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Pattern of Primary Resistance of Helicobacter pylori to Metronidazole or Clarithromycin in the United States
Michael S. Osato, PhD;
Rita Reddy, MS;
Siddharta G. Reddy, BS;
Rebecca L. Penland, BS;
Hoda M. Malaty, MD, PhD;
David Y. Graham, MD
Arch Intern Med. 2001;161:1217-1220.
ABSTRACT
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Background Therapy for Helicobacter pylori is generally
empiric despite the fact that resistance to metronidazole and clarithromycin
compromise therapeutic efficacy. The aim of this study was to aid clinicians
in choosing a course of therapy for H pylori infection
in the United States.
Methods The frequency of primary clarithromycin and metronidazole resistance
among H pylori isolated from patients enrolled in
US-based clinical trials between 1993 and 1999 was reviewed in relation to
patient age, sex, region of the United States, and test method (Etest and
2 agar dilution procedures).
Results Clarithromycin and metronidazole resistance rates were based on the
results of 3439 pretreatment Etest determinations and 3193 agar dilution determinations.
Sex and age were available on 900 and 823 individuals, respectively. Metronidazole
resistance was 39% by Etest and 21.6% by agar dilution (P<.001). Clarithromycin resistance was 12% by Etest and 10.6% by
agar dilution. Amoxicillin or tetracycline resistance was rare. Metronidazole
and clarithromycin resistance was more common in women than men (eg, 34.7%
vs 22.6% for metronidazole and 14.1% vs 9.7% for clarithromycin (P = .01 and P = .06, respectively). Antibiotic
resistance increased gradually up to age 70 years, then declined significantly
(P<.05) regardless of test method. Regional differences
in antimicrobial resistance did not occur.
Conclusions While age and sex had significant effects on resistance rates, regional
differences were not present. The high prevalence of resistance to metronidazole
and clarithromycin may soon require the performance of antimicrobial susceptibility
testing of H pylori isolates prior to initiating
treatment.
INTRODUCTION
ANTIBIOTIC treatment of Helicobacter pylori
infection is complex, requiring multiple antibiotics combined with acid suppressive
agents to ensure high cure rates. Treatment failure is attributed generally
to the lack of compliance with the drug regimen or to acquired antibiotic
resistance of the organism.1-2
Emergence of resistant H pylori has been documented
in various countries and threatens to compromise current treatment regimens.1-12
However, data on resistance are generally obtained from unique subsets of
populations over a short period. There are few data regarding the resistance
rates in the United States.
Investigations have shown that demographic factors play an important
role in the reported rates of antibiotic resistance in H pylori. Metronidazole resistance is higher in developing countries
and is especially high in immigrant populations,1, 3-7
in women,8, 10-11
and in areas where this agent is used routinely for the treatment of assorted
gastrointestinal maladies.7-8
There are few reports with regard to the role of demographic factors such
as age, sex, and region, on the prevalence of metronidazole and clarithromycin
resistance in H pylori from the United States.8-16
PATIENTS, MATERIALS, AND METHODS
POPULATION
The frequency of primary clarithromycin and metronidazole resistance
among H pylori isolated from patients enrolled in
17 US-based antibiotic treatment trials between 1993 and 1999 was reviewed
in relation to patient age, sex, region of the United States, and test method
(Etest and 2 agar dilution procedures). During the initial visit, 1 antral
and 1 corpus biopsy specimen was obtained from each patient for culture and
susceptibility testing. Only 1 isolate per individual was used to calculate
the overall resistance rates. The isolate selected was that from the antrum,
although the corpus isolate was used in a few instances if the antral site
yielded no growth of H pylori. Overall rates of clarithromycin
and metronidazole resistance were determined by dividing the total number
of resistant isolates by the total number of data points for that specific
test. Differences in results between techniques were determined by comparing
the responses between single isolates tested by both methods.
ETEST AND AGAR DILUTION PROCEDURES
Mueller-Hinton agar (Remel Laboratories, Lenexa, Kan) with 5% sheep
blood (BBL Becton Dickinson, Cockeysville, Md) was used as the base medium.
Metronidazole and clarithromycin Etest strips were aseptically placed onto
the dried surfaces of the inoculated plates. The Etest plates were incubated
under 12% carbon dioxide at 37°C for 4 days. The agar dilution procedure
was performed using Mueller-Hinton agar as the base medium to which 5% aged
(>2 weeks old) sheep blood and serial dilutions of metronidazole (Sigma Chemical
Co, St Louis, Mo) and clarithromycin (Abbott Laboratories, Abbott Park, Ill)
were added. The plates were poured on the day of testing. Fresh bacterial
suspensions were prepared in sterile saline and adjusted to an optical density
(OD) of 0.38 to 0.40 at 625 nm (approximately 6 x 108 cells/mL).
Five microliters of the adjusted inoculum was delivered to each plate by a
Steers type replicate plating device. The plates were incubated in anaerobic
jars with CampyPak Plus (BBL Becton Dickinson) gas generating envelopes for
72 hours at 37°C.
