Faecal microbiota transplantation for first or second Clostridioides difficile infection (EarlyFMT): a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Clostridioides difficile infection is an urgent antibiotic-associated health threat with few treatment options. Microbiota restoration with faecal microbiota transplantation is an effective treatment option for patients with multiple recurring episodes of C difficile. We compared the efficacy and safety of faecal microbiota transplantation compared with placebo after vancomycin for first or second C difficile infection. METHODS: We did a randomised, double-blind, placebo-controlled trial (EarlyFMT) at a university hospital in Aarhus, Denmark. Eligible patients were aged 18 years or older with first or second C difficile infection (defined as ≥3 watery stools [Bristol stool chart score 6-7] per day and a positive C difficile PCR test). Patients were randomly assigned (1:1) to faecal microbiota transplantation or placebo administered on day 1 and between day 3 and 7, after they had received 125 mg oral vancomycin four times daily for 10 days. Randomisation was done by investigators using a computer-generated randomisation list provided by independent staff. Patients and investigators were masked to the treatment group. The primary endpoint was resolution of C difficile-associated diarrhoea (CDAD) 8 weeks after treatment. We followed up patients for 8 weeks or until recurrence. We planned to enrol 84 patients with a prespecified interim analysis after 42 patients. The primary outcome and safety outcomes were analysed in the intention-to-treat population, which included all randomly assigned patients. The trial is registered with ClinicalTrials.gov, NCT04885946. FINDINGS: Between June 21, 2021, and April 1, 2022, we consecutively screened 86 patients, of whom 42 were randomly assigned to faecal microbiota transplantation (n=21) or placebo (n=21). The trial was stopped after the interim analysis done on April 7, 2022 for ethical reasons because a significantly lower rate of resolution was identified in the placebo group compared with the faecal microbiota transplantation group (Haybittle-Peto boundary limit p<0·001). 19 (90%; 95% CI 70-99) of 21 patients in the faecal microbiota transplantation group and seven (33%, 95% CI 15-57) of 21 patients in the placebo group had resolution of CDAD at week 8 (p=0·0003). The absolute risk reduction was 57% (95% CI 33-81). Overall, 204 adverse events occurred, with one or more adverse events being reported in 20 of 21 patients in the faecal microbiota transplantation group and all 21 patients in the placebo group. Diarrhoea (n=23 in the faecal microbiota transplantation group; n=14 in the placebo group) and abdominal pain (n=14 in the faecal microbiota transplantation group; n=11 in the placebo group) were the most common adverse events. Three serious adverse events possibly related to study treatment occurred (n=1 in the faecal microbiota transplantation group; n=2 in the placebo group), but no deaths or colectomies during the 8-week follow-up. INTERPRETATION: In patients with first or second C difficile infection, first-line faecal microbiota transplantation is highly effective and superior to the standard of care vancomycin alone in achieving sustained resolution from C difficile. FUNDING: Innovation Fund Denmark.

Psychometric methods for diagnosing and monitoring minimal hepatic encephalopathy -current validation level and practical use.

Hepatic encephalopathy (HE) is cerebral dysfunction caused by liver failure and inflicts 30-40% of patients with liver cirrhosis during their disease course. Clinically manifest HE is often preceded by minimal HE (MHE) - a clinically undetectable cognitive disturbance closely associated with loss of quality of life. Accordingly, detecting and treating MHE improve the patients' daily functioning and prevent HE-related hospital admissions. The scope of this review article is to create an overview of the validation level and usage of psychometric tests used to detect MHE: Portosystemic hepatic encephalopathy test, continuous reaction time test, Stroop EncephalApp, animal naming test, critical flicker frequency test, and inhibitory control test. Our work is aimed at the clinician or scientist who is about to decide on which psychometric test would fit best in their clinic, cohort, or study. First, we outline psychometric test validation obstacles and requirements. Then, we systematically approach the literature on each test and select well-conducted studies to answer the following questions:• Which percentage of patients with cirrhosis does the test deem as having MHE?• Is the test able to predict clinically manifest HE?• Is there a well-known test-retest variation and inter-observer variation?• Is the test able to detect a treatment response?• Is the test result affected by age, educational level, gender, or comorbidities?

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

INTRODUCTION: In non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu. METHODS: We studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants. RESULTS: Blood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50-100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils. CONCLUSION: In NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

Early loss of T lymphocyte 4-1BB receptor expression is associated with higher short-term mortality in alcoholic hepatitis.

