Marcello Menegatti

The Stool Antigen Test for Detection of Helicobacter pylori after Eradication Therapy

Context Standard treatment regimens do not eradicate infection in approximately 10% to 20% of people with ulcers or gastritis caused by Helicobacter pylori. Symptoms do not reliably identify patients who have persistent infection despite treatment. Although positive results on a urea breath test done 4 weeks after treatment reliably identify persistent infection, a noninvasive test that detects successful eradication earlier would be useful. Contribution This multicenter study shows that a positive finding on a stool antigen test done as early as 1 week after treatment identifies about 95% (range, 70% to 100%) of cases of persistent infection. Generalization Cautions Findings are from patients with dyspepsia who were referred for endoscopy; 20% of patients were still infected at 1 month despite eradication therapy. The Editors Noninvasive tests for Helicobacter pylori are important in primary care, both for initial diagnosis of H. pylori infection and for confirmation of eradication. Current guidelines recommend noninvasive testing and treatment of young dyspeptic patients without alarm symptoms (such as dysphagia or weight loss that suggest underlying malignant disease) in a primary care setting by using low-cost noninvasive tests (1, 2). Randomized, controlled trials have shown that a test and eradicate strategy toward H. pylori is effective in patients with dyspepsia seen in primary care settings who have not undergone investigations such as endoscopy or radiographic studies (3). Post-therapy testing is also growing in importance. Resistant strains of H. pylori are now widely prevalent in the United States and Europe, and eradication therapy with current regimens fails in 10% to 20% of patients (4, 5). Furthermore, some patients with ulcer disease remain symptomatic despite successful eradication of H. pylori and healing of the ulcer (6). In patients with persistent symptoms, testing for persistent H. pylori infection is important to direct further therapy. Routine testing to confirm eradication in patients with complicated ulcer disease, such as bleeding peptic ulcer, is necessary because the risk for rebleeding is greatly increased in patients with persistent infection (7). The choice of tests in the post-therapy setting is limited. Serologic tests are unreliable in determining eradication (8). Endoscopic tests (rapid urease test, histologic examination, or culture) are reliable, but endoscopy is expensive and inconvenient. Until recently, the only noninvasive test that reliably demonstrated whether eradication was successful was the urea breath test (9). This test has high sensitivity and specificity in the post-therapy setting but cannot be used until 4 weeks after treatment. Moreover, the breath test is still not widely available in the United States. The fecal antigen test is a relatively new noninvasive test for detection of H. pylori (10). This test detects the presence of infection by measuring the fecal excretion of H. pylori antigens. It has been approved by the U.S. Food and Drug Administration for detection of H. pylori before and after therapy. We sought to determine whether a stool antigen test administered at various times after treatment correctly identifies persons in whom H. pylori infection persists despite eradication therapy. Methods We prospectively studied 84 patients infected with H. pylori at six clinical centers (31 in Bologna, Italy; 29 in Amsterdam, the Netherlands; 9 in Rome, Italy; 8 in Lisbon, Portugal; 4 in Madrid, Spain; and 3 in Milwaukee, Wisconsin). The sample consisted of consecutive patients with dyspepsia (defined as pain or discomfort centered in the upper abdomen) who were referred by primary care physicians for upper endoscopy (11). Consenting patients were enrolled if they tested positive for H. pylori on endoscopic tests. Patients enrolled in this study have not been enrolled in other studies. Patients were excluded if they had taken proton-pump inhibitors, H2-receptor antagonists, nonsteroidal anti-inflammatory agents, or antibiotics in the 4 weeks before the study. Failure to return for follow-up endoscopy was an a priori exclusion criterion. All patients gave written informed consent, and the study was approved by the human subjects review committee or equivalent at each participating institution. At baseline, patients underwent endoscopy with biopsy sampling for histologic examination (two samples from the antrum and two from the corpus), culture (two samples from the antrum and two from the corpus), and a rapid urease test (one sample from the antrum). All patients were infected with H. pylori at baseline, as demonstrated by positive results on both rapid urease testing and histologic examination or a positive culture for H. pylori. Within 24 hours of the endoscopy, all patients underwent a 13C or 14C urea breath test. The breath test was chosen according to local availability and experience, but in all