Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates

cp60392 Module 1 Journal of Clinical Pathology 28/8/08 12:38:20 Topics: ; Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates M Romano, 1 M R Iovene, 2 M I Russo, 1 A Rocco, 3 R Salerno, 1 D Cozzolino, 1 A P Pilloni, 2 M A Tufano, 2 D Vaira, 4 G Nardone 3 1 Dipartimento Medico- Chirurgico di Internistica Clinica e Sperimentale, Gastroenterologia, Seconda Università di Napoli, Naples, Italy; 2 Microbiologia Clinica, Seconda Università di Napoli, Naples, Italy; 3 Dipartimento di Medicina Clinica e Sperimentale- Gastroenterologia, Università Federico II di Napoli, Naples, Italy; 4 Dipartimento di Medicina Interna e Gastroenterologia, Ospedale S. Orsola, Università of Bologna, Bologna, Italy Correspondence to: Professor G Nardone, Dipartimento di Medicina Clinica e Sperimentale, Unità di Gastroenterologia, via Pansini 5, 80131 Naples, Italy; nardone@unina.it Accepted 15 August 2008 Published Online First XXXX ABSTRACT Objectives: Helicobacter pylori infection is a major health problem worldwide, and effective eradication of the infection is mandatory. The efficacy of recommended eradication regimens is approximately 70%. In order to avoid treatment failure and the consequent development of secondary resistance(s), it is important to choose the most appropriate first-line treatment regimen. This choice should also be made based on the knowledge of the antimicrobial resistance peculiar to a given geographical area. We evaluated the prevalence of antimicrobialresistant H pylori strains isolated from naive patients and from patients with previous unsuccessful treatments. Methods: We studied 109 H pylori-infected subjects (Group 1) who had never received an eradication treatment and 104 H pylori-infected subjects (Group 2) who had failed one or more eradication treatments. Resistance to amoxicillin (AMO), tetracycline (TET), clarithromycin (CLA), metronidazole (MET) and levofloxacin (LEV) was determined using the epsilometer test. The significance of differences was evaluated by the x 2 test. Results: The prevalence of antimicrobial resistance was 0% versus 3.1% to AMO, 0% versus 2% to TET, 27% versus 41.3% to MET (p,0.05), 18% versus 45.8% to CLA (p,0.05) and 3% versus 14.6% to LEV (p,0.05) in Group 1 vs Group 2, respectively. In Group 2, there was an increased prevalence of H pylori strains resistant to multiple antimicrobials. Conclusions: This study confirms the high prevalence of H pylori strains resistant to CLA and MET, and indicates that unsuccessful treatments significantly increase resistance. Choosing eradication regimens other than standard triple therapy as a first-line therapy should be advisable in areas with high primary antimicrobial resistance prevalence. Helicobacter pylori is the major aetiological agent of chronic gastritis, peptic ulcer disease, gastric MALT lymphoma and gastric cancer. 1 2 Thus, once H pylori infection is diagnosed, it should be treated. In the last 20 years, standard triple therapy (ie, PPI, amoxicillin and clarithromycin/or metronidazole) has been largely adopted in various geographical regions worldwide and quoted as first-choice therapy by the Maastricht I and II consensus report. 3 4 However, standard triple therapy, like other available antimicrobial therapies for H pylori infection, has limited efficacy, because of side effects with poor compliance of patients to therapy and, more importantly, because of the development of antimicrobial resistance. The increasing prevalence of H pylori strains resistant to commonly used antimicrobials together with the lack of novel treatment options negatively affects eradication of H pylori infection, which is now approximately 70% and may lead to serious problems for the treatment of H pylori-related disorders in the near future. In recent years, the progressive decline in the eradication rate for H pylori has paralleled an increased rate of development of clarithromycin and metronidazole-resistant strains. 