Research in Microbiology 163 (2012) 109e113

www.elsevier.com/locate/resmic

Real-time PCR for Helicobacter pylori quantification and detection

of clarithromycin resistance in gastric tissue from patients
with gastrointestinal disorders
Mohammad Kargar a,*, Sadegh Ghorbani-Dalini b, Abbas Doosti c, Negar Souod a
b

a
Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
Department of Microbiology, Jahrom Branch, Young Researchers Club, Islamic Azad University, Jahrom, Iran
c
Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

Received 3 June 2011; accepted 16 November 2011

Available online 2 December 2011

Abstract
Helicobacter pylori gram-negative bacteria commonly infect the human gastrointestinal (GI) tract and are readily diagnosed by endoscopy.
H. pylori infection causes a broad range of host symptoms from discomfort to significant GI disorders (GIDs). Severity of the clinical manifestations depends mainly upon bacterial load. In this cross-sectional study, we investigated the affects of 23S rRNA point mutations on H. pylori
count in naturally infected human GI tissues. Two-hundred H. pylori patients with suspected GIDs were evaluated to determine bacteria
concentration and presence of four known 23S rRNA point mutations, causing clarithromycin resistance. Gastric biopsy specimens were
examined by rapid urease test and 16S rRNA-targeted PCR to identify H. pylori; then bacterial load was quantified by real-time PCR targeting
wild type and known 23S rRNA mutations. Eighty-two percent of the samples were confirmed as H. pylori-positive, having 104e1012 colonyforming units (CFU)/ml. The 106 load was most strongly associated with peptidyltransferase point mutations of the 23S rRNA gene A2144G
( p 0.033), A2143G ( p 0.005), A2143C ( p 0.005), and A2142G ( p 0.015). Thus, our findings indicated that dominant 23S rRNA
mutated H. pylori strains have the same growth rate as the wild type in a gastric environment.
2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Helicobacter pylori; Bacterial load; 23S rRNA; Point mutations; Real-time PCR

1. Introduction
Helicobacter pylori represents the most commonly diagnosed chronic gastric bacterial infection in the world (Oleastro
et al., 2003) and is a major cause of gastritis, peptic ulcer disease
and gastric cancer (Kargar et al., 2010). Severity of the clinical
manifestations of this infection is associated with bacterial
load (Kusters et al., 2006). The preferred eradication therapy is
a combination triple antibiotic consisting of metronidazole,
omeprazole and clarithromycin, which boasts a 90% success
* Corresponding author.
E-mail addresses: mkargar@jia.ac.ir (M. Kargar), ghorbanidalini@jia.ac.ir
(S. Ghorbani-Dalini), abbasdoosti@yahoo.com (A. Doosti), negarsouod@
yahoo.com (N. Souod).

rate. However, the efficacy of this therapeutic strategy drops to

between 0 and 50% when clarithromycin-resistant H. pylori
strains are present (Chisholm et al., 2001; Oleastro et al., 2003).
Clarithromycin acts to halt bacterial growth by physically
interacting with the 23S rRNA component of the bacterial
ribosome, a key component in the protein synthesis machinery.
Some H. pylori strains have evolved resistance to clarithromycin by mutation of the genetic sequence in the targeted
binding site. Specifically, single spontaneous point mutations
in the peptidyltransferase-coding region of the 23S rRNA gene
have been implicated in generation of the resistant phenotype
(Kargar et al., 2011a; Lascols et al., 2003). The four most
often observed mutations are: A2144G, A2143G, A2142G
and A2143C. Other mutations, such as A2142C, A2115G,
G2141A, A2142T and T2717C, have been reported but to

0923-2508/$ - see front matter 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2011.11.005