MIC DETERMINATIONS
Etest minimum inhibitory concentration (MIC) values were defined as
the intercept of the elliptical zone of inhibition with the graded Etest strip
per the instructions of the manufacturer. The MIC value for the agar dilution
method was defined as the lowest concentration of antibiotic that completely
inhibited visible growth.
MIC INTERPRETIVE CRITERIA AND COMPARISONS
Isolates were considered resistant if the MIC value was greater than
8 µg/mL for metronidazole and greater than 1 µg/mL for clarithromycin.17-18 Differences are reported in relation
to a variance in the respective MIC values of 2 log2 or higher
between the antral and corpus isolates, and as a change in the susceptibility
pattern from sensitive to resistant or vice-versa.
STATISTICAL ANALYSIS
The prevalence rates of clarithromycin and metronidazole resistance
were calculated for the total isolates. In addition, the age- and sex-specific
prevalence, as well as regional distribution and the year of the isolate collection
were calculated using each test independently. Mantel-Haenszel  2 statistics were applied to compare the magnitude of the difference
between rates of resistance to different antibiotics and within the same antibiotics
using different techniques. The data were analyzed using the SAS program,
version 5 (SAS Institute, Cary, NC, 1985).
RESULTS
The database consisted of 3439 isolates tested by Etest and 3193 isolates
tested by agar dilution. Of the 3193 agar dilution determinations, 2648 were
determined by the method approved by the National Committee on Clinical Laboratory
Standards. Differences in results between techniques were determined by comparing
the responses between single isolates tested by both methods in a subset of
samples: 188 specimens tested for metronidazole susceptibility and 332 isolates
tested for clarithromycin susceptibility.
EFFECT OF TEST METHOD
The test method greatly affected the results of susceptibility testing
for metronidazole (Table 1). Forty-two
percent of the metronidazole MIC values differed by 2 log2 or more
and resulted in a difference in susceptibility pattern in 17.6%. In contrast,
the difference in susceptibility pattern was only 3% with clarithromycin,
and thus the results with either method were combined for overall susceptibility.
The data with metronidazole are presented separately for both methods except
where noted.
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Table 1. Effect of Test Method on Frequency of Metronidazole Resistance:
Comparison of Etest vs Agar Dilution*
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RESISTANCE OVER A 7-YEAR PERIOD
The rates of resistance to clarithromycin varied among the years of
evaluation (2 = 12.855; P = .05)
(Figure 1). The overall prevalence
of clarithromycin resistance was 11.1% varying from 6.1% to 14.5% among the
years of evaluation.
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Figure 1. Frequency of clarithromycin resistance
rates by year of collection. Annual rate of clarithromycin resistance as determined
by combined Etest and agar dilution results. Data were combined because intratest
analysis revealed only a 3% change in susceptibility results between test
methods.
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The prevalence of metronidazole resistance differed depending on whether
the Etest or agar dilution was used. The prevalence of metronidazole resistance
with Etest was 39% (690/1768) compared with 25.2% (367/1459) determined by
the agar dilution methods (P<.001) (Table 2). The Etest consistently provided a higher estimate of the
prevalence of metronidazole resistance in all years of evaluation in which
more than 10 isolates were tested.
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Table 2. Frequency of Metronidazole and Clarithromycin Resistance Associated
With the Sex of the Patient
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EFFECT OF SEX
Women were more likely to have metronidazole-resistant H pylori than men (63% vs 35.1%, respectively, as determined by Etest
[P = .01] and 34.7% vs 22.6%, respectively, as determined
by agar dilution [P = .03]) (Table 2). The test method affected the overall rate of metronidazole
resistance for both men and women, but did not affect the comparative outcome
(ie, that the rate of metronidazole-resistant H pylori
was higher for women than for men).
Isolates from women were also more apt to be resistant to clarithromycin
than isolates from men (14.1% vs 9.7%), although the results were not significantly
different (P = .06) (Table 2).
EFFECT OF AGE
The age of the patient at the time of H pylori
recovery from the gastric biopsy specimens had a significant effect on both
the metronidazole and clarithromycin resistance rates. From ages older than
20 years, metronidazole resistance as determined by Etest increased to 50%
of the isolates tested until age 71 years or older, when the rate dropped
to 31% (P<.05). A similar pattern of increasing
resistance to metronidazole was seen with the agar dilution method (Figure 2). Helicobacter
pylori isolates from patients older than 70 years were also less likely
to be resistant to clarithromycin by either test method (P<.05).
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Figure 2. Frequency of metronidazole and
clarithromycin resistance as determined by agar dilution associated with the
age of the patient. The frequency of metronidazole and clarithromycin resistance
as determined by agar dilution with respect to each successive decade until
age 70 years. After age 70 years, there was a significant decrease in the
level of metronidazole and clarithromycin resistance.
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EFFECT OF GEOGRAPHIC REGION
We evaluated the effect of living in different regions of the United
States, and marked regional differences were not detected (Figure 3). The highest level of metronidazole resistance occurred
in the Southeast (27.5%; 88/320) and the lowest in the Northeast (22.1%; 91/412)
(P>.20). The highest rate of clarithromycin resistance
(13%; 67/516) occurred in the Northeast, while the lowest rate of clarithromycin
resistance was found in the West (8.3%; 44/533). However, none of the comparisons
was statistically significantly different.
