The RIVET RCT: Rifamycin SV MMX improves muscle mass, physical function, and ammonia in cirrhosis and minimal encephalopathy.

BACKGROUND: Minimal hepatic encephalopathy (MHE) negatively affects the prognosis of cirrhosis, but treatment is not standard. Rifamycin SV MMX (RiVM) is a nonabsorbable rifampin derivative with colonic action. METHODS: In a phase 2 placebo-controlled, double-blind randomized clinical trial patients with MHE were randomized to RiVM or placebo for 30 days with a 7-day follow-up. The primary endpoint was a change in stool cirrhosis dysbiosis ratio. Gut-brain (cognition, stool/salivary microbiome, ammonia, brain magnetic resonance spectroscopy), inflammation (stool calprotectin/serum cytokines), patient-reported outcomes (sickness impact profile: total/physical/psychosocial, high = worse), and sarcopenia (handgrip, bioelectric impedance) were secondary. Between/within groups and delta (post-pre) comparisons were performed. RESULTS: Thirty patients (15/group) were randomized and completed the study without safety concerns. While cirrhosis dysbiosis ratio was statistically similar on repeated measures ANOVA (95% CI: -0.70 to 3.5), ammonia significantly reduced (95% CI: 4.4-29.6) in RiVM with changes in stool microbial α/ß-diversity. MHE status was unchanged but only serial dotting (which tests motor strength) improved in RiVM-assigned patients. Delta physical sickness impact profile (95% CI: 0.33 = 8.5), lean mass (95% CI: -3.3 to -0.9), and handgrip strength (95% CI: -8.1 to -1.0) improved in RiVM versus placebo. Stool short-chain fatty acids (propionate, acetate, and butyrate) increased post-RiVM. Serum, urine, and stool bile acid profile changed to nontoxic bile acids (higher hyocholate/ursodeoxycholate and lower deoxycholate/lithocholate) post-RiVM. Serum IL-1ß and stool calprotectin decreased while brain magnetic resonance spectroscopy showed higher glutathione concentrations in RiVM. CONCLUSIONS: RiVM is well tolerated in patients with MHE with changes in stool microbial composition and function, ammonia, inflammation, brain oxidative stress, and sarcopenia-related parameters without improvement in cognition. RiVM modulates the gut-brain axis and gut-muscle axis in cirrhosis.

Alterations in gut virome are associated with cognitive function and minimal hepatic encephalopathy cross-sectionally and longitudinally in cirrhosis.

Cognitive dysfunction due to minimal hepatic encephalopathy (MHE) adversely impacts patients with cirrhosis and more precise therapies are needed. Gut-brain axis changes are therapeutic targets, but prior studies have largely focused on bacterial changes. Our aim was to determine linkages between individual cognitive testing results and bacteria with the virome using a cross-sectional and longitudinal approach. We included cross-sectional (n = 138) and longitudinal analyses (n = 36) of patients with cirrhosis tested using three cognitive modalities, which were psychometric hepatic encephalopathy score (PHES), inhibitory control test (ICT), Stroop, and all three. Stool metagenomics with virome and bacteriome were analyzed studied cross-sectionally and in a subset followed for development/reversal of MHE repeated at 6 months (longitudinally only using PHES). Cross-sectional: We found no significant changes in α/ß diversity in viruses or bacteria regardless of cognitive testing. Cognitively impaired patients were more likely to have higher relative abundance of bacteriophages linked with Streptococcus, Faecalibacterium, and Lactobacillus, which were distinct based on modality. These were also linked with cognition on correlation networks. Longitudinally, 27 patients remained stable while 9 changed their MHE status. Similar changes in phages that are linked with Streptococcus, Faecalibacterium, and Lactobacillus were seen. These phages can influence ammonia, lactate, and short-chain fatty acid generation, which are neuro-active. In conclusion, we found linkages between bacteriophages and cognitive function likely due to impact on bacteria that produce neuroactive metabolites cross-sectionally and longitudinally. These findings could help explore bacteriophages as options to influence treatment for MHE in cirrhosis.

A double-blind randomized placebo-controlled trial of albumin in outpatients with hepatic encephalopathy: HEAL study.

