ORIGINAL ARTICLE. Clin Microbiol Infect 2002; 8:

ORIGINAL ARTICLE The status of antimicrobial resistance of Helicobacter pylori in eastern Europe L. Boyanova 1, A. Mentis 2, M. Gubina 3, E. Rozynek 4, G. Gosciniak 5, S. Kalenic 6,V.Göral 7, L. Kupcinskas 8, B. Kantarçeken 9, A. Aydin 10, A. Archimandritis 11, D. Dzierżanowska 4, A. Vcev 12, K. Ivanova 13, M. Marina 13, I. Mitov 1, P. Petrov 13,A.Özden 14 and M. Popova 15 1 Department of Microbiology, Medical University of Sofia, Sofia, Bulgaria, 2 Hellenic Pasteur Institute, Athens, Greece, 3 Institute of Microbiology and Immunology, Ljubljana, Slovenia, 4 Children s Memorial Health Institute, Warsaw, Poland, 5 University of Medicine, Wroclaw, Poland, 6 Clinical Hospital Center Zagreb, Zagreb, Croatia, 7 D.U. School of Medicine, Diyarbakir, Turkey, 8 Kaunas Medical University, Kaunas, Lithuania, 9 Inonu University, Malatya, Turkey, 10 Ege University, Izmir, Turkey, 11 Medical School, University of Athens, Athens, Greece, 12 Clinical Hospital Osijek, Osijek, Croatia, 13 NCIPD, Sofia, Bulgaria, 14 Ankara University, Ankara, Turkey and 15 Executive Drug Agency, Sofia, Bulgaria Objective To evaluate the primary, secondary and combined resistance to five antimicrobial agents of 2340 Helicobacter pylori isolates from 19 centers in 10 countries in eastern Europe. Methods Data were available for centers in Bulgaria, Croatia, the Czech Republic, Estonia, Greece, Lithuania, Poland, Russia, Slovenia and Turkey. Susceptibility was tested by agar dilution (seven countries), E test (five countries) and disk diffusion (three countries) methods. Resistance breakpoints (mg/l) were: metronidazole 8, clarithromycin 1, amoxicillin 0.5, tetracycline 4, and ciprofloxacin 1 or 4 in most centers. Primary and post-treatment resistance was assessed in 2003 and 337 isolates respectively. Results for 282 children and 201 adults were compared. Results Primary resistance rates since 1998 were: metronidazole 37.9%, clarithromycin 9.5%, amoxicillin 0.9%, tetracycline 1.9%, ciprofloxacin 3.9%, and both metronidazole and clarithromycin 6.1%. Isolates from centers in Slovenia and Lithuania exhibited low resistance rates. Since 1998, amoxicillin resistance has been detected in the southeastern region. From 1996, metronidazole resistance increased significantly from 30.5% to 36.4%, while clarithromycin resistance increased slightly from 8.9% to 10.6%. In centers in Greece, Poland, and Bulgaria, the mean metronidazole resistance was slightly higher in adults than in children (39% versus 31.2%, P > 0.05); this trend was not found for clarithromycin or amoxicillin (P > 0.20). Post-treatment resistance rates exhibited wide variations. Conclusions In eastern Europe, primary H. pylori resistance to metronidazole is considerable, and that to clarithromycin is similar to or slightly higher than that in western Europe. Resistance to amoxicillin, ciprofloxacin and tetracycline was detected in several centers. Primary and post-treatment resistance rates vary greatly between centers. Keywords H. pylori, eastern Europe, metronidazole, clarithromycin, children, resistance Accepted 29 November 2001 Clin Microbiol Infect 2002; 8: 388 396 Corresponding author and reprint requests: L. Boyanova, Blvd Evlogui Georgiev 76-B, 1124 Sofia, Bulgaria Tel: þ359 2 431 404 Fax: þ359 2 51 66 727 E-mail: l.boyanova@lycos.com INTRODUCTION Resistance of Helicobacter pylori to metronidazole has been associated with a drop in efficacy of 15 50% for regimens involving nitroimidazoles; and clarithromycin resistance has a higher negative impact on treatment outcome, with a drop in ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

Boyanova et al Antimicrobial resistance of Helicobacter pylori in eastern Europe 389 efficacy from 92% to 50% in triple regimens including newer macrolides [1 4]. Recently, the resistance of H. pylori to amoxicillin, tetracycline and ciprofloxacin has been recognized [3,5 8]. While many studies have focused on H. pylori resistance patterns worldwide, there are still few reports concerning this topic in the eastern part of Europe. The aim of this study was to evaluate the primary, combined and post-treatment resistance prevalence to five antimicrobial agents of 2340 H. pylori isolates from 19 centers in 10 eastern European countries. MATERIALS AND METHODS The distribution of H. pylori