PDF meta-analysis of microbiome therapies for hepatic encephalopathy

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A meta-analysis of microbiome therapies for hepatic encephalopathy

Jie Gaoâ,b, Rui Nieâ,b, Hong Changâ,b, Wei Yangâ,b,c and Qian Renâ,b,c

Microbiome therapies may be reported to be effective in hepatic encephalopathy (HE). We thus did a meta-analysis of randomized controlled trials to assess the effect of microbiome therapies for HE. We systematically searched PubMed, Web of Science, EMBASE, and Cochrane Library for randomized controlled trials that compared the different treatments for HE including probiotics, symbiotics, and fecal microbiota transplant (FMT). Meta-analysis was performed to calculate pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Twenty-one studies met our inclusion criteria (N = 1746 participants). Probiotics, synbiotics and FMT significantly reversed minimal HE (MHE) (OR: 0.41, 95% CI: 0.19–0.90,

P = 0.03), reduced overt HE (OHE) development (OR, 0.41; 95% CI: 0.28–0.61 P < 0.00001)and the frequency of serious adverse events(SAEs) (OR:0.14, 95% CI: 0.04–0.47, P = 0.001), meanwhile decreased ammonia levels (WMD: −9.26, 95%

CI: −16.92 to −1.61; P = 0.02), NCT level (MD = −4.41, 95% CI: −0.87 to −0.22, P = 0.04) and hospitalization rates (OR, 0.38; 95% CI: 0.19–0.79, P = 0.009) compared with placebo/no treatment. Finally, we conclude that microbiome therapies were more effective in improving MHE and preventing progression to OHE, reducing the frequency of SAEs, and decreasing ammonia levels, NCT level, and hospitalization rates when compared to placebo/no treatment. Eur J Gastroenterol Hepatol 35: 927–937

Introduction

Hepatic encephalopathy (HE) represents a significant complication arising from severe acute or chronic liver insufficiency, predominantly characterized by alterations in personality, consciousness, cognition, and motor function. The manifestations of HE can span a spectrum from subtle (minimal HE) to severe (overt HE) and even coma [1,2]. HE, as a common complication of liver cirrhosis, imposes a considerable impact on patient’s quality of life and represents a substantial economic burden, ultimately serving as a primary reason for the hospitalization of individuals with liver cirrhosis [3]. Lactulose, a synthetic disaccharide consisting of galactose and fructose, is pres- ently the first-line treatment for HE [4]. Nonetheless, patient compliance with lactulose remains low due to its significant side effects, including diarrhea, abdominal distension, and flatulence. Alternative treatment options for HE includes rifaximin and L-ornithine-L- aspartic acid (LOLA). Oral rifaximin, particularly when combined with lactulose, has demonstrated a reduction in the recurrence of HE attacks and an improvement

in patients’ quality of life. However, the application of rifaximin is constrained by cost and long-term safety concerns. Despite the utilization of these drugs for pre- ventive treatment, a considerable number of patients continue to experience breakthrough HE attacks [5].

Consequently, the exploration of additional treatment strategies is necessary [6].

Recent evidence has established a close connection between HE pathophysiology and the gut microbiome [7–10]. The therapeutic role of microbiota in HE has undergone a transformative shift, driven by several landmark trials and substantial advancements in micro- biology. Microbiome therapies encompass probiotics, synbiotics, postbiotics, fecal microbiota transplants (FMTs), phages, and engineered probiotics, with multiple trials assessing the effects of probiotics, commensals, and FMT individually. A multitude of clinical studies have revealed alterations in the composition of gut microbes in patients with HE [8,11]. Preclinical investigations have demonstrated that interventions targeting the intestinal microbiome, such as probiotics, synbiotics, and FMT, can significantly reduce hyperammonemia and hypertoxine- mia levels in rats, decrease the central necrosis area and inflammatory reactions in liver lobules, and reduce the incidence of MHE [12–15]. Consequently, the potential of human intestinal microbiota as a therapeutic target for HE has garnered significant interest. Probiotics are sub- strates selectively utilized by host microorganisms, which confer a health benefit [16]. Synbiotics represent a combination of probiotics and prebiotics, where prebiotics are composed of fermentable dietary fibers that stimulate the growth and survival of probiotics [17]. FMT involves the transfer of processed stool from a healthy donor to a recipient [18].

