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Journal of Clinical Microbiology, April 2001, p. 1339-1344, Vol. 39, No. 4
Delft Diagnostic
Laboratory1 and R. de Graaf
Hospital,3 Delft, Department of Internal
Medicine, Bernhoven Hospital, Oss,4 and
Department of Microbiology, Bosch Medicentrum, Den
Bosch,5 The Netherlands; IPATIMUP
and Medical Faculty, University of Porto, Porto,
Portugal2; Division of Infectious
Diseases, Vanderbilt University School of Medicine, Nashville,
Tennessee6; and Department of
Medicine, New York University School of Medicine, New York, New
York7
Received 28 September 2000/Returned for modification 22 December
2000/Accepted 26 January 2001
Helicobacter pylori strains can be distinguished by
genotyping of virulence-associated genes, such as vacA and
cagA. Because serological discrimination between strain
types would reduce the need for endoscopy, 61 patients carrying
H. pylori were studied by vacA and
cagA genotyping of H. pylori in gastric biopsy
specimens and by detection of specific serum antibodies. Serological
responses to H. pylori were determined by
Helicoblot (versions 2.0 and 2.1). Antibodies to CagA also were determined by a rapid anti-CagA assay (Pyloriset screen CagA) as well as by two noncommercially
developed enzyme immunoassays, each using a recombinant CagA protein.
Assessment of performance of the Helicoblot assays
indicated substantial interobserver variation, with kappa values
between 0.20 and 0.93. There was no relationship between the
serological profiles on the Helicoblot and the genotypes
from the same patients, except for strong associations between the
presence of anti-CagA and the cagA-positive and
vacA s1 H. pylori genotypes. Detection of anti-CagA by the five different assays varied considerably, with kappa
values ranging from 0.21 to 0.78. Using the cagA genotype as the "gold standard," the sensitivity and specificity of the anti-CagA assays varied from 71.4 to 85.7% and from 54.2 to 100%, respectively. Thus, serological profiles of antibodies to H. pylori are heterogeneous and, with the exception of anti-CagA
antibodies, show no relation to the H. pylori vacA and
cagA genotypes. Detection of anti-CagA antibodies is
strongly dependent on the test used.
Helicobacter pylori
persistently colonizes the mucosa of the human stomach, causes chronic
gastritis, and is an important risk factor for gastroduodenal diseases,
such as peptic ulcers and gastric carcinoma (11). There is
increasing evidence that distinct variants of H. pylori
exist and that these may be associated with the pathogenicity of the
bacterium (41); several virulence-associated genes have
been identified (1, 8).
vacA encodes a vacuolating toxin that is released by
H. pylori and injuries epithelial cells (9,
21). vacA is present in all H. pylori
strains, and includes two variable regions (1). The
s region (encoding the signal peptide) is located at the 5' end
of the gene and exists as an s1 or s2 allele. Within type s1, several
subtypes (s1a, s1b, and s1c) can be distinguished (39).
The m region (middle) occurs as an m1 or m2 allele. The mosaic
combination of s- and m-region allelic types correlates with the
production of the cytotoxin and is thereby associated with virulence of
the bacterial strain (1).
cagA (cytotoxin-associated gene) is considered a marker for
the presence of the pathogenicity (cag) island of about 35 kbp (6). Carriage of cagA+ strains
increases the risk for the development of atrophic gastritis and
gastric cancer (4, 19). Several studies have shown
clinical relevance of specific antibodies to CagA using noncommercial
assays (3, 5, 7, 20, 29, 33-35, 44), whereas others
failed to confirm these findings (14, 17, 18, 23, 27, 45)
H. pylori can be diagnosed by analysis of gastric biopsy
specimens by urease tests, culture of the bacterium, histopathology, or
detection of bacterial DNA by PCR. Noninvasive diagnostic methods include the urea breath test and serological assays measuring antibodies to H. pylori in the serum (15, 22).
The distinct vacA and cagA genotypes of H. pylori can best be identified by molecular methods, using
cultured strains, or directly in gastric biopsy specimens, but this
requires endoscopy. Therefore, serological typing methods analyzing
specific antibodies to H. pylori if accurate, would be most
suitable for routine clinical use. The present study assessed
the relationship between the vacA and cagA
genotypes of H. pylori and the presence of specific
anti-Helicobacter antibodies, in particular, antibodies to CagA.
