Protocol for detecting Helicobacter suis infection in gastric biopsies and serum by PCR and ELISA.

Infection with Helicobacter suis, which causes many cases of gastric disease, is not reliably diagnosed. Here, we present a protocol for detecting H. suis infection. We describe steps for collecting gastric biopsies and sera from patients, preparing DNA for PCR, and targeting the H. suis-specific gene. We then define procedures for inoculating biopsies onto primary agar plates and transferring colonies to secondary agar plates. Finally, we detail whole-genome sequencing of bacteria and assess H. suis infection in sera with ELISA. For complete details on the use and execution of these protocols, please refer to Matsui et al.1.

Development of serological assays to identify Helicobacter suis and H. pylori infections.

Helicobacter suis, hosted by hogs, is the most prevalent gastric non-Helicobacter pylori Helicobacter species found in humans. Recent studies have suggested that H. suis infection has caused many cases of gastric disease, but the transmission route from hogs remains unclear. Diagnostic methods based on H. suis urease activity often yield negative results, and there is no reliable method for diagnosing H. suis infection in clinical practice without gastric biopsy specimens. This study presents the world's first use of whole-bacterial cell ELISA to simultaneously assess H. suis and H. pylori infections. The ELISAs showed high accuracy, with an area under the ROC curve of 0.96, 100% sensitivity, 92.6% specificity, 76.9% positive predictive value, and 100% negative predictive value for the H. suis test, and an area under the ROC curve of 0.92, 88.2% sensitivity, 87.5% specificity, 65.2% positive predictive value, and 96.6% negative predictive value for the H. pylori test.

Cost Analysis in Helicobacter pylori Eradication Therapy Based on a Database of Health Insurance Claims in Japan.

OBJECTIVE: Cost-benefit is an important consideration for Helicobacter pylori (H. pylori) eradication in Japan, where 1.5 million patients were reported to receive first-line eradication annually. This study aimed to identify the optimal cost-saving triple therapy regimen for H. pylori eradication in Japan. MATERIALS AND METHODS: This retrospective observational study used data from a large-scale, nationwide health insurance claims database (2015‒2018). Using success rates of first-line eradication, mean total costs of first-line and second-line eradications per patient were compared between regimens including a potassium-competitive acid blocker (P-CAB) or a proton pump inhibitor (PPI), and between two clarithromycin (CAM) doses (400 and 800 mg/day). Subgroup analyses by smoking habit or body mass index (BMI) were performed. RESULTS: Among propensity score (age, gender, CAM dose, disease name)-matched patients (P-CAB regimen, n=22,002; PPI regimen, n=22,002), total costs were lower with the P-CAB than the PPI regimen (Japanese yen [JPY] 12,952 vs 13,146) owing to significantly higher first-line eradication rates with the P-CAB regimen (93.6% vs 79.7%; p<0.001). For both regimens, even among current smokers or patients with BMI ≥25 kg/m2, eradication rates did not differ by CAM dose, and total costs were approximately JPY1000 lower with CAM 400 mg/day than with CAM 800 mg/day. CONCLUSION: High success rate of first-line eradication contributes to saving in total eradication costs by reducing costs of subsequent therapy, irrespective of patients' smoking status or BMI class. The combination of more potent acid-inhibitory medicine and low-dose CAM may be the optimal regimen in terms of efficacy and cost-benefit in Japan.

Isolation and characterization of Helicobacter suis from human stomach.

Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.

Endoscopic severe mucosal atrophy indicates the presence of gastric cancer after Helicobacter pylori eradication -analysis based on the Kyoto classification.