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M. Kargar et al. / Research in Microbiology 163 (2012) 109e113

a much lesser extent (Lascols et al., 2003; Van Der Ende et al.,
2001).
Determination of H. pylori density in gastric mucosa
biopsies is used to stratify patients for treatment and determine
prognosis; this is historically carried out in clinic by bacterial
culture and histological analysis of infected tissues. However,
both of these approaches are time-consuming and the histological observations are limited by their subjective nature
(Lascols et al., 2003). More recently, PCR-based diagnostics
have been introduced, including amplification of either the
ureC or 16S rRNA bacterial genes.
In this study we sought to develop a more quantitative realtime PCR-based diagnostic strategy based on the 23S rRNA
gene. The advantages of this approach were expected to be
threefold. First, by using whole samples of DNA isolated
directly from gastric biopsy tissues of infected individuals,
the presence of multiple H. pylori strains would be simultaneously detectable from a single sample. Second, the genomic
sequence of the H. pylori strains would reveal the presence or
absence of mutations associated with the clarithromycinresistant phenotype. Third, the obtained sequences would be
applicable to comparative analysis studies to determine the
relationship with associated bacterial properties (growth) and
the prevalence of certain point mutations.
2. Material and methods
2.1. Study design
Gastric biopsy specimens were obtained from 200 patients
from the Chaharmahal and Bakhtiari province who had been
referred to local hospitals for upper gastrointestinal tract
endoscopies to diagnose dyspeptic symptoms between June and
November 2009. All patients provided written informed consent
prior to endoscopy. Two antral samples and one corpus sample
were taken from each patient. H. pylori positivity was initially
determined by the standard rapid urease test (RUT) using the
Gastro Urease kit (Bahar-afshan, Co., Tehran, Iran). All of the
samples were transported to the laboratory under a strict cold
chain and stored at 70  C until further investigation. DNA was
isolated from each tissue by using the DNA extraction kit DNP
from CinnaGen (Tehran, Iran) according to the manufacturers
instructions. DNA was immediately subjected to molecular
analysis, including conventional PCR (for H. pylori diagnostic)
and real-time PCR (for H. pylori diagnostic, quantification and
identification of clarithromycin-resistant genotype).
2.2. Confirmation of H. pylori in gastric biopsy
specimens
PCR targeting the 16S rRNA gene was carried out to
confirm H. pylori in each specimen. The previously designed
and verified primer pair HP-1 and HP-2 was synthesized for
this purpose (Kargar et al., 2011b) (Table 1). PCR amplification of genomic DNA was performed in a reaction mixture of
25 ml which contained 2 ml biopsy-isolated DNA template,
1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates

Table 1
Oligonucleotide sequences used in this study.
Primer/probe

Sequence (50 / 30 )

Target

HP-1
HP-2
HP23S-1
HP23S-2
Pwt
P44G
P43G
P43C
P42G

CTGGAGAGACTAAGCCCTCC
ATTACTGACGCTGATTGTGC
CCACAGCGATGTGGTCTCAG
CTCCATAAGAGCCAAAGCCC
Cy5-GGGGTCTTTCCGTCT-BHQ2
TAMRA-GGTCCTTCCGTCTTG-Dabcyl
TET-GGTCTCTCCGTCTTG-Dabcyl
HEX-GGTCTGTCCGTCTTG-Dabcyl
FAM-GGTCTTCCCGTCTTG-Dabcyl

