JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1992, p. 176-180 0095-1137/92/010176-05$02.00/0 Copyright C) 1992, American Society for Microbiology
Vol. 30, No. 1
Evaluation of a Commercially Available Second-Generation Immunoglobulin G Enzyme Immunoassay for Detection of Helicobacter pylori Infection HERMAN GOOSSENS,l* YOURI GLUPCZYNSKI,2 ALAIN BURETTE,3 CHANTAL VAN DEN BORRE,l AND JEAN-PAUL BUTZLER' World Health Organization Collaborating Centre for Enteric Campylobacter, St-Pieters University Hospital, Hoogstraat 322, B-1000 Brussels,' Department of Microbiology, Brugmann University Hospital, B-1090 Brussels,2 and Gastroenterology Unit, Nouvelle Clinique de la Basilique, B-1080 Brussels,3 Belgium Received 15 July 1991/Accepted 23 October 1991 We evaluated a commercially available second-generation anti-H. pyloni immunoglobulin G enzyme immunoassay (EIA) (Cobas Core Anti-Helicobacter pyloni EIA; Roche S.A., Basel, Switzerland) for serodiagnosis of H. pylori infection. The results of the assay were assessed in relation to the results of bacterial culture, urease testing, and histological Giemsa stain of gastric biopsy specimens from 1,134 patients with a variety of symptoms relating to the upper gastrointestinal tract. H. pyloni was detected in biopsy specimens from 660 (58.2%) patients: 6 had a normal mucosa, 123 had chronic gastritis only, and 531 were found to have chronic active gastritis by histology; endoscopy showed duodenal and gastric ulcers in 137 and 64 patients of the last two groups, respectively. The test was evaluated with different age and ethnic groups. The prevalence, sensitivity, specificity, and positive and negative predictive values were, respectively, (i) for Belgian patients between 18 and 40 years old, 34, 93, 95, 91, and 96%; (ii) for Belgian patients more than 40 years old, 53, 96, 91, 93, and 95%; and (iii) for Mediterranean patients more than 17 years old, 87, 94, 70, 95, and 64%. All sera showing discordant immunoassay results compared with the results of histology and culture of biopsy specimens, as well as those with borderline immunoassay results, were tested further by immunoblotting. Among the EIA results considered false negative, we demonstrated an absence of seroconversion in 14 of 19 patients tested by immunoblotting. Among the EIA results considered false positive, immunoblotting showed the presence of specific antibodies in 28 of 37 patients tested. Among the borderline results obtained in the first assay with 22 patients' sera, a second assay showed positive results in 10 patients (8 were positive by immunoblotting) and negative reactions in 10 patients (9 were negative by immunoblotting), whereas 2 remained borderline. These data indicate that sera showing borderline immunoassay results must be tested again. In conclusion, this commercially available second-generation EIA, which is easy and quick to perform, was found highly reliable for the serodiagnosis of H. pylori infection.
patients from developing countries who frequently suffer from campylobacter infection. It has been generally agreed that there is a need for more highly purified and specific antigens in order to decrease backgrounds and increase specificity (10). We evaluated a commercially available second-generation immunoglobulin G (IgG) EIA (Cobas Core Anti-H. pylori EIA, article no. 07 34977; Roche S.A., Basel, Switzerland) to detect H. pylori infection. This second-generation assay uses purified antigens, unlike the first-generation test that used supernatant from a whole-cell sonicate. We observed false-positive reactions with the sonicated bacterial cells present in the first-generation EIA (results not published), which is one of the reasons that the manufacturers decided to change the nature of the antigenic material. The test was evaluated in clinically well-defined groups of dyspeptic patients, and the assay results were compared with the results of bacterial culture, urease testing, and Giemsa stain of gastric biopsy specimens.
Patients infected by Helicobacter pylori in their gastric mucosa elicit a specific systemic antibody response to the
bacterium (13). Early investigations included complement fixation and agglutination techniques (5, 6). More recent studies have shown that the enzyme-linked immunosorbent assay may be the technique of choice for detection of H. pylori-specific antibodies, because of its speed, low cost, simplicity, and reproducibility (1, 2, 7, 11, 13). Numerous enzyme immunoassays (EIAs) to detect H. pylori infection have been developed, including acid-glycine extracts, whole bacterial cells, formalin-treated cells, or sonicated bacterial cells. Some of these tests, such as the Pyloriset (Orion Diagnostica, Espoo, Finland), Pylori Stat (Whittaker Bioproducts, Walkersville, Md.), Malakit (Biolab, Limal, Belgium), and Helico-G (Porton-Cambridge, Maidenhead, United Kingdom), have been made commercially available. The flagellum proteins of H. pylori are well known to cross-react with antibodies produced against Campylobacter jejuni (9). The presence of these cross-reacting antigens could give false-positive reactions for H. pylori infection in those patients with recent campylobacter enteritis or in *
MATERIALS AND METHODS
Subjects. A series of 1,134 consecutive patients with a clinical indication for upper gastrointestinal endoscopy, originating from outpatient and inpatient referrals, was studied.
