Pylori-positive Samples Research Articles

Prevalence of cagA, cagM, vacA and oipA genes in isolates of Helicobacter pylori obtained from hospital patients in Northeast Brazil.

Helicobacter pylori is a major cause of gastrointestinal disorders such as chronic gastritis, peptic ulcers, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. It is estimated that around half of the world's population is infected with this pathogen, with underdeveloped countries reporting the highest frequencies. The genes cagA, cagM, vacA, and oipA are some of the most important virulence factors of H. pylori; however, there are no recent studies from Recife-PE demonstrating their frequency, and their relationship with severe gastric modifications. This work aims to use qualitative PCR to detect the virulence genes cagA, cagM, vacA, and oipA in H. pylori isolates obtained from patients in a public hospital in Recife (PE). We collected samples from the stomach's body and antrum of 147 patients, from which 71 (48%) tested positive for H. pylori. Among positive samples, the most frequently infected gender was female (44/71, 62%), and the most frequently infected age group was those above the age of 46 (31/71, 44%). Histological examination of H. pylori-positive samples revealed alterations other than chronic gastritis, including metaplasia and atrophy. The frequency of cagA, cagM, and oipA genes were identified in 84%, 56%, and 69% of the samples tested, respectively, as well as the vacA-s1m1 allelic combination (77%). However, there was no statistically significant variation in the occurrence of these genes, therefore they cannot be considered unique markers of severity in our setting. New research with larger samples and investigations of other genetic markers can aid uncover local risk factors and lead to a better understanding of H. pylori's pathogenesis.

Gastric microbiome changes in relation with Helicobacter pylori resistance.

Inadequate antimicrobial treatment has led to multidrug-resistant (MDR) bacteria, including Helicobacter pylori (H. pylori), which one of the notable pathogens in the stomach. Antibiotic-induced changes in the microbiota can negatively affect the host. This study aimed to determine the influence of H. pylori resistance on the diversity and abundance of the stomach microbiome. Bacterial DNA was extracted from biopsy samples of patients presenting dyspepsia symptoms with H. pylori positive from cultures and histology. DNA was amplified from the V3-V4 regions of the 16S rRNA gene. In-vitro E-test was used to detect antibiotic resistance. Microbiome community analysis was conducted through α-diversity, β-diversity, and relative abundance. Sixty-nine H. pylori positive samples were eligible after quality filtering. Following resistance status to five antibiotics, samples were classified into 24 sensitive, 24 single resistance, 16 double resistance, 5 triple resistance. Samples were mostly resistant to metronidazole (73.33%; 33/45). Comparation of four groups displayed significantly elevated α-diversity parameters under the multidrug resistance condition (all P <0.05). A notable change was observed in triple-resistant compared to sensitive (P <0.05) and double-resistant (P <0.05) groups. Differences in β-diversity by UniFrac and Jaccard were not significant in terms of the resistance (P = 0.113 and P = 0.275, respectively). In the triple-resistant group, the relative abundance of Helicobacter genera was lower, whereas that of Streptococcus increased. Moreover, the linear discriminant analysis effect size (LEfSe) was associated with the presence of Corynebacterium and Saccharimonadales in the single-resistant group and Pseudomonas and Cloacibacterium in the triple-resistant group. Our results suggest that the resistant samples showed a higher trend of diversity and evenness than the sensitive samples. The abundance of H. pylori in the triple-resistant samples decreased with increasing cohabitation of pathogenic bacteria, which may support antimicrobial resistance. However, antibiotic susceptibility determined by the E-test may not completely represent the resistance status.

Shotgun Metagenomics of Gastric Biopsies Reveals Compositional and Functional Microbiome Shifts in High- and Low-Gastric-Cancer-Risk Populations from Colombia, South America

