcoholic steatohepatitis (nash) (20) . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint a liver ct1 greater than 825ms has been shown to predict liver-related clinical outcomes (14) , comparably to histological fibrosis staging. ukb has released covid-19 test results from 16 march 2020 onwards, with the majority of sars-cov-2 positive tests coming from patients with symptoms in hospital due to the nature of uk testing. we accessed ukb to determine datasets related to liver health using application 9914 (determining the outcomes of people with liver disease). the ukb has approval from the north west multi-centre research ethics committee (mrec) and obtained written informed consent from all participants prior to the study. statistical analysis was performed using r software (version 3.6.1) with a p-value less than 0.05 considered statistically significant. descriptive statistics were used to summarise baseline participant characteristics. mean and standard deviation (sd) were used to describe normally distributed-continuous variables, median with interquartile range for non-normally distributed, and frequency and percentage for categorical variables. mean difference in biomarker values between those who tested positive for covid-19 and those who did not were compared using the mann-whitney-u test, and difference in the counts of the binary outcomes of the self-reported conditions were compared using chi-squared tests. to discriminate patients who tested positive from those who did not, we measured for count data computing the summary measures of risk and a chi-squared test for difference in the observed proportions from count data, presented in a 2 by 2 table with the function epi.2by2 from the library epir in r. subsequently, univariable logistic regression analysis was performed for all the potential predictors which included liver fat, ct1, age, sex, ethnicity, body mass index (bmi), self-reported diabetes and self-reported hypertension status. this was performed using . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint the glm function in r and the brglm2 tool that aims to reduce the bias observed when results are sparse. risk scores were extracted from the odds ratio estimates of having a positive rest as calculated in the logistic regression model with confidence intervals calculated with the wald's test. pearson's chi-square test of independence was used to investigate the relationship of the genetic variation and the result on the covid-19 test. participant characteristics for each of the groups evaluated in this study are reported in table1. in the symptomatic and tested population, male participants were more likely to test positive for covid-19. comparing those that tested positive to the untested population, being male, non-white-british, and diabetic all increased likelihood of being symptomatic and receiving a positive covid-19 test. the median age of those being symptomatic and testing positive was lower than in the untested population. table 2 . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint furthermore, the 32.7% of obese patients with liver fat ≥10% had a higher likelihood of being symptomatic and testing positive for covid-19 (or: 2.96, p=0.02). in contrast, the 37.2% of obese patients with normal liver fat (or: 0.36, p=0.09) did not (table3). the aim of this study was to explore the hypothesis that pre-existing liver disease increases the risk of having symptomatic covid-19. we report on a cohort of 42,146 participants from the ukb with complete liver imaging, of which 287 have covid-19 test results available with 79 participants testing positive. our study demonstrates that in addition to the previouslyreported risk factors of male gender, non-white-british ethnicity, and obesity (1-3), liver fat is also a significant risk factor for having symptomatic covid-19, with a person testing positive for covid-19 being 1.85 times more likely to have pre-existing severe fatty liver disease. to put that into context, 4743 people in this cohort of 42,146 had severe fatty liver disease, suggesting that 11% of the uk population carries this higher risk for covid-19. several risk factors have been identified from early studies in china (1), europe (2) and the us (3) that lead to severe infection, including increased age, male sex, non-caucasian ethnicity and the presence of pre-existing co-morbidities, such as cardiovascular and metabolic conditions, including diseases of the liver. unlike previous studies, our research does not find increasing age as a risk factor in this cohort, but this is likely an artefact of the limited age range in the ukb population and increasing likelihood of exposure to the virus in people of working age versus those that had recently retired. in line with our findings, previous studies have reported bmi as a risk factor for covid-19, but the mechanism(s) of excess weight in conferring additional risk remain unclear. in this study, more than a third of obese participants (37.2%) had normal liver fat and were not at increased . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint risk of being symptomatic and testing positive for covid-19. in contrast, obese patients with liver fat ≥10%, representing approximately one third of the obese population, did have significantly higher odds of being symptomatic and testing positive for covid-19. this highlights the important independent role of liver fat, in addition to obesity, in the severity of, and susceptibility to, covid-19. due to the nature of uk testing for covid-19 over the period these data were collected, only those symptomatic for the disease to the extent that hospitalisation was warranted will have had test results available. given the high prevalence of fatty liver disease in the general population, combined with the utility of lifestyle-interventions (22) , and new therapeutics that have demonstrated clear reversal of fatty liver disease in weeks, patients with high liver fat may benefit from going on calorie restriction diets and pharmacotherapeutic interventions. investigational therapies such as ngm282 and resmetirom have been shown to reduce liver fat in a matter of weeks (23, 24) , but need to be trialled specifically for utility in this context of use. more generally, public health measures should be considered to protect the 11% of the uk population that has liver fat greater than 10%, as their risk of experiencing covid-19 with sufficient severity to require hospitalisation has more than doubled. the main limitation of our study is the relatively small proportion of ukb participants for which testing results are available. this analysis was performed on the second release of covid-19 testing data and subsequent analyses as more data are released will provide additional power to investigate a larger number of exposures. this limitation is largely a result of uk policy at the time to only test those patients requiring hospitalisation; however, one benefit of this is policy is the ability to infer that those participants who tested positive had severe covid-19 disease requiring hospitalisation. this inference will not be possible in future releases of . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint covid-19 testing data following changes in the uk testing policy to support widespread testing. this study highlights the potentially important role of liver disease in covid-19 severity. as more data becomes available through the ukb, further investigations into the associations of liver health with other demographic characteristics and co-morbidities may reveal more detailed relationships, for example, between multiple risk factors and the development of severe covid-19. given the high prevalence of fatty liver disease, and the availability of new treatments specifically for fatty liver disease, these results have the potential to inform public health policy around the management of this at-risk population. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint abbreviations: covid-19, coronavirus disease. . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june 5, 2020. . . cc-by-nd 4.0 international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june 5, 2020. . https://doi.org/10.1101/2020.06.04.20122457 doi: medrxiv preprint epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study. the lancet liver injury in covid-19: management and challenges cell entry mechanisms of sars-cov-2 specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection. biorxiv non-alcoholic fatty liver diseases in patients with covid-19: a retrospective study covid-19 and drug-induced liver injury: a problem of plenty or a petty point? the nas and the histopathologic diagnosis in nafld: distinct clinicopathologic meanings. hepatol baltim md multiparametric magnetic resonance for the non-invasive diagnosis of liver disease multiparametric magnetic resonance imaging for the assessment of non-alcoholic fatty liver disease severity multiparametric magnetic resonance imaging predicts clinical outcomes in patients with chronic liver disease genomewide and mendelian randomisation studies of liver mri yield insights into the pathogenesis of steatohepatitis uk biobank: an open access resource for identifying the causes of a wide range of complex diseases of middle and old age characterisation of liver fat in the uk biobank cohort repeatability and reproducibility of multiparametric magnetic resonance imaging of the liver dallas steatosis index identifies patients with nonalcoholic fatty liver disease hepatic fat-content assessment using magnetic resonance-based methods opensafely: factors associated with covid-19-related hospital death in the linked electronic health records of 17 million adult nhs patients. medrxiv nutrition and nonalcoholic fatty liver disease: current perspectives. gastroenterol clin ngm282 improves liver fibrosis and histology in 12 weeks in patients with nonalcoholic steatohepatitis. hepatol baltim md mgl-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial key: cord-305956-l02xdq87 authors: alqahtani, saleh a; schattenberg, jörn m title: liver injury in covid-19: the current evidence date: 2020-05-26 journal: united european gastroenterol j doi: 10.1177/2050640620924157 sha: doc_id: 305956 cord_uid: l02xdq87 patients with novel coronavirus disease 2019 (covid-19) experience various degrees of liver function abnormalities. liver injury requires extensive work-up and continuous surveillance and can be multifactorial and heterogeneous in nature. in the context of covid-19, clinicians will have to determine whether liver injury is related to an underlying liver disease, drugs used for the treatment of covid-19, direct effect of the virus, or a complicated disease course. recent studies proposed several theories on potential mechanisms of liver injury in these patients. this review summarizes current evidence related to hepatobiliary complications in covid-19, provides an overview of the available case series and critically elucidates the proposed mechanisms and provides recommendations for clinicians. in the current pandemic coronavirus disease (covid19) , almost every country in the world has now registered covid-19 cases, and the confirmed cases have exceeded one million to date. while initial clinical studies, especially from china, the usa and italy, have highlighted the dominant clinical symptoms including fever, cough, fatigue and shortness of breath, the later research unveiled shreds of evidence on the extrapulmonary manifestations of the disease. these reports highlighted that beyond severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a complicated course of the disease or even viral infection itself can lead to involvement of other organs and multiorgan failure. the liver is the primary organ for detoxification and metabolism, and maintaining an optimal function is imperative to engage all available therapeutic modalities in the treatment of covid-19. abnormal liver function requires clinical evaluation, continuous surveillance and, potentially, specific therapy. to support clinical decision making and optimize the outcome in the treatment of covid-19, it will be crucial to clearly understand the possible mechanisms involved in liver injury. the current review summarizes the pathophysiology and potentially specific role of covid-19 in liver disease based on the available data and case series published, ahead of print and non-peer-reviewed preprints as of 2 april. the search strategy is detailed in the supplementary material online. emerging data from small clinical case studies have proposed that liver injury in covid-19 is frequently seen, but the extent and underlying mechanisms remain undetermined. figure 1 summarizes the pathophysiological findings, which are discussed below. the liver exerts a crucial function in host defense against microbes and is involved in most systemic infections as it receives both the portal and systemic circulation. certain viruses exert a direct cytopathic effect on hepatocytes and cholangiocytes although, in most cases, the pathogenesis seems multifactorial. yang et al. reported that sars-cov could cause direct cytopathic liver injury rather than inducing cellular stress from low oxygen supplies or cytokines as seen in sepsis. 1 autopsy studies in patients revealed that sars-cov was detectable in 41% of the liver tissue, with a maximum viral load of 1.6 â 10 6 copies/g of tissue. 2 the pathological findings of liver biopsy specimens from sars patients showed hepatocellular necrosis, mitoses, cellular infiltration and fatty degeneration. in a recent autopsy analysis of liver tissue from a patient with covid-19, moderate microvesicular steatosis and mild inflammation in the lobular and portal area was observed. however, this pattern of histological injury is not specific for one etiology but can also be observed during sepsis or drug-induced liver injury (dili). 3 similar to sars-cov, sars-cov-2 uses the angiotensin-2 converting enzyme (ace2) receptor protein to attack the host system. 4 the cell entry receptor, ace2, is widely expressed across the human body, including the lungs (type ii alveolar cells), gastrointestinal tract (esophageal epithelial cells and absorptive enterocytes of ileum and colon), hepatobiliary system (hepatocytes and cholangiocytes), cardiovascular system (myocardial cells), the renal system (proximal tubule cells and urothelial bladder cells) and the pancreas. 5 recent studies have observed that ace2 expression in the cell clusters of cholangiocytes was significantly higher than that in the hepatocytes population (59.7% vs. 2.6%). 6 the authors conclude that sars-cov-2 may directly bind to ace2 positive cholangiocytes, but not hepatocytes, to exert a cytopathic effect. cholangiocytes are involved in many aspects of liver physiology, including regeneration and adaptive immune response mechanisms, and the disruption of cholangiocyte function can cause hepatobiliary damage. this is supported by cholestatic markers, including gamma-glutamyl transferase (ggt), that can be found in some, but not all, case series of covid-19. [7] [8] [9] notably, a recent review reported unpublished data with ggt elevations in 54% of cases. 8 in a human organoid model of liver ductal organoids, permissiveness to sars-cov-2 infection was observed. here viral infection impaired the barrier and bile acid transporting functions of cholangiocytes through dysregulation of genes involved in tight junction formation and bile acid transportation, supporting the susceptibility of cholangiocytes in sars-cov-2-related liver injury. 10 activation of the immune system in covid-19 dysregulation of the innate immune response can be one aspect of liver injury in covid-19. patients with covid-19 exhibit marked activation of inflammatory markers, including abnormal levels of c-reactive protein (crp), lymphocytes, neutrophils and cytokines, in particular interleukin-6 (il-6). 8, [11] [12] [13] these mechanisms may contribute to pulmonary and extrapulmonary injuries 12, 14 and the control of cytokine dysregulation at an early stage could be beneficial to curb the disease progression. 15 hepatic inflammation involving activation of innate immune cells and the release of cytokines is a well-established driver of liver injury from various causes. 16 in some of the available case series of covid-19, a correlation between lymphopenia and liver injury was observed and crp !20 mg/l and a lymphocyte count <1.1 â 10 9 /l were independent risk factors for liver injury. notably, lymphopenia in covid-19 studies was reportedly observed in 63% to 70.3% of patients and those with lower lymphocyte counts more susceptible to fatal outcomes. 11 more than 20 publications to date reported abnormal levels of aminotransferases in patients with covid-19. [7] [8] [9] [11] [12] [13] [17] [18] [19] 21, [23] [24] [25] [26] [27] [29] [30] [31] [32] [33] 35, 36 a recent systematic review and meta-analysis on lft abnormalities provided a pooled elevation of aspartate aminotransferase (ast) in 33.3% and alanine aminotransferase (alt) in 24.1% of cases. 39 various investigators across different studies reported a correlation between the severity of covid-19 and the degree of liver dysfunction. 8, 11, 25 in one retrospective study, one patient experienced severe hepatitis with alt of 7 on the other hand, mild and moderate cases experienced only discrete abnormal lft values. these reports support the concept that the disease severity and an older age predispose to more severe liver injury from covid-19. based on these case series, patients with severe covid-19 and pre-existing liver conditions 8but also elderly patients 11 -should undergo surveillance and individually tailored therapeutic approaches for potential liver injury. a recently published article by bangash et al. argues after careful review of seven relevant studies that elevated alt and ast may not necessarily be of hepatic origin alone. the authors have given a timely reminder that it is common for other respiratory viruses to create similar lft elevations 28 and thus more prospective data related to the clinical relevance covid-19 and liver injury is required. the previous pathogenic coronaviruses, such as sars-cov and the middle east respiratory syndrome coronavirus (mers-cov), were also reported to manifest with elevated levels of alt and ast. 39 more generally, non-hepatotropic viral infections may affect the liver and induce hepatitis or fulminant acute liver failure. however, in the majority of cases, recovery from viral illness is often sufficient to resolve liver injury. 40 like in sars-cov and mers-cov infections, abnormal levels of albumin and lactate dehydrogenase (ldh) were also reported in sars-cov-2 infection, with the maximum of 98% and 76% of the patients affected as reported in the study by chen et al. 17 it is important to remember that ldh and ast elevation could be from muscle damage and not necessarily reflect liver injury. the current armory of therapeutic agents explored against sars-cov-2 includes several antiviral agents, supportive therapy and trials of alternative medicines in many regions of the world. given the fact that the liver is involved in the metabolism of many drugs, including nucleoside analogs and protease inhibitors that are currently used to treat covid-19, hepatotoxicity from these drugs can arise. a recent randomized controlled trial of lopinavir and ritonavir in severe covid-19 reported that elevated levels of ast, alt and total bilirubin occurred as adverse effects in a few patients. 41 another case series from wuhan reported that 55.4% of patients experienced liver injuries after treatment with lopinavir and ritonavir. 26 fan et al. published a retrospective study on covid-19 and observed that the utilization rate of this drug combination was significantly higher in patients with abnormal lfts compared with patients without lft elevations (56.1% vs. 25%, p ¼ 0.009). in this study, 47.3% of the discharged patients showed elevated lfts at baseline, and 23.7% developed abnormalities during hospitalization, suggesting emerging liver injury from drugs or during the course of the infection. importantly, lft elevation during the hospital stay was associated with prolonged length of hospitalization. 7 chloroquine, an old drug with a potential of repositioning for new treatment indications, has recently been tried in patients infected with sars-cov-2. after a profound success in inhibiting viral replication in vitro, concurrent clinical trials (>20) on chloroquine conducted at 10 hospitals across china have demonstrated superior efficacy in viral control. 42 the pharmacodynamic activity of this drug in covid-19 may involve the arresting of cytokine storms or the activation of cd8þ cells or by preventing endocytosismediated uptake of the virus. 43 importantly, hepatotoxicity related to chloroquine or hydroxychloroquine has rarely been reported. in severe cases of covid-19 with cytokine release, tocilizumab, an il-6 antagonist, which is humanized igg1 monoclonal antibody to the il-6 receptor, has been used as a potential therapy for sars-cov-2. in previous clinical trials for other indications tocilizumab was reported to cause mild elevations of lfts which were usually transient and commonly resolved within 2-6 weeks from exposure. 37 remdesivir is an experimental antiviral nucleotide analog with broad activity against coronaviruses 44 that is currently being trialed for sars-cov-2 infection. safety data from ongoing studies will guide on its use in patients, but so far no reports of liver toxicity have emerged. patients with pre-existing liver disease scare data has been published for covid-19 infection in patients with pre-existing liver disease. experience from previous episodes of coronavirus infection can guide on the extent of hepatic involvement and on the management of patients with pre-existing liver disease. in sars, the highest mortality rates were observed in the elderly and adults with underlying liver disease. 45 therefore, it has to be expected that the patients with covid-19 are also more vulnerable to hepatic injury. 9 in a case series from the zhejiang province, a prevalence of 11% of underlying liver disease was reported. about half of them experienced symptoms for more than 10 days after the illness onset. 21 in another study from wuhan, 9% of patients had the underlying liver disease of cirrhosis or hepatitis. 23 li et al., who investigated risk factors involved with hepatic injury, stated that two patients had presented with alcoholic liver disease at baseline. one of them had a moderate elevation of alt (120 u/l) within a week of hospitalization, while the other showed no such abnormalities. 11 in the initial cohort described from china, 2.7% exhibited hepatitis b virus infection with no mention of worsening outcomes. 26 therefore, the association of the pre-existing liver conditions with disease prognosis and outcomes in covid-19 will have to be evaluated by comprehensive data registries which recently started enrolling patients (e.g. covid-hep registry and secure-cirrhosis registry). management of post liver transplant recipients during the covid-19 pandemic presents a special challenge for clinicians because of the limited data available and the crucial need to continue immunosuppressive drugs in these patients, which puts them at risk for more severe courses of covid-19 infection and possible prolonged viral shedding. case reports from china did not reveal an increased mortality in organ transplant recipients. qin et al. reported the first case of sars-cov-2 infection in a patient with hepatocellular carcinoma who underwent liver transplantation. 29 lowering immunosuppression to the most acceptable level appears reasonable in infected liver transplant patients, in particular, in the setting of lymphopenia or clinical worsening of infection. 46 in addition clinicians have to be aware of drug-drug interactions in the transplant setting. in particular immunosuppressive drugs and ritonavir-boosted antiviral therapies exhibit relevant interactions through cyp34a which lead to increased levels of calcineurin and mtor inhibitors. accordingly, chloroquine-based regimes or remdesivir (compassionate use program only) appear to be safe, while boosted protease inhibitors should be avoided (see table 2 ). additionally, preventive strategies in those vulnerable patients include early and prolonged screening with polymerase chain reaction-based testing for patients with early symptoms, a contact history or infection. personal liver function abnormalities -predominantly ast elevation -in covid-19 appear to be frequent but not severe in most cases. direct viral hepatotoxicity, dili, 'bystander effects' during a systemic viral infection and potentially sepsis, or exacerbation of an underlying liver disease have to be considered. ex vivo studies offer that sars-cov-2 can selectively target the liver, in particular cholangiocytes through ace2, and thus hepatobiliary injury appears plausible. irrespective of the mechanisms involved in the hepatic injury of patients with covid-19, activation of the immune-mediated pathway seems to be critical. special high risk populations require close monitoring. these include the elderly population, patients with endstage liver disease and liver transplant recipients. symptomatic treatment with acetaminophen and avoidance of non-steroidal anti-inflammatory drugs in cirrhosis is recommended. cautious use of antiviral agents in patients with decompensated liver disease and drug-drug interactions in post liver transplant patients has to be considered. as emphasized by a recent position paper of the european study of liver disease, elective procedures and routine tests should be postponed according to the risk-benefit at the given time. on the other hand, emergency medical care needs to be done with appropriate measures to prevent infection. 46 declaration of conflicting interests sa has nothing to declare. jms has acted independently of this study as a consultant to boehringer ingelheim, galmed, genfit, gilead sciences, intercept pharmaceuticals, novartis, roche, siemens healthineers, and has received research funding from gilead sciences. clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome fatal severe acute respiratory syndrome is associated with multiorgan involvement by coronavirus pathological findings of covid-19 associated with acute respiratory distress syndrome sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor highly ace2 expression in pancreas may cause pancreas damage after sars-cov-2 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cause for concern perioperative presentation of covid-19 disease in a liver transplant recipient. hepatology 2020. epub ahead of print 27 clinical characteristics of non-icu hospitalized patients with coronavirus disease 2019 and liver injury. a retrospective study liver impairment in covid-19 patients: a retrospective analysis of 115 cases from a single center in wuhan city, china. liver int 2020. epub ahead of print 2 the clinical characteristics of pneumonia patients coinfected with 2019 novel coronavirus and influenza virus in wuhan a comparative study on the clinical features of covid-19 pneumonia to other pneumonias covid-19 and liver dysfunction: current insights and emergent therapeutic strategies clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia. radiology epub ahead of print bethesda (md): national institute of diabetes and digestive and kidney diseases clinical, laboratory and imaging features of covid-19: a systematic review and metaanalysis clinical manifestations, laboratory findings, and treatment outcomes of sars patients systemic viral infections and collateral damage in the liver a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies insights from nanomedicine into chloroquine efficacy against covid-19 prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper the authors received no financial support for the research, authorship, and/or publication of this article. j€ orn m schattenberg https://orcid.org/0000-0002-4224-4703 key: cord-342930-f7cw2ca6 authors: portincasa, piero; krawczyk, marcin; machill, antonia; lammert, frank; ciaula, agostino di title: hepatic consequences of covid-19 infection. lapping or biting? date: 2020-06-01 journal: eur j intern med doi: 10.1016/j.ejim.2020.05.035 sha: doc_id: 342930 cord_uid: f7cw2ca6 the outbreak of coronavirus disease 2019 (covid-19) starting last december in china placed emphasis on liver involvement during infection. this review discusses the underlying mechanisms linking covid-19 to liver dysfunction, according to recent available information, while waiting further studies. the manifestations of liver damage are usually mild (moderately elevated serum aspartate aminotransferase activities), and generally asymptomatic. few patients can still develop severe liver problems, and therapeutic options can be limited. liver dysfunction may affect about one-third of the patients, with prevalence greater in men than women, and in elderly. mechanisms of damage are complex and include direct cholangiocyte damage and other coexisting conditions such as the use of antiviral drugs, systemic inflammatory response, respiratory distress syndrome-induced hypoxia, sepsis, and multiple organ dysfunction. during new covid-19 infections, liver injury may be observed. if liver involvement appears during covid-19 infection, however, attention is required. this is particularly true if patients are older or have a pre-existing history of liver diseases. during covid-19 infection, the onset of liver damage impairs the prognosis, and hospital stay is longer. a novel coronavirus was reported to world health organization on dec 30, 2019, as the cause of a 2 cluster of pneumonia cases in china, city of wuhan, hubei province. the first name of 2019-3 ncov(human) was adopted on jan 7, 2020, lately changed to severe acute respiratory syndrome 4 coronavirus 2 (sars-cov-2). covid-19 infection became an outbreak throughout china on feb 5 11, 2020 and subsequently was identified as a global pandemic on march 11, 2020, spreading to 6 more than 120 countries, as a major threat to public health [1] [2] [3] . the covid-19 pandemic suddenly 7 represented an enormous burden of care [4] , and raised issues related to medical ethics [5] , since 8 specific therapies and/or vaccines are missing, to date. covid-19 may manifest in different ways. 9 many subjects may remain asymptomatic [6] , but the exact number is still unknown. specific 10 settings might facilitate the spread of infection e.g., in skilled nursing facility where more than half 11 of residents with positive test results were asymptomatic at the time of testing and most likely 12 contributed to transmission [7, 8] . the proposed 3-stage classification system of potential increasing 13 severity for covid-19 infection encompasses stage i (early infection), stage ii (pulmonary phase), 14 and stage iii (hyperinflammation phase) [9] . although the most frequent and critical clinical 15 presentation is secondary to the involvement of the lung (fever, cough), the infection by sars16 cov-2 virus may lead to a systemic and multi-organ disease [10] , also involving the gastrointestinal 17 tract (nausea/vomiting, or diarrhea) [11, 12] . the liver appears to be the second organ involved, 18 after the lung [13] [14] [15] . 19 the present paper explores the available evidences on liver involvement in patients with covid-19 20 infection, to provide a comprehensive understanding of the phenomenon, and to anticipate effective 21 follow-up. during covid-19 infection, patients can be asymptomatic or present clinical symptoms ranging 25 from fever, dry cough, headache to dyspnea and fatigue, to acute respiratory distress syndrome 26 6 (ards), shock, and cardiac failure [9, 16] . a nasopharyngeal swab is the collection method used to 1 obtain a specimen for testing. because the likelihood of the sars-cov-2 being present in the 2 nasopharynx increases over time, repeated testing is often used [17] . multi-organ involvement 3 secondary to covid-19 infection occurs in a subgroup of patients [10] . covid-19 infection can be 4 associated with myocardial injury [18] [19] [20] , heart failure [18] , vascular inflammation, myocarditis, 5 cardiac arrhythmias [19] , and hypoxic encephalopathy [21] . the progression and prognosis of 6 covid-19 infection is worse in the presence of diabetes mellitus [22, 23] . the case-fatality rate 7 increases with age (from 8% to 15% in the age range 70-79 years, and ≥80 years, respectively) and 8 with associated diseases, i.e., 11%. 7%, 6%, 6%, and 6% in patients with cardiovascular disease, 9 diabetes mellitus, chronic respiratory disease, hypertension, and cancer, respectively [ another study in zhejiang province [26] . gastrointestinal involvement could be the consequence of 15 covid-19-angiotensin-converting enzyme 2 (ace2) receptors at the enterocyte level (i.e. 16 glandular cells of gastric, duodenal and distal enterocytes), resulting in malabsorption, unbalanced 17 intestinal secretion and activated enteric nervous system, therefore diarrhoea) [28, 29] . in human 18 small intestinal organoids, sars-cov-2 rapidly infects the enterocytes and strongly induces a 19 generic viral response program, pointing to a marked viral replication in the intestinal epithelium 20 [30] . 21 notably, continuous viral rna shedding occurs into feces up to 11 days negativity of respiratory a study reported that the virus can be detected but not cultivated from stool (despite high rna 1 concentration), consistent with the lack of transmission [33] . in a case-control study from usa 2 (enrolling 278 covid-19 positive patients and 238 covid-19 negative patients), the presence of 3 gastrointestinal symptoms was predictive of covid-19 positivity, and symptoms were associated 4 with slower and less severe disease course [34] . when the virus binds to ace2 receptors [35] [36] [37] to enter the target cell [38] . receptors are well 10 expressed in epithelia of the lung, gastrointestinal tract, and vascular endothelium, also in the liver 11 [39]. this early period of covid-19 infection can evolve to the second stage of viral pneumonia.; (ii) extra-pulmonary systemic hyperinflammation syndrome occurs in the minority of infected 13 patients, and is characterized by the so-called "cytokine storm". at this moment, several cytokine 14 levels increase, namely interleukin (il)-2, il-6, il-7, il-10, and tumor necrosis factor (tnf)α. 15 additional inflammatory biomarkers include granulocyte-colony stimulating factor, interferon 16 (ifn)-γ inducible protein 10, monocyte chemoattractant protein 1, macrophage inflammatory patients with severe and fatal disease had significantly increased white blood cell count, and 24 decreased lymphocyte and platelet counts compared to non-severe disease and survivors. biomarkers of inflammation, muscle and cardiac injury, as well as liver and kidney function and year in 43% of the 99 covid-19 cases from wuhan [48] . this aspect deserves further attention. although the level of serum transaminases could be already elevated before the onset of covid-14 19, results from clinical reports and autopsy studies [26, 49, 50] suggest that liver dysfunction can 15 be an expression of a worse disease evolution, and that an isolated elevation of transaminases alone 16 is likely to be the indirect expression of a systemic inflammation. previous data from covid-19 outbreak in china found that 2-11% of patients had liver 18 comorbidities, 14-53% of patients presented with abnormal serum aminotransferases levels during 19 the disease, and that the rates of liver dysfunction were more present in subjects with the most 20 severe clinical presentation [26, 49] . in another large series of 417 chinese covid-19 patients, 21 abnormal liver tests (ast, alt, total bilirubin, ggt) were present in 76.3% of patients and 21.5% 16 china, in 6% to 22% and 21% to 28% of patients, respectively. in studies from wuhan, ast levels 17 were increased in 24% to 37% of patients, a proportion higher than in other chinese regions 18 (zhejiang), reporting a proportion of 16%. a gender difference might exist in this respect [62] , since 19 the prevalence of ast increase is higher in men than women, as documented by six case series (i.e., 20 average 66% vs. 35%, respectively). case reports and case series also suggest that the probability of 21 developing liver dysfunction increases with older age [61] . notably, the elevation of additional elements possibly concurring to liver damage are drug-related injury and the progression 14 of underlying liver diseases. 15 it is still under debate if these alterations can really be an expression of a clinically relevant liver 16 injury requiring particular attention in the management of the disease [13, 68]. in one study, patients 17 developing abnormal liver tests had higher risks of progressing to severe disease [51] , and the 18 finding is associated with longer hospital stay [62] . in addition, the more severe form of covid-19 19 infection is a predisposing condition to a more evident liver damage [10, 49, 69] , and therefore also promoted by systemic sepsis [71] . viral inclusions seem to be absent in the liver [57] , but this 2 possibility deserves further investigations, because of potential viral rna translocation from 3 intestine though portal blood. 4 another possibility is the direct damage from covid-19. cholangiocytes express ace2 receptors 5 (more that 20-fold than in hepatocytes). although cell damage can also occur at the level of bile covid-19 infection can progress to the inflammatory cytokine storm [75] , which involve both the 13 innate (toll-like receptors, tlrs) and the cellular adaptive immunity (killer t lymphocytes) [76, increased neutrophil counts and neutrophil to lymphocyte ratios, as well as hyperferritinemia [10] . elderly patients go worse, in this respect [79] . this sudden and immense immune hyperactivation 24 may result in multiple organ failure lungs but also to the liver, heart, and kidneys [75] . serum levels liver disorder in western industrialized countries, (prevalence ranging from 10 to 46% in the united 1 states [89-91]) and a median of 20% worldwide with a documented rising trend with time [92] . this 2 trend in north america and europe is the consequence of the rising prevalence of major risk factors 3 for nafld, including obesity, sedentary lifestyles, type 2 diabetes mellitus, dyslipidaemia, and 4 metabolic syndrome [92] [93] [94] [95] . however, lean non-alcoholic steatohepatitis (nash) can develop as 5 well [94] and is frequent in asia [96] . overall, factors contributing to nafld include the 6 environment, the gut microbiome, disrupted gluco-lipid metabolic pathways, metabolic was more frequent among those receiving lopinavir/ritonavir after hospital admission [62] . 14 remdesivir, a nucleoside analog prodrug developed by gilead sciences (usa), is effective against 15 covid-19 replication in vitro [116] and in infected patients [117] . this drug produced similar 16 effects on liver enzymes [118] . hydroxy chloroquine sulphate is also effective in vitro [116] and, in 17 covid-19 patients for short periods, appears to safe. rare case of fulminant hepatic failure have 18 been described with hydroxy chloroquine [119, 120] . acute liver injury is also possible after 19 azithromycin treatment, with a clinically evident presentation following about two weeks after drug 20 cessation, and after an average duration of treatment of 4 days [121] . several patients with 21 concomitant diseases (i.e. diabetes type 1 or 2, or hypertensive), undergo antihypertensive therapies 22 with ace inhibitors and angiotensin ii type i receptor blockers. in this context, a possibility is the 23 onset of ace2 overexpression. whether this condition will facilitate covid-19 infection and 24 penetrance, deserves further attention [122, 123] . there is no evidence, however, that ace 25 inhibitors will worsen the consequence of infection [123] . many patients with fever use antipyretic 1 agents, namely acetaminophen [124] . this drug might mediate, at least in part, the liver damage [57] . 2 patients with underlying metabolic abnormalities and nafld might be more exposed to drug-3 induced liver damage (dili) [99, 108]. as mentioned earlier, the cytokine mcp-1 is often increased 4 in covid-19 patients [42] and act as a further hit for steatohepatitis [125] . in addition, patients with 5 nafld/nonalcoholic steatohepatitis (nash) covid-19 infection, might be more susceptible to 6 dili, as well as to therapy with steatogenic drugs (amiodarone, sodium valproate, tamoxifen and world health organization. coronavirus disease 2019 (covid 19) association of coronavirus disease prominent changes in blood coagulation of patients 12 with sars-cov-2 infection abnormal 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management of patients 36 with autoimmune liver disease during covid-19 pandemia the covid-19 pandemic will have a long-lasting impact on the quality of 38 cirrhosis care covid-19 and 40 drug-induced liver injury: a problem of plenty or a petty point establishment of a new animal model of 1 azithromycin-induced liver injury and study the molecular pathological change during the process acute hepatocellular injury associated with azithromycin lopinavir/ritonavir 6 induces the hepatic activity of cytochrome p450 enzymes cyp2c9, cyp2c19, and cyp1a2 but 7 inhibits the hepatic and intestinal activity of cyp3a as measured by a phenotyping drug cocktail in 8 healthy volunteers hydroxychloroquine-induced toxic hepatitis in a patient with systemic lupus 10 erythematosus: a case report association of polymorphisms 12 of cytochrome p450 2d6 with blood hydroxychloroquine levels in patients with systemic lupus 13 in vitro evaluation of hepatotoxic drugs in human hepatocytes from 15 multiple donors: identification of p450 activity as a potential risk factor for drug-induced liver injuries expression and activities of cytochrome p450 enzymes in an age-dependent manner in mouse 19 liver remdesivir and chloroquine effectively inhibit 21 the recently emerged novel coronavirus (2019-ncov) in vitro first case of 2019 novel 23 coronavirus in the united states fulminant hepatic failure secondary to 26 hydroxychloroquine hydroxychloroquine, a less toxic derivative of 28 chloroquine, is effective in inhibiting sars-cov-2 infection in vitro clinical and 30 histologic features of azithromycin-induced liver injury are patients with hypertension and diabetes mellitus at 33 increased risk for covid-19 infection? the lancet misguided drug advice for covid-19 characteristics of and public health responses to the coronavirus disease 36 2019 outbreak in china inflammation in alcoholic and nonalcoholic fatty liver disease: friend or 38 foe? we confirm that there are no known conflicts of interest associated with this publication and there has been 3 no significant financial support for this work that could have influenced its outcome. 4 5 key: cord-348024-n8wn4och authors: lei, fang; liu, ye‐mao; zhou, feng; qin, juan‐juan; zhang, peng; zhu, lihua; zhang, xiao‐jing; cai, jingjing; lin, lijin; ouyang, shan; wang, xiaoming; yang, chengzhang; cheng, xu; liu, weifang; li, haomiao; xie, jing; wu, bin; luo, huiming; xiao, fei; chen, jing; tao, liang; cheng, gang; she, zhi‐gang; zhou, jianghua; wang, haitao; lin, jun; luo, pengcheng; fu, shouzhi; zhou, jihui; ye, ping; xiao, bing; mao, weiming; liu, liming; yan, youqin; liu, ling; chen, guohua; li, hongliang; huang, xiaodong; zhang, bing‐hong; yuan, yufeng title: longitudinal association between markers of liver injury and mortality in covid‐19 in china date: 2020-05-02 journal: hepatology doi: 10.1002/hep.31301 sha: doc_id: 348024 cord_uid: n8wn4och coronavirus disease 2019 (covid‐19) is a new infectious disease. to reveal the hepatic injury related to this disease and its clinical significance, we conducted a multicenter retrospective cohort study that included 5,771 adult patients with covid‐19 pneumonia in hubei province. we reported the distributional and temporal patterns of liver injury indicators in these patients and determined their associated factors and death risk. longitudinal liver function tests were retrospectively analyzed and correlated with the risk factors and death. liver injury dynamic patterns differed in alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), and total bilirubin (tbil). ast elevated first, followed by alt, in severe patients. alp modestly increased during hospitalization and largely remained in the normal range. the fluctuation in tbil levels was mild in the non‐severe and the severe group. ast abnormality was associated with the highest mortality risk compared to other indicators of liver injury during hospitalization. common factors associated with elevated liver injury indicators were lymphocyte count decrease, neutrophil count increase, and male gender. conclusion: the dynamic patterns of liver injury indicators and their potential risk factors may provide an important explanation for the covid‐19‐associated liver injury. because elevated liver injury indicators, particularly ast, are strongly associated with the mortality risk, our study indicates that these parameters should be monitored during hospitalization. this article is protected by copyright. all rights reserved coronavirus disease 2019 (covid-19) is a viral respiratory illness caused by a novel coronavirus (ncov). covid-19 is highly contagious, with more than 2.3 million cases and nearly 163 thousand deaths worldwide on april 21, 2020. (1, 2) the targeting organs of this coronavirus are believed to be the lungs and airways. however, patients often have evidence of damage to other organs, which significantly increases their mortality. (3, 4) the pathophysiological foundation of multiorgan damage may be associated with organ-specific immune response to disseminated coronavirus or secondary to hypoxemia, systemic cytokine storm, and medication. (5) in an attempt to determine the distributional and temporal patterns of liver injury in patients with covid-19 with a focus on clinical significance and determinants of this change, we conducted a multicenter retrospective cohort study that included 5,771 patients in hubei province. in this multicenter retrospective cohort study, patients with covid-19 were admitted to the following in the study. exclusion criteria, which applied to 1,258 patients, were as follows: age younger than 18 or older than 75 years; pregnancy; severe medical conditions, including liver cirrhosis (10 cases), chronic renal dysfunction (above chronic kidney disease stage 3 [ckd 3]), leukemia, cancer, acquired immune deficiency syndrome, acute myocardial infarction during hospitalization, acute pulmonary embolism (due to a long-term pulmonary embolism disease history, long-term bed rest, and coagulopathy) stoke, and acute pancreatitis; or transfer to other hospitals (fig. 1 ). chest computed tomography (ct) or throat-swab specimens were obtained from all patients upon admission. covid-19 was diagnosed by clinical manifestations, chest ct, or real-time polymerase chain reaction (rt-pcr) according to world health organization (who) interim guidance and the new coronavirus pneumonia prevention and control program (5th edition) published by the national this article is protected by copyright. all rights reserved health commission of china. at the time of admission, patients with fever or suspected respiratory infection, plus one of the following clinical manifestations including respiratory rate greater than 30 breaths/minute, severe respiratory distress, or oxygen saturation (spo 2 ) less than 90% on room air, were classified as severe cases. (6) staging of covid-19 and evaluation of organ damages were conducted by a team of physicians. this study was approved by the central institutional ethics committee and ratified by each hospital. a waiver of the requirement for documentation of informed consent was granted for analyzing existing data without interfering with patient treatment. the demographic, clinical characteristics, medical history, laboratory tests, radiological reports, therapeutic intervention, and outcome data were obtained from patients' electronic medical records. clinical symptoms, including fever, cough, fatigue, dyspnea, and comorbidities, were extracted. the laboratory examination included a complete blood count, c-reactive protein (crp), procalcitonin, ddimer, and serum biochemical tests for liver, kidney, heart, and coagulation dysfunction. medical history comprised coexistence of chronic obstructive pulmonary disease (copd), type 2 diabetes mellitus (t2dm), hypertension, coronary heart disease, cerebrovascular disease, chronic liver disease, ckd, cancer, and autoimmune diseases. the unilateral and bilateral lesions in chest ct scan images were recorded and analyzed. personal identification information (e.g., name and id) of the study subjects was anonymized and replaced with a coding system before data extraction. data were reviewed and confirmed by experienced physicians and double-checked to ensure accuracy. acute lung injury (ali), acute respiratory distress syndrome (ards), and septic shock were defined according to the who interim guideline for "clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected." (4) acute kidney injury was defined as an elevation in serum creatinine level ≥26.5 μmol/l within 48 hours. (7) acute liver injury was defined as the levels of serum alanine aminotransferase (alt) above 3-fold of the upper limit of normal (uln). increase in liver enzyme levels was defined as the levels of serum liver enzyme above the uln. cardiac injury was defined as the serum level of cardiac troponin i/t or hypersensitive troponin i/t above the uln. (8) clinical disseminated intravascular coagulation (dic) was defined according to the international society on thrombosis and haemostasis (isth) criteria for diagnosing this article is protected by copyright. all rights reserved dic. (9) the primary endpoint was recorded and evaluated in this longitudinal cohort, which was 28day all-cause death. data were reviewed and confirmed by two certified physicians to ensure accuracy. categorical variables were presented as frequency. continuous variables were described as median (interquartile range [iqr]). means for continuous variables were compared using independent group t tests when the data were normally distributed; otherwise, the mann-whitney test was used. proportions for categorical variables were compared using the chi-square test. proportions for categorical variables were compared using the chi-square test or fisher exact test. distribution of highest liver enzyme levels (e.g., alt, ast, alp, and tbil) by the severity of covid-19 was presented using kernel density estimation. dynamic changes of liver enzyme levels by the severity of covid-19 were presented using locally weighted scatterplot smoothing (loess). site was modeled as a random effect in the mixed-effect cox model. the mixed-effect cox proportional hazards regression models were used to investigate the relationship of liver enzyme levels with all-cause mortality among patients. the mixed-effect cox models were adjusted for age, gender, and coexisting chronic diseases, including hypertension, coronary artery disease, cerebrovascular disease, t2dm, and ckd. ordinal logistic regression analysis was applied to evaluate the association of baseline characteristics and medications happened before peaking of liver enzymes with the peak levels of inhospital liver enzymes in the longitudinal cohort, where the liver function markers were trichotomized. the parallel lines assumption of the model was also tested and met. the p values were 2-sided, and an alpha level of 0.05 was used to define statistical significance. all analyses were conducted using r version 3.6.3 (r foundation for statistical computing, vienna, austria) or spss version 23.0 (ibm, armonk, ny, usa). the selected baseline liver characteristics of the enrolled patients are shown in table 1 . a total of 5,771 adult patients with covid-19 were included in the analysis. the median age was 56 (iqr, 43-accepted article (79.4%) were non-severe cases and 1,186 (20.6%) were classified as severe cases. a total of 81 (1.4%) patients reported chronic liver disease. among these patients, 4 recorded fatty liver diseases and 77 viral hepatitis (supporting table s3 ). the median day for acute liver injury (alt >3 uln) occurs at day 17 (iqr, 13-23) after symptom onset (fig. 1 ). to determine the distribution and trajectory of parameters indicating liver functions in patients enrolled in the study, multiple results from liver function tests were recorded during hospitalization. less disperse in non-severe cases. these levels increased and grew more disperse as the disease became more severe. non-severe and severe case distribution had widest separation by ast level (fig. 2b ). loess models illustrated trajectory of alt, ast, alp, and tbil in non-severe and severe patients during hospitalization (fig. 2c) . the models suggested a significant elevation of ast level upon admission and was maintained at higher levels in the severe group than the non-severe group. although there was a significant difference in alt between the non-severe group and the severe group, the magnitude of increase in alt was not as dramatic as ast upon admission. however, it rapidly increased, surpassed the uln value, and reached its peak within 10 to 15 days after admission in the severe group. alp level was higher in the severe group than the non-severe group. both groups rose gradually during hospitalization but largely stayed in the normal range. the fluctuation in tbil levels was mild in the non-severe and the severe group. the dynamic changes in indicators suggested potential mechanisms of covid-19-associated liver injury. impaired liver function is closely related to mortality in covid-19 patients. the associations of increased alt, ast, alp, and tbil levels with all-cause mortality were assessed using mixedeffect cox model. hospital sites were treated as a random effect. age, gender, and comorbidities were adjusted as confounders. hazard ratios for the associations between alt, ast, alp, and tbil and this article is protected by copyright. all rights reserved all-cause mortality are depicted in table 2 . the kaplan-meier survival curves with elevation in alt, ast, alp, and tbil levels for all-cause mortality are illustrated in fig. 3 . among these liver enzymes, elevated ast was associated with the highest mortality risk. compared to the patients with ast in the normal range, all-cause mortality risk significantly increased 4.81-fold (95% confidence interval [ci], 3.38-6.86; p < 0.001) in patients with ast between 40 and 120 u/l and increased 14.87-fold (95% ci, 9.64-22.93; p < 0.001) in patients with ast above 120 u/l after adjusting for age, gender, and comorbidities. moreover, elevation of other enzymes and tbil were also significantly associated with adverse outcomes of covid-19 (table 2 and supporting table s4 -s5). given discharge might be a competing risk for death, we further analyzed the association of liver function with 28-day mortality of covid-19 using cumulative risk analysis, to further avoid the potential over-estimation of risk in the hazard estimates. in this model, ast ranging from 40 to 120 u/l and above 120 u/l were associated with significantly increased the risk of all-cause mortality (adjusted hr, 6.00; 95%ci, 4.29-8.39; p < 0.001 for 40-120 u/l; adjusted hr, 17.05; 95%ci, 11.21-25.93; p < 0.001 for ast >120 u/l) (supporting table s6) the ordinal regression analysis revealed the effects of age, sex, and coexisting disease-adjusted baseline characteristics and hospital medication on peak alt, ast, alp, and tbil levels in covid-19 patients from the longitudinal cohort (tables 3) . male gender, systemic corticosteroids application, lymphocyte count decrease, neutrophil count increase, and fever were factors positively associated with elevated alt levels ( table 3) . use of antifungal drugs, lymphocyte count decrease, chronic liver disease, systemic corticosteroids use, and male gender were the leading factors positively associated with elevated ast levels. alp levels were tightly associated with antifungal drug use, neutrophil counts increase, chronic liver disease, and male gender. antifungal, antiviral, systemic corticosteroids use, and platelet count reduction were main factors positively correlated with increased tbil levels. neutrophil count increase, lymphocyte count decrease, and male gender were common factors positively associated with elevated alt, ast, alp, and tbil levels during hospitalization (table 3) . this article is protected by copyright. all rights reserved multiorgan injury, which significantly increases mortality, is often evident in patients with covid-19. (10) this study presents trajectories of liver enzyme levels during hospitalization and depicts their clinical significance in a multicenter retrospective cohort-derived data set of 5,771 individuals. liver injury was mild and transient in non-severe patients. the major finding is that elevation of ast level was more frequent and significant than the increase of alt in severe patients on hospital admission, and ast levels had the highest correlation with mortality compared to other indicators reflecting liver injury. this is contradictory to other hepatitis-induced liver injury. increase of alp was more significant toward the latter phase of the disease but mainly stayed within the normal range. elevated alt, ast, alp, and tbil levels were associated with increased risks of mortality and, among these liver enzymes, elevated ast was associated with the highest mortality risk. common factors associated with elevated liver injury indicators were lymphocyte count decrease, neutrophil count increase, dyspnea, and male gender. the liver biopsy specimens of patients with covid-19 showed moderate microvascular steatosis and mild lobular and portal lesion. (5, 11) liver damage may be associated with organ-specific immune response to coronavirus or secondary to hypoxemia, systemic inflammation response, and medication. a previous study showed the angiotensin-converting enzyme 2 (ace2) receptor expression is very low in hepatocytes and only expressed in cholangiocytes in the liver, (12) suggesting that cholangiocytes might be a direct target for severe acute respiratory syndrome (sars)-coronavirus 2 (cov-2) invading the liver. however, in our study, alp, an index of cholangiocytes injury, mainly stayed within the normal range during hospitalization. elevation in alt and ast, the indicators of hepatocyte injury, was more common and severe in patients with covid-19. pathological analysis of liver tissue from a patient who died from covid-19 failed to demonstrate cholangiocyte damage and viral infiltration in the liver. the elevations of lactate dehydrogenase (ldh) and gamma-glutamyl transpeptidase (ggt), which also reflect bile duct injury, were not significant. (5) thus, the hypothesis of cholangiocytes mediating viral-associated injury needs further investigation. in this study, we observed ast elevation upon admissions, followed by an increase in alt. previous studies also reported that ast increase is more frequent than alt in severe patients upon admission. (3, 13) this article is protected by copyright. all rights reserved in which alt elevation is the primary manifestation of liver injury. (14) (15) (16) ordinal regression analysis also showed that ast elevation is positively correlated with the increase of neutrophil counts and the decrease of lymphocyte counts at baseline-pathological alterations that are proven indicators of disease severity. (17, 18) early elevation in ast and its association with indicators for disease severity suggest immune-mediated inflammation may play a critical role in liver impairment in severe patients with covid-19. mechanisms that mediate the early ast elevation in severe patients warrant further study. the combination of antibiotics, antivirals, and systemic corticosteroids is widely utilized in covid-19 treatment. (13, 19) antifungal medication is more likely to be applied in weaker patients. in this study, use of antifungal drugs, antibiotics, antivirals, and systemic corticosteroids exhibited a positive correlation with elevated liver enzymes. although these data could not prove the causal effect of drugs on liver damage, drug hepatotoxicity during the treatment of coronavirus infection needs to be considered. due to this emergent situation, there is an insufficient report and diagnosis on chronic liver disease. only 81 patients recorded history of chronic liver disease (4 with nonalcoholic fatty liver disease [nafld] and 77 with viral hepatitis), which are much lower than the prevalence in china. (20, 21) we are unable to assess whether coexistence of chronic liver comorbidities increases susceptibility to liver injury in sars-cov-2 infection. previous studies have shown that sars-cov-2 may aggravate liver injury in patients with viral hepatitis. (22, 23) therefore, liver injury in patients with chronic liver disease needs to be investigated and monitored. limitations exist in our research. this study was retrospective, and some cases did not have sufficient history of illness. the multiple tests for liver function were carried out at different time intervals for each patient. bias may occur due to the increased number of tests in patients with liver dysfunction. observational studies demonstrate association, not causation. whether abnormal liver function is caused by sars-cov-2 or inflammation needs to be further investigated by direct clinical evidence. in conclusion, the dynamic patterns of liver injury indicators and their potential risk factors may provide an important explanation for the covid-19-associated liver injury. because the increase of liver injury indicators is strongly associated with the risk of mortality, these parameters, especially ast, may need to be monitored during hospitalization. this article is protected by copyright. all rights reserved a novel coronavirus from patients with pneumonia in china coronavirus disease (covid-2019) situation reports clinical characteristics of coronavirus disease 2019 in china association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19 pathological findings of covid-19 associated with acute respiratory distress syndrome clinical management of severe acute respiratory infection when novel coronavirus (2019-ncov) infection is suspected: world health organization (ncov)-infection-is-suspected kidney disease: improving global outcomes (kdigo) acute kidney injury work group. kdigo clinical practice guideline for acute kidney injury accf/aha guideline for the management of heart failure: a report of the american college of cardiology/american heart association task force on clinical practice guidelines and the heart failure society of america towards definition, clinical and accepted article this article is protected by copyright. all rights reserved laboratory criteria, and a scoring system for disseminated intravascular coagulation clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china changes of serum hbv-dna in relation to serum transaminase level during acute exacerbation in patients with chronic type b hepatitis sars-associated viral hepatitis caused by a novel coronavirus: report of three cases sequential changes of serum aminotransferase levels in patients with severe acute respiratory syndrome clinical and immunologic features in severe and moderate coronavirus disease dysregulation of immune response in patients with covid-19 in wuhan, china intensive care during the coronavirus epidemic epidemiological feature of nafld from 1999 to 2018 in china accepted article this article is protected by copyright. all rights reserved unexpected rapid increase in the burden of nafld in china from liver injury during highly pathogenic human coronavirus infections clinical best practice advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld expert panel consensus statement author names in bold designate shared co-first authorship a/g ratio, median (iqr) abbreviations: a/g, albumin to globulin key: cord-279667-ikfduu2k authors: ronnje, louise; länsberg, john-kalle; vikhareva, olga; hansson, stefan r.; herbst, andreas; zaigham, mehreen title: complicated covid-19 in pregnancy: a case report with severe liver and coagulation dysfunction promptly improved by delivery date: 2020-09-04 journal: bmc pregnancy childbirth doi: 10.1186/s12884-020-03172-8 sha: doc_id: 279667 cord_uid: ikfduu2k background: it has been proposed that pregnant women and their fetuses may be particularly at risk for poor outcomes due to the coronavirus (covid-19) pandemic. from the few case series that are available in the literature, women with high risk pregnancies have been associated with higher morbidity. it has been suggested that pregnancy induced immune responses and cardio-vascular changes can exaggerate the course of the covid-19 infection. case presentation: a 26-year old somalian woman (g2p1) presented with a nine-day history of shortness of breath, dry cough, myalgia, nausea, abdominal pain and fever. a nasopharyngeal swab returned positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. her condition rapidly worsened leading to severe liver and coagulation impairment. an emergency caesarean section was performed at gestational week 32 + 6 after which the patient made a rapid recovery. severe covid-19 promptly improved by the termination of the pregnancy or atypical hellp (hemolysis, elevated liver enzymes and low platelet count) exacerbated by concomitant covid-19 infection could not be ruled out. there was no evidence of vertical transmission. conclusions: this case adds to the growing body of evidence which raises concerns about the possible negative maternal outcomes of covid-19 infection during pregnancy and advocates for pregnant women to be recognized as a vulnerable group during the current pandemic. the pandemic caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has exposed vulnerable populations to an unprecedented global health crisis. from the knowledge gained from previous human coronavirus outbreaks, it has been proposed that pregnant women and their fetuses are particularly at risk for poor outcomes [1] . the maternal and neonatal outcomes of pregnant women with covid-19 is limited to a handful of case reports which present diverse results. obstetricians are still learning about covid-19 presentation and progression in pregnancy and even though the majority of pregnancies infected by covid-19 have good outcomes, a recent systematic analysis [1] showed that up to 3% of pregnancies were associated with severe maternal morbidity. it was indicated that mothers with a complicated medical history could be at increased risk for severe outcomes. furthermore, experts are of the opinion that the clinical recommendations for managing covid-19 in pregnancy should be based on lessons learned from the current epidemic [2] which emphasizes the importance of presenting covid-19 cases associated with complex clinical management. we therefore present a case report of a young woman, pregnant in the third trimester, diagnosed with covid-19, showing severe liver and coagulation impairment. a 26-year-old somalian woman (gravida 2, para 1) who had been living in sweden for a year presented at the emergency department of skåne university hospital in malmö on april 17th, 2020 pregnant at 32 + 1 weeks of gestation. she was transferred to the infectious diseases department with suspicion of covid-19. a diagnostic test, based on quantitative real time polymerase chain reaction (qrt-pcr), from a nasopharyngeal swab, was positive for sars-cov-2. she had recently moved from stockholm to malmö. in 2015 she had a normal vaginal delivery in somalia. the patient had an appendectomy and a cholecystectomy in somalia. her medical history also included hypothyroidism, currently treated with 150 ug levothyroxine daily. the body mass index (bmi) on admission to prenatal care was 47 kg/ m 2 with length 163 centimetres (cm), weight 126 kg (kg). apart from the obesity, her pregnancy had been without complications. she had received an intramuscular injection of anti-d immunoglobulin at 28 + 5 weeks of gestation since she was rhesus d (rhd) negative and the fetus was rhd positive. on admission, the patient described a nine-day history of shortness of breath, dry cough, myalgia, nausea, abdominal pain and fever (fig. 1) . she had significant abdominal pain on admission but the surgeon did not find any signs of an acute abdominal event. the patient had also noticed reduced fetal movements for the last two days. obstetric examination including cardio-tocography (ctg) and an abdominal ultrasound showed no abnormalities. the patient's respiratory rate was 22 breaths/minute, oxygen saturation 95%, blood pressure 116/71, pulse 113 beats/minute and temperature 37.2 degrees celsius ( o c). the laboratory tests are shown in table 1 . the patient was given morphine, paracetamol and oxycodone for pain relief and also received thromboprophylaxis, dalteparin 7500 units /day subcutaneously. no additional oxygen was needed. on day 2 (18/4/2020), the patient was relatively stable apart from two short episodes of fever up to 38.9 o c. due to risk for preterm labour, the patient received 12 milligrams (mg) of betamethasone intramuscularly to aid fetal lung maturity. daily fetal monitoring using ctg showed no signs of fetal distress. the patient was discharged on day 3 (19/4/2020) with a planned obstetric follow-up including fetal growth assessment after recovery. she was prescribed dalteparin for four weeks. the patient returned to the emergency department the next day (20/4/2020) with a sore throat and severe difficulties in swallowing. apart from tachypnoea (25-35 breaths/minute) and tachycardia (118 beats/minute), other vital signs were normal. after examination, she was discharged with a prescription of betamethasone tablets for three days (6, 4 and 3 mg) for swallowing difficulties and potassium supplements for the hypokalaemia noted in the blood tests ( table 1) . the patient was readmitted to the infectious diseases department the next day (21/4/2020) (fig. 1) . her covid-19 symptoms (cough, myalgia, abdominal pain and fever) had worsened and she now presented with dyspnoea. at readmission, the patient's respiratory rate 42 breaths/minute, blood pressure 114/61, pulse was 120 beats/minute and temperature 38.9 o c. during episodes of coughing, her oxygen saturation fell to 86%, but with 5 l of oxygen on mask the saturation rose to 99%. laboratory tests are shown in table 1 . her condition deteriorated on day 2 (22/4/2020) of the readmission. in addition to the generalized pain and tenderness, the pain in her right upper abdomen had worsened. blood tests showed elevation of aspartate aminotransferase (asat), interleukin-6 (il-6) and ferritin concentrations. there was impaired coagulation as shown by a prolonged activated partial thromboplastin time (aptt), high d-dimer, falling platelet count and decreased level of anti-thrombin iii (table 1 ). hemolysis was indicated by a fall in the hemoglobin concentration and rising lactate dehydrogenase levels although haptoglobin concentrations only were slightly elevated (table 1) . despite her worsening condition, the patient felt active fetal movements and normal intermittent ctg controls were registered. intravenous antibiotic treatment with cefotaxime (2 g, 3 times daily) was initiated due to suspicion of concomitant bacterial infection (table 1 ). blood and urine cultures were taken but since the general condition of the patient had worsened, a decision was made to deliver by caesarean section (32 + 6 gestational weeks), on maternal indication. the operation was performed in spinal analgesia in an operating theatre with negative air ventilation. the local hospital guidelines were followed to prevent the spread of covid-19 [3] . an uncomplicated operation was completed within 40 min and the total blood loss was 200 millilitres (ml). after two hours in the post-operative unit, the patient returned to the ward and received thromboprophylaxis, dalteparin at a total dose of 10.000 units divided in two doses. a computed tomography (ct) lung scan, performed later the same day, showed bilateral diffuse, ground-glass opacities with both peripheral and perihilar distribution, but no signs of pulmonary embolism (fig. 2) . due to deranged liver values, the patient was unable to receive paracetamol and due to the covid-19 infection not able to receive ibuprofen [4] . the pain relief was managed by administering 2.5 ml of intravenous morphine as needed. however, the patient's condition worsened during the night and on examination, the patient was somnolent and lethargic but answered adequately when woken up (reaction level scale 2). the patient's pain was mostly localized to the upper right quadrant of the abdomen and epigastrium. her uterus was well contracted and there were no signs of postoperative complications such as bleedings and local infection. on examination, the patient had miotic pupils that reacted poorly to light stimulation. even though the patient only was given 7.5 mg of morphine over the course of 8 h, a morphine over-dose was suspected, and intravenous morphine was replaced by a combination of orally administered naloxone and oxycodone. post-operative mobilization was initiated one day after surgery where after the patient made a steady recovery. table 1 illustrates the drastic improvement in the patient's blood tests on day 3 (23/4/2020) of the readmission. the patient was discharged in good health on the 30th of april, 2020 with thromboprophylaxis planned for 6 weeks postpartum and a follow-up visit to the obstetrics clinic. a male baby was delivered with birth weight 2085 g (37th percentile), birth length 48 cm (99th percentile) and head circumference 33.5 cm (99th percentile). the cord was clamped immediately after birth and the baby was shown briefly to the mother before being taken to a neonatal resuscitation station. at 1-minute after delivery, the baby had a normal heart rate but was gasping and had absent tone and no spontaneous movements. positive pressure ventilation by a t-piece (neopuff) was given with peak inspiratory pressure (pip) 20 cmh 2 o and positive end expiratory pressure (peep) between 5 and 7 cmh 2 o intermittently during the first seven minutes of life. at ten minutes, the baby was spontaneously breathing, albeit grunting, through t-piece continuous positive airway pressure (cpap) with preductal oxygen saturation between 90-95% at fio 2 (fraction of inspired oxygen) 0.4. fine crackles could be heard on lung auscultation. intermittent intercostal retractions were also seen. skin colour, tone and reflex irritability improved gradually during the stabilisation process. apgar score; 1 min: 4 (appearance 1, pulse 2, reflex irritability 0, activity 1, respiration 0), 5 min: 6 (appearance 1, pulse 2, reflex irritability 1, activity 1, respiration 1), 10 min: 8 (appearance 2, pulse 2, reflex irritability 1, activity 1, respiration 2). vitamin k was given intramuscularly and a nasogastric tube was inserted. nasal cpap with peep 6 cmh 2 o was started. arterial umbilical cord blood gas showed mild combined respiratory and metabolic acidosis with ph 7.28, partial pressure of oxygen (po 2 ) 3.45 (kilopascal) kpa, partial pressure of carbon dioxide (pco 2 ) 6.97 kpa and base excess − 6.8. the baby was put in an incubator and transferred to the neonatal intensive care unit (nicu) and placed in an airborne infection isolation room (aiir) with negative pressure ventilation. it was given preterm formula supplemented with intravenous glucose infusion. venous blood gas was analysed at 4 h of age: ph 7.27, po2 5 kpa, pco2 7.3 kpa, be -1.8, hb 184 g/l and lactate 2.7 mmol/l. after parental consent, formula was changed to donated breast milk. the mother was supplied with a breast pump and instructed in its use. after the initial need of breathing support and supplemental oxygen during day one of life the baby has enjoyed an uneventful clinical course. nasopharyngeal swabs for sars-cov-2 detection were collected at 48 and 96 h of life and were found to be negative in both instances. we report a case of severe covid-19 during in third trimester pregnancy, which led to an emergency caesarean section and preterm delivery at 32 + 6 weeks of gestational age. this case adds to the growing body of evidence which raises concerns about the possible negative maternal outcomes of covid-19 infection during pregnancy [1, [5] [6] [7] [8] . whilst most mothers with covid-19 infection have mild symptoms which resolve without treatment, a number of cases have now emerged in the literature, where mothers have required intensive care admission, and one case requiring invasive ventilation with extracorporeal membrane oxygenation [8] . it has been suggested that pregnancy induced immune responses and cardio-vascular changes can exaggerate the course of the covid-19 infection [1] . mothers with prior medical conditions may be at higher risk for poor outcomes. in this case, there were several risk factors for preeclampsia such as somalian origin and high bmi [9] . our patient was severely obese (bmi 47 kg/m 2 ) and her condition deteriorated drastically 12 days after her initial symptoms. atypical hellp (haemolysis, elevated liver enzymes, and low platelet count) syndrome or mississippi class 3 [10] can present itself with platelets between 100 and 150 × 10 9 /l, aspartate aminotransferase (asat)/ alanine aminotransferase (alt) ≥ 0.68 µkat/l and lactate dehydrogenase (ldh) > 10.2 µkat/l but it is seldom associated with coagulation impairment [10] . in addition, the patient had remarkably high levels of asat (28 µkat/l), as compared to the other liver tests, which is not consistent with the generalized liver dysfunction seen in hellp or acute fatty liver of pregnancy [11] . liver injury has been reported by a number of studies in patients with severe covid-19 [12] [13] [14] [15] making this organ the most commonly affected besides the respiratory system. transient elevation of serum aminotransferases is often seen and a number of factors have been implicated for acute liver damage in severe covid-19, including severe hypoxemia due to acute respiratory failure, drug interactions, septic shock and multiorgan dysfunction [12] . on readmission, the plasma paracetamol concentration was well within the therapeutic reference interval (table 1 ) and liver injury secondary to a paracetamol overdose was therefore not suspected. although there is insufficient evidence for direct sars-cov-2 virus-related hepatocyte injury, liver dysfunction has been continuingly related to severe covid-19 infection [15] and intensive care admission [16, 17] . the patient also presented with elevated concentrations of several active-phase proteins (apps) including il-6, procalcitonin and ferritin, which may have indicated concomitant bacterial infection or severe systemic inflammation due to covid-19. subsequent blood and urine cultures were negative but the patient still received prophylactic treatment with broad spectrum antibiotics intravenously from day 1-5 of the readmission. the hyperactive immune responses characteristic of severe covid-19 can lead to stress-induced tissue injury and multiorgan impairment [18] . ruan et al. [19] found that elevated levels of il-6 were associated with a significantly increased risk of mortality in covid-19 patients but in this case the inflammatory markers (and liver enzyme tests) decreased significantly after the caesarean section. we can only speculate whether this indicated that severe covid-19 in pregnancy may improve promptly by the termination of the pregnancy or that the patient's condition was a combination of atypical hellp and covid-19 which subsequently improved after the caesarean section. considering the latter option, the patient showed some typical characteristics of hellp syndrome; including right upper abdominal pain, epigastric pain, nausea and vomiting but a normal blood pressure [20] . furthermore, she had no past obstetric history of preeclampsia or hellp. her platelet count was only marginally decreased and although subtle signs of haemolysis were present (decreased haemoglobin concentration and elevated ldh), haptoglobin concentration was not decreased [10] ( table 1 ). given that pregnancy itself is a hypercoagulable state, it has been suggested that covid-19 infection during pregnancy is associated with high risk of maternal thrombotic complications [15] [16] [17] . high levels of d-dimer in combination with elevated liver enzymes supports the likelihood that the patient's liver injury and coagulation dysfunction were secondary to the severe covid-19 infection. considering the neonate, we found no evidence for any vertical transmission of covid-19 between mother and the baby. however, there have been reported cases of early covid-19 detection in newborns, implying the potential risk of vertical transmission [21] , although in the vast majority of cases, no such evidence has been identified [22, 23] . since only a handful of cases have been reported in the literature, vertical transmission cannot be ruled out until systematic studies have been undertaken. our report has some limitations. we were unable to report a complete panel of coagulation tests due to errors in lab analysis. similarly, the presence of antiphospholipid antibodies to rule out antiphospholipid syndrome were not investigated. we did not evaluate the presence of sars-cov-2 in amniotic fluid, cord blood, or placental tissue which could further clarify the possibility of vertical transmission. in summary, we describe a severe case of maternal covid-19 during the third trimester of pregnancy which led to liver and coagulation impairment and preterm delivery. we believe these findings have important public implications both due to the severity of the disease progression but also due to the rapid nature of the improvement after delivery. atypical presentation of hellp could not be ruled out and the importance of a multidisciplinary team in the treatment and management of severe covid-19 during pregnancy is critical for positive patient outcome. pregnant women should be considered a vulnerable group in the population in which exposure to covid-19 should be avoided at all costs. maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies novel corona virus disease (covid-19) in pregnancy: what clinical recommendations to follow? coronavirus (covid-19) infection in pregnancy. information for healthcare professionals version 7: published thursday 9 are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? potentialmaternal and infant outcomesfrom (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records covid-19 in pregnancy: early lessons clinical manifestations and outcome of sars-cov-2 infection during pregnancy maternal deaths with covid19: a different outcome from mid to low resource countries? the spectrum of severe preeclampsia: comparative analysis by hellp (hemolysis, elevated liver enzyme levels, and low platelet count) syndrome classification acute fatty liver of pregnancy: pathophysiology, anesthetic implications, and obstetrical management characteristics and mechanism of liver injury in 2019 coronavirus disease coronavirus disease 2019 (covid-19): a perspective from china implications of covid-19 for patients with pre-existing digestive diseases clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china on the alert for cytokine storm: immunopathology in covid-19. arthritis rheumatol. 2020. accepted author manuscript clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china. intens care med revisiting hellp syndrome novel coronavirus infection in newborn babies under 28 days in china clinical characteristics and risk assessment of newborns born to mothers with covid-19 clinical characteristics of 19 neonates born to mothers with covid-19. front med publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors want to thank daisey lee for her contribution in editing fig. 2 authors' contributions lr: planning, data collection, analyzing and writing the manuscript. jkl: planning, data collection, writing the manuscript. ov: conception, planning, data collection and revising the manuscript. srh: conception, planning, data collection and revising the manuscript. ah: conception, planning, data collection and revising the manuscript. mz: conception, planning, data collection, writing and revising the manuscript. all authors have read and approved the manuscript. ethics approval and consent to participate ethical approval was not sought due to the nature of the case report. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. the patient provided written informed consent for the publication of potentially identifying images and clinical details on behalf of themselves and their child. the datasets used and analysed during the current study available from the corresponding author on reasonable request. a.h. is a board member and shareholder in amniotics ab, a start-up company developing treatment for severe lung disease in covid-19. s.r.h. holds patents for the diagnosis and treatment of preeclampsia. s.r.h. is also a cofounder of the company guard therapeutics international formerly named a1m pharma ab (https://guardtherapeutics.com). key: cord-332827-gll4nqdd authors: peixe, paula; calinas, filipe; tato marinho, rui title: hepatology in the covid era: another c virus, again challenging the liver date: 2020-04-30 journal: ge port j gastroenterol doi: 10.1159/000508116 sha: doc_id: 332827 cord_uid: gll4nqdd nan our way of life has changed! the world is changing dramatically. read the story! [1] . since the beginning of 2020, the worldwide spread of a new virus has made us rethink and reinvent new ways of living and caring for patients. doctors no longer have "the magic touch." in late 2019, a new type of severe pneumonia associated with a new type of coronavirus was first detected in the chinese province of hubei (people's republic of china). coronaviruses have existed for a long time, conditioning diseases in several animal species, but until 2003 (sars [severe acute respiratory syndrome]) there was no report of transmission to humans. the new coronavirus, initially called 2019n-cov and now sars-cov2, is the seventh coronavirus to infect humans, as identified by the chinese health authorities. it emerged as a human-transmitted zoonosis originating from the wild animal market in the city of wuhan. viruses with similar characteristics have been identified in bats, snakes, and pangolins, with an homology of genomic features showing an 89 and 82% nuclear acid sequence similarity with bat sars-covzxc21 and human sars-cov (wuhan patients) but the true transmitter of the disease has not yet been found [2] . the disease is called covid-19 (corona virus disease 2019, i.e., the year of the first case and virus identification). from the point of view of transmission, it occurs among humans through contamination by particles expelled through the airways (by coughing, sneezing, and talking) but also by fomites and interpersonal contact. aerosols and fecal-oral transmission are also considered potential sources of contamination. in addition to this community transmission, nosocomial transmission is important, since many symptomatic patients use health services where they can infect other people, particularly 2 doi: 10.1159/000508116 health professionals, when not adequately protected. the risk of infection for health professionals is 3 times more than in the general population [3] . the spread of covid-19 occurred in the beginning by contact with the original cases identified in wuhan but then, within < 30 days, it went from reaching a city to the whole country. the chinese new year season had a decisive impact as it corresponds to a period of the year when thousands of people are travelling. public transport, always full, favored the transmission of the infection at this time [4] . due to the ease of travel nowadays, cases quickly spread to all countries in the world. around 40 million flights worldwide each year is a terrific number for the capacity of disease transmission. it was possible to designate the transmission chain by identifying index cases. however, in each country, transmission started to occur locally and the links were lost. by march 2020, covid-19 had become widespread in all countries and the world health organization declared it a pandemic. today, about 2,400,000 cases and 165,000 deaths were reported worldwide (https://www.worldometers.info/coronavirus/ [accessed 2020abril19 18: 00]). understanding the pathophysiology of viral infection and the relationship between virus and host has become a matter of urgency. the scientific community has made an intense effort to identify these relationships, but most of the issues are still open. it was possible to identify the virus receptor in humans, namely, the angiotensin-converting enzyme 2 (ace2), a transcellular protein to which sars-cov2 binds. the cellular serine protease, transmembrane protease serine 2 (tmprss2), which cleaves the s protein of human coronaviruses onto the cell membrane, is critical for the fusion of viral cells and the cellular membranes in the host. the coexpression of these molecules seems to be crucial to the infection. ace2 is widely expressed in the human body, i.e., in the lungs, heart, vasculature, kidneys, brain, intestines, and testes. it is also expressed in hepatocytes and in cholangiocytes. the same is true for tmprss2 [5] [6] [7] . the ubiquity of distribution of ace2 and tmprss2 may explain the systemic involvement of the sars-cov2 infection, however, the disease activity is more significant at the respiratory level due to the expression of the molecules in these organs. patients may remain asymptomatic, without knowing exactly the percentage of infected people who do not have manifestations of the disease [8] . respiratory conditions manifesting as fever (41%), cough (56%), headache (25%), muscle pain (29%), and generalized weakness (24%) can have a mild and moderate evolution. severe cases with respiratory insufficiency eventually require ventilatory support (and, in some cases, extracorporeal life support); this has occurred in portugal in 17% of cases according to the records [9, 10]. gastrointestinal (gi) manifestation in covid-19 patients who report at least one gi tract symptom (nausea, vomiting, or diarrhea) varies from 5 to 11.4%. jin et al. [11] reported a rate of chronic liver disease of 10.81% in patients with gi symptoms, significantly higher than those without these symptoms (2.95%) (p = 0.004). they also reported that the rate of covid-19 patients with severe disease is significantly higher in those with gi symptoms (22.97 vs. 8.14%, p < 0.001). the impact of covid-19 on the liver is unclear. the analysis of this relationship must be done by examining 2 distinct aspects, namely liver damage caused by the new coronavirus/covid-19 and the impact of preexisting liver disease on covid-19. as already mentioned, the molecules involved in infectivity of the virus are also present in the cells of the liver (hepatocytes and cholangiocytes), so the liver is damaged by sars-cov2, as occurs with the other coronaviruses. the initial description of hepatic involvement emerged from the analysis of the first 99 cases, 43 of whom presented with an elevation of liver enzymes, alanine aminotransferase in 28% of patients, aspartate aminotransferase in 35%, and total bilirubin in 18%. the incidence of elevated serum liver biochemistry in hospitalized patients with covid-19 ranges from 14 to 53%. in a recent review, changes in liver test values were recorded in about 46% of patients at diagnosis (table 1) and more frequently during hospitalization (table 2 ) [12] . these changes were mild to moderate. only 1 patient had a significant hepatocellular lesion. there was no identification of a cholestatic pattern or liver failure [13] . zhan et al. (unpubl. data from their own center), reported the presence of elevated γ-glutamyl transferase (ggt) in 30 (54%) of 56 patients with covid-19 during hospitalization. such data have not been previously recorded [14] . ge recently, an association between the severity of co-vid-19 and the alteration of liver tests has been described, showing that patients with more severe or critical disease have significantly higher aminotransferase and bilirubin values than cases of mild-to-moderate disease. hypoalbuminemia and low platelets are also associated with more serious disease, but may be related to covid-19 instead of previous liver disease. following autopsy studies, liver changes included dark red hepatomegaly, hepatocyte degeneration with focal lobular necrosis and infiltration by neutrophils, infiltration by lymphocytes and monocytes in the portal spaces, and sinusoidal congestion with microthrombosis. despite these findings, no lesions of the bile ducts or liver failure characteristics were identified [15] . multiple mechanisms involved in liver injury have been described: immune-mediated damage, direct cytotoxicity, anoxia due to respiratory failure, drug-induced liver injury, and the reactivation of preexisting liver disease [16] . after recovery, liver enzyme values return to normal and no evolutive disease seems to occur. chronic liver disease has a significant burden worldwide that can be assessed by the joint contribution of cirrhosis (11th) and liver cancer (16th) as the cause of 3.5% of all deaths [17] . in published series, liver disease was not identified as a risk factor for sars-cov2 infection [11] [12] [13] [14] [15] . it is assumed that risk factors for the general population are also risk factors for patients with liver disease. however, covid-19 may indeed be more severe in liver patients. hepatic disease is associated with multiple factors including viral diseases, alcoholic disease, non-alcoholic fatty liver disease (nafld), and autoimmune pathology (particularly autoimmune hepatitis and primary biliary cholangitis and other cholangiopathies). special reference must be made to cirrhosis, hepatocellular carcinoma (hcc), and transplantation. despite this significant burden, the impact of cov-id-19 on liver disease and liver disease on covid-19 has not been evaluated and studies are needed to better understand the impact of these associations. despite this, some situations deserve a comment for the lessons that its analysis can give. the covid-19 pandemic started in china and rapidly spread to nearby countries. southeast asia has a high prevalence of hepatitis b virus (hbv) infection. in the study by guan et al. [15] 23 patients (n = 1,099) had hbv, according to positive tests for hbv surface antigen (hbs ag) with or without elevated liver enzymes. of these 23 patients, 22 had nonsevere disease (2.4 vs. 0.6%). no warning signs were triggered by the association of these infections. no reference has yet been made to hepatitis c virus (hcv) in the literature nor any comment has come from the usa. in the usa, the country with the largest number of covid-19 cases and with a relevant expression of hepatitis c, no statement has been issued by the centers of disease control and prevention or any group under surveillance, e.g., the veterans cohort. at this stage, it is urgent to modify the strategy regarding the approach to hcv, simplifying the necessary assessments for the institution of treatment and its availability. approximately 2 billion adults are obese or overweight and over 400 million have diabetes. these are risk factors for nafld and hcc [17] . diabetes, hypertension, and obesity associated with nafld are also dealt, alanine aminotransferase; ast, aspartate transaminase; ggt, γ-glutamyl-transpeptidase; alp, alkaline phosphatase; tbil, total bilirubin [12] . scribed as factors for a poor prognosis associated with covid-19. a preliminary report on nafld in patients with co-vid-19 calls attention to an increased risk of disease progression [6.6% (5/126) vs. 44.7% (34/76) p < 0.0001], a higher probability of abnormal liver function on admission and at discharge [70% (53/76) vs. 11.1% (14/126) p < 0.0001], and a longer time for viral clearance (17.5 ± 5.2 days vs. 12.1 ± 4.4 days p < 0.0001), when compared with non-nafld individuals [18] . the authors state that nafld patients also had a higher risk of progression to severe covid-19 and present an increased viral clearance time. with the global prevalence of nafld on the increase, it is worth noting that a significant number of patients may be at risk of developing severe covid-19. immune-mediated liver diseases, particularly autoimmune hepatitis, have not been mentioned as risk factors for covid-19, but the immunosuppressive treatment required has triggered fears about the risk of infection in patients. the analysis of previous outbreaks of human infection by coronaviruses (sars 2002 and mers 2012) did not record an increase in cases in patients under immunosuppression, or that when infected, the disease was more severe. recent data from the red zones in china and bergamo (italy) have shown neither an increase in the number of cases nor in their severity in immunosuppressed patients. even when infected, as long as there were no other risk factors, these patients had a mild or moderate course of covid-19 [19] . no analysis of the different types of drugs used in immunosuppression in liver diseases has been carried out. a warning has been issued about the use of corticosteroids in the context of sars-cov2 infection, where the most seriously ill patients most likely need corticosteroid therapy. the use of these drugs is associated with increased mortality from coronavirus pneumonia. no assessment has been made in patients with liver disease who were previously on stable treatment with corticosteroids and were infected; however, it is assumed that low doses have no impact on covid-19 (as described for patients in the wuhan observational study) [20, 21] . lleo et al. [22] drew attention to two relevant points: balancing the need to maintain treatment to prevent a serious episode that may require therapeutic measures and hospitalization (with an increased risk of nosocomial infection) and assessing the need for certain procedures, e.g., postponing liver biopsy until a period of less epidemic risk. there has been an expectation of canalicular lesions being caused by sars-cov2 due to the presence of ace2 in cholangiocytes. however, recent research has shown that ace2 has a canalicular specificity, which could explain the scarce documentation of the cholestatic lesions and the low number of cases in which ggt changes have been reported [13, 14] . the impact of sars-cov2 infection on cholestatic diseases such as primary biliary cholangitis or sclerosing cholangitis is unknown; further studies are needed to understand whether there is any clinical relevance [23, 24] . changes in liver tests, often associated with coagulation disorder and hypoalbuminemia, are commonly seen in patients with severe covid-19. these changes fluctuate according to the severity of the respiratory disease (pneumonia) and are not due to the direct effect of the virus on the liver. this may explain why there was no worse prognosis in a group of patients with cirrhosis and sars-cov2 infection. in a review of 7 published studies, it was possible to identify 42 patients with cirrhosis with mortality ranging from 0-2% [13] . despite this strange benign report, doctors should consider patients with chronic liver disease, liver cirrhosis, or liver cancer, to be a risk group as they have underlying immune dysfunction and a more reserved prognosis than other patients that go on to develop acute respiratory failure syndrome. for these reasons, patients with chronic liver disease need particular attention and have different specificities from other patients, especially if they are elderly and/or have comorbidities. data on sars-cov2 infection in cirrhotic and hcc patients are scarce. extensive records and targeted studies are needed to explore multiple open-ended questions such as the severity and mortality of covid-19 and episodes of acute-on-chronic or decompensation associated with the presence of this disease (ascites, hepatic encephalopathy, digestive bleeding, kidney dysfunction, and the risk of infection) or the response to treatment [25, 26] . regarding transplantation and cancer patients, there are also no warnings [16] . however, it is not yet possible to say whether transplantation-associated immunosuppression can alter the predisposition for the acquisition of sars-cov2 infection or how covid-19 evolves in these patients. a small published series showed a mortality rate of 27.8% (5/18) in a mixed group of solid organ-transplanted patients (liver recipients (33.3% [6/18] ) infected with sars-cov2 [27] . cancer patients undergoing chemotherapy may be at increased risk for illness and serious illness, especially if they have other risk factors. however, it is not known whether patients with liver cancer are at the same risk. there are still no data on decompensated cirrhosis, patients on the transplantation ge port j gastroenterol doi: 10.1159/000508116 list and, patients in the immediate posttransplant period or with rejection. decisions about transplantation should be considered on a case-by-case basis at this stage of the pandemic [28] . another concern is for patients with liver disease (either advanced or decompensated), namely, the use of drugs used off-label which have not been studied for use for the current indication or in fragile populations such as liver patients. multiple drugs have been referenced and are under study, but currently there is no known effective treatment [29] . during the covid-19 pandemic, recommendations have been issued to mitigate the transmission of sars-cov2, voluntarily isolation has been promoted, and frequenting public places has been advised against. patients associate hospitals and clinics with the risk of transmitting the virus. many patients have conditions that remain unchecked. however, it is necessary for patients to maintain regular follow-up and for situations that require intervention to be detected in a timely manner. on the other hand, the risk-versus-benefit of some interventions must also be weighed up and those without a negative impact should be postponed. a careful assessment will need to be made of the risk of episodes requiring hospitalization and the consequences associated with it or the risk of increased mortality if patients do not receive appropriate treatment or necessary treatment is delayed. scientific societies for the study of the liver, alone or in association, have been publishing guidelines for the approach and management of liver patients. basically, we can divide patients into outpatients and those patients who require hospitalization. stable outpatients with compensated liver disease should be supported in order to maintain the prescribed medication and recognize early warning signs and promote health literacy. teleconsultations are encouraged and consideration is given to carrying out diagnostic and follow-up exams. it is proposed that the blood samples and ultrasound examinations should, if possible, be postponed. if they are deemed necessary, they should be carried out in laboratories nearby avoiding the patient's movement, so as to minimize contacts and the risk of sars-cov2 transmission. patients must comply with all the protection and risk minimization rules proposed for the general population. the maintenance of surveillance of hcc has been discussed. in high-risk patients, this must be maintained so that lesions that could have an early diagnosis are not detected too late. patients with decompensated liver disease should minimize their contact with health-care personnel and, if possible, should not be admitted to hospital. if a patient (without covid-19) does have to be admitted, he or she must stay in a "clean" area in the hospital and the hospitalization time must be minimized (e.g., ambulatory paracentesis and outpatients albumin infusions can be performed). endoscopic exams, due to the extensive production of aerosols involved, are a high risk for the spread of sars-cov2, so they should only be performed in urgent cases. in the context of hepatology, the 2 main indications are upper gi bleeding, with particular attention to any related to portal hypertension or bile duct obstruction. the strategy for eradicating esophageal varices after an episode of rupture is not defined and should be considered on a case basis. endoscopic examinations for the stratification of portal hypertension should be postponed. it may be decided to start nonselective β-blockers, according to the baveno vi criteria, without endoscopy. liver biopsy should be deferred when not essential. follow-up of liver patients will be increased when collaboration with primary care doctors is possible [28, 29] . however, cirrhosis is a disease with inexorable evolution, so the need to focus all efforts on responding to the pandemic has consequences for these patients in the short, medium, and long term. in the short term, patient follow-up can be modified, i.e., seeing that there are fewer appointments and assessments and, potentially, crucial decisions can be delayed, such as the introduction of prophylactic therapy for the rupture of esophageal varices or the indication of resection or transplantation in cases of malignant liver nodules. a reduction of around 25% of donors has already been identified in this first quarter of the pandemic, and also a reduction in the number of liver transplants. in the medium term, when the social-distancing measures are withdrawn or reduced, it is expected that 3 problems will arise. the first is the identification of seriously ill patients and those who may have far exceeded the optimal time point for treatment decisions about a biologically more aggressive disease which evolves without adequate surveillance during the entire phase of mitigation. second, during the pandemic, there has been an increase in the consumption of alcohol and opioids. more people may develop liver disease and will appear as new patients, 6 doi: 10.1159/000508116 thus increasing the disease burden [30, 31] . the third problem relates to institutions and health professionals, who, also exhausted, will have to respond to an excessive number of patients in a short time, and their capacity to respond will likely be exceeded [32] . in the long term, irregular follow-up will be reflected in the health metrics, with an increase in the number of decompensations, hcc, patients on transplantation lists, and mortality [33] . modulation studies of sars-cov2 infection suggest that the pandemic will not end abruptly, but that there will be periods of covid-19 activity in the coming years [34] . we must prepare ourselves clinically and socially for the outcomes for liver patients as a consequence of changes in their follow-up due to covid-19. there is a huge gap in knowledge regarding sars-cov2/covid-19 and the relationship of both the virus and the disease to liver disease, concerning its different etiological aspects and different clinical forms. it is extremely important to acquire this knowledge in the present moment and for the future. the associação portuguesa para o estudo do fígado (apef; portuguese association for the study of the liver) supports all initiatives that favor the acquisition of this knowledge. this work does not require evaluation by an ethics committee. the authors declare that they have complied with the principles of ethics and the absence of plagiarism. severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection epidemiological, clinical and virological characteristics of 74 cases of coronavirus-infected disease 2019 (covid-19) with gastrointestinal symptoms death from covid-19 of 23 health 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highly pathogenic human coronavirus infections. liver int characteristics and mechanism of liver injury in 2019 coronavirus disease parekh dcovid-19 and the liver: little cause for concern risk factors for severity and mortality in adult covid-19 inpatients in wuhan covid-19 in solid organ transplant recipients: a single-center case series from spain clinical best practice advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld expert panel consensus statement care of patients with liver disease during the cov-id-19 pandemic: easl-escmid position paper alcohol use and misuse during the covid-19 pandemic: a potential public health crisis? lancet public health editorial: challenges to opioid use disorders during covid-19 a shift on the front line covid-19 pandemic will have a long-lasting impact on the quality of cirrhosis care projecting the transmission dynamics of sars-cov-2 through the postpandemic period the authors declare no conflicts of interest of any kind regarding this manuscript. there was no funding. all authors participated in the conception, writing and revision of the manuscript. the final version was approved by the authors. key: cord-290412-m6fesoyb authors: zhao, chang-qing; zhou, yang; ping, jian; xu, lie-ming title: traditional chinese medicine for treatment of liver diseases: progress, challenges and opportunities date: 2014-09-30 journal: journal of integrative medicine doi: 10.1016/s2095-4964(14)60039-x sha: doc_id: 290412 cord_uid: m6fesoyb abstract traditional chinese medicine (tcm) is commonly used in treating liver diseases worldwide, especially in china. the advantages of using tcm for treatment of liver diseases include: protecting hepatocytes, inhibiting hepatic inflammation and antifibrosis in the liver. in this article, we introduce tcm herbal preparations from the chinese materia medica (such as fuzheng huayu) that are typically used for the treatment of liver diseases. literature surrounding the mechanisms of tcm therapy for treatment of liver diseases is presented and discussed. we propose that side effects of herbal compounds are often under-appreciated, and that more care should be taken in the prescription of potentially hepatotoxic medicines. further, to deepen the understanding of tcm mechanisms, new techniques and methodologies must be developed. future studies will lead to the enhancement of clinical outcomes of tcm. as complementary and alternative therapies, tcms will play an expanding role in the future of liver disease treatment. in china, traditional chinese medicine (tcm), especially traditional chinese patent medicine, has been, and continues to be widely used to treat various diseases. even during the severe acute respiratory syndrome (sars) outbreak in 2003, tcm, combined with western medicines were used to control and eventually halt the spread of the disease. compared with western medicine alone, patients receiving treatment with western medicine and tcm had reduced hospital stays, pneumonia duration and mortality. early tcm treatment can also decrease glucocorticoid dosage needed in the treatment of sars [1] . before western medicine was introduced into china, the chinese health care system mainly depended on tcm. although tcm does not treat specific conditions, it treats patterns of illness such as those associated with infectious diseases [2] [3] [4] , cardiovascular and cerebrovascular diseases [5] [6] [7] [8] [9] [10] [11] , respiratory diseases [12] [13] [14] , digestive diseases [14] [15] [16] , urinary diseases [17] [18] [19] , reproductive diseases [20] and blood system diseases [21] , as well as fractures [22] , trauma [23] , ear, nose and throat diseases [24, 25] , skin diseases [26] and mental disorders [27] . tcm can improve the clinical symptoms, reverse some pathological changes and restore traditional chinese medicine for treatment of liver diseases: progress, challenges and opportunities the body's normal physiological function. since western medicine was introduced into china in the 16th century ce, most diseases listed above are treated mainly with western medicinal interventions. gradually, tcm has become an alternative medicine rather than mainstream medicine. even so, tcm therapy still has its advantages in some medical fields where western medicine has not been as effective, such as in liver diseases. this review will introduce tcm in the treatment of liver diseases. liver diseases are mainly classified into viral hepatitis, nonalcoholic fatty liver, alcoholic liver disease, autoimmune liver disease, schistosomiasis liver disease, drug-induced liver injury, hereditary liver disease, liver cirrhosis due to various causes and diverse liver tumors. tcm is widely applied in the treatment of liver diseases in china by both chinese medicine doctors and western medicine doctors because its ability to protect hepatocytes, inhibit hepatic inflammation and reduce fibrosis in the liver. in recent years, the application of tcm in liver cancer treatment has been increasingly widespread [28, 29] . it has been confirmed that tcm can not only reduce the toxic side effects of chemotherapy or radiotherapy, but also inhibit tumor growth and increase survival of patients with tumors [30] . although tcm has many uses in treating liver diseases, it cannot replace other treatment methods such as antiviral drugs, hormones, schistosomicide, surgical operation and transplantation. tcm can be applied to treat diseases in one of the two ways: treatment based on disease differentiation or syndrome differentiation [31] . for the disease differentiation approach, western medicine methods are typically employed to diagnose specific liver diseases. subsequently the appropriate tcm formula or patent drug is selected to treat the disease according to tcm's characteristics and advantages. the evaluation of the curative effect is based on the recovery of liver function or improvement in pathological changes. in the syndrome differentiation approach, tcm diagnosis of a patient's symptoms and signs is used to determine to which syndromes the patient belongs. the appropriate tcm formula or patent drug is then chosen to treat that tcm syndrome. the evaluation of the curative effect depends on the relief or elimination of the symptoms and signs. it is believed that the combination of two kinds of therapies can obtain greater curative effects for liver diseases [32] . 