cases a validated breath test analysis system was used. Cut-off values were determined according to the recommendations of the various manufacturers of these tests. Patients collected stool using a kit consisting of a plastic spoon that is used to scoop a small amount of stool from the toilet paper or toilet bowl into an airtight container. At all sites, the stool assay was performed by using the Premier Platinum HpSA test (Meridian Diagnostics, Inc., Cincinnati, Ohio). The assay is a microwell-based enzyme immunoassay that uses polyclonal antiH. pylori capture antibody adsorbed to microwells. Diluted patient samples and a peroxidase-conjugated polyclonal antibody were added to the wells and incubated at room temperature for 1 hour. A wash was performed to remove unbound material. Substrate was added and incubated for 10 minutes at room temperature. Color develops in the presence of bound enzyme. Stop solution was added, and the results were inspected spectrophotometrically at 450 nm within 15 minutes of adding the stop solution. Visual determination can also be used; this has been shown to have similar results (12). A positive control and a negative control are built into the test. The cut-off values were classified as negative (<0.140), indeterminate (0.140 to 0.159), or positive (>0.160). After completion of the baseline procedures, treatment was begun with ranitidine bismuth citrate (400 mg twice daily) or omeprazole (20 mg twice daily) in combination with amoxicillin (1 g twice daily) and clarithromycin (500 mg twice daily) for 7 to 10 days. Seven-day eradication therapy was used in Europe, where it is approved by the European Union and has been shown to be effective (5). Ten-day triple therapy with proton-pump inhibitors was used in the United States, where it is approved by the U.S. Food and Drug Administration. Patients collected stool for the stool antigen test on days 3, 7, 15, 21, 28, and 35 after completion of H. pylori eradication therapy. On day 35 after completion of eradication therapy, endoscopy was repeated and biopsy samples were again obtained for histologic examination, culture, and the rapid urease test, as performed at the baseline visit. The 13C or 14C urea breath test was repeated on day 35 by using the same method and cut-off values as at baseline. Patients were classified as being infected with H. pylori at baseline and having persistent infection on day 35 if culture of gastric biopsy specimens was positive for H. pylori or results of the rapid urease test and histologic examination were positive. All other patients were classified as negative. These criteria have been recommended by an expert panel for use in clinical trials of H. pylori eradication (13). At baseline, the sensitivity of the stool test and urea breath test were calculated by using the presence of infection (defined above) as the gold standard. At each time point after completion of therapy (days 3, 7, 15, 21, 28), predictive values were calculated by using continued infection on day 35 as the gold standard (positive result on culture or on rapid urease test and histologic examination). Trained investigators who were blinded to the results of the other diagnostic studies performed the stool assays. The first endoscopy procedure was performed before the stool and breath tests. Therapy was given on the basis of results on endoscopic testing. Endoscopists were blinded to the results of post-treatment stool studies and the breath test until all evaluations were completed. Long-Term Follow-up Patients in whom eradication of H. pylori was successful were eligible for entry into a long-term study evaluating the stool antigen test. For 6 months, stool antigen tests were done monthly and a urea breath test was obtained every 3 months. Statistical Analysis Statistical analysis was performed by using StatView for Windows, version 5.01 (SAS Institute, Inc., Cary, North Carolina). Results are presented as the mean (SD). Sensitivity, specificity, probabilities, and predictive values are presented with 95% exact binomial CIs. Equivocal stool tests are considered by inclusion in the denominator of sensitivity and specificity. Stool antigen concentrations at individual time points were compared by using theMannWhitney test with downward adjustment of the P values for repeated observations (14). Role of the Funding Source The manufacturer (Meridian Diagnostics, Inc.) provided the stool kits. The study had no other funding source. Collection, analysis, and interpretation of the data, including the decision to publish, were solely the decision of the authors; the manufacturer of the test had no role in this process. Results The mean age of the 84 study patients was 52 years (range, 18 to 81 years). Fifty-three patients were women, and 31 were men. Endoscopic findings were as follows: normal (7 patients), esophagitis (2 patients), erythema in the antrum (45 patients), erosions in the antrum (11 patients), erosive duodenitis (9 patients), duodenal ulcers (8 patients), gastric ulcer (2 p

