- Email: [email protected]
Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: High frequency of A2143G point mutation associated with clarithromycin resistance
Accepted Manuscript Title: Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: high frequency of A2143G point mutation associated with clarithromycin resistance Authors: Shima Mahmoudi, Setareh Mamishi, Maryam Banar, Sepideh Keshavarz Valian, Abbas Bahador, Mehri Najafi, Fatemeh Farahmand, Babak Pourakbari PII: DOI: Reference:
S2213-7165(17)30092-9 http://dx.doi.org/doi:10.1016/j.jgar.2017.04.011 JGAR 416
To appear in: Received date: Revised date: Accepted date:
5-2-2017 5-4-2017 11-4-2017
Please cite this article as: Shima Mahmoudi, Setareh Mamishi, Maryam Banar, Sepideh Keshavarz Valian, Abbas Bahador, Mehri Najafi, Fatemeh Farahmand, Babak Pourakbari, Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: high frequency of A2143G point mutation associated with clarithromycin resistance (2010), http://dx.doi.org/10.1016/j.jgar.2017.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: high frequency of A2143G point mutation associated with clarithromycin resistance
Shima Mahmoudi1, Setareh Mamishi1,2, Maryam Banar1, Sepideh Keshavarz Valian3, Abbas Bahador4, Mehri Najafi5, Fatemeh Farahmand5, Babak Pourakbari1*
1- Pediatric Infectious Diseases Research Center, Tehran University of Medical Sciences, Tehran, Iran 2-Department of Pediatric Infectious Disease, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3- School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 4- Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5-Department of Pediatric Gastroenterology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Corresponding author Babak Pourakbari, PhD No. 62, Dr. Gharib St., Pediatric Infectious Disease Research Center, Children Medical Center Hospital, Tehran,
Iran Tel\fax: +98 21 66428996 E-mail : [email protected] Highlights
The Antibiotic resistance of H. pylori is rising in developing countries In our study, the prevalence of H. pylori infection among children was 35.5% (32 positive cultures out of 90 biopsy specimens). The resistance rate to CLR was 21.8%, which was higher than the resistance rate detected by other studies in Iran The A2143G point mutation was detected in 71% of CLR resistant isolates (5 out of 7 CLR resistant isolates).
Abstract Objective: Increasing antibiotic resistance in Helicobacter pylori is a global concern and is associated with treatment failure. The aim of this study was to evaluate antibiotic resistance of H. pylori to different antibiotics including amoxicillin, erythromycin, ciprofloxacin, furazolidone, tetracycline, metronidazole and clarithromycin and to detection of A2143G mutation in clarithromycin resistant isolates by real-time PCR technique. Methods: Ninety patients with upper gastrointestinal symptoms were enrolled in this study. H. pylori were isolated from 32 specimens and the resistance rate of these strains to amoxicillin, erythromycin, ciprofloxacin, furazolidone, and tetracycline was tested by disc agar diffusion method. The resistance level to metronidazole and clarithromycin was determined by agar dilution method. The presence of A2143G point mutation in clarithromycin resistant isolates was determined using real-time PCR technique.
Results: The resistance rates to amoxicillin, erythromycin, ciprofloxacin, furazolidone, tetracycline, metronidazole and clarithromycin were 53%, 50%, 37.5%, 62.5%, 25%, 62.5% and 22%, respectively. The A2143G point mutation was detected in 71% of clarithromycin resistant strains (5 out of 7). Conclusions: The prevalence of H. pylori resistance to metronidazole, ciprofloxacin, erythromycin, amoxicillin and furazolidone in Iran is high. Determination of antibiotic susceptibility plays an important role in selecting of the appropriate anti H. pylori regimen.