European Journal of Gastroenterology & Hepatology 2023, 35:927–937 Keywords: fecal microbiota transplant, hepatic encephalopathy, microbiome therapies, probiotics, synbiotics

aLanzhou University, ^bDepartment of Gastroenterology, the First Hospital of Lanzhou University and ^cKey Laboratory for Gastrointestinal Diseases of Gansu Province, Lanzhou University, Lanzhou, China

Correspondence to Dr. Qian Ren, The First Hospital of Lanzhou University, Lanzhou University Medical Campus, Chengguan District, Lanzhou City, Gansu Province, China

Tel: +18209313260; e-mail: [email protected] Received 2 March 2023 Accepted 2 May 2023.

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Several meta-analyses have indicated that the adminis- tration of probiotics and synbiotics for HE can decrease intestinal ammonia production and improve quality of life, hospitalization rates, and mortality [19]. Some researchers postulate that FMT may contribute to the microbial restoration, including the enhancement of beneficial taxa diversity in patients with HE [20]. Liu et al.

discovered that probiotics reduced serum endotoxin and ammonia levels while alleviating neurocognitive impair- ment in patients with cirrhosis and MHE [21]. However, these findings remain inconsistent and possess limitations, such as restricting the analysis to a single type of microbial treatment (e.g. only probiotics or FMT). To further eluci- date the effect of probiotics, synbiotics, and FMT, as well as the resulting gut flora modulation on HE, a meta-analysis of available randomized controlled trials (RCT) was conducted.

Methods Search strategy

This systematic review was conducted according to the preferred reporting items for systematic reviews and meta-analyses guidelines. We established a protocol for the review, which was registered with PROSPERO prior to commencing the study (https://www.crd.york.ac.uk/

prospero/, CRD42022349109).

Eligibility criteria

Trials met the following criteria were included: (a) type of study: randomized controlled clinical trials (RCTs), (b) adults (defined as ≥18 years old) with HE, (c) in terms of intervention measures, the experimental group was treated with microbiome therapies, and the control group received placebo, no treatment, lactulose, (d) outcome indicators include the changes of intestinal flora, the reversal of MHE, the occurrence of OHE, serious adverse events, hospitalization rate, mortality and the reduction of serum total ammonia concentration.

Exclusion criteria: (a) repeatedly published literature;

(b) literature review; (c) non-RCT; (d) abstract and other non-full-text documents; (e) literature that cannot obtain complete outcome data; (f) animal experiments.

Study selection

The databases PubMed, ISI Web of Science, EMBASE, and Cochrane Library were used for the searching of the literature relating to the effect of probiotics, symbiotics, FMT, bacteriophage, engineered probiotics, postbiotic on HE in patients with cirrhosis. And the American clinical trial registration platform (clinicaltrials.gov) was searched. The retrieval time is from the establishment of the database to 16 July 2022. The retrieval adopts the combination of subject words and free words.

Quality assessment

All included RCTs were assessed for risk of bias according to the Cochrane Collaboration’s tool, using the following domains: sequence generation, allocation concealment,

blinding of participants, personnel and outcome asses- sors, incomplete outcome data, selective outcome reporting, and other potential threats to validity. We followed the pre-designed information extraction form, and data extraction, cross-check, in case of disagreement, discussed together or seek third-party opinions, and contact the author to obtain the required information if the information is not complete. Figures 1 and 2 show the risk of biased assessment of the included RCTs.

Study outcomes

The meta-analysis was performed using the Review Manager software (version 5.4, Cochrane Informatics

&Knowledge Management Department). Continuous outcome variables were assessed with weighted mean dif- ferences (WMDs) and 95% confidence intervals (CIs), and dichotomous outcomes were evaluated with aggregated odds ratios (ORs) and 95% CIs. Heterogeneity among included research results was adopted χ² Inspection and analysis (the inspection level is α = 0.1). At the same time, we judge the size of heterogeneity in combination with I².

I² < 50% indicates that there is no heterogeneity in the study, and use fixed effect model for analysis, otherwise use random effect model for analysis. The test level of meta-analysis is ɑ = 0.05.

