The first consensus report of the working party of the Asian Pacific Association for the Study of the Liver (APASL) set up in 2004 on acute-on-chronic liver failure (ACLF) was published in 2009. With international groups volunteering to join, the “APASL ACLF Research Consortium (AARC)” was formed in 2012, which continued to collect prospective ACLF patient data. Based on the prospective data analysis of nearly 1400 patients, the AARC consensus was published in 2014. In the past nearly four-and-a-half years, the AARC database has been enriched to about 5200 cases by major hepatology centers across Asia. The data published during the interim period were carefully analyzed and areas of contention and new developments in the field of ACLF were prioritized in a systematic manner. The AARC database was also approached for answering some of the issues where published data were limited, such as liver failure grading, its impact on the ‘Golden Therapeutic Window’, extrahepatic organ dysfunction and failure, development of sepsis, distinctive features of acute decompensation from ACLF and pediatric ACLF and the issues were analyzed. These initiatives concluded in a two-day meeting in October 2018 at New Delhi with finalization of the new AARC consensus. Only those statements, which were based on evidence using the Grade System and were unanimously recommended, were accepted. Finalized statements were again circulated to all the experts and subsequently presented at the AARC investigators meeting at the AASLD in November 2018. The suggestions from the experts were used to revise and finalize the consensus. After detailed deliberations and data analysis, the original definition of ACLF was found to withstand the test of time and be able to identify a homogenous group of patients presenting with liver failure. New management options including the algorithms for the management of coagulation disorders, renal replacement therapy, sepsis, variceal bleed, antivirals and criteria for liver transplantation for ACLF patients were proposed. The final consensus statements along with the relevant background information and areas requiring future studies are presented here. Updated on the basis of AARC data of >3300 cases enrolled into AARC registry prospectivelyACLF is distinct form Acute Decompensation of cirrhosisNewer sections on DILI-ACLF, AIH-ACLF, PVT/HVOTO–ACLFReversibility of Chronic Liver Disease in ACLFPortal and systemic hemodynamics and their relevance in ACLFAcute Portal Hypertension and Variceal progression in ACLFAARC score as a guide for treatment strategies in ACLFACLF in Children-first consensus on pediatric ACLF Liver failure is a common medical ailment and its incidence is increasing with the use of alcohol and growing epidemic of obesity and diabetes. It can present as acute liver failure (ALF) (in the absence of any pre-existing liver disease), acute-on chronic liver failure (ACLF) (an acute deterioration of known or unknown chronic liver disease), or an acute decompensation of an end-stage liver disease , 2. Eachwww.aclf.in). With the setting up of the APASL ACLF Research Consortium (AARC) in 2012, the collaborative research work, publications and protocol driven unified treatment had gained momentum. The retrospective and prospective data of patients from different centers were analyzed, and the completed patient records were utilized for defining predictors of mortality and grades of liver failure and incidence of other organ failures .1.9.Including organ failure in definition- for utility or futility?1.9.1.The terms “organ dysfunction” and “organ failure” need to be described more clearly based on the AARC database, used in APASL consensus [B2].1.9.2.Extrahepatic organ failure should not be included in definition of ACLF, as this would lead to missing out the potential therapeutic window for reversal of the ACLF syndrome (A1).1.9.3.> 1.5) (A1).Liver failure for definition of ACLF should include jaundice (serum bilirubin ≥ 5 mg/dL) and coagulation dysfunction . However, since the earlier (2014) definition had included mortality, the same definition was agreed.There is no consistent definition of ACLF in the literature. Each study done previously on ACLF has used its own definition, and there is no unanimity in these definitions in terms of criteria for liver failure, the acute event precipitating ACLF, and the diagnosis of underlying chronic liver disease. Since most of these studies were on patients who required liver support devices or liver transplantation, these studies were biased toward including sicker patients in the definition and patients having a mild disease were left out.A detailed analysis of the definition of liver failure and the need for the defined outcome of high 28-day mortality was taken into account. An estimated 33% mortality at 28 days was considered important. Having analyzed and defined the acute and chronic insults, the time frame and the criteria of liver failure, development and course of organ failure and sepsis, the APASL definition of ACLF of 2009 was reassessed. It was reported that this definition has been used in nearly 200 publications from the East and the West and has been found to be simple to use and with a high degree of predictive ability to define the outcome of a relatively homogenous group of liver failure patients with underlying chronic liver disease.“ACLF is an acute hepatic insult manifesting as jaundice (serum bilirubin ≥ 5 mg/dL (85 micromol/L) and coagulopathy (INR ≥ 1.5 or prothrombin activity < 40%) complicated within 4 weeks by clinical ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease/cirrhosis, and is associated with a high 28-day mortality.” .The consensus definition is:The spectrum of acute insult in the Asian region was revisited, while hepatitis B reactivation remains the predominant cause of acute hepatic insult in the East, from the global perspective. The trends showed an increase in the proportion of alcoholic hepatitis over the years. This was a bit unexpected for the Asian countries where alcoholic hepatitis is emerging as a major acute insult and shows the growing westernization of Asia. A review of the recent CANONIC study data showed that in the West the term precipitating event is generally used and probably details of events such as Hepatitis B or superadded hepatitis A and E are rarely encountered or recorded . SurprisAcute hepatic insults of infectious etiology included reactivation of hepatitis B virus (HBV) as the leading cause of ACLF in the Asian region –20. ReacDrug-induced liver injury (DILI) presenting as ACLF is an important entity less often addressed in the Global literature. Hepatotoxic drugs and complementary and alternative medicines (CAM) are important causes for acute and acute-on-chronic liver failure in the Asia–Pacific region .2.1.2.2.Diagnosis of ACLF-D is challenging as liver disease-related fluctuations in the liver function tests may be part of the natural history of the disease and may confound the diagnosis. Further, cirrhotic patients may not show marked derangements in transaminases .2.1.2.3.Those who develop ACLF-D are likely to have severe consequences including decompensation and death .2.1.2.4.Drugs responsible as acute insults, triggering ACLF-D in cirrhosis patients include anti-tubercular drugs, Complimentary and alternative medications, antiretroviral drugs and Methotrexate . More evidence is needed for drugs like azithromycin, azole antifungals and antimicrobials in cirrhotics .2.1.2.5.Risk of liver injury is proportional to the number of hepatotoxic drugs in anti-TB regimen .2.1.3.Autoimmune liver disease—distinction in presentation as ACLF and ALF.2.1.3.1.ACLF-AIH frequently presents as seronegative for autoantibodies or normal IgG levels [B2]. Seronegative AIH cases might be overlooked without histology .2.1.3.2.Diagnosis of ACLF-AIH requires liver biopsy (transjugular route preferred). Biopsy is more helpful in patients where etiology is not evident; antibodies and IgG are negative but there is a high index of suspicion  .2.1.3.3.Frequency/degree of fibrosis may define chronicity (ACLF or ALF), but fibrosis may progress in a few weeks from F0 to F1–2 .2.1.3.4.Corticosteroid therapy should be considered for a select group of patients presenting with ACLF-AIH .2.1.4.Acute variceal bleed.2.1.4.1.The frequency of acute variceal bleed (AVB) increases with severity of cirrhosis .2.1.4.3.Mortality in compensated cirrhosis increases with the development of ACLF in 90 day follow-up post-variceal bleed .2.1.4.4.Incidence of post-EVL ulcers in ACLF is higher than that in cirrhosis .2.1.4.5.Though infrequent, AVB can lead to ACLF in small proportion of Child A patients. Further studies are required in patients with Child B .2.1.5.Vascular liver diseases .2.1.5.1.In patients with cirrhosis, development of acute occlusive portal vein thrombosis (PVT) may precipitate ACLF in a small sub-group .2.1.5.2.In patients with cirrhosis or Budd–Chiari syndrome, development of acute hepatic vein thrombosis (PVT) may precipitate ACLF .2.1.5.3.Patients with Budd–Chiari syndrome (BCS) may infrequently present with or develop ACLF .2.1.5.4.Evaluation of thrombophilic disorders in patients with PVT or BCS and ACLF should be similar to those without ACLF .2.1.5.5.There is no evidence currently to suggest that non-cirrhotic portal fibrosis or EHPVO may present as ACLF .2.1.5.6.No data are available about the natural history or outcome of patients with PVT or BCS presenting with ACLF and no recommendations can be made for management of patients with vascular liver diseases and ACLF .Two aspects were carefully analyzed, what constitutes chronic liver disease, cirrhosis alone or non-cirrhotic chronic liver diseases and the etiology of the chronic liver disease.The degree of hepatic fibrosis and the functional hepatocellular mass remains heterogeneous in patients with the chronic hepatitis , 54. EveNAFLD is the leading cause of donor rejection in liver transplantation . ExperieAccurate and reliable assessment of underlying CLD in the setting of ACLF is important for the subsequent management and need for liver transplant in these patients. Diagnosis of chronic liver disease in the setting of ACLF is made by history, physical examination, and previously available or recent laboratory, endoscopic or radiological investigations . UltrasoA liver biopsy through the transjugular route may be of help when the presence of already underlying CLD and the cause of liver disease are not clear. The liver biopsy may highlight the etiology, stage of fibrosis, prognosis and outcome in patients with ACLF . In addiThere is a need to have reliable non-invasive tools to assess the severity of fibrosis in a previously