3 chinese materia medica is frequently used to treat liver diseases in acute stage of liver diseases, liver inflammation is prominent. materials listed in the chinese materia medica, especially those for heat-clearing and detoxifying, are often applied to protect the liver, inhibit inflammation, decrease activity of serum transaminase and reduce serum bilirubin [32] . meanwhile according to the symptoms and signs of each patient, a matching therapy, such as adjusting yin and yang, invigorating qi and blood, soothing the liver, regulating qi, clearing heat and removing dampness, is also applied. in the chronic stage of liver diseases, the symptoms of the disease are more complicated. treating the source of the disease (i.e., the virus) is one important and necessary approach. western medicine does well in inhibiting the viruses that cause hepatitis b (hbv) and hepatitis c (hcv). tcm has little effect in inhibiting the virus, but works well to protect liver function, inhibit inflammation, decrease activity of serum transaminase, reduce serum bilirubin, lower lipid levels [33] , promote diuresis [34] and relax the bowels [35] . depending on the stage of liver disease progression, or different syndrome classifications, tcm can be used to adjust yin and yang, invigorate qi and blood, sooth liver, regulate qi, clear heat and remove dampness [36] [37] [38] . zhang et al [39] summarized and provided a critical meta-analysis of randomized controlled trials (rcts) of tcm formulations for the treatment of chronic hepatitis b (chb) that were reported in china from 1998 to 2008. the results showed that (i) tcms (tcm formulations alone or in combination with interferon (ifn) or lamivudine (lam)) had a greater beneficial effect than ifn (p=0.000 3) and slightly better effect than lam (p=0.01) on normalization of serum alanine aminotransferase; (ii) tcms had a similar beneficial effect on antiviral activity when used in conjunction with inf or lam for chb, which was evidenced by the reduction of serum hbeag and hbv dna; (iii) treatment with tcms in conjunction with inf or lam resulted in improved liver function. when the liver tissue is damaged, its repair is accompanied by the formation of an extracellular matrix, also known as fibrosis. fibrosis is the common pathological process of many liver diseases, and is also reversible. antifibrotic effects are an important component in the treatment of various chronic liver diseases [40] . one famous hepatologist, professor hans popper, once said, "anyone who can stop or delay liver fibrosis would be able to cure most chronic liver diseases" [41] . the focus of western medicine scientific and medical research has been on discovering targets for antifibrotic therapy, and developing customized multi-drug regimens [42] . according to the tcm theory, diseases of liver zang will transmit to the spleen zang, thus in the course of treatment, the spleen zang should be strengthened before it is impaired. if the liver disease has been long-standing, kidney yin should be evaluated during the treatment because the liver zang and kidney zang are derived from the same source. in clinical practice, symptoms and signs of spleen-qi deficiency journal of integrative medicine www.jcimjournal.com/jim are frequently seen in patients with liver diseases. they always complain of fatigue, pain or weakness in the legs, abdominal fullness, right upper quadrant discomfort or pain, loose stool, pale tongue or swollen tongue (teeth-marked tongue) with whitish fur and weak pulse. patients with chronic liver disease additionally present symptoms and signs of kidney-yin deficiency, such as dry mouth, internal-heat, red (or red and dry) and uncoated tongue, and weak pulse. according to the theory of syndrome differentiation therapy, methods of invigorating spleen-qi and nourishing kidney-yin should be used to treat liver diseases. in tcm, the pathogenesis of liver diseases does not necessarily relate to the liver zang, it can also be related to the spleen and kidney. table 1 shows tcm functions matched with herbs and dosages commonly used in the treatment of liver diseases. herbs with different functions are selected according to syndrome differentiation. dosages within the recommended range are determined by the severity of the symptoms and signs. several patent drugs (chinese herbal formulas) for treatment clinical observations showed that fzhyc can effectively improve liver function and decrease the expression of fibrosis biomarkers such as serum hyaluronic acid, collagen type iv, procollagen type iii and laminin, in chronic liver disease patients with fibrosis or cirrhosis [43, 44] . fzhyc can also regulate immune function [45] , balance amino acid [46] and endocrine [47] metabolism and reduce portal hypertension [48] . the results of several multicenter rcts have confirmed that tcm can reverse liver fibrosis [49] [50] [51] . fzhyc was used to treat liver fibrosis in patients with chb for six months. fifty patients in the trial group were treated with fzhyc. a control group of 43 patients was treated with heluo shugan capsule, another chinese patent medicine. all patients received liver biopsies pre-and post-treatment. pathology results showed that in the treatment group, the stage of fibrosis was decreased by one or more in 52% of the patients; the remaining 48% of the patients in the trial group had no changes. however, in the control group only 23.3% patients had a decrease in fibrosis stage, 55.8% patients had no change and 20.9% of the patients had an increase in fibrosis stage [49] . in another study, fzhyc reversed the fibrosis at a rate of 57.9% early cirrhotic patients with chb [52] . to prevent esophageal variceal bleeding in cirrhotic patients, a multicenter randomized and placebo-controlled trial was carried out. the probability of survival in the fzhyc group was higher than that in the propranolol group (90.22% vs 70.92%, p=0.044 9). compared to the propranolol group the probability of esophageal variceal bleeding in the fzhyc group was significantly reduced (43.0% vs 23.9%, p=0.013 1). when the two treatments were taken together there was an even lower probability of bleeding which was also significantly lower than the propranolol alone (12.4% vs 43.0% p=0.008 6). in patients with small esophageal varices, treatment with fzhyc reduced the size of the varices. its effects may be related to the prevention of hepatic fibrosis, amelioration of liver function, and the decrease of et-1 concentration in the blood plasma [53] . a meta-analysis was conducted to evaluate the efficacy and safety of fzhyc combined with nucleoside antiviral drugs in treating fibrotic patients with chb. the analysis included seventeen rcts, with a total of 1 320 patients with chb, of which 636 were in control groups and 684 in trial groups. the meta-analysis showed that there was no significant improvement in serum hbeag level and hbv-dna copies. however, there was a statistically significant improvement in liver fibrosis and liver function after treatment for 24 or 48 weeks [54] . for the past six decades, many researchers have carried out extensive research to explore the mechanism of tcm in the treatment of liver diseases. it was found that tcm can improve hepatic microcirculation, scavenge oxygen free radicals, resist lipid peroxidation, promote bilirubin metabolism, accelerate synthesis of liver glycogen and protein, and increase the content of liver microsomal cytochrome p-450 [55] . these effects lead to a decrease in hepatocyte necrosis, inhibition of apoptosis and promotion of the hepatocyte regeneration [56] . we searched the literature for studies exploring the mechanism of fzhyc's antifibrotic activity. it has been shown that fzhyc can protect hepatocytes, resist lipid peroxidation and inhibit some cytokines [57, 58] . fzhyc can also inhibit the activation and proliferation of hepatic stellate cells (hscs), which play an important role in hepatic fibrogenesis and fibrosis [58] . fzhyc can also promote apoptosis of activated hscs, inhibit synthesis and secretion of collagen, inhibit angiogenesis and promote degradation of collagen [59] . studies showed that fzhyc achieved its antifibrotic activity through multiple signal pathways and targets [57, 58, [60] [61] [62] [63] , such as transforming growth factor β-1 (tgf-β1), smads, insulin-like growth factors-1 (igf-1), phosphatidylinositol 3-kinase (pi3k), extracellular signal-regulated kinase (erk), p38 mitogen-activated protein kinase (p38 mapk), rhoa/rho-associated coiled-coil forming protein kinase (rock) and renin-angiotensin system (ras) signaling pathways [64, 65] . the elevation of portal vein pressure is a common symptom of liver cirrhosis. the portal vein pressure is positively correlated with endothelin-1 (et-1) concentration in the liver tissue during the process of liver cirrhosis [66] . fzhyc can dramatically decrease cirrhosis-induced elevation of portal vein pressure by reducing et-1 levels in the liver tissue [67] . due to multiple ingredients in the chinese formula, the compound could not be used to incubate cells directly in vitro. only individual components or ingredients of herbal medicine are suitable for in-vitro research. to explore the underlying mechanisms of fzhyc's antifibrosis activity, we looked in the literature for research on salvianolic-acid b (sa-journal of integrative medicine www.jcimjournal.com/jim b), a component of danshen that is the main constituent herb of fzhyc. studies show that the effects of sa-b are just like fzhyc in the treatment of hepatic fibrosis and cirrhosis [64, 65, 68] . sa-b dramatically decreased the fibrosis level of rats with fibrotic liver, and also markedly decrease cirrhosis-induced elevated portal vein pressure and liver et-1 levels [16] . sa-b achieves its antihepatic fibrosis effect by inhibiting the erk and the p38 mapk pathways of tgf-b1 in hscs. it inhibits the erk pathway by inhibiting phosphorylation of mek. sa-b inhibits the p38 mapk pathway by blocking phosphorylation of mkk3/6 and inhibiting expression of myocyte enhancer factor 2 (mef2, a transcription factor), in hscs with or without tgf-b1 stimulation [69] . further, sa-b inhibits the crosstalk of the samd signaling pathway to the erk signaling pathway [63] . sa-b also significantly reduces et-1-activated hsc contractility by inhibiting rhoa/rock ii activation and the downstream mypt1 phosphorylation at thr696 [64] . although some positive effects of tcm in liver diseases are reported, we should also note the possibility of liver damage induced by some herbs during treatment. the perspective that herbs, as natural medicine, have no side effects is outdated and wrong. some chinese herbal medicines, which were used to treat liver diseases, have been reported to have hepatotoxicity in high doses or even in standard doses, such as heshouwu (polygonum multiflorum [70] . this potential toxicity should serve as a reminder that clinical practitioners should administer chinese medicine with as much caution as western pharmaceutical drugs. it is very important to take precautions against drug-induced liver injury when selecting herbs and their doses. generally speaking, applying herbs in accordance with the pharmacopeia is quite safe. for example, an aqueous solution of zhizi (gardenia jasminoides) extract, which has been reported to have hepatotoxicity, has no significant side effects on mice liver at medium dose (equivalent to 18 g dose for adults) and low dose (equivalent to 9 g dose for adults). the medium and low doses of zhizi extract did not obviously affect structure of the liver tissue or damage hepatocytes. we suggest that zhizi is not suitable for chronic use at a high dose, but short-term use at a modest dose (9 g per day) is still safe [71] . further, by appropriate preparation of herbal products, such as heshouwu, the hepatotoxicity can be reduced [72] . it is also important to note that in chinese medicine, herbs with similar names cannot be used interchangeably. for instance, using tusanqi (gynura segetum), which has severe hepatotoxicity, as a substitute for shensanqi (panax notoginseng) is dangerous, and they come from very different plants. 6 new techniques and methodologies are needed for studying tcm although tcm therapy is effective in the treatment of liver diseases, more research is required to understand the underlying mechanisms of action. at present, the pharmacological studies of chinese medicinal formulae are carried out extensively in vivo, however, the induced animal models cannot be relied on to fully mimic clinical pathogenesis of human patients. studies that examine the mechanisms behind tcm can only be conducted in vitro, are ill suited to the complex formulations of herbs used in chinese medicinal remedies and are restricted by existing research techniques and methodology [73] . current research technology is not designed to evaluate responses from multi-dimensional variables, like the herbal formulations used in tcm. this may be one of the reasons that the curative effects of tcm have been slow to receive approval among western medicinal practitioners. new research techniques and methodologies should be developed to evaluate the curative effects of tcm and to elucidate its mechanisms [74, 75] . we believe that as techniques and methodologies evolve to address the complex nature of tcm herbal formulations, a more mechanistic understanding of the use of tcm in treating liver diseases will emerge. these studies will thus lead to the improvement of clinical results and refinement of the contemporary practice of tcm. as a complementary and alternative therapy for the treatment of liver diseases, tcm is a powerful but underused tool in the present, and has great potential for future use. the authors have no conflicts of interest to declare. journal of integrative medicine (jim) is an international, peer-reviewed, pubmed-indexed journal, publishing papers on all aspects of integrative medicine, such as acupuncture and traditional chinese medicine, ayurvedic medicine, herbal medicine, homeopathy, nutrition, chiropractic, mind-body medicine, taichi, qigong, meditation, and any other modalities of complementary and alternative medicine (cam). article types include reviews, systematic reviews and meta-analyses, randomized controlled and pragmatic trials, translational and patient-centered effectiveness outcome studies, case series and reports, clinical trial protocols, preclinical and basic science studies, papers on methodology and cam history or education, editorials, global views, commentaries, short communications, book reviews, conference proceedings, and letters to the editor. • no submission and page charges • quick decision and online first publication for information on manuscript preparation and submission, please visit jim website. send your postal address by e-mail to jcim@163.com, we will send you a complimentary print issue upon receipt. effect of glucocorticoid with traditional chinese medicine in severe acute aespiratory syndrome (sars) interaction of a traditional chinese medicine (phy906) and cpt-11 on the inflammatory process in the tumor microenvironment artemisinin: discovery from the chinese herbal garden anti-infective and cytotoxic properties of bupleurum marginatum a review of the pharmacological mechanism of traditional chinese medicine in the intervention of coronary heart disease and stroke puerarin: a review of pharmacological effects potential benefits of chinese herbal medicine for elderly patients with cardiovascular diseases danqi pill protects against heart failure through the arachidonic acid metabolism pathway by attenuating different cyclooxygenases and leukotrienes b4 chinese medicine shensongyangxin is effective for patients with bradycardia: results of a randomized, double-blind, placebo-controlled multicenter trial effects of chinese herbal medicine yiqi huaju formula on hypertensive patients with metabolic syndrome: a randomized, placebo-controlled trial favorable circulatory system outcomes as adjuvant traditional chinese medicine (tcm) treatment for cerebrovascular diseases in taiwan randomized controlled multicenter clinical trial for integrated treatment of community-acquired pneumonia based on traditional chinese medicine syndrome differentiation prevention of allergic airway hyperresponsiveness and remodeling in mice by astragaliradix antiasthmatic decoction the anti-asthma herbal medicine ashmi acutely inhibits airway smooth muscle contraction via prostaglandin e2 activation of ep2/ep4 receptors clinical effects of the method for warming the middle-jiao and strengthening the spleen on gastric mucosa repair in chronic gastritis patients modified da-cheng-qi decoction reduces intra-abdominal hypertension in severe acute pancreatitis: a pilot study optimized project of traditional chinese medicine in treating chronic kidney disease stage 3: a multicenter double-blinded randomized controlled trial efficacy and safety of traditional chinese medicine (shenqi particle) for patients with idiopathic membranous nephropathy: a multicenter randomized controlled clinical trial tripterygium wilfordii hook f (a traditional chinese medicine) for primary nephrotic syndrome clinical effect of jiutengzhuyu tablets on promoting blood circulation in women with oviducal obstruction study on the efficacy and safety of xueyou mixture in treating hemophilia beneficial effects of traditional chinese medicine on the treatment of osteoporosis on ovariectomised rat models a traditional chinese medicine therapy warming meridians to nourish blood in treating chronic pain due to soft tissue injury of the neck and shoulder: a randomized controlled trial trial of chinese medicine wu-ling-san for acute low-tone hearing loss mechanism of traditional chinese medicine in the treatment of allergic rhinitis identifying core herbal treatments for urticaria using taiwan's nationwide prescription database effect of yi-gan san on psychiatric symptoms and sleep structure at patients with behavioral and psychological symptoms of dementia traditional herbal medicine in preventing recurrence after resection of small hepatocellular carcinoma: a multicenter randomized controlled trial treatment of middle/late stage primary hepatic carcinoma by chinese medicine comprehensive therapy: a prospective randomized controlled study clinical observation on the treatment of middle-late stage liver carcinoma by combined therapy of hepato-arterial chemo-embolising and chinese drugs for strengthening pi and regulating qi studies on treatment of fatty liver with traditional chinese medicine experience of treatment for posthepatitic cirrhosis based on tcm syndrome differentiation clinical study on effect of jiangan jiangzhi pill in treating nonalcoholic fatty liver diseases effects of baogan lishui decoction on patients with ascites due to cirrhosis: a report of 84 cases. guang ming zhong yi protective effect of compound tongfu granule on intestinal barrier in patients with cirrhosis of decompensation stage effects of method of regulating qi to dissipate blood stasis and phlegm with chinese herbs on expression of srebp-1c/fas in rats liver tissue with non-alcoholic steatohepatitis a prospective cohort study on the influence of high doses of herbs for clearing heat and resolving stasis on survival rates in patients with hepatitis b-related acute-on-chronic liver failure clinical observation of clearing heat and removing dampness, cooling blood and detoxicating therapy for acute on chronic liver failure in hepatitis b. beijing zhong yi yao contemporary clinical research of traditional chinese medicines for chronic hepatitis b in china: an analytical review liver cirrhosis procollagen iii levels in serum: do they provide additional information in liver disease? reversal of hepatic fibrosis -fact or fantasy? effect of fuzheng huayu 319 recipe on serological parameters of fibrosis in treating chronic hepatitis b. zhongguo zhong xi yi jie he za zhi multicenter clinical study about the action of fuzheng huayu capsule against liver fibrosis with chronic hepatitis b effect of ganping capsule on posthepatitic cirrhosis in adjusting abnormal immune function influence of fuzheng huayu capsules on abnormal amino acids spectrum in chronic liver diseases effect of fuzhenhuayu recipe in adjusting endocrine disorders in cirrhotic patient clinical research of ganping capsule against liver fibrosis in patients with chronic hepatitis b multicenter clinical study on fuzhenghuayu capsule against liver fibrosis due to chronic hepatitis b capsule oxymatrine in the treatment of liver fibrosis in patients with chronic virus hepatitis: a randomized, double blind clinical study on treatment of alcoholic liver disease by qinggan huoxue recipe results set of traditional chinese medicine basic theory research in 973 program a randomized controlled study of fuzheng huayu capsule for prevention of esophageal variceal bleeding in patients with liver cirrhosis meta-analysis on fuzhenghuayu capsule combined with nucleoside antiviral drug for chronic hepatitis b clinical frequently used chinese crude drugs with hepatoprotective effect research progress on mechanism of clinical hepatoprotective drugs effect of fuzheng huayu formula and its actions against liver fibrosis role of the igf-1/pi3k pathway and the molecular mechanism of fuzhenghuayu therapy in a spontaneous recovery rat model of liver fibrosis effects of fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats therapeutic efficacy of traditional chinese medicine 319 recipe on hepatic fibrosis induced by carbon tetrachloride in rats curative effects of fuzheng huayu capsules on hepatic fibrosis and the functional mechanisms: a review role of jak/stat pathway in ccl 4 -induced rat liver fibrosis model and molecular action mechanism of fuzheng huayu recipe in treatment of liver fibrosis salvianolic acid b inhibits erk and p38 mapk signaling in tgf-β1-stimulated human hepatic stellate cell line (lx-2) via distinct pathways. evid based complement alternat med salvianolic acid b lowers portal pressure in cirrhotic rats and attenuates contraction of rat hepatic stellate cells by inhibiting rhoa signaling pathway salvianolic acid b attenuates rat hepatic fibrosis via downregulating angiotensin ii signaling therapeutic potential of targeting the renin angiotensin system in portal hypertension traditional chinese medicine can improve liver microcirculation and reduce portal hypertension in liver cirrhosis salvianolic acid b inhibits hepatic stellate cell activation through transforming growth factor β-1 signal transduction pathway in vivo and in vitro effect of salvianolic-acid b on inhibiting mapk signaling induced by transforming growth factor-β1 in activated rat hepatic stellate cells facing the hepatotoxicity of chinese herbal medicine observation of treatment effect and hepatic toxicity of gardenia in mouse with acute liver injury effective components, toxic effects and research advances of present situation of modernization on compound prescriptions. zhongguo shi yan fang ji xue za zhi research status, problems and thinking of multi-component of chinese medicine. zhong yi xue bao discussion on research and development models in innovative chinese material medica key: cord-324529-xbrdtxnz authors: wang, ming; yan, weiming; qi, weipeng; wu, di; zhu, lin; li, weina; wang, xiaojing; ma, ke; ni, ming; xu, dong; wang, hongwu; chen, guang; yu, haijing; ding, hongfang; xing, mingyou; han, meifang; luo, xiaoping; chen, tao; guo, wei; xi, dong; ning, qin title: clinical characteristics and risk factors of liver injury in covid-19: a retrospective cohort study from wuhan, china date: 2020-10-07 journal: hepatol int doi: 10.1007/s12072-020-10075-5 sha: doc_id: 324529 cord_uid: xbrdtxnz background: coronavirus disease 2019 (covid-19) has rapidly become a major international public health concern. this study was designed to evaluate the clinical characteristics and risk factors of covid-19-associated liver injury. methods: a fraction of 657 covid-19 patients were retrospectively analyzed. clinical and laboratory data were derived from electronic medical records and compared between patients with or without liver injury. multivariate logistic regression method was used to analyze the risk factors for liver injury. results: among 657 patients, 303 (46.1%) patients had liver injury with higher rate in severe/critically ill patients [148/257 (57.6%)] than those in moderate cases [155/400 (38.8%)]. the incidence of liver injury was much higher in male [192/303 (63.4%)] than female [111/303 (36.6%)], and in severe/critical patients [148/303 (48.8%)] with percutaneous oxygen saturation ≤ 93% [89/279 (31.9%)] or peak body temperature ≥ 38.5 °c [185/301 (61.5%)] on admission. liver injury-related inflammations included increased white blood cells, neutrophils and decreased lymphocytes. more patients with liver injury than without had increased serum il-2r, tnfα, ferritin, hscrp, pct, esr, γ-gt, and ldh. multivariate regression analysis revealed that increasing odds of liver injury were related to male, higher serum hscrp (≥ 10 mg/l), and neutrophil-to-lymphocyte ratio (nlr) (≥ 5). moreover, more deceased patients (14/82 (17%)) had significantly elevated serum tbil than discharged patients [25/532 (4.7%)]. conclusion: liver injury is a common complication in covid-19 patients. the potential risk factors of liver injury include male, hscrp and nlr score. a close monitor of liver function should be warned in covid-19 patients, especially in severe/critical individuals. electronic supplementary material: the online version of this article (10.1007/s12072-020-10075-5) contains supplementary material, which is available to authorized users. the ongoing outbreak of coronavirus disease 2019 (covid -19) has been recently becoming a pandemic [1] . at present, it is believed that sars-cov-2 mainly invades the respiratory system. most patients have fever, cough, chest distress and dyspnea. furthermore, systemic hyperinflammation driving hypercoagulability is common in these patients [2] . however, accumulating evidences have suggested that some covid-19 patients have different degrees of liver dysfunction [3] [4] [5] [6] . of 99 patients with covid-19 enrolled in wuhan jinyintan hospital, among whom 43 patients with different degrees of abnormal liver function were admitted to intensive care unit (icu). one patient presented with obvious liver injury (alanine aminotransferase (alt) 7590 u/l, aspartate aminotransferase (ast) 1445 u/l) [3] . huang et al. showed that more patients admitted to icu had elevated ast levels [4] . wang et al. showed that patients admitted to icu had significantly higher alt levels [5] .moreover, guan et al. extracted the currently largest cohort regarding 1099 mainly moderate sars-cov-2 infected patients and showed 39.4% with severe disease had elevated ast and 28.1% had elevated alt, and the proportion was 18.2% and 19.8% in patients with non-severe disease [6] .given that the number of patients in these studies is relatively small, information about the clinical characteristics of liver injury in these patients is scarce. so far, to our knowledge, there are few reports that analyzed risk factors of liver injury in covid-19 patients. this current study aims to analyze the clinical characteristics of covid-19 patients with liver injury, and evaluate the potential risk factors for the development of liver injury. all 657 patients included in the study were admitted from january 13, 2020 to february 25, 2020 in tongji hospital. the clinical characteristics and outcomes (death or discharge) were monitored up to march 28, 2020. tongji hospital was urgently reconstructed and has been assigned by chinese government as a designated hospital for severe or critically ill covid-19 patients. according to the guidance provided by the chinese national health commission,all confirmed covid-19 patients were diagnosed by sars-cov-2 nucleic acid detection of respiratory tract specimens. the clinical data and laboratory data of patients were obtained from electronic medical records. the clinical data included sex, age, percutaneous oxygen saturation, heart rate, respiration rate, peak temperature during the course of the disease, duration of fever,comorbidities and treatment in the patients. the laboratory data included blood routine examination, liver function, renal function, coagulation function, creatine kinase (ck), creatine kinase-mb (ck-mb), hypersensitive cardiac troponin i (ctni), n terminal pro-brain type natriuretic peptide (nt-probnp), hypersensitive c-reactive protein (hscrp), erythrocyte sedimentation rate (esr), procalcitonin (pct), ferritin, cytokines (interleukin 1β (il-1β), interleukin 2 receptor (il-2r), interleukin 6 (il-6), interleukin 8 (il-8), interleukin 10 (il-10), tumor necrosis factor α (tnfα)),hepatitis b surface antigen and hepatitis c virus antibody examinations. all patients were classified as being moderate, severe, or critically ill according to the guidance for covid-19 (6th edition) released by the national health commission of china [7] (supp . table s1 ). liver injury was defined as serum level of alt or total bilirubin (tbil) greater than upper limit of normal value (uln). the uln of alt or tbil was 40u/l and 26 μmol/l, respectively, regardless of whether the patients had a history of chronic liver diseases. continuous variables were described as median and interquartile range (iqr), and tested using non-parametric mann-whitney u test. categorical variables were described as number and percentage, and tested using χ 2 test, fisher's exact test, as appropriate. multivariate logistic regression models were used to explore the risk factors for liver injury. wilcoxon signed-rank test was used for paired continuous variables. spss (version 22.0) was used for all analyses. p value < 0.05 was considered statistically significant. from january 13, 2020 to february 25, 2020, a fraction of 991 patients were enrolled from tongji hospital for this study. among them, 657 patients with confirmed sars-cov-2 infection were subsequently included for further analyses. as of march 28, 2020, 532 patients recovered and were discharged, 82 patients died and 43 patients remained hospitalized. as shown in table 1 , among 657 patients, 303 (46.1%) had liver injury. as shown in table 2 as shown in table 1 , liver injury appeared in 277 (42.2%) of 657 patients with alt elevation and in 32 patients (4.9%) with tbil elevation, 160 (24.4%) of 657 patients had γ-gt elevation. there were significant differences in laboratory findings between covid-19 patients with or without liver injury, including higher white blood cell, neutrophil and lower lymphocyte count in liver injury patients than those without. more [54/301 (17.9%)] patients with liver injury developed leukocytosis (white blood cell count ≥ 10 × 10 12 /l) than those [27/354 (7.6%)] without. there were no differences in nt-probnp and ctni levels between patients with or without liver injury ( table 2) . elevated hscrp (> 10 mg/l) appeared more frequent in patients with liver injury [230/274 (83.9%)] than those without [220/334 (65.9%)]. moreover, serum esr level was significantly higher in patients with liver injury compared with those without. in terms of cytokines, more patients with liver injury had elevated serum il-2r, il-6 and tnfα than those without. to further investigate the correlation of inflammation with liver injury, a series of inflammation-based scores including mgps, plr, nlr, and car, lmr, pni scores were calculated and compared (table 3 ). more patients with liver injury have mgps score over 2 points than those without. the plr, nlr, and car scores were significantly higher in patients with liver injury than those without. the lmr and pni scores were significantly lower in patients with liver injury than those without. the correlation between some surrogate markers of systemic inflammation response syndrome (sirs), such as il-2r, il-6, tnfα, hscrp, ferritin and pct, and alt, tbil and γ-gt levels, was investigated. and the results showed serum alt, tbil and γ-gt all correlated with hscrp and ferritin; meanwhile, tbil and γ-gt correlated with il-2r, tnfα and pct (supp . table s2 ). we also observed the effect of medications on liver injury. during hospitalization, some patients received one or more medications including antiviral drugs (oseltamivir, lopinavir/ritonavir, or arbidol), interferon inhalation, antibiotics, systemic glucocorticoid and non-steroidal anti-inflammatory drugs (nsaids the proportion of cases with γ-gt level above 1-2, 2-5 or greater than 5 times the uln was higher in severe/ critical patients than those in moderate patients. after considering the p value of univariate analysis, clinical significance and data integrity, 593 patients were enrolled and sex, metabolic disorder, viral hepatitis, hscrp, nlr and peak temperature were selected as variables of logistic regression models. the independent risk factors for liver injury were as follows: male (or 2.038, 95% ci 1.443-2.879, p < 0.001), hscrp ≥ 10 mg/l (or 1.733, 95% ci 1.118-2.687, p = 0.014), and nlr ≥ 5 (or 2.154, 95% ci 1.486-3.124, p < 0.001) ( table 4 ). the dynamic changes of hscrp and nlr with or without liver injury were further observed every 5 days after admission. both hscrp and nlr levels declined over the course of 15 days with higher values in patients with liver injury compared with those without (supp. figure s1 ). data were expressed as median (interquartile range, iqr) or n (%). p values were calculated by mann-whitney u test, χ 2 test, as appropriate. patients with at least one of the following three conditions are classified as metabolic disorders: 1. diabetes mellitus, 2. hypertension, 3. dyslipidemia to understand correlation of alt and tbil levels with disease progression, the dynamics of alt and tbil levels in 207 patients (21 deceased patients and 186 discharged patients) with complete data were analyzed from admission to the 15th day with an interval of every 5 days (fig. 1a, b) . during hospitalization, the alt levels of the discharged patients slightly increased from admission to day 5, then gradually decreased thereafter until discharge. whereas the alt levels of those deceased patients rapidly increased from the 5th day after admission until day 15. tbil levels of discharged patients stayed within normal range during the whole course, whereas it increased significantly in the deceased patients after admission, until day 15. the increase of alt from day 5 to day 15 was significantly higher in deceased patients than those in discharged patients (fig. 1c) . although the increases of tbil levels in deceased patients from day 5 to day 15 were notable, there was no statistical significance compared with discharged patients (fig. 1d) . furthermore, tbil levels of 1-2 or above 2 times the uln [7/82 (8.5%) and 7/82 (8.5%)] in deceased patients were markedly higher than those in discharged patients [24/532 (4.5%) and 1/532 (0.2%)]. the alt levels showed no significant differences between the two groups (table 5 ). the present study showed that liver injury was more prevalent in male, severe or critically ill patients with percutaneous oxygen saturation ≤ 93% or peak temperature ≥ 38.5 °c on admission, and comprehensively delineated the risk factors for covid-19-associated liver injury. liver injury-related inflammations included increased white blood cells, neutrophils and decreased lymphocytes. more patients with liver injury had increased serum il-2r, tnfα, ferritin, hscrp, pct, esr, γ-gt, and ldh. in addition, male, elevated hscrp (≥ 10 mg/l) and nlr (≥ 5) were risk factors for covid-19-associated liver injury. and patients with abnormal liver function tests (lft) had higher risks of progressing to severe disease. covid-19-associated liver injury is defined as any liver damage occurring during disease progression and treatment of covid-19 in patients with or without pre-existing liver diseases. in our cohort, there were 26 patients with elevated tbil alone. according to data analysis of these patients, the results showed that the bilirubin elevation was due to direct bilirubin elevation as main body (15 cases) or both direct bilirubin and indirect bilirubin elevation (8 cases) or indirect bilirubin elevation as main body accompanied by the rise of ast (3 cases). although all the 26 patients showed a degree of liver injury, hemolytic jaundice or gilbert's disease cannot be entirely excluded and requires further monitor in follow-up study. liver injury is not uncommon in previous studies in patients infected with severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) [8] . likewise, abnormal lfts were frequently seen in patients with covid-19, with incidence rates exceeding 50% in some studies. liver injury was presented in 30 out of 113 deceased patients from our previous reports [9] . however, patients with covid-19 rarely develop acute or acute-on-chronic liver failure [4, 6, 9] . taken together, these studies demonstrated that the majority of cases of covid-19-associated liver injury occurred in severe or critically ill patients. consistent with the above findings, current study in a relatively larger cohort of severe/critical patients further demonstrated liver injury occurred more frequently in these patients [148/257 (57.6%)] than in moderate ones [155/400 (38.8%)]. serum alt, tbil and γ-gt levels were markedly higher in severe and critically ill patients than in moderate patients, with a peak alt of 728 u/l and peak tbil of 174.1 μmol/l, respectively. the increase of alt from day 5 to day 15 after admission was significantly greater in deceased patients than in discharged patients, and the proportion of cases in deceased patients with serum tbil levels of 1-2 or above 2 times the uln was markedly higher than that in discharged patients. these results provided the concrete evidence that impaired liver function was more frequent in severe and critically ill patients. moreover, multivariate regression analysis showed that hscrp ≥ 10 mg/l, nlr ≥ 5 and male were the risk factors for liver injury in our study, which was consistent with lei's results [10] . as a traditional inflammatory marker, crp is an acute-phase protein used for the diagnosis and follow-up of infection or tissue damage. li et al. [11] showed crp was independently associated with hepatic injury in patients with covid-19. nlr, as a reliable marker of systemic inflammation and infection, has been considered as a predictor of bacterial infections, including pneumonia. liu et al. [12] reported that nlr might predict severe illness in the early stage of covid-19. zhang et al. [13] identified nlr as an independent risk factor for severe covid-19. current studies indicated that there might be a sex predisposition to covid-19, with men more prone to being affected likely due to much higher smoking rate in men [14] . underlying mechanisms involved in liver injury in patients with covid-19 are complex and interactive, including immune reconstitution in the presence of sars-cov-2, direct drug toxicity, systemic inflammation with liver involvement induced by cytokine storm or pneumoniaassociated hypoxia [15] . previous studies indicated that t lymphocytes particularly cd4 + t and cd8 + t cells decreased increase in tbil from day 5 to day 15 after admission. p values were calculated by mann-whitney u test, or wilcoxon signed-rank test, as appropriate in severe covid-19 patients [16] , and immune function dysregulation in some cases may be associated with the development of acute respiratory distress syndrome (ards), collateral damage to multiple organs, such as liver, kidney and heart as well as death [17] . the exaggerated immunemediated inflammation is known as "cytokine storm". similar to our study, accumulating evidence suggested that patients with severe covid-19 might have a cytokine storm syndrome. changes of inflammatory cytokines following sars-cov-2 infection may cause or contribute to liver damage. the underlying mechanism involved in cytokine accumulation in covid-19 and subsequent liver injury warrants further investigation. recent study [18] demonstrated no significant increase in alp and γ-gt in patients with covid-19. in contrast, among 657 cases in the present study, more severely/critically ill patients [83/257 (32.3%)] than moderate patients [77/400 (19.3%)] had elevated γ-gt level. previous pathological findings of covid-19 in the liver showed moderate microvesicular steatosis and active inflammation in the hepatic portal area but no viral inclusions identified in the liver tissue [19] . the latest research showed that angiotensin-converting enzyme 2 (ace2) was expressed in specific cholangiocytes in healthy liver tissues [20] and suggested liver injury in covid-19 patients may not be only due to hepatocyte damage but cholangiocyte dysfunction. in our study, elevated γ-gt and bilirubin in patients with covid-19 suggested that the virus may cause liver impairment by targeting cholangiocytes. the pathogenesis of covid-19-associated liver injury warrants further investigation. the impact of preexisting chronic liver diseases on the liver function of covid-19 patients remains not fully elucidated. ji et al. recently reported patients with nonalcoholic fatty liver disease (nafld) were at higher risk of disease progression, and developing abnormal liver function during hospitalization in covid-19 patients [21] . singh et al. [22] found covid-19 patients with preexisting liver disease, notably cirrhosis, were at higher risk for hospitalizations and mortality. guan et al. reported that patients with preexisting chronic hepatitis b did not have more severe disease compared to the overall population [6] . our study showed that 52 (7.9%) patients with underlying viral hepatitis (hepatitis b and hepatitis c)developed liver dysfunction, and more patients with underlying viral hepatitis developed liver injury than those without [31/303 (10.2%) vs 21/354 (5.9%)], which may be partly due to inflammatory responses and immune disorder of viral hepatitis. further multivariate regression analysis showed that underlying viral hepatitis and metabolic disorder were not independent risk factors for liver injury, which is consistent with recent report [23] , suggesting that liver injury cannot be entirely attributed to the preexisting chronic liver diseases in covid-19 patients. considering the administration of antivirals, antibiotics, nsaids and glucocorticoids in the treatment of patients, potential drug-induced liver injury (dili) deserves special concern. given data provided is limited, based on recent results, we cannot draw a definitive conclusion about whether lopinavir/ritonavir increase the risk of developing liver damage [18, 24, 25] . our study revealed a higher proportion of patients with liver injury had received systemic glucocorticoid. nevertheless, we found a slightly reduced risk of hepatic injury in patients treated with arbidol. this is likely due to the possibility that arbidol may effectively suppress sars-cov-2 replication, which may be a beneficial pathway to lower the risk of the subsequent viral-induced liver injury. it has recently been reported that arbidol combined with lopinavir/ritonavir achieved significantly higher rates of viral clearance and favorable clinical outcome compared with lopinavir/ritonavir used alone [26] . these findings further supported the hypothesis that effective antiviral therapy may prove useful in preventing or slowing the progression of the covid-19 disease. our study has some limitations. first, nearly one third of critically ill patients developed disorders of consciousness on admission, which might result in a loss of some information (particularly a detailed history and subjective symptoms). additionally, some physical examination and laboratory tests were not done in all the patients, and missing data or important tests might lead to bias of clinical characteristics. second, the impact of covid-19 in patients with preexisting chronic liver diseases, such as viral hepatitis, nafld, and alcohol-related liver disease, remains to be evaluated. third, in the early period of epidemic, owing to some environmental constraints, such as the explosive growth of the infected population and the shortage of medical supplies, the results may be difficult to interpret accurately. more intensive surveillance or individually tailored therapeutic approach is needed for severe cases, especially among patients with liver or other important organ dysfunction. in conclusion, immune-mediated inflammation may lead to covid-19-associated liver injury. the potential risk factors for liver injury include male, hscrp and nlr score. moreover, patients with abnormal lfts had higher risks of progressing to severe disease. attention should be paid to monitor liver function during the course of covid-19, especially in severely/critically ill patients. the data provided may be useful in helping us extend our understanding of the disease so that early effective intervention could be carried out promptly. appropriate intervention and reasonable supportive care are expected to reduce the inflammatory response and then further reduce liver injury of patients with covid-19. a novel coronavirus from patients with pneumonia in china coagulation abnormalities and thrombosis in patients with covid-19 epidemiological and clinical 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with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study we thank all the patients and their families involved in this study, as well as numerous doctors, nurses and civilians working together to fight against the sars-cov-2.author contributions mw and wmy share first authorship, and the order in which they are listed was determined by workload. qn, dx (dong xi), and wg designed the study and had full access to all data in the study and take responsibility for the integrity of data and the accuracy of the data analysis. mw and dx contributed to patient recruitment, data collection, data analysis, data interpretation, literature search, and writing of the manuscript. wmy, wpq, and dw contributed to patient recruitment, data collection, data analysis, and writing of the manuscript. lz, wnl, xjw, and km had roles in patient recruitment, data collection, and data interpretation. mn, dx (dong xu), hww, gc, hjy, hfd, myx, mfh, and tc had roles in the patient management, data collection, and clinical management. xpl contributed to data analysis, data interpretation, and writing of the manuscript. all authors had access to the study data and reviewed and approved the final manuscript.funding this work was funded by grants from the tongji hospital for pilot scheme project and partly supported by the chinese national thirteenth five years project in science and technology (2017zx10202201), national commission of health, and by chinese national major science and technology (2018zx09733001-002-006). open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-318755-fip8wj6y authors: el kassas, mohamed; alboraie, mohamed; al balakosy, amira; abdeen, nermeen; afify, shimaa; abdalgaber, mohammad; sherief, ahmed f; madkour, ahmad; abdellah ahmed, mohamed; eltabbakh, mohamed; salaheldin, mohamed; wifi, mohamed-naguib title: liver transplantation in the era of covid-19 date: 2020-05-12 journal: arab j gastroenterol doi: 10.1016/j.ajg.2020.04.019 sha: doc_id: 318755 cord_uid: fip8wj6y liver transplantation is considered the ultimate solution for patients with end-stage chronic liver disease or acute liver failure. patients with liver transplant need special care starting from preoperative preparation, surgical intervention ending with postoperative care. transplanted patients have to receive immunosuppressive therapy to prevent rejection. such a state of immune suppression could predispose to different types of infections in liver transplant recipients. currently, the world is suffering a pandemic caused by a new strain of the coronavirus family called covid-19. certain infection control precautions are needed to protect immunocompromised and vulnerable patients, including liver transplant candidates and recipients from acquiring covid-19 infection. restricting non-transplant elective surgical procedures, managing transplant patients in separate outpatient clinics, and in-patient wards can prevent transmission of infection both to patients and healthcare workers. telemedicine can help in the triage of patients to screen for symptoms of covid-19 before their regular appointment. management of immunosuppressive therapy and drug-drug interactions in liver transplant recipients infected with covid-19 should be cautiously practiced to prevent rejection and effectively treat the underlying infection. in this report, we are trying to summarize available evidence about different aspects of the management of liver transplant candidates and recipients in the era of covid-19. liver transplantation (ddlt) represented 20%. fifty-six percent of the reported cases were in egypt [8] . based on previous observations for sars and other related viruses, a theoretical risk of liver damage exists with covid-19 infection [9, 10] . however, available data only reported hepatic dysfunction in the form of abnormal levels of liver aminotransferases and slightly elevated bilirubin levels, mainly in critically ill patients [11] . on the other hand, reports during an influenza outbreak in germany in winter 2017/2018 showed increased organ failure scores of patients with liver cirrhosis where 5 out of 11 patients with liver cirrhosis developed acute liver failure during influenza infection [12] . no data available on the impact of covid-19 on decompensated liver disease patients awaiting ltx, but because of the known immunocompromised state of these patients, adequate protective measures should be maintained. although healthcare facilities are overwhelmed with management of covid-19 patients & health resources are being rapidly consumed, the american association for the study of liver diseases (aasld), recommended against postponing transplantation. moreover, they advised each program to consider its capability regarding intensive care unit (icu) beds, ventilators availability, and blood donation [10] . prioritization of transplant candidates is another problem that may face clinicians due to limited resources during the pandemic, as well as the exclusion of donors infected with covid-19 [10] . immunosuppression in the post-transplant recipients may be protective against cytokine storm induced by covid-19, which is responsible for the severe illness on the one hand. however, and on the other hand, recipients on immunosuppression may have more intense and prolonged shedding of the virus, increasing the risk of transmission to contacts, including healthcare workers [13] . this could emphasize the crucial role of implementing infection control measures to avoid losing candidates on the ltx waiting list because of the closed transplantation centers [14] . international societies like world health organization (who) and centre for disease control and prevention (cdc) are always confirming the necessity to use personal protection equipment (ppe) in addition to the restriction of outpatient and elective procedures as preventive measures against covid-19 [15] . limitations of aerosol-generating procedures like suction, endotracheal intubation, and advanced endoscopy are of major concern due to the fear of the possibility of disease transmission. further restrictions to prevent other routes of infections like feco-oral transmission, included colorectal surgeries and colonoscopies. currently, many interventional surgical societies, anesthesia, endoscopy, radiology, and intensive care have placed their statements, guidelines, and recommendations to adjust their practice to the current epidemic [16] . different reasons rationalized the delay or even cancellation of non-emergency procedures as they would consume ppe tools which are currently running short supply worldwide. the second reason that such elective procedures are postponed or canceled is to prevent unnecessary infections to medical staff and caregivers, which may be transmitted from asymptomatic covid-19 patients or their companions. also, they consider such procedures a further burden and workload on an already exhausted medical system. finally, occupying the operative theatres with such cases would warranty the need for mechanical ventilators that might be more beneficial and valuable if they are directed to rescue a covid-19 patient's life [17] . meticulous evaluation should be done before deciding for the priority of the procedure through detailed history taking, one by one consultation, temperature measurement, hand hygiene, and reporting of any suspected case of covid-19 (even if afebrile), and finally cleaning and disinfection protocols of premises. repeated physical examination and temperature measurement along with the revision of chest imaging like a computed tomography scan or a chest radiograph, and if covid-19 is suspected or confirmed, all non-emergency procedures would be delayed or canceled [18] . because of the rapidly changing situation of covid-19 infection worldwide, indications of ltx will need to be updated according to the emerging data. bearing in mind that any liver transplant related activity not only involves the donor and the recipient, but it involves many individuals, including doctors, paramedical staff, nurses, and health care workers. taking into consideration that there is a risk of the donor to recipient transmission of covid-19, from both deceased donors and living donors. the risk of donor-derived infection would depend upon donor exposure, infectivity in the incubation period, degree and duration of viremia, and viability of the virus within blood or specific organ compartments [13] . traditionally, the aasld/ast guidelines outline four major types of indications for ltx in the united states: acute liver failure, complicated cirrhosis, metabolic liver diseases, and systemic complications of chronic liver disease [19] . acute liver failure is the most important indication for emergency ltx. common causes of acute liver failure include acetaminophen overdose, acute viral hepatitis, drug-induced liver injury, mushroom poisoning, autoimmune hepatitis, wilson's disease, acute ischemic hepatitis (shock liver), and acute fatty liver of pregnancy [19] [20] [21] [22] . similarly, patients with cirrhosis and type 1 hepatorenal syndrome have a median survival of fewer than two weeks and should be urgently referred to a transplant center for expedited transplant evaluation, as should patients with other evidence for rapid hepatic decompensation [19, 23] . elective ltx is indicated in cases with cirrhosis associated with deteriorating hepatic synthetic function, renal function, and related complications like hcc [19, [24] [25] [26] [27] [28] [29] [30] [31] . to minimize the risk of covid-19 exposure in the hospital setting, ltx should be done judiciously as per the following criteria. in essence, non-urgent transplants should be postponed, and acute liver failure can be done according to the standard indications, acute on chronic liver failure decisions for transplantation should be based on individual center's experience [32] . despite being potentially transmitted by organ donation, recent recommendations suggest that ltx could be performed during the covid-19 pandemic [10] . as the organism is predominantly found in respiratory secretions, lungs are considered high-risk organs for transmission of covid-19infection if the donor is infected. however, all other organs could be considered at risk as the virus was isolated from blood in nearly 15% of cases [33] . these observations are supported by evidence from the sars epidemic in 2003 in which autopsy results could demonstrate the virus in almost all body organs [34] . two types of ltx techniques are available; the first is the conventional or standard method, which involves implantation of whole liver grafts ddlt, and this type is the most widely used in the usa and europe [19] . the other type is ldlt, and this is the only option available for patients living in some countries like egypt as cadaveric organ transplant is still banned there, and that is why some patients travel abroad seeking organ transplant elsewhere [35] . at present, the organ procurement organizations (opos) and hospitals having ldlt programs should screen potential donors for exposure and clinical symptoms suggestive of covid-19, and as more information builds up, the ideal approach would hopefully be developed [36] . although personal contact is the basis of the doctor-patient relationship, which allows rapid and accurate assessment of the patient's condition, unfortunately, it exposes physicians to the risk of infection and becomes a source of the virus spread. therefore, it is considered wise to substitute direct contact with patients with more distant interactions with the utilization of available technology. the same approach could also be applied to the post-operative period for the donor in the case of ldlt, aiming to be discharged as soon as possible from the hospital [37] . with the increasing number of infected cases and mortality caused by covid-19, a recent discussion for using organs from patients infected with covid-19 has led to several points of debate. for instance, from the clinical point of view, it could potentially be a life-saving procedure, especially that delays might result in more deterioration for the patient's need for the organ and even increase the risk of covid-19 exposure. on the other hand, it may transmit infection, which would even be more severe in transplant recipients, especially in the absence of effective therapy. financially speaking, it can be a possible source of income for hospitals despite reimbursements remain uncertain. it might be argued from the legal point that organs with hcv have already been transplanted after getting informed consent from patients. nevertheless, this disease has a different risk profile compared to hcv. as regards the ethical perspective, this procedure respects the wishes of donors and their families and ensures the autonomy of patients willing to do so. however, patients could be overwhelmed with responsibility for informed consent in the presence of little guidance [38] . the emergence of the covid-19 pandemic has posed extensive threats and problems to all the healthcare facilities, including ltx centers [39] . these effects are not only confined to donor or recipient issues but also extends to involve many other problems in the availability of healthcare resources [13] . we are faced with enormous challenges owing to the high communicability and virulence of the virus, the risk of introducing immunosuppressive therapies during this pandemic, and our utmost need for all the health care utilities. on the other hand, we do have a very long list of miserable patients waiting for ltx, which is the only available treatment option for this difficult to treat a group of patients, so judicious decisions and strict precautions became now mandatory [40] . streptococcal pneumonia and influenza vaccines are strongly recommended to all recipients prior to ltx, together with strict prophylaxis against complications of cirrhosis to reduce the number of hospital admissions [41] . it is also recommended to test for covid-19 in patients with acute decompensation or acute on top of chronic liver failure (aclf) [39] . for those on ltx lists, it is recommended to test both donors and recipients for covid-19 before ltx, putting into consideration that negative results do not totally exclude the infection. alternatively, computed tomography (ct) of the chest can be considered [42] . pre-procedure consent should include the potential hazard for the acquisition of nosocomial covid-19infection [39] . accepting only grafts with a low risk of delayed graft function to reduce complications and minimize the length of postoperative stay is also recommended [10] . diminishing exposure of health care workers as much as possible, through using online clinics and phone calls as a substitute to primary clinics can prevent unnecessary risk of infection. doctors may also talk to all patients by phone before their visits to rule out any possibility of covid-19 infection. deferring optional visits and restricting it only to urgent ones also help to prevent nosocomial infections [37] . modifications of the outpatient transplant clinics by widening the patients' waiting areas and following strict infection control precautions is of utmost importance [43] . workforce affairs are crucial; any member of the transplantation team suffering from any symptom suggestive of covid-19infection should be absent from work and self-isolate him/herself for fourteen days if the exposure occurred. moreover, a rapid test for the transplantation team is highly recommended [44] . post-operative care for ltx recipients during the pandemic of covid-19 is challenging. to guarantee the maximum benefits for both the patient and the graft, a multidisciplinary management team is usually involved in postoperative care of ltx recipients. such management includes infection control with extra care directed at preventing postoperative infections, including covid-19 infection [40] . liver transplant recipients should be admitted to separate wards where there is complete separation from covid-19 admission wards, along with the strict implementation of standard disinfection measures. it is recommended to limit surgical and medical rounds, requests for the image, and blood tests to the least required number [10] . follow up of liver transplant recipients is usually performed in tertiary referral hospitals, where covid-19 hotspots may be present [39] . it is thus recommended to limit in-person outpatient visits even in areas without significant covid-19 community spread [45] . hospital admission of liver transplant recipients should be considered only for patients suffering from major complications like rejection, decompensation, or vascular complications. during in-person outpatient follow up visits, liver transplant recipients should be evaluating in dedicated hepatology/liver transplant clinics away from clinics where confirmed or suspected cases of covid-19 are evaluated [10] . once the patient is clinically stable, it is recommended to perform his routine laboratory investigations, including drug levels at primary care facilities [39] . telemedicine can mitigate exposure of both patients and healthcare workers to covid-19 as it allows better physician-patient communication. development of covid-19 symptoms (fever, cough, shortness of breath, sore throat, diarrhea, the new loss of sense of taste or smell, contact with known covid-19 patients, history of recent travel) in a liver transplant recipient should prompt urgent referral for evaluation along with hepatic symptomatology surveillance and drug compliance assurance [37, 46] . epidemics can lead to a significant increase in demand for icu beds, so reducing the available beds. as an example, the sars outbreak in toronto led to closures of 35 icu beds for ten days, which represented 38 % of the tertiary-care university medical-surgical beds of icu in toronto [47] . many reasons represent a challenge in caring for patients with covid-19 with a high risk of exposure for icu staff. covid-19 is highly contagious, along with more than one route of transmission.the high exposure dose, long contact hours with cases, some procedures such as noninvasive ventilation, and the length of icu stay are representing the challenges for icu staff during the epidemic [48] . moreover, patients and staff from each sector of the icu should use different routes inside the icu to decrease the risk of infection [51] . another way to minimize the possibility of icu admissions is by applying the fast track protocols which can be applied safely for selected patients undergoing either living or ddlt [52] . the definition of fast-tracking in liver transplant still lacks consensus among different centers, ranging from early postoperative extubating in the operating room once the surgery is finished, to strategies that minimize postoperative ventilation time.generally, this term is reserved for early extubation, recovery in a post-anesthesia care unit (pacu), and direct transfer to the surgical ward avoiding an icu stay [53, 54] . patients who underwent ltx are usually poly medicated using many drug classes. the most important are immunosuppressive drugs. as most anti-covid-19 agents are investigational, drug-drug interactions are very critical in this situation in those fragile patients. possible drug-drug interactions between sars-cov-2 antiviral drugs and commonly used immunosuppressants for liver transplant recipients are presented in table 1 [55] [56] [57] . possible interactions with other drugs are summarized below: chloroquine and hydroxychloroquine act either as viral entry blockers or as immunomodulators. despite encouraging preliminary reports, side effects, and interactions with other medications are well known [55] . chloroquine has potential interactions with commonly used drugs like ampicillin, amlodipine, azithromycin, propranolol, and antacids [56] . chloroquine has significant drug interaction with ciclosporin as it increases levels of cyclosporin by decreasing its metabolism. also, chloroquine increases levels of tacrolimus by the same mechanism but to a lesser extent [56]. remdesivir is a nucleotide analog with broad-spectrum antiviral activity against single-stranded rna viruses [55] . it is an investigational drug that appears safe and may not affect other medications; however, remdesivir concentration can be affected by enzyme inducers like clarithromycin, rifampin, phenytoin, and phenobarbital [57] . it is also an investigational drug with antiviral activities through its selective inhibition of viral rna-dependent rna polymerase [55] . favipiravir increase concentration of pioglitazone, rosiglitazone, paracetamol, oseltamivir, and hormonal replacement therapy; however, no significant interactions with immunosuppressive medications nor steroid [57] . a fixed-dose combination for the prevention and treatment of hiv infection. the cytochrome p450 inhibitory effects of ritonavir prolong the half-life of lopinavir and extend its protease inhibitory action but, on the other hand, increases its liability for many drug interactions [55] . this combination is not recommended to be co-administer with many steroids forms, simvastatin, atorvastatin, domperidone, and sirolimus. there is also a potential interaction with ciclosporin, mycophenolate, tacrolimus, which makes its use in the setting of ltx is questionable. however, a recent case report confirmed successful covid-19 treatment by lopinavir/ritonavir in liver transplant recipients [58] . lopinavir/ritonavir can increase chloroquine and hydroxychloroquine concentrations. also, lopinavir/ritonavir induces qt interval prolongation that may potentiate chloroquine and hydroxychloroquine toxicity [57] . tocilizumab is a monoclonal antibody that targets the interleukin-6 receptor, which is the possible mediators of covid-19 induced inflammation and cytokine storms [55] . it has potential possibly minor interaction with ciclosporin, tacrolimus, and sirolimus. tocilizumab can reduce concentrations of calcineurin inhibitors with necessary drug level monitoring. its use with chloroquine and hydroxychloroquine may have additive toxicity. also, it may potentiate hematological toxicity of ribavirin and interferon-beta if used together as tocilizumab has a myelosuppressive effect [57] . ribavirin is an old antiviral drug used in the treatment of hcv for years, and that had a role in the treatment of sars. as it causes dose-dependent hemolytic anemia, ribavirin may potentiate the hematological toxicity of interferon-beta and tocilizumab. ribavirin had no significant interaction with immunosuppressive drugs [57] . one of the lessons learned from mers-cov infections is that host inflammatory responses play a major role in disease progression. this was the base of using interferon-beta in mers-cov and covid-19 infections [55] . in the setting of ltx, interferon-beta had no interactions with immunosuppressive drugs or steroids. nevertheless, it induces myelosuppression, so it should not be combined with tocilizumab. also, potential interaction with chloroquine and hydroxychloroquine may increase its toxicity [57] . patients with advanced liver disease and those after ltx represent vulnerable patient cohorts with an increased risk of infection and/or a severe course of covid-19 because of the immunosuppressed state they have [59] . as the innate immune response associated with increased serum interleukin-6 (il-6), interleukin-8 (il-8), and tumor necrosis factor-alpha (tnf-î±) levels may be the main driver for pulmonary injury due to covid-19, immunosuppression may be protective [33, 60] . coronaviruses have not shown to cause more severe disease in immunosuppressed patients. more interestingly, reviewing the mortality and morbidity reports published on coronaviruses outbreaks such sars that emerged in 2002, mers, and more recently covid-19, no fatalities were reported in patients undergoing transplantations, receiving chemotherapies or other immunosuppressive treatments [61] . risk factors for poor outcome include older age (post-transplant recipients aged >60 years old) male sex and presence of comorbidities (obesity, diabetes, heart disease, lung disease, kidney disease) [10] . available data on coronavirus before and during outbreaks suggest that immunosuppressed patients are not at increased risk of severe pulmonary disease compared to the general population; however, immunosuppression may prolong viral shedding in post-transplant patients with covid-19 if they are already infected [36, 60] . immunosuppressive therapy should be started in patients with liver disease with or without covid-19 who have strong indications for treatment (e.g., autoimmune hepatitis, liver transplant patients, and in cases of graft rejection). this should be done without compromising their transplant management as reducing the dosage or stopping immunosuppressants as this may precipitate acute rejection in a liver transplant recipient, so there is no need to reduce or stop immunosuppression for asymptomatic post-transplant patients without known covid-19 [10] . the reduction should only be considered under special circumstances (e.g., medication-induced lymphopenia, or bacterial/fungal superinfection in case of severe covid-19) after consultation of a specialist [62] . the potential role of corticosteroids for the prevention of progression of mild covid-19 to severe pneumonia is unknown. who recommends avoiding corticosteroids for the treatment of covid-19 unless indicated for another therapeutic purpose [63] . in patients with confirmed covid-19, we should minimize the dosage of prednisone, maintaining a sufficient dosage to avoid adrenal insufficiency [39] . similarly, it is also advised to reduce daily calcineurin inhibitor dosage; consider decreasing tacrolimus/cyclosporine by 50%, stop mycophenolate (cellcept/myfortic) and azathioprine especially in the setting of lymphopenia, fever, or worsening pneumonia attributed to covid-19. in the case of ground-glass opacities, pneumonia switching mammalian target of rapamycin (mtor) to calcineurin inhibitors (cni, e.g., tacrolimus) should be done given the possibility of pneumonitis with mtor; otherwise, we should stop all immunosuppression [59] . for outpatients on belatacept, switching to tacrolimus or cyclosporine should be considered after 28 days from the last dose, to avoid clinic visit. for inpatients on belatacept, we should not administer any further belatacept. twenty-eight days after the last dose, adding low dose cni should be considered. for cni intolerant patients, increasing daily prednisone dose from 5 mg to 7.5-10 mg daily might be considered. low dose prednisone (5 mg) in all patients who were receiving it before hospitalization can be maintained [59] . world health organization director-general's opening remarks at the media briefing on covid19-11 global organ transplant activities in 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transplantation in egypt: an emerging program. transplantation proceedings american society of transplantation. 2019-ncov (coronavirus): faqs for organ donation and transplantation virtually perfect? telemedicine for covid-19 covid-19 organ donation and transplantation town hall (donor issues and candidate concerns care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper covid-19: a global transplant perspective on successfully navigating a pandemic management of infections pre-and postliver transplantation negative nasopharyngeal and oropharyngeal swab does not rule out covid-19 characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china coronavirus disease (covid-19) outbreak: rights, roles and responsibilities of health workers, including key considerations for occupational safety and health cleaning and disinfection for community facilities telehealth seen as a key tool to help fight covid-19. the hospitalist identification and containment of an outbreak of sars in a community hospital early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia the critical care response to a hospital outbreak of middle east respiratory syndrome coronavirus (mers-cov) infection: an observational study intensive care management of liver transplanted patients liver transplantation in an icu dominated by covid-19 is a mandatory intensive care unit stay needed after liver transplantation? feasibility of fast-tracking to the surgical ward after liver transplantation a multicenter evaluation of safety of early extubation in liver transplant recipients fast tracking in liver transplantation. immediate postoperative tracheal extubation: feasibility and clinical impact individual risk management strategy and potential therapeutic options for the covid-19 pandemic covid-19 drug interactions successful treatment of severe covid-19 pneumonia in a liver transplant recipient massachusetts general hospital covid-19 treatment guidance coronaviruses and immunosuppressed patients. the facts during the third epidemic middle east respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission successful recovery of covid-19 pneumonia in a renal transplant recipient with long-term immunosuppression clinical management of severe acute respiratory infection (sari) when covid-19 disease is suspected: interim guidance (ncov)-infection-is-suspected accessed online key: cord-291851-xesef17i authors: wong, yu-jun; tan, malcolm; zheng, qishi; li, weiquan; kumar, rahul; fock, kwong-ming; teo, eng-kiong; ang, tiing-leong title: a systematic review and meta-analysis of the covid-19 associated liver injury date: 2020-08-31 journal: ann hepatol doi: 10.1016/j.aohep.2020.08.064 sha: doc_id: 291851 cord_uid: xesef17i introduction and objectives: the novel coronavirus disease 2019 (covid-19) has affected more than 5 million people globally. data on the prevalence and degree of covid-19 associated liver injury among patients with covid-19 remain limited. we conducted a systematic review and meta-analysis to assess the prevalence and degree of liver injury between patients with severe and non-severe covid-19. methods: we performed a systematic search of three electronic databases (pubmed/medline, embase and cochrane library), from inception to 24(th) april 2020. we included all adult human studies (>20 subjects) regardless of language, region or publication date or status. we assessed the pooled odds ratio (or), mean difference (md) and 95% confidence interval (95%ci) using the random-effects model. results: among 1543 citations, there were 24 studies (5961 subjects) which fulfilled our inclusion criteria. the pooled odds ratio for elevated alt (or = 2.5, 95%ci: 1.6-3.7, i(2) = 57%), ast (or = 3.4, 95%ci: 2.3-5.0, i(2) = 56%), hyperbilirubinemia (or = 1.7, 95%ci: 1.2-2.5, i(2) = 0%) and hypoalbuminemia (or = 7.1, 95%ci: 2.1-24.1, i(2) = 71%) were higher subjects in critical covid-19. conclusion: covid-19 associated liver injury is more common in severe covid-19 than non-severe covid-19. physicians should be aware of possible progression to severe disease in subjects with covid-19-associated liver injury. the novel coronavirus disease 2019 has affected more than 5 million patients globally, causing more than 300,000 death to date [1] . the symptoms of covid-19 range from mild respiratory symptoms to acute respiratory distress syndrome with multiorgan failure and death, particularly among the elderly with multiple comorbidities. extra-pulmonary symptoms such as covid-19 associated liver injury has been reported [2] [3] [4] . covid-19 associated liver injury is defined as any liver damage in patients with covid-19, with or without the pre-existing liver disease [5] . the possible mechanisms of covid-19 associated liver injury include immune-mediated damage and ischemic hepatitis secondary systemic inflammatory response syndrome in severe covid-19, drug-induced liver injury, as well as reactivation of underlying chronic liver disease [6] . besides, the direct virus-induced cytopathic effect has also been postulated as a result of viral replication within the infected hepatocytes [7] . the data on the prevalence and severity of covid-19 associated liver injury are conflicting. while growing evidence suggested a higher incidence of liver injury among severe covid-19, such findings are not consistent [8] [9] [10] . recent position statement highlighted our gaps in understanding this novel disease [11] [12] [13] . in particular, patients with non-alcoholic fatty liver disease may have co-existing comorbidities which put them at a higher risk of severe covid-19 [11] . furthermore, covid-19 associated liver injury is associated with prolonged hospitalization [6] . given this premise, it is of scientific interest to expand our understanding of the clinical outcome of patients with covid-19 associated liver injury. however, the severity of covid-19 patients was often poorly defined by including subjects ranging from tachypnea to intubation or death. as the literature in covid-19 is expanding exponentially, keeping up-to-date with scientific progress has become increasingly j o u r n a l p r e -p r o o f challenging for physicians. therefore, we systematically reviewed and summarized the existing literature on covid-19 associated liver injury among adult patients. our meta-analysis aims to compare the risks and clinical outcomes of covid-19 associated liver injury among adults with severe and non-severe covid-19. the objective of this meta-analysis is to compare the risk and clinical outcome of covid-19 associated liver injury between covid-19 patients with severe and non-severe covid-19. we included all adults with covid-19 associated liver disease, regardless of their underlying chronic liver disease or the severity of covid-19. only studies that report outcome data between severe and non-severe covid-19 were included, regardless of the pattern or the severity of the liver injury. our primary outcome was the pooled risk of serum alanine aminotransferase (alt) elevation in adult patients with severe and non-severe covid-19. our secondary outcomes were the pooled risk of the following parameters in adult patients with severe and non-severe covid-19: (1) serum aspartate aminotransferase (ast) elevation, (2) hyperbilirubinemia and (3) hypoalbuminemia. we also assessed the pooled mean difference (md) of gamma-glutamyl transferase (ggt) from the included studies. we defined the elevation of serum alt or ast as levels beyond 40 u/l. we defined hyperbilirubinemia as total bilirubin level higher than 17mmol/l. we defined hypoalbuminemia as serum albumin level below 40g/l. we conducted a comprehensive search of three electronic databases, pubmed/medline, embase and cochrane library (earliest inception to 24 th april 2020). keywords used in the search included a combination of "coronavirus disease 2019", "covid19", "sars-cov-2" and "coronavirus infection/complications [mesh]". we restricted our search to adult human studies. an experienced medical librarian helped with our literature search. in addition, we manually searched all references of selected articles for additional relevant articles. we used prisma (preferred reporting items for systematic reviews and meta-analyses) to report all studies identified using a pre-defined search protocol detailed in appendix 1. in this meta-analysis, we included all studies that met the following inclusion criteria: (1) population: adult patients infected with the covid-19, (2) reported outcome data on liver enzymes derangement (3) reported outcome data on the risk or severity of liver injury between severe and non-severe covid-19. all studies, j o u r n a l p r e -p r o o f regardless of language, geography, publication dates or publication status are included so long as they provided data relevant to this analysis. our exclusion criteria were: 1) any study with subjects < 21 years old, 2) case report or case series with less than 20 subjects, 3) review articles, editorial and guidelines. severe covid-19 was defined based on the definition used in the respective study. we further defined subjects as "critical covid-19" based on the need for admission to the intensive care unit, mechanical ventilation, or death. three authors (mt, jl, rk) independently reviewed all titles and abstracts of the studies identified in the primary search. studies that were duplicates or did not address the research question based on pre-defined criteria were excluded. we subsequently reviewed the full texts of all remaining articles to determine if they contained relevant information. any discrepancy in the article selection was resolved by consensus with a co-author (wyj). we extracted data on the demographic of study populations (age, gender, sample size, the proportion of subjects with baseline chronic liver disease and the use of lopinavir/ritonavir) as well as the pattern of covid-19 associated liver injury (alt, ast, bilirubin, albumin and ggt) from all included studies. the data from each study were independently extracted by into a standardized form by two authors (wyj, rk). we contact the corresponding author through email for any missing data. we did not encounter multiple reports from the same author or study population in this meta-analysis. all included studies were independently reviewed by 2 authors (mt, jl) to assess for quality and risk of bias using the subjects were followed-up = 0.5 points, < 50% subjects were followed-up = 0 point). the final decision on the overall risk of bias was made through discussion. all differences were resolved by discussions with third author (wyj). we considered studies with a score of ≥6, 3-4, < 3 as high-quality, medium-quality and low-quality, respectively. we used review manager software version 5.3 (the nordic cochrane centre, the cochrane collaboration, 2014) to perform our meta-analysis to estimate the pooled odds ratio (or), mean difference (md), and 95% confidence interval (95%ci). we used the random-effects model and validated our results using sensitivity analysis and heterogeneity assessment across the included studies. a p-value of less than 0.05 was considered to be statistically significant. the statistical heterogeneity was evaluated using the i 2 statistics. we defined substantial heterogeneity across study as low, moderate, substantial and considerable with a i 2 value of <30%, 31% to 60%, 61% to 74% and > 75% [15] . we performed subgroup analysis among subjects with critical covid-19 (defined as subjects needing admission to intensive care unit, mechanical ventilation, or death) to minimize the impact of heterogeneity in the definition of severe covid-19 on our results. as casecontrol studies are generally considered to have a higher risk of bias and more susceptible to selection and recall bias [16] , we performed subgroup analysis based on study design (cohort study versus case-control study). from an initial total of 1543 citations identified by using our search strategy, we identified a total of 112 studies. of these, 89 were excluded for the following reasons: no outcome data reported (n=44), case report or case series fewer than 20 subjects (n=25), review article (n=18) and pediatric study (n=2). we manually search the references of all the included studies and included one additional study [10] . a total of 24 studies met our inclusion criteria [10, . (figure 1 ). the characteristics of all included studies are presented in table 1 . most studies were retrospective (15 case-control studies and 8 cohort studies) in nature except for one prospective cohort study [24] . seven studies reported multicenter data, while the remaining reported single-center data. all studies were from asia. the definition of severe covid-19 was heterogeneous across all the included studies. the commonest definition of severe covid-19 was based on clinical criteria (n=15), followed by death (n=5) and icu admission (n=3). all studies were published as a full manuscript. overall, seven studies were considered to be of high quality, while the rest were considered to be medium quality (supplementary table 1) . twenty-four studies, with a total of 5952 patients (4024 in the severe group and 1928 in the non-severe group), were included in the final analysis [10, . the patients' characteristics were detailed in table 1 . the median age of subjects ranged from 38 to 71 years-old. the proportion of male ranged from 43% to 81%. the proportion of subjects with admission to an intensive care unit ranged from 1% to 100%. the prevalence of baseline chronic liver disease was low (ranged from 1% to 11%) [17-18, 20, 22-26, 28-33, 36, 38] . the liver injury occurred in 5%-76% of subjects with covid-19 [17-20, 22-26, 31-39] . j o u r n a l p r e -p r o o f twenty-three studies (5900 subjects) reported outcome data on alt [10, [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [35] [36] [37] [38] [39] . the mean level of alt is higher in severe covid-19 when compared to nonsevere group (31.9 u/l vs 23.5 u/l, 95%ci= 5.3-12.8, p<0.001), with high heterogeneity (i 2 =99%, p<0.001) (supplementary table 2) . the pooled risk of alt elevation was higher in severe covid-19 with moderate heterogeneity (or=2.8, 95%ci: 1.8-4.3, i 2 =63%) ( table 2) . when the stratified analysis was performed within the subgroup of patients with critical covid-19, the pooled risk of alt elevation remained higher among subjects with critical covid-19, with less heterogeneity (or=2.5, 95%ci=1.6-3.7, i 2 =57%) (figure 2) . twenty-one (5292 subjects) reported outcome data on ast [10, 17, [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [35] [36] [37] [38] . the mean level of ast is higher in severe covid-19 when compared to non-severe group (35.1 u/l vs 26.6 u/l, 95%ci= 8.7-14.2, p<0.001), with high heterogeneity (i 2 =99%, p<0.001) (supplementary table 2 ). the pooled risk of ast elevation was higher in severe covid-19 with moderate heterogeneity (or=3.4 95%ci: 2.3-5.1, table 2) . stratified analysis among subjects with critical covid-19 showed a higher pooled risk of ast elevation, with less heterogeneity (or=3.4, 95%ci: 2.3-5.0, i 2 =56%) (figure 3) . one study reported a higher risk of ast elevation in nonsurvivor (or=5.9, 95%ci=3.4-10.5, p<0.001) ( table 2) . [20] thirteen studies (3540 subjects) reported outcome data on serum bilirubin [17, 19-20, 23-26, 29, 32, 30, 36-38] . the mean level of bilirubin is higher in severe covid-19 j o u r n a l p r e -p r o o f when compared to non-severe group (12.2mmol/l vs 10.5 mmol/l, 95%ci= 1.5-3.5, p<0.001), with high heterogeneity (i 2 =98%, p<0.001) (supplementary table 2 ). the pooled risk of hyperbilirubinemia (serum bilirubin > 17mmol/l) is higher in subjects with severe (or=1.9, 95%ci: 1.1-3.1, i 2 =30%) ( table 2 ) and critical covid-19 (or=1.7, 95%ci: 1.2-2.5, i 2 =0%) (figure 4) . thirteen studies (3404 subjects) reported outcome data on serum albumin [19-20, 24-29, 32, 36-37, 39] . the mean level of albumin is lower in severe covid-19 when compared to non-severe group (37.2 g/l vs 40.1 g/l, 95%ci= -6.0, -2.9, p<0.001), with high heterogeneity (i 2 =100%, p<0.001) (supplementary table 2 ). the pooled risk of hypoalbuminemia (serum albumin <40g/l) was higher in subjects with severe covid-19 (or=8.8, 95%ci: 4.1-19.0, i 2 =46%) and critical covid-19 (or=7.1, 95%ci: 2.1-24.1, i 2 =71%) ( table 2) . three studies (699 subjects) reported outcome data on serum ggt [17, 20, 37] . there is a trend towards higher ggt is higher in severe covid-19 (68.5 u/l vs 36.8 u/l, p=0.07) and critical covid-19 (56.9 u/l vs 28.5 u/l, p<0.001), with high heterogeneity (i 2 =96% and 100% respectively, p<0.001) (supplementary table 2 ). the mean difference of ggt did not significantly differ in subjects with severe or critical covid-19 as compared to non-severe/non-critical covid-19 patients ( table 2 ). we performed a subgroup analysis based on the types of studies and the proportion of subjects with lopinavir/ritonavir usage. we performed a subgroup analysis based on study design by comparing a cohort study and case-control study. we found that the effect size for the casecontrol study was bigger as compared to cohort study for alt elevation, ast elevation, hyperbilirubinemia and hypoalbuminemia ( table 2) . the proportion of subjects using lopinavir/ritonavir (ranged from 14% to 100%) was reported in 12 studies [17-18, 20, 25-29, 32-34, 38] . we arbitrarily defined high lopinavir/ritonavir usage as a study with ≥ 80% of their subjects treated with lopinavir/ritonavir. among the four studies with high lopinavir/ritonavir usage [17, 25, 29, 33] , we observed a higher pooled odds risk for alt and ggt elevation ( table 2) table 4 ). we performed a sensitivity analysis to assess whether an individual study had a dominant effect on the overall pooled results. this was performed by serially removing one study at a time while repeating the analysis. the pooled risk of alt elevation ranged from 2.2 to 2.8, which is still within our reported 95%ci. besides, we also performed repeated analysis using the fixed-effect model. using the fixedeffect model, the level of ggt is higher in severe covid-19 as compared to nonsevere group (md=14.6, 95%ci: 14.1-17.2, i 2 =96%). all the remaining findings were similar in the sensitivity analysis (supplementary table 2 ). we assessed heterogeneity based on the percentages of i 2 for each reported outcome. we consider the heterogeneity on the observed risk and severity of covid-19 associated liver injury as moderate and substantial, respectively. this heterogeneity likely the result of a different definition of severe disease used in various studies. there was no evidence of publication bias based on visual inspection of the funnel plot ( figure 5) . covid-19 associated liver injury is defined as any liver damage in patients covid-19 patients, with or without the pre-existing liver disease [5] . while the exact j o u r n a l p r e -p r o o f mechanism remains unknown, several potential mechanisms have been postulated [6] [7] [8] [9] . severe systemic inflammatory response during severe covid-19 can lead to immune-mediated damage or ischemic hepatitis from the severe systemic inflammatory response. current treatment options such as lopinavir/ritonavir, hydroxychloroquine and remdesivir are potentially hepatotoxic and may cause druginduced liver injury (dili) [11] . other immunosuppressive alternative such as tocilizumab or steroid can result in reactivation of underlying chronic hepatitis b. while direct cytotoxicity due to viral replication