Fecal Microbiota Transplantation in Alcohol-Associated Acute on Chronic Liver Failure: An Open-label Clinical Trial


 Background: Severe alcoholic hepatitis (SAH) presenting as acute-on-chronic liver failure (ACLF) carries a high short-term mortality. Alteration of gut microbiota is a crucial component implicated in its pathogenesis, whose modulation has been suggested as a potential therapeutic tool. We evaluated the safety of fecal microbiota transplantation (FMT) and its efficacy in improving short-term survival and clinical severity scores in patients with SAH-ACLF.Methods: Thirty-three patients [13 in the FMT arm;20 in the standard of care arm (SOC] with SAH-ACLF were included in this open-label study. A single FMT session was administered as a freshly prepared stool suspension from pre-identified healthy family member stool donors through a nasojejunal tube. Patients were followed up on days seven, twenty-eight, and ninety. Results: Survival at twenty-eight and ninety days was significantly better in the FMT arm (100% versus 60%, P=0.01; 53.84% versus 25%, P=0.02). Hepatic encephalopathy resolved in 100% versus 57.14% (FMT versus SOC, P=0.11) patients, while ascites resolved in 100% versus 40% survivors (P=0.04). Major adverse event rates, including spontaneous bacterial peritonitis and gastrointestinal bleeding, were similar in both groups (P=0.77; P=0.70). Median IL1beta decreased by21.39% (IQR -73.67-7.63) in the FMT group, whereas it increased in the SOC by 27.44% (IQR -0.88-128.11) (P=0.01). Percentage changes in bilirubin and ALT between baseline and day seven emerged as predictors of ninety-day mortality.Conclusion: FMT is safe, improves short-term and medium-term survival, and leads to improvement in clinical severity scores in patients with SAH-ACLF.

(PAMPs), which bind to hepatic pattern recognition receptors (PRRs), and activates the immune cascade, leading to hepatic in ammation and injury. [9][10][11] Modulation of gut ora emerges as a prudent therapeutic strategy, and fecal microbiota transplantation (FMT) appears promising in this regard based on initial work. [12][13][14][15] Hence, in this open-label controlled study, we aimed to determine the safety and e cacy of FMT in improving survival and liver function and mitigating systemic in ammation in patients with SAH presenting as ACLF.

Materials And Methods:
Study Conduct: The study was an investigator-initiated open-label non-randomized trial conducted at a tertiary care referral hospital with a specialized liver unit carried out between January 2019 to December 2019. The study protocol conformed to the 1975 Declaration of Helsinki's ethical guidelines as re ected in a priori approval by the Institutional Ethics Committee (Reference number NK/4720/MD/779). Written informed consent was obtained from each patient or close relative and FMT donor prior to inclusion. All patients were allocated into either the FMT or standard medical therapy (SOC) groups.

Patients:
The trial recruitment occurred between February and September 2019. Patients aged eighteen to sixty years with a clinical diagnosis of severe alcoholic hepatitis 16 with a modi ed Maddrey's discriminant function (mDF) of equal to or greater than 32, with acute-on-chronic liver failure (ACLF) [17][18] were eligible.
Exclusion criteria were other causes of liver disease, uncontrolled infections, uncontrolled upper gastrointestinal bleeding, grade 3 or 4 hepatic encephalopathy, more than two organ failures, 18 malignancy, intestinal paralysis, or perforation.
All patients received standard medical therapy comprising supportive care and nutritional support. Nonabsorbable antibiotics were avoided for the treatment of hepatic encephalopathy in the FMT group. Stool softening agents were continued. IV antibiotics were continued as per the treating physician's decision in the event of active sepsis based on culture and sensitivity. Complications arising during the study were managed as per standard protocols 19 following admission to the liver intensive care unit. All patients underwent detailed history, physical examination, and relevant laboratory investigations. They also underwent a pre-allocation upper gastrointestinal endoscopy to determine the status of varices. The severity of liver disease was determined by Chronic Liver Failure Consortium Organ Failure (CLIF-C OF), Chronic Liver Failure-Sequential Organ Failure Assessment (CLIF-SOFA), CLIF Consortium Acute-On-Chronic Liver Failure (CLIF-C ACLF, where applicable), Child-Turcotte-Pugh (CTP), mDF, model for endstage liver disease (MELD) and MELD sodium (MELD-Na) scores calculated at baseline and subsequently on days seven, twenty-eight and ninety post inclusion. Percentage changes in severity scores were calculated as delta change using the formula 100*(score on follow-up day-baseline score)/baseline score.
Cytokines (IL1beta and IL6) were measured in all patients' plasma using Human IL1beta/IL6 PicoKine ELISA kits (Boster Biological Technology) according to the manufacturer's protocol. Kit ranges were 3.9-250 picograms per milliliter (pg/mL) for IL1beta and 4.69-300 pg/mL for IL6. The plates were read at 450 nm. Absorbance was converted to pg/mL using a standard curve prepared with recombinant human IL1beta and IL6. Measures were taken at baseline and one month post inclusion. All other laboratory parameters were measured at baseline and on days seven, twenty-eight and ninety post inclusion.

