Gedgaudas, Rolandas

Rising antimicrobial resistance of Helicobacter pylori is a public health challenge. Genomic-based susceptibility testing allows for the identification of resistance-associated mutations, complementing conventional diagnostics and advancing towards pathogen-based personalised therapies. Our study aimed to identify genes and mutations involved in antimicrobial resistance in H pylori and evaluate the extent to which these markers can be used as predictors of phenotypic resistance against clarithromycin and levofloxacin.

Pratarmė. Mieli studentai, Gastroenterologija yra viena įdomiausių ir greičiausiai besiplėtojančių medicinos sričių. Dėl mokslo ir technologijų pažangos XXI a. gastroenterologija apima vis platesnį kepenų ir virškinamojo kanalo ligų, gydymo metodų spektrą. Tai neabejotinai viena įvairiapusiškiausių specialybių, kur akivaizdi klinikinių įgūdžių, molekulinių ir ultragarsinių tyrimų bei sudėtingų endoskopinių intervencijų sąsaja. Uždegiminės žarnyno ligos, retos virškinamojo kanalo ligos, mikrobiotos ir kepenų transplantacija yra tik nedidelė dalis sričių, kurios yra patikėtos gydytojams gastroenterologams. Šis Lietuvos sveikatos mokslų universiteto Medicinos akademijos Medicinos fakulteto Gastroenterologijos klinikos (toliau - Gastroenterologijos klinika) kolektyvo parengtas vadovėlis yra skirtas studentams, siekiantiems susipažinti su gastroenterologijos pagrindais. Jau beveik 30 metų Gastroenterologijos klinika aktyviai dalyvauja rengiant tarptautines diagnostikos ir gydymo gaires, yra prestižinių tarptautinių mokslo projektų ir konsorciumų dalyvė, o 2020 m. klinika tapo ir asocijuota Europos retų kepenų ligų tinklo nare. Mūsų klinikos mokslinių tyrimų rezultatai yra publikuojami prestižiniuose mokslo leidiniuose: Nature, Lancet, New England Journal of Medicine, Nature Genetics ir kituose. Rengdami šį vadovėlį stengėmės perteikti visą klinikos sukauptą ilgametę pedagoginę ir klinikinę patirtį bei naujausius mokslo pasiekimus. Tikimės, kad šis vadovėlis ne tik suteiks Jums pagrindinių žinių apie gastroenterologiją, bet ir paskatins rinktis šią specialybę rezidentūros studijose. Autorių vardu prof. Juozas Kupčinskas

Introduction: Patients with gastric intestinal metaplasia (GIM) are at an increased risk of developing gastric cancer (GC). The histological Operative Link on Gastric Intestinal Metaplasia (OLGIM) classification aids in risk stratification. The recently developed endoscopic grading of GIM (EGGIM)may accurately predict the OLGIM stage. This study aimed to evaluate the diagnostic accuracy of EGGIM in assessing histological GIM using data from two large, prospective European consortia. Aims & Methods: Interim data were derived from the Artificially Intelligent Diagnostic Assistant (AIDA) and Towards Gastric Cancer Screening Implementation in the European Union (TOGAS) consortia, both part of a European Horizon programme focused on gastric cancer prevention. Patients in AIDA underwent oesophagogastroduodenoscopy (OGD) for GIM surveillance or treatment of neoplastic lesions, while participants in TOGAS (aged 50–74) were offered an OGD during screening colonoscopy or polyp surveillance. Procedures were performed at eight academic centres by both expert and non-expert endoscopists, using high-resolution white-light endos -copy and virtual chromoendoscopy. The EGGIM score (range 0–10) was based on the extent of GIM (0: 0%, 1: ≤30%, 2: >30% of mucosa) across five anatomical sites: the lesser and greater curvatures of the antrum and corpus, and the incisura. Targeted biopsies were taken if GIM was suspect-ed; otherwise, random biopsies were collected according to the Sydney system to determine the OLGIM stage. Healthy controls and GIM patients with an EGGIM score ≥1 but OLGIM stage 0 were also included. ROC curve and Spearman analyses assessed the diagnostic and ordinal correlation between EGGIM and OLGIM stages III–IV. Results: A total of 252 patients were included (median age 60 years, IQR9; 57.4% female): 91 from the TOGAS study (75 cancer screening, 16 polyp surveillance) and 161 from AIDA (117 GIM surveillance, 21 neoplasia treatment, 23 healthy controls). OLGIM staging identified 89 (35.3%) patients in stage 0, 74 (29.4%) in stage I, 49 (19.4%) in stage II, 35 (13.9%) in stage III, and 5 (2.0%) in stage IV. The area under the ROC curve for EGGIM in detecting OLGIM III/IV was 0.90 (95% CI 0.79–0.93, p < 0.001), with an optimal cut-off at EGGIM ≥5 (sensitivity 85%, specificity 89%).Conclusion: Our findings prospectively validate the EGGIM score across two large European cohorts. This scoring system may serve as a practical tool to streamline gastric cancer surveillance by reducing the need for biopsies and enhancing endoscopic risk stratification.

