Fecal microbiota transplantation in alcohol-associated acute-on-chronic liver failure: an open-label clinical trial

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. 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 7, 28, and 90. Survival at 28 and 90 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 by 21.39% (IQR − 73.67 to 7.63) in the FMT group, whereas it increased in the SOC by 27.44% (IQR − 0.88 to 128.11) (p = 0.01). Percentage changes in bilirubin and ALT between baseline and day 7 emerged as predictors of 90-day mortality. FMT is safe, improves short-term and medium-term survival, and leads to improvement in clinical severity scores in patients with SAH–ACLF. NCT03827772 available from http://clinicaltrials.gov/ct2/show/NCT03827772 CTRI Reference number: CTRI/2019/02/017538 dated 7 February 2019.


Introduction
Alcoholic liver disease has become one of the foremost causes of chronic liver disease across the world [1]. Alcoholic steatohepatitis is a fulminant entity in this broad spectrum, carrying 1-month mortality rates of 20-50% [2]. No robust data for a treatment to substantially improve outcomes currently exist for severe alcoholic hepatitis (SAH) amounting to acute-on-chronic liver failure (ACLF), with most therapy aimed at supportive care. The benefits accrued by corticosteroids, the current standard of care, are modest [3]. Early liver transplantation, albeit demonstrated to improve survival in a select few, is still of limited applicability owing to stringent recipient selection criteria, the pretransplant requirement of alcohol abstinence at most centers, donor availability, and financial constraints [4].
Intestinal dysbiosis has been accepted as a pathogenic mechanism in alcoholic hepatitis. Rodent and human studies reveal ethanol-induced alterations in gut microflora [5][6][7]. Rodent studies also demonstrate the transmissibility of hepatic inflammation by transplanting dysbiotic flora from patients with severe alcoholic hepatitis (SAH) [8]. Gut dysbiosis and the resultant gut microbial alteration form an important pathophysiological pathway in SAH. Dysbiosis disrupts the intestinal epithelial barrier, increases intestinal permeability with trans-epithelial translocation of pathogen-associated molecular patterns (PAMPs), which bind to hepatic pattern recognition receptors (PRRs), and activates the immune cascade, leading to hepatic inflammation and injury [9][10][11].
Modulation of gut flora 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 efficacy of FMT in improving survival and liver function and mitigating systemic inflammation in patients with SAH presenting as ACLF.

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 and December 2019. The study protocol conformed to the 1975 Declaration of Helsinki's ethical guidelines as reflected 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 18-60 years with a clinical diagnosis of severe alcoholic hepatitis [16] with a modified Maddrey's discriminant function (mDF) of equal to or greater than 32, with acute-on-chronic liver failure (ACLF) [17,18] were eligible. ACLF was defined as per the Asian Pacific Association for the study of the liver (APASL) or Chronic Liver Failure (CLIF) Consortium Acute-on-Chronic Liver Failure in Cirrhosis (CANONIC) criteria [17,18]. Patients with only alcohol as the acute insult 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. After satisfying the selection criteria, patients were allocated to either receive FMT or standard medical care. The allocation to FMT was dependent upon approval and consent of the patient and designated family member, followed by a screening of the family member to be suitable for participation as a stool donor. In the event of failure to identify a suitable donor, the patient was given standard medical treatment. The physician administering FMT was aware of the treatment being administered, as the nature of the intervention meant that it was not possible to make an identical placebo.
All patients received standard medical therapy, which comprised of nutritional recommendations of a saltrestricted (< 2 gm/day) and a high-protein diet (1.5-2 g/kg/ day) with a targeted caloric intake of 35-40 kcal/kg/day. Intravenous albumin (as per standard recommendations), diuretics (for ascites), lactulose (for hepatic encephalopathy), intravenous antibiotics (in the event of active sepsis), and multivitamins any episode of suspected variceal bleed was managed with proton pump inhibitors, vasoconstrictors, and endoscopic intervention as indicated. All complications arising during the study were managed as per standard protocols [19] following admission to the liver intensive care unit. The potentially poorer response to glucocorticoids in patients with SAH-ACLF along with the concerns about increased susceptibility to infections precluded the use of the same as a component of standard medical therapy in our study, in accordance with our institutional policy [20][21][22]. All patients underwent detailed history-recording, 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 end-stage liver disease (MELD), and MELD sodium (MELD-Na) scores calculated at baseline and subsequently on days 7 (as the standard of care in ACLF), 28 and 90 post-inclusion. Values obtained post-antibiotic administration prior to the FMT were considered to represent the baseline (day 0) in the FMT group. 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 day 28 post-inclusion. All other laboratory parameters were measured at baseline and on days 7, 28, and 90 post-inclusion.