BACKGROUND: Gastric cancer after Helicobacter pylori (HP) eradication is a crucial clinical challenge today as HP eradication therapy is widely performed. Detecting gastric cancer after HP eradication tends to be difficult with normal white-light endoscopy. In the present study, we aimed to identify easily-evaluated endoscopic findings that indicate the presence of gastric cancer after HP eradication so that endoscopists can consider additional detailed examinations at the site. METHODS: We analyzed the endoscopic images of 43 patients who underwent endoscopic submucosal dissection for early gastric cancer after HP eradication and 119 patients with an HP eradication history who underwent esophagogastroduodenoscopy for a medical checkup. Endoscopic findings were evaluated according to the Kyoto classification of gastritis (atrophy, intestinal metaplasia, enlarged folds, nodularity, and diffuse redness) and map-like redness. RESULTS: Patients with gastric cancer had significantly higher total Kyoto risk scores; more atrophy, intestinal metaplasia, and diffuse redness; and a significantly higher prevalence of map-like redness compared with those without gastric cancer, in the univariate analyses. We used logistic regression analysis with forward selection based on the likelihood ratio to develop a model using atrophy and diffuse redness. Receiver operating characteristic analysis showed that a score of A2 in the Kyoto classification of gastritis (open-type atrophic pattern in the Kimura-Takemoto classification) was an endoscopic marker for the presence of post-HP-eradication gastric cancer. CONCLUSIONS: Endoscopic severe gastric mucosal atrophy is useful to screen patients for gastric cancer after HP eradication.

Questionnaire-Based Survey on Gastrointestinal Bleeding and Management of Antithrombotic Agents during Endoscopy Among Asian Countries.

OBJECTIVE: Guidelines on the management of antithrombotic therapy for endoscopic procedures vary among countries. Differences in the management of antithrombotic agents for endoscopic procedures between Western and Eastern countries have already been reported. However, no study has investigated the differences among Asian countries. The aim of this study was to examine the differences in the etiology of gastrointestinal bleeding and management of antithrombotic agents during endoscopic procedures between Japan and other Asian countries (OAC). METHODS: Questionnaires regarding gastrointestinal bleeding in clinical practice and management of antithrombotic agents during endoscopy were distributed to members of the International Gastroenterology Consensus Symposium Study Group. We analyzed the questionnaire answers and compared the results between Japan and OAC. RESULTS: The cause of and treatment methods for gastrointestinal bleeding differed between Japan and OAC. In Japan, the trend was to continue drugs at the time of biopsy and endoscopic therapy. Even in cases of discontinuation, the drug withdrawal period was as short as <3 days. Thrombotic complications caused by the withdrawal of antithrombotic agents were observed more frequently in Japan (34.78%) than in OAC (22.46%; p = 0.016). CONCLUSION: Due to differences in guidelines and complications associated with discontinuation of drugs, the antithrombotic withdrawal period in Japan tended to be shorter than that in OAC.

A Caucasian American patient with celiac disease diagnosed in Japan and successfully treated with a gluten-free diet.

We report the case of a 33-year-old Caucasian American man diagnosed with celiac disease in Japan. He presented to a community hospital because of chronic watery diarrhea and weight loss for 6 months. The laboratory data showed low serum albumin and serum cholesterol. A colonoscopy was normal. He was referred to our hospital for further work-up. Serum tissue transglutaminase immunoglobulin A (IgA) and endomysial antibody were positive. The HLA type was DQ2. Esophagogastroduodenoscopy (EGD) revealed nodular and mosaic-patterned mucosa from the bulb to the second part of the duodenum. The histopathological findings were consistent with Marsh type 3c of the modified Marsh classification for celiac disease. The patient was instructed to follow a gluten-free diet (GFD). Six months after the initiation of the GFD, his symptom and the levels of serum albumin and cholesterol were improved, and the serum tissue transglutaminase IgA and endomysial antibody became negative. However, EGD showed little improvement. Capsule endoscopy also revealed mosaic-patterned mucosa, nodular mucosa, and scalloping of the folds of the duodenum and proximal small intestine. There was no definite improvement in histopathological findings. Collectively, the GFD was effective in this patient with celiac disease, but it should be maintained to achieve endoscopic and histopathologic healing.

Questionnaire-Based Survey on Diagnostic and Therapeutic Endoscopies and H. pylori Eradication for Elderly Patients in East Asian Countries.

