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Address for correspondence. Cyriac Abby Philips M.D., D.M., The Liver Institute, Center of Excellence in GI Sciences, Ground Floor, Phase II, Rajagiri Hospital, Chunangamvely, Aluva, Ernakulam, Kerala 683 112, India.
Clinical and Translational Hepatology, The Liver Institute, Center of Excellence in GI Sciences, Rajagiri Hospital, Chunangamvely, Aluva, Ernakulam, Kerala, IndiaMonarch Liver Laboratory, The Liver Institute, Center of Excellence in GI Sciences, Rajagiri Hospital, Chunangamvely, Aluva, Ernakulam, Kerala, India
Monarch Liver Laboratory, The Liver Institute, Center of Excellence in GI Sciences, Rajagiri Hospital, Chunangamvely, Aluva, Ernakulam, Kerala, IndiaDepartment of Gastroenterology and Advanced GI Endoscopy, Center of Excellence in GI Sciences, Rajagiri Hospital, Chunangamvely, Aluva, Ernakulam, Kerala, India
Healthy donor fecal microbiota transplantation (FMT) was preliminarily shown to have clinical benefits in hepatic encephalopathy (HE), severe alcohol-associated hepatitis (SAH), and alcohol use disorder. However, the long-term outcomes of FMT and the gut microbiota (GM) changes in patients with SAH are unknown.
Patients with SAH who underwent FMT (N = 35) or standard of care (SoC, N = 26) from May 2017 to June 2018 were included, and their stored stool samples were analyzed prospectively. Clinical outcomes, including infections, hospitalizations, critical illness, alcohol relapse, and survival, were evaluated. Metagenomic analysis was undertaken to identify the relative abundances (Ras) and significant taxa at baseline and post-therapy (up to three years) among survivors between the two groups.
At follow-up, the incidences of ascites, HE, infections, and major hospitalizations were significantly higher in the SoC than in the FMT group (P < 0.05). Alcohol relapse was lower (28.6% versus 53.8%), and the time to relapse was higher in the FMT than in the SoC group (P = 0.04). Three-year survival was higher in the FMT than in the SoC group (65.7% versus 38.5%, P = 0.052). Death due to sepsis was significantly higher in the SoC group (N = 13/16, 81.2%; P = 0.008). GM analysis showed a significant increase in the RA of Bifidobacterium and a reduction in the RA of Acinetobacter in the FMT group. Beyond one to two years, the RA of Porphyromonas was significantly higher and that of Bifidobacterium was lower in the SoC than in the FMT group.
In terms of treatment for patients with SAH, healthy donor FMT is associated with significantly lesser ascites, infections, encephalopathy, and alcohol relapse (with a trend toward higher survival rates) than SoC, associated with beneficial GM modulation. Larger controlled studies on FMT are an unmet need.
Gut microbiota (GM) modulation via healthy donor fecal microbiota transplantation (FMT) was preliminarily shown to have clinical benefits in various liver disease conditions. These benefits include amelioration of hepatic encephalopathy (HE) in patients with cirrhosis, improved survival in patients with severe alcohol-associated hepatitis (SAH) and alcoholic hepatitis (AH)-related acute-on-chronic liver failure (ACLF), and improved quality of life and abstinence in patients with alcohol use disorder.
Nevertheless, the long-term clinical outcomes and the GM changes in patients with SAH who undergo FMT are unknown. In this study, we present data on retrospectively analyzed clinical outcomes, alcohol relapse, and prospective analysis of stored stool microbiota of patients with SAH who underwent healthy donor FMT and compared the data to those of patients with SAH who underwent standard of care (SoC).
Patients and methods
Patients with SAH who underwent FMT (100 mL of freshly processed stool samples via a nasoduodenal tube for seven days; the FMT group) or SoC (40 mg of oral prednisolone once daily for 28 days; the SoC group) from May 2017 to June 2018 were retrospectively included in this study. Only those patients with ‘definite’ alcohol-associated hepatitis as defined by Singal et al. were included for analysis.
