- Email: [email protected]
Clarithromycin-Resistant Genotypes and Eradication of Helicobacter Pylori
Clarithromycin-Resistant Genotypes and Eradication of Helicobacter Pylori Ruggiero Francavilla, MD, PhD,* Elena Lionetti, MD,* Stefania Castellaneta, MD, Marcella Margiotta, PhD, Domenico Piscitelli, MD, Lorenzo Lorenzo, PhD, Luciano Cavallo, MD, and Enzo Ierardi, MD Objective To compare the eradication rates among the different point mutations and the efficacy of triple therapy and a sequential regimen according to genotypic resistance.
Study design Post hoc retrospective cohort study in a tertiary referral center for pediatric gastroenterology in southern Italy. All 168 children who were positive for Helicobacter pylori were enrolled. Patients had received clarithromycin-based 7-day triple therapy (73 children) or 10-day sequential therapy regimen (95 children). Realtime polymerase chain reaction for assessing clarithromycin resistance was performed on sections of paraffinembedded gastric biopsy samples. Results H pylori eradication was achieved in 16 of 32 (50%) children with the A2143G mutation, in 8 of 10 patients with either A2142G or A2142C strains (80%), and in 112 of 116 children with susceptible strains (88.9%). The presence of A2143G mutation was associated with a lower cure rate compared with the rate in the absence of this mutation (50% vs. 89%; P = .001). The sequential regimen achieved a higher cure rate than triple therapy in patients with A2143G mutant strains (80% vs nil; P < .001). Conclusions The A2143G mutation confers higher risk of treatment failure. Sequential regimen has higher efficacy than standard therapy, even in children with A2143G mutatant strains. (J Pediatr 2010;157:228-32).
H
elicobacter pylori (H pylori) infection plays a critical role in the pathogenesis of chronic active gastritis and peptic ulcer disease both in children and adults1,2 and is considered a serious risk factor for the development of gastric carcinoma.3 Younger age at onset has been suggested to increase the risk of developing cancer later in life.4 Standard treatment for H pylori infection, recommended in US and European guidelines has shown a progressive decrease in efficacy.5,6 Recent review studies report eradication rates below 75%.7,8 Clarithromycin resistance is considered the most important cause of eradication failure both in adults and children.9 In Western countries, 3 point mutations (A2143G, A2142G, A2142C) in domain V of 23S ribosomal RNA, are responsible for more than 90% of cases of clarithromycin resistance in adults.10 The role of these mutations on H pylori treatment outcome in children is unknown. Three years ago, we investigated, in children, the efficacy of a novel sequential regimen consisting of a simple dual therapy given for the first 5 days followed by a triple therapy for the remaining 5 days, showing a high cure rate as compared with standard triple therapy (97% vs 75%)11; this result was confirmed on a larger series in 2008.12 Moreover, in adults, it has been demonstrated that this regimen may be effective even in clarithromycin-resistant strains.13 Our aim was to evaluate the role of different point mutations in the success of eradication therapy and to compare the efficacy of standard triple therapy and the sequential regimen in patients with these mutations.
Methods A post hoc retrospective cohort study was performed. All children with proven H pylori infection, diagnosed between 2002 and 2007 at the Department of Pediatric Gastroenterology of the University of Bari and previously enrolled in randomized studies, were identified. Only children at their first eradicating attempt either with standard 7-day triple therapy [omeprazole (1 mg/kg/ day) plus amoxicillin (50 mg/kg/day) and clarithromycin (15 mg/kg/day)] or with the 10-day sequential therapy [omeprazole (1 mg/kg/day) plus amoxicillin (50 mg/kg/day) for 5 days followed by omeprazole (1 mg/kg/day) plus clarithromycin (15 mg/kg/day) and tinidazole (20 mg/ From the Department of Biomedicina dell’Eta` Evolutiva kg/day)] were considered eligible. (R.F., L.C.), University of Bari, Italy; Centro To assess H pylori status, patients underwent endoscopy during which biopsy Interdipartimentale Gastroenterologia ed Epatologia Eta` Evolutiva (R.F., M.M., L.L., L.C.), Italy; the Department of specimens were collected for histology (2 from the antrum and 2 from the corpus Paediatrics (E.L.), University of Catania, Italy; the Department of Paediatrics (S.C.), San Paolo Hospital, of the stomach), and rapid urease test (one sample from the antrum) (CP test, Bari, Italy; the Department of Pathology, Universita` degli Studi di Bari (D.P.), Bari, Italy; and the Department of Medical Sciences (E.I.), University of Foggia, Italy.
