Hepatology Communications, VOL. 3, NO. 5, 2019  Effect of a Multistrain Probiotic on Cognitive Function and Risk of Falls in Patients With Cirrhosis: A Randomized Trial Eva Román,1-3,6* Juan Camilo Nieto,3* Cristina Gely,3 Sílvia Vidal,3,4 Marta Pozuelo,5 Maria Poca,1,6 Cándido Juárez,3,4 Carlos Guarner,1,6 Chaysavanh Manichanh,5,6 and Germán Soriano1,6 Probiotics can modulate gut microbiota, intestinal permeability, and immune response and could therefore improve cognitive dysfunction and help avoid potential consequences, such as falls, in patients with cirrhosis. The aim of this study was to evaluate the effect of a multistrain probiotic on cognitive function, risk of falls, and inflammatory re- sponse in patients with cirrhosis. Consecutive outpatients with cirrhosis and cognitive dysfunction (defined by a Psychometric Hepatic Encephalopathy Score [PHES] < −4) and/or falls in the previous year were randomized to receive either a sachet of a high-concentration multistrain probiotic containing 450 billion bacteria twice daily for 12 weeks or placebo. We evaluated the changes in cognitive function (PHES); risk of falls (Timed Up and Go [TUG] test, gait speed, and incidence of falls); systemic inflammatory response; neutrophil oxidative burst; intestinal barrier integrity (serum fatty acid–binding protein 6 [FABP-6] and 2 [FABP-2] and zonulin and urinary claudin-3); bacte- rial translocation (lipopolysaccharide-binding protein [LBP]); and fecal microbiota. Thirty-six patients were included. Patients treated with the probiotic (n = 18) showed an improvement in the PHES (P = 0.006), TUG time (P = 0.015) and gait speed (P = 0.02), and a trend toward a lower incidence of falls during follow-up (0% compared with 22.2% in the placebo group [n = 18]; P = 0.10). In the probiotic group, we observed a decrease in C-reactive protein (P = 0.01), tumor necrosis factor alpha (P = 0.01), FABP-6 (P = 0.009), and claudin-3 (P = 0.002), and an increase in poststimulation neutrophil oxidative burst (P = 0.002). Conclusion: The multistrain probiotic improved cognitive function, risk of falls, and inflammatory response in patients with cirrhosis and cognitive dysfunction and/or previ- ous falls. (Hepatology Communications 2019;3:632-645). I ntestinal dysbiosis, gut barrier failure, bacte- diseases.(1) These factors have also been implicated in rial translocation, and subsequent inflammatory worsening of liver failure and portal hypertension and, response are key factors in the progression of liver consequently, in the development of complications, Abbreviations: CRP, C-reactive protein; FABP, fatty acid–binding protein; HRQOL, health-related quality of life; IL, interleukin; LBP, lipopolysaccharide-binding protein; NHP, Nottingham Health Profile; PHES, Psychometric Hepatic Encephalopathy Score; PMA, phorbol 12-myristate 13-acetate; TUG, Timed Up and Go test. Received October 9, 2018; accepted January 30, 2019. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep4.1325/suppinfo. Supported by the Instituto de Salud Carlos III (Madrid, Spain) (PI12/00629), Mendes S.A. (Lugano, Switzerland) and Actial Farmaceutica LDA (Funchal, Portugal). ClinicalTrials.gov NCT01686698. *These authors contributed equally to this work. © 2019 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep4.1325 Potential conflict of interest: Dr. Soriano received research and travel grants from Mendes, Actial, and Grifols. 632 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. including cognitive dysfunction, after advanced effect of probiotics on the risk of falling in these chronic liver disease or cirrhosis is established.(1-3) patients has not been previously evaluated. Cognitive dysfunction, or minimal hepatic enceph- The aim of this study was to evaluate the effect of alopathy, is a risk factor for overt hepatic encepha- a multistrain probiotic on cognitive function and the lopathy, worsening in health-related quality of life risk of falls in patients with cirrhosis who also have (HRQOL), traffic accidents, and mortality in patients cognitive dysfunction and/or previous falls. We also with cirrhosis.(4-6) In addition, these patients are more studied potential mediators of these effects, particu- predisposed to accidental falls.(7,8) larly the systemic inflammatory response, the gut bar- Falls are particularly important in patients with cir- rier, bacterial translocation, and intestinal microbiota. rhosis, because they have a greater risk of fracture than the general population.(9) Moreover, falls are a signif- icant cause of complications, mortality, and HRQOL impairment.(6-9) In addition to their negative conse- Patients and Methods quences for the patient, falls have implications for the Between February 2013 and March 2016, we per- patient’s relatives and are an economic and social bur- formed a double-blind placebo-controlled randomized den for the community.(10) Moreover, individuals with trial at Hospital de la Santa Creu i Sant Pau, a ter- previous falls are frequently predisposed to recurrent tiary care hospital in Barcelona, Spain. The protocol falling,(7,8,11) thus supporting the growing concept of conformed to the 1975 Declaration of Helsinki and frailty in patients with cirrhosis(12) and the need for Guidelines for Good Clinical Practice in Clinical Trials preventive measures.