After granting informed consent, all the patients underwent liver biopsy for definitive diagnosis of SAH. For FMT, fresh donor stool samples (minimum mass = 30 g) were obtained, processed, and infused within 6 h of collection. In brief, 100 mL of sterile normal saline was added to each stool sample and homogenized for 2 min using a hand blender. The resulting suspension was strained, filtered thrice, and delivered through a nasoduodenal tube placed under fluoroscopic guidance one day prior. Each patient was kept nil orally for at least 4 h prior to stool instillation and 2 h after FMT (Figure 1). Only patients who completed the FMT protocol and those who were corticosteroid responders (i.e., with Lille model score <0.45 at the end of one week) were considered for study inclusion. Since this was a retrospective study, we included only steroid responders within the study time with long-term follow-up data were included and analyzed. Those lost to follow up and without complete documentation were excluded. In this regard, only per-protocol analysis was undertaken within the retrospective design. All patients were initiated on maximally tolerated beta-blocker therapy in the presence of clinically significant portal hypertension, and for those with ascites, a lowest tolerated and effective dose of diuretics was continued. In those who developed HE after completion of treatment, rifaximin was added as secondary prophylaxis. Additionally, in those who developed infections or acute kidney injury on follow up and required admission, intravenous antimicrobial therapy, albumin infusions with or without terlipressin were provided. Stool samples were collected at the start of FMT or corticosteroid treatment, within 24 h of admission. Post-treatments follow up samples were collected in each patient at each outpatient follow up or admission between 1–2 years and 2–3 years and only the latest follow-up stored samples between the specified periods were sent for sequencing and further analysis. All stool samples were stored at −80 °C for prospective analysis. All patients were followed up for events such as the development of ascites (defined as grade 2 and above), HE (defined as grade 2 and above, or overt in nature), non-critical infections (those which did not require admission and intravenous antimicrobial management), critical infections (those requiring hospital admission and intravenous antimicrobials), and need for hospitalization (defined as any liver-related or portal hypertension event requiring admission to the hospital). The study protocol was approved by the Institutional Review Board of the hospital and was performed in conformance with the Helsinki declaration of 1975 and its pertinent revisions. All participants (or their immediate family members) provided informed consent for the procedure and the use of their de-identified stored fecal samples for future research.
Statistical analysis was performed using MedCalc Statistical Software (Ostend, Belgium). Data are presented as mean and standard deviation or as median and range. The Shapiro–Wilk test (for small sample sizes) was used to test the normality of continuous data between groups. One-way analysis of variance (ANOVA) was used to test for differences at baseline between the means of investigational variables of groups. P-values < 0.05 were considered significant. Before ANOVA, Levene's test for equality of variances was performed. If Levene's test was positive (i.e., P < 0.05), logarithmic transformation was applied to the data. When ANOVA revealed significant differences (i.e., P < 0.05), post-hoc analysis was used for comparison between subgroups using Tukey–Kramer test to look for significant differences between groups at baseline. “N-1” Chi-squared test was used to compare significance in proportions between groups. The probability of patients surviving up to the study endpoints was calculated using the Kaplan–Meier method and graphically represented by the survival time curve. Log-rank test was used to compare the survival curves, and P-values < 0.05 were considered significant. Using an Illumina MiSeq next-generation sequencer (Illumina, CA, USA), we performed fecal 16S rRNA amplicon sequencing at the V3–V4 region of bacterial DNA extracted from approximately 200 mg of collected stool samples. Bacterial DNA was extracted using a validated protocol modification of the commercially available QIAmp DNA Stool Mini Kit1 (Qiagen, Venlo, The Netherlands) to identify bacterial communities, which were classified taxonomically according to the Greengenes Database (version 13.8). The Quantitative Insights into Microbial Ecology (version 1.9.1) and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (version 1.1.1) were used to ascertain quantitative and qualitative microbial communities. The multivariable biomarker discovery method known as linear discriminant analysis effect size, which combines the Kruskal–Wallis and pairwise Wilcoxon tests, was used to identify significantly different microbial communities between groups at baseline and post-therapy (at one to two years and at two to three years) among survivors between groups. We used default significance (i.e., an alpha value of 0.05) and a linear discriminant analysis threshold of 2.0 at all taxonomic levels between time points and between groups.
Following a retrospective review of hospital records from May 2017 to June 2018, patients with SAH who underwent healthy donor FMT (N = 35; the FMT group) and those that are corticosteroid responders (N = 26; the SoC group) were included in this study. All the patients were men matched for age, liver disease and AH severity, and a number of portal hypertension and infection episodes at admission. Total bilirubin levels and chronic liver failure consortium scores were significantly higher in the FMT group than in the SoC group, possibly due to steroid ineligibility factors. In contrast, there were no significant differences in the incidence of extrahepatic organ failure and the grade of ACLF between the two groups at baseline (Table 1).