Stool donors
Stool donors were chosen from among healthy close family members aged 18-60 years. They were first screened to rule out systemic illness. Exclusion criteria were enteric infections within the previous month, antibiotic use in the previous 2 months, a history of abnormal bowel motions, substance abuse, inflammatory 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 immunodeficiency virus (HIV) type 1 and 2 antibodies, Venereal Disease Research Laboratory (VDRL), stool analyses for ova, cysts, parasites, Clostridium difficile 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 mg thrice daily, Ciprofloxacin 500 mg twice daily, and Amoxycillin 500 mg orally thrice daily for 5 days to reduce the host bacterial load and facilitate donor microbiota colonization, discontinued at least 12 h before the fecal transplant. Six hours before the procedure, 30 g of fresh stool was collected from the pre-identified stool donors, and homogenized with a hundred milliliters of sterile normal saline in a blender for 2-4 min, followed by filtration 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 4 h before FMT, and a liquid diet was commenced 2 h following FMT.
The SOC group did not receive pre-procedure antibiotics or FMT. Both groups were followed up on days 7, 28, and 90 post-baseline (day 0).

Clinical outcomes
The primary outcome was survival at 28 and 90 days postbaseline. 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% confidence interval) or median with interquartile range. Shapiro-Wilk test of normality was used to identify normally distributed variables. Inter-group comparisons of quantitative variables were performed using the Mann-Whitney U test. Within-group analysis of quantitative variables at discrete time points was performed using the Wilcoxon matched-pair signed rank test. A value of p < 0.05 was considered significant. We did not perform multiple correction testing as the post hoc tests were considered exploratory. 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 identified on univariate analyses, and the variables with p < 0.10 were subjected to multivariate logistic regression analysis. For pertinent variables, we carried the last value forward, and since the variables thus obtained had a normal distribution as per Shapiro-Wilk test, we performed a one-way repeated-measures ANOVA to determine withingroup differences, using a conservative (Greenhouse-Geisser) F test. Bonferroni's multiple comparison post hoc tests were used as needed. However, as there are a large proportion of missing values which had to be carried forward from the last visit owing to the patients' death, this has been included as a supplementary analysis (supplementary table 4 and supplementary figures). All statistical tests were two-sided and performed at a significance level of alpha = 0.05.

Study patients
Fifty-one patients with SAH-ACLF aged between 18 and 60 years were screened for enrollment. Thirty-three patients (64.7%) fulfilled the eligibility criteria and were included in the study. Eighteen patients (35.3%) were excluded (Fig. 1). Baseline patient classification as per ACLF definition and organ failures is tabulated in Supplementary Table 1.