C-UBT PCR rRNA
C-area breath test Polymerase chain reaction Ribosomal RNA
*Contributed equally to this work. The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright Ó 2010 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.02.007
228
Vol. 157, No. 2 August 2010 Yamanouchi Pharma S.p.A., Carugate, Italy). Endoscopies were always performed by the same physicians (S.C., R.F.) (endoscope model GIF XP20; Olympus, Tokyo, Japan) after sedation with intravenous midazolam. All histologic examinations were carried out by the same observer (D.P.) using hematoxylin-eosin staining for assessment of gastritis and tissue Gram strain for detection of H pylori. Within 24 hours of endoscopy, patients completed a standard 13C-urea breath test (13C-UBT) after overnight fasting (Automated Breath 13 Carbon Analyzer, Europe Scientific, Ltd., Crawley, West Sussex, United Kingdom). 13C-UBT was validated in children of our geographic area by our group.14 In the case of assumption of medications such as antibiotics or antacids, 13C-UBT was deferred for at least 4 weeks. Patients were considered H pylori positive if 2 of the 3 tests (histology, rapid urease test, 13C-UBT) were positive. Investigators considered infections to be eradicated if 13 C-UBT was negative 8 weeks after the end of treatment, and treatment was considered to have failed if the test result was positive. Adherence to therapy was good (>95%) in all cases irrespective of treatment; no patient discontinued therapy due to side effects; and no major side effects were reported. The protocol was approved by the local ethics committee. Assessment of Clarithromycin Resistance The main 3-point mutations responsible for sustaining clarithromycin resistance (A2142C, A2142G, and A2143G) were investigated by molecular analysis with a novel method (TaqMan real-time PCR) first used for detection of Mycobacterium tuberculosis15 and, thereafter, for H pylori DNA sequencing on bacterial isolates16 and paraffin-embedded biopsy samples.17 This method was successfully used in a previous retrospective investigation.17 Real-time PCR, for genotyping resistance, was performed in a single center where 10-mm sections of paraffinembedded samples were collected. The methods were validated by comparing TaqMan real-time PCR detection of bacterial DNA with rapid urease test, 13C-UBT, and histology with 100% sensitivity and specificity. To evaluate the lower limit of detection of the real-time PCR protocol, serial dilutions of H pylori DNA, extracted from paraffin-embedded sections and bacterial culture, were processed. Serial dilutions, ranging from 100 ng to 100 fg of DNA, were performed before real-time PCR, and a linearity of amplification was obtained until 500 fg. The amplification efficiency of DNA, from paraffin-embedded sections, was identical to that obtained from DNA samples of bacterial colonies.18 Statistical Analysis Sample size was determined before commencing the study, based on data available in the literature. In actual fact, few data are available in children as far as concerns the eradication rate in patients with primary clarithromycin-resistant strains, with only 2 studies reporting an eradication rate of 0% after standard triple therapy,19,20 whereas with the sequential regimen, the infection was eradicated in 82% of
adult patients (whereas no data are available in children).13 Assuming an extraordinarily higher eradication rate for the triple therapy (30%) and a relatively poor success rate for the sequential regimen (50%), in children with primary clarithromycinresistant strains, we calculated that at least 73 patients per group were needed to detect a statistically significant difference with 0.9 power and an a level of 0.05 (2-sided). Eradication rates by H pylori clarithromycin-resistant strain mutation (A2142C, A2142G, and A2143G) were compared using the Fisher exact test or c2 test, as appropriate. Clinical characteristics in the different groups were compared using Student t test for unpaired data (age) and the c2 test (sex and clinical symptoms distribution and eradication rates). All differences were considered to be statistically significant at a 5% probability level. Statistical analysis was performed using Number Cruncher Statistical System (version 11.0) program for Windows (Kaysville, Utah).
Results Of 253 patients with H pylori infection observed, 77 were not included in the investigations, as they did not meet the inclusion criteria; the remaining 176 patients were enrolled. A further 8 patients were excluded because they failed to attend for the post-treatment 13C-UBT. Therefore, the final study sample comprised 168 patients with H pylori; all were 13 C-UBT positive at the time of the diagnosis (Figure). Seventy-three (30 males, 41%) had been treated with standard 7-day triple therapy and 95 (45 males, 48%) had received the 10-day sequential treatment regimen (Table I). Infection was eradicated in 50 (68.5%) children receiving standard triple therapy and in 86 (90.5%) receiving sequential treatment (P < .003). Clarithromycin resistance was assessed in biopsy specimens obtained prior to treatment in all 168 patients, and
Figure. Flow diagram of participants through each stage. 229
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Table I. Baseline demographic and clinical characteristics of patients
Mean age (range), years Sex (M/F) Dysmotility-like/ulcer-like dyspepsia Clarithromycin-susceptible strains Clarithromycin-resistant strains
Triple therapy n = 73
Sequential therapy n = 95
9.9 (4.3-16) 30/43 54/19 58 (80%) 15 (20%)
11.0 (3.3-16) 45/50 74/21 68 (72%) 27 (28%)