Table 1Baseline Investigation Parameters Between Groups.
Fecal microbiota transplantation (N = 35)
Standard of care (N = 26)
0.7 to 1.2
11 to 13
25–75P, third quartile; CLIF-C, chronic liver failure consortium score; Creat, serum creatinine (mg/dl); CTP, Child Turcotte Pugh score; DLP, dyslipidemia; DM, diabetes mellitus; Hb, hemoglobin (g/L); HTN, hypertension; INR, international normalized ratio; MDF, Maddrey's discriminant function; MELD, model for end stage liver disease; Na, serum sodium (mmol/L); PC, platelet count (× 103 per microliter); SD, standard deviation; TB, total bilirubin (mg/dl); TLC, total leucocyte count (× 103 per microliter).
At follow-up, the incidences of ascites (34.3% versus 73.1%, P = 0.003), HE (20% versus 69.2%, P < 0.001), non-critical (20% versus 65.4%, P < 0.001) and critical (17.1% versus 53.8%, P = 0.003) infections, and need for hospitalization (42.8% versus 69.2%, P = 0.04) were found to be significantly lower in the FMT group than in the SoC group. Further, the incidence of alcohol relapse was lower (28.6% versus 53.8%, P = 0.04) and time to relapse was higher (median [range] = 436 [160–676] days versus 197 [64–412] days, P = 0.002) in the FMT group than in the SoC group. However, there were no significant differences in the proportion of patients with alcohol relapse who developed recurrent SAH between the two groups.
At the end of three years of follow-up, we observed a trend toward improved survival in the FMT group as the rate of mortality was higher in the SoC group than in the FMT group (hazard ratio [95% confidence interval] = 2.14 (0.99–4.58), P = 0.0504, Figure 2). The most common cause of death was acute variceal bleeding in the FMT group and infection in the SoC group. A summary of the follow-up outcomes is shown in Table 2.
Table 2Summary of the follow-up clinical outcomes among patients between both groups.
Fecal microbiota transplantation (N = 35)
Standard of care (N = 26)
Acute kidney injury
Significant fibrosis on liver biopsy (≥ grade 3)
0 + 1
2 + 3
ON FOLLOW UP
Acute kidney injury
Acute variceal bleeding
Need for critical care
Non-critical infections Lung SBP SSTI UTI
7 (20%) 1 0 2 4
17 (65.45) 7 4 2 4
Critical infections Blood Lung SBP SSTI UTI
6 (17.1%) 0 2 2 1 1
14 (53.8%) 2 7 4 1 0
Alcohol relapse Time to relapse (days) (Median; min - max)
10 (28.6%) 436 (160–676)
14 (53.8%) 197 (64–412)
Recurrence of alcohol-related hepatitis Cause of death AVB AVB+sepsis HCC+sepsis Liver failure/MOF Sepsis
GM analysis of the two groups at baseline (FMT group: N = 25; SoC group: N = 18), follow-up between one and two years (FMT group: N = 23; SoC group: N = 16), and follow-up between two and three years (FMT group: N = 28; SoC group: N = 11) revealed remarkable changes in the RAs of bacterial phyla in the FMT group but not in the SoC group. In the FMT group, the RA of Proteobacteria decreased, while the RAs of Actinobacteria and Bacteroides increased. In the SoC group, the RAs of bacterial communities at the phylum level did not change significantly, except for the emergence of Fusobacteria in survivors beyond one year but within two years of follow-up. High-dimensional multivariate biomarker discovery analysis at the genus level revealed a significant increase in the RA of Bifidobacterium and a decrease in the RA of Acinetobacter in the FMT group. Among survivors on corticosteroid therapy, the RA of Leptotrichia, which belongs to the Fusobacteria phylum, was significantly increased between the first and second years of follow-up. Furthermore, the RAs of Erwinia (Enterobacteriaceae) and Porphyromonas (Bacteroidetes) were significantly higher and the RA of beneficial Bifidobacterium was lower in the SoC group than in the FMT group at one to two years of follow-up. In patients who survived beyond two years, the RA of beneficial Bifidobacterium was higher in the FMT group than in the SoC group (Figure 3).