BACKGROUND & AIMS: Even after recovery from overt hepatic encephalopathy (HE), minimal HE (MHE), which impairs quality of life (QoL), can persist. A double-blind, placebo-controlled randomized clinical trial was performed to determine the impact of albumin vs. saline on MHE and QoL in individuals with prior HE already on standard of care. METHODS: Outpatients with cirrhosis and prior HE, MHE and hypoalbuminemia already on treatment for HE were included. Patients on regular IV albumin infusions were excluded. Participants were randomized 1:1 to receive either weekly infusions of 25% IV albumin 1.5 g/kg or saline over 5 weeks. MHE was defined using either psychometric hepatic encephalopathy score (PHES), Stroop or critical clicker frequency. MHE, QoL (based on sickness impact profile [SIP] total, physical, psychosocial domain) and serum markers (inflammation, endothelial dysfunction, and ischemia-modified albumin) were compared between baseline, the final infusion visit (end-of-drug [EOD]) and 1-week post final infusion (end-of-study [EOS]). RESULTS: Forty-eight (24/group) participants were randomized and balanced (including by HE medication use) at baseline. Adverse events were similar, with MELD and ammonia remaining stable between/within groups. Albumin levels increased and ischemia-modified albumin decreased only in the albumin group at EOD and EOS vs. baseline. PHES and Stroop MHE reversal and improvement were greater in the albumin group at EOD and persisted at EOS. SIP total and psychosocial, but not physical, domain improved only in the albumin group at EOD and EOS vs. baseline. A significant reduction in IL-1ß and endothelial dysfunction markers was also observed in the albumin group. CONCLUSION: In a double-blind, placebo-controlled trial of outpatients with cirrhosis, prior HE and current MHE, albumin infusions were associated with improved cognitive function and psychosocial QoL, likely due to amelioration of endothelial dysfunction. CLINICAL TRIALS REGISTRATION: www. CLINICALTRIALS: gov NCT03585257. IMPACT AND IMPLICATIONS: Even after recovery from overt hepatic encephalopathy (HE), minimal HE (MHE), which impairs quality of life, can persist. We found that intravenous albumin infusions were associated with improved cognitive function and psychosocial quality of life, likely owing to amelioration of endothelial dysfunction, compared to placebo in outpatients with prior HE and current MHE. In patients who continue to demonstrate cognitive dysfunction and impaired quality of life despite standard of care therapy for HE, albumin infusions could be considered if these results are validated.

Stool microbiota are superior to saliva in distinguishing cirrhosis and hepatic encephalopathy using machine learning.

BACKGROUND & AIMS: Saliva and stool microbiota are altered in cirrhosis. Since stool is logistically difficult to collect compared to saliva, it is important to determine their relative diagnostic and prognostic capabilities. We aimed to determine the ability of stool vs. saliva microbiota to differentiate between groups based on disease severity using machine learning (ML). METHODS: Controls and outpatients with cirrhosis underwent saliva and stool microbiome analysis. Controls vs. cirrhosis and within cirrhosis (based on hepatic encephalopathy [HE], proton pump inhibitor [PPI] and rifaximin use) were classified using 4 ML techniques (random forest [RF], support vector machine, logistic regression, and gradient boosting) with AUC comparisons for stool, saliva or both sample types. Individual microbial contributions were computed using feature importance of RF and Shapley additive explanations. Finally, thresholds for including microbiota were varied between 2.5% and 10%, and core microbiome (DESeq2) analysis was performed. RESULTS: Two hundred and sixty-nine participants, including 87 controls and 182 patients with cirrhosis, of whom 57 had HE, 78 were on PPIs and 29 on rifaximin were included. Regardless of the ML model, stool microbiota had a significantly higher AUC in differentiating groups vs. saliva. Regarding individual microbiota: autochthonous taxa drove the difference between controls vs. patients with cirrhosis, oral-origin microbiota the difference between PPI users/non-users, and pathobionts and autochthonous taxa the difference between rifaximin users/non-users and patients with/without HE. These were consistent with the core microbiome analysis results. CONCLUSIONS: On ML analysis, stool microbiota composition is significantly more informative in differentiating between controls and patients with cirrhosis, and those with varying cirrhosis severity, compared to saliva. Despite logistic challenges, stool should be preferred over saliva for microbiome analysis. LAY SUMMARY: Since it is harder to collect stool than saliva, we wanted to test whether microbes from saliva were better than stool in differentiating between healthy people and those with cirrhosis and, among those with cirrhosis, those with more severe disease. Using machine learning, we found that microbes in stool were more accurate than saliva alone or in combination, therefore, stool should be preferred for analysis and collection wherever possible.

Multiple bacterial virulence factors focused on adherence and biofilm formation associate with outcomes in cirrhosis.

