JOURNAL OFCLINICALMICROBIOLOGY, Nov. 2010, p. 4266–4268 Vol. 48, No. 11
0095-1137/10/$12.00 doi:10.1128/JCM.01034-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
High Prevalence of Clarithromycin Resistance and
cagA
,
vacA
,
iceA2
,
and
babA2
Genotypes of
Helicobacter pylori
in Brazilian Children
䌤
Gabriella T. Garcia,
1,2Katia R. S. Aranda,
1Manoel E. P. Gonc¸alves,
3Silvia R. Cardoso,
3Kiyoshi Iriya,
4Neusa P. Silva,
5and Isabel C. A. Scaletsky
1*
Departamento de Microbiologia, Imunologia e Parasitologia,1Departamento de Pediatria,2and Disciplina de Reumatologia,5
Universidade Federal de Sa˜o Paulo, Instituto da Crianc¸a,3and Departamento de Patologia Me´dica,4Faculdade de
Medicina, Universidade de Sa˜o Paulo, Sa˜o Paulo, Brazil
Received 28 May 2010/Returned for modification 2 August 2010/Accepted 26 August 2010
We isolated 45Helicobacter pyloristrains from 217 child patients. Resistance to clarithromycin, metronida-zole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence ofcagA,vacA s1m1,
iceA2, andbabA2genes.
Helicobacter pyloriinfection is found worldwide and consti-tutes a public health concern in many countries. Previous ep-idemiological studies have shown a high prevalence ofH. pylori
infection in Brazil (2, 20, 24). 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 (25). However, antibiotic resistance is frequently associated with eradication failure (3, 16). Resistance to metronidazole and clarithromycin is population dependent, and several studies suggest that clarithromycin resistance is higher in strains iso-lated from children than in strains isoiso-lated from adults (10). In Brazil, the prevalence of clarithromycin-resistant strains in adults is reported to be from 7 to 10% (15, 18). However, little is known about the prevalence of clarithromycin-resistantH. pyloriinfection in Brazilian children.
The primary aims of this study were to determine the prev-alence of clarithromycin-resistantH. pyloristrains in children, to identify those isolates via rapid methodology, and to exam-ine the severity of gastritis caused by the antibiotic-resistantH. pyloriisolates. Metronidazole, amoxicillin, and tetracycline re-sistance was also studied. Furthermore, the study aimed to genotype thevacAandiceAgenes and to detect thecagAgene in gastric biopsy specimens, since recent studies found a high frequency ofcagA-positive andiceA2-positive strains as well as a strain with the vacAsignal region genotype s1and middle region sequencem1among pediatricH. pyloriisolates in Brazil (6, 7, 11, 23). This is also the first investigation ofbabA2gene prevalence in Brazilian children.
A total of 217 consecutive child patients, aged 1 to 18 years (mean age, 10 years) (105 girls and 112 boys), who underwent
upper gastrointestinal endoscopy for the evaluation of dyspep-tic symptoms at the outpatient clinic of Pediatric Gastroenter-ology at the Instituto da Crianc¸a, Faculdade de Medicina da Universidade de Sa˜o Paulo, during 2008 and 2009 were in-cluded. The study was approved by the Ethics Committee of the University Hospital. Patients previously treated for H. pyloriinfections 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 (4).
Antral gastric specimens were transported in sodium thio-glycolate broth (Difco, Detroit, MI) in an ice bath and ground before submission to DNA extraction and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis with primers specific to the H. pylori 23S rRNA gene (17). The QIAmp tissue kit (Qiagen) was used for DNA extraction. Point mutations related to clarithromycin resistance in the 23S rRNA amplicon were investigated in allH. pylori isolates by PCR-RFLP using BsaI and MboII enzymes (27). ThevacA,
cagA, iceA, and babA2genotypes were detected by PCR, as described elsewhere (1, 9, 21, 26, 28). In each experiment,H. pyloristrain 26695 (ATCC 700392) was used as the positive-control strain.
H. pyloristrains were cultured on Belo Horizonte medium (22) under microaerophilic atmosphere at 37°C for 3 to 7 days, and the isolates were identified by Gram staining and biochem-ical tests for oxidase, catalase, and urease production. Resis-tance to clarithromycin, metronidazole, amoxicillin, and tetra-cycline was determined by the disc diffusion method (Oxoid), and MICs were determined by the Etest according to the manufacturer’s recommendations (AB Biodisk, Solna, Swe-den). An isolate was considered resistant to clarithromycin or tetracycline if the MIC was⬎1 mg/liter and to metronidazole or amoxicillin if the MIC was⬎4 mg/liter (19).
