TITLE: Multistrain probiotic and lactulose in the treatment of minimal hepatic encephalopathy

  ABSTRACT.BACKGROUND:
Some evidence has shown benefits of probiotics in the management of minimal hepatic encephalopathy (MHE). We evaluated the efficacy of a multistrain probiotic compound, alone and in combination with lactulose, in the treatment of MHE.

ABSTRACT.MATERIALS AND METHODS:
This study has two parts. First, consecutive adult patients with MHE were randomized to receive lactulose (30–60 mL/day) + probiotic (200 million colony forming units of seven bacteria species/day) (Gp-LPr) or lactulose + placebo (Gp-L). In second part, a non-randomized group of patients received probiotic alone (Gp-Pr). Medication duration was for 2 weeks and patients were followed-up for another 8 weeks. Improvement in MHE status was assessed by psychometric hepatic encephalopathy score (PHES). Development of overt encephalopathy, hospitalization, and death were considered as secondary outcomes.

ABSTRACT.RESULTS:
Sixty patients (80% male, mean age 38.4 ± 9.6 years) completed the intervention. PHES significantly improved after medication in all the three groups (Gp-LPr: −3.8 ± 3.9 to −1.6 ± 3.0; Gp-L: −4.8 ± 4.1 to −1.6 ± 2.9; and Gp-Pr: −4.9 ± 3.7 to −2.1 ± 2.5, P < 0.001). After 8 weeks follow-up, improvement was maintained in Gp-LPr and Gp-Pr, but there was deterioration in those who did not receive probiotics (Gp-L: PHES score reversed to −4.8 ± 4.2). Two patients (one each in Gp-L and Gp-Pr) experienced overt encephalopathy. One patient was hospitalized due to worsening of ascites (Gp-LPr) and one due to spontaneous bacterial peritonitis (Gp-L). Side effects were mild and not significantly different among the groups.

ABSTRACT.CONCLUSION:
Lactulose and probiotics are effective for the treatment of MHE; however, probiotics, but not lactulose, have long-term effects. More studies are required before suggesting probiotics for the standard treatment of MHE.

BODY.INTRODUCTION:
Hepatic encephalopathy (HE) is a common and usually reversible neurocognitive syndrome occurring in patients with cirrhosis. It manifests as a spectrum of changes from minimal HE (MHE), which is a state of low-level cognitive dysfunction detectable in up to 70% of the patients, to an overt HE, which has the risk of cerebral edema and death.[1] Although MHE is an underdiagnosed problem, it can affect daily functioning and impair attention span and reaction time.[2] Studies have shown that MHE increases the risk of motor vehicle accidents[3] and falls.[4]  The pathogenesis of MHE is multifactorial and the exact mechanisms causing brain dysfunction are still unknown. Increased blood ammonia is present in about 90% of the patients and is considered to play a major role.[5] Ammonia is produced mainly in the gastrointestinal tract by enterocytes from glutamine and by colonic bacterial catabolism of nitrogenous sources. Then, it enters the circulation via the portal vein and interferes with brain function at different sites.[67] Another possible source of ammonia is the urea digested by Helicobacter pylori in the stomach, although the role of H. pylori in HE and effects of its eradication are still under investigation.[89]  Regarding the role of ammonia in the pathophysiology of HE, several therapeutic strategies aimed to lower plasma ammonia for improving HE. These strategies include reducing ammoniagenic substrates, inhibiting ammonia production, and increasing ammonia removal.[510] Besides restriction of dietary protein intake and administration of antibiotics and disaccharides (e.g. lactulose), alteration of the gut flora with probiotics/prebiotics has gained substantial attention in the management of HE.[1112] Studies on different probiotics have shown that modification of gut flora is associated with improvement in HE by lowering blood ammonia concentrations. Possible mechanism in this regard might be favoring the gut flora to colonization of acid-resistant, non-urease producing bacteria.[1112] However, there is still a lack of data for endorsing probiotics as effective therapy for MHE. Few reports are available on the efficacy of multistrain probiotics, and few studies have investigated the efficacy of probiotics in comparison/in combination with other treatments such as lactulose.[1112] Therefore, in a clinical trial, we aimed to evaluate the efficacy of a multistrain probiotic compound, lactulose, and combination of these therapies in the management of MHE. We hypothesized that probiotic compound is as effective as lactulose in the management of MHE and combination of these therapies is more effective than each of them alone.