isolates among centers is given in Table 1. Susceptibility testing was performed with an agar dilution method in centers in seven countries, with the E test in five countries, and with a disk diffusion method in three countries. Two methods were used in centers in five countries. Inocula of H. pylori, corresponding to McFarland turbidity standard 2 4, and bloodenriched Mueller Hinton agar were used. Other supporting media, such as brain heart infusion agar and Brucella agar enriched with blood, were used in only three centers using the disk diffusion method. Plates were incubated in micro-aerophilic conditions at 37 8C for 2 3 (exceptionally 5) days. The following resistance breakpoints were used: metronidazole 8 mg/l, clarithromycin 1 mg/l, amoxicillin 0.5 mg/l, tetracycline 4 mg/l, and ciprofloxacin 1 or 4 mg/l for most centers. Breakpoints for the disk diffusion method were 15 mm for metronidazole, 15 or 20 mm for clarithromycin, and 15 mm for amoxicillin. Statistical analysis was performed by chi-square without or with Yates correction, and chi-square among several groups. Prevalence of primary H. pylori resistance Data on the prevalence of primary H. pylori resistance in 1996 2000 were collected for 2003 clinical isolates, 1371of them being evaluated since 1998. Results obtained for centers in north central (Czech Republic and Poland), northeastern (Estonia, Russia and Lithuania), south central (Slovenia and Croatia) and southeastern (Bulgaria, Greece and Turkey) regions were compared [9 26] (pers. Table 1 Distribution of H. pylori isolates Study parameters MTZ CLA AMX TET CIP MET þ CLA 1. Primary resistance since 1996 No. of strains 2003 1973 1442 925 635 1041 No. of centers 119 18 17 9 8 8 No. of countries 110 9 8 6 5 5 2. Evolution of resistance since 1996 No. of strains 1353 1301 794 446 484 610 No. of strains isolated in 1996 97 515 461 388 204 250 287 No. of strains isolated in 1998 2000 838 840 406 242 234 323 No. of centers 10 10 12 3 5 4 No. of countries 5 5 5 2 3 3 3. Resistance in children/adults since 1998 No. of strains from children 282 282 282 168 168 243 No. of strains from adults 195 197 201 109 75 108 No. of centers 5 5 5 4 3 3 No. of countries 3 3 3 3 3 2 4. Post-treatment resistance No. of strains 120 218 116 No. of centers 4 6 3 No. of countries 3 5 3 Secondary resistance No. of strains 32 160 No. of centers 2 6 No. of countries 2 4 MTZ, metronidazole; CLA, clarithromycin; AMX, amoxicillin; TET, tetracycline; CIP, ciprofloxacin; MET þ CLA, metronidazole and clarithromycin.

390 Clinical Microbiology and Infection, Volume 8 Number 7, July 2002 comm.: A. Mentis; G. Gosciniak; M. Gubina; S. Kalenic; L. Boyanova; K. Ivanova and M. Marina; A. Aydin; V. Göral; A. Özden; E. Rozynek and D. Dzierżanowska). Evolution of primary H. pylori resistance from 1996 to 2000 The evolution of primary resistance was evaluated in 1353 H. pylori strains from centers in six countries (Table 1) [9,14 16,18,19,26] (pers. comm.: G. Gosciniak; M. Gubina; S. Kalenic; L. Boyanova; K. Ivanova and M. Marina; A. Aydin; V. Göral; A. Özden; E. Rozynek and D. Dzierżanowska). Resistance rates were compared between strains isolated in 1996 97 and those isolated in 1998 2000. Resistance prevalence was also calculated for the years at the beginning and the end of studies in different centers. Resistance patterns in children and adult patients Results obtained for children and adults in five centers in three countries since 1998 were compared [16 19] (pers. comm.: A. Mentis; G. Gosciniak; L. Boyanova; K. Ivanova and M. Marina; E. Rozynek and D. Dzierżanowska). Of 282 children aged 4 18 years, 52% were boys and 48% were girls. The origin of pediatric strains was as follows: 36 children in Greece (55.6% boys and 44.4% girls) aged 6 15 years (mean age 10.5 years) with symptoms of gastritis; 71 children in Bulgaria (46.5% boys and 53.5% girls) aged 4 17 years with gastritis (88.7%) or duodenal ulcer (11.3%); and 175 children in Poland (53.7% boys and 46.3% girls) aged 6 18 years with dyspeptic symptoms. Post-treatment and secondary resistance rates The prevalence of post-treatment resistance (detected after unsuccessful H. pylori eradication regardless of susceptibility status of the strain before treatment) and that of secondary resistance, defined as resistance acquired during therapy by previously susceptible