Introduction: Patient-derived organoid culture is a powerful and versa-tile system for investigating the molecular mechanisms underlying a wide range of gastrointestinal diseases, including inflammatory bowel disease(IBD) [1].In the context of IBD, intestinal organoids derived from patients retain key genetic, epigenetic, and disease-specific features, offering a unique plat-form to model individual responses to inflammatory triggers [2, 3].This approach also shows promise in predicting personalized responses to biologic and small-molecule therapies, advancing precision medicine strategies for IBD [4].Although intestinal organoids represent a major advancement in personalized ex vivo cellular modeling, the detailed effects of differentiation and inflammation on the cellular landscape remain poorly understood. Aims & Methods: In this study, we analyzed differentiated and undifferentiated intestinal epithelial organoids derived from both an IBD patient with active ulcerative colitis and a healthy donor (non-IBD) (n = 4). Inflammation was induced by treating organoids with the pro-inflammatory cytokines TNF-α (10 ng/mL) and IFN-γ (10 ng/mL). Organoids were dissociated using TrypLE Express Enzyme supplemented with Y27632, and viable cells were enriched by fluorescence-activated cell sorting (FACS) using7-AAD staining. Cell viability and concentration were assessed using the Countess 3 automated cell counter and Trypan Blue staining. Single-cell suspensions were then processed using the 10x Genomics GEM-X Universal 3’ Gene Expression application, and libraries were sequenced on the NovaSeq X Plus platform. Primary data processing was performed using Cell Ranger (10X Genomics), followed by downstream visualization in Loupe Browser (10X Genomics) and an in-house pipeline. Results: The mean number of recovered cells per sample was 1,957, with an average of 142,015 reads per cell and 27,127 genes detected across all samples. Single-cell transcriptome analysis revealed five distinct clusters in undifferentiated organoids (both treated and untreated), and six to seven clusters in differentiated organoids (treated and untreated). The majority of cells expressed the epithelial marker EPCAM, confirming their epithelial origin. Inflammatory stimulation led to a significant upregulation of TNF-α and IFN-γ gene expression (p < 0.05) in both differentiated and undifferentiated samples. Furthermore, differentiated organoids displayed a significant increase in specific epithelial subtypes, including Paneth cells, enteroendocrine cells, and proliferative cells (p < 0.05), compared to their undifferentiated counterparts. Conclusion: To conclude, single-cell transcriptomic profiling of patient-derived intestinal organoids revealed a dynamic and heterogeneous cellular landscape shaped by both differentiation and inflammatory signals. The observed upregulation of inflammatory markers underscores the organoid model’s utility in mimicking inflammatory responses. Additionally, the potential expansion of distinct epithelial subpopulations in differentiated conditions highlights the model’s ability to faithfully recapitulate the complexity of the intestinal epithelium.

