JCM Accepts, published online ahead of print on 8 September 2010 J. Clin. Microbiol. doi: /jcm

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1 JCM Accepts, published online ahead of print on September 0 J. Clin. Microbiol. doi:./jcm.0- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved High Prevalence of Clarithromycin Resistance and caga, vaca, icea, and baba Genotypes of Helicobacter pylori in Brazilian Children Gabriella Garcia, 1, Katia R. S. Aranda, 1 Manoel E. P. Gonçalves, Silvia R. Cardoso, Kiyoshi Iriya, Neusa P. Silva, and Isabel C. A. Scaletsky, 1* 1 Departamento de Microbiologia, Imunologia e Parasitologia, Departamento de Pediatria, and Disciplina de Reumatologia, Universidade Federal de São Paulo, and Instituto da Criança and Departamento de Patologia Médica, Faculdade de Medicina, Universidade Estadual de São Paulo, São Paulo, Brasil *Corresponding author. Mailing address. Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu,, º andar, 00-0, São Paulo, SP, Brasil. Phone: --. Fax: scaletsky@unifesp.br. Downloaded from on January 1, 01 by guest 1

2 ABSTRACT We isolated Helicobacter pylori strains from 1 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in %, 1%, %, and 0% of strains, respectively. The A1G mutation was the most prevalent (%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of caga, vaca s1m1, icea, and baba genes. Downloaded from on January 1, 01 by guest

3 Helicobacter pylori infection is found worldwide and constitutes a public health concern in many countries. Previous epidemiological studies have shown a high prevalence of H. pylori infection in Brazil (, 1, ). H. pylori infection, generally acquired in childhood, persists asymptomatically for decades in most individuals. Amoxicillin, tetracycline, metronidazole, and clarithromycin are frequently used, combined with proton pump inhibitors or bismuth salts, for the treatment of H. pylori infections (). However, antibiotic resistance is frequently associated with eradication failure (, 1). Resistance to metronidazole and claritromycin is population dependent, and several studies suggest that clarithromycin resistance is higher in strains isolated from children than from adults (). In Brazil, the prevalence of clarithomycin-resistant strains in adults is reported to be from to % (1, 1). However, little is known about the prevalence of clarithromycinresistant H. pylori infection in Brazilian children. The primary aim of this study was to determine the prevalence of clarithromycin-resistant H. pylori strains in children, to identify those isolates via rapid methodology, and to examine the severity of gastritis caused by the antibiotic-resistant H. pylori isolates. Metronidazole, amoxicillin and tetracycline resistance were also studied. Furthermore, the study aimed to genotype the vaca and icea and detect the caga gene in gastric biopsy specimens, since recent studies found a high frequency of caga-positive, icea-positive, and the vaca signal region genotype s1 and middle region sequence m1 strains among pediatric H. pylori isolates in Brazil (,, 1, ). This is also the first investigation of baba gene prevalence in Brazilian children. A total of 1 consecutive child patients, aged from 1-1 years (mean age, years) ( girls and boys) who underwent upper gastrointestinal endoscopy for evaluation of dyspeptic symptoms at the outpatient clinic of Pediatric Gastroenterology at the Child Institute of the Faculdade de Medicina da Universidade de São Paulo during 00 and 00, were included. The study was Downloaded from on January 1, 01 by guest

4 approved by the Ethics Committee of the University Hospital. Patients previously treated for H. pylori infections were not included. Gastric biopsy specimens were processed for histological examination and evaluated according to the updated Sydney System of classification and grading of gastritis (). Antral gastric specimens were transported in sodium thioglycolate broth (Difco, Detroit, Mich.) in an ice bath and ground before submission to DNA extraction and PCR-restriction fragment length polymorphism analysis with primers specific to the H. pylori S rrna gene (1). QIAmp Tissue Kit (Qiagen) was used for DNA extraction. Point mutations related to clarithromycin resistance in the S rrna amplicon were investigated in all H. pylori isolates by PCRrestriction fragment length polymorphism using BsaI and MboII enzymes (). The vaca, caga, icea, and baba genotypes were detected by PCR, as described elsewhere (1,,,, ). In each experiment, H. pylori strain (ATCC 00) was used as the positive control strain. H. pylori strains were cultured on Belo Horizonte medium () under microaerophilic atmosphere at C for to days and the isolates were identified by Gram staining and biochemical tests for oxidase, catalase, and urease production. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was determined by disc diffusion method (Oxoid, USA) and MICs were determined by the E-test according to the manufacturer s recommendations (AB Biodisk, Solna, Sweden). An isolate was considered resistant to clarithromycin or tetracycline if the MIC was >1 mg/l and to metronidazole or amoxicillin if the MIC was > mg/l (0). Data were analyzed by the -tailed χ test and Fisher exact test. P value < 0.0 was considered statistically significant. H. pylori was isolated in (0.%) of the 1 children; 1 (.%) of the strains were clarithromycin-resistant, (1.%) were metronidazole-resistant, and (.%) were amoxicillin-resistant. All cultured H. pylori strains were susceptible to tetracycline (Figure 1). No histological differences were observed between biopsies with antibiotic-resistant strains and those with susceptible Downloaded from on January 1, 01 by guest

