Helicobacter pylori eradication is the most important target in the treatment of peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and prevention of gastric cancer development (1). Antimicrobial resistance, specially to clarithromycin, is the main cause of the eradication failure (2). H pylori infection is acquired during the first 5 years of age, but there are far fewer indications for eradication in infected children than in adult patients (3). As a consequence, there is little consensus on testing H pylori susceptibility to antimicrobials, mainly when retreatment is required because of failure in the first-line regimen (1).
Standard triple therapy (clarithromycin or metronidazole and amoxicillin) based on susceptibility test increases eradication rates (4), and it remains the first choice in regions with proven low clarithromycin resistance rates (1). An effective anti—H pylori therapy with susceptible strains should always reach 100% eradication per protocol (5), but in developing countries, even an efficient eradication regimen shows lower rates and children usually present rates 10% lower than adults using the same triple therapy (3).
The efficacy of H pylori treatment differs according to the geographical region (6). In developing countries, the resistance rate to antimicrobials is higher because of an increased use of these drugs. Then, antimicrobial choice should be determined according to resistance pattern to predict the range of possible outcomes (5,7). Thus, the growing emergence of resistant strains shows the need to know primary and secondary resistance rates in each region by routine susceptibility tests (8).
H pylori eradication requires an approach similar to those used in other infectious diseases, which is based on susceptibility tests, regional susceptibility, or both. Nevertheless, antimicrobials are empirically prescribed (9).
In Brazilian adults, metronidazole resistance ranges from 52.9% to 55% (10,11), resistance to furazolidone occurs in 4% and resistance to amoxicillin occurs in 29% (12). Tetracycline presented resistance rates of 9% (11) and clarithromycin of 9.8% (10). No studies exist regarding antimicrobial susceptibility in infected children, and the resistance pattern must be known to assist in choosing the antimicrobial schedule and to predict the probability of eradication failure (8). This is mainly because unlike American children, studies show low eradication rates using triple therapy in Brazilian children (13,14)
The aim of the present study was to assess the resistance rate of H pylori strains isolated in gastric biopsies from children and adolescents to clarithromycin, amoxicillin, furazolidone, tetracycline, and metronidazole, the most commonly used antimicrobials in eradication treatment.
From February 2008 to August 2009, 77 consecutive H pylori isolates, 71 strains obtained from patients without previous eradication treatment for H pylori infection, and 6 strains from patients in whom previous eradication treatment had failed were obtained from children and adolescents (range 3–20 years, mean age 11.1 ± 3.9 years, median 10.8 years; M/F: 1:1.08), 42 (54.5%) whites, and 35 (45.5%) nonwhite, who were subject to endoscopic examination to evaluate dyspeptic symptoms at Hospital São Paulo, Universidade Federal de São Paulo, and Cândido Fontoura Children's Hospital (Table 1). The findings were antral nodularity in 62 (80.5%) patients, duodenal ulcer in 5 (6.5%), antral erosive gastritis in 4 (5.2%), normal endoscopy in 3 (3.8%), gastric erythema in 3 (3.8%), and erosive esophagitis and gastric polyp (1.3%).
Previous use of proton pump inhibitors or antimicrobials, at least for 1 month, or associated chronic disease, were exclusion criteria. The study was approved by the Federal University of São Paulo ethics committee, and informed consents were signed.
Biopsy Sampling and Bacterial Strains
Two antral biopsy specimens were collected and transported to the microbiology laboratory in brain-heart infusion (BHI) broth plus glycerol (10%) preserved at a temperature of 4°C. Gastric biopsy specimens were homogenized and 5-μL solution was inoculated into selective BHI agar base (DIFCO, Lawrence, KS) containing 7% to 10% defibrinated sheep blood, vancomycin (10 mg/L), trimethoprim (5 mg/L), cefsulodin (5 mg/L), and amphotericin B (5 mg/L) (H pylori selective medium, Dent Supplement Oxoid, Basingstoke, Hampshire, UK). All of the plates were incubated for 10 days at 37°C in a microaerobic atmosphere (10% CO2, 85% N2, 5% O2) at 95% humidity (Microaerobac, Probac do Brasil, São Paulo, Brazil). H pylori INCQS 00380—ATCC 43504 strain was used as control.
Isolates that produced a bacterial growth demonstrating typical morphologic features by dark-field microscopy and produced urease, oxidase, and catalase were stored at −70°C in BHI broth containing 30% glycerol.