Data were analyzed by the two-tailed2test and Fisher exact test.Pvalues of⬍0.05 were considered statistically significant.
H. pyloriwas isolated in 45 (20.7%) of the 217 children; 12 (26.7%) of the 45 strains were clarithromycin resistant, 6 (13.3%) were metronidazole resistant, and 2 (4.4%) were amoxicillin resistant. All culturedH. pyloristrains were
suscep-* Corresponding author. Mailing address: Departamento de Micro-biologia, Imunologia e Parasitologia, Universidade Federal de Sa˜o Paulo, Rua Botucatu 862, 3° andar, 04023-062 Sa˜o Paulo, SP, Brazil. Phone: 55-11-55764537. Fax: 55-11-55724711. E-mail: scaletsky @unifesp.br.
䌤Published ahead of print on 8 September 2010.
tible to tetracycline (Fig. 1). No histological differences were observed between biopsy specimens with antibiotic-resistant strains and those with susceptible strains. PCR-RFLP was per-formed with all 12 clarithromycin-resistant isolates: 8 had the 23S rRNA A2143G point mutation, and 4 had the 23S rRNA A2142G mutation.
Among the 45 H. pylori-infected children, 13 had mild chronic gastritis, 28 had moderate chronic gastritis, 2 had marked chronic gastritis, and 2 had normal gastric mucosa. The percentage ofH. pylori-infected children with chronic gas-tritis was 95.5% (43 patients), while 4.4% of the children (2 patients) had normal mucosa (P⬍0.001).
vacAwas detected in all 45H. pylori-positive gastric biopsy specimens. ThevacAgenotypess1m1,s2m2, ands1m2ors2m1
were found in 57.7, 33.3, and 4.4% of the specimens, respec-tively. TheiceA1allele was detected in 9 (20%) and theiceA2
allele in 31 (68.9%) of the samples. Of the 45H. pylori-positive biopsy specimens, 28 (62%) werecagApositive and 38 (84.4%) werebabA2positive. Correlation of histopathology results with
vacA, cagA, and iceA genotypes showed that vacA s1m1-,
cagA-, andiceA2-positive strains were more frequently found in patients with moderate and marked gastritis (77%) than in patients with mild gastritis (23%) (P⬍0.001). Interestingly, in Slovenian children,vacA s1and cagAwere also shown to be associated with more pronounced chronic gastritis (12). In contrast, in Korean children, althoughvacA s1m1 cagA iceA1
was the predominant genotype, no association with gastritis severity was observed (14).
In conclusion, we found a high incidence of clarithromycin-resistantH. pyloristrains (27%) in Brazilian children. Further-more, we found an association between clarithromycin resis-tance and either thevacA s1m1(P⫽0.007) or theiceA2(P⫽
0.038) genotype. The high level of clarithromycin resistance among strains from children compared to adults (15, 18) sug-gests the importance of susceptibility testing, especially in Bra-zilian children. All together, these data stress the relevance of susceptibility testing and genotyping for establishing antibiotic treatment in pediatricH. pyloriinfection.
In our study, PCR-RFLP proved to be a rapid and accurate method for the detection of clarithromycin resistance gene mutation directly in gastric biopsy samples. 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 (5, 13, 29).
Our data also demonstrate an association betweenH. pylori
infection and gastritis in Brazilian children. In addition, we confirmed the reported association of infection withvacA s1m1 cagA iceA2-positiveH. pyloristrains and gastritis severity (6, 11, 23). Furthermore, a high frequency ofbabA2was found among
H. pyloriisolates. Previous studies of adults in Brazil reported a high prevalence ofbabA2-positive strains from patients with different upper gastrointestinal diseases (8). The high inci-dence ofbabA2inH. pyloriBrazilian isolates suggests that this gene could be a useful marker for identifying patients with a high risk ofH. pyloriinfection in Brazil.
Gabriella T. Garcia and Katia R. S. Aranda contributed equally to this work.
This work was supported by the Conselho Nacional de Desenvolvi-mento Cientifíco e Tecnolo´gico and Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo.
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