(7-9) and was approved by the clinical research ethics com- Probiotics can modulate gut microbiota, the intes- mittee (Comitè d’Ètica d’Investigació Clínica-CEIC) tinal barrier, and the immune response and could at our institution. Monitoring was performed by the therefore decrease pathological bacterial translocation Research Institute (IIB-Sant Pau) according to good and ameliorate immune system alterations in cirrho- clinical practice recommendations. All patients received sis.(1,13-15) These changes could not only improve liver information regarding their participation in the study function and help prevent bacterial infections and and signed an informed consent form. The protocol other complications, but also ameliorate cognitive was registered at ClinicalTrials.gov (NCT01686698). dysfunction and avoid its consequences, such as overt hepatic encephalopathy, traffic accidents, deterioration PATIENT SELECTION of HRQOL, and falls.(1,13,14) Studies have shown that certain probiotics improve We included consecutive outpatients with cirrhosis the proinflammatory state,(1,13,15) liver function,(1) who had cognitive dysfunction and/or falls during the portal hemodynamics,(1) cognitive function,(1,16) previous year and were visited at the nursing outpa- incidence of overt hepatic encephalopathy,(1,16) and tient office. Patients were selected by the nurses and HRQOL(1) in patients with cirrhosis. However, the hepatologists involved in the study. Cirrhosis was ARTICLE INFORMATION: From the 1 Department of Gastroenterology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; 2 Escola Universitària d’Infermeria EUI-Sant Pau, Barcelona, Spain; 3 Institut de Recerca IIB-Sant Pau, Barcelona, Spain; 4 Department of Immunology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain; 5 Fundació Hospital Universitari Vall d’Hebron-Institut de Recerca, Barcelona, Spain; 6 CIBERehd, Instituto de Salud Carlos III, Madrid, Spain. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: German Soriano, M.D., Ph.D. 08041 Barcelona, Spain Department of Gastroenterology E-mail: gsoriano@santpau.cat Hospital de la Santa Creu i Sant Pau Tel: +34-93-556-5920 Mas Casanovas, 90 Fax: +34-93-556-5608 633 ROMÁN, NIETO, ET AL. Hepatology Communications, May 2019 diagnosed by means of clinical, analytical, and ultraso- L. acidophilus DSM 24735, L. delbrueckii subsp bul- nographic findings or by liver biopsy. The presence of garicus DSM 24734, and L. plantarum DSM 24730, cognitive dysfunction was defined as a Psychometric at a total dose of 450 billion live bacteria per 4.4 g Hepatic Encephalopathy Score (PHES) of less than sachet with maltose and silicon dioxide as excipients. −4,(17,18) and previous falls were assessed using a The probiotic mixture was manufactured by Danisco- described questionnaire.(7,8) Exclusion criteria were DuPont (Madison, WI) and is currently marketed hospitalization during the previous month, hepato- under the brand Vivomixx (MENDES, S.A., Lugano, cellular carcinoma or any other neoplasia, acute or Switzerland) in Europe and Visbiome (ExeGi chronic overt hepatic encephalopathy, neurologic dis- Pharmaceuticals, Rockville, MD) in the United ease, active alcohol intake (in the previous 3 months), States. The placebo contained maltose and silicon clinically significant cognitive impairment, inability to dioxide as inactive agents and was formulated as iden- perform psychometric tests, severe comorbidities, life tical in appearance to the active agent. Patients were expectancy of less than 6 months, any treatment with instructed to keep the study product at 4ºC in the nonabsorbable disaccharides, laxatives, antibiotics refrigerator at home and to take one sachet diluted in and/or antivirals in the previous 3 months, and refusal one glass of water, milk, or juice at room temperature to participate in the study. twice daily (every 12 hours) over 12 weeks. STUDY DESIGN OUTCOMES AND ENDPOINTS Patients were randomized by a hepatologist, other At baseline and at the end of treatment, we assessed than those who selected the patients, to take either cognitive function according to PHES and the risk of a probiotic (probiotic group) or a placebo (placebo falls by means of the Timed Up and Go (TUG) test group). Randomization was performed by means of and gait speed. The main endpoints were cognitive a computer-generated sequence using blocks of four function and risk of falls, and the secondary endpoints and consecutively numbered opaque sealed envelopes. were fall incidence, HRQOL, systemic inflammatory All patients were treated for 12 weeks and assessed at response, gut barrier, bacterial translocation, and fecal baseline and at 12 weeks (end of treatment) for clini- microbiota. cal and analytical data, complications of cirrhosis, side effects, adherence, cognitive function, risk of falls, sys- CLINICAL AND ANALYTICAL temic inflammatory response, and biomarkers of intes- DATA tinal barrier and bacterial translocation. A subgroup of patients was evaluated for fecal microbiota. Additional At baseline, week 6, week 12 (end of treatment), visits were performed at week 6 and week 20 (8 weeks and week 20 (end of study), we recorded demographic, after the end of treatment) to check patients’ clinical clinical, and analytical data. We also recorded param- and analytical (routine standard analysis) status, side eters that can influence the predisposition to fall, effects, adherence (only at week 6), and incidence of including comorbidity, muscular strength, visual acuity falls. Patients were instructed to return the boxes with assessed by the Snellen test, and walking problems.