BACKGROUND & AIMS: Altered gut microbiota is associated with poor outcomes in cirrhosis, including infections and hepatic encephalopathy (HE). However, the role of bacterial virulence factors (VFs) is unclear. Aim: Define association of VFs with cirrhosis severity and infections, their linkage with outcomes, and impact of fecal microbiota transplant (FMT). METHODS: VF abundances were determined using metagenomic analysis in stools from controls and cirrhosis patients (compensated, HE-only, ascites-only, both and infected). Patients were followed for 90-day hospitalizations and 1-year death. Stool samples collected before/after a placebo-controlled FMT trial were also analyzed. Bacterial species and VFs for all species and selected pathogens (Escherichia, Klebsiella, Pseudomonas, Staphylococcus, Streptococcus, and Enterococcus spp) were compared between groups. Multi-variable analyses were performed for clinical biomarkers and VFs for outcome prediction. Changes in VFs pre/post-FMT and post-FMT/placebo were analyzed. Results: We included 233 subjects (40 controls, 43 compensated, 30 HE-only, 20 ascites-only, 70 both, and 30 infected). Decompensated patients, especially those with infections, had higher VFs coding for siderophores, biofilms, and adhesion factors versus the rest. Biofilm and adhesion VFs from Enterobacteriaceae and Enterococcus spp associated with death and hospitalizations independent of clinical factors regardless of when all VFs or selected pathogens were analyzed. FMT was associated with reduced VF post-FMT versus pre-FMT and post-placebo groups. CONCLUSIONS: Virulence factors from multiple species focused on adhesion and biofilms increased with decompensation and infections, associated with death and hospitalizations independent of clinical factors, and were attenuated with FMT. Strategies focused on targeting multiple virulence factors could potentially impact outcomes in cirrhosis. PRESENTATIONS: Portions of this manuscript were an oral presentation in the virtual International Liver Congress 2021. ABBREVIATIONS: VF: virulence factors, HE: hepatic encephalopathy, FMT: Fecal microbiota transplant, PPI: proton pump inhibitors, LPS: lipopolysaccharides, VFDB: Virulence factor database, OTU: operational taxonomic units, SBP: spontaneous bacterial peritonitis, UTI: urinary tract infections, MRSA: methicillin resistant Staphylococcus aureus, VRE: vancomycin-resistant Enterococcus, MAAsLin2: Microbiome Multivariable Associations with Linear Models, LPS: lipopolysaccharides, AKI: acute kidney injury.

Distinct gut microbial compositional and functional changes associated with impaired inhibitory control in patients with cirrhosis.

Most cirrhosis etiologies, such as alcohol, hepatitis C, and obesity, involve behavior that require the loss of inhibitory control. Once cirrhosis develops, patients can also develop cognitive impairment due to minimal hepatic encephalopathy (MHE). Both processes could have distinct imprints on the gut-liver-brain axis. Determine the impact of inhibitory control versus traditional cirrhosis-related cognitive performance on gut microbial composition and function. Outpatients with cirrhosis underwent two tests for MHE: inhibitory control test (MHEICT, computerized associated with response inhibition) and psychometric hepatic encephalopathy score (MHEPHES, paper-pencil HE-specific associated with subcortical impairment) along with stool collection for metagenomics. MHEICT/not, MHEPHES/not, and discordant (positive on one test but negative on the other) were analyzed for demographics, bacterial species, and gut-brain modules (GBM) using multi-variable analyses. Ninety-seven patients [47 (49%) MHEPHES, 76 (78%) MHEICT, 41 discordant] were enrolled. MHEPHES/not: Cirrhosis severity was worse in MHEPHES without differences in alpha/beta diversity on bacterial species or GBMs. Pathobionts (Enterobacteriaceae) and γ-amino-butryic acid (GABA) synthesis GBM were higher in MHEPHES. MHEICT/not: We found similar cirrhosis severity and metagenomic alpha/beta diversity in MHEICT versus not. However, alpha/beta diversity of GBMs were different in MHEICT versus No-MHE patients. Alistipes ihumii, Prevotella copri, and Eubacterium spp. were higher, while Enterococcus spp. were uniquely lower in MHEICT versus no-MHE and discordant comparisons. GBMs belonging to tryptophan, menaquinone, GABA, glutamate, and short-chain fatty acid synthesis were also unique to MHEICT. Gut microbial signature of impaired inhibitory control, which is associated with addictive disorders that can lead to cirrhosis, is distinct from cirrhosis-related cognitive impairment.

Impact of Antibiotic Resistance Genes in Gut Microbiome of Patients With Cirrhosis.

BACKGROUND AND AIMS: Cirrhosis is associated with changes in intestinal microbiota that can lead to hepatic encephalopathy (HE) and infections, especially with antibiotic-resistant organisms. However, the impact of gut microbial antibiotic resistance gene (ARG) burden on clinical outcomes is unclear. The aims of the study were to determine the impact of ARGs in cirrhosis-related gut metagenome on outcomes and disease progression, study the effect of rifaximin on ARG burden, and compare ARGs in cirrhosis with chronic kidney disease (CKD) and diabetes. METHODS: In outpatients with cirrhosis who underwent metagenomics, we evaluated change in ARG abundances with progression and their multivariable impact on 90-day hospitalizations and deaths over 1 year. We also studied ARGs pre- and 8 weeks post-rifaximin in patients with compensated cirrhosis in an open-label trial. Finally, ARGs from CKD and diabetes studies were compared with cirrhosis on machine learning. RESULTS: A total of 163 patients with cirrhosis (43 compensated, 20 ascites-only, 30 HE-only, 70 both) and 40 controls were included. ARG abundances were higher in cirrhosis versus controls and worsened with advancing cirrhosis severity; 44 patients were hospitalized and 14 died. ARG abundances were associated with hospitalizations and mortality while controlling for cirrhosis complications, medications, and demographics. Rifaximin trial: ARG abundance patterns were minimally affected in 19 patients post-rifaximin. CKD/diabetes comparison: ARG abundance patterns in cirrhosis are distinguishable on machine learning and include more gram-positive ARGs. CONCLUSIONS: Cirrhosis is associated with high gut microbial ARG gene burden compared with controls, which worsens with disease progression and may be different from CKD and diabetes. ARGs are not affected by rifaximin and are associated with hospitalizations and death.