ABSTRACT Along with Helicobacter pylori infection, the gastric microbiota is hypothesized to modulate stomach cancer risk in susceptible individuals. Whole metagenomic shotgun sequencing (WMS) is a sequencing approach to characterize the microbiome with advantages over traditional culture and 16S rRNA sequencing including identification of bacterial and non-bacterial taxa, species/strain resolution, and functional characterization of the microbiota. In this study, we used WMS to survey the microbiome in extracted DNA from antral gastric biopsy samples from Colombian patients residing in the high-risk gastric cancer town Túquerres (n = 10, H. pylori-positive = 7) and low-risk town of Tumaco (n = 10, H. pylori-positive = 6). Kraken2/Bracken was used for taxonomic classification and abundance. Functional gene profiles were inferred by InterProScan and KEGG analysis of assembled contigs and gene annotation. The most abundant taxa represented bacteria, non-human eukaryota, and viral genera found in skin, oral, food, and plant/soil environments including Staphylococus, Streptococcus, Bacillus, Aspergillus, and Siphoviridae. H. pylori was the predominant taxa present in H. pylori-positive samples. Beta diversity was significantly different based on H. pylori-status, risk group, and sex. WMS detected more bacterial taxa than 16S rRNA sequencing and aerobic, anaerobic, and microaerobic culture performed on the same gastric biopsy samples. WMS identified significant differences in functional profiles found between H. pylori-status, but not risk or sex groups. H. pylori-positive samples were significantly enriched for H. pylori-specific genes including virulence factors such as vacA, cagA, and urease, while carbohydrate and amino acid metabolism genes were enriched in H. pylori-negative samples. This study shows WMS has the potential to characterize the taxonomy and function of the gastric microbiome as risk factors for H. pylori-associated gastric disease. Future studies will be needed to compare and validate WMS versus traditional culture and 16S rRNA sequencing approaches for characterization of the gastric microbiome.

Detection of Helicobacter pylori and its Antimicrobial Susceptibility Pattern from Gastric Biopsy Specimens

Background: Early detection of Helicobacter pylori (H. pylori) infection is essential for its treatment. Resistance to amoxicillin, clarithromycin and metronidazole has been on the increase in many countries. Phenotypic resistance is correlated with treatment failure. So, there is an urgent need to explore sensitivity of other antibiotics, such as levofloxacin to combat H. pylori infection. Objective: The study was aimed to detect H. pylori from gastric biopsy samples and its susceptibility profile to commonly used antimicrobial drugs. Methods: Gastroduodenal biopsy specimens were collected from 143 adult dyspeptic patients during March 2018-February, 2019, who attended the outpatient department of gastroenterology, Bangabandhu Sheikh Mujib Medical University (BSMMU) and Dhaka Medical College Hospital (DMCH), for endoscopy. H. pylori was identified by rapid urease test (RUT), ureC gene PCR, histological staining (Giemsa) and culture. From culture isolates antimicrobial susceptibility of clarithromycin, levofloxacin, amoxicillin and metronidazole were detected by disk diffusion method. Results: The highest rate of H. pylori infection was found in the age group between 41-50 years (25.5%). According to case definition, H. pylori positive cases were 47 (32.9%) and H. pylori negative cases were 96 (67.1%). Thirty five H. pylori positive samples were subjected to culture and only 10 (28.6%) were positive. Among 10 culture positive H. pylori isolates, clarithromycin exhibited 20% resistance, levofloxacin 30%, metronidazole 30% and no resistance found to amoxicillin. Conclusion: PCR based assays can be an alternative rapid approach for the detection of H. pylori. In this study, levofloxacin showed high resistance, a further larger study is required to confirm this finding. Bangladesh Med Res Counc Bull 2022; 48(1): 3-9

Role of BTLA/HVEM network in development of gastric cancer

The immunopathological mechanism underlying intestinal metaplasia and gastric cancer remain incompletely understood. Regarding the role of B- and T-lymphocyte attenuator (BTLA) / herpesvirus entry mediator (HVEM) in tumorigenesis, this research was conducted to determine the BTLA/HVEM expression in development of gastric cancer. Gastric biopsy and peripheral blood was drawn from 32 non-ulcer dyspepsia (NUD) as control group, 19 intestinal metaplasia (IM), and 63 gastric cancer (GC). BTLA/HVEM expression were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction. Soluble HVEM (sHVEM) and anti-Helicobacter pylori IgG antibody were assessed by ELISA. Our result showed that BTLA mRNA and protein were significantly increased in advanced stages of gastric cancer. HVEM was higher only at the protein level in the GC group. The sHVEM concentration was also higher in the GC group than in the NUD groups. In addition, we observed H. pylori-positive samples had a lower H-score of HVEM than H. pylori-negative ones. These results suggest that BTLA/HVEM/sHVEM inhibitory pathway is involved in immune regulation and progression of gastric cancer. Therefore, this inhibitory pathway might be a therapeutic target to further immunotherapy of gastric cancer.