Introduction: The European Union has signalled gastric cancer screening and prevention as a major health priority. The TOGAS consortium (TO-wards GAstric cancer Screening in the European Union; togas.lu.lv), fund-ed in response to this call, investigates different strategies for primary and secondary prevention of gastric cancer in Europe. One pilot study aims at determining the yield of having an oesophagogastroduodenoscopy (OGD) on the same day of a colonoscopy for colorectal cancer screening or polyp surveillance. Aims & Methods: This is the first interim data on n=602 individuals recruited in 8 centres across Europe (France, Germany, Ireland, Latvia, Lithuania, the Netherlands, Portugal, and Spain). Individuals of the age of 50-74years, attending for screening or surveillance colonoscopy, were invited to participate if they did not have a past diagnosis of gastric preneoplastic conditions requiring surveillance. Standardization of endoscopic procedures was assured by a rigorous protocol including all quality parameters defined by ESGE. This included standards for image documentation and biopsy sampling. Demographics, comorbidities, medication and the previous surgical and family history were recorded. Patients with marked preneoplastic changes (i.e. glandular atrophy or intestinal metaplasia [IM] at OLGA/OLGIM stage 3 or 4, and/or EGGIM score >5) were registered as requiring further endoscopic surveillance. Mann-Whitney U-test was applied for comparison of age between groups and Chi2 test for comparison of categorical variables (significance for p<0.05).Results: The mean age of participants was 62 years (SD +/- 8.0), with55.8% being male. Of 245 individuals who underwent an OGD prior to inclusion, 93.1% of these procedures were done >3years before and 60.4%were done at a tertiary centre. We identified 6 (1.0%) malignant/neoplastic lesions, 4 gastric cancers/dysplastic lesions and 2 oesophageal carcinomas. A positive H. pylori status was diagnosed in 34.2%. Endoscopically, a diagnosis of preneoplastic conditions was made in 217 participants(36.0%). Of these, 84 (14.0%) showed gastric atrophy, 45 (7.5%) gastric IM and 75 (12.5%) both conditions combined. Of those with gastric atrophy, this was classified as multifocal in 44 (27.6%) individuals, of those with IM in 45 (37.5%). OLGA stages 3 and 4 were confirmed for 1.9% and 0.8%,OLGIM stages 3 and 4 for 2.3% and 0.4%. Benign ulcers were diagnosed in28 (4.7%) participants. The majority being gastric ulcers in 19 (3.2%) individuals, duodenal ulcers in 9 (1.5%), and oesophageal ulcers in 2 (0.3%).An additional 13 (2%) participants were diagnosed with Barrett’s oesophagus. It is of note that also 12 (2.0%) colorectal cancers were identified in the cohort. A positive H. pylori status was associated with the need for gastric surveillance (p=0.031).Conclusion: This interim analysis of the first European pilot study on a combined screening for upper and lower GI tumours shows that this approach is feasible. Regarding the detection of upper GI malignancy and preneoplastic conditions, our findings are in line with previous systematic reviews, but we also demonstrate a reasonable rate of non-malignant findings that are also of relevance when defining the best screening strategy in Europe.

Fecal microbiota transplantation is an effective treatment method for recurrent Clostridioides difficile infection. Widely used enteric tube and colonoscopy methods demonstrate excellent efficacy and safety results. Recent data suggest that new fecal microbiota transplantation methods using oral capsules may provide a less invasive approach. In this study, we aimed to compare primary fecal microbiota transplantation efficacy as well as short- and long-term safety of two different administration routes: oral capsules and enteric tube.

Microbiome analysis has become a crucial tool for basic and translational research due to its potential for translation into clinical practice. However, there is ongoing controversy regarding the comparability of different bioinformatic analysis platforms and a lack of recognized standards, which might have an impact on the translational potential of results. This study investigates how the performance of different microbiome analysis platforms impacts the final results of mucosal microbiome signatures. Across five independent research groups, we compared three distinct and frequently used microbiome analysis bioinformatic packages (DADA2, MOTHUR, and QIIME2) on the same subset of fastQ files. The source data set encompassed 16S rRNA gene raw sequencing data (V1-V2) from gastric biopsy samples of clinically well-defined gastric cancer (GC) patients (n = 40; with and without Helicobacter pylori [H. pylori] infection) and controls (n = 39, with and without H. pylori infection). Independent of the applied protocol, H. pylori status, microbial diversity and relative bacterial abundance were reproducible across all platforms, although differences in performance were detected. Furthermore, alignment of the filtered sequences to the old and new taxonomic databases (i.e., Ribosomal Database Project, Greengenes, and SILVA) had only a limited impact on the taxonomic assignment and thus on global analytical outcomes. Taken together, our results clearly demonstrate that different microbiome analysis approaches from independent expert groups generate comparable results when applied to the same data set. This is crucial for interpreting respective studies and underscores the broader applicability of microbiome analysis in clinical research, provided that robust pipelines are utilized and thoroughly documented to ensure reproducibility.IMPORTANCEMicrobiome analysis is one of the most important tools for basic and translational research due to its potential for translation into clinical practice. However, there is an ongoing controversy about the comparability of different bioinformatic analysis platforms and a lack of recognized standards. In this study, we investigate how the performance of different microbiome analysis platforms affects the final results of mucosal microbiome signatures. Five independent research groups used three different and commonly used bioinformatics packages for microbiome analysis on the same data set and compared the results. This data set included microbiome sequencing data from gastric biopsy samples of GC patients. Regardless of the protocol used, Helicobacter pylori status, microbial diversity, and relative bacterial abundance were reproducible across all platforms. The results show that different microbiome analysis approaches provide comparable results. This is crucial for the interpretation of corresponding studies and underlines the broader applicability of microbiome analysis.

