Role of antimicrobial susceptibility testing on ef cacy of triple therapy in Helicobacter pylori eradication

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1 Aliment Pharmacol Ther 2000; 14: 1639±1643. Role of antimicrobial susceptibility testing on ef cacy of triple therapy in Helicobacter pylori eradication S. TORACCHIO*, L. CELLINI, E.DICAMPLI, G.CAPPELLO*,M.G.MALATESTA*,A.FERRI*, A. F. CICCAGLIONE*, L. GROSSI* L. MARZIO* *Gastroenterology Unit and Department of Biochemical Sciences, G. D'Annunzio University, Chieti-Pescara, Italy Accepted for publication 28 July 2000 SUMMARY Background: Helicobacter pylori treatment failure may be due to resistance to macrolides and 5-nitroimidazoles. Aim: To test whether a preliminary in vitro susceptibility test of H. pylori to tinidazole and clarithromycin and a consequent speci c regimen could improve the eradication rate. Methods: A total of 109 consecutive H. pylori-positive patients with dyspeptic symptoms were included. At endoscopy, biopsy from the antrum was obtained for H. pylori culture and antimicrobial susceptibility testing. Fifty-six patients were treated with omeprazole, tinidazole and clarithromycin for 10 days (group OTC) and 53 patients received therapy on the basis of the susceptibility test (group SUSC). Treatment success was evaluated by the 13 C-urea breath test 1 month after the end of therapy. Results: Eight patients dropped out. Overall primary resistance to clarithromycin, tinidazole and both antibiotics was 13%, 33% and 4%, respectively. In group OTC, H. pylori was eradicated in 81% and 75% of patients by per protocol and intention-to-treat analysis, respectively. Per protocol and intention-to-treat eradication rates for group SUSC were 98% and 91% (P < 0.05 vs. group OTC). Conclusions: These data show that in H. pylori infection, antibiotic therapy based on the results of culture and susceptibility testing gives, in comparison to standard therapy, a signi cant improvement in eradication rate. INTRODUCTION Optimal therapy for Helicobacter pylori infection consists of a proton pump inhibitor in combination with metronidazole (or tinidazole) and clarithromycin. This therapy presents eradication rates of H. pylori from 70% up to 90% and few side-effects. 1, 2 However, antibiotic resistance jeopardizes the success of this regimen. 3, 4 A large number of studies have demonstrated the prevalence of H. pylori resistance to metronidazole and clarithromycin in various countries. In a recent European survey, resistance ranged from 10% to 50% for Correspondence to: Professor L. Marzio, G. D'Annunzio University, Casa di Cura Pierangeli, P.zza L. Pierangeli 1, Pescara, Italy. marzio@unich.it metronidazole and 0% to 15% for clarithromycin, whilst resistance to amoxicillin has also been reported but appears to be uncommon. 5, 6 Several studies have evaluated the relationship between pre-treatment antibiotic resistance and eradication rate using different standard treatments and performing a susceptibility test before the initiation of the therapeutic regimen. These studies used standard treatments without taking into consideration the results of the susceptibility test; the impact of antibiotic resistance on eradication rate was calculated retrospectively. 7±10 To date there are no prospective studies that use culture and antibiotic susceptibility tests to choose a speci c therapy for H. pylori eradication. The aim of this study has therefore been to examine whether a preliminary in vitro susceptibility test of Ó 2000 Blackwell Science Ltd 1639