primary clarithromycin resistance was detected in 42 patients (25%). As shown in Table I, patients receiving either triple or sequential treatment did not differ by age, sex, and clinical symptoms (ulcer-like/dysmotility-like dyspepsia)21 and the presence of a clarithromycin-resistant strain. In the 42 resistant isolates, the most frequently identified mutation was A2143G (n = 32; 76.2%), followed by A2142G (n = 9; 21.4%); none harbored the A2142C mutation alone. In 1 child, a double mutation (A2142C and A2142G) was found. The prevalence of these point mutations was distributed equally between males and females (25% vs 20%), patients with ulcer-like or dysmotility-like dyspepsia (24% vs 20%), and children treated with triple or sequential regimen (20% vs 28%). After treatment, H pylori was eradicated in 136 patients (81%), and treatment failed in 32 cases (19%). Overall, the infection was cured in 112 of the patients with clarithromycinsusceptible and in 24 patients with clarithromycin-resistant strains [88.9%, (95% CI, 79% to 95%) vs 57.1% (95% CI, 46% to 75%); P < .001]. Sequential regimen was more effective than triple therapy in the clarithromycin-resistant but not in the clarithromycin-susceptible strains, meaning that clarithromycin resistance significantly reduces the cure rate of triple therapy but not of sequential regimen (Table II). The eradication rate was evaluated taking into account the 2 different mutations observed (A2143G and A2142G); the child with double mutation was included with the A2142G group because a dominant mode of inheritance was assumed. Harboring the A2143G mutation determines the lowest eradication rate; however, in this subset of patients, the sequential regimen achieved a higher eradication rate compared with triple therapy (84% vs nil; P < .001; Table III). Overall, compliance with therapy was good (>95%), and no patient discontinued therapy due to side effects. No major side effects were reported; 13 patients (13.7%) receiving sequential treatment and 8 patients (10.9%) receiving standard triple therapy reported mild to moderate gastrointestinal side effects (P = 1.0).
Discussion Clarithromycin is the most powerful antibiotic currently available for H pylori infection, and its use in first-line therapy regimens is endorsed by current guidelines5,6; however, H pylori clarithromycin resistance is increasing worldwide, raising the failure rate of eradicating regimens. 230
Vol. 157, No. 2 Data herein reported clearly show that sequential therapy may overcome clarithromycin resistance achieving, a high success rate even in children harboring an antibiotic resistant strain. The Maastricht III Consensus Report recommends, as first choice treatment in children, a triple therapy using a proton pump–inhibiting agent with clarithromycin and amoxicillin or metronidazole given twice daily for 7 to 14 days.6 However, the eradication failure rate exceeds 30%7,8 and clarithromycinresistant strains represent the main cause, both in adults and in children.9 Several authors19,20 have reported complete treatment failure when a standard triple therapy using clarithromycin was administered in the presence of resistant strains in children, and Dore at al22 reported a dramatically reduced efficacy (approximately 55%) in adults; a similar trend was found by us with triple therapy achieving an overall cure rate of only 14% in clarithromycin resistant strains. Recently, in an uncontrolled study, we found that sequential therapy achieved an overall eradication rate of 85% in children,12 confirming our previous finding of a higher efficacy than with triple therapy.11 The overall higher success rates for sequential therapy seem to apply to clarithromycinresistant strains. Indeed, in this subset of patients, although reduced, the eradication rate after the sequential regimen was 6-fold higher than that achieved by standard triple therapy (13%). Data presented herein are consistent with a recent study in adults.13 Clarithromycin resistance of H pylori is caused by point mutations in three adjacent 23S rRNA nucleotides10 (A2142G, A2143G, and A2142C) that cause decreased affinity of the ribosomes for several macrolides; the first 2 mutations are the most frequently found mutations in patients of our geographic area.13 Our study, as another,23 reveals that the A2143G is the main genetic mutation leading to H pylori clarithromycin resistance in vivo. We also showed that the outcome of H pylori therapy is markedly affected by the A2143G mutation, with a very low eradication rate (50%), which occurred in the presence of this mutation and was achieved exclusively in children treated with the sequential regimen; clarithromycin resistance associated with the A2142G mutation appears to be clinically insignificant because the eradication rate reaches 80% in both mutatant and wild-type strains, irrespective of the type of treatment. Recent studies on H pylori infection in children, using antibiotic-susceptibility testing, have demonstrated a higher cure rate when H pylori strains are sensitive to the antibiotic Table II. Helicobacter pylori eradication rates in standard therapy and sequential therapy groups according to clarithromycin resistance Triple therapy n = 73
Sequential therapy n = 95
P
Resistant strains
2/15 22/27 .001 13% (95% CI, 2-51%); 81% (95% CI, 52-92%) Susceptible strains 48/58 64/68 .8 83% (95% CI, 64-92%) 94% (95% CI, 82-98%)
Francavilla et al
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Table III. Eradication rates after standard triple therapy and sequential treatment according to mutations in resistant strains Susceptible strains Triple therapy (n = 73) Sequential therapy (n = 95) Overall (n = 168)
48/58 83% (95% CI 72-87%) 64/68 94% (95% CI 84-95%) 112/126e 88.9% (95% CI, 79-95%)
Resistant strain A2143G
Resistant strain A2142G
a
0/13
2/2b
16/19c 84% (95% CI, 49-94%) 16/32f 50% (95% CI, 28-72%)
6/8d 75% (95% CI, 30-95%) 8/10g 80% (95% CI, 37-96%)
a vs c: P < .001.; b vs d: P = .1.; e vs (f + g): P < .001.