undiagnosed CLD. Ultrasound and CT abdomen may pick up CLD. However, to assess the degree of fibrosis in an unshrunken liver would require other radiological modalities. Transient elastography is a good modality to detect hepatic fibrosis . HoweverThe second issue was about the etiology of chronic liver disease and cirrhosis in the Asian pacific region. The experts reviewed the data from the AARC database and the etiologic profile of cirrhosis in ACLF was found to be similar to etiology of cirrhosis in general in the respective countries , 67, 68.Recommendations2.2.Defining the underlying CLD:2.2.1.Cirrhotic and non-cirrhotic chronic liver diseases qualify as chronic liver diseases .2.2.2.The common underlying chronic liver diseases include alcohol, hepatitis B, hepatitis C, NAFLD-related chronic liver disease or cirrhosis of the liver .2.2.3.Chronic hepatitis and/or significant fibrosis without cirrhosis should be taken as a chronic liver disease, if such a patient presents as ACLF .2.2.4.NAFLD-related chronic hepatic injury; NASH, if associated with significant fibrosis, should be taken as a chronic liver disease in ACLF .2.2.5.Patients with known previous decompensation with jaundice, HE, and ascites should be excluded .2.2.6.Diagnosis of chronic liver disease and cirrhosis in the setting of ACLF is made by history, physical examination, laboratory, endoscopic or radiological investigations .2.2.7.A liver biopsy through the transjugular route may be helpful when the presence of underlying chronic liver disease and/or the cause of chronic liver disease and/or the acute insult is not clear .Comorbidities also influence the outcome of ACLF as far as the disease and outcome are concerned. The presence of co-morbidities like obesity, sarcopenia and other metabolic risk factors like diabetes mellitus, hypertension and dyslipidemia have a bearing on the outcome of patients with cirrhosis . HoweverRecommendations2.3.Impact of comorbidities and obesity.2.3.1.The presence of overweight or obesity and dyslipidemia increases the severity of liver disease in ACLF patients .2.3.2.The presence of overweight or obesity increases the short-term (30 day) mortality in patients with ACLF .2.3.3.There is need to compare the development and natural history of ACLF in patients with NASH versus NASH cirrhosis .The epidemiology of acute insult has changed significantly in the past 5 years. In the recent AARC data, alcohol has now emerged as the most common etiology for acute insult (49%) as well as for underlying chronic liver disease in contrast to previuos data of HBV predominance. DILI and autoimmune etiologies have shown increasing trend; however, HAV/HEV had decreasing trend. HBV infection-induced ACLF as well as HAV/HEV-induced ACLF is now showing a decreasing trend over time, whereas alcohol and herbs, drugs and supplements (HDS)-induced ACLF show an increasing trend. The unknown causes for acute insult and chronic injury constitute only 5–15% cases of ACLF in the East in contrast to the West, where these are seen in about 40% of ACLF patients.Recommendations2.4.Changing trends for the etiology of acute insult and chronic injury.2.4.1.Alcohol is now the commonest etiology for acute hepatic insult as well as for the underlying chronic liver disease in the Asian continent .2.4.2.DILI and autoimmune etiologies have shown increasing trend .2.4.3.HBV infection–reactivation of hepatitis B-induced ACLF as well as acute HAV/HEV-induced ACLF shows a decreasing trend over time in certain regions, whereas alcohol and herbs, drugs and supplements (HDS)-induced ACLF show an increasing trend .2.4.4.The unknown causes for acute insult and chronic injury constitute only 5–15% cases of ACLF in the East in contrast to the West, where these are seen in about 40% of ACLF patients .The two disease entities look similar and are often misunderstood. The experts reviewed the literature and presented their data. The data from the AARC database were presented. The discussion revolved around the following main issues:Acute decompensation occurs in a cirrhotic with or without prior decompensation and is often associated with a precipitant   . The pro < 0.01] .Development of ascites represents a state of acute portal hypertension in ACLF patients. This rapid rise in portal pressure is a result of severe hepatic inflammation and ongoing liver failure. Highly activated stellate cell population, cytokine storm and ongoing hepatic parenchymal necrosis perpetuate the portal hypertension syndrome. The use of non-selective beta-blockers has been shown to be effective in reducing the mortality and risk of variceal bleed in ACLF patients.ACLF is a unique disease entity and is distinct from acute decompensation by considering only those patients who have one type of AD and in a specified time frame of 28 days; this includes patients who develop after a hepatic insult, jaundice and coagulopathy followed by development of acute portal hypertension in the form of ascites and or HE Table .Table 3DRecommendations2.5.ACLF is distinct from acute decompensation (AD): differentiating AD and ACLF.2.5.1.Natural history and outcome of ACLF.2.5.1.1.The main etiologies for ACLF are alcohol-related injury, viral hepatitis, drug-induced liver injury, and autoimmune liver disease. In the Asian Pacific region, in only about 5–10% of the cases, the acute insult is unidentifiable .2.5.1.2.Age and the presence of cirrhosis are independent risk factors for mortality in ACLF .2.5.1.3.Portal hypertension with an HVPG greater than 18 mmHg and/or variceal bleeding, presence of complications including ascites, SBP and encephalopathy are independent predictors for mortality .2.5.1.4.Starting NSBBs is safe in ACLF and its use is associated with improved short-term survival .2.5.1.5.Appropriate management has key impact on the outcomes of ACLF, early (within 2 weeks) anti-HBV treatment for HBV-ACLF, corticosteroid therapy for alcoholic ACLF and AIH-ACLF are worthwhile options .2.5.2.Natural history and outcome of acute decompensation.2.5.2.1.2.5.2.2.Patients with AD who have or progress to develop extrahepatic organ failure have high short-term mortality .2.5.2.3.Early evaluation of potential predictors and precipitating agents can help in managing these patients .Acute decompensation (AD) is currently defined as acute occurrence of decompensating events  in a patient with CLD .2.5.3.Acute decompensation—differentiating from ACLF for clarity in definition.2.5.3.1.AD develops in a patient with chronic liver disease/cirrhosis, with or without prior decompensation, and is often associated with an identifiable precipitant and develops in less than 3 months .2.5.3.2.Any decompensation preceding jaundice strongly favors AD .2.5.3.3.Absence of repeated episodes of decompensation differentiates ACLF as a unique syndrome . This has implication on the management decisions and prognostication, including reversibility of the syndrome.2.5.3.4.Long-term survival, reversal and/or recovery of hepatic reserve has been documented with ACLF .2.5.4.5.The differentiating features between different presentations of AD and the ACLF need to be studied carefully by expanding the AARC database .Since the previous consensus statement, new data and insights into the liver histopathology have become available. The main questions that were addressed in the current consensus meeting were: (1) Is liver biopsy feasible and safe in ACLF, (2) Can liver biopsy help to differentiate ACLF from ALF and chronic liver disease, (3) Are there any histological predictors of outcome in ACLF, such as need for liver transplantation or mortality and (4) Are there any differences in regenerative response in sequential biopsies of survivors and non-survivors?Percutaneous liver biopsy is generally not feasible in patients with ACLF due to coagulopathy and ascites. Transjugular liver biopsy (TJLB), on the other hand, is considered relatively safe and can help assess stage of fibrosis and severity of hepatic injury. For example, severity of alcoholic hepatitis in alcoholic liver disease-related ACLF could be assessed only by liver biopsy . It can Mini-laparoscopic liver biopsy is another alternative for getting liver biopsy in patients of advanced cirrhosis with acceptable bleeding risk. More data needed on this modality and comparison with TJLB are lacking at present. However it can be considered in areas with poor access to TJLB and biopsy is definitely needed for the decision- making .Differentiating ALF and chronic hepatitis with flare is based on findings of fibrous bands (spurs and bridges) and ductular proliferation. Features of cholestasis and bile duct proliferation are more common in patients with acute injury . Differentiation between cirrhosis with acute deterioration and compensated cirrhosis is based on the presence of necrosis and features of acute hepatitis in the former group of patients . It was Liver histopathology could be very useful in prognosticating the outcome in a patient with ACLF , 81. Thep < 0.001) and CLD (p < 0.001).Liver regeneration is considered to play an important role in ACLF as prognosis can be improved if the critical threshold of functional liver cell mass is regained. Decompensated cirrhosis is considered irreversible owing to loss of regeneration potential. Liver histology can provide morphological evidence supporting these concepts and for assessing regenerative potential and prognosis . In thisThere is significant relationship between HSCs and presence of HPCs, indicating a possible dynamic role of HSCs in liver regeneration and pathobiology of ACLF . Liver bRecommendations2.6.Role of liver histology in ACLF.2.6.1.2.6.2.Liver biopsy is helpful in patients where the presence and stage of underlying chronic liver disease and/or the cause of chronic liver disease are not clear . Biopsy can help identify unsuspected/multiple etiologies, i.e., primary and concomitant etiologies.A liver biopsy through the transjugular route is helpful to diagnose/confirm the cause of acute injury .2.6.3.The need of liver biopsy in ACLF should be individualized, especially in alcoholic hepatitis, severe autoimmune hepatitis, and flare of Wilson disease .2.6.4.Liver biopsy indicates the stage of fibrosis and is helpful in the prognosis and outcome in patients with ACLF (B 1). It can help in distinguishing ACLF from decompensated cirrhosis .2.6.5.Certain histologic parameters are predictors of prognosis of ACLF, like ductular bilirubinostasis, eosinophilic degeneration, and parenchymal extinction .2.6.6.It can help in distinguishing ACLF from decompensated cirrhosis .2.6.7.Standardization of liver biopsy assessment is essential for a uniform approach to the diagnosis and treatment for CLD