OBJECTIVES: In alcoholic hepatitis (AH), dysfunctional T lymphocytes may contribute to the high mortality from infections. T lymphocyte activation is governed by the expression of co-stimulatory receptors such as 4-1BB balanced by inhibitory receptors such as Programmed Death receptor 1 (PD-1). 4-1BB expression is unaccounted for in AH, while PD-1 is elevated. We characterized expression of 4-1BB and PD-1 and the associated T lymphocyte functional status in AH and investigated whether these were associated with short-term mortality. METHODS: Thirty-five patients with AH (at diagnosis and days 7 and 90) were compared with healthy controls (HC). Spontaneous and in vitro stimulated receptor expression were quantified by flow cytometry, and plasma proteins by ELISA. RESULTS: At diagnosis, the patients showed increased stimulated 4-1BB responses of CD4+ T lymphocytes. Also, the frequencies of PD-1+ T lymphocytes both with and without co-expressed 4-1BB were increased. Further, interferon-gamma was predominantly produced in T lymphocytes co-expressing 4-1BB. A decrease in the frequency of spontaneous 4-1BB+ T lymphocytes and an increase in soluble 4-1BB during the first week after diagnosis were associated with higher mortality at day 90 in AH. PD-1 expression showed no systematic dynamics related to mortality. CONCLUSIONS: We found an increased stimulated 4-1BB response of T lymphocytes in AH and early loss of these lymphocytes was associated with a higher short-term mortality. This suggests a role of T lymphocyte 4-1BB expression in the progression of AH.

Diminished Non-Classical Monocytes in the Blood Associate with Disease Severity in Alcoholic Hepatitis.

OBJECTIVE: Alcoholic hepatitis (AH) holds a high mortality, and vast macrophage infiltration of the liver is involved in the progressive liver injury. No efficient medical treatment exists, and macrophages may be a future treatment target. Here, we examine associations between non-classical monocyte subsets and cell surface markers of migration with disease activity in patients with severe AH. METHODS: We analyzed samples from two cohorts of patients with AH. Cohort 1 included 15 AH patients, followed for 30 days, and 8 healthy controls (HCs). Cohort 2 included 23 AH patients, followed for 90 days, and 9 HCs. Peripheral blood mononuclear cells (PBMCs) from both cohorts were analyzed by flow cytometry. Liver biopsies from cohort 2 were analyzed by RNA sequencing. RESULTS: Circulating non-classical monocytes in all but absent in patients with AH compared to HC in both cohorts (both p<0.0001). The frequency of non-classical monocytes was significantly associated with Maddrey's discriminant function (mDF) (r=-0.79, p=0.0008, cohort 1), Child-Pugh score (CP) (r=-0.56, p=0.03, cohort 1), Model for End-Stage Liver Disease (MELD) (r=-0.54, p=0.02, cohort 2) and C-reactive protein (CRP) (r=-0.51, p=0.027, cohort 2). The surface expression of CD11b was increased on non-classical monocytes in patients with AH compared to HC (p<0.0001) (cohort 1). The mRNA expression of CD11b was increased in liver biopsies in patients with AH compared to HC (cohort 2) (p<0.0001). CONCLUSION: In this study, we describe an almost complete depletion of circulating non-classical monocytes in the blood in two independent cohorts of patients with AH, which may be associated with a possible harmful recruitment of these cells to the liver. These results contribute to a better understanding of the disease, which hopefully can lead to therapies that target the acute inflammatory response leading to severe AH.

No Effect in Alcoholic Hepatitis of Gut-Selective, Broad-Spectrum Antibiotics on Bacterial Translocation or Hepatic and Systemic Inflammation.

INTRODUCTION: In alcoholic hepatitis (AH), translocation of gut bacteria may drive hepatic macrophage activation and systemic inflammation. We investigated the effect of oral non-absorbable, broad-spectrum antibiotic treatment on bacterial translocation and liver and systemic inflammation in AH. METHODS: We consecutively recruited 31 patients with AH. Fourteen were given vancomycin 500 mg, gentamycin 40 mg, and meropenem 500 mg once daily for 7 days. Seventeen patients were a reference group receiving standard-of-care. Circulating markers of bacterial translocation and inflammation were measured at baseline, by day 7 and 90. Gut bacteriome profiling was performed before the intervention and at day 7. RESULTS: At study entry, blood lipopolysaccharide-binding protein was multifold higher than normal, remained unchanged at day 7, but decreased at day 90 (P < 0.001) with no difference between the study groups. The macrophage activation markers sCD163 and sCD206 showed the same pattern (P < 0.001, day 90), still without group differences. The systemic inflammation markers tumor necrosis factor-alpha, interleukin (IL)-6, IL-8, and IL-10 showed similar dynamics without group differences. There was no difference in 90-day mortality (total of 6 deaths) between the groups. The remnant gut bacteriome was markedly diversified by the intervention with growth of bacterial species rare for human flora. DISCUSSION: In patients with AH, gut-targeted antibiotic treatment does not change markers of bacterial translocation and liver and systemic inflammation. This suggests that bacterial translocation is less important once the inflammatory process is established or that bacteriome reduction is less important than composition.