Corresponding author. 176
VOL. 30, 1992
These patients included 544 men and 590 women with an age range of 13 to 94 years (mean, 52.6 years). Patients studied included 855 Belgians, 257 Mediterraneans (i.e., 99 Moroccans, 66 Italians, 33 Greeks, 31 Spaniards, 19 Turks, 8 Yugoslavians, and 1 Portugese), and 22 patients of other nationalities. The patients had a variety of symptoms relating to the upper gastrointestinal tract, most commonly epigastric pain or dyspepsia or both. Patients taking antimicrobial agents and/or bismuth salts within the last month before endoscopy were excluded from the study. Patients receiving H2-receptor antagonists were included in the study, since these agents are not active against H. pylori (4). Endoscopy and sampling procedures. A blood sample was taken from each patient for serological tests immediately before the endoscopy procedure, and sera were stored at -20°C until assayed. All endoscopic examinations were carried out by the same endoscopist (A.B.). The presence of hyperemia, erosions, ulcerations, atrophy, or hypertrophy of the mucosa was recorded. Four gastric biopsy specimens were randomly taken from both the antrum and the corpus. From each area, two biopsy samples were immediately placed in 10% buffered formalin for histological examination, and those for microbiological examination were dipped with a sterile cotton swab in a semisolid agar transport medium (Portagerm; bioMerieux, Marcy l'Etoile, France). One antral specimen was placed into a 2% urea-buffered broth for the rapid detection of H. pylori urease activity. Different sterile biopsy forceps with a cup size of 5 by 5 mm were used to collect antral and body biopsy samples to exclude the possibility of sample contamination. Between patients the flexible endoscopes (including all of the channels) and biopsy forceps were carefully cleaned and disinfected by immersion in a 2% glutaraldehyde (Cidex; Surgikos Ltd, Livingstone, Scotland) solution for at least 15 min, rinsed in water, and dried. Histology. Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin and eosin to grade the severity of gastritis and with Giemsa stain to detect H. pylori. The classification of gastritis was made in accordance with the Sydney system for the classification of gastritis (12). Both the mononuclear and the polymorphonuclear infiltrates were graded separately-on the basis of the extent of inflammatory cell infiltration-as absent, mild, moderate, or severe. Four different scores (from 0 to 3) were attributed separately to each type of inflammatory cell infiltrate. Other types of mucosal changes, e.g., gland atrophy, intestinal metaplasia, and degenerative and regenerative changes of the epithelium, were rated separately but were not used as evidence of inflammation. The histological preparations were always examined by the same pathologist, who was unaware of the patients' clinical status and of the results of the other tests. Microbiology. Gastric mucosal biopsy specimens were inoculated directly onto two culture plates: (i) a Columbia agar plate (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 10% horse blood and (ii) the same medium made selective by the addition of vancomycin (10 mg/liter), colistin (2,500 U/liter), amphotericin B (10 mg/ liter), and trimethoprim (5 mg/liter). Agar plates were incubated in a special incubator at 37°C under microaerobic atmosphere and were inspected after 3, 5, and 7 days. Bacterial colonies were identified as H. pylori on the basis of growth characteristics, colonial morphology, Gram stain examination, catalase and oxidase reactions, and rapid urease
production.
Identification of H. pylori-positive patients by invasive
EVALUATION OF A NEW EIA FOR H. PYLORI
177
procedures was based on culture and/or histology results; a positive urease biopsy test only was not considered sufficient for diagnosis. Serology. The anti-H. pylori IgG EIA (Cobas Core Anti-H. pylori EIA) is a second-generation two-step EIA for the detection of IgG antibodies to H. pylori in human serum, based on a set of fast protein liquid chromatography-purified cell surface antigens including the native urease enzyme (3). The test was performed according to the manufacturer's instructions. In the first step, the patients' samples and controls were diluted with the specimen dilution buffer and incubated with beads coated with purified H. pylori-specific antigens. Specific antibodies were bound to the beads. After removal of the unbound material by a washing step, the antigen-antibody complex on each bead was detected with peroxidase-conjugated goat anti-human IgG antibody. After the removal of unbound conjugate, the beads were incubated with a substrate solution containing tetramethylbenzidine and hydrogen peroxide. A blue color developed in proportion to the amount of H. pylori-specific IgG bound to the beads. The enzymatic reaction was stopped by the addition of acid, and the A450 values were determined. The cutoff value was determined by adding 0.07 to the mean value of the absorbance of the negative controls. Values within a zone of ±10% of the cutoff value were reported as borderline. Results below the 10% zone of the cutoff value were considered negative for anti-H. pylori antibodies, and specimens for which the absorbances were greater than the 10% zone of the cutoff value were considered reactive for anti-H. pylori. Intra- and interrun variations of the EIA were evaluated by repeated testing of (i) sera from 20 patients without H. pylori antibodies and whose gastric biopsy specimens were negative for H. pylori and (ii) sera from 30 patients with H. pylori antibodies and with the organisms present in their stomach. All sera showing (i) an absence of IgG antibodies to H. pylori but the presence of the bacterium in gastric biopsy specimens by Giemsa stain and/or culture, (ii) the presence of IgG antibodies but an absence of the bacterium in biopsy specimens, or (iii) borderline results were tested again. These three groups of sera were further analyzed for H. pylori IgG antibodies by immunoblotting. The antigen preparation was a whole-cell preparation of the type strain H. pylori CCUG 18818. The method of Towbin et al. (15) formed the basis of the Western blot (immunoblot) procedure. The nitrocellulose paper strips were incubated with a 1:25 dilution of the serum for 16 h at room temperature. Sera were diluted in 0.1% bovine serum albumin in buffered saline. Peroxidase-conjugated goat anti-human IgG (United States Biochemical Corp.) was used as the conjugate. Immunoblotting profiles were compared with profiles obtained with sera from 20 patients positive for H. pylori by IgG EIA as well as Giemsa stain and/or culture of biopsy specimens; immunoblotting profiles were also compared with profiles obtained with sera from 15 asymptomatic blood donors without H. pylori antibodies as shown by IgG EIA. The presence of more than four dense bands or a band at approximately 120 kDa was considered positive by immunoblotting. Bands at between 45,000 and 66,000 Da were regarded as nonspecific because they were usually present in the blood donors' sera. Statistical analysis. For statistical analysis we used the Epi-info version 5.01A (Centers for Disease Control and World Health Organization program). Comparison of mean values was performed by using the Kruskal-Wallis one-way
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GOOSSENS ET AL.