In this study, which is the largest study with the longest follow-up of patients with SAH who underwent FMT, it was found that, compared to SoC, GM modulation improves cumulative (proportional) survival. In addition to a significant reduction in the number of liver-related and portal hypertension events such as ascites, HE, and critical infections in the short-term and the long-term, FMT was found to be associated with a low risk of repeated alcohol misuse and a long time to relapse. These beneficial clinical outcomes were found to be associated with marked changes in intestinal bacterial communities that demonstrate a change of pathogenic species at baseline toward favorable genera such as Bifidobacterium, which are known to promote homeostasis in terms of host immunity, metabolism, and organ function.
In an open-label pilot study, Philips et al. performed fresh FMT through a nasoduodenal tube once a day for seven days on patients with SAH who were steroid-ineligible. Compared to matched historical controls, at one year of follow-up, FMT improved patient survival, which is associated with a reduction in the RAs of potentially pathogenic species and with an increase in the RAs of non-pathogenic species, such as Enterococcus villorum, Bifidobacterium longum, and Megasphaera elsdenii, 6–12 months post-therapy.
In another open-label retrospective study, the same group of researchers reported that three-month survival was higher in patients who underwent FMT than in patients who underwent corticosteroid, pentoxifylline, and nutritional therapy. Intestinal microbiome analysis revealed that patients with SAH have significantly high RAs of Veillonella, Dialister, Lentisphaera, and Victivallis species at baseline. Post FMT, there was an increase in the RAs of beneficial Roseburia and Micrococcus at one month that is associated with a reduction in the lipopolysaccharide synthesis pathways.
Like these two studies, our study shows that FMT improves survival in patients with SAH. This improvement is associated with beneficial changes in the GM related to a reduction in the number of portal hypertension, liver-related, and infection episodes.
Bajaj et al. demonstrated long-term safety and sustained improvement in clinical and cognitive function parameters in patients with cirrhosis who underwent FMT. In their study, the most notable improvement was in the prevention of recurrence of HE and liver-related hospitalizations, which is associated with improved cognition, in patients who underwent FMT compared to patients who underwent SoC. Patients who underwent SoC had 10 hospitalizations, while patients who underwent FMT had only one hospitalization. The majority of hospitalizations in patients who underwent SoC were liver-related and include HE, infection, and ascites.
Although our study included patients with decompensated cirrhosis, our findings are consistent with the reported outcomes of patients who underwent FMT in the long term.
Patients with cirrhosis, especially those with advanced complications of portal hypertension and severe liver disease, have alarmingly high levels of antimicrobial exposure due to repeated hospitalizations and interventions. These high levels of antimicrobial exposure increase the treatment difficulty of multi-drug resistant infections in patients with cirrhosis. Bajaj et al. reported that, in patients with decompensated cirrhosis, FMT reduces antibiotic resistance gene expression and abundance as revealed by metagenomic analysis, whereas antibiotic pre-intervention increases quinolone resistance.
In real-world scenarios, these study findings may correlate with low rates of infection episodes and hospitalizations as we found that patients with SAH who underwent FMT have low rates of critical infections, non-critical infections, and hospitalizations; thus, these patients have a low need for antimicrobial treatment. In the current study, patients undergoing FMT had significantly higher CLIF-C scores and total bilirubin levels at baseline than those on SoC. This is probably because FMT was inherently considered a salvage option in those who were ineligible for steroids and not ideal candidates for (or unwilling) liver transplantation in the short-term. In these patients, the commonest cause for steroid ineligibility was the presence of active infections, and hence, more severe jaundice and higher Chronic Liver Failure—Consortium scores. Nonetheless, Child-Pugh, model for end-stage liver disease, and AH severity scores were comparable between groups at baseline.
In a randomized trial by Bajaj et al., FMT was shown to be safe and associated with a short-term reduction in alcohol craving and consumption with changes in beneficial intestinal bacterial communities compared with placebo in patients with alcohol-associated cirrhosis and alcohol use disorder. FMT was also shown to be associated with a reduction in alcohol misuse-related events over six months in these patients.
These reports are consistent with our study findings. In our study, we found that the rate of alcohol relapse was lower and the time to relapse was longer in the FMT group than in the SoC group. However, there were no significant differences in the proportion of patients with alcohol relapse who developed recurrent SAH between the two groups. GM analysis revealed an abundance of beneficial bacterial taxa that may be associated with reduced alcohol misuse in patients who underwent FMT. A relationship between bacterial communities in the gut and brain function has been demonstrated in animal and human experiments. Modulation of GM geared toward a favorable profile that includes short-chain fatty acid (SCFA)-producing taxa has been shown to improve gut–brain axis-associated outcomes in multiple studies. These reports in published literature support our clinical findings and the microbial changes observed in patients with SAH who underwent FMT.