5 A systematic review of literature on eradication therapy indicated a 53% decrease in eradication rates of H pylori infection if clarithromycin resistance was present, and a clarithromycin-containing regimen was used. 6 Therefore, because clarithromycin resistance seems to be the most important limiting factor for treatment failure, 7 8 the prevalence of this resistance should be recognised before choosing the H pylori eradication regimen. The overall rate of clarithromycin resistance in Europe is 10%, ranging from 4% in northern European countries to 18.5% in southern European countries. The different prevalence in clarithromycin resistance depends mainly on the consumption of macrolides in the corresponding countries as expressed by measuring the daily dose of macrolides per 1000 inhabitants. 9 The recent Maastricht III consensus has recommended that the threshold of clarithromycin resistance at which this antibiotic should not be used or a susceptibility test performed is 15 20%. 10 Should this be the case, quadruple therapy represents the first-choice therapy. Failure of first-line therapy may contribute to the development of secondary resistance or resistance to multiple antimicrobials. This further hampers the efficiency of eradication therapy and strongly limits the number of effective antimicrobials available for eradication. This study, therefore, sought to investigate the prevalence of H pylori strains resistant to the antimicrobials commonly used for eradication (ie, clarithromycin, metronidazole, amoxicillin, levofloxacin and tetracycline). To this purpose, we studied H pylori clinical isolates obtained from H pylori-infected patients who had never been treated and H pylori isolates obtained from patients with one or more previous unsuccessful treatments. PATIENTS AND METHODS We consecutively enrolled 109 H pylori-infected patients (59 men and 50 women, age range 42 65 years) (Group 1) who had never received an J Clin Pathol 2008;000:1 5. doi:10.1136/jcp.2008.060392 1

cp60392 Module 1 Journal of Clinical Pathology 28/8/08 12:38:21 Topics: < = eradication treatment and 104 H pylori positive patients (56 men and 48 women, age range 44 68 years) (Group 2) who had failed one or more eradication treatments, referred to the Gastroenterology Units of the University of Naples because of dyspeptic symptoms. For histology, we took two biopsy specimens from the antrum, two from the body and one from the angulus; diagnosis and grading of gastritis were made according to the updated Sidney system. 11 One biopsy specimen was taken from the antrum and one from the body for a rapid urease test. Diagnosis of H pylori infection was based on positivity to both histology and 13 C urea breath test. Patients gave their written informed consent to enter the study, which was approved by the Ethical Committee of the Second University and the Federico II University of Naples, Italy. Bacterial culture and antimicrobial susceptibility testing Biopsy specimens were cultured in Helicobacter-selective agar at 37uC in microaerophilic atmosphere (O 2 5%, CO 2 5%, 12 13 Campygen Oxoid) for 3 6 days as previously described. H pylori was identified by Gram staining and by oxidase and catalase and urease reactions. All isolates identified as H pylori were subcultured in Helicobacter-selective agar at 37uC in a microaerophilic atmosphere for 3 6 days. From each subculture, a suspension in Brucella broth (2 ml) supplemented with fetal bovine serum (2%), yielding a viable count of 108 to 109 colonyforming units/ml, equivalent to 3 4 McFarland turbidity standard, was prepared. From each suspension, 100 ml was transferred onto the surface of a Mueller-Hinton agar plate with 5% sheep blood and streaked with a cotton swab. Subsequently, two antibiotic strips (E test) were applied onto the surface of each dried agar plate. After incubation, in a microaerophilic atmosphere at 37uC for 3 6 days, the concentration shown in the E test strip that was closest to the intersection point with growth on the plate (ie, the lowest concentration of drug inhibiting visible bacterial growth) was defined as minimal inhibitory concentration (MIC). E test strips containing amoxicillin (AMO), levofloxacin (LEV), tetracycline (TET), clarithromycin (CLA) and metronidazole (MET) were used. MICs are expressed in mg/l. Statistical analysis We set the power of the study to 0.8, I type error = 0.05 and control to case 1:1. The aim was to detect a minimal difference of 10% between the two groups. Data were analysed with the SPSS package for Windows (SPSS, Chicago). The significance of differences was assessed by x 2 test, and the level of significance was set at