Liver function and scores of severity
Baseline liver function parameters were comparable between both groups (Table 1). Bilirubin reduced significantly from 17.08 mg/dL at baseline to 13.58 mg/dL on day 7 post-FMT (p = 0.01). INR reduced significantly from 2.14 at baseline to 1.34 on day 90 post-FMT (p = 0.01). Corresponding changes in the SOC group were not statistically significant ( Table 2). A detailed description of laboratory parameters in both groups at different time points of follow-up is depicted in Supplementary Table 2. There was no significant difference in laboratory parameters between pre-antibiotic day (− 6) and baseline (day 0) in the FMT group (Supplementary Table 2).
The total leucocyte count (TLC) was significantly higher at baseline in the SOC group as compared with the FMT group (Table 1). It reduced in surviving patients of both groups. Baseline TLC or percentage change (delta TLC) did not emerge a predictor of mortality in either the FMT (p = 0.16) or the SOC group (p = 0.36, Table 3).
CTP and MELD scores reduced significantly from baseline on day 7 post-FMT (p = 0.005; p = 0.01) ( Table 2). CLIF-SOFA and MELD-Na scores reduced significantly from baseline on day 28 post-FMT (p = 0.008; p = 0.01) ( Table 2). CLIF-C OF score reduced significantly from baseline on days 28 and 90 post-FMT (p = 0.005; p = 0.01) ( Table 2). mDF score reduced significantly from day 28 to day 90 post-FMT (p = 0.01) ( Table 2). Corresponding changes in severity scores in the SOC group were not statistically significant.
Inter-group comparison revealed that CTP, CLIF-C OF, and CLIF-SOFA scores were significantly lower on day 7 post-inclusion in the FMT group as compared with the SOC group (p = 0.002; p = 0.02; p = 0.01) ( Table 2).
Although total leucocyte counts were significantly lower at baseline and on days 7 and 28 in the FMT group as compared with the SOC group (p = 0.05; p = 0.02; p = 0.02) ( Table 2), these counts or percentage changes between baseline and day 7 did not predict 90-day mortality (p = 0.48; p = 0.24; 95% CI 0.99-1.04).
Supplementary analysis after carrying the last value forward revealed that INR, MELD, MELD-Na, CLIF-SOFA, and CLIF-C OF scores reduced significantly over time in the FMT group (within-subjects effects p = 0.008; p = 0.005; p < 0.001; p < 0.001; p < 0.001), without a corresponding significant reduction in the SOC group (supplementary table 4 and supplementary figures).

Organ failures
At baseline, seven (53.8%) patients had organ failures in the FMT group as compared with 13 (65%) in the SOC group (p = 0.52) ( Table 1). One (20%) of the five patients with a single organ failure and both (100%) with two organ failures at baseline (patient 1-liver and circulation; patient 2-liver and coagulation) died during the 90-day follow-up in the FMT group, while 7 (58.3%) patients with a single organ failure and the only patient with two organ failures at baseline (liver and coagulation) died during the 90-day follow-up in the SOC group. The presence of organ failures at baseline did not predict 90-day mortality (p = 0.21; 95% CI 0.07-1.79) ( Table 3).

Resolution of hepatic encephalopathy and ascites
Grading of hepatic encephalopathy (HE) was performed using the West Haven classification system. HE was present at baseline in 6 (46.15%) patients in the FMT group and 14 (70%) in the SOC group (Table 1). Within 7 days of FMT, complete resolution occurred in both patients with grade 1 and in two with grade 2 HE at baseline. Worsening of HE grading from baseline occurred on day-7 evaluation in three (21.4%) patients in the SOC group and none in the FMT group. HE resolved by day 28 in all 6 (100%) patients in the FMT group as compared to 8 (57.14%) in SOC group (p = 0.11). Four patients in the SOC group who had HE at baseline died by day 28.
Ascites was present at baseline in all patients recruited in the study (Table 1). Ascites resolved by day 90 in 100% of surviving patients in the FMT group as compared to 40% in the SOC group (p = 0.04).

Antibiotic usage
Three (23.07%) patients in the FMT group and 4 (20%) in the SOC group received intra-venous antibiotics as they were recovering from infections (p = 1.00). One of these patients died and two survived in the FMT group, while two died and two survived in the SOC group at 90-day follow-up (p = 0.99; p = 0.24). Baseline intravenous antibiotic use did not predict 90-day mortality (p = 0.21; 95% CI 0.05-1.89). Baseline oral (thereby including all those in the FMT group) or intravenous antibiotic use did not predict 90-day mortality on multivariate analysis (p = 0.40; 95% CI 0.03-3.98) ( Table 3).

