Previous Article | Next Article 
Journal of Clinical Microbiology, April 2001, p. 1217-1220, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1217-1220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
PCR-Based Diagnosis of Helicobacter
pylori Infection and Real-Time Determination of Clarithromycin
Resistance Directly from Human Gastric Biopsy Samples
Stephanie A.
Chisholm,1
Robert J.
Owen,1,*
E. Louise
Teare,2 and
Seth
Saverymuttu3
Laboratory of Enteric Pathogens, Central
Public Health Laboratory, London NW9 5HT,1
Public Health Laboratory, Chelmsford CM2
OYX,2 and Broomfield Hospital,
Chelmsford CM1 7ET,3 United Kingdom
Received 6 September 2000/Returned for modification 20 November
2000/Accepted 12 January 2001
 |
ABSTRACT |
A novel PCR detection assay that amplifies the Helicobacter
pylori-specific vacuolating cytotoxin gene (vacA) and
thus enables rapid diagnosis of infection is described. Additionally, a
real-time probe hybridization melting point analysis assay to detect
all three mutations in the 23S rRNA gene associated with clarithromycin resistance was applied directly to antral gastric biopsy samples. Comparison with culture and an alternative PCR assay targeting the 16S
rrn gene showed that the vacA assay was
sensitive and specific when tested on biopsy samples from 121 patients.
Clarithromycin susceptibilities could be determined in the majority
(92.3%) of culture-positive gastric biopsy samples analyzed, four of
which generated melting peaks indicative of clarithromycin resistance by either an A
G or AC mutation. The presence of the mutations correlated with the clarithromycin disk diffusion sensitivities of
matched cultures. This PCR-based system was simple to perform and could
be completed in 3 to 4 h, thereby overcoming the delays associated
with conventional culture methods for H. pylori
identification and susceptibility testing.
| |
INTRODUCTION |
Helicobacter pylori, a
major cause of chronic gastritis, is strongly associated with the
development of gastric and duodenal ulcers and has been linked with
gastric adenocarcinoma and B-cell mucosa-associated lymphoid tissue
lymphoma (15, 17, 18). Infection can be eradicated in up
to 90% of patients using current combination triple therapies, of
which the macrolide antibiotic clarithromycin is a key component
(5). Rates of resistance to clarithromycin of 1 to 9%
have been reported in several European countries and the United States,
with even higher rates in some countries, such as France and Belgium
(24). The development of clarithromycin resistance in
H. pylori is recognized as a significant contributing factor
in treatment failure (8, 14), and the mechanism is
attributed to single point mutations in the peptidyltransferase region
of the 23S rRNA gene (25). Adenine residues at either position 2143 or 2144 can mutate. Transition to guanine (A2143G and
A2144G) is the most common mutation type, with the transversion mutation to cytosine (A2143C) less common (16, 21, 22,
25). Although culture of gastric biopsy samples allows further
H. pylori strain analysis, including determination of
antibiotic susceptibility, tests can take up to 2 weeks to complete.
The aim of the present study was to develop a PCR-based system using
conventional and real-time techniques enabling same-day diagnosis of
H. pylori infection and determination of clarithromycin resistance.
| |
MATERIALS AND METHODS |
Gastric biopsy samples and strain isolation.
Two sets of
gastric biopsy samples were used in this study. First, we examined a
series of preserved (
20°C) gastric biopsy samples from 39 dyspeptic
patients attending an open-access endoscopy clinic in Chelmsford during
1995 and 1996. These biopsy samples were confirmed positive for
H. pylori by both culture and histology, and the patients
were also confirmed seropositive for H. pylori (19). Second, we performed a prospective study to assess
the clinical applicability of the assays in which two biopsy samples per patient were obtained over a 3-month period in 1999 from the gastric antra of 121 dyspeptic patients referred for endoscopy by
general practitioners. One biopsy sample was used directly for culture
of H. pylori on Columbia base agar containing 10% (vol/vol)
defibrinated horse blood at 36°C under microaerobic conditions (90%
CO2, 4% O2, 4% N2, 2%
H2). For antibiotic susceptibility testing, approximately
107 CFU of H. pylori and a disk containing 2 µg of clarithromycin were applied to a blood agar plate.
Clarithromycin susceptibility was recorded after microaerobic
incubation for 48 h at 36°C. The second biopsy sample was stored at
20°C until required for DNA extraction.