used19-24; however, the facilities to perform H pylori culture and to test antibiotic susceptibility are not widely available, H pylori is not easily isolated,12 and costs limit applicability in routine practice. In our opinion, the high eradication rate of the sequential regimen even in clarithromycinresistant strains has important implications on therapy of infected children, favoring the use of the sequential regimen as practical and cost-effective, especially in areas where antibiotic susceptibility tests are not easily available. Finally, considering that the prevalence of clarithromycin-resistant H pylori strains may exceed 40%,25 the use of a therapy with a higher efficacy for such resistant strains would markedly reduce the overall cost associated with management of clarithromycin resistance in clinical practice. Although the reasons for superiority of the sequential regimen for resistant strains is not yet clear, it is possible to speculate that the ability of amoxicillin to destroy the bacterial wall could impair the adenosine triphosphatase–dependent pumps on the bacterial membrane responsible for the clarithromycin resistance resulting in an intracellular entrapment of the macrolide.26 The high intracellular macrolide concentration could overcome the rescue attempt engineered by the bacterium to prevent fatal clarithromycin-ribosomal binding. Therefore, the first course of sequential regimen based on amoxicillin administration could act as an ‘‘induction’’ therapeutic phase, favoring the efficacy of the clarithromycintinidazole therapy that immediately follows, even in some clarithromycin-resistant strains. We are aware of the limits of this study mainly due to the limited number of children harboring the resistant strains; however, the difference in eradication rates according to the therapeutic regimen used, if confirmed by larger independent studies, represents an interesting result that may be of help in overcoming clarithromycin resistance. Finally, most of the data on sequential regimes have been derived from Italian studies, and experience in other countries would be welcome to confirm that this approach is appropriate in other settings. n The authors thank Dr O. Burattini, Dr G. Leone, and Dr S. Fico for clinical support, and Mrs N. Iavernaro for expert technical collaboration. Submitted for publication Aug 29, 2009; last revision received Nov 24, 2009; accepted Feb 4, 2010.
Reprint requests: Dr Ruggiero Francavilla, Clinica Pediatrica ‘‘B. Trambusti,’’ Piazza Giulio Cesare, 11–Policlinico, Bari, Italy. E-mail: rfrancavilla@bioetaev. uniba.it.
References 1. Goodwin CS, Mendall MM, Northfield TC. Helicobacter pylori infection. Lancet 1997;349:265-9. 2. Helicobacter pylori in Peptic Ulcer Disease. NIH Consensus Statement 1994;12:1-23. 3. Uemura N, Okamoto S, Yamamoto S. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001;345:784-9. 4. Blaser MJ, Chyou PH, Normura A. Age at establishment of Helicobacter pylori infection and gastric carcinoma, gastric ulcer and duodenal ulcer risk. Cancer Res 1995;55:562-5. 5. Howden CW, Hunt RH. Guidelines for the management of Helicobacter pylori infection: Ad Hoc Committee on Practice Parameters of the American College of Gastroenterology. Am J Gastroenterol 1998;93: 2330-8. 6. Malfertheiner P, Megraud F, O’Morain C, Bazzoli F, El-Omar E, Graham D, et al. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007;56:772-81. 7. Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Med 2002;347:1175-86. 8. Oderda G, Shcherbakov P, Bontems P, Urruzuno P, Romano C, Gottrand F, et al. European Pediatric Task Force on Helicobacter pylori: results from the Pediatric European Register for Treatment of Helicobacter pylori (PERTH). Helicobacter 2007;12:150-6. 9. Me´graud F. Resistance of Helicobacter pylori to antibiotics. Aliment Pharmacol Ther 1997;11(S1):43-53. 10. Vakil N. Helicobacter pylori treatment: a practical approach. Am J Gastroenterol 2006;101:497-9. 11. Francavilla R, Lionetti E, Castellaneta SP, Magista` AM, Boscarelli G, Piscitelli D, et al. Improved efficacy of 10-Day sequential treatment for Helicobacter pylori eradication in children: a randomized trial. Gastroenterology 2005;129:1414-49. 12. Francavilla R, Lionetti E, Cavallo L. Sequential treatment for Helicobacter pylori eradication in children. Gut 2008;57:1178. 13. De Francesco V, Margiotta M, Zullo A, Hassan C, Troiani L, Burattini O, et al. Clarithromycin-resistant genotypes and eradication of Helicobacter pylori. Ann Intern Med 2006;144:94-100. 14. Rutigliano V, Ierardi E, Francavilla R, Castellaneta S, Margiotta M, Amoruso A, et al. Helicobacter pylori and nonulcer dyspepsia in childhood: clinical pattern, diagnostic techniques, and bacterial strains. J Pediatr Gastroenterol Nutr 1999;28:296-300. 15. Wada T, Maeda S, Tamaru A, Imai S, Hase A, Kobayashi K. Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR. J Clin Microbiol 2004;42:5277-85. 16. Lascols C, Lamarque D, Costa JM, Copie-Bergman C, Le Glaunec JM, Deforges L, et al. Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H pylori isolates from gastric biopsy specimens by real-time PCR. J Clin Microbiol 2003;41:4573-7.