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing was performed by the agar dilution method in accordance with Clinical Laboratory Standards Institute protocols (2006, M7-A5). For antimicrobial agents without a Clinical Laboratory Standards Institute–recommended breakpoint, cutoff value was selected based on the literature. Five milliliters of frozen isolates were subcultured onto BHI agar containing 10% defibrinated sheep blood and incubated for 3 days at 37°C under microaerophilic conditions. The colonies were suspended in BHI broth and adjusted to McFarland 4 turbidity standard (approximately 1 × 108 cfu/mL), then inoculated onto Mueller-Hinton agar plates using a multipoint replicating device delivering 2 μL of inoculum.
The final concentrations of clarithromycin (Abbott Laboratories, Chicago, IL), tetracycline, furazolidone, and amoxicillin (Sigma Aldrich Chemie, Steinheim, Germany) ranged from 0.015 to 64 μg/mL. Metronidazole (Sigma Aldrich Chemie) concentration ranged from 0.015 to 256 μg/mL. The plates were incubated at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2 at 95% humidity) for 72 hours. Isolates were considered resistant if the minimum inhibitory concentration (MIC) was ≥2 μg/mL to amoxicillin and furazolidone, ≥4 μg/mL to tetracycline, and ≥8 μg/mL to metronidazole. Clarithromycin isolates were considered resistant with MIC ≥2 μg/mL, and intermediary if MIC was 0.5 μg/mL.
Statistical analysis of categorical data from resistant and sensible H pylori isolates was performed by χ2 test. Differences with P < 0.05 were considered statistically significant. Susceptibility and resistance rates were calculated by proportion and expressed as percentage. MIC 50 and MIC 90 were determined as minimum concentration of antimicrobial that is capable to inhibit the development of 50% and 90% of bacteria isolates, respectively.
Resistance was observed in 49.3% (38/77) of H pylori strains; almost all of them (40.2%) presented resistance to metronidazole, 19.5% to clarithromycin, and 10.4% to amoxicillin. Furazolidone and tetracycline showed no resistance (0%). Fourteen (18.2%) H pylori isolates showed multiple resistance: 6 of 14 (43%) to clarithromycin and metronidazole; 5 of 14 (36%) to amoxicillin and metronidazole; 2 of 14 (14%) to amoxicillin, clarithromycin, and metronidazole; and 1 of 14 (7%) to clarithromycin and amoxicillin. All strains resistant to amoxicillin presented multiple resistance.
Resistance rates, MIC range, MIC 50, and MIC 90 are shown in Table 2. MIC 90 was near the cutoff value (≤1 μg/mL) to clarithromycin and 3 log2 over recommended cutoff (64 μg/mL) to metronidazole; however, MIC 50 was within the antimicrobial susceptibility patterns of tested antibiotics (Table 2).
Secondary resistance was observed in 3 of 6 (50%) patients who had been treated previously: 2 had used clarithromycin, amoxicillin, and proton pump inhibitor; one of them showed resistance to clarithromycin and another to amoxicillin and metronidazole; and the third patient had used doxycycline and furazolidone and was resistant to clarithromycin and metronidazole. There were no association of resistant H pylori strain with race, sex, age (cutoff 12 years), and familial peptic disease (P > 0.05) (Table 1).
In the present study, 5 of the most used antimicrobials for H pylori eradication in Brazilian children were evaluated (13–15). Our results showed resistance to 3 antimicrobials: metronidazole, clarithromycin, and amoxicillin. As expected in a developing country, metronidazole presented the highest resistance rate, that is, 40.2%, probably caused by disseminated consumption to intestinal parasitic and gynecological infections. This result is similar to Brazilian adults (10,11) and children in other developing countries (16). Unfortunately, metronidazole should not be prescribed empirically when resistance is >40% (1); however, metronidazole is a prodrug that needs activation by nitroreductase enzymes, and the antimicrobial action depends on the oxireduction process; H pylori has several nitroreductases that can activate the drugs (17). Then, in vitro resistance cannot impede the use of metronidazole because resistance could be beaten by a higher dosage (17), but it needs further study.
Clarithromycin is one of the most widely used antimicrobials to eradicate H pylori infection, and resistance is the main reason for eradication failure in adults (18) and children (19). Previous local studies using clarithromycin in empirical antimicrobial treatment showed lower eradication rates comparing with triple therapy without clarithromycin (13–15). Our results showed a high resistance rate of H pylori to clarithromycin (19.5%), similar to European children. Megraud (20) observed primary resistance rate of 12.4% to 23.5% and Koletzko et al (21) reported a global resistance to clarithromycin of 24% (primary resistance in 20% and secondary in 42%) in multicenter studies. Our rate of resistance to clarithromycin probably results from the disseminated use of this antimicrobial for respiratory infectious diseases. Considering that clarithromycin should not be prescribed empirically if resistance rate ranges from 15% to 20% (1), and 1 study showed that no eradication was achieved when strains are resistant (8), clarithromycin should be precluded in empirical eradication therapy in Brazil; however, the rate of primary resistance to clarithromycin depends on the assessed population and the period of the study. A recent study showed that resistance rates to clarithromycin were stable in Belgian children, probably because of the decreasing prescription of macrolides (22).
