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Journal of Clinical Microbiology, December 1999, p. 4071-4080, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Performance Criteria of DNA Fingerprinting Methods for Typing
of Helicobacter pylori Isolates: Experimental
Results and Meta-Analysis
Christophe
Burucoa,*
Vincent
Lhomme, and
Jean
Louis
Fauchere
Laboratoire de Microbiologie A, CHU La
Milétrie, 86021 Poitiers, France
Received 19 February 1999/Returned for modification 15 June
1999/Accepted 20 September 1999
 |
ABSTRACT |
Typing systems are used to discriminate between isolates of
Helicobacter pylori for epidemiological and clinical
purposes. Discriminatory power and typeability are important
performance criteria of typing systems. Discriminatory power refers to
the ability to differentiate among unrelated isolates; it is
quantitatively expressed by the discriminatory index (DI).
Typeability refers to the ability of the method to provide an
unambiguous result for each isolate analyzed; it is quantitatively
expressed by the percentage of typeable isolates. We evaluated the
discriminatory power and the typeability of the most currently used DNA
fingerprinting methods for the typing of H. pylori
isolates: ribotyping, PCR-based restriction fragment length
polymorphism (PCR-RFLP) analysis, and random amplified
polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance
criteria. A meta-analysis of typeability and discriminatory power was
conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA
sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis
demonstrated the optimal typeability (100%) and the excellent
discriminatory powers of PCR-based typing methods: RAPD
analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis,
DIs, 0.70 to 0.97). Chromosome restriction-based typing methods
(ribotyping and PFGE) are limited by a low typeability (12.5 to 75%)
that strongly decreases their discriminatory powers: ribotyping, DI,
0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of
ribotyping and PFGE for the typing of H. pylori isolates.
We recommend the use of PCR-based methods.
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INTRODUCTION |
Helicobacter pylori
colonizes human gastric mucosa, causes chronic gastritis and ulcers,
and is a major risk factor for the development of gastric cancers
(15, 17, 26). Discrimination between closely related
isolates of H. pylori is needed for epidemiologic and
clinical purposes (23). Precise methods of strain
characterization are necessary to monitor H. pylori
infections (familial or nosocomial transmissions, treatment failures,
relapses, and cocolonizations). On the basis of the high degree of
genomic variability of isolates within the H. pylori
species, various molecular techniques have been developed to
differentiate clinical isolates (23). Some of these are
based on the restriction patterns of the chromosomal DNA, such as
restriction enzyme analysis (6, 8, 9, 38, 46, 67, 76),
ribotyping (5, 11, 21, 24, 37, 40, 43, 44, 47-50, 52, 54, 56, 58,
68), and pulsed-field gel electrophoresis (PFGE) (27, 57,
64-66, 73). Others are based on PCR, such as sequencing of PCR
products (1, 18, 33), restriction fragment length
polymorphism (RFLP) analysis of PCR products (PCR-RFLP analysis)
(1, 4, 9, 12, 16, 18-20, 22, 29-31, 33, 35, 36, 41, 42, 45, 52,
56, 58, 60-62, 67, 74), repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR) (13, 25, 72), and random
amplified polymorphism DNA (RAPD) analysis (2, 3, 7, 14, 16, 32,
33, 39, 55, 59, 67, 69-71, 73, 75, 76).
The techniques most frequently reported to be used for typing are
PCR-RFLP analysis, RAPD analysis, and ribotyping. These techniques are
increasingly used to type H. pylori isolates, but their
performances, particularly their typeabilities and discriminatory powers, have not been completely and explicitly evaluated. Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed (63). A good fingerprinting method requires an excellent typeability in order to be able to type
all the isolates studied. Discriminatory power refers to the
ability of the method to differentiate among unrelated isolates (63). To be sure that two isolates with the same fingerprint are really genetically linked, the discriminatory power must be high.
The discriminatory powers of methods used to type H. pylori
have most often been assessed subjectively or by considering the number
of types obtained by each method. Although this allowed some level of
discrimination, the comparison of methods is difficult because several
values, generally, the size and the number of groups of isolates, are
compared. In 1988, Hunter and Gaston (28) used a single
numerical index of discrimination, the discriminatory index (DI), based
on Simpson's index of diversity, to compare the discriminatory powers
of several methods used to type Candida albicans and some
enteric species. The calculation is based on the probability that two
unrelated isolates would be classified as the same type. Since that
time, the DI has widely been used to compare the discriminatory powers
of typing methods, and the determination of this index is now
recommended in guidelines for the evaluation of typing methods
(63). It was never used, however, to evaluate H. pylori typing methods.
Special attention should be paid to the selection of an appropriate set
of test strains for evaluation of the typeability and discriminatory
power of a typing system. The rationale for most of the reported typing
studies is that different isolates of an epidemiologic cluster or
successive isolates from a single patient may be clonally related, that
is, are directly derived from a common parental strain. The aims of
such studies are mainly to group the isolates rather than to
distinguish them. Several studies have compared many epidemiologically
related isolates thought to be clonal and a few (less than 10)
unrelated isolates (2, 5, 6, 9, 14, 20-22, 27, 32, 44, 50,
52-57, 60, 67, 70, 71, 76). These studies have confirmed that the typing systems tested have good concordance in terms of the epidemiologic relatedness of the isolates. However, because in these
studies the isolates are often epidemiologically related, such
strategies are not appropriate for evaluation of the typeability and
the discriminatory power of a typing method (63). A large test population of isolates correctly identified to the species level
must be selected to reflect the diversity of the species as much as
possible (63). The test population should include isolates
that are unrelated epidemiologically on the basis of detailed clinical
and epidemiological data. Thus, studies evaluating the typeabilities
and discriminatory powers of typing system needs to be done with
collections of isolates from unrelated patients.
Typeability and discriminatory power can be expressed
quantitatively (28, 63). In reported studies in which enough
unrelated isolates have been typed, some of these criteria were
evaluated qualitatively but were never expressed quantitatively and
thus could not be compared from one study to another. Furthermore, the
performances of more than two different typing techniques were never
compared by using the same H. pylori isolates.
To evaluate the discriminatory power and the typeability of ribotyping,
PCR-RFLP analysis, and RAPD analysis as methods for the typing of
H. pylori isolates, we studied 39 unrelated H. pylori isolates from different geographic origin and three
familial isolates (from a mother and her son and daughter) considered
genetically related. We also performed a meta-analysis of typeability
and discriminatory power using raw data from previously reported
studies of H. pylori typing systems.
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MATERIALS AND METHODS |
Bacterial isolates.
The 42 H. pylori isolates
were isolated between 1984 and 1995 from gastric tissues collected
during endoscopy of 42 dyspeptic patients of different geographic
origins. These patients included 4 patients from Venezuela (kindly
provided by N. Muñoz), 3 patients from the United States (kindly
provided by M. J. Blaser), and 35 patients from different areas of
France. Among these 42 isolates, 39 were unrelated and 3 were familial
isolates isolated from the mother, the son, and the daughter of a
French family.
The isolates were identified to the species level by use of standard
criteria, including colony morphology, Gram staining result, and
biochemical test results (positivity for urease, catalase, and
oxidase). Until they were used, the isolates were frozen at 
70°C in
10% glycerol.
Extraction of total DNA.