16S rRNA
23S rRNA
Wild type
A2144G
A2143G
A2143C
A2142G

(dNTPs) mixture, 0.2 mM of each primer and 1 U of Taq DNA

polymerase (CinnaGen, Co., Tehran, Iran). The following
thermal-cycling conditions were used: initial denaturation at
95  C for 5 min; 30 cycles of 95  C for 1 min, 58  C for 1 min
and 72  C for 1 min; and a final extension at 72  C for 5 min.
The resulting Hp16S fragment (expected size of 109 base
pairs) was visualized after electrophoresis on a 1.5% agarose
gel stained with ethidium bromide.
2.3. Identification of the clarithromycin resistance
genomic mutation
TaqMan real-time PCR was performed with the primer pair
HP23S-1 and HP23S-2 and either modified probes Pwt, P44G,
P43G and P43C (previously reported by Pina et al.,) or newly
designed probe P42G (according to the 23S rRNA gene
sequence from GenBank accession no. U27270) in order to
identify wild type, A2144G, A2143G, A2143C and A2142G
genotypes, respectively (Table 1).
Real-time PCR analysis was performed on a Rotor-Gene
6000 (Corbett Research, Sydney, Australia). Briefly, the
25 ml PCR mixture containing 2 ml of extracted DNA, 200 mM
dNTPs (CinnaGen, Co., Tehran, Iran), 0.2 mM (each) primer
HP23S-1 and HP23S-2, 0.1 mM probe Pwt, 0.2 mM probe
P44G, 0.1 mM probe P43G, 0.1 mM probe P43C and 0.75 mM
probe P42G (Bioneer, Daejeon, Korea), 1.5 mM MgCl2, and
1.5 U of Taq polymerase in PCR buffer (CinnaGen, Co.,
Tehran, Iran), was denatured at 95  C for 5 min; amplification
was carried out in 45 cycles of 95  C for 30 s and 58  C for
40 s. The fluorescence reading for each sample was taken at
the annealing step on different channels. All samples were run
in duplicate and positive and negative controls were included
in each assay. Data were analyzed with instrument compliant
software (ver1.7; Corbett Research). In order to test the
specificity of the primers, purified DNA from non-H. pylori
bacterial strains was used as template. The cycle threshold
(Cq) value was calculated for each channel as the number of
cycles at which fluorescence exceeded the threshold limit,
which was set at the top of the second derivative fluorescence
curve and expressed as fractional cycle numbers.
2.4. Determination of bacterial load by real-time PCR
Standard curves were generated by PCR of serial dilutions.
After DNA extraction, one 10-fold serial dilution of H. pylori

M. Kargar et al. / Research in Microbiology 163 (2012) 109e113

DNA was made (ranging from 5  102 to 5  107 bacteria per

1 ml). Bacterial quantification was determined by TaqMan
probe-based real-time PCR, as described above. Positive
controls included H. pylori J99 for wild type and strains with
our mutation (previously identified by sequencing); the negative control included H. pylori with another mutation (for
example, a strain with the A2143G mutation was used as
control for non-A2143G mutation strains).
2.5. Statistical analysis
Comparisons of intergroup differences were conducted by
the chi-square test using the SPSS for Windows statistical
software (version 17; SPSS, Inc., Chicago, IL, USA). p-values
less than 0.05 were considered as statistically significant.
3. Results
3.1. Direct detection of H. pylori in gastric biopsy
samples
RUT results showed that the majority (164 of 200; 82%) of
gastric biopsy samples were H. pylori-positive. All RUTpositive samples that were checked by means of 16S rRNA
gene PCR (n 164) were also positive according to the
diagnostic PCR assay.
3.2. Determination of the cut-off for real-time PCR Cq
values
Among the 164 biopsy specimens, detection of fluorescence was positive in at least one of the different channels for
143 (87.2%) cases. Sixteen biopsy specimens displayed more
than one Cq value, indicating mixed genotypes. Cq values
ranged from 21.4 to 43.6. Cq values less than 35 were determined as pure samples containing a single genotype, while Cq
between 35 and 40 indicated mixed samples. Detection of
fluorescence over 40 cycles was considered as indicative of
a false-positive.
3.3. Development of TaqMan real-time PCR
When DNA from cultured H. pylori was amplified, linearity
was achieved over a 5-log range of input DNA amounts, from
5  102 to 5  107 bacteria/ml. PCR identified 40 bacteria by
detection of 80 copies of the 23S rRNA gene fragment, since
there are two gene copies in the genome of H. pylori (Alm
et al., 1999; Tomb et al., 1997).
The amplification efficiencies obtained with DNA prepared
from gastric biopsy specimens were identical to those obtained
with DNA samples prepared from bacterial colonies. Thus, the
sensitivity of our assay, for DNA samples prepared either from
cultures or from gastric biopsy specimens, could quantitatively
detect up to 400 bacteria or 800 copies of the amplicon per
PCR reaction, and could also qualitatively determine the
presence of up to 40 bacteria or 80 copies per sample. When
we examined the reproducibility of our assay by using 10-fold