p,0.05. RESULTS The endoscopic, histological and serological findings in all 150 patients are summarised in table 1. Both groups showed comparable (ie, not statistically different) prevalence of endoscopic and histological alterations. Also, the prevalence of anti-caga positivity was not statistically different in the two groups of patients. Culture and in vitro antimicrobial susceptibility testing was unsuccessful in nine patients of Group 1 and in eight patients in Group 2. MIC values for each antimicrobial tested in Group 1 and Group 2 are shown in table 2. Table 3 shows the prevalence of antimicrobial resistance in Group 1 (100 subjects) and in Group 2 (96 subjects). In particular, the prevalence of antimicrobial resistance (n%) of H pylori in Group 1 (n = 100) was 0 (0%) to amoxicillin, 0 (0%) to tetracycline, 27 (27%) to metronidazole, 18 (18%) to clarithromycin and 3 (3%) to levofloxacyn. Prevalence of antimicrobial resistance (n%) in Group 2 (n = 96) was 3 (3.1%) to amoxicillin, 2 (2.1%) to tetracycline, 40 (41.3%) to metronidazole, 44 (45.8%) to clarithromycin and 14 (14.6%) to levofloxacin. The prevalence of resistance to clarithromycin, metronidazole or levofloxacin was significantly higher in patients who had already been treated compared with those who had never received an eradication treatment (table 3). Five subjects in Group 1 were resistant to both clarithromycin and metronidazole, whereas in Group 2 (ie, following unsuccessful eradication treatment), the prevalence of multiple resistance was much higher, with 21 subjects being resistant to both clarithromycin and metronidazole, five subjects to clarithromycin, metronidazole and levofloxacin, two subjects to clarithromycin and levofloxacin, two subjects to metronidazole and levofloxacyn, one subject to all antimicrobials but tetracycline and one subject to all of the antimicrobials tested (table 4). DISCUSSION This study shows that in a restricted area of southern Italy, there is an increased prevalence of H pylori strains which are resistant to commonly used antimicrobials. Also, previous treatment failures seem to greatly increase the rate of antimicrobial resistance of H pylori clinical isolates and favour the development of H pylori strains which are resistant to more than one antimicrobial. The management of H pylori-related gastroduodenal diseases strictly relies on the eradication of the infection. This is accomplished in 70 80% of cases through a therapeutic regimen based on the association between a proton-pump inhibitor (PPI) and two antimicrobials (ie, clarithromycin and amoxicillin) as first-line therapy. 10 14 Lack of 100% efficacy of commonly used eradication regimens depends on several factors. The location of the bacterium within the stomach (ie, the mucus lining the surface epithelium or the surface of mucous cells) provides a challenge for antimicrobial therapy. Also, the gastric lumen is a hostile environment for antimicrobial therapy because the drug must penetrate a thick mucous and may need to be active at a ph value below neutral. Moreover, compliance to therapy is hampered by the frequent occurrence of antimicrobial-related side effects. Finally, the number of H pylori strains resistant to common antimicrobials is increasing. 15 Therefore, choosing the most appropriate first-line therapy is crucial in order to eradicate the infection and to avoid the emergence of secondary antimicrobial resistance. Our study performed in an Italian region with a high prevalence of H pylori infection indicates that the primary resistance to the antimicrobials which are routinely used in the treatment of the infection is becoming unacceptably high. In particular, clarithromycin resistance is approximately 20%, thus suggesting that, at least in this geographical area, this antimicrobial should not be included in the first-line therapy of the infection. The reasons for high clarithromycin resistance are not completely known. One may hypothesise that because of its efficacy and good tolerability, this antimicrobial is largely used especially in the treatment of upper-airways infections in childhood, thus increasing the likelihood of resistance. The clinical effect of clarithromycin resistance is essentially complete loss of any anti-h pylori effect, and the outcome of therapy can generally be predicted on the basis of what could be expected if only the other antimicrobial(s) in the regimens were used. 16 Based on this, in the Maastricht III consensus conference on H pylori, it was stated that in areas with a clarithromycin 2 J Clin Pathol 2008;000:1 5. doi:10.1136/jcp.2008.060392