Stored isolates of H. pylori were regrown on Columbia agar with 5% sheep blood. The
plates were soaked with an inoculum of a no. 5 McFarland standard and
were incubated for 2 days at 37°C under microaerobic conditions.
Bacterial cells were harvested from two plates and were suspended on
ice in 1 ml of lysis buffer (10 mM Tris-HCl [pH 8.5], 100 mM EDTA,
1% sodium dodecyl sulfate, 0.05 mg of pronase per ml). The mixture was
incubated for 60 min at 37°C. The sample was extracted with an equal
volume of phenol-chloroform-isoamyl alcohol (25:24:1), and after
centrifugation, the aqueous phase was collected. RNase A (50 µg/ml)
was added, and the mixture was incubated for 60 min at 60°C and then
ethanol precipitated. After centrifugation (13,000 × g) the pellet was resuspended in 20 µl of sterile distilled water.
The DNA concentrations and the quality of the DNA in the samples were
estimated after agarose gel electrophoresis with DNA
standards.
PCR-RFLP analysis.
PCR-RFLP analysis was performed as
described by Foxall et al. (20) and Owen et al.
(52). Four techniques were used to compare the 42 isolates:
restriction of a 2.4-kb fragment containing the ureA and
ureB genes by HaeIII, BamHI, or
HindIII and restriction of a 1.1-kb fragment containing
the ureC (now glmM) gene by Sau3A or
HaeIII and HindIII.
The PCR was carried out according to the instructions supplied with the
reagents (Eurogentec, Seraing, Belgium) in a Perkin-Elmer
GeneAmp PCR
system 2400 thermal cycler (Perkin-Elmer Cetus, Norwalk,
Conn.) in 100 µl containing 1 µl of chromosomal DNA (~20 ng),
3 mM
MgCl
2, each primer at a concentration of 0.2 µM, 2.5 U of
Eurotaq DNA polymerase (Eurogentec), each deoxynucleoside triphosphate
(Eurogentec) at a concentration of 0.2 µM, 10 mM Tris-HCl (pH
8.3),
and 50 mM
KCl.
To amplify the 2.4-kb fragment containing the
ureA and
ureB genes, we used two oligonucleotides with recognition
sequences
of 5'-AGGAGAATGAGATGA-3' (base pairs 308 to 322)
and 5'-ACTTTATTGGCTGGT-3'
(base pairs 2718 to 2703),
respectively (
34). To amplify the
1.1-kb fragment containing
the
ureC gene, we used two oligonucleotides
with recognition
sequences of 5'-TTTGGGACTGATGGCGTGAGGGGTAA-3'
(base pairs 10 to 35) and 5'-GGACATTCAAATTCACCAGGTTTTGAG-3' (base
pairs
1142 to 1116), respectively (
34).
After an initial denaturation of target DNA at 95°C for 5 min,
thermal cycling for each set of primers was 95°C for 1 min,
50°C
for 1 min, and 72°C for 1 min for a total of 35 cycles. The
final
cycle included extension for 5 min at 72°C.
Ten microliters of the reaction products was analyzed by
electrophoresis on a 0.8% (wt/vol) agarose gel at 110 V for 45
min.
Prior to digestion for RFLP analysis, 90 µl of the PCR product was
transferred to a fresh tube, and DNA was precipitated by
adding 2 volumes (approximately 180 µl) of ethanol (95%; vol/vol).
After 10 min of gentle mixing, the samples were centrifuged at
14,000 ×
g for 15 min. The DNA pellet was washed with 70% ethanol,
dried, and resuspended in 10 µl of sterile distilled
water.
For unique restriction, 5 µl of the concentrated DNA was added to
11.5 µl of sterile distilled water and 15 U (1.5 µl) of
enzyme
(
HaeIII,
BamHI, or
Sau3A) with 2 µl
of the appropriate
restriction buffer, giving a final volume of 20 µl. This mixture
was incubated at 37°C for 3 h. For mixed
restriction, DNA that
had already been digested with the first enzyme
was precipitated,
washed, dried, and resuspended in 20 µl of the
second buffer consisting
of 16.5 µl of sterile distilled water, 15 U
(1.5 µl) of the second
enzyme (
HindIII), and 2 µl of
the appropriate restriction buffer.
This mixture was incubated at
37°C for 3
h.
The digested PCR products (20 µl) were analyzed by submarine gel
electrophoresis at 110 V for 45 min through a 1.8% (wt/vol)
agarose
gel. pBR328-
BglI + pBR328-
HinfI (DNA
molecular weight
marker VI; Boehringer Mannheim) was used as a size
marker in all
gels. Calculation of the sizes of the DNA bands produced
by DNA
fingerprinting was performed with Taxotron software (P. A. D.
Grimont, Institut Pasteur, Paris,
France).
Digested PCR products yielding identical numbers of bands of the same
size were considered identical and were grouped in the
same PCR-RFLP
type designated by a numeral. Four numerals were
assigned to each
isolate on the basis of its four PCR-RFLP types.
A similar analytical
procedure was applied to the RAPD analysis
profiles and to the
ribotyping patterns
(ribopatterns).
Ribotyping.
DNA samples (~2 µg) were digested at 37°C
for 4 h with 30 U of the HaeIII restriction enzyme
(Eurogentec), which in previous studies (5, 21, 50, 53) gave
the most clearly resolved patterns for analysis. DNA fragments were
separated by electrophoresis through 0.8% agarose gels (length, 13 cm)
at 35 V for 16 h in TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA
[pH 8.3]). After electrophoresis, the gels were stained with ethidium
bromide and photographed. The DNA fragments were then transferred to
nylon membranes (Hybond-N; Amersham) by capillarity and hybridized with an acetylaminofluorene (AAF)-labeled rRNA (AAF-rRNA; Eurogentec) under
the conditions specified by the manufacturer. A molecular weight marker
(Raoul 1; Eurogentec) was loaded onto the agarose gel and was
specifically hybridized with the AAF-labeled pBR322, which is
visualized as AAF rRNA.
RAPD analysis.
The PCR was carried out as described above
for PCR-RFLP analysis. Three arbitrary primers were used: primers 1254 (5'-CCGCAGCCAA-3'), 1247 (5'-AAGAGCCCGT-3'), and
1281 (5'-AACGCGCAAC-3') (3). The cycling program
was 1 cycle of 94°C for 2 min, 37°C for 1 min, and 72°C for 4 min
and 29 cycles of 94°C for 2 min, 37°C for 3 min, and 72°C for 7 min. After PCR, 20 µl of the PCR products was electrophoresed in 2%
agarose gels containing 1× Tris acetate running buffer.
pBR328-BglI + pBR328-HinfI (DNA molecular
weight marker VI; Boehringer Mannheim) was used as a size marker in all gels.
Typeability.
Typeability refers to the ability of a method
to provide an unambiguous result for each isolate analyzed. The
typeability of each typing system was defined as the percentage of
typeable isolates among the 40 unrelated isolates tested (39 unrelated isolates and 1 of the 3 familial isolates). A typeable isolate is an
isolate for which the typing system can provide a readable result
consisting of a pattern of several well-defined bands.
Discriminatory power.
The discriminatory power of each
fingerprinting method was estimated both by the number of identified
types among the 40 unrelated isolates and by the DI (28).
The DI is the probability that two isolates randomly chosen from a
population of unrelated isolates will be distinguished by that typing
method. The DI was calculated by the following equation:
where
N is the total number of isolates,
s
is the total number of types, and
nj is the number of
isolates belonging to the
jth type. Thus, this index can be
calculated from the distribution
of types, i.e., the number of isolates
of each
type.