BACKGROUND: Gastrointestinal endoscopy and Helicobacter pylori(H. pylori) eradication therapy are commonly performed even among the elderly population. The aim of this study was to understand the way endoscopists viewed the application of endoscopy and H. pylori eradication in the elderly of East Asian countries. METHODS: Self-administered questionnaires containing 13 questions on endoscopy and H. pylori eradication in the elderly were distributed to major institutions in Japan, South Korea, China, Indonesia, and the Philippines. RESULTS: Two hundred and fifteen endoscopists (111 in Japan, 39 in China, 24 in Korea, 21 in Indonesia, and 20 in the Philippines) participated in this study. In the institutions where these endoscopists were associated, around 50% of patients undergoing endoscopy were above the age of 60 years. The participating endoscopists indicated that the necessity of screening esophagogastroduodenoscopy and colonoscopy was lower in populations aged over 81 than the other age groups. They hesitated to perform therapeutic endoscopy, such as endoscopic submucosal dissection or endoscopic retrograde cholangiopancreatography, more often in patients over 85. They also hesitated to perform H. pylori eradication in patients aged over 81, especially in Japan. CONCLUSION: Endoscopists had significantly different attitudes regarding the indications for screening or therapeutic endoscopy and H. pylori eradication therapy in younger and elderly populations in East Asian countries.

[Clinical characteristics of patients with pneumatosis cystoides intestinalis].

Pneumatosis cystoides intestinalis (PCI) is a relatively rare disease in which multilocular or linear pneumatic cysts develop under the mucosa or serosa of the intestinal wall. We conducted a retrospective analysis to investigate the clinical characteristics of 68 patients with PCI. Hepatic portal venous gas (HPVG) was present in 9 patients, 8 of which had underlying intestinal tract necrosis. In most patients, PCI was mild and asymptomatic and resolved spontaneously. The main treatment strategy for PCI is conservative therapy. However, in cases complicated by HPVG, the presence of underlying intestinal tract necrosis must be considered in order to promptly determine whether emergency surgery is required.

Changes in the first line Helicobacter pylori eradication rates using the triple therapy-a multicenter study in the Tokyo metropolitan area (Tokyo Helicobacter pylori study group).

BACKGROUND AND AIM: Helicobacter pylori (H. pylori) infection is a strong risk factor for the development of gastric cancer. In 2013, the Japanese government approved H. pylori eradication therapy in patients with chronic gastritis as well as peptic ulcer. However, the continuing decline in eradication rates for first-line H. pylori eradication therapies is an urgent problem. In this study, we investigated changes in the first-line eradication rate from 2001 to 2010. METHODS: Eradication rates for 7-day triple therapy [proton pump inhibitor (rabeprazole 20 mg, lansoprazole 60 mg, or omeprazole 40 mg)+amoxicillin 1500 mg + clarithromycin (CAM) 400 or 800 mg, daily] were collated from 14 hospitals in the Tokyo metropolitan area. The urea breath test was used for the evaluation of eradication. The cut-off value was less than 2.5%. RESULTS: The yearly eradication rates (intention to treat/per protocol) were 78.5/79.5% (2001, n=242), 71.2%/72.9% (2002, n=208), 67.8%/70.5% (2003, n=183), 75.6%/84.6% (2004, n=131), 56.4%/70.5% (2005, n=114), 70.5%/75.8% (2006, n=271), 67.4%/82.0% (2007, n=135), 64.0%/76.3% (2008, n=261), 60.5%/74.3% (2009, n=329), and 66.5%/78.8% (2010, n=370), respectively. Examination of eradication rates according to CAM dosage revealed an eradication rate of 65.6% (383/584) for CAM 400 mg daily, and 68.5% (1124/1642) for CAM 800 mg daily, with no significant difference seen between dosages. CONCLUSION: In recent years, eradication rates for first-line triple therapy have obviously decreased, but no noticeable decrease has occurred after 2001.

[The practical assessment of H. pylori eradication].

Helicobacter pylori (H. pylori) gastritis has been approved by Ministry of Health, Labour and Welfare as an additional indication for H. pylori eradication in Japan on Feburary 21, 2013. Diagnostic methods of H. pylori infection have been divided into direct (invasive) and indirect (non-invasive). Invasive tests requiring endoscopic biopsy include culture, histology and rapid urease test (RUT). Non-invasive tests not requiring endoscopic biopsy include measurement of H. pylori antibody(serum, urine), urea breath test(UBT) and stool antigen test. Assessment of the efficacy of H. pylori eradication therapy should be performed at least 4 weeks after the completion of treatment. UBT and monoclonal stool antigen test are both recommended for the assessment of H. pylori eradication. When the results obtained is doubtful for assessment of H. pylori eradication, it is preferable to perform another test or follow-up.