Perspectives of Inpatients With Cirrhosis and Caregivers on Using Health Information Technology: Cross-sectional Multicenter Study.

BACKGROUND: Health information technology (IT) interventions to decrease readmissions for cirrhosis may be limited by patient-associated factors. OBJECTIVE: The aim of this study was to determine perspectives regarding adoption versus refusal of health IT interventions among patient-caregiver dyads. METHODS: Inpatients with cirrhosis and their caregivers were approached to participate in a randomized health IT intervention trial requiring daily contact with research teams via the Patient Buddy app. This app focuses on ascites, medications, and hepatic encephalopathy over 30 days. Regression analyses for characteristics associated with acceptance were performed. For those who declined, a semistructured interview was performed with themes focused on caregivers, protocol, transport/logistics, technology demands, and privacy. RESULTS: A total of 349 patient-caregiver dyads were approached (191 from Virginia Commonwealth University, 56 from Richmond Veterans Affairs Medical Center, and 102 from Mayo Clinic), 87 of which (25%) agreed to participate. On regression, dyads agreeing included a male patient (odds ratio [OR] 2.08, P=.01), gastrointestinal bleeding (OR 2.3, P=.006), or hepatic encephalopathy admission (OR 2.0, P=.01), whereas opioid use (OR 0.46, P=.03) and alcohol-related etiology (OR 0.54, P=.02) were associated with refusal. Race, study site, and other admission reasons did not contribute to refusing participation. Among the 262 dyads who declined randomization, caregiver reluctance (43%), perceived burden (31%), technology-related issues (14%), transportation/logistics (10%), and others (4%), but not privacy, were highlighted as major concerns. CONCLUSIONS: Patients with cirrhosis admitted with hepatic encephalopathy and gastrointestinal bleeding without opioid use or alcohol-related etiologies were more likely to participate in a health IT intervention focused on preventing readmissions. Caregiver and study burden but not privacy were major reasons to decline participation. Reducing perceived patient-caregiver burden and improving communication may improve participation. TRIAL REGISTRATION: ClinicalTrials.gov NCT03564626; https://www.clinicaltrials.gov/ct2/show/NCT03564626.

Fecal Microbiota Transplant in Cirrhosis Reduces Gut Microbial Antibiotic Resistance Genes: Analysis of Two Trials.

Antibiotic resistance leads to poor outcomes in cirrhosis. Fecal microbiota transplant (FMT) is associated with reduction in antibiotic resistance gene (ARG) burden in patients without cirrhosis; however, the impact in cirrhosis is unclear. We aimed to study the effect of capsule and enema FMT on ARG abundance in fecal samples, which were collected during two published FMT trials in patients with cirrhosis on rifaximin, lactulose, and proton pump inhibitors. ARGs were identified using metagenomics and mapped against the Comprehensive Antibiotic Resistance Database. Changes in ARG abundance were studied within/between groups. The capsule FMT trial involved a one-time FMT or placebo capsule administration with stool collection at baseline and week 4 postintervention. Antibiotics+enema FMT included preprocedure antibiotics followed by FMT enema versus standard-of-care (SOC). Stool was collected at baseline, postantibiotics, and day 7/15 postintervention. Both trials included 20 patients each. There was no safety/infection signal linked to FMT. In the capsule trial, beta-lactamase (OXY/LEN) expression decreased post-FMT versus baseline. Compared to placebo, patients who were post-FMT had lower abundance of vancomycin (VanH), beta-lactamase (ACT), and rifamycin ARGs; the latter was associated with cognitive improvement. No changes were seen within patients treated with placebo. In the antibiotics+enema trial for postantibiotics at day 7 versus baseline, there was an increase in vancomycin and beta-lactamase ARGs, which decreased at day 15. However, quinolone resistance increased at day 15 versus baseline. Between SOC and FMT, day 7 had largely lower ARG (CfxA beta-lactamase, VanW, and VanX) that continued at day 15 (cepA beta-lactamase, VanW). No changes were seen within the SOC group. Conclusion: Despite differences in routes of administration and preintervention antibiotics, we found that ARG abundance is largely reduced after FMT compared to pre-FMT baseline and non-FMT groups in decompensated cirrhosis.

Gut Microbial Signature of Hepatocellular Cancer in Men With Cirrhosis.