cp60392 Module 1 Journal of Clinical Pathology 28/8/08 12:38:21 Topics: Table 1 Endoscopic, histological and serological characteristics of patients in naïve (Group 1) and previously treated (Group 2) patients n (%) n (%) Endoscopy Erosions 52 49 Erythema 43 45 Duodenal ulcer 3 2 Histology Chronic gastritis Mild 46 40 Moderate 40 39 Severe 14 17 Activity Mild 50 51 Moderate 25 27 Severe 25 18 Atrophy Mild 10 13 Moderate 5 8 Severe 4 6 Metaplasia Mild 9 10 Moderate 4 5 Severe 1 1 Helicobacter pylori density Mild 39 40 Moderate 43 45 Severe 18 11 Anti-CagA positive 64 60 resistance between 15% and 20%, clarithromycin should not be used unless a pretreatment antimicrobial susceptibility testing is performed. In this regard, we have previously demonstrated that, at least in patients undergoing an upper endoscopy, performing culture and pretreatment antimicrobial susceptibility testing increases the efficacy of first-line therapy and is also cost-saving, decreasing the need for re-treatment and retesting. 17 Our study also confirms a high prevalence of primary resistance to metronidazole which is as high as 27%. This figure increases to 40% in patients who have already been treated for H pylori infection without achieving eradication of the infection. However, increasing the dosage of metronidazole administered (i.e from 1 to 1.5 g/day) generally improves the results of therapy when treating metronidazole-resistant H pylori strains. Levofloxacin-based triple therapy has recently been used and suggested as a successful rescue treatment following failure of Table 3 Prevalence of antimicrobial resistance in Group 1 (naïve Helicobacter pylori patients) and Group 2 (Helicobacter pylori-infected patients with previous unsuccessful treatments) Resistance n (%) Resistance n (%) p Value Amoxicillin 0 (0) 3 (3.1) NS Tetracycline 0 (0) 2 (2.1) NS Metronidazole 27 (27) 40 (41.3) 0.03 Clarithromycin 18 (18) 44 (45.8),0.0001 Levofloxacyn 3 (3) 14 (14.6) 0.04 first-line therapy. 18 19 Interestingly, in our study, primary resistance to levofloxacin is approximately 3%, thus making this antimicrobial suitable to be used in first-line therapy as an alternative to clarithromycin in areas with a high prevalence of primary resistance to this antimicrobial. Secondary resistance develops as the result of failed therapy. In fact, in our study, there is a statistically significant increase in the resistance toward all of the antimicrobials tested following one or more treatment failures. In particular, there was a 1.48- fold increase in metronidazole resistance, a 2.4-fold increase in clarithromycin resistance and a 4.6-fold increase in levofloxacin resistance. Moreover, there was the appearance of amoxicillinand tetracycline-resistant strains, even though the prevalence was as low as 3% and 2%, respectively. More interestingly, following unsuccessful eradication treatments, there was the appearance of several H pylori strains resistant to more than one antimicrobial. In particular, one patient was resistant to four antimicrobials and one other patient to all of the antimicrobials tested. This indicates that other effective antimicrobials or drugs which interfere with metabolic pathways necessary for the survival of the bacterium are needed. Recently, potential developments in anti-h pylori treatment have been made, suggesting that antimicrobial peptides, 20 essential oils, 21 porphyrins 22 and probiotics 20 may be promising but still far from being used in clinical practice. Also, sequential therapy using PPI and amoxicillin for 5 days followed by PPI plus clarithromycin and tinidazole for 5 more days. > 23 This regimen has been reported to achieve a near 95% success rate, 23 also in children. 