within infected hepatocytes has been postulated, viral inclusions for sars-cov-2 has not been found in the liver [40] . the current data on the severity and prevalence of covid-19 associated liver injury remains conflicting due to the heterogeneity in study populations. to address these issues, we systematically reviewed the current literature on liver injury in covid-19 and performed a meta-analysis on the severity and risk of covid-19 associated liver injury in these patients. at the point when this meta-analysis was conducted, most studies did not specifically report the prevalence of covid-19 patients with baseline chronic liver disease. none of the included studies reported outcomes of covid-19 patients based on their underlying liver disease. as all included studies reported a low incidence of chronic liver disease (cld), our findings will be more reflective of the general population. our findings were consistent with current literature that adult patients with severe covid-19 have a higher risk of liver injury. most liver injuries were mild. to date, there is only one reported case of death from liver failure in covid-19 patients without pre-existing liver disease. in a recently published international registry of covid-19 among patients with chronic liver disease, only 12% of death was j o u r n a l p r e -p r o o f attributed to liver disease [41] . that having said, the mortality risk among patients with the pre-existing liver disease did increase with the severity of underlying liver disease (13% in chronic liver disease, 24% in child-pugh's a cirrhosis, 43% in child-pugh's b cirrhosis and 63% in child-pugh c cirrhosis) [41] . based on our analysis, the pooled or of alt elevation was 2.5, ast elevation was 3.4, and hyperbilirubinemia was 1.7 among covid-19 patients who were critically ill. we found that indirect markers for liver injury, such as hypoalbuminemia increased by seven-fold in patients with severe covid-19. while we are mindful that hypoalbuminemia could be confounded by factors such as systemic inflammation, malnutrition and the timing of presentation to hospital, similar findings were reported in other studies [42] [43] . therefore, physicians should be alert about potential clinical deterioration (intubation, icu admission and death) when covid-19 associated liver injury was observed. in this meta-analysis, the pooled risk of liver injury was higher among studies with high usage of lopinavir/ritonavir. in a recent retrospective study involving 417 covid-19 patients [17] , lopinavir/ritonavir was associated with 7-fold higher risk of liver injury. in this meta-analysis, we found a higher level of ggt and alt elevation in studies with high lopinavir/ritonavir usage, suggesting that lopinavir/ritonavir might be hepatotoxic ( table 2) . although lopinavir/ritonavir did not significantly increase the risk of liver injury in a recent open-labelled randomized trial, this trial excluded subjects with abnormal baseline liver functions. we are unable to exclude preferential treatment of severe covid-19 patients using lopinavir/ritonavir from this meta-analysis. based on our findings, physicians may consider monitor for liver injury when lopinavir/ritonavir is being used for covid-19 patients. this meta-analysis adds value to the current literature by summarizing the prevalence and pattern of liver injury in adults with severe covid-19. prior to this, the prevalence of covid-19 associated liver injury was confounded by the heterogeneity of study population, and study design as pertinent information such as the proportion of subjects' baseline chronic liver disease or icu admission were often not reported [5] . in this meta-analysis, we found that the severity of most covid-19 associated liver injury was considered mild, suggesting liver failure is uncommon among critical covid-19. nevertheless, physicians should be mindful of potential deterioration among covid-19 patients liver injury as it is associated with icu admission, mechanical ventilation and death. there are several strengths in our meta-analysis. to our best knowledge, this is the most comprehensive description of the liver manifestation of covid-19 in comparison. earlier meta-analysis had either included fewer studies evaluating liver manifestation among covid-19 patients [43] [44] [45] [46] [47] [48] [49] or included a wide range of "severe" covid-19 patients ranging from the presence of tachypnoea to death [43] [44] [45] [46] . in this meta-analysis, we conducted a systematic search of the literature using a predefined inclusion and exclusion criteria; including a large number of studies to allow assessment of publication bias and subgroup analysis, detailed extraction of data on study outcomes; subgroup analysis to define a more homogenous study population j o u r n a l p r e -p r o o f for sensitivity analysis; rigorous evaluation of study quality; and the use of various statistical methods to evaluate the validity of our findings. our meta-analysis was limited by the heterogeneity in the study population of included studies. we attempted to mitigate this limitation by performing subgroup analysis based on the characteristics of each study. most of the included studies were limited by their retrospective nature of study design and small sample size. because of this, we cannot estimate the incidence and comparative risk of druginduced liver injury across different treatment with high confidence. while liver injury is more likely to occur in adults with critical covid-19, we cannot be certain of its prognostic value as most of the studies were retrospective in nature. in conclusion, covid-19 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disease 2019 and prevalence of chronic liver disease: a meta-analysis liver injury is associated with severe coronavirus disease 2019 (covid-19) infection: a systematic review and metaanalysis of retrospective studies aga institute rapid review of the gastrointestinal and liver manifestations of covid-19, meta-analysis of international data, and recommendations for the consultative management of patients with covid-19 and liver dysfunction: a systematic review and meta-analysis of retrospective studies markers of liver injury and clinical outcomes in covid-19 patients: a systematic review and meta-analysis liver profile in covid-19: a meta-analysis manifestations and prognosis of gastrointestinal and liver involvement in patients with covid-19: a systematic review and meta-analysis covid-19" and hasabstract[text] and humans[mesh])) or ("coronavirus disease 2019" and hasabstract[text] and humans[mesh])) or ("sars-cov-2" and hasabstract[text] and humans[mesh]) filters: abstract; humans 565 #5 search "covid19" filters: abstract abbreviations: *mean (sd); nr=not reported; cld = chronic liver disease; ct = computer tomography; nr=not reported j o u r n a l p r e -p r o o f key: cord-340710-dmow5p7k authors: lagana, stephen m.; kudose, satoru; iuga, alina c.; lee, michael j.; fazlollahi, ladan; remotti, helen e.; del portillo, armando; de michele, simona; de gonzalez, anne koehne; saqi, anjali; khairallah, pascale; chong, alexander m.; park, heekuk; uhlemann, anne-catrin; lefkowitch, jay h.; verna, elizabeth c. title: hepatic pathology in patients dying of covid-19: a series of 40 cases including clinical, histologic, and virologic data date: 2020-08-13 journal: mod pathol doi: 10.1038/s41379-020-00649-x sha: doc_id: 340710 cord_uid: dmow5p7k the novel coronavirus sars-cov-2 (coronavirus disease 19, or covid-19) primarily causes pulmonary injury, but has been implicated to cause hepatic injury, both by serum markers and histologic evaluation. the histologic pattern of injury has not been completely described. studies quantifying viral load in the liver are lacking. here we report the clinical and histologic findings related to the liver in 40 patients who died of complications of covid-19. a subset of liver tissue blocks were subjected to polymerase chain reaction (pcr) for viral ribonucleic acid (rna). peak levels of alanine aminotransferase (alt) and aspartate aminotransferase (ast) were elevated; median alt peak 68 u/l (normal up to 46 u/l) and median ast peak 102 u/l (normal up to 37 u/l). macrovesicular steatosis was the most common finding, involving 30 patients (75%). mild lobular necroinflammation and portal inflammation were present in 20 cases each (50%). vascular pathology, including sinusoidal microthrombi, was infrequent, seen in six cases (15%). pcr of liver tissue was positive in 11 of 20 patients tested (55%). in conclusion, we found patients dying of covid-19 had biochemical evidence of hepatitis (of variable severity) and demonstrated histologic findings of macrovesicular steatosis and mild acute hepatitis (lobular necroinflammation) and mild portal inflammation. we also identified viral rna in a sizeable subset of liver tissue samples. the pandemic caused by the novel coronavirus sars-cov-2 (covid-19) has resulted in significant pulmonary morbidity and mortality, but has also raised many questions regarding involvement of other organ systems such as the liver. hepatologists and liver pathologists were therefore interested to find that researchers from china demonstrated liver function test (lft) abnormalities in~44% of covid-19 patients and that these abnormalities seemed to correlate with the severity of pulmonary disease [1, 2] . histologic reports from china were limited. a small case series reported the presence of fat in hepatocytes, with macrovesicular steatosis depicted in the accompanying photomicrograph (although described in the text as microvesicular fat) [3] . a more recent study performed core needle biopsies on the livers of four patients and reported nonspecific findings, attributed to preexisting disease or perimortem injury [4] . our group recently described moderate acute hepatitis in a liver allograft recipient with concomitant acute cellular rejection [5, 6] . nonetheless, the spectrum of histological liver injury in covid-19 remains unknown. a central question has been whether the virus specifically infects the liver, or is the liver injury entirely related to the "cytokine storm" that some patients exhibit, or is a combination of these factors present. the angiotensin converting enzyme receptor 2 (ace2) has been suggested to be the main mode of entry for the virus into the cell. it is found abundantly on cholangiocytes, but only rarely on hepatocytes; though it may be upregulated on hepatocytes at times of physiologic stress [7] . ace2 is also expressed on endothelial cells, such as those found in the portal vasculature [8] . following the sars-cov outbreak of 2002, virus was reported in liver and features of acute hepatitis (referred to as "bystander hepatitis") were described [9] . early in the course of the pandemic, new york city was the global epicenter for cases of, and deaths from, covid-19. our volume of autopsies increased sharply, and most of the cases were due to sequelae of covid-19. the lung findings predominated, as expected; however,~2/3 of covid-19 patients in our healthcare system had abnormal lfts [10] . therefore, we characterized the liver findings (clinically and pathologically) in 40 autopsy cases. we also investigated whether viral ribonucleic acid (rna) could be detected in liver tissue by polymerase chain reaction (pcr). following approval from the columbia university irving medical center institutional review board, we reviewed lung and liver sections from 40 consecutive autopsies from patients who died of illness related to covid-19. all liver slides were reviewed by an experienced liver pathologist (sml) and any findings (or potential findings), which are uncommonly identified in routine practice (e.g., phlebosclerosis), were reviewed amongst the liver pathology group for consensus. sections were typical autopsy sections (~2 cm × 2 cm) taken from representative appearing area(s), formalin fixed and paraffin embedded (ffpe). hematoxylin and eosin (h&e) was the only stain routinely employed, though if further stains were used for clinical purposes, they were reviewed as deemed relevant. pcr was performed on selected cases for which additional tissue blocks of liver were available. rna from samples was extracted using quick-rna ffpe miniprep (zymo research). quantitative rt-pcr was performed on extracted rna using taqman 4x master mix and sars-cov-2 primer/ probe sets (idt) to detect presence of virus per the cdc recommendations [11] . each assay included a standard curve to determine the viral load (log 10 copies/ml). demographic and clinical data were collected on all cases as available, specifically: age, gender, length of hospital stay, body mass index (bmi), hypertension, diabetes mellitus, chronic kidney disease, and chronic heart disease. in addition, charts were interrogated for evidence of known or suspected chronic liver disease, including viral hepatitis, alcohol-related liver disease, nonalcoholic fatty liver disease (nafld), autoimmune liver disease, transplantation status, and/or cirrhosis. laboratory values were also investigated (initial and peak), including aspartate aminotransferase (ast), alanine aminotransferase (alt), total bilirubin (tb), albumin (initial and nadir), creatinine, ferritin, ddimer, c-reactive protein (crp), and interleukin-6 (il-6). certain therapeutic interventions were investigated including administration of corticosteroids, hydroxychloroquine, and tocilizumab (il-6 inhibitor). continuous variables were expressed as median (25th-75th percentile). categorical variables were shown as fraction (percentage). clinical and pathologic features among those with and without detectable virus by pcr and among those with and without acute lung injury (ali) were compared using fisher's exact test for categorical variables and mann-whitney u test for continuous variables. in addition, differences in the type of therapeutic intervention and length of stay stratified by the distribution and extent of steatosis and lobular or portal inflammation were assessed using fisher's exact test or kruskal-wallis test as appropriate. patients with missing data were excluded from the analysis. all statistical analyses were performed using r (version 3.6.1). lung findings were simplified for the purposes of this report into "ali" and "no ali." ali comprised the histologic spectrum of diffuse alveolar damage (dad) with an acute (exudative) phase demonstrating hyaline membranes with or without an organizing (proliferative) phase exhibiting interstitial fibroblastic proliferation as well as a single case with predominantly fibrin, compatible with acute and fibrinous pneumonia. we sequentially examined the liver sections of the first 44 covid-19 autopsies at our institution; however, four were excluded for severe autolysis resulting in a cohort of 40 patients. the overall median (iqr) age was 70 (66-80) years and 29 (70%) were men. twenty-three patients were hispanic, five were african american, and two were caucasian (the remaining ten were unknown). seven of the patients had died on arrival, and had either no or limited clinical data. the median length of stay was 8.5 days. twenty-two patients (55%) received steroids during their admission, 19 (47.5%) received hydroxychloroquine, and six (15%) received tocilizumab (this cohort was encountered before remdesivir was widely used, and none of these patients received it). patient characteristics and known comorbidities are summarized in table 1 . there were two patients with evidence of chronic liver disease, one with alcohol-related cirrhosis and one with a history of liver transplant for autoimmune-related liver disease and acute cellular rejection at the time of admission. five patients had imaging evidence of nafld on admission. in addition, one patient with imaging evidence of steatosis also had an isolated anti-hepatitis b (hbv) core antibody positive with low-level hbv dna detected. initial and peak laboratory values including liver enzymes and inflammatory markers are displayed in table 2 . the median initial and peak ast and alt were 1-3 times the upper limit of normal, while median tb values were in the normal range. kidney dysfunction was common with a median peak creatinine of 2.64 mg/dl (upper limit of normal = 0.98 mg/dl for females and 1.30 mg/dl for males). median peak levels of inflammatory markers including crp (268 mg/l, upper limit of normal = 10 mg/l), ferritin (1810 ng/ml, upper limit of normal = 150 ng/ml for females, 400 ng/ml for males), d-dimer (9.6 μg/ml, upper limit of normal = 0.8 μg/ml), and il-6 (>315 pg/ml, upper limit of normal = 5 pg/ml) were all markedly elevated. there were no significant associations between laboratory values and any specific histological feature (data not shown). grossly, two livers showed fibrosis and one had abscesses, the remaining livers showed varying degrees of steatosis, congestion, and ischemia, but no other significant gross pathology. histologically, the most frequently encountered findings were macrovesicular steatosis, mild acute hepatitis, and minimal-to-mild portal inflammation. various less frequent findings were also observed. the findings are described below and major findings are summarized in table 3 . as the focus of this study is liver pathology, the lungs were considered only in the context of how the pulmonary findings may relate to liver injury. overall, 29 of 40 patients (73%) had evidence of ali histologically (the gross appearance of the lungs was not reviewed for this study). the patients with ali did not differ from those without with respect to any of the laboratory variables we cataloged, or to any histologic findings (data not shown). there was, however, a trend toward increased serum crp in the ali group (282 mg/l vs. 218 mg/l, p = 0.07). cardiac disease, n = 28 10/28 (36%) all statistics are presented as median (interquartile range) or n (%). bmi body mass index. a chronic liver disease n based on patients in whom we had preadmission data. all statistics are presented as median (interquartile range). alt alanine aminotransferase, ast aspartate aminotransferase, crp c-reactive protein, tb total bilirubin. a total of 20 cases (50%) showed features of acute hepatitis, defined as the presence of lobular necroinflammation, fig. 1b . these foci contained lymphocytes and rare histiocytes. plasma cells were rare. sixteen of these cases were of mild severity (80%) and four were of moderate severity (20%). no severe hepatitis (e.g., submassive necrosis, massive hepatic necrosis, bridging necrosis) was encountered. four patients without necroinflammation showed rare, individual apoptotic hepatocytes. given the focality of the finding (one or two such cells in one or two sections of liver), the significance of this observation is uncertain. six cases with lobular necroinflammation also demonstrated occasional individual apoptotic hepatocytes, fig. 1c . lobular mitoses were seen in three cases (8%). two of these cases also had acute hepatitis, whereas one occurred in an allograft which did not demonstrate active hepatitis. there were no associations between lobular necroinflammation and length of stay (p = 0.87), hydroxychloroquine treatment (p = 0.73), or tocilizumab administration (p = 0.17). twenty patients (50%) had portal inflammation. three cases had interface hepatitis (equivalent to batts-ludwig grade 2, fig. 1d) , whereas 17 had only minimally increased portal mononuclear cells (lymphocytes and few portal macrophages). one of the cases with interface occurred in an allograft with recent severe rejection with autoimmune features (acr-aih). eosinophils and neutrophils were rare, and not prominent in any case. an occasional plasma cell was present, but in none of the cases were they significant enough to invoke consideration of autoimmune hepatitis or drug induced liver injury with autoimmune features (dili-aih), with the exception of the acr-aih case. no case demonstrated fig. 1 common autopsy findings. a an example of marked steatosis involving all three zones (×100). this patient had a body mass index of 25. b a typical focus of lobular necroinflammation, comprised predominantly of lymphocytes with admixed apoptotic debris (×600). c a single apoptotic hepatocyte (arrow, ×600). panel d is taken from one of the rare cases with interface hepatitis (×200). all images depict hematoxylin and eosin stained slides. lymphoid aggregates. overall, 6 of the 20 patients did not have evidence of lobular/acute hepatitis. of these one was the acr-aih patient and two came into the emergency room in cardiopulmonary arrest, and without history in our system. there were no associations between portal inflammation and length of stay (p = 0.86), hydroxychloroquine treatment (p = 0.20), or tocilizumab administration (p = 0.10). in cases with acute, severe cardiac dysfunction areas of congestion (right heart failure) and ischemia (left heart failure) are common regardless of etiology. in our series, 32 (78%) patients exhibited congestion and 16 (40%) exhibited centrilobular ischemic necrosis. none of the cases had diffuse vascular pathology; however, focal findings were identified. phlebosclerosis, reminiscent of veno-occlusive disease (vod) was present in six cases, fig. 2a . in five cases, this occurred in a portal venule, but one case had involvement of the central vein. portal arterioles were abnormal in nine cases. three of these had arteriolar muscular hyperplasia, fig. 2b . each case with muscular hypertrophy of the portal arterioles also had venous phlebosclerosis. four cases had hyalinosis of the vessel wall (fig. 2b) . two cases had fibrinoid necrosis with endothelial apoptosis (fig. 2c) . sinusoidal microthrombi were present in six cases (fig. 2d) . c4d immunohistochemistry (ihc) was performed on ten cases and was negative (either entirely or almost entirely negative in all cases). three cases had granulomatous inflammation. one case had portal and lobular granulomas reminiscent of "fibrin ring" morphology, fig. 3a . this patient had been treated with a number of potentially hepatotoxic medications throughout the hospital course including hydroxychloroquine and tocilizumab for severe covid-19, as well as amiodarone for atrial fibrillation and ceftriaxone and piperacillin/tazobactam for infection. one patient had multiple necrotizing granulomas forming grossly appreciable abscesses and had structures suggestive of schistosoma eggs. the final case demonstrated portal nonnecrotizing granulomas in two portal tracts and resembled primary biliary cholangitis (pbc). however, the patient was male, had no known history of pbc, and had a normal alkaline phosphatase. two cases had pale ovoid sinusoidal inclusions, which at low power resembled apoptotic hepatocytes. upon close examination, it became evident that these inclusions were present in the sinusoidal spaces, fig. 3b . we stained these with cd61, which was positive (fig. 3b inset) , supporting that these aggregates were rich in platelets. we chose to refer to these structures as "thrombotic bodies." pcr was performed on 20 autopsy livers and was positive in 11 (55%) ranging from 10 copies to 9254 copies/μl rna. the longest interval between initial diagnosis by nasopharyngeal swab and death was 25 days and the median was 13 days. there was no relationship between time from initial diagnosis (positive nasopharyngeal swab) and likelihood of pcr positivity in the liver at autopsy (p = 0.51). we investigated whether pcr positivity was associated with any laboratory parameters (table 4 ). median peak ast was higher among patients with a positive pcr compared to negative (239 vs. 86 u/l, respectively) though this did not reach statistical significance (p = 0.063). in addition, peak ferritin (3623 vs. 1014 ng/ml, p = 0.048) and peak creatinine (4.50 vs. 2.02 mg/dl, p = 0.025) were significantly higher among pcr positive patients. there were no significant correlations between pcr positivity and any histologic finding including congestion (supplementary table 1 ). here, we present the hepatic findings in 40 autopsies of patients who died of complications of covid-19. excluding perimortem changes (congestion and ischemia) the main findings were hepatic steatosis (75%), mild acute hepatitis (50%), and portal inflammation (50%). we determined that 55% of patients who died of covid-19 had virus in the liver detected by pcr, albeit the majority at very low levels. alt alanine aminotransferase, ast aspartate aminotransferase, crp c-reactive protein, tb total bilirubin. *wilcoxon rank-sum tests were performed. macrovesicular steatosis was common (75%) of patients. this is consistent with other studies, for example, a study performed by a working group affiliated with the centers for disease control found steatosis in 50% of autopsy livers (4/8) [12] . a high proportion of our patients were diabetics, and that could account for some of the steatosis we encountered. however, the distribution of fat in our series was not typical of nafld. ten cases had only mild steatosis, but had appreciable periportal fat. only 2 of 32 patients in whom bmi was recorded had a bmi > 35 (median 26.5). a total of 2 of 40 patients had ballooning and mallory-denk bodies, indicative of alcoholic or nonalcoholic steatohepatitis. ischemia, viral hepatitis (e.g., hepatitis b, hepatitis c, human immunodeficiency virus), medications (corticosteroids), malnutrition, and various other acute insults are known to cause fatty liver [13] . steroid administration was frequent in our cohort, and this could lead to steatosis, though no direct association was seen in this cohort [14] . the facts that steatosis was not correlated to bmi or diabetes, and that there were many cases of "mismatched fat" are surprising if preexisting nafld explains the entirety of the histology. we did not have alcohol intake history for every patient, however alcoholic steatosis often demonstrates features of steatohepatitis with ballooning, neutrophilic satellitosis, and mallory-denk bodies. features of frank steatohepatitis were present in only two cases. therefore, it would seem likely that the steatosis in some of these cases developed during the course of their covid-19 illness. the etiology may be multifactorial with corticosteroid administration, hypoxia, malnutrition, and direct viral effects all plausible considerations (though neither corticosteroid use nor length of stay, a surrogate for malnutrition, was statistically associated with amount or distribution of steatosis). a series of over 300 covid-19 cases in china investigated the relationship between steatosis and neutrophil to lymphocyte ratio (nlr). a higher ratio suggests more severe systemic inflammation. this study showed that steatosis and nlr were associated with severity of illness, and also that the effects of steatosis and high nlr compounded to predict the most severe cases [15] . the exact mechanism/pathobiology of hepatic steatosis in covid-19 is worthy of further investigation. mild acute hepatitis, defined as lobular necroinflammation, was present in 50% of cases. this climbs to 60% if cases with no inflammation, but with focal apoptotic hepatocytes are included. in general, individual apoptotic hepatocytes are considered as evidence of acute hepatitis, but in this series, such cases showed such infrequent hepatocyte apoptosis that the significance is uncertain. the degree of necroinflammatory activity was generally mild, but was moderate in four cases. the necroinflammatory foci consisted of one to several dead and dying hepatocytes with few accompanying lymphocytes and histiocytes. plasma cells were only rarely encountered. the cohort of histologically defined acute hepatitis cases did not demonstrate higher serum ast/alt levels than the cohort without lobular necroinflammation. indeed, laboratory values were essentially identical in the histologic acute hepatitis and nonhepatitis groups. there was, however, a trend toward significance when we queried if those with pcr positivity were more likely to have evidence of acute hepatitis (44% of pcr negative patients vs. 64% of pcr positive patients did; p = 0.08). it will be interesting to investigate this variable in a larger number of cases. we have previously reported moderate acute hepatitis in a liver allograft recipient (distinct from the allograft recipient in this report) whose biopsy demonstrated large clusters of apoptotic hepatocytes with abundant lymphohistiocytic inflammation [5, 6] . none of the cases in this series were as profoundly affected as that case. the role of immunosuppression and allograft recipient status on liver injury associated with covid-19 will need further study. portal inflammation was generally minimal and was not suggestive of any specific etiology. this finding is similar to what was reported in a series of 12 autopsies in europe, which described "minimal lymphoplasmacellular portal infiltrate" [16] . approximately 70% of patients in our series with portal inflammation had mild acute (lobular) hepatitis. the few patients with portal inflammation, but without acute hepatitis, were a heterogenous group including an allograft recipient and several patients who arrested on arrival in the emergency room without available medical records, so it is difficult to interpret the significance of isolated portal inflammation. there was no evident bile duct injury, with the exception of the allograft with recent history of severe rejection. ace2 receptor is present on cholangiocytes, so we were certainly interested in identifying bile duct injury, but it was not observed [17] . cholestasis, however, was common (38%). four patients had ductular cholestasis, suggesting sepsis. lobular cholestasis without injury to the bile ducts is suggestive of a hepatocellular insult; e.g., at the bile canaliculus. it is, however, possible that the cholangiocytes are functionally impaired, but that the injury is not evident by h&e morphology. further investigation by electron microscopy or genomic/proteomic methods may prove enlightening. the potential role of endothelial injury in covid-19 continues to evolve. virus has been demonstrated in endothelial cells, and one paper reported lymphocytic endotheliitis in liver [8, 17] . in our series, however, few vascular abnormalities were histologically identified. a minority of our cases had a vascular abnormality (portal arteriole hyperplasia, vod-like phlebosclerosis, sinusoidal thrombi), and these changes were very focal and sparse in any one histologic section. in our patient cohort, therefore, vascular injury did not appear to be a major form of injury. while we cannot exclude these findings being meaningful (if they are present throughout the entire liver), we do not believe that vascular injury is the major mechanism of injury in the liver in covid-19 patients. this contrasts with a case series from italy, which describes vascular pathology not frequently encountered in our cohort [18] . the "thrombotic bodies" we observed are likely the same "microthrombi" identified in multiple organs in a recent series of seven autopsies [19] . this study includes the largest cohort of liver tissue subjected to pcr. the results are interesting for several reasons. at the most superficial level, it is valuable to know that slightly more than half of patients had detectable virus in liver. pcr positivity was not significantly associated with ast/alt elevations, though there was a trend for association between pcr positivity and higher peak ast (86 u/ l vs. 239 u/l, p = 0.063). as additional cases are subjected to pcr, further clarity may be obtained. there was a statistically significant elevation of ferritin and trends toward increased d-dimer and albumin nadir in the positive group. there was also statistically significantly increase in serum creatinine in the pcr positive cohort. the overall impression is that the pcr positive group had a slightly more severe inflammatory state. a detail worthy of further exploration is the variation of pcr positivity in liver. nine cases had low viral loads (and the possibility of serum positivity cannot be excluded), whereas two cases had high viral loads. time between initial diagnosis and death was not a factor associated with pcr positivity (p = 0.51). the peak creatinine elevation in the pcr positive group (4.50 vs. 2.02; p = 0.025) is a noteworthy finding, as it raises the question of whether some patients have a more disseminated phenotype and others perhaps a more anatomically (airway) restricted one. further studies will be useful to determine if this is true, and if so, if it is clinically relevant. this study has weaknesses, including that being an autopsy study, this paper describes patients with severe disease. as the epidemic evolves, it will be important to gather data on liver injury in patients with non-lethal covid-19. we did not investigate pathogenesis on the cellular level. we attempted to validate in-situ hybridization (ish) protocols and ihc for sars-cov-2, but nonspecific marking of lipofuscin, and reaction with endogenous alkaline phosphatase in liver tissue from non-covid-19 patients who died years ago was nearly ubiquitous (sometimes strong and diffuse). thus, for technical reasons, we cannot report these tissue preserving methods at present. in our clinical experience, as well as in the published literature, both ish and ihc can be used to detect virus in lung, particularly during the acute phase of dad, and occasionally in other organs, such as placenta [20, 21] . it will be important and interesting to attempt to identify virus in specific cells in the liver and to determine whether the ace2 receptor is upregulated in hepatocytes in patients with severe covid-19. nonetheless, based on the pattern of injury observed and the results of the pcr analysis, sars-cov-2 seems to involve the liver, and is associated with, possibly causal of, macrovesicular steatosis and acute hepatitis. here, we have provided a description of the histologic, clinical, and virologic characteristics related to the liver in patients who died of sequelae of covid-19. we have shown that patients dying of complications of covid-19 often have abnormal liver enzymes, steatosis, mild acute hepatitis, and viral rna in liver. while more work is needed to further elucidate the possibly multifactorial mechanisms of liver injury in patients with severe covid-19, given the lack of association between steatosis and known nafld risk factors, and between hepatitic findings and known drug administration, these data suggest virally mediated liver injury based on clinical and histologic observations. conflict of interest the authors declare that they have no conflict of interest. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pattern of liver injury in adult patients with covid-19: a retrospective analysis of 105 patients pathological findings of covid-19 associated with acute respiratory distress syndrome pathological study of the 2019 novel coronavirus disease (covid-19) through postmortem core biopsies covid-19 associated hepatitis complicating recent living donor liver transplantation a case of an infant with sars-cov-2 hepatitis early after liver transplantation hepatic consequences of covid-19 infection. lapping or biting? endothelial cell infection and endotheliitis in covid-19 sars-associated viral hepatitis caused by a novel coronavirus: report of three cases acute liver injury in covid-19: prevalence and association with clinical outcomes in a large us cohort 2019-ncov) real-time rt-pcr diagnostic panel. cdc-006-00019, cdc/ddid/ncird/division of viral diseases pathology and pathogenesis of sars-cov-2 associated with fatal coronavirus disease, united states fatty liver and the forensic pathologist functional proteomic analysis of corticosteroid pharmacodynamics in rat liver: relationship to hepatic stress, signaling, energy regulation, and drug metabolism detrimental effects of metabolic dysfunction-associated fatty liver disease and increased neutrophil-to-lymphocyte ratio on severity of covid-19 postmortem examination of patients with covid-19 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis liver histopathology in severe covid 19 respiratory failure is suggestive of vascular alterations megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in covid-19: a case series in situ detection of sars-cov-2 in lungs and airways of patients with covid-19 detection of sars-cov-2 in formalin-fixed paraffin-embedded tissue sections using commercially available reagents key: cord-292501-2jv7xkfn authors: jiang, saiping; wang, rongrong; li, lu; hong, dongsheng; ru, renping; rao, yuefeng; miao, jing; chen, na; wu, xiuhua; ye, ziqi; hu, yunzhen; xie, minghua; zuo, minjuan; lu, xiaoyang; qiu, yunqing; liang, tingbo title: liver injury in critically ill and non-critically ill covid-19 patients: a multicenter, retrospective, observational study date: 2020-06-23 journal: front med (lausanne) doi: 10.3389/fmed.2020.00347 sha: doc_id: 292501 cord_uid: 2jv7xkfn background: liver injury commonly occurs in patients with covid-19. there is limited data describing the course of liver injury occurrence in patients with different disease severity, and the causes and risk factors are unknown. we aim to investigate the incidence, characteristics, risk factors, and clinical outcomes of liver injury in patients with covid-19. methods: this retrospective observational study was conducted in three hospitals (zhejiang, china). from january 19, 2020 to february 20, 2020, patients confirmed with covid-19 (≥18 years) and without liver injury were enrolled and divided into non-critically ill and critically ill groups. the incidence and characteristics of liver injury were compared between the two groups. demographics, clinical characteristics, treatments, and treatment outcomes between patients with or without liver injury were compared within each group. the multivariable logistic regression model was used to explore the risk factors for liver injury. results: the mean age of 131 enrolled patients was 51.2 years (standard deviation [sd]: 16.1 years), and 70 (53.4%) patients were male. a total of 76 patients developed liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%) with a median occurrence time of 10.0 days. critically ill patients had higher and earlier occurrence (81.5 vs. 51.9%, 12.0 vs. 5.0 days; p < 0.001), greater injury severity (p < 0.001), and slower recovery (50.0 vs. 61.1%) of liver function than non-critically ill patients. multivariable regression showed that the number of concomitant medications (odds ratio [or]: 1.12, 95% confidence interval [ci]: 1.05–1.21) and the combination treatment of lopinavir/ritonavir and arbidol (or: 3.58, 95% ci: 1.44–9.52) were risk factors for liver injury in non-critically ill patients. the metabolism of arbidol can be significantly inhibited by lopinavir/ritonavir in vitro (p < 0.005), which may be the underlying cause of drug-related liver injury. liver injury was related to increased length of hospital stay (mean difference [md]: 3.2, 95% ci: 1.3–5.2) and viral shedding duration (md: 3.0, 95% ci: 1.0–4.9). conclusions: critically ill patients with covid-19 suffered earlier occurrence, greater injury severity, and slower recovery from liver injury than non-critically ill patients. drug factors were related to liver injury in non-critically ill patients. liver injury was related to prolonged hospital stay and viral shedding duration in patients with covid-19. clinical trial registration: world health organization international clinical trials registry platform, chictr2000030593. registered march 8, 2020. the mean age of 131 enrolled patients was 51.2 years (standard deviation [sd]: 16.1 years), and 70 (53.4%) patients were male. a total of 76 patients developed liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%) with a median occurrence time of 10.0 days. critically ill patients had higher and earlier occurrence (81.5 vs. 51.9%, 12.0 vs. 5.0 days; p < 0.001), greater injury severity (p < 0.001), and slower recovery (50.0 vs. 61.1%) of liver function than non-critically ill patients. multivariable regression showed that the number of concomitant medications (odds ratio [or]: 1.12, 95% confidence interval [ci]: 1.05-1.21) and the combination treatment of lopinavir/ritonavir and arbidol (or: 3.58, 95% ci: 1.44-9.52) were risk factors for liver injury in non-critically ill patients. the metabolism of arbidol can be significantly inhibited by lopinavir/ritonavir in vitro (p < 0.005), which may be the underlying cause of drug-related liver injury. liver injury was related to increased length of hospital stay (mean difference [md]: 3.2, 95% ci: 1.3-5.2) and viral shedding duration (md: 3.0, 95% ci: 1.0-4.9). since december 2019, a newly recognized acute respiratory illness, now officially named coronavirus disease-19 (covid19) , has become widespread globally and accounts for considerable human morbidity and mortality over 200 countries, areas, and territories worldwide (1) (2) (3) (4) . the novel coronavirus is identified and designated as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (5) by the international committee on taxonomy of viruses. as of may 30, 2020, more than five million covid-19 cases had been diagnosed worldwide, and almost 360,000 deaths had been reported (2) . covid-19 has been officially declared a pandemic by the world health organization (6) due to the ongoing outbreak globally. the common clinical manifestations of covid-19 include fever, cough, and shortness of breath (4, 7, 8) . according to the latest epidemiological studies, ∼16-53% of patients with covid-19 experienced different degrees of liver injury (4, (7) (8) (9) (10) (11) (12) (13) , and some patients have developed severe liver injury. the coagulant function abnormality induced by liver injury may cause serious bleeding, especially in critically ill patients who are receiving continuous renal replacement or extracorporeal membrane oxygenation. liver function deterioration can lead to liver failure and even death. therefore, liver injury in patients with covid-19 needs close attention. although some studies have reported the incidence of liver injury in patients with covid-19 (8, 14, 15) , there are limited data describing the course of liver injury occurrence, such as liver injury onset, progression, and recovery, during an entire hospitalization period, particularly in patients with different disease severity. studies on the causes and risk factors of liver injury during sars-cov-2 infection are still limited and controversial. according to current research, liver injury in covid-19 is associated with several main factors, such as sars-cov-2 infection, treatment with potentially hepatotoxic drugs, virally induced cytotoxic t cells, and dysregulated innate immune response (16) . moderate microvesicular steatosis and mild lobular activity are observed in the liver tissue of patients with covid-19 (17) . a preliminary study indicates that sars-cov-2 may directly bind to ace2-positive cholangiocytes to dysregulate liver function. however, systematic study of the causes and the abbreviations: covid-19, coronavirus disease 2019; sars-cov-2, severe acute respiratory syndrome coronaviruses 2; dili, drug-induced liver injury; rucam, a roussel uclaf causality assessment method; alt, alanine aminotransferase; ast, aspartate aminotransferase; alp, alkaline phosphatase; tbl, total bilirubin. risk factors of liver injury in specific populations, such as critically ill and non-critically ill patients with covid-19, is still lacking. additionally, little is known about the correlations between liver injury and some important clinical outcomes, such as length of hospital stay and duration of sars-cov-2 shedding. therefore, a further in-depth study is needed. here, we conducted a multicenter, retrospective, observational study to explore liver injury in critically ill and non-critically ill patients with covid-19 in zhejiang province, china. this study aims to reveal the course of occurrence, risk factors, and correlations with clinical outcome of liver injury in specific covid-19 populations. our study may be helpful in understanding the pathogenesis of liver injury in patients with covid-19, preventing liver injury, and optimizing individual therapeutic treatment. this retrospective observational study was conducted in three tertiary hospitals designated to treat patients with covid-19 in zhejiang province, china. the study was launch by the first affiliated hospital of zhejiang university, which is a university-affiliated tertiary hospital with 2m500 beds and over 100,000 discharged patients per year. sars-cov-2 infection was confirmed using real-time polymerase chain reaction (4, 7) by the local designated hospitals. from january 19, 2020, to february 20, 2020, patients diagnosed with covid-19 were enrolled in this study. patients were excluded on the basis of the following criteria: (1) pregnancy in women, (2) the clinical electronic medical records were reviewed, and epidemiological, clinical, demographic, laboratory, and outcome data were collected for all included patients. a standard case report form was used to record data, including sex, age, chronic medical illness, laboratory data, systemic antiviral agents (i.e., lopinavir/ritonavir, arbidol, fapilavir, and darunavir/cobicistat), potentially hepatotoxic concomitant drugs (18-21) (i.e., corticosteroids, quinolones, statins, immunosuppressive drugs, and non-steroidal anti-inflammatory drugs [nsaids]), number of concomitant drugs, length of hospital stay, and duration of viral shedding. clinical data were followed up until march 10, 2020. topical drugs were not included in concomitant medications. missing data were obtained by direct communication with doctors responsible for the treatment of the patient and their families. all data were verified by three researchers. the severity of covid-19 was defined as non-severe (mild or moderate pneumonia), severe (severe pneumonia), and critically ill during admission in accordance with the diagnostic and treatment guidelines for sars-cov-2 pneumonia of the chinese national health committee (version 6) (22) . in accordance with the severity of covid-19, the patients were divided into non-critically ill (non-severe and severe disease severity) and critically ill groups. non-severe cases included patients with mild and moderate covid-19. the clinical symptoms of mild cases were mild, and there was no sign of pneumonia on imaging. moderate covid-19 refers to fever and respiratory symptoms with radiological findings of pneumonia. severe covid-19 refers to cases meeting any of the following criteria: (1) respiratory distress, (2) oxygen saturation, and (3) arterial partial pressure of oxygen (pao2)/fraction of inspired oxygen (fio2) ≦300 mmhg or cases with chest imaging that showed obvious lesion progression within 24-48 h >50%. critically ill refers to cases meeting any of the following criteria: (1) respiratory failure necessitating mechanical ventilation, (2) shock, and (3) combination with organ failure and admission to an intensive care unit. liver injury was defined as any increase above the normal range for alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), or total bilirubin (tbl). the degree of liver injury was classified as mild, moderate, or severe ( table 1) in accordance with the common terminology criteria for adverse events (version 5) (23). mild, moderate, and severe liver injuries were defined as the occurrence of adverse event grades 1, 2, and 3 (i.e., the increase in alt, ast, alp, or tbl, table 1 ), respectively. the recovery rate of liver function was defined as the decrease in the number of patients with liver injury at discharge divided by the number of patients with liver injury during treatment. viral clearance was defined as the presence of two consecutive negative results with qpcr detection over an interval of 24 h. the metabolic interactions between lopinavir/ritonavir and arbidol was tested in human hepatic microsomes. the metabolic reaction was performed in 0.1 ml incubation mixture containing 0.5 mg microsome protein. the reaction was started by adding 1 mm nicotinamide adenine dinucleotide phosphate and statistical tests were performed using spss19.0 (www.spss.com) and r 3.5.1 (r core team, www.r-project.org). continuous variables were presented as mean (standard deviation [sd]) or median (interquartile range [iqr] ) and compared between and within non-critically ill and critically ill groups by using the student's t-tests or the mann-whitney u-test, as appropriate. categorical variables were presented as frequency (percentage) and assessed using the pearson χ 2 or fisher's exact test (cell size < 5). the occurrence time of liver injury was defined from the time a patient was admitted to hospital until liver injury occurred. the occurrence time of liver injury was portrayed by the kaplan-meier plot and compared between patients in critically ill and non-critically ill groups with a log-rank test. liver injury after admission and at discharge, abnormal liver function indicators, and recovery rate of liver function were compared between the critically ill and non-critically ill groups. univariable and multivariable logistic regression models were used to explore the risk factors for liver injury. demographic data, laboratory test indicators, disease severity, antiviral agents, and potentially hepatotoxic concomitant drugs were investigated. the factors that showed a significant association (95% confidence interval [ci]: does not include one) after univariate logistic regression analysis were entered into the multivariable logistic regression analysis. clinical outcomes (i.e., length of hospital stay and duration of viral clearance) were compared between patients with or without liver injury within each group. p < 0.05 was considered statistically significant. the demographic and clinical characteristics of the patients are shown in table 2 . nineteen ineligible patients were excluded, and the clinical data of 131 patients with confirmed covid-19 were collected (figure 1) . the sample sizes of included the baseline characteristics of patients in critically ill and noncritically ill groups were significantly different (p < 0.05) for the parameters age, prevalence of cardiovascular and cerebrovascular diseases, laboratory data (i.e., leucocytes, neutrophils, albumin, serum creatinine, and c-reactive protein), and treatments (i.e., number of concomitant medications and use of glucocorticoids and antiviral agents). the incidence of liver injury over the study period is shown in figure 2 . during the treatment, 76 patients (58.0%) had liver injury (mild, 40.5%; moderate, 15.3%; severe, 2.3%, table 3 ). the median occurrence time of liver injury was 10.0 days. the percentage of liver injury was reduced to 24.4% at discharge, and liver function returned to normal levels in 57.9% of patients with liver injury. however, none patients with severe liver injury returned to the normal liver function at discharge ( table 3) . the kaplan-meier survival curves for liver injury in different groups are presented in figure 2 . the incidence of liver injury was significantly different between patients in critically ill and non-critically ill groups (figure 2 , median: 12.0 day vs. 5.0 days, p < 0.001) over the study period. as shown in table 3 , 81.5% of the patients in the critically ill group developed liver injury, compared with 51.9% in the non-critically ill group. the severity of liver injury in the critically ill group was greater (p < 0.001) than that in the non-critically ill group. alt and ast levels were more commonly elevated in critically ill patients (p < 0.05) than in the non-critically ill group, whereas no statistical difference was observed in the abnormal alp and tbl levels ( table 3) . the recovery rate of liver function in the non-critically ill group was higher than that in the critically ill group (61.1 vs. 50.0%). univariable and multivariable logistic regression models were used to explore the risk factors for liver injury in the critically ill and non-critically ill groups. however, given the limited sample size in the critically ill group, the statistical power was insufficient, and the multivariable logistic regression model was not conducted in this group. the comparison between patients with or without liver injury in the critically ill group showed that the patients with liver injury had lower lymphocyte numbers, received more concomitant medications, and had higher serum creatinine levels on admission, but the differences were not statistically different (supplementary table s1 ). in the non-critically ill group, the univariate logistic analyses showed that the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were significantly associated with liver injury ( table 4) . after the multivariable regression analysis, the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were determined to be independent risk factors for liver injury. the patients who received the combination treatment of lopinavir/ritonavir and arbidol had 3.58 times the odds (95% ci: 1.44-9.52) of liver injury than patients who did not receive the aforementioned treatment. for every increase in concomitant medication, the odds of liver injury increased by 12.1% (95% ci: 4.9%−21.2%, table 3 ). lopinavir/ritonavir combined with arbidol was shown to be a risk factor of liver injury. we inferred that the metabolic interaction between arbidol and lopinavir/ritonavir may increase drug concentrations and may thus lead to a higher risk of liver injury. the metabolic interactions were tested in human hepatic microsomes in vitro, and the results showed that the metabolism of arbidol can be significantly inhibited after exposure to different concentrations of lopinavir/ritonavir (p < 0.005, figure 3) , whereas arbidol had no effect on the metabolism of lopinavir/ritonavir (p > 0.05, figure 3 ). the average length of hospital stay was 16.6 (sd: 5.7) days and was statistically longer in patients with liver injury than in patients without liver injury (md: 3.2, 95% ci: 1.3-5.2). the mean duration of viral clearance in patients with liver injury was 13.6 days, which was 3 days longer than that in patients with normal liver function (95% ci:1.0-4.9). within the non-critically ill and critically ill groups, the length of hospital stay and duration of viral clearance tended to increase in patients developing liver injury ( table 5) . covid-19 is a newly identified illness that has spread around the world and has become a global health crisis (2, 4, 7, 24) . epidemiological studies have demonstrated that liver injury can occur in patients with covid-19 (7, 8, 10, 12) and may be related to sars-cov-2 infection or therapeutic drugs (9, 17) . here, the onset, progression, recovery, risk factors, and correlation with clinical outcomes of liver injury in patients with varying severities of covid-19 were investigated. the results show that the liver injury in critically ill patients with covid-19 occurred more frequently and earlier, developed more seriously, and recovered more slowly than that in non-critically patients. drug factors, including the combination treatment of lopinavir/ritonavir and arbidol and the number of concomitant medications were independent risk factors for liver injury in non-critically ill patients with covid-19, which may be due to drug interactions at the metabolic level. liver injury was found to be related to prolonged hospital stay and delayed virus eradication in all enrolled patients. consistent with previous studies (7, 8, 25) , this study has found that the high incidence of liver injury in patients with covid-19 is related to disease severity. additionally, the progression and the recovery process of liver injury during the entire hospital stay were investigated. our results indicated that the liver injury in critically ill patients with covid-19 happened earlier and recovered more slowly than that in non-critically ill patients. a higher rate of liver injury was presented in critically ill patients over the study period. our further data showed that the severity of liver injury was also related to covid-19 disease severity. the incidences of mild liver injury were similar in different groups, whereas moderate and severe liver injuries occurred more frequently in critically ill patients than in noncritically ill patients. sars-cov-2 may directly dysregulate liver function by binding to ace2-positive cholangiocytes (26) . our results demonstrated that rather than tbl, alt and ast were the most elevated indicators in critically ill patients with covid-19. hepatocyte injury can be caused by immune interactions that involve virally induced cytotoxic t and kupffer cells (16) . we speculated that besides the direct damage by sars-cov-2, virus-induced cytokine storm may also play an important role in critically ill patients with liver injury. particularly, unlike other studies, this study has found that drug factors rather than disease severity may play a more important role in the liver injury of non-critically ill patients with covid-19. the combination treatment of lopinavir/ritonavir and arbidol was an independent risk factor. lopinavir/ritonavir and arbidol are recommended as potential drugs for sars-cov-2 infection by the chinese national health committee. existing evidence shows that elevated serum aminotransferase and jaundice occur in patients receiving lopinavir/ritonavir containing antiretroviral regimens (18) and that arbidol may induce an increase in transaminase (27). lopinavir and arbidol are mainly metabolized by cytochrome p3a (cyp3a) (28) . ritonavir is a potent cyp3a inhibitor. our results demonstrated that lopinavir/ritonavir can significantly inhibit the metabolism of arbidol, thereby leading to increased arbidol serum concentration. in addition, tbl was the major elevated indicator in non-critically ill patients, indicating that arbidol may have novel adverse reactions in jaundice. therapeutic drug monitoring may be useful in optimizing the regimens in covid-19 patients receiving the combination treatment of lopinavir/ritonavir and arbidol. the number of concomitant medications was another independent predictor of liver injury. concomitant drugs can affect the metabolism of other drugs through induction, inhibition, or substrate competition (20) . we considered that an increased number of concomitant drugs may lead to complex drug interactions and can increase the risk of liver injury. our study also found that the liver injury in patients with covid-19 was related to prolonged viral shedding and hospital stay durations. we inferred that liver injury would lead to immune dysfunction, thereby causing a delay in virus clearance. the prolonged hospital stay can be explained by the need for increased time for liver function recovery or the failure of virus eradication. in the critically ill group, the length of hospital stay and the duration of viral shedding in patients with liver injury were only numerically but not statistically higher than those in patients without liver injury. we speculated that the durations of hospital stay and viral shedding should be influenced by complex factors in critically ill patients with covid-19, and liver injury may not be the only factor affecting the clinical outcomes. our study has some limitations. first, this study is a retrospective, non-randomized clinical observational trial. our cohort is a convenience sample of patients with covid-19 admitted to three hospitals in zhejiang, china. however, this study reflects real-world clinical practices and provides relevant data about liver injury in patients with covid-19. second, the treatments among the three centers were highly consistent because all patients were from zhejiang province. antiviral agents were administered to all patients with covid-19 but limited to a few kinds (e.g., lopinavir/ritonavir and arbidol). therefore, our results cannot be extrapolated to antiviral agents that are not involved in this study, and large controlled studies are necessary to explore the potential risks of liver injury by other antivirals. finally, univariable and multivariable logistic regression models were planned to be used to explore the risk factors for liver injury in the critically ill and non-critically ill groups. however, considering the limited sample size in the critically ill group, the statistical power was insufficient, and the multivariable logistic regression model was not conducted in this group. the trends in risk factors can be reflected through data comparison to a certain extent. focusing on such a population with an expanded sample size would be challenging but would be interesting future research. liver injury has occurred widely in patients with covid-19. critically ill patients suffered higher incidence, earlier occurrence, greater injury severity, and slower recovery from liver injury. drug factors were independent risk factors for liver injury of non-critically ill patients, and drug interaction based on the cyp450 enzymes and concomitant drugs should be closely monitored. liver injury was related to prolonged hospital stay and viral shedding duration in patients with covid-19. therefore, special attention to liver injury during sars-cov-2 infection is recommended. healthcare workers should closely monitor the medications used during hospitalization and adjust and optimize the drug treatment in a timely manner. all datasets presented in this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by the ethics committee of the first affiliated hospital, college of medicine, zhejiang university (reference number: 2020iit[71]). written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. this study followed the statement of strengthening the reporting of observational studies in epidemiology. tl, yq, and xl participated in the conception and design of this study. sj and xl were the project managers and coordinated patient recruitment. dh, ll, rw, rr, yr, jm, nc, xw, zy, and mz coordinated all analyses in the project. sj, ll, rw, and dh were involved in the acquisition, analysis, or interpretation of data. mz and yh conducted the in vitro experiments on drug interaction. data analysis was done by dh, jm, zy, and nc. drafting of the manuscript was done by ll, rw, and xw. all authors contributed to the critical review and final approval of the manuscript. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia map production: who health emergencies programme epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 available online at clinical characteristics of coronavirus disease 2019 in china clinical features of patients infected with 2019 novel coronavirus in wuhan liver injury in covid-19 management and challenges epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of covid-19 related liver damage liver injury during highly pathogenic human coronavirus infections covid-19 and the liver: little cause for concern pathological findings of covid-19 associated with acute respiratory distress syndrome available online at interaction of glucocorticoids with fxr/fgf19/fgf21-mediated ileumliver crosstalk easl clinical practice guidelines: drug-induced liver injury drug-induced liver injury national health commission and state administration of traditional chinese medicine. diagnosis and treatment protocol for novel coronavirus pneumonia (trial version 6). (2020) available online at covid-19 in a designated infectious diseases hospital outside hubei province pecific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection efficacy and safety of arbidol in treatment of naturally acquired influenza pharmacokinetics, metabolism, and excretion of the antiviral drug arbidol in humans we thank the medical and nursing staff at all collaborating centers and especially all patients participating in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmed. 2020.00347/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 jiang, wang, li, hong, ru, rao, miao, chen, wu, ye, hu, xie, zuo, lu, qiu and liang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-288721-3bv3aak6 authors: schneider, annika; kurz, sandra; manske, katrin; janas, marianne; heikenwälder, mathias; misgeld, thomas; aichler, michaela; weissmann, sebastian felix; zischka, hans; knolle, percy; wohlleber, dirk title: single organelle analysis to characterize mitochondrial function and crosstalk during viral infection date: 2019-06-11 journal: sci rep doi: 10.1038/s41598-019-44922-9 sha: doc_id: 288721 cord_uid: 3bv3aak6 mitochondria are key for cellular metabolism and signalling processes during viral infection. we report a methodology to analyse mitochondrial properties at the single-organelle level during viral infection using a recombinant adenovirus coding for a mitochondrial tracer protein for tagging and detection by multispectral flow cytometry. resolution at the level of tagged individual mitochondria revealed changes in mitochondrial size, membrane potential and displayed a fragile phenotype during viral infection of cells. thus, single-organelle and multi-parameter resolution allows to explore altered energy metabolism and antiviral defence by tagged mitochondria selectively in virus-infected cells and will be instrumental to identify viral immune escape and to develop and monitor novel mitochondrial-targeted therapies. mitochondria are crucial for cellular energy metabolism, critically involved in the coordination of signalling processes within cells and orchestrate induction of apoptotic cell death 1, 2 . besides this, cell-autonomous defence mechanisms during viral infection link innate immune sensing of infection and inflammation at the level of mitochondria 3, 4 . the research in the recent years has expanded our knowledge about the different roles of mitochondria. for the different functions mitochondrial shape and motility, but also size, are important and are highly dynamic processes 5 . mitochondrial shape and size are continuously changed during the dynamics of mitochondrial fusion and fission and mitochondrial turnover is controlled by mitophagy 5 . viruses modify the host cell to create an ideal ambience, which includes metabolic support for viral gene expression and replication. such modifications of cellular metabolism and structure of viruses can also affect mitochondria. there are more and more reports about viruses known to influence mitochondrial dynamics. viruses known to enhance mitochondrial fission are hepatitis b virus (hbv), hepatitis c virus (hcv) and epstein-barr virus [6] [7] [8] [9] . viruses, which interfere with or enhance mitophagy are hbv, hcv and measles virus [6] [7] [8] 10 . sars coronavirus is reported to enhance the fusion of mitochondria 11 . but the influence of viral infection on mitochondrial membrane potential and stress response has not been addressed in detail because of methodological constraints. so far, analysis of mitochondria and their functions relied mostly on bulk analysis of mitochondrial populations analysed ex vivo. in infected tissues where both, infected and non-infected cells are simultaneously present, it is very difficult to discriminate between mitochondria from infected versus healthy non-infected cells. this may be achieved by serial tissue sections analysed by electron microscopy, where viral particles could be visualized. however, this is a very time demanding process yielding results with little statistical power. we therefore aimed to develop a technology, where high numbers of single mitochondria and their function can be analysed in the context of viral infection in order to characterize changes induced by viral infection. we chose the liver, and more specifically hepatocytes, as viral infection increases size and mitochondrial fragility of liver mitochondria. we first aimed to determine the influence of viral infection of the liver on the size of liver mitochondria by flow cytometry. to that end, we established a reference curve using polystyrene microparticles with defined sizes (0.88 µm, 1,34 µm and 3 µm). forward scatter analysis of these polystyrene microparticles revealed clear demarcation of the differently sized microparticles and a direct linear correlation of forward scatter results with microparticle size (r 2 = 0.99) ( fig. 2a) , consistent with earlier reports that forward scatter measurements directly correlate with microparticle size down to 0.5 µm 20, 21 . the flow cytometric analysis revealed that mitochondria isolated from healthy non-infected liver ranged in size from 0.8 µm up to 1.4 µm (fig. 2b ) assuming that isolated mitochondria are spherical in morphology, which is indicated by electron microscopy (see fig. 1b ). since mitochondria from hepatocytes are much larger than those from non-parenchymal liver cells or immune cells, we assume that mitochondria ≥0.8 µm in size are derived from hepatocytes. mitochondria purified from virus-infected livers had a slightly higher mean size compared to healthy liver (1.04 ± 0.06 µm compared to 0.97 ± 0.04 µm, respectively) and ranged in size from 0.8 µm up to 3 µm (fig. 2b) . infection with recombinant replication-deficient adenoviruses is a well-established preclinical model system to study hepatotropic infections [22] [23] [24] . however, to confirm the results we repeated the experiments by infection with wildtype replication-competent lymphocytic choriomeningitis virus (lcmv). also after lcmv-infection, we detected an increase of mitochondrial size confirming the results obtained after adenoviral infection (supp. fig. 1a ). in order to investigate whether innate immunity generated during viral infection, was responsible for this increase in mitochondrial size, we induced a type i interferon response by application of poly i:c 25 . flow cytometric analysis of mitochondria isolated after poly i:c application did not reveal any differences in their size compared to the control groups suggesting other mechanisms (supp. fig. 1b) . the exact determination of the size of single mitochondria now opened the possibility to use this information for further analysis. next, we evaluated mitochondrial functionality by determining the mitochondrial membrane potential using the potentiometric dilc 1 (5) fluorescence dye. dose titration experiments of the dilc 1 (5) dye demonstrated a dose-dependent increase in fluorescence intensity in purified mitochondria (supp. fig. 1c ). upon addition of the electron chain uncoupling agent cccp, we found a profound reduction in dilc 1 (5) fluorescence (fig. 2c ) demonstrating that flow cytometric determination of changes in dilc 1 (5) fluorescence reflected mitochondrial membrane potential. by flow cytometric analysis we observed a significant decrease in the mitochondrial membrane potential of mitochondria isolated from virus-infected vs. healthy livers after either adenoviral or lcmv infection compared to healthy controls ( fig. 2d and supp. fig. 1d ). in contrast, we did not detect changes in the membrane potential after innate immune stimulation by poly i:c (supp. fig. 1e ). yet, the size of mitochondria may influence dilc 1 (5) signal intensity. indeed, we found a direct correlation between mitochondrial size and dilc 1 (5) staining ( fig. 2e and supp. fig. 1f ) suggesting that larger mitochondria purified from virus-infected livers should show higher dilc 1 (5) fluorescence intensity. we therefore compared mitochondria with the same size isolated from healthy or virus-infected livers. such direct comparison demonstrated that mitochondria of the same size from healthy vs. virus-infected livers showed a remarkable decrease in the membrane potential of mitochondria from infected livers (fig. 2f ) and suggested that viral infection caused changes in mitochondrial functionality. mitochondria also function to take up calcium from the cytosol and thereby coordinate cellular function 26 , which can also serve as a stress test. when challenged with high concentrations of calcium (100 µm), mitochondria isolated from virus-infected livers are much more fragile shown by time-dependent loss of membrane potential and change of their morphology indicated by decrease in side-scatter (fig. 2f ). this accurately detects mitochondrial swelling after loss of membrane potential following ca 2+ challenge which is also detected by bulk analysis with a classical stress test by adding ca 2+ and detection of loss of membrane potential by rh123-fluorescence and swelling by measuring optical density at 540 nm (supp. fig. 1g ) 18 . consistent with the loss of membrane potential and changes in side-scatter signals, we detected loss of mitochondrial integrity after www.nature.com/scientificreports www.nature.com/scientificreports/ calcium challenge. number of viable mitochondria detected per second by flow-cytometry declined after calcium challenge, consistent with loss of mitochondrial integrity, and did so much faster in samples from virus-infected livers (fig. 2f ). comparing mitochondria with different sizes, it became evident that larger mitochondria are more fragile and disappeared more rapidly after ca 2+ -challenge (fig. 2f ). taken together, here we detected an increase in size and a decrease in membrane potential as well as mitochondrial fragility of liver mitochondria after viral infection. however, since both, non-infected as well as infected hepatocytes are present in livers after adenoviral infection (see fig. 1b ), current protocols for isolation and analysis yield a mixture of mitochondria derived from healthy as well as infected hepatocytes. this makes it necessary to develop a methodology, by which mitochondria from healthy and virus-infected hepatocytes can be separated in order to characterize changes in mitochondrial function specifically in virus-infected cells. we generated a recombinant adenovirus expressing the fluorescent protein dsred fused to a mitochondrial localization sequence (ad-cmv-mitorl) that accumulates and selectively labels mitochondria within infected cells (supp. fig. 2 ). we combined this mitochondrial labelling in infected cells with a multispectral flow cytometric single organelle measurement of isolated mitochondria. upon infection of hepatocytes with ad-cmv-mitorl in vitro we detected mito-dsred-fluorescence in mitochondria using confocal microscopy (fig. 3a) . since ad-cmv-mitorl also codes for luciferase, we detected in vivo bioluminescence of the liver after infection, thus www.nature.com/scientificreports www.nature.com/scientificreports/ confirming successful infection of hepatocytes in vivo (fig. 3b ). this allowed us to test whether mitochondria from ad-cmv-mitorl-infected hepatocytes (mito-dsred + mitochondria) could be distinguished from those of non-infected hepatocytes (mito-dsred − mitochondria) within the same liver. after density-gradient purification, mitochondria isolated from virus-infected livers were counterstained with mitotracker green and analysed by flow cytometry allowing discrimination of mito-dsred + mitochondria from mito-dsred − mitochondria from the same liver (fig. 3c ). mito-dsred + mitochondria from virus-infected hepatocytes had a mean size of 1.19 ± 0.06 µm as compared to mito-dsred − mitochondria from healthy hepatocytes with a mean size of 0.96 ± 0.01 µm (fig. 3d ). this confirmed the results obtained from mitochondria isolated from non-infected livers and further demonstrated a more pronounced size difference when mitochondria from virus-infected could be distinguished at the single organelle level from those of healthy hepatocytes. this was most likely related to a relative underestimation of size for mitochondria from virus-infected livers due to contamination with mitochondria from non-infected cells that are smaller than hepatocyte mitochondria. yet, we cannot formally exclude that mito-dsred localizing to mitochondria after infection with ad-cmv-mitorl may have contributed to the size difference. the almost identical forward scatter results and size of mito-dsred − mitochondria compared to mitochondria isolated from non-infected livers (fig. 3d) indicated that there was no influence of viral infection in neighbouring hepatocytes on mitochondrial size after isolation. differences in size of mitochondria may have also an influence on other parameters detected by flow cytometry and we therefore systematically measured mitochondrial autofluorescence from 450 to 800 nm using a spectral flow cytometer. as expected, we detected increased fluorescence at 590 nm in mito-dsred + mitochondria, where the maximum of dsred fluorescence emission (590-650 nm) is expected 27 . interestingly, we detected higher autofluorescence signals between 500 and 550 nm as well as above 650 nm in mito-dsred + mitochondria (fig. 3e) . as the strength of autofluorescence may be influenced by the size of mitochondria, we analysed autofluorescence signals against the size of isolated mitochondria (fig. 3f ). we found that autofluorescence intensity between 430 and 550 nm directly correlated with mitochondrial size, which may explain the higher autofluorescence observed in larger mito-dsred + mitochondria. together, these data demonstrate the usefulness of single-organelle analysis by flow cytometry in combination with in vivo mitochondrial labelling in virus-infected hepatocytes to exactly determine physical parameters such as size or autofluorescence. mitochondrial crosstalk enables changes in membrane potential. since discrimination of mitochondria isolated from virus-infected compared to non-infected hepatocytes was reliably achieved using flow cytometry, we proceeded to test for changes in mitochondrial functionality upon infection. we assumed that the difference in membrane potential detected between mitochondria isolated from virus-infected livers compared to non-infected livers (see fig. 2 ) has previously been underestimated, and that our method would allow to more specifically discriminate mitochondria from virus-infected hepatocytes compared to non-infected hepatocytes. we determined whether mito-dsred + differed from mito-dsred − mitochondria with respect to dilc 1 (5) fluorescence intensity. to our surprise, we found that the dilc 1 (5) signal was similar for all sizes of mito-dsred + compared to mito-dsred − mitochondria (fig. 4a ). since dilc 1 (5) fluorescence was homogenous in all mitochondria isolated from ad-cmv-gol infected livers (see fig. 1 ), although they consisted of a mixture of mitochondria from infected and non-infected hepatocytes, we wondered whether there was an exchange of molecules between mitochondria. therefore, we mixed dilc 1 (5)-labelled mitochondria with non-labelled mitochondria and by time-dependent flow cytometric analysis found that dilc 1 (5) fluorescence decreased in pre-labelled and increased in un-labelled mitochondria reaching an equilibrium of intermediate fluorescence intensity within 30 seconds (fig. 4b) . however, mito-dsred was not exchanged between mitochondria, because we found clearly distinct dsred staining of mitochondria isolated from ad-cmv-mitorl-infected livers, and mito-dsred − mitochondria showed the same absent dsred fluorescence intensity as mitochondria isolated from non-infected livers. in order to further evaluate mitochondrial functionality, we challenged mitochondria with ca 2+ as stress test and performed time kinetic measurements of dilc 1 (5) fluorescence and side-scatter of mito-dsred + and mito-dsred − mitochondria isolated from ad-cmv-mitorl infected livers. remarkably, the differences in mitochondrial characteristics observed when comparing mitochondria isolated from infected livers to mitochondria from non-infected livers (see fig. 2f ) where not present any more when comparing mito-dsred + to mito-dsred − mitochondria originating from the same liver. in fact, loss of dilc 1 (5) fluorescence, decrease in side scatter and mitochondrial events were the same for mito-dsred + mitochondria as compared to mito-dsred − mitochondria (fig. 4c) . when in direct physical contact with mito-dsred + mitochondria, also mito-dsred − mitochondria showed the same fragility as mitochondria from virus-infected hepatocytes. there, was still a small difference in the large mitochondrial group after calcium stimulation and flow cytometric analysis of the ssc and dilc 1 (5) which could be explained by the fact that 5 to 10 minutes after calcium stimulation the number of events was drastically reduced. only approximately 10% from the initial number of events are still detectable (shown by number of events/s). because of the statistical variation the conclusions at later time points has to be taken with caution. interestingly, also mixing of dilc 1 (5) labelled mitochondria isolated from either ad-cmv-golor lcmv-infected with those from healthy uninfected livers yielded in rapid loss of mitochondrial membrane potential to that measured in mitochondria from infected livers (fig. 4d and supp. fig. 2 ) taken together these data demonstrate that mitochondria which are in close physical proximity exchange information leading to changes in mitochondrial membrane potential but not in mitochondrial size. here, we describe the influence of viral infection on the phenotype and function of mitochondria employing a new methodology combining spectral flow cytometry with virus-encoded markers to simultaneously evaluate multiple mitochondrial parameters at the level of single organelles. most studies involve confocal microscopy to detect mitochondria, which is also available in an automated manner to quantify large datasets of mitochondria 28 . www.nature.com/scientificreports www.nature.com/scientificreports/ while most of these microscopic studies are performed in cell cultures to explore mitochondrial dynamics at the level of single cells, there are only few reports specifically detecting tagged mitochondria in tissues for ex vivo or in vivo analysis 29, 30 . since in vivo microscopic analysis of mitochondria requires a complex experimental setup, is rather time consuming and does not allow for analysis of large numbers of mitochondria, we aimed to establish a methodology to evaluate mitochondria directly ex vivo following isolation from virus-infected tissue. so far, most of available methods analyse properties of mitochondria ex vivo at the level of mitochondrial populations, www.nature.com/scientificreports www.nature.com/scientificreports/ such as extracellular flux analysis, western blot analysis, calcium uptake or swelling assays. beyond visualization by microscopy, flow cytometry has emerged as technology to characterize mitochondria 18, 31, 32 . however, mitochondria isolated from virus-infected tissues can be derived from both, virus-infected cells as well as healthy cells, which may skew the experimental results. we therefore generated recombinant adenoviruses containing a mito-dsred expression cassette to selectively label mitochondria of infected cells. fusion of a fluorescent marker to mitochondrial target sequences has previously been reported to reliably and specifically label mitochondria as shown by confocal microscopy 33, 34 . we combined virus-encoded mito-dsred labelling of mitochondria to separate mitochondria of virus-infected cells from those originating from healthy cells, with the power of multi-parameter analysis by spectral flow cytometry. using this methodology, we provide evidence that mitochondria can be reliably separated from virus-infected cells and that viral infection led to an increase in size as well as a decrease of mitochondrial membrane potential. such changes in biophysical and functional properties of mitochondria were not triggered by innate immunity following recognition of infection through microbe-associated pattern recognition receptors indicating other reasons for these changes, which still have to be defined. time kinetic measurements of single mitochondria by flow cytometry further allowed us to detect a previously unknown mitochondrial cross-talk that involves rapid exchange of small molecules like the potentiometric dye dilc 1 (5) . such exchange of molecules among mitochondria required physical contact, occurred within seconds and did not include mitochondrial matrix-embedded proteins. this indicates a dynamic regulation of mitochondrial properties by cell autonomous mechanisms that require further investigation. taken together, the combination of mitochondrial labelling through mito-dsred together with single organelle analysis using spectral flow cytometry is ideally suited to further unravel biophysical and functional properties of mitochondria as well as mechanisms and consequences of mitochondrial interconnectivity in virus-infected cells. given the important role of mitochondria in cellular metabolism, anti-viral defence, cell signalling and cell death, the multiparametric analysis of single mitochondria opens new avenues to explore these complex mitochondrial functions in more detail in virus-infected cells. mice. c57bl/6 j mice were purchased from charles river (sulzfeld, germany). mice were maintained under specific pathogen-free (spf) conditions in the central animal facility of the klinikum rechts der isar, in accordance with the guidelines of the federation of laboratory animal science association. animal experiments were approved by the animal care commission of bavaria. male mice between the ages of 6-10 weeks were used. the expression cassette for cloning into recombinant adenovirus consists of the genes for the fluorescent protein dsred linked to a mitochondrial targeting sequence and cbg99-luciferase separated by p2a linker sites from the porcine teschovirus 1 followed by a bgh poly(a) signal. gene expression was driven by the ubiquitous minimal cmv-promoter (ad-cmv-mitorl). ad-cmv-gol generation has been reported before 23 . recombinant second generation serotype 5 adenoviruses were generated using the gateway ® technology from thermofisher as described before 23 . briefly, expression cassettes with cmv promotor, dsred linked to the mitochondrial targeting site, cbg99-luciferase and the bgh poly(a) signal were synthesized (eurofins genomics, germany) and cloned into gateway ® pentr ™ 11 dual selection vector (thermofisher scientific, germany). recombination of pentr ™ with expression cassette into pad/pl-dest ™ gateway ® vector (thermofisher scientific, germany) was performed in vitro via the lr clonase ® enzyme mix (thermofisher scientific, germany). the obtained pad/pl-dest ™ with expression cassette was linearized using the paci restriction enzyme and the resulting adenoviral dna was transfected with lipofectamine 2000 (thermofisher scientific, germany) into hek293 cells (crl-1573 ™ ; atcc, usa). cell debris and supernatant were harvested when complete detachment of the cells occurred. this suspension was freeze/thawed, centrifuged and used for further infection of hek293 cells. cells from several cell culture dishes were harvested and resuspended in tris-buffer and freeze/thawed three times. cell debris was removed by centrifugation and supernatant purified by a two-step cscl gradient ultracentrifugation. the band containing adenovirus was harvested and dialyzed. virus titer was determined via adenovirus hexon titration. hek293 cells were infected with serial dilutions of purified adenovirus. after 35 to 40 hours, cells were fixed with methanol, and virus infected cells were stained with anti-hexon antibody (anti-hexon 2297hrp, acris, germany) and detected via dab (dako, usa). the infected cells were counted and the titer was calculated. bioluminescence imaging. imaging of luciferase expression in infected mice was monitored by ivis lumina lt-series iii instrument (perkinelmer las, germany). five minutes before measurement mice have been anesthetized with 2.5% isofluran and treated intraperitoneally with 100 mg/kg bodyweight d-luciferin-k-salt (pjk gmbh, germany). isolation of mitochondria from murine liver tissue. heparin/nacl (300 u/150 µl) was injected i.p. into the mouse 5 minutes prior to preparation. mice were sacrificed and livers were perfused via portal vein for 1 minute with pbs to remove blood. liver was removed and weighed, and the liver was rinsed with isolation buffer www.nature.com/scientificreports www.nature.com/scientificreports/ (220 mm mannitol, 80 mm sucrose, 10 mm hepes, 1 mm edta, ph 7.4). the whole isolation procedure was performed on ice and in ice-cold isolation buffer. the tissue was rinsed with 1 ml isolation buffer and cut with a blunt end scissor into small pieces. the liver fragments were resuspended in 1 ml isolation buffer supplemented with 0.5% bsa and protease inhibitor (protease inhibitor cocktail, edta-free, roche, switzerland) per 0.1 gram of weighted organ and homogenized in a potter-elvehjem with 3 strokes at 800 rpm. the homogenate was transferred to cooled 50 ml falcon and centrifuged at 600 x g for 10 minutes to remove nuclei, intact cells and cellular debris. the supernatant was transferred to a glass tube and centrifuged at 4000 x g for 10 minutes to sediment mitochondria. the received crude pellet was gently dislodged with a glass pestle from the side of the glass tube. mitochondrial purification by density gradient centrifugation. mitochondria were purified as previously described 35, 36 . in brief, a discontinuous percoll density gradient was used for mitochondrial purification. crude mitochondria were resuspended in ip-buffer (300 mm sucrose, 5 mm tes, 0.2 mm egta, ph 6.9), loaded on a percoll density gradient (60%, 30% and 18% diluted in ipp buffer: 300 mm sucrose, 10 mm tes, 0.2 mm egta, 0.1% w/v bsa, ph 7.2) and separated at 9000 × g for 10 minutes. the phase containing mitochondria between 60% and 30% percoll-layer was recovered with a glass pipette and transferred to a 30 ml glass tube, resuspended in 15 ml ip-buffer and centrifuged for further 10 minutes at 9000 × g. the pellet was washed again in 10 ml ip-buffer and centrifuged at 9000 × g for 10 minutes to get rid of remaining percoll. the supernatant was removed and mitochondrial pellet was dislodged from the side of the glass tube. the received mitochondria were resuspended in 100 µl ip-buffer and kept on ice. determination of protein concentration. the protein content in the mitochondrial preparations was determined using the dc tm protein assay kit (bio rad laboratories, germany). the assay was performed according to the manufacturer´s protocol. four different bsa-dilutions reaching from 0.25 mg/ml to 1.5 mg/ml in ip-buffer were used as standards. the optical density was measured at 750 nm with a multiplate reader (infinitem100 pro, tecan, germany). determining mitochondria by flow cytometry. mitochondria were diluted to 10 µg protein per µl in ice-cold mitochondrial staining buffer msb (0.2 m saccharose, 10 mm mops-tris, 5 mm succinate, 1 mm phosphoric acid, 10 µm egta). the different mitochondrial probes were diluted in msb, mixed with the mitochondrial dilution in a 1:1 ratio and incubated at room temperature for 20 minutes. the cell permeable carbocyanine-based mitotracker green probe (mtg, 200 nm), which contains a mildly thiol-reactive chloromethyl moiety, was used to selectively stain all undamaged mitochondria regardless of the membrane potential. dilc1(5) (100 nm), a cationic carbocyanine dye, was used to measure the membrane potential of isolated mitochondria. mitochondria were pelleted at 9000 x g for 2 minutes and washed once in ice cold pbs. mitochondrial pellet was resuspended in msb to a final concentration of 10 µg/µl and stored on ice for analysis. immediately before analysis, samples were diluted in ice-cold and filtered pbs to the final analysis concentration of 0.05 µg/µl. samples were analysed using the spectral cell analyzer sp6800 (sony biotechnology inc, japan). the sample flow rate was set to record about 1500 events per second. as mitochondrial uncoupling by the protonophore cccp is well known to dissipate mitochondrial membrane potential (mmp), 5 µm cccp (sigma-aldrich, st. louis, missouri, usa) was used as a positive control for membrane potential dependence of diic 1 (5) (biotium, hayward, usa). the mitochondrial permeability transition (mpt), a process characterized by a large increase of permeability of the inner mitochondrial membrane (imm), leading to an influx of solutes with a molecular weight less than 1.5 kda and water into the mitochondrion, is a ca 2+ -induced process. the influx of solutes and water leads to swelling of mitochondria. in mpt-measurements 100 µm ca 2+ in msb was added to induce swelling and samples were analyzed immediately after administration and every following 5 minutes for 45 minutes in total. cyclosporina (sigma-aldrich, st. louis, missouri, usa) inhibiting mpt and thereby reversing the effect of ca 2+ , was added at a concentration of 5 µm. mitochondrial size was determined using polystyrene particle size standard beads (flow cytometry grade, spherotech) in three sizes: 0.88 μm, 1.34 μm and 3 μm. beads of each size were separated via ultrasound, vortexed and 20000 beads/size were added per ml filtered pbs. immediately before analysis, mitochondria were diluted in bead mixture to the final analysis concentration of 0,05 µg/ml. data were analysed using flowjo software (version 10, flowjo, oregon, usa). western-blot. 30 µg of protein per sample was loaded onto 4-20% mini-protean ® tgx stain-free ™ precast gels (bio rad laboratories, münchen) and separation was performed within a gel chamber filled with 1x sds electrophoresis buffer at 100 v for 1 to 2 hours. after separation, proteins were blotted using the trans-blot ® turbo ™ mini pvdf transfer packs (bio rad laboratories, germany). proteins were transferred onto membranes at 2.5 a for 30 minutes using the trans-blot turbo ™ (bio rad laboratories, germany). membranes were blocked with 10% milk in tbs-t (tbs + 0.1% tween-20) for 1 hour at room temperature, washed three times with tbs-t and incubated with primary antibodies in 5% bsa in tbs-t overnight at 4 °c. the membranes were washed three times with tbs-t and incubated for 4 hours at room temperature with hrp-coupled secondary antibodies in 10% milk powder in tbs-t. blots were washed three times and developed using cheluminate-hrp picodetect (applichem gmbh, germany), which was evenly distributed on the membrane. the luminescence was detected for up to 20 minutes using the imaging-system chemidoc tm xrs (bio rad laboratories, germany). to visualize www.nature.com/scientificreports www.nature.com/scientificreports/ several proteins on the same blot, primary and secondary antibodies were removed by incubating membranes for 45 minutes at 50 °c in stripping buffer containing ß-mercaptoethanol. subsequently membranes were washed three times with tbs-t and incubated as previously described with primary and secondary antibodies. following primary antibodies were used: adenine nucleotide translocator (ant) (santa cruz biotechnology usa), cytochrome-c-oxidase (cox iv), cytochrome-c (cyt-c), glyceraldehyde 3-phosphate dehydrogenase (gapdh), glucose-regulated-protein 78 (grp78), histon 2b (h2b), voltage dependent anion channel (vdac) (all cell signaling technology, usa), lysosome-associated membrane protein 2 (lamp2) (thermo fisher scientific, usa), peroxisomal membrane protein 70 (pmp70) (origene technologies, usa), following secondary antibodies were used: rabbit anti-goat hrp (santa cruz biotechnology, usa), mouse anti-rabbit hrp, goat anti-mouse hrp (jackson immunoresearch, uk). histology. mouse livers were fixed for 48 hours in 4% paraformaldehyde. dehydrated livers (leica asp300s, germany) were embedded in paraffin. serial 2 µm-thin sections were prepared with a rotary microtome (hm355s, thermofisher scientific, usa) and subjected to histological and immune-histochemical analysis. hematoxylin-eosin (he) staining was performed on deparaffinized sections with eosin and mayer's haemalaun according to standard protocol. immunohistochemistry was performed using a bondmax rxm system (leica, wetzlar, germany, all reagents from leica) with primary antibody against egfp (a-11122, diluted 1:500 in antibody diluent, invitrogen, thermofisher scientific, usa). slides were deparaffinized, pre-treated with epitope retrieval solution 1 for 30 minutes. bound antibody was detected with a polymer refine detection kit without post primary reagent and visualized with dab as a dark brown precipitate. counterstaining was done with hematoxyline. electron microscopy. tissues were fixed in 2.5% electron microscopy grade glutaraldehyde in 0.1 m sodium cacodylate buffer ph 7.4 (science services, munich, germany), postfixed in 2% aqueous osmium tetraoxide 37 , dehydrated in gradual ethanol (30-100%) and propylene oxide, embedded in epon (merck, darmstadt, germany) and cured for 48 hours at 60 °c. semithin sections were cut and stained with toluidine blue. ultrathin sections of 50 nm were collected onto 200 mesh copper grids, stained with uranyl acetate and lead citrate before examination by transmission electron microscopy (zeiss libra 120 plus, carl zeiss nts gmbh, oberkochen, germany). pictures were acquired using a slow scan ccd-camera and item software (olympus soft imaging solutions, münster, germany). statistics. student's t tests were calculated using graphpad prism software. significance was set at p < 0.05 and denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001. all results are expressed as the mean ± standard deviation (sd). the data within this manuscript are available from the corresponding author upon reasonable request. mitochondrial control of cellular life, stress, and death mitochondrial signaling pathways: a receiver/integrator organelle mechanisms of mavs regulation at the mitochondrial membrane identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 mitochondria: dynamic organelles in disease, aging, and development hepatitis b virus disrupts mitochondrial dynamics: induces fission and mitophagy to attenuate apoptosis hepatitis c virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence hepatitis c virus induces the mitochondrial translocation of parkin and subsequent mitophagy epstein-barr virus latent membrane protein-2a alters mitochondrial dynamics promoting cellular migration mediated by notch signaling pathway mitophagy enhances oncolytic measles virus replication by mitigating ddx58/rig-i-like receptor signaling sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/ traf3/traf6 signalosome living in the liver: hepatic infections correlated morphometric and biochemical studies on the liver cell. i. morphometric model, stereologic methods, and normal morphometric data for rat liver bioluminescence imaging allows measuring cd8 t cell function in the liver isolation of mitochondria from cultured cells and liver tissue biopsies for molecular and biochemical analyses analysis of mitochondria by flow cytometry real-time flow cytometry analysis of permeability transition in isolated mitochondria flow cytometric analysis of isolated liver mitochondria to detect changes relevant to cell death measurement of mitochondrial mass by flow cytometry during oxidative cell sizing: a light scattering photometer for rapid volume determination overcoming limitations of microparticle measurement by flow cytometry tnf-induced target cell killing by ctl activated through cross-presentation outcome of anti-viral immunity in the liver is shaped by the level of antigen expressed in infected hepatocytes perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis reduced type i interferon production by dendritic cells and weakened antiviral immunity in patients with wiskott-aldrich syndrome protein deficiency calcium uptake mechanisms of mitochondria biochemistry, mutagenesis, and oligomerization of dsred, a red fluorescent protein from coral deep analysis of mitochondria and cell health using machine learning multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo in vivo imaging of disease-related mitochondrial dynamics in a vertebrate model system flow cytometry of isolated mitochondria during development and under some pathological conditions why to compare absolute numbers of mitochondria analysis of mitochondrial dynamics and functions using imaging approaches strategies for imaging mitophagy in high-resolution and high-throughput a semi-automated method for isolating functionally intact mitochondria from cultured cells and tissue biopsies progressive stages of mitochondrial destruction caused by cell toxic bile salts a chrome-osmium fixative for electron microscopy this work was funded by the deutsche forschungsgemeinschaft (dfg, german research foundation) -projektnummer 272983813 -trr 179 to d.w and p.k. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-44922-9.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-300187-fr6tme32 authors: kearns, shawn title: infectious hepatopathies in dogs and cats date: 2009-11-26 journal: top companion anim med doi: 10.1053/j.tcam.2009.06.004 sha: doc_id: 300187 cord_uid: fr6tme32 this article serves to review the various infectious diseases that affect the liver primarily or as a part of systemic infection. although bacterial infections are probably the most common cause of infectious hepatitis, the clinician should be aware of other potential organisms and other commonly involved systems. therefore, this article includes a description of common bacterial, mycobacterial, viral, fungal, protozoal, parasitic, and rickettsial diseases in dogs and cats. alimentary flora circulates to the liver under various clinical conditions. these bacteria are extracted by kupffer cells, killed by neutrophils, or excreted in bile in healthy clinical states. a low-flow, low-pressure perfusion of hepatic sinusoids may allow superior removal of bacteria by phagocytes, and pressure differentials in the biliary system may limit retrograde access of enteric organisms. 2, 4, 5 changes in this sinusoidal flow may decrease the effectiveness of phagocytosis when portal flow is compromised. bowel disease, cholestasis, immunosuppression, and altered gut motility result in altered portal circulation, and the subsequently unchecked bacterial access to the liver may result in bacterial hepatitis or cholangiohepatitis. common isolates implicated in bacterial hepatitis and cholecystitis include escherichia coli, enterococcus spp, bacteroides spp, streptococcus spp, and clostridium spp. 6 cultures can be obtained by liver aspirate, liver biopsy, and cholecystocentesis. a combination of liver and gall bladder samples (fig 1) may increase the likelihood of identification of the offending organism(s). surgical or laparoscopic biopsies may be more rewarding for culture growth compared with aspirates. 6 in suspected cases, broad-spectrum antibiotics for common enteric isolates should be initiated pending specific culture results. focal micro-and macro-abscesses have also been documented in dogs and cats. 7, 8 predisposing causes include alterations in blood flow, trauma, ascending biliary infections, liver lobe torsions, 9 immunocompromised clinical states, 10 and neoplasia. 7, 11 microabscesses are often identified in association with extrahepatic infection and sepsis. 7, 12 ultrasound has greatly enhanced the early diagnosis of hepatic abscesses. 