Genomic DNA was extracted from both sets of gastric biopsy samples
using a modification of a previously described method
(12). Briefly, the thawed biopsy samples were homogenized
in Griffith's tubes containing 400 µl of sterile saline (0.85%
[wt/vol]), transferred to 1.5-ml screw-cap Eppendorf tubes, and
centrifuged (10,000 × g for 2 min) and the supernatant
was discarded. Pellets were resuspended in extraction buffer (20 mM
Tris-HCl [pH 8.0], 0.5% [vol/vol] Tween 20) and proteinase K (0.5 mg/ml), vortexed for 2 to 5 s, and incubated at 56°C for 1 h and then 100°C for 10 min. DNA extracts were stored at 20°C
until required.
Conventional PCR assays.
A PCR assay targeted at the 16S
rRNA gene of H. pylori was performed with primer pair Hp1
and Hp2 (Table 1) by a modification of
the protocol described previously (9). Briefly, the
50-µl PCR mixture, containing 5 µl of extracted DNA, 200 µM
(each) deoxynucleoside trisphosphates (dNTPs) (Gibco BRL, Paisley,
Scotland), 0.4 µM (each) primer (MWG Biotech, Milton Keynes,
England), 1.5 mM MgCl2, and 1 U of Taq
polymerase (Gibco BRL) in PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM
KCl, 0.2% [vol/vol] glycerol), was held for 5 min at a denaturation
temperature of 95°C, followed by 35 cycles of 30 s each at a
denaturation temperature of 95°C, an annealing temperature of 60°C,
and an elongation temperature of 72°C and by 5 min at 72°C.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Details of primers and probe system used in PCR-based
assays for H. pylori detection and mutation identification
|
|
A novel PCR assay to detect the
vacA gene was developed
using primers designed from in silico analysis of the 30 complete
sequences of
vacA currently deposited in GenBank. All
alignments
were performed with GeneBase, version 1 (Applied Maths,
Kortrijk,
Belgium). A 229-bp fragment of
vacA at the 3' end
of the gene
was amplified with the primer pair VAC3624F and VAC3853R
(Table
1) in a 50-µl reaction mixture containing PCR buffer, 2.0 mM
MgCl
2, 200 µM (each) dNTP, 0.3 µM (each) primer, 1 U of
Taq polymerase
(Gibco BRL), and 5 µl of extracted DNA.
Following denaturation
at 95°C (5 min), the
vacA fragment
was amplified through 35 cycles
as follows: 95, 53, and 72°C for
30 s each; extension was continued
at 72°C for 5 min. Aliquots
of each PCR product were electrophoresed
in a 1% (wt/vol) agarose gel
(Ultra Pure; Gibco BRL) in Tris-borate-EDTA
buffer (90 mM Tris-HCl, 90 mM boric acid, 0.002 M EDTA), and stained
in ethidium bromide at 0.5 µg/ml.
Real-time PCR assay and probe melting point hybridization
analysis.
Point mutations in the 23S rRNA gene associated with the
acquisition of clarithromycin resistance were detected using a
modification of the previously described assay developed for the
LightCycler (BioGene Ltd., Kimbolton, England) (7). The
reaction mixture was prepared as follows: 200 µM (each) dNTP, 0.4 U
of platinum Taq polymerase (Gibco BRL), Idaho Technology
buffer (50 mM Tris-HCl [pH 8.3], 2 mM MgCl2) (Biogene)
adjusted to give a final MgCl2 concentration of 3 mM, SYBR
Green 1 (BioGene) diluted 1/10,000, 5 pmol of each primer (Table 1),
and 5 pmol of probe (Table 1). DNA (1 µl) was added to a 9-µl
reaction mixture, and the PCR was performed as described previously
(7), but with the modification that the amplification
stage was increased to include 75 cycles.
| |
RESULTS |
Detection of H. pylori directly in gastric biopsy
samples.