Amoxicillin is another key antimicrobial in the standard triple therapy to eradicate H pylori. In the literature, the resistance rate ranged from 0% to 2% (20–27). Our study showed a resistance rate of 10.4%, which surprisingly is not higher considering its wide use in pediatric practice. Moreover, in vitro resistance does not necessarily affect eradication rates; even when genetic markers, for example, pbp 1A (“penicillin-binding protein”) is present, mutation is present. There are some possible reasons: the low frequency of responsible mutations for resistance, wide variations in pbp 1A mutation, and the necessity of “cooperative” mutations to express amoxicillin resistance (28–30); therefore, even when susceptibility tests demonstrate resistance, amoxicillin can be used in the eradication schedule. Amoxicillin, however, demonstrates other difficulties in susceptibility tests. The amoxicillin susceptibility test should be performed rapidly after obtaining the biopsy because amoxicillin-resistant strains often change to susceptible strains by freezing at −80°C. The 10.4% rate could, therefore, be higher than observed. Another phenomenon that needs attention is β-lactam antimicrobial tolerance, which occurs because there is a difference between the MIC and the minimum bactericidal concentration (MBC). In this situation, a serum concentration of antimicrobial can inhibit the bacteria but cannot kill them. Considering this, the MBC also should be evaluated in amoxicillin (31).
The H pylori strains in our study showed total susceptibility to tetracycline and furazolidone, but these antimicrobials are not generally prescribed for children. No resistance to tetracycline was observed in some studies (23,24,32), and low rates were observed in others (26,27,33); however, tetracycline presents adverse events mainly in tooth calcification. The degree of exposure, number of courses, total dosage, and timing of tooth development may affect the risk of adverse event occurrence (34); however, doxycycline can replace tetracycline (15) with less occurrence of tooth discoloration, even in children (mean age 4 years) (35).
Studies also presented low resistance rate to furazolidone in Brazilian (4%) (12), Spanish (1.8%) (36), and Korean adults (2%) (37); however, there are no studies on furazolidone resistance in children, probably because it is not recommended for children younger than 8 years. Furazolidone is commonly used to replace nitroimidazoles, and it is commercially available in some developing countries such as Iran, Pakistan, Mexico, and Brazil, and in some Asian countries such as China (38). European countries (European Medicines Agency) and the United States (FDA) had banned furazolidone because of the adverse effects. Unfortunately, furazolidone seems to be an option to retreatment, mainly in developing countries where resistance to metronidazole is too high. More studies are needed to link the adverse effects (genotoxic and carcinogenic effects) to the dose and time prescribed to H pylori infection; furazolidone presents a higher risk of complications in long-term use (39). Furthermore, the risks of genotoxicity and carcinogenic effects need to be evaluated against the risk of carcinogenic effects of H pylori infection in high-risk patients. Despite the adverse effects, and considering the cost-benefit ratio compared with conventional triple therapy, furazolidone should be an option in developing countries, where this antimicrobial could expand the range of “intention-to-treat” treatments.
In this study, there was no clinical difference between the group of patients infected by resistant strains and the group infected by susceptible strains, but differences are usually observed between these groups when sex, age, and region of study were evaluated (20,40,41). It is probable that the small sample in this study has confounded the statistical analysis. The present study was restricted to 2 centers in São Paulo City, so clinical condition and antimicrobial resistance status may not be representative of the Brazilian population. Further prospective surveillance of H pylori resistance is essential to ensure that appropriate data are available to guide the choice of therapy, particularly in high-risk populations.
In summary, we have observed high resistance to clarithromycin and metronidazole in clinical H pylori isolates and this resistance can exclude these antimicrobials in empirical eradication treatment in Brazil. Furazolidone and tetracycline presented no resistance. These antimicrobials usually are not prescribed to children, mainly because of adverse effects. When the risks and benefits are properly assessed, these 2 antimicrobials and their derivatives can be used in empirical eradication schedules in developing countries where resistance rates are high. Amoxicillin is a good choice in standard triple therapy because of its low resistance rate despite its wide use in pediatric patients.
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