Keywords: Helicobacter pylori, antibiotic resistance, A2143G mutation, Iran. Introduction Helicobacter pylori (H. pylori) is a Gram-negative curved microaerophilic bacterium that can survive and colonize in the acidic condition of gastric mucosa (1, 2). It is one of the most common human bacterial infections (3). It is the main cause of chronic gastritis and is responsible for peptic ulcer disease (PUD), mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma (4, 5). The prevalence of H. pylori infection in developing countries is more than in developed countries, and studies show that its prevalence in Iran is about 54% (6, 7). Infection of this bacterium usually occurs in childhood (8) and plays a decisive role in infection rates in adults; therefore, evaluation of H. pylori infection in children is important (4) and can help to implement appropriate preventive and therapeutic programs (6). The current therapy regimen for eradication of H. pylori infections consists of one or two antibiotics such as clarithromycin, amoxicillin, or metronidazole in combination with a Proton Pomp Inhibitor (PPI) (2, 9). One of the most important antibiotics in this regimen is clarithromycin and occurrence of point mutations such as the transition of Adenine to Guanine at
position 2143 (A2143G) in the bacterial 23SrRNA gene is responsible for the emergence of clarithromycin resistant isolates (10). The aim of this study was to evaluate antibiotic resistance of H. pylori isolates, and determine A2143G mutation in clarithromycin resistant strains by real-time PCR. Materials and Methods Patients and specimens Between April 2014 and March 2015, one gastric biopsy specimens was taken from 90 patients with upper gastrointestinal symptoms who were referred to children’s medical center hospital, Tehran, Iran. Ethical approval was obtained by Tehran University of Medical Sciences. The patients’ demographic data including name, sex, age, symptoms and treatment history were collected and sampling was accomplished with their consent. One part of the specimen was used for Rapid urease test (RUT) (11) and another section was added into BHI broth (Merck, Germany) and was transferred to the microbiology laboratory within 3 hours. Culture The isolation of H. pylori strains from biopsy specimens were performed according to the method of Milani et al. with some modifications (12). At first, specimens were crushed between two sterile glass slides, then the resulting extracts was cultured on Brucella agar (Merck, Germany) supplemented with 7% defibrinated sheep blood (Jihad daneshgahi of Tehran University, Tehran, Iran), 5% fetal bovine serum (FBS) (Jihad daneshgahi of Tehran University, Tehran, Iran), vancomycin (6mg/L, Sigma), trimethoprim (5 mg/L, Sigma) and amphotricin B (2mg/L, Sigma). Then plates were incubated in the microaerophilic conditions (6% O2, 10% CO2 and 84% N2) using the gas pack incubation method (Merck, Germany) at 37°C for 3-5 days.
Isolates were identified as H. pylori based on colony morphology, gram staining and positive reactions of urease, catalase and oxidase tests. Antibiotic Susceptibility Test Resistance of strains to amoxicillin (AMX), erythromycin (ERY), ciprofloxacin (CIP), furazolidone (FUR) and tetracycline (TET) antibiotics was assessed by using the disc agar diffusion method. Bacterial suspension equivalent to McFarland turbidity standard no. 3 (approximately 1.0×109 CFU/ml) were prepared in sterile normal saline and spread on brucella blood agar plates (without antibiotics) which had been enriched with 5% FBS. After plates dried, antibiotic disks were placed and plates were incubated in microaerophilic atmosphere at 37°C for 72 h, then the zones of inhibition were measured and the sensitive and resistant isolates were determined based on CLSI criteria (13). The minimum inhibitory concentrations (MIC) of the clarithromycin (CLR) and metronidazole (MTZ) antibiotics were determined using agar dilution method (13). Various concentrations of metronidazole (ranging from 2 to 32 mg/ml) and clarithromycin (ranging from 0.125 to 4 mg/ml) were added to brucella agar supplemented with 5% defibrinated sheep blood and 5% FBS. Bacterial suspension with turbidity equivalent to McFarland standard no. 2 was prepared in sterile normal saline and 3 μl of them was inoculated on the plates and MICs were determined after 3 days incubation. A strain was considered as resistant to metronidazole if the MIC value was > 8 mg/ml and MIC > 1 was indicative of resistance to clarithromycin. H. pylori ATCC43504 was used as quality control of the two tests. TaqMan real-time PCR A TaqMan real-time PCR assay was used to detect A2143G point mutation in CLR resistant H. pylori isolates. DNA was extracted from fresh isolates using boiling method (14). An 86-bp
fragment of the 23S rRNA gene of H. pylori was amplified and coupled with detection of the product by probe hybridization (15). The assay was accomplished in a final volume of 20 μl containing: 2 μl of PCR buffer, 0.2 unit of Taq DNA polymerase, 0.5 μl of dNTP , 0.5 μl of MgCl2 , 0.5 μl of each primer, 0.5 μl of probe, 1 μl of template DNA and 14.3 μl of dH 2O. The thermal cycler conditions were a first denaturation cycle at 95°C for 10 min, followed by 40 cycles of 20 s at 95 °C, 20 s at 61°C and extension at 72°C for 20 s. An ABI step one real-time PCR system (Applied Biosystems, Foster City, California, USA) was used and acquired results were evaluated on the basis of the melting curves. Statistical analysis Descriptive statistics were used to summarize all demographic characteristics of the patients. Categorical data were tested using the χ2 test for dependence and a P-value <0.05 considered significant. All statistical analyses were done using statistical packages SPSS 16.0 (SPSS Inc. Chicago,
IL).