Data extraction

Two researchers independently screened the literature, extracted data and cross-checked it. During literature screening, we first read the title and abstract of the arti- cle and then evaluated the full text to determine whether it meets the inclusion criteria after excluding obviously irrelevant literature. Any disagreement between them over the eligibility of studies will be resolved through discussion with a third reviewer. The contents of data extraction include (1) basic information included in the study:

title, first author’s name and publication time; (2) baseline characteristics, intervention means and duration of intervention of the subjects; (3) outcomes: the changes of intestinal flora, the reversal of MHE, the occurrence of OHE, serious adverse events, hospitalization rate, mortality, and the reduction of serum total ammonia concentration.

Results

The basic characteristics of the included studies

Through the search strategy, we have retrieved 755 articles in PubMed, Web of science, EMBASE, and the Cochrane library database. By screening the title and abstract, 694 articles were excluded because they were in-vitro or in-vivo experiments, reviews, and unrelated topics. The remaining 61 articles were read in full text, 9 articles were non-RCTs, and 17 articles did not meet the inclusion criteria for the study. 11 studies were excluded because they did not measure the outcome of interest. Three studies were excluded because of duplication. Finally, 21 studies were included. The selection process for the articles is shown in Fig. 3. The characteristics of included trials are summarized in Table 1. Twelve RCTs evaluated probiotics, symbiotics, and FMT versus placebo/no treatment

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[10,21–28,30,31,41]. Six RCTs investigated probiotics/

symbiotics versus lactulose [32–37], 2 investigated probiotics versus rifaximin and lactulose [40], 1 investigated probiotics versus rifaximin and LOLA [38], and 1 investigated probiotics versus lactulose and LOLA [39].

Twenty-two RCTs with a total of 1761 patients met our specified criteria. Of these, fifteen evaluated the effect of probiotics [10,22,24,25,27,28,32,33,35–41], three of synbiotics [21,26,34] and four of FMT [23,29–31] on HE.

Changes in intestinal microflora

Two trials reported an increase in Lachnospiraceae and Ruminococcaceae after FMT, which was closely associated with the production of short-chain fatty acids [23,31]. Four studies reported a statistically significant increase in beneficial bacteria such as Lactobacillus and Bifidobacterium by taking probiotics [21,33,40,41]. It was not possible to conduct a meta-analysis on the reduction of changes in intestinal microflora due to the clinical heterogeneity in the reporting outcomes.

Serious adverse events

The primary outcomes were safety and tolerability of probiotics, symbiotics and FMT compared to placebo related to serious adverse events (SAEs consisting of hospitalizations and ER visits). Three trials evaluated the frequency of serious adverse events [23,27,30]. One reported the number of AEs [37]. A combined analysis of data from three studies illustrated that probiotics and FMT significantly reduced the frequency of serious adverse events.

(OR: 0.14, 95% CI: 0.04–0.47, P = 0.001)The frequency of serious adverse events was 10.2% (5/49) in the probiotic and FMT group, and 48% (15/31) in the placebo group. In the study in which AEs were reported, 10 patients experienced adverse events related to the study drug, and none of these events were considered serious.

The adverse events that occurred with lactulose were diarrhea in all 7 patients, and abdominal pain reported in 3 patients treated with probiotics (Fig. 4).

Reversal of MHE

In Night studies [21,22,24,25,33,35,38,39,41], MHE in probiotics, synbiotics and FMT treatment group was improved compared with placebo(OR: 0.41, 95% CI:

0.19–0.90, P = 0.03). There was significant heterogeneity between high risk of bias, the results were similar (OR:

0.36, 95% CI: 0.15–0.84, P = 0.02). Probiotics showed no significant improvement of MHE in comparison with lactulose (OR: 1.16, 95% CI: 0.75–1.80, P = 0.50) [33,35,37,39,40] (Fig. 5).

Progression to overt hepatic encephalopathy

Seven of 12 trials showed significantly reduced OHE development when probiotics, synbiotics and FMT were used as the intervention compared with placebo/no treatment (OR, 0.41; 95% CI: 0.28–0.61 P < 0.00001) [24,25,31–

33,39,41]. In comparison with lactulose, probiotics had no significant effect on the prevention of OHE (OR: 1.32, 95% CI: 0.77–2.29, P = 0.32) [32,33,37,39] (Fig. 6).