DI depends on the number of types and on the homogeneity of the
frequency distribution of isolates into types; ideally, each
isolate
should have a different type (DI = 1). For the purpose
of
calculation, nontypeable isolates were grouped together. According
to
recent guidelines, a typing system should achieve a DI of >0.95
for
reliable assessment of the clonal relatedness of isolates
(
63).
Meta-analysis.
To compare our results to published results
for typeability and discriminatory power, we conducted a meta-analysis.
The English-language medical literature from 1990 to 1998 was searched,
using PubMed Medline, for articles about H. pylori DNA
typing, H. pylori fingerprinting, RAPD analysis, PCR-RFLP
analysis, PFGE, REP-PCR, and ribotyping. Related articles proposed by
PubMed were also searched. Only studies that supplied primary data and
that used typing methods similar enough to be statistically grouped by
typing methods were included in the meta-analysis.
For each typing method typeability was calculated as the percentage of
typeable isolates among all the unrelated isolates
studied in the
literature. Familial isolates and isolates obtained
consecutively from
the same patient were excluded. Reference strains
tested in numerous
studies were considered only one time for the
meta-analysis. A pooled
typeability was calculated for each typing
method. The pooled
typeability consists of the percentage of typeable
isolates among the
unrelated isolates of all the reported studies
that used the same
technique.
The DI was retrospectively calculated for the unrelated isolates of
each study when primary data were presented and when more
than 10 unrelated isolates were tested. For each typing method,
a pooled DI was
calculated as the mean DI for all reported studies
by using the same
method, but the calculation was weighted by
the number of isolates
tested in each
study.
| |
RESULTS |
Raw data for the 42 isolates tested are presented in Table
1. The discriminatory powers and
typeabilities of the eight techniques tested in this study are
presented in Table 2.
Typeability.
Among the 40 unrelated isolates, 10 were not
typeable by ribotyping because DNA samples were not cleaved by
HaeIII (typeability = 30 of 40 = 75%). The
PCR-RFLP analysis protocols successfully amplified 2.4- and 1.1-kbp
fragments of the ureAB genes and the ureC gene,
respectively, from all the 40 unrelated H. pylori isolates examined. Each of these two PCR products were submitted to two restriction protocols, generating four PCR-RFLP patterns for each of
the 40 isolates. All 40 unrelated isolates could be typed by these four
methods (typeability of PCR-RFLP = 100%). The three primers used
for RAPD analysis generated DNA fingerprints for all 40 unrelated
H. pylori isolates examined (typeability of RAPD analysis = 100%).
Discriminatory power.
The eight patterns obtained for each
isolate by the eight tested techniques are given in Table 1 (for each
technique, a particular pattern is designated by a numeral).
For ribotyping, all 30 typeable isolates were easily distinguished
within 30 ribopatterns. By considering the 40 unrelated
isolates tested
and gathering the 10 noncleavable isolates into
one particular group,
the DI was 0.94. The ribopatterns each had
5 to 10 bands. The majority
of the isolates exhibited ribopatterns
with two common bands, at
approximately 800 and 1,000 bp (Fig.
1).
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FIG. 1.
Ribopatterns (HaeIII digests) of DNAs from 12 H. pylori isolates (isolates 11-D, 94-60, 94-48, 94-55, 94-47, 93-37, 93-38, 92-4, 86-6, 85-B, V17, V3). Different problems
with typeability are shown: DNA was not cleaved by HaeIII
(lanes 6, 7, and 9), insufficient intensities of DNA bands (lanes 3 and
11), degradation of chromosomal DNA (lane 10), and incomplete digestion
of chromosomal DNA (lane 12). Lanes M, molecular size marker Raoul 1 (bands of 48.5, 18.5, 14.9, 10.6, 9.0, 7.4, 5.6, 4.4, 4.0, 3.6, 2.9,
2.3, 1.8, 1.4, 1.2, 1, 0.9, 0.7, and 0.6 kbp, from top to bottom,
respectively).
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For PCR-RFLP analysis, four techniques were tested. PCR with the two
primers specific for
ureAB amplified a 2.4-kbp fragment
from
within the urease A and B genes of the 40 unrelated isolates.
Restriction enzyme analysis of these PCR products with
HaeIII
yielded two to three bands, depending on the isolate,
with sizes
of between 250 bp and 2 kbp. A 650-bp fragment was most
commonly
found. Twenty different profiles were identified among the 40
unrelated isolates, with a DI of 0.96. Mixed restriction analysis
with
BamHI and
HindIII of the 2.4-kpb fragment
amplified from
within the
ureAB genes yielded two to four
bands ranging from
200 to 2,000 bp. A 500-bp fragment was found to be
present in
the majority of the patterns. This mixed restriction allowed
the
40 unrelated isolates to be clustered into seven groups on the
basis of the presence of one, two, or three sites and their relative
positions. The DI was 0.71. PCR with the two
ureC-specific
primers
amplified a 1.1-kbp fragment from within the
ureC
genes of the
40 unrelated isolates. Mixed restriction analysis with
HaeIII
and
HindIII of the 1.1-kpb fragment
yielded two to eight bands
ranging from 8 to 1,000 bp. This mixed
restriction allowed the
40 unrelated isolates to be clustered into 11 groups on the basis
of the presence of one to seven sites and their
relative positions.
The DI was 0.89. Restriction enzyme analysis of
these PCR products
with
Sau3A yielded three to six bands,
with sizes of between 8
and 600 bp (Fig.
2), depending on the isolate. This RFLP
analysis
with
Sau3A yielded 27 fragment profiles among the
40 unrelated
isolates, giving a DI of 0.97. The DIs of all PCR-RFLP
analyses
combined are given in Table
3.
The highest DI (0.99) was obtained
by combining the results of two
single restriction techniques:
digestion of
ureC with
Sau3A and digestion of
ureAB with
HaeIII.
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FIG. 2.
PCR-RFLP patterns of the amplified ureC gene
products from 12 H. pylori isolates (isolates 93-32, 93-33, 93-34, 93-35, 93-37, 93-38, 93-40, 93-41, 93-42, 94-47, 94-48, and
94-55) whose amplified DNAs were digested with Sau3A. Two
unrelated isolates (isolates 93-34 and 93-35 [lanes 3 and 4, respectively]) yielded identical pattern. Lanes M, molecular size
marker pBR328-BglI + pBR328-HinfI (bands of
2,176, 1,766, 1,230, 1,033, 653, 517, 453, 394, 298, 234, 220, and 154 bp, from top to bottom, respectively).
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For RAPD analysis, the profiles generated with the three primers
contained several bands (three to nine bands for primer 1254
and
two to five bands for primers 1247 and 1281) of various intensities.
The bands ranged from 400 to 2,200 bp in size (Fig.
3). We did
not observe any bands in lanes
in which amplified blanks were
run. With primer 1254 or primer 1247, the 40 unrelated isolates
were gathered in 38 banding patterns (Table
1). Primer 1281 yielded
40 distinct RAPD patterns among the 40 unrelated isolates examined.
The DI was 0.99 for primers 1254 and 1247, and the DI was 1 for
primer 1281.
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FIG. 3.