24 Alternatively, in subjects undergoing an upper endoscopy, pretreatment susceptibility testing might prove to be of use. 17 In conclusion, this study indicates that in our region there is a large prevalence of H pylori strains resistant to clarithromycin and metronidazole. Therefore, in this setting, choosing quadruple therapy or levofloxacin-based triple therapy as a first-line eradication regimen should be advisable. Also, our study indicates that secondary resistance, even to multiple antimicrobials, is becoming a major problem in the management of Table 2 Group 2 Comparative activity of the antimicrobials tested against Helicobacter pylori isolates in Group 1 and E test range (mg/l) MIC50 (mg/l) MIC90 (mg/l) E test range (mg/l) MIC50 (mg/l) MIC90 (mg/l) Amoxicillin 0016 to 256 0.032 0.50 0016 to 256 0.64 0.50 Metronidazole 0016 to 32 0.50.32 0016 to 32 4.0.32 Clarithromycin 0016 to 256 0.125 32 0016 to 256 1.0 32 Tetracycline 0016 to 256 0.094 1.0 0016 to 256 0.64 4.0 Levofloxacyn 0002 to 32 0.38 0.75 0002 to 32 0.250 3.0 MIC50 values ranged from 0.032 mg/ml for amoxicillin to.32 mg/ml for metronidazole. MIC50, minimal inhibitory concentration required to inhibit the growth of 50% of organisms; MIC90, minimal inhibitory concentration required to inhibit the growth of 90% of organisms. J Clin Pathol 2008;000:1 5. doi:10.1136/jcp.2008.060392 3

cp60392 Module 1 Journal of Clinical Pathology 28/8/08 12:38:25 Topics: Table 4 Prevalence of multiple antimicrobial resistance in Group 1 (ie, naïve Helicobacter pylori patients) and Group 2 (ie, Helicobacter pyloriinfected patients with previous unsuccessful treatments) Resistance n (%) Resistance n (%) MET+CLA 5 (5) 21 (21.8) CLA+LEV 0 2 (2.1) MET+LEV 0 2 (2.1) MET+CLA+LEV 0 5 (5.2) MET+CLA+LEV+AMO 0 1 (1.04) MET+CLA+LEV+AMO+TET 0 1 (1.04) AMO, amoxicillin; CLA, clarithromycin; LEV, levofloxacin; MET, metronidazole; TET, tetracycline. H pylori infection and suggests that the very low efficiency of rescue treatments might be due to increased secondary resistance to almost all of the antimicrobials available for H pylori eradication. This further strengthens the concept that the best rescue regimen should be a near to 100% efficient first-line therapy. Competing interests: None. Ethics approval: Ethics approval was provided by the Ethical Committee of the Second University and the Federico II University of Naples, Italy. Patient consent: Obtained. REFERENCES 1. Zarrilli R, Ricci V, Romano M. Molecular response of gastric epithelial cells to Helicobacter pylori-induced damage. Cell Microbiol 1999;1:93 9. 2. Romano M, Ricci V, Zarrilli R. Helicobacter pylori-related gastric carcinogenesis implications for chemoprevention. Nat Clin Pract Gastroenterol Hepatol 2006;3:622 32. 3. Malfertheiner P, Megraud F, O Morain C, et al. Current European concepts in the management of Helicobacter pylori infection: the Maastricht Consensus Report. The European Helicobacter pylori Study Group. Eur J Gastroenterol Hepatol 1997;9:1 2. 4. Malfertheiner P, Megraud F, O Morain C, et al. European Helicobacter pylori Study Group (EHPSG). Current concepts in the management of Helicobacter pylori infection: the Maastricht 2 200 Consensus Report. Aliment Pharmacol Ther 2002;16:167 80. 5. Megraud F. H pylori antibiotic resistance: prevalence, importance, and advances in testing. Gut 2004;53:1374 84. 6. Vergara M, Catalan M, Gisbert JP, et al. Meta-analysis: role of Helicobacter pylori eradication in the prevention of peptic ulcer in NSAID users. Aliment Pharmacol Ther 2005;21:1411 18. 7. Megraud F, Lamouliatte H. Review article: the treatment of refractory Helicobacter pylori infection. Aliment Pharmacol Ther 2003;17:1333 43. 8. McMahon BJ, Hemmessy TW, Bensler JM, et al. The relationship among previous antimicrobial use, antimicriobial resistance, and treatment outcomes for Helicobacter pylori infections. Ann Intern Med 2003:139:463 9. 9. Cars O, Molstad S, Melander A. Variation in antibiotic use in the European Union. Lancet 2001;357:1851 3. 10. Malfertheiner P, Megraud F, O Morain C, et al. Current concepts in the management of Helicobacter pylori infection: the Maasstricht III Consensus Report. Gut 2007;6:772 81. 