Variation in number of cagA EPIYA-C phosphorylation motifs between cultured Helicobacter pylori and biopsy strain DNA.

Abstract

The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after entering the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in the variable 3'-region. Four different cagA EPIYA motifs have been defined according to the surrounding amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the corresponding DNA extracts it has been suggested that genotyping assays preferentially should be performed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific culturing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed using total DNA isolated from biopsy specimens.

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Short communication
Variation in number of cagA EPIYA-C phosphorylation motifs between cultured
Helicobacter pylori and biopsy strain DNA
Anneli Karlssona, Anna Rybergb, Marjan Nosouhi Dehnoeib, Kurt Borcha, Hans-Jürg Monsteinb,⇑
aDivision of Surgery, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, S-581 85 Linköping, Sweden
bDivision of Clinical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, County Council of Östergötland, S-581 85
Linköping, Sweden
a r t i c l ei n f o
Article history:
Received 28 June 2011
Received in revised form 20 October 2011
Accepted 25 October 2011
Available online 9 November 2011
Keywords:
Gastroduodenal diseases
Helicobacter pylori
cagA EPIYA-C motif variation
Gastric biopsy H. pylori strains
Cultured H. pylori strains
Amplicon sequencing
a b s t r a c t
The Helicobacter pylori cagA gene encodes a cytotoxin which is activated by phosphorylation after enter-
ing the host epithelial cell. Phosphorylation occurs on specific tyrosine residues within EPIYA motifs in
the variable 30-region. Four different cagA EPIYA motifs have been defined according to the surrounding
amino acid sequence; EPIYA-A, -B, -C and -D. Commonly, EPIYA-A and -B are followed by one or more
EPIYA-C or -D motif. Due to observed discrepancies in cagA genotypes in cultured H. pylori and the cor-
responding DNA extracts it has been suggested that genotyping assays preferentially should be per-
formed directly on DNA isolated from biopsy specimens. Gastric biopsies randomly selected from a
Swedish cohort were homogenised and used for both direct DNA isolation and for H. pylori specific cul-
turing and subsequent DNA isolation. In 123 of 153 biopsy specimens, the cagA EPIYA genotypes were in
agreement with the corresponding cultured H. pylori strains. A higher proportion of mixed cagA EPIYA
genotypes were found in the remaining 30 biopsy specimens. Cloning and sequencing of selected cagA
EPIYA amplicons revealed variations in number of cagA EPIYA-C motifs in the mixed amplicons. The study
demonstrates that culturing of H. pylori introduces a bias in the number of EPIYA-C motif. Consistent with
other H. pylori virulence genotyping studies, we suggest that cagA EPIYA analysis should be performed
using total DNA isolated from biopsy specimens.
? 2011 Elsevier B.V. All rights reserved.
1. Introduction
HelicobacterpyloriisamicroaerophilicGram-negativebacterium
that chronically infects the gastric mucosa. It is recognised as a hu-
man pathogen associated not only with chronic gastritis (Marshall
and Warren, 1984), but also with peptic ulcer (Cover and Blaser,
1992) andgastric cancer(Parsonnetet al., 1991). Initially,classifica-
tion of H. pylori was based on the combination of morphologicaland
biochemicalcharacteristicsandgrowthrequirements(Marshalland
Warren,1984).Geneticcriteriahavebecomeincreasinglyimportant
in the identification and characterisation of H. pylori. The cagA gene
is a commonly used molecular marker of H. pylori virulence (Oleas-
troetal.,2009;vanDoornetal.,1998).TheCagAcytotoxinisdirectly
injected into epithelial cells via a type IV secretion system (Akopy-
