Role of compliance in Helicobacter pylori eradication treatment: Results of the European Registry on H. pylori management.

BACKGROUND: Adherence to Helicobacter pylori (H. pylori) eradication treatment is a cornerstone for achieving adequate treatment efficacy. OBJECTIVE: To determine which factors influence compliance with treatment. METHODS: A systematic prospective non-interventional registry (Hp-EuReg) of the clinical practice of European gastroenterologists. Compliance was considered adequate if ≥90% drug intake. Data were collected until September 2021 using the AEG-REDCap e-CRF and were subjected to quality control. Modified intention-to-treat analyses were performed. Multivariate analysis carried out the factors associated with the effectiveness of treatment and compliance. RESULTS: Compliance was inadequate in 646 (1.7%) of 38,698 patients. The non-compliance rate was higher in patients prescribed longer regimens (10-, 14-days) and rescue treatments, patients with uninvestigated dyspepsia/functional dyspepsia, and patients reporting adverse effects. Prevalence of non-adherence was lower for first-line treatment than for rescue treatment (1.5% vs. 2.2%; p < 0.001). Differences in non-adherence in the three most frequent first-line treatments were shown: 1.1% with proton pump inhibitor + clarithromycin + amoxicillin; 2.3% with proton pump inhibitor clarithromycin amoxicillin metronidazole; and 1.8% with bismuth quadruple therapy. These treatments were significantly more effective in compliant than in non-compliant patients: 86% versus 44%, 90% versus 71%, and 93% versus 64%, respectively (p < 0.001). In the multivariate analysis, the variable most significantly associated with higher effectiveness was adequate compliance (odds ratio, 6.3 [95%CI, 5.2-7.7]; p < 0.001). CONCLUSIONS: Compliance with Helicobacter pylori eradication treatment is very good. Factors associated with poor compliance include uninvestigated/functional dyspepsia, rescue-treatment, prolonged treatment regimens, the presence of adverse events, and the use of non-bismuth sequential and concomitant treatment. Adequate treatment compliance was the variable most closely associated with successful eradication.

Comparison of the management of Helicobacter pylori infection between the older and younger European populations.

The prevalence of Helicobacter pylori remains high in the older population. Specific age-related peculiarities may impact the outcomes of H. pylori treatment. The aim of the study was to evaluate the diagnostics and effectiveness of H. pylori eradication between the younger and older European populations. "European Registry on H. pylori Management (Hp-EuReg)" data from 2013 to 2022 were analyzed. Patients were divided into older (≥ 60 years) and younger (18-59 years) groups. Modified intention-to-treat (mITT) and per-protocol (PP) analysis was performed. 49,461 patients included of which 14,467 (29%) were older-aged. Concomitant medications and penicillin allergy were more frequent among the older patients. Differences between younger and older populations were observed in treatment duration in first-line treatment and in proton pump inhibitors (PPIs) doses in second-line treatment. The overall incidence of adverse events was lower in the older adults group. The overall first-line treatment mITT effectiveness was 88% in younger and 90% in the older patients (p < 0.05). The overall second-line mITT treatment effectiveness was 84% in both groups. The effectiveness of the most frequent first- and second-line triple therapies was suboptimal (< 90%) in both groups. Optimal efficacy (≥ 90%) was achieved by using bismuth and non-bismuth-based quadruple therapies. In conclusion, the approach to the diagnostics and treatment of H. pylori infection did not generally differ between younger and older patients. Main differences were reported in the concurrent medications, allergy to penicillin and adverse events both in first- and second-line treatment. Optimal effectiveness rates were mostly achieved by using bismuth and non-bismuth-based quadruple therapies. No clinically relevant differences in the effectiveness between the age groups were observed.

Analysis of Clinical Phenotypes through Machine Learning of First-Line H. pylori Treatment in Europe during the Period 2013-2022: Data from the European Registry on H. pylori Management (Hp-EuReg).

The segmentation of patients into homogeneous groups could help to improve eradication therapy effectiveness. Our aim was to determine the most important treatment strategies used in Europe, to evaluate first-line treatment effectiveness according to year and country. Data collection: All first-line empirical treatments registered at AEGREDCap in the European Registry on Helicobacter pylori management (Hp-EuReg) from June 2013 to November 2022. A Boruta method determined the "most important" variables related to treatment effectiveness. Data clustering was performed through multi-correspondence analysis of the resulting six most important variables for every year in the 2013-2022 period. Based on 35,852 patients, the average overall treatment effectiveness increased from 87% in 2013 to 93% in 2022. The lowest effectiveness (80%) was obtained in 2016 in cluster #3 encompassing Slovenia, Lithuania, Latvia, and Russia, treated with 7-day triple therapy with amoxicillin-clarithromycin (92% of cases). The highest effectiveness (95%) was achieved in 2022, mostly in Spain (81%), with the bismuth-quadruple therapy, including the single-capsule (64%) and the concomitant treatment with clarithromycin-amoxicillin-metronidazole/tinidazole (34%) with 10 (69%) and 14 (32%) days. Cluster analysis allowed for the identification of patients in homogeneous treatment groups assessing the effectiveness of different first-line treatments depending on therapy scheme, adherence, country, and prescription year.