RAPD patterns of the amplified DNAs of 12 H. pylori isolates (isolates 85-B, 86-G, 92-3, 92-4, 92-7, 92-8, 92-9, 92-10, 92-11, 92-16, 93-25, and 93-31) obtained by RAPD analysis
with primer 1254. Two pairs of unrelated isolates (lanes 2 and 3 and
lanes 6 to 10, respectively) yielded identical patterns. Lanes M,
molecular size marker pBR328-BglI + pBR328-HinfI (bands of 2,176, 1,766, 1230, 1033, 653, 517, 453, 394, 298, 234, 220, and 154 bp, from top to bottom,
respectively).
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Meta-analysis.
The typeabilities and DIs of the different
techniques presented in the literature and determined in our study are
presented in Tables 4 to 6.
Table
4 shows the low typeabilities of
chromosomal restriction-based techniques:
HaeIII ribotyping
(78%),
NotI PFGE (66%),
NruI PFGE (51%),
ApaI PFGE (56%), and
KpnI PFGE (12.5%).
HindII
ribotyping has good typeability (100%). The DIs
of all ribotyping
and PFGE techniques are less than the recommended
value of 0.95,
indicating a low discriminatory power.
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TABLE 4.
Typeabilities and DIs of chromosomal restriction-based
techniques (ribotyping and PFGE) determined with data from
selected studies
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Table
5 shows the excellent
typeabilities of PCR-RFLP methods (100%). The higher
DIs were obtained by amlplification of
ureC (1.1 kb)
digested with
Sau3A (DI = 0.97) and by amplificaiton
of
ureAB digested with
HaeIII (DI = 0.96).
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TABLE 5.
Typeabilities and DIs of PCR-RFLP analysis of
ureAB (2.4 kb) digested with HaeIII,
ureC (820 or 1,100 bp) digested with Sau3A, or
ureC (820 bp or 1.1 kbp) digested with HhaI
determined with data from selected studies
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Table
6 shows the excellent typeabilities
and DIs obtained by RAPD analysis (typeability = 100%; DI = 1) and REP-PCR (typeability
= 100%; DI = 0.99).
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TABLE 6.
Typeabilities and DIs of RAPD analysis with primer 1254 or primer 1281 and REP-PCR with data from selected studies
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DISCUSSION |
Typeability.
The excellent typeabilities of both PCR-RFLP and
RAPD analyses as assessed from the results of our study are confirmed
by the meta-analysis in which the pooled typeability is 100% for the
PCR-based techniques (Tables 5 and 6). The low typeability of
ribotyping with HaeIII observed in our study (75%) is
confirmed by the meta-analysis (78%). Despite this low typeability,
HaeIII is the most commonly used enzyme since it gave the
best-resolved patterns for analysis and the best discriminatory power
(21). In the literature, the typeability of ribotyping is
variable according to the restriction enzyme used (5, 47, 48, 50,
51, 53, 58). The meta-analysis of published studies in which
ribotyping was used yielded pooled typeabilities of 78% when
HaeIII was used and 100% when HindIII was
used (Table 4). In studies in which two enzymes were tested, some
isolates, from which DNA was not digested with HaeIII could
be well digested with HindIII (5, 48, 58). A
good typeability could then be obtained, but two or three consecutive
assays were required. Moreover, as shown in Fig. 1, several other
problems can occur with this technique: insufficient DNA yield
(lanes 3 and 11), DNA degradation (lane 10), and noncomplete digestion
of chromosomal DNA (lane 12). Because of these problems several assays
were required to achieve a readable result. PFGE analysis of DNA from
H. pylori isolates is based on the same principle
as ribotyping: the generation of restriction fragments from the
chromosomal DNA (23). The meta-analysis of published studies
in which PFGE was used yielded low typeabilities, ranging from 12.5 to
66% (Table 4). The low typeabilities for both these two restriction
techniques (ribotyping and PFGE) could be due to protection from
restriction endonuclease digestion by the production of endogenous
methylases (66).
Discriminatory power.
The highest DIs were obtained for RAPD
analysis: a DI of 1 with primer 1281, which yielded 40 distinguishable
patterns among the 40 unrelated isolates, and DIs of 0.99 with primers
1254 and 1247, which yielded 38 distinguishable patterns. The
meta-analysis of published studies in which RAPD analysis with primers
1254 and 1281 was used confirmed our results, yielding pooled DIs of 1 for four and five studies, respectively (Table 6). Interestingly, two
primers (primers 1247 and 1254) yielded different but dependent patterns in our study. Each primer gave 38 patterns for the 40 unrelated isolates. The banding pattern obtained with one of the primers was clearly different from the one obtained with the other primer, although the two pairs of indistinguishable isolates were the
same with these two primers (Table 1, RAPD patterns 1 and 3). These two
pairs of isolates (isolates 86-G and 92-3 and isolates 92-8 and 92-16)
were easily distinguishable by the other typing systems (ribotyping,
PCR-RFLP analysis, and RAPD analysis with primer 1281). This
observation highlights the possibility of achieving a false identity by
RAPD analysis, even if two primers are used.
With another primer, Cho et al. (
7) distinguished only 11 RAPD types among 108 unrelated isolates. The calculated DI was
low:
0.83. This discrepancy in the discriminatory powers of RAPD
typing
systems highlights the difficulty in appreciating the level
of
discrimination when this technique is applied for the first
time in a
laboratory. Since the interlaboratory reproducibility
of RAPD analysis
is very low (
10,
41,
69), it is impossible
to predict the
discriminatory power of this typing method even
if the technical
conditions tend to reproduce the published conditions.
This problem of
reproducibility is of great importance since a
single variation in one
of the multiple technical conditions of
RAPD analysis could change the
discriminatory power of this typing
method.
In our study the DIs of PCR-RFLP methods ranged from 0.71 to 0.97 (Table
2). The higher DIs were obtained with the
Sau3A
restriction digest of the PCR-amplified
ureC genes (DI = 0.97)
and the
HaeIII restriction digest of the
PCR-amplified
ureAB genes
(DI = 0.96), and the DIs were
greater than the recommended value
of 0.95 (
28,
63).
The meta-analysis allowed us to compare the DIs of three PCR-RFLP
techniques. For PCR-RFLP analysis of
ureAB (2.4 kb) digested
with
HaeIII, the DIs of six published studies ranged from
0.87
to 0.99, with a pooled DI of 0.96 (Table
5). For PCR-RFLP analysis
of
ureC digested with
Sau3A, the experimental
results were discordant
from the published results. Results form the
three published studies
could be analyzed, yielding a pooled DI of
0.86. The experimental
results yielded a DI of 0.97. This discrepancy
may be explained
by differences in the fragments amplified within the
ureC gene:
we amplified a 1,100-bp fragment of
ureC, while a 820-bp fragment
was amplified in the three
previously described studies whose
data were analyzed. A similar
discrepancy was observed for PCR-RFLP
analysis of
ureC
digested with
HhaI for four published studies,
in which
820-bp fragments of
ureC were amplified (
22,
60-62),
and the study of Li et al. (
36), in which a
fragment of 1.1
kb was amplified (Table
5). The amplification of this
longer
fragment of the
ureC gene allowed a better
discrimination of isolates
when both
Sau3A and
HhaI restrictions were
used.