Results Ninety children with chronic abdominal pain were included in this study (Table1). Among them, H. pylori were isolated from 32 individuals (35.5%) with average age of 9.4 years. The distribution of female and male within H. pylori positive patients were 17 female (53%) and 15 male (47%) and there was no relationship between H. pylori infection and gender (p>0.5). Thirty- four samples were RUT positive (38%). The antimicrobial resistance of 32 H. pylori isolates to TET, CIP, AMX, ERY and FUR were determined by disc agar diffusion method and results are listed in Table 2. The highest resistance rate was observed against FUR (62.5%) and the lowest resistance rate was seen against TET (25%). The MIC values of MTZ and CLR were determined by agar dilution method. Resistance to CLR was found in 7 isolates (22%) and 20 isolates (62.5%) were resistant to MTZ. seven strains were resistant to both CLR and MTZ (double resistant) . The presence of A2143G point mutation was assessed in CLR resistant isolates by TaqMan Real-time PCR. This mutation was detected in 5 (71%) out of 7 resistant strains. Discussion The Antibiotic resistance of H. pylori is rising in developing countries (9), which can affect the effectiveness of current therapeutic regimens and decrease their eradication rates (16). Given the role of H. pylori infections in developing gastric malignancies, controlling of its infections and monitoring of antibiotic resistance is important (16, 17). In this study, we investigated the resistance of H. pylori isolates to AMX, TET, CIP, ERY, FUR, MTZ and CLR by disk agar diffusion and agar dilution methods.
In our study, the prevalence of H. pylori infection among children was 35.5% , which was similar to the rate reported from China (18). However, it was higher than the prevalence rate in Belgium (19) and lower than those reported in Iran (6, 20), Portugal (21) and Brazil (22) (Table 3). As is evident, the prevalence of H. pylori infection in developing countries is higher than developed countries, which can be due to the use of effective combination therapies, higher level of hygiene and restricted use of antibiotics in these communities (9). The resistance rate to CLR was 22%, which was higher than the resistance rate detected by other studies in Iran (12, 23-25), Brazil (26), South Korea (27) and the Netherlands (28) and was lower than the rate reported from Portugal (29) (Table 4). Several factors may be involved in the emergence of resistance to CLR including extensive use of CLR for the treatment of respiratory infections in children (26), applying monotherapy regimen and cross-reactivity between CLR and ERY (members of macrolides) (30). The overall resistance to MTZ was 62.5% and was lower than the results of previous studies in Iran (12, 25) and China (16). The resistance rate to MTZ was higher than the rates reported by other studies in Iran(23, 24), South Korea (27), Europe (28, 29, 31, 32) and Brazil (26) (Table 4).. It seems that the extensive use of MTZ for the treatment of genital and parasitic infections led to the emergence of MTZ resistance in H. pylori strains (17). Resistance rate to AMX in this study was 53%. This rate was lower than the rates reported by Rafeey et al., in Iran (25). However, the resistance rate to AMX in our study was much higher than the results of some studies conducted in Iran (12, 23, 24), Europe (29, 31), Brazil (26), South Korea (27) and China (16) (Table 4).Frequent use of AMX for the treatment of sick children and consumption of antibiotics without a prescription can be the reason for the high level resistance to AMX observed in our study (26).
The rate of resistance to TET in this study was 25%, which was higher than the results of some reports in Iran (12, 23, 25), Brazil (26) and Europe (31) and lower than the results of one study in Iran (24) and South Korea (27) (Table 4).. TET is not prescribed to Iranian children, especially because of its adverse effects on color and calcification of children tooth (26). Probably emergence of resistance to this antibiotic is related to acquisition of genetic elements containing multidrug resistance genes.