and acute insult .2.6.8.Noninvasive tools to measure liver stiffness and biomarkers may help in identifying patients with advanced fibrosis. Studies are needed to validate the performance of these tests in the setting of ACLF .Acute liver failure is generally defined as development of HE within 4 weeks of onset of jaundice , 2. SincThe clinical presentations in ACLF is varied and depends upon the severity of acute insult and degree of underlying chronic liver disease. In the published reports, patients included as having ACLF had severe jaundice associated with organ failure manifested as either HE or hepatorenal syndrome (HRS) , 2.Defining the liver failure in ACLF, therefore, required a detailed consideration of all the existing liver failure scores and the criteria defining liver failure in the organ failure scores such as SOFA and APACHE II. The two main variables are bilirubin and coagulopathy.Serum bilirubin analysis of the AARC data revealed that patients with a bilirubin between 5 and 10 mg/dl also had substantial mortality ranging around 38%. The data for patients below this level were, however, not collected as per the initial definition, but is likely to yield mortality rates much below 33%. On the other hand, in the CANONIC study, the level of bilirubin for hepatic failure was taken as 12 mg/dl so as to determine 15% mortality at 28 days. If these criteria were applied to the ACLF patients in the Asian region, a much higher mortality was observed in this cohort. After detailed discussion, the original value of > 5 mg/dl was accepted as the cutoff for bilirubin for defining liver failure ) and coagulopathy (INR > 1.5 or prothrombin activity < 40%) are mandatory parameters to assess liver failure .3.2.3.3.Liver failure score (AARC score) which includes total bilirubin, INR, grade of HE, plasma lactate and serum creatinine reliably predicts the disease severity and outcome .Ascites and/or encephalopathy as determined by physical examination also reliably reflect significant hepatic functional impairment .3.4.Grading of liver failure as per AARC score I (5–7), II (8–10), III (11–15) effectively prognosticates and guides the therapy .3.5.The assessment of coagulation system by global coagulation methods (viscoelastic technique/thrombin generation test) may be considered as a useful tool for assessing coagulation anomalies in ACLF patients .Sepsis is a syndrome of systemic inflammatory response of the host to an identifiable infection. The systemic inflammatory response syndrome (SIRS) is defined by the presence of at least two of the following criteria: (1) altered temperature, (2) elevated respiratory rate or hyperventilation, (3) tachycardia, and (4) altered white blood cell count  . Sepsis Due to the hyperdynamic circulation and complications of portal hypertension, the currently accepted clinical definition of SIRS and sepsis may not be entirely applicable to patients with cirrhosis or ACLF. Hence, a high index of suspicion is required for making a clinical diagnosis of sepsis in these patients. Liver failure initiates and predicts the development of SIRS. New onset SIRS in the first week is an important determinant of early sepsis, organ failure, and survival Fig. . Prompt Consideration of sepsis as an acute insult in the absence of liver failure is confusing and with limited scientific basis. Sepsis is a consequence rather than the cause for development of ACLF. The APASL definition does not include sepsis as a primary cause for liver failure, but in the Western definition, sepsis is considered as the most common precipitant. The inclusion of sepsis in the definition is likely to be associated with concomitant multi-organ involvement, poor prognosis and would be unlikely to provide a targeted therapy or a definitive therapy such as liver transplant.SIRS is the inflammatory response to the damaged organ in the host. It could be a result of sterile inflammation or an occult infection –95. In fThe current paradigm regarding the host immune response to sepsis is debated and is a matter of great interest in clinical trials as well as basic science. Two theories have been proposed to describe the host response to sepsis. According to the most accepted theory, both pro-inflammatory and anti-inflammatory responses occur early and simultaneously in sepsis although the net initial effect of these competing processes is typically manifested by an early, dominant, hyperinflammatory phase characterized by shock, fever and hypermetabolism , 97. SubAccording to the second theory, there is rapid and sustained upregulation of genes that regulate the innate immune response and the simultaneous downregulation of genes that regulate the adaptive immune response. There is protracted, unabated inflammation driven by the innate immune system with resultant organ dysfunction and failure .Whether sepsis is the cause or a result of liver failure was debated at length. The fact that patients who presented with no SIRS or subsequently developed SIRS or sepsis over a period of 1–2 weeks indicates that infection and sepsis develop after liver failure and unabated inflammation leads to immune paresis and provides an opportunity for infections and sepsis to occur. Non-hepatic infections are also common in patients with ACLF , 102. NeBacterial infection is present in about 1/3rd of ALCF patients at presentation to a tertiary referral hospital, and this further increases by first week . The AARInvasive fungal infection is not uncommon in ACLF patients. However, data are minimal. The diagnosis of invasive fungal infection can be proven, probable or possible, depends on mycological evidence, and clinical evidence. Invasive pulmonary aspergillosis (IPA) is increasingly recognized as a cause of morbidity and mortality in patients with ACLF. Prophylaxis of invasive fungal infection may be applicable with echinocandins in selected patients. Prophylaxis with fluconazole followed by echinocandins needs to be evaluated in ACLF patients. Predictors of poor progression (risk factors) include diabetes, AKI, ICU admission, and admission for bacterial infection, prolonged antibiotic therapy (> 5 days from admission), prior h/o hospitalization. Biomarkers such as galactomanan or B–D Glucan can be used for supporting diagnosis if there is invasive fungal infection in ACLF. Administration of albumin is recommended in patients with SBP for preventing from type-1 HRS and reducing mortality. The role of albumin in preventing or treating other infections in ALCF is not clear.Recommendations4.0.Sepsis in ACLF.4.1.There is a central role of inflammation and dysbalance of innate and adaptive immune responses in ACLF patient .4.2.It is difficult to differentiate SIRS from early sepsis in cirrhosis .4.3.Bacterial infection is present in about 1/3rd of ALCF patients at presentation to a referral hospital, and this may increase in the first week .4.4Patients who do not have sepsis have low 28-day mortality [B2]. Patients with accompanying sepsis at baseline or who develop sepsis at day 4 have high mortality .4.5Organ dysfunction and organ failure are significantly higher in infected cohort and this is attended with high short-term mortality .4.6Bacterial infection (BI) is an important factor to predict development of organ failure in ACLF. The most frequent infections are SBP, pneumonia, UTI, and Bacteremia .4.7Second infection (2nd hit) is associated with poor outcome in patients with ACLF .4.8Hospitalized patients with ACLF should be monitored closely for the presence of infections to enable early diagnosis and treatment. Routine examination of blood and body fluids is recommended .4.9The acute phase proteins, such as C-reactive protein and procalcitonin, have been proven to be reliable biomarkers associated with infection and are recommended for screening for the presence of bacterial infections .4.10As soon as bacterial infection is suspected or diagnosed, broad spectrum antimicrobial agents alone or in combination should be started and thereafter, the therapy should be adjusted according to the results of the antibiotic sensitivity test results .4.11.Empirical antibiotic therapy should be based on environment, local bacterial resistance profiles, severity and type of infection. To optimize the empirical antibiotic treatment, it is quite important to distinguish among community acquired, health care associated and nosocomial infections .4.12.Invasive fungal infection is not uncommon in ACLF patients. These can be proven, probable or possible, depending on mycological and clinical evidences . Biomarkers such as galactomanan or B-D glucan can be used for supporting the diagnosis .4.13.Invasive pulmonary aspergillosis (IPA) is increasingly recognized as a cause of morbidity and mortality in patients with ACLF. Voriconazole plasma concentration monitoring may ensure the safety and efficacy of ACLF patients with Invasive aspergillosis infection .4.14.Prophylaxis of invasive fungal infection can be done using echinocandins in selected patients (B2). Prophylaxis with fluconazole followed by echinocandins needs to be evaluated in ACLF patients .4.15.Predictors of poor progression (risk factors) of fungal infections in ACLF are the presence of diabetes, AKI, ICU admission, and admission with bacterial infection, prolonged antibiotic (> 5 days pre-admission) and prior hospitalization .4.16.The value of qSOFA and Sepsis-3 criteria is unclear to assess severity of infection in patients with ACLF.4.17.Administration of albumin is recommended in patients with SBP to prevent development of type-1 HRS and reduce mortality (A2). The role of albumin in preventing or treating other infections in ALCF is not clear .4.18.Patients who respond to treatment for bacterial infection have significantly reduced mortality .The concept to differentiate organ dysfunction from organ failure is useful in assessing the degree of organ damage and predicting the probability of disease progression or regression; prioritizing the patient for liver transplantation and the likelihood of futility of care or high probability of death.The data from AARC database were carefully analyzed with respect to defining organ dysfunction and organ failure for each of the extrahepatic organs.Renal failure in patients of ACLF is considered to be a complex and challenging condition that is associated with an ominous prognosis. Further, kidneys are one of the most frequent extrahepatic organs involved in patients with ACLF. The EASL-CLIF consortium has defined ACLF in which kidney dysfunction is used as a defining condition . Hence, In patients with decompensated cirrhosis, the main abnormality causing renal dysfunction is systemic and splanchnic vasodilatation secondary to portal  