8 greater than 50% of solitary abscesses are polymicrobial. antimicrobial treatment should be directed at both anaerobes and aerobes regardless of whether anaerobic cultures are negative if a polymicrobial hepatic infection is documented. 2 bacterial isolates in hepatic abscesses are similar to those identified in diffuse bacterial hepatic disease. however, clinically rare isolates including klebsiella, listeria, salmonella, brucella, yersinia pseudotuberculosis, actinomyces, nocardia, and pasturella have also been documented. 13 focal abscesses may require surgical drainage and antibiotic therapy. treatment in all cases must be implemented for a minimum of 6 to 8 weeks. leptopirosis is an extremely common nonenteric bacterial infection in the canine liver. leptospires are thin, filamentous, spiral-shaped motile bacteria with a lipopolysaccharide outer envelope. direct transmission occurs via contact with infected urine, venereal and placental tissues, or fluids. indirect transmission can occur through contaminated water sources, soil, food, or bedding. the organism can stay stable for several months with the right environmental conditions. the organism initially penetrates the mucous membranes and rapidly multiplies after entry into the vascular space. dissemination and replication occur in many tissues, including the liver. however, the organism tends to persist in the kidney and can be shed for weeks to months after infection. certain serovars are more frequently associated with hepatic involvement and include leptospira icterohaemorrhagiae and l. pomona. young dogs (ͻ6 months of age) seem to develop signs of hepatic dysfunction more frequently in disease outbreaks. 14 profound hepatic dysfunction may occur without significant histologic changes because of subcellular damage produced by bacterial toxins. the endothelial damage, subsequent thrombosis, and possible disseminated intravascular coagulation seen in acute disease may contribute to hepatic damage. chronic hepatitis has been reported as a sequelae to leptospiral infection. 15, 16 diagnosis is usually made based on clinical signs and serologic titers. however, leptospirosis polymerase chain reaction (pcr) performed be-fore treatment may increase testing sensitivity given vaccinal interference and delayed seroconversion in the acute phase. 17 penicillins are the treatment of choice in the acute phase and must be followed by appropriate antibiotics to eliminate the carrier state. alternatively, doxycycline may be used for both the acute and carrier states. bartonella spp are gram-negative fastidious bacteria and are well adapted for the intracellular environment. a recent case report documented b. henselae and b. clarridgeiae dna in the liver of 2 dogs with granulomatous inflammation. both had a positive clinical response to azithromycin and demonstrated biochemical reduction in hepatocellular enzymes. 18 another dog with peliosis hepatitis (a rare vascular condition characterized by multiple, randomly distributed blood-filled cavities throughout the liver) had b. henselae dna amplified from multiple hepatic specimens by pcr. 19 helicobacter canis has been isolated from the liver of a single dog with hepatitis. 20 helicobacter spp have also been amplified from hepatic tissue in cats with cholangiohepatitis. further studies are required to determine whether these organisms are associated with inflammatory liver disease. these organisms are difficult to culture, and this failure may reflect the fastidious nature of these bacteria. pcr positivity may reflect the presence of intestinal helicobacter from the enterohepatic circulation or transient colonization rather than a true disease association. 21 francisella tularensis (tularemia) is a pleomorphic, gramnegative, nonspore-forming bacillus. this disease frequently occurs as a result of exposure to ticks or wildlife. macrophages are the primary host cells, and bacteremia with multiorgan involvement is common. lungs, spleen, liver, and skin are common sites for embolic spread, resulting in microabscesses and granulomatous disease. puppies and young cats appear more susceptible to infection, and dogs are generally more resistant to infection. clinical findings include depression, oral/lingual ulceration, regional or generalized lymphadenomegaly, hepatosplenomegaly, panleukopenia with severe toxic neutrophil changes, hyperbilirubinemia, and bilirubinuria. [22] [23] [24] examination for evidence of microscopic agglutinating antibody is most frequently used for diagnosis, although indirect fluorescent antibody testing may be useful as well. 24 aminoglycosides are the primary treatment in humans. however, tetracyclines (doxycycline), chloramphenicol, and quinolones are commonly used in dogs and cats. unfortunately, clinical relapse is common with these antibiotics. tyzzer's disease (clostridium piliforme) is caused by a flagellated, spore-forming, gram-negative intracellular parasite. although spores have been identified in rodent species, interspecies transmission via ingested feces has not been documented. however, spontaneous disease has been documented in dogs and cats. [25] [26] [27] [28] colonization of the liver results in multifocal, periportal hepatic necrosis and may result from a currently unidentified toxin. 29 minimal inflammation may be present despite extensive necrosis. 30 death usually occurs within 24 to 48 hours once the organism is in the liver. [31] [32] [33] [34] [35] [36] [37] rhodococcus is a soil-borne pleomorphic, gram-positive bacteria normally associated with suppurative infections in figure 1 . fine-needle aspirate and cytology from the gallbladder of a cat with cholangiohepatitis. the aspirate consists predominantly of bacteria of mixed type. the bacteria are frequently present in chains (black arrow). also, note dark brown-staining amorphous material (bile pigment: yellow arrow). the finding of bacteria in cytologic specimens of bile is considered abnormal. the following organisms were cultured from the bile: escherichia coli, streptococcus pneumoniae, an anaerobic bacterial rod, prevotella oralis, and a gram-positive rod that could not be classified. courtesy of the pathology department, angell animal medical center, boston, massachusetts. domestic livestock. inhalation from soil or wound inoculation are the suspected routes of transmission. disseminated infection and death have been reported in a single dog. 38 clinical reports are rare in cats. mycobacterium spp are aerobic, nonspore-forming, nonmotile bacteria with a wide host affinity and pathogenic potential. they are typically classified based on growth in culture and by the pathologic production of tubercles or granulomatous disease. mycobacterium tuberculosis and m. bovis are the most pathogenic, and humans are reservoirs for these species. aerosolized organisms in sputum are considered the primary mode of transmission. however, m. bovis can be acquired via uncooked meats and wildlife reservoirs. mycobacterial disease is often subclinical in dogs and cats, but signs may be associated with granuloma formation in various organs. 39, 40 nontuberculous mycobacterium, including those in the mycobacterium avium complex, are saprophytic opportunistic organisms primarily implicated in disseminated disease in cats [41] [42] [43] [44] [45] and occasionally in dogs. 46 -53 no clear associations have been identified with retroviral diseases. canine and feline breeds with potentially increased susceptibility include basset hounds, 51 miniature schnauzers, 53 siamese, 45 and abyssinians. 42 dogs with m. avium complex-induced disease will often demonstrate extensive granulomatous disease of the intestine, spleen, liver, and mesenteric lymph nodes. animals undergoing immunosuppressive drug therapy with inhibition of cell-mediated immunity may be at risk for disseminated disease, including renal transplant patients. 43 acidfast cytology can demonstrate bacilli, although false negatives can occur. negative bacterial images may be identified on routine stains (fig 2) . pcr may provide greater sensitivity and safety than culture. 52 combination therapies are often required, because organisms build resistance quickly, particularly with disseminated disease. although not a risk for immunocompetent individuals, dogs and cats infected with saprophytic mycobacterium pose a risk for immunodeficient people. mycobacterium lepraemurium was considered the main causative agent for feline leprosy until recently. however, m. visibilis has been associated with feline multisystemic granulomatous mycobacteriosis, resulting in diffuse cutaneous disease and widespread dissemination to multiple internal organs. 54 organisms responsible for disseminated fungal infections include histoplasma capsulatum, coccidioides immitis, blastomyces dermatitides, aspergillosis sp, cryptococcussp, and sporothrix schenckii. most are dimorphic, saprophytic, opportunistic fungi that exist in the mycelial stage in the environment. spores are produced in the mycelial stage and be-come pathogenic on inhalation, ingestion, or inoculation. dissemination occurs via the hemolymphatic system. specific environmental conditions are required for the individual organisms, and this dictates their geographic range. histoplasma capsulatum is located primarily in the temperate and subtropical areas of the world. organisms are phagocytized by mononuclear cells and replicate intracellularly once they are inhaled and converted to the yeast phase. the primary clinical signs in dogs are associated with the gastrointestinal system (diarrhea, tenesmus, mucous, fresh bloods in stools). clinical signs in cats are vague. dissemination to other visceral organs (including the liver) has been documented in both species. [55] [56] [57] clinically affected animals are usually young (1-4 years of age). diagnosis is usually achieved with fine-needle aspirate or exfoliative cytology of affected organs. aspergillosis is primarily associated with rhinitis. however, several reports have documented systemic infections in german shepherds and in non-shepherd breeds. aspergillus terreus 58 -62 and a. deflectus 63, 64 have been most frequently implicated in systemic infection. predisposing factors include optimal climatic conditions, access to a partial strain, or subtle defects in mucosal immunity. 65 disseminated aspergillosis has also been documented in cats. 66, 67 neurologic deficits, spinal column pain, urinary system disorders, and respiratory pathology are the primary presenting clinical signs. prototheca is a saprophytic, achlorophyllous alga found in the southeastern united states. three species of prototheca have been identified, but p. zopfii is the only one associated with disseminated disease. the organism is associated with sewage, slime flux of trees, and animal waste. transmission generally occurs through ingestion or penetration of injured skin or mucosa. disease can develop with diminished host resistance or concurrent diseases. 68 concomitant large intestinal diarrhea and ocular signs should prompt clinical consideration of prototheca infection. dissemination via blood or lymph to other organs including the liver is common. various stages of development of the organism may be identified on cytology or histopathology. urine culture and sediment are also useful in organism identification. 69 this disease is invariably fatal, although disease progression may be delayed with various antifungal and antibacterial agents. 70 -73 coccidioides immitus is a dimorphic fungus with preference for the alkaline sandy soil environment found in the lower sonoran life zone in the southwestern united states, western mexico, and central and south america. mycelia are produced during rainfall, but arthroconidia develop with soil drying and become airborne under dry and windy conditions. inhalation is the primary mode of infection in dogs and cats. the spherule (tissue parasitic form) undergoes division with eventual rupture. the severity and extent of clinical disease depend on immunocompetence and range from a mild, asymptomatic, pulmonic form to severe, life-threatening disseminated disease. dissemination most commonly involves the axial and appendicular skeleton and overlying skin. tissues from abdominal viscera, the central nervous system (cns), pericardium, myocardium, and prostate can also be involved. 74, 75 cytology or histology may reveal spherules, although diagnosis is often made based on history, clinical signs, and positive serology. antigens for sero-testing commonly use tube precipitin and complement fixation with agar gel immunodiffusion. sporothrix schenckii causes a chronic granulomatous disease of worldwide distribution. infection is usually the result of trauma and inoculation with infective conidiophores. the skin is the primary target organ. however, disseminated disease has been reported, particularly in the cat. no clear dissemination pattern has been identified because of low case numbers, but affected organs include the internal lymph nodes, liver, lungs, eyes, bone, muscles, and cns. 76, 77 diagnosis is frequently made by cytology. blastomyces dermatitidis is found primarily in mississippi, missouri, the ohio river valley, the mid-atlantic states, and some canadian provinces. growth of the organism requires sandy, acidic soil with some proximity to water. preferred sites for dissemination include the skin, eyes, bones, and lymph nodes, although dissemination to the liver has been reported. 78, 79 cryptococcus neoformans has a worldwide distribution. inhalation may be the primary mode of infection, and sites of infection tend to be areas of the body with cooler temperatures, including the respiratory passages and subcutaneous tissues. the fungus is occasionally disseminated to the kidneys and rarely to the liver. 80 treatment of most disseminated fungal infections involves the use of triazoles, including itraconazole and fluconazole, as well as amphoterocin b. 59,81-85 clinical signs may resolve in many cases, but relapses occur and patients with severe clinical illness generally have a poor prognosis. leishmania, transmitted by the sandfly (lutzomyia in the new world, phlebotymus in the old world), frequently causes cutaneous and visceral lesions in the dog. promastigotes transmitted by the female sandflies become amastigotes in the vertebrate and are phagocytized by mononuclear cells. the organism travels through hemolymph organs to remote dermal sites and other organs. clinical signs will not develop in all exposed animals, and the immune response at the time of infection appears important in determining development of disease. leishmania infection should be considered in dogs from endemic areas with marked hyperglobulinemia or in those with a travel history to endemic areas. mild increases in liver enzymes are often noted. however, unlike the kidneys, the liver is not a primary target organ. infection can be associated with chronic hepatitis. 86 definitive diagnosis is made by demonstration of organisms on cytology or histopathology, or by serology, culture, or pcr. amphotericin b in a soybean oil lipid emulsion has been intravenously administered for higher clinical cure success rates and greater numbers of negative posttreatment parasitologic tests compared with other treatments. other less successful treatment options include allopurinol and the pentavalent antimonials. 87 hepatozoon canis is a worldwide protozoal disease reported in domestic dogs and is most prevalent in subtropical and temperate climates. the primary vector is the rhipicephalus sanguineous tick, which is primarily located in warm and temperate regions. transmission occurs through ingestion of ticks containing mature protozoal oocyts. sporozoites are released in the intestine on tick ingestion and penetrate the gut wall, invade mononuclear cells, and disseminate. target organs include the bone marrow, spleen, and lymph nodes but can involve other internal organs such as the liver, kidney, and lungs. 88, 89 the most striking clinicopathologic abnormality is leukocytosis with evidence of parasitemia of the white cells on peripheral blood smears. clinical findings can range from incidental hematologic findings to severe life-threatening illness. hepatitis, glomerulonephritis, and pneumonitis have all resulted from h. canis infection. 90 coinfections with other protozoal diseases (toxoplasma, leishmania, and babesia spp) or other tick-borne diseases (ehrlichia spp) and immunosuppressive states can predispose animals to clinical illness. the hepatitis is associated with developing meronts within the liver and their associated neutrophilic and mononuclear inflammation. hepatozoon has also been documented in felines. [91] [92] [93] microscopic detection of gamonts in peripheral blood smears is the most frequently used diagnostic test. imidocarb is the treatment of choice in dogs. subcutaneous or intramuscular injections are administered every 14 days until gamonts are no longer visualized in the leukocytes. a new species, hepatozoon americanum, was identified in 1997, with the amblyomma maculatum tick as its definitive host. 94 this emerging disease has spread to the north and the east since its initial identification in the gulf coast region. clinical signs are often severe, even in the absence of other diseases or in the presence of immunosuppression. waxing and waning clinical signs are attributed to repeated cycles of asexual reproduction and pyogranulomatous inflammation. the primary site of infection for the merozoites is the cardiac and skeletal muscle. however, single zoites can enter circulation and reproduce asexually at distant locations. 95 diagnosis is most often made with muscle biopsy, although a recent study has identified promise in the use of pcr testing. 96 an enzyme-linked immunosorbent assay has been developed with sporozoites as the antigen. 97 no treatment effectively eliminates the tissue stages of h. americanum. however, treatment with trimethoprim-sulfadiazine, clindamycin, and pyrimethamine followed by long-term administration of decoquinate resulted in extended survival times and an excellent quality of life. 98 the microsporidial parasite encephalitozoon cuniculi is an obligate intracellular protozoan. infection likely occurs by inhalation or ingestion of spores from contaminated urine or feces shed by infected hosts. the organism undergoes asexual reproduction or binary fission after infecting host cells and ruptures, leading to infection of new cells or shedding of resistant spores into the environment. typical organs of localized infection include the kidney, liver, lungs, and brain with resultant granulomatous inflammation. 99, 100 cats and older dogs are not commonly affected, and renal disease predominates in young dogs. cytological examination of fluids (particularly urine) is important in making a diagnosis in animals with disseminated disease as other tests are commercially unavailable. cytauxzoon felis is a tick-borne protozoal disease of domestic and wild cats. the bobcat is the natural reservoir in north america and is usually asymptomatic despite persistent erythroparasitemia. the tissue phase of infection consists of the development of large schizonts in mononuclear phagocytes. the schizonts line the lumens of vessels in most organs, eventually leading to vessel occlusion. merozoites are released into blood or tissue fluid once the host cells rupture and infect red blood cells. late-stage parasitemia can often be detected on blood films at about 1 to 3 days before death. most clinical signs, including those associated with liver abnormalities, are due to schizont-associated mechanical obstruction. however, parasite by-products may also be toxic, pyogenic, and vasoactive. the anemia is regenerative but mild in comparison with clinical icterus. this may be useful in differentiating this infection from hemotropic mycoplasmas. demonstration of piroplasms in wright's-stained or giemsa-stained blood films most frequently provides a definitive diagnosis. histopathology reveals schizont-laden mononuclear phagocytes in the veins of the lungs, liver, and spleen. the prognosis is generally considered poor, but different geographic strains may have varying virulence. 101 treatment with diminazene or imidocarb has been somewhat successful. 102 toxoplasma gondii is an obligate intracellular coccidian parasite that infects almost all warm-blooded animals. do-mestic cats are the definitive hosts and excrete the infective oocyts. three stages of the life cycle are considered infectious, including oocyst sporozoites, tissue cyst tachyzoites, and tissue cyst bradyzoites. transmission can occur through ingestion of oocysts or tissue cysts and via congenital transmission. other reported modes include lactation, transfusions, and transplantation. 103 a higher frequency of disease is reported in dogs and cats fed raw meat or those in a rural/ feral environment. the extra-intestinal life cycle is the same in all hosts, and sporozoites encyst in the intestinal lumen, penetrate cells, and divide into tachyzoites. the tachyzoites can form cysts in the cns, muscle, and visceral organs, and may persist for the life of the host. clinical signs were diverse in 100 cats with histologically confirmed toxoplasmosis, and more than 90% had pulmonary, cns, and liver manifestations. 104, 105 in dogs, disseminated infection is most often associated with canine distemper, other infections including ehrlichiosis and immunosuppression, or vaccination with live attenuated vaccines. 106 clinical cases in cats have been seen with steroids, cyclosporine use, hemotropic mycoplasms, and viral disease. [107] [108] [109] liver and lung involvement is associated with quicker mortality than other organ involvement. tachyzoites may be detected on cytology of various organs and body fluids. however, diagnosis is most frequently based on clinical signs, serology (immunoglobulin g, immunoglobulin m), and response to treatment. clindamycin is the treatment of choice. neospora caninum is a protozoan similar to toxoplasma. dogs and coyotes are considered definitive hosts, and deer and cattle are intermediate hosts. the predominant mode of transmission is transplacental in the dog, and clinical signs are usually secondary to exacerbation of a congenital infection. acute phases of infection include widespread dissemination to many organs, including the liver, whereas chronically infected animals are restricted to muscular and neuronal sites. 103 serology and muscle biopsy often provide a diagnosis, although tachyzoites may be detected in other parasitized tissue or body fluid. 110 sarcocystis canis is an apicomplexan protozoan with no particular geographic distribution. infection results in disseminated disease, including protozoal hepatitis. 111, 112 many reports involve puppies, suggesting the presence of congenital infection. however, the life cycle is still unknown. sarcocystis canis is the only sarcocystis species known to form schizonts in canine tissue. infectious canine hepatitis (ich) is caused by adenovirus type 1. this is the only virus with primary tropism for the liver. 113 infection leads to severe hepatic necrosis and can also cause ocular and renal changes. the virus localizes in the tonsils after oronasal exposure, spreads to regional lymph nodes, and disseminates via the thoracic duct. hepatic parenchymal cells and vascular endothelial cells are the prime targets of viral localization, and injury leading to centrilobular to panlobular hepatic necrosis ranges from self-limiting to fatal. most affected dogs are less than 1 year of age and unvaccinated. severely affected dogs can become moribund and die within hours of disease onset and with few predictive clinical signs. if patients survive the acute phase, they may develop clinical signs including vomiting, diarrhea, and abdominal pain. 114, 115 those that survive may go on to develop chronic hepatitis and fibrosis, likely secondary to self-perpetuating liver inflammation rather than chronic infection. 116 diagnosis is frequently made based on clinical signs and serology, although the virus can be isolated in cell cultures. this disease is rarely encountered because of the high efficacy of vaccination. canine acidophil hepatitis is believed to be caused by a viral agent. however, the specific agent is not yet identified. disease has been reproduced via injections of sterile liver homongenates from spontaneously affected animals. acute infections can lead to acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. diagnosis is made on histology because acidophils are scattered throughout lesions. this disease has only been reported in great britain. 117, 118 canine herpesvirus causes tissue necrosis and localized mucosal or generalized systemic infections in young or immunocompromised animals. the virus only infects dogs because of specific cell-surface receptors. replication occurs via viral dna synthesis within the host nucleus. transmission occurs through direct contact with mucosal secretions from the respiratory or genital tract of animals. factors predisposing to infection in puppies include hypothermia and a poorly developed immune system. newborns can acquire disease in utero, during passage through the birth canal, during contact with infected littermates, from oronasal secretions of the dam, and from fomites. puppies less than 1 week of age are more susceptible to generalized fatal infections. dissemination leads to hemorrhagic necrosis in several organs including the adrenal glands, kidney, liver, lungs, and spleen. clinical signs include loss of interest in nursing, loss of body weight, soft yellow-green feces, abdominal discomfort, and dullness. a marked increase in alanine aminotransferase is often noted on biochemistry profile. definitive diagnosis is by viral isolation. 119 feline leukemia virus is a single-stranded retrovirus that replicates in many tissues. clinical illness is generally related to the hematopoietic system and the immune system. feline leukemia virus has also been associated with icterus and various inflammatory and degenerative liver diseases including focal liver necrosis. 120 feline infectious peritonitis (fip) is a feline coronavirus that has undergone frequent rna mutations, resulting in an ability to enter and replicate in macrophages. an immunemediated vasculitis occurs if the virus is not eliminated. affected cats develop signs related to target organ lesions (kidney, liver, cns, intestine) or due to fluid redistribution. abnormal liver enzymes can occur because of hepatitis, hepatic lipidosis, or prehepatic sequalae of vasculitis, erythrocyte destruction, and hypoxia. hyperbilirubinemia is common and usually secondary to vasculitis in the liver. 121 histopathology is required for definitive diagnosis but is sup-ported by history, physical examination, and laboratory findings. a new pcr test may also prove useful in the diagnosis of fip. 122 treatment is generally unrewarding. conflicting information exists on the usefulness of feline recombinant interferon, although it may be beneficial for a subpopulation of fip-infected cats. 123, 124 rickettsial diseases the most common agents encountered in dogs with clinical evidence of liver involvement include the ehrlichia sp, rickettsia rickettsii, and borrelia burgdorferi. these organisms can infect either hepatocytes or endothelial cells. hepatic involvement in erhlichia infections occurs in more than 80% of human patients, leading to mild transient increases in transaminases. 125 liver injury may be related to organism proliferation in hepatocytes and stimulation of immunologic and nonspecific inflammatory mechanisms. rocky mountain spotted fever is vasculotropic in nature and can cause moderate increases in transaminases. experimental evidence with borrelia suggests direct hepatic invasion by the spirochetes in conjunction with cellular and humoral immunologic mechanisms. 126 an association with borrelia was observed and confirmed with liver biopsy in 2 dogs. lesions were consistent with lobular dissecting hepatitis and mixed multifocal inflammation leading to focal pyogranulomas in the other. 2 chronic cholangitis associated with liver fluke infestation in endemic areas is primarily observed in cats and less frequently in dogs. most infections are due to opistorchus and metorchis, which require 2 intermediate hosts. the first hosts are water snails, and the second hosts include a wide variety of fish with encysted metacercariae. the final host acquires infection by ingestion of fish, and the young liver flukes migrate to the liver through the bile ducts. this results in bile duct thickening and dilation. rarely, cysts may be formed as well. 127 a slight to moderate inflammation may be seen both within the ducts and in the portal areas. although eosinophils may be present, they are usually limited in numbers. the number of liver flukes and eggs within the dilated bile ducts varies markedly, and often only limited evidence of liver flukes or eggs is identified. platynosomum concinnum is a trematode of the feline biliary system. terrestrial snails, lizards, toads, and terrestrial isopods act as intermediate hosts based on geographic location. disease is most prevalent in the tropical and subtropical climates. clinical cases involve adult indoor or indoor-outdoor cats. the severity of clinical signs is proportional to the number of adult flukes as well as to the duration of parasitemia. early diagnosis can be difficult. however, diagnosis is easier when eggs have been identified in the bile. 128 treatment of p. concinnum and liver fluke infections is best accomplished with praziquantel. there are many infectious diseases that ultimately affect the liver. few, however, have primary tropism for hepatic tissue. testing should be directed based on signalment, geographic locale, and primary presenting complaint. cytology and/or histopathology of the liver will most frequently provide a definitive diagnosis in clinical situations with liver involvement. the prognosis is guarded with many disseminated infections. toxic, metabolic, infectious, and neoplastic liver diseases infectious diseases of the dog and cat detection of portal and systemic bacteremia in dogs with severe induced hepatic disease and multiple portosystemic shunts diseases of the gallbladder and biliary tree cholangitis/cholangiohepatitis complex in the cat bacterial culture results from liver, gallbladder, or bile in 248 dogs and cats evaluated for hepatobiliary disease: 1998-2003 hepatic abscesses in cats: 14 cases hepatic abscesses in 13 dogs: a review of the ultrasonographic findings, clinical data and therapeutic options liver lobe torsion and liver abscess in a dog hepatic abscesses associated with diabetes mellitus in two dogs hepatocellular carcinoma with secondary abscessation in a cat hepatic abscesses in dogs hepatic abscesses in dogs: 14 cases (1982-1994) infectious diseases of the dog and cat chronic active hepatitis in dogs associated with leptospires chronic hepatitis associated with leptospiral infection in vaccinated beagles clinical application of a polymerase chain reaction assay for diagnosis of leptospirosis in dogs detection of bartonella henselae and bartonella clarridgeiae dna in hepatic specimens from two dogs with hepatic disease peliosis hepatis in a dog infected with bartonella henselae helicobacter canis isolated from a dog liver with multifocal necrotizing hepatitis association of helicobacter with cholangiohepatitis in cats feline tularemia on tularemia in a cat acute tularemia in three domestic cats bacillus piliformis infection in an adult dog tyzzer's disease in kittens with familial primary hyperlipoproteinaemia tyzzer's disease in a puppy tyzzer's disease in puppies infectious diseases of the dog and cat the liver in systemic disease. an innocent bystander naturally occurring tyzzer's disease as a complication of distemper and mycotic pneumonia in a dog tyzzer's disease in a dog tyzzer's disease in an adult cat naturally occurring tyzzer's disease in a cat naturally occurring tyzzer's disease in a puppy tyzzer's disease complicated with distemper in a puppy tyzzer's disease in puppies vapa-negative rhodococcus equi in a dog with necrotizing pyogranulomatous hepatitis, osteomyelitis, and myositis mycobacterium tuberculosis complex infection in a dog putative transmission of mycobacterium tuberculosis infection from a human to a dog disseminated mycobacterium avium infection in a cat disseminated mycobacterium avium infection in young cats: overrepresentation of abyssinian cats disseminated mycobacterium avium complex infection following renal transplantation in a cat tuberculosis in cats disseminated mycobacterium avium complex infection in three siamese cats fatal mycobacteriosis with hepatosplenomegaly in a young dog due to mycobacterium avium a case of disseminated tuberculosis in a dog caused by mycobacterium avium-intracellulare systemic mycobacterium smegmatis infection in a dog disseminated mycobacterium avium infection in a dog disseminated mycobacterium avium complex infection in a dog tuberculosis in five basset hounds systemic mycobacterium avium infection in a dog diagnosed by polymerase chain reaction analysis of buffy coat disseminated mycobacterium avium infection in three miniature schnauzer litter mates histologic and genotypic characterization of a novel mycobacterium species found in three cats atypical histoplasma capsulatum infection in a dog disseminated histoplasmosis in dogs: 12 cases (1981-1986) disseminated histoplasmosis in cats: 12 cases (1981-1986) disseminated aspergillosis in two dogs in israel long-term survival of four dogs with disseminated aspergillus terreus infection treated with itraconazole disseminated aspergillus terreus infection in a dog disseminated aspergillosis in a dog with diskospondylitis and neurologic deficits disseminated aspergillosis in a dog systemic mycosis due to aspergillus deflectus in a dog disseminated aspergillosis attributable to aspergillus deflectus in a springer spaniel canine disseminated aspergillosis systemic aspergillosis and mucormycosis in 23 cats feline disseminated aspergillosis altered immune function in a dog with disseminated protothecosis urinary tract manifestations of protothecosis in dogs more than meets the eye: subretinal aspirate from an acutely blind dog disseminated protothecosis causing acute blindness and deafness in a dog disseminated protothecosis in a dog infectious diseases of the dog and cat deep mycotic infections in cats disseminated coccidioidomycosis in a dog pathology of sporotrichosis in 10 cats in rio de janeiro disseminated sporotrichosis in a cat ocular changes in a cat with disseminated blastomycosis legendre am: blastomycosis, in greene ce fatal disseminated cryptococcosis and concurrent ehrlichiosis in a dog disseminated opportunistic fungal disease in dogs: 10 cases infectious diseases of the dog and cat cryptococcosis coccidioidomycosis and paracoccidioidomycosis infectious diseases of the dog and cat chronic hepatitis associated with canine leishmaniosis (leishmania infantum): a clinicopathological study of 26 cases initial and long term efficacy of a lipid emulsion of amphotericin b desoxycholate in the management of canine leishmaniasis hepatozoon canis infection in two dogs canine hepatozoonosis in oklahoma hepatozoon canis infection feline hepatozoonosis granulomatous cholangiohepatitis in a cat due to a protozoan parasite resembling hepatozoon canis hepatozoon species infection in domestic cats: a retrospective study a new hepatozoon species from dogs: description of the causative agent of canine hepatozoonosis in north america characterization of stages of hepatozoon americanum and of parasitized canine host cells diagnosis of canine hepatozoon spp. infection by quantitative pcr an indirect enzymelinked immunosorbent assay for diagnosis of american canine hepatozoonosis treatment of dogs infected with hepatozoon americanum: 53 cases (1989-1998) mammalian microsporidiosis experimental encephalitozoonosis in neonatal dogs cats surviving natural infection with cytauxzoon felis: 18 cases (1997-1998) administration of diminazene aceturate or imidocarb dipropionate for treatment of cytauxzoonosis in cats toxoplasmosis and neosporosis infectious diseases of the dog and cat acute primary toxoplasmic hepatitis in an adult cat shedding toxoplasma gondii oocysts fatal toxoplasmosis in five cats acute toxoplasmosis following renal transplantation in three cats and a dog feline immunodeficiency virus predisposes cats to acute generalized toxoplasmosis neonatal toxoplasmosis in littermate cats histologically confirmed clinical toxoplasmosis in cats: 100 cases (1952-1990) neospora caninum associated with septic peritonitis in an adult dog fatal cutaneous and visceral infection in a rottweiler dog associated with a sarcocystis-like protozoan fatal hepatic sarcocystosis in a puppy with eosinophilia and eosinophilic peritoneal effusion canine viral diseases viral hepatitis of dogs (rubarth's disease) infectious canine hepatitis (hepatitis contagiosa canis use of polymerase chain reaction and immunohistochemistry for detection of canine adenovirus type 1 in formalin-fixed, paraffin-embedded liver of dogs with chronic hepatitis or cirrhosis a new transmissible agent causing acute hepatitis, chronic hepatitis, and cirrhosis in dogs persistent hepatitis and chronic fibrosis induced by canine acidophil cell hepatitis virus canine herpesvirus frequency and significance of feline leukemia virus infection in necropsied cats feline infectious peritonitis and feline enteric coronavirus feline infectious peritonitis: typical findings and a new pcr test use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis effect of feline interferonomega on the survival time and quality of life of cats with feline infectious peritonitis ehrlichial diseases of humans: emerging tickborne infections host-pathogen interactions in the immunopathogenesis of lyme disease severe cholestatic liver disease secondary to liver fluke key: cord-284693-mgpxnnk0 authors: jothimani, dinesh; venugopal, radhika; vij, mukul; rela, mohamed title: post liver transplant recurrent and de novo viral infections date: 2020-09-26 journal: best pract res clin gastroenterol doi: 10.1016/j.bpg.2020.101689 sha: doc_id: 284693 cord_uid: mgpxnnk0 survival following liver transplantation has changed dramatically owing to improvement in surgical techniques, peri-operative care and optimal immunosuppressive therapy. post-liver transplant (lt) de novo or recurrent viral infection continues to cause major allograft dysfunction, leading to poor graft and patient survival in untreated patients. availability of highly effective antiviral drugs has significantly improved post-lt survival. patients transplanted for chronic hepatitis b infection should receive life-long nucleos(t)ide analogues, with or without hbig for effective viral control. patients with chronic hepatitis c should be commenced on directly acting antiviral (daa) drugs prior to transplantation. daa therapy for post-lt recurrent hepatitis c infection is associated with close to 100% sustained virological response (svr), irrespective of genotype. de novo chronic hepatitis e infection is an increasingly recognised cause of allograft dysfunction in lt recipients. untreated chronic hev infection of the graft may lead to liver fibrosis and allograft failure. similarly, cmv and ebv can reactivate leading to systemic illness following liver transplantation. with covid-19 pandemic, post-transplant patients are at risk of sars-co-v2 infection. majority of the lt recipients require hospitalisation, and the mortality in this population is around 20%. early recognition of allograft dysfunction and identification of viral aetiology is essential in the management of post-lt de novo or recurrent infections. optimising immunosuppression is an important step in reducing the severity of allograft damage in the treatment of post-transplant viral infections. viral clearance or control can be achieved by early initiation of high potency antiviral therapy. liver transplantation (lt) is the only curative therapy for patients with decompensated cirrhosis, with an one year and 5-year survival of around 90% and 70%, respectively 1 survival following olt has improved over the years from a 1 and 5 year survival of 70% and 50% to 90% and 70%, respectively. significant improvement in surgical techniques, peri-operative care and immunosuppression therapy has translated in to improved survival. however, in this review, we focus on the prevalence, natural history and clinical outcomes of hepatitis c (rhcv), hepatitis b (rhbv), hev and other viral infections such as cmv, hsv and ebv in lt recipients. in addition, given the current covid-19 pandemic, we discuss impact of sars-cov-2 in the liver allograft and their outcomes. recurrent hepatitis c (rhcv) infection of the liver allograft is universal in untreated patients transplanted for this indication. allograft colonisation by viral particles occurs at the time of portal reperfusion inducing liver damage as early as 72 hours post-liver transplantation. a study on hcv viral kinetics demonstrated an initial sharp decline in the rna levels within 24 hours of reperfusion followed by a rapid increase to nearly 20 fold higher than the pretransplant values during the first week 6 . untreated rhcv may progress with necroinflammation, leading to allograft injury and fibrosis. in a study to evaluate the natural course of 183 hcv liver transplanted patients using protocol liver biopsies, fibrosis score progressed from 1.2 to 2.2 over 10 year period. in addition, patients with severe fibrosis at 1 j o u r n a l p r e -p r o o f year due to rhcv was associated with poor survival. furthermore, donor age >33 years and hcv genotype 1 or 4 developed rapid fibrosis 7 . post-transplant immunosuppression accelerates rhcv related liver damage with fibrosis progression at the rate of 0.3%-0.8% per year leading to cirrhosis and graft loss in 20-40% of patients within 5 years 8, 9 . up to 40% patients with rhcv develop hepatic decompensation within a year of cirrhosis 10 . these patients follow an accelerated course with a three year survival of only 10% following hepatic decompensation unlike 60% in those with native cirrhotic liver. over all patients transplanted for hcv have a lower 3 and 5 year survival of 73% and 67%, respectively 11 . rarely (<10%), rhcv can present in a severe form, fibrosing cholestatic hepatitis (fch), characterized by bilirubin >6 mg/dl, alp and ggt >5x upper limit normal, high hcv rna typically occurring in the first 6 months post liver transplantation. liver histology characterised by severe cholestasis, hepatocyte ballooning spotty necrosis and kupffer cell hypertrophy. progressive fch is associated with severe allograft dysfunction leading to graft failure leading to death within 12 months. in summary, hcv lt recipients had worst long term survival until 5 years ago. hcv viral clearance described as sustained virological response (svr) defined as undetectable hcv rna at 24 weeks following antiviral therapy, but recently reduced to 12 weeks (svr12). svr has a major implication in disease progression in rhcv. similar to chronic hepatitis c in pre-transplant patients, achieving svr in post-transplant patients results in stability of the disease or even regression of fibrosis in 75% of patients 12 . whereas, failure to achieve svr results in worsening of fibrosis. unlike, in patients with chronic liver disease, fibrosis in rhcv progresses at faster pace. in patients untreated rhcv, fibrosis score ≥2 progress rapidly to cirrhosis and graft failure than patients with absent or mild fibrosis 13 . in a 10 year follow up italian study of 358 lt patients for hcv, patients who did not achieve svr had the worst 10-year survival 39.8% compared to 84.7% in those with svr 14 . a number of studies evaluated recipient, donor and virus related risk factors associated with rhcv. advanced recipient age, diabetes mellitus, severe liver disease (child pugh >10), il-28b polymorphism, high hcv rna >10 7 iu/ml, ischemic/reperfusion injury, cmv, donor age >65 years, cold ischaemic time over 8 hours and warm ischemia over 90 minutes, marginal graft, dcd donor, higher immunosuppression in particular high dose corticosteroids for acute cellular rejection, use of anti-thymocyte globulin were significantly associated with rhcv in the liver allograft 15, 16 . calcineurin inhibitors (cni) such as tacrolimus and cyclosporine are the most commonly used immunosuppressive drugs in lt recipients. ideal immunosuppressive regimen in hcv lt recipients to avoid rejection and at the same time to reduce the risk of rhcv is not established. corticosteroid was considered to be the main drug associated with increased risk of rhcv. in particular, use of steroid boluses and rapid tapering has been shown to reactivate hepatitis c in the graft 17 . various immunosuppressive modifications were adopted in the past to reduce rhcv of the allograft. low dose slow steroid tapering has shown to reduce rhcv in the graft. a study by berenguer et al., showed 29% rhcv in patients with cni and tapering steroids over 9-12 months compared to 48% in the historical controls 18 . studies attempted to eliminate corticosteroids using il-2receptor antagonist induction regimens to reduce rhcv. in a prospective randomised multi-centre trial evaluation dacluzimab induction followed by tacrolimus and mmf (n=153) vs standard treatment rhcv occurred in 33% vs 40% (p not significant) 19 with increasing demand for cadaveric organs, dcd organ harvest has been increasingly utilized. studies showed increased rhcv in patients undergoing lt from a dcd compared to donation after brain death (dbd) liver, probably related to ischaemic reperfusion injury 22 . interestingly, a large case control study on hcv patients who received dcd liver showed no significant differences in the hcv rna titres (p=0.7), severe rhcv with fibrosis 8% vs 15%, p=0.38) and graft loss (5% vs 9%, p=0.6) between the two groups at 12 months posttransplant 23 . previously, interpretation of abnormal lfts in patients transplanted for hcv infection was difficult to distinguish between a rejection episode and rhcv infection, in the absence of liver biopsy. therefore, management of graft dysfunction in hcv transplant recipients was a challenge. this led to the development of non-invasive markers of liver fibrosis. fibroscan using transient elastography technique plays a major role in the assessment of disease severity in these patients 24 . liver histology is definitive in the diagnosis and management of rhcv. acute hepatitis c in the liver allograft shows mild lobular activity with mononuclear inflammatory infiltrate, necroinflammatory foci and apoptotic bodies. nodular lymphoid aggregates may also be present. fch is characterised histologically by irregular portal expansion, portal fibrosis with immature pericellular/sinusoidal fibrous bands, extensive hepatocyte ballooning and degeneration, bile ductular reaction, marked canalicular and intracellular bilirubinostasis, j o u r n a l p r e -p r o o f and mild to moderate mononuclear inflammation. confluent or bridging necrosis may be present. pre-lt hcv rna level strongly predicts post-transplant rhcv severity. therefore, treatment should be aimed at making the patient virus free prior to liver transplantation.. svr has consistently shown to improve several aspects of hcv related complications. achieving svr in patients awaiting liver transplantation has shown to decrease the rate of disease progression, symptomatic improvement, improvement in meld score and importantly improves post-transplant survival 25, 26 treatment of hcv in the previous era: a decade ago, the treatment of hcv in patients with decompensated cirrhosis was nearly impossible. in the absence of advanced liver disease, the use of peg ifn and ribavirin achieved 50-70% svr depending on the genotype. unfortunately, both these drugs are contraindicated in patients with decompensated cirrhosis due to higher adverse events, like anaemia and sepsis. subsequent introduction of first generation directly acting antiviral (daa) therapy, protease inhibitors telaprevir and boceprevir achieved svr around 70% given with peg ifn and ribavirin. however, protease inhibitors were effective only against hcv genotype 1 and similarly these were contraindicated in advanced liver disease. both these drugs were discontinued in 2015. treatment of hcv in patients with decompensated cirrhosis changed significantly following the introduction of sofosbuvir, the second generation daa with high efficacy, shorter treatment course, better safety profile and importantly interferon free regime. sofosbuvir with ribavirin for up to 48 weeks in 61 hcv hcc patients awaiting liver transplantation, showed an undetectable hcv rna at the time of lt was associated with j o u r n a l p r e -p r o o f 70% svr12 in the post-transplant period. moreover, patients with undetectable hcv rna at least 4 weeks prior to lt never developed rhcv. adverse events leading to drug discontinuation was noted in 2 patients (sepsis and kidney injury) which was unrelated to sofosbuvir 27 . the success of this therapy changed the perspective of hcv management in patients undergoing liver transplantation. subsequent studies were conducted in hcv patients with decompensated cirrhosis with second generation daa. solar-1 was a phase 2 open label multi-centre study on 12 or 24 weeks of ledipasvir (ns5a polymerase inhibitor), sofosbuvir and ribavirin in patients with advanced cirrhosis (child pugh b or c, n=108). svr12 for child pugh b was 87% and 89%, and for child pugh c was 86% and 87%, with 12 and 24 weeks treatment irrespective of previous therapy. adverse events occurred in 23% of patients but only 4% discontinued treatment 28 . similarly, solar-2 study was conducted across european and new zealand sites on hcv child pugh b (n=56) and child pugh c (n=51) with svr12 of 87% and 96%, and 85% and 78% for 12 and 24 weeks, respectively. adverse events occurred in 11-50% of patients, predominantly in child pugh c, including 3 (12%) patients death. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance in patients with decompensated cirrhosis. with the higher viral eradication rates, meld score improved in these patients over a period of time; however, in many of the study centres patients have undergone liver transplantation in view of shorter waiting times in different parts of the study sites 29 .data from our liver unit showed a weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin in patients with decompensated cirrhosis (n=60) with an svr12 of 83% 30 . patients with child pugh a and b had a higher svr12 than c, 94%, 94% and 56%, respectively. likewise, genotype 1b had a better svr12 than genotype 1a (100% vs 76%). in patients with decompensated cirrhosis, meld score improved by -0. higher rates of svr with daa has shown to reduce hcv related death by 74% 31 . achieving svr has shown to reduce the chance of liver disease progression with improvement in portal pressure 32 . in several cases there were significant improvement in meld score leading to delisting of patients waitlisted for lt. in a study involving 409 patients with child b and c cirrhosis, the mean meld declined by 0.85 within 6 months compared to untreated patients (p<0.0001) and they encountered reduced episodes of hepatic decompensation (3.7% vs 10%, p=0.009) in patients with svr 33 . importantly, a cohort study from srtr database showed 32% reduction in the hcv lt waitlist after they cleared hcv with daa therapy 34 . in the previous decade, management of post-transplant rhcv has been posed several difficulties. introduction of peg-ifn and ribavirin in the treatment of rhcv slightly improved clinical outcome. unfortunately, these drugs were associated with significant side effects including graft rejection. therefore, treatment was recommended only in patients with moderate and severe rhcv. fatigue, diarrhoea and anaemia occurred in 30%, 28% and 20% of patients 37 . initial studies with sofosbuvir and ribavirin combination therapy for post-transplant rhcv showed poor drug tolerance, however, the main adverse event was anaemia related to ribavirin in 62% of patients, and subsequent hepatic decompensation related to the low haemoglobin 38 . solar 1 and solar 2 also evaluated the efficacy of the combination of sofosbuvir and ledipasvir also included post liver transplantation rhcv patients. solar-1 study involving 12 or 24 weeks of ledipasvir, sofosbuvir and ribavirin in post-lt rhcv (n=229), the svr12 was 96%, 96%, 86%, 60%, 100% in patients with no cirrhosis, child a, child b, child c and fch patients with 12 weeks and, 98%, 96%, 88%, 75% and 100% for 24 weeks therapy. adverse events such as mild hyperbilirubinemia and anaemia however, mostly managed by reducing median dose of ribavirin to 600 mg daily 37 . solar-2 trial evaluated 226 post-transplant rhcv patients with ledipasvir sofosbuvir combination however, with child pugh score above 13 were excluded. svr12 was 93%, 100% and 95% in patients with no cirrhosis, child a and child b cirrhosis with 12 weeks j o u r n a l p r e -p r o o f therapy and 100%, 96%, 100% with 24 weeks therapy. these drugs were well tolerated with 5% adverse events, 2% discontinuation rates, and 2% viral relapse rates 39 .the success of solar trial with all oral daa in this challenging population opened the avenue for patients post lt rhcv. these two studies clearly demonstrated high svr rates in patients with 12 weeks of this combination therapy with effective hcv clearance post-transplant rhcv, than the previous generation drugs. ally-1 study also recruited patients with post-transplant rhcv infection (n=53) to assess the efficacy of 12 weeks daclatasvir 60 mg daily in combination with sofosbuvir 400 mg twice a day and weight based ribavirin. svr12 was 94% in lt recipients. viral relapsed in 3 posttransplant patients and were subsequently treated with 24 weeks of daclatasvir, sofosbuvir and ribavirin. there was no significant drug interactions with cni requiring dose adjustments 30 . interestingly, a real world analysis of daclatasvir and sofosbuvir showed 87-100% svr12 in patients with child b and c cirrhosis, and 58% of decompensated cirrhotic patients showed improvement in meld score 40 . a large multicentre study on daclatasvir in combination with sofosbuvir or simeprevir with or without ribavirin for 24 weeks was carried out on 97 lt recipients with severe rhcv including 37% patients with fch and 31% with cirrhosis. svr12 with dac + sof was 91% and dac + smv was 72%. there was a significant improvement in child pugh score from 6.8 to5.7 (p<0.001) and meld score from 12.1 to 9.7 (p<0.001) following svr. however, these scores worsened in 13% patients despite antiviral therapy 41. introduction of pangenotypic velpatasvir-sofosbuvir combination further simplified hcv therapy with maximal efficacy. a phase 3 study on 624 patients with hcv increased svr to 99% including in cirrhotic patients 42 . higher alt was shown to accelerate disease progression and cirrhosis. alt>100 u/l was shown to predict cirrhosis at 5 years due to rhcv (35% vs 6%). similarly, serum bilirubin >3.5 mg/dl was strongly associated with development of cirrhosis following rhcv (prieto 1999 hepatology, rosen 1999 transplantation) 43, 44 . practice of protocol liver biopsies at regular intervals post transplantation were carried out in the past. presence of moderate to severe lobular inflammation was associated with disease progression (30% vs 0.10%) compared to no or minimal inflammation over 5 years post transplantation. hcv rna titres has a direct correlation with the severity of post-transplant rhcv infection. in a study by shakel et al., 45 .peak hcv rna in the first year of untreated patients was associated with poor patient survival. a level of less than 10 7 , 10 7 -10 8 and >10 8 49 .in a recent innovative approach non-liver recipients of hcv positive donors were prescribed a short 7 day course of newer daa glecaprevir/pibrentasvir along with ezetimibe (hcv entry inhibitor) showed 100% viral clearance at week 12 50 . in summary, long term survival of hcv lt recipients has improved dramatically following second generation daa. with current svr close 100%, excellent safety profile daa therapy are increasingly used in decompensated cirrhosis and for post-transplant rhcv treatment. most centres treat hcv decompensated cirrhosis because it is associated with significant improvement in meld and may help reducing transplant waitlist. there was a debate j o u r n a l p r e -p r o o f whether to treat these patients while waiting for transplant or to treat in the posttransplant period, because some studies observed an increased risk of hcc following svr. in the management of rhcv, the timing of antiviral therapy is not well established. most experts recommend starting daa after first 3 months with stable immunosuppression onboard. a study by pellicelli et al., showed significant adverse events including hepatic decompensation and 25% mortality in those with advanced disease following treatment with daclatasvir and sofosbuvir for post-transplant rhcv 51 . similarly, forns et al, provided compassionate access sofosbuvir and ribavirin to 104 patients with severe rhcv with life expectancy less than 12 months. svr was 59% and much higher (73%) in patients with severe rhcv. however, severe adverse event occurred in 47% of patients including 13% mortality 52 . therefore, it is advisible to commence antiviral therapy prior to hepatic decompensation. post-transplant rhcv management previously included allograft biopsy to assess the severity of rhcv before commencing anti-viral therapy. however, the necessity of biopsy may be arguable with the currently available daa. abnormal liver enzymes in the presence of high hcv rna level may suffice to commence daa. retransplantation for graft failure secondary to rhcv had much worse outcomes. retransplantation for rhcv related graft failure is associated with prolonged hospitalization, increased cost and reduced survival. however, outcome and patients selection differed amongst various studies. in a multi-centred study from the us showed 1 and 3 year survival of hcv retransplantation was 69% and 49% with no difference in survival compared to other indications 53 .however, the currently available daa, the need for retransplantation likely to have reduced. hepatitis b is the major cause of chronic liver disease across the world affecting 350 million population, with higher prevalence in asian and african countries despite universal vaccination. hepatitis b was a considered an absolute contraindication in the initial years of liver transplantation due to early graft reinfection and poor survival benefit (less than 50%, j o u r n a l p r e -p r o o f therefore, most liver transplant centres adopted lifelong entecavir or tenofovir to reduce viral relapse following liver transplantation, particularly in low risk patients such as those with decompensated cirrhosis and low viral load (<100 copies/ml), alf presentation. low dose and short course hbig with antivirals may be useful in patients with high hbv dna, hbeag positive patients. tenofovir alafenamide (taf) is the most recently introduced formulation of tenofovir, to overcome renal and bone related adverse events related to tenofovir disoproxil fumarate. the efficacy is similar to its prodrug tenofovir disoproxil fumarate with a good safety profile. it is recommended in patients with chronic kidney disease and osteoporosis. data in posttransplant patients with taf is scarce. small case series found switching from tdf to taf hepatic involvement occurs in 14-53% of patients with covid-19, particularly in severe cases 73 . ace2 receptor is highly expressed in cholangiocytes (59.7%), vascular endothelial cells. interestingly, only 2% hepatocytes express ace2 and no expression observed in sinusoidal endothelial cells 72 . in a recent multicentre study on covid-19 patients with underlying cirrhosis (n=50), 97% required hospitalization and 71% required respiratory support. the number of patients with meld>15 increased from 13% to 26% (p=0.037) and aclf occurred in 28% of patients. in that study the 30-day mortality was 34% 74 . sars-cov-2 in decompensation in patients is associated with higher mortality 75 . analysis of apasl covid-19 liver injury spectrum study (apcolis) on 228 patients (43 cirrhotics), hepatic decompensation occurred in 9% and aclf in 11.6% of patients. child pugh score >9 predicted higher mortality (roc 0.94, hr 19.2). in patients with decompensated cirrhosis, mortality was 33% compared to 16.3% in compensated cirrhosis 76 to the non-transplant setting, however, chronic presentation defined as detectable hev rna or hev igm in the serum for 6 months, is exclusively observed in the post-transplant patients 86 . chronic hev following in transplant recipients was first recognised by kamar et al, including in lt patients. these patients continued to have allograft dysfunction following an acute infection and persistent positive hev rna in serum or stool for 10 to 24 months 87 . exact prevalence of hev seroprevalence in lt recipients is unknown. tests based on hev rna or hev igg level, a retrospective analysis of frozen sera showed 1% to 16% seroprevalence of hev in lt recipients 88 . in a french study, hev seroprevalence (hev igg) was observed in 12.9% of patients during pre-lt evaluation, interestingly one third of these patients became hev negative in the post-transplant period 89 late onset cmv disease occurs after completion of prophylaxis. especially in (r-/d+) recipients and its incidence is 25% which when compared to 8.3% in preemptive treatment 3-6 months reduces the advantage of universal prophylaxis. some studies report mitigation of this by extending the prophylaxis to 200 days, but data are insufficient 118 varicella zoster, that causes chicken pox remains latent in the dorsal root ganglion. this can reactivate following immunosuppression leading to herpes zoster, characterized by painful vesicular rash along the nerve distribution, usually restricted to one side. the 1-year incidence is 3% whereas the 5-and 10-year incidences are 14% and 18% respectively 146 human herpes virus 6 (hhv-6) belongs to beta herpesviridae subfamily under roseolovirus genus. this is a common name for 2 similar viruses hhv-6a and hhv-6b, the latter accounting in most cases 149 . asymptomatic exposure to hhv-6 occur in childhood such that 90-95% adult population are seropositive 150 . the circular dna of the virus integrates with the host genome and may remain latent for several years in the mononuclear cells 151, 152 . hhv-6 infection occurs as a result of reactivation in the post-transplant state. occasionally, donor derived infections or rarely through blood products have been reported 153 . incidence of hhv6 varies between 14% to 82% 154 , commonly occurring in the first 2-8 weeks after liver transplantation. sporadic cases were reported as early as 10 days and rarely after 5 years following liver transplantation 155 . in a prospective study of 51 lt patients, 11 (21.5%) developed hhv-6b reactivation, of which 4 had fever and abdominal pain 156 . reactivation rates are less when patients are covered up for cmv prophylaxis with ganciclovir. most hhv-6 reactivation are asymptomatic with low viral load 156 and they usually do not require treatment. clinically, hhv-6 reactivation presents with fever, and skin rash and raised liver enzymes 158, 159 . histologically, hhv-6 hepatitis may mimic acute cellular rejection with elevated liver enzymes and portal lymphocytic infiltration and confluent periportal necrosis on liver 153, 158 . in 170 lt patients with graft hepatitis, high levels of intra-hepatic hhv-6 dna and hhvantigenemia was significantly associated with decreased graft survival 160 . hhv-6 j o u r n a l p r e -p r o o f encephalitis though rare in lt recipients occurs usually within 4-6 weeks of transplantation. hhv-6a is neurotrophic and is detected in plasma as well as csf. brain imaging shows characteristic signal intensity in medial temporal lobes involving amygdala and hippocampi. hhv-6 infection may also present as post-transplant colitis in some 161 . it can also occur as co infection with cmv where hhv-6 antigenemia precedes cmv antigenemia 162 to summarise, hhv-6 infection after liver transplantation is rare, but its reactivation is associate with significant increase in graft failure, mortality, hepatitis c progression and cmv disease 169 . hhv infections in lt patients can be treated successfully with cmv antivirals ganciclovir, cidofovir and foscarnet. post lt viral infections can cause significant allograft dysfunction. early recognition, diagnosis and systematic approach can ameliorate the infective process and preserve allograft function. newly evolving less familiar viral infections such as covid-19 should also be considered in the differential diagnosis of post-transplant viral infections. management j o u r n a l p r e -p r o o f of post-transplant viral infection has improved over the last decade due to significant changes in immunosuppression protocols with optimal usage, and introduction of effective anti-viral drugs with high genetic barrier for resistance. these high potency anti-viral drugs have translated in to better long-term allograft and patient survival. • patients with decompensated cirrhosis due to chronic hepatitis b or hepatitis c infection should be commenced on high efficacy antiviral therapy • pathogenesis of covid-19 in post-liver transplant patients requires larger studies. • ideal immunosuppressive regimen to reduce the chance of recurrent or de novo viral infection needs to be studied. j o u r n a l p r e -p r o o f liver transplantation around the world. curr opin organ transplant hepatitis b virus epidemiology, disease burden, treatment, and current and emerging 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in european patients with the hepatitis b surface antigen lamivudine plus low-dose hepatitis b immunoglobulin to prevent recurrent hepatitis b following liver transplantation transmission of hepatitis b infection from hepatitis b core antibody--positive liver allografts is prevented by lamivudine therapy failure of reactivation of hepatitis b after liver transplantation in hepatitis b surface antigen-negative, core antibody-positive recipients rapid early hdv rna decline in the peripheral blood but prolonged intrahepatic hepatitis delta antigen persistence after liver transplantation a pneumonia outbreak associated with a new coronavirus of probable bat origin world health organization. situation report -120 evaluating new evidence in the early dynamics of the novel coronavirus covid-19 outbreak inwuhan, china with real time domestic traffic and potential asymptomatic transmissions. medrxiv specific ace2 expression in cholangiocytes may causeliver damage after 2019-ncov infection 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the united states and other countries prevalence and clinical consequences of hepatitis e in patients who underwent liver transplantation for chronic hepatitis c in the united states hepatitis e virus and chronic hepatitis in organ-transplant recipients prevalence of hepatitis e virus infection in pediatric solid organ transplant recipients--a single-center experience prevalence of hepatitis e virus infection in liver transplant recipients hepatitis e virus infection without reactivation in solid-organ transplant recipients, france. emerg infect dis hepatitis viruses and liver transplantation: evolving trends in antiviral management liver transplant from a donor with occult hev infection induced chronic hepatitis and cirrhosis in the recipient factors associated with chronic hepatitis in patients with hepatitis e virus infection who have received solid organ transplants ribavirin for chronic hepatitis e virus infection in transplant recipients sofosbuvir inhibits hepatitis e virus replication in vitro and results in an additive effect when combined with ribavirin sofosbuvir monotherapy fails to achieve hev rna elimination in patients with chronic hepatitis e -the hepnet sofe pilot study management of cmv infection and disease in transplant patients european association for the study of the liver. electronic address: easloffice@easloffice.eu. easl clinical practice guidelines: liver transplantation randomised trial of efficacy and safety of oral ganciclovir in the prevention of cytomegalovirus disease in liver-transplant recipients. the oral ganciclovir international transplantation study group dynamics of cytomegalovirus replication during preemptive therapy with oral ganciclovir who among cytomegalovirus-seropositive liver transplant recipients is at risk for cytomegalovirus infection? liver transpl cytomegalovirus hepatitis in liver transplantation: prospective analysis of 93 consecutive orthotopic liver transplantations infections and allograft rejection 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recipients. transplantation mycophenolate mofetil increases cytomegalovirus invasive organ disease in renal transplant patients selection and use of immunosuppressive therapies after liver transplantation: current german practice use of everolimus-based immunosuppression to decrease cytomegalovirus infection after kidney transplant antiviral drug resistance of human cytomegalovirus epstein-barr virus infections: biology, pathogenesis, and management lymphoproliferative disorders and de novo malignancies in intestinal and multivisceral recipients posttransplant lymphoproliferative disease following liver transplantation occurrence of post-transplant lymphoproliferative disorders among over thousand adult recipients: any role for hepatitis c infection? associations between ebv serostatus and organ transplant type in ptld risk: an analysis of the srtr national registry data in the united states diagnosis of post-transplant lymphoproliferative disorder in solid organ transplant recipients 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as distinct viruses update on human herpesvirus 6 biology, clinical features, and therapy latent human herpesvirus 6 infection of human monocytes/macrophages the prevalence of chromosomally integrated human herpesvirus 6 genomes in the blood of uk blood donors et pediatric liver transplant patients hhv-6a, 6b, and 7: persistence in the population, epidemiology and transmission human herpesvirus-6 infection after liver transplantation clinical impact of human herpesvirus 6 infection after liver transplantation in syncytial giant-cell hepatitis detection of hhv-6 in over a thousand samples: new types of infection revealed by an analysis of antigenemia related to cmv infection after liver transplantation co-infection and clinical impact of human herpesvirus 5 and 6 in liver transplantation human herpesvirus-6 in liver transplant recipients: role in pathogenesis of fungal infections, neurologic complications, and outcome and cytomegalovirus dna in liver donor biopsies and their correlation with hla matches and acute cellular rejection acute hepatitis with periportal confluent necrosis associated with human herpesvirus 6 infection in liver transplant patients encephalitis caused by human herpesvirus-6 in a liver transplant recipient treating hhv-6 infections diagnosis and clinical management hhv-6 in liver transplantation: a literature review key: cord-261608-4sjlg0p0 authors: trejo-paredes, camila; mohammed, turab jawaid; salmon, adrian title: covid-19-induced liver injury: a clinical distraction? date: 2020-10-31 journal: chest doi: 10.1016/j.chest.2020.08.901 sha: doc_id: 261608 cord_uid: 4sjlg0p0 nan a 51-year-old male with a history of hypertension and diabetes was admitted with fever and breathlessness secondary to covid-19 and started on hydroxychloroquine (hcq) and azithromycin. his routine blood work, including a complete metabolic panel, was unremarkable. he was intubated for progressive hypoxia, and given the elevated ferritin-5088 ng/dl and d-dimer >2500mg/l, he was started on high-dose heparin infusion for anticoagulation. his ferritin and d-dimer continued to uptrend, and on day 10, he was noted to have a total bilirubin of 8.4 mg/dl. highest peak values noted, aspartate aminotransferase-229, alanine aminotransferase-167, alkaline phosphatase-162, total bilirubin-22, and direct bilirubin-15.5. tests for hepatitis a, b, c, hsv, ebv, hemolysis workup, and imaging studies were unremarkable. after a week, the liver enzymes and bilirubin down trended and returned to baseline spontaneously. no clear etiology was ascertained and was presumed multifactorial likely from viral hepatitis, sepsis or drug-induced cholestasis. his hospital course was also complicated by acute renal failure requiring renal replacement therapy, coagulopathy, and encephalopathy which coincided with liver injury trailing behind the peak of inflammatory markers. he underwent tracheostomy and percutaneous endoscopic gastrostomy tube placement prior to discharge after 7 weeks. emerging data support the hypothesis that liver injury in covid-19 is often the result of sars-cov-2 directly binding to ace2+ cholangiocytes, leading to cholangiohepatitis. in addition, cytokine storm may further exacerbate the hepatic injury in covid-19 (2). although our patient lacked any known risk factors for liver injury and preexisting liver disease, he developed a significant hyperbilirubinemia and transaminitis without sequalae. hepatic congestion in ventilated patients, shock liver and particularly, drug-induced liver injury (dili) remains an important consideration in covid-19 patients. initiating antiviral therapy and curtailing cytokine dysregulation at an early stage could be beneficial to curb the disease progression (3). conclusions: covid-19 induced viral hepatitis is now being increasingly identified as a self-resolving complication and the physician should be mindful of it and in the right setting, it may only be a clinical distraction. we should be cognizant of other potential causes of liver injury in covid-19 patients like concurrent infection, sepsis-induced and dili. liver injury in covid-19: the current evidence clinical characteristics and mechanism of liver damage in patients with severe acute respiratory syndrome covid-19 and the liver: little cause for concern. the lancet gastroenterology & hepatology disclosures: no relevant relationships by turab jawaid mohammed, source¼web response no relevant relationships by adrian salmon, source¼web response no relevant relationships by camila trejo-paredes american college of chest physicians key: cord-025168-be7zube4 authors: saleh, mahshid; taher, mohammad; sohrabpour, amir ali; vaezi, amir abbas; nasiri toosi, mohsen; kavianpour, maria; ghazvinian, zeinab; abdolahi, shahrokh; verdi, javad title: perspective of placenta derived mesenchymal stem cells in acute liver failure date: 2020-05-24 journal: cell biosci doi: 10.1186/s13578-020-00433-z sha: doc_id: 25168 cord_uid: be7zube4 acute liver failure (alf) is a life-threatening disease and is determined by coagulopathy (with inr ≥ 1.5) and hepatic encephalopathy as a result of severe liver injury in patients without preexisting liver disease. since there are problems with liver transplantation including lack of donors, use of immunosuppressive drugs, and high costs of this process, new therapeutic approaches alongside current treatments are needed. the placenta is a tissue that is normally discarded after childbirth. on the other hand, human placenta is a rich source of mesenchymal stem cells (mscs), which is easily available, without moral problems, and its derived cells are less affected by age and environmental factors. therefore, placenta-derived mesenchymal stem cells (pd-mscs) can be considered as an allogeneic source for liver disease. considering the studies on mscs and their effects on various diseases, it can be stated that mscs are among the most important agents to be used for novel future therapies of liver diseases. in this paper, we will investigate the effects of mesenchymal stem cells through migration and immigration to the site of injury, cell-to-cell contact, immunomodulatory effects, and secretory factors in alf. liver is one of the largest vital organs in human body that controls various biological processes, including the production of multiple hormones, storage of glycogen, neutralization of toxins and drugs, control of metabolism, metabolism of urea, and synthesis of plasma protein. typically, most physiological features of liver function are controlled by liver cells or hepatocytes; therefore, the loss of hepatocytes is the main cause of liver failure. several diseases related to malfunction of the liver are caused by damage to or loss of hepatocytes, including viral hepatitis, fatty liver disease, drug and toxin-induced liver injury, hepatocellular carcinoma, and hepatic abnormalities associated with autoimmunity and cirrhosis [1] . (aspartate aminotransferase), alt(alanine transaminase), tbil (total bilirubin indirect level), alb (albumin)). liver failure is divided into three forms as follows. alf within 48 h to several days with jaundice, coagulopathy and encephalopathy; acute-on-chronic liver failure (aclf) with a background of chronic liver disease leading to rapid progression of liver injury and associated with jaundice and ascites; and clf occurring within months to years [7] . alf is an unpredictable and potentially catastrophic condition often encountered in intensive care units, with more than 2500 cases reported each year in the united states. the progression potential of acute hepatic dysfunction toward multi-organ failure demands rapid diagnosis and management of the disease. due to a set of hepatic and non-hepatic complications, alf indirectly leads to immediate follow-up for liver transplantation [8] . alf, formerly known as fulminant hepatic failure, means the development of hepatocellular disorders such as coagulopathy and encephalopathy with inr ≥ 1.5 in patients without a history of liver disease within 26 weeks. more than half of the cases of alf progression require liver transplantation and significant improvements have been reported in the last decade after liver transplantation. alf mortality is usually due to intracranial hypertension (ich) and infection [9] [10] [11] ). however, patients with varying degrees of hemodynamic disorders and renal failure have also been reported [12, 13] . clinically, the patients show coagulopathy, jaundice and hepatic encephalopathy. the period between the onset of the first clinical symptoms and hepatic encephalopathy is crucial in determining the prognosis of these patients [14, 15] . there are obvious differences in the development mechanisms of early alf. the three main factors determining the prognosis of this disease include metabolic problems leading to the loss of liver cells, secretion of toxins and mediators from the liver tissue, and capacity of the remaining hepatocytes to repair the liver [15, 16] . common treatments are therapies that are often meant to improve the complications of acute liver failure (alf). multiple organ failure (mof) and severe infection are the most prevalent factors of mortality in these patients. therefore, management of treatment for alf patients should focus on the handling and prevention of infection [17] . alf patients with severe hepatic encephalopathy, those with renal failure and patients who have any of sirs criteria use broad-spectrum antibiotics [18] . application of vasoconstrictors and dialysis reduce the incidence of cerebral edema [19] . in case of hepatic encephalopathy, the patient is transferred to icu and ventilator devices are used to regulate the level of blood gases patients with alf have qualitative and quantitative coagulation abnormalities. in control of bleeding and during invasive procedures, there is indication for ffp and platelet administration [20] . to prevent gastrointestinal bleeding in alf, patients admitted to icu are treated with h2 blockers or proton pump inhibitors (ppi) [21] . patients with alf are at risk of hypovolemia for a number of reasons, including poor oral fluid intake, vomiting, and vasodilation, in which case bolus fluids are used and level of fluids is frequently maintained if necessary to keep serum sodium levels and prevent fluid overload [22] . in addition to the mentioned treatments, 10-20% glucose is administered when glycemic target is 140 mg/dl and na level is 135-145 mmol/l, as well as n-acetylcysteine and stress ulcer prophylaxis agents [17] . a wide variety of factors cause alf (table 1) [23] [24] [25] . the most common causes of this disease are viral infections and drug-induced liver inflammation. in asia and parts of europe, mainly viral hepatitis agents are involved and acetaminophen is the predominant factor in countries such as usa and australia [26] . impaired function of both humoral and innate immunity is implicated in the pathophysiology of alf [27] . the mechanism of alf begins with necrosis of hepatocytes [28] . oxidative stress is triggered when liver injury is caused by factors such as viral infections, alcohol consumption, drug intoxication, autoimmune diseases, herbal remedies and many other factors [29] [30] [31] . oxidative stress results in the production of reactive oxygen species, which in turn activates the janus kinase (jnk) signaling pathway [32] and generates damageassociated molecular patterns (damps), followed by liver inflammation. liver inflammation is a major factor in immunopathology of several hepatic diseases [33, 34] . damps activate hepatic macrophages (kupffer cells (kcs)) and induce the formation of inflammasome [32, 35] that eventually leads to the secretion of il-1, il-18, and caspase 1 [32] . damps are detected by kupffer cells [33, 36] that express a large number of damp receptors, including tlr4, tlr9, and rage [36] . kcs are activated in this process and release inflammatory cytokines such as tnfα, oxygen radicals, and chemokines such as ccl2 under the effect of inflammatory signals. the presence of inflammatory factors mobilizes inflammatory cells such as neutrophils and monocytes and thereby increases inflammation [33, 34] . hepatic encephalopathy (he) is a function of neurotoxins that reach the brain through the bloodstream [37] . various factors such as blood ammonia levels, infection, necrotic liver, toxins, and systemic inflammatory response syndrome (sirs) can lead to he [38] . in normal conditions, the ammonia produced in the body is efficiently excreted by the liver through the urea cycle and glutamine synthesis and thus a small amount of ammonia remains in hepatic vessels. in alf, ammonia levels rise in the hepatic vein, and the liver loses the ability to release ammonia from the hepatic veins. the muscles and brain begin ammonia detoxification through glutamine synthesis. therefore, both of these tissues are considered as an ammonia scavenging and glutamine releasing organs [39] . tissue damage is the first factor triggering sirs reaction. as explained above, the injury leads to the release of inflammatory mediators such as damps, tnfα, il-6, and il-18. inflammatory cells such as lymphocytes and monocytes reach the damage site and enhance the inflammatory response. coagulation factors as well as primary and secondary homeostasis also become involved and result in sirs reaction [38] . these reactions are associated with the development of he [40, 41] , bacteremia [42] and, in some cases, infection [41, 43] . compensatory anti-inflammatory response syndrome (cars) occurs in reaction to sirs, leading to the secretion of anti-inflammatory factors (including il-10 and spli) from hepatic macrophages during the early stages. this reaction is meant to alleviate the inflammatory status [44, 45] . both of these reactions eventually lead to dysregulation of the immune system and defective immune responses to microbial agents [46, 47] . mscs are fusiform non-hematopoietic cells capable of adhering to plastic surfaces, which can be isolated from various tissues, including placenta, umbilical cord, bone marrow, adipose, and other tissues [48] . despite their morphological and phenotypical similarities, mscs have different regeneration potentials [49] , which is due to the microenvironment and cellular niches affecting their fate [50] . the number of stem cells in many adult tissues is small and isolation of them is associated with several risks; for example, the cells exhibit a limited capacity for differentiation and proliferation after removal from the body, making it difficult to produce large numbers of stem cells [51] . in comparison to adipose tissue and bm, in which mscs are affected by donor's age, placenta is a rich source of stem cells [52] and high differentiation capacity and pluripotentiality of placental cells are related to their origin [53] . pd-mscs have a higher proliferative potential than bm-mscs [54] which reduces the number of passages to reach a large number of cells as well as the risk of cell aging [55, 56] . among mcss, pd-mscs have a higher potential for in vitro proliferation and differentiation of hepatocytes [57] . human bm-msc cells are involved in neovasculogenesis and synergize with endothelial colony forming cells (ecfcs) to create microvessels in vivo [58, 59] . bm-msc cells serve as the gold standard for bone and cartilage repair [60] . adipose tissue-derived mesenchymal stem cells (ad-mscs) are isolated from adipose tissue by liposuction, are capable of differentiation to hepatocytelike cells in the presence of hgf, fgf-1, and fgf-4 factors and participate in the regeneration of hepatocytes and vasculogenesis [61] . wharton's jelly mesenchymal stem cells (wj-mscs) exhibit stemness and pluripotential properties and have been shown to generate various types of neurons and connective tissue cells [62, 63] . umbilical cord-derived mesenchymal stem cells (uc-mscs) have been recognized as low-immunogenicity cells because of their immunomodulatory properties. uc-mscs are involved in neovascularization and differentiation into hepatocyte-like cells [64, 65] . umbilical cord blood has always been considered as a source of hematopoietic stem cells (hscs) [66] . the phenotypic characteristics of uc-mscs are consistent with bm-msc cells [67] . dental tissue-derived mesenchymal stem cells (dp-msc) have limited differentiation capacity relative to bm-mscs [68] . dental pulp stem cells (dpscs) are dental stem and progenitor cells that are capable of selfrenewal and differentiation, which differentiate into neurons and adipocytes in addition to odontogenic cells [69, 70] . the definition of mscs according to international society for cell therapy (isct) is as follows: mscs are (1) able to bind plastic surfaces, (2) able to differentiate into all three classes of chondrocytes, adipocytes and osteocytes in vitro, and (3) capable of expressing cd73, cd90, and cd105 markers but not hematopoietic markers like cd45, cd14, cd19, cd34, and hla-dr [71] . mscs release numerous factors such as vascular endothelial growth factor (vegf), insulin-like growth factor 1 (igf-1), basic fibroblast growth factor (bfgf), nerve growth factor (ngf), transforming growth factor beta-1 (tgf-b1), placental growth factor (pgf), stromal cell-derived factor 1 (sdf-1/cxcl12), monocyte chemoattractant protein-1 (mcp 1/ccl2), hepatocyte growth factor (hgf), interleukin-6 (il-6), il-8, il-10, il-13, g-csf and gm-csf [72] [73] [74] [75] . there are various tissue-specific factors in mscs depending on the tissues from which mscs are isolated. for example, factors such as hgf, bfgf, and il-6 are mainly secreted by mscs isolated from placental tissue or interferon-γ (ifn-γ), tumor necrosis factor α (tnfα), interleukin-1 alpha (il-1α), and interleukin-1 beta (il-1β) secreted by mscs from menstrual blood-derived stem cells (menscs) [76, 77] . hence, it can be said that the selection of mesenchymal stem cells extracted from tissues is an important consideration in the treatment of diseases with respect to the secretory factors they produce. embryonic stem cells are isolated from embryonic tissues, especially multiple extraembryonic tissues. tissues such as amniotic fluid, wharton's jelly, amnion, chorion, embryonic membrane and placenta have mscs. the placenta is one of the largest organs with an essential role in the development of the fetus, which plays a role in the secretion of nutrients for the fetus and immune protection (tolerance) of it. it has recently been observed that pd-msc are a new alternative source of mscs for regenerative therapies [78] . studies have shown that pd-mscs possess self-renewal capacity, have multilineage differentiation, lack ethical problems, are accessible, abundant, and show strong immunosuppressive effects [79] [80] [81] ). in addition, placental tissue derived from the fetus is voluminous and can be easily manipulated to increase the number of mscs, which exceeds the number of mscs present in bone marrow and adipose tissue [81, 82] . another advantage of these placental stem cells is that we do not require an invasive method to isolate them, whereas invasive methods are needed to isolate adult mscs [78] . typically, pd-mscs can maintain a high proliferative capacity in culture medium for at least 20 passages [83] . some studies have recently suggested the differentiation of pd-mscs into hepatocyte-like endodermal cells [57, 84] . investigations have shown that many perinatal resources of mscs such as amniotic membrane (am), chorionic plate (cp), parietal decidua [85] , and umbilical cord (uc) have advantages relative to adult sources, including bone marrow (bm) [86] [87] [88] . the mscs isolated from these tissues have their own characteristics as follows. vcam1 is a biomarker of chorionic plate with unique immunosuppressive activity that plays an important role in immune responses [86] . cp-derived mesenchymal cells copiously secrete hgf and vcam1. parietal decidua derived mesenchymal stem cells (dmscs) [85] show a high secretion of ang1 and vegf but the lowest secretion of tgfβ1. umbilical cord (uc) derived mscs have a high secretion level of igf1 and amniotic membrane (am) derived mscs highly release peg2 and tgfβ1 [89] . considering the above statements, we show in this research that amniotic membrane-derived mesenchymal stem cells may be effective in treatment of premature ovarian aging due to overexpression of peg2 and tgfβ1, cp-derived mscs could be used for angiogenic therapy because of pro-angiogenic activity, and parietal decidua derived mscs [85] might be useful for the treatment of vital organ ischemia, and uc-mscs may be used for other therapies because of secreting a large number of factors [90] . most animal and human studies on mscs have indicated therapeutic effects of these cells. however, there is evidence for low engraftment of mscs due to shortterm viability after injection [77, 91] . mscs are trapped in the lung after injection and a lower number of these cells may reach their destination [92] . therefore, the reduction of cell loss during migration is an advantage of topical over intravenous injection [93] . several studies have indicated that a single injection of mscs is safe for the patient and does not stimulate the immune system, but re-injection of mscs may lead to the generation of alloantibodies [94] . in addition, the fbs that is used to grow mscs could induce an immune response in the patient [95] . in general, mscs show a dual behavior when faced with tumors and pd-mscs are no exception in this regard. for example, some in vitro studies have indicated that uc-mscs increase the expression of proliferating cell nuclear antigen (pcna) [96] , induce the proliferation promoting genes like epgn/mzt2a, downregulate transcription factors associated with the suppression of tumor development such as tal1/fos/egr1/klf10, which stimulates different tumor populations [97] . pursuant to this dual role of pd-mscs, one study introduces the antitumor role of these cells in a particular type of tumor but suggests a promoter role in another type. wj-mscs have an antitumor role in the face of squamous cell carcinoma in vitro, but stimulate the growth of cancer in vivo [98] . clinicians have observed that a number of patients with alf may recover spontaneously and that the clinical outcome of these patients largely depends on the balance between loss and repair of hepatocytes [99] . the damaged hepatocytes are rapidly replaced by normal hepatocytes in moderate disease, but in case of severe injury and widespread death of hepatocytes, the repair capacity of remaining hepatocytes may not be complete and lead to the deployment of liver progenitor cells (lpc) that act as hepatocytes [100] . in most alf patients, these progenitor cells are insufficient to repair and replace hepatocytes, eventually leading to the adoption of limited therapeutic approaches by physicians [101] . today, liver transplantation is the only way to treat liver failure patients. however, liver transplantation has failed for a number of reasons such as lack of proper organs, high costs, and the administration of immunosuppressive agents for long periods of time. other treatment strategies include bioartificial liver with less hepatocytes and drug therapy [102] . hepatic failure is a disastrous consequence of liver loss, in which the repair of residual hepatocytes is not performed in a timely and appropriate manner, resulting in increased mortality [103] . massive hepatic necrosis in acute liver failure [97] is caused by sudden loss of hepatocytes due to a variety of acute injuries induced by hepatotoxic drugs, immune system attack, and viral infections [104] [105] [106] . while most hepatocytes are completely destroyed in alf, the circulating bone marrow-derived cells and endogenous hepatocyte progenitor cells can rapidly regenerate the liver [107] . cell-based therapies have been promising in regenerative medicine. mscs can be important sources of alternative therapy because of various properties such as self-renewal, proliferation and differentiation [108] . the precise mechanism of mscs in alf is not completely understood [109] . according to several studies, it can be stated that placenta-derived mesenchymal stem cells (pd-msc) are able to affect the liver damages in several ways: 1. pd-mscs are recruited to the damaged area by vcam-1 and vla-4 adhesion molecules [104, 110, 111] affecting the remaining hepatocytes through cell-cell contact and secretion of tgf-α, egf, hgf, and vegf tropic factors [112, 113] . increase treg cells, modulating the immune system as well as suppressing activated t-cells, nk cells, b-cells and il-10 production [113, 114] . 3. pd-mscs decrease the inflammation of hepatocytes and prevent their apoptosis by suppressing tnfα and ifnγ, which leads to the regeneration of hepatocytes by releasing hgf, il-6, paf and vegf [115, 116] . 4. mscs are capable of secreting various angiogenic factors, including vegf, sdf-1α, and mmp1, which promote angiogenesis [117] [118] [119] . 5. in addition to their immunomodulatory properties, mcss differentiate into vascular cells and pericytes in vivo [117] . they also have the potential to differentiate into hepatocyte-like cells both in vivo and in vitro, leading to improvement of liver damage ( fig. 1) [115, 120] . a majority of studies have used the intravenous route to inject mscs, after which most mscs are trapped in lungs in the early stages [121, 122] . after 24 h, mscs move toward other organs (especially the liver and spleen) and settle in them [123] . they also migrate to damaged tissues [123] . for instance, in a study on patients with cirrhosis, mscs labeled with 111 in-oxine were detected in the liver after 48 h (through radioactivity assay) where they remained for 10 days [124] . elimination of mscs may be related to the immune system, which does not rule out the functional effect of these cells. one study has reported that phagocytosis of dead mscs induces the production of regulatory macrophages modulating the immune response by producing il-10 factors [125, 126] . moreover, a small fraction of these cells that have been spared elimination could be responsible for the therapeutic effects [126] . mscs play a critical role in liver regeneration because of their ability to produce and regulate platelet-activating factor (paf), hepatocyte growth factor (hgf) and vascular endothelial growth factor (vegf) [104] . several studies have demonstrated the significance of mscs in liver diseases. mscs have been used in various investigations on alf in both animal models [127, 128] and clinical trials [129, 130] . nevertheless, the precise mechanism of the function of these cells remains unclear. since mscs are able to move to the site of injury and inflammation [131] as well as being capable of proliferating and differentiating into hepatocytes [132, 133] , they play an essential role in regenerative therapies. mscs show immunomodulatory feature because they do not express stimulatory molecules or hla ii [134] and are therefore a good source for allogeneic and autologous transplantation. several studies have shown that mscs secrete tropic factors and can be effective in reducing inflammation, fibrosis and apoptosis of liver cells as well as repairing damaged tissue by stimulating angiogenesis [74] . high migration ability is a major advantage of pd-mscs. migration involves the movement of mscs toward damaged and inflamed sites through interactions between mscs with cytokines and adhesion molecules secreted from the injured tissue environment [135] . migration of mscs has been investigated in both animal [136] and in human studies [137] . for example, various researches have revealed that mscs express adhesion molecules and integrins such as vcam-1 and vla-4, which are composed of cd29 and cd49d components. compared with bm-derived mesenchymal stem cells (bm-mscs), placental mscs express a higher level of vla-4 and animal studies have indicated mscs binding to endothelial cell surface markers such as p-selectin and vcam-1, which is indicative of the high implantation capacity of pd-mscs into damaged tissue [111, 138] . a clinical trial of cirrhotic patients showed that 111 in-oxinelabeled mscs were trapped in the lungs in the early hours after injection through peripheral blood and that they left there after 48 h and migrated to the liver and spleen, remaining in these tissues for 10 days [137] . there are various mechanisms in the creation of an immunologically safe environment by placenta for the fetus [139] . this feature is a strong advantage for pd-msc cell therapy in allogeneic transplantation, which prevents graft rejection, stabilizes the transplant and drives mscs, including bm-mscs and amniotic fluidderived mscs (af-mscs), toward the site of injury [140] . embryonic-derived mscs are also capable of migrating to the placenta and blood brain barrier (bbb) [141] . it can be argued that the beneficial effects of mscs in liver diseases (including alf) are not limited to hepatocyte repair, but rather the tropical factors released by fig. 1 mesenchymal stem cells and its effects on acute liver failure them modulate the deleterious effects of the immune response [142] . the immunosuppressive effects of mscs on the secretion of tnfα and ifn γ prevent from apoptosis of hepatocyte cells and reduce hepatic inflammation, and the suppression of these cytokines appears to be systemic [143] . mscs in mice with alf suppress activated t-cells, decreasing the inflammatory cytokines tnfα, γ ifn and il-4 and exerting their immunosuppressive effects by increasing il-10 levels [143, 144] . cells such as natural killer t (nkt) are of high importance in the pathogenesis of alf and are immunomodulatory targets mediated by mscs along with dendritic cells (dcs), macrophages and t-cells [143, 145] . hgf is one of the most important factors in the repair of hepatic tissue, which is secreted by mscs. hepatocyte growth factor is an effective mitogen for hepatic tissue repair that is dependent on c-met receptor during tissue damage [146] . the hgf/c-met signaling pathway is essential for liver repair and implantation of mscs in the affected area [147] . many studies have reported the protective effects of hgf/c-met signaling pathway on liver injury [148, 149] . hgf as well as other factors like tnfα and egf is considered a mitogenic factor associated with hepatocyte proliferation [112, 150] . on the other hand, hgf together with ngf factor secreted by mscs induces apoptosis of hepatocyte stellate cells (hsc), indicating the antifibrotic property of these cells [151] [152] [153] . many studies have shown that angiogenesis plays a crucial role in hepatic repair so that the injection of antiangiogenic factors such as anti-vegf inhibits hepatic repair [154, 155] but factors such as bfgf enhance it [156] . vegf boosts angiogenesis and contributes to the healing process [157] . angiogenesis is essential for wound healing, regeneration and organogenesis [158] . il-6 binds to gp80 and gp130 receptors, which activate the jak pathway and in turn phosphorylate tyrosines in the intracellular domain of gp130, subsequently activating the mapk pathway and stat 1 and 3 transcription factors that lead to hepatocyte proliferation [159] [160] [161] . recent experiments on animal models have shown that il-6 and tnf-2 are involved in regeneration of liver mass [162] . limited information is available on the repair mechanism of mscs in various diseases; therefore, further in vivo studies provide a broad perspective for mscs use in clinical practice. choosing the right cell, determining the proper dose, selecting the appropriate injection site and timely injection can help improve the function and implantation of mscs in the target tissue, and they can be highly important and applicable for further research in the future. in this review paper, we concluded that pd-mscs can be considered as a good allogeneic source for alf in future because of their safety, easy accessibility, lack of immune system stimulation, secretion of appropriate factors for liver tissue and healing properties. mscs: mesenchymal stem cells; pd-mscs: placenta-derived mesenchymal stem cells; alf: acute liver failure; aclf: acute-on-chronic liver failure; clf: choronic liver failure; ich: intracranial hypertension; damps: damage-associated molecular patterns; sirs: systemic inflammatory response syndrome; cars: compensatory anti-inflammatory response syndrome; isct: international society for cell therapy; vegf: vascular endothelial growth factor; igf-1: insulin-like growth factor 1; bfgf: basic fibroblast growth factor; ngf: nerve growth factor; tgf-b1: transforming growth beta-1; ifn-γ: interferon-γ; tnf-α: tumor necrosis factor α; il-1α: interleukin-1 alpha; il-1β: interleukin-1 beta; pgf: placental growth factor; sdf-1/cxcl12: stromal cell-derived factor 1; mcp 1/ccl2: monocyte chemoattractant protein-1; hgf: hepatocyte growth factor; g-csf: granulocyte-colony stimulating factor; gm-csf: granulocytemacrophage colony stimulating factor; am: amniotic membrane; cp: chorionic plate; uc: umbilical cord; lpc: liver progenitor cells; paf: platelet-activating factor; vla-4: very late antigen 4; bbb: blood brain barrier; hsc: hepatocyte stellate cells; jak: janus kinase. prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti-human monoclonal antibodies introduction to the revised american association for the study of liver diseases position paper on acute liver failure liver transplantation : official publication of the american association for the study of liver diseases and the 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stellate cells proliferation by mesenchymal stem cells and the possible mechanisms nerve growth factormediated paracrine regulation of hepatic stellate cells by multipotent mesenchymal stromal cells expression and role of vascular endothelial growth factor in liver regeneration after partial hepatectomy in rats liver regeneration is an angiogenesis-associated phenomenon endothelial-directed hepatic regeneration after partial hepatectomy isolation and characterization of bipotent liver progenitor cells from adult mouse angiogenesis is crucial for liver regeneration after partial hepatectomy liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase cascade activation is a key signalling pathway involved in the regulation of g(1) phase progression in proliferating hepatocytes interleukin-6-type cytokine signalling through the gp130/jak/stat pathway acutephase response factor, increased binding, and target gene transcription during liver regeneration publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. key: cord-342808-yonbowkb authors: francque, sven title: innovative liver research continues during the current pandemic date: 2020-05-24 journal: jhep rep doi: 10.1016/j.jhepr.2020.100121 sha: doc_id: 342808 cord_uid: yonbowkb nan whilst the covid-19 pandemic runs its devastating course, heavily impacting on the health and quality of life (qol) of many, the scientific community continues to work hard to reconcile the need for new knowledge to fight this unprecedented threat to individual and global health, the high clinical needs, as well as the continuous need to invest in research for the non-covid-19related diseases that continue to impact on our populations. the editorial team of jhep reports is therefore pleased to present the next issue of the journal on schedule. first, we tackle the covid-19 issue by providing a key position paper that comprehensively summarises our current knowledge on how covid-19 impacts on the liver, how preexisting liver disease might impact on covid-19 infections and how to continue to ensure care for patients with acute and chronic liver disease in the current context. 