Gastroenterology and Hepatology from Bed to Bench

Aim: This systematic review and meta-analysis evaluated the subtyped Blastocystis sp. isolated from humans in Iran.

Background: Blastocystis sp. is an anaerobic intestinal protozoan that infects humans as well as domestic and wild animals, i.e. mammals, amphibians, reptiles, and arthropods.

Methods: A comprehensive search for papers published before April 2022 was undertaken utilizing English and Persian databases. The following MeSH keywords were used in the electronic search: (Blastocystis sp.) AND (molecular OR subtype) AND (prevalence OR epidemiology) AND Iran. The quality of the included studies was evaluated. Thereafter, a random-effects meta-analysis was conducted to estimate the pooled prevalence and odds ratios regarding the included studies.

Results: A total of 32 studies comprised of five case-control studies and 27 cross-sectional studies met the eligibility criteria. The overall pooled prevalence of subtyped Blastocystis sp. in Iran was estimated to be 10% (95% confidence interval: 6 to 15%). Eight subtypes of Blastocystis sp. (ST1- ST7 and ST9) were identified in our study, of which ST3 was the most common subtype (0.04); 0.02-0.07). The difference in subtypes between two case and control groups in reported studies was not significant, but the odds ratio of infection by ST3 (0.98; 95% CI, 0.30 to 3.20) was higher in cases.

Conclusion: The current systematic review showed that with the exception of ST8 and ST12, all human Blastocystis sp. subtypes reported in the world are found in different parts of Iran.

Aim: This study aimed to identify key genes, non-coding RNAs, and their possible regulatory interactions during gallbladder cancer (GBC).

Background: The early detection of GBC, i.e. before metastasis, is restricted by our limited knowledge of molecular markers and mechanism(s) involved during carcinogenesis. Therefore, identifying important disease-associated transcriptome-level alterations can be of clinical importance.

Methods: In this study, six NCBI-GEO microarray dataseries of GBC and control tissue samples were analyzed to identify differentially expressed genes (DEGs) and non-coding RNAs {microRNAs (DEmiRNAs) and long non-coding RNAs (DElncRNAs)} with a computational meta-analysis approach. A series of bioinformatic methods were applied to enrich functional pathways, create protein-protein interaction networks, identify hub genes, and screen potential targets of DEmiRNAs and DElncRNAs. Expression and interaction data were consolidated to reveal putative DElncRNAs:DEmiRNAs:DEGs interactions.

Results: In total, 351 DEGs (185 downregulated, 166 upregulated), 787 DEmiRNAs (299 downregulated, 488 upregulated), and 7436 DElncRNAs (3127 downregulated, 4309 upregulated) were identified. Eight genes (FGF, CDK1, RPN2, SEC61A1, SOX2, CALR, NGFR, and NCAM) were identified as hub genes. Genes associated with ubiquitin ligase activity, N-linked glycosylation, and blood coagulation were upregulated, while those for cell-cell adhesion, cell differentiation, and surface receptor-linked signaling were downregulated. DEGs-DEmiRNAs-DElncRNAs interaction network identified 46 DElncRNAs to be associated with 28 DEmiRNAs, consecutively regulating 27 DEGs. DEmiRNAs-hsa-miR-26b-5p and hsa-miR-335-5p; and DElnRNAs-LINC00657 and CTB-89H12.4 regulated the highest number of DEGs and DEmiRNAs, respectively.

Conclusion: The current study has identified meaningful transcriptome-level changes and gene-miRNA-lncRNA interactions during GBC and laid a platform for future studies on novel prognostic and diagnostic markers in GBC.

Aim: This systematic review examined the diet’s impact on the human gut microbiota to identify potential consequent health outcomes.

Background: The extreme macronutrient profile of the ketogenic diet (KD) instigates compositional shifts in the gut’s microbial community.

Results: The results provide strong evidence for a persistent reduction in Bifidobacterium abundance following KD adherence. A reduced abundance of key Firmicutes butyrate-producing bacteria was found to be a likely impact, although two studies with extended intervention periods indicate this may be time-limited. Studies investigating short-chain fatty acids (SCFA’s) indicate KD reduces total fecal SCFA’s, acetate, and butyrate.

Conclusion: Changes to microbial communities resulting from KD adherence are potentially detrimental to colonic health. The persistent reduction in Bifidobacterium abundance was concerning, with obesity, type-2 diabetes, and depression highlighted as potential consequent risks. For nutrition and healthcare professionals, the findings emphasize the importance of considering KDs microbial effects and resulting health implications at an individual level.