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17. De Francesco V, Margotta M, Zullo A, Hassan C, Valle ND, Burattini O, et al. Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real time polymerase chain reaction. Aliment Pharmacol Ther 2006;23:429-35. 18. De Francesco V, Margiotta M, Zullo A, Hassan C, Giorgio F, Burattini O, et al. Prevalence of primary clarithromycin resistance in Helicobacter pylori strains over a 15 year period in Italy. J Antimicrob Chemother 2007;59:783-5. 19. Faber J, Bar-Meir M, Rudensky B, Schlesinger Y, Rachman E, Benenson S, et al. Treatment regimens for Helicobacter pylori infection in children: is in vitro susceptibility testing helpful? J Pediatr Gastroenterol Nutr 2005;40:571-4. 20. Kalach N, Benhamou PH, Campeotto F, Bergeret M, Dupont C, Raymond J. Clarithromycin resistance and eradication of Helicobacter pylori in children. Antimicrob Agents Chemother 2001;45:2134-5. 21. Rasquin-Weber A, Hyman PE, Cucchiara S, Fleisher DR, Hyams JS, Milla PJ. Childhood functional gastrointestinal disorders. Gut 1999; 45(Suppl 2):II60-8. 22. Dore MP, Leandro G, Realdi G, Sepulveda AR, Graham DY. Effect of pre-treatment antibiotic resistance to metronidazole and clarithromycin
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on outcome of Helicobacter pylori therapy: a meta-analytical approach. Dig Dis Sci 2000;45:68-76. Raymond J, Burucoa C, Pietrini O, Raymond J, Burucoa C, Pietrini O, et al. Clarithromycin resistance in Helicobacter pylori strains isolated from French children: prevalence of the different mutations and coexistence of clones harboring two different mutations in the same biopsy. Helicobacter 2007;12:157-63. Street ME, Caruana P, Caffarelli C, Magliani W, Manfredi M, Fornaroli F, et al. Antibiotic resistance and antibiotic sensitivity based treatment in Helicobacter pylori infection: advantages and outcome. Arch Dis Child 2001;84:419-22. Caristo E, Parola A, Rapa A, Vivenza D, Raselli B, Dondi E, et al. Clarithromycin resistance of Helicobacter pylori strains isolated from children’ gastric antrum and fundus as assessed by fluorescent in-situ hybridization and culture on four-sector agar plates. Helicobacter 2008;13:557-63. Roberts MC. Resistance to macrolide, lincosamide, streptogramin, ketolide, and oxazolidinone antibiotics. Mol Biotechnol 2004;28: 47-62.
Francavilla et al
Study design Post hoc retrospective cohort study in a tertiary referral center for pediatric gastroenterology in southern Italy. All 168 children who were positive for Helicobacter pylori were enrolled. Patients had received clarithromycin-based 7-day triple therapy (73 children) or 10-day sequential therapy regimen (95 children). Realtime polymerase chain reaction for assessing clarithromycin resistance was performed on sections of paraffinembedded gastric biopsy samples. Results H pylori eradication was achieved in 16 of 32 (50%) children with the A2143G mutation, in 8 of 10 patients with either A2142G or A2142C strains (80%), and in 112 of 116 children with susceptible strains (88.9%). The presence of A2143G mutation was associated with a lower cure rate compared with the rate in the absence of this mutation (50% vs. 89%; P = .001). The sequential regimen achieved a higher cure rate than triple therapy in patients with A2143G mutant strains (80% vs nil; P < .001). Conclusions The A2143G mutation confers higher risk of treatment failure. Sequential regimen has higher efficacy than standard therapy, even in children with A2143G mutatant strains. (J Pediatr 2010;157:228-32).
H
elicobacter pylori (H pylori) infection plays a critical role in the pathogenesis of chronic active gastritis and peptic ulcer disease both in children and adults1,2 and is considered a serious risk factor for the development of gastric carcinoma.3 Younger age at onset has been suggested to increase the risk of developing cancer later in life.4 Standard treatment for H pylori infection, recommended in US and European guidelines has shown a progressive decrease in efficacy.5,6 Recent review studies report eradication rates below 75%.7,8 Clarithromycin resistance is considered the most important cause of eradication failure both in adults and children.9 In Western countries, 3 point mutations (A2143G, A2142G, A2142C) in domain V of 23S ribosomal RNA, are responsible for more than 90% of cases of clarithromycin resistance in adults.10 The role of these mutations on H pylori treatment outcome in children is unknown. Three years ago, we investigated, in children, the efficacy of a novel sequential regimen consisting of a simple dual therapy given for the first 5 days followed by a triple therapy for the remaining 5 days, showing a high cure rate as compared with standard triple therapy (97% vs 75%)11; this result was confirmed on a larger series in 2008.12 Moreover, in adults, it has been demonstrated that this regimen may be effective even in clarithromycin-resistant strains.13 Our aim was to evaluate the role of different point mutations in the success of eradication therapy and to compare the efficacy of standard triple therapy and the sequential regimen in patients with these mutations.