111

serial dilutions of purified H. pylori DNA, no significant

differences between runs were observed.
3.4. Determination of mutant strain prevalence in
infected tissues by real-time PCR
A total of 164 gastric biopsy samples were positive for H.
pylori DNA by real-time PCR, and real-time PCR was able to
detect the genotype associated with clarithromycin susceptibility or resistance in 159 of the 164 biopsies (97.0%) tested.
One-hundred and five (64.0%) of the samples were determined
to be pure wild type and 59 (36.0%) were found to carry
resistance mutations. Thirty-eight (23.2%) of the samples were
determined to have only a single mutation, and the specific
point mutations were distributed as follows: 3 samples with
A2144G (1.8%), 13 A2143G (7.9%), 7 A2143C (4.3%) and 15
A2142G (9.15%). When only one type of the 23S rRNA gene
was detected, the sample was considered to be a pure infection. Sixteen samples (9.8%) were determined to be mixes of
different mutations and wild type DNA. Only five of the
samples (3%) yielded ambiguous or negative results with these
probes.
3.5. Quantification of bacterial density
After the genotype was defined, quantification of the
bacterial load was directly performed on samples using the
TaqMan real-time-PCR assay. One-hundred-and-fifty-nine H.
pylori-positive DNA samples were evaluated and bacterial
density was found to range from 1.5  104 to 5.9  1012. In
order to evaluate the relationship between bacterial concentration and point mutations, samples were divided into nine
groups of different bacterial loads, i.e. 104 (n 6), 105
(n 11), 106 (n 87), 107 (n 59), 108 (n 9), 109 (n 2),
1010 (n 2), 1011 (n 2) and 1012 (n 2) bacteria/ml of
sample (Table 2). Distribution of mixed genotypes in the same
samples is presented in Table 3.
Comparison between the genotypes present and the bacterial load revealed a statistically significant relationship
between wild type and 106 ( p < 0.001) and 107 ( p < 0.001)
bacterial density. Moreover, the four common clarithromycinresistant genotypes were each significantly associated with 106
density: A2144G ( p 0.033), A2143G ( p 0.005), A2143C
( p 0.005), and A2142G ( p 0.015).
4. Discussion
In this study, we developed an easy-to-use, real-time PCR
technique to amplify the H. pylori 23S rRNA gene with TaqMan probes in order to rapidly and accurately detect infection.
In addition, this sequence-based technique is capable of
identifying clarithromycin-resistant strains by quantitatively
detecting known mutations in the more prevalent alleles of this
gene. In particular, the genotypes associated with the wild
type, clarithromycin sensitivity and clarithromycin resistance
(via four well-defined point mutations) can be revealed.
Finally, this technique is capable of quantifying the H. pylori

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M. Kargar et al. / Research in Microbiology 163 (2012) 109e113

Table 2
Number of samples with loads of each H. pylori genotype.
Bacterial
count

Wild type

A2144G

A2143G

N(%)

N(%)

N(%)

N(%)

pa

10
105
106
107
108
109
1010
1011
1012

1
7
39
51
9
2
2
2
2

0.260
0.732
0.00b
0.00b
0.06
1.00
1.00
1.00
1.00

0
0
4
0
0
0
0
0
0

1.00
1.00
0.033b
0.300
1.00
1.00
1.00
1.00
1.00

2
2
18
4
0
0
0
0
0

0.179
0.687
0.005b
0.041
0.0357
1.00
1.00
1.00
1.00

2 (13.33)
1(6.67)
12 (80)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0.067
1.00
0.005b
0.03
1.00
1.00
1.00
1.00
1.00