hypertension which leads to decreased effective arterial blood volume and activation of neurohormonal systems, the rennin–angiotensin aldosterone (RAAS), the sympathetic nervous system and non-osmotic release of antidiuretic hormone, resulting in sodium and water retention –106. TheSerum creatinine is used for defining and staging AKI in patients with cirrhosis. However, serum creatinine is influenced by various factors, which affect its production and excretion, as well as those that impact its measurement and, therefore, is unreliable in patients with severe liver dysfunction including ACLF. The AKI criteria seem more relevant as they rely on a dynamic change rather than a static measurement of serum creatinine. A lower value of serum creatinine is more relevant in patients with ACLF. Serum creatinine above 0.7 mg/dl (as derived from the AARC score) has a sensitivity of 78% and specificity of 36% for prediction of 30-day mortality in patients with ACLF. For the diagnosis of kidney failure, the conventional cutoff of 1.5 mg/dl even though had a low sensitivity of 48% but had a specificity of 99.8% for 30-day mortality. Kidney failure (serum creatinine ≥ 1.5 mg/dl) was seen in 22% of ACLF patients at baseline and developed in another 30% within a month. The majority of patients of ACLF developed new episodes of AKI in the first 2 weeks (11%). Further, the KDIGO definition for AKI (incorporating the urine output criteria) has been shown to be more accurate in determining AKI course in critically ill cirrhotics; however, this needs to be evaluated for patients with ACLF .Apart from the severity, the course of AKI was seen to be an important predictor of clinical outcomes. Almost 80% of the patients, who did not present with AKI at baseline or developed within the first 7 days, survived 1 month. However, in patients who presented with AKI, resolution of AKI was associated with a better survival. Progression of AKI was associated with highest mortality of 75%. Development of new AKI as well as non-resolution or persistence of AKI was associated with almost 50% mortality at 1 month in patients with ACLF .N-GAL, Kim-1, IL-18 and l-FABP to differentiate functional AKI or HRS from structural AKI, i.e., ATN patients with ACLF .5.1.1.2.AKIN criteria should be used for the diagnosis and prognostication of AKI in ACLF patients .5.1.1.3.5.1.1.4.In the absence of baseline serum creatinine, AKI should be diagnosed based on the cutoff value of serum creatinine. A cutoff value of 1.1 mg/dl is a reliable marker of significant renal dysfunction and 1.5 mg/dl of kidney failure in patients with ACLF .5.1.2.Course of renal failure in ACLF.Urine output should be considered to assess the stage and course of AKI in patients with ACLF admitted to the ICU .5.1.2.1.AKI is more common and rapidly progressive in patients with ACLF as compared to decompensated cirrhosis and is associated with significantly worse outcome .5.1.2.2.Patients with ACLF and AKI have high 30-day mortality. The course of AKI is an important determinant of clinical outcomes .5.1.2.3.Progression of AKI has the worst outcome and at the same time resolution is associated with improved survival. Both persistence and new development of AKI are associated with a high short-term mortality in patients with ACLF .5.1.2.4.Serum creatinine levels 1.1–1.5 mg/dl or AKIN I are also associated with significant mortality in ACLF patients .5.1.3.Treatment of renal failure in ACLF.5.1.3.1.All attempts should be made to prevent development of new AKI in patients with ACLF .5.1.3.2.Patients with ACLF should be stratified based on the PIRO  score for identifying patients at risk of AKI development .5.1.3.3.N-Acetylcysteine), bilirubin reduction, avoidance of nephrotoxic drugs, aggressive management of circulatory failure and maintaining a high mean arterial pressure may prevent AKI development and progression in patients with ACLF .Targeting the components of PIRO, i.e., combating systemic inflammation with anti-inflammatory strategies . Terlipressin use in ACLF should be carefully monitored for adverse effects .5.1.3.5.Severity of AKI, MELD score and ACLF grade predicts therapeutic response to terlipressin and albumin in patients with HRS-AKI in ACLF .5.1.3.6.New treatments should be explored for patients with AKI-ACLF looking at systemic inflammation as a potential target .5.1.3.7.Patients with ACLF with AKI persistence should be considered for additional pharmacologic interventions to prevent AKI progression and enhance AKI resolution .5.1.3.8.Initiate RRT emergently when life-threatening changes in volume overload, hyperkalemia, hypernatremia and worsening metabolic acidosis not responding to conservative management . The threshold for initiating RRT should be lowered when AKI occurs as part of multi-organ failure , or in non-oliguric patients if the daily fluid balance cannot be maintained.5.1.3.9.Biomarkers of tubular damage, urine NGAL and IL-18 need to be evaluated for a role in patients with ACLF to determine the need for early RRT or artificial liver support .The development of HE within 4 weeks of onset of jaundice is part of the criteria for defining acute-on-chronic liver failure (ACLF) , 2. In tThe pathophysiology of HE is complex, and impairment of brain energy and development of brain edema appear to be central in the pathogenesis of encephalopathy , 118. Inp < 0.001). l-Ornithine l-Aspartate (LOLA) for Hepatic Encephalopathy has a conflicting role as far as HE and ammonia reduction in patients of cirrhosis is concerned. Few recent meta-analysis showed a positive role of LOLA in reduction of ammonia as well as improvement in encephalopathy. Hence, LOLA can be considered as a potentially beneficial therapy for ACLF patients with HE and/or hyperammonemia .5.2.3.Inflammation plays a major role in the pathogenesis of HE in patients with ACLF and is associated with cytokine storm .5.2.4.Management of HE in hospitalized patients requires admission to the high dependency or intensive care unit and includes—(1) identification and treatment of precipitating factors, including infections, and (2) specific measures for decreasing hyperammonemia and systemic inflammation. Large volume plasmapheresis or albumin dialysis and identification and embolization of portosystemic shunts may be required in refractory patients .5.2.5.Ammonia is a simple surrogate marker for HE in ACLF and correlates with severity of HE/cerebral failure .5.2.6.Dynamic change in ammonia level correlates well with clinical course of HE .5.2.7.Ammonia-targeted therapy needs further trials and validation .5.2.8.Lactulose, rifaximin, NH3-lowering strategies remain the main therapy for HE in patients with cirrhosis .In ACLF, alterations in primary and secondary hemostasis result in rebalanced coagulation, which leads to either bleeding or thrombotic episodes –125. In p < 0.001); however, no effect on platelets has been noted. ACLF cases with sepsis at presentation show increased INR and low platelets. Studies indicate the decline of platelets in first week associated with increased chances of organ failure and short-term mortality .5.3.2.Traditional coagulation measures, including prothrombin time (PT), activated partial thromboplastin time (aPTT), international normalized ratio (INR), fibrinogen levels and bleeding time (BT) do not measure bleeding risk in ACLF .5.3.3.Coagulopathy assessment and management in ACLF should be guided based on global coagulation assessment system [ROTEM/TEG/SONOCLOT] .5.3.4.d-dimer assay, respectively .Patients need to be characterized as bleeding or thrombosis phenotype by clinical assessment of major bleeding and 5.3.5.Global viscoelastic tests (TEG/Sonoclot/ROTEM) provide a more physiologic assessment of coagulation and should be considered to guide blood transfusion requirements in liver transplantation [1A] and other major surgery [2B] and invasive procedures [2C]. Application of global viscoelastic testing with ACLF in the ICU setting requires more data [2C].p < 0.019) and higher HVPG was associated with increased risk of variceal hemorrhage and mortality at 30 (p < 0.02) and 90 days (p < 0.001) .5.4.2.2.NSBBs may reduce systemic inflammation and may have beneficial effects in ACLF patients over and above their portal hemodynamic effects .5.4.2.3.ACLF patients should be started or continued on NSBBs, if there are no contraindications .5.4.3.PICD incidence, presentation, diagnosis and management.5.4.3.1.PICD is a result of significant derangement of systemic and splanchnic hemodynamics with a decrease in effective arterial blood volume, which is triggered by large volume paracentesis (> 5 L) .5.4.3.2.PICD occurs in about 80% of ACLF patients when a large volume paracentesis is performed without additional therapeutic management. However, the incidence is reduced when volume expansion with albumin is used .5.4.3.3.Terlipressin, a vasopressin analog, is partially effective and has been shown to have a synergistic effect with albumin in preventing PICD .5.4.3.4.Non-selective β-blockers, such as propranolol, have been suggested to increase the risk of PICD and mortality in cirrhotic patients with refractory ascites. There is lack of data in patients with ACLF .The disease severity assessment is needed for prognostication and to guide the therapy –146. Disp < 0.001). In large UK and US cohort of severe autoimmune hepatitis showed that not the baseline MELD/UKELD but the use of corticosteroid and no improvement in MELD/UKELD scores within 7 days had a poor outcome and suggested early consideration of other strategies including liver transplant .6.1.2.The cumulative mortality increases with rise in AARC score in the first week in Grade I (5–7), II (8–10) and III (11–15), respectively .6.1.3.Treatment recommendations for ACLF should be based on AARC score. A trend of AARC score within first week can predict the need of liver transplant. Score of  < 10 at presentation or decrease in score below 10 by the end of first week is associated with higher chance of survival .6.1.4.Patients with AARC Score > 10 should be listed for LT. Transplant evaluation based on the AARC score needs prospective studies .6.1.5.AARC-ACLF score be estimated at admission in patients diagnosed to have ACLF as per AARC-ACLF definition .6.1.6.AARC-ACLF score should also be estimated on Day 4 and Day 7 to predict the course of illness and prognosis .6.1.7.The TPPM model has a superior predictive value for HBV ACLF outcomes than MELD and CLIFF-SOFA models .6.1.8.AARC score holds good in predicting the outcome in critically ill ACLF patients ).6.1.9.Lactate should be used for defining the severity of the ACLF syndrome. Lactate clearance compared to baseline lactate is a better indicator