isolates, were assessed in 337 isolates from six centers within five countries [19,20,25,27,28] (pers. comm.: G. Gosciniak; S. Kalenic; L. Boyanova; A. Vcev). The data encompass the resistance rates in H. pylori after failure of eradication using different triple or double treatment regimens in separate centers (Table 1). RESULTS Prevalence of primary H. pylori resistance Overall mean rates of primary H. pylori resistance in 1998 2000 are listed in Table 2. During the last 3 years, there were no significant differences in resistance rates to metronidazole or clarithromycin between centers situated in central versus eastern countries (P > 0.10) and between those in northern versus southern countries (P > 0.20). Although, in 1996, all H. pylori isolates were metronidazole susceptible in centers in Slovenia and no clarithromycin resistance was found in six centers within three countries, the susceptibility patterns have changed since 1998. During the last 3 years, metronidazole resistance was found everywhere, ranging from 18.5% to 56.6% (and to 62.5% in one center using a disk diffusion method); and clarithromycin resistance was observed in all but one center. Resistance rates to clarithromycin varied widely from 0% to 3.7% to 14.4% (and to 18.7% in one center using a disk diffusion method). From 1998, resistance to amoxicillin, ciprofloxacin and tetracycline was found only in centers in the southeastern part of Europe. In one center, amoxicillin resistance was observed only in 1996 and not in 1997 98; and in another center resistance to tetracycline and ciprofloxacin was found in 1997 and not in the following 2 years. When present, resistance to amoxicillin, ciprofloxacin and tetracycline was rare, with mean rates for centers in separate countries ranging from 1% to 4.3% for amoxicillin, 3.3% to 6.9% for tetracycline, and 5.2% to 6.2% for ciprofloxacin. From 1998, the overall mean resistance prevalences for amoxicillin 0.5 mg/l, ciprofloxacin 1 mg/l and tetracycline 4 mg/l, detected by both an agar dilution method and E test, were 0.5% (including four of 888 isolates), 3.3% (nine of 270 isolates), and 1% (six of 606 isolates), respectively. In addition, resistance to amoxicillin, ciprofloxacin and tetracycline was detected in 4.3% (five of 115 isolates), 5.2% (six of 115 isolates) and 6.9% (eight of 115 isolates), respectively, but only by modified disk diffusion methods. Evolution of primary H. pylori resistance With regard to ten centers within five countries, primary metronidazole resistance increased significantly from 30.5% to 36.4% (P < 0.05) in the group as a whole between 1996 97 and 1998 2000,

Boyanova et al Antimicrobial resistance of Helicobacter pylori in eastern Europe 391 Table 2 Primary resistance rates of H. pylori in 1998 2000 Antimicrobial agent and methods No. of centers No. of countries No. of strains Mean rate (%) of resistance 95% CI All methods a Metronidazole 18 10 1371 37.9 35.3 40.5 Clarithromycin 18 9 1337 9.5 7.9 11.1 Amoxicillin 17 8 1003 0.9 0.3 1.5 Tetracycline 9 6 721 1.9 0.9 2.9 Ciprofloxacin 8 5 385 3.9 2.0 5.8 Metronidazole þ 8 5 754 6.1 4.4 7.8 clarithromycin Agar dilution method Metronidazole 7 5 824 34.5 31.2 37.8 Clarithromycin 5 3 705 9.5 7.3 11.7 Amoxicillin 5 3 463 0.6 0 1.3 Tetracycline 5 3 456 1.3 0.3 2.3 Ciprofloxacin 3 2 182 4.9 1.8 8.0 Metronidazole þ 5 3 475 6.7 4.7 9.0 clarithromycin E test Metronidazole 7 5 378 34.7 29.9 39.5 Clarithromycin 7 9 378 8.7 5.9 11.5 Amoxicillin 7 8 378 0.3 0 0.8 Tetracycline 2 6 150 0 0 1.3 Ciprofloxacin 4 5 88 0 0 2.1 Metronidazole þ clarithromycin 1 5 196 4.6 1.7 7.5 a Agar dilution method, E test and modified disk diffusion method. while resistance rates to other agents and combined resistance to metronidazole and clarithromycin were relatively stable (Figure 1). A significant increase in primary metronidazole resistance was detected in centers in Russia, Slovenia and Croatia, and was not found in those in Bulgaria and Poland. Clarithromycin resistance increased markedly only in one center in Moscow. The same trends in evolution of resistance were observed when the prevalence was recalculated Figure 1 Incidence of mean overall primary H. pylori resistance in 1996 2000. MTZ, metronidazole; CLA, clarithromycin; AMX, amoxicillin; TET, tetracycline; CIP, ciprofloxacin; MET þ CLA, metronidazole and clarithromycin.

