Detection of Helicobacter pylori in Stool Specimens by PCR and Antigen Enzyme Immunoassay

A highly sensitive seminested PCR assay to detect Helicobacter pylori DNA in feces was developed. PCR with stool specimens and a novel antigen enzyme immunoassay (EIA) for H. pylori detection in feces were evaluated as diagnostic tools and in follow-up with samples from 63 infected and 37 noninfected persons. Infected individuals received eradication therapy followed by endoscopic follow-up 35 days after the start of treatment. At that time, a second stool specimen was obtained from 55 of these patients. Before eradication, the sensitivity of PCR was 93.7% and that of EIA 88.9%. Specificities were 100 and 94.6%, respectively. Of the 55 follow-up specimens, 41 originated from patients from whom H. pylori had been eradicated. Of these, 21 were still positive by PCR and 13 were positive by EIA, indicating that 1 month may be too short a period for follow-up evaluation of stool specimens by these tests.     

Evaluation of a Helicobacter pylori Stool Antigen Test for the Diagnosis and Follow-Up of Infections in Children

 The aim of this study was to evaluate the performance of a newly developed enzyme immunoassay kit (HpSA) for detecting Helicobacter pylori antigens in the stool of children. This study was comprised of 58 children referred to various endoscopy units for evaluation of gastrointestinal symptoms and upper gastroduodenal endoscopy and 11 children for post-therapy follow-up. In the first group, 23 children were diagnosed as positive for Helicobacter pylori using bacteriological and/or histological methods. Stool antigens were detected in 20 of these positive patients, for a sensitivity of 86.9% and a negative predictive value of 91.9%. Since only one false-positive reaction was observed with the HpSA kit, the specificity was 97.1% and the positive predictive value 95.2%. Results obtained for post-therapy follow-up were also promising. The HpSA assays were negative for the eight children whose infections were eradicated after therapy, and a positive result was obtained for two of three patients who had a persistent infection.     

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection

Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a “gold standard” for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The test's positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection.     

Application of a Stool Antigen Test To Evaluate the Incidence of Helicobacter pylori Infection in Children and Adolescents from Tehran, Iran

Helicobacter pylori infection is acquired mainly in childhood, especially in developing countries, where a low-cost, rapid diagnostic technique which is reliable for all age groups may be useful for the management of H. pylori infection. For this purpose, we used an HpSA test (Equipar) to detect H. pylori infection in children and adolescents from Tehran, Iran. Thirty-five children who were positive or negative for H. pylori infection by endoscopy-based tests were used as positive and negative controls for the HpSA test. Stools were collected from 430 randomly selected children and adolescents (4 to 18 years old) from southwest, near the center, and northwest of Tehran. A questionnaire that included presence of recurrent abdominal pain (RAP), family history of infection and/or peptic ulcer disease (PUD), and income of parents was completed. A good agreement was found between the results of endoscopy-based tests and those of the HpSA test; the sensitivity and specificity of the Equipar-HpSA test were 100% and 83.4%, respectively. Among 430 children and adolescents, 47% were positive by the HpSA test, of whom 82% had RAP. No difference in incidence was observed between the two sexes; the various categories of age showed an increasing incidence, ranging from 24% (ages 4 to 6) to 58% (ages 16 to 18). The rate of infection in children and adolescents from the southwest was significantly higher (70%) than the rate in those from the northwest (32%), and a family history of H. pylori infection or PUD was observed in 59% of the HpSA positive subjects. The HpSA test is a useful test to detect H. pylori infection in children and adolescents from developing countries.     

Short-Term Follow-Up by Serology of Patients Given Antibiotic Treatment for Helicobacter pylori Infection