Our results showed that the majority (36 of
39) of the DNA samples derived from stored gastric biopsy
samples (set 1) from confirmed H. pylori-positive patients
were positive by the diagnostic PCR assays for both the 16S
rrn and the vacA targets (Table
2). Both assays were of equal sensitivity
(92.3%) in this biopsy set. DNA samples obtained from biopsy samples
received during the prospective study (set 2) showed that 17 (14.0%)
of the 121 antral gastric biopsy samples were confirmed both culture
positive and histology positive for H. pylori. The
conventional PCR assay targeting vacA amplified the specific
product of 229 bp in 15 of 17 (88.2%) of the culture-positive biopsy
samples, as well in one sample from a patient who was culture negative.
The 16S rRNA PCR assay confirmed the presence of H. pylori
in 14 of 17 (82.3%) of these biopsy samples, although three
culture-negative biopsy samples also generated the expected PCR product
of 109 bp. The sensitivities of the vacA and 16S rRNA assays
were 89.5, and 85.0%, respectively, and their specificities were 99.0 and 98.1%, respectively (Table 2).
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Comparison of performances of PCR-based detection assays
targeting the H. pylori vacA and 16S rRNA genes with
culture from the two sets of gastric biopsy samples
|
|
Direct detection of mutations conferring clarithromycin
resistance.
The LightCycler PCR assay to detect point mutations in
the 23S rRNA gene was applied to determine clarithromycin
susceptibilities on DNA from 142 gastric biopsy samples from both sets
of patients, comprising 56 culture-positive and 86 culture-negative
biopsy samples. Analysis of DNA from 48 of the 56 culture-positive
biopsy samples and from one culture-negative biopsy sample produced
melting peaks characteristic of a clarithromycin-sensitive (wild-type) genotype (Table 3). Repeat PCR analyses
were necessary on five of these samples to generate sufficient product
to allow mutation detection. Antibiotic susceptibility testing by disk
diffusion of matched cultures of H. pylori confirmed that
strains isolated from 47 of 48 of these biopsy samples were
clarithromycin sensitive, while one was a mixed population of
clarithromycin-sensitive and -resistant phenotypes. DNA from three
biopsy samples generated melting peaks indicative of a
clarithromycin-resistant phenotype with an AG mutation (Table 3).
Disk diffusion results on the two available matched cultures of
H. pylori confirmed that one of these was clarithromycin
resistant. The other was sensitive to clarithromycin, which suggested
that the in vivo infection might be a mixture of both
clarithromycin-resistant and -sensitive forms even though only the
latter form was isolated on culture. The remaining culture-positive
biopsy sample produced a melting peak indicative of clarithromycin
resistance due to an AC mutation. Disk diffusion testing of the
matched culture confirmed clarithromycin resistance, and the
LightCycler assay when applied to this culture generated a melting peak
(AC mutation) identical to that of the corresponding biopsy sample.
Finally, LightCycler PCR was not successful for the four remaining
culture-positive biopsy specimens (PCR negative), and therefore 23S
rRNA genotypes could not be determined (Table 3).
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Performance of PCR-based probe melting point
hybridization assay to determine clarithromycin susceptibility of
H. pylori directly from 142 gastric biopsy samples
compared with that of the culture-based disk diffusion test
|
|
| |
DISCUSSION |
The results of the study showed that H. pylori DNA
could be detected by PCR directly in human gastric biopsy samples after frozen storage with high specificity and sensitivity. Our approach was
to use two target genes for detection to reduce uncertainties associated with use of a single target. The 16S rrn primers
have been applied extensively to a range of clinical samples and are established as sensitive and specific (6, 9, 11). But as
such DNA sequences encoding rRNA are highly ubiquitous, there is a
possible risk of nonspecific products, particularly when analyzing DNA
extracted from mammalian tissue (3). We therefore developed a novel PCR assay that targeted the vacA gene,
which is a species-specific and highly conserved locus in H. pylori (1, 4). Although vacA has a mosaic
structure and some regions such as the midregion are highly diverse,
the assay was designed to target a conserved region based on in silico
comparisons of vacA sequences in GenBank.