[Gastric hyperplastic polyps and H. pylori infection, their relationship and effects of eradication therapy].

Gastric hyperplastic polyp is frequently found in the stomach. The polyp almost never occurs in normal gastric mucosa and is most commonly associated with chronic gastritis induced by H. pylori infection. Most gastric hyperplastic polyps disappear after eradication of H. pylori. Eradication therapy is safe, non-invasive, and reduces medical cost in comparison with polypectomy. It is now expected that H. pylori infection rate may decrease in the future, and H. pylori eradication treatment in Japan may become more common. Therefore, cases of gastric hyperplastic polyp and gastric cancer may decrease.

Lactobacillus reuteri tablets suppress Helicobacter pylori infection--a double-blind randomised placebo-controlled cross-over clinical study.

UNLABELLED: We studied the effect of Lactobacillus reuteri strain SD2112 Tablets Reuterina (ERINA Co., Inc.), in suppressing H. pylori urease activity and to use the urea breath test (UBT) as a marker for the burden of infection. Method 1: Assessment of UBT and H. pylori density. Subjects were 33 H. pylori-positive patients from whom were obtained gastric biopsy specimens by upper gastrointestinal endoscopy. The correlation between UBT and H. pylori density was investigated. Individual UBT was established for each patient. Patients were divided by H. pylori density was 3 groups: Group I (low-density), Group II (moderate-density), and Group III (high-density). The individual UBTs were then correlated to the established H. pylori quantity. Method 2: Assessment of suppressive effect of L. reuteri on H. pylori urease activity. Subjects were 40 asymptomatic volunteers with an UBT exceeding 15 per thousand, randomly allocated to four groups: Subjects in Group A underwent active treatment for 4 weeks (period 1) and placebo treatment for the following 4 weeks (period 2). These in Group B underwent treatment in reverse order. Those in Group C underwent placebo. Group D consisted of volunteers with negative UBT undergoing active treatment for the full 8 weeks. Result 1: UBT was 11.6+/-2.0 per thousand, 22.1+/-2.6 per thousand, and 35.4+/-7.6 per thousand in Groups I, II, and III, showing UBT that increased significantly (I vs. II: p< 0.01 and I vs. III: p<0.05) based on H. pylori density. Result 2:Significant differences were seen in the decrease in UBT before versus after medication in Groups A and B. In Group A, lower UBT was maintained until the end of the full 8-week period. The overall decrease in UBT due to medication with L. reuteri Tablets was 69.7+/-4.0% (p<0.05). CONCLUSION: Administration of L. reuteri Tablets [Reuterina (ERINA Co.,Inc.)] significantly decreased UBT in H. pylori-positive subjects, demonstrating that L. reuteri suppresses H. pylori urease activity and H. pylori density.

[Generalization of Helicobacter pylori eradication therapy and its future in Japan].

After official acceptance of eradication therapy for Helicobacter pylori infected peptic ulcer disease in 2000, this treatment has been generalized as ulcer therapy in Japan. In 2003 the consensus statement of the Japanese Society for Helicobacter Research (JSHR) for Helicobacter pylori infection was presented. According to this statement MALT lymphoma was recommended to treat H. pylori infection because of the efficacy of its out come. Atrophic gastritis was also advised to treat H. pylori infection for the purpose of preventing gastric cancer development. The extra alimentary disease such as idiopathic thrombocytopenic purpura was still under evaluation. In diagnostic method the importance of drug susceptibility test is rising because of the increase of drug resistant H. pylori strain in Japan. The false positive case of urea breath test, the accuracy of new Japanese original serology tests and the stool antigen test are also under investigation. The standard regimen of H. pylori eradication therapy in Japan is proton pomp inhibitor (PPI) + amoxicilin (AMPC) + clarithromycin (CAM). The problem of this regimen is the decrease of eradication rate. The reason of this trend might be increase of CAM resistant strain. JSHR recommended a rescue regimen as PPI + AMPC + metronidazole for the patient whose first eradication therapy was unsuccessful result. The eradication of this second line regimen was reported as about 90%.

[Evaluation of 13C-urea breath test to confirm eradication of Helicobacter pylori].