of patient status .6.1.10.There is limited role of Renal Replacement Therapy (RRT) & Extra Corporeal Liver Support System (ECLS) to treat hyperlactetemia .6.2.Organ dysfunction and organ failure in ACLF for early risk stratification.6.2.1.Organ dysfunction and failure should not be used in the definition, but for prognostication in patients with ACLF .6.2.2.Liver failure should be defined by a combination of bilirubin and INR and should be classified as mild/moderate and severe based on AARC as all patients have liver failure .6.2.3.Liver, kidney and brain remain organs of utility in patients with ACLF .6.2.4.Organ dysfunction and failure for brain should be based on AARC score .6.2.4.Respiratory and circulatory failure may be considered as organs of futility in patients with ACLF which may contraindicate liver transplant .6.2.5.For the diagnosis of organ dysfunction for kidneys in patients with ACLF as for patients with cirrhosis, AKIN criteria should be used .6.2.6.Kidney failure should be defined based on serum creatinine above 1.5 mg/dl as per the AARC score .6.2.7.The mortality in ACLF increases with the number of organ failures .The presence of high HBV DNA [> 10(5) copies/ml/or > 2 × 10 (4) IU/ml] is highly sensitive and specific for the diagnosis . Early aBesides patients who present with ACLF, it is worthwhile that prophylactic therapy should be considered for HBsAg-positive patients undergoing chemotherapy . There aRecommendations7.1.Antiviral strategies in ACLF HBV reactivation.7.1.1Nucleos(t)ide analogs should be started immediately in all HBV-infected patients at presentation while waiting for confirmation by HBV DNA level. Potent antiviral drugs, such as tenofovir, tenofovir alafenamide or entecavir, should be used .7.1.2.Assessment of reduction of HBV DNA level at day 15 after nucleos(t)ide analogs is encouraged; if < 2 log reduction, it suggests poor prognosis ..A characteristic feature of ACLF is its rapid progression, the requirement for multiple organ support and a high incidence of short- and medium-term mortality of 50–90%. The 28-day mortality rate was 15 times higher in patients with ACLF as compared to other chronic liver disease (CLD) patients , 158. PaMost important aspect in managing this group of sick patients is to decide the time frame and an algorithm. If the patient is too sick from the beginning and needs early LT without waiting for spontaneous recovery, this should be decided soon enough. On the other hand, patients who are salvageable and need time for recovery should be monitored closely in a time frame before deterioration so that they either recover or can undergo LT at an appropriate time. The third group is of those who need bridge therapy observed while on supportive care and bridging therapy, to define non-response and emergency LT or for transplant-free survival.Every ACLF patient at admission needs to be assessed for disease severity score, presence of SIRS with or without sepsis, HE and number of organ dysfunction/failure. The baseline MELD > 28, AARC Score > 10, advanced HE in the absence of overt sepsis or multi-organ failure can be considered for early LT . In the P/F ratio < 150] or HE requiring ventilator support > 72 h .7.2.2.LT should be offered early in the course of ACLF. When indicated, early liver transplantation avoids complications of sepsis and multi-organ failure .7.2.3.SIRS, sepsis, HE, liver failure, extrahepatic organ dysfunction/organ failure are important prognostic factors .7.2.4.per se should not be a contraindication for liver transplantation, except if cardiac or pulmonary support is needed or there is rapidly progressive organ failure at day 4 or 7 .Organ failure 7.2.5.APASL AARC model seems to be better in patient selection for liver transplantation as it enrolls patients early enough, before organ failure. However, the AARC score needs to be validated in large and varied populations and also its capability to predict post LT outcome .7.2.6.9/L .Patients with HBV reactivation with intermediate MELD should be assessed for early transplant if cirrhosis, bilirubin > 10 mg/dL, PT < 40% and platelet < 100 × 107.2.7.Steroid ineligible patients with severe alcoholic hepatitis should be listed on priority for liver transplant. Selective use of liver transplantation can be lifesaving for medically refractory alcoholic hepatitis .7.2.8.Liver transplantation should be reserved for severe alcoholic hepatitis patients with low risk of recidivism as assessed by a formal psychosocial evaluation .7.2.9.Transplant evaluation can be started based on liver specific dynamic scores by the end of first week of medical treatment or earlier .7.2.10.Allocation of cadaveric livers should depend on maturity of cadaveric program in the country .7.2.11.Patients with advanced ACLF also have good outcome after LT .The hepatocellular injury in ACLF is driven to a large extent by a “cytokine burst”, with elevated levels of multitude of cytokines, small molecular weight toxins, and vasoactive substances which are known to accumulate secondary to the failing liver . There iExtracorporeal liver support therapies are used to bridge the liver until recovery or liver transplantation in patients with ALF and ACLF. Various randomized controlled trials in patients with ACLF have shown improvement in HE, hepatorenal syndrome, circulatory dysfunction and immune dysfunction without improvement in transplant-free survival –182. In In a large proportion of patients with ACLF, however, liver transplant is not feasible, due to lack of an organ, a donor, severity of the illness or other social challenges. There are few alternatives at present to liver transplant. There have been promising results of the use of growth factors in such patients. Garg et al.  have shoHowever, despite the encouraging results and two randomized controlled clinical trials, it was felt that the use of these agents should be undertaken only under protocols and more data are required before recommending routine use of these agents.Recommendations7.3.1.Plasma exchange appears to be a promising and effective bridging therapy in patients with ACLF to liver transplant or spontaneous regeneration 7.3.2.Plasma exchange can be safely undertaken in patients with ACLF in specialized liver units .7.3.3.Plasmapheresis may be considered as a specific therapy for patients with Wilson’s disease and patients with severe flare of autoimmune liver disease (deemed unsuitable for steroids) .7.3.4.Combination of PE with therapies to potentiate liver regeneration should be evaluated in patients with ACLF .An extensive PubMed search using keywords ‘Acute-on-chronic liver failure in children; ACLF in children; Pediatric acute-on-chronic liver failure; Pediatric ACLF’, returned only 5 studies, from 3 Indian centers –199. TheOnly 8.7% of the pediatric ACLF cohorts were ≤ 3 years. Metabolic liver diseases (MLD) account for majority of CLD in this age group. On analysis of MLD data, it was found that only 3/63 (4.8%) could fulfill the definition of ACLF but were labeled as ALF as there is some overlap in the 2 definitions , 199. ChThere is no separate definition of pediatric ACLF. The major issues with the current definition in children are: 1) clinical identification of hepatic encephalopathy is often difficult and/or delayed specially those below 3 years of age, (2) clinical ascites may be difficult to identify especially in younger children with organomegaly, (3) some pediatric liver disease may present with liver failure without jaundice. The current ACLF definition requires jaundice to be followed by clinical ascites or HE within 4 weeks  clinical. Pediatrp < 0.0005) [Theoretically, pediatric ACLF should have a better outcome than adults as the 2 commonest CLD, i.e., Wilson disease and autoimmune liver disease presenting as pediatric ACLF have specific medical therapy, better hepatic reserve (due to shorter duration of illness) and lesser incidence of co-morbidities. Outcome has been defined at different time points in the published pediatric literature –204. WilThe proposal for a definition of ACLF in children by the APASL is the first such attempt to address the issue of ACLF in children. Hope this will be enriched by further large and multicentric studies in future.Recommendations8.0.Acute-on-chronic liver failure in children.8.1.Pediatric ACLF is not uncommon .8.2.The most common underlying liver diseases presenting as ACLF in children are Wilson disease and autoimmune liver disease .8.3.The most common acute precipitating events are viral  hepatitis and flare of underlying disease/hepatotoxic drugs .8.4.Standardized definitions of disease flare, as a precipitating event need to be further updated .8.5.The existing definition of ACLF can be used to diagnose ACLF in children. However, there is an urgent need to generate more multicentric data from prospective studies.8.6.Clinical diagnosis of hepatic encephalopathy, though difficult, is important for diagnosis/prognosis of pediatric ACLF. For diagnosis of HE in older children, West Haven scale can be used. For children less than 3 years, modified HE assessment scale can be used .8.7.Clinical and/or radiological ascites can be used for defining ACLF in children .8.8.Short-term (28-day) outcome is poor in approximately 33% of pediatric ACLF subjects .8.9.One or more extrahepatic organ failures are seen in around half of children with ACLF .8.10.Acute kidney injury and grade 3-4 HE are most common extrahepatic organ failures in pediatric ACLF.8.11.Half the cases of ACLF at presentation have AKI. The presence of AKI increases the risk of poor outcome by several folds .8.12.AARC-ACLF model is an easy, bedside, dynamic prognostic model for pediatric ACLF .8.13.A score more than or equal to 11 needs urgent listing and evaluation for liver transplant .8.14.Pediatric modification of these scores may be useful .In summary, the field of ACLF has moved very rapidly in the past 5 years. The availability of large volume of published data from the East and the West has allowed reassessing the initial definitions. The need for having a well-defined homogenous population of patients, with wellcharacterized acute and chronic insult and which would reflect the term acute-on-chronic liver failure, is at the core. Attempts to abrogate, ameliorate or reverse the ongoing injury would allow return of hepatic synthetic functions and reversal of the liver damage. Early predictors of mortality and non-reversibility of the condition would pave way to offer priority liver transplantation to such patients. An attempt to converge the thoughts from the East and West is possibly the only way forward to achieve more scientific and timely interventions for such seriously sick patients.