Helicobacter pylori serology and in particular enzyme-linked immunosorbent assays for the measurement of immunoglobulin G (IgG) antibody titers form an accurate means of diagnosing H. pylori infection in patients before treatment. H. pylori serology is of limited value in monitoring treatment because of the slow decline in antibody titers. In the present study we aimed to measure the most suitable moment after antibiotic treatment at which serology should be used to monitor treatment. Sixty-four patients who had nonulcer dyspepsia and H. pylori infection and who underwent upper gastrointestinal endoscopy because of persistent dyspeptic symptoms were included in the study. H. pylori cure was confirmed by histology and culture 5 weeks after the completion of the antibiotic treatment. Serological examination was performed before therapy and at 5 weeks, 10 weeks, and 1 year after the completion of antibiotic treatment. Diagnostic performance was assessed by receiver-operating characteristic analysis. The areas under the receiver-operating characteristic curves of the H. pylori antibody titers at 5 weeks, 10 weeks, and 1 year after the completion of treatment were 0.53 (95% confidence interval [CI], 0.36 to 0.69), 0.60 (95% CI, 0.43 to 0.76), and 0.78 (95% CI, 0.63 to 0.93), respectively. The areas under the receiver-operating characteristic curves of the changes in H. pylori IgG antibody titers at 5 weeks, 10 weeks, and 1 year after the completion of treatment in comparison with the pretreatment titers were 0.85 (95% CI, 0.72 to 0.97), 0.96 (95% CI, 0.89 to 1.0), and 1.0 (95% CI, not estimable), respectively. We conclude that serology forms a useful means of monitoring treatment in patients with nonulcer dyspepsia and H. pylori infection as early as 10 weeks and maybe even sooner after the completion of treatment for the infection.     

Posttreatment follow-up of Helicobacter pylori infection using a stool antigen immunoassay.

The Helicobacter pylori stool antigen enzyme immunoassay (HpSA) was evaluated during posttreatment follow-up of patients in a country with a very high prevalence of H. pylori infection. From among 273 dyspeptic individuals (18 to 55 years) initially recruited from a shantytown in Lima, Peru, 238 participants who met the inclusion criteria and were suspected to be H. pylori positive based on (14)C urea breath test (UBT) results underwent endoscopy. Participants with endoscopy-proven infections received standard eradication therapy and were monitored by UBT and HpSA at 1 month following treatment and at 3-month intervals for 9 months posttreatment. A second endoscopy was performed if UBT results showed evidence of treatment failure or H. pylori recurrence. Biopsy results were considered the "gold standard" in all analyses. Among patients who underwent endoscopy, HpSA had a pretreatment sensitivity of 93%. Two-hundred thirty patients completed the treatment regimen, of whom 201 (93%) were considered to have had successful treatment outcomes based on a negative follow-up UBT. Thirty-two patients with UBT-defined treatment failures or H. pylori recurrences at any point during the 9-month follow-up underwent a second endoscopy. In the posttreatment setting, HpSA had an overall sensitivity of 73% and a specificity of 67%. Agreement between UBT and HpSA diminished throughout the follow-up. Among 14 participants in whom HpSA remained positive at 1 month following treatment despite UBT evidence of treatment success, 12 (86%) became HpSA negative within 3 months posttreatment. Although this study confirmed the validity of the HpSA in the initial assessment of dyspeptic patients, the test demonstrated a reduced overall accuracy in the detection of treatment failures and H. pylori recurrences during 9 months of posttreatment follow-up. Furthermore, in some patients it may take up to 3 months after successful eradication for antigen shedding to diminish to levels within the negative HpSA range.     

Helicobacter pylori Infection in Infants and Toddlers in South America: Concordance between [13C]Urea Breath Test and Monoclonal H. pylori Stool Antigen Test

Accurate noninvasive tests for diagnosing Helicobacter pylori infection in very young children are strongly required. We investigated the agreement between the [¹³C]urea breath test ([¹³C]UBT) and a monoclonal ELISA (HpSA) for detection of H. pylori antigen in stool. From October 2007 to July 2011, we enrolled 414 infants (123 from Brazil and 291 from Peru) of ages 6 to 30 months. Breath and stool samples were obtained at intervals of at least 3 months from Brazilian (n = 415) and Peruvian (n = 908) infants. [¹³C]UBT and stool test results concurred with each other in 1,255 (94.86%) cases (kappa coefficient = 0.90; 95% confidence interval [CI] = 0.87 to 0.92). In the H. pylori-positive group, delta-over-baseline (DOB) and optical density (OD) values were positively correlated (r = 0.62; P < 0.001). The positivity of the tests was higher (P < 0.001; odds ratio [OR] = 6.01; 95% CI = 4.50 to 8.04) in Peru (546/878; 62.2%) than in Brazil (81/377; 21.5%) and increased with increasing age in Brazil (P = 0.02), whereas in Peru it decreased with increasing age (P < 0.001). The disagreement between the test results was associated with birth in Brazil and female gender but not with age and diarrhea. Our results suggest that both [¹³C]UBT and the stool monoclonal test are reliable for diagnosing H. pylori infection in very young children, which will facilitate robust epidemiological studies in infants and toddlers.     