The gut microbiome is altered in cirrhosis. Recent evidence has suggested a key role for the gut microbiota in the progression of cirrhosis and the development of hepatocellular carcinoma (HCC). We studied the differences in the microbial composition in patients with cirrhosis with prior and future HCC in the context of other complications (eg, infections, hepatic encephalopathy). The following 2 cohorts were recruited prospectively: the prior HCC cohort, in which outpatients with HCC within 2 years were age-matched, sex-matched, and Model for End-Stage Liver Disease (MELD) score-matched with those without HCC; and the future HCC cohort, in which patients were followed for 2 years and divided into future HCC versus no HCC after age, sex, and MELD-score matching and other complications were also recorded. Microbiota composition and predicted function were analyzed with ribosomal RNA sequencing and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PiCRUST)and compared between (1) prior HCC versus none and (2) future HCC versus none, and in the future cohort, comparisons were also made between those patients who developed (1) HCC only versus without complications, (2) HCC only versus non-HCC complications only, and (3) HCC + other complications versus non-HCC complications only. A total of 142 men (76 total in the prior cohort [38 with/38 without HCC] and 66 total in the future cohort [33 with/33 without future HCC]) were included. The groups had similar etiology, lactulose/rifaximin/proton pump inhibitor use, diabetes mellitus, and non-HCC complications. Microbial diversity was similar between prior HCC/not or future HCC/not. On DESeq2 higher Clostridium sensu stricto and Anaerotruncus were significantly associated with protection from HCC, whereas the reverse was seen with Raoultella and Haemophilus regardless of prior/future HCC comparisons. Functions focused on urea cycle, bioenergetics, tryptophan, and toluene metabolism were different between the groups. Rothia was specific for other complications. Despite age, sex, and MELD-score matching and accounting for other complications, gut microbiota composition and the predicted function are different in men with cirrhosis with and without prior HCC and can be extended toward future HCC development.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Sex is associated with differences in gut microbial composition and function in hepatic encephalopathy.

BACKGROUND & AIMS: Altered microbiota can affect the gut-liver-brain axis in cirrhosis and hepatic encephalopathy (HE), but the impact of sex on these changes is unclear. We aimed to determine differences in fecal microbiota composition/functionality between men and women with cirrhosis and HE on differing treatments. METHODS: Cross-sectional stool microbiome composition (16s rRNA sequencing) and microbial functional analyses were performed in men and women with cirrhosis, and controls. Patients with HE on rifaximin+lactulose (HE-Rif), patients with HE on lactulose only (HE-Lac) and those with cirrhosis without HE (No-HE) were compared to controls using random forest classifier. Men and women were also compared. RESULTS: A total of 761 individuals were included, 619 with cirrhosis (466 men, 153 women) and 142 controls (92 men, 50 women). Men were older and more frequently used proton pump inhibitors (PPIs), but model for end-stage liver disease score, No-HE (n = 319), HE-lac (n = 130) and HE-Rif (n = 170) proportions were similar. PPI/age-adjusted AUC of differentiation between controls vs. all cirrhosis, and controls vs. No-HE were higher within women than men, but the adjusted AUC for No-HE vs. HE-Rif was higher in men. Control vs. HE-Rif differentiation was similar across sexes. Men vs. women were different in all cirrhosis, No-HE and HE-Lac but not HE-Rif on PERMANOVA and AUC analyses. Autochthonous taxa decreased and pathobionts increased with disease progression regardless of sex. In men, Lactobacillaceae were higher in HE-Lac but decreased in HE-Rif, along with Veillonellaceae. Pathways related to glutamate and aromatic compound degradation were higher in men at all stages. Degradation of androstenedione, an estrogenic precursor, was lower in men vs. women in HE-Rif, likely enhancing feminization. CONCLUSIONS: There are differences in gut microbial function and composition between men and women with cirrhosis, which could be implicated in differential responses to HE therapies. Further studies linking these differences to sex-specific outcomes are needed. LAY SUMMARY: Patients with cirrhosis develop changes in their brain function, and men often develop feminization with disease progression. However, the interaction between sex, microbiota and disease severity is unclear. We found that as disease progressed in men, their microbial composition began to approach that observed in women, with changes in specific microbes that are associated with male hormone metabolism.

A Randomized Clinical Trial of Fecal Microbiota Transplant for Alcohol Use Disorder.

BACKGROUND AND AIMS: Alcohol use disorder (AUD) is associated with microbial alterations that worsen with cirrhosis. Fecal microbiota transplant (FMT) could be a promising approach. APPROACH AND RESULTS: In this phase 1, double-blind, randomized clinical trial, patients with AUD-related cirrhosis with problem drinking (AUDIT-10 > 8) were randomized 1:1 into receiving one placebo or FMT enema from a donor enriched in Lachnospiraceae and Ruminococcaceae. Six-month safety was the primary outcome. Alcohol craving questionnaire, alcohol consumption (urinary ethylglucuronide/creatinine), quality of life, cognition, serum IL-6 and lipopolysaccharide-binding protein, plasma/stool short-chain fatty acids (SCFAs), and stool microbiota were tested at baseline and day 15. A 6-month follow-up with serious adverse event (SAE) analysis was performed. Twenty patients with AUD-related cirrhosis (65 ± 6.4 years, all men, Model for End-Stage Liver Disease 8.9 ± 2.7) with similar demographics, cirrhosis, and AUD severity were included. Craving reduced significantly in 90% of FMT versus 30% in placebo at day 15 (P = 0.02) with lower urinary ethylglucuronide/creatinine (P = 0.03) and improved cognition and psychosocial quality of life. There was reduction in serum IL-6 and lipopolysaccharide-binding protein and increased butyrate/isobutyrate compared with baseline in FMT but not placebo. Microbial diversity increased with higher Ruminococcaceae and other SCFAs, producing taxa following FMT but not placebo, which were linked with SCFA levels. At 6 months, patients with any SAEs (8 vs. 2, P = 0.02), AUD-related SAEs (7 vs. 1, P = 0.02), and SAEs/patient (median [interquartile range], 1.5 [1.25] vs. 0 [0.25] in FMT, P = 0.02) were higher in placebo versus FMT. CONCLUSIONS: This phase 1 trial shows that FMT is safe and associated with short-term reduction in alcohol craving and consumption with favorable microbial changes versus placebo in patients with alcohol-associated cirrhosis with alcohol misuse. There was also a reduction in AUD-related events over 6 months in patients assigned to FMT.