Ammonia levels

Nine trials evaluated the effect of probiotics, synbiotics and FMT on ammonia levels compared with placebo/

no treatment [21,22,24,25,32–34,39,41]. Four studies compared the effects of probiotics, prebiotics and FMT Fig. 1. Risk of bias graph: review authors judgments about each risk of

bias item presented as percentages across all included trials.

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with lactulose in reducing ammonia levels [32,33,35,37].

Probiotics, synbiotics and FMT had no significant effect on ammonia levels when compared with placebo (WMD:

−9.26, 95% CI: −16.92 to −1.61; P = 0.02). There was

significant heterogeneity between the trials(I² = 88%, P < 0.00001). After excluding the study with a high risk of bias, the results were similar (WMD: −9.87, 95% CI:

−17.91 to −1.82; P = 0.02). Compared with probiotics,

Fig. 2. Risk of bias summary: review authors’ judgments about each risk of bias item for each included trial.

Fig. 3. PRISMA flowchart showing study identification and selection process. PRISMA, preferred reporting items for systematic reviews and meta-analyses.

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Table 1. Characteristics of the human studies reviewed

Study Age (years)

Total sample size

(number of

patients) Intervention group Comparison

Length of

treatment Primary outcome Bajaj 2014 [¹⁰] T:52 ± 8

C:58.4 ± 4.3 37 Lactobacillus LG Placebo 8 weeks MELD score, ALT,

AST, Albumin, Child score, Ammonia, NCT, Endotoxin, Inflammatory

Cytokine, Hospitalizations,

OHE Pereg 2011

[²²]

T:63.2 ± 10.5

C:65.9 ± 8.4 40 Capsules containing 4 freeze- dried bacteria probiotic yogurt (Lactobacillus acidophilus, La)

Placebo 6 months Albumin, ALT, AST, Bilirubin, Am monia,

INR, CTP Bajaj et al.

2019 [²³]

T:52 ± 8

C:54 ± 4 25 Ctobacilluscasei, Lactobacillus bulgaricus, Bifidobacterium, and

Streptococcus thermophiles

No treatment 60 days OHE, NCT, BDT, DST

Dhiman 2014 [²⁴]

T:48.0 (45.2–50.8)

C:50.1 (47.6–52.5) 130 VSL#3, lyophilized probiotic preparation granulated powder with

4 Lactobacillus species

Placebo 6 months OHE, Hospitalizations, Liver functions,

Inflammatory markers, Biochemical markers, Reversal

of MHE Lunia 2014

[²⁵]

T:8.5 ± 10.5

C:49.4 ± 11.5 160 VSL#3 (B. breve, B. longum, B. infantis, L. acidophilus, L. plantarum, L.

paracasei, L. bulg aricus, and S.

thermophiles)

No treatment 5 months Albumin, ALT, AST, Bilirubin, Ammonia,

MELD score, CTP, SIBO, OHE, Mortality

Ammonia levels Malaguarnera

2007 [²⁶]

T:46.0 ± 11.0

C:45.0 ± 12.0 60 B. longum + FOS Placebo 3 months BDT, MMSE, TMT

Liu 2004 [²¹] T1 : 55.0 ± 12.0 T2 : 53.0 ± 10.0 C:57.0 ± 12.0

55 T1:symbiotic (4 freeze-dried bacteria (Pediacoc cus pentoseceus, Leuconostoc mesenteroides, L.

paracasei, and L. plantarum) T2:1 sachet/day of only the bioactive, fermentable fibers described above

Placebo 1 month Serum Endotoxin,

Ammonia, Liver functions, NCT, CTP

Yossef 2020 [²⁷]

T:65.6 ± 11.4

C:62.9 ± 9.2 40 Probiotic formulation of B. subtilis strain HU58

Placebo 1 month Ammonia, SAEs

Xia 2018 [²⁸] T:41.5 ± 12.9

C:43.8 ± 10.3 67 Mixture containing Clostridium butyricum combined with B. infantis

Placebo 3 months ALT, AST, Albumin, Child score, Ammonia, NCT, DST Bajaj 2019 [²⁹] T:63.3 ± 4.2

C:64.2 ± 6.2 20 FMT was prepared from one stool sample from one healthy donor stool

sourced from a universal stool bank

Placebo 6 months SAEs, Cognitive function, Intestinal

barrier changes Bajaj 2018 [³⁰] Mean age:

65 ± 6.4 20 90 ml (27 g of stool) of the FMT material from the donor was administered via

enema with the patients

Placebo 15 days SAEs, Cognitive

function, Intestinal barrier changes Bajaj 2021 [³¹] T:64.5 ± 5.1

C:62.9 ± 9.8 20 90 ml of FMT enema Placebo 6 months SAEs, Cognitive

function, Intestinal barrier changes,

Hemoglobin, Bilirubin, ALT, AST,

Albumin Agrawal 2012

[³²]

T:45.4 ± 11.7 C1 : 41.7 ± 10.7 C2 : 46.0 ± 11.2

243 3 capsules/day containing 112.5 × 10¹¹ CFU/capsule of viable lyophilized bacteria (4 strains of Lactobacillus (L.

casei, L. plantarum, L. acidophilus, and L. bulgaricus], 3 strains of Bifidobacterium (B. longum, B. breve,

and B. infantis) and S. thermophiles

C1:Lactulose

C2:No treatment 12

months Recurrence of HE, Hospitalizations, Hemoglobin, Bilirubin, ALT, AST, Albumin, Child score,

MELD, Ammonia, CFF Ziada 2013

[³³]

T:50.3 ± 7.8 C1 : 48.8 ± 8.2 C2 : 51.2 ± 7.5

90 L. acidophilus 1 × 10⁶ CFU/capsule C1:Lactulose C2:Placebo

1 month Albumin, ALT, AST, Bilirubin, Creatinine,

Ammonia Malaguarnera

2010 [³⁴]

Mean age:

50.1 ± 9.4 125 B. longum + FOS C: Lactulose 2 months Ammonia, Liver

function, Sharma 2008

[³⁵]

T:43.5 ± 12.1 C1 : 39.5 ± 13.0

C2 : 43.7 ± 10.0

105 Lyophilized probiotics (each capsule:

Streptococcus faecalis, 60 million;

Clostridium butyricum, 4 million;

Bacillus mesentericus, 2 million; and Lactobacillus, 100 million)

C1:Lactulose C2:Lactulose+Probiotic

1months ALT, AST, Bilirubin, Albumin, M ELD, CFF Child score,

P300ER P Shavakhi

2014 [³⁶]

Mean age:

38.4 ± 9.6 60 Lyophilized probiotics [Lactobacillus strains (L. casei, L. rhamnosus, L.

acidophilus, and L. bulgaricus)], Bifidobacterium strains (B. breve and

B. longum) and S. thermophiles in a total of 1 × 10⁸ CFU per capsule.

C1:Lactulose+Probiotic

C2:Lactulose+Placebo 10 weeks OHE, Reversal of MHE, PHES, Mortality

(Continued )

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lactulose significantly reduced ammonia level, but there was no significant difference between the microbiome and lactulose group (WMD: −4.42 95% CI: −8.86–0.01;

P = 0.05) and the heterogeneity between the studies was NS (P = 0.49 I² = 0%) (Fig. 7).

Hospitalizations

Six studies compared hospitalization rates among patients who were treated with microbiome therapies versus no treatment/placebo [23–25,31,32,39]. The hospitalization rate was 21% (45/215) in the microbiome therapies group, and 40% (85/215) in the placebo group.

Probiotics, synbiotics and FMT significantly reduce the hospitalization rate (OR, 0.38; 95% CI: 0.19–0.79, P = 0.009). In contrast, there was no difference in hospitalization between the probiotic and lactulose groups (OR: 1.02, 95% CI: 0.52–2.00, P = 0.96) [32,36,39]

(Fig. 8).

Effect of NCT values

NCT is commonly used for the clinical diagnosis of sub- clinical HE. Three studies compared the effect on NCT values between patients who were treated with microbiome therapies versus placebo. There was a significant difference in NCT values between the probiotic and

placebo (MD = −4.41, 95% CI: −0.87 to −0.22, P = 0.04) (Fig. 9).

Discussion

The presented study constitutes the most up-to-date meta-analysis, encompassing individual references not featured in previously published meta-analyses [27,28,39]. This analysis includes 21 randomized control trials and assesses the efficacy of microbial therapy in comparison to both placebo and lactulose. The meta-analysis evaluates the overall therapeutic effects of probiotics, synbiotics, and FMT, and examines the safety of using microbial therapy, an aspect not evaluated in prior meta-analyses.