11. Dixon MF, Genta RM, Yardley JH, et al. Participants in the International Workshop on the Histopathology of Gastritis, Houston 1994: classification and grading of gastritis. The updated Sydney system. Am J Surg Pathol 1996;20:1161 81. Study highlights What is current knowledge: c H pylori eradication is becoming increasingly mote difficult to achieve due to increasing prevalence of antimicrobial resistant H pylori strains. c Prevalence of antimicrobial resistant H pylori strains vary from region to region. c First-line treatment should take into account the prevalence of antimicrobial-resistant H pylori strains in a given area. What is new here: c Failure to eradicate H pylori infection is associated with a significant increase in the prevalence of H pylori strains resistant to the most commonly used antimicrobials. c Eradication failures lead to the development of H pylori strains with multiple antimicrobial resistance. c Regimens other than standard triple therapy should be considered as first-line treatment in areas with high primary resistance to clarithromycin or metronidazole. 12. Iovene MR, Romano M, Pilloni AP, et al. Prevalence of antimicrobial resistance in eighty clinical isolates of Helicobacter pylori. Chemotherapy 1999;45:8 14. 13. Megraud F and Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007;20:280 322. 14. Chey WD, Wong BCJ, the Practice Parameters Committee of the American College of Gastroenterology. American College of Gastroenterology guideline on the management of Helicobacter pylori infection. Am J Gastroenterol 2007;102:1 18. 15. Gerrits MM, van Vliet AHM, Kuipers E, et al. Helicobacter pylori and antimicrobial resistance: molecular mechanisms and clinical implications. Lancet Infect Dis 2006;6:699 709. 16. Huang J, Hunt RH. The importance of clarithromycin dose in the management of Helicobacter pylori infection: a meta-analysis of triple therapies with a proton pump inhibitor, clarithromycin and amoxicillin or metronidazole. Aliment Pharmacol Ther 1999;13:719 29. 17. Romano M, Marmo R, Cuomo A, et al. Pretreatment antimicrobial susceptibility testing is cost-saving in the eradication of Helicobacter pylori. Clin Gastroenterol Hepatol 2003;1:273 8. 18. Gisbert JP, Morena F. Systematic review and meta-analysis: levofloxacin-based rescue regimens after Helicobacter pylori treatment failure. Aliment Pharmacol Ther 2006;23:35 44. 19. Saad R, Shoenfeld P, Hyungjin MK, et al. Levofloxacin-based triple therapy versus bismuth-based quadruple therapy for persistent Helicobacter pylori infection: a metaanalysis. Am J Gastroenterol 2006;3:488 96. 20. Colado MC, Gonzalez A, Gonzalez R, et al. Antimicrobial peptides are among the antagonistic metabolites produced by bifidobacterium against Helicobacter pylori. Int J Antimicrob Agents 2005;25:385 91. 21. Bergonzelli GE, Donnicola D, Porta N, et al. Essential oils as components of a dietbased approach to management of Helicobacter pylori infection. Antimicrob Agents Chemother 2003;47:3240 6. 22. Stojiljkovic I, Evavold BD, Kumar V. Antimicrobial activity of porphyrins. Expert Opin Invest Drugs 2001;10:309 20. 23. Vaira D, Zullo a, Vakil N, et al. Sequential therapy versus standard triple-drug therapy for Helicobacter pylori eradication: a randomized trial. Ann Int Med 2007;146:556 63. 24. Francavilla R, Lionetti E, Castellaneta SP, et al. Improved efficacy of 10-day sequential treatment for Helicobacter pylori eradication in children: a randomized trial. Gastroenterology 2005;129:1414 19. 4 J Clin Pathol 2008;000:1 5. doi:10.1136/jcp.2008.060392

cp60392 Module 1 Journal of Clinical Pathology 28/8/08 12:38:25 Topics: Authors Queries Journal: Journal of Clinical Pathology Paper: cp60392 Title: Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates Dear Author During the preparation of your manuscript for publication, the questions listed below have arisen. Please attend to these matters and return this form with your proof. Many thanks for your assistance Query Reference Query Remarks 1 You have opted not to pay a fee to make your article free online (Unlocked). If you wish to change your mind, please indicate this clearly and provide invoice address and contact details when you return your proofs. This is the last point in the production process where you can choose to Unlock your paper; the cost of this service is 1,200/$2,220/J1,775 (+VAT) and further details can be found at http://jcp.bmj.com/info/unlocked.dtl 2 I have define MIC50 and MIC90 in table 2 footnote. Please check 3 In table 2: 0016 to 256 -- Why the leading zeros ( 00 )? Please clarify 4 Sentence Also, sequential therapy using PPI and amoxicillin for 5 days followed by PPI plus clarithromycin and tinidazole for 5 more days. does not make sense. Please check and reword. J Clin Pathol 2008;000:1 5. doi:10.1136/jcp.2008.060392 5