The double restriction techniques were less discriminatory: 0.71 for
BamHI-
HindII restriction digests of the
PCR-amplified
ureAB genes and 0.89 for
HaeIII-
HindIII restriction digests of
the
PCR-amplified
ureC gene (Table
2). As observed for RAPD
analysis,
correlations between PCR-RFLP types were observed. In fact, a
triplet of isolates and three pairs of isolates were indistinguishable
by three of the four PCR-RFLP techniques used (Table
1). An
epidemiological
link between these isolates was not likely, since two
of these
indistinguishable isolates came from two different geographic
areas and also because all isolates of the triplet and the three
pairs
were sampled over a 2-year interval. Moreover, these isolates
were
clearly distinguished with the other typing systems. A genetic
link
within the urease operon could explain these correlations
between the
restriction patterns of fragments amplified into functionally
linked
genes.
Some studies that used the PCR-RFLP method with other genes or with
other enzymes could not be compared with others but are
of interest.
Moore et al. (
42) used three PCR-RFLP typing systems
based
on restriction digestion of the PCR-amplified
ureC (1.1
kb)
gene. With 21 unrelated isolates, a DI of 0.71 could be calculated
for
HindIII restriction, a DI of 0.84 could be calculated
for
AluI restriction, and a DI of 0.38 could be calculated
for
PvuII
restriction. Li et al. (
36) digested a
PCR-amplified
ureC fragment
(1.1 kb) and obtained a DI of
0.91 for
MboI and a DI of 0.77 for
AluI. Fujimoto
et al. (
22) applied three PCR-RFLP techniques
to 25 unrelated isolates. PCR-RFLP analyses with the
ureC-amplified
portion (820 bp) digested with
HhaI,
MboI, or
MseI yielded DIs
of
0.87, 0.92, and 0.89, respectively. Forbes et al. (
19)
amplified
a portion of
flaA from 49 unrelated isolates. By
using four restriction
enzymes, the DIs obtained were 0.47 for
MspI, 0.29 for
HindIII,
0.88 for
MboI, and 0.80 for
AluI. Salaün et al.
(
58) amplified
flaA and restricted it with
MboI and
HaeIII, yielding DIs of 0.87
and 0.19, respectively. Restriction analysis of urease genes seems
to be more
discriminatory than restriction analysis of flagellin
genes.
Results from only three previously described studies that used REP-PCR
could be analyzed, yielding a pooled DI of 0.99. This
excellent
discriminatory power must be confirmed by more studies
with this typing
method.
The low discriminatory power of
HaeIII ribotyping observed
in our study (DI = 0.94) may be explained by the low typeability
of this typing system. The 10 untypeable isolates, grouped together
into one type, decreased the DI to just under the recommended
value of
0.95 (
28,
63). The meta-analysis of data from previously
published studies confirmed the limited value of the DI of
HaeIII
ribotyping (Table
4). The pooled DI was 0.94, which
was identical
to our result and which was less than the recommended
value of
0.95. As described by Fraser et al. (
21), the
meta-analysis
confirmed the low discriminatory power of
HindIII ribotyping (Table
4). Despite an excellent
typeability, the pooled DI was 0.92.
In the same way, the low
discriminatory power of PFGE calculated
in the meta-analysis is
explained by the very low typeability
of this technique (Table
4). The
higher DIs were obtained with
the
NotI enzyme, but with a
pooled DI of 0.88.
This study focused on two performance criteria: typeability and
discriminatory power. Performance criteria also include reproducibility
and stability (
63). Reproducibility refers to the ability of
a technique to yield the same result when the same isolate is
tested
repeatedly (
63). Stability refers to the ability of a
typing
system to recognize the clonal relatedness of strains derived
in vitro
or in vivo from a common ancestor strain (
63). The
reproducibilities and stabilities of
H. pylori typing
systems
have been already evaluated in many studies (
3,
4,
19-22,
33,
56,
66-69). RAPD analysis, for which reproducibility is
critical if technical conditions are not standardized, was especially
evaluated for its reproducibility (
3,
10,
33,
41,
69).
In this study, evaluation of the reproducibilities and stabilities of
the methods were conducted, with the only goal being
to confirm
acceptable performance. For this purpose, we tested
three clonally
related familial isolates (isolates 93-28, 92-1,
and 95-93) and
serially subcultivated strains of isolate 92-1.
Our study confirmed the
good reproducibilities and stabilities
of the three typing techniques
(ribotyping, PCR-RFLP analysis,
and RAPD analysis). Since
reproducibility was tested with only
a few isolates and since technical
conditions were not exhaustively
tested, our evaluation is only
qualitative. Our opinion is that
the PCR-RFLP analysis is the more
easily reproducible technique
tested. For ribotyping, the main
difficulty in terms of reproducibility
was obtaining an identical
amount of well-digested total DNA.
Different amounts of digested DNA
yielded differences in the intensities
of the bands constituting
ribopatterns. The reproducibility of
RAPD analysis has been
investigated by several authors (
10,
32,
33,
41,
69) and
should not represent a problem if
the technique is standardized,
including the use of standard methods
of DNA preparation, the use of
consistent volumes and concentrations
of reagents, consistent use of
the same thermostable DNA polymerase,
use of the same thermal cycler,
and use of a standard procedure
for visualization of the fingerprint.
We have observed that when
these factors known to contribute to
variability were rigidly
controlled, the reproducibilities of the RAPD
patterns were excellent
over the limited period of the
study.
Since the three familial isolates, collected over 3 years from three
persons who had not lived together for 10 years, yielded
the same
profiles by all the techniques tested, the stabilities
of these markers
are excellent. This absence of independent evolution
of isolates with a
common origin over several years was already
detected in studies with
familial isolates (
5,
7,
68).
Summary.
Our experimental results and the meta-analysis showed
that PCR-based typing methods (PCR-RFLP analysis, RAPD analysis, and REP-PCR) have optimal typeabilities. Two PCR-RFLP techniques (PCR-RFLP analyses of ureAB digested with HaeIII and
ureC digested with Sau3A) yield excellent
discriminatory powers. The low interlaboratory reproducibility of
typing by RAPD analysis does not allow an a priori evaluation of
its discriminatory power. For this rapid and convenient method,
determination of the concordance of the results with those obtained
with another typing system is necessary. The excellent
discriminatory power of REP-PCR should be confirmed. Chromosome
digestion methods (ribotyping and PFGE) are limited by their very
low typeabilities, which strongly decrease their discriminatory
powers, and therefore, they are not recommended for use in the
typing of H. pylori isolates. We recommend the use of
the PCR-RFLP method.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the Ligue contre
le Cancer, a grant from the Université de Poitiers, and a grant
from the Région Poitou-Charentes.
We are grateful to J. V. Solnick (Department of Internal Medicine,
School of Medicine, University of California, Davis) for helpful
discussions and for reviewing the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Microbiologie A, CHU La Milétrie, BP 577, 86021 Poitiers, France.
Phone: (00-33) 549443889. Fax: (00-33) 549443888. E-mail:
c.burucoa{at}chu-poitiers.fr.
| |
REFERENCES |
| 1.
|
Akashi, H.,
T. Hayashi,
H. Koizuka,
T. Shimoyama, and T. Tamura.
1996.
Strain differentiation and phylogenic relationships, in terms of base sequence of the ureB gene, of Helicobacter pylori.
J. Gastroenterol.
31(Suppl. 9):16-23.
|
| 2.
|
Akopyants, N. S.,
K. A. Eaton, and D. E. Berg.
1995.