ants et al., 1998; Covacci et al., 1993; Yamazaki et al., 2003). In the
host cell, CagA localises to the plasma membrane and undergoes
phosphorylation on specific tyrosine residues within repeating
penta amino acid Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs, present at
the C-terminus of the protein (Backert et al., 2001; Hatakeyama,
2003;Higashietal.,2002).The30-endregionofcagAwherethetyro-
sine phosphorylation sites are located are highly polymorphic
(Covacci et al., 1993; Tummuru et al., 1993; Yamaoka et al., 1998;
Yamazaki et al., 2005). Four different CagA EPIYA motifs, EPIYA-A,
-B, -C,and -D, have beendefined based on the aminoacid sequences
surrounding the EPIYA residue (Higashi et al., 2002; Jones et al.,
2009; Panayotopoulou et al., 2007; Sgouras et al., 2009; Yamazaki
et al., 2005). CagA proteins nearly always possess an EPIYA-A and
an EPIYA-B, followed by various number of EPIYA-C repeats in
Western-type(Yamazakietal.,2005)orEPIYA-DmotifsinEastAsian
typestrains(Panayotopoulouetal.,2007;Sgourasetal.,2009).Ithas
beensuggestedthattheconsiderablevariationinnumberofrepeat-
ingEPIYA-Cor-DmotifsdeterminesthebiologicalactivityofCagAin
phosphorylation-dependent as well as phosphorylation-indepen-
dent ways (Costa et al., 2009; Higashi et al., 2002). Furthermore, it
has been shown that the number of CagA EPIYA-C motifs is an
important risk factor for cancer amongst Western strains (Basso
et al., 2008; Batista et al., 2011). A high number of H. pylori CagA
EPIYA-C phosphorylation sites increase the risk of gastric cancer,
but not duodenal ulcer (Basso et al., 2008; Batista et al., 2011;
1567-1348/$ - see front matter ? 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.meegid.2011.10.025
⇑Corresponding author. Address: Department of Clinical Microbiology, Faculty of
Health Sciences, Linköping University, County Council of Östergötland, S-581 85
Linköping, Sweden. Tel.: +46 (0)13 1032475; fax: +46 (0)13 1034596.
E-mail address: hans-jurg.monstein@liu.se (H.-J. Monstein).
Infection, Genetics and Evolution 12 (2012) 175–179
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Infection, Genetics and Evolution
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Chuangetal.,2011),andBatistaandco-workersfurthershowedthat
mixed strain infection was significantly more frequent in patients
with gastric cancer than in those with gastritis.
Most studies on the H. pylori cagA gene have been carried out on
DNAisolatedfromculturedH.pyloriisolatesorfrommucosalbiopsy
specimens (Fujimoto et al., 1994; Gunn et al., 1998; Lopez-Vidal
etal.,2008;Morales-Espinosaetal.,1999;Yamaokaetal.,1998).Dif-
ferentPCR-basedassayshavebeendescribedformoleculartypingof
EPIYA phosphorylation motifs both in gastric biopsy specimens
(Gunn et al., 1998; Monstein et al., 2010; Rota et al., 2001) and in
co-cultured H. pylori isolates (Argent et al., 2005). Some studies
established a correlation between genotypes and disease outcome,
while other studies did not (Acosta et al., 2010; Ahmad et al.,
2009; Sgouras et al., 2009; Shokrzadeh et al., 2009). The question
arises whether the choice of different PCR-based assays used in
thevariousstudiescontributestotheinconsistentresults,orifother
factors may contribute to the result outcome. One such factor may
be the occurrence of mutations, selection of a single strain from a
sample containing mixed strains, or both, when culturing H. pylori
strains (Kraft and Suerbaum, 2005; Marshall et al., 1998). It is still
debated whether or not molecular genotyping of cagA should be
performed on cultured H. pylori strains or biopsy DNA (Gunn et al.,
1998; Kim et al., 2009; Park et al., 2003).
Herein, we compare the number of cagA EPIYA genotypes
between 153 biopsy total DNA and the corresponding DNA isolated
from cultured H. pylori strains using a recently described improved
PCR-based strategy (Monstein et al., 2010; Ryberg et al., 2008).