2 1640 S. TORACCHIO et al. H. pylori to tinidazole and clarithromycin, and a related speci c therapy could improve the eradication rate in patients affected by H. pylori infection. MATERIALS AND METHODS Patients and study protocol A total of 109 consecutive H. pylori-positive patients with dyspeptic symptoms and without previous H. pylori treatment were included in this clinical study. All patients underwent endoscopy with two biopsies from the antrum. One biopsy was drawn for rapid urease test (CP-test, Yamanouchi Pharma S.p.A., Milan, Italy), and one biopsy for bacterial culture was placed in 0.4 ml of Brucella Broth (BB, Biolife Italiana, Italy). The H. pylori status was also determined by 13 C-urea breath test. This test was considered positive if the delta over baseline at 30 min (that is the difference between d-value at 30 min and d-value at baseline) was ³ 5%. All patients were consecutively assigned to one of the following regimens according to a predetermined randomization schedule: OTC (standard triple therapy); or SUSC (speci c triple therapy according to the susceptibility test results). At 1 month after completion of the drug regimen, the 13 C-urea breath test was repeated to evaluate treatment success. Exclusion criteria included age: < 16 years or > 80 years; prior eradication treatment; treatment with proton pump inhibitors (omeprazole, lansoprazole, pantoprazole), H 2 -blockers (cimetidine, ranitidine, nizatidine, famotidine, roxatidine), and/or antibiotics during the 4 week before the study; gastrointestinal malignancy; severe concomitant diseases; history of allergy to any of the substances used in the study; previous gastric surgery; pregnancy or lactation; alcohol abuse; drug addiction; and chronic use of corticosteroids or non-steroidal anti-in ammatory drugs. Patients gave their written informed consent to the study, which was approved by the Ethics Committee of G. D'Annunzio University. Randomization schedule This was a randomized, parallel group study. Patients who met the inclusion/exclusion criteria were randomized according to a predetermined randomization schedule to receive standard or personalized therapy. The patients were randomly assigned with equal allocation to the treatment groups as determined by the treatment number. Treatment numbers were assigned in ascending order. Patients were recruited with the following schedule: patients examined in the unpaired days of the week (Monday, Wednesday, Friday) in the gastroenterological division and who met the inclusion/exclusion criteria were assigned to group OTC, patients examined in the paired days of the week (Tuesday, Thursday and Saturday) were assigned to group SUSC. The number of patients (male and female) in each group was calculated to be 120, which provided at least 80% power to detect a 20% difference in proportion between personalized therapy and standard therapy at an a ˆ 0.05 signi cance level. An interim analysis was planned when the number of patients recruited was greater than 25%, 50% and 75% of randomization in both groups. Bacterial culture Antral biopsy specimens were processed within 1 h. Biopsies were trimmed with a rotor, homogenized and cultured in Chocolate agar plus 1% of IsoVitaleX (CA, Becton Dickinson & Co., Cockeisville, MD) and Campylobacter selective medium (CSM, Unipath Ltd, Basingstoke, UK). Plates were incubated in a microaerophilic atmosphere (CampyPack Jar-Oxoid Ltd, Basingstoke, UK) at 37 C for 7 days. Typical H. pylori colonies were identi ed by standard methods. 11 Susceptibility tests Tinidazole (Sigma, Milan, Italy) and clarithromycin (Abbott Laboratories, North Chicago, IL) were reconstituted following the manufacturer's instructions. To evaluate the tinidazole and clarithromycin resistance, blood agar media were added with 5 lg/ml and 1 lg/ ml of tinidazole and clarithromycin, respectively. The antibiotic concentrations were used following the cut-off levels related to the breakpoint for resistance. For antibiotic-resistant H. pylori isolates the minimal inhibitory concentrations (MIC) of clarithromycin and tinidazole were also determined. Clarithromycin and tinidazole MICs were determined by the agar dilution method using Mueller±Hinton agar (Biolife, Italy) with 7% laked horse blood containing clarithromycin and tinidazole in twofold dilutions ranging from 0.3 to 40 lg/ml and 0.62±80 lg/ml, respectively. 12 Plates were prepared immediately prior to inoculation. The