(7,8) the used and unused sachets at the week 6 and week 12 visits to assess the adherence. The blinding was COGNITIVE FUNCTION maintained for participants and the study team until the end of the study and the analysis of the results. Cognitive function was evaluated by the PHES, a neuropsychological battery widely used to assess cognitive function in patients with cirrhosis,(17) vali- STUDY PRODUCT dated for the Spanish population,(18) using the com- The probiotic formulation was a mixture of eight puter program of the Spanish Network of Hepatic strains, namely, Streptococcus thermophilus DSM Encephalopathy (www.redeh.org). Patients were con- 24731, Bifidobacterium breve (B. breve) DSM 24732, sidered to show minimal hepatic encephalopathy or B. longum DSM 24736, B. infantis DSM 24737, cognitive dysfunction when they showed a PHES Lactobacillus paracasei (L. paracasei) DSM 24733, score of less than −4.(17,18) 634 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. RISK OF FALLS (IL) 10 (BD Pharmingen, San Diego, CA), and IL-6 (ImmunoTools, Friesoythe, Germany) in serum The risk of falling was assessed using the TUG using specific enzyme-linked immunosorbent assays test and gait speed.(8,19,20) The TUG test measures (ELISA). To determine neutrophil oxidative burst the time the patient needs to stand up from a chair, (reactive oxidants), we incubated 105 peripheral blood walk 3 m, turn around, walk back, and sit down in cells for 20 minutes either with 200 ng/mL phorbol the chair without support. Gait speed was measured 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. according to the time taken to walk 5 m. Gait speed Louis, MO) or without stimulus at 37ºC. The forma- is generally considered as abnormal and representative tion of reactive oxidants was monitored by the oxi- of increased risk of falling when it is 1 m/second or dation of dihydrorhodamine 123 (Sigma-Aldrich) to slower.(19) rhodamine by flow cytometry. INCIDENCE OF FALLS INTESTINAL BARRIER AND Falls were prospectively assessed at each visit BACTERIAL TRANSLOCATION during the study using a described specific question- naire(7,8) and a revision of clinical records. The num- The integrity of the intestinal barrier was assessed ber of falls, severity of injuries, and health care needed indirectly by the determination of fatty acid–binding were also recorded. Injuries were categorized as con- protein 6 (FABP-6; Cloud-Clone Corp., Houston, tusion, wound or fracture, and health care needed was TX), FABP-2 (Cloud-Clone Corp.), and zonulin classified as primary care, emergency room care, or (Bioss Antibodies Inc., Woburn, MA) in serum, and hospitalization.(7,8) claudin-3 in urine (Elabscience, Houston, TX)(23,24) using specific ELISA. We determined lipopoly- saccharide-binding protein (LBP) concentration HAND GRIP MUSCULAR in serum using specific ELISA (Biometec GmbH, STRENGTH Greifswald, Germany) according to the manufactur- Sarcopenia, a factor related to the predisposition to er’s instructions. fall,(11) was evaluated by hand grip muscular strength using a dynamometer (KERN MAP-BA-s-0910) fol- FECAL MICROBIOME ANALYSIS lowing the manufacturer’s instructions. We analyzed the fecal microbiome in the second half of patients included in the study. Patients pro- HRQOL vided fecal samples at baseline and at 12 weeks (end To evaluate HRQOL, we used the Nottingham of treatment) to assess changes in their microbiomes. Health Profile (NHP), a general questionnaire that The methods for sample collection, genomic DNA has been used in patients with cirrhosis.(21) The NHP extraction, and high-throughput DNA sequenc- has two parts. The first part includes 38 items that ing have been described(25) and are detailed in the cover six domains, with scores ranging from 0 to 100. Supporting Information. The second part consists of seven questions about limitations due to health problems in relation to seven STATISTICAL ANALYSIS functional activities of daily life: occupation, house- work, social life, family life, sexual function, hobbies, To compare the two groups at baseline, we used and holidays. Higher scores or percentages indicate the Fisher’s exact test for categorical variables. The worse quality of life. We used the validated Spanish Student t test (if the data distribution was normal) version of the NHP.