2. Materials and methods
2.1. Study subjects and tissue collection
Frozen (?80 ?C) gastric biopsy specimens from a gastroscopic
screening study in a randomly selected cohort of the population of
Linköping, Sweden (Borch et al., 2000), were used. The study was
approved by the local ethical committee in Linköping, Sweden
(Dnr. 98007) and conducted in accordance with the Helsinki decla-
ration. From this cohort, 71 individuals with H. pylori infection were
selected and gastroscopic biopsies from antrum, corpus or bulbus
duodeni were analysed. A total of 153 gastric biopsy specimens
from71individuals(59corpus,57antrum,37bulbusduodeni)were
homogenised by grinding. For 51 of the individuals, biopsies from
more than one location were included. The homogenates were then
divided into two parts. Approximately one part was used for direct
automated DNA isolation and whole genome amplification by
means of multiple displacement amplification (MDA), generating
total MDA-DNA (cellular and bacterial DNA), using a Illustra
GenomiPhiV2DNAkit (GE-Healthcare,Uppsala,Sweden)according
to the manufacturer’s instruction.The other part of the homogenate
was used for bacterial culturing using established clinical routine
procedures (Redeen et al., 2011). Subsequent, bacterial DNA was
extracted, followed by multiple displacement amplification gener-
ating H. pylori MDA-DNA (providing equal genotyping conditions
for biopsy and cultured H. pylori strain derived DNA). In both cases,
DNA was isolated using the BioRobot M48 and MagAttract DNA
Mini M48 kit following the manufacturer’s instruction (Qiagen,
Hilden, Germany).
2.2. cagA EPIYA motif sequence analysis
The 30-end of the cagA gene encoding the EPIYA motifs, was
amplified using MDA-DNA derived from biopsy specimens and cul-
tured H. pylori strains. Primers used were M13-cagA.epiya.SE (TGT
AAA ACG ACG GCC AGT CCC TAG TCG GTA ATG GRT TRT CT) and
T7-cagA.epiya.AS (TAA TAC GAC TCA CTA TAG GGT GTG GCT GTT
AGT AGC GTA ATT GTC) (Monstein et al., 2010), tagged with a uni-
versal M13 uni (-21) or T7 sequence, respectively (in italics). PCR
was performed in a final reaction volume of 20 ll, including
10 pmol of each primer, 1 ll of MDA-DNA, and 1? HotStarTaq Mas-
ter mix (Qiagen, Hilden, Germany) using PCR conditions as follows:
95 ?C for 15 min; 30 cycles of 95 ?C for 20 s, 55 ?C for 20 s, 72 ?C for
40 s; and final extension at 72 ?C for 10 min. Prior to DNA sequence
analysis, amplicons were analysed by capillary gel electrophoresis
(CGE) using a QIAxcel system and a QIAxcel DNA Screening kit (Qia-
gen, Hilden, Germany). The cagA EPIYA amplicons were sequenced
using a M13 uni (-21) sequencing primer at a customer sequencing
service (Eurofins MWG Operon, Ebersberg, Germany). The obtained
DNA sequences were analysed using the CLC Bioinformatics DNA
Workbench version 5.5 (CLC-Bio, http://www.clcbio.com). CagA
empty site was verified as described previously (Monstein et al.,
2010).
2.3. Cloning and sequence analysis of cagA amplicons
Amplicons derived from MDA-DNA of five biopsies (Nos. 125C,
242C, 310C, 346A, 346C) (Table 3) were selected and cloned using
a TOPO-TA cloning kit (pCR 2.1-TOPO vector) according to the pro-
tocol (Invitrogen, Carlsbad, USA). One to ten white colonies of each
isolate were picked and used directly in a confirmatory cagA EPIYA
PCR amplification assay as described above. The amplicons were
sequenced using M13-cagA.epiya.SE (described in Section 2.2) as
sequencing primer at a custom sequencing service (Eurofins
MWG Operon).