5 strains. PCR-RFLP was performed with all 1 clarithromycin-resistant isolates: had the S rrna A1G point mutation, and had the S rrna A1G mutation. Among the H. pylori-infected children, 1 had mild chronic gastritis, had moderate chronic gastritis, had marked chronic gastritis, and had normal gastric mucosa. The percentage of H. pylori-infected children with chronic gastritis was.% ( patients), while.% of the children ( patients) had normal mucosa (P < 0.001). vaca was detected in all H. pylori-positive gastric biopsy specimens. The vaca genotypes s1m1, sm, and s1m or sm1 were found in.,., and.% of the specimens, respectively. The icea1 allele was detected in (0%) and the icea allele in 1 (.%) of the samples. Of the H. pyloripositive biopsy specimens, (%) were caga positive and (.%) were baba positive. Correlation of histopathology results with vaca, caga, and icea genotypes showed that vaca s1m1, caga, and icea positive strains were more frequently found in patients with moderate and marked gastritis (%) in comparison to patients with mild gastritis (%) (P < 0.001). Interestingly, in Slovenian children, vaca s1 and caga were also shown to be associated with more pronounced chronic gastritis (1). Incontrast, in Korean children, although vaca s1m1, caga, icea1 was the predominant genotype, no association with gastritis severity was observed (1). In conclusion, we found a high incidence of clarithromycin-resistant H. pylori (%) in Brazilian children. Furthermore we found an association between clarithromycin resistance and either vaca s1m1 (P = 0.00) or icea (P = 0.0) genotypes. The high level of clarithromycin resistance among strains from children compared to adults (1, 1) suggests the importance of susceptibility testing, especially in Brazilian children. Altogether, these data stress the relevance of susceptibility testing and genotyping for establishing antibiotic treatment in pediatric H. pylori infection. In our study, PCR-RFLP proved to be a rapid and accurate method for the detection of clarithromycin-resistance gene mutation directly in gastric biopsies. Downloaded from on January 1, 01 by guest

6 Only a few groups have studied mutations involved in clarithromycin resistance in strains obtained from children, and their results are similar to those obtained in our study (, 1, ). Our data also demonstrate an association between H. pylori infection and gastritis in Brazilian children. In addition, we confirmed the reported association of infection with vacas1m1, caga, icea-positive H. pylori strains and gastritis severity (, 1, ). Furthermore, a high frequency of baba was found among H. pylori isolates. Previous studies of adults in Brazil reported a high prevalence of baba-positive strains from patients with different upper gastrointestinal diseases (). The high incidence of baba in H. pylori Brazilian isolates suggests that this gene could be a useful marker for predicting the high-risk patients of H. pylori infection in Brazil. ACKNOWLEDGMENTS Gabriella T. Garcia and Katia R. S. Aranda contributed equally to this work. This work was supported by the Conselho Nacional de Desenvolvimento Cientifíco e Tecnológico and Fundação de Amparo a Pesquisa do Estado de São Paulo. REFERENCES 1. Atherton, J. C., T. L. Cover, R.J. Twells, M. R. Morales, C. J. Hawkey, and M. J. Blaser. 1. Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori. J. Clin. Microbiol. :-.. Braga, A. B., A. M. Fialho, M. N. Rodrigues, D. M. Queiroz, A. M. Rocha, and L. L. Braga. 00. Helicobacter pylori colonization among children up to years: results of a community-based study from northeastern Brazil. J. Trop. Pediatr. :-. Downloaded from on January 1, 01 by guest

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10 status and clinical outcome: studies in four different countries. J. Clin. Microbiol. :-.. Yang, Y. J., J. C. Yang, Y. M. Jeng, M. H. Chang, and Y. H. Ni Prevalence and rapid identification of clarithromycin-resistant Helicobacter pylori isolates in children. Pediatr. Infect. Dis. J. 0:-. LEGEND FIGURE 1. Distribution of MICs for the H. pylori strains. Downloaded from on January 1, 01 by guest

11 Number of isolates < MIC (mg/l) Clarithromycin Metronidazole Amoxicilin Tetracycline > Downloaded from on January 1, 01 by guest