Adaptative mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets.
Infect. Immun.
63:116-121[Abstract].
|
| 3.
|
Akopyanz, N.,
N. O. Bukanov,
T. U. Westblom,
S. Kresovich, and D. E. Berg.
1992.
DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting.
Nucleic Acids Res.
20:5137-5142[Abstract/Free Full Text].
|
| 4.
|
Akopyanz, N.,
N. O. Bukanov,
T. U. Westblom, and D. E. Berg.
1992.
PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori.
Nucleic Acids Res.
20:6221-6225[Abstract/Free Full Text].
|
| 5.
|
Bamford, K. B.,
J. Bickley,
J. S. A. Collins,
B. T. Johnston,
S. Potts,
V. Boston,
R. J. Owen, and J. M. Sloan.
1993.
Helicobacter pylori: comparison of DNA fingerprints provides evidence for intrafamilial infection.
Gut
34:1348-1350[Abstract/Free Full Text].
|
| 6.
|
Beji, A.,
P. Vincent,
I. Darchis,
M. O. Husson,
A. Cortot, and H. Leclerc.
1989.
Evidence of gastritis with several Helicobacter pylori strains.
Lancet
i:1402-1403.
|
| 7.
|
Cho, M.-J.,
W.-K. Lee,
Y.-S. Jeon,
K.-H. Kim,
S.-H. Kim,
S.-C. Bai,
K.-H. Rhee,
Y.-O. Kim,
H.-S. Yoon, and N.-S. Kim.
1995.
Intrafamilial transmission of Helicobacter pylori detected by random amplified polymorphic DNA fingerprinting.
Mol. Cells
5:508-513.
|
| 8.
|
Clayton, C. L.,
H. Kleanthous,
J. C. Dent,
C. A. M. McNulty, and S. Tabaqchali.
1991.
Evaluation of fingerprinting methods for identification of Helicobacter pylori strains.
Eur. J. Clin. Microbiol. Infect. Dis.
10:1040-1047[Medline].
|
| 9.
|
Clayton, C. L.,
H. Kleanthous,
D. D. Morgan,
L. Puckey, and S. Tabaqchali.
1993.
Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis.
J. Clin. Microbiol.
31:1420-1425[Abstract/Free Full Text].
|
| 10.
|
Davin-Regli, A.,
Y. Abed,
R. N. Charrel,
C. Bollet, and P. de Micco.
1995.
Variations in DNA concentrations significantly affect the reproducibility of RAPD fingerprint patterns.
Res. Microbiol.
146:561-568[Medline].
|
| 11.
|
Desai, M.,
D. Linton,
R. J. Owen,
H. Cameron, and J. Stanley.
1993.
Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rRNA and a species-specific DNA probe.
J. Appl. Bacteriol.
75:574-582[Medline].
|
| 12.
|
Desai, M.,
D. Linton,
R. J. Owen, and J. Stanley.
1994.
Molecular typing of Helicobacter pylori isolates from asymptomatic, ulcer and gastritis patients by urease gene polymorphism.
Epidemiol. Infect.
112:151-160[Medline].
|
| 13.
|
Dore, M. P.,
M. S. Osato,
D. H. Kwon,
D. Y. Graham, and F. A. K. El-Zaatari.
1998.
Demonstration of unexpected antibiotic resistance of genotypically identical Helicobacter pylori isolates.
Clin. Infect. Dis.
27:84-89[Medline].
|
| 14.
|
Dubois, A.,
D. E. Berg,
E. T. Incecik,
N. Fiala,
L. M. Heman-Ackah,
G. I. Perez-Perez, and M. J. Blaser.
1996.
Transient and persistent experimental infection of nonhuman primates with Helicobacter pylori: implications for human disease.
Infect. Immun.
64:2885-2891[Abstract].
|
| 15.
|
Dunn, B. E.,
H. Cohen, and M. J. Blaser.
1997.
Helicobacter pylori.
Clin. Infect. Dis.
10:720-741.
|
| 16.
|
Dzierzanowska, D.,
A. Gzyl,
E. Rozynek,
E. Augustynowicz,
U. Wojda,
D. Celinska-Cedro,
M. Sankowska, and T. Wadström.
1996.
PCR for identification and typing of Helicobacter pylori isolated from children.
J. Physiol. Pharmacol.
47:101-114[Medline].
|
| 17.
|
The EUROGAST Study Group.
1993.
An international association between Helicobacter infection and gastric cancer.
Lancet
341:1359-1362[Medline].
|
| 18.
|
Evans, D. G.,
D. J. Evans,
H. C. Lampert, and D. Y. Graham.
1995.
Restriction fragment length polymorphism in the adhesin gene hpaA of Helicobacter pylori.
Am. J. Gastroenterol.
90:1282-1288[Medline].
|
| 19.
|
Forbes, K. J.,
Z. Fang, and T. H. Pennington.
1995.
Allelic variation in the Helicobacter pylori flagellin genes flaA and flaB: its consequences for strain typing schemes and population structure.
Epidemiol. Infect.
114:257-266[Medline].
|
| 20.
|
Foxall, P. A.,
L.-T. Hu, and H. L. T. Mobley.
1992.
Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates.
J. Clin. Microbiol.
30:739-741[Abstract/Free Full Text].
|
| 21.
|
Fraser, A. G.,
J. Bickley,
R. J. Owen, and R. E. Pounder.
1992.
DNA fingerprints of Helicobacter pylori before and after treatment with omeprazole.
J. Clin. Pathol.
45:1062-1065[Abstract/Free Full Text].
|
| 22.
|
Fujimoto, S.,
B. Marshall, and M. J. Blaser.
1994.
PCR-based restriction fragment length polymorphism typing of Helicobacter pylori.
J. Clin. Microbiol.
32:331-334[Abstract/Free Full Text].
|
| 23.
|
Ge, Z., and D. E. Taylor.
1998.
Helicobacter pylori molecular genetics and diagnostic typing.
Br. Med. Bull.
54:31-38[Abstract/Free Full Text].
|
| 24.
|
Georgopoulos, S. D.,
A. F. Mentis,
C. A. Spiliadis,
L. S. Tzouvelekis,
E. Tzelepi,
A. Moshopoulos, and N. Skandalis.
1996.
Helicobacter pylori infection in spouses of patients with duodenal ulcers and comparison of ribosomal RNA gene patterns.
Gut
39:634-638[Abstract/Free Full Text].
|
| 25.
|
Go, M. F.,
K. Y. Chan,
J. Versalovic,
T. Koeuth,
D. Y. Graham, and J. R. Lupski.
1995.
Cluster analysis of Helicobacter pylori genomic DNA fingerprints suggests gastroduodenal disease-specific associations.
Scand. J. Gastroenterol.
30:640-646[Medline].
|
| 26.
|
Graham, D. Y., and M. F. Go.
1993.
Helicobacter pylori: current status.
Gastroenterology
105:279-282[Medline].
|
| 27.
|
Hirschl, A. M.,
M. Richter,
A. Makristathis,
P. M. Prückl,
B. Willinger,
K. Schütze, and M. L. Rotter.
1994.
Single and multiple strain colonization in patients with Helicobacter pylori-associated gastritis: detection by macrorestriction DNA analysis.
J. Infect. Dis.
170:473-475[Medline].
|
| 28.
|
Hunter, P. R., and M. A. Gaston.
