New Rapid Helicobacter Pylori Blood Test Based on Dual Detection of FliD and CagA Antibodies for On-Site Testing

Abstract

Helicobacterpylori is the most prevalent bacterial infection, affecting half of the world’s population, with a high morbidity and mortality rate.1Hooi J.K.Y. et al.Gastroenterology. 2017; 153: 420-429Abstract Full Text Full Text PDF PubMed Scopus (1614) Google Scholar,2Malfertheiner P. et al.Gut. 2017; 66: 6-30Crossref PubMed Scopus (1987) Google Scholar Several invasive and noninvasive testing procedures are available, and their selective use serves the specific needs of diverse clinical scenarios. For gastric cancer prevention, mass screening is necessary and requires a noninvasive, rapid, accurate and cost-effective test. For this purpose H pylori serology currently seems to be the preferred noninvasive diagnostic method. Population-based testing and treatment for H pylori is cost effective in high-risk countries, but less effective in low- and medium-risk countries.3Liou J.M. et al.Gut. 2020; 69: 2093-2112Crossref PubMed Scopus (177) Google Scholar,4Lansdorp-Vogelaar I. et al.Best Pract Res Clin Gastroenterol. 2013; 27: 933-947Crossref PubMed Scopus (61) Google Scholar Many serologic tests are available on the market, with inconsistent performance often being observed. Therefore, international guidelines recommend considering only serologic tests with high accuracy that have been validated in the respective local populations. To date, no rapid point-of-care test (POCT) has reached a sufficient degree of accuracy. We evaluated the diagnostic performance of a new H pylori POCT for whole blood, serum or plasma, based on the detection of 2 highly specific and sensitive antibodies (Figure 1). The test uses the flagellar filament capping protein (FliD), which is an essential element of H pylori functional flagellar assembly and exists in every H pylori strain,5Khalifeh G.M. et al.Int J Med Microbiol. 2013; 303: 618-623Crossref PubMed Scopus (37) Google Scholar and the cytotoxin-associated gene A (CagA).6Huang J.Q. et al.Gastroenterology. 2003; 125: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar The test development is described in the Supplementary Methods. Materials from 2 different prospective trials were used: ERA-Net PathoGenoMics – trial (Number 80/11) and ERANET-Bavarian study (Number 19-277) (Table 1). A total of 111 patients (72% female) underwent esophagogastroduodenoscopy for different clinical reasons. Patients were prospectively enrolled from July 2011 to December 2012 (ERA-Net PathoGenoMics) and from December 2020 to June 2021 (ERANET-Bavarian study), respectively. The following exclusion criteria were defined: cancer, stomach surgery, upper abdominal irradiation, immunosuppressive therapy, oral anticoagulation, and antibiotic therapy in the 2 weeks before study entry. Ongoing or past proton pump inhibitor therapy was not defined as an exclusion criteria, because most patients suffering from upper gastrointestinal complaints had already been treated with proton pump inhibitors.Table 1Demographic Characterization of the Studied CohortGender, n (%)Age, y (range)Culture available, n (%)Histopathologic staining available, n (%)Female, 80 (72.1)52.1 (19–86)80 (100)80 (100)Male, 31 (27.9)47 (20–69)30 (97)31 (100) Open table in a new tab Serum and whole blood samples were used for the analyses. The optimal readout time was determined to be between 20 and 25 minutes. The results of the lateral flow tests were rated in a blinded manner by an expert panel of 5 health care professionals. H pylori detection in histology and culture was used as the reference method. The new rapid FliD/CagA POCT demonstrated a sensitivity of 100% and a specificity of 87.9%. The accuracy of the test was 93.7%. In 53 H pylori–infected patients, the dual antigen test positivity rate for the FliD or CagA combination was 100%. Of the 58 H pylori–negative patients, the dual antigen test detected 12.1% as positive for either FliD or CagA antibodies. In 87.9% of the patients, the dual antigen test confirmed the absence of H pylori infection. Comparing the results of the 5 medical professionals, the accuracy of the test ranged from 91.5% to 95.1%, the sensitivity from 98.1% to 100%, and the specificity from 89.1% to 94.3% (Table 2).Table 2Results of Serologic FliD and CagA Analysis in Helicobacter Pylori–Positive and -Negative SubjectsGender, nCulture, n (%)Histopathologic staining, n (%)FLiD+, n (%)CagA+, n (%)FLiD+ and CagA+, n (%)FLiD+ or CagA+, n (%)Female (n = 80)41 positive (51.3)41 positive (51.3)40 (98)24 (58.6)23 (56.1)41 (100)Male (n = 31)11 positive (35.5)12 positive (38.7)12 (100)8 (66.7)8 (66.7)12 (100)Female (n = 80)39 negative (48.7)39 negative (48.7)4 (10.3)0 (0)0 (0)4 (10.3)Male (n = 31)19 negative (61.3)19 negative (61.3)3 (15.8)0 (0)0 (0)3 (15.8)CagA, cytotoxin-associated gene A; FLiD, flagellar filament capping protein. Open table in a new tab CagA, cytotoxin-associated gene A; FLiD, flagellar filament capping protein. The use of serology as a noninvasive test for the detection of H pylori infection in clinical settings is restricted because of its lower accuracy compared with standard noninvasive tests, such as urease breath test or fecal antigen test. Furthermore, it has the limitation of not discriminating between current and past infections.2Malfertheiner P. et al.Gut. 2017; 66: 6-30Crossref PubMed Scopus (1987) Google Scholar To date, POCTs have failed to reach the level of accuracy required for responsible and safe use in the clinical management of patients with dyspepsia. The new rapid FliD/CagA POCT presented here shows unprecedented performance with an accuracy of 93.7% as compared with culture and histology reference methods. Data regarding the half-life of FliD antibodies are lacking, so further studies are needed that include patients after successful eradication in long-term serologic monitoring. Such studies could clarify whether the test is also suitable for discriminating between current and past H pylori infections based on the time lag between eradication therapy and the disappearance of FliD antibodies. The long-term persistence of CagA antibodies has been best documented in subjects with gastric cancer, where antibodies are still detectable even after gastrectomy.6Huang J.Q. et al.Gastroenterology. 