Diagnosis of Penicillium marneffei Infection by Quantitation of Urinary Antigen by Using an Enzyme Immunoassay

Penicillium marneffei is a major cause of opportunistic infection in patients with AIDS in north and northeastern Thailand. A method for the quantitation of P. marneffei antigen in urine was developed by using fluorescein isothiocyanate-labelled purified rabbit hyperimmune immunoglobulin G in an enzyme-linked immunosorbent assay. This method was evaluated with 33 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects, 248 hospitalized patients without penicilliosis) from the same area in which penicilliosis is endemic. Urinary antigen was found in all 33 (100%) patients with penicilliosis, with a median titer of 1:20,480. With undiluted samples, 67 (27%) of 248 hospital patients and 3 (6%) of 52 healthy controls were reactive. At a cutoff titer of 1:40, the urine antigen detection assay had a diagnostic sensitivity of 97% and specificity of 98% (positive predictive value, 84%; negative predictive value, 99.7%). This test offers a valuable and rapid method for the diagnosis of penicilliosis in patients with AIDS and could be a useful addition to conventional diagnostic methods in areas in which penicilliosis is endemic.     

Effect of Low-Dose Antigen Exposure on Development of Immunity to Helicobacter pylori Infection in Mice

The effect of low-dose antigen exposure on the development of immunity to Helicobacter pylori infection was studied in outbred mice. Animals that were primed with a subinfectious number of H. pylori bacteria exhibited significantly lower bacterial loads after challenge with an infectious dose of pathogen (versus controls, P < 0.05).     

Evaluation of Helicobacter pylori Immunoglobulin G (IgG), IgA,and IgM Serologic Testing Compared to Stool Antigen Testing

The utility of Helicobacter pylori serology was evaluated in 4,722 specimens and compared to stool antigen detection. Immunoglobulin M (IgM) sensitivity (6.8%) was unacceptably low. Key performance differences were observed in IgG specificity, IgA sensitivity, and specificity between adults and children that may warrant differentiating optimal serologic cutoff values by age.Helicobacter pylori causes gastrointestinal disease in both children and adults (10). Noninvasive diagnostic tests include the [¹³C]urea breath test, serology, and stool antigen testing (HpSA). Numerous studies have evaluated these diagnostic tests but have been limited by a small sample size or restriction to either children or adults (3-9, 11-17, 19, 20). The clinical utility of serologic testing in both children and adults has been debated; moreover, it has not been established whether positive cutoff levels need to be adjusted for age (4, 5, 8, 13, 19). Immunoglobulin A (IgA) and IgG serologic tests are possibly less reliable in children than adults, but this has not been definitively established (13). Some investigators have supported the use of IgM as an indicator of active disease (2), while others have found IgM to have little diagnostic utility (7, 18). Because of the conflicting data, we performed a large-scale study on H. pylori serology to analyze its utility and differences in performance in children and adults.Paired results of H. pylori serology (IgG, IgA, and/or IgM) and HpSA from October 1998 to January 2009 were analyzed in tests performed within 2 months of each other. HpSA was performed using the Premier Platinum HpSA Plus enzyme immunoassay according to the manufacturer''s instructions (Meridian Bioscience, Inc., Cincinnati, OH). The cutoff optical density at 450 nm was <0.100 for a negative result and ≥0.100 for a positive result.Serology has been performed with in-house enzyme-linked immunosorbent assay (ELISA) kits used since 1998. The IgG and IgA ELISAs were validated against the Enteric Products Inc. (Stony Brook, NY) ELISA. The IgM ELISA was validated against the MRL (now Focus Diagnostics, Cypress, CA) IgM ELISA. H. pylori antigens (CagA and VacA; Micro Detect, Inc., Tustin, CA) were used to coat microtiter plates at 1.0 μg/ml. Samples were diluted 1:101 for IgG and IgA and 1:51 for IgM and then reacted at room temperature for 30 min. After washing, diluted horseradish peroxidase-conjugated anti-human IgG, IgA, or IgM was reacted for 30 min at room temperature. After washing again, the wells were developed with tetramethylbenzidine for 30 min and the absorbance was measured at 450 nm. The cutoffs (in index values) for IgG and IgA were ≤1.7 for a negative result, 1.8 to 2.2 for an equivocal result, and ≥2.3 for a positive result. For IgM the cutoffs were ≤0.8 for a negative result, 0.9 to 1.1 for an equivocal result, and ≥1.2 for a positive result.Statistical analyses were performed using SAS software, version 9.1 (SAS Institute Inc., Cary, NC). The study was approved by the Institutional Review Board of the University of Utah (no. 7275).For all tests performed over the 11-year period, including nonpaired samples, the positivity rate of HpSA (12.1% [10,440/86,284]) was significantly lower (P < 0.001) than those for H. pylori IgG (35.6% [155,370/413,222]) and IgA (32.7% [60,091/166,997]), and IgM was significantly less often positive (4.3% [5,320/120,135]) than the other three tests (P < 0.001) based on the binomial test.There were 4,722 paired serology and HpSA results for 2,730 women (57.8%) and 1,992 men (42.2%). Eighty-eight percent of these tests were collected within 2 weeks of each other. Using HpSA as the gold standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated with 95% confidence intervals for IgG, IgA, and IgM and according to age group: children (≤17 years) and adults (≥18 years) (Table ​(Table1).1). IgG demonstrated the highest sensitivity (87.6%) and lowest specificity (61.0%) and was significantly more specific in children (82.6%) than adults (46.2%). In children, IgA was significantly more specific than adults (95.8% versus 48.8%) but also less sensitive (29.6% versus 73.8%). Overall, IgM demonstrated low sensitivity (6.8%) but high specificity (95.8%) with no statistical difference between children and adults.