1(5)
1(5)
14 (70)
4 (20)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)

0.483
1.00
0.015b
0.21
0.602
1.00
1.00
1.00
1.00

Total

115

a
b

(0)
(0)
(100)
(0)
(0)
(0)
(0)
(0)
(0)

A2142G

(0.87)
(6.08)
(33.91)
(44.35)
(7.83)
(1.74)
(1.74)
(1.74)
(1.74)

A2143C

N(%)

(7.69)
(7.69)
(69.23)
(15.38)
(0)
(0)
(0)
(0)
(0)

26

15

20

p value.
Statistically significant.

bacterial load, including the loads of the resistant mutants,

directly from gastric biopsy specimens.
The biopsy specimens used in this study were not fresh;
they required approximately seven days of storage from
excision until processing by the laboratory. A principal
advantage of PCR-based techniques is that they do not require
viable bacteria, in contrast to culture-based techniques. Thus,
when using this newly developed technique for H. pylori
diagnosis, the transport conditions from the clinic to the lab
are not as critical and shipments may be more cost-effective
(Burucoa et al., 2008).
Previous H. pylori investigations by Wang and colleagues
sought to determine the patterns of competitive growth and of
individual growth of different clarithromycin-resistant mutant
strains from clinical samples. Their study defined the order of
preference for competitive accumulation in vitro as:
A2142G > A2143G >>> A2142C > A2143C (A2143T). If
the same is true in vivo, then once an A-to-G transient mutation occurs (spontaneously or pharmacologically induced), the
other types of mutation that exist in the same environment, if
any, are likely to be replaced after a certain period of time.
Indeed, Wang et al. (1999) reported that A-to-C and A-to-T
mutants were only isolated when an A-to-G mutant had not yet
appeared at that particular gastric niche. Their results provide

a rational explanation for the genomic profile of mutations

observed in clinical isolates. These findings were confirmed in
a study by Van Der Ende et al. (2001), who noted that the
growth rates of H. pylori isolates that carried the A2142G,
A2143G or A2142C mutation did not differ from that of the
wild type, whereas H. pylori isolates with other 23S rDNA
mutations grew more slowly.
When regarding the point mutations conferring resistance
to clarithromycin, we found that A2143G was dominant
(n 26), followed by A2142G, A2143C and A2144G mutations, respectively. Although the prevalence of the A2143G
mutation varies between different regions, e.g. Spain 79.4%
(Agudo et al., 2011), Brazil 74% (Lins et al., 2010) and
Tunisia 88% (Mansour et al., 2010), it is the dominant mutation in many studies.
We used real-time PCR for simultaneous identification of H.
pylori, clarithromycin-resistance-related point mutations and
direct quantification of bacterial density in the affected gastric
mucosa. We were also interested in determining why some
clarithromycin resistance point mutations are more prevalent in
clinical strains than are other mutations. We first considered the
validity of Wangs proposal that A-to-G mutations were more
common in H. pylori (Wang et al., 1999), and found that to be
the case in our sample cohort, because 50 samples were A-to-G

Table 3
Distribution of bacterial load in mixed samples.
Genotype

Load
Wild

A2144G

A2143G

A2143C

A2142G

A2143G/Wild
A2143G/Wild
A2143G/Wild
A2143C/Wild
A2142G/Wild
A2143G/A2143C/Wild
A2144G/A2143G/A2143C/Wild
A2143G/A2143C
A2143G/A2142G
A2143G/A2142G
A2143G/A2143C/A2142G
A2143G/A2143C/A2142G