3 SUSCEPTIBILITY TESTING AND H. PYLORI THERAPY 1641 bacterial suspensions were prepared in Brucella broth and adjusted to a McFarland standard no. 1 (approximately CFU/mL) and applied with a multipoint replicator (delivering 1 ll of sample, per spot). Minimal inhibitory concentration was de ned as the lowest concentration of drug inhibiting visible bacterial growth. H. pylori ATCC was used as standard control strain. Therapeutic regimens Group OTC was treated with a 10-day course of triple therapy (omeprazole 20 mg b.d., tinidazole 500 mg b.d., clarithromycin 500 mg b.d.), independently from the antibiogram results. Group SUSC was treated with a 10-day course of personalized therapy according to the susceptibility test. Omeprazole 20 mg b.d., tinidazole 500 mg b.d. and clarithromycin 500 mg b.d. was prescribed if H. pylori strain was sensitive to clarithromycin and tinidazole. Omeprazole 20 mg b.d., tinidazole 500 mg b.d. and amoxicillin 1 g b.d. was prescribed if H. pylori strain was clarithromycinresistant. Omeprazole 20 mg b.d., amoxicillin 1 g b.d. and clarithromycin 500 mg b.d. was prescribed if H. pylori strain was tinidazole-resistant. Omeprazole 20 mg b.d. and amoxicillin 1 g b.d. was prescribed if H. pylori strain was resistant to both antibiotics. Speci c therapy was prescribed at each individual after completion of the susceptibility testing, which took usually 14±16 days. Statistics Both intention-to-treat and per protocol analyses were used for the assessment of the eradication rates of H. pylori in the two groups. Eradication rates of H. pylori were compared using the v 2 -test. The signi cance level was set at P < RESULTS A total of 109 patients were enrolled into the randomized parallel group study, with 56 patients (mean age 58 years, range 42±74 years, 34 female, 22 male) in group OTC and 53 patients (mean age 50 years, range 19±81 years, 24 female, 29 male) in group SUSC. Of these, 101 patients completed a course of treatment and underwent follow-up 13 C-UBT. Eight patients (four patients in each group) were excluded from analysis for the following reasons: one patient refused a follow-up with 13 C-UBT, two patients suspended therapy because of diarrhoea and vomiting and ve patients received antibiotics during the study period. The resistance and susceptibility spectrum of H. pylori isolates against clarithromycin and/or tinidazole is reported in Table 1. Table 2 shows the eradication rate after standard therapy in group OTC and speci c therapy in group SUSC, and the relationship with resistance to antibiotics. Only one patient with combined antibiotic resistance failed to respond to personalized therapy. Table 1. The overall primary resistance and susceptibility of H. pylori strains to clarithromycin and tinidazole No. of pts CLAs and TINs TINr CLAr CLAr and TINr Group OTC (42%) 20 (39%) 8 (15%) 2 (4%) Group SUSC (59%) 13 (27%) 5 (10%) 2 (4%) CLA, clarithromycin; TIN, tinidazole; r, resistant; s, sensitive. Table 2. H. pylori eradication after standard therapy in group OTC and speci c therapy in Group SUSC Group OTC No. of pts treated No. (%) of pts with H. pylori eradication Group SUSC No. of pts treated No. (%) of pts with H. pylori eradication CLAs-TINs (100%) (100%) TINr (80%) (100%) CLAr 8 4 (50%) 5 5 (100%) CLAr-TINr 2 0 (0%) 2 1 (50%) CLA, clarithromycin; TIN, tinidazole; pts, patients; r = resistant; s = sensitive.