Meta-analysis of mucosal microbiota reveals universal microbial signatures and dysbiosis in gastric carcinogenesis

The consistency of the associations between gastric mucosal microbiome and gastric cancer across studies remained unexamined. We aimed to identify universal microbial signatures in gastric carcinogenesis through a meta-analysis of gastric microbiome from multiple studies. Compositional and ecological profiles of gastric microbes across stages of gastric carcinogenesis were significantly altered. Meta-analysis revealed that opportunistic pathobionts Fusobacterium, Parvimonas, Veillonella, Prevotella and Peptostreptococcus were enriched in GC, while commensals Bifidobacterium, Bacillus and Blautia were depleted in comparison to SG. The co-occurring correlation strengths of GC-enriched bacteria were increased along disease progression while those of GC-depleted bacteria were decreased. Eight bacterial taxa, including Veillonella, Dialister, Granulicatella, Herbaspirillum, Comamonas, Chryseobacterium, Shewanella and Helicobacter, were newly identified by this study as universal biomarkers for robustly discriminating GC from SG, with an area under the curve (AUC) of 0.85. Moreover, H. pylori-positive samples exhibited reduced microbial diversity, altered microbiota community and weaker interactions among gastric microbes. Our meta-analysis demonstrated comprehensive and generalizable gastric mucosa microbial features associated with histological stages of gastric carcinogenesis, including GC associated bacteria, diagnostic biomarkers, bacterial network alteration and H. pylori influence.

Loss of p53 Expression in Gastric Epithelial Cells of Helicobacter pylori-Infected Jordanian Patients.

Background Around half of the global population is chronically infected with the stomach bacterium Helicobacter pylori, making it one of the most common chronic infections worldwide. H. pylori induces the production of reactive oxygen species, DNA damage, and accelerates the degradation of the tumor suppressor protein p53, which may lead to cancer development. In this study, we investigated the relationship between H. pylori infection and the expression of p53 in gastric mucosa in a group of patients from Jordan. Methods In this retrospective case-control study, the epithelium of gastric glands in subjects chronically infected with H. pylori was examined for the expression of p53. Paraffin-embedded gastric biopsy samples from the archives for 50 Jordanian patients diagnosed with chronic H. pylori infection and 25 samples free of H. pylori infection and any other gastric abnormalities were selected. Samples were analyzed for the presence of H. pylori as well as p53 expression levels in the mucosa and submucosa by immunohistochemical analyses and Western blotting. Results H. pylori was detected in the gastric tissues of infected individuals (n = 50); whereas, no H. pylori infection was detected in uninfected healthy individuals (n = 25) using immunohistochemistry. In contrast to the noninfected samples of gastric mucosa, no nuclear p53 expression was detected in the infected samples using immunohistochemistry. In addition, the levels of p53 in H. pylori-positive samples detected by Western blotting were significantly lower than those in the negative individuals. Conclusion Our data reveal that p53 protein expression decreased in gastric mucosa of patients infected with H. pylori. The loss of this tumor suppressor may play a role in the increased risk for tumor initiation associated with H. pylori carriage.

Helicobacter pylori Is Present at Quantifiable Levels in Raw Vegetables in the Mediterranean Area of Spain

Vegetables are one of the main foodstuffs consumed in the Mediterranean diet. However, raw vegetables have been associated with relevant foodborne outbreaks worldwide. Accurate knowledge of the microbiological quantitative risks associated with these matrices is crucial in order to define effective control measures, avoiding the survival and dissemination of foodborne pathogens through the different food chain stages. The aim of the present study is the assessment of the prevalence of Helicobacter pylori (a unique carcinogenic biological agent recognized to date) on leafy vegetables (spinach, lettuce, and chard) by means of the detection of the specific pathogenicity vacA gene. A real-time quantitative polymerase chain reaction (qPCR) optimized approach was used to detect H. pylori-positive samples and the concentration of this pathogen (with a limit of detection equal to 10 cells). One hundred raw vegetable samples were acquired in markets corresponding to the Spanish Mediterranean area. Sliced vegetable leaves were homogenized and centrifuged, and DNA was extracted from the homogenates. qPCR results confirmed 20 out of 100 H. pylori-positive samples, with melting temperature (Tm) values in the range of 84.8–86.5 °C (TmvacA H. pylori = 85 °C). Amplicons were cut, purified, and sequenced to confirm the homology with the H. pylori vacA gene. A total of 17 out of 100 vegetable samples (12/45 (26.6%) lettuce, 2/21 (9.5%) spinach, and 3/34 (8.8%) chard samples) were finally confirmed as H. pylori-positive. Contamination levels were in the range of 1.5 ± 0.3 to 2.5 ± 0.1 log10 cycles (36–335 CFU/g leafy vegetables). Our results show that H. pylori is detected by qPCR at levels close to infectious doses in fresh vegetables, thus posing a food safety hazard.

Characterization of Domain V Mutations in Clinical Isolates of Helicobacter pylori in Pakistan and Their Effect on Clarithromycin MIC.