3
2
1
1
1
1
1
2
1
1
1
1

106
107
107
107
107
105
105
e
e
e
e
e

e
e
e
e
e
e
106
e
e
e
e
e

106
106
107
e
e
105
104
107
106
106
105
104

e
e
e
106
107
106
104
106
e
e
104
105

e
e
e
e
e
e
e
e
107
106
104
105

M. Kargar et al. / Research in Microbiology 163 (2012) 109e113

mutations. We next considered the proposal by Van Der Ende

et al. (2001) that evaluation of only a single isolated colony as
representative of the entire H. pylori population infecting an
individual might lead to a patient being diagnosed as
clarithromycin-sensitive when, in fact, they are infected with
clarithromycin-resistant strains that would survive treatment.
Our sequencing-based strategy did indeed identify numerous
patients (21 samples) with mixed genotypes of H. pylori, validating the idea that all patient samples should be examined for
diagnosis and for planning treatment.
Several studies by others to determine the 23S rRNA
genotype of H. pylori infections in Europe revealed that
A2144G, A2143G and A2143C were prevalent in this mainly
Caucasian population (Marais et al., 1999; Pina et al., 1998;
Russmann et al., 2001). Our study focused on individuals in
Iran and showed that this population was afflicted by the same
three mutant strains, but also the A2142G mutant.
In conclusion, our data show that wild type H. pylori strains
reach a bacterial load of 106 to 107 bacteria/ml of sample in
naturally-infected human gastric tissues. Strains with clarithromycin resistance causing mutations reach a load of 106,
indicating that this mutant strain is as infective as the wild
type, but more robust in terms of survival under conditions of
standard antibiotic treatment. Nonetheless, the dominant
mutated strains have the same growth rate as wild type in
culture and biopsy samples in human infections, further suggesting that a mechanism of natural selection force is at play.
Acknowledgments
The authors are grateful to the Islamic Azad University,
Jahrom and Shahrekord branches, for their executive support
of this project.
References
Agudo, S., Perez-Perez, G., Alarcon, T., Lopez-Brea, M., 2011. Rapid
detection of clarithromycin resistant Helicobacter pylori strains in Spanish
patients by polymerase chain reaction-restriction fragment length polymorphism. Rev. Esp. Quimioter 24, 32e36.
Alm, R.A., Ling, L.S., Moir, D.T., King, B.L., Brown, E.D., Doig, P.C.,
Smith, D.R., Noonan, B., Guild, B.C., deJonge, B.L., Carmel, G.,
Tummino, P.J., Caruso, A., Uria-Nickelsen, M., Mills, D.M., Ives, C.,
Gibson, R., Merberg, D., Mills, S.D., Jiang, Q., Taylor, D.E., Vovis, G.F.,
Trust, T.J., 1999. Genomic-sequence comparison of two unrelated isolates
of the human gastric pathogen Helicobacter pylori. Nature 397, 176e180.
Burucoa, C., Garnier, M., Silvain, C., Fauchere, J.L., 2008. Quadruplex realtime PCR assay using allele-specific scorpion primers for detection of
mutations conferring clarithromycin resistance to Helicobacter pylori. J.
Clin. Microbiol. 46, 2320e2326.
Chisholm, S.A., Owen, R.J., Teare, E.L., Saverymuttu, S., 2001. PCR-based
diagnosis of Helicobacter pylori infection and real-time determination of

113

clarithromycin resistance directly from human gastric biopsy samples. J.