The posterior tibial nerve was stimulated to obtain the maximal double-twitch force of the calf muscles with (59.4 and 108.4 N) and without multidirectional transverse load. Compared to the unloaded condition, the rate of force development (RFD) was reduced by 5.0 ± 8.1% (p = 0.048) and 6.9 ± 10.7% (p = 0.008) for the 59.4 and 108.4 N load, respectively. No significant reduction  in maximum muscle force (mF) was found for the lower load (59.4 N), but application of the higher load (108.4 N) resulted in a significant reduction of mF by 4.8 ± 7.0% (p = 0.008). Mean pressures induced in this study  are within the pressure range reported for compression garments. Taking the results of the present study into account, a reduction in maximum muscle force would be expected for compression garments with pressures ≥26.3 mm Hg. However, it should be noted that the loading condition (sling vs. compression garment) differs and that compression garments may influence other mechanisms contributing to force generation. For example, wearing compression garments may enhance sport performance by enhanced proprioception and reduced muscle oscillation. Thus, superposition of several effects should be considered when analyzing the impact of compression garments on more complex sport performance.It has been demonstrated that unidirectional transversal muscle loading induced by a plunger influences muscle shape and reduces muscle force. The interaction between muscle and transversal forces may depend on specific neuromuscular properties that change during a lifetime. Compression garments, applying forces from all directions in the transverse plane, are widely used in sports for example to improve performance. Differences in the loading direction  may have an impact on force generating capacity of muscle and, thus, on muscle performance. The aim of this study was to examine the effect of multidirectional transversal loads, using a sling looped around the calf, on the isometric force during plantarflexions. Young male adults (25.7 ± 1.5 years,  These neighboring tissues may transfer forces to the muscle, modifying muscle architecture  direction. Examination of the interaction of muscles with external forces is important for a better understanding of movement generation and control. Myofascial force transmission via fascial connections between neighboring muscles has been shown to influence longitudinal muscle force  induced by a plunger reduced muscle work and force (up to 15%) performed in the longitudinal direction as well as the rate of force development (RFD) (up to 35%). Unidirectional transverse muscle loading may also be experienced during sitting. In contrast, compression garments  induce external forces that act on the muscle from all directions in the transverse plane . These different loading situations  lead to different passive muscle deformations and may subsequently lead to different contraction dynamics.Studies on isolated rat muscles ,b, 2016 Experimental studies examining the impact of multidirectional transverse loading on muscle force and deformation are rare. RFD in young adults. This was achieved by a sling that was looped around the human calf and loaded with two different weights (59.4 and 108.4 N) during electrically induced plantarflexions. It was hypothesized that multidirectional transversal muscle loading leads to a reduction in longitudinal muscle force.Muscle deformations during contraction are strongly influenced by muscle architecture as well as active and passive muscle properties, which change dramatically during aging . Thus, mFifteen male young adults  participated in this study. All subjects were informed about the risks of the experiments and gave their written consent. The study protocol was approved by the ethical committee of the university hospital of Tuebingen and conducted according to the latest declaration of Helsinki.Figure 1). The lift height Δh of the load was calculated as the difference between its maximal height during contraction and its initial height in the passive state when the passive muscle was compressed. Work performed to lift the load liftW was calculated by multiplying Δh and the gravitational force .We measured the force generated by the calf muscles during a double-twitch stimulation applied to the posterior tibial nerve. The double-twitch force was used as it is highly reproducible  and commFigure 1). The left foot was placed and secured beside the force plate. The stimulation electrodes were attached on the skin and the subject was immobilized in this position  with shoulder barriers and with a hip belt. Subjects then performed a further warm-up consisting of 10 submaximal (increasing from about 30 to 90% maximum voluntary contraction) isometric plantarflexions. First stimulation was performed about 5 min after the warm up.At first the subjects performed a standardized warm-up consisting of 5 min of running on a treadmill (with 12 km/h), 3 sets of 10 calf raises , and 10 repetitive calf jumps . Afterward, the subjects were instructed to lie prone in a calf press apparatus with the right foot attached to a 3D force plate  covering the gastrocnemius muscle belly for the most part (about 70%). The longitudinal distance of the proximal border of the sling to the popliteal fossa (where the cathode was fixed) was about 7 cm. An aluminum box was hung on both ends of the sling for storage of the metal weights (5 or 10 kg). The draw wire sensor was connected via a hook to the bottom of the box. Due to the pulling force (3 N) of the draw wire sensor, and the deadweight of the aluminum box (0.5 kg) and sling (0.25 kg), loading of the box with 5 and 10 kg resulted in transverse loads of 59.4 and 108.4 N, respectively. We performed two series of loaded experiments  each consisting of 5 paired stimuli. In between the two series a rest time of 1 min was set without transverse muscle loading. Finally, five double-twitches every 10 s were performed without transverse muscle loading (post-test) to assess possible conditional changes during the experiment.The experiments started with five paired stimuli, applied to the posterior tibial nerve every 10 s at rest for the pretest baseline. Then, a leather sling  was looped around the calf at its most prominent bulge (mF) and the RFD using custom-made Matlab scripts . RFD was calculated as the force difference between 0.1 and 0.9 mF divided by the corresponding required time. The mean and standard deviation of a stimulation series  was assessed and used for further statistical analyses.A moving average filter (window length: 11 samples) was used for smoothing the force and kinematic data. The resultant force was calculated from the measured 3D force components for each double-twitch and used to quantify the maximal double-twitch force (t-test for dependent samples was performed to test for differences between 59.4 and 108.4 N load. One-way repeated measures ANOVA (rANOVA) were conducted for the force parameters m, RFD(F) to test for differences between conditions . The level of significance was set at p < 0.05. Significant main effects or interactions of the rANOVA were probed using Bonferroni post-hoc tests. Effect size was determined using partial eta squared (ηp2) and classified as follows: low ηp2 = 0.01, medium ηp2= 0.06, and large ηp2 = 0.14  and the mean of 5 unloaded reference contractions. The force-time curves indicate reduced RFD and reduced maximal force.Multidirectional transverse muscle loading affected the force generation during plantarflexion. RFD . The post hoc tests revealed a significant reduction of RFD by 5.0 ± 8.1% and 6.9 ± 10.7% between pretest and 59.4 N load and between pretest and 108.4 N load , respectively. There were no significant differences between pre- and post-test as well as between 59.4 and 108.4 N loading.The statistical analysis showed significant effects of transverse loading for mF. The post-hoc test revealed a significant reduction in mF by 4.8 ± 7.0% (p = 0.008) for the higher load (108.4 N). The reduction in mF of 3.2 ± 4.8% induced by the lower load (59.4 N) was not significant . Furthermore, there were no significant differences between the 59.4 and 108.4 N load (p = 0.141) as well as between pre- and post-test.The rAnova revealed significant effects of muscle loading for t-test for dependent samples showed no significant differences between the lift heights (p = 0.064). The work performed to lift the load was higher (p < 0.001) for the 108.4 N load (616 ± 213 mJ) compared to the 59.4 N load (273 ± 70 mJ).Due to problems with the data acquisition of the lift height for one subject, only data of 14 subjects have been analyzed. During plantarflexion contractions, the load was lifted by 4.6 ± 1.2 mm and by 5.6 ± 2.0 mm for the lower (59.4 N) and the higher (108.4 N) load, respectively. A RFD were decreased by 4.7% and 6.9% in the loaded (108.4 N) condition, respectively, compared to the unloaded condition. In general, our observations are consistent with experiments applying unidirectional transversal forces on isolated rat muscles  resulted in a more pronounced reduction of mF (4.8–12.8%) and RFD (20–35%)  . This miimF = 11.2 N; imF. Applying a similar normalization for the transversal forces in this study  used in rat experiments by the maximum isometric GM muscle force  compared to the unidirectional action of forces in z-direction in the plunger experiments. This impacts the muscle shape and architecture during passive muscle loading and changes at least the initial conditions of a contraction. Moreover, uni- and multidirectional transverse muscle loading may result in different deformations of the myofilament grid which might have impact on force generation capacity of cross bridges  plane  and it is unclear if these forces were transmitted to the underlying M. soleus (SOL) as the depth of compression was not ascertained in this study. Therefore, specific contributions of GAS and SOL to calf deformations (and thus lifting height of the load) and reduction in longitudinal muscle force are unknown. In principle, transmission of transversal forces between neighboring muscles is possible . However, more precise examinations of 3D muscle architectures of calf muscles and their deformations during contraction are required to gain a better understanding of 3D interactions of synergistic muscles with each other as well as with external transverse forces.Comparison of results of unidirectional and multidirectional transverse muscle loading is even more difficult as different specimens  have been observed. For a single muscle, the reduction in muscle force can be mainly described by a simple model approach using a lever to convert transverse force and length change  into longitudinal force and length , 2018. Ipossible  allowingn = 4) of leopard frogs. The authors constructed small rigid plastic tubes, fitted to the diameter of the muscle, and placed them around the muscle. Small reductions of about 5% in maximum isometric force were reported, but were not significant (from 1.66 ± 0.36 N to 1.58 ± 0.30 N). Furthermore, the amount of muscle shortening and work output was reduced significantly during isotonic contractions against 25% imF in the constraint condition. There are only two studies that have applied multidirectional forces in the transverse plane to the muscle, but with deviating experimental approaches. Measured mean forces of contractions without transverse muscle loading evoked by electrical stimulation are 165 ± 30 N. Considering a gearing of 2.8  with a mean value of 19.9 mm Hg (0.26 N/cm2). These pressures are within the range of mean pressures (transverse load divided by contact area between muscle and sling) of 0.19 ± 0.02 and 0.35 ± 0.03 N/cm2 induced in the present study with loads of 59.4 and 108.4 N, respectively. Taking these results into account, pressure induced by compression garments might be in a range where no effect on muscle force can be expected . Since we found a significant decrease in muscle force of 4.8% for the higher transverse load , a decrease in muscle force might be expected for compression garments that induce a higher pressure (≥0.35 N/cm2∼26.3 mm Hg). However, results concerning the impact of compression garments on acute sport performance are controversial, as stated above. Regarding this, it should be noted that compression garments may influence other mechanisms contributing to force generation (which might be negligible in the current study) and counteract a potential reduction in muscle force induced by muscle compression. Wearing compression garments may enhance sport performance through enhanced proprioception  on muscle performance. For example, compression garments are increasingly used by elite and recreational athletes, as they are thought to enhance performance. However, the results concerning increased acute sport performance are controversial . Kraemeroception , increasoception , and redoception  during roception . FurtherIn addition to compression garments, slings, or elastic bandages, increased stiffness of the ECM restricts muscle deformation. The mechanical properties of the ECM change with age, exercise, or illness . AlnaqeeTS, NS, JW, and DR conceived and designed the experiments. ME performed the experiments. TS, NS, and ME analyzed the data. TS and DR prepared the figures. All authors interpreted the results, edited, revised, and drafted the manuscript, and approved the final version of manuscript.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.