A recent study showed that metabolites arising from the tryptophan-kynurenine pathway (TKP) were central to the interface between intestinal bacteria, host immune response, and brain functions. Analysis of GM revealed that SCFA producers, as well as bacterial metabolites, including butyrate and medium-chain fatty acids, were associated with metabolites of the TKP. The authors concluded that targeting the glutamatergic neurotransmission through the modulation of the kynurenine pathway, via GM modulation, might represent an interesting alternative for beneficially modifying alcohol-related behavior.
In our study, Bifidobacterium a key SCFA producer was significantly higher post-FMT, substantiating this proof of concept.
It is important to note that, in the Kaplan–Meier analysis in our study, the survival curves intersected between three and six months of follow-up. This is indicative of a modification effect between the two groups that may be due to abstinence from alcohol. During the initial three to six months post-treatment, patients in both groups maintained alcohol abstinence. This abstinence may explain the intersection of curves observed in the survival analysis. However, in the long-term, GM modulation and alcohol misuse were significantly less in the FMT group than in the SoC group and may have resulted in the worsening dysbiosis or absence of beneficial GM changes from baseline and the poor long-term clinical outcomes in the SoC group. This explanation also supports the fact that the emergence or resurgence of alcohol-associated GM dysbiosis drives clinical events in the long-term in patients with SAH.
Prior studies on fecal transplantation in healthy volunteers demonstrated that FMT led to significant long-term changes of the GM at one year with a shift toward donor microbiota composition, representing the relative safety of the procedure in the absence of long-term adverse events.
Similarly, another study analyzed the long-term effects of FMT in recurrent Clostridium difficile infection among 45 patients followed on average of 3.8 years. The authors found that FMT was a durable, safe, and acceptable treatment option for patients with recurrent C. difficile infection also in long-term with potential benefits over antimicrobial treatment.
In a study from Atlanta, Georgia, authors followed up long-term (22 months) clinical outcomes of FMT treatment in patients with recurrent C. difficile infection and found that 82% of patients had the durable cure of C. difficile infection and those with recurrence had more post-FMT antibiotic exposure.
In the current study, we demonstrate similar long-term outcomes and durability of GM in SAH patients treated with seven days of nasoduodenal route FMT. On the contrary, in those receiving steroids, the need for hospitalization and episodes of infections and thus repeated requirements for antimicrobial therapy were higher, which could have been due to lack of GM modulation and worsening of dysbiosis. We did not identify any long-term adverse events in our follow-up patients directly related to FMT which is in line with current published literature on FMT.
Our study is limited by its retrospective single-center design and its small sample size. Therefore, randomized controlled trials with larger sample sizes should be conducted to verify our findings. Further, our study did not consider confounding factors (specifically unmeasured and unmeasurable; residual confounders) such as dietary habits and environment, which are difficult to quantify and may have affected the GM. Moreover, rifaximin, which was administered more to patients who underwent SoC to prevent secondary episodes of overt HE, may have differentially modified gut microbial communities in these patients compared to patients who underwent FMT. Nevertheless, even in the long-term, beneficial bacterial taxa were significantly more predominant in the FMT group than in the SoC group. However, we did not specifically assess GM in the subgroup of patients with relapse of alcohol consumption.
In conclusion, we present the largest series on the longest clinical outcomes and the associated GM changes in patients with SAH who underwent FMT (Figure 4, visual abstract). Our study reveals that, in the long-term, patients with SAH who undergo healthy donor FMT have significantly fewer HE episodes, critical infections, hospitalizations, and alcohol relapse with a trend toward improved survival than corticosteroids responders. Further, clinically relevant outcomes are associated with beneficial GM modulation in patients who undergo FMT. However, it is necessary to conduct larger controlled studies on FMT.
The authors acknowledge the services of Gene-Path-Dx Lab, Pune, Maharashtra, India and Helical Bio, Ann Arbor, Michigan, USA for their support regarding Amplicon 16S RNA sequencing and bioinformatics analysis.
No grant or financial support was taken for this research.
☆This study has been chosen for presentation at The Presidential Plenary—Clinical and Translational Hepatology session of The Liver Meeting 2021, the annual meeting of the American Association for the Study of Liver Diseases (AASLD) and has also been conferred the Best of Liver Meeting 2021 honor, by the AASLD.