Clin. Microbiol. 39, 1217e1220.
Kargar, M., Baghernejad, M., Doosti, A., 2010. Role of NADPH-insensitive
nitroreductase gene to metronidazole resistance of Helicobacter pylori
strains. DARU 18, 137e140.
Kargar, M., Baghernejad, M., Doosti, A., Ghorbani-Dalini, S., 2011a. Clarithromycin resistance and 23S rRNA mutations in Helicobacter pylori
isolates in Iran. Afr. J. Microbiol. Res. 5, 853e856.
Kargar, M., Ghorbani-Dalini, S., Doosti, A., Baghernejad, M., 2011b.
Molecular assessment of clarithromycin resistant Helicobacter pylori
strains using rapid and accurate PCR-RFLP method in gastric specimens in
Iran. Afr. J. Biotechnol. 10, 7675e7678.
Kusters, J.G., van Vliet, A.H.M., Kuipers, E.J., 2006. Pathogenesis of Helicobacter pylori infection. Clin. Microbiol. Rev. 19, 449e490.
Lascols, C., Lamarque, D., Costa, J.M., Copie-Bergman, C., Glaunec, J.M.L.,
Deforges, L., Soussy, C.J., Petit, J.C., Delchier, J.C., Tankovic, J., 2003.
Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H. pylori isolates from
gastric biopsy specimens by real-time PCR. J. Clin. Microbiol. 41,
4573e4577.
Lins, A., Klima, R.A., Magalhaes, M., 2010. Clarithromycin-resistant Helicobacter pylori in Recife, Brazil, directly identified from gastric biopsies
by polymerase chain reaction. Arq. Gastroenterol. 47, 379e382.
Mansour, K.B., Burucoa, C., Zribi, M., Masmoudi, A., Karoui, S., Kallel, L.,
Chouaib, S., Matri, S., et al., 2010. Primary resistance to clarithromycin,
metronidazole and amoxicillin of Helicobacter pylori isolated from
Tunisian patients with peptic ulcers and gastritis: a prospective multicentre
study. Ann. Clin. Microbiol. Antimicrob. 9, 22. Available: http://www.annclinmicrob.com/content/9/1/22.
Marais, A., Monteiro, L., Occhialini, A., Pina, M., Lamouliatte, H.,
Megraud, F., 1999. Direct detection of Helicobacter pylori resistance to
macrolides by a polymerase chain reaction/DNA enzyme immunoassay in
gastric biopsy specimens. Gut 44, 463e467.
Oleastro, M., Menard, A., Santos, A., Lamouliatte, H., Monteiro, L.,
Barthelemy, P., Megraud, F., 2003. Real-time PCR assay for rapid
and accurate detection of point mutations conferring resistance to
clarithromycin in Helicobacter pylori. J. Clin. Microbiol. 41,
397e402.
Pina, M., Occhialini, A., Monteiro, L., Doermann, H.P., Megraud, F., 1998.
Detection of point mutations associated with resistance of Helicobacter
pylori to clarithromycin by hybridization in liquid phase. J. Clin. Microbiol. 36, 3285e3290.
Russmann, H., Adler, K., Haas, R., Gebert, B., Koletzko, S., Heesemann, J.,
2001. Rapid and accurate determination of genotypic clarithromycin
resistance in cultured Helicobacter pylori by fluorescent in situ hybridization. J. Clin. Microbiol. 39, 4142e4144.
Tomb, J.F., White, O., Kerlavage, A.R., Clayton, R.A., Sutton, G.G.,
Fleischmann, R.D., Ketchum, K.A., Klenk, H.P., et al., 1997. The complete
gene sequence of the gastric pathogen Helicobacter pylori. Nature 388,
539e547.
Van Der Ende, A., Van Doorn, L.J., Rooijakkers, S., Feller, M., Tytgat, G.N.J.,
Dankert, J., 2001. Clarithromycin-susceptible and -resistant Helicobacter
pylori isolates with identical randomly amplified polymorphic DNA-PCR
genotypes cultured from single gastric biopsy specimens prior to antibiotic
therapy. J. Clin. Microbiol. 39, 2648e2651.
Wang, G., Rahman, M.S., Humayun, M.Z., Taylor, D.E., 1999. Multiplex
sequence analysis demonstrates the competitive growth advantage of the
A-to-G mutants of clarithromycin-resistant Helicobacter pylori. Antimicrob. Agents Chemother. 43 (3), 683e685.