2003; 125: 1636-1644Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar The reading of the test results is laboratory-free, rapid, and consistent, based on a colorimetric detection of the antibody response against the 2 selected H pylori antigens. The POCT offers a new opportunity in the management of patients presenting with dyspeptic symptoms in a nonspecialized outpatient setting. The high sensitivity limits the chances of an unrecognized H pylori infection and identifies patients with a positive test who require a further confirmatory test before initiating eradication therapy. Upper gastrointestinal endoscopy is recommended for adults older than 50 years of age and for subjects experiencing alarm symptoms.7Brenner H. et al.Am J Epidemiol. 2004; 159: 10Crossref Scopus (175) Google Scholar In the event of a negative H pylori test, “symptomatic” treatment may be offered as a first-line treatment option.8Chey W.D. et al.Aliment Pharmacol Ther. 2004; 19: 1-8Crossref PubMed Google Scholar, 9Palsson O.S. et al.Rome IV diagnostic questionnaires and tables for investigators and clinicians..Gastroenterology. 2016; (S0016-5085(16)00180-3. https://doi.org/10.1053/j.gastro.2016.02.014)Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 10Moayyedi P. et al.Lancet. 2000; 355: 1665-1669Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar In addition, serologic H pylori testing is the method of choice for proton pump inhibitor patients who cannot interrupt their therapy. In our exploratory study, detection of FliD antibodies alone was possible in all but 1 patient with H pylori infection based on the reference requirement for 2 positive (invasive) H pylori tests. In clinical management, patients who test positive for both antigens on site in general practice require a confirmatory positive test before commencing H pylori eradication therapy. Patients younger than 50 years of age having no alarm symptoms and a negative family history of gastrointestinal cancer with a negative POCT may be assigned to empirical symptom-related therapies. At present, the test is a novel first-line diagnostic tool for H pylori infection. Its use as a POCT can be applied in different clinical settings. Until more data become available, patients with a positive result require a further confirmatory test for current infection. It must be emphasized that a negative POCT can reliably exclude H pylori infection. The authors thank Volker Wedershoven and Michaela Buhler, both from ImevaX GmbH, for their contribution and input to the test development. They also thank Dr. Frank Gessler and Dr. Sibylle Pagel- Wieder for their support and contribution to the laboratory work during the development of the point-of-care test. The authors thank Dr. Lukas Macke and Dr. Riccardo Vasapolli for their contributions as read-out board members. The rapid test is based on an immunochromatographic lateral flow assay and contains cellulose nanobeads (Asahi Kasei, Tokyo, Japan) available in 2 different colors, each conjugated to a specific Helicobacter pylori antigen, FliD or CagA, respectively. For the preparation of the nitrocellulose membrane, recombinant H pylori FliD and CagA antigens and an anti-CagA-specific monoclonal antibody were used as test capture lines or the control, respectively. Recombinant CagA protein was purchased from Mikrogen (Neuried, Germany). The monoclonal antibody (anti-CagA) for the control was obtained from HyTest (Turku, Finland). Although the recombinant FliD used for the development of the laboratory-scale prototype was an in-house product as previously described (5), a similar antigen was later purchased from Mikrogen for use in the final assay layout. A comprehensive exploratory and elaborate development was carried out at Miprolab (Göttingen, Germany), which was followed by a preliminary characterization of the test performance. Briefly, the capture reagents for the test lines and the control line were dispensed separately on a nitrocellulose membrane using a dispensing module. Following application of the test lines and the control line, the membrane was dried under defined conditions in a climate chamber, which allowed the reagents to be immobilized onto the membrane. Cellulose nanobead conjugates were prepared according to the manufacturer’s instructions. The conjugate pad was saturated with cellulose nanobead conjugates and then dried under defined conditions in a climatic chamber. The lateral flow test materials were assembled as shown in Figure 1. The test strip placed in the test cassette consists of a backing card (adhesive backing) on which the nitrocellulose membrane, the conjugate pad, the sample pad, and the absorbent pad were placed. The sample pad was attached to the bottom of the test strip, overlapping the conjugate pad by 2 mm. The conjugate pad was fixed to the bottom of the backing card, overlapping the membrane by 2 mm. The absorbent pad was fixed to the other side of the membrane in a similar manner as the conjugate and sample pads. After assembling all materials onto the backing card, the test strips were mounted into plastic housings and sealed in aluminum foil pouches with desiccant until use.

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