This study aimed to evaluate the effectiveness of the 13C-urea breath test (UBT) for assessment of Helicobacter pylori eradication after treatment. One hundred twenty six patients were enrolled with 85 receiving proton pomp inhibitor based triple therapy. They were underwent upper gastrointestinal endoscopy with biopsies for diagnosis and assessment of H. pylori infection using culture, histology, rapid urease test (RUT) and 13C-UBT. Assessment of eradication needs to be performed 4 weeks or more after completion of treatment. Breath samples were taken 15 minutes after the ingestion of 100 mg 13C-urea. Breath samples were analyzed on a mass spectrometer system. The gold standard for H. pylori infection was a positive culture or positive histology + positive RUT; negative for infection was defined as negative results of all three biopsy tests. Based on ROC curves, the most appropriate cut-off value for diagnosis of H. pylori infection was identified as 2.5/1000, which provided 96.2% sensitivity, 100% specificity, and 96.8% accuracy as judged by the gold standard. However, when confirming the eradication of H. pylori, it was 3.5/1000, which provides for 100%, 95.8%, and 96.5%, respectively. Ten patients (11.8%) had delta13C values that were 2.5-5.0/1000 4-12 weeks after therapy. Eight patients were considered cured of H. pylori infection, and 2 were considered to still have H. pylori infection following 13C-UBT, serology, and H. pylori specific antigen test. The false-positive rate of 13C-UBT was 9.4% (8/85). When the grey zone of 13C-UBT was set at a level of 2.5 to 5.0/1000 (2.5 > : negative, 5.0 < or = : positive) after eradication therapy, the sensitivity and specificity of 13C-UBT was 100% and 98.4% compared to the gold standard. It was concluded that to avoid false-positive results of 13C-UBT, the grey zone of 13C-UBT needs to be set at a level of 2.5 to 5.0/1000; thus improving the accuracy of test for the assessment of eradication of H. pylori infection.

Evaluation of Helicobacter pylori stool antigen test before and after eradication therapy.

BACKGROUND AND AIM: The Helicobacter pylori stool antigen (HpSA) test is useful for initial diagnosis of H. pylori infection, but there is disagreement regarding its diagnostic accuracy after eradication therapy. The aim of the present study was to evaluate the diagnostic accuracy of the HpSA test before and after eradication therapy. METHODS: One hundred and thirty-six patients underwent upper gastrointestinal endoscopy with biopsies for the diagnosis of H. pylori infection using culture, histology and the rapid urease test. Fifty-four H. pylori-positive patients were treated with 1-week triple therapy. Six to 10 weeks after the end of therapy, the patients underwent re-endoscopy and received the same biopsy-based methods. In addition, the 13C-urea breath test was performed. The HpSA test was performed before and 6-10 weeks after the end of therapy. In 23 patients, the HpSA test was also performed at the end of therapy. RESULTS: Before therapy, the sensitivity and specificity of the HpSA test was 98.3% (95% confidence interval (CI): 95.9-100%) and 95.0% (95% CI: 75.1-99.9%), respectively. At the end of therapy, the HpSA tests were all negative both for eradication and non-eradication patients. The sensitivity and specificity of the HpSA test after eradication therapy were 90% (95% CI: 55.5-99.7%) and 97.7% (95% CI: 93.3-100%), respectively. CONCLUSIONS: The HpSA test is a useful method for the diagnosis of H. pylori infection before and after eradication therapy.

Helicobacter pylori and acetylsalicylic acid synergistically accelerate apoptosis via Fas antigen pathway in rabbit gastric epithelial cells.

The mechanism for gastric epithelial cellular apoptosis by both H. pylori and NSAIDs remains unknown. Normal gastric epithelial cells, collected from rabbit stomach, were cultured with viable H. pylori and/or an acetylsalicylic acid for 24 hr as an in vitro model of gastric epithelial cell injury. The Fas antigen expression rate in the gastric epithelial cells was measured using a FACScan. In group E, in which H. pylori inoculation of epithelial cells was followed by treatment with an NSAID, the rate of Fas antigen expression was significantly higher than the rate observed after treatment with either H. pylori or NSAID alone. Despite the fact that drug treatment alone did not significantly increase the Fas expression rate vs control cells. The results suggest that H. pylori and NSAID induce gastric cell injury as apoptosis via the Fas antigen pathway in a synergistic manner and that this effect is particularly strong when NSAID treatment follows H. pylori infection.