TABLE 1. Correlation between IgG Cobas Core Anti-H. pylori EIA results and detection of H. pylori organisms with Giemsa stain and culture of biopsy specimens from 1,134 symptomatic patients No. of patients
With EIA results':
H. pylori negative (n = 474)
-
+
B
(n = 660)
-
+
B
Histological Normal Chronic gastritis only Mild Moderate Severe Chronic active gastritis Mild Moderate Severe
300 130 121 8 1 44 30 11 3
283 108 103 5 0 38 26 9 3
13 20 16 3 1 6 4 2 0
4 2 2 0 0 0 0 0 0
6 123 57 60 6 531 244 222 65
0 8 4 4 0 20 10 7 3
6 114 52 56 6 496 227 206 63
0 1 1 0 0 15 7 8 0
Endoscopicb Normal Duodenal ulcer Gastric ulcer Gastric cancer
126 10 25 4
118 10 24 4
6 0 1 0
2 0 0 0
26 137 64 5
2 4 4 1
24 131 60 3
0 2 0 1
Diagnosis
H. pylori positive
With EIA results:
-, negative; +, positive; B, borderline. b Other patients were found to have various types of diffuse erythematous lesions of the stomach.
a
analysis variance or the Wilcoxon signed-rank test. Results were considered significantly different for P values of 17 yrs old
18-40 yrs old
yrs old
207 73
630 349
245 222
34 93 95 91 96
53 96 91 93 95
87 94 70 95 64
No. of patients Total With H. pylori IgG antibodies
Prevalence (%) Sensitivity Specificity (%) Positive predictive value (%) Negative predictive value (%)
>40
the mean optical densities of sera from patients with chronic active gastritis were statistically significantly higher than those from patients with chronic gastritis only (P = 0.009). Mean optical densities of sera from patients suffering from gastric ulcer were also statistically significantly higher than those from patients with duodenal ulcer (P = 0.03). However, no statistically significant differences between mean optical densities of sera from patients with duodenal and gastric ulcer in relation to the degree of histological lesions were found by multivariate analysis. This indicates that histological differences between both groups might be responsible for this statistically significant difference. Immunoblot. Immunoblots were performed on three types of serum. (i) One type included sera from 19 of the 28 patients with no H. pylori IgG antibodies in the Roche EIA but with H. pylori present in their gastric biopsy specimens by Giemsa stain and/or culture. Immunoblots confirmed the absence of IgG H. pylori antibodies in 14 of the 19 patients tested, whereas in 5 patients immunoblots were considered positive. Moreover, for three patients (two patients negative by Roche EIA and immunoblotting and one patient negative by Roche EIA and positive by immunoblotting), we obtained a second serum specimen. For two patients (both negative by Roche EIA and immunoblotting) we obtained five and six subsequent serum specimens. Endoscopy with histology, Giemsa stain, urease testing, and culture of biopsy specimens was carried out simultaneously. H. pylori was still present in the stomach, and serological results (Roche EIA and immunoblotting) showed identical results for all five
patients. (ii) A second type included those 22 serum specimens showing borderline results in the first assay. Table 2 reports the second IgG Cobas Core Anti-H. pylori ETA assay and in immunoblotting, in relation to the detection of H. pylori in the patients' biopsy specimens. Among the 10 serum specimens which were negative with the second EIA, the absence of IgG antibodies was confirmed by immunoblotting in nine patients. Among the 10 serum specimens which were positive with the second EIA, the presence of IgG antibodies was confirmed in eight patients. Two serum specimens remained borderline; one on the results with these sera in
was considered positive and the other was negative by immunoblotting. (iii) A third type of serum included sera from those patients showing a positive result with the Roche EIA but for whom H. pylori could not be detected in their biopsy
179
specimens by Giemsa stain, urease testing, and culture. Sera from 37 of 39 patients were available for immunoblotting, and the presence of specific H. pylori IgG antibodies was confirmed in 28 patients. A subsequent serum specimen was obtained for six patients (all positive by the Roche EIA in the first and second assays as well as by immunoblotting). The second serum specimen was again positive in the Roche EIA, and immunoblotting confirmed the presence of H. pylori-specific antibodies. Giemsa stain, urease testing, and culture of biopsy specimens were still negative. DISCUSSION The overall proportion (58%) of biopsy specimens positive for H. pylori determined by cultural and histological techniques was of the same order as in most other reports, in which it ranged from 42 to 66% (8, 16). In the present study, and in common with other workers (8, 16), we have found that there was a clear association between the presence of H. pylori and chronic active gastritis. The presence of H. pylori was also strongly correlated with a diagnosis of duodenal ulcer (93%) and somewhat less with gastric ulcer (72%). On the other hand, 98% of the patients with normal histology were not infected by H. pylori (Table 1). We found an excellent correlation between the presence of H. pylori and raised IgG antibody by using the IgG Cobas Core EIA (Table 1). The EIA was evaluated in different age and ethnic groups (Table 3). Although very few workers evaluated serological tests taking into consideration the variation of prevalence in different populations, we believe that this is relevant because particularly the negative predictive value will depend upon the prevalence of antibodies in the population studied. One major role for H. pylori serology might be screening younger dyspeptic patients to eliminate unnecessary endoscopies (14). This is another reason that we considered two age profiles in the Belgian patients when assessing the H. pylori EIA. The test performed excellently in both Belgian age groups, with sensitivities, specificities, and positive and negative predictive values of 90% or more (Table 3). However, in the Mediterranean population, the negative predictive value decreased to 64%, which might be related to the high prevalence (87%). It was interesting to note that the specificity of the assay is also lower in the Mediterranean group of patients. However, there is no evidence that the strains of H. pylori differ antigenically in this group. These data clearly indicate that it is difficult to compare the performance of this new EIA from Roche with that of other commercially available tests that have been reported in the literature. Moreover, the evaluation of such tests will also depend on the sensitivity of the culture and Giemsa stain for the detection of H. pylori from gastric biopsy specimens. The only way to assess correctly H. pylori serology is to evaluate it with the same serum specimens, from histologically and bacteriologically well-defined groups of patients. False-positive EIA results with the Roche test were obtained with 39 serum specimens, although immunoblotting confirmed the presence of IgG anti-H. pylori antibodies in 28 patients. This may be explained in several ways. It is possible that the active site of infection was missed because H. pylori has a patchy distribution. These patients may have had a previous infection with H. pylori, and their antibody levels still were raised; or these patients may still have taken antibiotics prior to endoscopy, because they ignored this, forgot to mention it during the interview, or had not remembered having taken antibiotics within 1 month prior to
180
GOOSSENS ET AL.