Serum Levels of Metabolites Produced by Intestinal Microbes and Lipid Moieties Independently Associated With Acute-on-Chronic Liver Failure and Death in Patients With Cirrhosis.

BACKGROUND & AIMS: Inpatients with cirrhosis have high rates of acute-on-chronic failure (ACLF) development and high mortality within 30 days of admission to the hospital. Better biomarkers are needed to predict these outcomes. We performed metabolomic analyses of serum samples from patients with cirrhosis at multiple centers to determine whether metabolite profiles might identify patients at high risk for ACLF and death. METHODS: We performed metabolomic analyses, using liquid chromatography, of serum samples collected at time of admission to 12 North American tertiary hepatology centers from 602 patients in the North American Consortium for the Study of End-Stage Liver Disease sites from 2015 through 2017 (mean age, 56 years; 61% men; mean model for end-stage liver disease score, 19.5). We performed analysis of covariance, adjusted for model for end-stage liver disease at time of hospital admission, serum levels of albumin and sodium, and white blood cell count, to identify metabolites that differed between patients who did vs did not develop ACLF and patients who did vs did not die during hospitalization and within 30 days. We performed random forest analysis to identify specific metabolite(s) that were associated with outcomes and area under the curve (AUC) analyses to analyze them in context of clinical parameters. We analyzed microbiomes of stool samples collected from 133 patients collected at the same time and examined associations with serum metabolites. RESULTS: Of the 602 patients analyzed, 88 developed ACLF (15%), 43 died in the hospital (7%), and 72 died within 30 days (12%). Increased levels of compounds of microbial origin (aromatic compounds, secondary or sulfated bile acids, and benzoate) and estrogen metabolites, as well as decreased levels of phospholipids, were associated with development of ACLF, inpatient, and 30-day mortality and were also associated with fecal microbiomes. Random forest analysis and logistic regression showed that levels of specific microbially produced metabolites identified patients who developed ACLF with an AUC of 0.84 (95% confidence interval [CI] 0.78-0.88; P = .001), patients who died while in the hospital with an AUC of 0.81 (95% CI 0.74-0.85; P = .002), and patients who died within 30 days with an AUC of 0.77 (95% CI 0.73-0.81; P = .02). The metabolites were significantly additive to clinical parameters for predicting these outcomes. Metabolites associated with outcomes were also correlated with microbiomes of stool samples. CONCLUSIONS: In an analysis of serum metabolites and fecal microbiomes of patients hospitalized with cirrhosis at multiple centers, we associated metabolites of microbial origin and lipid moieties with development of ACLF and death as an inpatient or within 30 days, after controlling for clinical features.

Minimal Hepatic Encephalopathy and Mild Cognitive Impairment Worsen Quality of Life in Elderly Patients With Cirrhosis.

BACKGROUND & AIMS: Patients with cirrhosis are growing older. The overlap between minimal hepatic encephalopathy (MHE) and predementia mild cognitive impairment (MCI) could affect quality of life (QOL). We investigated the performance of elderly patients with cirrhosis on tests for MHE and MCI and their effects on QOL. METHODS: We recruited outpatients with cirrhosis (n = 109) and without cirrhosis (controls, n = 100), 65 years or older, at 4 centers (derivation cohort). All study participants were assessed for psychometric hepatic encephalopathy score (PHES), EncephalApp score, and QOL. MCI was tested in patients with cirrhosis using the repeatable battery for assessment of neuropsychological status and assigned to the following groups: unimpaired, MCI only, MHE only, and MCI+MHE. We created adjusted norms to detect MHE using PHES and EncephalApp scores from the controls. Findings were validated using data from a separate cohort of 77 patients with cirrhosis (mean age, 69.49 ± 4.36 y; 72% men) at the same study sites. RESULTS: Controls were older but were more educated, performed better cognitively, and had better QOL. Among patients with cirrhosis, age, education, model for end-stage liver disease score, EncephalApp score, and QOL were similar, but PHES and repeatable battery for assessment of neuropsychological status differed among sites. In the derivation cohort, the presence of MHE, with or without MCI, was associated with poor QOL, which was lowest in the MCI+MHE group. When we adjusted for age, sex, and education, 49% of patients with cirrhosis had MHE based on the EncephalApp and 8% had MHE based on the PHES. A similar pattern (49% MHE based on EncephalApp and 6% MHE based on PHES) was found in a validation cohort. CONCLUSIONS: In a multicenter study of patients with cirrhosis (>65 y) and controls, the presence of MHE, regardless of MCI, was associated with poor cognition and QOL. We created adjusted norms that defined the high sensitivity of EncephalApp for the detection of MHE in older individuals and validated it in a separate cohort.