According to the results of this meta-analysis, microbiome therapies may play a crucial role in the management of HE. In several analyses, the clinical impact of microbiome therapies was found to be comparable to that of lactulose, and superior to the use of placebo. For example, the application of probiotics and synbiotics or FMT treatment led to an increase in beneficial flora in the gut and restored intestinal flora diversity compared to placebo or no treatment. Furthermore, these therapies significantly reversed MHE, reduced OHE and the frequency of serious adverse events, and decreased ammonia levels, NCT levels, and hospitalization rates,

Study Age (years)

Total sample size

(number of

patients) Intervention group Comparison

Length of

treatment Primary outcome Mouli 2015

[³⁷]

T:39.6 ± 11.4

C:44.2 ± 10.4 120 VSL#3 (B. breve, B. longum, B. infantis, L. acidophilus, L. plantarum, L.

paracasei, L. b ulgaricus, and S.

thermophiles)

C: Lactulose 2 months Ammonia, PHES, OHE, Reversal of MHE

Sharma 2014 [³⁸]

T:33.87 ± 13.2 C1 : 43.9 ± 12.5 C2 : 42.0 ± 11.4 C3 : 38.0 ± 11.8

124 5 × 10¹²CFU of lyophilized probiotics (L. acidophilus, L. rhamnosus, L.

plantarum, L. casei, B. longum, B.

infantis, B. breve, Sacchromyces boulardi, and S. thermophiles)

C1:LOLA C2:Rifaximin C3:Placebo

2 months PHES, OHE, Reversal of MHE

Mittal 2011 [³⁹]

T:44.2 ± 11.8 C1 : 41.2 ± 11.9 C2 : 43.8 ± 10.9 C3 : 42.1 ± 8.7

160 1.10 × 10 ¹⁰ CFU of probiotics C1:standard treatment for cirrhosis C2:lactulose C3:LOLA

3 months Ammonia, CTP score, MELD score, NCT,

FCT, BDT Manzhalii

2022 [⁴⁰]

T:48.85 ± 1.93 C1 : 48.92 ± 1.64 C2 : 49.07 ± 1.76

45 Probiotics (2,5-25 109 colony forming units cfu/g), 1 capsule (QD), and then twice a day (bid) were given according to the protocol for the first

4 days for 1 month

C1:Lactulose C2:Rifaximin

1 month Ammonia, Stroop

Fig. 4. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment in reducing serious adverse events. CI, confidence interval.

Table 3 (Continued )

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without affecting overall mortality. This review also demonstrated that probiotics exhibit similar efficacy in reversing MHE, preventing the development of OHE, and reducing ammonia levels and hospitalization rates as lactulose.

Previous meta-analyses comparing probiotics and synbiotics with current guideline-recommended drugs for the treatment of HE (e.g. lactulose, rifaximin) demonstrated that probiotics and synbiotics were as effective as lactulose in altering intestinal flora (i.e. increasing beneficial

Fig. 5. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment, lactulose on the reversal of minimal hepatic encephalopathy.

CI, confidence interval.

Fig. 6. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment or lactulose on the development of overt hepatic encephalopathy. CI, confidence interval.

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flora and decreasing SIBO), reversing MHE, preventing the development of OHE, and reducing ammonia levels [19,42]. Additionally, probiotics reversed MHE and pre- vented the development of OHE compared to placebo/no treatment, while synbiotics significantly reduced ammonia levels compared to placebo/no treatment. Bajaj et al.

discovered that the levels of ammonia-producing bacteria Alcaligenaceae in the intestine increased under HE condi- tions. Consequently, the successful use of probiotics for the restoration of microbiota considerably reduced bac- terial urease activity, ammonia uptake by the intestine, and the intensity of the inflammatory process and endotoxemia, due to the reduced absorption of indoles, phe- nols, and thiols [43]. Lactulose is considered the standard treatment for HE due to its efficacy. However, lactulose may not be the best therapy for long-term HE treatment because of its side effects, cost, and relatively poor adher- ence. Among the patients in this study, only 2/30 (6.7%) discontinued treatment due to poor tolerance, but more patients experienced different adverse effects, primarily flatulence and nausea, at 29.2% and 20.8%, respectively [44,45]. Similar results were previously obtained with lactulose treatment, such as diarrhea, flatulence, and nausea.