(22) or the Mann-Whitney U test (if the distribution was not normal) were used for quantitative variables. To compare the changes between baseline and the end of SYSTEMIC INFLAMMATION treatment in each group, we used the Student t test if We determined serum C-reactive protein (CRP), the data distribution was normal and the Wilcoxon tumor necrosis factor alpha (TNF-α), interleukin test if the distribution was not normal. Normality was 635 ROMÁN, NIETO, ET AL. Hepatology Communications, May 2019 assessed by the Shapiro–Wilk test. Correlations were COGNITIVE FUNCTION assessed by the Spearman test. Data are expressed as frequencies, percentages, and mean ± SEM. A Cognitive function evaluated by the PHES two-sided P value of less than 0.05 was considered improved at the end of treatment (12-week visit) statistically significant. The methods used for statis- compared with baseline results in the probiotic group tical analysis of changes in the fecal microbiome are (−1.29 ± 0.72 to −0.35 ± 0.82; P = 0.006). No changes described in the Supporting Information. were observed in patients in the placebo group Sample size was calculated according to previous (Fig. 2). data on the effect of the probiotic on cognitive func- tion in patients with cirrhosis.(16) We used the com- RISK OF FALLING AND puter program GRANMO version 7.12, released in INCIDENCE OF FALLS 2012 and developed by the Institut Municipal d’In- vestigació Mèdica, Barcelona, Spain. We considered a We observed a decrease in TUG (11.38 ± 0.57 to resolution rate of minimal hepatic encephalopathy of 10.00 ± 0.49 seconds; P = 0.015) and an increase 55% in patients receiving the probiotic versus 10% in in gait speed (0.90 ± 0.05 to 1.12 ± 0.10 m/second; patients receiving placebo. With a calculated 10% of P = 0.02) at the end of treatment (12-week visit) in patients lost during follow-up, an alpha error of 0.05, comparison to the baseline results in the probiotic and a power of 0.80, 17 patients per group would be group, whereas we did not observe changes in the necessary to show statistically significant differences placebo group (Fig. 2). in the effect on cognitive function between the pro- The incidence of falls throughout the 20 weeks biotic and the placebo. Statistical analysis was per- of the study was 0 of 18 (0%) in the probiotic group formed using the program released in 2017 (IBM and 4 of 18 (22.2%) in the placebo group (P = 0.10). SPSS Statistics for Windows, Version 25.0; IBM Regarding the severity of injuries due to falls, one fall Corp, Armonk, NY). caused a wound, whereas the others involved contu- sions only. The patients were treated at primary health care centers, and one was seen at the emergency room. Results No hospitalizations were required. PATIENT CHARACTERISTICS HAND GRIP MUSCULAR From February 2013 to March 2016, 279 patients STRENGTH with cirrhosis were evaluated for eligibility at the No statistically significant differences were found nursing outpatient office (Fig. 1). Of the patients, 158 between baseline and end-of-treatment results either had a PHES of at least −4 and no previous falls, and in the probiotic group (20.76 ± 2.27 to 20.62 ± 2.05 kg; therefore did not fulfill the inclusion criteria. Of the P = 0.78) or in the placebo group (20.98 ± 2.28 to remaining 121 patients, 85 met one or more exclusion 20.24 ± 2.05 kg; P = 0.12). criteria. Finally, 36 patients were randomized to one of two groups: 18 to the probiotic group and 18 to HRQOL the placebo group. As indicated in Table 1, the two groups had similar clinical and analytical characteris- At baseline, patients in both groups showed a rea- tics at baseline. In the probiotic group, 1 patient died sonably well-preserved HRQOL, as seen from the low during the treatment period and therefore was not values in each domain from the first part of the NHP evaluated at the 12-week visit (end of treatment), and and in the functional activities of daily life in the sec- we could not determine gait speed and TUG test at ond part of the questionnaire (Supporting Fig. S1). the end of treatment in another patient due to hip We did not observe statistically significant changes at enthesitis. In the placebo group, 1 patient died after the end of the treatment in either group, but there was the end of treatment but before the 20-week fol- a trend to an improvement in the domain of social low-up visit. isolation in the probiotic group (P = 0.05). 636 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. FIG. 1. Flowchart of the study. After the end of treatment (week 12), patients were followed up until week 20. One patient in the placebo group died during this period, at week 16. LIVER AND RENAL FUNCTION group (P = 0.002), without significant changes in rest- ing oxidative burst. No changes were observed in the As indicated in Table 2, no variations in liver or placebo group (Fig. 3). renal function were found at the end of treatment in comparison to baseline values in either group. INTESTINAL BARRIER AND BACTERIAL TRANSLOCATION SYSTEMIC INFLAMMATORY Regarding the intestinal barrier, there was a RESPONSE decrease in serum FABP-6 (P = 0.009) and urinary There was a decrease in serum CRP (7.62 ± 2.64 claudin-3 (P = 0.002) in the probiotic group at the to 3.45 ± 0.98 mg/L; P = 0.01) and TNF-α (P = 0.01) end of treatment without changes in serum FABP-2 at the end of treatment in the probiotic group, with- or zonulin. There were no variations in the placebo out significant changes in IL-6 or IL-10. Neutrophil group. We observed a not statistically significant trend oxidative burst after stimulation with PMA increased toward a decrease in LBP in the probiotic group significantly at the end of treatment in the probiotic (P = 0.13) (Fig. 4). 