Altered profile of human gut microbiome is associated with cirrhosis and its complications.

BACKGROUND & AIMS: The gut microbiome is altered in cirrhosis; however its evolution with disease progression is only partly understood. We aimed to study changes in the microbiome over cirrhosis severity, its stability over time and its longitudinal alterations with decompensation. METHODS: Controls and age-matched cirrhotics (compensated/decompensated/hospitalized) were included. Their stool microbiota was quantified using multi-tagged pyrosequencing. The ratio of autochthonous to non-autochthonous taxa was calculated as the cirrhosis dysbiosis ratio (CDR); a low number indicating dysbiosis. Firstly, the microbiome was compared between controls and cirrhotic sub-groups. Secondly, for stability assessment, stool collected twice within 6months in compensated outpatients was analyzed. Thirdly, changes after decompensation were assessed using (a) longitudinal comparison in patients before/after hepatic encephalopathy development (HE), (b) longitudinal cohort of hospitalized infected cirrhotics MELD-matched to uninfected cirrhotics followed for 30days. RESULTS: 244 subjects [219 cirrhotics (121 compensated outpatients, 54 decompensated outpatients, 44 inpatients) and 25 age-matched controls] were included. CDR was highest in controls (2.05) followed by compensated (0.89), decompensated (0.66), and inpatients (0.32, p<0.0001) and negatively correlated with endotoxin. Microbiota and CDR remained unchanged in stable outpatient cirrhotics (0.91 vs. 0.86, p=0.45). In patients studied before/after HE development, dysbiosis occurred post-HE (CDR: 1.2 to 0.42, p=0.03). In the longitudinal matched-cohort, microbiota were significantly different between infected/uninfected cirrhotics at baseline and a low CDR was associated with death and organ failures within 30days. CONCLUSIONS: Progressive changes in the gut microbiome accompany cirrhosis and become more severe in the setting of decompensation. The cirrhosis dysbiosis ratio may be a useful quantitative index to describe microbiome alterations accompanying cirrhosis progression.

Diagnosis of covert hepatic encephalopathy without specialized tests.

BACKGROUND & AIMS: Covert hepatic encephalopathy (CHE) impairs quality of life (QOL) and can be difficult to diagnose. Patient-administered methods that do not require specialized tests or equipment might increase rates of detection. We performed a longitudinal study to determine whether demographic data and responses to a validated QOL questionnaire, the Sickness Impact Profile (SIP), can identify patients with CHE. METHODS: Patients with cirrhosis without prior overt HE were recruited from outpatient liver clinics at the Virginia Commonwealth University Medical Center, from August 2008 through February 2012. We performed cognitive tests on 170 patients (mean age, 55 y; mean model for end-stage liver disease score, 9; 50% with hepatitis C-associated and 11% with alcohol-associated cirrhosis). Patients also were given the SIP questionnaire (136 questions on 12 QOL topics, requiring a yes or no answer) at enrollment, at 6 months, and at 12 months. The proportion of patients that responded "yes" to each question was compared between those with and without CHE. Patient variables (noncognitive), demographics (age, education, sex, alcoholic etiology), and SIP questions that produced different responses between groups were analyzed by logistic regression and receiver operating characteristic analyses. RESULTS: Based on cognitive test results, 93 patients (55%) had CHE when the study began. They had a higher proportion of "yes" responses to 54 questions on the SIP questionnaire, across all categories. We developed a formula to identify patients with CHE based on age, sex, and responses to 4 SIP questions (a SIP CHE score). Baseline SIP CHE scores greater than 0 identified patients with CHE with 80% sensitivity and 79% specificity. Of the 98 patients who returned for the 6-month evaluation, 50% had CHE (the SIP CHE identified these patients with 88% sensitivity). Of the 50 patients who returned for the 12-month evaluation, 32% had CHE (the SIP CHE score identified these patients with 81% sensitivity). CONCLUSIONS: We developed a system to identify patients with CHE based on age, sex, and responses to 4 SIP questions; this formula identified patients with CHE with more than 80% sensitivity over a 12-month period after the initial enrollment. Patient-administered CHE screening strategies that do not include specialized tests could increase the detection of CHE and improve therapy.

The Stroop smartphone application is a short and valid method to screen for minimal hepatic encephalopathy.