Combined antrum and corpus biopsy protocol improves Helicobacter pylori culture success.

BACKGROUND: Helicobacter pylori (H. pylori) causes chronic gastritis, peptic ulcer disease, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma. Eradication rates have fallen, mainly due to antimicrobial resistance. Consensus guidelines recommend that first-line treatment is based on the local prevalence of antimicrobial resistance and that rescue therapies are guided by antimicrobial susceptibility testing (AST). However, H. pylori culture is challenging and culture-based AST is not routinely performed in the majority of hospitals. Optimisation of H. pylori culture from clinical specimens will enable more widespread AST to determine the most appropriate antimicrobials for H. pylori eradication. AIM: To determine whether dual antrum and corpus biopsy sampling is superior to single antrum biopsy sampling for H. pylori culture. METHODS: The study received ethical approval from the joint research ethics committee of Tallaght University Hospital and St. James's Hospital. Patients referred for upper gastrointestinal endoscopy were invited to participate. Biopsies were collected in tubes containing Dent's transport medium and patient demographics were recorded. Biopsies were used to inoculate Colombia blood agar plates. Plates were incubated under microaerobic conditions and evaluated for the presence of H. pylori. Statistical analyses were performed using Graphpad PRISM. Continuous variables were compared using the two-tailed independent t-test. Categorical variables were compared using the two-tailed Fisher exact test. In all cases, a P value less than 0.05 was considered significant. RESULTS: In all, samples from 219 H. pylori-infected patients were analysed in the study. The mean age of recruited patients was 48 ± 14.9 years and 50.7% (n = 111) were male. The most common endoscopic finding was gastritis (58.9%; n = 129). Gastric ulcer was diagnosed in 4.6% (n = 10) of patients, while duodenal ulcer was diagnosed in 2.7% (n = 6). Single antrum biopsies were collected from 73 patients, whereas combined antrum and corpus biopsies were collected from 146 patients. There was no significant difference in age, sex or endoscopic findings between the two groups. H. pylori was successfully cultured in a significantly higher number of cases when combined antrum and corpus biopsies were used compared to a single antrum biopsy [64.4% (n = 94/146) vs 49.3% (36/73); P = 0.04)]. CONCLUSION: Combined corpus and antrum biopsy sampling improves H. pylori culture success compared to single antrum biopsy sampling.

Molecular Detection of Antibiotic-Resistant Helicobacter pylori.

Antimicrobial susceptibility testing (AST) for H. pylori is essential to accurately assess the prevalence of antibiotic resistance in each population. Antibiotic resistance rates form the basis of local guidelines for H. pylori treatment and AST may also be used as a personalized medicine approach to tailor therapy. This chapter provides an update on global antibiotic resistance rates and describes molecular mechanisms that confer H. pylori antibiotic resistance. An overview on the advantages and limitations of molecular AST using both invasive and noninvasive approaches is also provided.

An Overview of Helicobacter pylori Infection.

Helicobacter pylori (H. pylori) represents one of the most widespread bacterial infections globally. Infection causes chronic gastritis and increases the risk of peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. The pioneering discovery of H. pylori by Marshall and Warren in the early 1980s has initiated fervent research into H. pylori as a pathogen ever since. This chapter aims to provide an overview of our understanding of H. pylori infection and its management, with a focus on current options for diagnosis, the challenges associated with H. pylori eradication, and the need for alternative therapeutic strategies based on furthering our understanding of host: H. pylori interactions.

Purification of Total RNA from Stomach Tissue Biopsies.

In order to further our understanding of the physiological consequences of Helicobacter pylori infection , analysis of clinical tissue specimens is required. To this end, RNA is frequently isolated from stomach biopsies of H. pylori-infected patients and compared to samples from uninfected controls to monitor gene expression using molecular methods such as reverse-transcription real-time PCR, microarrays, and next-generation sequencing. The successful purification of sufficient quantities of high-quality RNA is essential for accurate and reproducible downstream analysis. This chapter describes the key steps for high-quality RNA purification from human tissue samples, including sample collection and storage, tissue disruption and lysis, RNA purification, and assessment of RNA yield and quality.