On the other hand, 37.5% of our isolates were resistant to CIP, which was higher than the results of previous studies conducted by Rafeey et al., and Milan et al., from Iran(12, 25). The rate of resistance to CIP in our study was also higher than the rate of resistance in Portuguese and South Korean children (27, 29) (Table 4). These results represent a rising trend of resistance to CIP in Iran. Possibly the widespread use of fluoroquinolones in the treatment of H. pylori infections, is the main cause of increasing levels of resistance to this group. Frequency of ERY resistance in our study was 50%, which was higher than the results of previous studies performed in Iran (12, 23) and South Korea (27). ERY is a macrolide antibiotic and high level resistance to this drug may be related to cross-reactivity with other macrolides. At the present work, the resistance level to FUR was 62.5%, which was higher than those reported by Maleknejad et al., (24), Rafeey et al., (25) and Fallahi et al., (23) in Iran. The resistance level to FUR in other countries such as Brazil, China and South Korae was lower than our study (16, 26, 27) (Table 4). Due to the genotoxicity and carcinogenic effects of FUR, its consumption in Europe and
USA is banned and it is available in some developing countries such as Iran (26) and it is probable that the high resistance observed in our study is due to the excessive intake of this medicine in Iran. According to the results, it can be stated that the antibiotic resistance pattern of H. pylori varies between different geographic areas and depends on the different factors such as population density, age, race and availability of antibiotics (33). CLR belongs to the macrolide family and inhibits protein synthesis in bacteria through binding to the peptidyl transferase of 23SrRNA (10). Occurrence of some point mutations such as A2143G, A2142G and A2142C in bacterial 23SrRNA gene leads to the emergence of CLR resistance in H. pylori (34). In the present study, the A2143G point mutation was detected in 71% of CLR resistant isolates (5 out of 7 CLR resistant isolates). Our report is in consistent with previous studies in which A2143G mutation was detected in 73.68% (35) and 71.4% [34] of CLR resistant isolates. However, in the study conducted by Eghbal et al., (10) and Vala et al., (36) A2143G mutation was detected in 100% of the CLR resistant isolates. These data demonstrated the importance of A2143G point mutation in the development of CLR resistance in H. pylori. Because the presence of other point mutations (A2142G and A2142C) and other resistance mechanisms such as RNA methylation and efflux pumps were not evaluated in this study, the observed difference between phenotypic and genotypic resistance of CLR can be explained. Based on the guidelines proposed in the Conference of Maastricht III (37), the first-line of treatment against H. pylori infections that is used in our hospital is AMX, omeprazole, and CLR for 7 days.
This study has some limitations. First, its sample size (n=90) was small compared to other studies and biopsy specimens were obtained from the admitted patients of one hospital (i.e. CMC hospital). Therefore, the results should be analyzed cautiously when compared with other studies and are not generalizable to the entire city of Tehran. Second, the presence of other point mutations in H. pylori 23srRNA gene (i.e. A2142G and A2142C) that are associated with CLR resistance was not evaluated in this study. The use of Realtime PCR method which is a highly sensitive technique can be the strength point of our survey. Conclusion According to the results of our study, resistance of H. pylori isolates to MTZ, CIP, ERY, FUR and AMX in our study was higher than other countries, which could be the reason for treatment failure in Iran. These results indicate the necessity of determination the antibiotic resistance patterns of H. pylori before prescribing drugs, which can play an important role in preventing the spread of drug resistance in this bacterium and leads to selection of appropriate treatment regimen. A majority of the CLR resistant isolates showed A2143G mutation in 23S rRNA gene that indicates the importance of this mutation.
Declarations Funding: This study was supported by a grant (grant number: 92-01-88-19590) from Tehran University of Medical Sciences to Dr. Babak Pourakbari. Competing Interests: None Ethical Approval: Ethical approval was obtained by Tehran University of Medical Sciences.