2.4. 16S rDNA pyrosequencing analysis
For detection and verification of the presence of H. pylori DNA,
the 16S rDNA variable V3 region was amplified using primers
bHJ.HP.JBS.V3.SE (Biotin-CCT AGG CTT GAC ATT GAN AGA A) and
B-V3.AS (ACG ACA GCC ATG CAG CAC CT). PCR amplification was
performed in the same concentrations and conditions as described
in Section 2.2. Prior to sequencing, amplicons were analysed by
CGE using QIAxcel DNA High Resolution kit (Qiagen, Hilden, Ger-
many). Pyrosequencing was carried out using a PyroMark Gold
Q24 kit following the manufacturer’s instruction (Qiagen, Hilden,
Germany). Obtained DNA sequences were aligned and compared
with catalogued H. pylori 26695 [GenBank:NC000915], H. pylori
J99 [GenBank:AE001439], H. pylori Shi470 [GeneBank:CP001072],
and H. pylori P12 [GeneBank:CP001217] sequences using the CLC
Bioinformatics DNA workbench version 5.5 (CLC-Bio, http://
www.clcbio.com).
3. Results
3.1. Overall comparison between biopsy DNA and cultured H. pylori
results
A total of 153 gastric biopsy specimens from 71 individuals
were investigated for cagA EPIYA genotypes. 123 of the samples re-
vealed equal cagA genotypes between biopsy MDA-DNA and the
corresponding cultured H. pylori MDA-DNA. Multiple (two or
more) cagA EPIYA amplicons of different sizes were detected in
16 of these 123 biopsies (Table 1; Fig 1). DNA sequencing of the
single amplicons revealed the presence of different cagA EPIYA mo-
tifs; EPIYA-ABC in 52, -ABCC in 23, -ABCCC in one, -AB in two, -AC
in one, -ACC in one, and -AABC in one of the 123 samples. In 26
biopsies, no cagA amplicons were generated, which was verified
by cagA empty site PCR (Table 1).
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3.2. Variations between biopsy DNA and cultured H. pylori
CGE and sequencing of amplicons derived from biopsy MDA-
DNA and the corresponding cultured H. pylori MDA-DNA revealed
different cagA EPIYA genotypes in 30 of 153 biopsies. In these 30
biopsies, multiple cagA EPIYA amplicons were observed in 21 of
the biopsy MDA-DNA, whereas the corresponding cultured H. pylori
MDA-DNA revealed single amplicons of cagA EPIYA -AB, -ABC or -
ABCC genotypes (Table 2). Two of the 30 biopsy MDA-DNA samples
(Nos. 120C and 290C) yielded single amplicons of cagA EPIYA-ABCC
genotype (Table 2), whereas the corresponding cultured H. pylori
MDA-DNA yielded multiple amplicons. In one sample (No. 152A),
multiple amplicons were generated using biopsy MDA-DNA, how-
ever no amplicon was generated using MDA-DNA derived from
the corresponding cultured H. pylori MDA-DNA. In five biopsies
(144A, 144B, 162B, 228C and 309C), both biopsy MDA-DNA and
the corresponding cultured H. pylori MDA-DNA displayed multiple
amplicons with different size patterns (Fig 1; Table 2).
3.3. Cloning and sequence analysis of selected mixed amplicons
derived from biopsy DNA
Cloningoffiveselectedsampleswithmultipleamplicons(gastric
biopsy DNA Nos. 125C, 242A, 310C, 346A, 346C) and subsequent
sequencingconfirmedconsiderablevariationsinthe numberofEPI-
YA-C motifs within each sample (Table 3). In one case (sample no.
310C),fivedifferentcagAEPIYA-Cgenotypes(ABCC,ABCCC,ABCCCC,
ABCCCCC and ABCCCCCC) were identified (Table 3). Similar varia-
tions in the number of EPIYA-C motifs were observed in the other
cloned amplicons. Only one cagA EPIYA-ABCC genotype could be
established from cultured H. pylori isolate No. 242A, since cloning
of the amplicon yielded only one colony (Table 3).