UNLABELLED: Minimal hepatic encephalopathy (MHE) detection is difficult because of the unavailability of short screening tools. Therefore, MHE patients can remain undiagnosed and untreated. The aim of this study was to use a Stroop smartphone application (app) (EncephalApp_Stroop) to screen for MHE. The app and standard psychometric tests (SPTs; 2 of 4 abnormal is MHE, gold standard), psychometric hepatic encephalopathy score (PHES), and inhibitory control tests (ICTs) were administered to patients with cirrhosis (with or without previous overt hepatic encephalopathy; OHE) and age-matched controls from two centers; a subset underwent retesting. A separate validation cohort was also recruited. Stroop has an "off" state with neutral stimuli and an "on" state with incongruent stimuli. Outcomes included time to complete five correct runs as well as number of trials needed in on (Ontime) and off (Offtime) states. Stroop results were compared between controls and patients with cirrhosis with or without OHE and those with or without MHE (using SPTs, ICTs, and PHES). Receiver operating characteristic analysis was performed to diagnose MHE in patients with cirrhosis with or without previous OHE. One hundred and twenty-five patients with cirrhosis (43 previous OHE) and 134 controls were included in the original cohort. App times were correlated with Model for End-Stage Liver Disease (Offtime: r = 0.57; Ontime: r = 0.61; P < 0.0001) and were worst in previous OHE patients, compared to the rest and controls. Stroop performance was also significantly impaired in those with MHE, compared to those without MHE, according to SPTs, ICTs, and PHES (all P < 0.0001). A cutoff of >274.9 seconds (Ontime plus Offtime) had an area under the curve of 0.89 in all patients and 0.84 in patients without previous OHE for MHE diagnosis using SPT as the gold standard. The validation cohort showed 78% sensitivity and 90% specificity with the >274.9-seconds Ontime plus Offtime cutoff. App result patterns were similar between the centers. Test-retest reliability in controls and those without previous OHE was good; a learning effect on Ontime in patients with cirrhosis without previous OHE was noted. CONCLUSION: The Stroop smartphone app is a short, valid, and reliable tool for screening of MHE.

Asymmetric dimethylarginine is strongly associated with cognitive dysfunction and brain MR spectroscopic abnormalities in cirrhosis.

BACKGROUND & AIMS: Asymmetric dimethylarginine (ADMA) is an inhibitor of nitric oxide synthase that accumulates in liver disease and may contribute to hepatic encephalopathy (HE). We aimed at evaluating the association of ADMA with cognition and brain MR spectroscopy (MRS) in cirrhosis. METHODS: Cirrhotic patients with/without prior HE and non-cirrhotic controls underwent cognitive testing and ADMA determination. A subgroup underwent brain MRS [glutamine/glutamate (Glx), myoinositol (mI), N-acetyl-aspartate (NAA) in parietal white, occipital gray, and anterior cingulated (ACC)]. Cognition and ADMA in a cirrhotic subgroup before and one month after transjugular intrahepatic portosystemic shunting (TIPS) were also tested. Cognition and MRS values were correlated with ADMA and compared between groups using multivariable regression. ADMA levels were compared between those who did/did not develop post-TIPS HE. RESULTS: Ninety cirrhotics (MELD 13, 54 prior HE) and 16 controls were included. Controls had better cognition and lower ADMA, Glx, and higher mI compared to cirrhotics. Prior HE patients had worse cognition, higher ADMA and Glx and lower mI compared to non-HE cirrhotics. ADMA was positively correlated with MELD (r=0.58, p<0.0001), abnormal cognitive test number (r=0.66, p<0.0001), and Glx and NAAA (white matter, ACC) and negatively with mI. On regression, ADMA predicted number of abnormal tests and mean Z-score independent of prior HE and MELD. Twelve patients underwent TIPS; 7 developed HE post-TIPS. ADMA increased post-TIPS in patients who developed HE (p=0.019) but not in others (p=0.89). CONCLUSIONS: A strong association of ADMA with cognition and prior HE was found independent of the MELD score in cirrhosis.

Modulation of the fecal bile acid profile by gut microbiota in cirrhosis.

BACKGROUND & AIMS: The 7α-dehydroxylation of primary bile acids (BAs), chenodeoxycholic (CDCA) and cholic acid (CA) into the secondary BAs, lithocholic (LCA) and deoxycholic acid (DCA), is a key function of the gut microbiota. We aimed at studying the linkage between fecal BAs and gut microbiota in cirrhosis since this could help understand cirrhosis progression. METHODS: Fecal microbiota were analyzed by culture-independent multitagged-pyrosequencing, fecal BAs using HPLC and serum BAs using LC-MS in controls, early (Child A) and advanced cirrhotics (Child B/C). A subgroup of early cirrhotics underwent BA and microbiota analysis before/after eight weeks of rifaximin. RESULTS: Cross-sectional: 47 cirrhotics (24 advanced) and 14 controls were included. In feces, advanced cirrhotics had the lowest total, secondary, secondary/primary BA ratios, and the highest primary BAs compared to early cirrhotics and controls. Secondary fecal BAs were detectable in all controls but in a significantly lower proportion of cirrhotics (p<0.002). Serum primary BAs were higher in advanced cirrhotics compared to the rest. Cirrhotics, compared to controls, had a higher Enterobacteriaceae (potentially pathogenic) but lower Lachonospiraceae, Ruminococcaceae and Blautia (7α-dehydroxylating bacteria) abundance. CDCA was positively correlated with Enterobacteriaceae (r=0.57, p<0.008) while Ruminococcaceae were positively correlated with DCA (r=0.4, p<0.05). A positive correlation between Ruminococcaceae and DCA/CA (r=0.82, p<0.012) and Blautia with LCA/CDCA (r=0.61, p<0.03) was also seen. Prospective study: post-rifaximin, six early cirrhotics had reduction in Veillonellaceae and in secondary/primary BA ratios. CONCLUSIONS: Cirrhosis, especially advanced disease, is associated with a decreased conversion of primary to secondary fecal BAs, which is linked to abundance of key gut microbiome taxa.