References 1. Khademi F, Poursina F, Hosseini E, Akbari M, Safaei HG. Helicobacter pylori in Iran: A systematic review on the antibiotic resistance. Iranian journal of basic medical sciences. 2015;18(1):2. 2. Khodadad A, Farahmand F, Najafi M, Shoaran M. Probiotics for the treatment of pediatric helicobacter pylori infection: a randomized double blind clinical trial. 2013. 3. Pourakbari B, Mirsalehian A, Maleknejad P, Mamishi S, Azhdarkosh H, Daryani NE, et al. Evaluation of a new antigen for diagnosis of Helicobacter pylori infection in stool of adult and children. Helicobacter. 2011;16(1):42-6. 4. Kim N. Prevalence and Transmission Routes of H. pylori. Helicobacter pylori: Springer; 2016. p. 3-19. 5. Sayehmiri F, Kiani F, Sayehmiri K, Soroush S, Asadollahi K, Alikhani MY, et al. Prevalence of cagA and vacA among Helicobacter pyloriinfected patients in Iran: a systematic review and meta-analysis. The Journal of Infection in Developing Countries. 2015;9(07):686-96. 6. Ghasemi-Kebria F, Ghaemi E, Azadfar S, Roshandel G. Epidemiology of Helicobacter pylori infection among Iranian children. Arab Journal of Gastroenterology. 2013;14(4):169-72. 7. Moosazadeh M, Lankarani KB, Afshari M. Meta-analysis of the prevalence of helicobacter pylori infection among children and adults of Iran. International journal of preventive medicine. 2016;7. 8. Mamishi S, Eshaghi H, Mahmoudi S, Bahador A, Hosseinpour Sadeghi R, Najafi M, et al. Intrafamilial transmission of Helicobacter pylori: genotyping of faecal samples. British journal of biomedical science. 2016;73(1):38-43. 9. Shokrzadeh L, Alebouyeh M, Mirzaei T, Farzi N, Zali MR. Prevalence of multiple drug-resistant Helicobacter pylori strains among patients with different gastric disorders in Iran. Microbial Drug Resistance. 2015;21(1):105-10. 10. Eghbali Z, Mojtahedi A, Ansar MM, Asl SF, Aminian K. Detection of 23SrRNA Mutations Strongly Related to Clarithromycin Resistance in Helicobacter pylori Strains Isolated From Patients in the North of Iran. Jundishapur journal of microbiology. 2016;9(2). 11. Pourakbari B, Ghazi M, Mahmoudi S, Mamishi S, Azhdarkosh H, Najafi M, et al. Diagnosis of Helicobacter pylori infection by invasive and noninvasive tests. Brazilian Journal of Microbiology. 2013;44(3):795-8. 12. Milani M, Ghotaslou R, Somi MH, Rafeey M, Akhi MT, Nahaei MR, et al. The status of antimicrobial resistance of Helicobacter pylori in Eastern Azerbaijan, Iran: comparative study according to demographics. Journal of Infection and Chemotherapy. 2012;18(6):848-52. 13. CLSI MfDASTfBTGAasp-. 14. Queipo-Ortuño MI, Colmenero JDD, Macias M, Bravo MJ, Morata P. Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clinical and Vaccine Immunology. 2008;15(2):293-6. 15. De Francesco V, Zullo A, Ierardi E, Giorgio F, Perna F, Hassan C, et al. Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits. Journal of antimicrobial chemotherapy. 2010;65(2):327-32. 16. Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, et al. Antibiotic resistance of Helicobacter pylori isolated in the Southeast Coastal Region of China. Helicobacter. 2013;18(4):274-9.
17. Khademi F, Faghri J, Poursina F, Esfahani BN, Moghim S, Fazeli H, et al. Resistance pattern of Helicobacter pylori strains to clarithromycin, metronidazole, and amoxicillin in Isfahan, Iran. Journal of Research in Medical Sciences. 2013;18(12). 18. Yu Y, Su L, Wang X, Wang X, Xu C. Association between Helicobacter pylori infection and pathological changes in the gastric mucosa in Chinese children. Internal Medicine. 2014;53(2):83-8. 19. Mana F, Vandebosch S, Deyi VM, Haentjens P, Urbain D. Prevalence of and risk factors for H. pylori infection in healthy children and young adults in Belgium anno 2010/2011. Acta Gastro Enterol Belg. 2013;76:381-5. 20. Soltani J, Amirzadeh J, Nahedi S, Shahsavari S. Prevalence of helicobacter pylori infection in children, a population-based cross-sectional study in west iran. 2013. 21. Bastos J, Peleteiro B, Pinto H, Marinho A, Guimarães JT, Ramos E, et al. Prevalence, incidence and risk factors for Helicobacter pylori infection in a cohort of Portuguese adolescents (EpiTeen). Digestive and Liver Disease. 2013;45(4):290-5. 