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Journal of Clinical Microbiology, May 1998, p. 1271-1276, Vol. 36, No. 5
Delft Diagnostic Laboratory, Delft, The
Netherlands1;
IPATIMUP2 and
Faculty of
Pharmacy,4
University of Porto, Porto,
Portugal; and Innogenetics N.V., Industriepark Zwijnaarde, Zwijnaarde,
Belgium3
Received 10 September 1997/Returned for modification 30 December
1997/Accepted 30 January 1998
The present report describes an analysis of two virulence genes of
Helicobacter pylori. Parts of the cagA gene, as
well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers
and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip
containing multiple specific probes for the vacA s region (s1a, s1b, and s2 alleles), the vacA m region (m1 and m2
alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric
biopsy specimens, and surgical specimens from patients living in
Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA
was detected in 84 and 73% of the Portuguese and Dutch patients,
respectively. vacA typing results, as determined by reverse
hybridization, were completely concordant with those of sequence
analysis. Most Portuguese patients (72%) contained type s1b, whereas
most Dutch patients (61%) contained type s1a (P < 0.001). The method is also very effective at detecting the presence of
multiple genotypes in a single biopsy specimen. The prevalence of
multiple strains in Portuguese patient samples was significantly higher
(29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of
ulcers or gastric carcinoma and the presence of vacA type
s1 (s1a or s1b; P = 0.008) and cagA
(P = 0.003) genes.
Helicobacter pylori is an
important human pathogen that causes chronic gastritis and that is
associated with the development of peptic ulcer disease, atrophic
gastritis, and gastric malignancies (6). The establishment
and maintenance of H. pylori colonization in the stomach
depend on several host and bacterial factors. Expression of specific
host antigens, such as the Lewis-type blood group antigens, may play an
important role (2, 23). H. pylori itself secretes
several enzymes, such as urease, that facilitate its survival in the
acidic environment of the stomach (6).
The genetic variability among H. pylori strains is
relatively high (16) and can be assessed by various methods,
such as restriction fragment length polymorphism analysis and PCR
fingerprinting (17, 19, 28, 29). Analysis of this overall
genomic variability is useful for epidemiological studies, but this
genomic variability is not related to the pathogenicity of H. pylori strains. Recently, specific bacterial genes that are
associated with phenotypic strain virulence have been described
(5, 7, 22). Approximately 50 to 60% of the H. pylori strains contain the cytotoxin-associated (cagA)
gene and consequently produce the 128-kDa cagA protein (10). The presence of cagA is associated with
duodenal ulceration, gastric mucosal atrophy, and gastric cancer
(7). cagA is part of a larger genomic entity,
designated the pathogenicity (cag) island (9),
which contains multiple genes that are related to the virulence and
pathogenicity of the H. pylori strain. Therefore, the
presence of cagA can be considered a marker for this genomic pathogenicity island and is associated with more virulent H. pylori strains.
Another virulence factor, produced by approximately 50% of the
H. pylori strains, is a cytotoxin that induces the formation of vacuoles in mammalian cells in vitro and that leads to cell death
(20). This toxin is encoded by the vacA gene,
which is present in virtually all H. pylori strains
(11, 13, 25). Recently, the existence of different allelic
variants in two parts of this gene has been described (3,
12) (Fig. 1). The N-terminal signal
(s) region occurs as either an s1a, s1b, or s2 allele. The middle (m)
region is present as an m1 or an m2 allele. This mosaic structure of
the vacA gene (Fig. 1) accounts for the differences in the
cytotoxin production between strains. The s1/m1 type strains produce
high levels of toxin, s1/m2 type strains produce low to moderate levels
of toxin, whereas s2/m2 strains do not produce any active toxin at all.
Strains containing s2/m1 have not yet been found. The vacA s
and m regions appear to have different clinical relevance
(4). vacA s1a strains are associated with greater
antral mucosal neutrophil and lymphocyte infiltrates than type s1b or
s2 strains. vacA type m1 strains are associated with greater
gastric epithelial damage than type m2 strains. Duodenal ulcer disease
appears to be more prevalent in patients infected or colonized with
type s1a strains than in patients colonized with type s1b and s2
strains.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Typing of Helicobacter pylori vacA Gene
and Detection of cagA Gene by PCR and Reverse
Hybridization
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (15K):
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FIG. 1.
Mosaic structure of the vacA gene comprising
the variable s and m regions. The locations of the various primer
target sites are indicated by arrows, and the expected amplimer sizes
are shown. Primer sets A through F were described by Atherton et al.
(3). Primer sets G and H were described in the present study
and permit general amplification of the vacA s and m regions
with single primer sets.