endoscopy. Cross-reaction could have occurred between H. pylori antigens and other antibodies in the patients' sera. Low specificity of serology may also be explicable by the insensitivity of microscopy and culture. Finally, failure to detect H. pylori in biopsy specimens, despite the presence of H. pylori IgG antibodies, could have been due to intestinal metaplasia and/or atrophy of the gastric mucosa of the stomach. This may in turn explain the lower prevalence of H. pylori in elderly patients. It is also conceivable that some specific antibody reactivity is lost on immunoblotting because of a loss of three-dimensional conformation during sodium dodecyl sulfate-polyacrylamide gel electrophoresis but is detectable by EIA. Of the 28 serum specimens showing false negative serological results, 19 were available for immunoblotting, and the absence of H. pylori antibodies was confirmed in 14 of these serum specimens. Among these 14 serum specimens, immunoblots were also carried out with the strains isolated from the patients' biopsy specimens in 13 patients (results not shown), which excludes false-negative immunoblots because of possible antigenic differences between H. pylori strains. Furthermore, an absence of systemic IgG antibodies with the presence of H. pylori in the stomach was confirmed with subsequent serum and biopsy specimens from 4 of these 14 patients, excluding the possibility of a serum specimen taken in the early phase of H. pylori infection. These results clearly suggest that in some patients, no systemic IgG antibodies will occur, although H. pylori can be detected from the gastric biopsy specimens. Evans et al. (2) also reported an absence of seroconversion in two patients. Borderline serological reactions were obtained with only 22 of 1,134 (1.9%) serum specimens tested. However, conclusive results were obtained with 20 serum specimens on repeated testing, and this correlated well with the immunoblot results (Table 2). Therefore, it is advisable to repeat the Roche EIA if borderline results are obtained in the first assay.
In conclusion, we evaluated a second-generation H. pylori IgG EIA from Roche S.A. in patients with upper gastrointestinal disorders. The test is easy and quick to perform and was found highly reliable in relation to histological diagnosis and endoscopic findings, as well as immunoblotting. The production of such excellent commercial serological tests will further stimulate research on the exact role of H. pylori serology in serodiagnosis and monitoring of anti-H. pylori therapy. REFERENCES 1. Bolton, F. J., and D. N. Hutchinson. 1989. Evaluation of three Campylobacter pylori antigen preparations for screening sera from patients undergoing endoscopy. J. Clin. Pathol. 42:723726.
J. CLIN. MICRtOBIOL. 2. Evans, D. J., D. G. Evans, D. Y. Graham, and P. D. Klein. 1989. A sensitive and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology 96:1004-1008. 3. Gassner, D., and H. Dreismann. 1990. Rapid purification of urease from Helicobacter pylori and incorporation of the enzyme into a second generation serologic test. Rev. Esp. Enferm. Dig. 78(Suppl. I):34. 4. Glupczynski, Y., A. Burette, J. F. Nyst, C. De Prez, E. De Koster, and M. Deltenre. 1989. Campylobacter pylori-associated gastritis: attempts to eradicate the bacteria by various antibiotics and anti-ulcer regimens. Acta Gastroenterol. Belg. 51:329337. 5. Jones, D. M., J. Eldridge, and A. J. Fox. 1986. Antibody to the gastric Campylobacter-like organisms (Campylobacter pyloridis)-clinical correlations and distribution in the normal population. J. Med. Microbiol. 22:57-62. 6. Jones, D. M., A. M. Lessells, and J. Eldridge. 1984. Campylobacter-like organisms on the gastric mucosa: culture, histological and serological studies. J. Clin. Pathol. 37:1002-1006. 7. Loffeld, R. J. L. F., E. Stobberingh, J. A. Flendrig, J. P. Van Spreeuwel, and J. W. Arends. 1989. Diagnostic value of an immunoassay to detect anti-Campylobacter pylori antibodies in non-ulcer dyspepsia. Lancet i:1182-1185. 8. Musgrove, C., F. J. Bolton, A. M. Krypczyk, J. M. Temperley, S. A. Cavius, W. Gowen, and D. N. Hutchinson. 1988. Campylobacter pylori: clinical, histological and serological studies. J. Clin. Pathol. 41:1316-1321. 9. Newell, D. G. 1987. Identification of the, outer membrane proteins of Campylobacter pyloridis and aiitigenic cross-reactivity between C. pyloridis and C. jejuni. J. Gen. Microbiol. 7:570575. 10. Newell, D. G., and A. Stacey. 1989. Antigen for the serodiagnosis of Campylobacter pylori infections. Gastroenterol. Clin. Biol. 13:37B-41B. 11. Perez-Perez, G. I., B. M. Dworkin, J. F. Chodos, and M. J. Blaser. 1988. Campylobacter pylori antibodies in humans. Ann. Intern. Med. 109:11-17. 12. Price, A. B. 1991. The Sydney system: histological division. J. Gastroenterol. Hepatol. 6:209-222. 13. Rathbone, B. J., J. I. Wyatt, B. W. Worsley, S. E. Shires, L. K. Trejdosiewicz, R. V. Heatley, and M. S. Losowsky. 1986. Systemic and local antibody responses to gastric Campylobacter pyloridis in non-ulcer dyspepsia. Gut 27:642-647. 14. Sobala, G. M., J. E. Crabtree, J. A. Pentith, B. J. Rathbone, T. M. Shallcross, J. I. Wyatt, M. F. Dixon, R. V. Heatley, and A. T. R. Ascon. 1991. Screening dyspepsia by serology to Helicobacter pylori. Lancet 338:94-96. 15. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 16. von Wulffen, H., J. Heesemann, G. H. Butzow, T. L6ning, and R. Laufs. 1986. Detection of Campylobacter pyloridis in patients with antrum gastritis and peptic ulcers by culture, complement fixation test, and immunoblot. J. Clin. Microbiol. 24:716-720.