PurposeClarithromycin is commonly prescribed for H. pylori infection. Domain V mutations are responsible for clarithromycin resistance. This study was aimed to characterize the clarithromycin resistance and its associated mutations in clinical isolates of H. pylori in Pakistan.Materials and MethodsInfection was diagnosed in 93 patients’ biopsies using culture, rapid urease test, 16S rRNA, and vacA gene multiplex PCR. Clarithromycin resistance was assessed by the agar dilution method. Mutations were detected by PCR-RFLP using 46 (1.4 kb) domain V fragments. Sequencing was executed for 13 domain V fragments, of which 12 showed unusual amplicon size (1.2 kb) and 01 had a new MboII RFLP pattern.ResultsA total of 48 (83%) strains were obtained from 58 (62.3%) PCR H. pylori-positive samples. Resistance (MIC ≥ 0.001 mg/mL) and intermediate resistance phenotype (MIC = 0.0005 mg/mL) was observed in 22 (46%), and 10 (21%) isolates, respectively. The primary resistance was found in 23 (39.6%) samples. PCR-RFLP detected A2142G, A2143G, and double mutations in 19, 04, and 01 resistant strain, respectively. Sequencing of 10 amplicons obtained from intermediated resistant strains and 03 amplicons from resistant strains showed 138 new mutations. Among them, T2182C was also seen in 04 intermediated resistant isolates, whereas A2142G, A2143G, and A2143C were observed in resistant isolates. The new MboII RFLP pattern in an intermediated resistant strains was due to A1761G mutation.ConclusionH. pylori domain V mutations showed extensive diversity. Multiple mutations in domain V may give endurance to H. pylori against clarithromycin. Further investigations on the molecular mechanism of antibiotic resistance in H. pylori seem crucial at this stage.

VIRULENCE PROFILE OF HELICOBACTER PYLORI DETECTED IN GASTRIC BIOPSIES OF PATIENTS UNDERGOING ENDOSCOPY IN UPPER EGYPT

Background: Helicobacter pylori (H. pylori) is a common cause of gastric ulcers and is a risk factor for gastric carcinoma. Little data are available about the characters of H. pylori causing infections in patients from Upper Egypt. Therefore, the aim of this study was to investigate the frequency of H. pylori infection in Egyptian patients undergoing endoscopy and complaining of persistent upper gastrointestinal symptoms directly in biopsy specimens using PCR technique and to study the associated virulence-related genes in H. pylori positive samples. Materials and Methods: One hundred and twenty gastric biopsy specimens were collected from Egyptian patients admitted to Assiut University Hospital and complaining of persistent upper gastrointestinal symptoms. H. pylori DNA was extracted for molecular identification by PCR. Positive samples were further analyzed to investigate the presence of different virulence-related genes and gene combinations. Results: H. pylori was detected in 92/120 (76.6%) of collected biopsies. All strains carried the vacA s1 subtype. The prevalence of the virulence genes cagA, cagE, iceA1, iceA2 and oipA were 79 (84.8%), 41(44.5%), 69 (75%), 44 (47.8%), and 55(59.7%) of the H. pylori positive samples, respectively. Genetic analysis showed that H. pylori were grouped into 29 different genotype combinations. The s1/m1/i1/d1/c1/cagA/iceA1/oipA genotype was the most predominant. Conclusion: Our results show a high frequency of H. pylori infections among Egyptian patients with gastrointestinal symptoms. The s1/m1/i1 vacA hybrid is the most prevalent subtype. The distribution of the different virulence-related genes shows alarming rates of cagA, cagE, iceA1, iceA2 and oipA. Careful monitory of H. pylori infections and investigation of their genetic characters should be carried out to control the spread of the virulent strains.

Evaluation of Helicobacter pylori Genotypes in Obese Patients with Gastric Ulcer, Duodenal Ulcer, and Gastric Cancer: An Observational Study

Introduction: Obesity is a well-known risk factor for a variety of gastrointestinal disorders (GID). Helicobacter pylori is associated with different GID, such as gastric cancer and chronic gastritis. In this study, we investigated the prevalence of dominant genotypes in H. pylori isolated from obese patients diagnosed with gastric ulcer, duodenal ulcer, and gastric cancer. Methods: A total of 222 H. pylori-positive samples were collected from patients with obesity. GID and gastric cancer were identified by endoscopy and histopathology, respectively. Three biopsy specimens from the gastric antrum were obtained from each patient for culture tests, histological examination, and identification of vacuolating cytotoxin A (vacA) (vacA s1, vacA s2, vacA m1, vacA m2, vacA s1m1 vacA s1m2, vacA s2m1, and vacA s2m2), cagA, cagE, iceA1, oipA, dupA, and babA2 using polymerase chain reaction. Results: vacA, cagE, cagA, iceA1, oipA, dupA, and babA2 genes were detected in 222 (100%), 171 (77%), 161 (72.5%), 77 (34.6%), 77 (34.6%), 137 (61%), and 69 (31%) patients with obesity, respectively. Our findings revealed that vacA, iceA1, oipA, and babA2 were significantly associated with a higher risk of GID, while cagE, cagA, and dupA indicated no correlation with the development of GID. Also, in the combination of s- and m-region genotypes, s1m2 (79%) was the most frequently identified genotype in patients with obesity. A significant association was also found between cagA and the presence of vacA genotypes (except for vacA m1 and babA2). Conclusions: This study indicated the high prevalence of different virulence genes in H. pylori isolated from obese patients and supported the significant role of H. pylori in the development of GID.