The aim of the present study was to develop an efficient DNA-based detection and typing system for the virulence-associated cagA and vacA genes of H. pylori either in cultured isolates or directly in gastric biopsy specimens. The method was evaluated by comparison of the results obtained by the method with those obtained by direct sequence analysis of PCR products obtained from a number of patients from Portugal and The Netherlands. Finally, the clinical relevance of vacA and cagA genotyping was investigated with patients with functional dyspepsia, peptic ulcers, or gastric carcinomas.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Patients. A total of 103 Dutch (n = 48) and Portuguese (n = 55) patients were studied. Biopsy specimens were obtained from 48 consecutive patients who underwent standard gastroscopy procedures in a Dutch community hospital and were used directly for PCR. Of these patients, 23 had nonulcer dyspepsia (NUD), 18 had a duodenal ulcer, and 7 had a gastric ulcer.
Gastric biopsy specimens were also obtained from a total of 35 consecutive Portuguese patients. H. pylori was cultured from specimens from 20 of these patients, who had duodenal ulcer disease. The remaining 15 biopsy specimens were from patients with functional dyspepsia and were not used for culture but were used directly for PCR. Surgical specimens were obtained from 20 consecutive Portuguese patients with gastric carcinoma and were processed directly for PCR. Biopsy specimens were transported in Portagerm medium (BioMérieux, Marcy l'Etoile, France) and were stored at
70°C. In all cases parallel biopsy specimens were processed for
routine histopathological examination and/or the CLO test (Delta-West,
Bentley, Australia).
Culture of H. pylori from gastric biopsy specimens. Gastric biopsy specimens were collected from the transport medium and were plated onto agar medium (Gelose; BioMérieux), and the plates were incubated at 35°C under microaerobic conditions (5% O2, 15% CO2, 80% N2) for 5 days.
Isolates of H. pylori were identified by Gram staining as well as by oxidase, catalase, urease, gamma-glutamyltransferase, and nitrate reductase reactions (Rapidec pylori; BioMérieux) and were stored at70°C in Trypticase soy broth containing 15% glycerol.
DNA isolation. DNA was isolated from the cultured bacteria by harvesting cells from a plate in phosphate-buffered saline followed by centrifugation. The pelleted cells were resuspended in 200 µl of proteinase K solution (10 mM Tris-HCl [pH 7.8], 5 mM EDTA, 0.5% sodium dodecyl sulfate [SDS], 50 mg of proteinase K per ml), and the mixture was incubated at 55°C for 30 min. The DNA was extracted with phenol-chloroform-isoamyl alcohol by standard procedures (26). DNA was precipitated by the addition of 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol. After centrifugation, the DNA pellet was washed with 70% ethanol and was dissolved in 50 µl of TE buffer (10 mM Tris-HCl [pH 8.3], 0.1 mM EDTA).
Gastric biopsy specimens were homogenized in 200 µl of proteinase K solution with a sterile micropestle (Eppendorf, Hamburg, Germany). DNA was isolated from the homogenate by the same protocol described above for the cultured isolates.PCRs. PCRs were performed in a volume of 100 µl containing 10 mM Tris-HCl [pH 9.0], 50 mM KCl, 2.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, a 200 µM concentration of the deoxynucleoside triphosphates, 0.25 U of SuperTaq (SphaeroQ, Leiden, The Netherlands), and 10 to 50 pmol of both forward and reverse primers. The reaction mixtures were covered with mineral oil, and PCR was performed in a BioMed-60 thermocycler, comprising 2 min of preincubation at 95°C, followed by 40 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min at 74°C. Final extension was performed for 5 min at 74°C. vacA typing with allele-specific PCR primers was performed as described earlier (3). PCR products were visualized by electrophoresis on 2% agarose gels by standard procedures (26).
PCR primers. For analysis of the vacA s region, the following primers were used for PCR (see also Table 1 and Fig. 1). Primers VA1F and VA1R have been described earlier (3) and generate a fragment of 259 bp for s1a/b variants and a fragment of 286 bp for s2 variants. Primers VA1F and VA1XR generate a fragment of 176 bp for s1a/b variants and a fragment of 203 bp for s2 variants.
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Sequence analysis. The PCR products were used for direct sequence analysis as described previously (18). Briefly, PCR products, containing a biotin moiety on one of the primers, were immobilized onto streptavidin-coated paramagnetic particles (Dynabeads; Dynal, Oslo, Norway). The PCR product was denatured by alkaline treatment, and each strand was used in separate sequencing reactions with the T7 sequencing system (Pharmacia, Uppsala, Sweden). The sequences were read manually and were analyzed with PC-Gene software (Intelligenetics, Mountain View, Calif.).