637 ROMÁN, NIETO, ET AL. Hepatology Communications, May 2019 TABLE 1. BASELINE CLINICAL AND ANALYTICAL for all phylogenetic levels and all bacterial groups). CHARACTERISTICS OF PATIENTS FROM THE PLACEBO AND PROBIOTIC GROUPS Sequence data were deposited in the National Center for Biotechnology Information database, with access Placebo Probiotic number PRJNA499079. Group Group (n = 18) (n = 18) P Age (years) 64.0 ± 2.6 65.8 ± 3.1 0.65 CORRELATIONS AMONG Sex (male/female) 8 (44.4)/10 6 (33.3)/12 0.73 INTESTINAL BARRIER, (55.6) (66.7) INFLAMMATORY RESPONSE, Etiology (%) 0.90 COGNITIVE FUNCTION, AND Alcohol 10 (55.6) 9 (50) Hepatitis C virus 3 (16.7) 3 (16.7) RISK OF FALLING Hepatitis B virus 1 (5.6) 1 (5.6) Including all patients from the two groups at base- Hepatitis C virus and alcohol 2 (11.1) 1 (5.6) line, we observed mild but significant correlations Other 2 (11.1) 4 (22.2) between FABP-6 and LBP (r = 0.54; P = 0.002); Previous decompensations (%) 13 (72.2) 16 (88.9) 0.40 FABP-6 and CRP (r = 0.40; P = 0.01); LBP and Previous hepatocellular carcinoma (%) 2 (11.1) 1 (5.6) 1.00 CRP (r = 0.42; P = 0.01); CRP and poststimulated Child-Pugh score 6.0 ± 0.4 5.9 ± 0.2 0.42 MELD score 9.3 ± 0.8 9.0 ± 0.9 0.78 neutrophil oxidative burst (r = −0.43; P = 0.01); post- Minimal hepatic encephalopathy (%) 6 (33.3) 4 (22.2) 0.71 stimulated oxidative burst and TUG test (r = −0.37; Previous falls (%) 15 (83.3) 15 (83.3) 1.00 P = 0.03); and TUG test and PHES score (r = −0.41; Severe deficit visual acuity* (%) 0 0 1.00 P = 0.01). Walking problems† (%) 4 (22.2) 2 (11.1) 0.66 Modified Charlson comorbidity score 2 2.8 ± 0.3 2.5 ± 0.4 0.46 ADVERSE EVENTS, Body mass index (kg/m ) 28.9 ± 1.7 26.1 ± 0.9 0.27 COMPLICATIONS OF CIRRHOSIS, Diabetes mellitus (%) 6 (33.3) 5 (27.8) 1.00 Diuretics (%) 10 (55.6) 11 (61.1) 1.00 AND MORTALITY Beta blockers (%) 7 (38.9) 8 (44.4) 1.00 The incidence and number of adverse events and Proton pump inhibitors (%) 10 (55.6) 9 (50) 1.00 severe adverse events during treatment and follow-up Antidepressants (%) 2 (11.1) 2 (11.1) 1.00 were similar in the two groups (Supporting Table Serum sodium (mmol/L) 138.7 ± 0.8 140.1 ± 0.8 0.35 Serum creatinine (µmol/L) 76.1 ± 6.2 80.5 ± 8.1 0.67 S1). Ten patients (55.5%) in the probiotic group and Serum bilirubin (µmol/L) 21.1 ± 4.6 19.2 ± 2.6 0.88 12 patients (66.6%) in the placebo group (P = 0.73) Serum albumin (g/L) 36.8 ± 1.4 37.7 ± 0.8 0.49 presented 13 and 21 adverse events, respectively. INR 1.18 ± 0.04 1.19 ± 0.06 0.44 Specifically, in the probiotic group, 2 patients pre- Mean arterial pressure (mm Hg)‡ 91.3 ± 3.4 94.7 ± 4.3 0.54 sented complications of cirrhosis (1 patient died at week 6 due to respiratory infection and spontaneous Note: Data are expressed as number (%) of patients and mean ± bacterial peritonitis [SBP], and another presented standard error of the mean. *Less than 3 of 10 using Snellen number chart. grade 2 hepatic encephalopathy). In the placebo † Use of any walking-aid device. group, 3 patients presented complications of cirrhosis ‡ Millimeters of mercury. (1 patient was diagnosed with hepatocellular carci- Abbreviations: INR, international normalized ratio; and MELD, Model for End-Stage Liver Disease. noma, 1 patient developed ascites, and another died at week 16 due to SBP). Overall mortality during the study was therefore 2 of 36 (5.5%), 1 patient in each FECAL MICROBIOTA group. Regarding infections, 4 patients from the pro- We analyzed the changes in the fecal microbiota biotic group presented five infections, and 5 patients in the second half of patients included in the study from the placebo group presented five infections (9 patients from the probiotic group and 8 patients (Supporting Table S1). No adverse events attributable from the placebo group). We did not observe signifi- to the study product were observed. Overall adherence cant changes at the end of the treatment in compar- was considered satisfactory, as patients took 95.8% of ison with baseline data in either group at a phylum, the assigned sachets (97.2% in the probiotic group genus, or species level (false discovery rates > 0.21 and 94.4% in the placebo group). 