The performance of both diagnostic PCR assays in relation to culture
and histology was established in an analysis of 39 known culture-positive biopsy samples, and we found that H. pylori
could be detected in both assays with comparably high sensitivity
(92.3%). Further analyses based on 121 biopsy samples (comprising
culture-positive and -negative samples) demonstrated that both assays
were highly specific, with the vacA assay giving a
specificity of 99.0% compared with 98.1% for the 16S rrn
assay. There were minor differences between the sensitivity results for
the two sets of biopsy samples, with the vacA assay being
the most sensitive (89.5 versus 85.0%). The exact reason for the lower
sensitivities observed is unclear although for this set of biopsy
samples culture was performed on one biopsy sample at the primary
diagnostic laboratory and a second biopsy sample was frozen and
subsequently transported to our laboratory for molecular analyses.
Reduced sensitivities could have been due to differences in transport
conditions that occurred with some specimens. We have evidence (data
not shown) suggesting that H. pylori DNA in gastric biopsy
samples could be rapidly degraded if transport conditions were
suboptimal due to delays before freezing, repeated freezing and
thawing, and use of an inappropriate suspending medium. Alternatively
H. pylori colonization may not be uniformly distributed
across the gastric mucosa and thus biopsy samples taken from different
sites may occasionally give conflicting results. Overall, however, the
new vacA PCR assay generally performed better than the 16S
rrn assay. Nonspecific bands were amplified occasionally
from some gastric biopsy DNA extracts (Fig.
1), but these were significantly larger than the expected specific 229-bp product of the vacA assay,
and therefore reporting a false-positive result was unlikely.
Generation of nonspecific DNA bands could theoretically reduce the
sensitivity of the vacA assay due to competitive
coamplification of specific and nonspecific products. However, this did
not appear to be a problem, as strong specific signals were observed
regardless of any nonspecific amplification. The exact origin of these
bands is not known. They were generated occasionally in both
culture-positive and culture-negative biopsy samples but not from DNA
extracted from a pure culture of the infecting strain of H. pylori (data not shown). It is therefore most likely that
human DNA was amplified. A PCR-based approach to diagnostic testing has
the advantage over existing non-culture-based tests in that it is
simple to perform and can provide additional genotypic
information about the infecting strain, including markers associated
with antibiotic susceptibilities. In addition, although the initial
cost of equipment is high, the cost of reagents and consumables for
each test is extremely low in comparison with corresponding costs for
other methods such as the urea breath test and stool antigen test.
View larger version (108K):
[in this window]
[in a new window]
|
FIG. 1.
Examples to illustrate the quality of vacA
and 16S rRNA PCR products from H. pylori culture-positive
biopsy samples. Shown are PCR products generated by assays targeting
vacA (lanes 1 to 5) and 16S rrn (lanes 6 and 7).
Lane 8, 123-bp molecular weight marker.
|
|
Clarithromycin is a key component of most current triple-therapy
regimens for treatment of H. pylori infection; however,
resistance can dramatically decrease the chances of successful
eradication. The identification of specific point mutations (namely,
A2143G, A2144G, or A2143C) in the 23S rRNA gene (16, 21, 22,
25) has enabled development of molecular tests that allow
determination of clarithromycin resistance directly from biopsies
(2, 10, 12, 13, 20). The main limitations are that most
assays could not detect the A2143C mutation (2, 13, 20)
and some were of low sensitivity (20), while others
required multiple PCRs (10, 12, 23). The LightCycler assay
we describe is simpler and more rapid. All three common mutations were
detectable directly from a gastric biopsy sample in a single reaction
tube in under 1 h, so avoiding the associated delay of
culture-based susceptibility testing. Most assays provided information
on the clarithromycin susceptibility of the infecting strain that
corresponded accurately with culture-based disk diffusion results.