1988.
Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity.
J. Clin. Microbiol.
26:2465-2466[Abstract/Free Full Text].
|
| 29.
|
Hurtado, A., and R. J. Owen.
1994.
Identification of mixed genotypes in Helicobacter pylori from gastric biopsy tissue by analysis of urease gene polymorphisms.
FEMS Immunol. Med. Microbiol.
8:307-313[Medline].
|
| 30.
|
Hurtado, A.,
R. J. Owen, and M. Desai.
1994.
Flagellin gene profiling of Helicobacter pylori infecting symptomatic and asymptomatic individuals.
Res. Microbiol.
145:585-594[Medline].
|
| 31.
|
Ito, A.,
T. Fujioka,
T. Kubota, and M. Nasu.
1996.
Molecular typing of Helicobacter pylori: differences in pathogenicity among diverse strains.
J. Gastroenterol.
31:1-5[Medline].
|
| 32.
|
Jorgensen, M.,
G. Daskalopoulos,
V. Warburton,
H. M. Mitchell, and S. L. Hazell.
1996.
Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification from sequential and multiple biopsy specimens.
J. Infect. Dis.
174:631-635[Medline].
|
| 33.
|
Kansau, I.,
J. Raymond,
E. Bingen,
P. Courcoux,
N. Kalach,
M. Bergeret,
N. Braimi,
C. Dupont, and A. Labigne.
1996.
Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique.
Res. Microbiol.
147:661-669[Medline].
|
| 34.
|
Labigne, A.,
V. Cussac, and P. Courcoux.
1991.
Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity.
J. Bacteriol.
173:1920-1931[Abstract/Free Full Text].
|
| 35.
|
Li, C.,
D. A. Ferguson,
T. Ha,
D. S. Chi, and E. Thomas.
1993.
A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates.
J. Clin. Microbiol.
31:2157-2162[Abstract/Free Full Text].
|
| 36.
|
Li, C.,
T. Ha,
D. S. Chi,
D. A. Ferguson,
C. Jiang,
J. J. Laffan, and E. Thomas.
1997.
Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.
J. Clin. Microbiol.
35:3021-3025[Abstract].
|
| 37.
|
Linton, D.,
M. Moreno,
R. J. Owen, and J. Stanley.
1992.
16S rrn gene copy number in Helicobacter pylori and its application to molecular typing.
J. Appl. Bacteriol.
73:501-506[Medline].
|
| 38.
|
Majewski, S. I. H., and C. S. Goodwin.
1988.
Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation.
J. Infect. Dis.
157:465-471[Medline].
|
| 39.
|
Marshall, D. G.,
A. Chua,
P. W. Keeling,
D. J. Sullivan,
D. C. Coleman, and C. J. Smyth.
1995.
Molecular analysis of Helicobacter pylori populations in antral biopsies from individual patients using randomly amplified polymorphic DNA (RAPD) fingerprinting.
FEMS Immunol. Med. Microbiol.
10:317-323[Medline].
|
| 40.
|
Marshall, D. G.,
D. C. Coleman,
D. J. Sulivan,
H. Xia,
C. A. O'Morain, and C. J. Smyth.
1996.
Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori using short oligonucleotide probes containing repetitive sequences.
J. Appl. Bacteriol.
81:509-517[Medline].
|
| 41.
|
Meunier, J. R., and P. A. D. Grimont.
1993.
Factors affecting reproducibility of random amplified polymorphic DNA fingerprinting.
Res. Microbiol.
144:373-379[Medline].
|
| 42.
|
Moore, R. A.,
A. Kureishi,
S. Wong, and L. E. Bryan.
1993.
Categorization of clinical isolates of Helicobacter pylori on the basis of restriction digest analyses of polymerase chain reaction-amplified ureC genes.
J. Clin. Microbiol.
31:1334-1335[Abstract/Free Full Text].
|
| 43.
|
Morgan, D. D., and R. J. Owen.
1990.
Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts.
Mol. Cell. Probes
4:321-334[Medline].
|
| 44.
|
Nwokolo, C. U.,
J. Bickley,
A. R. Attard,
R. J. Owen,
M. Costas, and I. A. Fraser.
1992.
Evidence of clonal variants of Helicobacter pylori in three generations of a duodenal ulcer disease family.
Gut
33:1323-1327[Abstract/Free Full Text].
|
| 45.
|
Ohta-Tada, U.,
A. Takagi,
Y. Koga,
S. Kamiya, and T. Miwa.
1997.
Flagellin gene diversity among Helicobacter pylori strains and IL-8 secretion from gastric epithelial cells.
Scand. J. Gastroenterol.
32:455-459[Medline].
|
| 46.
|
Owen, R. J.,
J. Fraser,
M. Costas,
D. Morgan, and D. R. Morgan.
1990.
Signature patterns of DNA restriction fragments of Helicobacter pylori before and after treatment.
J. Clin. Pathol.
43:646-649[Abstract/Free Full Text].
|
| 47.
|
Owen, R. J.,
J. Bickley,
M. Costas, and D. R. Morgan.
1991.
Genomic variation in Helicobacter pylori: application to identification of strains.
Scand. J. Gastroenterol. Suppl.
181:43-50.
|
| 48.
|
Owen, R. J.,
J. Bickley,
A. Lastovica,
J. P. Dunn,
P. Borman, and C. Hunton.
1992.
Ribosomal RNA gene patterns of Helicobacter pylori from surgical patients with healed and recurrent peptic ulcers.
Epidemiol. Infect.
108:39-50[Medline].
|
| 49.
|
Owen, R. J.,
C. Hunton,
J. Bickley,
M. Moreno, and D. Linton.
1992.
Ribosomal RNA gene restriction patterns of Helicobacter pylori: analysis and appraisal of HaeIII digests as a molecular typing system.
Epidemiol. Infect.
109:35-47[Medline].
|
| 50.
|
Owen, R. J.,
G. D. Bell,
M. Desai,
M. Moreno,
P. W. Gant,
P. H. Jones, and D. Linton.
1993.
Biotype and molecular fingerprints of metronidazole-resistant strains of Helicobacter pylori from antral gastric mucosa.
J. Med. Microbiol.
38:6-12[Abstract/Free Full Text].
|
| 51.
|
Owen, R. J.,
M. Desai,
N. Figura,
P. F. Bayeli,
L. Di Gregorio,
M. Russi, and R. A. Musmanno.
1993.
Comparisons between degree of histological gastritis and DNA fingerprints, cytotoxicity and adhesivity of Helicobacter pylori from different gastric sites.
Eur. J. Epidemiol.
9:315-321[Medline].
|
| 52.
|
Owen, R. J.,
J. Bickley,
A. Hurtado,
A. Fraser, and R. E. Pounder.
1994.
Comparison of PCR-based restriction length polymorphism analysis of urease genes with rRNA gene profiling for monitoring Helicobacter pylori infections in patients on triple therapy.
J. Clin. Microbiol.
32:1203-1210[Abstract/Free Full Text].
|
| 53.
|
Prewett, E. J.,
J. Bickley,
R. J. Owen, and R. E. Pounder.
1992.
DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum.
Gastroenterology
102:829-833[Medline].
|
| 54.
|
Rautelin, H.,
W. Tee,
K. Seppälä, and T. U. Kosunen.
1994.
Ribotyping patterns and emergence of metronidazole resistance in paired clinical samples of Helicobacter pylori.