Cognition and hospitalizations are linked with salivary and faecal microbiota in cirrhosis cohorts from the USA and Mexico.

BACKGROUND & AIMS: Gut microbiota are affected by diet and ethnicity, which impacts cognition and hospitalizations in cirrhosis. AIM: Study interactions of diet with microbiota and impact on hospitalizations and cognition in American and Mexican cohorts. METHODS: Controls and age-balanced patients with compensated/decompensated cirrhosis were included and followed for 90-day hospitalizations. A subset underwent minimal hepatic encephalopathy (MHE) testing. Parameters such as dietary, salivary and faecal microbiota (diversity, taxa analysis, cirrhosis dysbiosis ratio CDR:high = good) between/within countries were analysed. Regression analyses for hospitalizations and MHE were performed. RESULTS: In all, 275 age-balanced subjects (133 US [40 Control, 50 Compensated, 43 Decompensated] and 142 Mexican [41 Control, 49 Compensated, 52 Decompensated]) were enrolled. MELD/cirrhosis severity was comparable. Diet showed lower protein and animal fat intake in all decompensated patients, but this was worse in Mexico. Diversity was lower in stool and saliva in decompensated patients, and worse in Mexican cohorts. Prevotellaceae were lower in decompensated cirrhosis, particularly those with lower animal fat/protein consumption across countries. Hospitalizations were higher in Mexico vs the USA (26% vs 14%, P = .04). On regression, Prevotellaceae, Ruminococcaceae and Lachnospiraceae lowered hospitalization risk independent of MELD and ascites. MHE testing was performed in 120 (60/country and 20/subgroup) subjects and MHE rate was similar. MELD and decompensation increased while CDR and Prevotellaceae decreased the risk of MHE. CONCLUSIONS: Changes in diet and microbiota, especially related to animal fat and protein intake and Prevotellaceae, are associated with MHE and hospitalizations in Mexican patients with cirrhosis compared to an American cohort. Nutritional counselling to increase protein intake in cirrhosis could help prevent these hospitalizations.

Metabolomics and microbial composition increase insight into the impact of dietary differences in cirrhosis.

BACKGROUND & AIMS: Dietary changes can modulate gut microbiota and interact with cirrhosis. Our prior study demonstrated that microbial diversity was higher in cirrhotics from Turkish vs the USA, which was associated with lower risk of 90-day hospitalizations. We aimed to define gut microbial functional and metabolomic changes to increase insight into benefits of the Mediterranean compared to Western diets. METHODS: In all, 139 Turkish (46 controls/50 compensated/43 decompensated) and 157 American subjects (48 controls/59 compensated/50 decompensated) were studied. Turkish subjects consumed a modified Mediterranean diet with daily fermented milk intake, whereas Americans consumed a Western diet. Predicted gut microbial functionalities and plasma metabolomics were compared between/within countries. Correlation network differences between microbiota and metabolites in cirrhotics from Turkey vs the USA were evaluated. RESULTS: Predicted microbial function showed lower amino acid, bioenergetics and lipid pathways, with functions related to vitamin B, glycan, xenobiotic metabolism, DNA/RNA synthesis, in cirrhotics from Turkey compared to the USA. Plasma metabolomics demonstrated higher relative lactate levels in Turkey vs the USA. The metabolite changes in decompensated cirrhosis, compared to controls, showed similar trends in Turkey and the USA, with reduced lipids and phosphocholines. Phosphocholines were significantly lower in patients hospitalized in 90 days (P = .03). Correlation networks in cirrhotics demonstrated linkage differences between beneficial taxa, Blautia and Oscillispira, and lactate and unsaturated lipids, in Turkey compared to American patients. CONCLUSIONS: A modified Mediterranean diet was associated with altered plasma metabolomics and beneficially alters microbiota functionality and correlations compared to Western diet in cirrhosis. These altered diet-microbial interactions could potentially affect the 90-day hospitalization risk.

Neuroinflammation in Murine Cirrhosis Is Dependent on the Gut Microbiome and Is Attenuated by Fecal Transplant.