Nevertheless, DNA from one biopsy sample generated a melting peak
suggesting an AG mutation while the matched culture was
clarithromycin sensitive. That was possibly indicative of a mixed
infection, as a similar discrepancy between mutation detection and
phenotypic susceptibility testing was reported previously
(13). Three culture-positive biopsy samples that were also
positive by the diagnostic PCR assays were negative for the
clarithromycin susceptibility assay. The precise reason for this was
unclear, but the sensitivity may have been affected by sampling error,
as the target copy number contained in a volume as small as 1 µl of
DNA could be extremely low in some biopsy samples, particularly if
degradation of specific DNA has indeed occurred. Sensitivity may be
improved by modification of the assay to a nested-PCR format.
Alternatively, given that the method of DNA extraction was extremely
simple, we speculate that the differences in sensitivity may be due to
the presence of low levels of substances inhibitory to the PCR, which
may affect the efficiency of the LightCycler assay to a greater extent
than that of the conventional PCR assays.
In conclusion, application of our PCR-based approach not only allowed
same-day diagnosis of H. pylori infection but also supplied further strain information concerning clarithromycin susceptibility. Rapid provision of such information could have a significant impact on
patient management in terms of turnaround times, appropriate antibiotic
prescription, and ultimately treatment outcome.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Helicobacter
Reference Unit, Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Ave., Colindale, London NW9 5HT, United Kingdom. Phone: (44) 20 8200 4400. Fax: (44) 20 8905 9929. E-mail: rowen{at}phls.nhs.uk.
| |
REFERENCES |
| 1.
|
Atherton, J. C.,
P. Cao,
R. M. Peek, Jr.,
M. K. Tummuru,
M. J. Blaser, and T. L. Cover.
1995.
Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration.
J. Biol. Chem.
270:17771-17777[Abstract/Free Full Text].
|
| 2.
|
Bjorkholm, B.,
R. Befrits,
B. Jaup, and L. Engstrand.
1998.
Rapid PCR detection of Helicobacter pylori-associated virulence and resistance genes directly from gastric biopsy material.
J. Clin. Microbiol.
36:3689-3690[Abstract/Free Full Text].
|
| 3.
|
Chong, S. K.,
Q. Lou,
J. F. Fitzgerald, and C. H. Lee.
1996.
Evaluation of 16S rRNA gene PCR with primers Hp1 and Hp2 for detection of Helicobacter pylori.
J. Clin. Microbiol.
34:2728-2730[Abstract].
|
| 4.
|
Cover, T. L.
1996.
The vacuolating cytotoxin of Helicobacter pylori.
Mol. Microbiol.
20:241-246[CrossRef][Medline].
|
| 5.
|
de Boer, W. A., and G. N. Tytgat.
2000.
Regular review: treatment of Helicobacter pylori infection.
BMJ
320:31-34[Free Full Text].
|
| 6.
|
Dowsett, S. A.,
L. Archila,
V. A. Segreto,
C. R. Gonzalez,
A. Silva,
K. A. Vastola,
R. D. Bartizek, and M. J. Kowolik.
1999.
Helicobacter pylori infection in indigenous families of Central America: serostatus and oral and fingernail carriage.
J. Clin. Microbiol.
37:2456-2460[Abstract/Free Full Text].
|
| 7.
|
Gibson, J. R.,
N. A. Saunders,
B. Burke, and R. J. Owen.
1999.
Novel method for rapid determination of clarithromycin sensitivity in Helicobacter pylori.
J. Clin. Microbiol.
37:3746-3748[Abstract/Free Full Text].
|
| 8.
|
Goodwin, C. S.
1997.
Antimicrobial treatment of Helicobacter pylori infection.
Clin. Infect. Dis.
25:1023-1026[Medline].
|
| 9.
|
Ho, S. A.,
J. A. Hoyle,
F. A. Lewis,
A. D. Secker,
D. Cross,
N. P. Mapstone,
M. F. Dixon,
J. I. Wyatt,
D. S. Tompkins, and G. R. Taylor.
1991.
Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals.
J. Clin. Microbiol.
29:2543-2549[Abstract/Free Full Text].
|
| 10.
|
Maeda, S.,
H. Yoshida,
H. Matsunaga,
K. Ogura,
O. Kawamata,
Y. Shiratori, and M. Omata.
2000.