Abstract 5439: Distinct genomic profile in H. pylori associated gastric cancer

Abstract Background: Gastric cancer is one of the most common and deadly cancer types. Currently 4 subtypes have been identified with unique molecular alterations: Epstein-Barr virus (EBV)-positive, microsatellite instability (MSI), chromosomal instability (CIN), and genomic stable (GS) tumors. However, many gastric tumors are associated with the bacterium Helicobacter pylori but the genomic landscape of this subgroup of tumors remains largely unknown. Methods: Targeted-sequencing covering 425 genes were performed retrospectively on 1759 gastric tumor tissues and matched normal blood samples. The mean sequencing depth of tumors was 1159X. Nonsynonymous mutations, copy-number variation (CNV) and MSI status were called from human DNA reads; non-human DNA reads were mapped to NCBI microbial reference genome using Kraken and significant species were identified. Results: Overall, 37 (2.67%) samples were EBV-positive, 200 (11.37%) samples were H. pylori-positive, and 10 (0.57%) samples were positive for both. Other notable species identified in tumor samples were E. coli and C. acnes. No bacteria or viral reads were found in normal blood. Among the rest, 59 (3.35%) samples were MSI, 380 (21.6%) were CIN, and 1017 (57.82%) were GS. H. pylori-positive samples tend to be GS (85.5%, p &amp;lt; 0.001) and microsatellite stable (95%, p = 0.04). The most common mutations identified in H. pylori-positive tumors were TP53 (47%), CDH1 (12%) and ARID1A (8.5%). The top CNV events were amplification in MCL1 (14.5%), TERC (9%), CCNE1 (8.5%), PTPRD (8.5%), and MYC (7.5%). Compared to other GS tumors, mutation in AKT3 (p = 0.002) and amplification of TERC (p = 0.008) as well as MCL1 (p = 0.0.029) were significantly enriched in H. pylori-positive tumors. There was no difference in patients' sex or age among subtypes (p &amp;gt;= 0.05). Conclusion: This study characterized the genomic landscape of H. pylori positive gastric tumors using targeted-sequencing, which allowed direct comparison with other subtypes of gastric cancer. Identification of DNA reads from infectious agents in the tumor samples indicates that deep sequencing platform has promising utility in uncovering the characteristics of microbial environment of tumor. Co-infection between H. pylori and EBV occurred at a low frequency. H. pylori-positive tumors were GS and microsatellite stable with unique molecular features. Future study examining difference in treatment response among these subtypes of gastric cancer are warranted. Citation Format: Ao Wang, Hua Bao, Pengfei Hu, Yong Wu, Jinfeng Zhang, Xue Wu, Xiaonan Wang, Yang Shao. Distinct genomic profile in H. pylori associated gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5439.

Clostridium difficile-associated disease and Helicobacter pylori seroprevalence: A case-control study.

Helicobacter pylori inhabits the stomach and causes persistent inflammation, with changes in gastric acidity. However, it is unclear whether the presence of Hpylori plays a role in Clostridium difficile-associated disease (CDAD). The study's aim was to examine relationships of Hpylori seroprevalence and serum pepsinogens (PGs), as markers of gastric inflammation, with CDAD. A case-control study was conducted among 49 CDAD cases and 54 controls (median age 82years). Using enzyme-linked immunosorbent assays, sera were tested for Hpylori IgG antibody, and PGI and PGII levels. Helicobacter pylori-positive samples were tested for IgG antibody to recombinant cytotoxin-associated gene A (CagA) virulent protein. Logistic regression models were fitted. Cases and controls were comparable in age (P=.5) and sex distribution (females 62% vs 57%, P=.6). Helicobacter pylori IgG seroprevalence was 47%, of whom 23% were CagA seropositives. Among cases compared to controls, 43% vs 28% were Hpylori seropositive but lacking CagA IgG antibody: adjusted odd ratio (OR) 3.43 (95% confidence intervals [CI] 1.29-9.10); 18% vs 4% were positive for CagA phenotype: adjusted OR 9.32 (95% CI 1.61-53.76). This association was not affected by PG levels. Helicobacter pylori infection, especially with CagA virulent phenotype, might predispose to Cdifficile infection in elderly patients.