Helicobacter pylori Virulence Factor Genotyping.

Helicobacter pylori (H. pylori) infection causes chronic gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. Bacterial, host, and environmental factors influence the progression of disease from superficial gastritis to cancer. H. pylori is genetically diverse, and expression of its specific virulence factors has been linked to increased risk of more severe pathologies. Described in this chapter is a protocol for detecting important H. pylori virulence factors by firstly extracting DNA from culture material or stomach tissue biopsies, followed by PCR amplification and agarose gel electrophoresis.

Three paralogous clusters of the miR-17~92 family of microRNAs restrain IL-12-mediated immune defense.

MicroRNAs (miRNAs) have been widely implicated in immune regulation, but evidence for the coordinated function of paralogous miRNA clusters remains scarce. Here, by using genetically modified mice with individual or combined cluster deficiencies, we found that three paralogous clusters of the miR-17~92 family of miRNAs collectively suppressed IL-12 production in macrophages. Accordingly, miR-17~92 family miRNAs deficiencies resulted in heightened production of IL-12 and thus enhanced the host defense against intracellular pathogen Listeria monocytogenes in vivo. Mechanistically, different members of the miR-17~92 family of miRNAs acted on a common target, PTEN, to inhibit IL-12 expression by modulating the PI3K-Akt-GSK3 pathway. In addition, the expression of miR-17~92 family miRNAs was collectively inhibited by the transcription factor RBP-J, and RBP-J-associated macrophage functional defects were genetically rescued by deleting three clusters of miR-17~92 family miRNAs on a RBP-J null background. Thus, our results illustrated key roles of three clusters of miR-17~92 family miRNAs in cooperatively controlling IL-12-mediated immune responses and identified miR-17~92 family miRNAs as functional targets of RBP-J in macrophages.

Shared changes in angiogenic factors across gastrointestinal vascular conditions: A pilot study.

BACKGROUND: Neovascularisation is common to a variety of gastrointestinal (GI) disorders with differing aetiologies and presentations; usually affecting adults above 60 years. Shared angiogenic factors modulated by disease specific elements could be a common denominator and represent novel diagnostic and therapeutic targets. As yet, assessment of angiogenic factors across several GI vascular disorders associated with recurrent bleeding and anaemia has not been reported. AIM: To assess serum levels of angiogenic factors in several intestinal vascular disorders. METHODS: A case control study was performed in Tallaght University Hospital in patients with endoscopically proven small bowel angiodysplasia (SBA), portal hypertensive gastropathy (PHG), gastric antral vascular ectasia (GAVE) and non-bleeding, non-anaemic controls. Using enzyme-linked immunosorbent assay, concentrations of Angiopoietin 1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) were measured from 2 serum tubes of blood following informed consent. The relative expression of Ang-1 and Ang-2 and Ang-1/2 ratio was calculated and compared between groups. Statistical analysis was applied using a t-test, and a P value of < 0.05 was considered significant. RESULTS: To date 44 samples were tested: 10 SBA, 11 PHG, 8 GAVE and 15 controls. Mean age 60 (range 20-85) years and 20 (45%) were males. Controls were significantly younger (49 years vs 66 years, P = 0.0005). There was no difference in VEGF levels between the groups (P = 0.6). SBA, PHG and GAVE Ang-1 levels were similar and were significantly lower than controls, (P = 0.0002, 95%CI: 241 to 701). Ang-2 levels were statistically higher in PHG and GAVE groups compared to controls (P = 0.01, 95%CI: 77.8 to 668) and as a result, also had a lower Ang-1/2 ratios compared to controls. While SBA Ang-2 levels were higher than controls, this did not reach statistical significance. Neither age nor haemoglobin level, which was similar between disease groups, could explain the difference. In addition, the median Ang-1/Ang-2 ratio for all patients was found to be significantly lower compared to controls, 8 vs 28 respectively, P = 0.001, 95%CI: -27.55 to -7.12. CONCLUSION: Our novel pilot study suggests common alterations in Ang-1 and Ang-2 levels across several GI vascular disorders. Differences in Ang-1/Ang-2 ratios among vascular disorders compared to controls suggest disease-specific modulation.

The colonic macrophage transcription factor RBP-J orchestrates intestinal immunity against bacterial pathogens.