Methods A post hoc retrospective cohort study was performed. All children with proven H pylori infection, diagnosed between 2002 and 2007 at the Department of Pediatric Gastroenterology of the University of Bari and previously enrolled in randomized studies, were identified. Only children at their first eradicating attempt either with standard 7-day triple therapy [omeprazole (1 mg/kg/ day) plus amoxicillin (50 mg/kg/day) and clarithromycin (15 mg/kg/day)] or with the 10-day sequential therapy [omeprazole (1 mg/kg/day) plus amoxicillin (50 mg/kg/day) for 5 days followed by omeprazole (1 mg/kg/day) plus clarithromycin (15 mg/kg/day) and tinidazole (20 mg/ From the Department of Biomedicina dell’Eta` Evolutiva kg/day)] were considered eligible. (R.F., L.C.), University of Bari, Italy; Centro To assess H pylori status, patients underwent endoscopy during which biopsy Interdipartimentale Gastroenterologia ed Epatologia Eta` Evolutiva (R.F., M.M., L.L., L.C.), Italy; the Department of specimens were collected for histology (2 from the antrum and 2 from the corpus Paediatrics (E.L.), University of Catania, Italy; the Department of Paediatrics (S.C.), San Paolo Hospital, of the stomach), and rapid urease test (one sample from the antrum) (CP test, Bari, Italy; the Department of Pathology, Universita` degli Studi di Bari (D.P.), Bari, Italy; and the Department of Medical Sciences (E.I.), University of Foggia, Italy.
C-UBT PCR rRNA
C-area breath test Polymerase chain reaction Ribosomal RNA
*Contributed equally to this work. The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright Ó 2010 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.02.007
228
Vol. 157, No. 2 August 2010 Yamanouchi Pharma S.p.A., Carugate, Italy). Endoscopies were always performed by the same physicians (S.C., R.F.) (endoscope model GIF XP20; Olympus, Tokyo, Japan) after sedation with intravenous midazolam. All histologic examinations were carried out by the same observer (D.P.) using hematoxylin-eosin staining for assessment of gastritis and tissue Gram strain for detection of H pylori. Within 24 hours of endoscopy, patients completed a standard 13C-urea breath test (13C-UBT) after overnight fasting (Automated Breath 13 Carbon Analyzer, Europe Scientific, Ltd., Crawley, West Sussex, United Kingdom). 13C-UBT was validated in children of our geographic area by our group.14 In the case of assumption of medications such as antibiotics or antacids, 13C-UBT was deferred for at least 4 weeks. Patients were considered H pylori positive if 2 of the 3 tests (histology, rapid urease test, 13C-UBT) were positive. Investigators considered infections to be eradicated if 13 C-UBT was negative 8 weeks after the end of treatment, and treatment was considered to have failed if the test result was positive. Adherence to therapy was good (>95%) in all cases irrespective of treatment; no patient discontinued therapy due to side effects; and no major side effects were reported. The protocol was approved by the local ethics committee. Assessment of Clarithromycin Resistance The main 3-point mutations responsible for sustaining clarithromycin resistance (A2142C, A2142G, and A2143G) were investigated by molecular analysis with a novel method (TaqMan real-time PCR) first used for detection of Mycobacterium tuberculosis15 and, thereafter, for H pylori DNA sequencing on bacterial isolates16 and paraffin-embedded biopsy samples.17 This method was successfully used in a previous retrospective investigation.17 Real-time PCR, for genotyping resistance, was performed in a single center where 10-mm sections of paraffinembedded samples were collected. The methods were validated by comparing TaqMan real-time PCR detection of bacterial DNA with rapid urease test, 13C-UBT, and histology with 100% sensitivity and specificity. To evaluate the lower limit of detection of the real-time PCR protocol, serial dilutions of H pylori DNA, extracted from paraffin-embedded sections and bacterial culture, were processed. Serial dilutions, ranging from 100 ng to 100 fg of DNA, were performed before real-time PCR, and a linearity of amplification was obtained until 500 fg. The amplification efficiency of DNA, from paraffin-embedded sections, was identical to that obtained from DNA samples of bacterial colonies.18 Statistical Analysis Sample size was determined before commencing the study, based on data available in the literature. In actual fact, few data are available in children as far as concerns the eradication rate in patients with primary clarithromycin-resistant strains, with only 2 studies reporting an eradication rate of 0% after standard triple therapy,19,20 whereas with the sequential regimen, the infection was eradicated in 82% of
adult patients (whereas no data are available in children).13 Assuming an extraordinarily higher eradication rate for the triple therapy (30%) and a relatively poor success rate for the sequential regimen (50%), in children with primary clarithromycinresistant strains, we calculated that at least 73 patients per group were needed to detect a statistically significant difference with 0.9 power and an a level of 0.05 (2-sided). Eradication rates by H pylori clarithromycin-resistant strain mutation (A2142C, A2142G, and A2143G) were compared using the Fisher exact test or c2 test, as appropriate. Clinical characteristics in the different groups were compared using Student t test for unpaired data (age) and the c2 test (sex and clinical symptoms distribution and eradication rates). All differences were considered to be statistically significant at a 5% probability level. Statistical analysis was performed using Number Cruncher Statistical System (version 11.0) program for Windows (Kaysville, Utah).