Cirrhosis and hepatic encephalopathy (HE) is associated with an altered gut-liver-brain axis. Fecal microbial transplant (FMT) after antibiotics improves outcomes in HE, but the impact on brain function is unclear. The aim of this study is to determine the effect of colonization using human donors in germ-free (GF) mice on the gut-liver-brain axis. GF and conventional mice were made cirrhotic using carbon tetrachloride and compared with controls in GF and conventional state. Additional GF mice were colonized with stool from controls (Ctrl-Hum) and patients with cirrhosis (Cirr-Hum). Stools from patients with HE cirrhosis after antibiotics were pooled (pre-FMT). Stools from the same patients 15 days after FMT from a healthy donor were also pooled (post-FMT). Sterile supernatants were created from pre-FMT and post-FMT samples. GF mice were colonized using stools/sterile supernatants. For all mice, frontal cortex, liver, and small/large intestines were collected. Cortical inflammation, synaptic plasticity and gamma-aminobutyric acid (GABA) signaling, and liver inflammation and intestinal 16s ribosomal RNA microbiota sequencing were performed. Conventional cirrhotic mice had higher degrees of neuroinflammation, microglial/glial activation, GABA signaling, and intestinal dysbiosis compared with other groups. Cirr-Hum mice had greater neuroinflammation, microglial/glial activation, and GABA signaling and lower synaptic plasticity compared with Ctrl-Hum mice. This was associated with greater dysbiosis but no change in liver histology. Pre-FMT material colonization was associated with neuroinflammation and microglial activation and dysbiosis, which was reduced significantly with post-FMT samples. Sterile pre-FMT and post-FMT supernatants did not affect brain parameters. Liver inflammation was unaffected. Conclusion: Fecal microbial colonization from patients with cirrhosis results in higher degrees of neuroinflammation and activation of GABAergic and neuronal activation in mice regardless of cirrhosis compared with those from healthy humans. Reduction in neuroinflammation by using samples from post-FMT patients to colonize GF mice shows a direct effect of fecal microbiota independent of active liver inflammation or injury.

Variability and Lability of Ammonia Levels in Healthy Volunteers and Patients With Cirrhosis: Implications for Trial Design and Clinical Practice.

INTRODUCTION: Ammonia levels are used to assess hepatic encephalopathy, but their levels are highly variable in clinical practice. METHODS: We studied factors associated with variation in ammonia values in cirrhotic patients without previous hepatic encephalopathy and healthy volunteers (HVs). RESULTS: Ammonia increased by 12% and 18% at 1 and 2 hour, respectively, after a protein meal in 64 cirrhotic patients (P < 0.001). In 237 HVs, ammonia levels varied significantly between sites (P < 0.0001). New site-specific ammonia upper limits based on HV levels using a strict analysis protocol differed from routinely used values. Correlation between paired fresh samples was high (r = 0.83) but modest between fresh and frozen samples (r = 0.62). DISCUSSION: Sample handling, processing, and protein intake impact ammonia levels across sites.

Microbial functional change is linked with clinical outcomes after capsular fecal transplant in cirrhosis.

BACKGROUNDHepatic encephalopathy (HE) is associated with poor outcomes. A prior randomized, pilot trial demonstrated safety after oral capsular fecal microbial transplant (FMT) in HE, with favorable changes in microbial composition and cognition. However, microbial functional changes are unclear. The aim of this study was to determine the effect of FMT on the gut-brain axis compared with placebo, using microbial function based on bile acids (BAs), inflammation (serum IL-6, LPS-binding protein [LBP]), and their association with EncephalApp.METHODSTwenty cirrhotic patients were randomized 1:1 into groups that received 1-time FMT capsules from a donor enriched in Lachnospiraceae and Ruminococcaceae or placebo capsules, with 5-month follow-up for safety outcomes. Stool microbiota and BA; serum IL-6, BA, and LBP; and EncephalApp were analyzed at baseline and 4 weeks after FMT/placebo. Correlation networks among microbiota, BAs, EncephalApp, IL-6, and LBP were performed before/after FMT.RESULTSFMT-assigned participants had 1 HE recurrence and 2 unrelated infections. Six placebo-assigned participants developed negative outcomes. FMT, but not placebo, was associated with reduced serum IL-6 and LBP and improved EncephalApp. FMT-assigned participants demonstrated higher deconjugation and secondary BA formation in feces and serum compared with baseline. No change was seen in placebo. Correlation networks showed greater complexity after FMT compared with baseline. Beneficial taxa, such as Ruminococcaceae, Verrucomicrobiaceae, and Lachnospiraceae, were correlated with cognitive improvement and decrease in inflammation after FMT. Fecal/serum secondary/primary ratios and PiCRUST secondary BA pathways did not increase in participants who developed poor outcomes.CONCLUSIONGut microbial function in cirrhosis is beneficially affected by capsular FMT, with improved inflammation and cognition. Lower secondary BAs in FMT recipients could select for participants who develop negative outcomes.TRIAL REGISTRATIONClinicaltrials.gov NCT03152188.FUNDINGNational Center for Advancing Translational Sciences NIH grant R21TR002024, VA Merit Review grant 2I0CX001076, the United Kingdom National Institute for Health Research Biomedical Facility at Imperial College London, the British Heart Foundation, Wellcome Trust, and King's College London.