Macrophages play pleiotropic roles in maintaining the balance between immune tolerance and inflammatory responses in the gut. Here, we identified transcription factor RBP-J as a crucial regulator of colonic macrophage-mediated immune responses against the enteric pathogen Citrobacter rodentium. In the immune response phase, RBP-J promoted pathogen clearance by enhancing intestinal macrophage-elicited Th17 cell immune responses, which was achieved by maintenance of C/EBPß-dependent IL-6 production by overcoming miRNA-17∼92-mediated suppressive effects. RBP-J deficiency-associated phenotypes could be genetically corrected by further deleting miRNA-17∼92 in macrophages. In the late phase, noneradicated pathogens in RBP-J KO mice recruited abundant IL-1ß-expressing CD64+Ly6C+ colonic macrophages and thereby promoted persistence of ILC3-derived IL-22 to compensate for the impaired innate and adaptive immune responses, leading to ultimate clearance of pathogens. These results demonstrated that colonic macrophage-intrinsic RBP-J dynamically orchestrates intestinal immunity against pathogen infections by interfacing with key immune cells of T and innate lymphoid cell lineages.

Helicobacter pylori resistance to current therapies.

PURPOSE OF REVIEW: Helicobacter pylori eradication rates have fallen in recent years, mainly because of the emergence of antibiotic-resistant infections. Indeed the WHO has recently designated clarithromycin-resistant H. pylori infection a high priority for antibiotic resistance research and development. This review aims to discuss the most up-to-date information on the methods to detect H. pylori antibiotic resistance, the recent data on resistance rates, and the most appropriate treatment strategies to overcome antibiotic resistance. RECENT FINDINGS: There has been active research into the development and assessment of genotypic diagnostic assays for both the invasive and noninvasive detection of antibiotic-resistant infection. There are regional variations in the prevalence of H. pylori antibiotic resistance. Primary resistance rates in general are on the rise and high rates of clarithromycin resistance (>15%) have been reported in many parts of the world. SUMMARY: Optimizing antimicrobial susceptibility testing by both invasive and noninvasive means is crucial to accurately evaluate resistance rates for the optimization of both regional and personalized H. pylori treatment strategies.

Can bacterial virulence factors predict antibiotic resistant Helicobacter pylori infection?

AIM: To evaluate the association between virulence factor status and antibiotic resistance in Helicobacter pylori (H. pylori)-infected patients in Ireland. METHODS: DNA was extracted from antral and corpus biopsies obtained from 165 H. pylori-infected patients. Genotyping for clarithromycin and fluoroquinolone-mediating mutations was performed using the Genotype HelicoDR assay. cagA and vacA genotypes were investigated using PCR. RESULTS: Primary, secondary and overall resistance rates for clarithromycin were 50.5% (n = 53/105), 78.3% (n = 47/60) and 60.6% (n = 100/165), respectively. Primary, secondary and overall resistance rates for fluoroquinolones were 15.2% (n = 16/105) and 28.3% (n = 17/60) and 20% (n = 33/165), respectively. Resistance to both antibiotics was 12.4% (n = 13/105) in treatment-naïve patients, 25% (n = 15/60) in those previously treated and 17% (n = 28/165) overall. A cagA-positive genotype was detected in 22.4% (n = 37/165) of patient samples. The dominant vacA genotype was S1/M2 at 44.8% (n = 74/165), followed by S2/M2 at 26.7% (n = 44/165), S1/M1 at 23.6% (n = 39/165) and S2/M1 at 4.8% (n = 8/165). Primary clarithromycin resistance was significantly lower in cagA-positive strains than in cagA-negative strains [32% (n = 8/25) vs 56.3% (n = 45/80) P = 0.03]. Similarly, in patients infected with more virulent H. pylori strains bearing the vacA s1 genotype, primary clarithromycin resistance was significantly lower than in those infected with less virulent strains bearing the vacA s2 genotype, [41% (n = 32/78) vs 77.8% (n = 21/27) P = 0.0001]. No statistically significant association was found between primary fluoroquinolone resistance and virulence factor status. CONCLUSION: Genotypic H. pylori clarithromycin resistance is high and cagA-negative strains are dominant in our population. Less virulent (cagA-negative and vacA S2-containing) strains of H. pylori are associated with primary clarithromycin resistance.

Differential modulation of Helicobacter pylori lipopolysaccharide-mediated TLR2 signaling by individual Pellino proteins.