Patients.
Gastric (antral) biopsy specimens and serum
samples were obtained from patients, undergoing upper endoscopy for
routine clinical indications. Biopsy specimens were tested for H. pylori by a rapid urease assay (CLO test; Delta-West). A total of
61 patients were randomly selected from several hundred patients in The
Netherlands to ensure that each of the vacA s1,
vacA s2, vacA m1, vacA m2, cagA-negative, and cagA-positive genotypes was
sufficiently represented in the test group.
Genotyping of H. pylori.
The vacA and
cagA status of H. pylori was determined directly
in gastric biopsy specimens, as described earlier (38,
39). Briefly, total DNA was isolated from the specimens, and
vacA s and m regions as well as part of cagA were
simultaneously amplified by multiplex PCR. PCR products were hybridized
to subtype-specific probes by reverse hybridization on a line probe
assay (LiPA), detecting vacA s1a, s1b, s1c, s2, m1, m2a, and
m2b and cagA. This assay has been extensively evaluated and
showed high accuracy for detection of distinct genotypes
(38-40).
Serological assays.
Serum samples were analyzed by various
commercially available and noncommercial assays. Immunoglobulin G
antibody profiles were determined by Helicoblot versions
2.0 and 2.1 (Genelabs Diagnostics, Singapore, Singapore) and were
performed according to the manufacturer's instructions. The
Helicoblot assays are based on Western blot analysis of
whole-cell H. pylori antigens. Interpretation of the serologic reactivity is restricted to antigens of various molecular masses. Version 2.0 contains antigens of 19.5, 26.5, 30, 35, 89 (VacA),
and 116 (CagA) kDa. Version 2.1 contains antigens of 19.5, 30, 35, 37, 89 (VacA), and 116 (CagA) kDa. For Helicoblot 2.1, the
criteria for H. pylori seropositivity are as follows: (i) positive result for the 116-kDa (CagA) band, where CagA has to be
present with one or more bands at the following positions: 89 (VacA),
37, 35, 30 (UreA), and 19.5 kDa together, or with current infection
marker (CIM); (ii) presence of any one band at 89, 37, or 35 kDa, with
or without the current infection marker; (iii) presence of both the 30- and 19.5-kDa bands, with or without the CIM.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1339-1344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Assessment of Helicobacter pylori vacA
and cagA Genotypes and Host Serological Response
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Statistical analyses. All statistical analyses were performed using SPSS version 8.0 for Windows (SPSS, Inc., Chicago, Ill.). To assess the interobserver variation for interpretation of the Helicoblot assays, and to determine the agreement between the different anti-CagA assays, Cohen's kappa values were calculated. kappa values represent the degree of agreement between any pair of observations, and the values can be interpreted as follows: 0 to 0.2 (poor), 0.21 to 0.40 (fair), 0.41 to 0.60 (moderate), 0.61 to 0.80 (good), 0.81 to 0.99 (very good), and 1 (perfect agreement).
Relationships between serological profiles and genotypes were calculated by the chi-square test with Bonferroni correction to increase the stringency of the analysis, since multiple comparisons are being made.| |
RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genotypes of H. pylori strains in the gastric biopsy
specimens.
Gastric biopsy specimens and serum samples were
obtained from 61 H. pylori-positive patients living in The
Netherlands. DNA was isolated from the biopsy specimens, and the
H. pylori vacA and cagA genotypes were determined
by multiplex PCR and LiPA (Table 1). All
samples could be completely genotyped. In seven (11.5%) patients,
multiple vacA genotypes were detected, indicating the presence of multiple H. pylori strains in the biopsy
specimens. Of the 31 s1 strains, 29 were s1a, one was s1b, and one was
s1c. Twenty (64.5%) of the 31 s1 strains also were cagA
positive, whereas only 2 (8.7%) of the 23 s2 strains contained
cagA (P < 0.001).
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Serological responses to H. pylori antigens.