4 1642 S. TORACCHIO et al. Table 3. H. pylori eradication rates Per protocol and intention-to-treat analyses show that the overall percentage of H. pylori eradication is signi cantly increased in the patients receiving speci c therapy, compared to the standard therapy (Table 3). DISCUSSION Eradication rates Per protocol Group OTC 42/52 (81%) Group SUSC 48/49 (98%)* Intention-to-treat Group OTC 42/56 (75%) Group SUSC 48/53 (91%)** *P < 0.01 vs. group OTC; **P < 0.05 vs. group OTC. Our study shows that, whilst standard triple therapy with omeprazole, tinidazole and clarithromycin eradicates H. pylori infection in 81% of patients, personalized therapy in patients affected by H. pylori infection signi cantly enhances the eradication rate to values that are close to 98%. It is also demonstrated that the main cause of failure of this therapy is due to antibiotic resistance. Omeprazole combined with two antimicrobials has proven to be highly effective in eradicating H. pylori and it has therefore been suggested as a rst-line option therapy. 13, 14 Clinical studies indicate that 7 or 10 days of triple therapy with omeprazole, tinidazole and clarithromycin gives eradication rates ranging from 70% to 90% of treated cases a value which is similar to the one obtained in the present study in our group OTC. 1, 2 Treatment failure in naive patients has been advocated either to the presence of antibiotic-resistant organisms or to features of the infection that make it inaccessible to the antibiotics despite a sensitivity shown in the in vitro test or both. 3, 4 From our results, the rst hypothesis seems the more likely, since treatment failure in patients treated with standard therapy was found only in those with resistance to clarithromycin or tinidazole or both. Indeed, in vitro susceptibility testing does not predict a treatment failure in all cases evaluated. In our group treated with standard therapy, the eradication rate was actually superior to the one that could be predicted on the basis of susceptibility test. This may be due to the presence in vivo of an active metabolite of clarithromycin and tinidazole with additive or synergistic properties when combined with the parent compound. 15±17 The use of a personalized therapy prescribed on the basis of the results of susceptibility testing allowed us to reduce treatment failure to 0% of cases, except in patients with H. pylori resistant to both antibiotics. This suggests once more that antibiotic resistance is the main cause of treatment failure, whilst features due to the infection seem less implicated. Several authors have emphasized the importance of the use of the routine pre-treatment susceptibility testing in the treatment of H. pylori infection. Indeed, most of the studies performed on this topic analysed the role of culture and susceptibility testing of H. pylori strain only after eradication failure with the classic triple therapy or after a failed second attempt to select a third-line treatment. 14, 18, 19 In a recent meta-analytical approach, Dore et al. identi ed 49 clinical treatment trials that provided results in relation to H. pylori susceptibility to clarithromycin and metronidazole. 10 In all studies examined, a standard therapy was used and the results of pre-therapy antibiotic susceptibility testing was utilized only to analyse treatment failure. Recently, Breuer & Graham described a decision analysis model which compares the outcome for treatment of H. pyloripositive ulcer patients for two different strategies: one with routine pre-treatment susceptibility testing and the other without antibiotic susceptibility testing. 20 Their decision model reports similar cure rates, that is between 95% and 99%, for both strategies. Our prospective study shows a 17% difference in eradication rate, regardless of whether or not a personalized therapy is used, and emphasizes the importance of the susceptibility testing in the treatment of H. pylori infection, questioning the usefulness of the decision model presented by the above cited authors. In our cases with primary resistance to both clarithromycin and tinidazole, standard therapy failed to eradicate, whilst personalized therapy was successful in one case out of two. So far, in cases with combined resistance to antibiotics, several eradication regimens have been proposed empirically, with a successful treatment rate from 60% to 70%. 21 These data suggest that new drugs should be tested in vitro to improve eradication rates in these speci c cases. Susceptibility testing has been performed in our study on cultures of gastric biopsies taken during upper gastrointestinal endoscopy; this is therefore mandatory