Introduction: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, predominantly diagnosed in patients with chronic liver diseases. The absence of specific biomarkers and the typically asymptomatic progression of the disease lead to delayed diagnosis.Patients with liver disease, including HCC, exhibit alterations in the gut microbiome and increased bacterial permeability, factors that may influence disease progression and patient outcomes. In recent years, there has been a growing interest in exploring circulating microbiome signatures as potential indicators of liver disease stutus, providing a new avenue for understanding HCC.Aims & Methods: Aims: To identify the signatures of the circulating blood microbiome associated with hepatocellular carcinoma, response to treatment and disease prognosis.Methods: This study conducted a post-hoc analysis of the SORAMIC clinical trial utilizing peripheral blood plasma samples. For this analysis, blood plasma samples from a cohort of 71 patients with HCC were examined(25 treated with Sorafenib and 46 with SIRT/Sorafenib). Peripheral blood samples were collected at two time points: baseline (prior to the initiation of anti-tumor therapy) with all 71 patients included, and post-therapy (7 to 9 weeks after treatment) with 39 patients included. Additionally, peripheral blood samples were obtained from 52 control subjects, who were endoscopically diagnosed with colonic diverticulosis, to compare against the patient group. The composition of the circulating blood microbiome was assessed through 16S rRNA gene sequencing, targeting the V1-V2 hypervariable regions. Subsequent analyses focused on identifying associations with potential novel biomarkers for diagnosis, prognosis, and response to treatment.Results: Patients with HCC had significantly diminished β-diversity of the circulating blood microbiome as compared to controls. Significant differences were also observed in between microbial communities of these two cohorts based on Bray-Curtis dissimilarity index. Patients with HCC had significantly higher levels of Proteobacteria and Bacteroidetes and lower levels of Firmicutes and Campylobacterota. Differential abundance analysis revealed that families of Gordoniaceae, Cryomorphaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae were significantly enriched in patients with HCC. At the genus level, patients with HCC had higher levels of Gordonia, Fluviicola, Burholderia, Limnohabitans and Escherichia/Shigella and lower levels of Corynebacterium. HCC patients, who responded to treatment with SIRT/Sorafenib showed an increase in blood bacterial diversity, enrichment of families Burkholderiales incertae sedis,Arcobacteraceae, Peptostreptococcaceae, Moraxellaceae, genera Sphaero-tilus, Aliacrobacter, Rhodoluna, Giesbergeria, Microbacterium, Rombout-sia, Arcicella, Pseudorhodoferax and depletion in families Gordoniaceae,Cryomorphaceae and genera Cloacibacterium, Gordonia, Limnohabitans,Fluviicola after the treatment. Levels of Acinetobacter, Enhydrobacter and Micrococcus in the blood before the HCC treatment were associated with patient prognosis.Conclusion: Patients with HCC exhibit a unique circulating blood microbiome profile characterized by reduced bacterial diversity. Response to treatment is linked to an increase in α-diversity and changes in the circulating microbiome profile. Specific members of the circulating micro-biome show potential prognostic capabilities, suggesting their utility as biomarkers for predicting disease progression and treatment outcomes in HCC.