TABLE 1.

H. pylori IgG, IgA, and IgM serology performance using H. pylori stool antigen as the gold standard^a

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.     

Objective Interpretation of the Rapid Urease Test for Helicobacter pylori Infection Using Colorimetry

BackgroundThe rapid urease test (RUT) is a major diagnostic tool for detecting Helicobacter pylori infection. This study aimed to establish an objective method for measuring the color changes in the RUT kit to improve the test’s diagnostic accuracy.MethodsA UV-visible spectrophotometer was selected as the colorimeter; experiments were conducted in three stages to objectively identify the color changes in the RUT kit.ResultsFirst, the urea broth solution showed an identifiable color change from yellow to red as the pH increased by 0.2. The largest transmittance difference detected using the UV-visible spectrophotometer was observed at a 590-nm wavelength. Second, the commercialized RUT kit also showed a gradual color change according to the pH change detected using the UV-visible spectrophotometer. Third, 13 cases of negative RUT results with a biopsy specimen and 16 of positive RUT results were collected. The transmittance detected using the UV-visible spectrophotometer showed a clear division between the positive and negative RUT groups; the largest difference was observed at a 559-nm wavelength. The lowest transmittance in the negative RUT group was 64, while the highest in the positive RUT group was 56, at the 559-nm wavelength. The UV-visible spectrophotometry reading showed a consistency of 92.7% compared with that of manual reading.ConclusionA transmittance of 60 at a 559-nm wavelength detected using UV-visible spectrophotometer can be used as a cutoff value for interpreting RUT results; this will help develop an automatic RUT kit reader with a high accuracy.     

Cytokine Expression in Pediatric Helicobacter pylori Infection

Helicobacter pylori infection is one of the most common gastrointestinal infections worldwide and almost invariably causes chronic gastritis in the infected host. A predominant Th1 profile has been demonstrated in H. pylori-infected mucosa from adults, but no previous study has evaluated in situ cytokine expression in children. We therefore examined expression of proinflammatory, anti-inflammatory, and regulatory cytokines by immunohistochemistry in cryopreserved antral biopsy specimens from 10 H. pylori-infected and 10 uninfected children and correlated expression of cytokines with histology scores. Concomitant expression of interleukin-8 (IL-8), gamma interferon (IFN-γ), IL-4, transforming growth factor β, and tumor necrosis factor alpha was seen in 8/10 H. pylori-infected cases and in 5/10 noninfected cases; all H. pylori-infected subjects showed staining for at least two of the cytokines. The proportion of epithelial cytokine-specific staining did not differ significantly between the groups, either in surface or glandular epithelium. Furthermore, no significant differences were noticed between intraepithelial or lamina propria lymphocyte staining in the groups. There was, however, a tendency of higher numbers of IFN-γ- and IL-8-positive cells in the H. pylori-infected group. IFN-γ and IL-8 lamina propria lymphocyte expression correlated significantly with antrum chronic inflammation, but there was no correlation between histology scores and epithelial cytokine expression. When the same techniques were used, the cytokine response appeared to be smaller in H. pylori-infected children than in adults, and there was no clear Th1 dominance. These results therefore suggest a different mucosal immunopathology in children. It remains to be determined whether the gastric immune response is downregulated in children with H. pylori infection and whether this is relevant to the outcome of infection.     