1 this document is a joint effort of the european association for the study of the liver and the european society of clinical microbiology and infectious diseases and gives guidance regarding the burning issues. patients with pre-existing medical conditions are regarded as populations at risk of a severe disease course, but it is currently unclear to what extent chronic liver diseases should be considered as risk factors. patients with advanced liver disease as well as liver transplant patients likely represent vulnerable patient cohorts with an increased risk of infection and/or a severe course of covid-19. furthermore, an unusual allocation of healthcare resources is currently required in virtually all countries around the world. this may negatively impact the care of patients with chronic liver disease who continue to require medical attention. this position paper offers guidance on how to balance the need to avoid nosocomial dissemination of the virus to patients and healthcare providers, whilst at the same time maintaining a high standard of care for patients who require immediate and continued medical attention. the question of whether nucleos(t)ide analogues (nucs) can be safely discontinued in certain patients with chronic hepatitis b remains a matter of debate. one of the factors that could potentially allow for the safe withdrawal of nucs is the depletion of cccdna that might occur with long-term treatment. in the current issue of jhep reports, lai et al. report that viral rebound occurred in most cases following nuc cessation in a series of 19 patients with undetectable intrahepatic cccdna (covalently closed circular dna). 2 to what extent the determination of cccdna on liver biopsy can reliably reflect liver cccdna depletion is obviously questionable, but this study adds to the growing knowledge on how to handle patients following successful nuc treatment. van hees et al. 3 also reported a >50% rebound rate after treatment cessation in caucasian patients with hbeag seroconversion, with potentially fatal outcomes. the study of lai et al. adds a note of caution regarding nuc cessation, but unfortunately it does not fill the unmet need of identifying reliable predictors for the safe discontinuation of nucs. whilst hepatitis c has been successfully tackled in the last decade, hepatitis b and hepatitis b-d coinfection continue to challenge the hepatology community, as many knowledge gaps persist and viral eradication is rarely achieved in hepatitis b, whilst the treatment of hepatitis d is even more challenging. the complex interaction between the virus and host immune system is incompletely understood, which hampers the development of efficacious treatments. 4 the development of animal models that reliably mimic the infection in patients is particularly challenging. usai et al. present their results on a large and comprehensive study on the immunological mechanisms driving disease progression in hepatitis b-d coinfection. 5 in an adeno-associated vector-mediated mouse model of hepatitis b-d the authors surprisingly found a limited role of different immune cell populations (e.g. t cells or natural killer cells) in the induction of liver damage. by contrast, interferon-and tumour necrosis factor alpha (tnf-a)-mediated pathways appeared to be of crucial importance. inhibition of tnf-a by etanercept ameliorated hepatitis-d-induced liver damage. although the conclusion that pharmacological inhibition of tnf-a represents an attractive strategy to control hepatitis-d-induced acute liver damage is premature, the findings are innovative and substantially contribute to our understanding of the immunology of hepatitis-d-related disease, which should allow further exploration of the manipulation of these pathways in the quest for an effective treatment. although distinct diseases, alcoholic and non-alcoholic fatty liver disease (ald and nafld) share multiple features. not only the histological picture, but also several pathophysiological mechanisms overlap, e.g. the impact of alcohol on metabolic pathways including sterol regulatory element-binding protein 1c or peroxisome proliferator-activated receptor-a that are key metabolic pathways in the pathophysiology of nafld, 6 or the role of genetic factors. furthermore, in many patients both the drivers of nafld (such as overweight and diabetes) and the use of alcohol are concomitantly present. this can impact on the management of ald and nafld, elegantly summarised in the review by louvet et al. 7 one of the aspects that is poorly touched upon in the (medical) management of patients, is their perceived qol. several tools have been developed over time to capture the impact of the disease on this important aspect of disease burden, which is particularly challenging in the context of nafld, as the aforementioned comorbidities intrinsically affect qol. sweeney et al. extensively reviewed the literature on qol impairment in the specific population of patients with nafld-related compensated cirrhosis, concluding that those patients suffer significantly from lower qol and poorer physical health. 8 they also reviewed the available patient-reported outcome measures (proms) that are increasingly important in the design of clinical trials and patient management policies. they highlight the current limitations of the proms that have been used so far and conclude that there is a need for a standardised and structured approach to these measures in clinical trials of patients with nafld-related cirrhosis, as well as in daily clinical practice. rare liver diseases ask for collaborative efforts to provide high quality data that can solve clinical research questions. the european union has actively promoted this by recognising european reference networks (ern) for rare diseases, including rare liver for rare liver diseases. a group of centres, some from this ern, report on their pooled series of patients with autoimmune hepatitis with normal igg levels, which corresponds to 10% of their patient population. 9 although these studies inevitably struggle with their retrospective nature and with selection bias (reinforced in this study by the fact that igg levels are part of the diagnostic criteria for autoimmune hepatitis), the findings of hartl et al. are insightful and substantially contribute to our understanding of this clinical entity. it appears that these patients differ little in their characteristics at diagnosis compared to patients with a typical elevation of igg, but have a higher, albeit with 24% still rather low, chance of remaining in stable remission after cessation of all treatment. the issue of whether to stop any immunosuppressive treatment and the selection of patients who might qualify for this, is still ill-defined and hence the study by hartl et al. is an important contribution to help settle this clinical challenge. wilson's disease is an even rarer condition, and acute liver failure in this disease is usually confined to childhood or young adulthood. in this issue of jhep reports, shribman et al. not only report on a very rare case of acute liver failure as a primary manifestation of wilson's disease in a patient over 60 years of age, but also report the experience of the transplant community in the united kingdom with liver transplantation in this specific setting over the past 2 decades. 10 only 8 such cases of acute liver failure as the first presentation of wilson's disease in patients older than 40 years, or 1 in 1,250 listed for transplantation, could be identified. although rare, this implies that this cause of acute liver failure should be part of the differential diagnosis of acute liver failure at any age. budd-chiari syndrome is another rare disease with multiple challenges in clinical management. one of these is the frequent occurrence of benign regenerative lesions that need to be discriminated from malignant lesions, especially in case of mild elevations of alpha-foetoprotein that frequently accompany these benign lesions. as the contrast dynamics in budd-chiari syndrome are altered compared to a classical cirrhotic condition (especially impacting on the venous wash-out that is an important criterion in imaging-based diagnosis of hepatocellular carcinoma), and as the regenerative lesions in budd-chiari syndrome have features of focal nodular hyperplasia and hence show hyperenhancement in the arterial phase of contrast-enhanced imaging, the classical imaging criteria do not always suffice to reliably rule out malignancy in focal liver lesions of patients with budd-chiari syndrome. in a retrospective analysis of 26 patients with suspected liver lesions (a large single centre series for this condition), van wettere et al. report on the accuracy of mri with hepatobiliary excretion contrast agent in differentiating hepatocellular carcinoma lesions, which were all homogenously hypointense in the hepatobiliary excretion phase, from the benign lesions, which were almost all hyperintense, at least in the periphery of the lesion, or also homogenously. 11 diagnostic accuracy was very high, especially in combination with an alphafoetoprotein level >15 ng/ml. this important observation is of great relevance in the management of budd-chiari syndrome. from the same group, the current issue of jhep reports offers a comprehensive state-of-the-art overview of the role of imaging in the diagnosis of liver tumours, focusing mainly but not exclusively, on hepatocellular carcinoma. besides a detailed and critical appraisal of what has become standard of care, gregory et al. shed light on the future role of imaging in the clinical management of patients with a large variety of liver tumours. 12 of particular interest is the assessment of tumour response to various therapies. this goes beyond the assessment of viable tumour tissue, for some years the cornerstone of treatment response assessment in hepatocellular carcinoma. the response to immunotherapy is particularly challenging due to specific radiological images that appear in line with dynamics of treatment-induced alterations. 3d volumetry and radiomics offer new perspectives, the latter working on voxel level, mathematically analysing and manipulating their binary signatures in complex models, allowing to go beyond size or human-eye based semantic descriptors. also, quantitative and functional imaging provide insight into microscopic tumour changes that may be used as early predictors of response. this elegant review will guide clinicians through the fascinating innovations that imaging is offering in diagnosing these various liver tumours and in assessing treatment responses (to therapies with different modes of action, which require different corresponding imaging modalities). we hope you enjoy the issue! care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper rebound of hbv dna after cessation of nucleos/tide analogues in chronic hepatitis b patients with undetectable covalently closed circular dna stopping nucleos(t)ide analogue treatment in caucasian hepatitis b patients after hbeag seroconversion is associated with high relapse rates and fatal outcomes treating chronic hepatitis delta: the need for surrogate markers of treatment efficacy tnf-alpha inhibition ameliorates hdv-induced liver damage in a mouse model of acute severe infection ppara gene expression correlates with severity and histological treatment response in patients with non-alcoholic steatohepatitis combined alcoholic and non-alcoholic steatohepatitis health-related quality of life and patient-reported outcome measures in nash-related cirrhosis features and outcome of aih patients without elevation of igg liver transplantation for late-onset presentations of acute liver failure in wilson's disease: the uk experience over 2 decades hepatobiliary mr contrast agents are useful to diagnose hepatocellular carcinoma in patients with budd-chiari syndrome evaluation of liver tumour response by imaging key: cord-340576-dabcs3w5 authors: nishikawa, hiroki; enomoto, hirayuki; nishiguchi, shuhei; iijima, hiroko title: liver cirrhosis and sarcopenia from the viewpoint of dysbiosis date: 2020-07-24 journal: int j mol sci doi: 10.3390/ijms21155254 sha: doc_id: 340576 cord_uid: dabcs3w5 sarcopenia in patients with liver cirrhosis (lc) has been attracting much attention these days because of the close linkage to adverse outcomes. lc can be related to secondary sarcopenia due to protein metabolic disorders and energy metabolic disorders. lc is associated with profound alterations in gut microbiota and injuries at the different levels of defensive mechanisms of the intestinal barrier. dysbiosis refers to a state in which the diversity of gut microbiota is decreased by decreasing the bacterial species and the number of bacteria that compose the gut microbiota. the severe disturbance of intestinal barrier in lc can result in dysbiosis, several bacterial infections, lc-related complications, and sarcopenia. here in this review, we will summarize the current knowledge of the relationship between sarcopenia and dysbiosis in patients with lc. in the gastrointestinal mucosa, various immune cells including macrophages, dendritic cells, etc., are constantly present [1, 2] . paneth cells, which are a type of intestinal epithelial cell, secrete antimicrobial peptides and are responsible for intestinal innate immunity by eliminating pathogens and by symbiosis with resident bacteria [3] . however, the barrier mechanism formed in the mucus layer may be incomplete, and indigestible proteins, bacteria, viruses, etc., along with nutrient components, can always enter the tissue across the mucus barrier [1, 2] . in that case, a secondary barrier consisting of macrophages, t cells, and b cells present in the lamina propria will respond to their invasion (the biological barrier, table 1 and figure 1 ). the foreign substances passing through the lamina propria can enter the bloodstream and reach the liver via the portal vein. a large number of kupffer cells (liver macrophages) are present in the sinusoidal blood vessels of the liver (the final barrier) to create a unique immune system [4] . in this way, the gastrointestinal tract and liver cooperate to participate in biological defense (gut-liver axis) [4, 5] . on the other hand, it has been revealed that various intestinal bacteria inhabit the colon, and they play an important role in maintaining homeostasis of the human body (the environmental barrier, table 1 and figure 1 ) [6] . the majority of bacteria in the colon are tightly attached to the outer side of the mucus layer, and the inner side of the mucus layer forms a barrier which limits bacterial contact with the epithelium (the physical barrier, table 1 and figure 1 ) [7] . dysbiosis refers to a state in which the diversity of gut microbiota (gm) is decreased by decreasing the bacterial species and the number of bacteria that compose the gm [8] [9] [10] [11] . analysis of gm at the gene level using a next-generation sequencer has come to the fore, and thus gm in patients with various table 1 . three types of intestinal barrier. gut microbiota antimicrobial peptide immune cells mucus layer tight junction in individuals with chronic liver diseases (clds), metabolic or nutritional dysfunctions including protein-energy malnutrition (pem) or muscle abnormalities are frequently found, which can be related to disabilities, poor quality of life, or mortality [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . liver cirrhosis (lc) involves a hypermetabolic state with increasing demand for calories and protein [23] . in addition, the energy metabolism of lc patients is in a hypercatabolic state, and when fasting early in the morning, they are in the same degree of starvation as when a healthy person fasts for 2-3 days [31, 32] . when liver function worsens, the detoxification of harmful substances such as ammonia can be reduced [32] . branched-chain amino acids (bcaas) are often excessively consumed in skeletal muscles to detoxify harmful substances in patients with decreased liver function [20, 32] . in lc patients, it is difficult to adequately supplement bcaas with diet intake alone [32] . in lc patients, sarcopenia, which is defined by decline in muscle mass and strength and/or physical activity, can occur because the excessive consumption of bcaas makes it difficult to synthesize the protein required for muscle mass increase [20] . sarcopenia is one of the most common consequences seen in patients with lc [20, 27, [33] [34] [35] [36] [37] [38] [39] . in japan's aging population, cld is also a crucial public health issue because aging is also closely linked to sarcopenia [40] [41] [42] . how sarcopenia is related to adverse consequences requires looking at sarcopenia as a systemic disorder [22, 43, 44] . lc-related complications themselves such as hepatocellular carcinoma (hcc), ascites, spontaneous bacterial peritonitis (sbp), varices, hepatic encephalopathy (he), and acute or chronic liver failure (aclf) can cause sarcopenia [22, 40] . clinical and research interest in sarcopenia in clds has thus been growing internationally. in 2016, the japanese society of hepatology (jsh) created their own criteria for the assessment of sarcopenia in in individuals with chronic liver diseases (clds), metabolic or nutritional dysfunctions including protein-energy malnutrition (pem) or muscle abnormalities are frequently found, which can be related to disabilities, poor quality of life, or mortality [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . liver cirrhosis (lc) involves a hypermetabolic state with increasing demand for calories and protein [23] . in addition, the energy metabolism of lc patients is in a hypercatabolic state, and when fasting early in the morning, they are in the same degree of starvation as when a healthy person fasts for 2-3 days [31, 32] . when liver function worsens, the detoxification of harmful substances such as ammonia can be reduced [32] . branched-chain amino acids (bcaas) are often excessively consumed in skeletal muscles to detoxify harmful substances in patients with decreased liver function [20, 32] . in lc patients, it is difficult to adequately supplement bcaas with diet intake alone [32] . in lc patients, sarcopenia, which is defined by decline in muscle mass and strength and/or physical activity, can occur because the excessive consumption of bcaas makes it difficult to synthesize the protein required for muscle mass increase [20] . sarcopenia is one of the most common consequences seen in patients with lc [20, 27, [33] [34] [35] [36] [37] [38] [39] . in japan's aging population, cld is also a crucial public health issue because aging is also closely linked to sarcopenia [40] [41] [42] . how sarcopenia is related to adverse consequences requires looking at sarcopenia as a systemic disorder [22, 43, 44] . lc-related complications themselves such as hepatocellular carcinoma (hcc), ascites, spontaneous bacterial peritonitis (sbp), varices, hepatic encephalopathy (he), and acute or chronic liver failure (aclf) can cause sarcopenia [22, 40] . clinical and research interest in sarcopenia in clds has thus been growing internationally. in 2016, the japanese society of hepatology (jsh) created their own criteria for the assessment of sarcopenia in clds [40] . in the jsh criteria for sarcopenia, age limitation is excluded because clds can cause secondary sarcopenia due to pem, which can occur regardless of age. in addition, the measurement of walking speed was abolished due to the difficulty of measuring it in daily clinical practice, and it was decided to use only grip strength for the evaluation of muscle strength (cutoff values: <26 kg in males and <18 kg in females). furthermore, since computed tomography (ct) is frequently used in cld patients, a standard value for ct was set for measuring muscle mass, and it was decided to use the bioimpedance analysis method (cutoff values: <7.0 kg/m 2 in males and <5.7 kg/m 2 in females) and/or ct method at the l3 level (cutoff values: <42 cm 2 /m 2 in males and <38 cm 2 /m 2 in females) to evaluate muscle mass [40] . in japan, a lot of debate regarding sarcopenia in clds has taken place based on the jsh criteria. here in this review, we will summarize the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. lc patients with hyperammonemia are often encountered in routine clinical practice. most of the ammonia produced in vivo is derived from the digestive tract. the organs that metabolize ammonia include the liver, skeletal muscles, brain, and kidneys. of these, the only organ with sufficient capacity to detoxify ammonia produced in the body into urea is the liver, which has a urea cycle [45] . lc patients manifest the characteristics of low levels of bcaas due to pem and elevated blood ammonia level due to an impaired urea cycle caused by zinc deficiency, etc. [46] . ammonia suppresses phosphorylation of eukaryotic initiation factor 2α and mammalian target of rapamycin complex1 (mtorc1) signal through general control nonderepressible 2 which is an amino acid deficiency sensor, and directly suppresses protein synthesis in skeletal muscles. bcaa suppresses these reactions and promotes muscle protein synthesis, but l-leucine (one of bcaas) is consumed due to ammonia metabolism in skeletal muscles [47] . when the l-leucine level is decreased, protein synthesis in skeletal muscles becomes unsuccessful [48] . in lc patients, muscle proteolysis is stimulated via the activation of the ubiquitin-proteasome pathway [49, 50] . persistent chronic inflammation in lc can cause the marked elevation of the pro-inflammatory cytokines including tumor necrosis factor-alpha (tnf-α) and il-1, -6, which in turn stimulate muscle autophagy [51] . the inflammation-inducible ubiquitin-proteasome system can be linked to muscle atrophy through activation of muscle atrophy-related genes [52] . myostatin suppresses muscle satellite cell proliferation and differentiation. elevated myostatin levels in skeletal muscles can cause sarcopenia in lc patients [53] . hyperammonemia has been demonstrated to elevate muscle myostatin expression via tlr-independent nuclear factor kappa beta activation in an animal model [54] . as serum and skeletal muscle ammonia levels are often elevated in lc because of portosystemic shunts or impaired ureagenesis, significant increase of myostatin expression in skeletal muscles can be observed [55] . the decrease in serum free testosterone levels, bcaa, and insulin-like growth factor-1 levels also result in elevated myostatin levels in lc [56, 57] . lc patients are often involved in gonadal dysfunction, which can also result in hypermyostatinemia in skeletal muscles [57] . in our previous report, we demonstrated that elevated serum myostatin level can be associated with hyperammonemia (correlation coefficients; r = 0.5856 in males and r = 0.3922 in females), hypoalbuminemia (correlation coefficients; r = −0.3844 in males and r = −0.3945 in females), and poor outcomes [58] . in addition, we found a close inverse correlation between serum myostatin level and psoas muscle mass as assessed by ct at the l3 level in lc patients (median psoas muscle index (high vs. low serum myostatin group); 4.84 cm 2 /m 2 vs. 6.37 cm 2 /m 2 (p < 0.0001) in males and 3.87 cm 2 /m 2 vs. 4.25 cm 2 /m 2 (p = 0.0175) in females) [58] . sarcopenia in lc patients can contribute to increase risk of minimal or overt he. hanai et al. reported that in their 120 lc patients sarcopenia (hazard ratio (hr) = 3.31, 95% confidence interval (ci) = 1.19-9.42; p = 0.02) and serum bcaa levels <327 nmol/ml (hr = 2.98, 95% ci = 1.08-8.34) were found to be independent adverse predictive factors for the incidence of minimal he [59] . a recent meta-analysis (6 studies, comprising 1795 patients) reported that sarcopenia was closely linked to the presence of he (hr = 2.74, 95% ci = 1.87-4.01) [60] . on the other hand, chronic use of proton pump inhibitors (ppis) can alter the gm and can be a risk factor for he in lc patients [61, 62] . skeletal muscle is considered to be a metabolic organ, which consumes a lot of energy and plays an important role in supporting exercise capacity and regulating body fat mass and blood glucose levels. skeletal muscles take up glucose and control blood glucose levels and play an important role in storing glucose as glycogen [63, 64] . skeletal muscle has also been found to have a role as an endocrine organ secreting myokine (physiologically active substance) and regulating organ function throughout the body [65, 66] . skeletal muscle decline can be associated with insulin resistance and disturbance of gm (muscle-gut axis) [66, 67] . microorganisms in the gut exert their functions mainly through enzyme pathways for the purpose of digesting complex dietary carbohydrates and proteins [68, 69] . gm provides the bcaas including valine, leucine, and isoleucine, and particularly glycine, which is necessary for the synthesis of glutathione. glutathione has an auxiliary role in protecting cells from reactive oxygen species such as free radicals and peroxides [68, 69] . intake of a high fat diet can cause dysbiosis, which may be linked to the development of colorectal cancer (crc) [70] . microbial metabolites from the intestines have been demonstrated to act as nutrients or metabolic modulators in skeletal muscles [71] . short-chain fatty acids (scfas) include organic acids such as butyrate (4 carbon atoms), propionate (3 carbon atoms), and acetate (2 carbon atoms), which are produced by the gm. acetate and propionate pass into the bloodstream and are taken up by the peripheral organs and the liver, where they can act as substrates for gluconeogenesis and lipogenesis [69, 72] . butyrate has a role in providing energy for cell metabolism and regulates apoptosis, cell differentiation, and chemical modification of nuclear proteins and nucleic acid through action on numerous cells [72, 73] . butyrate is the most important energy source for intestinal epithelial cells and exerts excellent physiological effects such as an anti-inflammatory action. butyrate has been reported to have a significant effect on skeletal muscles [74, 75] . butyrate can help prevent skeletal muscle mass loss and maintain skeletal muscle mass via anti-inflammatory effects and activation of regulatory pathways, leading to atp increase and suppression of muscle protein catabolism and apoptosis [73, 76] . the relationship between gm and lipid metabolism in clds depends on the degree of liver damage [77] . a negative correlation between hepatic venous pressure gradient (hvpg) and butyrate levels in lc patients can be found [78] . acetate has also been reported to have the following effects: (1) decrease in intestinal ph, (2) suppression of ammonia-producing bacterial growth, and (3) suppression of absorption of intestinal ammonia [79, 80] . these observations may account for the high prevalence of sarcopenia in lc patients. the frequency of sarcopenia in lc patients is reported to be 10-70% [40] . the function of gm and its associated immune regulation mechanism have been elucidated, and it has been revealed that the intestine, which acts as a barrier at the forefront of the living body, affects the whole body by controlling substance permeation from the gastrointestinal tract into the systemic circulation [71] . it has been revealed that the tight junction (tj) between intestinal epithelial cells regulates the immune response to invasion of bacteria and foreign antigens from the intestinal tract in cooperation with intestinal-associated lymphoid tissue and the intestinal neuroendocrine network (the physical barrier, table 1 and figure 1 ) [81] [82] [83] . the major role of epithelial cells in contact with the outside is to protect the host from foreign antigens by forming epithelial cell sheets (tightly adhering cells) with tjs [84] . furthermore, it has also been revealed that the gm is deeply involved in the development and maintenance of the intestinal epithelial barrier [81] [82] [83] . tj was identified as a 47kda protein and named zonulin. zonulin enhances intestinal permeability, is involved in innate immunity, and is strongly suggested to be associated with the development of autoimmune diseases such as type 1 diabetes [81] [82] [83] . cumulative evidence has highlighted the relevance of increase in intestinal permeability (i.e., leaky gut syndrome) and consequent bacterial translocation in the development of clds. particularly, in recent hypotheses regarding patients with non-alcoholic fatty liver disease (nafld), intestinal permeability impairment, dietary habits, and gut dysbiosis are considered to be the main pathogenic triggers [85] [86] [87] . leaky gut is associated with chronic inflammation [87] . in advanced liver diseases, intestinal permeability can be enhanced by portal hypertension, which consequently leads to increased bacterial translocation that further damages liver function. furthermore, these pathogenic mechanisms are implicated in most lc-related complications, such as sbp, hepatorenal syndrome, severe ascites, he, sarcopenia, and hcc [85] [86] [87] . in lc rats, intestinal bacteria such as gram-negative bacilli in mesenteric lymph nodes were more likely to be detected compared with control, and the same strain of bacteria was detected in ascites [88, 89] . therefore, bacterial translocation is considered to be an etiology of the early stage of sbp. sarcopenia could worsen as liver disease progresses. hanai et al. reported that in patients with child-pugh class a, b, and c, the rate of decrease of skeletal muscle per year was 1.3%, 3.5%, and 6.1% [90] . cirrhosis to dysbiosis ratio (cdr, described later) decreases with the worsening of liver function [91, 92] . considering this evidence, the severity of sarcopenia in lc can be closely associated with the severity of dysbiosis. dysbiosis in lc can cause: (1) decreased bacterial diversity [91] , (2) decreased scfa (energy source in human body) production [93] , (3) collapse of tj and subsequent increased intestinal permeability (leaky gut syndrome) [94] , (4) antioxidant dysfunction [95] , and (5) endotoxemia [96, 97] . these can be associated with anabolic resistance, chronic inflammation, mitochondrial dysfunction, oxidative stress, and insulin resistance, which can lead to lc progression and subsequent development of sarcopenia in lc patients [25] . the ratio of beneficial to potentially harmful bacterial taxa, or the cdr (autochthonous to non-autochthonous taxa ratio), has been proposed as an index of alterations in the gm [92] . examples of benign and autochthonous gut taxa include lachnospiraceae, ruminococcaceae, veillonellaceae, and clostridiales incertae sedis xiv, while pathogenic gut taxa include enterobacteriaceae and bacteroidaceae [92] . a low cdr may suggest a decrease in beneficial bacteria and/or an excessive abundance of harmful taxa. altered bacterial function has also been demonstrated in lc patients compared with healthy controls [2, 12] . in other words, a deficit of autochthonous non-pathogenic bacteria and an excessive growth of potentially pathogenic bacteria are common characteristics in lc patients [2, 12, 92] . progressive alterations in the gm were found in worsening lc, such that the cdr was significantly decreased with liver disease progression [45] . in contrast, cdr and the gm were unchanged in patients without disease progression (i.e., stable liver disease) [92] . bajaj et al. demonstrated that in hospitalized patients with lc (n = 180), dysbiosis of the gm as assessed by cdr, etc., on admission can be associated with elevated risk of extra-hepatic organ failure, aclf, and mortality, independent of baseline clinical characteristics [98] . another study reported a significant fungal dysbiosis in lc patients [99] . in their results, the gm in lc patients altered differentially with antibiotics and ppi use, and stool bacterial/fungal profiles predicted 90-day hospitalizations well in lc patients [99] . small intestine bacterial overgrowth (sibo) is common in lc patients as a result of intestinal motility disorders and delayed transit times, and exacerbation of lc is associated with sibo [100] . in a previous study, the multivariate analysis showed that sibo (hr = 8.10, p = 0.002) and ascites (hr = 4.56, p = 0.022) were independently associated with the occurrence of malnutrition [100] . the severity of sibo can be linked to the severity of lc status [101] . increased intestinal permeability may help bacteria move into the systemic circulation. sibo has been implicated as an important risk factor in the etiology of both sbp and he in lc patients [102, 103] . thus, sibo is deeply involved in the progression of cld, which may be linked to sarcopenia [102, 103] . bile acids (bas) are synthesized from cholesterol in the liver and metabolized by the gm into secondary bas (e.g., deoxycholic acid (dca)). there is a positive correlation between abundance of ruminococcaceae (benign bacteria) and dca [104] . most of the bas that reach the ileum are reabsorbed and repeat gut-liver circulation, but some bas reach the colon and are converted by the gm (secondary bas). secondary bas regulate functions related to glucose and fat metabolism in the liver [105, 106] . in mice with dysbiosis, the expression levels of proteins in the liver involved in glycogen metabolism, cholesterol biosynthesis, and ba biosynthesis were altered, and these changes were recovered by supplementation with secondary bas [105] . atrophic change of skeletal muscle was confirmed in rats lacking the ba receptor tgr5 expressed in skeletal muscle, indicating that tgr5 enhances skeletal muscle hypertrophy and skeletal muscle cell differentiation [107] . in lc patients, a decreased conversion of primary to secondary fecal bas due to dysbiosis can be found [104, 108] . preventive effects of secondary bas on sarcopenia in lc patients are currently unknown. inoue et al. demonstrated informative data with regard to dysbiosis in patients with hepatitis c virus (hcv) as summarized below [91] : (1) even in hcv carriers with normal liver function (persistent normal alanine aminotransferase (pnalt)), alterations in gm already appeared. (2) as the liver function worsened from pnalt or chronic hepatitis to lc or hcc, the proportion of resident bacteria in the gm decreased, the types of bacteria that compose the gm decreased, and the ph of feces increased. these results mean that dysbiosis of the gm was occurring. (3) as the liver function worsened, streptococcus salivarius, which is a genus of streptococci, increased abnormally in the gm. it is possible that these bacteria decomposed urea in the intestinal tract to produce ammonia, and the ph of feces elevated. avoiding proliferation of such ammonia-producing bacteria may lead to the prevention or treatment of hyperammonemia seen in lc patients. (4) early interventions for gm (administration of probiotics, administration of appropriate antibiotics, oral care, etc.) may suppress the progression of hepatitis c and the development of hcc [91] . a recent study reported an increase in potentially pathologic bacteria and a decrease in potentially beneficial bacteria or genes in patients with hepatitis b virus, which is similar to data in patients with hcv [109] . in recent years, the association between dysbiosis and alcoholic hepatitis associated with excessive drinking has been receiving more attention [110] . in alcoholic liver injury, intestinal permeability is increased (i.e., leaky gut), and pathogen-associated molecular patterns (pamps) represented by endotoxin (lipopolysaccharide (lps)) derived from bacteria reach the liver through the portal vein and cause liver damage by activating kupffer cells [110] . endotoxin is mainly present in the cell wall of gram-negative bacteria. intestinal sterilization with antibiotics and probiotics such as lactobacillus can suppress alcoholic liver injury, and in lps receptor cd14 and toll-like receptor (tlr) 4 knockout mice, the onset of liver injury by chronic alcohol administration is suppressed [111, 112] . in addition, it has been suggested that the onset and progression of non-alcoholic steatohepatitis (nash) are associated with intestinal endotoxin [113] . in patients with nafld, low dose endotoxin can overreact to cause nash [113] . gm can be affected by aging. odamaki et al. demonstrated using fecal samples from 367 healthy japanese persons between 0 and 104 years that certain transition types of gm were enriched in infants (e.g., bifidobacterium), adults (e.g., lachnospiraceae), elderly individuals (e.g., bacteroides), and both infant and elderly subjects (e.g., enterobacteriaceae) [114] . on the other hand, flemer et al. reported in their prospective study that the gm in crc patients differs significantly from that of healthy persons throughout the colon [115] . coronavirus disease-19 (covid-19) has been rapidly becoming a global challenge. a recent study reported that in patients with covid-19, fecal gm alterations were associated with covid-19 severity [116] . additionally, covid-19 is likely to be accompanied by liver damage, and caution is required especially in lc patients for the disease progression caused by covid-19 [117] . covid-19 patients with liver disease had significantly higher mortality rates than those without (hr = 3.0, p = 0.001) [117] . interestingly, ren et al. reported that gm markers validated strong diagnosis potential for the early stage of hcc (area under the receiver operating characteristic curve = 0.8064) [118] . one possible factor which alters gm is the taking of antibiotics. while antibiotics are effective for the treatment and prevention of bacterial infections, they can cause dysbiosis [119] . rifaximin, which is a poorly-absorbable rifamycin-based antibiotic, acts on gm that are a source of ammonia to reduce ammonia production, thereby improving hyperammonemia in he [120, 121] . rifaximin has an effect of inhibiting bacterial rna synthesis, and the antibacterial activity of rifaximin covers a broad spectrum of bacteria [120, 121] . rifaximin has a favorable safety profile for long-term administration compared with oral systemic antibiotics [121] . kaji et al. demonstrated in their 20 decompensated lc patients that 4 weeks rifaximin therapy improved hyperammonemia and reduced endotoxin activity in direct correlation with the decline in serum ammonia levels, without impact on the composition of gm [97] . rifaximin also acts favorably on the serum pro-inflammatory cytokine profile and fecal secondary ba levels [122, 123] . rifaximin seems to alter the secondary to primary ba ratio in compensated lc patients, which can be associated with reduction in endotoxemia and reduction in harmful metabolite levels [104] . in addition, rifaximin appears to be effective and safe for the treatment of sibo [124] . the clinical activity of rifaximin may be attributed to the effects on metabolic function of the gm, rather than an alteration in relative bacterial abundance [125] . a recent meta-analysis (5 studies, comprising 555 patients) reported that rifaximin therapy may be effective in preventing sbp in patients with lc and ascites compared with systemic absorbed antibiotics and compared with placebo [126] . flamm et al. demonstrated that in patients with model for end-stage liver disease score 12 or greater and international normalized ratio 1.2 or greater, rifaximin group (n = 140) reduced the relative risk of any first complication (he, sbp, variceal bleeding, acute kidney injury, or hepatorenal syndrome) experienced during the study period by 59% [hr = 0.41, 95% ci = 0.25-0.67; p < 0.001] vs. placebo group (n = 159) [127] . kumar et al. reported that in rats with port-systemic shunts, the increase in skeletal muscle myostatin expression, suppressed mtorc1 function, and hyperammonemia-related stress response (i.e., autophagy markers) were reversed by ammonia-lowering therapy, concluding that it can lead to the improvement in skeletal muscle phenotype and function [128] . however, the preventive effects of rifaximin on sarcopenia incidence or progression in lc patients remain unclear. table 2 demonstrates randomized controlled trials (rcts) published since 2010 regarding the effects of rifaximin on outcomes in patients with decompensated lc . rcts with the improvement of sarcopenia as a primary endpoint are not found. in our hypothesis, rifaximin treatment in lc patients with sarcopenia potentially has an impact on the improvement of sarcopenia through the improvement of hyperammonemia and subsequent hypermyostatinemia in skeletal muscles. further exams with regard to the effect of rifaximin on sarcopenia in lc patients will be required to confirm these results. l-carnitine therapy, which is also an ammonia-lowering therapy, can improve sarcopenia in lc patients [154, 155] . probiotics are defined as microorganisms that have positive effects on the human body, or drugs and foods containing them. probiotics may act on the gm, intestinal epithelial cells, immunocompetent cells present in the intestinal mucosa, etc. [85, 156] . many neurotoxic substances are derived from the gm, and the usefulness of probiotics for improving the composition of gm has been investigated as a treatment for he [85, 156] . probiotics enhance the expression of tj-related proteins with improvement of dysbiosis and improve the intestinal barrier function [157, 158] . a previous meta-analysis demonstrated that probiotics reduce the risk of hospitalization and the progression to overt he to the same extent as lactulose in patients with minimal he, but do not affect mortality [125] . on the other hand, another systematic review comparing probiotics with placebo or no treatment summarized as follows: (1) there was no significant difference in mortality from any cause. (2) the non-recovery rate and the incidence of adverse events including he were lower in the probiotics group, but the effect on hospitalization was unclear. (3) quality of life was slightly improved in the probiotics group. (4) in comparison of probiotics and lactulose, the effects on mortality rate from any cause, non-recovery rate, incidence of adverse events including he, hospitalization, and quality of life were unable to be assessed due to the low quality of evidence [159] . table 3 demonstrates rcts published since 2010 regarding the effects of probiotics on outcomes in patients with decompensated lc [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] . rcts with the improvement of sarcopenia as a primary endpoint are not found. the effects of probiotics on sarcopenia in lc patients are unclear as well as those of rifaximin. in mice given probiotics (lactobacillus paracasei ps23), aging-related muscle mass decline and muscle strength decline significantly improved [173] . across a range of chronic diseases, several guidelines recommend exercise training. it has been found that exercise can change the composition of the gm, which leads to an intestinal flora with a beneficial metabolic system [174] . clarke et al. showed that the gm of rugby players was highly diverse, clearly different from normal healthy subjects, and that there was a high positive correlation between the diversity of gm and protein intake [175] . athletes presented a higher level of scfa-producing bacteria and bacterial genes related to nutritional metabolism compared with sedentary controls [176] . aerobic and resistance training revealed improved gm composition and functionality in rats with nafld [174] . however, only a few reports have shown exercise-related alterations on the gm in humans, and most evidence comes from non-randomized studies. huber et al. reported that in 41 nafld patients receiving an 8-week exercise program, increased metagenomic richness of the gm (i.e., increased diversity) was observed [177] . to date, there are no clinical studies or rcts looking specifically at exercise and the gm in patients with lc with sarcopenia, although exercise can decrease hvpg in lc patients [178] . if the improvement of the gm in lc patients with sarcopenia by exercise is confirmed, future treatment strategies for lc patients with sarcopenia and dysbiosis will be changed. interactions between dietary nutrients and gm promote host nutrition and health via various signaling pathways, and to maintain and promote human health, beneficial bacteria should be dominant in the gm [7] . in lc patients, these interactions can be disturbed due to pem, amino acid imbalance, dysbiosis, etc., which can cause sarcopenia. sarcopenia is a public health problem that cannot be overlooked. as mentioned earlier, skeletal muscle is considered to be a metabolic organ. when understanding the pathophysiology of lc, we must always keep in mind the relationship between organs including skeletal muscles and the digestive tract, that is, the organ network. in this article, we overviewed the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. a summarized scheme is shown in figure 2 . in the past decade, marked advances have been made in this research field. our current research questions are whether or not rifaximin, probiotics, or exercise training can improve sarcopenia in lc through the improvement of the gm. to the best of our knowledge, appropriate rcts to address these research questions cannot be found. future research is eagerly awaited. across a range of chronic diseases, several guidelines recommend exercise training. it has been found that exercise can change the composition of the gm, which leads to an intestinal flora with a beneficial metabolic system [174] . clarke et al. showed that the gm of rugby players was highly diverse, clearly different from normal healthy subjects, and that there was a high positive correlation between the diversity of gm and protein intake [175] . athletes presented a higher level of scfaproducing bacteria and bacterial genes related to nutritional metabolism compared with sedentary controls [176] . aerobic and resistance training revealed improved gm composition and functionality in rats with nafld [174] . however, only a few reports have shown exercise-related alterations on the gm in humans, and most evidence comes from non-randomized studies. huber et al. reported that in 41 nafld patients receiving an 8-week exercise program, increased metagenomic richness of the gm (i.e., increased diversity) was observed [177] . to date, there are no clinical studies or rcts looking specifically at exercise and the gm in patients with lc with sarcopenia, although exercise can decrease hvpg in lc patients [178] . if the improvement of the gm in lc patients with sarcopenia by exercise is confirmed, future treatment strategies for lc patients with sarcopenia and dysbiosis will be changed. interactions between dietary nutrients and gm promote host nutrition and health via various signaling pathways, and to maintain and promote human health, beneficial bacteria should be dominant in the gm [7] . in lc patients, these interactions can be disturbed due to pem, amino acid imbalance, dysbiosis, etc., which can cause sarcopenia. sarcopenia is a public health problem that cannot be overlooked. as mentioned earlier, skeletal muscle is considered to be a metabolic organ. when understanding the pathophysiology of lc, we must always keep in mind the relationship between organs including skeletal muscles and the digestive tract, that is, the organ network. in this article, we overviewed the current knowledge of the relationship between dysbiosis and sarcopenia in patients with lc. a summarized scheme is shown in figure 2 . in the past decade, marked advances have been made in this research field. our current research questions are whether or not rifaximin, probiotics, or exercise training can improve sarcopenia in lc through the improvement of the gm. to the best of our knowledge, appropriate rcts to address these research questions cannot be found. future 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treatment of patients with hepatic encephalopathy lactobacillus paracasei ps23 decelerated age-related muscle loss by ensuring mitochondrial function in samp8 mice beneficial effects of exercise on gut microbiota functionality and barrier integrity, and gut-liver crosstalk in an in vivo model of early obesity and non-alcoholic fatty liver disease the microbiome of professional athletes differs from that of more sedentary subjects in composition and particularly at the functional metabolic level improvement of non-invasive markers of nafld from an individualised, web-based exercise program changes in hepatic venous pressure gradient induced by physical exercise in cirrhosis: results of a pilot randomized open clinical trial key: cord-009987-biop7gyd authors: ali, muhammad; khan, tariq; fatima, kaneez; ali, qurat ul ain; ovais, muhammad; khalil, ali talha; ullah, ikram; raza, abida; shinwari, zabta khan; idrees, muhammad title: selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions date: 2017-10-19 journal: phytother res doi: 10.1002/ptr.5957 sha: doc_id: 9987 cord_uid: biop7gyd insight into the hepatoprotective effects of medicinally important plants is important, both for physicians and researchers. main reasons for the use of herbal medicine include their lesser cost compared with conventional drugs, lesser undesirable drug reactions and thus high safety, and reduced side effects. the present review focuses on the composition, pharmacology, and results of experimental trials of selected medicinal plants: silybum marianum (l.) gaertn., glycyrrhiza glabra, phyllanthus amarus schumach. & thonn., salvia miltiorrhiza bunge., astragalus membranaceus (fisch.) bunge, capparis spinosa (l.), cichorium intybus (l.), solanum nigrum (l.), sapindus mukorossi gaertn., ginkgo biloba (l.), woodfordia fruticosa (l.) kurz, vitex trifolia (l.), schisandra chinensis (turcz.) baill., cuscuta chinensis (lam.), lycium barbarum, angelica sinensis (oliv.) diels, and litsea coreana (h. lev.). the probable modes of action of these plants include immunomodulation, stimulation of hepatic dna synthesis, simulation of superoxide dismutase and glutathione reductase to inhibit oxidation in hepatocytes, reduction of intracellular reactive oxygen species by enhancing levels of antioxidants, suppression of ethanol‐induced lipid accumulation, inhibition of nucleic acid polymerases to downregulate viral mrna transcription and translation, free radical scavenging and reduction of hepatic fibrosis by decreasing the levels of transforming growth factor beta‐1, and collagen synthesis in hepatic cells. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. liver disorders have been classified in the high priority areas of health care. according to an estimate by the world health organization, approximately 500 million people of the world are suffering from a severe form of liver disorders, that is, chronic hepatitis (al-asmari et al., 2014) . medicine of herbal origin may serve as a feasible therapy for the prevailing liver problems because of their safety, easier availability, cost effectiveness, and environment friendliness (izzo, hoon-kim, radhakrishnan, & williamson, 2016) . medicinal plants have acquired importance in healthcare system throughout the world for their proven and effective therapeutic properties (helmstädter & staiger, 2014 ). an estimated 80% of the world's population is relying on medicines that contain compounds of herbal origin (ekor, 2013) . the international union for conservation of nature has suggested that approximately 50,000 to 80,000 flowering plants are used for medicinal purposes (chen, li, ren, & hu, 2016) . many factors regarding these medicines are important. herbal medicines are claimed to both treat and prevent diseases, which adds to a deep belief that these abbreviations: alt, alanine aminotransaminase; asp, angelica sinensis polysaccharides; ast, aspartate transaminase; egf, epidermal growth factor; hbv, hepatitis b virus; lbps, lycium barbarum polysaccharides; wf4, woodfordia fruticosa flower extract. treatments are safe because they are "natural and gentle" and therefore, a harmless alternative to the conventional medicine. moreover, the latter may sometimes cause disappointing results and undesirable side effects in patients (izzo et al., 2016) . in addition, the less expensive herbal products are often not subject to strict regulations and medication prescribed by a physician or other qualified practitioners (hunter & hegele, 2017) . although medicinal plants have been used globally, their wider usage is limited to a few countries like japan, india, china, pakistan, thailand, iran, and some african countries (bahmani et al., 2014; iwu, 2014; li, 2016; sivasankari, anandharaj, & gunasekaran, 2014) . other countries are also encouraging the use of plant-based medicinal products in their healthcare systems. for example, natural health product regulations of canada for the plant-based product in healthcare encourages usage of modern technology and evidencebased scientific support towards promoting medicinal plants and the associated products (tomlinson & akerele, 2015) . a major concern of scientists investigating herbal treatments is that the chemical composition of the plants contributing to their biological effects is mostly undetermined (ling et al., 2009 ). herbs and herbal medicines have been used for the treatment of liver diseases for a long time (dhiman & chawla, 2005) . there are many herbs having ingredients that are potential sources of medicine for the treatment of liver diseases having various modes of actions and bioactivities (babu, bhuvaneswar, sandeep, ramaiah, & rajendra, 2017; gnanadesigan, ravikumar, & anand, 2017; pereira, barros, & ferreira, 2016) . however, several of them are well-studied for their bioactive components and the mechanism of hepatoprotective activity. in the current review, we have selected some of these compounds for which elaborate detail about hepatoxicity is available in literature in the form of either in vivo studies, study into biochemical parameters and bioactive compounds. this article highlights the possible ways of inducing hepatoxicity in mice models and encompasses the mechanisms in which certain medicinally important plants perform their hepatoprotective activity. the article further aims to summarize studies conducted on the composition, pharmacology, and nature of the selected plants in the light of possible mechanism deduced from experimental trials. a thorough search was conducted on the electronic literature databases, google scholar, pubmed, scopus, and web of science. literature was retrieved using the key words and phrases "hepatitis c", "hepatoprotective activity", "mechanism of action", "medicinal plants", "herbal", and "treatment". about 100 relevant articles were extracted after a narrow search for a combination of the keywords and subsequent analysis per the inclusion criteria. there were two sets of criteria applied to articles for inclusion in this manuscript. according to the first, "general criteria", articles selected for this manuscript were those which (a) reported plants and their parts that were traditionally applied to hepatitis and liver disorders and any other type of hepatoprotective activity; (b) reported extract or pure compounds important for their hepatoprotective role; and (c) attempted to explain the mechanism of hepatoprotective action of these plants. the 2nd criteria were used for selecting those plants that are discussed in detail (shown in figure 3 ). for this purpose, seventeen plants were selected for which recent articles were available that (a) studied in vitro and in vivo hepatoprotective activities of herbal products, (b) reported active compounds from the plant, and (c) described the mechanism of action herbal hepatoprotective products. the plants described in detail were selected if literature available for them fulfilled at least two of the above 3-point criteria. each of the selected plants was discussed; mainly focusing its hepatoprotective activities, active compounds, and possible mechanism of action. in addition, featured hepatoprotective herbal combinations have been deliberated. toxicity and quality control issues associated with these herbs/herbal products have been debated. two of the authors independently reviewed all the full-text articles obtained during the electronic search. data from the eligible articles were extracted; all the disagreements were discussed and were referred to a third reviewer (one of the author) for a final decision. all the data were extracted in two tables (tables 1 and 2) , and the mechanisms of action were explained in respective subheadings and demonstrated through four different figures (figures 1-3, and 4 ). the figures were constructed in chembiodraw ultra (version 14.0) software package. furthermore, the plant database "plant list" was used for the taxonomic categorization of all the documented plant species (theplantlist, 2013) . the prerequisite to screen/study any medicinal compound for its hepatoprotective activity is to develop a model (animal model or cell culture model) in which hepatic injury is induced (salehi, karegar-borzi, karimi, & rahimi, 2016) . several studies have manipulated mice models to induce hepatotoxicity and then treat those induced liver diseases using herbs and herbal products (figure 1 ). this approach provides insight into how hepatitis and other liver diseases are caused. however, for many plants, their mechanism of action against hepatotoxic agents is not well-documented. the common prototype applied for hepatoprotective drug screening is the carbon tetrachloride (ccl 4 ) induced hepatic injury (rodrigues et al., 2016) . as ccl 4 have been reported for its damaging effects on the liver because on metabolism by p450, it produces free radicals (johnston & kroening, 1998) . these free radicals cause lipid peroxidation by binding to dna, proteins, or lipids (yasuda, izumi, shimada, kobayakawa, & nakanishi, 1980) . the degree of hepatic injury is evaluated by the higher level of biochemical parameters that is ascribed to the production of trichloromethyl free radicals which eventually causes lipids peroxidation present in cellular (dusheiko, 1996) calotropis procera (aiton) dryand. crude hydro-ethanol solution extract prevents of the depletion of gsh levels. c. procera contains flavonoids thus it also performs the antioxidant activity (mcomish et al., 1994) clerodendrum abilioi r. fern. ethanol extract decreased the serum enzyme alt, ast, alp, tgl, and total cholesterol and considerably increased the glutathione level (chamberlain, adams, saeed, simmonds, & elliott, 1997) ficus carica l. leaves crude petroleum ether extract reduction in the levels of alt and ast. the petroleum ether extract of ficus leaves repair the damaged liver cell (gond & khadabadi, 2008) glycyrrhiza uralensis -glycyrrhizin glycyrrhizin administered in plc/prf/5 cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes (sato et al., 1996) momordica dioica roxb. ex willd. alkaloids, phenolic compounds, glycosides, flavonoids oral administration of the extract significantly normalized and restored the elevated serum enzymatic levels of ast, alt, salp, and total bilirubin. its hepatoprotective activity is due to the antioxidant and free radical scavenging activity. (surai, 2015) nelumbo nucifera gaertn. leaves catechin glycoside, myricitrin-3-o-glucoside, hyperin, isoquercitrin, quercetin-3-o-rhamnoside, astragalin lotus leaf extract possess significant hepatoprotective and antioxidant activity in ccl 4 -induced toxicity rat model. free radicalscavenging and antioxidant activity due to the presence of some flavonoids and phenolic compounds results in the hepatoprotective activity. (theplantlist, 2013) paeonia lactiflora pall. and a. membranaceus (fisch.) bunge. -progression of ccl 4 -induced hepatic fibrosis was inhibited in rates by decreasing the level of tumor growth factor-β1 and inhibit collagen synthesis (sun et al., 2007) s. miltiorrhiza bunge. roots -s. miltiorrhiza could reverse the ccl 4 -induced fibrosis treatment by decreasing the levels of transforming growth factor-β1, procollagens i and iii, and metalloproteinase-1 and decreasing the levels of metalloproteinase-13 in liver of the affected rates (wasser et al., 1998) s. miltiorrhiza bunge. roots s. miltiorrhiza polysaccharides protects liver against immunological injury by adjusting the levels of alanine aminotransferase, aspartate aminotransferase, nitric oxide, tumor necrosis factor and interleukin-1 (zein et al., 1996) solanum nigrum l. total decoction crude aqueous extract inhibited thioacetamide-induced collagen (α1) and transforming growth factor-β1 mrna levels in the liver of mice with thioacetamide-induced liver fibrosis (hsieh et al., 2008) s. nigrum l. and cichorium intybus l. crude plant extract protect dna against oxidative damage in the reaction mixture containing calf thymus dna and free radical generating system (sultana et al., 1995) (continues) membrane (chen, yu et al., 2016) . figure 2 shows the different strategies applied for studying the in vivo effects of induced hepatotoxicity in mice models. the leaves are marked by distinct white "milky" veins that give the plant its common name (theplantlist, 2013) . historically, s. marianum was used medicinally to treat disorders of the gallbladder, spleen, and liver, but the most important medicinal application of s. marianum is its use as a hepatoprotective herbal treatment and as supportive treatment for chronic inflammatory liver disorders such as hepatitis, cirrhosis, fatty infiltration, and some other forms of liver damages due to toxic chemicals, poisonous mushrooms, and alcohol (freitag et al., 2015) . the most important component extracted from s. marianum is silymarin (lu, lu, chen, zhang, & wu, 2007; wu, wang, & que, 2006) , which is used to treat a variety of liver disorders, including chronic and acute viral or drug/toxin-induced hepatitis, alcoholic liver disease, and liver cirrhosis (lu et al., 2007) . silymarin is a combination of different ingredients with silibinin as the most active among them (surai, 2015) . silymarin has been approved for clinical studies in treating the hepatitis c virus infection (ferenci et al., 2008) . there are many studies on the mechanism of hepatoprotective effects of silymarin. recently, tunca et al. (2009) showed that silymarin has a protective action on pyridine-induced hepatic injury in syrian hamsters. the study concluded that it decreases the metabolic activation of pyridine (by decreasing the cytochromes p450 1a1 protein concentration) and control the elevation of inducible nitric oxide synthase expression. all these factors play a protective role in liver injury. in another study, farghali, kamenikova, hynie, and kmonickova (2000) concluded that in addition to inhibition of lipid peroxidation, the hepatoprotective activity against thioacetamide-induced hepatotoxicity (khatri, garg, & agrawal, 2009) tephrosia purpurea (l.) pers. decreased serum aspartate aminotransaminase (35% and 31%), alanine aminotransaminase (50% and 42%), gamma glutamyl transpeptidase (56% and 49%), alkaline phosphatase (46% and 37%), total bilirubin (61% and 48%), and liver mda levels (65% and 50%), and significant improvement in liver glutathione (73% and 68%) when compared with thioacetamide-damaged rats. (hosseinzadeh & nassiri-asl, 2015) vitex negundo l. administration of ethanol solution extract of vitex leaf caused a significant decrease in tb, ast, alt, and alp levels in rats. (abdulkarim et al., 1998) zanthoxylum armatum dc. bark berberine elevated serum enzymatic levels of serum transaminases, alkaline phosphatase. total bilirubin was considerably restored to a normal level. (cha et al., 1991) note. gpx = glutathione peroxidase; mda = malondialdehyde; ast = aspartate transaminase; alt = alanine aminotransaminase; ccl 4 = carbon tetrachloride; sod = superoxide dismutase; gsh = glutathione; tb = total bilirubin; alp = alkaline phosphatase hbsag = hepatitis b surface antigen; tgl = triglyceride lipase. inhibition of the increased intracellular ca 2 i plays a critical role in the hepatoprotective effect of silymarin. although, upadhyay, kumar, and singh (2007) showed that silymarin restores the changes in the expression and activity of cytochrome p450 (cyp) enzymes (cyp1a1, cyp1a2, and cyp2e1), glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. glycyrrhizin administered in plc/prf/5 cells suppressed the secretion of hbsag into the culture medium and concluded that glycyrrhizin modifies the intracellular transport and the surface nature of the hepatocytes glycyrrhizin administered intraperitoneally inhibits the lipopolysaccharide-and d-galactosamine-induced liver injury by preventing inflammatory responses and il-18 production in mice glycyrrhizin inhibited anti-fas antibody-induced hepatitis in mice by acting upstream of cpp32-like protease administration of glycyrrhizin or glycyrrhetinic acid, significantly suppressed α2 (i) collagen gene promoter activation and progression of liver fibrosis induced by repeated ccl 4 injections in transgenic mice (liew, erali, page, hillyard, & wittwer, 2004; martell et al., 1992; ogata, alter, miller, & purcell, 1991; sato et al., 1996) phyllanthin phyllanthus amarus schum. et thonn. phyllanthin help in restoration of antioxidant potential of rat hepatocytes, level of gsh, and sod and gr activities reduced by ethanol (chirdchupunseree & pramyothin, 2010) p-methoxy benzoic acid capparis spinosa l. the compound alleviated the enzyme levels increased as result of administration of ccl 4 , and pcl (gadgoli & mishra, 1999) silymarin silybum marianum (l.) gaertn. silymarin attenuated the rifampicinand/or pyrogallol-induced hepatotoxicity by restoring the alterations in the expression and activity of cyp1a2 and cyp2e1, glutathione-s-transferase, glutathione reductase and glutathione peroxidase, and lipid peroxidation in male swiss albino mice. silymarin suppresses n-nitrosodiethylamine induced hepatocarcinogenesis by modulating the antioxidant defense status of the animals (farghali et al., 2000; upadhyay et al., 2007) note. hbsag = hepatitis b surface antigen; cpp32 = 32-kda putative cysteine protease; ccl 4 = carbon tetrachloride; gsh = glutathione, sod = superoxide dismutase; gr = glutathione reductase; cyp = cytochrome p450; pcl = paracetamol. g. glabra is a member of the glycyrrhiza genus (isbrucker & burdock, 2006) , an ancient genus that contains the most commonly used herbs in chinese traditional medicine (hosseinzadeh & nassiri-asl, 2015) . glycyrrhiza species are considered among the most important herbaceous plants for a diverse array of pharmacological activities (hosseinzadeh & nassiri-asl, 2015) . chemical structures of (1) cryptotanshinone, (2) phyllanthin, (3) quercetin, (4) glycyrrhizin, (5) silymarin, and (6) p-methoxybenzoic acid, also known as p-anisic acid. all the images were adopted from ncbi-pubchem with the compound ids; 160254, 358901, 5280343, 14982, 7073228, and 7478, respectively (pubchem, 2017) (mao et al., 2016) . one of the bioactive compounds from p. amarus is phyllanthin, which is a lignan compound and is traditionally applied in the treatment of many liver diseases (hanh, sinchaipanid, & mitrevej, 2014) . it was shown to have hepatoprotective effects on ethanol-induced oxidative damage in primary culture of rat hepatocytes through its antioxidant activity especially the activities of superoxide dismutase (sod) and glutathione reductase (chirdchupunseree & pramyothin, 2010) . previously, naaz, javed, and abdin (2007) 3.9 | c. intybus l. c. intybus l., commonly known as chicory, belongs to the lactuceae family and is typical mediterranean plant indigenous to western asia, europe, north america, and egypt, which varies in perianth color from white, red to blue (norbaek, nielsen, & kondo, 2002 3.10 | s. nigrum l. s. nigrum l., commonly known as "black nightshade", is a species in the family solanaceae ( ccl 4 -induced hepatic necrosis. this hepatoprotective effect might be due to its adjustment of antioxidant activity, detoxification enzymes, and its free radical scavenger effects. in another study, hsieh, fang, and lina (2008) induced liver fibrosis by administering thioacetamide in mice and treated them with distilled water and s. nigrum extract via oral administration for 12 weeks. this treatment alleviated the hepatic hydroxyproline and α-smooth muscle actin protein levels in mice and inhibited thioacetamideinduced collagen and transforming growth factor-β1 mrna levels in the liver. histological examination of liver also confirmed that this extract reduced the degree of fibrosis caused by thioacetamide treatment which is the probable reason for the reduction of hepatic fibrosis. 3.11 | s. mukorossi gaertn. s. mukorossi gaertn., commonly known as ritha or aritha, is abundantly found in india. its fruit is reported to have expectorant, purgative, antidotal, and abortifacient effects. additionally, it is used in epilepsy, extreme salivation, and chlorosis (suhagia, rathod, & sindhu, 2011) . the saponins extracted from this plant are spermicidal (in vitro) and due to this property, it has been used in contraceptive cream (rastogi & mb, 1999) . pharmacological studies of s. mukorossi have shown their potential effect as hepatoprotective agents (upadhyay & singh, 2012) . to assess the hepatoprotective activity of the s. mukorossi, wistar male rats were treated with ccl 4 . administration of ccl 4 to normal rats increased the serum levels of alt, ast, alp, and bilirubin. these enzymes eventually cause damage to the hepatic cells. the ccl 4 -treated liver cells cultured on petri plates were treated with the extracts of s. mukorossi and were reported to alleviate the levels of these enzymes. when histopathological studies of the ccl 4 -treated rats were performed, they showed that it also causes the demolition of architectural configuration of target cells. however, rats that were treated with s. mukorossi presented normal lobular structural design, which shows its reparative properties and thus its hepatoprotective effects. 3.12 | g. biloba l. g. biloba l. belongs to family ginkgoaceae (theplantlist, 2013) . it is one of the significant herbs of the chinese traditional medicine. g. biloba leaf extract has been reported to have therapeutic activities against age-related memory deficit problems, including alzheimer's and dementia; cardioprotective, antiasthmatic, antidiabetic, hepatoprotective, photoprotective effects, dna repair mechanism, antioxidant, and antiinflammatory activities (mohanta, tamboli, & zubaidha, 2014) . g. biloba has been associated with a strong hepatoprotective activity through numerous studies (parimoo et al., 2014) . g. biloba amplifies cellular antioxidant protection system consisting of glutathione peroxidase, glutathione s-transferase, glutathione reductase, nonprotein thiols, catalase, and antioxidant enzymes (sod). the binding of an individual part of herbal tracks to that of phosphatidylcholine produces phytosome having better efficacy compared with traditional herbal extracts (naik, pilgaonkar, & panda, 2006) . rifampicin is an antibiotic widely used in tuberculosis chemotherapy. it has been reported to cause hepatoxicity, the reason of which is unknown as it is always given in combined form with other antibiotics such as isoniazid and ethambutol. wistar albino rats were treated with rifampicin that caused hepatoxicity in them. their blood samples were taken, and assays of their blood samples were performed to know the levels of sgpt, sgot, and alp. the elevated levels of sgot, sgpt, and alp show liver damage as these enzymes escape from the liver into the blood in case of liver damage. with parallel treatment through ginkoselect phytosome® and the standard drug silymarin, the markers enzymes levels in serum were nearly at a normal level or marginally elevated. this suggests the hepatoprotective quality of g. biloba plant. it also elevates total protein levels and albumin, which shows its hepawith activities against chronic hepatic fibrosis (nitha, prabha, ansil, & latha, 2014) . it also shows antiinflammatory, antibiotic, antileprosy, and antihelminthic properties (arya et al., 2015; shoaib et al., 2016; syed & khan, 2016) . in an experiment designed to check the hepatoprotective effect of w. fruticosa flower extract (wf4), albino wistar rats were administered with ccl 4 which resulted in the increased level of alp, ast, alt, and lactate dehydrogenase. these enzymes leak from serum into the blood. thus, ccl 4 damage causes loss of enzymes which are responsible for drug metabolism (chandan et al., 2008) . these rats were administered with wf4. the extract reversed the elevated lipid peroxidation and regulated the liver glucose-6-phosphate and gsh levels. these results are in line with former information for other hepatoprotective agents . 3.14 | v. trifolia l. v. trifolia l., known generally as chaste tree, is a high-value medicinal plant that belongs to the family verbenaceae. its leaves are effective as plaster against pains, infections, and fever. its fruits are used in curing amenorrhea, and the flowers are effective against fever (chan, baba, chan, kainuma, & tangah, 2016) . the active constituents of this plant are essential oil (kvasnicka, biba, sevcik, voldrich, & kratka, 2003) , viterifolins, and diterpenes. it also possesses some important pharmacological qualities, that is, antipyretic (rani & sharma, 2013) , antibacterial (lawitz et al., 2014) , antiallergic, and antiasthmatic properties (lawitz & gane, 2013) . medical practitioners use this plant in the treatment of acute jaundice. however, literature study suggested that this plant is not well screened for its hepatoprotective activity. nonetheless, the tribal groups of western ghats use this plant leaf extracts in treating jaundice, and these results give some scientific evidence of hepatoprotective activity. 3.16 | s. chinensis (turcz.) baill s. chinensis (turcz.) baill is widely used in traditional and modern chinese medicine for the treatment of many disorders including insomnia, respiratory failure, and weakness. moreover, mental health improving ability along with fatigue reduction property is also validated for s. chinensis in russian medicine (szopa, ekiert, & ekiert, 2017) . in general, dibenzocyclooctadiene lignans found in s. chinensis are known to exhibit potent hepatoprotective activity (zheng et al., 2017) . in one of the study of individual lignin, gomisin a was found responsible for the acceleration of hepatocytes proliferation and increase hepatic flow (panossian & wikman, 2008) . furthermore, elevation of mitochondrial glutathione concentration was found to be linked with γ-schisandrin hepatoprotective mechanism. the increase in vitamin c concentration in the liver of test animals upon treatment with γ-schisandrin also validates its hepatoprotective ability. another individual lignin, schisandrin b was also found to counter oxidative harm to liver tissues (thandavarayan et al., 2015; xin et al., 2017) . in one scientific study, the hepatoprotective mechanism against acetaminophen-induced liver injury of six schisandra lignans (deoxyschisandrin, schisantherin a, schisandrin b, gomisin a, schisandrin c, and schisandrin) was elucidated. the hepatoprotective ability of these lignins was found to be associated with inhibition of cytochrome-mediated bioactivation (jiang et al., 2015) . furthermore, another mechanistic study investigated the hepatoprotective effect of schisandra polysaccharide in nonalcoholic fatty liver disease mice models. the results demonstrate potential down regulation of hepatic lipogenesis genes and lxrα/srebp-1c/ fas/acc and srebp-2/hmgcr signaling pathways in the liver (wang, song et al., 2016) . 3.17 | c. chinensis lam. c. chinensis lam. also known as chinese dodder is a parasitic plant having diverse traditional medicinal uses as a tonic, sex enhancer, and abortion preventer (zheng, dong, & she, 1998) . studies also have scientifically validated the hepatoprotective activity of c. chinensis (donnapee et al., 2014) . yen, wu, lin, and lin (2007) chinensis seeds ethanol solution extract was found to be more effective in rats with acetaminophen-induced hepatotoxicity (yen, wu, lin, cham, & lin, 2008) . the mechanism of hepatoprotective potential as demonstrated by ethanol solution extract of c. chinensis is proposed to be the elevated activities of antioxidant enzymes. 3.18 | l. barbarum l. l. barbarum l. berries are very famous in traditional chinese medicine for the treatment of inflammation, cancer, eye disorders, throat infection, and anemia. the use of these berries has been validated as food and also has gained great importance due to its significant antioxidant potential (cheng et al., 2015) . the major active components of l. barbarum berries are l. 3.19 | a. sinensis (oliv.) diels a. sinensis (oliv.) diels is reported in chinese herbal medicine for the treatment of cardiovascular disease, anemia, and hepatic disorders (bunel, antoine, nortier, duez, & stévigny, 2015) . the a. sinensis polysaccharides (asp) extracted from a. sinensis roots having the average molecular weight of 72,900 da is regarded as a potential active component of a. sinensis that exhibits a wide range of pharmacognostic properties (hsu, tsai, & tsai, 2014) . the hepatoprotective potential of asp in ccl 4 -induced liver injury and via using ischemia/reperfusion rat is widely established (zhang et al., 2010) . wang, wen, li, zhang, & yang (2016) the number of hepatoprotective products from plants is ever increasing. in addition to the hepatoprotective role of a general class of phenolics and flavonoids, many studies have defined specific compounds for their preferable role in hepatitis and other liver disorders (shehab et al., 2015) . some of the important plants and their products are highlighted in figure 3 . among the many different compounds, few are distinguished for their promising role in liver inflammation. we have, therefore, selected six important compounds for their hepatoprotective role (table 2) . quercetin, for instance, a major flavonol commonly found most of the plants, is a potent hepatoprotective agent. the first basis of quercetin-based potency has been attributed to the antioxidant activity of this compound. one of the specific mechanisms of quercetin supplementation was established through ethanol-induced cytotoxicity which affects the activity of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. for instance, quercetin supplementation restored the glutathione reductase activity which was affected by ethanol that in turn reduced the glutathione content of liver. quercetin supplementation has also been attributed to hepatoprotection against metals, pesticides, drugs, toxins, and viruses (miltonprabu et al., 2016) . rhein (4, 5-dihydroxyanthraquinone-2-carboxylic acid) is another important hepatoprotective compound extensively found in medicinal herbs, such as rheum palmatum l., cassia tora l., polygonum multiflorum thunb., and aloe barbadensis miller. the mechanism of action through which rhein acts has been described as modulation of cyp enzymes in rat liver, attenuation of total cholesterol and triglyceride levels in serum, and amelioration of glucose and lipid metabolism. similarly, rhein downregulated the levels of serum alt, hyaluronic acid, procollagen type iii, and liver malondialdehyde and inhibited the expression of transforming growth factor beta 1 and alpha-smooth muscle actin in tetrachloride/ethanol-induced liver fibrosis rats . similarly, flavonoids, lignans, terpenoids, and steroids from vitex negundo l. have also been shown to demonstrate hepatoprotective activities (zheng et al., 2015) . extracts containing these compounds have been shown to improve biochemical and functional parameters and thus alleviate ccl 4 -induced damage in liver rats. negundoside, for instance, which is a glycoside, has been demonstrated to reduce calcium-mediated toxicity and ccl 4 -induced oxidative stress through regulation of calcium homeostasis and decreasing the production of ros and lipid peroxidation (zheng et al., 2015) . table 2 with their structures given in figure 4 . the present literature is not sufficient to assess the safety of most of the hepatoprotective and liver regenerative herbs and herbal products, as most of the studies focus on their antihepatotoxic effects only. however, some previous experiments on rats show that no adverse effects were observed by administering intraperitoneal injection of the a. membranaceus extracts at 0.5 g/kg for 30 days, whereas large doses (1 g/kg) of a. membranaceus root extracts resulted in mutagenicity in mice when injected directly into the stomach lining ). in another study, sato et al. (1996) observed no significant toxicity of various concentrations of glycyrrhizin on plc/prf/5 cells in vitro. gadgoli and mishra (1999) reported that p-methoxy benzoic acid extracted from c. spinosa was nontoxic at 1 mg/ml when applied to rat hepatocytes in vitro. this supports the claims made in the traditional system of medicine. similarly, silymarin is shown to have a lack of toxicity and side effects even at high doses (upadhyay et al., 2007) . isbrucker and burdock (2006) reported that no-observed-effect levels for purified glycyrrhizin are in the range of 15-229 mg/ kg/day and concluded that current levels of consumption of licorice extract products and glycyrrhizinate are safe. although, several herbals show potential activity for the treatment of acute and chronic liver diseases premarketing drug-testing, and pharmacovigilance is needed as with any other drug. so far, herbals to treat chronic liver diseases should not be recommended outside clinical trials as the evidence supporting its use is insufficient (stickel, patsenker, & schuppan, 2005) , and publications relevant to the cytotoxicity of medicinal plants should be encouraged (mukhtar et al., 2008) . moreover, there are issues like approval of the plant products/extracts as a drug from regulatory agencies such as the food and drug administration or any other equivalent agencies. extensive literature survey of hepatoprotective plants clearly indicates that herbal drugs have an enormous potential for the treatment of liver diseases. in this article, we reviewed the scientific merit of selected plants studied for their hepatoprotective mechanism of action. the major hepatoprotective mechanism identified by the majority of the studies is through combating the oxidative stress that damages the liver. we have summarized the effect of extracts and compounds from different herbs on liver injury considering changes in their biochemical parameters. we also presented the possible data available in the literature for different plants regarding their phytochemical constituents. we, therefore, conclude that herbs and herbal preparations are among the most important sources of hepatoprotective and liver regeneration medicines. however, further research is needed to identify, characterize, and standardize the active ingredients, useful compounds, and their preparations for the treatment of liver diseases. moreover, a combination of the traditional herbal medicines with the modern and conventional medicine may be one of the best options for the treatment of liver disorders and other diseases and infections, soon. the importance of medicinal plants can be determined from world health organization's estimates, which states that up to 80% of the world's population fulfill their healthcare needs from medicinal plants (mukhtar et al., 2008) . there has been a significant rise in using overthe-counter medicinal plant products containing powerful medicinal drugs and are believed to have to produce progressive effects with reduced side effects. however, therapeutic failures or adverse effects have been observed in many cases as pharmacological mechanisms of the herbal mixtures/preparations are not well-studied. the most important concern involving the use of medicinal plants is to identify and standardize the exact method of preparation of an extract, identification of active ingredients and details of administration (yip & kwan, 2006) . in this relationship, the screening and characterization of other undiscovered herbal products in traditional medicine is needed. the integration of the therapeutic use of traditional chinese medicinal knowledge with the synthetic and traditional oriental medicinal knowledge is a key area of research (cho & leung, 2007) . however, medicinal plants cultivated in different geographical regions are believed to differ in therapeutic effects in different diseases and infections. for example, a. membranaceus used in chinese traditional 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delivery system effects of salvianolic acid a on oxidative stress and liver injury induced by carbon tetrachloride in rats lycium barbarum polysaccharide attenuates alcoholic cellular injury through txnip-nlrp3 inflammasome pathway schisandrin b attenuates the inflammatory response, oxidative stress and apoptosis induced by traumatic spinal cord injury via inhibition of p53 signaling in adult rats the protective effect of tinoridine against carbon tetrachloride hepatotoxicity nanoparticles formulation of cuscuta chinensis prevents acetaminopheninduced hepatotoxicity in rats hepatoprotective and antioxidant effects of cuscuta chinensis against acetaminopheninduced hepatotoxicity in rats salvia miltiorrhiza bunge and its active component cryptotanshinone protects primary cultured rat hepatocytes from acute ethanol-induced cytotoxicity and fatty infiltration molecular identification of astragalus membranaceus at the species and locality levels hepatitis c virus genotypes in the united states: epidemiology, pathogenicity, and response to interferon therapy. collaborative study group systematic review of the renal protective effect of astragalus membranaceus (root) on diabetic nephropathy in animal models extraction, chemical analysis of angelica sinensis polysaccharides and antioxidant activity of the polysaccharides in ischemia-reperfusion rats levistilide a inhibits angiogenesis in liver fibrosis via vascular endothelial growth factor signaling pathway phytochemical and pharmacological profile of vitex negundo modern study of traditional chinese medicine schisantherin a protects against liver ischemia-reperfusion injury via inhibition of mitogen-activated protein kinase pathway selected hepatoprotective herbal medicines: evidence from ethnomedicinal applications, animal models, and possible mechanism of actions key: cord-309795-2kozsv4z authors: dewidar, bedair; kahl, sabine; pafili, kalliopi; roden, michael title: metabolic liver disease in diabetes – from mechanisms to clinical trials date: 2020-06-20 journal: metabolism doi: 10.1016/j.metabol.2020.154299 sha: doc_id: 309795 cord_uid: 2kozsv4z abstract non-alcoholic fatty liver disease (nafld) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (nash) and fibrosis/cirrhosis and may lead to end-stage liver failure or hepatocellular carcinoma. nafld is tightly associated with the most frequent metabolic disorders, such as obesity, metabolic syndrome, and type 2 diabetes mellitus (t2dm). both multisystem diseases share several common mechanisms. alterations of tissue communications include excessive lipid and later cytokine release by dysfunctional adipose tissue, intestinal dysbiosis and ectopic fat deposition in skeletal muscle. on the hepatocellular level, this leads to insulin resistance due to abnormal lipid handling and mitochondrial function. over time, cellular oxidative stress and activation of inflammatory pathways, again supported by multiorgan crosstalk, determine nafld progression. recent studies show that particularly the severe insulin resistant diabetes (sird) subgroup (cluster) associates with nafld and its accelerated progression and increases the risk of diabetes-related cardiovascular and kidney diseases, underpinning the critical role of insulin resistance. consequently, lifestyle modification and certain drug classes used to treat t2dm have demonstrated effectiveness for treating nafld, but also some novel therapeutic concepts may be beneficial for both nafld and t2dm. this review addresses the bidirectional relationship between mechanisms underlying t2dm and nafld, the relevance of novel biomarkers for improving the diagnostic modalities and the identification of subgroups at specific risk of disease progression. also, the role of metabolism-related drugs in nafld is discussed in light of the recent clinical trials. finally, this review highlights some challenges to be addressed by future studies on nafld in the context of t2dm. nonalcoholic fatty liver diseases (nafld) is currently defined by lipid deposition that exceeds more than 5% of hepatocytes, as assessed from liver biopsy, and/or by more than 5 .6% hepatocellular fat content per liver weight, as assessed from magnetic resonance (mr) methods, in the absence of significant alcohol consumption and other causes of fatty liver [1, 2] . nafld, which affects about 25% of the population [3] , comprises a broad range of abnormalities ranging from simple fatty liver (steatosis) to non-alcoholic steatohepatitis (nash), characterized by inflammation, necrosis, and hepatocellular ballooning, and progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma (hcc) [2] . some gene variants promote risk of nafld by altering lipid droplet formation and de novo lipogenesis (dnl), such as variants of patatin-like phospholipase domain-containing protein 3 (pnpla3) and glucokinase regulatory protein [4] , or by decreasing very-low-density lipoproteins (vldl) export as shown for a missense mutation (e167k) in transmembrane 6 superfamily member 2 (tm6sf2) [5] . nevertheless, nafld is tightly associated with common acquired metabolic diseases such as obesity and type 2 diabetes (t2dm). the mutual relationship between both diseases is illustrated by several epidemiological data. the prevalence of steatosis and nash has been estimated to be 50 and 56%, respectively, in t2dm [6] . the age-adjusted relative risk of nafld is about 5.36fold higher in t2dm compared to healthy humans [7] . t2dm is also an emerging risk factor for nash progression to advanced fibrosis, cirrhosis and hcc [8, 9] . diabetes was even a better predictor for hcc development in people with nash and cirrhosis compared to other metabolic risk factors such as hyperlipidemia, body mass index (bmi) and hypertension [10] . recently, a consensus panel has proposed to rename nafld a metabolic-dysfunction-associated fatty liver disease (mafld) based on the presence of overweight/obesity, t2dm and evidence of so-called "metabolic dysregulation" [11] . future will tell, if this will help to better understand the multiple relationships between nafld and t2dm. in this context, nafld per se associates with more than double risk of incident diabetes pointing to specific liver-related mechanisms [12, 13] . moreover, multicenter studies in skandinavia and germany have recently found that diabetes can be stratified into subtypes j o u r n a l p r e -p r o o f the circulation. on the contrary, a recent study demonstrated that obesity-induced insulin resistance preceded inflammation in adipose tissues of mice [29] . indeed, adipose tissue inflammation might be a protective feedback mechanism as its local inhibition in mice induced ectopic lipid accumulation in liver, glucose intolerance, and systemic inflammation [30] . besides released inflammatory cytokines and ffa, adipose tissue could still communicate with the liver and muscle through secretion of different adipokines such as adiponectin and leptin. persons with nash have lower serum adiponectin compared to those with nafld with or without normal liver enzymes [31] . by contrast, the circulating levels of leptin were higher in people with nafld and t2dm, probably due to increased leptin resistance, and were associated with disease severity [32] . increased ffa influx to skeletal muscle promotes accumulation of intramyocellular lipid (imcl). reduced mitochondrial oxidation contributes as well to imcl as shown in aging and insulin-resistant humans [33] . consequently, skeletal muscle exhibits insulin resistance, which often precedes the onset of t2dm and insulin resistance in the liver [34] . lipid intermediate metabolites, in particular sn 1,2 diacylglycerols (dag), link imcl to skeletal muscle insulin resistance through activation of protein kinase c-theta (pkcθ) resulting in its translocation from cytoplasm to the plasma membrane [35] . muscles of insulin resistant humans with obesity and t2dm showed increased dag content and pkcθ activity as compared to healthy humans [35] . mutation studies highlighted serine amino acid residue (ser1101) of irs1 to be a substrate for activated pkcθ [36] . as a consequence of skeletal muscle insulin resistance, postprandial energy storage shifts from glycogen synthesis in the muscle into triacylglycerol (tag) in the liver, promoting nafld development [25] . gut microbiome is increasingly recognized as a modulator of liver pathogenesis through what is called the "gut-liver axis" [37] . distinctive alterations of gut microbiome were reported in humans having nash and t2dm [38] . the intestinal microbiome alters host metabolism by modulating the production of short-chain fatty acids (scfa) e.g. butyrate, acetate, and propionate, which have beneficial effects on insulin sensitivity, lipid and glucose metabolism [38] . also, intestinal dysbiosis could associate with increased intestinal permeability permitting translocation of bacterial lipopolysaccharide (lps) into the systemic circulation, j o u r n a l p r e -p r o o f 6 which could induce fat deposition in the liver, nash progression, weight gain, and diabetes [39] . moreover, intestinal microbiome could suppress the expression of fasting-induced adipocyte factor (fiaf) in intestinal epithelium, which functions as an inhibitor of circulating lipoprotein lipase, resulting in increased tag storage in the peripheral tissues [22] . also, ethanol-producing microbiome could increase blood alcohol concentration in nash, which is metabolized in the liver generating high levels of reactive oxygen species (ros). the last mechanism could explain the histological similarity between nash and alcoholic steatohepatitis [22] . furthermore, microbiota metabolize liver-derived primary bile acids into secondary bile acids. the latter are reabsorbed into bloodstream and may act as signaling molecules via a variety of receptors including farnesoid x receptor (fxr), which regulates the transcription of different metabolic genes involved in bile acid synthesis, transport, lipogenesis, and glucose homeostasis [40] . insulin resistance in both skeletal muscle and adipose tissues initiates liver steatosis by providing precursors and substrates for dnl and mitochondrial β-oxidation e.g. glucose, ffa and glycerol [25] . although reesterification of ffa derived from diet and adipose tissue is the dominant contributor to tag pool in the liver (59%), it did not increase in people with nafld. on the other side, dnl-derived ffa contribute by about 26%, which is severalfold higher as compared to individuals without nafld (10%) [41] . later, insulin resistance of the liver develops, resulting in increased gluconeogenesis and elevation of endogenous glucose production (egp) from the liver, which contributes to fasting hyperglycemia in individuals with t2dm [25] . insulin signaling inhibits typically hepatic gluconeogenesis through akt-induced phosphorylation of forkhead box (foxo1) and induces lipogenesis through activation of sterol regulatory element-binding proteins (srebp1c) and mammalian target of rapamycin complex (mtorc1) pathways. during hepatic insulin resistance, insulin does not suppress gluconeogenesis efficiently, while dnl is preserved or even increased. to explain this discrepancy, pathway-selective hepatic insulin resistance was postulated, which means that only one arm of insulin signaling is defective i.e. akt/foxo1 leading to reduced insulin-mediated suppression of gluconeogenesis, whereas insulin-activated srebp-1c/mtorc1 pathway remains intact and activates lipogenesis [28] . actually, dnl was reduced after induction of hepatic insulin resistance by feeding mice with j o u r n a l p r e -p r o o f 7 a high-fat diet (hfd) [42] , which challenges the concept of selective hepatic insulin resistance and suggest the existence of alternate mechanisms that contribute to increased dnl and gluconeogenesis in insulin-resistant liver, for example, hyperinsulinemia, insulinindependent re-esterification of adipose tissue-derived ffa, and increased acetyl coa generation from β-oxidation of ffa, which allosterically activates pyruvate carboxylase enzyme, leading to enhanced gluconeogenesis [25] . in addition, increased blood glucose level can activate carbohydrate response element-binding protein (chrebp) signaling pathway, thereby stimulating expression of several glycolytic genes, which provide additional metabolic precursors for dnl [43] . in line, chrebp overexpression induced stearoyl-coa desaturase 1 (scd1) expression, an enzyme responsible for the biosynthesis of monounsaturated fatty acids (mufa), resulting in increased liver fat content [44] . pkc epsilon (pkcε) is crucial in mediating hepatic insulin resistance and once activated by dag, it translocates to the cell membrane, and phosphorylates specific threonine residue (thr1160 in human and thr1150 in mouse) on insulin receptor leading to destabilization of the active configuration of insulin receptor kinase and inhibition of its tyrosine kinase activity [45] (figure 2 ). in general, both hyperglycemia and toxic lipids such as ceramides, dag, ffa, and cholesterol can induce deleterious effects on liver cells (glucolipotoxicity), which might initiate nafld progression from simple steatosis to nash and fibrosis via various mechanisms, including cell death, oxidative stress, endoplasmic reticulum (er) stress and mitochondrial disorders [46] . alterations in the activity and abundance of oxidative phosphorylation (oxphos) proteins and antioxidant enzymes were described in various animal models of nafld [47] . impaired hepatic mitochondrial function was evident as well in t2dm and nash [48, 49] . in a mouse model of choline-deficient diet-induced nafld, mitochondrial oxphos was increased at 12 weeks but lost at a later time point [50] . also, the higher maximal respiration rate of liver mitochondria was severalfold higher in insulin-resistant obese individuals with fatty liver as compared to lean individuals [51] . these studies highlight the flexibility of mitochondria to adapt to increased metabolic inputs to keep energy homeostasis and to protect against the harmful effects of increased ffa and tag in the liver. in nash, mitochondrial flexibility was lost, which was associated with increased ros production and exhaustion of protective antioxidant enzymes [51] (figure 2 ). whether loss of mitochondrial flexibility is a cause or consequence for nafld progression remains obscure. depletion of atp due to j o u r n a l p r e -p r o o f mitochondrial disorders, together with hyperglycemia and lipid overload could be inducers for another signaling pathway, termed unfolded protein response "upr", which is adaptive response to resolve unfolded or misfolded proteins and to restore er homeostasis [52, 53] . prolonged upr stress can activate jnk and nf-kb signaling pathways, which are involved in insulin resistance, liver steatosis, and inflammation [52] . furthermore, er stress could increase insulin resistance through induction of lipin-2 expression, which is a phosphatase enzyme that catalyzes biosynthesis of dag leading to activation of dag/pkcε axis [54] . interestingly, upr could also increase liver steatosis through activation of srebp-1c signaling pathway [53] . elevated ros and upr are well-identified pathways that could induce hepatocytes death in nash. hepatocytes apoptosis and necroptosis are the main forms of cell death in human steatohepatitis and diet-induced mouse models of nash [55] . the key fibrogenic liver cells, hepatic stellate cells (hscs), usually exist in a quiescent state and get activated by engulfment of apoptotic bodies, the inflammatory milieu, or damage-associated molecular patterns (damps) released from stressed and dying hepatocytes [56] . interestingly, ffa-mediated lipotoxic effects stimulate hepatocytes to release extracellular vesicles (evs) with distinctive micrornas (mirna) profile that increase the expression of fibrogenic genes in the surrounding hscs [57] (figure 3 ). free cholesterol