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AIM: To determine the effectiveness of a third, culture-guided, treatment of H. pylori infection after two unsuccessful attempts. PATIENTS AND METHODS: Forty-two consecutive patients with a diagnosis of peptic ulcer were included in an open prospective and multicenter study. After two unsuccessful attempts at eradication (demonstrated by positive urea breath test), all patients underwent endoscopy and H. pylori infection was confirmed by urease test, histology and culture (Pylori-Agar, Bio Merieux, France). Antibiotic susceptibility to metronidazole, amoxicillin, tetracycline and clarithromycin was defined by E-test. Thirty-nine patients received a two-week quadruple culture-guided therapy defined by the protocol, which considered sensitivity data and previous allergies to antibiotics (one culture was contaminated, one patient refused treatment and one was allergic to tetracycline and amoxicillin and was resistant to metronidazole and clarithromycin). Compliance was monitored by pill counting and eradication was defined as a negative urea breath test six weeks after the end of treatment. RESULTS: Sensitivity data were obtained in 41 patients. Intention-to-treat analysis revealed that overall eradication was achieved in 60% (24/40). Eighteen strains (43.9%) were resistant to metronidazole, 21 (51.2%) were resistant to clarithromycin and 8 (19.5%) were resistant to both drugs. None of the strains were resistant to amoxicillin or tetracycline. We used mainly two kinds of quadruple therapy in the 39 patients. Despite good compliance with treatment based on omeprazole (20 mg/12 h), bismuth subcitrate (120 mg/6 h), tetracycline (500 mg/4 h) and clarithromycin (500 mg/ 12 h) (OBTC) eradication was achieved in only 9 of 19 patients (47.4%; CI: 24.4-71.1) (one patient failed to attend the urea breath test). Nineteen clarithromycin-resistant patients received amoxicillin (1,000 mg/12 h) instead of clarithromycin (OBTA) and this treatment was effective in 14 (73.7%; CI: 48.8-90.9). Eradication was achieved in one patient who was allergic to amoxicillin and resistant to clarithromycin and metronidazole and who received ciprofloxacin (500 mg/8 h) instead of clarithromycin (OBTCipro). No clinical factors associated with eradication failure were found. CONCLUSIONS: Despite the use of two-week, high-dose, quadruple and culture-guided combinations of drugs, a third treatment was frequently unsuccessful. The lowest eradication rate was obtained in patients with H. pylori strains sensitive to all antibiotics; therefore, we believe that other factors could influence eradication rates. New prospective and randomized studies are needed in this subgroup of patients to find effective treatments.