Some authors rejected the use of lactulose as the standard treatment for MHE due to its cost and poor long-term tolerability, advocating for a safe and well-tolerated alternative [46]. As probiotics are safe, natural, well-tolerated, and suitable for long-term use, probiotic therapy is considered an ideal treatment strategy for HE and has been gaining acceptance worldwide in recent years. Several large meta-analyses have found that probiotics improve HE symptoms, reverse minimal HE, reduce significant HE episodes, and lower ammonia levels. Concurrently, studies have demonstrated that probiotics can alleviate neurological cognitive function [47,48]. The present meta-analysis

also discovered that blood ammonia and NCT levels of MHE patients tended to decrease significantly following the application of microecological preparations. This may be related to the effect of balancing the intestinal microbiota to treat bacteria and reduce the production of harmful substances, as well as enhancing the intestinal barrier function of liver disease patients and reducing toxin absorption. Furthermore, probiotics did not impact patients’ quality of life or mortality and were not superior to lactulose in all outcomes. These findings are consistent with the present analysis. No meta-analysis results are available regarding FMT. The three trials that included FMT in this paper were conducted by the same study group (Bajaj et al.), randomly assigning patients to the oral 1-day FMT capsule group and the placebo capsule group [20,23]. FMT in both trials was from the same donor. The primary outcome of both trials assessed the safety of FMT and found no safety concerns except for a small reversible increase in Model for End-Stage Liver Disease (MELD) scores after broad-spectrum antibiotic treatment. A systematic review of FMT concluded that FMT is effective in treating and reducing the recurrence of HE [49]. Additionally, arterial ammonia levels were significantly reduced after FMT treatment. Studies evaluating FMT for HE have been small and designed to assess safety, not efficacy. Clinical efficacy results remain inconclusive; however, certain positive aspects have been observed. In published trials involving a single donor and a one-time dose of FMT, the efficacy may have been influ- enced by donor variability. To assess clinical improvement in HE using different FMT dosing strategies, it may be necessary to conduct larger trials.

This meta-analysis presents several notable limitations. First, only articles published in English were considered, potentially excluding relevant studies in other

Fig. 7. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment or lactulose in the reduction of ammonia levels. CI, confidence interval.

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languages. Second, the majority of the studies in the meta-analysis were conducted in India and the USA, which could limit the applicability of the results to other regions. The small sample sizes of the included trials may have also affected the meta-analysis outcomes, particularly for the RCT of fecal flora transplantation for HE.

Additionally, the three trials that involved FMT were conducted by the same research group, possibly intro- ducing some bias. Large heterogeneity was observed in studies regarding blood ammonia levels, MHE reversal rates, and hospitalization rates in patients with HE, which could be attributed to inconsistencies in patients’

underlying diseases, dosages, and regimens. The types and doses of probiotics, synbiotics, and FMT transplant flora used in the studies were inconsistent, potentially affecting the findings’ reliability. The results of this study should be validated through additional, higher-quality, and larger-sample RCTs. Nevertheless, this is the first systematic review to provide valuable information on the improvement of gut flora and the reduced incidence of adverse events in patients with HE treated with microbial therapy, offering clinical implications to sup- port this population.

In conclusion, microbial therapy may serve as an effective approach to combating HE. The meta-analysis results suggest that microbial therapy is more effective than no treatment/placebo in reducing adverse event incidence, preventing OHE development in patients with underlying MHE, and decreasing NCT levels and hospitalization rates. However, this review underscores the necessity for larger, high-quality RCTs with longer follow-up periods to investigate the role of gut microbes in intestinal flora changes, blood ammonia reduction, HE prevention and/or reversal, neurological cognitive function mitigation, and quality of life improvement. Further studies examining the effects of fecal flora transplantation on HE are also warranted.

Acknowledgements

We sincerely thank Prof. Jinhui Tian for many help in data processing on this project.

This study was supported by the Research Funds of the Frist Hospital of Lanzhou University (ldyyyn2019-74), and the Higher Education Innovation Foundation of Gansu Province (2021B-024).

Fig. 8. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment or lactulose in reducing hospitalization rate. CI, confidence interval.

Fig. 9. Forest plot displaying the effect of microbiome therapies versus placebo/no treatment in reducing NCT level. CI, confidence interval.

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Conflicts of interest

There are no conflicts of interest.

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