Detection of clarithromycin-resistant Helicobacter pylori strains by a preferential homoduplex formation assay.
J. Clin. Microbiol.
38:210-214[Abstract/Free Full Text].
|
| 11.
|
Mapstone, N. P.,
D. A. Lynch,
F. A. Lewis,
A. T. Axon,
D. S. Tompkins,
M. F. Dixon, and P. Quirke.
1993.
Identification of Helicobacter pylori DNA in the mouths and stomachs of patients with gastritis using PCR.
J. Clin. Pathol.
46:540-543[Abstract/Free Full Text].
|
| 12.
|
Marais, A.,
L. Monteiro,
A. Occhialini,
M. Pina,
H. Lamouliatte, and F. Megraud.
1999.
Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens.
Gut
44:463-467[Abstract/Free Full Text].
|
| 13.
|
Matsuoka, M.,
Y. Yoshida,
K. Hayakawa,
S. Fukuchi, and K. Sugano.
1999.
Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure.
Gut
45:503-507[Abstract/Free Full Text].
|
| 14.
|
Megraud, F., and H. P. Doermann.
1998.
Clinical relevance of resistant strains of Helicobacter pylori: a review of current data.
Gut
43(Suppl. 1):S61-S65[Free Full Text].
|
| 15.
|
NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease.
1994.
Helicobacter pylori in peptic ulcer disease.
JAMA
272:65-69[Abstract/Free Full Text].
|
| 16.
|
Occhialini, A.,
M. Urdaci,
F. Doucet-Populaire,
C. M. Bebear,
H. Lamouliatte, and F. Megraud.
1997.
Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.
Antimicrob. Agents Chemother.
41:2724-2728[Abstract].
|
| 17.
|
Parsonnet, J.
1994.
Gastric adenocarcinoma and Helicobacter pylori infection.
West J. Med.
161:60[Medline].
|
| 18.
|
Parsonnet, J.,
S. Hansen,
L. Rodriguez,
A. B. Gelb,
R. A. Warnke,
E. Jellum,
N. Orentreich,
J. H. Vogelman, and G. D. Friedman.
1994.
Helicobacter pylori infection and gastric lymphoma.
N. Engl. J. Med.
330:1267-1271[Abstract/Free Full Text].
|
| 19.
|
Peters, T. M.,
R. J. Owen,
E. L. Teare, and C. Goodbourn.
1997.
Detection of Helicobacter pylori by PCR on culture- and histology-negative gastric biopsies from seropositive patients.
PHLS Microbiol. Dig.
14:227-229.
|
| 20.
|
Sevin, E.,
D. Lamarque,
J. C. Delchier,
C. J. Soussy, and J. Tankovic.
1998.
Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR.
Microbiol. Lett.
165:369-372[CrossRef].
|
| 21.
|
Stone, G. G.,
D. Shortridge,
J. Versalovic,
J. Beyer,
R. K. Flamm,
D. Y. Graham,
A. T. Ghoneim, and S. K. Tanaka.
1997.
A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori.
Antimicrob. Agents Chemother.
41:712-714[Abstract].
|
| 22.
|
Taylor, D. E.,
Z. Ge,
D. Purych,
T. Lo, and K. Hiratsuka.
1997.
Cloning and sequence analysis of two copies of a 23S rRNA gene from Helicobacter pylori and association of clarithromycin resistance with 23S rRNA mutations.
Antimicrob. Agents Chemother.
41:2621-2628[Abstract].
|
| 23.
|
Trebesius, K.,
K. Panthel,
S. Strobel,
K. Vogt,
G. Faller,
T. Kirchner,
M. Kist,
J. Heesemann, and R. Haas.
2000.
Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation.
Gut
46:608-614[Abstract/Free Full Text].
|
| 24.
|
Tytgat, G. N.
1997.
Antimicrobial therapy for Helicobacter pylori infection.
Helicobacter
2(Suppl. 1):S81-S88.
|
| 25.
|
Versalovic, J.,
D. Shortridge,
K. Kibler,
M. V. Griffy,
J. Beyer,
R. K. Flamm,
S. K. Tanaka,
D. Y. Graham, and M. F. Go.
1996.
Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.
Antimicrob. Agents Chemother.
40:477-480[Abstract].
|
Journal of Clinical Microbiology, April 2001, p. 1217-1220, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1217-1220.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Sugimoto, M., Wu, J.-Y., Abudayyeh, S., Hoffman, J., Brahem, H., Al-Khatib, K., Yamaoka, Y., Graham, D. Y.
(2009). Unreliability of Results of PCR Detection of Helicobacter pylori in Clinical or Environmental Samples. J. Clin. Microbiol.
47: 738-742
[Abstract]
[Full Text]
-
Burucoa, C., Garnier, M., Silvain, C., Fauchere, J.-L.
(2008). Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori. J. Clin. Microbiol.
46: 2320-2326
[Abstract]
[Full Text]
-
Lottspeich, C., Schwarzer, A., Panthel, K., Koletzko, S., Russmann, H.
(2007). Evaluation of the Novel Helicobacter pylori ClariRes Real-Time PCR Assay for Detection and Clarithromycin Susceptibility Testing of H. pylori in Stool Specimens from Symptomatic Children. J. Clin. Microbiol.
45: 1718-1722
[Abstract]
[Full Text]
-
Megraud, F., Lehours, P.
(2007). Helicobacter pylori Detection and Antimicrobial Susceptibility Testing. Clin. Microbiol. Rev.
20: 280-322
[Abstract]
[Full Text]
-
Nishizawa, T., Suzuki, H., Umezawa, A., Muraoka, H., Iwasaki, E., Masaoka, T., Kobayashi, I., Hibi, T.
(2007). Rapid Detection of Point Mutations Conferring Resistance to Fluoroquinolone in gyrA of Helicobacter pylori by Allele-Specific PCR. J. Clin. Microbiol.
45: 303-305
[Abstract]
[Full Text]
-
Lawson, A. J., Elviss, N. C., Owen, R. J.
(2005). Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales. J Antimicrob Chemother
56: 282-286
[Abstract]
[Full Text]
-
Schabereiter-Gurtner, C., Hirschl, A. M., Dragosics, B., Hufnagl, P., Puz, S., Kovach, Z., Rotter, M., Makristathis, A.
(2004). Novel Real-Time PCR Assay for Detection of Helicobacter pylori Infection and Simultaneous Clarithromycin Susceptibility Testing of Stool and Biopsy Specimens. J. Clin. Microbiol.
42: 4512-4518
[Abstract]
[Full Text]
-
Megraud, F
(2004). H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut
53: 1374-1384
[Full Text]
-
Chisholm, S. A., Owen, R. J.
(2004). Frameshift mutations in frxA occur frequently and do not provide a reliable marker for metronidazole resistance in UK isolates of Helicobacter pylori. J Med Microbiol
53: 135-140
[Abstract]
[Full Text]
-
Lascols, C., Lamarque, D., Costa, J.-M., Copie-Bergman, C., Le Glaunec, J.-M., 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: 4573-4577
[Abstract]
[Full Text]
-
Owen, R. J., Sharp, S. I., Chisholm, S. A., Rijpkema, S.
(2003). Identification of cagA Tyrosine Phosphorylation DNA Motifs in Helicobacter pylori Isolates from Peptic Ulcer Patients by Novel PCR-Restriction Fragment Length Polymorphism and Real-Time Fluorescence PCR Assays. J. Clin. Microbiol.
41: 3112-3118
[Abstract]
[Full Text]
-
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: 397-402
[Abstract]
[Full Text]
-
Menard, A., Santos, A., Megraud, F., Oleastro, M.
(2002). PCR-Restriction Fragment Length Polymorphism Can Also Detect Point Mutation A2142C in the 23S rRNA Gene, Associated with Helicobacter pylori Resistance to Clarithromycin. Antimicrob. Agents Chemother.
46: 1156-1157
[Full Text]
-
Owen, R J
(2002). Molecular testing for antibiotic resistance in Helicobacter pylori. Gut
50: 285-289
[Abstract]
[Full Text]
Top
Abstract
Introduction