638 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. FIG. 2. Cognitive function assessed by the PHES (A) and risk of falls assessed by TUG test (B) and gait speed (C) at baseline and at the end of treatment (12 weeks) in the placebo group (n = 18) and in the probiotic group (n = 17 for PHES and n = 16 for TUG and gait speed). Data are expressed as mean ± SEM. P is not significant for any parameters in the placebo group. TABLE 2. LIVER AND RENAL FUNCTION AT BASELINE AND AT THE END OF TREATMENT (WEEK 12) IN THE PLACEBO GROUP AND THE PROBIOTIC GROUP Placebo Group (n = 18) Probiotic Group (n = 17) End of Treatment End of Treatment Baseline (week 12) P Baseline (week 12) P Serum sodium (mmol/L) 138.7 ± 0.8 139.2 ± 0.7 0.47 139.7 ± 0.8 139.9 ± 0.8 0.84 Serum creatinine (µmol/L) 76.1 ± 6.2 75.3 ± 6.6 0.69 74.0 ± 5.2 75.0 ± 6.7 0.66 Serum bilirubin (µmol/L) 21.1 ± 4.6 23.2 ± 5.3 0.10 18.2 ± 2.6 18.6 ± 2.5 1.00 Serum albumin (g/L) 36.8 ± 1.4 37.3 ± 1.3 0.23 37.9 ± 0.8 38.1 ± 0.7 0.54 INR 1.18 ± 0.04 1.19 ± 0.04 0.58 1.20 ± 0.06 1.15 ± 0.05 0.69 AST (U/L) 43.5 ± 4.7 46.1 ± 4.9 0.43 38.6 ± 5.7 36.9 ± 5.4 0.80 ALT (U/L) 36.4 ± 5.1 41.3 ± 6.5 0.76 30.7 ± 8.1 36.3 ± 9.3 0.06 GGT (U/L) 92.7 ± 16.1 110.0 ± 22.2 0.34 68.4 ± 15.4 55.2 ± 10.4 0.05 Note: Data are expressed as mean ± SEM. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma-glutamyltransferase; and INR, inter- national normalized ratio. Discussion probiotic treatment, however, the final results of the TUG test and gait speed indicated an improvement The main finding of the present study is that treat- in the risk of falls in the probiotic group, but not in ment with a multistrain probiotic improved cognitive the placebo group.(19,20) Moreover, there was a not function and decreased the risk of falling in patients significant decrease in the incidence of accidental falls with cirrhosis who had cognitive dysfunction and/or throughout the study in patients from the probiotic previous falls. group in comparison with the placebo group (0% ver- Because of the strict exclusion criteria to avoid sus 22.2%), although the study was not designed with confounding factors, the patients included were highly the statistical power to demonstrate differences in this selected and in a stable clinical situation, as can be clinical endpoint. observed by their low Child-Pugh and Model for End- The cause of the predisposition of patients with Stage Liver Disease scores and the low prevalence of cirrhosis to falling is probably multifactorial, but cog- abnormal PHES results. Most of them were included nitive dysfunction (or minimal hepatic encephalopa- in the study because of previous falls, and their TUG thy) appears to play an important role.(8,26) Cognitive test and gait speed values were clearly impaired and function improved in patients from the probiotic indicative of risk of falling.(19,20) At the end of the group, as shown by the increase in the PHES results 639 ROMÁN, NIETO, ET AL. Hepatology Communications, May 2019 FIG. 3. Parameters of systemic inflammatory response at baseline and at the end of treatment (12 weeks) in the placebo group and in the probiotic group: CRP (A), TNF-α (B), neutrophil resting oxidative burst (C), IL-6 (D), IL-10 (E), and neutrophil oxidative burst after PMA stimulation (F). Data are expressed as mean ± SEM. P is not significant for any parameters in the placebo group and for IL-6, IL-10, and neutrophil resting oxidative burst in the probiotic group. Abbreviations: MFI, mean fluorescence intensity; Rho, rhodamine. at the end of the treatment. This finding is in agree- related to the PHES results, but to more accurate ment with studies reporting an improvement in cog- tests of executive function, such as the Wisconsin nitive function in patients with cirrhosis treated with Card Sorting Test.(30) According to these data, the probiotic used here(16,27) as well as in studies using impaired PHES could be a surrogate marker of the other probiotics.(1,13,28) underlying brain alterations that predispose patients Most patients in our study had a baseline PHES with cirrhosis to falling.(30) We consider that the of at least −4, which is above the limit to diagnose improvement in the PHES observed in our study in minimal hepatic encephalopathy. Therefore, we could patients treated with the probiotics may have clinical be skeptical about the clinical significance of the relevance, especially considering that it was associated improvement in the PHES observed in the probiotic with an improvement in the tests to assess the risk of group. However, Giménez-Garzó et al.(29) recently falls. Recently, Pinto-Sanchez et al.(31) reported that demonstrated that patients with a normal PHES may treatment with the probiotic B. longum NCC3001 exhibit different types of neuropsychological alter- in patients with irritable bowel syndrome produced ations that are mild but have clinical consequences. changes in the cerebral activation pattern evaluated These data are in line with previous studies showing a by functional magnetic resonance imaging (MRI). It lack of correlation between the incidence of falls and would be interesting to use this technique to study the degree of impairment in the PHES.