392 Clinical Microbiology and Infection, Volume 8 Number 7, July 2002 Figure 2 Resistance of H. pylori to metronidazole in children versus adults in 1998 2000. only for the years at the beginning and end of studies in different centers. Resistance patterns in children and adult patients since 1998 In all centers within Poland, Greece and Bulgaria, metronidazole resistance was slightly more prevalent in adults than in children (39% versus 31.2%, P > 0.05), significant differences being found only in Bulgaria (Figure 2). Reverse trends were observed for clarithromycin resistance (Figure 3). No significant differences were present between pediatric and adult patients in the group of centers as a whole and nor in separate institutions. The mean prevalences of H. pylori resistance in children were: metronidazole 31.2% (95% CI: 25.8 36.6%), clarithromycin 11.7% (7.9 15.5%), amoxicillin 0.7% (0 1.7%), tetracycline 1.2% (0 2.8%), ciprofloxacin 1.8% (0 3.8%), and metronidazole clarithromycin 5.3% (2.5 8.1%). Post-treatment and secondary resistance rates The overall mean rates of post-treatment and secondary H. pylori resistance were: 56.7% (95% CI: 47.8 65.6%) and 56.2% (38.7 73.7%) for Figure 3 Primary H. pylori resistance to clarithromycin in children versus adults in 1998 2000.

Boyanova et al Antimicrobial resistance of Helicobacter pylori in eastern Europe 393 Figure 4 Post-treatment and secondary H. pylori resistance rates. MTZ, metronidazole; CLA, clarithromycin. metronidazole, and 11.9% (7.6 16.2%) and 3.8% (0.8 6.8%) for clarithromycin, respectively. Amoxicillin resistance was found in 1.7% (95% CI: 0 4.1%) of all isolates after treatment failure (Figure 4). DISCUSSION In the east of Europe, the overall mean rate of primary resistance of H. pylori to metronidazole is considerable and that to clarithromycin is similar or slightly higher than that in most western countries [1,5]. Several reports have focused on the discrepancy, usually around 10 22%, between results for metronidazole obtained by agar dilution and those obtained by other techniques [2,29,30]. Mégraud has recommended the use of an agar dilution method for metronidazole, whereas any technique seems acceptable for clarithromycin [4]. In this study, when only results of the agar dilution method were taken into account, the global primary resistance rate to metronidazole since 1998 was similar (34.5%) to that obtained by E test (34.7%, P > 0.20) and significantly lower than the resistance prevalence evaluated by disk diffusion methods (42%, P < 0.02). However, additional national, regional and methodological factors (e.g. incubation of 3 5 days in one center) may be associated with the higher resistance prevalence found by those who used modified disk diffusion methods. Although it is generally accepted that clarithromycin 1 mg/l and metronidazole 8 mg/l correspond to resistance breakpoints for H. pylori [1,30,31], MIC interpretive standards for other antimicrobial agents are still lacking. Therefore, the resistance breakpoint for amoxicillin (0.5 mg/ L) was chosen according to cut-off values used in the multicenter study of Glupczynski et al. [5] and other data [30]. A tetracycline breakpoint of 4 mg/ L was used, as this concentration is recommended by the NCCLS [32] for determining susceptible strains in anaerobic microbiology. The cut-off level for ciprofloxacin (1 mg/l) was chosen because sensitive H. pylori strains have exhibited MICs 0.5 mg/l and resistant strains have shown MICs > 2 mg/l [33]. Disk diffusion methods are usually considered inappropriate for slow-growing bacteria, because the gradient of drug concentration in agar after prolonged incubation is not reproducible. However, this technique seems applicable for testing the susceptibility of H. pylori to antibiotics when there is a clearly bimodal population (e.g. clarithromycin) [4]. Several authors have reported the modified disk diffusion method to be a reliable technique for H. pylori susceptibility testing, with inhibitory zones indicating resistance being <16 mm for metronidazole and 20 mm for clarithromycin [34,35]. The breakpoints for H. pylori resistance to clarithromycin were 15 mm in most centers and 20 mm in one center, the latter cut-off value being more reliable according to a recent study [35]. As different interpretive inhibitory zone diameters and different supporting media were used in several centers to determine H. pylori resistance to clarithromycin,