BODY.MATERIALS AND METHODS.PATIENTS AND SETTINGS:
This study has two parts; a randomized, placebo-controlled, comparative study with two arms; and an open-label study with one arm which are described as follow. The study was conducted on adult patients with MHE who referred consecutively to the gastroenterology clinic of a university hospital in Isfahan city (Iran) between June and October 2012. Cirrhosis was diagnosed histologically (unless biopsy was contraindicated) and on clinical and radiological grounds. Diagnosis of MHE was based on the Conn's modification of the Parsons-Smith classification (grade 1 and above).[13] Patients with overt HE, known brain lesions, active gastrointestinal bleeding, active ongoing infection, renal impairment (serum creatinine >2 mg/dL), electrolyte abnormalities (serum sodium <130 or >150 meq/dL, serum potassium <3.0 or >5.5 meq/dL), and those who received HE treatments such as lactulose and antibiotics or consumed benzodiazepines, narcotics, opioids, or alcohol in the preceding 8 weeks were not included into the trial. Considering type I error (α) = 0.05, study power = 0.8, and expecting 0.8 difference in psychometric hepatic encephalopathy score (PHES) between the two groups, sample size was calculated as 20 patients in each group. The study was approved by the ethics committee of Isfahan University of Medical Sciences and informed consent was obtained from the patients or their families. Also, the study was registered at Iranian Registry of Clinical Trials (IRCT201211012417N9).

BODY.MATERIALS AND METHODS.INTERVENTION:
Using a table of random numbers generated by random allocation software,[14] patients were randomized into two groups of lactulose + probiotic (Gp-LPr) and lactulose + placebo (Gp-L). Another non-randomized group of patients who received probiotic alone (Gp-Pr) were separately included for further comparisons; this group did not receive placebo of lactulose. All patients received routine treatment for cirrhosis, including diuretics, β-blockers, endoscopic treatment, and a salt-restricted diet but not protein-restricted diet in those with ascites. For Gp-LPr and Gp-L, lactulose syrup was administered as 30–60 mL/day in divided doses for a stool frequency of two to three soft defecations per day. For Gp-LPr and Gp-Pr, a multistrain probiotics compound, the Balance® (Protexin Co., Somerset, UK), was administered twice daily after meal. Balance capsules contains seven bacteria species including Lactobacillus strains (L. casei, L. rhamnosus, L. acidophilus, and L. bulgaricus), Bifidobacterium strains (B. breve and B. longum), and Streptococcus thermophiles. Total viable count is 1 × 108 colony forming units (CFU)/per capsule. Other Ingredients are fructo-oligosaccharides (FOS) as prebiotic, magnesium stearate, and hydroxypropyl methyl cellulose. These mentioned interventions were continued for 14 consecutive days and compliance was assessed with pill and bottle count.

BODY.MATERIALS AND METHODS.ASSESSMENTS:
All patients were assessed by an experienced gastroenterologist throughout the study. Demographic data and disease characteristics including causes of cirrhosis were gathered from patients' documents. Education level was considered as years of being on education. Severity of cirrhosis was assessed with the Child–Pugh classification which is a scoring system widely used to determine the severity and prognosis of liver diseases, mainly cirrhosis: Grade A = 5-6, Grade B = 7-9, and Grade C = 10-15.[15]  Primary endpoint was improvement in MHE status and was assessed by applying the PHES at baseline, 14 days after starting the intervention (14th day), and then at 8 weeks follow-up (10th week). The PHES is a set of neuropsychological tests including the line tracing test, digit symbol test, serial dotting test, and the number connection test. These tests are used in the diagnosis and grading of MHE and examine visual perception, visuo-spatial orientation, visual construction, motor speed and accuracy, concentration, attention, and memory. Subjects could achieve between +6 and −18 points. The PHES has a high sensitivity and specificity to detect MHE[1617] and overt HE[18] and is proposed by the 11th World Congress of Gastroenterology for diagnosis and monitoring of MHE.[19]  Secondary outcomes were development of overt HE, admission in hospital for any other complication of cirrhosis, or death. Participants, attending physician, and the outcome assessor were blinded to the Gp-L and Gp-LPr arms, but were aware of the Gp-Pr arm.

BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES:
Data were analyzed with the SPSS software for Windows, version 16.0. Descriptive data are presented as mean ± SD or number (%). All continuous variables had normal distribution; therefore, analysis of variance (ANOVA) was applied for comparing continuous variables among the three groups and paired t-test was applied for evaluating changes in each group. Chi-square test was used for comparison of categorical variables. A P value of <0.05 was considered significant in all analyses.