Serological profiles were determined by Helicoblot 2.0 and
2.1. Results were independently interpreted by three persons, the individual reactivity (positive, negative, or dubious) to each of the
indicated antigens was scored, and average kappa values were calculated
for each of the antigens on the blots (Table
2). For Helicoblot 2.0, the
average kappa values of the 19.5-, 26.5-, 30-, 35-, 89-, and 116-kDa
antigens ranged between 0.457 (19.5 kDa) and 0.692 (26.5 kDa). For
Helicoblot 2.1, the average kappa values of the 19.5-, 30-, 35-, 37-, 89-, and 116-kDa antigens ranged between 0.325 (37 kDa) and
0.646 (116 kDa). The average kappa value for the CIM in
Helicoblot 2.1 was 0.822. A consensus score (Table 2) also
was determined for the reactivity of each serum sample to the
individual antigens. If antibody reactivity was scored positive by two
or more observers, it was considered a positive reaction. As such,
negative and dubious scores were grouped together. The results of
Helicoblot 2.0 and 2.1, using the interpretation criteria
provided in the manufacturer's instructions, showed a high degree of
discordance. Helicoblot 2.0 yielded positive results with
52 (85.2%) of the 61 patients tested, whereas Helicoblot 2.1 yielded positive results with 57 (93.4%) of the patients tested. The positivity rate for individual antigen bands on
Helicoblot 2.0 varied from 26.2% (19.5 kDa) to 83.6%
(26.5 kDa). For Helicoblot 2.1, the positivity rates varied
between 54.1% (116 kDa) and 75.4% (19.5 kDa). Antibodies to the
89-kDa antigen, representing the vacuolating toxin VacA, were detected
in 39.3% of the sera by using Helicoblot 2.0 and 59.0% of
the sera by using Helicoblot 2.1. Antibodies to the CIM
(which was applied as an additional and separate line on the
Helicoblot 2.1 membrane) were found in 52 (85.2%) of the
sera.
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Anti-CagA antibodies.
Antibodies to CagA also were measured by
the Pyloriset screen rapid assay and two independent anti-CagA EIAs
based on recombinant CagA proteins (Table
3). The detection rate of anti-CagA
differed considerably between the five different assays, and kappa
values, measuring interassay agreement, ranged from 0.213 to 0.783. The agreement between the two versions of the Helicoblot with
respect to anti-CagA (116 kDa) was only 0.557.
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= 0.736), but this was not
significantly higher than that for the Pyloriset screen test (0.632)
and the Vanderbilt EIA (0.696). Using the cagA genotype as
the "gold standard," the sensitivity of the assays ranged from
71.4% (Helicoblot 2.0 and Pyloriset) to 85.7% (DDL EIA).
The specificity of the assays ranged from 54.2%
(Helicoblot 2.1) to 100% (Vanderbilt EIA). The positive predictive values varied between 66.6% (Helicoblot 2.1)
and 100% (Vanderbilt EIA), and the negative predictive values were
between 68.4% (Helicoblot 2.1) and 87.9% (DDL EIA) (Table
4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Serological methods are valuable tools for detecting the presence of H. pylori but do not allow direct characterization of the H. pylori strain. The present study compared the serum antibody responses to H. pylori antigens with the H. pylori vacA and cagA genotypes in antral biopsy specimens. That the vacA and cagA genotypes could be determined for all 61 patients by the extensively validated PCR LiPA method facilitated this analysis (37-39, 42). Specimens from approximately 10% of the patients contained multiple genotypes, which is in agreement with earlier findings (37).
Serum antibodies to several H. pylori antigens, identified
by their molecular weight, were determined by two different versions of
the Western blot-based Helicoblot assay, which generate
particular serological profiles. The reactivity toward each antigen was
assessed visually, and signal intensities may vary considerably.
Consequently, interobserver variation of both the 2.0 and 2.1 versions
was substantial. Of all the individual antigens, an additional antigen
line, designated the CIM, which is of unknown clinical relevance,
performed best ( = 0.822), but antibodies to the CIM were found
in only 85.2% of the H. pylori-positive patients. That
the CIM was present on Helicoblot 2.1 as a separate line at
a clearly defined position probably limited erroneous interpretations.