(8) In addition, the effect of probiotics on brain function in patients the alterations in brain white matter tracts observed with cirrhosis. by magnetic resonance–diffusion tensor imaging in The lack of statistically significant improvements patients with cirrhosis and previous falls were not in HRQOL measured by the NHP could be due to 640 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. FIG. 4. Parameters of intestinal barrier and bacterial translocation at baseline and at the end of treatment (12 weeks) in the placebo group and in the probiotic group: FABP-6 (A), FABP-2 (B), zonulin (C), urinary claudin-3 (D), and LBP (E). Data expressed as mean ± SEM. Higher values indicate more impaired intestinal barrier. P is not significant for any parameters in the placebo group and for FABP-2, zonulin, and LBP (P = 0.13) in the probiotic group. the fact that patients presented a relatively well-pre- the treatment, and this was associated with a signifi- served baseline HRQOL that was therefore difficult cant decrease in serum TNF-α. Taking into consid- to ameliorate. Another possible explanation is that eration the importance of a proinflammatory state, the improvements in physical and cognitive func- and CRP in particular, in the prognosis of patients tions were insufficient to improve the multifactorial with cirrhosis,(2,32) we consider this one of the most HRQOL in patients with cirrhosis. Nevertheless, we relevant findings in our study. Specifically, systemic observed a not significant trend to improvement in inflammation is related to neuroinflammation, which the domain of social isolation in the probiotic group. in turn is a main factor involved in the pathophysi- We aimed to explore the potential mechanisms ology of covert and overt hepatic encephalopathy.(1-3) by which the probiotic may have improved cogni- In common bile duct ligated mice, D’Mello et al.(33) tive function and decreased the risk of falling, such showed that treatment with the same probiotic mix- as modulation of systemic inflammation, improve- ture as that used here reduced systemic inflammation, ment of the intestinal barrier, and changes in bacterial improved activation of brain microglia and cere- translocation and gut microbiota.(1,3,13,14) bral monocyte infiltration, and ameliorated sickness Regarding systemic inflammation, baseline serum behavior. In patients with cirrhosis treated with the CRP levels were slightly increased in the two groups, same probiotic, a decrease in systemic inflammation, suggesting a mild proinflammatory state, as may an improvement in cognitive function, and a decrease have been expected considering the characteristics in the recurrence rate of hepatic encephalopathy have of the patients included in the study. However, only been reported.(1,16) patients in the probiotic group presented a decrease In the setting of the proinflammatory state, patients in CRP levels to almost normal values at the end of with cirrhosis present alterations in the neutrophil 641 ROMÁN, NIETO, ET AL. Hepatology Communications, May 2019 oxidative burst that can contribute to worsening prog- observe any significant effect on gut permeability, as nosis and increase the risk of bacterial infections.(34,35) assessed by a panel of markers including lactulose/ Among these alterations, the impaired production of mannitol ratio and fecal zonulin, or inflammatory reactive oxygen species after stimulation is especially biomarkers. Liu et al.(42) evaluated patients under- relevant.(35) This exhaustion of neutrophils is consid- going colorectal surgery treated with a three-strain ered to be mainly due to the maintained proinflam- probiotic and reported a decrease in serum zonulin, matory state in the setting of repeated translocation a parameter that was unchanged in our study. These of intestinal bacteria or their products (i.e,. endo- discrepancies between studies may be explained by the toxin) as a consequence of the impaired intestinal bar- differences in study populations, the probiotic product rier.(35) In our study, patients receiving the probiotic used,(13,14,39) and the assessment parameters. It should showed an improvement in neutrophil oxidative burst be pointed out that the biomarkers we used to eval- after stimulation. This finding is probably related to uate the intestinal barrier have not been validated in the decrease in the proinflammatory state and may cirrhosis. Although previous data in other settings, help prevent infections and improve prognosis in such as intestinal ischemia, intense exercise, human these patients.(34,35) Interestingly, Stadlbauer et al.(36) immunodeficiency virus infection or chronic hepatitis, observed that treatment with a single species probiotic suggest they could be surrogate parameters to assess (Lactobacillus casei Shirota) restored neutrophil phago- the integrity of the intestinal barrier,(23,43-45) studies in cytic capacity in patients with compensated alcoholic patients with cirrhosis are largely lacking. cirrhosis. This effect was probably a consequence of Regarding bacterial translocation, we quantified an improvement in pathological bacterial transloca- LBP in blood and failed to demonstrate signifi- tion, as suggested by the decrease in the neutrophil cant changes after probiotic treatment. However, we toll-like receptor 4 expression after stimulation with should again emphasize that the patients were in a endotoxin in patients treated with the probiotic.(36) In stable situation and showed relatively well-preserved a more recent study, Horvath et al.