Beta-defensins and analogs in Helicobacter pylori infections: mRNA expression levels, DNA methylation, and antibacterial activity.

Antimicrobial peptides can protect the gastric mucosa from bacteria, but Helicobacter pylori (H. pylori) can equally colonize the gastric apparatus. To understand beta-defensin function in H. pylori-associated chronic gastritis, we investigated susceptibility, human beta-defensin mRNA expression, and DNA methylation changes to promoters in the gastric mucosa with or without H. pylori infection. We studied the expression of HBD2 (gene name DEFB4A), HBD3 (DEFB103A), and HBD4 (DEFB104) using real-time PCR in 15 control and 10 H. pylori infection patient gastric specimens. This study demonstrates that H. pylori infection is related to gastric enhancement of inducible HBD2, but inducible HBD3 and HBD4 expression levels remained unchanged. HBD2 gene methylation levels were overall higher in H. pylori-negative samples than in H. pylori-positive samples. We also assessed antimicrobial susceptibility using growth on blood agar. The H. pylori strain Tox+ was susceptible to all defensins tested and their analogs (3N, 3NI). These results show that HBD2 is involved in gastritis development driven by H. pylori, which facilitates the creation of an epigenetic field during H. pylori-associated gastric tumorigenesis.

Antibiotic Resistance and Genotypes of Helicobacter pylori Strains in Patients with Gastroduodenal Disease in Southeast Poland.

The aim of this study was to investigate genetic diversity of Helicobacter pylori virulence markers to predict clinical outcome as well as to determine an antibiotic susceptibility of H. pylori strains in Poland. Gastric biopsies from 132 patients with gastrointestinal disorders were tested for presence of H. pylori with the use of rapid urease test, microbial culture, and polymerase chain reaction (PCR) detection. The genetic diversity of 62 H. pylori positive samples was evaluated by detection of cagA and PCR-typing of vacA and iceA virulence-associated genes. Most common H. pylori genotypes were cagA(+)vacAs1m2 (27.4%) and cagA(−)vacAs2m2 (24.2%). In logistic regression analysis, we recognized the subsequent significant associations: gastritis with ureC, i.e., H. pylori infection (p = 0.006), BMI index (p = 0.032); and negatively with iceA1 (p = 0.049) and peptic ulcer with cagA (p = 0.018). Thirty-five H. pylori strains were cultured and tested by E-test method showing that 49% of strains were resistant to at least one of the tested antibiotics. This is the first study that reports the high incidence and diversity of allelic combination of virulence genes in gastroduodenitis patients in Poland. Genotyping of H. pylori strains confirmed the involvement of cagA gene and vacAs1m1 genotype in development and severity of gastric disorder.

Novel high resolution melt curve assay for the analysis of predominance of Helicobacter pylori clarithromycin resistance.

Clarithromycin resistance is the most common cause of Helicobacter pylori treatment failure and it is attributed to three point mutations, A2142G, A2142C and A2143G, within the 23S rRNA gene. We aimed to determine the prevalence of H. pylori clarithromycin resistance using a novel high resolution melt assay. A total of 151 stool samples were collected from treatment-naïve patients with general gastric discomfort who also performed 13CO2 breath tests. Stool antigen tests were also performed on 126 of the 151 stool samples collected. Bacterial DNA was extracted from the stool and analyzed by comparing it with four reference plasmids incorporating the three mutations and the wild type (WT) sequences. The melt assay detected 106 H. pylori positive samples, of which 54 had a WT sequence, and 52 had a point mutation associated with clarithromycin resistance, including A2142G in 10, A2142C in 13, A2143G in 18 and heterozygosity (multiple peaks) in 11. Compared with the gold standards (13CO2 breath and stool antigen tests), the melt assay had a sensitivity of 100% and 99% and a specificity of 82% and 78%, respectively. Therefore, our stool-based molecular assay is able to identify H. pylori infection and clarithromycin resistance. It could be used for screening prior to administration of clarithromycin eradication therapy.

Initial Reverse Transcription is Essential for Reliable Helicobacter pylori 23S rDNA Real-Time PCR Diagnostics from Fecal Samples.