BODY.RESULTS:
A total of 97 patients were evaluated during the study period. Twenty patients did not enter the trial according to the criteria of inclusion and eight patients were not willing to participate. After randomization, two patients from the Gp-L, four patients from the Gp-LPr, and three patients from the Gp-Pr declined to receive intervention. Finally, 60 adult patients with cirrhosis (80% male, mean age 38.4 ± 9.6 years) started the trial and completed the intervention [Figure 1]. Etiologies of cirrhosis included viral hepatitis (73.3%), autoimmune hepatitis (18.3%), and other etiologies (8.3%). Patients among the three groups were similar regarding demographic data and disease characteristics [Table 1].  Figure 1Study flowchart demonstrating patient flow throughout the study procedures   Table 1 Comparison of demographic data and disease characteristics among the three groups   The PHES score at baseline, 14th day, and 10th week are presented in Table 2. Patients among the three groups were similar regarding baseline PHES scores. At 14th day, a significant improvement in HE status was observed in all the three groups (paired t-test P < 0.001). During the follow-up period, one patient from each of the Gp-LPr and Gp-L was lost to follow-up. After 8 weeks follow-up, there was a deterioration in the Gp-L (−1.6 ± 2.9 to −4.8 ± 4.2), while the status of the Gp-LPr and Gp-Pr arms did not change significantly [Figure 2].  Table 2 Comparison of psychometric hepatic encephalopathy scores among the three groups   Figure 2Trend of changes in psychometric hepatic encephalopathy scores from baseline to 10th week among the three groups. PHES, psychometric hepatic encephalopathy score  Secondary outcome variables and reported side effects are presented in Table 3. None of the patients died during the study period. Variceal bleeding plus overt HE was observed in one patient of the Gp-L and one patient of the Gp-Pr. In the Gp-LPr, one patient was hospitalized for worsening of ascites and in the Gp-L, one patient was hospitalized for spontaneous bacterial peritonitis. Common side effects included abdominal pain and bloating, but in none of the patients, these side effects resulted in discontinuing the trial. Patients among the three groups were not significantly different regarding secondary outcomes and side effects [Table 3].  Table 3 Comparison of secondary outcome variables and side effects among the three groups 

