Rapid detection of clarithromycin resistant Helicobacter pylori strains in Spanish patients by polymerase chain reaction-restriction fragment length polymorphism.

INTRODUCTION: The aim of this study was to characterize the mutations types present in the 23S rRNA gene related to H. pylori clarithromycin-resistance strains in Spain and evaluate a novel PCR-RFLP method for detection of the most frequent point mutation in our population. METHODS: Gastric biopsies were obtained by endoscopy from patients with gastric symptoms. H. pylori was cultured according to standard microbiological procedures and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux) according to the manufacturer instructions. Analyses for point mutations in 23S rRNA gene strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using BsaI enzyme to detect restriction sites that correspond to the mutation (A2143G). RESULTS: We found 42 out of 118 (35.6%) strains resistant to clarithromycin by E-test. E-test results were confirmed for the presence of point mutation in 34 (88.1%) of these strains. Mutation A2143G was found in 85.3% of the strains. Analyses with the restriction enzyme BsaI was able to confirm the presence of A2143G mutation. There were 8 H. pylori strains resistant to clarithromycin by E-test but without any point mutation in the 23 rRNA gene. CONCLUSIONS: We conclude that PCR-RFLP is a reliable method to detect clarithromycin-resistance H. pylori strains in countries with a high prevalence of clarithromycin-resistance as Spain. It may be useful before choosing regimens of H. pylori eradication.

[High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolates obtained from Spanish children].

OBJECTIVE: To determine the primary and secondary resistance to several antimicrobial agents in Spanish Helicobacter pylori clinical isolates obtained from paediatric patients from January 2002 to June 2006. METHODS: Samples were collected from gastric biopsies of symptomatic paediatric patients and H. pylori cultured according to standard microbiological procedures. Resistance was determined by E-test. Strains were considered resistant if minimal inhibitory concentration (MIC) > or = 2 mg/l for amoxycillin, > or = 4 mg/l for tetracycline, > or = 8 mg/l for metronidazole, > or = 1 mg/l for clarithromycin, MIC > or = 4 mg/l for ciprofloxacin, MIC > or = 32 mg/l for rifampicin and intermediate if MIC = 0.5 mg/l for clarithromycin, and MIC = 2 mg/l for ciprofloxacin. RESULTS: A total of 101 patients were included: 38 males and 63 females (sex ratio M/F: 0.6). Average age was 10 years (range: 4-18 years). All strains were susceptible to amoxycillin, tetracycline and rifampicin, 35.7% were resistant to metronidazole, 54.6% to clarithromycin and 1.8% to ciprofloxacin. 2.0% were intermediate to clarithromycin and 1.8% to ciprofloxacin. Double resistance to metronidazole and clarithromycin rated at 17.2%. Thirty-five patients (34.7%) had a history of treatment failure, and were considered as secondary H. pylori. Primary resistance rates to metronidazole and clarithromycin were 32.8% and 49.2%, respectively, and secondary resistance rates were 41.2% and 70.6%, respectively. CONCLUSIONS: Resistance to clarithromycin (56.6%) was higher than to metronidazole (35.7%) in the H. pylori strains studied. Clarithromycin resistance was very high even in strains from paediatric patients not previously treated for H. pylori infection.

High percentage of clarithromycin and metronidazole resistance in Helicobacter pylori clinical isolates obtained from Spanish children / Alto porcentaje de resistencia a claritromicina y metronidazol en aislamientos clínicos de Helicobacter pylori procedentes de pacientes pediátricos

Introducción. Determinar la resistencia primaria y secundaria a varios agentes antibióticos en aislamientos clínicos de Helicobacter pylori procedentes de pacientes pediátricos desde enero de 2002 a junio de 2006. Métodos. Las muestras fueron biopsias gástricas procedentes de pacientes pediátricos sintomáticos. H. pylori fue cultivado acorde con los estándares de los procedimientos microbiológicos. La resistencia a antibióticos fue determinada mediante E-test. Los aislamientos fueron considerados resistentes si la CMI ≥ 2 mg/l para amoxicilina, ≥ 4 mg/l para tetraciclina, ≥ 8 mg/l para metronidazol, ≥ 1 mg/l para claritromicina, ≥ 4 mg/l para ciprofloxacino y ≥ 32 mg/l para rifampicina, y fue considerado intermedio si la concentración mínima inhibitoria (CMI) = 0,5 mg/l para claritromicina, y CMI = 2 mg/l para ciprofloxacino. Resultados. De los 101 pacientes incluidos en el estudio: 38 fueron hombres y 63 mujeres. La media de edad fue de 10 años. Todas las cepas fueron sensibles a amoxicilina, tetraciclina y rifampicina. El 35,7% de los aislamientos fueron resistentes a metronidazol, el 54,6% fue- ron resistentes a claritromicina y el 1,8% a ciprofloxacino. El 17,2% de los aislamientos tuvieron doble resistencia a metronidazol y claritromicina. Treinta y cinco pacientes (34,7%) tuvieron un fallo en el tratamiento frente a H. pylori, previamente. La resistencia primaria a metronidazol y claritromicina fue del 32,8 y 49,2%, respectivamente, y la resistencia secundaria fue de un 41,2 y 70,6%, respectivamente. Conclusión. La resistencia a claritromicina (56,6%) fue más alta que a metronidazol (35,7%) en los aislamientos clínicos de H. pylori estudiados. La resistencia a claritromicina fue alta incluso en los pacientes que no habían tenido tratamientos previos frente a H. pylori (AU)

Objective. To determine the primary and secondary resistance to several antimicrobial agents in Spanish Helicobacter pylori clinical isolates obtained from paediatric patients from January 2002 to June 2006. Methods. Samples were collected from gastric biopsies of symptomatic paediatric patients and H. pylori cultured according to standard microbiological procedures. Resistance was determined by E-test. Strains were considered resistant if minimal inhibitory concentration (MIC) ≥ 2 mg/l for amoxycillin, ≥ 4 mg/l for tetracycline, ≥ 8 mg/l for metronidazole, ≥ 1 mg/l for clarithromycin, MIC ≥ 4 mg/l for ciprofloxacin, MIC ≥ 32 mg/l for rifampicin and intermediate if MIC = 0.5 mg/l for clarithromycin, and MIC = 2 mg/l for ciprofloxacin. Results. A total of 101 patients were included: 38 males and 63 females (sex ratio M/F: 0.6). Average age was 10 years (range: 4-18 years). All strains were susceptible to amoxycillin, tetracycline and rifampicin, 35.7% were resistant to metronidazole, 54.6% to clarithromycin and 1.8% to ciprofloxacin. 2.0% were intermediate to clarithromycin and 1.8% to ciprofloxacin. Double resistance to metronidazole and clarithromycin rated at 17.2%. Thirty-five patients (34.7%) had a history of treatment failure, and were considered as secondary H. pylori. Primary resistance rates to metronidazole and clarithromycin were 32.8% and 49.2%, respectively, and secondary resistance rates were 41.2% and 70.6%, respectively. Conclusions. Resistance to clarithromycin (56.6%) was higher than to metronidazole (35.7%) in the H. pylori strains studied. Clarithromycin resistance was very high even in strains from paediatric patients not previously treated for H. pylori infection (AU)