Prevalence of peptic ulcer in Helicobacter pylori positive blood donors.

This study aimed to determine the importance of raised antibodies to Helicobacter pylori in an asymptomatic population. A total of 128 asymptomatic blood donors who were seropositive for H pylori and consented to endoscopy were investigated. These subjects were from a population of 1010 blood donors screened for antibodies to H pylori. A questionnaire was completed to determine if any subjects had complained of symptoms, and they subsequently had endoscopy. Altogether 121 of 128 were positive for H pylori by histology and urease test and/or culture and all 121 had chronic active gastritis on histology. Twenty five of these subjects had peptic ulcer (20 duodenal, five gastric), a further 21 had erosive duodenitis, and two were found to have gastric cancer. H pylori associated peptic ulcer disease and duodenitis occur more frequently than previously recognised and this suggests that H pylori infection, even if asymptomatic, is of far greater clinical relevance than originally thought.

What is the role of Helicobacter pylori in complicated ulcer disease

The role of Helicobacter pylori in the pathogenesis of duodenal and gastric ulcer and ulcer recurrence is widely known. Bleeding, perforation, and obstruction represent the most serious, potentially life-threatening manifestations of ulcer disease (hemorrhage in 15%-20%). The lifetime prevalence of perforation and obstruction is much lower (approximately 5% and 2%, respectively). Despite improved diagnostic and therapeutic options, bleeding-related mortality rates remain at 6%-7% in the United States and between 4% and 14% in Europe. H. pylori and nonsteroidal anti-inflammatory drugs are now recognized as the two primary causes of ulcer disease. Eradication of H. pylori in patients with uncomplicated ulcers results in recurrence rates of < 10%, suggesting that eradication of H. pylori in patients with bleeding ulcers may virtually prevent recurrence of both the disease and its complications. Although the prevalence of H. pylori infection is almost 100% in duodenal and 80%-90% in gastric ulcer patients not using nonsteroidal anti-inflammatory drugs, the prevalence of the organism in bleeding duodenal and gastric ulcers does not reach 70% and 60%, respectively, which may be due to false-negative test results.

Invasive and non-invasive tests for Helicobacter pylori infection

There are two general ways in which a diagnosis of infection by Helicobacter pylori can be made: by using either an invasive or non‐invasive procedure. The invasive procedures involve an endoscopy and biopsy. A biopsy is essential because often the mucosa may appear macroscopically normal but nevertheless be inflamed. A biopsy is obtained by histological examination, culture, polymerase chain reaction or detection of the presence of urease activity in biopsy material.

Blood tests in the management of Helicobacter pylori infection. Italian Helicobacter pylori Study Group

There are three main types of blood test available for the management of Helicobacter pylori infection: those that detect an antibody response; tests of the pathophysiological state of the stomach; and those that indicate an active infection. Enzyme linked immunosorbent assay (ELISA) based kits are the most numerous of the commercially available tests. Originally the kits used crude antigen preparations but many of the newer kits use a more purified antigen preparation giving increased specificity but a lower sensitivity. The sensitivity, specificity, and predictive values of the tests can also be affected by the population under test and coexistent disease in the patients. Near patient test kits are based on either latex agglutination or immunochromatography. Generally, they have low sensitivities compared with laboratory tests. Commercial western blotting kits have also been developed and are used to detect the presence of specific virulence markers. The exact role of serology in the management of Helicobacter infection has still to be defined, although there is evidence that, used as a screening procedure, it can reduce endoscopy cost and workload. Gastrin and pepsinogen blood concentrations may provide valuable information on the pathophysiological state of the stomach--for example, the presence of inflammation or gastric atrophy. A combination of serology and serum concentrations of gastrin and pepsinogen may be used effectively to detect serious gastroduodenal disease in patients.