Accurate diagnosis of Entamoeba histolytica is important, as it is known as a causative agent for both invasive intestinal and extra-intestinal amoebiasis. Amoebiasis has a worldwide distribution, especially in developing countries, and it is responsible for up to 100,000 fatal cases annually. A number of diagnostic methods, including microscopy, culture, antigen detection, molecular based methods, and serological assays have been proposed to assist in diagnosing amoebiasis. The present study aimed to gather new data and review the available diagnostic tests of both intestinal and extra-intestinal amoebiasis and to highlight pitfalls and challenges of each of them. A broad literature of electronic databases was conducted and covered articles published up to March 2022. For laboratory diagnosis of intestinal amoebiasis, direct microscopy stool examinations and cultures should be held as the high-performance diagnostic strategies. Molecular and immunological-based assays are also recommended as complementary tests. To diagnose extra-intestinal infection, the use of serological tests is still considered the method of choice. However, serodiagnosis requires further improvement for the accurate differential diagnosis of active infection from past infections.

Aim: This study aimed to evaluate hepatic steatosis index (HSI) as a non-invasive tool in diagnosing and predicting NASH and to compare it with abdominal ultrasound as the gold standard tool.

Background: Non-alcoholic steatohepatitis (NASH) is a type of non-alcoholic fatty liver disease (NAFLD) with inflammation which can progress to liver cirrhosis. Rapid and valid screening can remarkably prevent the progression of this disease.

Methods: A total of 464 people were included in the present study based on inclusion criteria. Patients were evaluated for (BMI), AST, ALT, and ALP indices. The liver echogenicity of patients was evaluated by abdominal ultrasound technique, and patients were classified.

Results: The results showed that out of 464 people included in the study, 32.33% represented fatty liver. It was found that 79.1% of patients were female. There was no significant difference between the two groups in terms of age. Furthermore, it was found that ALT, AST, and ALP levels were significantly increased in the two groups of patients compared to the control group. It was determined that out of 150 patients, 75.3% were grade I and 24.7% were grade II NASH cases; no grade III cases were observed. The mean HSI for the NASH- group was 35.51±5.72 and for the NASH+ group was 42.84±5.70, a significant difference. The receiver operating characteristic (ROC) curve also showed that the area under the curve (AUC) of HSI was 0.833 (95% CI, 0.796-0.870) for detecting NASH patients. At the cutoff point of > 37.0, the sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were 84.0% (95% CI, 77.2 -89.0), 70.1% (95% CI, 64.8 -74.9), 90.2%, and 57.3%, respectively. Pearson correlation showed a direct and significant correlation between ultrasound data and HSI values.

Conclusion: Overall, the present study results showed that HSI as a non-invasive and non-imaging tool can diagnose NASH.

Aim: The TBS-derived image processing method, based on the observer's diagnosis, has been developed in the current investigation. Image parametrization is proposed for both novel description and convergent shreds of evidence.  

Background: Condensed X-ray images of the esophageal timed barium swallow (TBS) provide substantial implications for elucidating the pathophysiological dimensions of esophageal motility disorders.

Methods: Through the simultaneous study on TBS and high-resolution manometry (HRM) findings, we performed a retrospective cohort study on 252 patients from March 2018 to October 2019. Interventions, irrelevant information, and insufficient patient data were excluded. Only subjects with adequate data and acceptable test accuracy were considered for participation. We reviewed 117 Dicom (digital imaging and communications in medicine) X-ray images from patients with confirmed diagnoses of achalasia type II, esophagogastric junction outflow obstruction (EGJOO), or non-achalasia.

Results: The results suggested a cut-off level of 47% in DDi (dilated diameter index) as a sign of the dilated body. In achalasia type II patients (n=66 images), the mean DDi was 55.6%. Our method presented a sensitivity of 95% and a specificity of 93% compared to images of the non-achalasia findings. The mean DDi in EGJOO patients was 50.4%, according to the 27 images. Moreover, results from EGJOO patients provided a sensitivity of 85% and specificity of 87%.

Conclusion: TBS is an efficacious method and a prominent component in the process of achalasia diagnosis. Standard parametrization might develop radiological exports proposed by DDi. Our method could assist in obtaining a non-invasive medical diagnosis and help advance diagnostic reports to identify achalasia subtypes somewhat earlier. To the best of our knowledge, this interface is an innovative parametrization for TBS image review.

Aim: This study is an attempt to screen the key immune elements that participate during HBV infection and the related pathways that are modulated.

Background: The pathogenesis of Hepatitis B virus and the corresponding clinical manifestations in the host are primarily immune-mediated.

Methods: This study utilizes a PCR array to screen immune-related genes that are differentially expressed in the presence of the virus in HBV replicating HepG2.2.15 cells as compared to control HepG2 cells. The significantly up-regulated genes were subjected to bioinformatic analysis utilizing GSEA and STRING. Additionally, ELISA was used to corroborate the levels of Alpha 1 antitrypsin (AAT) from patients’ sera.