In this study, an RIL  population derived from the cross of Zhonghuang 24 (ZH 24) and Huaxia 3 (HX 3) (160 lines) was tested using hydroponic cultivation. Relative root elongation (RRE) and apical Al3+ content (AAC) were evaluated for each line, and a significant negative correlation was detected between the two indicators. Based on a high-density genetic linkage map, the phenotypic data were used to identify quantitative trait loci (QTLs) associated with these traits. With composite interval mapping (CIM) of the linkage map, five QTLs that explained 39.65% of RRE and AAC variation were detected on chromosomes (Chrs) Gm04, Gm16, Gm17 and Gm19. Two new QTLs, qRRE_04 and qAAC_04, were located on the same region of bin93-bin94 on Chr Gm04, which explained 7.09% and 8.98% phenotypic variation, respectively. Furthermore, the results of the expression analysis of candidate genes in the five genetic regions of the QTLs showed that six genes  exhibited significant differential expression between the Al3+ treatment and the control of two parents. The results of qRT-PCR analysis indicated that Glyma.04g218700 was upregulated by Al3+ treatment with the hundreds-fold increased expression level and may be a candidate gene with potential roles in the response to aluminum stress. Therefore, our efforts will enable future functional analysis of candidate genes and will contribute to the strategies for improvement of aluminum tolerance in soybean.Aluminum (Al Thus, 25 μΜ AlCl3 and 24 h of treatment were selected to obtain the widest separation in the RIL population.To explore the appropriate AlClve lines . Moreove12 RILs with a mean of 71.26 ± 16.92% and CV of 23.74%  (The ANOVA results demonstrated significant phenotypic differences among the RILs in RRE as well as AAC P<0.01), but no significant differences among the three replications. The RRE results for all the lines showed a continuous distribution ranging from 34.78 to 103.60% among all the 160 F, but no % Tables . The ave% Tables . In addi% Tables . The lin< 0.001) .12 lines under Al3+ stress  explained by 7.09%-8.52% and LOD values ranging from 2.73 to 3.32  . Three Q to 3.32 . Two QTLectively . The com92.30 cM , which ihttps://www.soybase.org/) was used to investigate effective candidate genes associated with aluminum tolerance. The analysis showed that 66 annotated genes were mapped on the regions of the five QTLs . A total of 54 genes were detected in Gm04_bin93-bin94, from 45290936 bp to 46017212 bp. There were 6, 2 and 4 annotated genes in the three short intervals on Chr. Gm16 (Gm16_bin8), Gm17 (Gm17_bin93) and Gm19 (Gm19_bin19), respectively. To analyze the functional annotation of each gene, the AgriGO toolkit (http://bioinfo.cau.edu.cn/agriGO/index.php) was used to perform gene ontology (GO) analysis [).The Soybase database , hematoxylin staining and root dry weight have been widely used as indicators for the assessment of Alultivars –62. Heman barley , which i160 RILs . Indeed,chanisms . Previouenotypes , and simenotypes , 64. Undchanisms . The sigus lines , which mMoreover, the estimated heritability of RRE was 92.59% indicating the trait tolerant to Al stress using RRE index had high selection efficiency. The heritability of AAC was 64.90% lower than that of RRE, which might be related to the effect of genotype-environment interactions.3+ tolerance using different mapping populations. The two populations named Young × PI 416937 and Kefeng No. 1 × Nannong 1138–2 are favored by researchers. Various QTLs associated with Al3+ resistance were identified by improving the genetic linkage map established by RFLP and SSR markers using the same RIL populations [12 generations with diversified segregation, and there were considerable differences between the two parents in terms of Al3+ tolerance traits. Moreover, high-density genetic maps have been constructed using the RAD-seq technology with SNP markers and have been applied to multiple traits [A practical way to study the genetics of quantitative traits is to construct a genetic linkage map and map QTLs with segregation of populations based on those traits. Several previous studies have detected QTLs for Alulations , 34, 66.e traits , 36. The3+ resistance in soybean. As a result, a total of five Al3+ tolerance QTLs explaining 39.65% of the total variation were mapped on four chromosomes with narrow intervals (qRRE_17) is close to the SSR marker Satt186 in the genome of Williams 82 version 1.01, which was associated with Al3+ tolerance in previous studies [qRRE_04, qRRE_16, qAAC_04, qAAC_19) are novel loci. Bianchihall et al. [et al. [3+ tolerance on Chr. Gm16 and Gm19, which could not be detected here  and Glyma.04g216100 (Trihelix), encoding two transcription factors, were also reported to respond to multiple forms of abiotic stress [Glyma.04g220600, was recorded to encode a peroxidase (POD) activated by reactive oxygen species (ROS), which may contribute to Al3+ resistance by inducing the production of ROS in plant roots [Glyma.04g212800 gene upregulated by aluminum stress encodes a GDP-mannose transporter with an ambiguous relationship between GDP-mannose transport and aluminum stress. Thus, these 6 genes were identified as candidate genes for aluminum stress tolerance based on the potential response to other forms of abiotic stress  were mapped on four chromosomes  with comprehensive genetic effects of 39.65%. The QTLs qRRE_04 and qAAC_04 could be detected by the same markers in a genetic region between 90.50 and 92.30 cM, indicating a new QTL on Chr.04 for the Al3+ tolerance trait of soybean. In addition, 66 annotated genes were predicted in the regions of the five QTLs, with six genes showing significantly different expression after Al3+ exposure by qRT-PCR. Glyma.04g218700upregulated by Al3+ treatment with the highest expression level may be a candidate gene with potential roles in the response to aluminum stress. Therefore, our efforts will enable future functional analysis of candidate genes and will contribute to the strategies for improvement of aluminum tolerance in soybean.In summary, an RIL population derived from ZH 24 × HX 3 was used to investigate the quantitative inheritance of RRE and AAC for AlS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig3+ treatment. There were 15 differentially expressed genes between the conditions with and without Al3+ treatment, with the soybean gene β-Tubulin as the internal reference.RT-PCR analysis was carried out using the two parents ZH 24 or HX 3 under the conditions with or without Al(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 TableA total of 53 out of 66 predicted genes could be amplified successfully, and the other 13 predicted genes could not be amplified, had very short sequences or were not true genes.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


For patients with recurrent flares of gout, tophi, urate crystal arthropathy, and renal stones, urate-lowering therapies  are the first-line treatment. Due to the widespread use of these ULTs , assessment of the associated renal risk is essential. Accordingly, we performed a disproportionality analysis of reported cases of acute renal failure (ARF) associated with allopurinol and febuxostat.We carried out a case/non-case study of the World Health Organization’s VigiBase® pharmacovigilance database between January 1, 2008, and December 31, 2018. The frequency of reports of ARF as a standardized Medical Dictionary for Regulatory Activities query for allopurinol and febuxostat was compared with that of all other reports for the two drugs and quoted as the reporting odds ratio (ROR) [95% confidence interval (CI)]. The results’ stability was assessed in a series of sensitivity analyses (notably after the exclusion of putative competing drugs).Among 3509 “suspected drug” notifications for febuxostat and 18,730 for allopurinol, we identified respectively 317 and 1008 cases of ARF. Acute renal failure was reported significantly more frequently for febuxostat and allopurinol than for other drugs . For both drugs, the ROR was higher in women than in men, respectively 11.60 [9.74–13.82] vs. 3.14 [2.69–3.67] for febuxostat and 4.45 [4.04–4.91] vs. 2.29 [2.11–2.50] for allopurinol. The sensitivity analyses confirmed the disproportionality for these two ULTs.Acute renal failure was reported respectively 5.7 and 3.3 times more frequently for febuxostat and for allopurinol than for other drugs. Due to the potential consequences of ARF, physicians should take account of this disproportionality signal when prescribing the ULTs febuxostat and allopurinol. Gout is the most common type of inflammatory arthritis. Although the reported prevalence of gout varies greatly from one country to another ~ 0.1 to ~ 10%), a value above 1% is found in North America, Europe, and most developed countries  to ~ 10%. In viewThe oldest and most frequently prescribed ULT is allopurinol, which was first marketed in 1966. The second most frequently prescribed ULT is febuxostat, which has been authorized in France since 2008. In both cases, the mechanism of action is based on xanthine oxidase (XO) inhibition  for febuxostat and allopurinol vs. all other drugs  for febuxostat (authorized in 2008), we also performed a ROR by year of notification from 2011 onwards , 29. We Of the 9,066,403 ICSRs in adult patients of known age and gender reported to VigiBase® between January 1, 2008, and December 31, 2018, 3509 concerned febuxostat and 18,730 concerned allopurinol. Respectively 317 and 1008 ICSRs for febuxostat and allopurinol matched the SMQ for ARF . The cases predominantly occurred in men . The mean ± standard deviation age was 68.0 ± 15.0 for cases of ARF linked to febuxostat and 67.0 ± 14.5 for those linked to allopurinol. With regard to the outcome, the mortality rate was 6.9% and 9.6% for cases reported for febuxostat and allopurinol, respectively. Most of the notified cases of ARF came from Europe and North America for febuxostat and from Europe for allopurinol.Febuxostat was the sole “suspected drug” in 58.0% of the ICSRs with ARF in febuxostat-treated patients. The corresponding value for allopurinol was 44.3%. In the other ICSRs, the other “suspected drugs” in febuxostat-treated patients were anti-anemics or antihemorrhagics (11.4%), diuretics (10.3%), lipid-lowering agents (6.9%), immunosuppressive therapies (6.6%), calcium channel blockers (5.9%), antigout drugs (5.5%), and NSAIDs (3.0%) Table . The othThe RORs [95%CI] for ARF due to febuxostat and allopurinol were respectively 5.67 [5.05–6.36] and 3.25 [3.05–3.47] . The ROR was even greater when we excluded ICSRs involving the drugs mostly associated with ARF; the values were 6.34 [5.65–7.11] for febuxostat and 3.64 [3.42–3.88] for allopurinol (Table The ROR was 26.93 [25.49–28.44] for the positive control (ibuprofen) and 0.40 [0.34–0.47] for the negative control .In the present study, we detected a disproportionality signal for ARF and the ULTs febuxostat and allopurinol. The presence of this signal was confirmed in a variety of sensitivity analyses .The purpose of studying the disproportionality of spontaneous ICSRs is to generate pharmacovigilance alerts concerning unknown or underestimated ADRs. Until the present case/non-case study, the risk of AKI associated with allopurinol and febuxostat had been underestimated. In fact, renal and urinary tract disorders were reported  during an extended phase III febuxostat clinical trial . HoweverThe main known risk factors for AKI are underlying kidney disease, diabetes, cardiovascular disease, and acute situations such as dehydration . AdverseA significant ROR was found for each subgroup studied in our secondary analyses. Women had a higher ROR than men—especially for febuxostat. A number of recent literature results are controversial; although most clinical studies predominantly include male patients –47, recePatients with gout are often treated with drugs known to induce AKI, such as diuretics, ACEIs, angiotensin II receptor antagonists, and NSAIDs. To assess the stability of our results, we performed a sensitivity analysis in which these drugs were excluded. The RORs were still significant for both drugs and were even higher than in the primary analysis.Our study suffers from the inherent limitations of all pharmacovigilance studies , 31 and In contrast, our study has several important strengths linked to its case/non-case design , 31. FirWe reported that ARF is respectively 5.7 and 3.3 more frequently reported with febuxostat and allopurinol than with other drugs. Due to the potential consequences of AKI in terms of mortality and renal impairment , 54, phyAdditional file 1: Table S1. MedDRA terms included in the standardized MedDRA query “acute renal failure”. Table S2. Calculation of the ROR. Table S3. Reporting odds ratios for the risk of ARF induced by febuxostat and by allopurinol in the overall study population, by year.