could directly sensitize hscs to the action of tumor growth factor (tgf)-β, a potent fibrogenic cytokine [58] . treatment of human and rat immortalized hscs cell lines with saturated fatty acid increased the expression of various profibrogenic genes [59] . macrophages aggregate as well around dead hepatocytes forming a crown-like structure, a phenomenon that exists only in persons with nash but not in those with simple steatosis [60] . recruitment of more inflammatory cells from systemic circulation is facilitated by "find me" signals that are released from dead cells [61] . inhibition of inflammatory monocytes recruitment via inhibition of c-c chemokine receptors type 2 and type 5 (ccr2/ccr5) suppressed fibrogenesis and steatohepatitis in murine nash [62] . macrophages can modulate also hepatic insulin resistance and favor tag accumulation in hepatocytes through secretion of il-1β that downregulates peroxisome proliferator-activated receptor (ppar) α, a key transcriptional pathway involved in fatty acid oxidation [63] . liver sinusoidal cells (lsecs) are fenestrated cells that exist in close vicinity to hepatocytes, j o u r n a l p r e -p r o o f macrophages and hscs, which, under physiological conditions, regulate lipid transport, maintain the quiescence of kupffer cells, resident liver macrophages, and hscs [64] . at early stage of nafld, lsecs lose their fenestrae, a process termed capillarization, which could favor liver steatosis and initiate hscs activation [64] (figure 3 ). during nash, lsecs display a pro-inflammatory phenotype that promotes steatohepatitis [64] . autophagy is a self-degradative process which is used by the cells to remove misfolded proteins and damaged organelles [65] . singh et al. showed that the cells can use autophagic process as lipolytic mechanism to mobilize lipids from intracellular lipid store, which is termed in this case "macrolipophagy" [66] . various in vitro and in vivo studies showed that autophagy was decreased in fatty hepatocytes [67] . impairment of autophagy by palmitic acid in macrophages induces inflammatory il-1β production [68] . on the other side, selective loss of autophagic activity reduced liver fibrogenesis in cultured hscs [69] and protected against diet-induced insulin resistance [70] . the results highlight that the net effect of autophagy on nafld might depend on the tissue or type of the cells that show autophagic dysfunction and the stage of nafld. the liver biopsy is considered the gold standard for nafld diagnosis, especially for distinguishing steatosis from inflammation and fibrosis [2] . nevertheless, this technique has several limitations not only resulting from the invasive procedure and rare post-interventional complications, but also due to the small tissue volume, which might not be representative for the whole liver and may not take into account inhomogeneous distribution of nafldassociated alterations. novel imaging modalities such as ultrasound-or mr-based techniques are of increasing value, as recently reviewed [71] , but are still not generally available, so that there is an urgent need for noninvasive screening tools. non-invasive detection of nash and fibrosis remains challenging today. biomarker-based panels such as aspartate aminotransferase (ast)/platelets count ratio index, fibrosis-4 (fib-4) index, fibrotest, fibrospect ii, and nafld fibrosis score (nfs) offer variable diagnostic efficacy for assessing different stages of liver fibrosis [73, 74] . although combination of these panels could enhance their predictive values [73] , they still suffer from limited accuracy even compared to the alanine aminotransferase (alt) and ast, particularly in t2dm [75] . in this regard, transient elastography looks like a promising alternative in diabetes clinics for detection of liver fibrosis in nafld [74] . numerous biomarkers have been developed to specifically track features of nash progression and fibrosis. cytokeratin (ck) 18, an intermediate filament protein that is cleaved during cell death to ck18 m30 and ck18 m65, has been intensively investigated as a surrogate of nash-associated liver cell damage, but a recent meta-analysis of 25 studies reported a maximum sensitivity of 0.75 for nash diagnosis [76] and suboptimal diagnostic value was shown in t2dm [75] . the ecm turnover marker, type iii procollagen, can offer a diagnostic efficacy of 0.81 and 0.88 to detect moderate and advanced liver fibrosis in t2dm, respectively [77] . in 2016, the european associations for the study of the liver, obesity and diabetes (easl-easo-easd) jointly released recommendations for diagnosis and monitoring of disease severity in persons with suspected nafld and metabolic risk factors [78] . people with metabolic risk factors, such as t2dm, should undergo assessment by abdominal ultrasound, serum transaminases and fibrosis markers (e. g. fib-4, nfs). elevated transaminases or steatosis plus abnormal fibrosis test shall require referral to a specialist. this strategy has raised the question of a possible overreferral when adhering to these guidelines [79] . however, recent analyses show that a refined strategy of specific combining indices such as fli and fib-4 could reduce the number of people with t2dm for further diagnostic work up to a reasonable size [80] . this retrospective analysis also found that certain non-invasive biomarkers are consistently associated with different patterns of diabetes-related complications. analyses of the plasma metabolomic of insulin-resistant individuals with nafld suggested that bile salts, e. g. glycocholate, taurocholate, and glycochenodeoxycholate allow to detect nash in one study [81] , while another study reported an association with insulin resistance j o u r n a l p r e -p r o o f but not with liver necroinflammation [82] . also amino acids, specifically a glutamate-serineglycine index, was associated with hepatic insulin resistance and transaminases and discriminated individuals with fibrosis grade 3-4 from those with grade 0-2 independently of bmi [83] . the diagnostic performance of a serum-based lipidomic analysis was substantially lower for nafld detection in t2dm compared to healthy individuals [84] . nevertheless, certain sphingolipid species were recently found to be increased in insulin-resistant nash and to correlate with hepatic oxidative stress and inflammation [85] . one circulating small noncoding rna, mirna-122, was found to be more than 5.7fold in steatosis and further doubled in nash [86] . this mirna was also higher in t2dm with nafld than in those without nafld and provided better prediction of nafld when combined with ldl and waist-to-hip ratio [87] . moreover, extracellular vesicles (ev), which among other cargo also transport mirna, may be promising biomarkers for nafld as shown by higher serum levels in people with nafld, obesity and diabetes [107] . recently, metagenomics data derived from gut microbiota alterations allowed to detect advanced fibrosis in 86 nafld patients, of whom 23% had t2dm, with a robust diagnostic accuracy (auroc: 0.936) [88] . the current guidelines recommend only lifestyle modification and -for certain groupsbariatric or metabolic surgery to treat nafld [1, 2] . although the numerous activities in this field, no pharmacological treatment is currently approved or expecting approval for the use in nafld with or without concomitant t2dm (table 1 and 2), except for obeticholic acid (oca). marketing authorization application for oca has been submitted to the u.s. food and drug administration (fda) and european medicines agency (ema), awaiting fda decision by june 2020 [89] . against this background, current guidelines and recommendations primarily advise lifestyle modification (healthy nutrition and exercise) and weight loss in overweight/obese persons. the easl-easo-easd guidelines recommend to consider pharmacological treatment in people with nash when combined with fibrosis and in those with less severe disease, but high risk conditions for disease progression such as t2dm [78] . the american association for the study of liver diseases (aasld) recommends to limit pharmacological treatment to those with biopsy-proven nash and fibrosis [1] . j o u r n a l p r e -p r o o f weight management and physical exercise are key to the treatment of both nafld and t2dm. a proof-of-concept study showed that a hypocaloric diet with weight loss of ~8 kg within 12 weeks not only normalized fasting egp and thereby hyperglycemia, but decreased hepatic tag content down to normal concentrations in obese t2dm humans with nafld [90] . subsequent studies in larger cohorts extended these results by demonstrating that a very low-caloric diet with weight loss of about 15% rapidly normalized liver fat content in 90 in dividuals with t2dm, which persisted for one year if weight loss was maintained [91] . interestingly, mediterranean, low-saturated fat and high plant-based protein diets also improve steatosis in nafld combined with t2dm despite minor or no relevant weight loss [92, 93] . in addition, physical activity and exercise training interventions can also decrease liver fat content, which may not be exclusively depending on concomitant weight loss [94] . although the beneficial effects of structured behavioral treatment to improve histological endpoints, likely extend beyond reduction of hepatic fat content to ameliorating the grade and stage of inflammation and fibrosis [95] , only a minority of the people manages to adhere to dietary weight loss and exercise programs, which raises the issue of other therapeutic approaches [94] . weight-loss inducing drugs could be an attractive option for persons with nafld with a bmi > 30 kg/m 2 or >27 kg/m 2 in the presence of at least one metabolic comorbidity, as an adjunct treatment to lifestyle modifications [96, 97] . currently, the most-popular weight-loss inducing medications associated with at least 5% body weight decrease in one year as compared to placebo are orlistat, the fixed combination of phentermine and topiramate or naltrexone and bupropion, and the glucagon-like peptide-1 (glp-1) receptor agonist (glp-1ra), liraglutide [97] . of these drugs, orlistat treatment failed to improve liver histology when compared to placebo [98] , while no reports are available for topiramate, naltrexone, bupropion and phentermine regarding hepatic endpoints in humans with nafld [99] . only bariatric or metabolic surgery is a therapeutic option to induce sustained weight loss partricularly in people with combined nafld and t2dm. in obese humans, bariatric surgery resulted in 85% resolution of nash within one year [101] and in 77% complete remission of t2dm [102] . surgical weight loss improves glucose metabolism and insulin sensitivity by different mechanisms such as increased glp-1 secretion [103] and epigenetic modification [104, 105] . several antihyperglycemic drug classes were or are currently being investigated in clinical trials and preclinical models to evaluate their efficacy for people with nafld and with or without t2dm. metformin reduces body weight, hepatic gluconeogenesis -by yet unclear mechanisms [106] , and the risk of macrovascular complications, which is still controversially discussed due to lack of optimally designed clinical trials [116] . despite its action on hepatic metabolism, former small-scale studies reported conflicting results [109, 110] (table 1 ) and recent metaanalysis failed to demonstrate any effect of metformin on liver histology [111] . nevertheless, epidemiological, observational and preclinical studies suggest that metformin may reduce the risk of hcc and also in t2dm, possibly by promoting apoptosis, but controlled clinical trials are not available [112] . dpp4 degrades incretins such as glp-1 so that dpp-4i treatment increases the postprandial levels of endogenous glp-1, which leads to lower glucose peaks after meals [113, 114] . in individuals with nafld with prediabetes or recent onset diabetes, sitagliptin did not improve liver steatosis or liver fibrosis compared to placebo as assessed by mr-based techniques [115] . in line, another recent trial reported no benefits for sitagliptin versus placebo on liver fibrosis or steatohepatitis in t2dm [116] (table 1) . in contrast, a moderate reduction in liver fat content was observed with vildagliptin in individuals with t2dm [117] . j o u r n a l p r e -p r o o f 14 the actions of endogenous glp-1 are mimicked by glp-1 receptor agonists (glp-1ra), which effectively reduce blood glucose levels and also reduce the risk for cvd in t2dm [118] . in individuals with nafld and t2dm, liraglutide in combination with metformin reduced liver, subcutaneous, and visceral fat [119] . also, liraglutide improved hepatic steatosis measured by mr-based methods as well as resolved biopsy-proven nash without worsening of fibrosis [100, 120] (table 1) . however, a recent subanalysis did not detect an effect of liraglutide on liver fat content quantified by 1 h-mrs [121] (table 1) . respective trials with semaglutide and dulaglutide are still waiting for results (nct02970942, nct03648554). in an animal model of t2dm, exenatide also counteracted hcc development by suppression of stat3-regulated genes [122] . glucose-dependent insulinotropic polypeptide (gip) represents the second important incretin with proposed beneficial effects on peripheral energy metabolism [123] . tirzepatide, a novel dual agonist for gip and glp-1 receptors with probably greater efficacy than glp-1ra [124] , decreased transaminases and surrogate markers of liver injury paralleled by increased circulating adiponectin levels in people with t2dm [125] . a clinical trial on nash resolution is currently ongoing in persons with nash with or without t2dm (nct04166773). sglt2i inhibit reabsorption of glucose in the proximal renal tubule resulting in glucosuria and calory loss, thus effectively reducing blood glucose level and body weight in t2dm. moreover, sglt2is show beneficial effects on cardiovascular risk and progression of nephropathy [118, 126] . most, but not all recent randomized controlled trials (rcts) showed that sglt2i treatment resulted in reduction in liver fat content compared to placebo [127] [128] [129] [130] [131] (table 1) . in an open-label pilot study in liver-biopsy proven cohort of nash with t2dm, treatment with empagliflozin for 6 months reduced liver steatosis, ballooning and fibrosis and induced nash resolution in approximately half of those persons [132] . animal models further suggested anti-inflammatory, anti-oxidant and pro-apoptotic actions of sglt2i with cardio-protective effects as well as inhibition of nash progression and tumor growth of hcc [133, 134] . the dual sglt1/2i, licogliflozin, is expected to block both intestinal and renal glucose (re)absorption [135] and currently investigated to evaluate its efficacy on resolution of nash as monotherapy and as combination therapy with the fxr agonist, tropifexor, in people with nash and fibrosis (nct04065841). first results hint at improvement of transaminases and reduction of steatosis by licogliflozin in nash [136] . ppars is a family of nuclear receptors composed of multiple isoforms with wide tissue distribution [137] . the ppar-γ agonist pioglitazone had convincing efficacy on nash resolution in individuals with and without t2dm, but with conflicting results on fibrosis [138] [139] [140] (table 1) . of note, pioglitazone has been withdrawn in many european countries because of its disadvantageous safety profile [231] . however, pioglitazone exerts beneficial effects on cardiovascular outcomes in persons with t2dm and a history of cvd [141] . it has been proposed that non-ppar-γ dependent mechanisms, as the inhibition of the mitochondrial pyruvate carrier (mpc), might mediate the beneficial effects of pioglitazone on nafld [142] . however, a novel drug with ppar-γ sparing effects and mpc binding activity did not improve histological components of nash in individuals with t2dm compared to placebo [143] . elafibranor is a dual agonist for ppar-α and ppar-δ without ppar-γ stimulation [144] . based on a post-hoc analysis, nash was resolved without worsening of fibrosis in a higher percentage of people with and without t2dm in the elafibranor group as compared to the placebo [145] and a follow-up study on these findings has been initiated ( table 2) . recent announcements on an interim analysis state that elafibranor treatment failed to resolve nash and improve fibrosis when compared to placebo [146] . saroglitazar is another dual ppar-α/γ agonist with higher affinity for ppar-α. in a mouse model of nash, saroglitazar reduced liver steatosis, inflammation and prevented fibrosis development and a respective clinical trial is ongoing [147] ( table 2) . lanifibranor is a pan-ppar agonist for ppar-α, β, and γ receptors focused on treatment of liver fibrosis and nash (nct03008070). in an animal model of liver fibrosis, lanifibranor decreased hepatic collagen deposition [148] . several other strategies for pharmacological targeting of nash have emerged over the last few years; however, these strategies are not specifically designed for t2dm, but for the whole nafld collective. they comprise antiinflammatory drugs, but also modulators of other pathways, which are briefly summarized in the following sections. j o u r n a l p r e -p r o o f fxr can be activated by bile acids in a negative feedback mechanism to suppress bile acid synthesis [40] . oca is a potent semisynthetic and selective fxr agonist approved for treatment of primary biliary cholangitis [149] . oca treatment resulted in histologic improvement of nash in people with and without t2dm compared to placebo [150, 151] ; however, concerns were raised about oca-induced changes in plasma lipoprotein profile [152] . tropifexor, another potent fxr agonist [153] , is currently being tested in combinatorial approaches of nash treatment (cenicriviroc and licogliflozin; nct03517540). oltipraz is a synthetic dithiolethione with antisteatotic effects by inhibiting the activity of lxr-α. it activates adenosine monophosphate activated protein kinase (ampk) and inactivates s6k1, affecting lxr-α thus reducing lipogenesis and increasing lipid oxidation [154] . a recent 24-week phase 2 clinical trial found decreased steatosis measured by 1 h-mrs with oltipraz compared to placebo treatment [154] and a respective phase 3 clinical trial is ongoing ( table 2 ). chemokine receptors type 2 (ccr2) and type 5 (ccr5) are expressed on various inflammatory and fibrogenic cells [155] . cenicriviroc is a ccr2/crr5 dual antagonist that reduced insulin resistance, liver inflammation and fibrosis in diet-induced models of nash [62] . in recent rcts in a nash cohort with fibrosis, cenicriviroc treatment did not improve nas but may reduce liver fibrosis [156, 157] . currently, there is an ongoing clinical trial to evaluate the effects of cenicriviroc on fibrosis in nash ( table 2) . both people with obesity, metabolic syndrome or t2dm as well as those with nafld are at increased risk for dyslipidemia. statins, inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme a (hmg-coa) reductase, are generally safe and have unmet efficacy to decreased serum ldl and prevent cardiovascular outcomes, despite slightly increasing the risk of t2dm [2] . use of statins associated with a 46% lower relative risk of hepatic decompensation and mortality in cirrhosis and a trend towards lower fibrosis progression in non-cirrhotic liver diseases [158] . however, the data on liver histology ist limited [159] so that statins are not currently recommended for the management of nafld [2] . the inhibitor of intestinal j o u r n a l p r e -p r o o f cholesterol absorption, ezetimibe, decreased the histological nas, but not consistently liver fat content in an analysis of the few available studies [160] . fenofibrate, a ppar-α agonist, does not affect or even increase liver fat content or volume [161, 162] . nevertheless, the combination of statins with certain anti-nash drugs, such as oca, could be beneficial to counteract drug-related increases in ldl during long-term treatment. inhibition of dnl in nafld may be achieved by inhibition of acc as this enzyme catalyzes the conversion of acetyl coa into malonyl coa, which acts as a substrate for fatty acid synthesis and inhibitor for fatty acid β-oxidation [163] . in a recent clinical trial in individuals with nash with and without t2dm, treatment with the dual acc1 and acc2 inhibitor gs-0976 decreased liver steatosis without improvement of fibrosis [164] . the major concern of this strategy is that decreased dnl in nafld might channel lipids towards other harmful directions, e.g. increased blood lipids [163] . scd1 is a key enzyme for hepatic lipogenesis that catalyzes the conversion of saturated fatty acids to mufa and its downregulation protected mice from high carbohydrate-induced liver steatosis [165] . a clinical trial with aramchol, an inhibitor of scd1, showed a reduction in liver fat content, resolution of nash and improvement of liver fibrosis in persons with nafld with prediabetes or t2dm [166] . these results paved the way for a respective phase 3 clinical trial ( table 2) . mitochondrial uncoupling describes any process that uncouples the electron transport from atp synthesis in mitochondria [167] . 2,4-dinitrophenol (dnp) was widely used for the treatment of obesity before its discontinuation due to life-threatening serious adverse events [168] . to overcome the side effects of dnp, improved formulas of dnp were recently developed to decrease the toxic to effective dose ratio (dnp-methyl ether (dnpme) and controlled-release mitochondrial protonophore (crmp)) [169, 170] . oral crmp targets mainly the liver due to the first-pass effect and decreased hepatic insulin resistance, hepatic steatosis and liver fibrosis in a methionine-choline deficient rat model of nash [170] . crmp was also effective in nonhuman primates with diet-induced nafld for reduction of hepatic steatosis and egp [171] . thyroid hormone deficiency has been associated with nafld development [172] . thyroid hormone analogues decreased liver fat, liver transaminases, and inflammatory and fibrosis markers in animal models of nash [172] . resmetirom is a liver-targeted highly selective thr-β agonist which showed efficacy in reducing liver fat content in nash with and without t2dm [173] . currently, there is ongoing clinical trial for evaluation of long-term outcomes of resmetirom and its efficacy on nash resolution ( table 2 ). vitamin e is a potent antioxidant [174] . in nash without t2dm, vitamin e improved nas without worsening of fibrosis [139] . despite concerns regarding adverse effects of long term vitamin e usage, treatment with vitamin e for ≥2 years reduced the risk of liver failure in a nash cohort with advanced fibrosis and with or without t2dm [175] . therapeutic manipulation of intestinal microbiome is still in its infancy. rodent data showed efficacy for fecal microbiota transplantation in improving nafld in a diet-induced nash model [176] . also, modulation of the intestinal microbiota by antibiotic treatment reduced liver transaminases in nafld [177] . the use of prebiotics and probiotics in obese nafld/nash is currently not supported by high-quality clinical studies with mr-or biopsy-based endpoints [178]. the evidence for shared pathophysiological mechanisms between t2dm and nafld will help to develop strategies for detecting and treating both diseases and preventing their leading complication, cvd. altered lipid and energy metabolism, insulin resistance, low-grade inflammation and intestinal dysbiosis represent key targets. in addition to weight loss by lifestyle modification or bariatric surgery, glp-1ra and sglt2i are promising antihyperglycemic concepts with beneficial effects on nafld and cvd. in addition, many j o u r n a l p r e -p r o o f metabolism-based drugs are currently studied comprising ppar agonists, endocrine dual coagonists to modulators of hepatic metabolism or microbiota. nevertheless, several roadblocks need to be overcome to reduce the burden of nafld in t2dm. first, there is still lack of preclinical animal models that encapsulate essential features of human nash and diabetes. second, available biomarkers lack diagnostic efficacy to identify nafld progression. innovative strategies such as cluster analysis already enabled detection of a diabetes subtype (sird) with high risk of nafld [14] . combination with computational integration of multiomics data shall identify specific disease signatures and pave the way to precision medicine and targeted management of t2dm and nafld. finally, studies on so-called endpoints are scarce, which may be due to the need of long-term studies to evaluate liver-related mortality, to the current neglect to accept cvd morbidity and mortality as nafld outcome and to ongoing discussions on the relevance of surrogate markers. the research of the authors is supported in part by grants from the german federal ministry phase iii registered interventional trials on "clinicaltrials.gov" for nafld as accessed on 10 th april 2020. bl, baseline; ccr2/5, c-c chemokine receptors type 2 and type 5; fxr, farnesoid x receptor; hba 1c , glycated haemoglobin; lxr, liver x receptor; mpc, mitochondrial pyruvate carrier; na, data not available; nafld, non-alcoholic fatty liver disease; nfs, nafld fibrosis score; ppar, peroxisome proliferator-activated receptor; nash, non-alcoholic steatohepatitis; scd, stearoyl-coa desaturase; sglt, sodium-glucose cotransporter; thr, thyroid hormone receptor; t2dm, type 2 diabetes. *only placebo-controlled, randomized clinical trials are 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reduces hepatic de novo lipogenesis and steatosis in patients with nonalcoholic steatohepatitis hepatic stearoyl-coa desaturase-1 deficiency protects mice from carbohydrate-induced adiposity and hepatic steatosis one-year results of the global phase 2b randomized placebo-controlled arrest trial of aramchol, a stearoyl coa desaturasemodulator in nash patients n mitochondrial uncoupling and lifespan emerging pharmacological targets for the treatment of nonalcoholic fatty liver disease, insulin resistance, and type 2 diabetes reversal of hypertriglyceridemia, fatty liver disease and insulin resistance by a liver-targeted mitochondrial uncoupler controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats controlledrelease mitochondrial protonophore (crmp) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates nonalcoholic fatty liver disease and hypercholesterolemia: roles of thyroid hormones, metabolites, and agonists mgl-3196) for the treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial nonalcoholic fatty liver disease and diabetes: part ii: treatment vitamin e improves transplant-free survival and hepatic decompensation among patients with nonalcoholic steatohepatitis and advanced fibrosis total fecal microbiota transplantation alleviates high-fat diet-induced steatohepatitis in mice via beneficial regulation of gut microbiota efficacy of rifaximin on circulating endotoxins and cytokines in patients with nonalcoholic fatty liver disease key: cord-271635-tydlyc1q authors: abdel-hamid, nabil m.; abass, shimaa a.; mohamed, ahmed a.; muneam hamid, daniah title: herbal management of hepatocellular carcinoma through cutting the pathways of the common risk factors date: 2018-11-30 journal: biomedicine & pharmacotherapy doi: 10.1016/j.biopha.2018.08.104 sha: doc_id: 271635 cord_uid: tydlyc1q abstract hepatocellular carcinoma (hcc) is considered the most frequent tumor that associated with high mortality rate. several risk factors contribute to the pathogenesis of hcc, such as chronic persistent infection with hepatitis c virus or hepatitis b virus, chronic untreated inflammation of liver with different etiology, oxidative stress and fatty liver disease. several treatment protocols are used in the treatment of hcc but they also associated with diverse side effects. many natural products are helpful in the co-treatment and prevention of hcc. several mechanisms are involved in the action of these herbal products and their bioactive compounds in the prevention and co-treatment of hcc. they can inhibit the liver cancer development and progression in several ways as protecting against liver carcinogens, enhancing effects of chemotherapeutic drugs, inhibiting tumor cell growth and metastasis, and suppression of oxidative stress and chronic inflammation. in this review, we will discuss the utility of diverse natural products in the prevention and co-treatment of hcc, through its capturing of the common risk factors known to lead to hcc and shed the light on their possible mechanisms of action. our theory assumes that shutting down the risk factor to cancer development pathways is a critical strategy in cancer prevention and management. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. the mortality rate due to hepatocellular carcinoma (hcc) increased rapidly during the past decade. unluckily, the clinically satisfactory and successful treatment for hcc patient is still absent [1] . several risk factors are involved in hepatocarcinogenesis like non-alcoholic fatty liver disease (nafld), hepatitis b virus (hbv) and hepatitis c virus (hcv) infection, alcoholism, obesity, aflatoxin b1, iron accumulation and diabetes [2] . there are several protocols used in the treatment of hcc, including, surgical resection, ablation, chemotherapy and embolization. the use of these methods is limited due to their side effects and the development of resistance to the available chemotherapy and their complexities. due to the limited treatment options to hcc, other than surgery and the poor prognosis of the disease, there is a critical need for additional therapies to enhance the survival or the quality of life. complementary and alternative medicine (cam) is considered as one way that may improve the anticancer drug efficacy and reduce their toxic effects [3] . the use of herbal medicines can be traced back to more than 4000 years ago in ancient china and egypt. over recent decades, an increasing number of herbal products, including medicinal herbs and phytochemicals, have been used for treating chronic liver diseases worldwide due to cost effectiveness, higher safety margins, long-lasting curative effects and few adverse effects. according to the previous studies, medicinal herbs and phytochemicals could protect the liver by several mechanisms such as eliminating the virus, blocking fibrogenesis, inhibiting oxidative injury and suppressing tumorigenesis [4] . in this review, we discuss several factors that lead to hcc development, focusing on the role of different herbal medicines that used in the treatment of hcc by alleviating these risk factors. is similar to the fatty liver damage that caused by alcoholism, but, it happens in non-alcohol abuse people. nafld is characterized by the accumulation of triglycerides within hepatocytes, which is usually associated with metabolic syndrome and obesity [5] . the prevalence of nafld was established by the histological features found in ∼70% of obese individuals suffering from steatosis, while steatosis was present in ∼ 35% of lean individuals. nafld was also present in about18.5% of obese individuals and in about 2.7% of leans [6] . fatty liver is also found in about 13-22% of lean non-alcoholic individuals through several studies based on ultrasound imaging [7, 8] . nafld is present in 20-35% of adult individuals and 5-17% of children in the western world [9] . nafld is considered as one of the important reasons leading to chronic liver diseases in hong kong (∼27.3%) [10] and china (∼15%) [11] . this is because of the high-fat content in the modern diet and individual's lifestyle. nafld is considered as one of the most important reasons that cause chronic liver disease in developing and developed countries. nafld increases the risk of hepatocarcinogenesis similar to other pot cirrhotic liver diseases. hcc is now the end stage as a leading cause of obesity-related cancer deaths in middle-aged men in the usa [12] . an increasing number of case reports showed that hcc arises in non-cirrhotic individuals suffering from nafld [13] . other hcc risk factors may be synergistically involved in hcc development besides nafld, such as alcoholic liver injury and chronic hepatitis c. several mechanisms are involved in nafld-related hcc development ( fig. 1 ) [14] [15] [16] . obesity participates in increasing the risk of cancer development through a low-grade, chronic inflammatory impact [17, 18] . the expansion of adipose tissue stimulates the generation of pro-inflammatory cytokines such as tumor necrosis factor (tnf) and interleukin-6 (il-6) [19] . tnf and il-6 derived from adipose tissue play an important role in hcc development. this role has been supported in an experimental model, assuming that obesity enhances the growth of diethylnitrosamine-induced malignant liver tumor in mice [20] . the prevalence of hcc due to nafld is increasing around the world [21] , where, 4-22% of hcc patients in western countries are attributable to nafld [22] . in asia, viral hepatitis remains endemic, so that, 1-2% of hcc patients are attributed to nafld [23, 24] . wolfberry, a famous traditional chinese supplement or drug, is the fruit part of lycium barbarum plant, family solanaceae. it has valuable benfits in both eyes and liver [26] . the most important part of wolfberry is the lycium polysaccharide portion(lpp), which has a numerous biological actions, like immunoregulation, antioxidant effect, neuroprotection, control of glucose metabolism and anti-tumor activities. the lymphocyte number, interleukin-2 and immunoglobulin g level, were found to be increased upon the intake of polysaccharide juice in human beings in one of the clinical trials. it was reported also that it increases the levels of serum antioxidants and decreases the lipid peroxide level [27, 28] . in the liver, lpp was found to inhibit hepatocyte proliferation and induce apoptosis of hepatoma cells, which indicate its possible application as anti-tumor [29, 30] . another study demonstrated that lpp causes a restoration of the activities of antioxidant enzymes and reduction of oxidative stress products caused by high-fat diet induced liver injury [31] . the co-treatment of lpp with ethanol administration markedly enhanced the liver injury in an alcohol-induced liver injury rat model by decreasing the oxidative stress and the accumulation of lipid in the liver [32] . in acute liver injury, lpp was found to keep the normal hepatic histology, decrease the hepatic inflammation/chemoattraction, stimulate the partial regeneration of the liver through the nuclear factor kappa b (nf-κb)-dependent pathway, and reduce the oxidative stress when used prior ccl 4 intoxication in mice [33] . lpp is helpful in nafld due to its useful properties in decreasing the inflammation and the oxidative stress. the co-treatment with lpp, orally, in nafld in rats, showed a significant improvement in the hepatic histology, reduction in the fibrosis, oxidative stress, inflammation, accumulation of fats and apoptosis, through modulating the transcriptional factors nf-κb and activator protein-1 (ap-1). furthermore, the uptake of lpp for long-term did not have any unwanted side effects on the liver of healthy rats. so lpp can be useful in nafld treatment with minimal side effects [25] . green tea (camellia sinensis) leaves, has been reported to be used in the prevention of liver diseases. the origin of green tea is china, which then distributed to asian countries, including korea, vietnam and japan. in the last years, green tea also spread to western countries that traditionally consume black tea. the beneficial or therapeutic properties of green tea extracts have been reported by several studies. the major polyphenol of green tea, epigallocatechin-3-gallate (egcg), was used in ccl 4 -treated mice and showed a significant therapeutic potential in hepatic damage, inflammation and oxidative stress induced by ccl 4 in a dose-dependent manner at both biochemical and histological levels [34] . it was also reported that co-treatment of the whole green tea extract with alcohol administration showed an effective reduction of the hepatic oxidative stress and reduced form of nicotinamide adenine dinucleotide phosphate (nadph) oxidase systems in experimental alcohol-induced liver injury [35] . green tea extract also showed an enhancement of the nash features, such as oxidative stress, inflammation and lipid accumulation in obese mice [36] . a recent study also demonstrated that green tea extract showed significant improvement in inflammatory, chemical, metabolic, and radiological parameters of nafld patients who were dyslipidemic and non-diabetic [37] . it also showed an improvement in the liver enzymes of nafld patients in another study [38] . numerous in vitro studies demonstrated the chemoprevention and anti-tumor properties of green tea in hcc [39] . the growth and cell proliferation of hcc-derived cells are inhibited by egcg by induction of apoptosis [40] . the human hcc cell line, hepg2, growth was also inhibited by egcg, through suppressing the phosphorylation of insulinlike growth factor-1 receptor (igf-1r) and reducing the activation of its signaling molecules like extracellular signal-regulated kinase (erk), stat-3, akt and gsk-3β [41] . drinking of egcg caused a significant inhibition of liver cell adenoma development in contrast to the egcguntreated control group. this is related to decreased phosphorylation of erk, akt and igf-1r proteins, activation of the hepatic amp-activated kinase protein and the treatment of liver steatosis. the administration of egcg in drinking water also caused a significant reduction of serum levels of insulin, igf-1, igf-2, tumor necrosis factor (tnf)-α, and free fatty acid. it also caused a decrease in the hepatic expression of tnf-α, interleukins(il-1β,il-6), and il-18 mrnas [42] . the development of glutathione s-transferase placental form (gst-p + ) foci was also inhibited after the administration of egcg in drinking water through the reduction of hepatic fibrosis, triglyceride content, inflammation, oxidative stress and inhibition of excessive hepatocyte proliferation [43] . resveratrol (rsv; 3, 5 4′-trihydroxystilbene), a phytoalexin that extracted from red grapes, is considered as one of the most accepted and recognized herbal derivatives worldwide as it has a powerful antioxidant and anti-inflammatory properties [44] . recently, rsv has been found to be helpful in the treatment of nafld. when rsv is given after nafld induction by using high fat-diet, it causes a reduction of lipogenic genes as srebp-1c and fas [45] . the treatment with rsv also reduced the inflammation and oxidative stress in rats [46] . rsv causes a dysregulation in the metabolism of lipids in nafld through sirtuin 1 (sirt1) pathway [47] and the up-regulation of hepatic low-density lipoprotein receptor [48] . rsv increases apoptosis in hcc cells, which is associated with the reduction of hexokinase 2 expression. additionally, rsv improved the inhibition of cell growth induced by sorafenib in aerobic glycolytic hcc cells. the inhibition of hexokinase 2 by rsv can be considered to be a new trend to treat hcc and prevent its progression [49] . rsv was also found to decrease the expression of myosin light chain kinase (mlck), which inhibited liver tumorigenesis and promoted cell apoptosis in hcc rats induced by dena. the expression of mlck was found to be higher in hcc rats than normal rats, which is responsible for the proliferation and anti-apoptotic properties [50] . the latin word "inflammation" means set a light or ignite, which describes exactly its effect on cancer. inflammation increases the resistance to chemotherapy and promotes oncogenes and genes that convert healthy cells to tumors. it also stimulates cancer cell spreading and improves the cancer cells' ability for angiogenesis. because cancer is defined as an inflammation, the anti-inflammatory drugs can be useful in treating cancers as the relation between cancer development and inflammation has been appreciated [51, 52] . the hepatic damage may be due to either chronic or acute inflammation. in response to inflammation, several kinds of hepatic cells are activated, such as hepatic stellate cells (hscs), liver sinusoidal endothelial cells (lsecs), kupffer cells (kcs) and dendritic cells (dcs) to produce several types of cytokines, immune mediators and chemokines. one of the important pro-inflammatory cytokines is interleukin-6 (il-6) that can inhibit apoptosis and tissue inflammation [53] . the inflammatory response mediates a defense mechanism against the microbial infection and stimulates tissue regeneration and repair. there is a relation between the inflammation and cancer development as chronic inflammation stimulates the development of dysplasia. about 15% of cancer occurrence is associated with microbial infection. in immunocompetent patients, chronic inflammation, like hepatitis b and c viral infection or human papilloma virus may result in the development of hepatocellular and cervical carcinoma, respectively [54] . cancer also may result from an opportunistic infection like kaposi's sarcoma, which results from human herpes virus (hhv)-8 infection. inappropriate immune responses to microbes may also lead to cancer development as gastric cancer, which may result from chronic inflammation due to helicobacter pylori colonization. the long-standing inflammatory bowel disease may lead to colon cancer. long-term exposure to asbestos, silica and cigarette smoke, may lead to chronic irritation and subsequent inflammation that result in cancer development [51, 52] . the promotion of tumor cells requires both the survival of the initiated cells and their expansion. numerous inflammatory mediators like chemokines, eicanosoids, and cytokines have the ability to stimulate the proliferation of both untransformed and tumor cell [51] . inflammation plays an important role in tumor growth through angiogenesis mediation. it also plays a critical role in other aspects of tumor progression like metastasis and tissue invasion. matrix metalloproteinases and their inhibitors are important for remodeling of extracellular matrix and angiogenesis, which enhances vascular invasion of migrating cells [55] .the mechanisms contributed to the role of inflammation in hepatocarcinogenesis was showed in fig. 2 [56] . xanthorrhizol (xnt), a sesquiterpenoid complex, obtained from curcuma xanthorrhizza rhizome, family zingiberaceae. the anti-inflammatory effect of xnt was reported for the first time in lipopolysaccharide-mediated mouse leukemic monocyte macrophage cell raw 264.7. it caused a significant reduction in the activities of inducible nitric oxide synthase (inos) and reduced cyclooxygenase-2 (cox-2), through the inhibition of nitric oxide (no) and prostaglandin e2 (pge 2) production [57] . xnt was also found to inhibit pro-inflammatory cytokine interleukin-6 (il-6), tnf-α, inos and cox-2 in activated microglial cells [58] . the anti-inflammatory property of xnt was also postulated in another study as it blocks the inflammatory and neurogenic pain response in pain test that induced by formalin in rats [59] . xnt has also anticancer activities, which may be due to its antiinflammatory effect by inhibiting the activity of nf-kb, cox-2 and inos release [60] . it also inhibited the tumor development and formation in different in vivo studies. it decreased the expression of cox-2, ornithine decarboxylase and suppressing nf-kb signaling activity. an in vivo study also demonstrated that xanthorrhizol has anti-metastatic activity through inhibiting matrix metallopeptidase 9 and cox-2 in a mice lung metastasis model [61] . it has a potent anti-proliferative effect on hepg2 cells through apoptosis induction via bcl-2 family members [62] . berberine is a small alkaloid molecule isolated from coptidis rhizome. it possesses anti-inflammatory and anticancer activities [63] . it down-regulates numerous hepatic pro-inflammatory genes such as il-6, serum amyloid a3 (saa3), nf-kb and tnf-α. these genes play a vital role in steatohepatitis development [64] . it was reported that berberine has an anti-inflammatory activity for hepatic cells in different animal models. it has also been showed to decrease tnf-α and cox-2 expression in cyclophosphamide-induced hepatotoxicity in a rat model [65] . it also has the ability to inhibit tnf-α and il-6 production in hepg2 cells. the anti-inflammatory effect of berberine may be due to the inhibition of erk1/2 activation [66] . its anti-inflammatory effect was postulated through the inhibition of lps-induced inflammatory response in macrophages [64] . berberine has anti-cancer activity on the human hcc cell lines through the induction of apoptosis and inhibition of tumor cell proliferation [67] . berberine induces both cell death and apoptosis in hepg2 cells. this is related to the down-regulation of cd147, which is highly expressed in hcc cells [68] . it was shown to selectively inhibit the human hepatocellular cancer cell growth through the induction of ampk-mediated caspase-dependent mitochondrial pathway cell apoptosis in addition to suppressing p53 [69] . the expression of p53 was found to be up-regulated by berberine through suppression of mdm2, the inner p53 inhibitor, at the post-transcriptional level [70] . the combination of berberine and vincristine showed a helpful effect against hepatoma cell lines through the potentiation of the pro-apoptotic effect of the single drug [71] . alpinia officinarum, known as lesser galangal, belongs to family zingiberaceae. it has a variety of pharmacological actions as antioxidant, anti-inflammatory, antimicrobial, antiemetic and cytotoxic properties [72] . different mechanisms are involved in the anti-inflammatory effect of alpinia officinarum, including the regulation of nf-κb, mapks pathway and the inhibition of prostaglandin e2 synthase and cox-2, the important enzymes involved in the inflammation. this effect is usually due to its content of diarylheptanoids and flavonoids [73] . two compounds of alpinia officinarum rhizome extract, galangin and 5-hydroxy-7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-3-heptanone, exhibited antioxidant and anti-inflammatory activities due to their phenolic content. these compounds showed a high affinity toward cox-2 active site through a molecular docking study [74] . our recent study also demonstrated that five compounds isolated from the alpinia officinarum rhizome extract showed a powerful anti-inflammatory effect on hepg2 cells stimulated by lipopolysaccharide. these compounds are galangin, isorhamnetin, two diarylheptanoids and kaempferide. these compounds down-regulated the gene expression of il-6, il-1β, pro-inflammatory cytokines and tnf-α in a dose-dependent manner. this indicates that these isolated compounds may be a promising treatment for other inflammatory diseases [75] . alpinia officinarum rhizome extract and its components showed anticancer activities against numerous cancer cell lines, such as breast, neuroblastoma, lung, and liver [76] [77] [78] . our previous study suggested that alpinia officinarum rhizome extract can be used as a promising natural chemopreventive agent against hcc in rats. it is also a helpful chemosensitizing agent when used in combination with cisplatin in the treatment of hcc. additionally, alpinia officinarum rhizome extract improved hepatic functions and decreased alpha-fetoprotein concentration in experimental hcc model. it also protected the hepatic tissue of the treated rats [79] . oxidative stress (os) occurs when the body is exposed to either a harmful exogenous or endogenous per-oxidative stimuli. one of these stimuli is free radicals, including reactive nitrogen species (rns) and reactive oxygen species (ros). free radicals are produced during numerous oxidation-reduction (redox) reactions that happened in the cells. os is usually associated with the development of various diseases as cardiovascular and nervous system diseases, diabetes and cancer through the induction of oxidative damage of dna and abnormal expression of proteins [80] . os may be a risk factor for hcc as it elevates hepatocyte dna oxidative damage [81] . chronic viral infections may increase the production of ros and rns by causing inflammation and necrosis of hepatocytes that accompany immune cell infiltration [82] . the main cause of dna damage is mutation, caused by increasing ros and the failure in dna repair resulting in an increase in the mutation of cancer-related genes, meaning that chronic inflammation is considered the main risk factor for hcc [83, 84] . increasing os and the degree of dna damage has elevated the incidence of hcc related to viral infection [85] . the elevated level of transforming growth factor beta (tgfβ) and also tnf-α in patients with chronic hepatitis c is mainly related to oxidative stress. tgf-β is used as an indicator of how tissues are damaged and the extent to which they are injured [86] . ros is elevated due to increased oxidative stress, which stops the electron transport chain in damaged mitochondria. the excessive release of tnf-α not only causes severe damage to the mitochondrial respiratory chain but also causes damage to cytochrome oxidase. the high level of ros increases lipid peroxidation and inhibits the respiratory electron transport chain [87] . ros not only alter the mitochondrial metabolic activity but also affects the apoptotic pathways, changes the membrane permeability and causes damage to mitochondrial dna [88] . telomere shortening is accelerated by os that leads to an increase in the cytoplasmic migration of reverse transcriptase telomerase subunits [89] . damaging of the cells, mainly by os, causes the release of a unique type of dna that is called free circulating dna (fcdna), produced as oxides released by dna from dead cells. it can be used as an indication for a wide group of diseases like cancer. fc dna also can be present by low value in normal cells [90] . during the exposure to os, different types of genes and immune system mediator expression is altered. among these mediators, is micro rna (mirna), which is a small non-coding rna molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in rna silencing and post-transcriptional regulation of gene expression. mirna dysfunction is related to different types of cancer including hcc [91] . the mechanisms of hcc development caused by os can be summarized in fig. 3 [89]. 4.1.1. vitamin c and diallyl sulfide vitamin c, (l-ascorbate, l-ascorbic acid), is known by its antioxidant activity and the ability to capture free radical with singlet oxygen such as oh − or hoo − and superoxide ion o 2 − and produce dehydroascorbate. vitamin c activity is related to its free electron that interacts with electron deficient ions. several in vitro studies reported the antioxidant activity of vitamin c [92] . in vivo studies also showed the antioxidant properties of vitamin c in a dose-dependent manner. vitamin c administration protects guinea pigs, that do not form vitamin c, and osteodystrophy syndrome rats from oxidative stress when given oxidizing agents as endotoxin [93] and carbon tetrachloride [94] . it also protects rats exposed to cigarette smoke [95] . parquet is highly toxic nitrogen herbicide and has strong oxidizing stress. the administration of vitamin c reduces oxidative stress before (not after) parquet administration [96] . vitamin c has a promising role in cancer treatment through its selective cytotoxic activity for diverse cancer cell lines [97, 98] . in 1970s, a clinical study was performed and proved that ascorbate has an important role in increasing the survival of cancer patients at late stages [99] . combination of methotrexate with vitamin c is promising in cancer treatment through the reduction of h 2 o 2 produced from methotrexate in hep3b cells [100] . it was shown that vitamin c helped in the treatment of hepatotoxicity induced by dena in smp30 ko mice [101] . garlic contains diallyl sulfide (das) as a major active constituent. das is characterized by its anti-inflammatory activity through the modulation of cytokines as it inhibits the activity nf-kb [102] . das' anti-inflammatory activity is related to the nuclear factor erythroid 2-related factor 2 (nrf2) transcription activation and it also has antioxidant activity. nrf2 is an emerging regulator of cellular resistance to oxidants [103] . a combination of vitamin c and das was shown to offer several benefits, as inhibition of circulatory tnf-α and il-6 in dena-induced hcc in rats [104] and increases the sensitivity to chemotherapy as cisplatin in the treatment of hcc [105] . ginger (zingiber officinale) is one of the predominant herbaceous plants. it is a perennial plant and the main active part is the rhizome. ginger not only used as a condiment but also it has antiemetic and anticancer activity [106] . ginger has strong antioxidant and cell protection activity. this action of ginger is due to its potent active constituents as sesquiterpenoids, tannin, gingerols, shogaols and anthocyanin. several in vitro and in vivo studies documented the antioxidant activity of ginger. the protective effect of ginger was showed against several toxic agents, like bromobenzene and cisplatin [107] . another study displayed the chemopreventive effect of ginger against cancer [108] . ginger has a great activity in the treatment of experimental cancer in a rat model. it decreased the level of growth factors and αfetoprotein (liver tumor marker) after giving rats a daily dose about 50 mg/kg of ginger extract [109] . the anticancer effect of ginger is due to it's proapoptotic and anti-inflammatory properties. the anti-inflammatory activity of ginger was confirmed by inhibiting nf-κb and tnf-α after administration of 100 mg/kg of ginger in hcc rat model [110] . ginger contains other different constituents as clavatol, pinostrobin, and geraniol. these active components were detected by gas chromatography and mass spectrometry. ginger was found to inhibit the proliferation of cells in hepg-2 cell line (ic50, 900 μg/ml) [111] . one of the most popular active components in ginger is 6-shogaol which showed an anticancer effect against hepatoma cell line through the activation of ros-mediated caspase-dependent apoptosis in a multidrug resistance [112] . one of the most popular cultivated plants is broccoli which is distinguished by its high content of the antioxidant content. the most active antioxidant components in this plant are vitamins, flavonoids and carotenoids. isothiocyanates, the hydrolytic product of glucosinolate, is considered one of the antioxidant components, which motivates dna protection from damage through its antioxidant activity [113, 114] . the antioxidant property of broccoli may be direct by contributing in biochemical, cellular and physiological steps that inhibit free radical production, or indirect by inducing phase ii enzymes that have a protective effect against os [115] . the antioxidant activity of broccoli was observed in the human colon mucosa that exposed to oxidative stress [115] . broccoli showed a potential anticancer activity due to its high content of glucosinolates [116] . broccoli also contains a distinct component, sulforaphane, which is characterized by its activity as antioxidant and its ability to protect dna from breaking down by highly reactive electrophiles through increasing the antioxidant system activity and inhibition of inflammation [117] . antioxidant activity of sulforaphane is related to certain pathways, including the reduction of inflammation through inhibiting nf-κb and overexpression of transcription of nrf2, which has a very important role in keeping healthy cells and protect them from toxic chemicals and lifestyle-related factors [118] . previous in vivo studies registered that sulforaphane has abroad activity against different types of liver diseases related to toxic chemicals [119, 120] , consumption of alcohol [121] and using high calorie food [122] . broccoli has a major role in the suppression of different types of cancers, including liver hep-g2 and colon cancer. sulforaphane has different pathways related to its anticancer effect as it has anti-inflammatory, proapoptotic and cell cycle arresting action [123] . hbv and hcv infections are considered the chief reasons for hcc. usually, there are no symptoms for people with chronic infection but lately; cirrhosis and hcc are developed [124] . treating and overcoming hcv and hbv infection can help in the prevention of hcc development as they are oncogenic viruses [125] . the association between hcv infection and hcc varies worldwide. in western countries and africa, hcv is considered as the main cause of hcc, it also contributes to 80% to 90% of hcc cases in japan [126, 127] . as well as,80% of patients infected with hcv can progress to chronic hepatitis, with about 20% developing cirrhosis [128] . the hcv -related liver cirrhosis can increase the risk of liver cancer, with17-fold higher risk of developing hcc than in chronic hepatitis c infection alone, although this risk differs and depends on the degree of liver fibrosis caused by hcv [129] . the elevation of the risk of hcc development in patients infected with hcv arises from chronic inflammation, which results from the progression of liver fibrosis and cirrhosis. these inflammations cause alteration of the architecture of hepatocyte and defects of both cellular functions and the microcirculation of liver. hcv rna does not integrate into the host genome. alternatively, hcv viral proteins like hcv core protein and their induced host response have been involved in reactive oxygen species (ros) production, apoptosis, activation of transcription and modulation of immunity through up-regulation of tnf-α, il-6, and il-1, which participate in the transformation into malignancy [130] . hepatitis b virus (hbv) is a circular genome. chronic hbv infection can be confirmed by the presence of serum hbsag for a period not less than 6 months [131] . about 10%-25% of hepatitis b patients have a high risk of hcc development during their life. chronic hepatitis differs than other causes of hcc, as hcc occurs in the absence of cirrhosis [132] . after tobacco smoking, hbv is considered the second environmental carcinogen that affects individuals, resulting in about 55% of all hcc cases around the world [133] . the association of chronic hbv infection and hcc that now widely recognized was first explained by beasley and colleagues in 1981 [134] in taiwanese patients with positive serum hb surface antigen (hbsag). serum hbsag can be detected in 24%-27% of patients with hcc in japan, 41% of patients with hcc in the united states and 70% of patients with hcc in china in the absence of other risk factors [135] . once hbv arrives at the liver cell, transcription of messenger viral rnas occurs, followed by a translation into viral proteins and then, synthesis of dna of the virus. dna of the virus is then capable of integrating into the host genome in infected hepatocytes. cancer can be facilitated via this process through numerous ways, like rapid cell cycling of hepatocytes and viral dna integration into the host genome which causes instability and it may insert into, or adjacent to, genes that code proteins required for cancer development. it also leads to a chronic inflammation with fibrosis and proliferation of hepatocyte, which ultimately result in cirrhosis and cancer development [136] [137] [138] . andrographis paniculata nees (a. paniculata) is a medicinal plant, which belongs to family acanthaceae. it is used in japan, india, korea, china and other asian countries for a long time in the treatment of several diseases like inflammations, viral and bacterial infections and high blood pressure [139] . the most abundant di-terpene lactone found in the leaves and stems of a. paniculata is andrographolide. the andrographolide treatment was found to reduce the replication of hcv markedly and have a synergistic effect with the clinical trial drug psi-7977 or current antiviral drugs like telaprevir and ifn-α when used in combination to treat hcv. the mechanism of action of andrographolide fig. 3 . the mechanism of hcc development due to oxidative stress [89] . may be due to its ability to induce the p38 mapk/nrf2/ho-1 pathway, where, mapk stands for mitogen activated protein kinase and ho-1 is heme oxygenase-1. it was shown that andrographolide can be used as natural product or potential drug that is helpful in the treatment of hcv [140] . the treatment with a. paniculata aqueous extract was found to enhance the activity of hepatic enzymes and normalizes histopathological changes of malignant hepatic tissue induced by hexachlorocyclohexane [141] . it showed indirect and direct effects on tumor cells by inducing apoptosis and cancer cell necrosis, improving body's own immune system against tumor cells and inhibiting cell-cycle arrests and cancer cells proliferation [142] . the ethanolic extract of a. paniculata showed a cytotoxic effect against diverse human cancer cell lines like pc-3 (prostate), hepg2 (hepatoma), colon 205 (colonic) cancer cells and jurkat (lymphocytic) [143] . the inhibitory effect of andrographolide and its analogs on tumor cells may be due to their ability to depress cyclin-dependent kinase and induce the expression of inhibitory proteins of the cell cycle that result in blocking the cell cycle progression at g0/g1 [144] . andrographolide causes induction of apoptosis by several mechanisms including, the activation of caspase cascade, the release of cytochrome c from mitochondria and the activation of proapoptotic bcl-2 family members bax conformational change [145] . it also causes activation of ros-dependent c-jun nh 2 -terminal kinase (jnk) resulting in the activation of tumor suppressor p53 and thereby increasing p53 phosphorylation and protein stabilization [146] . silymarin is extracted from milk thistle seeds, silybum marianum l. gaertn., which belongs to asteraceae family [147] . the extract of silymarin contains silibinin, which consists of a mixture of two flavonolignans called silybin a (sa) and silybin b (sb). it has diverse pharmacological activities including, antioxidant, antiproliferative, immunomodulatory, antiviral activities and antifibrotic in different tissues and organs [148] [149] [150] . silymarin and its component silibinin possess antiviral activity against hcv infection in cell culture. their antiviral activity is due to their ability to block the entry of the virus, the synthesis of viral rna and protein, viral fusion, virus transmission and the activity of hcv n5sb rna dependent rna polymerase [148, 149, 151, 152] . one study showed that the treatment with a soluble form of silibinin in the form of daily intravenous injection causes a significant inhibition of hcv viral loads by 3-4 logs in 1-2 weeks in previous ifn non-responder patients [153] . several studies reported that silymarin has a potential anticancer effect against hcc. in a dose-dependent manner, silymarin inhibits the population growth of the human hepatocellular cancer cells (hepg2) as it elevates the percentage of apoptotic cells [154] . the antiproliferative activity of silymarin was also reported by another study without affecting the nontumor hepatic cells. in the g0/g1 phase, silymarin caused an increase in the percentage of cells, while in the s-phase it decreased the cell percentage associated with down-regulation of cyclin e, cyclin d1, phospho-rb and cdk4 and up-regulation of p53, retinoblastoma protein (rb), p27kip1, and p21cip1 [155] . silymarin also showed in vivo preventive and therapeutic efficiency against liver cancer. ramakrishnan et al. [156] reported that silymarin has a protective effect against dena-induced hcc in rats. silymarin also showed a potent preventive effect against spontaneous hcc in hbv x protein transgenic mouse model. oral silymarin in a dose-responsive manner causes a restoration of the early stage hepatic damage and fatty changes that lead to the recovery of hepatic tissue [157] . the oral administration of silibinin showed a significant reduction of hcc xenograft growth by inducing histone acetylation, apoptosis and expression of sod1 and inhibiting cell cycle progression, cell proliferation (ki-67 expression), erk and pten/p-akt signaling [158] . glycyrrhiza glabra, a perennial herb, originates from south-western and central asia and the mediterranean region. it showed numerous pharmacological activities like antioxidant, anti-inflammatory and immunomodulatory activities. the main constituent of glycyrrhiza glabra root is glycyrrhizin 1-9%, w/w [159] . glycyrrhizin has anti-viral, antiinflammatory, hepatoprotective and anti-tumor activities [160] . the antiviral activity was reported for glycyrrhizin and other components that isolated from glycyrrhiza species against different viruses, such as herpes simplex, hiv, severe acute respiratory syndrome, influenza virus, coronavirus, enteroviruses and hepatic viruses [161] [162] [163] . glycyrrhizin has been reported to be used in the treatment of hepatic diseases like chronic hepatitis c and b [164] . a preparation that contains glycyrrhizin was reported to reduce hepatic steatosis in transgenic mice expressing the full-length hcv poly-protein [165] . it was shown to have an inhibitory effect on hcv core gene expression and hcv fulllength viral particle both at protein and rna level and have a synergistic effect with interferon [166] . glycyrrhizin and other components of glycyrrhiza glabra showed antitumor activity in different kinds of cancers such as skin, liver and breast cancer, through inhibition of cellular proliferation, development and growth of cancer cells [167] . glycyrrhizic acid, a major bioactive component of the extract of glycyrrhiza glabra, has the ability to inhibit hcc occurrence in dena-treated mice [168] . another study showed that glycyrrhiza glabra extract has a potent effect in the treatment of hcc induced by dena/ccl4 in rats and this effect is more potent than the effect of cisplatin alone or cisplatin combined with glycyrrhiza glabra, so that cisplatin has several side effects and glycyrrhiza glabra is not associated with that side effects [169] . apoptosis, or programmed cell death, has a great interest in the field of oncology [170] . the recognition of each pathway of apoptosis is very important not only in understanding cancer development but also in the prevention and treatment of the disease. the normal tissue homeostasis maintained by keeping the balance between the proliferation of cells and their death. the imbalance between these two processes may lead to dysregulated clonal expansion, the cause of all neoplastic diseases [171, 172] . the mechanism of action of numerous cytotoxic agents includes apoptosis. several experimental approaches aimed to stimulate apoptosis that leads to the improvement of therapeutic response. numerous natural products play a vital role in the regulation of cellular proliferation and differentiation. the chemopreventive and chemotherapeutic activities of natural products may be due to their role in mediating different pathways involved in cancer development and progression [173] . 6.1. herbals with cytotoxic activity 6.1.1. nigella sativa nigella sativa (n. sativa), an annual flowering plant, originates from south and southwest asia and northern africa is grown almost all over the world [174] . n. sativa and its main constituent thymoquinone (tq) possess numerous therapeutic and pharmacological activities like antiinflammatory [175] , anticancer [176] , antioxidant [177] and immunomodulatory activities [178] . it has cytotoxic activity, as the ethyl acetate column chromatographic fraction of the ethanolic extract of n. sativa showed a cytotoxic effect against diverse cell lines such as molt4, hep g2 and ll/2 [179] . another in vitro studies showed about 50% cytotoxicity of a crude methanolic extract of n. sativa against dalton's lymphoma ascites (dla), ehrlich ascites carcinoma (eac) and sarcoma-180 cells (s-180 cells) [180] . its anti-cancer activity is due to its ability to exhibit powerful pro-apoptotic, anti-proliferative, anti-mutagenic, anti-metastatic and anti-oxidant effects. it also can inhibit tumor initiation and progression and has an anti-inflammatory and immunomodulatory effect. n. sativa can regulate signaling pathways like p53, inos and caspases [181] . the anti-tumor activity of n. sativa was reported in several in vivo and in vitro studies. a decoction that consists of seeds of n. sativa, smilax glabra rhizome, and hemidesmus indicus roots showed a significant improvement in the hepatocarcinogenesis induced by dena(4-6 g/kg/day) in rats [182] . the ethanolic extract of n. sativa showed a marked enhancement of dena-induced histopathological variations of the hepatic tissue [183] . another in vivo study showed that the administration of a methanolic extract of n. sativa in hcc albino rat model showed modulation of glucoregulatory enzymes [184] . aqueous extract of n. sativa showed in vitro antiproliferative activity and morphological changes like membrane damage and cell shrinkage in hepg2 cells, which lead to dna damage, cell death and a decrease in cell proliferation [185] . the mechanisms that explain the pharmacological effects of nigella sativa can be summarized in fig. 4 [186]. illicium verum (i. verum) hook belongs to family illiciaceae. it is commonly known as chinese star anise or star anise. it is an aromatic evergreen tree that originates from pakistan, china and other asian countries. due to its low toxic effects to humans, it was classified as "food and medicine" in 2002 by the ministry of health, people's republic of china [187] . the main active constituents that present in i. verum are sesquiterpenoids, monoterpenoids, lignans, phenylpropanoids, volatile compounds, and flavonoids. it also contains tannins, bitter principles and essential oils. these essential oils include transanethole, limone, α-pinene, β-phellandrene, farnesol, safrol and α-terpineol [188] . it also possesses antimicrobial, antioxidant, antifungal, analgesic, anti-inflammatory, sedative, insecticidal and anticonvulsive activities [189] . its cytotoxic activity was also reported in several studies [190] [191] [192] . it showed numerous mechanisms that involved in cell death such as apoptosis induction, scavenging of free radicals and tumor metastasis inhibition [191] . it was reported that alcoholic extract of i. verum showed a significant in vitro antiproliferative activities [190] . similar in vitro study also showed a marked inhibition of cell proliferation by its alcoholic extract by promoting apoptosis, growth inhibition and modulating the pro-apoptotic gene expression like bax and p53 [193] . separately, the cytotoxic effect of i. verum extract was studied in liver cancer model,it exhibited a significant anticancer outcome in hepatic tissue of rats with a significant improvement of tumor burden (decrease of nodule incidence, multiplicity, size, volume and liver weight). it also up-regulated phase ii detoxifying enzymes (glutathione-s-transferase) and decreased oxidative stress by restoration of superoxide dismutase activity [194] . sex has a vital role in hcc development as males are more diagnosed for hcc than females, with a ratio of 2:1-4:1. this may be due to the higher susceptibility of males to be infected with viral hepatitis, smoking, consuming higher amounts of alcohol and have a higher body mass index than females. the higher level of testosterone is associated with advanced hepatic fibrosis in males infected with hcv and hcc in hepatitis b carriers [195, 196] . another potent hepatocarcinogen is aflatoxin, which produced by aspergillus species found on corn, grains, soybeans or peanuts that stored in warm humid conditions [197] . several genetic and metabolic diseases are associated with hcc development such as wilson's disease, hemochromatosis, α-1 antitrypsin disease, glycogen-storage disease types i and ii, porphyrias and tyrosinemia [198] . other factors are also reported to be associated with the marked elevation of hcc development such as cigarette smoking and prolonged use of contraceptive pills [199] . diabetes mellitus is now considered as an independent risk factor for hcc [200, 201] . it causes liver cell damage through hyperinsulinemia and insulin resistance [202] . hyperinsulinemia induces hcc through inflammation, cellular proliferation and apoptosis inhibition. as well, the increase in insulin levels can lead to a decline in the synthesis of insulin growth factor binding protein 1 by the liver, which is supposed to cause an increase in the bioavailability of insulin-like growth factor 1, in addition to the fig. 4 . the cellular and molecular mechanisms of n. sativa that explain its pro-apoptotic, antiproliferative, cytotoxic, anti-oxidant, anti-metastatic, anti-mutagenic and nk-mediated effects. (lsa: lipid-bound sialic acid, tsa: total sialic acid, tnfα: tumor necrosis factor α, mda: malondialdehyde, afp: α-fetoprotein, il-6: interleukin-6, ros: reactive oxygen species, no: nitric oxide, gsh: glutathione, t-pa: tissue-type plasminogen activator, ifn-γ: interferon-γ, u-pa: urokinase-type plasminogen activator, pai-1: plasminogen activator inhibitor type 1) [186] . increase in apoptosis inhibition and cellular proliferation [203] . insulin has also been related to increased oxidative stress and the ros production, participating in dna mutation [204] . efficient storage of cereals liable to aspergillus attack, continuous inspection of both male and female sex hormones in suspected individuals, managing storage diseases as wilson's and hemochromatosis and controlling diabetes mellitus and other leading diseases will certainly contribute to a decline in hcc liability among risky individuals. the plants and their activities mentioned in this review could be summarized in table 1 : hcc is a prevalent disease in many countries around the world. it is highly related to the increase in deaths rate. the development of hcc passes through several intermediate steps such as molecular and transcriptional events that end finally to malignant transformation of hepatocytes. several factors are involved in these steps including; nafld, hcv, hbv, oxidative stress, chronic inflammation, some inborn metabolic errors, environmental toxins and some drugs, etc. accumulating evidence suggested that many dietary and natural products could be potential sources for prevention and treatment of liver cancer. these natural products (summarized in table 1 ) and their active ingredients can inhibit the liver cancer development and progression, through cutting the roads in front of the known leading risk factors for hcc. we here call urge the ministry of health in each country to establish records of liver patients, especially liver tumors. we recommend the use of these plants side by side to recent chemical medications and after stopping these chemicals, as a maintenance therapy to avoid hcc progression and decrease its global incidence. we also draw attention of specialists in the food industry to add some of these natural products in different recipes to reduce the probabilities of liver diseases infections. none. none. the anti-inflammatory, anti-oxidant, cytotoxic and immunomodulatory [175, 176, 177, 178, 179] illicium verum cytotoxic activity, antioxidant, anti-inflammatory, induction of apoptosis, and inhibition of tumor metastasis [189, 190, 191, 192] therapeutic applications of herbal medicines for cancer patients molecular pathogenesis of hepatic fibrosis and current therapeutic approaches traditional herbal medicine: a review of potential of inhibitory hepatocellular carcinoma in basic research and clinical trial current status of herbal medicines in chronic liver disease therapy: the biological effects, molecular targets and future prospects prevalence of nonalcoholic fatty liver disease in the united states: the third national health and nutrition examination survey fatty liver hepatitis (steatohepatitis) and obesity: an autopsy study with analysis of risk factors prevalence of and risk factors for hepatic steatosis in northern italy frequency of abnormalities detected by abdominal ultrasound among japanese adults prevalence of fatty liver in children and adolescents prevalence of non-alcoholic fatty liver disease and advanced fibrosis in hong kong chinese: a population study using proton-magnetic resonance spectroscopy and transient elastography epidemiology of non-alcoholic fatty liver disease in china risk of cancer in patients hospitalized with fatty liver: a danish cohort study non-alcoholic fatty liver disease as a risk factor for hepatocellular carcinoma: mechanisms and implications nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace obesity, inflammation, and insulin resistance inflammation and metabolic disorders lipid homeostasis, lipotoxicity and the metabolic syndrome dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing il-6 and tnf expression liver transplantation in patients with nonalcoholic steatohepatitis-related hepatocellular carcinoma hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis hepatocellular carcinoma in japanese patients with nonalcoholic fatty liver disease, alcoholic liver disease, and chronic liver disease of unknown etiology: report of the nationwide survey recent advances in the herbal treatment of non-alcoholic fatty liver disease use of anti-aging herbal medicine, lycium barbarum, against aging-associated diseases. what do we know so far? lycium barbarum (goji) juice improves in vivo antioxidant biomarkers in serum of healthy adults immunomodulatory effects of a standardized lycium barbarum fruit juice in chinese older healthy human subjects hot water-extracted lycium barbarum and rehmannia glutinosa inhibit proliferation and induce apoptosis of hepatocellular carcinoma cells effect of lycium barbarum polysaccharide on human hepatoma qgy7703 cells: inhibition of proliferation and induction of apoptosis chemical characterization of lycium barbarum polysaccharides and its inhibition against liver oxidative injury of high-fat mice the effect of lycium barbarum polysaccharide on alcohol-induced oxidative stress in rats lycium barbarum polysaccharides protect mice liver from carbon tetrachloride-induced oxidative stress and necroinflammation green tea polyphenols prevent toxin-induced hepatotoxicity in mice by down-regulating inducible nitric oxide-derived prooxidants depression by a green tea extract of alcohol-induced oxidative stress and lipogenesis in rat liver green tea extract protects against nonalcoholic steatohepatitis in ob/ob mice by decreasing oxidative and nitrative stress responses induced by proinflammatory enzymes therapeutic benefits of green tea extract on various parameters in non-alcoholic fatty liver disease patients, pak the effect of green tea extract supplementation on liver enzymes in patients with nonalcoholic fatty liver disease chemopreventive and therapeutic potential of tea polyphenols in hepatocellular cancer green tea constituent (-)-epigallocatechin-3-gallate inhibits hep g2 cell proliferation and induces apoptosis through p53-dependent and fasmediated pathways egcg inhibits activation of the insulin-like growth factor (igf)/igf-1 receptor axis in human hepatocellular carcinoma cells chemopreventive potential of green tea catechins in hepatocellular carcinoma epigallocatechin-3-gallate suppresses hepatic preneoplastic lesions developed in a novel rat model of non-alcoholic steatohepatitis therapeutic potential of resveratrol: the in vivo evidence resveratrol improves non-alcoholic fatty liver disease by activating amp-activated protein kinase resveratrol inhibits nonalcoholic fatty liver disease in rats resveratrol inhibits the expression of srebp1 in cell model of steatosis via sirt1-foxo1 signaling pathway alleviative effects of resveratrol on nonalcoholic fatty liver disease are associated with up regulation of hepatic low density lipoprotein receptor and scavenger receptor class b type i gene expressions in rats by reducing hexokinase 2, resveratrol induces apoptosis in hcc cells addicted to aerobic glycolysis and inhibits tumor growth in mice resveratrol down-regulates myosin light chain kinase, induces apoptosis and inhibits diethylnitrosamine-induced liver tumorigenesis in rats inflammation and cancer: back to virchow? inflammation and cancer stats in cancer inflammation and immunity: a leading role for stat3 infections as a major preventable cause of human cancer new functions for the matrix metalloproteinases in cancer progression role of nonresolving inflammation in hepatocellular carcinoma development and progression, npj precis suppressive effect of natural sesquiterpenoids on inducible cyclooxygenase (cox-2) and nitric oxide synthase (inos) activity in mouse macrophage cells xanthorrhizol inhibits 12-o-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-kappa b evaluation of the antinociceptive activity and acute oral toxicity of standardized ethanolic extract of the rhizome of curcuma xanthorrhiza roxb xanthorrhizol: a review of its pharmacological activities and anticancer properties antiproliferative property and apoptotic effect of xanthorrhizol on mda-mb-231 breast cancer cells xanthorrhizol induced dna fragmentation in hepg2 cells involving bcl-2 family proteins advances in the study of berberine and its derivatives: a focus on anti-inflammatory and anti-tumor effects in the digestive system berberine suppresses proinflammatory responses through ampk activation in macrophages berberine mitigates cyclophosphamide-induced hepatotoxicity by modulating antioxidant status and inflammatory cytokines berberine inhibits inflammatory response and ameliorates insulin resistance in hepatocytes berberine induces autophagic cell death and mitochondrial apoptosis in liver cancer cells: the cellular mechanism berberine induces cell death in human hepatoma cells in vitro by downregulating cd147 berberine induces selective apoptosis through the ampkmediated mitochondrial/caspase pathway in hepatocellular carcinoma berberine inhibits p53-dependent cell growth through induction of apoptosis of prostate cancer cells the combinational effect of vincristine and berberine on growth inhibition and apoptosis induction in hepatoma cells alpinia: the gold mine of future therapeutics a review on the pharmacological activities and phytochemicals of alpinia officinarum (galangal) extracts derived from bioassayguided fractionation and isolation isolates of alpinia officinarum hance as cox-2 inhibitors: evidence from anti-inflammatory, antioxidant and molecular docking studies isolates from alpinia officinarum hance attenuate lps-induced inflammation in hepg2: evidence from in silico and in vitro studies diarylheptanoids from the rhizomes of alpinia officinarum and their anticancer activity diarylheptanoids derived from alpinia officinarum induce apoptosis, s-phase arrest and differentiation in human neuroblastoma cells galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-bid mitochondrial pathway chemosensitizing effect of alpinia officinarum rhizome extract in cisplatin-treated rats with hepatocellular carcinoma autophagy, mitochondria and oxidative stress: crosstalk and redox signalling survival rate in patients with hepatocellular carcinoma: a retrospective analysis of 389 patients hepatitis, cirrhosis, and hepatoma radical causes of cancer bagging survival trees viralinduced human carcinogenesis: an oxidative stress perspective role of oxidative stress and dna damage in human carcinogenesis hesperidin alleviates acetaminophen induced toxicity in wistar rats by abrogation of oxidative stress, apoptosis and inflammation is mitochondrial dna content a potential biomarker of mitochondrial dysfunction? mitochondrion oxidative stress and liver cancer: etiology and therapeutic targets circulating tumor cells and circulating tumor dna oxidative dna damage correlates with cell immortalization and mir-92 expression in hepatocellular carcinoma vitamin c as an antioxidant: evaluation of its role in disease prevention endotoxin increases oxidative injury to proteins in guinea pig liver: protection by dietary vitamin c the effect of vitamin c on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production vitamin c supplementation on hepatic oxidative stress induced by cigarette smoke in vivo dual effects of vitamin c on paraquat-induced lung damage: dependence on released metals from the damaged tissue pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer vitamin c enhances anticancer activity in methotrexatetreated hep3b hepatocellular carcinoma cells preventive effects of vitamin c on diethylnitrosamine-induced hepatotoxicity in smp30 knockout mice garlic (allium sativum l.) modulates cytokine expression in lipopolysaccharide-activated human blood thereby inhibiting nf-kappab activity diallyl sulfide enhances antioxidants and inhibits inflammation through the activation of nrf2 against gentamicin-induced nephrotoxicity in wistar rats abdel-bakey, polyol profile as an early diagnostic and prognostic marker in natural product chemoprevention of hepatocellular carcinoma in diabetic rats vitamin c and diallyl sulfide as chemosensitizers to cisplatin in treating hepatocellular carcinoma cancer chemoprevention effects of ginger and its active constituents: potential for new drug discovery hepatoprotective, antioxidant, and ameliorative effects of ginger (zingiber officinale roscoe) and vitamin e in acetaminophen treated rats aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells ginger ingredients inhibit the development of diethylnitrosoamine induced premalignant phenotype in rat chemical hepatocarcinogenesis model ginger extract (zingiber officinale) has anti-cancer and anti-inflammatory effects on ethionine-induced hepatoma rats induction of apoptosis by ginger in hep-2 cell line is mediated by reactive oxygen species 6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism antioxidant functions of sulforaphane: a potent inducer of phase ii detoxication enzymes phenolic compounds in brassica vegetables antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion increasing antioxidant content of broccoli sprouts using essential oils during cold storage protective effect of sulforaphane against oxidative stress: recent advances sulforaphane-rich broccoli sprout extract improves hepatic abnormalities in male subjects protective effect of sulforaphane pretreatment against cisplatin-induced liver and mitochondrial oxidant damage in rats sulforaphane increases the survival rate in rats with fulminant hepatic failure induced by dgalactosamine and lipopolysaccharide sulforaphane induces nrf2 and protects against cyp2e1-dependent binge alcohol-induced liver steatosis sulforaphane attenuates obesity by inhibiting adipogenesis and activating the ampk pathway in obese mice antiproliferative effects and metabolism of sulforaphane and glucoraphanin from broccoli sprouts in human colon and liver cancer cells hepatitis b virus infection predictive value of antiviral effects in the development of hepatocellular carcinoma in the general korean population with chronic hepatitis b the global health burden of infection-associated cancers in the year 2002 high prevalence of multinodular hepatocellular carcinoma in patients with cirrhosis attributable to multiple risk factors management of hepatocellular carcinoma alcohol and hepatocellular carcinoma: the effect of lifetime intake and hepatitis virus infections in men and women epidemiology of hepatocellular carcinoma reactivation of hepatitis b: pathogenesis and clinical implications current management of hepatocellular carcinoma hepatocellular carcinoma and hepatitis b virus. a prospective study of 22 707 men in taiwan 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human colorectal carcinoma lovo cells by andrographolide 123 poster critical role of pro-apoptotic bcl-2 family members in andrographolide-induced apoptosis in human cancer cells andrographolide sensitizes cancer cells to trail-induced apoptosis via p53-mediated death receptor 4 upregulation hepatoprotective and antiviral functions of silymarin components in hcv infection inhibition of t-cell inflammatory cytokines, hepatocyte nf-kappab signaling, and hcv infection by standardized silymarin identification of hepatoprotective flavonolignans from silymarin anticancer potential of silymarin: from bench to bed side silymarin inhibits in vitro t-cell proliferation and cytokine production in hepatitis c virus infection differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy silymarin inhibited proliferation and induced apoptosis in hepatic cancer cells synergistic anti-cancer effect of baicalein and silymarin on human hepatoma hepg2 cells suppression of n-nitrosodiethylamine induced hepatocarcinogenesis by silymarin in rats chemopreventive effect of silymarin on liver pathology in hbv x protein transgenic mice runx3 directly interacts with intracellular domain of notch1 and suppresses notch signaling in hepatocellular carcinoma cells studies on mechanism of action of glycyrrhizin against hepatitis a virus replication in vitro therapeutic basis of glycyrrhizin on chronic hepatitis b review of pharmacological effects of glycyrrhiza sp. and its bioactive compounds glycyrrhizic acid inhibits virus growth and inactivates virus particles glycyrrhizin, an active component of liquorice roots, and replication of sarsassociated coronavirus herbal medicine in the treatment of liver diseases a glycyrrhizin-containing preparation reduces hepatic steatosis induced by hepatitis c virus 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zeamais adults biological activities and chemical constituents of illicium verum hook fruits (chinese star anise) investigation of the antioxidant activity of illicium verum extracts in vitro screening for cytotoxic activity of herbal extracts anticancer attributes of illicium verum essential oils against colon cancer, south afr a new flavane acid from the fruits of illicium verum in vitro screening for cytotoxic activity of herbal extracts chemo-preventive effect of star anise in n-nitrosodiethylamine initiated and phenobarbital promoted hepato-carcinogenesis a cohort study of serum testosterone and hepatocellular carcinoma in shanghai, china el-serag, higher serum testosterone is associated with increased risk of advanced hepatitis c-related liver disease in males global burden of aflatoxin-induced hepatocellular carcinoma: a risk assessment hepatocellular carcinoma: a review el-serag, oral contraception and the risk of hepatocellular carcinoma the association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence diabetes and cancer: a consensus report liver disease in patients with diabetes mellitus an evaluation of the role of insulin-like growth factors (igf) and of type-i igf receptor signalling in hepatocarcinogenesis and in the resistance of hepatocarcinoma cells against druginduced apoptosis the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, preferentially forms dna adducts at codon 249 of human p53 gene, a unique mutational hotspot in hepatocellular carcinoma key: cord-277535-u283k70i authors: vaja, rakesh; rana, meenal title: drugs and the liver date: 2020-09-22 journal: nan doi: 10.1016/j.mpaic.2020.07.001 sha: doc_id: 277535 cord_uid: u283k70i the liver is a major organ with multiple functions. a number of drugs are metabolized by the liver during phase 1 and 2 reactions which include complex processes involving cytochrome p450 enzymes. genetic and acquired variability in cytochrome p450 activity may have profound effects on pharmacokinetics. additionally, drugs can also modify how the liver functions and cause dysfunction or even failure of the organ both by a direct effect on the liver or by alteration in liver blood flow. it is important to recognize the signs and symptoms of liver failure in patients and identify possible causes including drug interactions. furthermore, once a patient has been recognized to be suffering with liver dysfunction or failure drug choice and dosing regime will need to be rationalized. paracetamol overdose can have severe and life threatening consequences for patients due to its effect on liver function. it is the leading cause of acute liver failure in the uk, 1 correct and early management is crucial and will be discussed within this article. the liver receives approximately 30% of cardiac output. uniquely it receives both arterial blood from the hepatic artery and venous blood from the portal veins. the portal vein supplies 70e75% of hepatic blood flow but only 50% of oxygen supply, the remaining blood flow and oxygen supply being from the hepatic artery. anatomically the liver is divided into two lobes and further into functional lobules based around a central vein, which contains blood from the hepatic arterial and portal venous circulations. blood arriving to the liver flows into the sinusoids which are spaces lined by hepactocytes. blood then drains towards the centre of the lobule and the central vein then hepatic vein to return blood back to the heart via the inferior vena cava. it is the portal veins taking blood directly from the gut to the liver which allows for first pass metabolism, making the liver susceptible to ingested drugs as they are absorbed from the gastrointestinal tract and transported to the liver. the liver has a broad range of functions categorized in table 1 . the liver metabolises a wide range of drugs the end result being to produce water soluble compounds which can be excreted in the bile. this results from phase 1 reactions mediated by cytochrome p450 including oxidation, reduction and hydrolysis reactions. this is followed by phase 2 reactions which are conjugative. the cytochrome p450 family are a group of enzymes found mainly in the liver, which perform oxidation and reduction reactions (phase 1) using iron to enhance the water solubility of drugs to aid excretion. cyp450 enzymes are so named as they are bound to membranes within the cell and contain a haem pigment that absorbs light at a wavelength of 450nm when exposed to carbon monoxide. after reading this article you should: c understand the mechanisms of drug metabolism by the liver c have an appreciation of alterations to drug choice and dosing regimens in patients with liver disease due to their altered pharmacokinetics c know the management of a patient with paracetamol overdose there are many different isoforms of cyp450, classified according to their amino acid sequencing into families, subfamilies and individual genes. their importance can be seen in certain subgroups that lack particular genes. an example pertinent to anaesthesia is deficiency in cyp2d6 which metabolises codeine to morphine, these patients therefore find codeine ineffective. conversely there is a small subgroup of people of saudi arabian and ethiopian decent with very high expression of 2d6 who metabolize codeine into vast amounts of morphine. refer to table 2 for more details. an individual more detailed breakdown of cyp450 genes is beyond the scope of this article. some drugs can induce or inhibit cyp450 enzymes which have the sequential effect on the metabolism of other drugs, either increasing or reducing it respectively. possibly the most important example is cyp3a4 which metabolises many substrates and is induced by rifampicin, carbamazepine, phenytonin and dexamethasone. of interest to anaesthesia this will increase metabolism of opioids, benzodiazepines and local anaesthetics. another well cited example is the increased metabolism of the oral contraceptive pill and its reduction in efficacy. for a more exhaustive list of substrates, inducers and inhibitors see table 3 (see table 4 ). a number of non-cytochrome p450 dependent reactions occur in the liver eg oxidation of dopamine and alcohol and hydrolysis of amides and esters (eg lignocaine and pethidine respectively). a predominant rise in aspartate aminotransferase (ast) and alanine aminotransferase (alt) signals hepatocellular injury or death. this can be caused by drug reactions or toxicity (e.g. paracetamol), viral hepatitis, autoimmune hepatitis, alcoholic hepatitis, ischaemic hepatitis secondary to profound hypotension, and rare causes such as wilson's disease. an obstructive pattern has a rise predominantly in alkaline phosphatase (alp) and gamma-glutamyltransferase (ggt), these are canalicular enzymes and suggest cholestasis. this is caused by obstruction, either calculi or tumour (primary biliary, pancreatic or metastases), and liver disease such as primary biliary cirrhosis. pharmacological causes include antibiotics, anabolic steroids and oral contraceptives. a mixed pattern can be seen in sepsis, some drug reactions, cholangitis, congestive cardiac failure and alcoholic liver disease. halothane hepatitis can cause raised liver enzyme assays, raised bilirubin and jaundice. an isolated rise in unconjugated bilirubin may be attributed to gilbert's syndrome or haemolysis. the sars-cov-2 virus uses angiotensin-converting enzyme 2(ace-2) receptors to gain entry into cells. 3 liver particularly in the ductal region has abundance of these receptors, hence may be susceptible to sars-cov-2 3 elevated lft have been reported widely in hospitalized patients with covid-19, the range of elevation is highly variable from 14 to 58%. 4 surprisingly, the pattern of elevation mimics hepatocyte damage (ast/alt higher than bilirubin or alp) rather than cholangiocytic damage, as would have been expected given the density of ace-2 receptors is higher in the ductal system. 4 additionally low albumin has been seen and is a marker of severe disease. 5 one study reported longer hospital stay in patients with elevated lft. 6 an international registry has suggested that has many as 25% of patients with covid-19 may present with hepatic decompensation in absence of respiratory symptoms. 7 the above findings hold important implications for anaesthetists, especially in preop assessments. most drugs given in anaesthesia and intensive care are given intravenously, thus having a bioavailability of 1. however some maybe given orally or nasogastrically and absorbed enterally. the absorption will be affected by delayed gastric emptying or reduced by diarrhoea and increased gastric transit time seen in liver failure. additionally if vasopressors are used there maybe splanchnic vasoconstriction with associated reduced absorption. volume of distribution is a theoretical calculated volume within which a dose of a drug is dissolved. hepatic dysfunction can cause fluid retention and will increase the volume within which drugs are present, particularly those which usually remain in the plasma, thus increasing their volume of distribution and reducing their plasma concentration. 2 in liver disease, protein synthesis may be reduced. these proteins are important as binding sites for drugs and as such alter the amount of free drug available, volume of distribution, half life and duration of action. an important example is albumin. hypo-albuminaemia will increase the proportion of free drug which is active, therefore doses of highly protein bound drugs may need to be reduced, for example phenytoin and benzodiazepines, aspirin and warfarin. 8 another protein produced by the liver, a 1 acid glycoprotein binds basic drugs such as carbamazepine, propanolol, alprenolol and imipramine as well as steroids. bilirubin can also compete for protein binding sites, so raised levels can increase amount of free drugs, the effect however is less in vivo than in vitro. 1 problems with absorption of enterally delivered drugs have been described. once absorbed these drugs undergo the "first pass effect" by the liver before reaching the systemic circulation. in liver failure the degree of metabolism will be reduced, therefore the extraction ratio will also be reduced and more drug will reach the systemic circulation, thus increasing bioavailability. prevalence of ultrarapid metabolisers metabolism of drugs in liver disease depends on liver blood flow. this can be reduced in a cirrhotic liver as portovenous shunting in the form of varices which are created and blood is diverted directly into the systemic circulation by-passing the liver. thus first pass metabolism is reduced. drug metabolism by the liver may also be reduced by the use of vasopressors on intensive care which reduce liver blood flow due to varying degrees of splanchnic vasoconstriction. the phase 1 and 2 reactions performed by the liver are affected and metabolism and thus extraction ratios are reduced. drugs can be divided into those with high extraction ratios >0.7, for example fentanyl and morphine and low extraction ratios <0.3 such as lorazepam, diazepam and methadone. most drugs have low extraction ratios <0.3 that is they have poor permeability and are metabolized by the liver but poorly extracted, therefore clearance is limited by reduced metabolism not by blood flow. those with high extraction ratios >0.7 are highly permeable and clearance is dependent on blood flow. 9 hepatic dysfunction is not uncommon within the intensive care setting affecting 11e54% of critically ill patients depending on definitions used. 2 there is currently no tool akin to renal clearance to indicate degree of liver dysfunction. 9 therefore clinicians use liver function blood tests, international normalized ratio (inr), serum albumin and clinical scores such as the child pugh score act as a surrogate for function. more recently the model for end stage liver disease (meld score) and the meld-na have been used to more accurately predict the severity of liver dysfunction. 10, 11 although their correlation with pharmacokinetic function not well understood. due to the alterations discussed in pharmacokinetics in liver dysfunction drug choices, dosages and frequency may need to be rationalized and altered accordingly. for example the induction dose and maintenance dose, for either anaesthesia or sedation, needs to be reduced. historically inhalational agents, particularly halothane have been implicated in causing hepatitis. the risk is related to the generation of trifluroacteyl chloride (tfa) by metabolism of agents, which is implicated in toxicity 12 around 20% of administered dose of halothane is metabolized by the liver, more specifically by cytochrome p450. this is a relatively high percentage when compared to more modern inhalational agents, for example, 0.2% isoflurane, 0.02% desflurane and 3% sevoflurane. even though sevoflurane undergoes 3% metabolism it does not generate tfa and hence is not linked to immune mediated injury 13 sevoflurane metabolism produces fluoride which is not linked to hepatotoxicity. 14 inhalational agents themselves cause a dose dependent reduction in hepatic blood flow (hbf). isoflurane and sevoflurane result in relatively lower reduction in hbf at 1 mac as compared to desflurane. 15 as long as hypotension is avoided and above effects are kept in mind desflurane is probably the safest choice of inhalational agent due to its' low rate of metabolism and rapid and predictable emergence from anaesthesia. 