Vol. 30, No. 1
Evaluation of a Commercially Available Second-Generation Immunoglobulin G Enzyme Immunoassay for Detection of Helicobacter pylori Infection HERMAN GOOSSENS,l* YOURI GLUPCZYNSKI,2 ALAIN BURETTE,3 CHANTAL VAN DEN BORRE,l AND JEAN-PAUL BUTZLER' World Health Organization Collaborating Centre for Enteric Campylobacter, St-Pieters University Hospital, Hoogstraat 322, B-1000 Brussels,' Department of Microbiology, Brugmann University Hospital, B-1090 Brussels,2 and Gastroenterology Unit, Nouvelle Clinique de la Basilique, B-1080 Brussels,3 Belgium Received 15 July 1991/Accepted 23 October 1991 We evaluated a commercially available second-generation anti-H. pyloni immunoglobulin G enzyme immunoassay (EIA) (Cobas Core Anti-Helicobacter pyloni EIA; Roche S.A., Basel, Switzerland) for serodiagnosis of H. pylori infection. The results of the assay were assessed in relation to the results of bacterial culture, urease testing, and histological Giemsa stain of gastric biopsy specimens from 1,134 patients with a variety of symptoms relating to the upper gastrointestinal tract. H. pyloni was detected in biopsy specimens from 660 (58.2%) patients: 6 had a normal mucosa, 123 had chronic gastritis only, and 531 were found to have chronic active gastritis by histology; endoscopy showed duodenal and gastric ulcers in 137 and 64 patients of the last two groups, respectively. The test was evaluated with different age and ethnic groups. The prevalence, sensitivity, specificity, and positive and negative predictive values were, respectively, (i) for Belgian patients between 18 and 40 years old, 34, 93, 95, 91, and 96%; (ii) for Belgian patients more than 40 years old, 53, 96, 91, 93, and 95%; and (iii) for Mediterranean patients more than 17 years old, 87, 94, 70, 95, and 64%. All sera showing discordant immunoassay results compared with the results of histology and culture of biopsy specimens, as well as those with borderline immunoassay results, were tested further by immunoblotting. Among the EIA results considered false negative, we demonstrated an absence of seroconversion in 14 of 19 patients tested by immunoblotting. Among the EIA results considered false positive, immunoblotting showed the presence of specific antibodies in 28 of 37 patients tested. Among the borderline results obtained in the first assay with 22 patients' sera, a second assay showed positive results in 10 patients (8 were positive by immunoblotting) and negative reactions in 10 patients (9 were negative by immunoblotting), whereas 2 remained borderline. These data indicate that sera showing borderline immunoassay results must be tested again. In conclusion, this commercially available second-generation EIA, which is easy and quick to perform, was found highly reliable for the serodiagnosis of H. pylori infection.
patients from developing countries who frequently suffer from campylobacter infection. It has been generally agreed that there is a need for more highly purified and specific antigens in order to decrease backgrounds and increase specificity (10). We evaluated a commercially available second-generation immunoglobulin G (IgG) EIA (Cobas Core Anti-H. pylori EIA, article no. 07 34977; Roche S.A., Basel, Switzerland) to detect H. pylori infection. This second-generation assay uses purified antigens, unlike the first-generation test that used supernatant from a whole-cell sonicate. We observed false-positive reactions with the sonicated bacterial cells present in the first-generation EIA (results not published), which is one of the reasons that the manufacturers decided to change the nature of the antigenic material. The test was evaluated in clinically well-defined groups of dyspeptic patients, and the assay results were compared with the results of bacterial culture, urease testing, and Giemsa stain of gastric biopsy specimens.
Patients infected by Helicobacter pylori in their gastric mucosa elicit a specific systemic antibody response to the
bacterium (13). Early investigations included complement fixation and agglutination techniques (5, 6). More recent studies have shown that the enzyme-linked immunosorbent assay may be the technique of choice for detection of H. pylori-specific antibodies, because of its speed, low cost, simplicity, and reproducibility (1, 2, 7, 11, 13). Numerous enzyme immunoassays (EIAs) to detect H. pylori infection have been developed, including acid-glycine extracts, whole bacterial cells, formalin-treated cells, or sonicated bacterial cells. Some of these tests, such as the Pyloriset (Orion Diagnostica, Espoo, Finland), Pylori Stat (Whittaker Bioproducts, Walkersville, Md.), Malakit (Biolab, Limal, Belgium), and Helico-G (Porton-Cambridge, Maidenhead, United Kingdom), have been made commercially available. The flagellum proteins of H. pylori are well known to cross-react with antibodies produced against Campylobacter jejuni (9). The presence of these cross-reacting antigens could give false-positive reactions for H. pylori infection in those patients with recent campylobacter enteritis or in *
MATERIALS AND METHODS
Subjects. A series of 1,134 consecutive patients with a clinical indication for upper gastrointestinal endoscopy, originating from outpatient and inpatient referrals, was studied.