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Figure 3. Regional resistance patterns for Helicobacter pylori isolates. CR indicates clarithromycin
resistance; MR, metronidazole resistance. All data are given as
percentages. Regional information was available for 1741 isolates for clarithromycin
resistance and 1165 isolates for metronidazole resistance. No significant
regional differences in resistance to metronidazole or clarithromycin occurred.
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COMMENT
Treatment outcomes of anti–H pylori therapies
are primarily dependent on compliance with the regimen and the presence of
antibiotic-resistant H pylori. Because antimicrobial
testing is not widely available for H pylori in most
regions, empiric therapy has been the rule. We used the antibiotic susceptibility
data from clinical trials in patients with duodenal ulcers performed in the
United States between 1993 and 1999 to evaluate the prevalence and rate of
emergence of resistance to clarithromycin and metronidazole in the United
States. Interlaboratory variation was eliminated as a variable because all
testing was performed in a single laboratory using standard procedures and
established end points.
Metronidazole resistance was present and stable in approximately 35%
of the isolates tested, while clarithromycin resistance was present in approximately
11% (higher in some populations, eg, women). It seems unlikely that the United
States is not involved in the worldwide trend of increasing clarithromycin
resistance.13-17
Women and the young adults were found to harbor resistant H pylori more often than men and older patients. This may be attributed
to the use of metronidazole-containing treatment regimens used by women to
treat gynecologic infections. The frequency of either metronidazole or clarithromycin
resistance was lower in those older than 70 years than in those of middle
age. We can only conjecture as to the reason for this finding, but confounding
demographic factors are suspected. There were no significant regional differences
in antibiotic resistance, suggesting that such differences can be ignored
currently when choosing an antibiotic regimen to treat H pylori infection.
While the proportion of samples with resistant H pylori was higher with the Etest than with agar dilution, the difference
was particularly striking only with metronidazole. It is important to emphasize
that neither of these test methods (Etest and agar dilution) has been clinically
verified with metronidazole, and only the presence of clarithromycin resistance
generally predicts a treatment failure. As a general rule, clarithromycin
resistance results in that antibiotic effectively "dropping out," with the
results approximating those expected with the other components of the therapy
(eg, a proton pump inhibitor plus clarithromycin treatment would yield almost
no cures with clarithromycin-resistant H pylori,
and the combination of a proton pump inhibitor, amoxicillin, and clarithromycin
would yield what would be expected with the proton pump inhibitor and amoxicillin
alone).19 In contrast, high-dose metronidazole
therapy can often overcome resistance when part of a multidrug regimen.19
Although not part of this investigation, 3 clinical H pylori isolates were found to be amoxicillin resistant (Etest MIC>16
µg/mL).20 The rates for amoxicillin and
tetracycline resistance among H pylori in the United
States are sufficiently low (<0.0005% of 6470 isolates; unpublished observation,
1999) that antimicrobial susceptibility testing for these drugs is currently
not needed clinically. However, data should be gathered to monitor the level
of emergent resistance in the general population.
Triple and quadruple antibiotic treatment regimens used for initial
therapy still achieve eradication in more than 80% of infected patients. However,
pretreatment resistance is a growing problem and is the main factor responsible
for treatment failure with regimens using these compounds. Continued increases
in the level of pretherapy antibiotic resistance might necessitate pretreatment
antibiotic susceptibility testing in many regions. Retreatment owing to failed
initial therapy is associated with higher costs and possibly higher levels
of adverse effects related to salvage therapies.21
Knowledge of the antibiogram of the infecting H pylori
strain, especially for metronidazole and clarithromycin, will have an immediate
beneficial outcome on the type and course of treatment.
AUTHOR INFORMATION
Accepted for publication January 23, 2000.
Research was supported in part by Hilda Schwartz and the Veterans Affairs
Medical Center, Houston, Tex.
Data were obtained from clinical trials supported by the following pharmaceutical
companies: Abbott Laboratories, Abbot Park, Ill; Tap Pharmaceuticals Inc,
Deerfield, Ill; Pfizer Inc, New York, NY; Astra-Merck Inc, Wayne, Pa; Wyeth-Ayerst
Pharmaceuticals, Philadelphia, Pa; and Glaxo-Wellcome Inc, Research Triangle
Park, NC.
Reprints: Michael S. Osato, PhD, Gastroenterology Microbiology Laboratory,
Department of Medicine, Veterans Affairs Medical Center, MS 111D, 2002 Holcombe
Boulevard, Houston, TX 77030 (e-mail: mosato{at}bcm.tmc.edu).
From the Departments of Medicine (Drs Osato, Malaty, and Graham and
Mr S. G. Reddy and Mss Reddy and Penland) and Molecular Virology and Microbiology
(Dr Graham), Veterans Affairs Medical Center, Baylor College of Medicine,
Houston, Tex.
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