Dogs are naturally exposed to numerous pathogenic serogroups. Leptospirosis vaccines are claimed to afford a clinical protection restricted to the serogroups of which they are composed.Dogs exhibiting liver and kidney injury were suspected of having leptospirosis. The purpose of this study was to compare the microscopic agglutination test (MAT) results in naive and vaccinated dogs experiencing leptospirosis outcomes. Only MAT-positive animals were included in the study.Over five years, 3 512 dogs were suspected of having leptospirosis. For each case, biochemical parameter results were recorded. Leptospirosis involvement was investigated by MAT performed against 6 major serogroups . MAT-positive results confirmed leptospirosis cases in 147 naïve dogs and in 580 fully vaccinated dogs. Serological titres of agglutinating antibodies were related to the severity of liver and kidney failure.The most prevalent outcome of leptospirosis in unvaccinated dogs was liver failure (57.8%) compared to 51.7% for kidney disease, but the most severe onset (90.8%) was found among the cases of acute kidney injury compared to the severe (42.3%) hepatitis cases. In dogs vaccinated by bivalent Icterohaemorrhagiae and Canicola bacterins, hepatitis decreased from 57.8 to 46.5% and acute kidney injury from 51.7 to 21.6%. The decrease was shown in leptospirosis cases induced by field strains belonging to the six most prevalent serogroups, including the 4 serogroups heterologous to the vaccine.Leptospira spp infection regardless of the MAT-prominent serogroup and limited the disease severity in the remaining cases.Common vaccination was efficient in decreasing hepatitis and kidney failure induced by field  The first step was to determine the liver and kidney injury patterns of each of the 6 major serogroups based on the MAT results of unvaccinated dogs. Therefore, comparison of the cases recorded in vaccinates and naïve cases allowed a global estimation of the serological and clinical impacts triggered by the usual whole cell bacterins.22.1A form was distributed among veterinary practitioners to collect clinical data of pets suspected of having leptospirosis.•Delay of sampling since the onset of the disease•Delay of sampling since the last leptospirosis vaccination .The template, necessary to confirm or reject a leptospirosis case by MAT results, was prepared by the laboratory:2.2•Liver injury (L): results of APL, ALT and biliary acids, icterus…•Acute kidney injury (AKI): blood urea (BUN) and creatinine…•Gastroenteritis, vomiting.•Other symptoms relevant to leptospirosis, including bleeding, pulmonary distress or reproductive disorders.Three grades of liver injury were defined by comparison with normal values  13, 14], 1413, 12.3cterohaemorrhagiae and Canicola), and Australis (AUS), Autumnalis (AUT), Grippotyphosa (GRIP) and Sejroë (SEJ). A panel of 16 serovars , routinely used for these 6 serogroups, were used in this study.As previously described , 5, the A titre of 1:40 was considered positive. Nevertheless, in vaccinated animals, Icterohaemorrhagiae and Canicola reciprocal titres from 40 to 320 were considered as post-vaccination titres. The prominent serogroup was designed by the serovar exhibiting the highest titre, regardless of the positive serovar.2.4The completion of the data sheet was necessary for any case confirmation.(1)Vaccination status (vaccinated for leptospirosis or not);(2)Time lapse between the vaccination and the sampling (less or more than 6 months prior);(3)Delay between the sampling time and the onset of the first clinical outcome (less or more than 10 days prior).(4)MAT Icterohaemorrhagiae and Canicola reciprocal titres ≥40 in unvaccinated and ≥320 in vaccinated,).(5)MAT titres for other serogroups ≥40.A leptospirosis case was first excluded if the data related to the 5 selected biochemical and epidemiological factors were not relevant to clinical leptospirosis, and in a second step if MAT results were negative. Confirmation was serologically assessed by positive MAT results according to the algorithm previously defined 2.5The data forms and MAT results were stored by Access 2000 software.1-x2).The results were treated by Pearson's chi-squared tests. Results were significant for P ≤ 0.05. Confidence intervals were expressed for P = 0.05 . Sera samples provided by 580 vaccinates exhibited 1 506 positive results (mean = 2.60). However, when positive results for Icterohaemorrhagiae and Canicola were retrieved because they could be attributed to the previous vaccination performed in these animals, this mean dropped to 1.09.The seroprevalence of each serogroup was estimated by using the threshold dilution of 1:40, but has to be evaluated according to these numerous responses.3.2.1In the group of unvaccinated dogs, the highest seroprevalence of 77.6% (CI 95% = 70.9–84.3) was shown by Icterohaemorrhagiae , followeThe natural incidence of each serogroup could be roughly estimated by the highest reciprocal titres (≥320). The highest seroincidence  among th−3), to Sejroë (P = 8.10−5), Grippotyphosa (P = 1.10−11), Autumnalis (P = 3.10−12) and Canicola (P = 8.10−13).Icterohaemorrhagiae infection was the most frequent compared to Australis  for Icterohaemorrhagiae and 61.89% (CI 95% = 57.9–65.9) for Canicola. The 4 other serogroup seroprevalences were 35.9% for Sejroë (CI 95% = 32.0–39.8), 30.0% for Australis (CI 95% = 26.3–33.7), 22.9% for Grippotyphosa (CI 95% = 19.5–26.3) and 20.3% for Autumnalis (CI 95% = 17.0–23.6).Leptospira vaccination.Similarly, incidence estimated by reciprocal high titres had to be studied separately from the other serogroups for Icterohaemorrhagiae and Canicola (≥320), which could be related to the previous 3.2.2.1High titres for Icterohaemorrhagiae and Canicola were found in 58.4% and 12.6% of the dogs, respectively. However, the titres against Icterohaemorrhagiae and Canicola decreased with the time elapsed since the last vaccination . They de3.2.2.2Conversely to the low titres, high titres against the other serogroups could not have been elicited by a previous vaccination and therefore expressed the incidence. For Australis, high titres were shown in 17.9% of cases (CI 95% = 15.1–20.7), followed by 11.2% for Sejroë (CI 95% = 9.1–13.8) and Grippotyphosa and Autumnalis with 6.9% (CI 95% = 4.8–8.4) and 6.9% (CI 95% = 4.5–8.1), respectively .Fig. 4Hi3.3Among the 147 leptospirosis cases of unvaccinated dogs, liver damage (L) was recorded in 85 dogs  and acute kidney injury (AKI) in 76 dogs . Both symptoms were recorded simultaneously in 14 dogs.Severe hepatitis (42% of hepatic cases) was shown by 24.5% of the dogs (36/147). Severe kidney damage (90% of AKI cases) was shown by 46.9% of the 147 dogs. In naïve dogs, high titres against Icterohaemorrhagiae were related to 16 out of the 36 severe hepatitis cases, whereas high titres to Canicola were only related to 3 severe AKI cases. Whatever the other focused serogroup, high titres were mostly related to severe AKI cases. Other detected clinical aspects of leptospirosis were generally associated with one or two of these symptoms. The ratio of severe cases of liver injury and AKI was dependent from the serogroup . All theAmong the 580 confirmed cases in fully vaccinated dogs (V+), liver injury was recorded in 281 dogs  and AKI in 148 dogs . The remaining dogs exhibited a broad panel of symptoms, including fever, lameness, respiratory distress, gastroenteritis and bleeding.The third grade of liver damage (54% among the hepatitis cases) was recorded in 151/580 dogs  and grades III to V kidney damage (78% among the AKI) were recorded in 116/580 dogs . Among the vaccinated dogs, 84 exhibiting the third grade of liver damage (14.5%) showed prominently high Icterohaemorrhagiae titres and 21 (3.6%) showed high Canicola titres. Icterohaemorrhagiae-related severe kidney damage was seen in 71 dogs (12.2%) and in 8 (1.4%) cases related to Canicola.4Leptospira species throughout France Several limits of this study performed in privately owned pets exposed to field strains of pathogenic Clinical picture was the first selection filter. The clinical recording of cases in vaccinates and naïve dogs was implemented by the same practitioner population. To minimise recording bias, this study only focused on the easy to quantify biochemical parameters of the well-known issues among various aspects of polysystemic leptospirosis , 21, 22.Our purpose was to estimate the global protection elicited by bacterins in vaccinates exposed to the most pathogenic serogroups encountered in France highlighted by MAT results of sick unvaccinated pets . The MATSera provided by unvaccinated dogs gave positive results to a mean of 2.36 different serogroups, highlighting the high level of cross-reactivity within the 6 field serogroups. As expected vaccinated dog samples exhibited a higher mean of seroreactive serogroups (2.60). New antibodies raised towards the infectious strain are added to the post-vaccine agglutinating antibodies (Icterohaemorrhagiae and Canicola). They increased the cross-agglutinations Icterohaemorrhagiae remained the major infectious serogroup as shown by the highest incidence in naïve dogs (44.2%), whereas Canicola appeared as the least infectious with 3.4%. Similar lower exposure (approximately minus 40%) was confirmed in vaccinates even if the previous vaccination allowed serological result increase (Icterohaemorrhagiae incidence 58% and Canicola 12%). These results are in accordance with previous data , 29, 30.−5). Moreover, the severe form of AKI  significantly dropped in vaccinates from 46.9 to 20.0% (P = 6.10−6). The specificity of that protection was questionable in naturally exposed vaccinates [Protection triggered by bacterins is claimed to be serogroup-specific , 7, 8. Tccinates , 32. It It was hypothesised that natural exposure of vaccinates could not be higher than that of naïve dogs. However, high titres against Autumnalis and Grippotyphosa increased in vaccinated dogs compared to naïve dogs . It has Conversely, severe liver and kidney damage cases related to Australis and Sejroë high titres decreased . TherefoL. biflexa is unable to afford any protection [Leptospira spp share many other common and specific antigens Leptospira spp remains to be identified It is well known that the widely cross-agglutinating saprophytic otection , 10, 34,Leptospira spp regardless of their composition.In conclusion, whole cell bacterins afford a significant protection against field strains belonging to homologous serogroups as well as heterologous pathogenic Geneviève André-Fontaine: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.Laetitia Triger: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare the following conflict of interests: Genevieve Andre-Fontaine is an inventor holding patents related to leptospirosis. The founding sponsors had no role in the collection, analysis or data interpretation, in writing the manuscript or in the decision to publish the study. The other author declares no conflict of interest.No additional information is available for this paper.