Natural Competence Promotes Helicobacter pylori Chronic Infection

Animal models are important tools for studies of human disease, but developing these models is a particular challenge with regard to organisms with restricted host ranges, such as the human stomach pathogen Helicobacter pylori. In most cases, H. pylori infects the stomach for many decades before symptoms appear, distinguishing it from many bacterial pathogens that cause acute infection. To model chronic infection in the mouse, a human clinical isolate was selected for its ability to survive for 2 months in the mouse stomach, and the resulting strain, MSD132, colonized the mouse stomach for at least 28 weeks. During selection, the cagY component of the Cag type IV secretion system was mutated, disrupting a key interaction with host cells. Increases in both bacterial persistence and bacterial burden occurred prior to this mutation, and a mixed population of cagY⁺ and cagY mutant cells was isolated from a single mouse, suggesting that mutations accumulate during selection and that factors in addition to the Cag apparatus are important for murine adaptation. Diversity in both alleles and genes is common in H. pylori strains, and natural competence mediates a high rate of interstrain genetic exchange. Mutations of the Com apparatus, a membrane DNA transporter, and DprA, a cytosolic competence factor, resulted in reduced persistence, although initial colonization was normal. Thus, exchange of DNA between genetically heterogeneous H. pylori strains may improve chronic colonization. The strains and methods described here will be important tools for defining both the spectrum of mutations that promote murine adaptation and the genetic program of chronic infection.     

Evaluation of a Monoclonal Antibody-Based Test for Detection of Helicobacter pylori-Specific Antigen in Stool Samples from Mice

A test using monoclonal antibodies for detection of antigen in stool samples was compared with culture and histology for noninfected (n = 25), Helicobacter pylori-infected (n = 25), and Helicobacter felis-infected (n = 6) mice. Sensitivity and specificity were 96%. The monoclonal antibody-based test is therefore a noninvasive technique that is able to diagnose H. pylori infection in mice.     

A Novel Line Immunoassay Based on Recombinant Virulence Factors Enables Highly Specific and Sensitive Serologic Diagnosis of Helicobacter pylori Infection

Helicobacter pylori colonizes half of the world''s population, and infection can lead to ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. Serology is the only test applicable for large-scale, population-based screening, but current tests are hampered by a lack of sensitivity and/or specificity. Also, no serologic test allows the differentiation of type I and type II strains, which is important for predicting the clinical outcome. H. pylori virulence factors have been associated with disease, but direct assessment of virulence factors requires invasive methods to obtain gastric biopsy specimens. Our work aimed at the development of a highly sensitive and specific, noninvasive serologic test to detect immune responses to important H. pylori virulence factors. This line immunoassay system (recomLine) is based on recombinant proteins. For this assay, six highly immunogenic virulence factors (CagA, VacA, GroEL, gGT, HcpC, and UreA) were expressed in Escherichia coli, purified, and immobilized to nitrocellulose membranes to detect serological immune responses in patient''s sera. For the validation of the line assay, a cohort of 500 patients was screened, of which 290 (58.0%) were H. pylori negative and 210 (42.0%) were positive by histology. The assay showed sensitivity and specificity of 97.6% and 96.2%, respectively, compared to histology. In direct comparison to lysate blotting and enzyme-linked immunosorbent assay (ELISA), the recomLine assay had increased discriminatory power. For the assessment of individual risk for gastrointestinal disease, the test must be validated in a larger and defined patient cohort. Taking the data together, the recomLine assay provides a valuable tool for the diagnosis of H. pylori infection.