New immunological assays for the diagnosis of Helicobacter pylori infection

There are several types of immunological tests available for the diagnosis and management of Helicobacter pylori infection. Most commercially available serological kits use the enzyme linked immunosorbent assay (ELISA) test format. Originally the kits used crude antigen preparations although many of the newer kits use a more purified antigen preparation, with often increased specificity but lower sensitivity. Near patient test kits are based either on latex agglutination or immunochromatography. Generally they have low sensitivities compared with laboratory tests. Western blotting, ELISA, and recombinant immunoblot assays (RIBA) have also been developed into commercially available kits and can be used to indicate the presence of specific virulence markers. An antigen detection kit has been developed for the detection of Helicobacter pylori in faeces. Immunological reagents have also been combined with other diagnostic modalities to develop immunohistochemical stains and DNA immunoassays. Helicobacter pylori is now recognised as the cause of gastritis and most cases of peptic ulcer disease (PUD); its long term carriage increases the risk of gastric adenocarcinoma sixfold and it is designated as a class I carcinogen. H pylori has also been implicated as a cause of gastric mucosa associated lymphoid tissue lymphomas. Its relation to non-ulcer dyspepsia remains controversial. Additionally, long term carriage of the organism may be associated with short stature in young girls and, in the general population, as a possible risk factor for the development of vasospastic disorders and possibly skin immunopathology such as urticaria. With the recognition of H pylori as an important human pathogen, it has become one of the growing number of organisms to have its complete genome sequence mapped. Serology is an important method of determining colonisation status and can be used for diagnosis, as a screening procedure, or to follow the efficacy of eradication regimens. Most serological assays are in the ELISA format although some are based on the latex agglutination reaction. These latter are used principally as near patient assays. Most assays detect IgG in serum although some detect serum IgA. More recently developed assays detect IgA in saliva and the production of affinity purified antibodies has led to the development of an antigen detection assay for faecal specimens. Serological reagents have also been used in immunocytochemistry and to speed up the detection of amplified products of the polymerase chain reaction (PCR)-DNA immunoassays.

Effect of Helicobacter pylori eradication on development of dyspeptic and reflux disease in healthy asymptomatic subjects

Background and aim: There are few data on the course of Helicobacter pylori infection in asymptomatic subjects. The aim of this study was to assess the effect of eradication therapy on the development of dyspeptic and gastro-oesophageal reflux disease in a cohort of asymptomatic individuals observed over a prolonged period. Methods: A total of 169 blood donors infected with H pylori who had volunteered for studies on eradication in 1990 formed the cohort. To be included in this cohort subjects had to have no symptoms, as determined by a validated symptom questionnaire at the baseline visit. Eighty eight subjects were infected with H pylori while 81 had successfully undergone eradication therapy. Subjects were followed up (annually) using the same symptom questionnaire and in 2000 they underwent repeat endoscopy. Results: Thirteen subjects developed symptoms during follow up. The incidence of symptoms in H pylori positive subjects was 1.893/100 person-years of follow up and in H pylori negative individuals 0.163/100 person-years of follow up. H pylori infected subjects were significantly more likely to develop symptoms (log rank test, p=0.003) as well as those infected with CagA positive strains (log rank test, p=0.017). The development of symptomatic gastro-oesophageal reflux disease was no different in individuals with and without eradication (odds ratio 0.57 (95% confidence interval 0.26–1.24); p=0.163). Conclusions:H pylori eradication prevents the development of dyspeptic symptoms and peptic ulcer disease in healthy asymptomatic blood donors and is not associated with an increase in the incidence of symptomatic gastro-oesophageal reflux disease.