The primer sequences were analyzed for specificity by using the Blast program at the National Institute of Health Data libraries (1).LiPA. Oligonucleotide probes (see Table 2) for specific identification of the different vacA types or subtypes were deduced from the sequence alignments. Probes were selected from those regions that showed consistent type or subtype-specific heterogeneity, as described earlier by Atherton et al. (3). For reverse hybridization, the probes were tailed with poly(dT) and were immobilized as parallel lines on nitrocellulose strips as described earlier (27).
Ten microliters of PCR product was denatured by the addition of 10 ml of 400 mM NaOH and 10 mM EDTA in a plastic trough. After incubation at ambient temperature for 10 min, 1 ml of prewarmed hybridization solution (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% SDS) was added and a reverse hybridization line probe assay (LiPA) strip was submerged. The troughs were incubated in a shaking water bath at 50 ± 0.5°C for 1 h. The strips were washed with 1 ml of 2× SSC-0.1% SDS for 30 min at 50°C. Subsequently, the strips were rinsed three times in phosphate buffer, and conjugate (streptavidin, alkaline phosphatase) was added. After 30 min of incubation at room temperature, the strips were rinsed again and 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate substrate was added. Positive hybridizations were visible as purple probe lines. Interpretation of the results was performed visually.Statistical analysis. Data were analyzed by the chi-square test.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Amplification and sequence analysis of vacA. In order to investigate the variability of the vacA s region, amplification products from 34 isolates, comprising fragments of 259 bp (s1) or 286 bp (s2), generated by the primer set VA1F-VA1R as described by Atherton et al. (3) were directly sequenced. On the basis of the size of the PCR products and the homology to the prototype sequences, all sequences could be classified as either s1 or s2. Within the s1 group, s1a and s1b subtypes could be recognized. The sequences of representative vacA s1 and s2 variants are presented in Fig. 2. On the basis of sequence analysis a new conserved reverse primer (VA1XR) was chosen. Amplification with VA1F and VA1XR yielded PCR products of 176 and 203 bp for type s1 and type s2, respectively.
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LiPA. In order to confirm PCR amplification of fragments from the cagA and the vacA gene, a reverse hybridization assay that allows specific detection of the various PCR products in a single step was developed. Oligonucleotide probes (Table 2) were immobilized on a nitrocellulose strip as parallel lines. The outline and examples of the LiPA strip are presented in Fig. 4. For each of the allelic variants within the s region (s1a, s1b, and s2) and m region (m1 and m2), probes were developed and used on the strip. In order to exclude cross-reactivity or false-negative results due to unexpected mutations, two separate probes were used for each allelic variant. Samples from more than 50 individual patients were typed by LiPA as well as by direct sequence analysis. Identification of the vacA s1a, s1b, s2, m1, and m2 types was completely concordant between both methods. No cross-reactivity was observed between any of the different variants on the LiPA strip. All PCR fragments hybridized to both probes of a particular vacA subtype.
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Clinical evaluation. In order to evaluate the PCR-LiPA system further, a total of 103 H. pylori-positive clinical specimens were tested by the PCR-LiPA. The genotyping results are presented in Table 3. vacA typing of 20 Dutch strains was performed with allele-specific primers as described previously (3). This yielded results identical to those of sequencing and PCR-LiPA, although aspecific amplification products were observed, making interpretation of the results on the gel difficult in four cases.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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H. pylori infections can cause serious clinical problems, such as atrophic gastritis, gastric or duodenal ulcers, and gastric cancer. Therefore, it is important that accurate diagnostic methods are available.
There is increasing evidence that the genetic variability of H. pylori may also have clinical importance. Three different genes that are associated with bacterial pathogenicity have been described, i.e., cagA, vacA, and iceA (5, 8). The present study describes the development of a rapid method for analysis of the vacA and cagA genes. This method was evaluated by testing clinical samples from Portuguese and Dutch patients.
cagA detection. PCR primers for the detection of the cagA gene were aimed at the 5' part of the gene, which appears to be the most conserved region (10). There are indications of sequence variability of the cagA gene in isolates of different geographic origins (24), and this may lead to underestimation of the true prevalence of cagA-positive H. pylori strains. This requires more extensive sequence analysis of H. pylori strains of different geographic origins.
vacA detection and typing. Analysis of the vacA gene is more complicated. The number of available vacA sequences in the data libraries is very limited. The PCR-based typing system of the s and m regions described by Atherton et al. (3) is based on a number of separate allele-specific PCRs for the detection of the s1/s2, s1a, s1b, s2, m1, and m2 alleles, respectively. This requires multiple PCRs for each strain, and in some cases, formation of aspecific PCR products made interpretation of the results on the gel difficult. To discriminate between types m1 and m2, two type-specific primer sets have been described (3). The target regions for these primer sets do not comprise the highly variable insertion or deletion site of the m region but are located more downstream in vacA. Since aberrant amplification products are often encountered or no amplification products are obtained, these primers were not satisfactory (21), which may be due to the fact that they were based on sequence information from H. pylori isolates obtained in North America.