Taken together, our results demonstrate that the reproducibility of the
visual scoring of the Helicoblot results is limited, in
part due to the low intensity of some signals and due to variation
between different strip batches, preventing accurate alignment with the
provided template. Scanning of blots and computer-assisted
interpretation may improve the performance of these assays. Although
Helicoblot 2.0 and 2.1 contain different antigens,
reactivities against the shared 19.5-, 30-, 35-, 89-, and 116-kDa
proteins differed considerably. This may be due to differences in
H. pylori strains used in both assays, in batches of strips,
and in observers. Therefore, to adequately compare serological results
from various patients or use for follow-up studies, strips from the
same batch should be used and need to be interpreted by the same observer.
The only significant associations were found between the presence of anti-CagA on the Helicoblot and the vacA s1 and cagA-positive genotypes. Since strains lacking cagA do not produce the CagA antigen, the lack of anti-CagA antibodies in hosts carrying cagA-negative strains was expected. The association we found between colonization with vacA s1 strains and presence of anti-CagA antibodies confirms earlier findings, since most cagA-positive strains also have the vacA s1 genotype (41).
The 89-kDa antigen represents the mature vacuolating toxin VacA (9). Although antibodies to VacA might have been expected in all H. pylori-positive patients, in our patient group only 39.3 to 59.0% of the sera contained antibodies to the 89-kDa antigen, confirming earlier studies (12, 32), but with no significant association with the vacA genotype of H. pylori. This result may indicate that most antibodies against VacA recognize conformational epitopes (25), whereas the Helicoblots only present denatured, linear epitopes, in contrast to studies using purified VacA protein (31).
The great majority of cagA-positive strains produce the CagA protein (26). Therefore, if cagA-positive strains are present, the immune system usually will have been exposed to the CagA antigen, especially since CagA is injected into gastric epithelial cells by a type IV secretion apparatus encoded by the cag pathogenicity island (28). Absence of anti-CagA antibodies in such patients may be due to sequence variation in cagA, resulting in different epitopes (24, 43). Several cagA variants have been described that have particular geographical distribution (36, 40). The anti-CagA assays we studied are based on different CagA proteins, which may contain different B-cell epitopes. The Vanderbilt and DDL EIAs were both based on recombinant CagA protein, derived from Western H. pylori strains. No information is available on the source of CagA used for the Helicoblot 2.0, 2.1, and Pyloriset assays. However, specimens from patients from different parts of the world appear able to recognize a single recombinant CagA protein (16, 30).
The presence of antibodies may reflect recent past carriage, and therefore, antibodies may be found when the bacteria are no longer present in the stomach. However, none of the patients in the present study had been treated, and all were H. pylori positive at the time of investigation, as determined by PCR. Similarly, it is possible that cagA-positive strains were present in the stomach but were not detected due to sampling error, since each antral gastric biopsy specimen only represents a very small sample of the entire gastric mucosa.
The five assays comprised three different test formats, i.e., Western blot (Helicoblot), rapid immunoassay using a lateral flow system (Pyloriset screen CagA), and microtiter EIA (DDL and Vanderbilt EIAs), and were not in full agreement with each other (Table 3). Although different assay formats may influence the test performance of anti-CagA measurements (2, 13), using the cagA genotype as the standard, both Helicoblot assays show limited sensitivity and specificity. In contrast, the DDL assay was most sensitive, while the Vanderbilt assay was most specific (Table 4).
In conclusion, measurement of anti-CagA antibodies by different test formats revealed considerable differences in detection rates. Both in-house EIAs and the Pyloriset screen CagA showed good agreement, but the Helicoblots were not highly reproducible or accurate. Therefore, in future studies in which anti-CagA measurements are used, investigators should include evaluation of the serological assays used in the population studied.
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ACKNOWLEDGMENTS |
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We thank Hans Pottel for statistical advice.
This work was supported in part by grant R01 DK53707 from the National Institutes of Health and by the Medical Research Service of the Department of Veterans Affairs.
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FOOTNOTES |
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* Corresponding author. Mailing address: Delft Diagnostic Laboratory, R. de Graafweg 7, 2625 AD Delft, The Netherlands. Phone: 31-15-2604581. Fax: 31-15-2604550. E-mail: L.J.van.Doorn{at}ddl.nl.
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Abstract
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Discussion
References
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Journal of Clinical Microbiology, April 2001, p. 1339-1344, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1339-1344.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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