Results: The expressions of 31% of the studied genes were significantly up-regulated (> 2-fold, p<0.05) in HepG2.2.15 cells compared to controls, and this included the SERPINA1, FN1, IL1R2, LBP, LY96, LYZ and PROC genes. When they were clustered based on biological processes, signaling pathways, and disease progression, the genes related to biotic stimulus, complement-coagulation cascades, and fibrosis, respectively, showed the highest (p<0.05) enrichment. Analysis of patients’ sera by ELISA revealed that the serum AAT (SERPINA1) levels were significantly higher in asymptomatic carriers and in patients with chronic liver disease than in controls (p<0.05). Moreover, SERPINA1 levels were also positively correlated with the levels of serum ALT (r=0.4495, p<0.05) among HBV infected patients.

Conclusion: The current study highlights the key immune elements and pathways that are modulated during HBV infection and proposes the possible use of AAT as a non-invasive immunological biomarker to follow the progression of liver disease.

Aim: This study aimed to detect gene signatures in RNA-sequencing (RNA-seq) data using Pareto-optimal cluster size identification.

Background: RNA-seq has emerged as an important technology for transcriptome profiling in recent years. Gene expression signatures involving tens of genes have been proven to be predictive of disease type and patient response to treatment.

Methods: Data related to the liver cancer RNA-seq dataset, which included 35 paired hepatocellular carcinoma (HCC) and non-tumor tissue samples, was used in this study. The differentially expressed genes (DEGs) were identified after performing pre-filtering and normalization. After that, a multi-objective optimization technique, namely multi-objective optimization for collecting cluster alternatives (MOCCA), was used to discover the Pareto-optimal cluster size for these DEGs. Then, the k-means clustering method was performed on the RNA-seq data. The best cluster, as a signature for the disease, was found by calculating the average Spearman's correlation score of all genes in the module in a pair-wise manner. All analyses were performed in the R 4.1.1 package in virtual space with 100 Gb of RAM memory.

Results: Using MOCCA, eight Pareto-optimal clusters were obtained. Ultimately, two clusters with the greatest average Spearman's correlation coefficient scores were chosen as gene signatures. Eleven prognostic genes involved in HCC's abnormal metabolism were identified. In addition, three differentially expressed pathways were identified between tumor and non-tumor tissues.

Conclusion: These identified metabolic prognostic genes help us to provide more powerful prognostic information and enhance survival prediction for HCC patients. In addition, Pareto-optimal cluster size identification is suggested for gene signature in other RNA-Seq data.

Aim: This study investigated the association between methylation status and expression levels of BTG2, PPP1CA, and PEG3 genes in colon cancer.

Background: Aberrant DNA methylation is one of the most important epigenetic modifications in the development of cancer. Evidence indicates that hypermethylation of various tumor suppressor genes could be a potential mechanism of colon tumorigenesis.

Methods: The expression levels of BTG2, PPP1CA, and PEG3 genes were evaluated in HT-29/219, HCT116, SW48, SW742, SW480, and LS180 cell lines using quantitative Real-Time PCR. The methylation status of BTG2 and PPP1CA was determined by methylation-specific PCR (MSP) method, and the methylation pattern of PEG3 was evaluated by bisulfite sequencing PCR (BSP). To investigate the effect of methylation on the expression of these genes, all colon cancer cell lines were treated by 5-Azacitidine (5-Aza) and/or Trichostatin A (TSA).

Results: The expression levels of BTG2, PPP1CA, and PEG3 were highly heterogeneous and quantitatively correlated to their promoter methylation status in the studied colon cancer cell lines. Treatment by 5-Aza and/or TSA increased the expression of the above-named genes in colon cancer cell lines.

Conclusion: Overall, it seems that BTG2, PPP1CA, and PEG3 act as tumor suppressor genes in colon cancer, and methylation is a potential mechanism for their loss of expression. Therefore, these genes may be considered as suitable targets for demethylation approaches and, eventually, colon cancer treatment. Combined treatment by 5-Aza and TSA may be a promising therapeutic strategy for colon cancer treatment. Further studies may contribute to confirm these results.

Aim: This study aimed to perform a head-to-head comparison of changes during NASH progression throughout 6-11 weeks of an experiment to supply a faster nutritional model in mimicking NASH to decrease the duration and cost of in vivo studies.

Background: New therapies are urgently needed because of the growing prevalence of non-alcoholic steatohepatitis (NASH) and the lack of an effective treatment approach. Currently, dietary interventions are the most efficient options.

Methods: This study compared features of NASH in a murine model using protocol that combined special nutritional regimes based on the combination of 21.1% fat, 41% sucrose, and 1.25% cholesterol with weekly intraperitoneal injections of carbon tetrachloride (CCl4). Male C57BL/6J mice received either special compositions + CCl4 (NASH group) or standard chow diet (healthy control group) for 11 weeks. Liver histopathology based on hematoxylin and eosin (H&E) and Masson’s Trichrome (TC) staining and biochemical analyses were used to assess disease progression.