Helicobacter pylori and type 1 diabetes mellitus in children.

BACKGROUND Helicobacter pylori is a recognized gastroduodenal pathogen and H. pylori infection is one of the most common bacterial infections, usually acquired during childhood. However, diabetes mellitus is characterized by an increased susceptibility to infections. METHODS We compared the prevalence of H. pylori infection as well as cytotoxin-associated gene A-CagA-and vacuolating cytotoxin gene A-VacA-positivity in 103 children and adolescents with type 1 diabetes mellitus and in 236 nondiabetic children. We used a novel Recombinant ImmunoBlot Assay-Strip (RIBA SIA) with individual band for whole H. pylori lysate and recombinant CagA and VacA. RESULTS H. pylori-positive subjects, both diabetics and controls, were significantly older than negative subjects. In the whole group of diabetic patients the prevalence of each of the three reactivities was higher than in control subjects, reaching significance only for lysate. Only diabetic patients over 12 years of age, with a longer disease duration, had a higher prevalence of positive cases, although not significantly so. CONCLUSIONS In the first few years of disease, diabetic children do not differ from the nondiabetic population. Subsequently they show an H. pylori seroprevalence tendentially higher than that of controls of the same age. Therefore, H. pylori infection acquired in childhood and lasting several years, could be one of the causes of chronic atrophic gastritis, which is more frequent in longstanding diabetes mellitus.

Anti-CagA Reactivity in Helicobacter pylori-Negative Subjects (A Comparison of Three Different Methods)

Emerging evidence suggests that infection byCagA-positive Helicobacter pylori strains is related tothe development of more serious gastroduodenal diseases,thus conferring to the determination of anti-CagA antibodies a relevant clinical significance inserological screenings. The detection of anti-CagApositivity in sera negative for anti-H. pyloriantibodies raises the question of whether thisapparently nonsense result is merely due to a falsepositive reaction. To address this issue, we comparedthree different methods for the detection of anti-CagAantibodies. In all, 272 selected sera from patients with precisely defined H. pylori status(positive or negative concordance of five tests, ie,histology by Giemsa in both antrum and corpus, rapidurease test, culture, [13C]urea breath test,IgG ELISA) were tested for anti-CagA reactivity by threedifferent techniques (western immunoblotting, ELISA, andrecombinant immunoblotting assay). In order to assessthe sensibility and specificity of each tests, we considered as “true” anti-CagApositive sera those with two out of three positiveresults. Sera from 70% of H. pylori-positive patientsand 10% from H. pylori-negative patients turned out to be “true” positives foranti-CagA antibodies. The three methods showed similarexcellent results, in terms of both sensitivity andspecificity, always over 93%. It is confirmed that aproportion of patients with a negative conventionalserology against H. pylori possess anti-CagA antibodiesin their sera. In this paper we demonstrate that it canhappen even in patients without any biological signs of actual H. pylori infection. The possibilitythat this can be due to a false positive laboratoryresult is very likely ruled out by the accuracy of thethree methods used. The clinical management of these patients needs further study on largerseries.

Peptic ulcer disease and Helicobacter pylori infection

The most significant epidemiologic findings over this review period have been the isolation of Helicobacter pylori from feces and the results of the Eurogast Study firmly linking colonization by H. pylori to the development of gastric carcinoma. With respect to the mechanisms of pathogenesis, the purification of the cytotoxin and an investigation of its mode of action are also important. Similarly, the demonstration of the effect of H. pylori on somatostatin and gastrin-releasing peptide levels open further avenues of research. Numerous serologic tests are currently available, and an increasing use in screening procedures to reduce endoscopy workloads can be anticipated. The current regimen giving the highest eradication rate is bismuth, metronidazole, and tetracycline for 2 weeks, although the use of omeprazole with antibiotics is becoming more prevalent. Finally, the development of a vaccine is likely to be a major research area for the future.