(37) reported that liver function. Therefore, patients presented low levels treatment with a different multispecies probiotic in of LBP at baseline, suggesting a low degree of bacte- outpatients with cirrhosis produced an immune mod- rial translocation that would be difficult to improve ulation consisting of an increase in neutrophil resting with any treatment. We cannot rule out the possibility oxidative burst and serum neopterin, a marker of mac- that the probiotic treatment produced a decrease in rophage activation. bacterial translocation of enterobacteria, suggested by Because probiotics can improve the intestinal bar- the slight decrease in LBP. This hypothesis should be rier, we assessed several biomarkers of gut barrier confirmed by sequencing analysis of the composition integrity. We observed a decrease in serum FABP-6 of blood bacterial DNA.(46) Such analysis was not and urinary claudin-3 in the probiotic group at the conceptualized and therefore not performed in the end of treatment, suggesting a possible improvement present study. Using a different probiotic, Lactobacillus in the intestinal barrier,(23) without significant changes GG, Bajaj et al.(15) found that endotoxemia and in serum FABP-2 and zonulin. Interestingly, FABP-2 serum TNF-α decreased and fecal microbiota dysbi- is considered to reflect the intestinal barrier in the osis improved in patients with cirrhosis with minimal whole gut, whereas FABP-6 is more specific of the hepatic encephalopathy. ileum.(23) Therefore, our findings suggest a more spe- We studied the fecal microbiota by 16S ribosomal cific effect of the probiotic in this area. Previous stud- DNA sequencing in a subgroup of patients and failed ies have demonstrated a beneficial effect of certain to demonstrate significant changes at the end of probiotics on the intestinal barrier in several experi- the treatment in either group. This finding must be mental and clinical settings.(38,39) This finding has also considered with caution due to the small number of been reported in models of experimental cirrhosis, in patients studied (9 in the probiotic group and 8 in the which several probiotics improved the intestinal bar- placebo group) and the potential confounding factors, rier, decreased bacterial translocation, and ameliorated such as inadvertent changes in life style. Moreover, the proinflammatory state.(40,41) However, in the study the analysis of the fecal microbiota does not neces- by Horvath et al.(37) in patients with cirrhosis treated sarily reflect changes in upper areas of the gut, where with a different probiotic mixture, the authors did not these changes could have more physiopathological 642 Hepatology Communications, Vol. 3, No. 5, 2019 ROMÁN, NIETO, ET AL. consequences in the setting of cirrhosis.(47) Another the present study that could suggest an improvement possible explanation is that the probiotic treatment in the intestinal barrier after probiotic treatment are did not produce a change in the microbiota compo- consistent with previous data.(23,38-45) sition but in its functions, as has been reported with As future research, a study with a larger number rifaximin(48) and with the same probiotic as that used of patients and with a longer treatment protocol is here in other settings.(49) warranted to assess the usefulness of the probiotic in Adverse events were similar in the two groups, decreasing the incidence of falls. Moreover, it would and no side effects attributable to the probiotic were be interesting to analyze the intimate mechanisms observed, confirming previous studies and suggesting determining the improvement in the risk for falls that this probiotic is safe in this setting.(16,27) through accurate neurological tests and MRI-based We hypothesize that the probiotic ameliorated techniques(30,31) as well as the changes in the microbi- the intestinal barrier and modulated the crosstalk ota function after probiotic treatment.(48,49) between intestinal bacteria and the immune system. We conclude that this multistrain probiotic As a consequence, there was a decrease in systemic improved cognitive function, decreased the risk of falls, inflammation, and probably in neuroinflammation and modulated the systemic inflammatory response in (not evaluated in the present study), and therefore an patients with cirrhosis and cognitive dysfunction and/ improvement in cognition and neural circuits involved or previous falls. This probiotic mixture could be use- in gait, which finally would decrease the risk of falls. ful in the prevention of falls and complications, espe- The correlations between the parameters of the intes- cially hepatic encephalopathy and bacterial infections, tinal barrier, the inflammatory response, cognitive in patients with cirrhosis. function, and the risk of falling observed in the pres- ent study further support this hypothesis. Acknowledgment: We thank Carolyn Newey for the Our study has several limitations. 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Rifaximin exerts beneficial effects Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep4.1325/suppinfo.   645