Introduction: In the gut, intestinal epithelial cells form a structural barrier, delineating the intestinal lumen from the lamina propria. Within the lumen, microbes exert essential functions in maintaining gastrointestinal homeostasis and functionality. Regardless of commensal or pathogenic nature of the bacteria, their interaction with gut epithelium is inevitable. During ulcerative colitis (UC), both mucosal barrier and microbial compo -sition are disturbed. However, it is still unclear whether naturally occurring gut bacteria might influence the control or management of the protective lining of cells in the colon. Aims & Methods: The aim of the study was to identify which faecal microbiota components remain unaltered during UC and assess the interplay of these bacteria with colonic epithelial cells.The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (protocol no. BE-2-31). Faecal microbiota was analysed by employing 16S rRNA-gene v1-v2 region sequencing on stool samples of active and quiescent UC patients (n=47) and non-IBD controls (n=25).Functional analysis was performed using colonic epithelial organoid-derived monolayers of UC patients (n=9) and non-IBD controls (n=8). Mono -layers were co-cultured with Escherichia coli (ATCC 25922) and Phocaeicola vulgatus (ATCC 8482) bacteria type strains. The interplay between bacteria and colonic epithelial cells was assessed by analysing the expression of gene markers for epithelial barrier integrity (ZO1), pathogen recognition(TLR4) and stress induction (HSPA1A, HSPB1).Results: First, 16S rRNA-gene sequencing revealed a persistent bacterial imbalance in both active and quiescent UC patients compared to non-IBD controls. Specifically, Chao1, Shannon, and Simpson α-diversity indices were significantly lower (p Wilcoxon < 0.05) in both active and quiescent UC patient groups when compared to non-IBD controls. Microbial community clusters, assessed by Bray-Curtis dissimilarity index, also differed significantly between UC patients and non-IBD controls (p PERMANOVA < 0.05). Subsequent co-occurrence and differential abundance analyses identified that in contrast to healthy individuals, UC patients exhibited statistical significance (p adj. Wilcoxon < 0.05) in the relative abundance of 5 genera, including Alistipes, Mediterraneibacter, Paraprevotella, etc., while 35 gen-era were found to be present at comparable levels (p adj. Wilcoxon ≥ 0.05). Of them, the most abundant ones were Phocaeicola, Collinsella, Roseburia,Holdemanella, and Bacteroides. Functional analysis, utilizing co-cultures of commensal bacteria and colonic epithelial cells of active UC patients and non-IBD controls, showed that both E. coli and P. vulgatus did not induce pathogen-pattern recognition and stress responses.Interestingly, both species demonstrated potential to provoke diverse tight junction-related cellular reactivity (however, due to high variation inpatient-to-patient responses, results did not reach statistical significance(padj. Wilcoxon ≥ 0.05)). More precisely, a contact with E. coli and P. vulgatus tended to increase the expression of ZO1 in non-IBD epithelial cells (FC = 3.59 and FC = 2.27,respectively) compared to untreated cells, while an opposite trend was observed in epithelial cells of UC patients (FC = 0.67 and FC = 0.75, respec -tively).Conclusion: Diverse colonic epithelial cell responses to distinct commensal bacteria species imply the contribution of stably abundant gut microbiota to the impairment of mucosal barrier integrity during UC.

Introduction: Liver cirrhosis is a late stage of liver fibrosis characterized by progressive deterioration of liver function, leading to potentially life-threatening complications. Recent studies focusing on bacterial alterations of gut microbiota have shown their association with liver disease pathogenesis, decompensation, and hospitalization rates. Recently, there has been increasing recognition of the potential role of gut fungal changes in liver cirrhosis, however, further investigation in this field is warranted.Aims & Methods: The aim of this study was to determine gut fungi composition changes in patients with liver cirrhosis and to assess whether these changes are related to liver disease severity. For the analysis were used DNA samples, extracted from the liver cirrhosis patients’ and healthy controls’ stool samples. Internal transcribed spacer (ITS) was amplified and sequenced using MiSeq Illumina sequencing machine. Bioinformatical and statistical analysis were performed to reveal the fungal profile of each study group.Results: Forty-five patients with liver cirrhosis and 45 healthy controls were included in the study. No significant differences in age or gender were observed between the cirrhosis and control groups. Cirrhosis patients showed significantly reduced fungal richness compared to the control cohort. However, no difference was observed in the Shannon alpha diversity index. No significant alterations in fungal compositions were noted concerning gender and etiology within the cirrhosis group. Patients who had Child-Pugh B and C liver cirrhosis had a significant shift in stool fungi composition compared to those in the Child-Pugh A group. Patients with Child C liver cirrhosis had a significantly increased fungi abundance compared with the Child A group. In accordance with the MELD score,patients with score ≥15, had significantly increased richness and fungal diversity. Furthermore, an elevated abundance of the Candida genus was observed in liver cirrhosis patients group, while Saccharomyces genus was increased in the control group.Conclusion: Patients with liver cirrhosis had reduced richness in stool fungal composition and an increased abundance of the Candida genus when compared with healthy controls. Moreover, patients with worse liver function had significant shifts in fecal fungal composition, suggesting that fungal alterations may be associated with cirrhosis progression.