Corresponding author. 176
VOL. 30, 1992
These patients included 544 men and 590 women with an age range of 13 to 94 years (mean, 52.6 years). Patients studied included 855 Belgians, 257 Mediterraneans (i.e., 99 Moroccans, 66 Italians, 33 Greeks, 31 Spaniards, 19 Turks, 8 Yugoslavians, and 1 Portugese), and 22 patients of other nationalities. The patients had a variety of symptoms relating to the upper gastrointestinal tract, most commonly epigastric pain or dyspepsia or both. Patients taking antimicrobial agents and/or bismuth salts within the last month before endoscopy were excluded from the study. Patients receiving H2-receptor antagonists were included in the study, since these agents are not active against H. pylori (4). Endoscopy and sampling procedures. A blood sample was taken from each patient for serological tests immediately before the endoscopy procedure, and sera were stored at -20°C until assayed. All endoscopic examinations were carried out by the same endoscopist (A.B.). The presence of hyperemia, erosions, ulcerations, atrophy, or hypertrophy of the mucosa was recorded. Four gastric biopsy specimens were randomly taken from both the antrum and the corpus. From each area, two biopsy samples were immediately placed in 10% buffered formalin for histological examination, and those for microbiological examination were dipped with a sterile cotton swab in a semisolid agar transport medium (Portagerm; bioMerieux, Marcy l'Etoile, France). One antral specimen was placed into a 2% urea-buffered broth for the rapid detection of H. pylori urease activity. Different sterile biopsy forceps with a cup size of 5 by 5 mm were used to collect antral and body biopsy samples to exclude the possibility of sample contamination. Between patients the flexible endoscopes (including all of the channels) and biopsy forceps were carefully cleaned and disinfected by immersion in a 2% glutaraldehyde (Cidex; Surgikos Ltd, Livingstone, Scotland) solution for at least 15 min, rinsed in water, and dried. Histology. Formalin-fixed, paraffin-embedded tissue sections were stained with hematoxylin and eosin to grade the severity of gastritis and with Giemsa stain to detect H. pylori. The classification of gastritis was made in accordance with the Sydney system for the classification of gastritis (12). Both the mononuclear and the polymorphonuclear infiltrates were graded separately-on the basis of the extent of inflammatory cell infiltration-as absent, mild, moderate, or severe. Four different scores (from 0 to 3) were attributed separately to each type of inflammatory cell infiltrate. Other types of mucosal changes, e.g., gland atrophy, intestinal metaplasia, and degenerative and regenerative changes of the epithelium, were rated separately but were not used as evidence of inflammation. The histological preparations were always examined by the same pathologist, who was unaware of the patients' clinical status and of the results of the other tests. Microbiology. Gastric mucosal biopsy specimens were inoculated directly onto two culture plates: (i) a Columbia agar plate (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 10% horse blood and (ii) the same medium made selective by the addition of vancomycin (10 mg/liter), colistin (2,500 U/liter), amphotericin B (10 mg/ liter), and trimethoprim (5 mg/liter). Agar plates were incubated in a special incubator at 37°C under microaerobic atmosphere and were inspected after 3, 5, and 7 days. Bacterial colonies were identified as H. pylori on the basis of growth characteristics, colonial morphology, Gram stain examination, catalase and oxidase reactions, and rapid urease
production.
Identification of H. pylori-positive patients by invasive
EVALUATION OF A NEW EIA FOR H. PYLORI
177
procedures was based on culture and/or histology results; a positive urease biopsy test only was not considered sufficient for diagnosis. Serology. The anti-H. pylori IgG EIA (Cobas Core Anti-H. pylori EIA) is a second-generation two-step EIA for the detection of IgG antibodies to H. pylori in human serum, based on a set of fast protein liquid chromatography-purified cell surface antigens including the native urease enzyme (3). The test was performed according to the manufacturer's instructions. In the first step, the patients' samples and controls were diluted with the specimen dilution buffer and incubated with beads coated with purified H. pylori-specific antigens. Specific antibodies were bound to the beads. After removal of the unbound material by a washing step, the antigen-antibody complex on each bead was detected with peroxidase-conjugated goat anti-human IgG antibody. After the removal of unbound conjugate, the beads were incubated with a substrate solution containing tetramethylbenzidine and hydrogen peroxide. A blue color developed in proportion to the amount of H. pylori-specific IgG bound to the beads. The enzymatic reaction was stopped by the addition of acid, and the A450 values were determined. The cutoff value was determined by adding 0.07 to the mean value of the absorbance of the negative controls. Values within a zone of ±10% of the cutoff value were reported as borderline. Results below the 10% zone of the cutoff value were considered negative for anti-H. pylori antibodies, and specimens for which the absorbances were greater than the 10% zone of the cutoff value were considered reactive for anti-H. pylori. Intra- and interrun variations of the EIA were evaluated by repeated testing of (i) sera from 20 patients without H. pylori antibodies and whose gastric biopsy specimens were negative for H. pylori and (ii) sera from 30 patients with H. pylori antibodies and with the organisms present in their stomach. All sera showing (i) an absence of IgG antibodies to H. pylori but the presence of the bacterium in gastric biopsy specimens by Giemsa stain and/or culture, (ii) the presence of IgG antibodies but an absence of the bacterium in biopsy specimens, or (iii) borderline results were tested again. These three groups of sera were further analyzed for H. pylori IgG antibodies by immunoblotting. The antigen preparation was a whole-cell preparation of the type strain H. pylori CCUG 18818. The method of Towbin et al. (15) formed the basis of the Western blot (immunoblot) procedure. The nitrocellulose paper strips were incubated with a 1:25 dilution of the serum for 16 h at room temperature. Sera were diluted in 0.1% bovine serum albumin in buffered saline. Peroxidase-conjugated goat anti-human IgG (United States Biochemical Corp.) was used as the conjugate. Immunoblotting profiles were compared with profiles obtained with sera from 20 patients positive for H. pylori by IgG EIA as well as Giemsa stain and/or culture of biopsy specimens; immunoblotting profiles were also compared with profiles obtained with sera from 15 asymptomatic blood donors without H. pylori antibodies as shown by IgG EIA. The presence of more than four dense bands or a band at approximately 120 kDa was considered positive by immunoblotting. Bands at between 45,000 and 66,000 Da were regarded as nonspecific because they were usually present in the blood donors' sera. Statistical analysis. For statistical analysis we used the Epi-info version 5.01A (Centers for Disease Control and World Health Organization program). Comparison of mean values was performed by using the Kruskal-Wallis one-way
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TABLE 1. Correlation between IgG Cobas Core Anti-H. pylori EIA results and detection of H. pylori organisms with Giemsa stain and culture of biopsy specimens from 1,134 symptomatic patients No. of patients
With EIA results':
H. pylori negative (n = 474)
-
+
B
(n = 660)
-
+
B
Histological Normal Chronic gastritis only Mild Moderate Severe Chronic active gastritis Mild Moderate Severe
300 130 121 8 1 44 30 11 3
283 108 103 5 0 38 26 9 3
13 20 16 3 1 6 4 2 0
4 2 2 0 0 0 0 0 0
6 123 57 60 6 531 244 222 65
0 8 4 4 0 20 10 7 3
6 114 52 56 6 496 227 206 63
0 1 1 0 0 15 7 8 0
Endoscopicb Normal Duodenal ulcer Gastric ulcer Gastric cancer
126 10 25 4
118 10 24 4
6 0 1 0
2 0 0 0
26 137 64 5
2 4 4 1
24 131 60 3
0 2 0 1
Diagnosis
H. pylori positive
With EIA results:
-, negative; +, positive; B, borderline. b Other patients were found to have various types of diffuse erythematous lesions of the stomach.