BACKGROUND: Eradication rates for current H. pylori therapies have fallen in recent years, in line with the emergence of antibiotic resistant infections. The development of therapeutic alternatives to antibiotics, such as immunomodulatory therapy and vaccines, requires a more lucid understanding of host-pathogen interactions, including the relationships between the organism and the innate immune response. Pellino proteins are emerging as key regulators of immune signaling, including the Toll-like receptor pathways known to be regulated by H. pylori. The aim of this study was to characterize the role of Pellino proteins in the innate immune response to H. pylori lipopolysaccharide. MATERIALS AND METHODS: Gain-of-function and loss-of-function approaches were utilized to elucidate the role of individual Pellino proteins in the Toll-like receptor 2-mediated response to H. pylori LPS by monitoring NF-ĸB activation and the induction of proinflammatory chemokines. Expression of Pellino family members was investigated in gastric epithelial cells and gastric tissue biopsy material. RESULTS: Pellino1 and Pellino2 positively regulated Toll-like receptor 2-driven responses to H. pylori LPS, whereas Pellino3 exerted a negative modulatory role. Expression of Pellino1 was significantly higher than Pellino3 in gastric epithelial cells and gastric tissue. Furthermore, Pellino1 expression was further augmented in gastric epithelial cells in response to infection with H. pylori or stimulation with H. pylori LPS. CONCLUSIONS: The combination of low Pellino3 levels together with high and inducible Pellino1 expression may be an important determinant of the degree of inflammation triggered upon Toll-like receptor 2 engagement by H. pylori and/or its components, contributing to H. pylori-associated pathogenesis by directing the incoming signal toward an NF-kB-mediated proinflammatory response.

Strategies for the Purification of Membrane Proteins.

Although membrane proteins account for approximately 30 % of the coding regions of all sequenced genomes and play crucial roles in many fundamental cell processes, there are relatively few membranes with known 3D structure. This is likely due to technical challenges associated with membrane protein extraction, solubilization, and purification. Membrane proteins are classified based on the level of interaction with membrane lipid bilayers, with peripheral membrane proteins associating noncovalently with the membrane, and integral membrane proteins associating more strongly by means of hydrophobic interactions. Generally speaking, peripheral membrane proteins can be purified by milder techniques than integral membrane proteins, whose extraction require phospholipid bilayer disruption by detergents. Here, important criteria for strategies of membrane protein purification are addressed, with a focus on the initial stages of membrane protein solublilization, where problems are most frequently are encountered. Protocols are outlined for the successful extraction of peripheral membrane proteins, solubilization of integral membrane proteins, and detergent removal which is important not only for retaining native protein stability and biological functions, but also for the efficiency of downstream purification techniques.

Molecular detection of Helicobacter pylori antibiotic resistance in stool vs biopsy samples.

AIM: To compare (1) demographics in urea breath test (UBT) vs endoscopy patients; and (2) the molecular detection of antibiotic resistance in stool vs biopsy samples. METHODS: Six hundred and sixteen adult patients undergoing endoscopy or a UBT were prospectively recruited to the study. The GenoType HelicoDR assay was used to detect Helicobacter pylori (H. pylori) and antibiotic resistance using biopsy and/or stool samples from CLO-positive endoscopy patients and stool samples from UBT-positive patients. RESULTS: Infection rates were significantly higher in patients referred for a UBT than endoscopy (overall rates: 33% vs 19%; treatment-naïve patients: 33% vs 14.7%, respectively). H. pylori-infected UBT patients were younger than H. pylori-infected endoscopy patients (41.4 vs 48.4 years, respectively, P < 0.005), with a higher percentage of H. pylori-infected males in the endoscopy-compared to the UBT-cohort (52.6% vs 33.3%, P = 0.03). The GenoType HelicoDR assay was more accurate at detecting H. pylori infection using biopsy samples than stool samples [98.2% (n = 54/55) vs 80.3% (n =53/66), P < 0.005]. Subset analysis using stool and biopsy samples from CLO-positive endoscopy patients revealed a higher detection rate of resistance-associated mutations using stool samples compared to biopsies. The concordance rates between stool and biopsy samples for the detection of H. pylori DNA, clarithromycin and fluoroquinolone resistance were just 85%, 53% and 35%, respectively. CONCLUSION: Differences between endoscopy and UBT patients provide a rationale for non-invasive detection of H. pylori antibiotic resistance. However, the GenoType HelicoDR assay is an unsuitable approach.

RBP-J-Regulated miR-182 Promotes TNF-α-Induced Osteoclastogenesis.

Increased osteoclastogenesis is responsible for osteolysis, which is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α-induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating the expression of microRNAs (miRNAs) important for this process. Using high-throughput sequencing of miRNAs, we obtained the first, to our knowledge, genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow-derived macrophages/osteoclast precursors during inflammatory osteoclastogenesis. Furthermore, we identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss- and gain-of-function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and B lymphocyte-induced maturation protein-1. Moreover, we identified that direct miR-182 targets, Foxo3 and Maml1, play important inhibitory roles in TNF-α-mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α-induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α-induced osteoclastogenesis. Our results provide a novel miRNA-mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.