13 there are two types of halothane hepatitis. type 1 which is mild, transient and has a relatively high incidence (25e30%). type 2 caused by oxidative metabolism of halothane in the liver leading to fever, jaundice, and dramatically elevated serum transaminases. the compounds synthesized by oxidation then bind to trifluoroacetylate proteins in the hepatic endoplasmic reticulum causing cellular dysfunction, it is thought to occur in genetically predisposed individuals. the committee on safety of medicines in 1986 recommended the avoidance of halothane in patients with a history of previous adverse reactions, those who had received halothane within 3 months unless clinically necessary, and those with a history of unexplained jaundice or pyrexia following previous halothane anaesthesia. iv anaesthetics: the induction agents have a marked effect on haemodynamics and may cause sudden precipitious fall in blood pressure. in clinical practice a standard induction dose need not be altered. however they should be titrated slowly to effect. there are no current recommendations on the use of tiva (total intravenous anaesthesia) in patients with liver disease. research is sparse and conflicting. some earlier reports suggested that inhalational anaesthesia results in smaller elevation of liver enzymes than tiva with propofol-fentanyl. 16 a more recent study however suggested slightly lower rate of elevation in lft after using tiva. 17 opiates morphine is metabolized by the liver to active metabolite morphine-6-glucoronide which has potent analgesic properties, and morphine-3-glucuronide, which has no analgesic properties but has adverse neurotoxic side effects such as confusion and respiratory depression. as both metabolites are excreted renally, they accumulate in renal failure. in liver failure morphine itself may accumulate as extraction ratio is reduced thereby enhancing further the effect of morphine. 13 therefore dose of morphine should be reduced. the same is true of fentanyl and alfentanil dose, as although there is no active metabolite they also rely on hepatic metabolism. remifentanil may be good choice as its metabolism is by plasma esterases and it has no active metabolites. a review of pain management in patients with liver disease by the american association for study of liver diseases (aalsd) in 2018 states in general most opioids have prolonged half life. their recommendations include increase the dosing intervals (6e12hr) and using immediate release preparations over extended release 18 in july 2013 the medicine and healthcare products regulatory agency (mhra) produced a drug safety update that restricted the use of codeine in children. 19 this was prompted by case reports of four children suffered serious harm following the administration of codeine in the immediate post-operative period. codeine should only be used to relieve acute moderate pain in children older than 12 years and only if it cannot be relieved by other painkillers such as paracetamol or ibuprofen alone. a significant risk of serious and life-threatening adverse reactions has been identified in children with obstructive sleep apnoea who received codeine after tonsillectomy or adenoidectomy (or both). codeine is now contraindicated in all children younger than 18 years who undergo these procedures for obstructive sleep apnoea. codeine is converted to morphine in the liver by the cyp2d6 enzyme. the extent of conversion of codeine to morphine depends on genetic variations of cyp2d6. people can be classified as: poor; intermediate; extensive; or ultra-rapid metabolisers. poor metabolisers convert very little codeine into morphine and therefore have little or no pain relief, ultra-rapid metabolisers or extensive metabolisers have an excessive amount of morphine in their blood following ingestion of codeine. ethnic origin is an important factor in genetic variability. up to 10% of caucasians are poor metabolisers whereas up to 29% of patients of african origin may be ultra-rapid metabolisers. (table 2) . this genetic variability leads to different plasma morphine concentrations in patients leading to different analgesic effects as well as side effects including respiratory depression. codeine is contraindicated in all patients of any age known to be cyp2d6 ultra-rapid metabolisers and should not be used by breastfeeding mothers because it can pass to the baby through breast milk and potentially cause harm. nsaids are contraindicated for systemic use in most liver disease patients, because of increased bioavalibilty, the high risk of precipitating gastrointestinal bleeding and renal failure. 20 pregabalin and gabapentin are not metabolized in the liver and can be considered for use. these drugs are renally excreted therefore patients with hepatorenal syndrome warrant cautious adminstration. 18, 20 gabapentin is considered as first line non opiod drug for analgesia. among the tricyclic antidepressants nortryptilline appear safer than amitriptyline and imipramine. 20 an increased dose of non-depolarising neuromuscular blockers (nmb) may be required in liver disease possibly due to altered pharmacology anaesthesia and intensive care medicine xxx:xxx protein binding and increased volume of distribution. however, those which are metabolized by the liver have a prolonged duration of action, atracurium as metabolized in the plasma has a more predictable duration of action. however, it is worth noting that in prolonged usage concentrations laudanosine from hoffmann degradation may accumulate with the potential to provoke epileptiform activity on electro encephalography (eeg). 13 this is of greater concern if the patient has concomitant renal failure or impaired blood brain barrier. the metabolism of succinylcholine may be prolonged due to reductions in pseudocholinesterase concentrations, but clinically this is of little significance. suggamadex is a unique reversal agent for amniosteriodal nmb which acts by chelating the nmb. it is not metabolized and is excreted almost exclusively unchanged by the kidneys within 24 h. data regarding use of sugammadex, in patients with liver dysfunction is limited. however as sugammadex is almost entirely excreted renally, no dose reduction is required in patients with mild to moderate liver dysfunction. in patients undergoing liver transplant sugammadex is able to reverse neuromuscular block maintained by rocuronium continuous infusion. 21 however it is important to note that the sugammadex recovery time in this population was found to be considerably longer than in other surgical settings, and should be considered in clinical practice. dexmedetomidine is a highly selective alpha-2 receptor agonist, with analgesic, anxiolytic and sedative. it is primarily metabolized in the liver and may have a prolonged half-life in patients with liver disease. dexmedetomidine has potential protective effects on the liver and intestine during hepatectomy and intraoperative use of dexmedetomidine during liver surgery is subject to ongoing research. 22 additionally patients with elevated bilirubin and bile salts secondary to jaundice may show bradycardias limiting its use. 23 paracetamol overdose paracetamol is the commonest drug taken in overdose in the uk to date, it can result in liver failure and in some cases fatal. 24 hepatocelluar necrosis can occur if as little as 7.5g of paracetamol is ingested. normal pathways of metabolism are saturated and hepatic glutathione stores are exhausted. patients are often initially asymptomatic for the first 24 h before reporting nausea, vomiting, right upper quadrant pain with progressive derangement of liver function tests (lfts) after 18 h. the mhra produced new guidelines for treatment of paracetamol overdose in 2012; the changes included an updated nomogram and a simplified treatment schedule. 25 the administration of acetylcysteine has previously been based on the rumack-matthew nomogram, which divided treatment groups into high and low risk. the dose was then calculated on a weight-based table, which increased the risk of drug error. the updated nomogram has a single treatment line (figure 1) 25 ; thereby eliminating the need for assessing whether the patient falls into the high-risk category. it also advises that in cases of staggered overdoses there should be no delay in the administration of acetylcysteine. another cause for concern was the adverse events that had been reported following the bolus dose of acetylcysteine, this has been addressed by increasing the duration over which it is infused, from 15 min to 60 min. acetylcysteine should be administered when the plasma paracetamol level is on or above a single treatment line joining points of 100mg/l at 4 h and 15mg/l at 15 h after ingestion. a baseline full blood count (fbc), urea and electrolytes (u&es), lfts and coagulation screen along with an arterial blood gas sample should also be done at the earliest opportunity. intravenous preparations of paracetamol were licensed in the uk in 2004 and it is routinely used in anaesthetic practice. however, there have been some concerns regarding the dosage and administration; especially in adults and children under 50kg, patients with pre-existing hepatic dysfunction and the elderly. children and infants weighing less than 10kg should receive the reduced dose of 7.5mg/kg not exceeding the daily dose of 30mg/ kg, whilst those >10kg can be prescribed up to 15mg/kg not exceeding the daily dose of 60mg/kg. both the mhra and the npsa have issued alerts, regarding the correct dose prescription as there have been reported cases of accidental overdose due to ml to mg conversion and the administration of 1g in adults weighing less than 50kg. the key issue is that with intravenous paracetamol plasma levels will peak immediately after administration. the traditional nomograms used to predict plasma levels after overdose refer to oral ingestion where the levels peak some hours after. indicators of severe paracetamol poisoning which is likely to require referral to a specialist liver centre include: inr of >2.0 at 24 h, >4 at 48 h or >6 at 72 hours; renal impairment (creatinine >200 micromol/l); hypoglycaemia; metabolic acidosis despite rehydration; hypotension despite resuscitation or encephalopathy. 26 the only other treatment in fulminant liver failure is transplantation. paracetamol as analgesic in chronic liver disease given the potential of paracetamol to cause liver injury, there is a common misconception that these patients should never take paracetamol. however, various studies have shown that if taken in appropriate doses, paracetamol is one of the safest analgesics for patients with cirrhosis. limiting the total daily dosage to 2e3 g/day with thrice daily dosing is generally recommended. 27 patients should be educated about over-the-counter and prescription medications that may also contain acetaminophen to avoid overdose. remdesvir: elevated liver enzymes are commonly observed in clinical trials of patients with remedevir. 28 the elevated values rarely warrant treatment discontinuation. current recommendations suggest that if the enzymes are elevated to 5 times or more above baseline, to discontinue the drug. tocilizumab: alt elevations are frequent but fulminant hepatitis is rare. the risk of re activation of hbv should be kept in mind if patient had chronic liver disease secondary to viral etiology. 28 steriods: low dose dexamethasone is probably safe in patients with chronic stable liver diease. however use of methylprednisolone in high doses may reactivate hbv and increase risk of spontaneous bacterial peritonitis (sbp) in severe cases. 28 hydoxychloroquine: data is limited but generally has not been associated with elevations in alt levels and is an extremely rare cause of drug induced liver injury. 28e31 changing patterns of causation and the use of transplantation in the united kingdom drug dosing considerations for the critically ill patient with liver disease sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor liver injury in covid-19: the current evidence hypoalbuminemia predicts the outcome of covid-19 independent of age and co-morbidity liver injury during highly pathogenic human coronavirus infections high mortality rates for sars-cov-2 infection in patients with pre-existing chronic liver disease and cirrhosis: preliminary results from an international registry differentiated effects of liver cirrohosis on albumin binding sites for diazepam, salicylic acid and warfarin anaesthesia for the patient with liver disease a model to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts meld-na (the new meld) and peri-operative outcomes in emergency surgery buggy donal j. pharmacology of anaesthetic agents ii: inhalation anaesthetic agents anaesthesia for patients with liver disease the effects of prolonged lowflow sevoflurane anesthesia on renal and hepatic function blood supply to the liver in the human after 1 mac desflurane in comparison with isoflurane and halothane the effect of isoflurane or propofol anaesthesia on liver injury after partial hepatectomy in cirrhotic patients comparison of the postoperative liver function between total intravenous anesthesia and inhalation anesthesia in patients with preoperatively elevated liver transaminase levels: a retrospective cohort study pain management in patients with cirrhosis analgesics in patients with hepatic impairment: pharmacology and clinical implications sugammadex versus neostigmine after rocuronium continuous infusion in patients undergoing liver transplantation dexmedetomidine reduces intestinal and hepatic injury after hepatectomy with inflow occlusion under general anaesthesia: a randomized controlled trial bile acids induce arrhythmias: old metabolite office for national statistics) paracetamol overdose: an evidence based flowchart to guide management the therapeutic use of analgesics in patients with liver cirrhosis: a literature review and evidence-based recommendations care of patients with liver disease during the covid-19 pandemic: easl-escmid position paper liver injury in covid-19: management and challenges covid-19: abnormal liver function tests pharmacokinetics in disease states modifying hepatic and metabolic function key: cord-323736-zup9cp6s authors: ko, sheung‐fat; chen, yi‐ling; sung, pei‐hsun; chiang, john y.; chu, yi‐ching; huang, chung‐cheng; huang, chi‐ruei; yip, hon‐kan title: hepatic (31)p‐magnetic resonance spectroscopy identified the impact of melatonin‐pretreated mitochondria in acute liver ischaemia‐reperfusion injury date: 2020-07-21 journal: j cell mol med doi: 10.1111/jcmm.15617 sha: doc_id: 323736 cord_uid: zup9cp6s acute liver ischaemia‐reperfusion injury (iri), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. however, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. this study tested the hypothesis that (31)p‐magnetic resonance spectroscopy ((31)p‐mrs) findings could provide reliable living images to accurately identify the degree of acute liver iri and melatonin‐pretreated mitochondria was an innovative treatment for protecting the liver from iri in rat. adult male sd rats were categorized into group 1 (sham‐operated control), group 2 (iri only) and group 3 (iri + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). by the end of study period at 72 hours, (31)p‐mrs showed that, as compared with group 1, the hepatic levels of atp and nadh were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. the liver protein expressions of mitochondrial‐electron‐transport‐chain complexes and mitochondrial integrity exhibited an identical pattern to (31)p‐mrs finding. the protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial‐damaged biomarkers displayed an opposite finding of (31)p‐mrs, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. microscopic findings showed that the fibrotic area/liver injury score and inflammatory and dna‐damaged biomarkers exhibited an identical pattern of cellular stress signalling. melatonin‐pretreated mitochondria effectively protected liver against iri and (31)p‐mrs was a reliable tool for measuring the mitochondrial/atp consumption in living animals. acute liver ischaemia-reperfusion injury (iri), which has been reported to commonly occur during liver resection and transplantation surgery is a crucial factor for predictive of poor outcome of liver transplantation. [1] [2] [3] studies have further displayed that several key factors contribute to the hepatic injury at the initiation and during the progression of liver iri, including those of elevation of anaerobic metabolism, dysfunction of mitochondria, insult of oxidative stress, overload of intracellular calcium, activation of liver kupffer cells, infiltration of immune cells and release of inflammatory cytokines. [2] [3] [4] [5] [6] although the underlying mechanisms of hepatic iri have been extensively investigated, the clinical practice to prevent iri is still limited. additionally, despite the state-of-the-art advance of critical care for patients with acute hepatic failure, the in-hospital morbidity and mortality remain unacceptably high. [7] [8] [9] [10] [11] [12] therefore, it is of utmost importance to both clinician and medical researchers to explore a safe and efficacious therapeutic modality for patients with acute liver iri refractory to conventional treatment. [13] [14] [15] [16] however, prior to ascertain a novel, safe and efficacious therapeutic modality for acute liver iri, the core property that induces this disease entity must be first clarified. it is well recognized that mitochondria are especially rich in liver and heart. the function of mitochondria in liver or other organs not only serves as the primary energy source but also plays a pivotal role in extensive oxidative metabolism and normal function of the liver. 17, 18 however, severely impaired mitochondrial function and activity have clearly identified in setting of liver ischaemia-reperfusion 4, 19, 20 with subsequent alternation in energy metabolism, generation of reactive oxygen species (ros), inflammatory cell infiltration and cellular apoptosis [21] [22] [23] [24] and ultimately irreversible damages to mitochondria, 16, 25 exhausting atp in ischaemia-reperfusion liver. 4, 16, 21, [25] [26] [27] these findings 4, 16, [19] [20] [21] [22] [23] [24] [25] [26] [27] highlight that the quantity (ie amount) and quality (functional integrity) of mitochondria play extremely important roles on acute liver iri. accordingly, these aforementioned issues 4, 16, 21, [25] [26] [27] raise the hypothesis that restoration of mitochondrial function through exogenous mitochondrial transfusion may be a potential strategy for the treatment of acute hepatic iri. melatonin, acting as a scavenger of free radicals for cell membrane stabilization, has been identified to have powerful ancient antioxidant 28 for suppressing the generation of oxidative stress. 29 basic researches have exhibited that melatonin enters mitochondria through oligopeptide transporters and specifically targets at the mitochondria where it seems to function as an apex antioxidant. 30 in addition to cell uptake from circulation, melatonin is produced in the mitochondria as well. 30 it is well recognized that oxidative damage is a result of free radicals produced in cells, especially in mitochondria. 31 recent study has further demonstrated the protective stabilization of mitochondrial permeability transition and mitochondrial oxidation during mitochondrial ca 2+ stress by melatonin's cascade metabolites. 32 thus, melatonin always plays a crucial role on protecting the mitochondrial number and functional integrity [30] [31] [32] through regulation of the oxidative stress and the preservation of atp/energy, resulting in protection of organs from iri. 16, 30, [33] [34] [35] [36] interestingly, previous studies 37, 38 16, 17, 21, 25, [28] [29] [30] [33] [34] [35] [36] this study tested the hypothesis that melatonin-pretreated exogenous mitochondria might offer an added benefit on protecting the liver against acute iri. additionally, this study further tested whether the 31 p-mrs examination could provide reliable living images to accurately identify the degree of atp consumption/depletion in hepatocytes, that is an indicator of acute liver ischaemia-reperfusion in rodent. whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. microscopic findings showed that the fibrotic area/ liver injury score and inflammatory and dna-damaged biomarkers exhibited an identical pattern of cellular stress signalling. melatonin-pretreated mitochondria effectively protected liver against iri and 31 p-mrs was a reliable tool for measuring the mitochondrial/atp consumption in living animals. the procedure and protocol have been described in our previous report 42 and were based on the other previous studies. 43, 44 in detail, the pathogen-free, adult male sprague dawley (sd) rats (n = 24) weighing 325-350 g were randomly divided into three groups: the procedure was described as follows: the left lobe of the liver was dissected free from the surrounding ligaments. hepatic ischaemia was induced by temporal obstruction of the vessels by placing a 4-0 silk loop around the hilar region of the left liver lobe in groups 2 and 3 animals, whereas sc animals received only laparotomy without undergoing hepatic ischaemia. reperfusion was started 60 minutes later when the hilar occlusion was released. all animals were killed by 72 hours after reperfusion. to elucidate the impact of acute liver ischaemia-reperfusion on hepatocyte damage and up-regulation of inflammatory reaction as well as the therapeutic effect of melatonin on the circulatory parameters, blood samples were collected before and after the experiments and plasma specimens were harvested for analyses of aspartate aminotransferase (ast) concentration, alanine aminotransferase (alt) and circulating levels of inflammatory cytokines. liver specimens were acquired and then stored at −80°c for individual study. tissues were also embedded in optimal cutting temperature compound or 4% buffered formaldehyde for cryo-sectioning and paraffin sectioning, respectively. for stabilization and attenuation of oxidative stress in isolated mitochondria, melatonin was given to donor rat (n = 3) at day 3 (50 mg), 2 (25 mg) and 1 (25 mg)/rat by intraperitoneal administration prior to acute liver ischaemia-reperfusion procedure. 16, 42 the procedure and protocol of liver mitochondrial isolation from donor sd rats have been previously described by our studies. 42, 45, 46 in detail, the rats (ie the donors) were fasted overnight prior to the mitochondrial isolation procedure, then killed and their gallbladders and livers carefully isolated and removed. immediately, the liver (3 g) was immersed in 50 ml of ice-cold ibc (10 mmol/l tris-mops, 5 mmol/l egta/tris and 200 mmol/l sucrose, ph 7.4) in a beaker, followed by rinsing the liver free of blood with ice-cold ibc. the liver was then minced with scissors in a beaker surrounded by ice. ibc was discarded during mincing and replaced with 18 ml of ice-cold fresh ibc. the liver was then homogenized with a teflon pestle. the homogenates were transferred to a 50-ml polypropylene falcon tube and centrifuged at 600 g for 10 minutes at 4°c. the supernatants were transferred to fresh tubes for centrifugation at 7000 g for 10 minutes at 4°c. the supernatants were discarded, and the pellets washed with 5 ml icecold ibc. again, the supernatants from the pellets were centrifuged at 7000 g for 10 minutes at 4°c. the supernatants were discarded and the pellets containing the mitochondria resuspended. the concentration of the mitochondrial suspensions was measured using the biuret method. each 10 mg of isolated mitochondria was labelled with 1 mol/l of mitotracker red cmxros (invitrogen, carlsbad, ca, usa) through incubation at 37°c for 30 minutes for tracing the mitochondria in the ischaemia-reperfusion liver. mitochondrial transfusion was performed for the study animals immediately after labelling (ie <3 hours after the isolation procedure). the liver mri examination was performed using a 9.4-t horizontalbore animal mr scanning system (biospec 94/20; bruker, ettingen, germany). this scanning system comprises a self-shielded magnet with a 20 cm clear bore and a bga-12s gradient insert (inner diameter: serial 31 p-mrs examinations were performed on each rat at baseline (preoperatively), before-surgery and day 1, 2 and 3 post-surgery. the 31 p-mrs was acquired using a simple pulse-acquisition sequence, with a pulse optimized to give a 90° excitation in the liver region with the added advantage of overtripping signal from the abdominal region. respiratory gating was not used as the spectra acquired were nonlocalized, and hence, the resulting spectrum was the average 31 p-mrs signal from the sensitive area of the coil, which was optimized to have the largest signal (90° pulse) in the liver. the 31 p-mrs data were collected with a tr of 3000 ms, 512 averages, and a spectral width of 8000 hz. data were analysed in the time cross via nmr data processing guide of paravision5.1 (bruker). the phosphomonoesters (pme), inorganic phosphate (pi), phosphodiester (pde), nadh and the three-nucleotide triphosphate (mainly adenosine triphosphate) peaks in the spectrum were fitted after application of 0.5-hz line broadening and manual phasing. after preparation of haematoxylin and eosin (he) stain, the degree of liver injury was determined according to our previous report with liver injury score defined as follows: 0 indicated no notable hepatocyte integrity impairment or sinusoidal distortion; 1 corresponded to mild hepatic injury with less than 25% of section involved; 2 indicated moderate hepatic injury with 25%-50% of section involved; 3 denoted severe hepatic injury with more than 50% involved. 42 for each animal, three liver sections were examined and three randomly selected high-power fields (hpfs) (ie the procedure and protocol of staining were based on our previous reports. 42, 45, 46 in detail, rehydrated paraffin sections ( the procedure and protocol for western blot analysis have been described in detail in our previous studies. 42, 45, 46 briefly, equal amounts (50 µg) of protein extracts were loaded and separated by sds-page using acrylamide gradients. after electrophoresis, the separated proteins were transferred to a polyvinylidene difluoride (pvdf) membrane (amersham biosciences, amersham, uk). nonspecific sites were blocked by incubation of the membrane in blocking buffer (5% 2000; cell signalling) was used as a secondary antibody for 1-hour incubation at room temperature. the washing procedure was repeated eight times within 1 hour. immunoreactive bands were visualized by enhanced chemiluminescence (ecl; amersham biosciences) and exposed to biomax l film (kodak, rochester, ny, usa). for the purpose of quantification, ecl signals were digitized using labwork software (uvp™ biospectrum™ imaging system, upland, ca, usa). quantitative data were expressed as mean ± sd. statistical analysis was adequately performed by anova followed by bonferroni's multiple comparison post hoc test. sas statistical software for windows version 8.2 (sas institute, arlington, va, usa) was utilized. a probability value <0.05 was considered statistically significant. first, to elucidate how the circulating levels of inflammatory biomarkers and the liver enzymes were augmented after ischaemia-reperfusion procedure, the elisa method was utilized for examination of these parameters. the result demonstrated that by 72 hours after ischaemia-reperfusion procedure, circulating levels of tnf-α, il-6 and mpo, three indicators of inflammation, were significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. consistently, serum levels of ast and alt, two indices of hepatocyte integrity, showed an identical pattern compared with that of inflammation. these findings imply that melatonin-pretreated mitochondria therapy attenuated circulatory inflammatory markers and protected the liver from acute iri (figure 1 ). next, to clarify whether mri was a sensitive and reliable non-invasive tool for determining the degree of liver iri, the phosphorus were substantially reduced in iri than in sc and iri + mito, and significantly reduced in iri + mito than sc. however, γ-atp level was only relatively reduced in iri as compared with the other two groups but it did not reach statistical significance, whereas the parameters of pme and pde did not differ among the three groups. these findings suggested that exogenous mitochondrial administration enriched the hepatocyte mitochondria ( figure 2 ). it is well known that mitochondrial integrity and the stability of mitochondrial-electron-transport-chain complexes (metcc) are fundamentally important for an effective-energy generation of atp in the cells. we therefore measured the protein expressions related to these two cardinal factors. consistently, the protein expressions of cytosolic cytochrome c, drp1 and cyclophilin d, three indices of mitochondrial-damaged biomarkers, were significantly higher in iri than in sc and iri + mito, and significantly higher in iri + mito than in sc, suggesting iri depleted hepatocyte endogenous mitochondria. on the other hand, the protein expressions of complex i, ii, iii and v, four indictors of metcc, and mitochondrial cytochrome c, an index of mitochondrial integrity, were significantly higher in sc than in iri and iri + mito, and significantly higher in iri + mito than in iri, suggesting that exogenous mitochondrial transfusion enriched the mitochondria in the injured hepatocytes ( figure 3a-h) . we further identified that the protein expressions of nox-1 and nox-2, two indicators of oxidative stress, were significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. on the other hand, the protein expressions of ho-1, nqo1 and nfr2, three indicators of antioxidants, were significantly progressively increased from groups 1 to 3, suggesting an intrinsic response to ischaemia-reperfusion stimulation that was enhanced by exogenous mito therapy ( figure 3i-m) . molecular level of inflammatory reaction has been crystal clearly identified to be augmented in setting of acute ischaemia-reperfusion. 16, 33, 36 here, we also found that the protein expressions of il-1ß, tnf-α, p-nf-κb, mmp-2 and mmp-9, five indices of inflammation, were significantly increased in iri than in sc and iri + mito, and significantly increased in iri + mito than in f i g u r e 2 mri findings of hepatic phosphorylated metabolism by 72 h after liver ischaemia-reperfusion injury induction. a, illustrating the hepatic 31 p-magnetic resonance spectra (ie 31 p-mrs) examination for identifying the atp (an indicator of energy storage) in hepatocytes of sc, iri and iri + mito animals, respectively. b-g, the relative level of each metabolites was subtracted from the β-atp value. the data are expressed as mean ± sd (n = 8 per group). one-way anova followed by post hoc tukey-kramer test was used for statistical analysis. symbols (*, †, ‡) indicate significant differences (at 0.05 level). atp, adenosine triphosphate; iri, ischaemia-reperfusion injury; nadh, nicotinamide adenine dinucleotide; pde, phosphodiester; pi, inorganic phosphate; pme, phosphomonoesters; sc, sham control sc. additionally, the protein expressions of pi3k, p-akt and p-mtor, three biomarkers of cellular stress signalling, displayed an identical pattern of inflammation among the three groups, suggesting that melatonin-pretreated mitochondria effectively suppressed inflammatory reaction ( figure 3n -u). to assess whether the therapy of melatonin-pretreated mitochondria would offer benefit on ameliorating the liver injury score, microscopic finding of haematoxylin and eosin-stained liver sections was performed in each animal. the result showed that the liver injury score was significantly increased in iri than in sc and iri + mito and significantly increased in iri + mito than in sc. additionally, the masson's trichrome stain displayed that the fibrotic area exhibited an identical pattern of liver injury score among the three groups. these findings implied that melatonin-pretreated mitochondria preserved the liver parenchymal architecture (figure 4 ). to examine the cellular level of inflammatory cell infiltrations in liver parenchyma, the if microscope was performed. consistent finally, we found that the cellular expressions of γ-h2ax, an indicator of dna-damaged marker, and mmp-9, an indicator of acute innate inflammatory reaction, were significantly higher in iri than in sc and iri + mito, and significantly higher in iri + mito than in sc ( figure 6 ). as a non-invasive and accurate instrument, the advantage of utilizing hepatic 31 p-mrs for detecting the metabolism of different liver disease entities has been extensively discussed. [39] [40] [41] however, there is still lacking data to address the situation of atp metabolism and consumption in hepatocytes in setting of iri with the exogenous mitochondrial supply. additionally, a non-invasive tool with precise diagnostic feature is in urgent need for our daily clinical practice to identify the degree of atp consumption and depletion in setting of iri, especially in acute liver iri, has not yet been reported. these issues encourage us to carry out this experimental study. one novel finding in the present study was that the 31 p-mrs tool clearly identified that the level of atp in hepatocytes of living animals was remarkably reduced in iri animals, suggesting that the mitochondria/atp, indicator of energy, was significantly depleted in hepatocytes. however, this parameter was remarkably reversed in iri animals treated by melatonin-pretreated mito. accordingly, our findings, in addition to extending the findings of previous studies, 39-41 pinpoint the exciting potential of studying metabolic processes in the human liver in vivo. accurate diagnosis of disease is of course very important, and the effective treatment of the disease is no doubted essential. plentiful data have shown that exogenous mitochondrial transfusion not only effectively rescued the function and integrity of endogenous mitochondria but also refreshed the depleted mitochondria in organ, resulting in preserving the ischaemia-reperfusion-related organ dysfunction. 16, 46 the most important finding in the present study was that melatonin-pretreated exogenous mitochondria not only refreshed the mitochondria (ie increased atp) in hepatocytes but also attenuated the anatomical-histopathological changes of liver parenchyma (ie ameliorated the liver injury score and fibrosis) and circulatory level of ast and alt (ie two indices of liver function/ hepatocyte damage). our findings, in addition to strengthening the plentiful studies have previously established that iri always elicits inflammatory reaction, dna damage, apoptosis and generation of oxidative stress, resulting in organ damage, tissue necrosis and unfavourable outcomes. [45] [46] [47] [48] [49] additionally, increased oxidative stress in circulation and intracellular/mitochondrial compartment notably down-regulated mitochondrial function and depleted energy-storage capacity. 16, 46 an essential finding in the present study was that these above-mentioned parameters of molecular-cellular perturbations [45] [46] [47] [48] [49] were substantially increased in iri animals as compared to sc animals. in this way, our findings, in addition to being consistent with the findings from previous studies, [45] [46] [47] [48] [49] could, once again, explain why the liver enzyme, fibrosis and liver injury score were markedly increased in iri animals. of importance was that melatonin-pretreated mito therapy significantly suppressed inflammatory reaction, dna damage, oxidative stress, cellular apoptosis and fibrosis. finally, figure 7 schematically summarized the innovative findings of our study to facilitate readers' understanding. conclusively, we first developed the 31 p-mrs technique for precise diagnosis of atp energy metabolism in the liver. second, we created an animal model of acute liver iri and measured the alternations of molecular-cellular perturbations in liver parenchyma and circulation as well as identified the ultrastructural changes of liver parenchyma. finally, we clearly proved that melatonin-pretreated exogenous mitochondria effectively protected the liver against iri mainly through refreshment of the endogenous mitochondria which were at the gate of death in iri hepatocytes. it is well known that melatonin is a dietary supplement that is very safe and non-toxic to humankind. some recent data have even shown that melatonin therapy effectively protected the lung against covid-19 infection mainly through regulating the immune system, that is immunomodulation. [50] [51] [52] [53] these mentioned issues [50] [51] [52] [53] and the results of our study raise the need of consideration melatonin could be utilized for those of acute liver iri patients shortly, especially in those of patients who are refractory to the conventional treatment. this study has limitations. first, although the results were attractive and promising, study period was relatively short (ie the study period was only 72 hours). accordingly, the long-term impact of melatonin-pretreated mito therapy on protecting the liver against iri is still currently unclear. second, despite the impact of melatonin on suppressing the cellular infiltration was clearly clarified, the therapeutic impact of this regimen on regulating the macrophage polarization (ie m1/m2 ratio) was not investigated. third, although extensive works were accomplished in the present study, the exact underlying mechanisms of melatonin-pretreated mito therapy on protecting the liver against iri remain regrettably uncertain. accordingly, based on our results, the proposed schematic mechanism of melatonin-pretreated mito therapy on safeguarding the liver in setting of iri only was illustrated in figure 7 . in conclusion, 31 p-mrs provided a reliable method for elucidating the level, activity and metabolic rate of atp in hepatocytes of living animals. besides, the results of the present study demonstrated that melatonin-pretreated mito therapy effectively protected the liver against iri in rat. we thank the molecular imaging core of the center for translational research in biomedical sciences, kaohsiung chang gung memorial f i g u r e 7 schematically proposed mechanism of mitochondrial transfusion for reducing acute liver ischaemiareperfusion injury in rat. 31 p-mrs, 31 p-magnetic resonance spectroscopy; atp, adenosine triphosphate; il-6, interleukin 6; iri, ischaemia-reperfusion injury; mito, mitochondria; mpo, myeloperoxidase; ros, reactive oxygen species; sc, sham control; tnf-α, tumour necrosis factor alpha hospital, kaohsiung, taiwan, for technical and facility supports on 9.4t animal mri. the authors confirm that there are no conflicts of interest. the data that support the findings of this study are available from the corresponding author upon reasonable request. https://orcid.org/0000-0002-6305-5717 liver ischaemia and reperfusion injury mechanisms of hepatic ischemia-reperfusion injury and protective effects of nitric oxide ischemia/reperfusion injury in liver surgery and transplantation: pathophysiology impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury reperfusion injury and reactive oxygen species: the evolution of a concept oxidative stress and acute hepatic injury hypoxic hepatitis: underlying conditions and risk factors for mortality in critically ill patients hypoxic hepatitis epidemiology of acute liver failure acute liver failure update on acute liver failure update in 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pretreatment enhances the therapeutic effects of exogenous mitochondria against hepatic ischemia-reperfusion injury in rats through suppression of mitochondrial permeability transition spironolactone effect in hepatic ischemia/reperfusion injury in wistar rats effects of apocynin on liver ischemia-reperfusion injury in rats combined therapy with ss31 and mitochondria mitigates myocardial ischemia-reperfusion injury in rats shock wave therapy enhances mitochondrial delivery into target cells and protects against acute respiratory distress syndrome early administration of cold water and adipose derived mesenchymal stem cell derived exosome effectively protects the heart from ischemia-reperfusion injury preactivated and disaggregated shape-changed platelets protect kidney against from ischemia-reperfusion injury in rat through attenuating inflammation reaction short-interval exposure to ambient fine particulate matter (pm2.5) exacerbates the susceptibility of pulmonary damage in setting of lung ischemia-reperfusion injury in rodent: pharmacomodulation of melatonin melatonin: roles in influenza, covid-19, and other viral infections lungs as target of covid-19 infection: protective common molecular mechanisms of vitamin d and melatonin as a new potential synergistic treatment melatonin inhibits covid-19-induced cytokine storm by reversing aerobic glycolysis in immune cells: a mechanistic analysis covid-19: melatonin as a potential adjuvant treatment hepatic 31 p-magnetic resonance spectroscopy identified the impact of melatonin-pretreated mitochondria in acute liver ischaemia-reperfusion injury key: cord-017603-wq4cgqs2 authors: shanmugam, naresh; dhawan, anil title: acute liver failure in children date: 2018-10-16 journal: pediatric hepatology and liver transplantation doi: 10.1007/978-3-319-96400-3_8 sha: doc_id: 17603 cord_uid: wq4cgqs2 “acute liver failure” (alf) and “fulminant liver failure” are terms used interchangeably to describe severe and sudden onset of liver cell dysfunction leading on to synthetic and detoxification failure across all age groups. considerable variations exist between alf in children and adults, in terms of aetiology and prognosis. encephalopathy is not essential to make a diagnosis of alf in children but when present has a bad prognosis. early recognition of alf and initiation of supportive management improve the outcome. liver transplantation remains the only definitive treatment when supportive medical management fails. trey and davidson coined the term "fulminant liver failure" 40 years ago to define onset of altered mental status within 8 weeks of initial symptoms of liver dysfunction in an otherwise healthy individual with no previous history of liver disease [1] . this definition was difficult to apply in children with alf as the disease process could start in utero and time quantification might not be possible and, also, encephalopa-thy might be difficult to diagnose. trying to address this issue, bhaduri and vergani defined alf in children as "a rare multisystem disorder in which severe impairment of liver function, with or without encephalopathy, occurs in association with hepatocellular necrosis in a patient with no recognized underlying chronic liver disease" [2] . this newer definition for children failed to differentiate between acute hepatitis and alf as "severe impairment of liver function" is very subjective and can vary from person to person. paediatric acute liver failure (palf) study group has come up with practical definition to select cases for their multicentre study. they used the following criteria to define acute liver failure (alf) in children: (1) hepatic-based coagulopathy defined as a prothrombin time (pt) ≥ 15 s or international normalized ratio (inr) ≥ 1.5 not corrected by vitamin k in the presence of clinical hepatic encephalopathy (he) or a pt ≥ 20 s or inr ≥ 2.0 regardless of the presence or absence of clinical hepatic encephalopathy (he), (2) biochemical evidence of acute liver injury and (3) no known evidence of chronic liver disease [3] . due to its simplicity and objectivity, palf definition is widely used in children. palf has used inr as a surrogate marker to denote overall liver synthetic inadequacy and an impending multiorgan failure. coagulopathy is not only a key criterion in diagnosing paediatric alf but also helps as prognostic marker. due to short half of several liver-based clotting factors, pt/inr functions as a dynamic marker of synthetic inadequacy due to loss of functioning hepatocytes in alf. factors ii, vii, ix and x depend on vitamin k for carboxylation of terminal glutamic acid residues to convert them into active form ( fig. 8.1 ). correction of coagulopathy by intravenous vitamin k differentiates between vitamin k deficiency due to decreased absorption and synthetic liver failure. isolated prolonged aptr is not due to liver disease, as factor vii in extrinsic pathway (fig. 8.1 ) has the shortest half-life of the • encephalopathy is not essential in the diagnosis of alf in children. • coagulopathy is being used as a prognostic marker in paediatric alf. • role of liver assist devices and hepatocyte transplant is still limited. • auxiliary liver transplantation if feasible should be offered where indicated as it provides a chance for native liver regeneration. vitamin k-dependent factors; therefore, it is the first factor depleted in alf and invariably affects inr. due to defective synthesis and impaired clearance of procoagulant/anticoagulant factors, inflammatory mediators, infection, etc., there could be a degree of intravascular coagulation (ic) that invariably exists in alf which can progress to fulminant disseminated intravascular coagulation. haemostasis in liver disease is best assessed using thromboelastography (teg). teg is a point-of-care assay using a specialized machine that assesses clot formation in whole blood, including plasmatic and cellular components. teg provides a graphical representation ( fig. 8.2 ) of assembly of a clot in whole blood and provides an assessment of overall haemostasis. the causes of alf vary with the age and geographical location. infectious aetiology predominates as a cause of alf in children in the developing countries, while drug-induced alf predominates in adults and is indeterminate in children in europe and north america [4] . [5] . though exact frequency of alf in the paediat-ric age group is unknown, overall annual incidence of alf in the usa was around 5.5 million population among all ages [6] . investigation of alf in children is outlined in table 8 .2. in developed countries drugs and toxins have become the most common identifiable cause of drug-induced acute liver failure in adults and children. drug-induced liver injury (dili) can be a dose-dependent response, an idiosyncratic reaction or a synergistic reaction when two medications are given together. it is essential to enquire about any indigenous/herbal medicine intake as some are potentially hepatotoxic [7] . acetaminophen is the most common drug associated with alf and is normally a dose-dependent hepatotoxic agent. acetaminophen is detoxified mainly by glucuronidation (40%), sulphation (20-40%) and n-hydroxylation (15%). a small fraction is metabolized via cytochrome p450 to yield n-acetyl-para-benzoquinoneimide (napqi), a toxic intermediate compound which irreversibly conjugates with the sulphhydryl group of glutathione and causes hepatocyte necrosis [8] . napqi forms acetaminophen-protein adducts, which acts as a specific biomarker for chronic acetaminophen-related toxicity. in acute acetaminophen poisoning, serum levels after 4 h of ingestion are useful in identifying high-risk patients. genetic polymorphism of cytochrome p450 isoenzymes could predispose affected people to acetaminophen toxicity. anti-tuberculosis drugs, particularly isoniazid, are associated with drug-induced alf. the mechanism of toxicity is similar to acetaminophen; oxidation via cytochrome p450 pathway results in toxic metabolites. the true incidence of idiosyncratic drug-induced liver injury (dili) is unknown; reports have suggested to up to 14 new cases/100,000/year [9] . around 8% of idiosyncratic dili developed alf [10] . dili is unpredictable, but genetic susceptibility of an individual to certain drugs and underlying mitochondrial cytopathies are proposed causes [11] . the councils for international organizations of medical sciences/ roussel uclaf causality assessment method (cioms/ rucam) scale is helpful in establishing causal relationship between offending drug and liver damage. using the scoring system, suspected drug could be categorized into "definite or highly probable" (score > 8), "probable" (score 6-8), "possible" (score 3-5), "unlikely" (score 1-2) and "excluded" (score ≤ 0) [12] . this scale is helpful in identifying druginduced hepatotoxicity even in newly marketed drugs and for a previously unreported older drug. chemotherapy drugs are known to produce veno-occlusive disease leading on to alf due to endothelial damage. few of the common drugs that cause alf are outlined in table 8 .2. water-borne viral hepatitis (hepatitis a and e) is the most common cause of alf in developing countries with poor sanitation facilities. following infection with hepatitis a virus, the risk of developing liver failure is 0.1-0.4%, and this further increases with underlying chronic liver disease. usually the disease runs a benign course with spontaneous recovery, but some might require liver transplantation [13] . with hepatitis e infection, the risk of developing alf in adults is 0.6-2.8% [14] . recent evidence suggests that case fatality due to hepatitis e-induced alf in pregnancy is similar to that of age-matched general population [15] . the alf due to hepatitis b virus (hbv) can occur at the time of acute infection, reactivation of chronic hbv infection or seroconversion from a hepatitis b e antigen-positive to a hepatitis b e antibody (hbeab)-positive state. superinfection or coinfection of hbv-infected patients with hepatitis delta virus (hdv) can cause liver failure. hepatitis c virus (hcv) infection has not been reported as a cause of alf, and herpes simplex virus can cause alf, of which herpes simplex virus 1 and 2 (hsv) is the predominant cause of viral-induced alf during the first month of life. babies presenting with fever, rash, lethargy, poor feeding and raised transaminase (in thousands) are usually suggestive of hsv hepatitis. disseminated neonatal herpes with liver failure carries high mortality. in stable neonates with alf due to hsv, liver transplantation has been successful. treatment with high-dose acyclovir should be initiated in all neonates with alf, until serology results are known. other members of herpes virus family such as cytomegalovirus, epstein-barr virus and varicella-zoster virus can cause alf. dengue virus causing alf is common in tropical countries, which was thought to be multifactorial due to direct viral injury, dysregulated immune response, hypoxic/ischemic injury secondary to shock, etc. [16] . neonatal haemochromatosis (nh) is the single most common cause of alf during the first month of life, due to massive iron deposition in the liver and extrahepatic tissues with sparing of the reticuloendothelial system. nh presents with jaundice, coagulopathy, moderately elevated alanine aminotransferase, high ferritin and raised iron saturation levels. the disease varies in severity; at one end of spectrum, it is associated with foetal death, while at the other end, spontaneous recovery is reported. the pattern of iron overloading is similar to hereditary haemochromatosis, but nh is entirely different condition affecting newborn, and so far no specific genetic mutation has been identified [17] . current hypothesis suggests nh to be an alloimmune process where maternal antibody is directed towards foetal liver cells resulting in hepatocyte loss [18, 19] . this hypothesis is supported by successful prevention of severe disease by antenatal and postnatal treatment with intravenous immunoglobulin. high serum ferritin is a non-specific marker and elevated in other causes of alf and so should not be used a marker for diagnosis. the diagnosis could be safely confirmed by labial salivary gland biopsy, showing extrahepatic iron deposits with reticuloendothelial system sparing [20] . metabolic disorders are important cause of alf in paediatric population particularly during infancy. galactosaemia, tyrosinaemia type i and fructosaemia are few of the metabolic disorders that could present as alf. tyrosinaemia type i is an inborn error of amino acid metabolism, due to absence of enzyme fumarylacetoacetase, the last enzyme in a series of five enzymes needed to break down tyrosine. this results in formation of intermediate compounds, maleylacetoacetic acid and fumarylacetoacetic acid, which is converted to succinylacetone, a toxin that damages the liver and kidneys. oral ntbc (nitro-4-trifluoromethylbenzoyl-1,3-cyclohexanedione) and phenylalanine-and tyrosine-free diet could help liver recovery, but some children might require lt. galactosaemia type 1 is autosomal recessive disorder with mutation in galactose-1-phosphate uridyl transferase (galt) gene located on chro-mosome 9p13. lactose-free diet should be started in any infant presenting with alf or hepatitis until the quantitative galt activity is available. galactose-free diet and supportive treatment may allow recovery of alf. rarely inborn errors of bile acid synthesis can present as alf. mitochondrial disorders are group of spontaneous or inherited disorders of mitochondrial proteins resulting in defective oxidative phosphorylation, fatty acid oxidation, urea cycle and other mitochondrial pathways [21] . deficiencies of complexes i, iii and iv, multiple complex deficiencies and mitochondrial dna (mtdna) depletion syndrome are associated with liver failure. diagnosis might be difficult due to particularly (mtdna) depletion syndrome where there is tissue-specific mitochondrial enzyme deficiency. these infants usually present with hypotonia, hypoglycaemia, feeding difficulties, seizures and deranged liver function. liver transplantation could be done in isolated liver-based mitochondrial disorders, and it is usually contradicted in multisystemic involvement [22] . sasaki et al. have reported 78% survival in a cohort of nine children with mitochondrial respiratory chain disorder, which included children with extrahepatic manifestation such as developmental delay and failure to thrive [23] . medium-chain acyl-coenzyme a dehydrogenases (mcad) are group of enzymes involved in β-oxidation of 6-12 carbon chain fatty acids in mitochondria. affected children could present with hypoketotic hypoglycaemia and recurrent liver failure, precipitated by otherwise minor illness. unless treated with dextrose supplementation, these episodes may quickly progress to coma and death. wilson's disease, an autosomal recessive disorder, could present as alf. the acute hepatic presentation is usually characterized by the presence of liver failure, coombs-negative haemolytic anaemia and low serum alkaline phosphatase. diagnosis might be difficult in acute presentation as blood test might show weakly positive autoantibodies, and tissue copper estimation might not be possible due to coagulopathy. new wilson index proposed by dhawan et al. used five parameters such as serum bilirubin, serum albumin, international normalized ratio, aspartate aminotransferase (ast) and white cell count (wcc) at presentation. based on serum levels, each parameter is graded from 0 to 4, with a total maximum score of 20. they identified a cutoff score of more than 11 for death without transplantation and proved to be 93% sensitive and 98% specific, with a positive predictive value of 88% [24] . haemophagocytic lymphohistiocytosis (hlh) is a type of haematological malignancy that could present as alf in children. hlh is due to paradoxical overactivation of natural killer cells and of cd8+ t-cell lymphocytes resulting in destruction of own haemopoietic cells. it could be familial (inherited) or acquired. familial hlh usually presents during infancy, while secondary hlh usually occurs after systemic infection or immunodeficiency, which can affect people at any age. familial hlh is an autosomal recessive disease resulting in reduced or defective production of cytoplasmic granules such as perforin in cytotoxic cells resulting in paradoxical overactivation. hlh presents with fever, cutaneous rash, hepatosplenomegaly, pancytopenia and, in severe cases, alf [25] . though rare, leukaemia or lymphoma could present with alf [26] . other causes: autoimmune hepatitis (aih), particularly type 2 (positive liver-kidney microsomal (lkm) antibody), can present with alf. alf due to aih with encephalopathy usually does not respond to any form of immunosuppression and needs urgent liver transplant [27] . in spite of extensive investigation, the diagnosis could not be found in some of the children (indeterminate). there is centre-to-centre variation in incidence of indeterminate alf, probably due to incomplete investigations, which has been highlighted by narkewicz et al. [28] . general investigation should include liver function tests, serum electrolytes, uric acid, lactate, cholesterol/triglyceride, amylase, serum amino acids, blood gas analysis, blood glucose levels, full blood count, blood grouping, coombs test coagulation studies (inr), urinary amino/organic acids and toxicology screen along with surveillance blood and urine cultures. in liver function tests, coagulation should be checked on regular basis that helps in monitoring the progression of disease. investigations to establish the underlying aetiology are listed in table 8 .2. detailed clinical history and physical examination give valuable clue of underlying diagnosis. this would provide guidance in choosing appropriate investigations. transplant-free survival is aetiology dependent. age of patient was not associated with outcome in adults [29] , while in children, neonates have worst prognosis (fig. 8.2) , probably due to predominance of certain aetiology in different age groups. prognostic scoring helps in predicting mortality and helps in listing appropriate patients for transplantation. for non-acetaminophen alf, several prognostic scoring systems are available for adults, but in children there are no universally accepted criteria for listing. to date, inr and factor v concentration remain the best indicators of mortality without transplantation in paediatric alf. bhaduri and mieli-vergani showed that the maximum inr reached during the course of illness was the most sensitive predictor of the outcome, with 73% of children with an inr less than 4 surviving compared with only 4 of 24 (16.6%) with an inr greater than 4 [2] . in children, a factor v concentration of less than 25% of normal suggests a poor outcome. a prognostic score incorporating serum bilirubin, serum albumin, international normalized ratio, aspartate aminotransferase (ast) and white cell count (wcc) is available predicting the outcome of decompensated wilson's disease [24] . in acetaminophen overdose adult criteria of inr > 6.5, creatinine > 300 μmol/l and hyperphosphatemia or metabolic acidosis arterial ph less than 7.3, after the second day of overdose in adequately hydrated patients, is used to list children for liver transplantation [30] . management of alf and its complications still remains a challenge. early diagnosis helps in initiation of investigation and safe transfer to a specialist centre. diagnostic algorithm for any child with abnormal liver function test is outlined in fig. 8 .3. all children with alf should be closely monitored in a quiet setting. vital parameters such as oxygen saturation, pulse, blood pressure and neurologic observations should be done on regular basis. prophylactic broad-spectrum antibiotics and antifungals should be started in all children, and acyclovir should be added in infants and neonates. hypoglycaemia should be avoided either by parenteral glucose or adequate enteral feeds. children with encephalopathy or an inr > 4 (regardless of encephalopathy) should be admitted to an intensive care unit for continuous monitoring. prophylactic histamine 2 blockers or proton-pump inhibitors should be started to all patients requiring mechanical ventilation [31] . coagulopathy is corrected only if the patient is already listed for transplant or prior to an invasive procedure. to correct coagulopathy, fresh frozen plasma could be given at a dose of 10 ml/kg and cryoprecipitate at 5 ml/kg (if fibrinogen is <1 g/l). factor vii concentrates improve the coagulopathy for a short period. platelet count should be maintained above 50 × 10 9 /dl, as thrombocytopenia is an important risk factor for haemorrhage. n-acetylcysteine (nac) is being increasingly used as a part of general supportive measure in non-acetaminopheninduced alf, as it enhances circulation and improves oxygen delivery. in a prospective, double-blind trial in adults with non-acetaminophen alf, nac usage is associated with significant improvement in transplant-free survival in patients with early (stage i-ii) coma [32] . a similar study in children failed to show any benefit, and paediatric acute liver failure study group does not recommend routine use of in non-acetaminophen-induced alf in children [33] . bowel cleansing agents and benzodiazepine antagonists are of no proven benefit [34] . elective intubation and mechanical ventilation should be considered for patients with grade 3/4 encephalopathy. apart from providing secure airway, sedation and controlled ventilation help in reducing sudden variation of intracranial pressure (icp). induction using suxamethonium and fentanyl and combination of morphine or fentanyl with a hypnotic such as midazolam for sedation is usually safe in children. normocapnia is to be maintained, as hypercapnia causes vasodilatation and increases cerebral congestion, while hypocapnia causes vasoconstriction and thus decreased blood flow to the brain. intravenous fluids should be restricted to two-thirds maintenance, with the idea of decreasing the possibility of development of cerebral oedema. ultrasonic cardiac output monitor (uscom), which is a non-invasive method to measure cardiac parameters, helps in decision-making regarding appropriate fluid regimens/inotropes even in small infants. in the presence of persistent hypotension, noradrenaline is the inotropic agent of choice. continuous filtration or dialysis systems should be considered when the urine output is less than 1 ml/kg/h to prevent acidosis and volume overload. the most serious complications of alf are cerebral oedema with resultant encephalopathy and intracranial hypertension, progressively leading on to brain herniation and death. systemic hypertension, bradycardia, hypertonia, hyperreflexia and in extreme cases decerebrate or decorticate posturing are clinical features of raised icp. ammonia-lowering measures such as dietary protein restriction, bowel decontamination or lactulose are of limited or no value in rapidly advancing encephalopathy. mannitol is an osmotic diuretic commonly used to treat intracranial hypertension. a rapid bolus of 0.5 g/ kg as a 20% solution over a 15-min period is recommended, and the dose can be repeated if the serum osmolarity is less than 320 mosm/l. hypertonic saline could be also used in emergency situation, where there is impending brainstem herniation. studies have shown mild cerebral hypothermia (32-35 °c) , sodium thiopental and hypernatremia (serum sodium > 145 mmol/l) improves cerebral perfusion. disease-specific management is outlined in table 8 .2. intravenous immunoglobulin (ivig) at a dose of 1 g/kg body weight given weekly from the 18th week until the end of gestation as antenatal prophylaxis to mothers whose previous pregnancy/child was affected with nh has been associated with milder phenotypic expression of the disease and 100% survival of babies [35] . evidence is accumulating towards the usefulness of high-dose ivig (1 g/kg), in combination with exchange transfusion resulting in significant decrease in the need for liver transplantation in nh. dietary intervention with restriction of phenylalanine and tyrosine together with oral medication, 2(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexenedione (ntbc), helps in normalization of liver function, but doesn't prevent long-term risk for development of hepatocellular carcinoma in children started beyond infancy. plasmapheresis is the removal or exchange of blood plasma. therapeutic plasmapheresis and therapeutic plasma exchange (tpe) are terms that are often used synonymously. tpe has been increasingly used over the past decade as a first-line and lifesaving treatment for various conditions classified by the american society for apheresis (asfa). in acute fulminant wilson's disease, it can rapidly remove significant amount of copper and, thereby, reduce haemolysis, prevent progression to renal failure and provide clinical stabilization. it has been reported to be used as a bridge to lt or can lead to elimination of the need for urgent lt. tpe is also helpful in stabilizing alf due to viral hepatitis, drug-induced hepatitis, etc. by removing albumin-bound toxins, large molecular weight toxins, aromatic amino acids, ammonia, endotoxin, indols, mercaptans, phenols, etc. simple liver assist devices detoxify blood by simple osmotic diffusion, while bioartificial liver support system which contains human or animal liver cells could perform complex synthetic function and detoxifying and detoxification. these devices have shown to decrease the toxins (ammonia, bilirubin, cytokines, etc.) but have no effect on mortality. successful use of these devices in children with alf as a bridge therapy, supporting liver function while the native liver regenerates, is not recommended outside research setting. liver transplant remains the only proven treatment that has improved the outcome of alf. appropriate patient selection and timing of transplantation are essential for graft and patient survival. several surgical techniques such split liver grafts, reduced grafts and auxiliary liver transplants are practiced, depending upon patient size, organ availability and surgical expertise available. auxiliary liver transplant is a surgical technique where the donor liver is placed alongside of native liver and the allograft supports the entire liver function, while the native liver regenerates. either left lateral segment or right lobe allograft could be used, based on recipient weight. once native liver regeneration is optimal [36] , then immunosuppression could be weaned and eventually stopped. in a series from king's college hospital, of the 20 children who received auxiliary liver transplantation for alf, immunosuppression was withdrawn successfully in 11 patients at a median time of 23 months after transplantation [37] . this would be an ideal option in alf due to indeterminate aetiology, as spontaneous regeneration of native liver remains a possibility. liver transplantation is indicated in alf due to liverbased disorders while contraindicated in haematological malignancies, uncontrolled sepsis, systemic mitochondrial/ metabolic disorders and severe respiratory failure (ards) [38] . relative contraindications are increasing inotropic requirements, infection under treatment, cerebral perfusion pressure of less than 40 mmhg for more than 2 h and a history of progressive or severe neurologic problems. hepatocyte transplantation, where hepatocytes are infused intraportally into the patient's liver, has been tried with variable success in certain liver-based metabolic disorders [39] . research is underway to use alginate-encapsulated hepatocytes, which could be injected intraperitoneally. this could act as a bridge until native liver regenerates. hepatocyte transplantation is not recommended outside research setting. improved intensive care management has greatly increased the alf survival. when compared to adult alf, the spectrum of underlying aetiology, management and outcome varies in paediatric alf. acyclovir should be started in all neonates with alf along with prophylactic antibiotics, until viral cultures are negative. liver transplantation is the only definitive treatment that improves survival in paediatric alf. wilson's disease and autoimmune liver disease presenting as alf usually do not respond to medical management and warrant liver transplantation. liver assist devices and hepatocyte transplantation are potential emerging therapies in paediatric alf. the management of fulminant hepatic failure fulminant hepatic failure: pediatric aspects. semin liver dis acute liver failure in children: the first 348 patients in the pediatric acute liver failure study group profile and outcome of first 109 cases of paediatric acute liver failure at a specialized paediatric liver unit in india neonatal liver failure: aetiologies and management-state of the art population-based surveillance for acute liver failure protective agents for acetaminophen overdose review article: drug-induced liver injury in clinical practice the characteristics and clinical outcome of drug-induced liver injury: a single-center experience drug-induced liver injury: a clinical update assessment of drug-induced hepatotoxicity in clinical practice: a challenge for gastroenterologists prognostic factors in paediatric acute liver failure global epidemiology and medical aspects of hepatitis e. forum (genova) a 20-year single-center experience with acute liver failure during pregnancy: is the prognosis really worse? dengue and its effects on liver neonatal hemochromatosis. genetic analysis of transferrin-receptor, h-apoferritin, and l-apoferritin loci and of the human leukocyte antigen class i region neonatal hemochromatosis: is it an alloimmune disease? novel mechanism of fetal hepatocyte injury in congenital alloimmune hepatitis involves the terminal complement cascade minor salivary gland biopsy in neonatal hemochromatosis disorders of the mitochondria liver transplantation for mitochondrial respiratory chain disorders: to be or not to be? liver transplantation for mitochondrial respiratory chain disorder: a single-center experience and excellent marker of differential diagnosis wilson's disease in children: 37-year experience and revised king's score for liver transplantation hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. pediatr blood cancer leukaemia presenting with fulminant hepatic failure in a child autoimmune paediatric liver disease pattern of diagnostic evaluation for the causes of pediatric acute liver failure: an opportunity for quality improvement results of a prospective study of acute liver failure at 17 tertiary care centers in the united states early indicators of prognosis in fulminant hepatic failure aasld position paper: the management of acute liver failure n-acetylcysteine on its way to a broader application in patients with acute liver failure intravenous n-acetylcysteine in pediatric patients with nonacetaminophen acute liver failure: a placebo-controlled clinical trial acute liver failure in children high-dose immunoglobulin during pregnancy for recurrent neonatal haemochromatosis clinical human hepatocyte transplantation: current status and challenges auxiliary liver transplantation for acute liver failure in children hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure cell therapy for liver disease: from liver transplantation to cell factory key: cord-018801-amet0wx4 authors: park, caroline; clark, damon title: care of the patient with liver failure requiring transplantation date: 2018-05-04 journal: surgical critical care therapy doi: 10.1007/978-3-319-71712-8_55 sha: doc_id: 18801 cord_uid: amet0wx4 patients undergo liver transplantation to address chronic liver failure, acute fulminant liver failure, or primary liver cancer. depending on acuity, patients with decompensated chronic or acute fulminant liver failure generally require preoperative intensive care unit admission to manage organ dysfunction. those with chronic liver failure are allocated an organ based on waiting list position determined by their local organ procurement organization (opo). this position is dependent upon blood type and model for endstage liver disease (meld) score. these patients thus are critically ill and require preoperative icu monitoring and care. patients with hepatocellular carcinoma (hcc) who require liver transplantation are given a meld exception and rarely require preoperative icu care. the patient’s ability to undergo liver transplant in the setting of hcc is determined by the milan criteria or the university of california, san francisco (ucsf) criteria. patients undergo liver transplantation to address chronic liver failure, acute fulminant liver failure, or primary liver cancer. depending on acuity, patients with decompensated chronic or acute fulminant liver failure generally require preoperative intensive care unit (icu) admission to manage organ dysfunction. those with chronic liver failure are allocated an organ based on waiting list position determined by their local organ procurement organization (opo). this position is dependent upon blood type and model for end-stage liver disease (meld) score. meld is determined by a weighting of serum bilirubin, creatinine, and international normalization ratio (inr). those with a high meld score have a greater risk of mortality and thus are given priority to transplantation. patients with a meld score of 40 have a 75% chance of death within 3 months. this is particularly important given the transplantation is typically performed on those with high meld scores in large urban areas such as ours in the greater los angeles area. these patients thus are critically ill and require preoperative icu monitoring and care. patients with hepatocellular carcinoma (hcc) who require liver transplantation are given a meld exception and rarely require preoperative icu care. the patient's ability to undergo liver transplant in the setting of hcc is determined by the milan criteria or the university of california, san francisco (ucsf) criteria. hcc patients with a single tumor <5 cm, up to three tumors <3 cm, absence of macroscopic vascular invasion, and absence of extrahepatic spread may undergo liver transplantation based on the milan criteria. altered level of consciousness is common in patients with decompensated cirrhosis. this is a result of portosystemic shunting and hepatocellular dysfunction/toxin production that results in hepatic encephalopathy. most severe or accelerated changes in the level of consciousness are the result of a precipitating event such as a significant upper gastrointestinal bleed and resultant uremia or a new infection. encephalopathy can be exacerbated by the administration of medications such as benzodiazepines that are generally avoided in the routine treatment of this patient population. those with low-grade encephalopathy, manifested by mild confusion and tremors, are administered agents such as lactulose to decrease ammonia absorption by acidifying bowel content and increase transit of the bowel contents. rifaximin is also commonly used to diminish the presence of urease and protease-splitting bacteria. it is important to remember to discontinue lactulose therapy in advance of anticipated liver transplantation to avoid intraoperative diarrhea and possible bowel distention. systemic and splanchnic arteriolar vasodilation is a wellknown physiologic derangement in patients with end-stage liver disease. as a result, these patients have hyperdynamic and low systemic vascular resistance cardiac profiles and often require vasopressor therapy-whether oral (e.g., midodrine) or intravenous (e.g., norepinephrine)-to maintain adequate mean arterial pressures (map) of >65 mmhg. it is important to consider that hypotension may be multifactorial, resulting from active hemorrhage, infection, and/or systemic inflammatory response (sirs). further, cardiac contractile function may be impaired in patients with longstanding cirrhosis or in those with associated ischemic cardiac disease. routine use of beta blockade as prophylaxis in those with varices may also cause hypotension. if hemor-rhage and sirs response have been addressed and ruled out, refractory hypotension could be a result of adrenal insufficiency; this is a common pathophysiology in end-stage liver disease patients. there is little additional role for invasive cardiac monitoring in cirrhotic patients for the diagnosis and treatment of shock over echocardiography and other noninvasive means. in general, the treatment of shock does not depart much from that in other patients. patients should be approached similarly with crystalloid resuscitation and early goal-directed therapy; there is little evidence that albumin should be the resuscitative fluid of choice. monitoring base deficit and lactate is as prognostic and useful to guide resuscitative efforts in patients with decompensated liver disease as it is for the general icu population. patients with advanced liver disease may have other associated pulmonary disorders, including hepatic hydrothorax, emphysema from alpha-1-antitrypsin deficiency, hepatopulmonary syndrome (hps), and portopulmonary hypertension (pph). hydrothorax is typically the result of tense abdominal ascites and should be addressed by paracentesis, thoracentesis, and total body volume management. draining of pulmonary effusions should be limited to those with impending respiratory failure due to the risk of infection and hemorrhage associated with invasive procedures and re-accumulation. definitive treatment of pulmonary effusions is with liver transplantation. hps is caused by intrapulmonary shunting and does not result in right heart failure. patients with hps will classically experience positional hypoxia. pph on the other hand results from pulmonary vascular vasoconstriction and can ultimately lead to thrombosis and fibrosis. in contrast to hps, right heart failure is typical in pph. it is important to distinguish between hps and pph. in general, liver transplantation is curative of the former and, until recently, was contraindicated in the latter. however, the introduction of many new agents and classes of agents to treat pulmonary hypertension may render liver transplantation possible in centers of excellence with careful pre-and intraoperative monitoring. although noninvasive monitoring is helpful and new modalities are being developed, the most expeditious and accurate modality to differentiate between hps and pph is right heart catheterization. hps, pph, and chf can all cause elevated pulmonary artery pressures. however, pulmonary artery wedge pressure (pawp) is low in hps and pph (and elevated in chf). pulmonary vascular resistance (pvr) is normal to decreased in hps but elevated in pph. thus, cardiac output is elevated in hps and normal to decreased in pph. one of the major causes of aki in patients with advanced cirrhosis and ascites is the phenomenon of hepatorenal syndrome (hrs) or acute renal failure without other etiology. there are two subtypes of hrs, types i and ii. the first type generally progresses with a rapid decrease in renal function and is characterized with doubling in serum creatinine within that period of time. it may be precipitated by spontaneous bacterial peritonitis, gastroenteritis with high-volume diarrhea, volume loss from gastrointestinal bleed, or largevolume paracentesis without appropriate volume repletion. type i hrs can be fatal and often leads to multi-system organ failure. type ii hrs demonstrates a more indolent course of renal failure, often precipitated by ascites refractory to diuretic treatment [1] . hrs develops due to splanchnic circulation vasodilation, intravascular hypovolemia, and renal vasoconstriction and is most often a diagnosis of exclusion after investigating for other causes of renal failure. the treatment approach includes strict intake and output monitoring, serum creatinine monitoring, and following changes from baseline or within the past 48 h. if the patient develops oliguria with elevated serum creatinine greater than 50% from a reference value or baseline serum creatinine level, suspect aki. multiple diagnostic variable are used to diagnose hrs in patients with cirrhosis ( fig. 55.1 ). these patients may require combined liver and renal transplant and/or intraoperative renal replacement therapy to assist with volume status, correcting acidosis, and electrolyte abnormalities. the mainstay of treatment for hrs is liver transplantation, after which a majority of patients demonstrate a return to adequate renal function. consideration should be given to a combined liver/kidney transplantation in those who have required dialysis for greater than 2 months, although this time period is controversial. a variety of approaches can be used to increase intravascular volume and map including albumin and vasopressors (norepinephrine, terlipressin). intermittent paracentesis may be necessary to manage third spacing of fluids in hepatorenal syndrome prior to liver transplant [2, 3] . type 1 hrs requires multiple therapeutic strategies and therapies; please refer to fig. 55 .2 for a detailed algorithm. the presence of infection in a cirrhotic patient quadruples mortality and worsens liver function. those with chronic liver failure are functionally immunosuppressed and are often colonized with multiresistant organisms. the most common infection in these patients is spontaneous bacterial peritonitis. strict attention should be paid to removing unnecessary catheters and avoiding intubation/mechanical ventilation, when possible. patients require prophylactic antibiotics for spontaneous bacterial peritonitis after a variceal bleed; however, there is little need for general broadspectrum antimicrobial therapy. patients with fulminant liver failure receive a meld exemption in listing-the appropriateness of transplantation may be determined by king's criteria (table 55 .1). in general, patients experience toxic necrosis of the liver on a baseline of normal function, most commonly due to intentional or inadvertent ingestion of large doses of acetaminophen. thus, the abnormalities noted are lactic acidosis, marked elevation of inr, and high-grade encephalopathy. ascites and hepatorenal syndrome are often not present given the acuity of presentation. creatinine, however, is often elevated due to atn. if time of ingestion is known, and within 8 h, n-acetylcysteine should be administered to prevent further toxicity in acute liver failure patients. if the patient fails medical management with progression of liver failure, liver transplantation will be required. the most significant risk of mortality to a patient with fulminant liver failure is that of cerebral edema and subsequent death from cerebral hernia-tion. intracranial monitoring remains controversial given severe coagulopathy, thrombocytopenia, and risk of infection. serum sodium of 145-150 meq/l should be maintained with hypertonic saline to decrease the amount of cerebral edema. if renal dialysis is required, a continuous mode is preferred to avoid rapid fluid shifts that may exacerbate cerebral edema. patients having undergone liver transplantation will require postoperative intensive care unit (icu) admission. close communication and coordination of care between the surgeons, anesthesiologists, intensivists, and nursing staff are essential to the management of the patient in this setting. liver transplantation typically entails a lengthy surgical procedure requiring significant amounts of blood product transfusion and risk of postoperative respiratory insufficiency. preoperatively, many of these patients have neurologic, cardiopulmonary, and renal dysfunction requiring fentanyl, a narcotic, is the first-line agent for the treatment of pain and agitation given its rapid onset and short duration of action in postoperative transplant patient. those who require additional sedation for agitation not controlled with narcotic analgesia benefit from the use of dexmedetomidine over benzodiazepines given a decreased risk of iatrogenic delirium and decreased length of mechanical ventilation [5] . dexmedetomidine is an alpha-2 adrenoreceptor agonist and should be used in caution with patients with hypotension and baseline bradycardia as it can exacerbate both conditions. with the use of spontaneous awakening trials (sats), richmond agitation and sedation score (rass), and confusion assessment method for the icu (cam-icu), patients have decreased episodes of delirium, duration of mechanical ventilation, and icu and hospital length of stay [6] . in this particular population, however, sustained delirium and encephalopathy may be the result of poor functioning of liver transplant graft, infection, intracranial hemorrhage or cerebral ischemia, seizures, and/or immunosuppressant toxicity. there should be a low threshold to pursue diagnostic ct scan of head, cultures including cerebral spinal fluid, and electroencephalography (eeg) in the posttransplant patient with change in mental status. encephalopathy due to cerebral edema associated with fulminant liver failure and elevated ammonia levels in patients with end-stage liver disease should be corrected with an adequately functioning liver transplant graft. if an intracranial monitor was placed preoperatively, it should be maintained until the inr is corrected and the liver is functioning well. in the past decade, early recovery after surgery or "fasttrack" programs have been implemented in a variety of disciplines, including hepatobiliary and colorectal patients after elective surgery with no worsening of postoperative outcomes and improvement in patient satisfaction. liver transplant patients may be eligible for fast-track extubation immediately postoperative in the operating room and upon arrival to the icu. patients that successfully undergo fasttrack extubation have been shown to benefit from decreased rates of re-intubation and tracheostomy along with improved survival [7] . patients that are likely not candidates for fasttrack extubation include those with preoperative acute liver failure, re-transplantation, child's c cirrhosis, and intraoperative red blood cell transfusion >6 units [7] . patients that require continued mechanical ventilation upon arrival to the icu should be placed on ventilator settings of tidal volume 6 ml/kg and fio2 < 0.4 [8] . patients who may exhibit transfusion-related lung injury after receiving a significant amount of blood products require ventilation strategies similar to patients with acute respiratory distress syndrome (ards); in this case, target tidal volumes of 6 ml/kg with supplemental oxygen and positive endexpiratory pressure (peep) [9] . infections of the lower respiratory tract require broad-spectrum antibiotics and antifungals until species and sensitivities are established. the liver transplant patients who remain hemodynamically stable and require minimal mechanical ventilation settings with resolved encephalopathy should undergo daily spontaneous breathing trials (sbt) and subsequent evaluation for possible extubation to reduce the duration of mechanical ventilation and icu length of stay [10] . early mobilization and aggressive chest physiotherapy are performed to prevent complications of atelectasis and inadequate ventilation. centers may opt to monitor patients intraoperatively with pulmonary artery catheters and/or transesophageal echocardiography. once stable and resuscitated, patients should be liberated from these devices. steroids are routinely administered as a part of early immunosuppression regimen after liver transplant and may require a prolonged course in treating hypotension secondary to adrenal insufficiency. liver transplantation patients remain at risk for postoperative hemorrhage due to thrombocytopenia, fibrinolysis, and deficiency of coagulation factors. abnormal coagulation tests and platelet count are not good predictors of bleeding; thus aggressive correction of these coagulopathies should be avoided. therapy should also include practical measures as avoiding hypothermia and persistent acidosis. aggressive correction of coagulopathy and thrombocytopenia may also put patients at higher risk of hepatic artery, portal vein, and deep vein thrombosis. typical target ranges include hemoglobin of 8 g/dl and platelet count >20 × 10^9/l [11] . thromboelastography (teg) may be useful in dictating guided blood product resuscitation in the post-liver transplant patients to decrease blood loss and transfusion requirements [12] . liver transplant patients with hemorrhage that are undergoing appropriate blood product resuscitation and become hemodynamically unstable or develop abdominal compartment syndrome should return immediately to the operating room. patients with advanced cirrhosis are often malnourished and as such are at higher risk for infections, worsening encephalopathy, and decompensation. though these patients may appear grossly overweight, their usual or dry weight is often masked by massive ascites and edema secondary to hypoalbuminemia. the american and european society for clinical nutrition and metabolism and the european society for clinical nutrition and metabolism (aspen [13] and espen [14] , respectively) have compiled an extensive set of guidelines, both of which provide a subset of consensus statements for patients with hepatic failure. the primary goals of nutrition for patients with hepatic failure include (1) identifying and assessing patients at risk for undernutrition, (2) calculating nutritional needs and incorporating adequate protein and high-calorie formulas, and (3) considering dobhoff placement if encephalopathy precludes voluntary enteral nutrition or short-term parenteral nutrition if unable to provide enteral feeds secondary to ileus or malabsorption. dry or usual weight may be difficult to ascertain given the chronicity of liver disease, thus complicating calculations for caloric needs. poor oral intake may be a result of underlying encephalopathy, gastroparesis, and overall decreased gastrointestinal motility. in prior years, protein restriction was emphasized to mitigate the effects on worsening hepatic encephalopathy. however, given the already reduced lean muscle mass of this vulnerable patient population, proteinrestricted diets can worsen hepatic failure. recommended protein intake is 1.2-1.5 g/kg/day, with a total energy intake of 35-40 kcal/kg/day. dobhoff placement is recommended if the patient is unable to meet his/her caloric needs per os; percutaneous endoscopic gastrostomy or open gastrostomy tube is otherwise not recommended given an increased risk of complications [14] . post-liver transplant acute kidney injury (aki) is a frequent event with reports of up to 52% of patients developing aki [15] . factors such as increased child-pugh score, preexisting diabetes, and large number of intraoperative transfusions increase the risk of aki in the post-liver transplant. the development of post-liver transplant aki leads to prolonged icu and hospital length of stay, increased mortality, and decreased duration of liver graft function [15] . in patients that develop aki post-liver transplantation, treatment includes the prevention of hypotension and decreased use of unnecessary blood products. the use of renal replacement therapy is reserved for patients that develop significant volume overload, uremia, and electrolyte abnormalities. the most effective treatment of postoperative liver transplant aki is prevention. preventive strategies include delayed initiation of calcineurin inhibitors, avoiding nephrotoxic agents such as iv contrast, and ensuring adequate control of hyperglycemia [15] . the most common cause of morbidity and mortality after liver transplantation is infection, accounting for 60% of the deaths after liver transplantation [16] . prolonged and complicated operations, multiple catheter insertions, immunosuppression, and large quantities of fresh frozen plasma can all increase the risk of infectious complications [17] . diagnosis of infections in this patient population may be difficult due to the lack of signs and symptoms such as fever, chills, cellulitis, and leukocytosis due to an immunosuppressed status. early postoperative infections in liver transplant patients are typically bacterial and related to the donor's status (previous infections from advanced cirrhosis), the surgical procedure itself, prolonged use of invasive catheters, and duration of mechanical ventilation. perioperative antibiotics are typically broad spectrum and may include third-generation cephalosporins. early removal of invasive catheters, early mobility, pulmonary toilet, vigilant monitoring of patient's surgical wounds and drains, and early discharge from the icu may decrease these infectious complications. liver transplant patients are at risk of developing opportunistic infections given the initial burst of immunosuppression with high-dose steroid therapy and, as such, should be initiated on prophylactic trimethoprimsulfamethoxazole (tmp-smx) to prevent pneumocystis carinii pneumonia and ganciclovir to prevent cytomegalovirus infection. besides surgical and coagulopathic bleeding, other postoperative complications can occur; these include postoperative hepatic artery thrombosis (3%) or portal vein thrombosis (< 1%) [11] . the resulting lack of blood flow and developing ischemia and necrosis from hepatic artery thrombosis present with signs and symptoms similar to fulminant liver failure patients with elevated liver serum tests, coagulopathy, and severe metabolic acidosis. doppler ultrasound of the hepatic artery and portal vein is routinely employed within the first 24-48 h after liver transplant to diagnose possible vascular complications prior to the development of ischemia and necrosis of the liver transplant graft. these patients are at high risk for continued ischemia and necrosis of the graft with the need for urgent relisting and re-transplantation. compared to patients with hepatic artery thrombosis, those with portal vein thrombosis do not present with such critical signs and symptoms as a rapid rise in liver function tests and disruption in synthetic function. although portal vein thrombosis leads to elevation in liver serum tests, signs and symptoms are less dramatic and may consist of mesenteric venous congestion, gastrointestinal hemorrhage, and the development of ascites. although these patients may require retransplantation, they can typically be managed with thrombectomy, shunt, or revision of the portal vein anastomosis. biliary duct complications, which include anastomosis stricture or leak, affect 5-25% of liver transplant patients and are often delayed diagnoses [18] . thrombosis of the liver transplant hepatic artery can also lead to nonanastomotic stricture [18] . biliary duct complications can be evaluated with ultrasound of the liver transplant graft looking for biloma and biliary duct dilation. similarly, internal to external drains placed during the liver transplantation may show biliary drainage during the first several postoperative days. elevated serum liver tests specifically bilirubin will elevate or fail to appropriately decrease after liver transplant, and the patients may develop signs and symptoms of infection. magnetic resonance cholangiopancreatography (mrcp) may be used as a noninvasive diagnostic modality to look for biliary anastomosis complications. endoscopic retrograde cholangiopancreatogram (ercp) can be used for the diagnosis of biliary anastomosis leak and stricture, in addition to possible treatment with sphincterotomy and/or biliary stent [19] . endoscopic treatment is often preferred over percutaneous management of biliary leaks and stricture. treatment options include endoscopic dilation and stenting and have excellent success rates approaching 75% [20] . surgical revision of the biliary anastomosis due to stricture or leak may be required in 10-20% of patients [18, 21] . the use of broad-spectrum antibiotics for treatment or prophylaxis is recommended due to the high risk of cholangitis and intra-abdominal sepsis [22] . 