394 Clinical Microbiology and Infection, Volume 8 Number 7, July 2002 tetracycline and ciprofloxacin, the results obtained by the disk diffusion technique must be interpreted with caution. The problem of combined H. pylori resistance is of utmost importance, as the treatment efficacy of triple regimens may be compromised [36,37]. The prevalence of double primary resistance to metronidazole and clarithromycin was worrying and oscillated around 4 6% in most centers, ranging from 0% to 9.5% [9,11,16,18] (pers. comm.: G. Gosciniak; L. Boyanova; K. Ivanova and M. Marina; E. Rozynek and D. Dzierżanowska). Although rare, primary resistance to other drugs, such as amoxicillin, ciprofloxacin and tetracycline, was detected in several centers. In centers within Slovenia and Lithuania, H. pylori isolates exhibited a low prevalence of resistance to all drugs studied. A high prevalence of clarithromycin resistance was detected in children at centers in Poland from 1998 and could be related to the actual macrolide consumption. Following the introduction of the newer macrolides in 1992 95 in this country, the consumption of these drugs (in kilograms of active substance) has increased more than threefold from 1996 to 2000. However, this association was not detected with children in Bulgaria, because the national consumption of macrolides (0.34 DDD/ 1000 inhabitants/day) and that of newer macrolides (0.08), such as roxithromycin, clarithromycin and azithromycin, was low in 1999. Recent studies involving children in France and Spain have shown considerable clarithromycin resistance rates in H. pylori in 28.3% and 21% of isolates, respectively [38,39]. Antimicrobial resistance rates in eastern Europe vary enormously among centers. This could reflect variables, such as regional or inter-hospital differences in protocol or procedures and national or regional differences in drug consumption, as well as methodological factors. In addition, considerable variations in post-treatment resistance rates to metronidazole (17.5 87.9%) and clarithromycin (1.8 50%) were found between centers as a result of different triple or double regimens used for H. pylori eradication. From 1996, a marked increase in the overall mean prevalence of H. pylori resistance was found only for metronidazole. However, the patterns of resistance evolution exhibited considerable differences among the centers studied. Global resistance patterns of H. pylori in children are worrying and worth extensive evaluation in multiple centres throughout Europe. Constant monitoring of both patterns and evolution of H. pylori resistance in eastern Europe, based on a complete standardization of susceptibility testing methods and interpretive breakpoints, is needed in order to counteract the serious consequences of H. pylori infection. ACKNOWLEDGMENTS This work was presented in part at the 11th European Congress of Clinical Microbiology and Infectious Diseases, Istanbul, Turkey, 1 4 April 2001. We express, with respect, our gratitude to Professor Francis Mégraud (C.H.U. Pellegrin, Bordeaux, France) for his kind assistance. REFERENCES 1. Megraud F, Lehn N, Lind T et al. Antimicrobial susceptibility testing of Helicobacter pylori in a large multicenter trial: the MACH 2 study. Antimicrob Agents Chemother, 1999; 43: 2747 52. 2. Megraud F. Helicobacter pylori resistance to antibiotics. In: Hunt RH, Tytgat GNJ, eds. Helicobacter pylori: basic mechanisms to clinical cure. Dordrecht: Kluwer, 1994: 570 83. 3. Pilotto A, O Morain C. Treatment of Helicobacter pylori infection. Curr Opin Gastroenterol 2000; 16(suppl 1): S44 51. 4. Megraud F. Resistance of Helicobacter pylori to antibiotics. Aliment Pharmacol Ther 1997; 11(suppl 1): 43 53. 5. Glupczynski Y, Megraud F, Andersen LP, Lopez- Brea M. Antibiotic susceptibility of H. pylori in Europe in 1998: results of the third multicenter study. Gut 1999; 45(suppl III): A105. 6. Hassan IJ, Stark RM, Greenman J, Millar MR. Activities of b-lactams and macrolides against Helicobacter pylori. Antimicrob Agents Chemother 1999; 43: 1387 92. 7. Dore MP, Graham DY, Sepulveda AR, Realdi G, Osato MS. Sensitivity of amoxicillin-resistant Helicobacter pylori to other penicillins. Antimicrob Agents Chemother 1999; 43: 1803 4. 8. Birac C, Bouchard S, Camou C, Lamouliatte H, Lamireau T, Megraud F. Six year follow-up of resistance to antibiotics of Helicobacter pylori in Bordeaux, France. Gut 1999; 45(suppl III): A107. 9. Koudryavtseva LV, Isakov VA, Ivanikov IO, Zaitseva SV. Evolution of H. pylori primary resistance to antimicrobial agents in Moscow (Russia) in 1996 1998. Gut 2000; 47(suppl 1): A8.

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