a
analysis variance or the Wilcoxon signed-rank test. Results were considered significantly different for P values of 17 yrs old
18-40 yrs old
yrs old
207 73
630 349
245 222
34 93 95 91 96
53 96 91 93 95
87 94 70 95 64
No. of patients Total With H. pylori IgG antibodies
Prevalence (%) Sensitivity Specificity (%) Positive predictive value (%) Negative predictive value (%)
>40
the mean optical densities of sera from patients with chronic active gastritis were statistically significantly higher than those from patients with chronic gastritis only (P = 0.009). Mean optical densities of sera from patients suffering from gastric ulcer were also statistically significantly higher than those from patients with duodenal ulcer (P = 0.03). However, no statistically significant differences between mean optical densities of sera from patients with duodenal and gastric ulcer in relation to the degree of histological lesions were found by multivariate analysis. This indicates that histological differences between both groups might be responsible for this statistically significant difference. Immunoblot. Immunoblots were performed on three types of serum. (i) One type included sera from 19 of the 28 patients with no H. pylori IgG antibodies in the Roche EIA but with H. pylori present in their gastric biopsy specimens by Giemsa stain and/or culture. Immunoblots confirmed the absence of IgG H. pylori antibodies in 14 of the 19 patients tested, whereas in 5 patients immunoblots were considered positive. Moreover, for three patients (two patients negative by Roche EIA and immunoblotting and one patient negative by Roche EIA and positive by immunoblotting), we obtained a second serum specimen. For two patients (both negative by Roche EIA and immunoblotting) we obtained five and six subsequent serum specimens. Endoscopy with histology, Giemsa stain, urease testing, and culture of biopsy specimens was carried out simultaneously. H. pylori was still present in the stomach, and serological results (Roche EIA and immunoblotting) showed identical results for all five
patients. (ii) A second type included those 22 serum specimens showing borderline results in the first assay. Table 2 reports the second IgG Cobas Core Anti-H. pylori ETA assay and in immunoblotting, in relation to the detection of H. pylori in the patients' biopsy specimens. Among the 10 serum specimens which were negative with the second EIA, the absence of IgG antibodies was confirmed by immunoblotting in nine patients. Among the 10 serum specimens which were positive with the second EIA, the presence of IgG antibodies was confirmed in eight patients. Two serum specimens remained borderline; one on the results with these sera in
was considered positive and the other was negative by immunoblotting. (iii) A third type of serum included sera from those patients showing a positive result with the Roche EIA but for whom H. pylori could not be detected in their biopsy
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specimens by Giemsa stain, urease testing, and culture. Sera from 37 of 39 patients were available for immunoblotting, and the presence of specific H. pylori IgG antibodies was confirmed in 28 patients. A subsequent serum specimen was obtained for six patients (all positive by the Roche EIA in the first and second assays as well as by immunoblotting). The second serum specimen was again positive in the Roche EIA, and immunoblotting confirmed the presence of H. pylori-specific antibodies. Giemsa stain, urease testing, and culture of biopsy specimens were still negative. DISCUSSION The overall proportion (58%) of biopsy specimens positive for H. pylori determined by cultural and histological techniques was of the same order as in most other reports, in which it ranged from 42 to 66% (8, 16). In the present study, and in common with other workers (8, 16), we have found that there was a clear association between the presence of H. pylori and chronic active gastritis. The presence of H. pylori was also strongly correlated with a diagnosis of duodenal ulcer (93%) and somewhat less with gastric ulcer (72%). On the other hand, 98% of the patients with normal histology were not infected by H. pylori (Table 1). We found an excellent correlation between the presence of H. pylori and raised IgG antibody by using the IgG Cobas Core EIA (Table 1). The EIA was evaluated in different age and ethnic groups (Table 3). Although very few workers evaluated serological tests taking into consideration the variation of prevalence in different populations, we believe that this is relevant because particularly the negative predictive value will depend upon the prevalence of antibodies in the population studied. One major role for H. pylori serology might be screening younger dyspeptic patients to eliminate unnecessary endoscopies (14). This is another reason that we considered two age profiles in the Belgian patients when assessing the H. pylori EIA. The test performed excellently in both Belgian age groups, with sensitivities, specificities, and positive and negative predictive values of 90% or more (Table 3). However, in the Mediterranean population, the negative predictive value decreased to 64%, which might be related to the high prevalence (87%). It was interesting to note that the specificity of the assay is also lower in the Mediterranean group of patients. However, there is no evidence that the strains of H. pylori differ antigenically in this group. These data clearly indicate that it is difficult to compare the performance of this new EIA from Roche with that of other commercially available tests that have been reported in the literature. Moreover, the evaluation of such tests will also depend on the sensitivity of the culture and Giemsa stain for the detection of H. pylori from gastric biopsy specimens. The only way to assess correctly H. pylori serology is to evaluate it with the same serum specimens, from histologically and bacteriologically well-defined groups of patients. False-positive EIA results with the Roche test were obtained with 39 serum specimens, although immunoblotting confirmed the presence of IgG anti-H. pylori antibodies in 28 patients. This may be explained in several ways. It is possible that the active site of infection was missed because H. pylori has a patchy distribution. These patients may have had a previous infection with H. pylori, and their antibody levels still were raised; or these patients may still have taken antibiotics prior to endoscopy, because they ignored this, forgot to mention it during the interview, or had not remembered having taken antibiotics within 1 month prior to