30-50% of patients with biliary stricture will have to undergo re-transplantation due to chronic biliary cirrhosis due to obstruction even with adequate treatment [19, 22] . primary graft nonfunction and hyperacute rejection can occur in the immediate or acute postoperative setting. primary graft nonfunction occurs in 2-14% of orthotopic liver transplants and typically presents similar to fulminant liver failure with significant metabolic acidosis, elevated liver enzymes, coagulopathy, and lack of bile production [11, 23] . intraoperative hemodynamic instability, reperfusion injury of the liver transplant graft, marginal livers, and advanced age of donors and recipient are factors that may lead to primary graft nonfunction. once diagnosed, the only treatment indicated is for relisting and liver re-transplantation. development of hyperacute rejection (har) after liver transplant is a rare complication that may develop intraoperatively or in the immediate postoperative period, which is antibody-mediated and due to abo crossmatch incompatibility. patients with har typically present with progressive encephalopathy and weakness, elevated bilirubin, severe coagulopathy, thrombocytopenia, metabolic acidosis, and shock. diagnosis is confirmed with doppler ultrasound that displays portal vein thrombosis and absence of biliary duct stricture. along with critical care supportive therapy, patients can be managed with plasma exchange for antibody removal and intravenous immunoglobulin [11] . overall, patients that develop har will need immediate relisting and re-transplantation. acute cellular rejection after liver transplant may occur within the first 6-8 weeks, and the patients are often out of the icu and no longer critically ill. patients with acute cellular rejection typically are not critically ill and may present with fever, weakness, and elevated liver function tests. prior to treating the patients for acute cellular rejection, one must rule out all possible acute infections that could account for the signs and symptoms given that the treatment of acute cellular rejection requires immunosuppression with pulse-dose glucocorticoids and adjustment of other immunosuppression medications. glucocorticoids are the first-line therapy for the prevention and treatment of acute cellular rejection. common glucocorticoids used for liver transplant include prednisone, hydrocortisone, and methylprednisolone with the first dose given while in surgery. intravenous hydrocortisone is typically administered in the immediate postoperative period until the patient is taking enteral nutrition and can transition to oral prednisone. most patients will undergo a glucocorticoid taper and either transitioned off of glucocorticoids or to a low maintenance dose, typically over a 6-month to 2-year period [24] . glucocorticoids have a significant number of side effects including poor wound healing, increased infection risks, hyperglycemia, and hypertension; these patients may need judicious adjustment of insulin sliding scale for hyperglycemia. glucocorticoid-free immunosuppression is possible and may be of benefit in patients with cirrhosis due to hepatitis c virus. [25, 26] calcineurin inhibitors (cni), including cyclosporine and tacrolimus, are used to prevent and treat acute rejection and liver transplant graft loss. both provide immunosuppression by inhibiting interleukin-2 and interferon-gamma production and require monitoring of blood levels to reach appropriate therapeutic levels. potential side effects including altered mental status, seizures, neuropathy, renal failure, electrolyte abnormalities, and others should be monitored for and treated appropriately. tacrolimus is currently the cni of choice and has demonstrated superiority in preventing acute rejection and graft loss with decreased mortality [27, 28] . posterior reversible encephalopathy syndrome (pres) is a rare syndrome and side effect of cni that is diagnosed with clinical exam and ct or mri. patients with pres most commonly present with seizures but may also have symptoms such as headache, delirium, and visual changes. head ct or mri typically demonstrates vasogenic edema of the parietal or occipital lobes; however mri may be more sensitive in diagnosing pres. treatment of pres most often involves discontinuing the offending cni and supportive care. mycophenolate mofetil (mmf, cellcept) is an antimetabolite that inhibits purine and pyrimidine synthesis with the active by-product of mycophenolic acid (mpa). mpa ultimately inhibits the proliferation of t lymphocytes for immunosuppression. mmf is typically used long term to reduce the dose or replace glucocorticoids. as such, the use of mmf will avoid common cni side effects, such as nephrotoxicity and neurotoxicity, though it can cause other side effects, including abdominal pain, nausea, vomiting, anorexia, diarrhea, and bone marrow suppression. mmf as a monotherapy after the acute phase of liver transplant has shown similar results to glucocorticoids and cni for prevention of chronic rejection and mortality [29, 30] . mammalian target of rapamycin (mtor) inhibitors (everolimus and sirolimus) inhibit the proliferation of lymphocytes. the use of mtor inhibitors allows for immunosuppression while avoiding renal dysfunction and has shown potential benefit in patients undergoing liver transplant for hcv. common complications of dyslipidemia and oral ulcers are typically easy to manage. hepatorenal syndrome pathogenesis of hepatorenal syndrome: implications for therapy systematic review of randomized trials on vasoconstrictor drugs for hepatorenal syndrome management of the critically ill patient with cirrhosis: a multidisciplinary perspective improved analgesia, sedation and delirium protocol associated with decreased duration of delirium and mechanical ventilation dexmedetomidine vs. midazolam or propofol for sedation during prolonged ventilation fast tracking in liver transplanatation: which patient benefits from this approach? a trial of intraoperative low-tidal-volume ventilation in abdominal surgery the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome protocolized versus non-protocolized weaning for reducing the duration of mechanical ventilation in critically ill adult patients immediate postoperative management and complications on the intensive care unit methods to decrease blood loss and transfusion requirements for liver transplantation guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition espen guidelines on enteral nutrition liver disease acute kidney injury following orthotopic liver transplantation: incidence, risk factors and effects on patient and graft outcomes causes of death in autopsied liver transplantation patients risk factors for cytomegalovirus and severe bacterial infections following liver transplantation: a prospective multivariate time-dependent analysis management of biliary complications after liver transplantation biliary complications after liver transplantation: the role of endoscopy efficacy of endoscopic and percutaneous treatments for biliary complications after cadaveric and living donor liver transplantation anastomotic biliary strictures after liver transplantation: causes and consequences nonanastomotic biliary strictures after liver transplantation, part 2: management, outcome and risk factors for disease progression improving the diagnostic criteria for primary liver graft nonfunction in adults utilizing standard and transportable laboratory parameters: an outcome-based analysis influence of steroids on hcv recurrence after liver transplantation: a prospective study a randomized, multicenter study comparing steroid free immunosuppression and standard immunosuppression for liver transplant recipients with chronic hepatitis c randomized, multicenter trial comparing tacrolimus plus mycophenolate mofetil to tacrolimus plus steroids in hepatitis c virus-positive recipients of living donor liver transplantation randomized controlled trial of tacrolimus versus microemulsified cyclosporine (tmc) in liver transplantation: poststudy surveillance to 3 years cyclosporin versus tacrolimus for liver transplanted patients corticosteroid-free strategies in liver transplantation four-year follow-up of mycophenolate mofetil for graft rescue in liver allograft recipients key: cord-018225-dozmy3lb authors: hawker, felicity h. title: the liver in critical illness date: 2008 journal: classic papers in critical care doi: 10.1007/978-1-84800-145-9_7 sha: doc_id: 18225 cord_uid: dozmy3lb the liver is in some ways the forgotten organ in intensive care practice. very many more laboratory and clinical studies have investigated the role, function, and support of the lung, heart, brain, and kidney in critical illness than have studied the liver. nevertheless, in the time of the greek scholars, there was already acknowledgement of the role of the liver in non-hepatic diseases such as systemic sepsis, and an understanding that such involvement confers a poorer prognosis – hence the inclusion of the wisdom of hippocrates in this compilation of classic papers. in the review article by matuschak and rinaldo, the reasons why liver dysfunction is associated with a poorer outcome in critical illness are explored, and the concept of the liver being a ‘driving force’ in multiple organ dysfunction is developed. in addition, jaundice without significant liver dysfunction is associated with left ventricular dysfunction, at least in the dog model developed by professor otto better and his colleagues in israel. this observation is relevant to the progressive resistance to inotropic and vasopressor agents in jaundiced critically ill patients. the liver is in some ways the forgotten organ in intensive care practice. very many more laboratory and clinical studies have investigated the role, function, and support of the lung, heart, brain, and kidney in critical illness than have studied the liver. nevertheless, in the time of the greek scholars, there was already acknowledgement of the role of the liver in non-hepatic diseases such as systemic sepsis, and an understanding that such involvement confers a poorer prognosis -hence the inclusion of the wisdom of hippocrates in this compilation of classic papers. in the review article by matuschak and rinaldo, the reasons why liver dysfunction is associated with a poorer outcome in critical illness are explored, and the concept of the liver being a 'driving force' in multiple organ dysfunction is developed. in addition, jaundice without significant liver dysfunction is associated with left ventricular dysfunction, at least in the dog model developed by professor otto better and his colleagues in israel. this observation is relevant to the progressive resistance to inotropic and vasopressor agents in jaundiced critically ill patients. one of the most devastating insults that can occur in critical illness is acute liver ischemia, resulting in the clinical syndrome of ischemic or hypoxic hepatitis. although relatively common, this syndrome has been difficult to study prospectively. consequently, the paper by jean henrion from belgium offers new insights into the hemodynamic associations of ischemic hepatitis, which is, of course, a circulatory disease, not a liver disease. commonly used 'liver function tests' assess damage to the liver rather than its function. an understanding of the complex relationship between critical illness and liver dysfunction has been hampered by the absence of a simple test that assesses at least one of the many functions of the liver. the megx test, developed by oellerich and co-workers in germany, allows dynamic assessment of liver function, and may prove useful in the evaluation of therapies to support liver function in critical illness. the complex issues involved in the management of patients with acute liver failure exemplify the many important functions of the healthy liver. with the advent of liver transplantation as the principle treatment for patients with fulminant hepatic failure, these patients are almost always managed in icus servicing liver transplant units. consequently, it is extremely important that intensive care practitioners in non-liver units understand the need to refer patients to transplant units, as well as the role of transplantation in acute liver failure and its indications. the seminal paper by o'grady and the group from king's college hospital, london, develops criteria for liver transplantation in acute liver failure. that a successful outcome is possible after emergency liver transplantation for acute liver failure is shown in the early series of cases reported from the french group from clichy and villejuif. when transplantation is not indicated, or while awaiting a donor, the principal treatment is supportive. the paper by harrison and co-workers, again from the king's liver unit, investigates the effects of n-acetylcysteine in patients with acute liver failure, and the findings of this study have resulted in widespread use of this agent in this setting. currently, there is still no safe, effective means of artificial liver support for patients awaiting transplantation or liver regeneration. the case report from saunders and co-workers from cape town, south africa, describes an early attempt to develop such support. finally, patients with cirrhosis and other forms of chronic liver disease present different management challenges for the intensive care specialist. the randomized controlled trial from the barcelona group shows that in patients with spontaneous bacterial peritonitis and cirrhosis, the outcome is improved by infusing albumin in addition to antibiotic therapy. there are many classic papers that have not been included in this small selection. the majority of papers have been chosen for the underlying ideas and variety rather than for scientific rigor. they are aimed at clinical intensive care specialists with enquiring minds who work in general intensive care units, rather than experts in liver units or intensive care units servicing liver transplant units. i have found the liver a fascinating organ to study. i trust this selection of papers will likewise stimulate your interest. hippocrates examines a number of questions with respect to jaundice associated with systemic infection. he concludes that jaundice is a result of impairment of the separative faculty of the liver; that is, impairment of its ability to purify all the humors. he further states that this may occur as a result of obstruction of the biliary vessels or of seething inflammations, in which case the blood is distributed through the whole body with a strong admixture of bile. this review article explores the role of the liver in the pathogenesis of the adult respiratory distress syndrome (ards) and progressive multiple systems organ failure. it is argued that the normal liver plays a pivotal role in aspects of host defence that are crucial to the protection of the lung and other organs in systemic sepsis. the authors note that the fixed hepatic macrophages, or kupffer cells, constitute nearly 90% of the body's reticuloendothelial cell mass. they further note that these cells are the major mechanism for clearance of systemic endotoxin and bacterial products. the strategic location of the liver, immediately downstream from the large gastrointestinal reservoir of bacteria and bacterial products, normally constitutes an effective first line in host control of systemic endotoxemia and bacteremia. however, the balance may be altered by an excess load of bacterial products in the gut, mesenteric or hepatic ischemia, and kupffer cell dysfunction, so that the phagocytic capacity of kupffer cells is exceeded, resulting in systemic spread of bacterial products. moreover, the authors describe the activation of kupffer cells by the phagocytic burst, resulting in the release of an array of inflammatory mediators that are 'exported' systemically, and result in acute pathophysiological reactions in distant organs. these inflammatory mediators also have effects on adjoining hepatic parenchymal cells. evidence is presented that the resulting hepatocyte damage may result in decreased clearance of some of these mediators, resulting in heightened and prolonged effects, and consequent amplification of multiple organ damage. furthermore, these inflammatory mediators -particularly interleukin-1 -induce changes in hepatic protein synthesis, characterized principally by the 'acute phase response'. thus, this review acknowledges the role of the liver in host defence but also proposes ways in which liver dysfunction might fuel multiple organ dysfunction -particularly ards. the liver plays a major role in host defence, but also has the capacity to augment the host response, with consequent adverse effects on other organs in some circumstances. this has led to the concept of the liver as 'the motor of multiple organ failure'. this review was among the first to provide a plausible concept of organ system interactions that may result in ards and multiple organ failure in sepsis. although some of the detail has been refined, the general principles remain accepted 15 years later. this paper synthesizes experimental findings from a number of disciplines -intensive care medicine, hepatology, immunology, and thoracic medicine -to arrive at a concept that was new at the time of publication, and which continues to be supported in the main by more recent evidence. this paper is not a systematic review. the general concepts in this review remain valid, and have led to a more concentrated focus on the gut and liver in the critically ill patient, both experimentally and in the clinical setting. although there have been major advances in understanding of relevant factors such as splanchnic blood flow, no 'liver-orientated therapy' has yet been shown to be of value in ards and multiple organ failure, and the treatment of liver dysfunction remains supportive. the "jaundiced heart": a possible explanation for postoperative shock in obstructive jaundice abstract patients with obstructive jaundice are susceptible to postoperative shock and kidney failure. the cause of these potentially fatal complications has not been fully clarified. the present study was designed to assess the role of myocardial dysfunction in the hemodynamic disturbance of obstructive jaundice. we studied the effect of isolated cholemia on left ventricular performance in five conscious dogs, before and 2 weeks after choledochocaval anastomosis, by using measurements of systolic time intervals (stis) and maximal dp/dt. mean left ventricular ejection time (lvet) decreased after cholemia from 159 +/-2.8 msec to 139 +/-2.6 msec (p < 0.005), while mean preejection period (pep) and mean pep/lvet were increased from 41 +/-8.5 msec to 87 +/-14 msec (p < 0.05) and from 0.39 +/-0.06 to 0.62 +/-0.1 (p < 0.01), respectively. during cholemia, stis were unchanged after intravenous administration of ouabain, whereas in the control period, there was shortening of mean pep from 71 +/-8.8 msec to 58 +/-7.6 msec (p < 0.05), and of q-s2 from 257 +/-12 msec to 235 +/-14 msec (p < 0.005) in response to ouabain. maximal dp/dt decreased after choledochocaval anastomosis from 4543 +/-593 mmhg/ sec to 3666 +/-648 mmhg/sec (p < 0.025). we conclude that cholemia in the dog is clearly associated with impaired left ventricular performance. the present data also support a previously published in vitro study from our laboratory showing that cholemia blunts the myocardial contractile response to sympathomimetic agents. the cardiodepressor effect of cholemia may explain the increased tendency of patients with obstructive jaundice to postoperative shock and renal failure. the effect of deep jaundice on left ventricular function was studied in five dogs. each dog was studied before and 2 weeks after choledochocaval anastomosis (cdca), thus each dog served as its own control. cdca results in severe jaundice with minimal liver damage. mean arterial blood pressure, end-diastolic left ventricular pressure, and maximal rate of increase in left ventricular pressure (dp/dt) were obtained by retrograde catheterization of the aorta and left ventricle. systolic time intervals (pre-ejection period (pep) and left ventricular ejection time (lvet)) were measured with simultaneous ecg and aortic pulse tracings. systolic time intervals were measured before and after intravenous injection of ouabain. the findings revealed that deep jaundice was associated with impaired left ventricular performance. there was a decrease in dp/dt without changes to arterial pressure, heart rate, or preload. moreover, there was an associated increase in mean left ventricular pep, and a decrease in lvet when compared with predicted values. although administration of ouabain in normal dogs (i.e. pre-cdca) was associated with decreased pep, no effect was observed in deeply jaundiced animals. 48 deep jaundice may have hemodynamic consequences even when liver function is relatively intact. it is associated with impaired left ventricular performance that is refractory to cardiac glycosides in this animal model. the hemodynamic manifestations of advanced liver failure with jaundice are well described, and are basically a 'high output-low resistance' state. this paper suggests that jaundice has its own effects on cardiac function independent of liver failure. the authors are clearly original thinkers who have devised an animal model to shed light on a clinical problem. the model is relevant to patients with jaundice associated with multiple organ failure, as well as obstructive jaundice this is a small animal study. the methods of assessment of left ventricular function are probably valid, but are not conventional. patients with multiple organ failure are frequently jaundiced, and in this setting, plasma bilirubin concentrations may be grossly elevated. such patients often also have severe hemodynamic compromise that can be refractory to inotropic agents. although this paper does not offer a solution to this problem, it does propose a plausible mechanism. the incidence of hypoxic hepatitis was prospectively studied for 1 year in a group of high-risk patients suffering from low cardiac output in a coronary care unit. hypoxic hepatitis, defined as an increase in serum aminotransferase activity of at least 20 times the upper limit of normal without any other cause for hepatic necrosis, was observed in 20 patients. this represents 2.6% of the 766 patients admitted to the unit during this period, and 21.9% of the 91 patients suffering from low cardiac output. clinical, biological, and hemodynamic data were compared between 20 patients with low cardiac output and hypoxic hepatitis, and 48 patients with low cardiac output but without hypoxic hepatitis who survived more than 24 hour. in these two groups of patients, hepatic blood flow was measured by galactose clearance at low concentration. patients with hypoxic hepatitis exhibited a higher central venous pressure (90% versus 38%, p < 0.001), as well as a lower hepatic blood flow (867 +/-377 ml/min versus 1429 +/-644 ml/min, p = 0.001). in conclusion, although it is considered a rare hepatic disorder, hypoxic hepatitis is frequent in patients with low cardiac output admitted to the coronary care unit, and is associated with a decrease in hepatic blood flow, and passive hepatic venous congestion. over a 1-year period in a belgian coronary care unit, 91 patients were identified as having a low cardiac output using clinical criteria. hypoxic (ischemic) hepatitis (hh) was said to be present if there was a clinical setting of circulatory failure, a sharp but transient increase in serum aminotransferase activities of at least 20 times the upper limit of normal, and after exclusion of other causes of hepatic necrosis. of the 91 patients, 20 developed hh (2.6% of admissions to the coronary care unit; 21.9% of patients with low cardiac output), 48 did not develop hh, and 23 died within 24 hours. hepatic blood flow was measured in the 20 patients with hh, and in 41 of the 48 surviving patients without hh, using the method of galactose clearance at low concentrations. when compared with patients who did not develop hh, patients with hh had lower hepatic blood flow (867 ± 377 ml/min vs 1429 ± 644 ml/min), higher central venous pressure (22.5 cmh 2 o vs 14 cmh 2 o), and a trend toward lower cardiac output (3.4 l/min vs 4.4 l/min). interestingly, only approximately 50% of patients in each group had a systolic arterial pressure below 90 mmhg. the overall mortality was high -55% in the hh group and 46% in the group without hh. the findings of this study show that ischemic hepatitis is common in environments where there are patients with low cardiac output states, such as coronary care units and intensive care units. the classic hemodynamic pattern is low cardiac output (resulting in reduced hepatic blood flow), and high right atrial pressure. hypotension is not a prerequisite. the mortality rate is >50% this is the first prospective study to determine the incidence of ischemic hepatitis in patients with severe cardiac disease and low cardiac output. it is also by far the most comprehensive study of hemodynamic measurements in patients with ischemic hepatitis, and the only one to measure liver blood flow. this is a large, prospective, observational study of 766 patients admitted to a single intensive care unit over a 1-year period. exclusion of the 23 patients who died within 24 hours of the episode of low cardiac output, before measurements of hepatic blood flow had been made, may have altered the findings, particularly regarding the incidence of ischemic hepatitis. the validity of the hepatic blood flow measurements themselves are open to some dispute -firstly because of the use of galactose clearance rather than indocyanine green clearance, and secondly because they were performed subsequent to the episode of low cardiac output, and therefore may not reflect the hemodynamic state at that time. ischemic hepatitis occurs in up to 5% of admissions to intensive care units and coronary care units, and is consequently more common than a number of other conditions that have attracted massive research interest. this paper describes the clinical setting, hemodynamic characteristics, and outcome of ischemic hepatitis, and consequently makes a major contribution to the body of knowledge on the subject. ischemic hepatitis is not a rare hepatic disorder, but rather it is a circulatory disorder, and therefore of great relevance to intensive care medicine. abstract a novel quantitative liver function test is described which is based on monoethylglycinexylidide (megx) formation after lidocaine bolus injection. following the administration of small single doses of lidocaine hydrochloride (1 mg/kg), monoethylglycinexylidide serum concentration-time curves were determined by a novel, highly sensitive fluorescence polarization immunoassay (fpia) in healthy volunteers, liver donors, and patients with liver cirrhosis. the fpia allowed rapid and reliable monoethylglycinexylidide determinations in serum and urine (between-days coefficient of variation: < 10.3%, recovery: 80-113%). monoethylglycinexylidide concentrations measured by fpia in 32 serum samples from patients correlated well with those determined by hplc. the monoethylglycinexylidide concentration in serum determined 15 minute after a lidocaine bolus injection proved to be a highly sensitive and specific indicator of hepatic dysfunction. average monoethylglycinexylidide concentrations in serum obtained 15 minute after lidocaine injection were substantially lower in patients with liver cirrhosis than in healthy volunteers. the average monoethylglycinexylidide concentrations in serum were also substantially lower in liver donors with ballooning or fatty changes of hepatocytes than in donors without relevant alterations of liver histology. by means of monoethylglycinexylidide formation in the liver donors, primary function of the transplanted liver was correctly predicted in 32/37 cases, and initial non-function in 4/6 cases. monoethylglycinexylidide (megx) is the first metabolite of lignocaine formed by oxidative de-ethylation by the hepatic cytochrome p-450 system. in this study, the appearance of megx measured by a fluorescence polarization immunoassay after a test dose of intravenous lignocaine was investigated as a liver function test. the findings showed that there was a satisfactory correlation between the measurement of megx by the novel fluorescence polarization immunoassay and by high performance liquid chromatography. the formation and elimination kinetics of megx were investigated for healthy volunteers, patients with histologically confirmed liver cirrhosis, and liver donors after a single intravenous bolus injection of lignocaine (1 mg/kg). in healthy volunteers, the peak megx concentration occurred 15 minutes after the bolus of lignocaine, whereas in patients with cirrhosis, its rate of formation was markedly decreased, and maximum concentrations were observed at about 4 hours. in liver donors the 15-minute megx concentration was lower in donors, with altered liver histology than in those with normal histology. patients with cirrhosis had the lowest 15-minute megx concentration of all the groups. conventional 'liver function tests' measure liver damage rather than liver function. the megx test assesses the liver's capacity to metabolize drugs (in this case lignocaine), and thus is a true 'liver function test'. the data presented in this study suggest that the 15-minute megx concentration varies with the adequacy of liver function. this paper is the first to describe and assess the formation of megx after a standard dose of lignocaine as a dynamic liver function test. novel approach. the test is not well validated in this report. because of its ease of use and rapid turnaround, the megx test has found widespread application for the real-time assessment of hepatic function. in liver transplantation, the megx test has found a place in selection of transplant candidates, and in monitoring of liver recipients early after transplantation. in intensive care patients, a rapid decrease in megx test values is associated with increased risk of developing multiple organ failure, and a poor outcome, and consequently may have a role in investigation of the role of the liver in the multiple organ failure syndrome. the successful use of orthotopic liver transplantation in fulminant hepatic failure has created a need for early prognostic indicators to select the patients most likely to benefit at a time when liver transplantation is still feasible. univariate and multivariate analysis was performed on 588 patients with acute liver failure managed medically during 1973-1985, to identify the factors most likely to indicate a poor prognosis. in acetaminophen-induced fulminant hepatic failure, survival correlated with arterial blood ph, peak prothrombin time, and serum creatinine -a ph < 7.30, prothrombin time > 100 seconds, and creatinine > 300 µmol/l indicated a poor prognosis. in patients with viral hepatitis and drug reactions, three static variables [etiology (non a, non b hepatitis or drug reactions), age < 11 and > 40 year, duration of jaundice before the onset of encephalopathy > 7 days], and two dynamic variables (serum bilirubin > 300 µmol/l, and prothrombin time > 50 seconds) indicated a poor prognosis. the value of these indicators in determining outcome was tested retrospectively in a further 175 patients admitted during 1986-1987, leading to the construction of models for the selection of patients for liver transplantation. the records of 588 patients with fulminant hepatic failure (fhf) and grade iii or iv encephalopathy admitted to king's college hospital, london, between 1973 and 1985, were scrutinized to identify simple parameters that might prove to be prognostic indicators. these parameters were examined by univariate and multivariate analysis to determine the factors most likely to be associated with poor prognosis. the analysis was performed in two groups, paracetamol-induced and non-paracetamol-induced, because of the higher incidence of renal failure and metabolic acidosis in the former group. the prognostic indicators so determined were then examined retrospectively, in a second group of 175 patients with fhf admitted during 1986-1987, to determine their sensitivity and specificity in prediction of a fatal outcome. models were then constructed for selection of patients for liver transplantation on the basis of an extremely poor prognosis with medical management. the criteria adopted for paracetamol-induced fhf were ph < 7.30 (irrespective of grade of encephalopathy), or prothrombin time >100 seconds and serum creatinine >300 µmol/l in patients with grade iii or iv encephalopathy. for fhf unrelated to paracetamol, the criteria determined were prothrombin time >100 seconds (irrespective of grade of encephalopathy), or any three of (irrespective of grade of encephalopathy): age <10 or >40 years, etiology (non-a, non-b hepatitis or idiosyncratic drug reactions), duration of jaundice before onset of encephalopathy > 7 days, prothrombin time > 50 seconds, and serum bilirubin > 300 µmol/l. this study has developed criteria that predict death in patients with fhf who are managed medically. in the era of liver transplantation, these criteria are also used to select patients for liver transplantation. all patients with fhf should be discussed with a transplant unit. patients who meet these criteria should be transplanted. this paper has changed practice. it allows decisions to be made about transplantation in fhf so that unnecessary transplantation can be minimized. because the criteria involve simple, easy to apply parameters that are readily available, the algorithm also permits decisions to transplant to be taken early, maximizing the period of time available to find a suitable donor, and allowing transplantation to be undertaken before the development of cerebral edema. this study is based on the largest number of patients in a single unit ever reported. data were collected over a period of 13 years, and it is possible that one or more aspects of management may have changed in that time, and may independently affect outcome. various models have been used to predict outcome in fhf, but the 'king's criteria' identified in this study are the most widely used. these criteria are based on the study of a huge number of patients treated medically, and, because so many patients are now transplanted, it is extremely unlikely that the study could ever be repeated. consequently, the criteria identified in this paper are likely to be used widely to select transplant candidates for the foreseeable future. orthotopic liver transplantation was done in 17 patients with fulminant hepatitis. the cause of the liver disease was infection with hepatitis b virus, or co-infection with hepatitis b virus and hepatitis d virus, or infection with hepatitis a virus in 6 patients; drug hepatotoxicity in 5; and indeterminate in 6. grafts from incompatible blood groups, steatotic grafts, or reduced-size grafts were used in 5, 4, and 4 patients, respectively. of the 17 patients, 5 died: 2 of early liver failure due to the poor quality of the graft, 1 presumably of accidentally transmitted acute infection with the human immunodeficiency virus, and 2 of decerebration occurring during or immediately after surgery. the 12 other patients were alive 2 to 15 months after transplantation. this paper reports the clinical details and outcome of 17 patients with acute liver failure who underwent orthotopic liver transplantation in the mid-1980s, in the hopital paul-brousse, villejuif, and the hopital beaujon, clichy, france. six patients had documented viral hepatitis, five had drug hepatotoxicity, and the cause was undetermined in the remaining six. despite compromises necessitated by emergency transplantation (abo and size incompatibility, and steatotic grafts), 12 patients survived. deaths were due to cerebral edema (2), early graft failure (2), and graft-transmitted infection. why it's important this paper was the first large case study to document the feasibility and success of liver transplantation for acute liver failure. this is a succinct review of 17 patients with acute liver failure who underwent liver transplantation, with appropriate demographic, etiological, clinical, and outcome information that remains relevant many years later. no case control data are reported. although it is stated that transplantation was undertaken for a rapidly deteriorating neurological condition and severe coagulation disorders, the outcome of patients who did not meet these criteria, or who met the criteria and could not be transplanted, is not reported. hence, although this paper describes the feasibility of liver transplantation in this population, it does not assess the value. liver transplantation, as described in this case series, remains the best chance of survival for the majority of patients with acute liver failure. although criteria for transplantation, and aspects of the surgical technique and postoperative immunosuppression have developed over the intervening years, orthotopic liver transplantation has not been superceded, and should be considered as an option in all patients with acute liver failure. abstract background. when administered early after an overdose of acetaminophen, intravenous acetylcysteine prevents hepatic necrosis by replenishing reduced stores of glutathione. how acetylcysteine improves the survival of patients with established liver damage induced by acetaminophen, however, is unknown. this study was undertaken to determine whether the beneficial effect of acetylcysteine under such circumstances could be due to enhancement of oxygen delivery and consumption. methods. we studied the effect of acetylcysteine on systemic hemodynamics and oxygen transport in 12 patients with acetaminophen-induced fulminant hepatic failure, and 8 patients with acute liver failure from other causes. the acetylcysteine was given in a dose of 150 mg/kg in 250 ml of 5% dextrose over a period of 15 minutes, and then in a dose of 50 mg/kg in 500 ml of 5% dextrose over a period of 4 hours; measurements were made before treatment began, and after 30 minutes of the regimen. results. in the patients with acetaminophen-induced liver failure, the infusion of acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml/min/m 2 (p = 0.0036), due to an increase in the cardiac index from 5.6 to 6.7 mm/m 2 (p = 0.0021). mean arterial pressure rose from 88 to 95 mmhg (p = 0.0054), despite a decrease in systemic vascular resistance from 1296 to 1113 dyn.sec.cm -5 per square meter (p = 0.027). there was an increase in oxygen consumption from 127 to 184 ml/min/m 2 (p = 0.0007) associated with an increase in the oxygen-extraction ratio from 16 to 21 percent (p = 0.022). the effects in the patients with acute liver failure from other causes were similar. conclusions. the increase in oxygen delivery and consumption in response to acetylcysteine may account for its beneficial effect on survival in patients with fulminant hepatic failure induced by acetaminophen. the effects of intravenous n-acetylcysteine on systemic hemodynamics and oxygen transport were studied in 12 patients with paracetamol-induced acute liver failure, and eight patients with acute liver failure from other causes. measurements were made before a bolus dose of 150 mg/kg of n-acetylcysteine given over 15 minutes, and 30 minutes after commencement of an infusion of 50 mg/kg of n-acetylcysteine given over 4 hours. the findings were that in patients with paracetamol-induced acute liver failure, infusion of n-acetylcysteine resulted in an increase in mean oxygen delivery from 856 to 975 ml/min/m 2 , resulting mainly from an increase in the mean cardiac index by approximately 20% (5.6 to 6.7 l/min/m 2 ). mean arterial pressure increased from 88 to 95 mmhg despite a decrease in the mean systemic vascular resistance from 1296 to 1113 dyn/sec/cm -5 . in patients with acute liver failure from other causes, the effects of n-acetylcysteine were similar but less marked, and there were significant changes (increases) only for oxygen consumption and oxygen extraction ratio. each patient had similar measurements performed before and during an infusion of prostacyclin at a rate of 5 ng/kg, and again when n-acetylcysteine and prostacyclin were infused simultaneously. the effects of prostacyclin were similar to those of n-acetylcysteine, but there was a decrease in mean arterial pressure rather than an increase, and a much smaller increase in oxygen extraction. four patients with acute liver failure secondary to paracetamol were studied after recovery. in this group, n-acetylcysteine had no effect on any hemodynamic or oxygen transport variable. however, with infusion of prostacyclin, there was an increase in cardiac index, decrease in systemic vascular resistance, and increased oxygen delivery without an increase in consumption. there is a characteristic high output-low resistance hemodynamic state in fulminant hepatic failure that may be associated with covert tissue hypoxia. intravenous infusion of n-acetylcysteine was associated with an increase in oxygen delivery and consumption, and also in tissue oxygen extraction in patients with fulminant hepatic failure. it is postulated that this may account for the beneficial effect of n-acetylcysteine on survival in patients with acute liver failure caused by paracetamol, and further, may be of benefit in patients with multiple organ failure from other causes, such as sepsis. this study is the justification for the widespread use of n-acetylcysteine in patients with acute liver failure, and it has also provoked investigation of possible clinical benefits in other conditions, such as septic shock. this is a comprehensive clinical study of a relatively homogeneous group of patients. because the fick method was used to calculate oxygen consumption from the cardiac output and the arteriovenous oxygen content difference, it is possible that an artifactual relationship between oxygen delivery and consumption was shown because of mathematical coupling. it is now generally accepted that oxygen delivery and consumption should be determined independently. although undoubtedly a classic study, the findings presented in this paper have not been confirmed in a similar more recent study (see related reference no. 1). in the latter study, oxygen consumption was measured independently of oxygen delivery, using a method based on respiratory gas analysis, and patients were studied over a longer period. nevertheless, the paper under review has been responsible in part for a resurgence of interest in n-acetylcysteine as a therapeutic agent, not only in patients with acute liver failure, but also in other groups of critically ill patients, such as those with sepsis, acute lung injury, and multiple organ failure. as with acute hepatic failure, published studies of other groups of critically ill patients have shown inconsistent findings, and the role of n-acetylcysteine in critically ill patients remains unclear. this paper marks the beginning of a long and still-continuing struggle to find a clinically relevant artificial liver device that can be used as a bridge to regeneration or transplantation in patients with acute liver failure. this is a report of original and far-sighted ideas that must have stretched the boundaries of knowledge and technology in the late 1960s. this is a case report only. it is possible that the improvement after cross-circulation may have been incidental, and the patient may have recovered spontaneously without it. the precise technique used in this report is most unlikely to be used again because of concerns about transmission of infection, and immunological considerations. however, more than 30 years later, there remains a need to develop a safe and effective artificial liver for use in patients with acute liver failure. abstract background: in patients with cirrhosis and spontaneous bacterial peritonitis, renal function frequently becomes impaired. this impairment is probably related to a reduction in effective arterial blood volume, and is associated with a high mortality rate. we conducted a study to determine whether plasma volume expansion with intravenous albumin prevents renal impairment and reduces mortality in these patients. methods: we randomly assigned 126 patients with cirrhosis and spontaneous bacterial peritonitis to treatment with intravenous cefotaxime (63 patients), or cefotaxime and intravenous albumin (63 patients). cefotaxime was given daily in dosages that varied according to the serum creatinine level, and albumin was given at a dose of 1.5 kg of body weight at the time of diagnosis, followed by 1 kg on day 3. renal impairment was defined as nonreversible deterioration of renal function during hospitalization. results: the infection resolved in 59 patients in the cefotaxime group (94%) and 62 in the cefotaxime-plus-albumin group (98%) (p = 0.36). renal impairment developed in 21 patients in the cefotaxime group (33%), and 6 in the cefotaxime-plus-albumin group (10%) (p = 0.002). eighteen patients (29%) in the cefotaxime group died in the hospital, as compared with 6 (10 percent) in the cefotaxime-plus-albumin group (p = 0.01); at three months, the mortality rates were 41 percent (a total of 26 deaths), and 22% (a total of 14 deaths), respectively (p = 0.03). patients treated with cefotaxime had higher levels of plasma renin activity than those treated with cefotaxime and albumin; patients with renal impairment had the highest values. conclusions: in patients with cirrhosis and spontaneous bacterial peritonitis, treatment with intravenous albumin in addition to an antibiotic reduces the incidence of renal impairment and death, in comparison with treatment with an antibiotic alone. the effect of intravenous albumin on renal impairment and mortality was studied in 126 patients with cirrhosis and spontaneous bacterial peritonitis. patients were randomly assigned to receive treatment with intravenous cefotaxime or cefotaxime and intravenous albumin. albumin was given at a dose of 1.5 g/kg body weight at the time of diagnosis, and 1 g/kg on day 3. the infection resolved in a similar number of patients in both groups. however, the incidence of renal impairment was very much lower in the cefotaxime plus albumin group (6 patients; 10%) than in the cefotaxime alone group (21 patients; 33%). furthermore, the mortality rates were lower in the cefotaxime plus albumin groups, i.e. 6 patients or 10% vs 18 patients or 29% for in-hospital mortality, and 14 patients (22%) and 26 patients (41%) at 3 months. the effects of whole bile and bile salts on the perfused heart severe sinus node dysfunction in obstructive jaundice obstructive jaundice blunts myocardial contractile response to isoprenaline in the dog: a clue to the susceptibility of jaundiced patients to shock? the megx test: a tool for the real-time assessment of hepatic function liver function and splanchnic ischemia in critically ill patients lignocaine metabolite formation: an indicator for liver dysfunction and predictor of survival in surgical intensive care patients assessment of prognosis in fulminant hepatic failure multivariate analysis of prognostic factors in fulminant hepatitis b early indicators of prognosis in fulminant hepatic failure: and assessment of the king's criteria liver transplantation in europe for patients with acute liver failure the effect of n-acetylcysteine on oxygen transport and uptake in patients with fulminant hepatic failure improved outcome of paracetamol-induced fulminant hepatic failure by late administration of acetylcysteine n-acetylcysteine increases liver blood flow and improves liver function in septic shock patients: results of a prospective, randomised, double-blind study molecular adsorbent recycling system (mars): clinical results of a new-membrane-based blood purification system for bioartificial liver support extracorporeal liver support: cell-based therapy for the failing liver key message complex forms of extracorporeal perfusion are possible in critically ill patients with acute liver failure and advanced encephalopathy. a focus on metabolic support (for example from functioning hepatocytes) rather than blood purification will most likely be successful spontaneous bacterial peritonitis bacterial infections in liver disease definition and diagnostic criteria of refractory ascites and hepatorenal syndrome in cirrhosis. international ascites club the case is reported of a 29-year-old woman who developed presumptive viral hepatitis. her jaundice initially resolved after 6 weeks, but recurred soon after and was accompanied by a flapping tremor. her neurological state deteriorated markedly to grade iv encephalopathy. she was treated with a regimen of daily exchange transfusion for 6 days, but despite some apparent improvement related to these procedures, this was not sustained. she duly became apneic, and after an unsuccessful seventh exchange transfusion, she underwent cross-circulation with a baboon. the baboon had been especially prepared by washing out the blood volume with lactate solution under hypothermia on bypass, and then replacing the blood volume with human blood compatible with the patient. after about 12 hours, spontaneous ventilation returned, and there was an improvement in conscious state and the treatment was terminated. she remained neurologically impaired for two further days, and then improved markedly to become fully conscious with no abnormal neurological signs. her improvement persisted until publication of the case report. patients treated with cefotaxime had higher levels of plasma renin activity than those treated with cefotaxime and albumin, and patients with renal impairment had the highest values.the authors conclude that in patients with cirrhosis and spontaneous bacterial peritonitis, treatment with intravenous albumin in addition to an antibiotic reduces the incidence of renal impairment and death in comparison with an antibiotic alone. spontaneous bacterial peritonitis is a common complication in patients with cirrhosis and ascites, and may be accompanied by renal impairment, development or worsening of encephalopathy, and increased risk of death. treatment with non-nephrotoxic antibiotics has been associated with improved outcome, but volume expansion with albumin in addition to antibiotics results in further improvement in survival and a reduced incidence of renal impairment. the most likely explanation is that albumin infusion prevents hypovolemia and the subsequent activation of vasoconstrictor systems. however, mechanisms unrelated to plasma expansion cannot be ruled out. this study provides strong evidence that intravenous albumin therapy should be given, in addition to antibiotic therapy, to patients with cirrhosis and spontaneous bacterial peritonitis. the effects of albumin therapy are not known in patients with organic nephropathy and serum creatinine concentration >265 µmol/l because these groups were excluded from the study. this is a well-designed, randomized, controlled trial from the group who virtually singlehandedly have defined the management of ascites in cirrhosis. there are few. further studies should investigate the effect of different doses of albumin, and whether other plasma expanders are equally effective. this study does not specifically involve patients treated in an intensive care unit. nevertheless, spontaneous bacterial peritonitis is responsible for many admissions of cirrhotic patients to the icu, as it is sometimes a silent precipitant of gastrointestinal bleeding, encephalopathy, and renal impairment, as well as overt sepsis. indeed, it should be excluded in every cirrhotic patient admitted to the icu. consequently, advances in its treatment are very relevant to the icu clinician. furthermore, despite the current uncertainty as to the place of albumin therapy in critically ill patients, it seems clear that it confers a survival advantage to patients with cirrhosis and spontaneous bacterial peritonitis. key: cord-033914-a9e3rncp authors: kauffman-ortega, e.; valdovinos-díaz, m.a. title: in memoriam ludwig van beethoven. clinical history and possible diagnoses of the genius of musical composition in silence() date: 2020-10-17 journal: nan doi: 10.1016/j.rgmxen.2020.10.006 sha: doc_id: 33914 cord_uid: a9e3rncp nan carried out one day after his death by dr. johann wagner and dr. karl von rokitansky, the father of modern pathologic anatomy. 1 ludwig van beethoven was born on december 16, 1770, in the small german city of bonn, situated on the banks of the rhine. he was the second of 7 children, of whom only 3 lived beyond infancy (ludwig, kaspar anton karl, and nikolaus johann). his paternal grandmother, josepha, and his father, johann van beethoven, suffered from alcohol use disorder, which led to his father's death when ludwig was 21 years old. complications from tuberculosis claimed the lives of his mother, maria magdalena keverich, and his younger brother, kaspar anton karl. during his childhood, ludwig contracted smallpox, which resulted in facial scarring, and he experienced recurrent respiratory infections, facilitated by underlying bronchial asthma. for the rest of his life, up to his death at the age of 56, he was tormented by numerous illnesses, the majority of unknown etiology. the childhood suffering he endured due to paternal violence and abuse, his mandatory role of providing for and supporting his family as an adolescent, the inner silence and confinement brought on by deafness, and his multiple health problems as an adult, interwoven with his immense commitment to music, resulted in beethoven's leaving us a musical legacy whose melodies, in addition to their beauty, have the power to embrace millions of souls (fig. 1 ). beethoven's deafness, one of the most interesting medical enigmas, with a still uncertain etiology, was first described in ludwig's personal correspondence with his close friend, franz gerhardt wegeler, in 1801. beethoven described a 3-year history of progressive bilateral hypoacusis to high frequency sounds, such as those produced by the violin, piccolo, and piano, that initially began in the left ear. the hypoacusis was associated with unbearable tinnitus, poor discrimination, and recruitment, characteristics that are consistent with sensorineural hearing loss. 2, 3 the hearing defect caused beethoven to have low self-esteem, emotional lability, and progressive isolation, leading to suicidal ideations (the heiligenstadt testament). he was irritable, combative, and arrogant. like his father and paternal grandmother, ludwig was a drinker, with important periods of alcohol consumption that began intermittently when he was 17 years old, particularly during his periods of depression. his favorite drink was hungarian wine, which at that time was of poor quality and tainted with lead to improve its aroma and flavor. the progression of his hearing loss culminated in complete deafness at 47 years of age. during that period of his life, beethoven composed the missa solemnis, the ninth symphony, which debuted in 1824, and his last sonatas for piano and string quartets, all of them important works that were written when he was completely deaf. the autopsy report described cranial thickness of twice the normal size, with a prominence of the frontal bone and irregularity of the zygomatic bones, thinning of the auditory nerves, predominantly of the left nerve, devoid of medullary substance, and a narrowed eustachian tube with retracted mucosa at the level of the bone portion. 1 numerous pathologies in the differential diagnosis have been proposed for beethoven's sensorineural hearing loss, and the most sustainable are: 1) lead poisoning, based on the presence of residuals of lead 100 times higher than normal in his hair and bones, according to an analysis performed in the united states in the mid 1990s, 4 2) cogan's syndrome, characterized by bilateral sensorineural hearing loss and interstitial keratitis secondary to vasculitis, albeit there is no evidence of vestibular dysfunction in beethoven's texts; that syndrome can be associated with idiopathic inflammatory bowel disease and reactive arthritis, 5 and 3) paget's disease is supported by the frontal bone prominence, tinnitus, and headache. other less probable diagnoses are otosclerosis, sarcoidosis, and syphilis. 1 after his mother's death, estimated to have occurred when he was between 17 and 22 years of age, beethoven began to have numerous gastrointestinal symptoms that would accompany him for the rest of his life. his clinical symptoms were characterized by generalized colicky abdominal pain with periods of exacerbation and remission. at times the pain was incapacitating, and he had changes in the frequency of bowel movements and stool consistency, with a predominance of watery diarrhea but no malabsorption or inflammatory features. he sometimes complained of hyporexia, headache, arthralgia, and meteorism. the pain episodes were exacerbated during periods of stress or depression and improved with analgesics, such as quinine and salicin, baths with cold or lukewarm river water (''danube baths''), or with alcohol consumption. the episodes of pain increased in frequency and intensity over time. between 34 and 37 years of age, beethoven presented with a purulent soft tissue infection in a toe, consistent with an abscess, that almost required amputation, followed by a submandibular abscess that was resolved in three months. at 53 years of age, he also presented with painful eye redness, most likely due to scleritis, uveitis, or interstitial keratitis. 6, 7 despite the gastrointestinal symptomatology described, beethoven had a robust physique. he was short, with broad shoulders, and had a short neck, round nose, dark skin, and a wide and prominent forehead. he leaned a bit forward when walking but had no signs of the chronic malnutrition that became apparent during the last years of his life, when he presented with complications associated with liver disease. at autopsy, the chest cavity and contents were normal, the stomach and intestines were distended with air, with no relevant macroscopic alteration, and the pancreas was large and hard. the main pancreatic duct was dilated and appeared ''as wide as a goosequill''. 1 the semiology of those signs and symptoms suggests that beethoven suffered from diarrhea-predominant irritable bowel syndrome, given that he had no alarm symptoms, such as gastrointestinal bleeding, or anatomopathologic findings of organic disease in the digestive tract. the description of the pancreas at autopsy is consistent with chronic pancreatitis, probably secondary to alcohol consumption. other less likely diseases in the differential diagnosis explaining his gastrointestinal symptomatology have been proposed, such as idiopathic inflammatory bowel disease, sarcoidosis, intestinal tuberculosis, whipple's disease, lead poisoning, and selective iga deficiency. the first manifestations of liver failure became evident in 1821, when beethoven was 51 years old. he presented with progressive jaundice, nausea, vomiting, abdominal pain, asthenia, and adynamia, related to a probable viral hepatitis or alcoholic hepatitis, that remitted after 3 months. in 1822, he had an episode of unilateral pleuritic chest pain, described as ''thoracic gout''. in 1826, he presented with complications that could be attributed to cirrhosis of the liver with portal hypertension, such as epistaxis that was probably thrombocytopenia-related, altered mental status, hepatic encephalopathy, and tense ascites. he underwent 4 paracentesis procedures, performed by his physician, dr. andreas ignaz wawruch, through laparotomy. twenty-two liters of ascites were drained, but no asepsis measures were utilized, nor was there adequate closure of the abdominal wall, and the patient developed an infected ascitic fistula and possible bacterial peritonitis. 8 despite numerous medical recommendations, beethoven, now emaciated, continued to eat and drink as he pleased, with intentions of composing a tenth symphony, a requiem, and music for goethe's faust. finally, in march of 1827, he presented with progressive jaundice, tense ascites, lower extremity edema, anuria, fever, and hepatic encephalopathy that resulted in his death. ludwig van beethoven died on march 26, 1827, at 56 years of age, in the city of vienna. the autopsy report described an emaciated, cachectic body, particularly the lower limbs, with multiple petechiae, and a distended abdomen. the abdominal cavity was filled with a turbid, grayish-brown fluid. the liver was half its normal size, hard, bluish-green, and had a nodular surface, characteristic of macronodular cirrhosis. there was sludge in the interior of the gallbladder. the spleen was more than twice its normal size, hard, and blackish. both kidneys were pale, and when sectioned, each calyx was full of calcareous concretions, consistent with papillary necrosis or kidney stones. 9 the probable cause of beethoven's death was acuteon-chronic liver failure. that syndrome is characterized by acute decompensation manifested by ascites, hepatic encephalopathy, coagulation alterations, bacterial infection, multiple organ failure, and elevated short-term mortality in patients with underlying cirrhosis of the liver. 10 even though 40% of patients do not have a recognizable precipitating factor, in beethoven's case, it can be attributed to an infectious process due to the ascitic fistula and secondary peritonitis, severe alcoholic hepatitis, or circulatory dysfunction after the paracentesis. alcohol consumption appears to be the most probable cause of beethoven's cirrhosis of the liver, despite the macronodular appearance described in the autopsy. although that finding is in contrast to micronodular cirrhosis (laennec's cirrhosis) described in patients with alcoholic liver cirrhosis, other diagnoses that have been proposed for explaining cirrhosis of the liver are hemochromatosis and autoimmune liver disease with primary sclerosing cholangitis associated with probable idiopathic inflammatory bowel disease. however, there is not sufficient evidence to confirm any of them. ' we music lovers and professionals of medicine, ''the most humanistic of the sciences and the most scientific of the humanities'', 12 celebrate your 250 years of existence and are eternally grateful for your legacy as a pianist, violinist, organist, composer, conductor, professor, idealist, and as a man who overcame enormous adversities to give full expression to his genius. beethoven forever! none. beethoven's autopsia revisitad: a patólogos rounds a final note a modern case sheds light on a classical enigma: beethoven's deafness the deafness of ludwig van beethoven: an immunopathy cogan syndrome: a retrospective review of 60 patients throughout half century lead and the deafness of ludwig van beethoven el genio de bonn atormentado por sus enfermedades: historia médica historical hepatology: ludwig van beethoven beethoven's renal disease based on his autopsy: a case of papillary necrosis acute-on-chronic liver failure rialp: traducción de joaquín esteban perruca the virtues in medical practice valdovinos-díaz * departamento de gastroenterología, instituto nacional de ciencias médicas y nutrición salvador zubirán the authors declare that they have no conflict of interests. key: cord-014770-cgtzlra1 authors: brandt, lawrence j.; farmer, richard d.; achord, james l.; friedman, lawrence s.; powers, cynthia i.; dorman, nancy j.; sullivan, donna c.; el-newihi, hussein m.; bank, simmy; ahn, john; devault, kenneth; thomas, eapen title: book reviews date: 1995 journal: dig dis sci doi: 10.1007/bf02065002 sha: doc_id: 14770 cord_uid: cgtzlra1 nan ad astra per aspera: this, the motto of kansas, teaches us that we achieved great things only by encountering and overcoming adversity. i felt as if i were achieving great things merely by successfully working my way through several of the chapters in this tome. pto, siology of the gastrointestinal tract is an awe-inspiring product--as it should be, and it was a daunting experience for this clinician to browse through it but, as one of my former professors said during an introductory seminar in medicine, "if you understand all you read, you are reading the wrong books." this is the right book! it is the right book for a library or research laboratory; it is the right book for a physiologist or an academically oriented clinician. anyone who has an interest in a particular problem in physiology will most likely find the answer herein; it may not, however, be readily apparent. thus, for example, if one wanted to know the recent advances made in understanding hunger and appetite control, the information is found under "gastric storage, satiety"--not an imponderable listing, but not too "user friendly" either. certain citations are missing or given short shrift. the physiology of the gastrointestinal tract in aging is covered in just 10 pages while aids is not discussed at all, despite severe associated derangements in the intestinal immune system. the gastric circulation is discussed fully, but blood flow of the colon is not mentioned and intestinal blood flow is treated in only nine lines within the enteric neuropeptide chapter. liver disease is covered only in two chapters on bile acids and the enterohepatic circulation, both by the same expert author (alan f. hofmann): one from the hepatobiliary perspective and one from the intestinal viewpoint. perhaps here then is my most significant critique. while i enjoyed studying the text, and learned a great great deal from each and every section that i read, i found little that was directly applicable to my everyday duties. rather, i felt as if i were on vacation in a land that spoke a vaguely familiar language, but not one with which i grew up. the growth of gastroenterologic knowledge during the 20th century. by joseph b. kirsner. lea & febiger, philadelphia, 1994, 522 pp., $75.00. as one approaches a book of this type and title, it might appear to be boring and anachronistic, especially in light of the rapidly paced, high-tech nature of the specialty of gastroenterology at the present time. happily, this is not so. the book is moderately sized and reflects very nicely the devotion of the authors and their enthusiasm for the exciting and evolving knowledge in gastroenterology during the 20th century. there are 31 chapters, each directed toward the historical background, incidence, and epidemiology and an emphasis on changes that have occurred in this aspect of the specialty during the past century. perhaps the initial response by a reader is of the usefulness of this book as background material in case one must give a talk relating to one of the subjects covered in the book. in addition to a very nice historical review of the subject covered, there are photographs and historical anecdotes relating to the specific field or disease. it is obviously not possible to review each contribution, but a number of highlights can be emphasized. in the section "history of acid, peptic disease," basil hirschowitz takes us from bismuth to billroth to black and back to bismuth. he provides a sharply focused review of peptic ulcer disease, including a review of the natural history and current status. likewise, the section "esophageal malignancies and premalignant conditions" is well written and well referenced and goes through the evolution of the concept of premalignant conditions. likewise, dr. kirsner, himself, describes the historical antecedent of inflammatory bowel disease including the contributions of crohn, bargen, and the evolution of our understanding of the diseases. it does seem, however, that the chapters, "gastrointestinal defenses against injury and inflammation" and "immunology of the gastrointestinal tract," should have been incorporated or integrated more along with the inflammatory bowel disease chapter. the chapter by dr. dienstag, "viral hepatitis," and by dr. lieber, "pathogenesis of cirrhosis," linked well with each other. chapters on important diseases and symptoms, appendicitis, diverticular disease, intestinal gas, and irritable bowel syndrome, were all well developed and likewise provide good background particularly if one were to lecture on one of these topics. one of the most interesting review chapters was by dr. starzl--"contribution of transplantation'--as this would certainly appear to be a subject for the immediate future and this serves as a very nice background for it. on the other hand, the chapters on gastrointestinal endoscopy and gastrointestinal radiology appear to be somewhat dated, undoubtedly because of their rapid and continuing evolution. nevertheless, the text is well written, well edited, and lends itself nicely to the reading of an individual chapter at a specific time (rather than reading the book from cover to cover, unless one's curiosity is piqued). in addition to the obvious historical aspects, which are covered very nicely, one can see somewhat into the future, by persons who are expert, knowledgeable and experienced, and it wilt certainly be of interest to see whether the predictions come to pass. the book seems to be particularly valuable for gastroenterologists in a teaching situation and also for internists, surgeons, and nurses who work in gastroenterology to illustrate how events of the past have led to the evolution and development of current activities and how these might lay the cornerstone for the future. for this reason, a hospital library would very likely wish to have a copy of this book as well. washington, dc this relatively small book is well presented. the organization is good and the imaging illustrations appear to me to be excellent, in contrast to the few histology illustrations, which are not good. there are 18 international contributors to nine chapters. eleven of the 18 contributors are radiologists experienced in ultrasound. the first five chapters are each written by radiologists specializing in ultrasound and are typically descriptive. the clinical correlations make up the differential diagnosis of the observations, which are extensive and well presented. the experienced clinician (and pathologist) will find fault with some of the statements made concerning clinical manifestations and presentations of disease but that would miss the more important points in the presentations. those doing diagnostic ultrasound will, i think, find these five chapters on the cutting edge of diagnostic techniques. the interest of the gastroenterologist lies in the last four chapters. these are written by gastroenterologists who are developing the techniques of intraluminal endoscopic ultrasonography, a topic that has captured the attention of a large number of clinicians. the presentation of this diagnostic method is, of course, descriptive but is appropriately conservative. nevertheless, it conveys the excitement and the promise of this new diagnostic tool. those gastroenterologists who are interested in getting into the field will profit from careful perusal of these four chapters. the cost of the book is a bit steep but is reasonable in view of the approximately 240 figures, an expensive process. i can recommend it to those who are interested in the field and suggest that it should be in institutional medical libraries for those who simply want to learn more about gastrointestinal ultrasound. jackson, mississippi in many ways, alcoholic liver disease is the final frontier of liver transplantation. with an estimated one to two million persons having alcoholic cirrhosis in the united states and a scarcity of donor livers limiting the number of liver transplants to about 3000 annually, the potential for patients with alcoholic liver disease to overwhelm the system is substantial. until recently, a lid was kept on the problem by the perception that results of liver transplantation in patients with alcoholic liver disease were significantly worse than those for patients with nonalcoholic liver disease. however, this conclusion has proved to be erroneous, and several centers have recently reported comparable results in carefully selected patients with alcoholic liver disease and those with nonalcoholic liver disease undergoing liver transplantation. this monograph by dr. lucey and his colleagues is therefore timely. drawing on their interest in and experience with liver transplantation in patients with alcoholic liver disease at the university of michigan and reviewing the literature carefully and critically, they have put together an elegant summary of the state of the art. in seven concise, beautifully orga-nized, and clearly written chapters, they explore important aspects of alcoholic liver disease as it relates to liver transplantation, including diagnostic difficulties, psychiatric assessment, medical assessment, peritransplant management, psychiatric follow-up, and the ethics of liver transplantation in alcoholics. by focusing on the issue at hand and ignoring related but peripheral matters that can be found in standard textbooks, such as the pathophysiology of alcoholic liver disease, the authors have produced a tightly knit and practical guidebook that can be read easily in an evening and that will be of enormous use to anyone with an interest in alcoholism, liver disease, or liver transplantation. a particular strength of this book are chapters by dr. beresford, a psychiatrist, on the diagnosis of alcoholism and the psychiatric assessment of alcoholic candidates for liver transplantation. with a scope that is worldly and perceptive, dr. beresford provides insight into the complexities and frailties of human behavior that underlie alcoholism. sprinkling his discussion with numerous enlightening case studies and clinical vignettes, he expertly discusses the difference between alcohol abuse and alcohol dependence, positive and negative risk factors for abstinence, the value of administering a pledge to an alcoholic, difficulties in confirming abstinence, and the important distinction between denial and ambivalence in the alcoholic's perception of his dependence. these discussions will be particularly enlightening to physicians who focus primarily on the medical aspects of alcoholic liver disease. a chapter by dr. lucey provides similarly perceptive insights into the medical aspects of liver transplantation in the alcoholic. he points out, for example, that, despite initial concerns, alcoholic cardiomyopathy has proven to be a less significant problem in patients undergoing liver transplantation than anticipated. in some cases, one may quibble with the opinions expressed by the authors. for example, prophylactic variceal band ligation in patients with varices that have not bled is dismissed, perhaps too hastily, as ineffective by extrapolation of studies of prophylactic variceal sclerotherapy. nevertheless, one must admire the forthrightness of the authors and their willingness to take a stand and defend it on virtually all relevant issues. their recommendations are often empirical, though always reasoned. the problem, of course, is that the data base is quite limited. even with their particular interest in alcoholic liver disease, they report on only 98 transplants in 90 alcoholics, and their principal study on assessing candidacy for liver transplantation is based on only 99 alcoholics, of whom 54 were deemed unsuitable for liver transplantation and in many cases lost to follow-up. still, this book is a superb introduction to a topic that is at the forefront of liver transplantation. moreover, the issue of liver transplantation in alcoholics is likely to become more contentious with time as greater constraints are placed on health-care expenditures. fittingly, the book concludes with a thoughtful consideration of the ethical aspects of liver transplantation in the alcoholic patient by drs. martin benjamin and jeremiah turcotte, who point out that it is no longer justifiable to exclude alcoholics from liver transplantation in the alcoholic will require considerable more study, debate, and thought for years to come. this book is an excellent start. lawrence s. friedman, md in their foreword to the 5th edition of margulis and burhenne's alimentary tract radiolog}" drs. alexander r. margulis and h. joachim burhenne state that their philosophy in the development of their textbook has been to create a resource that will stand as the "international cutting edge final word on alimentary tract radiology." this latest edition of the most comprehensive general textbook on radiology of the gastrointestinal system succeeds in its mission and proves itself a fitting successor to its four predecessors. the editors of the 5th edition, drs. patrick c. freeny and giles w. stevenson, have enlisted the assistance of 152 contributors from 11 countries in north america, europe, and asia. these men and women bring to their task a breadth of knowledge and experience in the field of gastrointestinal imaging that is remarkable in its scope. the textbook consists of 2154 pages organized into two volumes with 16 major subdivisions and 112 chapters. volume 1 presents sections on the history of alimentary tract, radiology, general anatomy and physiology, the hollow viscera, and miscellaneous disorders of the gastrointestinal tract, including infections and infestations, gastrointestinal manifestations of the acquired immunodeficiency syndrome, and the diagnosis and therapy of gastrointestinal hemorrhage. volume 2 deals with the solid viscera, the biliary tract, the peritoneum and retroperitoneum, the pediatric alimentary tract, interventional abdominal radiology, the acute abdomen, and abdominal trauma. the appropriate imaging procedures for the diseases specific to each organ--including plain films, fluoroscopically guided contrast examinations, ct, mri, ultrasonography, scintigraphy, arteriography, and endosonography--are discussed and illustrated. among the interventional techniques that are covered are: extracorporeal shock-wave lithotripsy; endoscopic intervention in calculus disease, chronic pancreatitis, and biliary duct neoplasm; percutaneous intervention in benign and malignant biliary disease; percutaneous biopsy of abdominal masses; percutaneous abscess drainage; percutaneous fluoroscopically guided gastrostomy; and therapeutic embolization for spontaneous gastrointestinal hemorrhage and hemorrhage after trauma. the text is supplemented with 46055 well-chosen illustrations of very good to exceptional quality. the index is 74 pages long and has detailed crossreferencing. this excellent textbook sets the standard against which other references devoted to radiology of the entire alimentary tract must be measured. it should be in the library of all gastrointestinal radiologists, all general radiologists performing gastrointestinal procedures, and all interventional radiologists. it is highly recommended to gastroenterologists. the book is expensive, but its cost is comparable to other radiology texts of similar magnitude. because of its expense, however, and the presumption, stated in the editors' preface, that a major revision will be required in four or five years, residents and fellows are advised to postpone purchase of this textbook until the completion of their training. this tenth monograph in the infectious disease and therapy series details scientific advanced concerning the etiology, epidemiology, pathophysiology, molecular biology, and immunology of viral agents causing diarrheal illness. the volume combines some basic reviews, such as chapters on the pathophysiology of viral diarrhea and gastrointestinal tract immunology, with detailed exploration of more focused topics, such as rotavirus proteins. this hardbound book is handsomely illustrated in black and white with excellent quality print. there are excellent photomicrographs of gastrointestinal tract histopathology and superb electron micrographs of various viruses which, in addition to some of the protein profiles and dna studies, are the outstanding features of this book. as seems inevitable in books authored by multiple contributors, the chapters varying quality and detail. particularly praiseworthy are the chapters on the natural history of human rotavirus infections and the rotavirus proteins, the latter providing fine detail with methodical approach and the most current references in the book. the chapter on enteric adenoviruses is also very current and covered many aspects of viral molecular biology including protein profiles, restriction fragment length polymorphisms, amino acid sequence comparisons, and genetic maps. the norwalk and norwalk-like viruses chapter provides an excellent synthesis of the literature with references dating into 1993 and contains a notably well-presented discussion of acquired immunity to these agents. rotavirus enthusiasts will be pleased to find at least 43% of the text devoted to this viral agent, including discussions of the natural history of human infections, viral proteins, detection assays, animal and non-group a rotaviruses, and vaccine-related issues. the section on jennerian approaches to vaccine development deals exclusively with rotaviruses. the chapter is well written but too brief and may surprise some readers by its failure to even mention live polio vaccines as an example and/or prototype to round out this introductory chapter. subsequent chapters cover current rotavirus vaccines that use animal viruses as possible human vaccine candidates. these chapters were informative, particularly the more up-to-date discussion of rhesus rotavirus-based human vaccines. in addition to rotaviruses, other viruses highlighted include norwalk agents, enteric adenoviruses, caliciviruses, toroviruses, and coronaviruses (human and animal). while interesting and one of the more clinically applicable sections of the book, the inclusion of a chapter on bacterial diarrhea in a volume dedicated to viral gastroenteritis is somewhat puzzling. similarly, the final chapter, which deals with the treatment of (predominantly bacterial) diarrheal diseases, may strike some readers as more of an afterthought than an integral part of a text focused on viral infections of the gastrointestinal tract. while this book has certain outstanding features, there are some shortcomings which warrant mention, most notably a paucity of references beyond 1990 in some chapters and the failure to recognize viral pathogens which, through gastrointestinal tract infection, cause significant disease other than diarrhea. some readers may be disappointed to find the enteroviruses (eg, poliovirus, echovirus, coxsackievirus) simply listed in a single table with no further recognition or discussion. hepatitis a virus is neglected altogether. human immunodeficiency virus receives short shrift in a single paragraph discussing immunology of the gastrointestinal tract. in all fairness, these omissions likely reflect a deliberate focus on viral gastroenteritis rather than an oversight implied by the broader scope of the book's title. perhaps a more descriptive title would be "viral gastroenteritis." the book is weighted toward animal models and should appeal to those interested in research concerning diarrheal disease caused by viral agents. clinical applications are limited, but the volume could be a valuable reference resource for physician-scientists with a strong interest in viral molecular biology. this atlas is written by a panel of national and international experts in the field of gastrointestinal motility. the book is divided into two sections. the first deals with the basic physiology and the available technology to study gut motility. this section reviews thoroughly various manometric devices as well as the different tests available for the clinician to study gut motility. these include the radiopaque markers and hydrogen breath tests used for large and small bowel transit measurement, respectively. scintigraphy, ambulatory ph monitoring, and ultrasound of the anal sphincter are also covered. the second section of the book deals with the clinical use and application of these tests in the clinical practice of gastroenterology. the effects of sleep and stress are detailed in two chapters. the final chapter in the book is devoted to the therapeutic aspects of gastrointestinal motility, including biofeedback training for fecal incontinence. this atlas is well balanced with enough text and extensive illustrations to help the reader understand the various technological aspects of studying gut motility. the numerous photographs of actual tracings and machines as well the different schematic repre-book reviews sentations made this valuable reference quite appealing and use friendly. this is a major strength in a book that deals with several topics not frequently covered in the basic training of a gastroenterologist. each chapter is preceded by a table of contents, which facilitates looking up a certain point. the bibliography that follows each chapter is both comprehensive and largely up to date. the size of the atlas is appealing, making it useful as a quick reference to the various tests described. as more tests of gastrointestinal motility are moving out of the confines of research laboratories and into our daily practice of gastroenterology, clinicians will find this atlas a helpful source to understand both the technological aspects of each test and their applications. gastroenterology fellows in training will also appreciate the wealth of diagrams and motility tracings in this atlas. technicians working in gastrointestinal motility laboratories will find this atlas invaluable for their daily practice. endoscopy is becoming only one of several tools made available for the clinician to tackle the complexity of gastrointestinal diseases, and this atlas is a valuable addition to the library of every gastroenterologist. hussein m. el-newihi, md jackson, mississippi clinical gastroenterology, 4th ed. edited by howard b. spiro. mcgraw-hill, new york, 1993 , 1235 pp., hardbound, $141.00. since the first edition of dr. howard spiro's, clinical gastroenterology, generations of physicians (today referred to as health-care workers) have learned from it the pearls and realities of gastroenterologic practice. as more editions were forthcoming, more and more pages illustrated the seemingly unsoundable depths of dr. spiro's erudition and fatherly advice. and now, dabit qui didit, for like the professional fundraiser's maxim, "he who has given, will give." once again, dr. spire has reached down into the charitable pockets of his experience and come up with a hearty 1200 plus pages of instinct, anecdote, and guidance, laced with a healthy dose of physiology, sophisticated diagnostics, and current nostrums--all the while, cautioning us "to mediate between the images and the patient" and "to listen to the story." hence, the beauty of this book. despite dr. spiro's need to enlist the aid of nine colleagues to write about half of the text, it still is flavored with redux of spiro. the book reads like the lyric of a storyteller whose main character is still the patient--with the patient's narrative woven into a clinical tapestry rich with pearls of wisdom and edged with science. ten parts, 57 sections, 332 divisions, countless subsections (at least for this review) and 1235 pages make up the volume. it is comprehensive, including the hollow viscera as well as the solid organs, adult diseases and congenital/metabolic disorders. the book does not pretend to be encyclopedic, and thus differs from the multivolume text in gastroenterology; nor is it a reference work written in the third person singular to maintain scientific objectivity. rather, it is to a large extent a personal approach to the practice of gastroenterologic medicine by a master--nuts and bolts with some chrome plating. illustrations are mainly roentgenographs, with black and white pathologic specimens. endoscopic photos are sequestered in a section inserted halfway through the volume. the latter galley is colorful and runs the gamut of common pathology seen throughout the gut. unfortunately, almost half of the photos are oriented upside down, not diminishing their teaching value, but distracting the purist's eye. reviewers are expected to criticize, for that is part of the job description. yes, the text paper is somewhat thinner than the weight i enjoy feeling as i languish over the text; yes, i wish there were specific sections on 1-1. pylori, so i did not have to search frantically in different sections to get a complete discussion of the organism; i would have liked a comprehensive discussion of aids and the gastrointestinal tract, rather than a passing reference to microsporidium, one of the most common causes of infectious diarrhea in these patients; and i wish there were a softening of such absolutisms as "cmv esophagitis does not occur in an immunocompetent host" or that "mesenteric angiography is of value (in bowel infarction) only to exclude the possibility that the superior mesenteric artery needs to be bypassed or cleaned out..." these and other minor carpings notwithstanding, i accept some "dogma"--even if i believe it to be wrong--from this great educator. at $141.00, the book is somewhat pricey, but i would pay that for the wisdom in the introduction alone. a great book for house staff, fellows, and young attending physicians who were not fortunate enough to grow up with the first three editions and those who rely too much on the data rather than on the patient's story. new york, 1994, hardbound, 205 pp., $49.50 . in a number of ways research with human subjects is more difficult than animal or basic research. if the project is well constructed, it usually takes two to five years to gather the data, perform a careful analysis, write the paper and, finally, see it in print. while molecular biology and other basic research methods have provided understanding of physiology and pathophysiologic processes, therapeutic clinical research must provide the final step in human applications. funding for clinical research has received short shrift in recent years. in some part, this is probably related to a dearth of well-trained md researchers. this small offering is based on seminars conducted at the university of texas southwestern medical school in dallas. its aim is to provide the basic principles of clinical research for those interested in the field. it addresses guidelines on possible conflicts of interest, informed consent, protocol preparation, and research grant applications. especially valuable are chapters 5 and 6, which cover various study designs and appropriate statistical approaches. these two chapters alone, comprising only 67 pages, justify the price. this book fills a need in all advanced training programs. it is intended to provide the basic needs and, if carefully read, serves its purpose admirably. in addition, it can serve every postgraduate as a source of reference when analyzing the clinical research published by others. i recommend it highly; for $49.50 it's a real bargain. it is a mere 15 years that the first textbook on the exocrine pancreas, by howat and arles, was published. since then, numerous books, monographs, proceedings of meetings and two international journals devoted solely to the pancreas have appeared. the present book, renamed simply the pancreas, from its former title, the exocrine pancreas, is clearly the most comprehensive text on the organ currently available. the addition of a chapter on endocrine-exocrine relationships of the pancreas in the new volume has all but perfected the book, making it impressive in content and range of subjects covered as well as size. whereas most previous texts have tended to have a clinical bent, the sections in this book relating to the biology of the gland, experimental models, and endocrine-exocrine relationships comprise 729 of the 1137 pages. these include the gross and fine developmental and comparative anatomy of the gland cell and molecular biology, gene expression of protein processing receptors, and electrophysiology of the acinar cells. hormonal and neural control of enzyme, lithostatine, and mucous secretion are all considered in detail, while experimental procedures encompass not only animal models but the cell and molecular biology of cell injury. current concepts of the enteroinsular axis, insulin-acinar relationships, â�¢ and the endocrine changes encountered in protein insufficiency are fully explored. the clinical sections, while more standard fare, have been revised and updated, and one would be hard pressed to find any subject of clinical pancreatology that has not been covered. one omission that may be mentioned is that it contains very little information regarding the use of endoscopic ultrasound in diagnosing pancreatic lesions, one of the rapidly growing areas in pancreatology today. both the medical and surgical aspects of pancreatic cancer as well as endocrine tumors are fully considered, and the penultimate chapter on congenital and hereditary diseases are particularly informative as they are rarely considered in detail in more recent books on the pancreas. needless to say, the editors have chosen authors who are household names in pancreatology with lives steeped in the biochemical or clinical aspects of the gland, and the list of contributors, of which there are 83, is as impressive as the book itself. despite the formidable scientific content of many chapters, the book is highly readable but has geared itself more to scientists and pancreatologists than general readership. it is just this wide net of content and expertise that makes it virtually essential in every library and in gastroenterological research and clinical departments. the quality of the text, the reproduction of the x-rays, figures, and tables are excellent, with ample bibliography and indexing. unfortunately, as the book became heftier, so did the price, which hopefully will not compromise access to it. simmy this book is designed as a review of the motor disorders of the gastrointestinal tract. it is divided into two sections. the first section consists of a series of chapters by an impressive collection of experts on gastrointestinal motility disorders, basic biochemistry mechanisms, and an overview of neuropeptides and pharmacology. the sequence of chapters then begins in the oropharynx and extends through to the rectum including discussion of pain management as well as psychiatric therapy for symptoms from gastrointestinal motor disorders. the second part of the book is a transcript of a series of interactive sessions. these sessions were conducted at the 1992 aga postgraduate course on gastrointestinal motility. the data were obtained using a computerized audience interactive system. the audience response and transcription of the panel discussions are provided. the chapters are well organized and presented in a logical sequence. they seem to follow the lecture format from the aga postgraduate course. the first three chapters discuss motility in general along with neuroregulation. this is, by necessity, an overview of those areas and adequate supplemental readings are suggested. a brief section on pharmacology then follows. once again, adequate references are provided for those who desire more in-depth study, the next several chapters begin at the oropharynx and proceed through the gastrointestinal tract, presenting a discussion of the motility of each area. the second half of the book is devoted to the question and answer sections from the postgraduate course. the panel discussions are interesting, although frequently not in-depth. it would have, perhaps, been better to have the moderator summarize the comments in more of a generalized statement. the editors and the american gastroenterological association should be congratulated for compiling the topics from their postgraduate course into this very useful book. it is a much more usable reference than the lecture handout guideline that was presented at the time of the course. one hopes similar efforts are forthcoming from subsequent aga postgraduate courses. i would recommend this book to gastroenterologists seeking an overview of motility and to gastroenterological trainees to provide a core of knowledge to assist in their training to assist in their as well as prepare for their board examination. the price is listed at $74.95 which is somewhat high for a 247-page paperback book but can be justified by the expertise of the individual chapter authors acute pancreatitis: diagnosis and therapy. edited by e. l. bradley. raven press, new york, 1994, 294 pp., $131.50 .this excellent book is a summary of the atlanta international symposium, the presentations of 40 experts who assembled in atlanta, georgia, in the fall of 1992 to discuss acute pancreatitis and its complications. these contributors represented six medical disciplines and 15 countries. about half were pancreatic surgeons. the book is organized based on the atlanta classification and divided into sections, such as severe acute pancreatitis, necrotizing pancreatitis, pancreatic abscess, acute pancreatic fluid collection and pseudocysts, and mild acute pancreatitis. each section has multiple authors. professor bradley has most succinctly written the last section, which is the summary.since each chapter is written by an international expert, there is variability in style and quality. dogmatic and controversial opinions are evident. the majority of authors are surgeons. these merely reflect the fact that it is basically a symposium proceedings.many chapters are excellent. these include the chapter on the atlanta classification by bradley, pathology of severe acute pancreatitis by kloppel, and contrast-enhanced ct by balthazar. internists and generalists would benefit from following the concepts put forward by barkin's group about medical therapy of severe acute pancreatitis. the whole section on the important topic of pancreatic abscess is outstanding. in general, the figures and tables are clear and of high quality.this is a fine monograph on an important topic. it should be of value to generalists as well as specialist in fields such as medicine, surgery, radiology, and emergency medicine--indeed all who encounter patients with acute pancreatitis. it covers all the relevant areas related to acute pancreatitis and so should be purchased by all libraries as a single authoritative reference source.eapen thomas, md johnson city, tennessee key: cord-029138-avfvpqs5 authors: croome, kristopher p.; taner, c. burcin title: the changing landscapes in dcd liver transplantation date: 2020-07-13 journal: curr transplant rep doi: 10.1007/s40472-020-00283-1 sha: doc_id: 29138 cord_uid: avfvpqs5 purpose of review: the transplant community continues to look for ways to help address the discordance between donor liver graft availability and patients on the liver transplant waiting list. donation after circulatory death (dcd) donor livers represents one potential means to help address this discordance. the present review describes the changing landscape of dcd liver transplantation (lt). recent findings: the number of dcd lts performed annually within the usa has continued to grow on an annual basis. importantly, national data has demonstrated that outcomes with dcd lt have been improving. this improvement has been driven by better understanding of how to successfully utilize these organs through better donor and recipient matching and careful evaluation of both hemodynamics during withdrawal of life support and the refinement of the procurement operation. summary: despite these improvements in outcome, ischemic cholangiopathy (ic) continues to be the achilles heel of dcd lt. emerging technologies such as various forms of machine perfusion may allow for reduction of complications and better prognostication of the risk associated with dcd liver grafts. the transplant community continues to look for ways to help address the discordance between donor liver graft availability and patients on the liver transplant waiting list. donation after circulatory death (dcd) donor livers represents one potential means to help address this discordance. initial reports examining the use of liver grafts from dcd described inferior longterm outcomes when compared with donation after brain death donors (dbd). these inferior results were ascribed to high rates of biliary complications, as well as increased rates of primary non-function and hepatic artery thrombosis [1] [2] [3] [4] . since those initial publications, there have been substantial developments in the understanding of how to effectively utilize dcd livers. more recent single center publications from high volume dcd programs have demonstrated equivalent outcomes between dcd and dbd liver transplantation (lt), with appropriate donor and recipient selection [5] [6] [7] . the present review describes the changing landscape of dcd lt. this article is part of the topical collection on liver transplantation prior to the development of the harvard criteria for brain death in 1968, all deceased organ donors were declared deceased using circulatory arrest criteria and thus represented the first dcd organ transplants performed [8] . following the acceptance of declaration of death according to neurologic criteria as a legal entity, most countries including the usa almost exclusively utilized dbd donors until the 1990s. resurgence of dcd lt was first described by groups from pittsburgh and wisconsin in the mid-1990s [9, 10] . excitement surrounded dcd lt resulted in rapid growth in the number of transplants performed from 1994 until 2007 (fig. 1 ). this initial excitement was tempered by multiple reports that described high rates of complications, specifically ischemic cholangiopathy (ic), resulting in graft failure or patient death following dcd lt [1] [2] [3] [4] . these results led to a contraction in the number of dcd lts performed from 2007 until 2012. as more data began to emerge demonstrating that acceptable results with dcd lt could be achieved with appropriate donor and recipient selection, the number of dcd lt performed annually has continued to grow from 2012 to 2018. in addition to increasing dcd lt volumes, a concomitant improvement in us national results with dcd lt has taken place. we previously demonstrated a sequential improvement in both graft and patient survival from 2003 to 2014 following dcd lt [11 •• ] (fig. 2) . moreover, changes in both recipient and donor characteristics consistent with published literature during the aforementioned time period were observed between eras. as with all innovations in transplant practice, there is undoubtedly a learning curve associated with the optimal utilization of liver grafts from dcd donors which has taken place as new data and analyses have become available. changes that were observed over the eras included: 1. decrease in proportion of dcd lt used for recipients: donor liver allocation in the usa has never been more contentious. following years of debate and legal challenges from all sides, a liver distribution system based on acuity circles went into effect on february 4, 2020 [12] . this acuity circle allocation system replaces donation service area (dsa) and regional boundaries previously used in liver organ distribution with a system based on distance between donor hospital and transplant hospital. this system will result in broader sharing of livers and substantially increase both the distances traveled and the costs for organ retrieval. it is also predicted that this system will reduce meld score disparity across the usa. in the newly adopted acuity system, livers from dcd donors are allocated earlier in the match sequence to transplant centers closer to the donor hospital compared with liver donor allocation for dbd donors < 70 years of age (fig. 3 the effects that allocation changes will have on dcd lts remain unknown. substantial variability in the utilization of dcd livers currently exists across the country [13 •• ] . this variability in dcd liver utilization has been shown to have no correlation with median meld score at transplant [13 •• ] . indeed, it is known that dcd utilization in the usa is driven by a few high-utilization centers [14] . likely, the new allocation changes will result in significant changes to behavior both from transplant centers as well as opos with regard to the pursuit and utilization of dcd livers. transplant center behavior has previously been shown to account at least partially for significant variability in median meld score at transplant both inter-and intra-regionally [15] . one question to be answered is whether transplant centers will be willing to travel substantial distances for dcd livers? a previous single-center study demonstrated that each successful dcd liver procurement required an average of 218 more miles of travel than each successful dbd liver procurement [16] . this difference was largely due to lower successful procurement proportion among dcd procurement episodes. only 49.7% of dcd liver offers resulted in a liver procured for transplant, compared with 92.3% of dbd. as a result, the authors of this paper suggested that current reimbursement policies poorly reflect increased surgeon travel (and time) expenditures between dcd and dbd liver offers. while several tools exist in order to predict the likelihood that a dcd donor will expire within 60 min, their predictive value is moderate at best, making the decisions on which dcd liver donors to pursue challenging [17] [18] [19] . historically, lt candidates with hcc have experienced a substantial advantage in deceased donor liver allocation with lower waitlist mortality/dropout within 1 year of listing compared with those candidates without a hcc diagnosis [20] . in addition to the recent implementation of the acuity circles system, deliberate steps have been taken to adjust hcc exception scores so that these transplant candidates are no longer so heavily advantaged. hcc patients no longer have a "ladder" model of increasing exception scores over time and instead are given an exception score of median meld at transplant minus 3 (mmat-3) for a 250 mile radius surrounding the listing center. undoubtedly this will result in decreased access to highquality dbd organs for patients with hcc. one potential option for this population may be increased utilization of dcd livers. this potential shift is congruent with the principle of using extended criteria organs in recipients with lower biological meld scores, such as many hcc patients, because of the perception that these recipients are better able to tolerate an extended criteria organ [21] . a previous publication demonstrated that after the implementation of the share 35 policy, more hcc patients have received livers from dcd donors [22] , potentially as the result of the highest quality organs being preferentially utilized by higher meld recipients with broader sharing. [12] wit and the importance of the procurement operation initial studies investigating lt using liver grafts from dcd donors linked prolonged donor warm ischemia time (dwit) to biliary complications and graft loss [3, 23, 24] . while there is a general consensus that prolonged dwit negatively effects outcome, there exists significant disagreement on what length of dwit is acceptable and even on how dwit is defined. as the collective experience with dcd lt increased, a concept of functional donor warm ischemic time (fdwit) arose from the notion that individual events during dcd procurement, such as variations in hemodynamics, mandatory wait period, time from incision to cannulation of the aorta and cross-clamp, all of which are included in total dwit, may have different impacts on the outcome of the liver graft [25, 26] . previous studies have defined the start of f-dwit based on different hemodynamic parameters (such as drop in mean arterial pressure (map) or sbp) or by a drop in oxygen saturation below a specific level. f-dwit terminates at the time of cold organ flush/cross-clamp. while the concept of f-dwit is ubiquitously accepted, no consensus on what parameters specifically define f-dwit exists [27] . previously published national and society guidelines for fdwit can be seen in table 1 [28] [29] [30] [31] . recently, a consensus conference on dcd lt transpired in venice, italy (january 30, 2020) in which attempts to more uniformly define and describe dwit and fdwit were made. the results of this consensus meeting have yet to be published. the importance of the procurement operation in order to achieve successful outcomes with dcd lt should not be understated. not only is it imperative to be able to quickly cannulate the donor vessels and begin cold perfusion of the liver, but judgement as to the timing and length of fdwit, flush quality, and overall liver appearance is paramount. judgement comes with experience and many of the high-volume transplant centers will only utilize dcd livers in cases where the procurement has been performed by a member (frequently an attending surgeon) of their team. this judgement on whether the liver is "usable" even more heavily relies on procuring surgeon experience when attempting to utilize extended criteria (ecd) dcd liver grafts. the fundamental techniques of the procurement operation have changed very little since the initial description of the "super rapid technique" [9] . many components of dwit are not modifiable since they occur prior to final pronouncement of donor death. alternatively, the time from final pronouncement of death until cold flush of the organs is largely within the procuring surgeon's control. the procuring surgeon should discuss the steps of the procedure with all assistants and operating room personnel prior to withdrawal of care. once the "no touch" time has transpired and the donor is declared deceased, efforts to cannulate and began cold flush in under 2 min should be made. donor hepatectomy time is the time from initiation of cold perfusion to the end of the hepatectomy and removal of the liver from the donor. as a general rule, extraction of the liver from the body of a dcd donor should be done as quickly and safely as possible. several recent reports have suggested that donor hepatectomy time (dht) may be associated with outcomes following dcd lt [32, 33] . in a study from the uk, dht > 60 min was associated with primary non-function (pnf) [32] . an abstract from the netherlands demonstrated that dht > 90 min was associated with both ic and early graft loss [34] .whether prolonged dht is itself a r