Results Of 253 patients with H pylori infection observed, 77 were not included in the investigations, as they did not meet the inclusion criteria; the remaining 176 patients were enrolled. A further 8 patients were excluded because they failed to attend for the post-treatment 13C-UBT. Therefore, the final study sample comprised 168 patients with H pylori; all were 13 C-UBT positive at the time of the diagnosis (Figure). Seventy-three (30 males, 41%) had been treated with standard 7-day triple therapy and 95 (45 males, 48%) had received the 10-day sequential treatment regimen (Table I). Infection was eradicated in 50 (68.5%) children receiving standard triple therapy and in 86 (90.5%) receiving sequential treatment (P < .003). Clarithromycin resistance was assessed in biopsy specimens obtained prior to treatment in all 168 patients, and
Figure. Flow diagram of participants through each stage. 229
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Table I. Baseline demographic and clinical characteristics of patients
Mean age (range), years Sex (M/F) Dysmotility-like/ulcer-like dyspepsia Clarithromycin-susceptible strains Clarithromycin-resistant strains
Triple therapy n = 73
Sequential therapy n = 95
9.9 (4.3-16) 30/43 54/19 58 (80%) 15 (20%)
11.0 (3.3-16) 45/50 74/21 68 (72%) 27 (28%)
primary clarithromycin resistance was detected in 42 patients (25%). As shown in Table I, patients receiving either triple or sequential treatment did not differ by age, sex, and clinical symptoms (ulcer-like/dysmotility-like dyspepsia)21 and the presence of a clarithromycin-resistant strain. In the 42 resistant isolates, the most frequently identified mutation was A2143G (n = 32; 76.2%), followed by A2142G (n = 9; 21.4%); none harbored the A2142C mutation alone. In 1 child, a double mutation (A2142C and A2142G) was found. The prevalence of these point mutations was distributed equally between males and females (25% vs 20%), patients with ulcer-like or dysmotility-like dyspepsia (24% vs 20%), and children treated with triple or sequential regimen (20% vs 28%). After treatment, H pylori was eradicated in 136 patients (81%), and treatment failed in 32 cases (19%). Overall, the infection was cured in 112 of the patients with clarithromycinsusceptible and in 24 patients with clarithromycin-resistant strains [88.9%, (95% CI, 79% to 95%) vs 57.1% (95% CI, 46% to 75%); P < .001]. Sequential regimen was more effective than triple therapy in the clarithromycin-resistant but not in the clarithromycin-susceptible strains, meaning that clarithromycin resistance significantly reduces the cure rate of triple therapy but not of sequential regimen (Table II). The eradication rate was evaluated taking into account the 2 different mutations observed (A2143G and A2142G); the child with double mutation was included with the A2142G group because a dominant mode of inheritance was assumed. Harboring the A2143G mutation determines the lowest eradication rate; however, in this subset of patients, the sequential regimen achieved a higher eradication rate compared with triple therapy (84% vs nil; P < .001; Table III). Overall, compliance with therapy was good (>95%), and no patient discontinued therapy due to side effects. No major side effects were reported; 13 patients (13.7%) receiving sequential treatment and 8 patients (10.9%) receiving standard triple therapy reported mild to moderate gastrointestinal side effects (P = 1.0).