Stool Donors:
Stool donors were chosen from among healthy close family members aged eighteen to sixty years. They were rst screened to rule out systemic illness. Exclusion criteria were enteric infections within the previous month, antibiotic use in the previous two months, a history of abnormal bowel motions, substance abuse, in ammatory bowel disorders, autoimmune illnesses, atopy, malignancy, diabetes, hypertension, hypothyroidism, anemia, or obesity. A pre-donation workup was performed, comprising of a hemogram, coagulogram, liver and kidney function tests, tests for hepatitis B surface antigen (HBsAg), total and IgM antibodies for hepatitis B core antigen (anti-HBc), anti-hepatitis C virus (HCV) antibodies, anti-hepatitis A virus (HAV) antibodies, Human immunode ciency virus (HIV) type 1 and 2 antibodies, Venereal Disease Research Laboratory (VDRL), stool analyses for ova, cysts, parasites, Clostridium di cile toxin, and culture for enteric pathogens.
FMT procedure: Post allocation into the FMT group, patients were administered pre-procedure antibiotics comprising of oral Metronidazole 500 milligrams thrice daily, Cipro oxacin 500 milligrams twice daily, and Amoxycillin 500 mg orally thrice daily for ve days to reduce the host bacterial load and facilitate donor microbiota colonization, discontinued at least twelve hours before the fecal transplant. Six hours before the procedure, thirty grams of fresh stool was collected from the pre-identi ed stool donors, homogenized with a hundred milliliters of sterile normal saline in a blender for 2-4 minutes, followed by ltration through gauze pieces. The product was instilled in a single setting through a nasojejunal tube placed under endoscopic guidance. Participants were kept nil per oral for four hours before FMT, and a liquid diet was commenced two hours following FMT.
The SOC group did not receive pre-procedure antibiotics or FMT. Both groups were followed up on days seven, twenty-eight, and ninety post inclusion.
Clinical Outcomes: The primary outcome was survival at twenty-eight and ninety days after commencement of treatment.
The secondary outcomes were the development of FMT-related adverse events, resolution of hepatic encephalopathy (HE) and ascites, improvement in liver disease severity scores (CLIF-C OF, CLIF-SOFA, CLIF-C ACLF where applicable, CTP, mDF, MELD and MELD-Na) and a change in IL levels.

Statistical Analysis:
All analyses were conducted using IBM SPSS STATISTICS (Version 23.0.0), Microsoft Excel, GraphPad Prism (Version 8.1.1), Stata, and SigmaPlot (Version 14) software. Categorical variables were expressed as n (%), quantitative variables were expressed as mean (95% con dence interval) or median with interquartile range. Kolmogorov Smirnov test of normality was used to identify normally distributed variables. Inter-group comparisons of quantitative variables were performed by using the student's t-test (if normally distributed) or the Mann-Whitney U test (if not normally distributed). Within-group analysis of quantitative variables at discrete time points was performed by using the ANOVA test (for parametric data) and Wilcoxon Signed Rank test (for non-parametric data). A value of P < 0.05 was considered signi cant. Survival curves were constructed for up to 90 days by Kaplan Meier analysis and compared by the log-rank method. Proportions were compared using Chi-squared or Fisher's exact test, whichever was applicable. Independent predictors of mortality were identi ed on univariate analyses, and the variables with P < 0.10 were subjected to multivariate logistic regression analysis. All statistical tests were two-sided and performed at a signi cance level of alpha = 0.05.

Results:
Study Patients: Fifty-one patients with SAH-ACLF aged between eighteen to sixty years were screened for enrolment. Thirty-three patients (64.7%) ful lled the eligibility criteria and were included in the study. Eighteen patients (35.3%) were excluded (Fig. 1).

Liver Function and Scores of Severity:
Baseline liver function parameters were comparable between both groups (Table 1). Bilirubin reduced signi cantly from 17.08 mg/dL at baseline to 13.58 mg/dL on day seven post FMT (P = 0.01). INR reduced signi cantly from 2.14 at baseline to 1.34 on day ninety post-FMT (P = 0.01). Corresponding changes in the SOC group were not statistically signi cant (