We have deduced novel primers for general amplification of the s and m regions from sequences obtained from European H. pylori strains. These primers allowed effective amplification of the s and m region sequences. Amplification of the s region was not possible for only one sample from a Dutch patient, probably due to additional sequence variation in the primer target regions.Reverse hybridization. Since visual inspection of PCR products on agarose gels provides only limited reliability, hybridization to allele-specific probes is obligatory for confirming the typing results. Therefore, allele-specific probes were selected for the development of a reverse hybridization assay. The multiparameter design of the LiPA was aimed at specific determination of the vacA and cagA status in a single hybridization step. The complete concordance between direct sequencing and LiPA results confirmed the high degree of specificity of the reverse hybridization method. Despite the high degree of genetic diversity of H. pylori, the differences between vacA s- and m-region alleles appear to be highly consistent and in almost all cases permitted the use of general PCR primers and allele-specific probes.
The reverse hybridization format also permits rapid and sensitive detection of multiple genotypes in a clinical specimen. This is an important advantage of direct analysis of DNA from a gastric biopsy specimen by PCR and allele-specific hybridization. Investigation of cultured H. pylori strains may considerably underestimate the prevalence of simultaneous infections with multiple strains due to strain selection during in vitro culture.Clinical evaluation. In order to evaluate the PCR-LiPA system, a number of biopsy specimens and isolates from patients in Portugal and The Netherlands comprising patients with NUD, peptic ulcers, or gastric carcinomas were tested.
The prevalence of cagA-positive samples appears to be slightly higher among Portuguese patients than among Dutch patients. This difference was not statistically significant and may be due to the inclusion of patients with gastric carcinomas in the Portuguese group. The majority of the Portuguese strains (72%) contained the s1b allele, whereas most of the Dutch strains (61%) contained type s1a. This highly significant difference is observed in both the NUD and the ulcer patients. Therefore, these results are likely to be indicative of the relative prevalence of vacA s types in both countries. The prevalence of the type s2 allele, as well as the type m1 and m2 alleles, does not appear to be different between strains from Portugal and those from The Netherlands. Since no gastric carcinoma patients from The Netherlands were studied, the prevalence of vacA types within this group could not be compared. The geographic distribution of distinct H. pylori genotypes remains largely unknown. The prevalence of more pathogenic bacterial genotypes in certain areas may have important epidemiological consequences and may be associated with the severity of H. pylori-related diseases in such regions (15). Since it has been demonstrated that H. pylori carries only a single copy of vacA (3, 11, 12), detection of multiple genotypes implies the presence of multiple strains in a clinical sample. The frequency of multiple genotypes in a single biopsy specimen appears to be much higher in Portugal than in The Netherlands (29 and 8%, respectively). This phenomenon may be related to the prevalence of H. pylori infections among the populations in each country. In Portugal, approximately 80% (14) of the adult population is infected, whereas in The Netherlands approximately 30% of the adult population is infected. The risk of coinfection or superinfection with multiple strains may be higher in countries with a high prevalence of H. pylori infection than in countries with a low prevalence. The presence of multiple H. pylori strains, as determined by random amplification of polymorphic DNA from cultured isolates, has been reported earlier (17, 19, 28, 29). In the present study, we confirmed these findings by directly analyzing gastric biopsy specimens for the H. pylori vacA gene. It is unknown whether infection with multiple strains increases the risk of serious clinical implications, such as the development of ulcers and gastric cancer. In conclusion, the present report describes the sequence analysis of the vacA s and m regions of Dutch and Portuguese H. pylori isolates. A rapid and simple PCR-reverse hybridization assay for vacA and cagA genotyping was developed. This assay may facilitate broader investigation of the clinical implications of the colonization with the various H. pylori genotypes. Furthermore, it offers an attractive tool for studying the variability of H. pylori isolates of different geographic origins.| |
ACKNOWLEDGMENTS |
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We thank Ger Scholte, An Lan Kam, Ricardo Sanna, Wink de Boer, and Peter Schneeberger for valuable contributions.
Part of this project was supported by PRAXIS XXI (project 1/2.1/SAU/1356/95).
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FOOTNOTES |
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* Corresponding author. Mailing address: R. de Graafweg 7, P.O. Box 5010, 2625 AD, Delft, The Netherlands. Phone: 31-15-2604577. Fax: 31-15-2604550. E-mail: L.J.van.Doorn{at}ddl.nl.
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Abstract
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Materials & Methods
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Discussion
References
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Journal of Clinical Microbiology, May 1998, p. 1271-1276, Vol. 36, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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