A sensitive, non-invasive molecular test for identifying Helicobacter pylori (H. pylori) in stool samples is important in the context of a "test-and-treat strategy". The goal of this study was to elucidate the benefit of an initial reverse transcription (RT) step for the sensitivity of H. pylori 23S rDNA Real-time PCR in stool samples. We compared a LightMix® Modular Helicobacter 23S rDNA PCR assay without (LightMix® PCR) and with an initial reverse transcription step (LightMix® RT-PCR) assay for rapid detection of Helicobacter pylori in fecal samples. For maximum sensitivity, all Lightmix® RT-PCR assays were performed in triplicate for each sample. Additionally, the LightMix® RT-PCR was compared to H. pylori AG ELISA. Direct detection of H. pylori in feces by Lightmix® PCR was less sensitive than LightMix® RT-PCR. Only 2 out of 44 stool samples (4.5%) from patients with diarrhea were positive for H. pylori in the LightMix® PCR assay. In contrast, a reverse transcription step prior Lightmix® PCR increased the assay sensitivity markedly (19 out of 44 positives, 43.2%). When re-testing 65 samples initially analyzed with H. pylori AG ELISA with LightMix® RT-PCR, the detection rate for H. pylori was similarly increased from 23.1% (15/65) with H. pylori AG ELISA to 43.1% (28/65) with H. pylori LightMix® RT-PCR. 21.5% of the 28 H. pylori positive samples were positive in 1/3, 32% in 2/3, and 46.5% in 3/3 triple RT-PCR approaches. When re-testing the 28 LightMix® RT-PCR positive samples by LightMix® PCR, only 17.9% (5/28) RT-PCR positive samples were positive in PCR. LightMix® RT-PCR is much more sensitive than Lightmix® PCR. In comparison to H. pylori AG ELISA, the LightMix® RT-PCR shows a markedly higher sensitivity. An initial reverse transcription step is crucial for reliable Helicobacter pylori 23S rDNA Real-time PCR diagnostics. The triple RT-PCR approach can additionally improve the detection of H. pylori in fecal samples. Thus, the method presented provides a highly sensitive, noninvasive assay to detect H. pylori in fecal samples with potential advantages compared to other H. pylori detection methods currently used in routine diagnostics.

Development and validation of a whole-cell ELISA for serologically diagnosing Helicobacter pylori infection in Brazilian children and adults: a diagnostic accuracy study.

Serological tests are practical, with low cost, but no noninvasive tests are available for diagnosing Helicobacter pylori (H. pylori) infection in Brazil. The aim here was to develop and validate enzyme-linked immunosorbent assay (ELISA) serological tests to detect anti-H. pylori immunoglobulin G antibodies, based on cultured strains from Brazilian patients. Cross-sectional, diagnostic accuracy study comparing a locally developed and validated ELISA and invasive tests among dyspeptic patients at two public hospitals in São Paulo, Brazil. An ELISA test was prepared using whole-cell antigen from 56 strains. After genotypic characterization, it was standardized and optical density (OD) cutoffs were determined based on the serum antibody response of 100 H. pylori-negative samples, compared with 82 H. pylori-positive samples. Validation was performed on 174 symptomatic patients. The optimal OD cutoffs established (for monoclonal and polyclonal tests, respectively) were 0.167 and 0.164; overall ELISA sensitivity: 84.3%, 78.9%; specificity: 88.6%, 90.6%; positive predictive value (PPV): 75.4%, 80%; negative predictive value (NPV): 93.1%, 81.8%; accuracy: 87.3%, 86.2%; child and adolescent ELISA sensitivity: 74.2%, 81.8%; specificity: 90.8%, 86.7%; PPV: 66.6%, 84.3%; NPV: 95.8%, 84.8%; accuracy: 88.5%, 84.6; adult ELISA sensitivity: 84.4%, 75%; specificity: 86.9%, 93%; PPV: 81.8%, 78.3%; NPV: 88.9%, 91.8%; accuracy: 85.9%, 88.5%. The polyclonal serological test developed using local strains presented better diagnostic performance among children and adolescents, while the monoclonal test was better among adults. The results from both tests suggest that these in-house serological tests could be used to detect anti-H. pylori antibodies in our population, for screening purposes.

Abstract A04: Microbial and immunologic characterization of gastroesophageal tissue biopsy samples: A multiparametric analysis