Colonic mucosal microbiome differs from stool microbiome in cirrhosis and hepatic encephalopathy and is linked to cognition and inflammation.

Although hepatic encephalopathy (HE) is linked to the gut microbiota, stool microbiome analysis has not found differences between HE and no-HE patients. This study aimed to compare sigmoid mucosal microbiome of cirrhotic patients to controls, between HE vs. no-HE patients, and to study their linkage with cognition and inflammation. Sixty cirrhotic patients (36 HE and 24 no-HE) underwent cognitive testing, stool collection, cytokine (Th1, Th2, Th17, and innate immunity), and endotoxin analysis. Thirty-six patients (19 HE and 17 no-HE) and 17 age-matched controls underwent sigmoid biopsies. Multitag pyrosequencing (including autochthonous genera, i.e., Blautia, Roseburia, Fecalibacterium, Dorea) was performed on stool and mucosa. Stool and mucosal microbiome differences within/between groups and correlation network analyses were performed. Controls had significantly higher autochthonous and lower pathogenic genera compared with cirrhotic patients, especially HE patients. HE patients had worse MELD (model for end-stage liver disease) score and cognition and higher IL-6 and endotoxin than no-HE. Mucosal microbiota was different from stool within both HE/no-HE groups. Between HE/no-HE patients, there was no difference in stool microbiota but mucosal microbiome was different with lower Roseburia and higher Enterococcus, Veillonella, Megasphaera, and Burkholderia abundance in HE. On network analysis, autochthonous genera (Blautia, Fecalibacterium, Roseburia, and Dorea) were associated with good cognition and decreased inflammation in both HE/no-HE, whereas genera overrepresented in HE (Enterococcus, Megasphaera, and Burkholderia) were linked to poor cognition and inflammation. Sigmoid mucosal microbiome differs significantly from stool microbiome in cirrhosis. Cirrhotic, especially HE, patients' mucosal microbiota is significantly different from controls with a lack of potentially beneficial autochthonous and overgrowth of potentially pathogenic genera, which are associated with poor cognition and inflammation.

Modulation of the metabiome by rifaximin in patients with cirrhosis and minimal hepatic encephalopathy.

UNLABELLED: Hepatic encephalopathy (HE) represents a dysfunctional gut-liver-brain axis in cirrhosis which can negatively impact outcomes. This altered gut-brain relationship has been treated using gut-selective antibiotics such as rifaximin, that improve cognitive function in HE, especially its subclinical form, minimal HE (MHE). However, the precise mechanism of the action of rifaximin in MHE is unclear. We hypothesized that modulation of gut microbiota and their end-products by rifaximin would affect the gut-brain axis and improve cognitive performance in cirrhosis. Aim To perform a systems biology analysis of the microbiome, metabolome and cognitive change after rifaximin in MHE. METHODS: Twenty cirrhotics with MHE underwent cognitive testing, endotoxin analysis, urine/serum metabolomics (GC and LC-MS) and fecal microbiome assessment (multi-tagged pyrosequencing) at baseline and 8 weeks post-rifaximin 550 mg BID. Changes in cognition, endotoxin, serum/urine metabolites (and microbiome were analyzed using recommended systems biology techniques. Specifically, correlation networks between microbiota and metabolome were analyzed before and after rifaximin. RESULTS: There was a significant improvement in cognition(six of seven tests improved, p<0.01) and endotoxemia (0.55 to 0.48 Eu/ml, p = 0.02) after rifaximin. There was a significant increase in serum saturated (myristic, caprylic, palmitic, palmitoleic, oleic and eicosanoic) and unsaturated (linoleic, linolenic, gamma-linolenic and arachnidonic) fatty acids post-rifaximin. No significant microbial change apart from a modest decrease in Veillonellaceae and increase in Eubacteriaceae was observed. Rifaximin resulted in a significant reduction in network connectivity and clustering on the correlation networks. The networks centered on Enterobacteriaceae, Porphyromonadaceae and Bacteroidaceae indicated a shift from pathogenic to beneficial metabolite linkages and better cognition while those centered on autochthonous taxa remained similar. CONCLUSIONS: Rifaximin is associated with improved cognitive function and endotoxemia in MHE, which is accompanied by alteration of gut bacterial linkages with metabolites without significant change in microbial abundance. TRIAL REGISTRATION: ClinicalTrials.gov NCT01069133.