3.4. 16S rDNA pyrosequencing
16S rDNA pyrosequencing revealed the presence of H. pylori
DNA in all biopsy specimens. DNA sequence comparison with cat-
alogued sequences revealed the presence of 16S rDNA V3 se-
quences corresponding to H. pylori 26695 in 80 of 153, H. pylori
27C
120B
120C
144A
144B
162B
228C
309C
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
Biopsy:
Culture:
single
single
multiple
single
single
multiple
multiple
multiple
multiple
multiple
multiple
multiple
multiple
multiple
multiple
multiple
Fig. 1. Superimposed electropherograms of cagA EPIYA amplicons with diverging
amplicon patterns derived from DNA isolated from eight selected gastric biopsy
samples (red), and from DNA isolated from the corresponding H. pylori cultures
(blue). First and last peak in each electropherogram indicates internal alignment
markers. Each peak between the alignment markers indicates the presence of one
cagA EPIYA genotype. Although multiple cagA EPIYA amplicons were detected in
biopsy total DNA and the corresponding DNA isolated from cultured H. pylori
strains, in five of the eight samples (228C, 144A, 144B, 162B, 309C) the size pattern
for each amplicon mix was unique. Single = one amplicon; multiple = two or more
amplicons.
Table 2
CagA EPIYA genotype differences between biopsy and culture H. pylori DNA.
aBiopsy no. CagA EPIYA genotype
BiopsyCulture
346A
1A
110C
120B
121A
121C
154C
201C
242A
273C
275C
281A
346C
26C
125A
125C
193C
273B
310C
352C
372C
152A
144A
144B
162B
228C
309C
120C
290C
27C
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strains
Mixed strainsb
Mixed strainsb
Mixed strainsb
Mixed strainsb
Mixed strainsb
ABCC
ABCC
ABC
AB
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABC
ABCC
ABCC
ABCC
ABCC
ABCC
ABCC
ABCC
ABCC
Empty site
Mixed strainsb
Mixed strainsb
Mixed strainsb
Mixed strainsb
Mixed strainsb
Mixed strains
Mixed strains
AB
aA, antrum; B, duodenum; C, corpus.
bBoth biopsy and culture contained multiple amplicons, however not identical
size patterns (Fig 1).
Table 1
CagA EPIYA genotypes revealed in biopsies.
CagA EPIYANo. of biopsy specimens Results (compared to culture)
Equal Not equal
Mixed strains
ABC
ABCC
ABCCC
AB
AABC
AABCC
AC
ACC
Empty site
37
55
28
1
2
1
1
1
1
26
16
52
23
1
2
1
21
3
5
1
1
1
26
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J99 in 28 of 153, H. pylori 26695/J99 in 34 of 153, and H. pylori
strain A in 9 of 153 biopsy specimens. In one biopsy each (Nos.
71C and 75C), the pyrogram revealed the presence of two 16S
rDNA V3 motifs corresponding to H. pylori 26695 and 26695/J99,
and 26695 and J99, respectively.
4. Discussion
Mutation and recombination occurring in the H. pylori genome
are considered to be responsible for generating strain diversity
(Kraft and Suerbaum, 2005). In this view, it is assumed that foun-
der strains of H. pylori, which initially colonise the gastric mucosa,
undergo microevolution of their genome structure over a relative
short period of time, generating H. pylori strains with highly similar
genomes that display minor genetic differences (Carroll et al.,
2004; Marshall et al., 1998). The general view is that microevolu-
tion occurs in most, if not all H. pylori strains. Therefore, it is con-
ceivable that adaptation over time of individual H. pylori strains to
different environmental conditions (biopsy specimen vs. cultured
strains) may in part be responsible for the observed discrepancies
reported in associating bacterial genotypes to diseases. Further-
more, a recent study has revealed that adaptive evolution may oc-
cur especially in host interaction genes, such as the cagA, resulting
in proteome diversification (Kawai et al., 2011).