Discussion Clarithromycin is the most powerful antibiotic currently available for H pylori infection, and its use in first-line therapy regimens is endorsed by current guidelines5,6; however, H pylori clarithromycin resistance is increasing worldwide, raising the failure rate of eradicating regimens. 230
Vol. 157, No. 2 Data herein reported clearly show that sequential therapy may overcome clarithromycin resistance achieving, a high success rate even in children harboring an antibiotic resistant strain. The Maastricht III Consensus Report recommends, as first choice treatment in children, a triple therapy using a proton pump–inhibiting agent with clarithromycin and amoxicillin or metronidazole given twice daily for 7 to 14 days.6 However, the eradication failure rate exceeds 30%7,8 and clarithromycinresistant strains represent the main cause, both in adults and in children.9 Several authors19,20 have reported complete treatment failure when a standard triple therapy using clarithromycin was administered in the presence of resistant strains in children, and Dore at al22 reported a dramatically reduced efficacy (approximately 55%) in adults; a similar trend was found by us with triple therapy achieving an overall cure rate of only 14% in clarithromycin resistant strains. Recently, in an uncontrolled study, we found that sequential therapy achieved an overall eradication rate of 85% in children,12 confirming our previous finding of a higher efficacy than with triple therapy.11 The overall higher success rates for sequential therapy seem to apply to clarithromycinresistant strains. Indeed, in this subset of patients, although reduced, the eradication rate after the sequential regimen was 6-fold higher than that achieved by standard triple therapy (13%). Data presented herein are consistent with a recent study in adults.13 Clarithromycin resistance of H pylori is caused by point mutations in three adjacent 23S rRNA nucleotides10 (A2142G, A2143G, and A2142C) that cause decreased affinity of the ribosomes for several macrolides; the first 2 mutations are the most frequently found mutations in patients of our geographic area.13 Our study, as another,23 reveals that the A2143G is the main genetic mutation leading to H pylori clarithromycin resistance in vivo. We also showed that the outcome of H pylori therapy is markedly affected by the A2143G mutation, with a very low eradication rate (50%), which occurred in the presence of this mutation and was achieved exclusively in children treated with the sequential regimen; clarithromycin resistance associated with the A2142G mutation appears to be clinically insignificant because the eradication rate reaches 80% in both mutatant and wild-type strains, irrespective of the type of treatment. Recent studies on H pylori infection in children, using antibiotic-susceptibility testing, have demonstrated a higher cure rate when H pylori strains are sensitive to the antibiotic Table II. Helicobacter pylori eradication rates in standard therapy and sequential therapy groups according to clarithromycin resistance Triple therapy n = 73
Sequential therapy n = 95
P
Resistant strains
2/15 22/27 .001 13% (95% CI, 2-51%); 81% (95% CI, 52-92%) Susceptible strains 48/58 64/68 .8 83% (95% CI, 64-92%) 94% (95% CI, 82-98%)
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Table III. Eradication rates after standard triple therapy and sequential treatment according to mutations in resistant strains Susceptible strains Triple therapy (n = 73) Sequential therapy (n = 95) Overall (n = 168)
48/58 83% (95% CI 72-87%) 64/68 94% (95% CI 84-95%) 112/126e 88.9% (95% CI, 79-95%)
Resistant strain A2143G
Resistant strain A2142G
a
0/13
2/2b
16/19c 84% (95% CI, 49-94%) 16/32f 50% (95% CI, 28-72%)
6/8d 75% (95% CI, 30-95%) 8/10g 80% (95% CI, 37-96%)
a vs c: P < .001.; b vs d: P = .1.; e vs (f + g): P < .001.
used19-24; however, the facilities to perform H pylori culture and to test antibiotic susceptibility are not widely available, H pylori is not easily isolated,12 and costs limit applicability in routine practice. In our opinion, the high eradication rate of the sequential regimen even in clarithromycinresistant strains has important implications on therapy of infected children, favoring the use of the sequential regimen as practical and cost-effective, especially in areas where antibiotic susceptibility tests are not easily available. Finally, considering that the prevalence of clarithromycin-resistant H pylori strains may exceed 40%,25 the use of a therapy with a higher efficacy for such resistant strains would markedly reduce the overall cost associated with management of clarithromycin resistance in clinical practice. Although the reasons for superiority of the sequential regimen for resistant strains is not yet clear, it is possible to speculate that the ability of amoxicillin to destroy the bacterial wall could impair the adenosine triphosphatase–dependent pumps on the bacterial membrane responsible for the clarithromycin resistance resulting in an intracellular entrapment of the macrolide.26 The high intracellular macrolide concentration could overcome the rescue attempt engineered by the bacterium to prevent fatal clarithromycin-ribosomal binding. Therefore, the first course of sequential regimen based on amoxicillin administration could act as an ‘‘induction’’ therapeutic phase, favoring the efficacy of the clarithromycintinidazole therapy that immediately follows, even in some clarithromycin-resistant strains. We are aware of the limits of this study mainly due to the limited number of children harboring the resistant strains; however, the difference in eradication rates according to the therapeutic regimen used, if confirmed by larger independent studies, represents an interesting result that may be of help in overcoming clarithromycin resistance. Finally, most of the data on sequential regimes have been derived from Italian studies, and experience in other countries would be welcome to confirm that this approach is appropriate in other settings. n The authors thank Dr O. Burattini, Dr G. Leone, and Dr S. Fico for clinical support, and Mrs N. Iavernaro for expert technical collaboration. Submitted for publication Aug 29, 2009; last revision received Nov 24, 2009; accepted Feb 4, 2010.
Reprint requests: Dr Ruggiero Francavilla, Clinica Pediatrica ‘‘B. Trambusti,’’ Piazza Giulio Cesare, 11–Policlinico, Bari, Italy. E-mail: rfrancavilla@bioetaev. uniba.it.
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