Adverse events
Adverse events included excessive flatulence which occurred in all 13 (100%) patients who received FMT as compared to one (5%) in the SOC group (p < 0.001). Gastroesophageal reflux occurred in 7 (53.8%) patients who received FMT as compared to 4 (20%) in the SOC group (p = 0.06). Both these persisted for 90 days post-procedure. Nausea occurred in 3 (23.1%) patients immediately post-FMT and resolved within 1 day. During the 90-day follow-up, upper gastro-intestinal bleed occurred in 3 (23.1%) patients who received FMT as compared to 7 (35%) in the SOC group (p = 0.70). Spontaneous bacterial peritoni-tis (SBP) occurred in 4 (30.76%) patients who received FMT as compared to 5 (25%) in the SOC group. Of these, 1 patient in the FMT group and 3 in the SOC group developed SBP within 28 days of inclusion. Three patients in the FMT group and 2 in the  Table 3).  Fig. 2. None of the patients received a liver transplant during the study period. Four (66.67%) out of 6 deceased patients in the FMT group and 5 (33.33%) out of 15 in the SOC group died due to refractory septic shock consequent to SBP. All the patients who developed SBP during followup died in both groups. However, the occurrence of SBP as cause of mortality was not significantly differ-ent between the two groups (p = 0.33). One (16.67%) deceased patient in the FMT group and 3 (20%) in the SOC group died due to massive upper gastrointestinal bleeding. One (16.67%) deceased patient in the FMT group and 4 (26.66%) in the SOC group died due to the worsening of organ failures and the development of refractory shock. Three (20%) deceased patients in the SOC group died due to pneumonia (Supplementary Table 1). Baseline parameters did not predict the 28-day or 90-day mortality. The percentage changes (depicted as delta) in bilirubin, total protein, ALT, MELD, MELD-Na, and CLIF SOFA scores between baseline and day 7 influenced mortality in univariate analyses with p < 0.10 (Table 3). In multivariate analyses, delta bilirubin and delta-ALT remained significant (p < 0.05). A rise in bilirubin predicted increased odds, and a fall in ALT predicted decreased odds of 90-day mortality (Table 3).

Interleukin analysis as a surrogate for systemic inflammation
IL-1beta and IL-6 levels were comparable at baseline in both groups (Table 1). Median IL-1beta value reduced from 20.42 (IQR 5.07-23.84) pg/mL at baseline to 9.25 (IQR 4.46-13.24) pg/mL on day 28 in the FMT group, while the corresponding IL-1beta value increased from 7.87 (IQR 5.75-15.67) pg/mL at baseline to 17.79 (IQR 12.80-47.56) pg/mL on day 28 in the SOC group (Fig. 3). Median change in the IL-1beta value was a decrease of 21 (Fig. 3).