5 SUSCEPTIBILITY TESTING AND H. PYLORI THERAPY 1643 when such a test is proposed. However, a routine use of endoscopy is not feasible since it is expensive and not well-tolerated by all patients. Other less invasive and less expensive methods need to be found for collecting specimens of H. pylori before this schedule is proposed for general application. 22 In conclusion, this study shows that H. pylori eradication may be signi cantly improved when antibiotic therapy is proposed on the basis of the results of in vitro susceptibility testing. ACKNOWLEDGEMENTS We are grateful to Abbott Laboratories, North Chicago, IL, for donating the clarithromycin powder. REFERENCES 1 Bazzoli F, Pozzato P. Therapy of H. pylori infection. J Physiol Pharmacol 1997; 48(Suppl. 4): S39±S46. 2 Pipkin GA, Williamson R, Wood JR. Review article: one-week clarithromycin triple therapy regimens for eradication of Helicobacter pylori. Aliment Pharmacol Ther 1998; 12: 823±37. 3 Graham DY. Antibiotic resistance in Helicobacter pylori: implications for therapy. Gastroenterology 1998; 115: 1272±7. 4 MeÂgraud F. Epidemiology and mechanism of antibiotic resistance in Helicobacter pylori. Gastroenterology 1998; 115: 1278±82. 5 MeÂgraud F. Antibiotic resistance in Helicobacter pylori infection. Br Med Bull 1998; 54: 207±16. 6 Debets-Ossenkopp YJ, Herscheid AJ, Pot RG, Kuipers EJ, Kusters JG, Vandenbroucke-Grauls CM. Prevalence of Helicobacter pylori resistance to metronidazole, clarithromycin, amoxycillin, tetracycline and trova oxacin in The Netherlands. J Antimicrob Chemother 1999; 43: 511±5. 7 Adamek RJ, Suerbaum S, Pfaffenbach B, Opferkuch W. Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillinðin uence on treatment outcome. Am J Gastroenterol 1998; 93: 386±9. 8 Realdi G, Dore MP, Piana A, et al. Pretreatment antibiotic resistance in Helicobacter pylori infection: results of three randomized controlled studies. Helicobacter 1999; 4: 106±12. 9 Pilotto A, Leandro G, Franceschi M, et al. The effect of antibiotic resistance on the outcome of three 1-week triple therapies against Helicobacter pylori. Aliment Pharmacol Ther 1999; 13: 667± Dore MP, Leandro G, Realdi G, Sepulveda AR, Graham DY. Effect of pretreatment antibiotic resistance to metronidazole and clarithromycin on outcome of Helicobacter pylori therapy: a meta-analytical approach. Dig Dis Sci 2000; 45: 68± MeÂgraud F, Bonnet F, Garnier M, Lamouliatte H. Characterization of `Campylobacter pyloridis' by culture, enzymatic pro- le, and protein content. J Clin Microbiol 1985; 22: 1007± National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial Susceptibility Test for Bacteria That Grow Aerobically, 4th edn. Approved Standard M7-A3. Villanova, Pa: NCCLS, Lee J, O'Morain C. Consensus or confusion: a review of existing national guidelines on Helicobacter pylori-related disease. Eur J Gastroenterol Hepatol 1997; 9: 527± The European Helicobacter pylori Study Group. Current European concepts in the management of Helicobacter pylori infection. The Maastricht Consensus Report. Gut 1997; 41: 8± Hardy DY, Swanson RN, Rode RA, Marsh K, Shipkowitz NL, Clement JJ. Enhancement of the in vitro and in vivo activities of clarithromycin against Haemophilus in uenzae by 14-hydroxyclarithromycin, its major metabolite in humans. Antimicrob Agents Chemother 1990; 34: 1407± Pendland SL, Piscitelli SC, Schreckenberger PC, Danziger LH. In vitro activities of metronidazole and its hydroxy metabolite against Bacteroides spp. Antimicrob Agents Chemother 1994; 38: 2106± Bergan T. Antibacterial activity and pharmacokinetics of nitroimidazoles. A review. Scand J Infect Dis Suppl 1985; 46: 64± de Boer WA, Tytgat GNJ. Regular review: treatment of Helicobacter pylori infection. Br Med J 2000; 320: 31±4. 19 Pajares-Garcia JM. Diagnosis of Helicobacter pylori: invasive methods. Ital J Gastroenterol Hepatol 1998; 30(Suppl. 3): S320±3. 20 Breuer T, Graham DY. Costs of diagnosis and treatment of Helicobacter pylori infection: when does choosing the treatment regimen based on susceptibility testing become cost effective? Am J Gastroenterol 1999; 94: 725±9. 21 Perri F, Festa V, Clemente R, Quitadamo M, Andriulli A. Rifabutin-based `rescue therapy' for Helicobacter pylori infected patients after failure of standard regimens. Aliment Pharmacol Ther 2000; 14: 311±6. 22 Ferguson DA Jr, Jiang C, Chi DS, Laffan JJ, Li C, Thomas E. Evaluation of two string tests for obtaining gastric juice for culture, nested-pcr detection, and combined single- and double-stranded conformational polymorphism discrimination of Helicobacter pylori. Dig Dis Sci 1999; 44: 2056±62.