It has been discussed that PCR-based genotyping directly from
biopsy specimens tend to underestimate the prevalence of H. pylori
specific virulence genes (Park et al., 2003; Secka et al., 2011). This
may be due to limited access of H. pylori DNA, inhibition of PCR
amplification due to high level of cellular genomic DNA, other PCR
inhibitorsorpotentnucleasesingastricbiopsyspecimens(Monstein
et al., 2005; Park et al., 2003; Thoreson et al., 1999). Whole genome
amplification by multiple displacement amplification (MDA) can be
used as a pre-PCR amplification step under conditions where PCR
amplifications normally are hampered due to presence of inhibitors
(Gonzalez et al., 2005) or where the amount of DNA is not sufficient
for analysis (Ryberg et al., 2008). In this view, our previous and
present studies have shown that PCR using MDA-DNA derived from
biopsy DNA provides a reliable source for multiple molecular
genotyping analysis (Monstein et al., 2010; Ryberg et al., 2008).
In this study, the majority of the cultured H. pylori cagA EPIYA-C
genotypes corresponded with the biopsy genotypes, but discrepan-
cies were observed in 30 of the 153 biopsies (20%; Table 2). Simi-
larly, Kim and co-workers showed that the inconsistent cagA
genotyping results between cultured H. pylori strain DNA and
biopsy DNA were 16% (Kim et al., 2009).
Different methodological approaches using either biopsy DNA
or cultured strains to verify the presence of mixed H. pylori strains
have shown conflicting results (Batista et al., 2011). Secka and
co-workers have suggested that both biopsy DNA and cultured H.
pylori should be analysed concomitantly (Secka et al., 2011). Park
and co-workers have suggested that studies identifying associa-
tions between virulence factors and disease outcome should be re-
stricted to sites with rare mixed H. pylori strain infection. However,
this might lead to false perception of the actual relationship of bac-
terial strains and disease outcome (Park et al., 2003). Furthermore,
they observed a higher proportion of mixed H. pylori strain infec-
tion in biopsy specimens (27%) compared to cultured H. pylori
strains (9%) (Park et al., 2003). Similarly, based on cagA EPIYA geno-
typing we detected a higher proportion of mixed H. pylori strains in
biopsy specimens (24%) compared to cultured H. pylori strains
(11%). Cloning of cagA amplicons and subsequent sequence analy-
sis was able to provide further information concerning the varia-
tion of cagA EPIYA genotypes. None of the methods described
provided information whether or not the genotype variations were
due to mixed H. pylori strain infection or arise within the stomach
from an ancestor H. pylori strain as suggested in an early study by
Yamaoka and co-workers (Yamaoka et al., 1999).
In view of a recent study (Sheu et al., 2009) where it was sug-
gested that H. pylori infection at different sites of the stomach in
the same patient could change the histological features in the an-
trum and the corpus, establishing of a correct number of cagA EPI-
YA-C motifs appears to be crucial for assessing links between H.
pylori strains and gastroduodenal diseases. However, it is still not
known whether or not certain threshold concentrations of individ-
ual H. pylori strains (quantification) present in biopsy specimens
have an impact on the disease outcome. Consequently, we believe
that it is important to genotype all H. pylori strain variations pres-
ent in a biopsy specimen. So far, molecular biology based methods
do not allow for an unequivocal discrimination between mixed H.
pylori strain infection or infection with an H. pylori founder strain
undergoing microevolution (Carroll et al., 2004; Kraft and Suer-
baum, 2005; Marshall et al., 1998). Consistently with other studies,
we recommend that molecular typing of total DNA (human and
bacterial DNA) isolated directly from biopsy specimens should be
performed. Moreover, the improved PCR-based strategy provides
a promising tool for high throughput molecular typing of H. pylori
strains in a clinical routine microbiology laboratory.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AK, AR, MND, KB, HJM participated in the conception, design,
data interpretation and drafting of the manuscript. AK, AR, MND
performed molecular genotyping. KB collected and selected the
biopsy specimens. All authors have read and approved to the
manuscript.
Acknowledgments
This study was supported by grants from the Research council
in the South-East of Sweden (FORSS), the ALF-program, and the
Molecular Biology Program at Clinical Microbiology, Laboratory
Centre-DC, University Hospital, Linköping, Sweden.
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5
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2
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2
aA, antrum; C, corpus.
bCGE, capillary gel electrophoresis.
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