Discussion
Patients with SAH-ACLF receiving a single session of FMT demonstrated significantly improved 28-and 90-day survival. Patients receiving FMT exhibited a more rapid resolution of liver dysfunction and improvement in clinical severity scores, and a reduction in pro-inflammatory 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 significantly improved bilirubin levels, CTP, MELD, MELD-Na scores and survival in eight patients over 1 year [12]. In another study comparing FMT with corticosteroids, nutritional therapy, and pentoxifylline, Philips et al. demonstrated improved survival at 3 months (75%, 38%, 29%, and 30%, respectively) [14]. While FMT was performed over 7 consecutive days without antibiotic pre-treatment in both studies, our study attempted to evaluate the efficacy 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. Future trials comparing single versus multiple FMT sessions may help determine the best possible dosing schedule of FMT in patients with SAH-ACLF.
Intestinal dysbiosis, particularly small intestinal bacterial overgrowth, has been implicated in the pathogenesis of hepatic encephalopathy in the setting of alcoholic liver disease [23,24]. Bajaj et al. found a higher abundance of non-autochthonous taxa in alcoholic cirrhotic patients with hepatic encephalopathy with a reduced relative abundance of autochthonous taxa, a phenomenon they referred to as a low cirrhosis dysbiosis ratio (CDR) [7]. In a randomized controlled trial, Bajaj et al. evaluated FMT as a means of alleviating this dysbiosis in patients with decompensated cirrhosis to mitigate recurrent hepatic encephalopathy. Neurocognitive test results improved significantly on day 20 after administration of a single session of FMT. Stool microbial analysis revealed an increase in beneficial taxa in the FMT arm with no microbiota change in the SOC arm [25]. The previously cited study by Philips et al. [12] observed resolution of hepatic encephalopathy in 71.4% of patients while demonstrating a corresponding decline in stool Proteobacteria and other pathogenic bacteria and metabolic pathways. The trend toward greater improvement in hepatic encephalopathy in patients receiving FMT in our study corresponds with the clinical outcomes of these studies.
Inflammation with a concomitant rise in inflammatory cytokines has long been implicated in setting the stage for ethanol-induced hepatic injury [10]. Elevated IL-1 and IL-6 levels have been demonstrated in patients with alcoholic hepatitis [26,27]. Proinflammatory cytokine reduction was also evaluated by Dhiman et al. [28] as an indirect marker of the therapeutic efficacy of gut-based therapies in mitigating systemic inflammation in patients with decompensated cirrhosis. The finding of a significant reduction in median IL1beta in the FMT group compared with the SOC group may point toward its role in reducing systemic inflammation in our patients. The decrease in interleukin levels as an indirect predictor of disease resolution needs to be studied further.
Percentage changes in bilirubin and ALT between baseline and day 7 emerged as predictors of 90-day mortality in our study. A rise in bilirubin predicted increased odds and a fall in ALT predicted decreased odds of 90-day mortality. The changes in bilirubin over 7 days have been validated as a component of the Lille model to predict response to corticosteroids and 6-month survival [29]. However, further studies with larger sample sizes are required to validate these findings and evaluate percentage change in ALT (delta-ALT) as a prognostic marker in alcoholic hepatitis and ACLF.
Total leucocyte counts serve as a surrogate of systemic inflammation, with increasing values associated with higher ACLF grades and greater mortality [30,31]. While the higher baseline TLC in our SOC group could be indicative of greater systemic inflammation, both baseline count as well as delta changes (delta TLC) did not predict mortality.
Infections have been reported with the use of FMT for recurrent C. difficile infection (CDI). A recently published update described the occurrence of ESBL producing Escherichia coli bacteremia in two patients after undergoing FMT [32]. The occurrence of SBP as a cause of mortality did not differ significantly between groups. However, the occurrence of SBP in patients receiving FMT remains an area of concern and larger trials may uncover disparities in SBP associated mortality. 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 due to the small sample size. Pre-FMT antibiotics were deemed a requirement to aid in donor microbiota colonization. Bajaj et al. have previously employed a preparative antibiotic regimen in the FMT arm of their randomized clinical trial demonstrating the safety and efficacy of FMT in improving cognitive scores in patients with cirrhosis and recurrent hepatic encephalopathy [25]. Mouse models also demonstrate improved donor FMT engraftment following preparative antibiotics [33]. While the precise role of FMT vis-à-vis antibiotics cannot be discerned with certainty based on our trial, the risk of antibiotic-resistant infections with antibiotic use precluded the inclusion of this step in our SOC group. Our study results may reflect the composite effects of preparatory antibiotics with FMT. This remains a limitation of our study. The impact and utility of a pre-FMT antibiotic schedule have been a matter of debate, and future studies should focus on refining the pre-FMT antibiotic strategy [34]. Second, 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. Additionally, patients who fall on the extreme spectrum of ACLF severity (ACLF grade III) are a group that needs to be evaluated in future studies. However, their enrollment in exploratory prospective trials remains a methodological challenge in view of their high intrinsic mortality. Our study's small sample size necessitates larger trials evaluating outcomes with FMT in SAH-ACLF. Finally, a key limitation of the study is the lack of co-relation between alteration in gut microbiota profiles and clinical outcomes. Future studies to document such co-relations and establishment donor flora engraftment will enrich the evidence for 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 significant improvement in shortand 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 placebocontrolled randomized controlled trials.
Author contributions AS and AR: data compilation and manuscript writing; RKD: concept, manuscript writing, and editing; MPK: manuscript editing and revision; NV: statistical analysis; AD: manuscript editing and revision; SG: data compilation; MC: data extraction; ST: manuscript revision.
Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Statement of human rights
The study has been approved by the institutional ethics committee and follows the ethical standards as laid down in the 1964 Declaration of Helsinki and revised in 2008.

Informed consent Obtained.
Consent for publication Yes.