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Results as *mean (95% con dence intervals) unless otherwise speci ed; CLIF-C ACLF = Chronic Liver  Table 2). CLIF-SOFA and MELD-Na scores reduced signi cantly from baseline on day twenty-eight post FMT (P = 0.008; P = 0.01) ( Table 2). CLIF-C OF score reduced signi cantly from baseline on days twentyeight and ninety post FMT (P = 0.005; P = 0.01) ( Table 2). mDF score reduced signi cantly from day twenty-eight to day ninety post FMT (P = 0.01) ( Table 2). Corresponding changes in severity scores in the SOC group were not statistically signi cant.
Inter-group comparison revealed that CTP, CLIF-C OF and CLIF-SOFA scores were signi cantly lower on day seven post inclusion in the FMT group as compared with the SOC group (P = 0.002; P = 0.02; P = 0.01) ( Table 2) Resolution of Hepatic Encephalopathy and Ascites: Grading of hepatic encephalopathy (HE) was performed using the West Haven classi cation system. HE was present at baseline in six of thirteen patients (46.15%) in the FMT group and fourteen of twenty patients (70%) patients in the SOC group (Table 1). Within seven days of FMT, complete resolution occurred in both patients with grade 1 and in two patients with grade 2 HE at baseline. Worsening of HE grading from baseline occurred on day-seven evaluation in three of the fourteen patients (21.4%) in the SOC group and none of the six patients (0%) in the FMT group. HE resolved by day twenty-eight in all six patients (100%) in the FMT group as compared with eight out of the fourteen patients (57.14%) in SOC group (P = 0.11). Four patients in the SOC group who had HE at baseline died by day twenty-eight.
Ascites was present at baseline in all patients recruited in the study (Table 1). Ascites resolved by day ninety in 100% of surviving patients in the FMT group as compared with 40% of the surviving patients in the SOC group (P = 0.04).

Adverse Events:
Adverse events included excessive atulence which occurred in all thirteen patients (100%) who received FMT as compared with one of twenty patients (5%) in the SOC group (P < 0.001 Baseline parameters did not predict the twenty-eight-day or ninety-day mortality. The percentage changes (depicted as delta) in bilirubin, total protein, ALT, MELD, MELD-Na, and CLIF SOFA scores between baseline and day seven in uenced mortality in univariate analyses with P < 0.10 (Table 4). In multivariate analyses, delta bilirubin and delta ALT remained signi cant (P < 0.05). A rise in bilirubin predicted increased odds, and a fall in ALT predicted decreased odds of ninety-day mortality (Table 3).    (Fig. 3).

Discussion:
Patients with SAH-ACLF receiving a single session of FMT demonstrated signi cantly improved twentyeight and ninety-day survival. Patients receiving FMT exhibited a more rapid resolution of liver dysfunction and improvement in clinical severity scores and a reduction in pro-in ammatory cytokines. Post FMT, ascites resolved in all survivors, and there was a trend toward greater improvement in hepatic encephalopathy.
A previous study showed that FMT signi cantly improved bilirubin levels, CTP, MELD, MELD-Na scores and survival in eight patients over one year. 12 In another study comparing FMT with corticosteroids, nutritional therapy, and pentoxifylline, Philips et al. demonstrated improved survival at three months (75%, 38%, 29%, and 30%, respectively). 14 While FMT was performed over seven consecutive days without antibiotic pre-treatment in both studies, our study attempted to evaluate the e cacy of a single FMT procedure following antibiotic pre-treatment to reduce host bacterial load and facilitate donor microbiota colonization. We found that FMT administered in a single session may improve both short-term and medium-term survival, a treatment that has been elusive in the management of these patients to date.
Intestinal dysbiosis, particularly small intestinal bacterial overgrowth, has been implicated in the pathogenesis of hepatic encephalopathy in the setting of alcoholic liver disease. 23 In ammation with a concomitant rise in in ammatory cytokines has long been implicated in setting the stage for ethanol-induced hepatic in ammation and injury. 10  Percentage changes in bilirubin and ALT between baseline and day seven emerged as predictors of ninety-day mortality in our study. A rise in bilirubin predicted increased odds and a fall in ALT predicted decreased odds of ninety-day mortality. The changes in bilirubin over seven days have been validated as a component of the Lille model to predict response to corticosteroids and six-month survival. 29 However, further studies with larger sample sizes are required to validate these ndings and evaluate percentage change in ALT (delta-ALT) as a prognostic marker in alcoholic hepatitis and ACLF.
Infections have been reported with the use of FMT for recurrent CDI.A recently published update described the occurrence of ESBL producing Escherichia coli bacteremia in two patients after undergoing FMT. 30 In our study, the occurrence of SBP did not differ signi cantly between groups. More stringent and uniform donor selection criteria need to be set in place for future studies, along with standardized methods of transplantation to minimize the risk of infection.
Our study has several limitations. Being an exploratory trial, we could enroll only 13 patients in the FMT arm, limiting our study's power. Secondly, being an open-labeled trial without a matched placebo, the study has an inherent risk of bias. However, given the intervention's nature, an appropriately matched placebo was not methodologically feasible, and future studies should be designed to address this limitation. Lastly, our clinical results are not corroborated with changes in gut microbiota composition and donor ora engraftment, which could have further substantiated the impact of FMT in SAH-ACLF.
In conclusion, single session FMT is safe in SAH-ACLF and leads to improvement in liver function and clinical severity scores along with signi cant improvement in short and medium-term survival in patients with SAH-ACLF with FMT administration. Correlation of clinical improvement with concomitant gut microbiota changes may further substantiate the role of FMT in SAH-ACLF in future placebo-controlled randomized controlled trials.  Patient ow and study design.

Figure 2
Kaplan-Meier curves showing overall survival according to study group. Interleukin levels at baseline (A), one month (B) and percentage change (C) according to study group.

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