BODY.DISCUSSION:
Studies have shown high prevalence of small intestinal bacterial overgrowth (SIBO)[20] and also alteration in the composition of the gastrointestinal bacterial flora among cirrhotic patients with MHE.[21] Probiotics are effective in the treatment of SIBO[22] and also can reduce the prevalence of harmful ammonia-producing bacteria in the gastrointestinal system[12] which provides rationale for the use of probiotics in the treatment of MHE. Our study showed that a multistrain probiotics compound containing Lactobacillus and Bifidobacterium strains and S. thermophiles can improve the neuropsychological status of patients with MHE. We found that combination of probiotics and lactulose does not increase the short-term efficacy of lactulose (or probiotics) while it might increase the side effects. However, administration of probiotics (alone or in combination with lactulose) was associated with maintenance of the achieved improvements. Indeed, the neuropsychological status deteriorated after discontinuing lactulose in patients who received lactulose alone. These results support the efficacy of probiotics for treatment of MHE.  Considering the role of gastrointestinal bacterial flora in the generation of ammonia and pathogenesis of MHE, several studies investigated if modulating the flora using prebiotics, probiotics, and synbiotics has beneficial effects on MHE. Lactulose is one of the most frequently used agents in the treatment of HE, and is effective as well as safe.[5] The underlying mechanisms by which lactulose improves HE is not clear. Lactulose converts to lactic and acetic acids and results in acidification of colonic contents. The low colonic pH then decreases passive non-ionic diffusion of ammonia and its systemic concentration. Also, lactulose is a kind of prebiotic that encourages the growth of beneficial bacteria (probiotics).[11] A meta-analysis by Shukla and colleagues on nine studies (with 349 patients) of prebiotics, probiotics, and synbiotics in the treatment of MHE found that administration of these agents was associated with significant improvement in MHE. This meta-analysis found lactulose as the most beneficial therapy, followed by probiotics and synbiotics. It also found that plasma ammonia level is decreased by probiotics/synbiotics, though there was heterogeneity in the results.[11] However, the results of this meta-analysis are affected by limitations of the included trials. Heterogeneity was present more in the studies on probiotics than in the studies on lactulose, and trial duration was longer in trials on lactulose. On the other hand, authors found that probiotics and synbiotics are better tolerated by patients than lactulose.[11] In contrast, the meta-analysis by Holte and colleagues on seven trials (393 patients) found that in comparison with placebo or lactulose, probiotics and synbiotics significantly improve HE, though they have no effects on major clinical outcomes such as preventing the progression of MHE to overt HE.[23] Another recent meta-analysis by McGee et al. on seven trials (with 550 patients) found that probiotics can reduce plasma ammonia level in MHE patients (the same as lactulose), but the efficacy of probiotics in altering clinically relevant outcomes such as mortality and quality of life was not approved.[12] Differences among these studies are mainly related to the included trials. Compared with these meta-analyses, we found that probiotics are as effective as lactulose in the short-term improvement of MHE, with the beneficial effects maintained for long term (unlike lactulose). Similar to previous studies, we found no difference among lactulose, lactulose in combination with probiotics, and probiotics with regard to major clinical outcomes such as mortality, overt encephalopathy, and hospitalization. These available data are in favor of using probiotics for the treatment of MHE. However, regarding the few studies available on the efficacy of probiotics in the treatment of MHE and a considerable heterogeneity among them with regard to the type of probiotics, duration of intervention (10-180 days), and outcome assessments, a clear conclusion cannot be obtained yet.[12]  An important finding of our study is that in contrast to lactulose, improvement achieved by 14 days of administration of probiotics was stable for up to 8 weeks. This finding is similar to the study by Loguercio et al., which showed that a 10-day administration of Enterococcus SF68 is as effective as lactulose in lowering blood ammonia, and contrary to lactulose, these effects persist after treatment withdrawal.[24] This finding highlights the modulation of gastrointestinal bacterial flora as one of the main underlying mechanisms by which probiotics can have long-term effects on MHE.[1112]  It must be noted that the probiotics compound that we used in our study was not a pure probiotic but indeed a synbiotic compound because it also contained FOS which is a prebiotic. In two separate studies, Malaguarnera et al. compared Bifidobacterium longum + FOS and placebo[25] or lactulose[26] and found significant improvement of MHE in terms of biochemical and neuropsychological tests. Studies have shown that FOS can enhance the effects of probiotics micro-organisms in the large intestine.[27]  As with many other conditions, the probiotic dose and duration needed to confer a health benefit is unknown for MHE. Studies showed that probiotics are well tolerated for long-term administration[2825] and it is possible to use them in the form of yogurt, which is supposed to be more tolerated by patients.[29] Thus, it is reasonable for future studies to assess the efficacy of probiotics at different dosages and with long-term administration.  There are some limitations of our study. We did not include a no-treatment or a pure placebo arm, and therefore, we cannot attribute the improvements in MHE exactly to the lactulose/probiotics. However, deterioration of MHE after discontinuing lactulose and maintenance of improvement in those who received probiotics can justify the role of treatments. Because we could not provide an appropriate placebo for lactulose, our study was not completely randomized and double blinded, which could affect our results. More importantly, our study sample size and duration were not enough to investigate clinically important outcomes such as mortality and incidence of overt HE.

BODY.CONCLUSIONS:
This study supports the efficacy of multistrain probiotics in the treatment of MHE. Probiotics are as effective as lactulose in the short-term improvement of MHE. However, the achieved improvement is maintained by probiotics and not by lactulose. Further well-designed studies with larger sample of patients and longer follow-ups on different probiotic types, dosages, and durations are warranted in this regard.

BODY.AUTHOR'S CONTRIBUTION:
ASh, MM, and HH generated the study idea and designed the study. HH, ET, ZhD, SF, and ShM gathered data. AGh conducted data analysis. SSh and AGh wrote manuscript draft. All authors studied, revised, and approved the manuscript.

Question: Compared to baseline what was the result of receive lactulose (30–60 mL/day) + probiotic (200 million colony forming units of seven bacteria species/day) (Gp-LPr) or probiotic alone (Gp-Pr) on psychometric hepatic encephalopathy score (PHES) after 8 weeks?
Evidence: After 8 weeks follow-up, there was a deterioration in the Gp-L (−1.6 ± 2.9 to −4.8 ± 4.2), while the status of the Gp-LPr and Gp-Pr arms did not change significantly [Figure 2].
Answer: significantly increased