Results: In C57BL/6J mice administered a high fat, high cholesterol, high sucrose diet and CCl4 for 8 weeks, steatohepatitis with pronounced hepatocyte ballooning, inflammation, steatosis, and fibrosis was observed. According to the NAFLD activity scoring system, the maximum NAS score was manifested after 8-9 weeks (NAS score: 6.75). Following this protocol also led to a significant increase in AST and ALT, total cholesterol, and total triglyceride serum levels in the NASH group.

Conclusion: Following the special nutritional regime based on high fat, cholesterol, and sucrose in combination with CCL4 injections resulted in a NASH model using C57BL/6J mice in a shorter time compared to similar studies. The obtained histopathological NASH features can be advantageous for preclinical drug testing.

Aim: The current study investigated the prevalence of Cryptosporidium spp. among children under 6 and adults over 60 years of age with diarrhea in the southwest of Iran.

Background: Cryptosporidiosis is an opportunistic parasitic infection caused by the species Cryptosporidium that causes gastrointestinal complications and diarrhea.

Methods: This cross-sectional study was conducted in Khuzestan province between January 2020 to December 2020. Out of 350 patients referring to medical centers with clinical signs of diarrhea, 57.4% were under six years of age and 42.6% were more than 60 years old. Fecal samples were examined using Modified Ziehl-Neelsen (MZN) staining and nested-PCR techniques.

Results: The overall prevalence of Cryptosporidium spp. infection in the study population was 0.9% as determined by microscopic and molecular methods (3/47).

Conclusion: The study results confirm the prevalence of parasitic infections as reported in previous studies in other regions of Iran. Preventive health measures are necessary.

Aim: The current study aimed to determine crucial genes targeted by toxin-A through network analysis.

Background: Clostridium difficile (C difficile) produces toxin-A and toxin-B and is known as a risk factor for hospital infection, especially after broad spectrum antibiotic therapy. Bioinformatics findings have led to the introduction of a set of genes and biological terms that are targeted by toxin-B in colon epithelia.

Methods: The significant differentially expressed genes (DEGs) of human intestinal Caco-2 cells treated by toxin-A versus control were retrieved from gene expression omnibus (GEO). The queried DEGs were analyzed using by protein-protein interaction (PPI) network analysis through STRING database and Cytoscape software v.3.7.2.

Results: Among 157 significant DEGs, JUN, VEGFA, CDKN1A, ATF3, SNAI1, DUSP1, HSPB1, MCL1, KLF4, FOSL1, HSPA1A, and SQSTM1 were determined as hubs and JUN, DUSP1, DUSP5, EZR, MAP1LC3B, and SQSTM1 were highlighted as bottlenecks.

Conclusion: JUN, DUSP1, and SQSTM1 are possible drug targets to prevent and treat C difficile infection.

Primary squamous cell carcinoma (SCC) of the liver is rare and has an extremely poor prognosis. It is very difficult to detect and is sometimes misdiagnosed. It has been reported that male sex, hepatic cyst, hepatolithiasis, hepatic teratoma, and liver cirrhosis may be associated with SCC of the liver. A 67-year-old woman was admitted to our hospital with anorexia, weakness, and right upper quadrant abdominal (RUQ) pain. Sonography and an abdominal computed tomography scan revealed a 36 × 34 cm mass in the liver. Pathological analysis of the sample suggested SCC. According to the negative radiographic findings in other major organs, the tumor was considered primary. The patient was treated with surgical resection and followed by palliative care. Our case died 5 months after the initial presentation.

Aim: The aim of the present study was to review in VigiBase the reports of serious hepatic disorders associated with the use of ivermectin for COVID-19 in adults.

Background: In the face of the global health emergency caused by SARS-CoV-2, ivermectin, among other drugs, has been repurposed in some Latin American countries to treat COVID-19. Studies are needed on the safety of ivermectin for this new indication. VigiBase is the WHO pharmacovigilance database that registers all individual case safety reports (ICSRs) from more than 130 countries.

Methods: We extracted the ICSRs of men or women aged ≥ 18 years and dated between 1 January 2020 and 7 March 2021 which included an association with the use of ivermectin.

Results: Of 1393 ICSRs associated with ivermectin, 60 (4.3%) were registered as "serious." Ivermectin had been used for COVID-19 in 25 of those cases. Among the latter, 6 experienced hepatic disorders (hepatitis, hepatocellular injury, cholestasis, increased alanine aminotransferase and/or aspartate aminotransferase levels, abnormal liver function tests).

Conclusion: The safety of the use of ivermectin should be studied more exhaustively, especially as regards the possibility of hepatic disorders developing when the drug is used for COVID-19.