J. Clin. Microbiol.
32:1079-1082[Abstract/Free Full Text].
|
| 55.
|
Raymond, J.,
E. Bingen,
N. Brahimi,
M. Bergeret, and N. Kalach.
1996.
Randomly amplified polymorphic DNA analysis in suspected laboratory Helicobacter pylori infection.
Lancet
i:975.
|
| 56.
|
Romero Lopez, C.,
R. J. Owen, and M. Desai.
1993.
Differentiation between isolates of Helicobacter pylori by PCR-RFLP analysis of urease A and B genes and comparison with ribosomal RNA gene patterns.
FEMS Microbiol. Lett.
110:37-44[Medline].
|
| 57.
|
Salama, S. M.,
Q. Jiang,
N. Chang,
R. W. Sherbaniuk, and D. E. Taylor.
1995.
Characterization of chromosomal DNA profiles from Helicobacter pylori strains isolated from sequential gastric biopsy specimens.
J. Clin. Microbiol.
33:2496-2497[Abstract].
|
| 58.
|
Salaün, L.,
C. Audibert,
G. Le Lay,
C. Burucoa,
J. L. Fauchere, and B. Picard.
1998.
Panmictic structure of Helicobacter pylori demonstrated by the comparative study of six genetic markers.
FEMS Microbiol. Lett.
161:231-239[Medline].
|
| 59.
|
Schütze, K.,
E. Hentschel,
B. Dragosics, and A. M. Hirschl.
1995.
Helicobacter pylori reinfection with identical organisms: transmission by the patients' spouses.
Gut
36:831-833[Abstract/Free Full Text].
|
| 60.
|
Shortridge, V. D.,
G. G. Stone,
R. K. Flamm,
J. Beyer,
J. Versalovic,
D. W. Graham, and S. K. Tanaka.
1997.
Molecular typing of Helicobacter pylori isolates from a multicenter U.S. clinical trial by ureC restriction fragment length polymorphism.
J. Clin. Microbiol.
35:471-473[Abstract].
|
| 61.
|
Stone, G. G.,
D. Shortridge,
R. K. Flamm,
J. Beyer,
A. T. Ghoneim, and S. K. Tanaka.
1997.
PCR-RFLP typing of ureC from Helicobacter pylori isolated from gastric biopsies during a European multi-country clinical trial.
J. Antimicrob. Chemother.
40:251-256[Abstract/Free Full Text].
|
| 62.
|
Stone, G. G.,
D. Shortridge,
R. K. Flamm,
J. Beyer,
D. Stamler, and S. K. Tanaka.
1997.
PCR-RFLP typing of ureC from Helicobacter pylori isolated in Argentina from gastric biopsies before and after treatment with clarithromycin.
Epidemiol. Infect.
118:119-124[Medline].
|
| 63.
|
Struelens, M. J., and the Members of the European Study Group on Epidemiological Markers (ESGEM), of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID).
1996.
Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems.
Clin. Microbiol. Infect.
2:2-11.
[Medline] |
| 64.
|
Takami, S.,
T. Hayashi,
H. Akashi,
Y. Tonokatsu,
T. Shimoyama, and T. Tamura.
1993.
Chromosomal heterogeneity of Helicobacter pylori isolates by pulse-field gel electrophoresis.
Zentbl. Batteriol. Parasitenkd. Infektkrankh. Hyg. Abt. 1 Orig.
280:120-127.
|
| 65.
|
Takami, S.,
T. Hayashi,
H. Akashi,
T. Shimoyama, and T. Tamura.
1994.
Genetic heterogeneity of Helicobacter pylori by pulse-field gel electrophoresis and re-evaluation of DNA homology.
Eur. J. Gastroenterol. Suppl.
6:S53-S56.
|
| 66.
|
Taylor, D. E.,
M. Eaton,
N. Chang, and S. M. Salama.
1992.
Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.
J. Bacteriol.
174:6800-6806[Abstract/Free Full Text].
|
| 67.
|
Taylor, N. S.,
J. G. Fox,
N. S. Akopyants,
D. E. Berg,
N. Thompson,
B. Shames,
L. Yan,
E. Fontham,
F. Janney,
F. M. Hunter, and P. Correa.
1995.
Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting.
J. Clin. Microbiol.
33:918-923[Abstract].
|
| 68.
|
Tee, W.,
J. Lambert,
R. Smallwood,
M. Schembri,
B. C. Ross, and B. Dwyer.
1992.
Ribotyping of Helicobacter pylori from clinical specimens.
J. Clin. Microbiol.
30:1562-1567[Abstract/Free Full Text].
|
| 69.
|
Tyler, K. D.,
G. Wang,
S. D. Tyler, and W. M. Johnson.
1997.
Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens.
J. Clin. Microbiol.
35:339-346[Medline].
|
| 70.
|
Van der Ende, A.,
E. A. J. Rauws,
M. Feller,
C. J. J. Mulder,
G. N. J. Tytgat, and J. Dankert.
1996.
Heterogeneous Helicobacter pylori isolates from members of a family with a history of peptic ulcer disease.
Gastroenterology
111:638-647[Medline].
|
| 71.
|
Van der Hulst, R. W. M.,
E. A. J. Rauws,
B. Köycü,
J. J. Keller,
F. J. W. ten Kate,
J. Dankert,
G. N. J. Tytgat, and A. Van der Ende.
1997.
Helicobacter pylori reinfection is virtually absent after successful eradication.
J. Infect. Dis.
176:196-200[Medline].
|
| 72.
|
Van Doorn, N. E.,
F. Namavar,
J. G. Kusters,
E. P. van Rees,
E. J. Kuipers, and J. de Graaff.
1998.
Genomic DNA fingerprinting of clinical isolates of Helicobacter pylori by REP-PCR and restriction fragment end-labelling.
FEMS Microbiol. Lett.
160:145-150[Medline].
|
| 73.
|
Wada, S.,
M. Matsuda,
M. Kikuchi,
T. Kodama,
I. Takei,
S. Ogawa,
S. Takahashi,
M. Shingaki, and T. Itoh.
1994.
Genome DNA analysis and genotyping of clinical isolates of Helicobacter pylori.
Cytobios
80:109-116[Medline].
|
| 74.
|
Wang, J.-T.,
J.-C. Sheu,
J.-T. Lin,
T.-H. Wang, and M.-S. Wu.
1993.
Direct DNA amplification and restriction pattern analysis of Helicobacter pylori in patients with duodenal ulcer and their families.
J. Infect. Dis.
168:1544-1548[Medline].
|
| 75.
|
Weel, J. F. L.,
R. W. M. Van der Hulst,
Y. Gerrits,
G. N. J. Tytgat,
A. Van der Ende, and J. Dankert.
1996.
Heterogeneity in susceptibility to metronidazole among Helicobacter pylori isolates from patients with gastritis or peptic ulcer disease.
J. Clin. Microbiol.
34:2158-2162[Abstract].
|
| 76.
|
Xia, H. X.,
H. J. Windle,
D. G. Marshall,
C. J. Smyth,
C. T. Keane, and C. A. O'Morain.
1995.
Recrudescence of Helicobacter pylori after apparently successful eradication: novel application of randomly amplified polymorphic DNA fingerprinting.
Gut
37:30-34[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, December 1999, p. 4071-4080, Vol. 37, No. 12
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
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