Abstract Gastric cancer carcinogenesis is associated with chronic inflammation, most commonly the result of Helicobacter pylori chronic infection in the stomach antrum. The development of gastric cancer in the context of chronic H. pylori infection is multifactorial, encompassing both bacterial factors and the altered immune microenvironment. However, a comprehensive analysis of the relation between inflammation and host microbial population in patient tissue samples has not previously been explored. We proposed an unbiased study to evaluate the relationships among microbiome composition, host immune response and genomic characterization from next-generation sequencing of gastric biopsy samples. Patients undergoing upper endoscopy without chronic inflammatory disease or chronic NSAID use were eligible for participation. Endoscopic biopsies from gastric fundus, body, and antrum were collected from patients with active H. pylori infection (n=21), prior infection (n=22) and no prior infection (n=26), and were sequenced at 10X to 30X coverage. In total, 77 gastric biopsies from 69 patients were freshly frozen for whole genome sequencing (WGS) and transcriptome (RNASeq) analysis. Detecting the microbiome from human biopsy sequencing data directly is challenging due to the low microbial content. A novel computational pipeline was developed to address this problem specifically (Zhang et al., Genome Biology 2015). A robust H. pylori signal was detected in samples from clinically verified H. pylori infected patients, and the results were further validated by qPCR. In our analysis population, in addition to identification of H. pylori, several bacteria associated with other cancers were also detected in several biopsy samples, such as Prevotella melaninogenica, Veillonella parvula and Fusobacterium nucleatum. H. pylori infection was associated with reduced microbial biodiversity compared to prior infection or control tissue (p=0.02). H. pylori active infection samples have a distinct non-H. pylori microbiome compared to prior infection and control samples. We also identified 5 patients with prior infection and 1 control patient with occult H. pylori infection (e.g., asymptomatic patients). To characterize the immune infiltration in the mucosal biopsy samples, we developed a 176-gene panel, collected from multiple published studies, to define the immune signatures. The expression profile of this immune gene panel was used evaluate the immune infiltration levels of multiple immune cell types. The result of unsupervised clustering revealed a much higher immune infiltration in H. pylori positive samples compared to uninfected samples, especially for CD8+, Th2 and Th17 cell populations. Two orthogonal experimental essays (ELISA and Flow Cytometry) were performed independently to verify the results. ELISA results confirmed the RNAseq-based expression profiling of inflammatory cytokines such as GRO, IL8, TNFa and SCD40L. Importantly, in 2 patients with prior H. pylori infection, the pro-inflammatory immune signature characteristic of H. pylori persisted. Finally, amongst H. pylori active infection patients, the biodiversity of the other bacteria present was inversely correlated with local immune infiltration (e.g., greater the bacterial diversity, the less robust the proinflammatory immune signature). In summary, this study established a methodology for microbiome and immune profiling of gastric biopsy samples. We have identified a proinflammatory immune response signature associated with H. pylori infection, confirmed and validated by orthogonal assays. Notably, in several samples from previously eradicated H. pylori infection, the proinflammatory immune signature persisted, suggesting a potential long-term impact of H. pylori infection on mucosal immunity, that might contribute to gastric tumorigenesis. Finally, we demonstrate that the biodiversity of the bacterial species correlates with the character of immune infiltration. Citation Format: Chao Zhang, Prashant Vijay Thakkar, Felice Schnoll-Sussman, Bridget McClure, Michelle Bigg, Greg Sonnenberg, Doron Betel, Manish Shah. Microbial and immunologic characterization of gastroesophageal tissue biopsy samples: A multiparametric analysis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A04.

The impact of Helicobacter pylori infection, eradication therapy and probiotic supplementation on gut microenvironment homeostasis: An open-label, randomized clinical trial.

BackgroundHelicobacter pylori (H. pylori) infection is associated with remodeling of gastric microbiota. However, comprehensive analyses of the impact of H. pylori infection, eradication therapy and probiotic supplementation on gut microbiota are still lacking. We aimed to provide evidence for clinical decision making.MethodsSeventy H. pylori-positive and 35 H. pylori-negative patients (group C) were enrolled. H. pylori-positive patients were randomly assigned to group A (14-day bismuth-containing quadruple therapy) and group B (quadruple therapy supplemented with Clostridium butyricum). Stool samples of group A and B were collected on day 0, 14 and 56 while stool samples of group C were collected on day 0. Gut microbiota was investigated by 16S rRNA sequencing.FindingsThe Sobs index (richness estimator) was significantly higher in H. pylori-positive samples than H. pylori-negative samples (p < .05). Several metabolic pathways were more abundant in H. pylori-positive communities while some disease-associated pathways had higher potential in H. pylori-negative community through KEGG pathway analysis. Abundances of most butyrate-producing bacteria significantly decreased, while several detrimental bacteria increased after eradication therapy. Probiotic supplementation was associated with improved gastrointestinal symptoms as well as increased Bacteroidetes:Firmicutes ratio.InterpretationWhile H. pylori infection may not be necessarily detrimental in all patients, eradication of H. pylori was associated with widespread changes in gut microbial ecology and structure. Probiotic supplementation could relieve more gastrointestinal symptoms by inducing alterations in gut microbiota and host immune responses. As such, the decision to eradicate H. pylori should be based on comprehensive analysis of individual patients.