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endoscopy. Cross-reaction could have occurred between H. pylori antigens and other antibodies in the patients' sera. Low specificity of serology may also be explicable by the insensitivity of microscopy and culture. Finally, failure to detect H. pylori in biopsy specimens, despite the presence of H. pylori IgG antibodies, could have been due to intestinal metaplasia and/or atrophy of the gastric mucosa of the stomach. This may in turn explain the lower prevalence of H. pylori in elderly patients. It is also conceivable that some specific antibody reactivity is lost on immunoblotting because of a loss of three-dimensional conformation during sodium dodecyl sulfate-polyacrylamide gel electrophoresis but is detectable by EIA. Of the 28 serum specimens showing false negative serological results, 19 were available for immunoblotting, and the absence of H. pylori antibodies was confirmed in 14 of these serum specimens. Among these 14 serum specimens, immunoblots were also carried out with the strains isolated from the patients' biopsy specimens in 13 patients (results not shown), which excludes false-negative immunoblots because of possible antigenic differences between H. pylori strains. Furthermore, an absence of systemic IgG antibodies with the presence of H. pylori in the stomach was confirmed with subsequent serum and biopsy specimens from 4 of these 14 patients, excluding the possibility of a serum specimen taken in the early phase of H. pylori infection. These results clearly suggest that in some patients, no systemic IgG antibodies will occur, although H. pylori can be detected from the gastric biopsy specimens. Evans et al. (2) also reported an absence of seroconversion in two patients. Borderline serological reactions were obtained with only 22 of 1,134 (1.9%) serum specimens tested. However, conclusive results were obtained with 20 serum specimens on repeated testing, and this correlated well with the immunoblot results (Table 2). Therefore, it is advisable to repeat the Roche EIA if borderline results are obtained in the first assay.
In conclusion, we evaluated a second-generation H. pylori IgG EIA from Roche S.A. in patients with upper gastrointestinal disorders. The test is easy and quick to perform and was found highly reliable in relation to histological diagnosis and endoscopic findings, as well as immunoblotting. The production of such excellent commercial serological tests will further stimulate research on the exact role of H. pylori serology in serodiagnosis and monitoring of anti-H. pylori therapy. REFERENCES 1. Bolton, F. J., and D. N. Hutchinson. 1989. Evaluation of three Campylobacter pylori antigen preparations for screening sera from patients undergoing endoscopy. J. Clin. Pathol. 42:723726.
J. CLIN. MICRtOBIOL. 2. Evans, D. J., D. G. Evans, D. Y. Graham, and P. D. Klein. 1989. A sensitive and specific serologic test for detection of Campylobacter pylori infection. Gastroenterology 96:1004-1008. 3. Gassner, D., and H. Dreismann. 1990. Rapid purification of urease from Helicobacter pylori and incorporation of the enzyme into a second generation serologic test. Rev. Esp. Enferm. Dig. 78(Suppl. I):34. 4. Glupczynski, Y., A. Burette, J. F. Nyst, C. De Prez, E. De Koster, and M. Deltenre. 1989. Campylobacter pylori-associated gastritis: attempts to eradicate the bacteria by various antibiotics and anti-ulcer regimens. Acta Gastroenterol. Belg. 51:329337. 5. Jones, D. M., J. Eldridge, and A. J. Fox. 1986. Antibody to the gastric Campylobacter-like organisms (Campylobacter pyloridis)-clinical correlations and distribution in the normal population. J. Med. Microbiol. 22:57-62. 6. Jones, D. M., A. M. Lessells, and J. Eldridge. 1984. Campylobacter-like organisms on the gastric mucosa: culture, histological and serological studies. J. Clin. Pathol. 37:1002-1006. 7. Loffeld, R. J. L. F., E. Stobberingh, J. A. Flendrig, J. P. Van Spreeuwel, and J. W. Arends. 1989. Diagnostic value of an immunoassay to detect anti-Campylobacter pylori antibodies in non-ulcer dyspepsia. Lancet i:1182-1185. 8. Musgrove, C., F. J. Bolton, A. M. Krypczyk, J. M. Temperley, S. A. Cavius, W. Gowen, and D. N. Hutchinson. 1988. Campylobacter pylori: clinical, histological and serological studies. J. Clin. Pathol. 41:1316-1321. 9. Newell, D. G. 1987. Identification of the, outer membrane proteins of Campylobacter pyloridis and aiitigenic cross-reactivity between C. pyloridis and C. jejuni. J. Gen. Microbiol. 7:570575. 10. Newell, D. G., and A. Stacey. 1989. Antigen for the serodiagnosis of Campylobacter pylori infections. Gastroenterol. Clin. Biol. 13:37B-41B. 11. Perez-Perez, G. I., B. M. Dworkin, J. F. Chodos, and M. J. Blaser. 1988. Campylobacter pylori antibodies in humans. Ann. Intern. Med. 109:11-17. 12. Price, A. B. 1991. The Sydney system: histological division. J. Gastroenterol. Hepatol. 6:209-222. 13. Rathbone, B. J., J. I. Wyatt, B. W. Worsley, S. E. Shires, L. K. Trejdosiewicz, R. V. Heatley, and M. S. Losowsky. 1986. Systemic and local antibody responses to gastric Campylobacter pyloridis in non-ulcer dyspepsia. Gut 27:642-647. 14. Sobala, G. M., J. E. Crabtree, J. A. Pentith, B. J. Rathbone, T. M. Shallcross, J. I. Wyatt, M. F. Dixon, R. V. Heatley, and A. T. R. Ascon. 1991. Screening dyspepsia by serology to Helicobacter pylori. Lancet 338:94-96. 15. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 16. von Wulffen, H., J. Heesemann, G. H. Butzow, T. L6ning, and R. Laufs. 1986. Detection of Campylobacter pyloridis in patients with antrum gastritis and peptic ulcers by culture, complement fixation test, and immunoblot. J. Clin. Microbiol. 24:716-720.