22. Pacheco SL, Ogata SK, Machado RS, da Silva Patrício FR, Pardo ML, Kawakami E. Diagnosis of Helicobacter pylori Infection by Means of Reduced-Dose 13C-Urea Breath Test and Early Sampling of Exhaled Breath. Journal of pediatric gastroenterology and nutrition. 2013;57(5):60711. 23. Fallahi G-H, Maleknejad S. Helicobacter pylori culture and antimicrobial resistance in Iran. The Indian Journal of Pediatrics. 2007;74(2):127. 24. Maleknejad S, Mojtahedi A, Safaei-Asl A, Taghavi Z, Kazemnejad E. Primary Antibiotic Resistance to Helicobacter pylori Strains Isolated From Children in Northern Iran: A Single Center Study. Iranian journal of pediatrics. 2015;25(6):e2661-e. 25. Rafeey M, Ghotaslou R, Nikvash S, Hafez AA. Primary resistance in Helicobacter pylori isolated in children from Iran. Journal of Infection and Chemotherapy. 2007;13(5):291-5. 26. Ogata SK, Godoy APO, da Silva Patricio FR, Kawakami E. High Helicobacter pylori resistance to metronidazole and clarithromycin in Brazilian children and adolescents. Journal of pediatric gastroenterology and nutrition. 2013;56(6):645-8. 27. Seo JH, Jun JS, Yeom JS, Park JS, Youn HS, Ko GH, et al. Changing pattern of antibiotic resistance of Helicobacter pylori in children during 20 years in Jinju, South Korea. Pediatrics International. 2013;55(3):332-6. 28. Mourad‐Baars PE, Wunderink HF, Luisa Mearin M, Veenendaal RA, Wit JM, Veldkamp KE. Low Antibiotic Resistance of Helicobacter pylori in The Netherlands. Helicobacter. 2015;20(1):69-70. 29. Oleastro M, Cabral J, Ramalho PM, Lemos PS, Paixão E, Benoliel J, et al. Primary antibiotic resistance of Helicobacter pylori strains isolated from Portuguese children: a prospective multicentre study over a 10 year period. Journal of antimicrobial chemotherapy. 2011;66(10):2308-11. 30. Golrokhi MM, Fakheri H, Haghshenas MR, Ahanjan M. The Determination of Antibiotic Resistance of Helicobacter Pylori Isolated from Patients Living in North of Iran (Sari). 2016. 31. Megraud F, Coenen S, Versporten A, Kist M, Lopez-Brea M, Hirschl A, et al. 853 Helicobacter pylori Resistance to Antibiotics in Europe and Its Relationship to Antibiotic Consumption. Gastroenterology. 2012;142(5):S-146. 32. Schwarzer A, Bontems P, Urruzuno P, Kalach N, Iwanczak B, Roma‐Giannikou E, et al. Sequential Therapy for Helicobacter pylori Infection in Treatment‐naïve Children. Helicobacter. 2015.
33. Gahbauer AM, Gonzales ML, Guglielmo BJ. Patterns of antibacterial use and impact of age, race/ethnicity, and geographic region on antibacterial use in an outpatient medicaid cohort. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2014;34(7):67785. 34. Liu Q, Qi D, Kang J, Jin Y, Liu W, Gao W, et al. Efficacy of real-time PCR-based detection of Helicobacter pylori infection and genotypic resistance-guided quadruple therapy as the first-line treatment for functional dyspepsia with Helicobacter pylori infection. European journal of gastroenterology & hepatology. 2015;27(3):221-5. 35. Mohammadi M, Doroud D, Mohajerani N, Massarrat S. Helicobacter pylori antibiotic resistance in Iran. World journal of gastroenterology. 2005;11(38):6009. 36. Vala MH, Eyvazi S, Goudarzi H, Sarie HR, Gholami M. Evaluation of Clarithromycin Resistance Among Iranian Helicobacter pylori Isolates by E-Test and Real-Time Polymerase Chain Reaction Methods. Jundishapur journal of microbiology. 2016;9(5). 37. 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(6):772-81.
Table 1. Demographic data of the patients Parameters
N
%
3 to 10 years
50
55.5
10 to 16 years
40
44.5
Female
53
59
male
37
41
90
100
Previous anti-H.pylori treatment (Recurrence)
10
11
Consumption of antibiotics before endoscopy
37
41
H. pylori positive
32
35.5
Age
Gender
Symptoms Abdominal pain History
Table2. Antibiotic susceptibility of the H.pylori isolates by disk diffusion method Isolates, N (%) Antimicrobial agent Tetracycline (TET)
Susceptible 24 (75)
Resistant 8 (25)
Ciprofloxacin (CIP)
20 (62.5)
12 (37.5)
Amoxicillin (AMX)
15 (46.9)
17 (53)
Erythromycin (ERY)
16 (50)
16 (50)
Furazolidone (FUR)
12 (37.5)
20 (62.5)
Table 3. Prevalence rate of H. pylori infection in different countries
2012 2013 2013 2013
Sample size 21160 194 294 516
Infection rate (%) 66.20 50.50 64.20 11
2013
129
41.10
2014 2017
17731 90
32.10 35.5
Authors (reference)
Country (city)
Year
Bastos et al., )12( Ghasemi-Kebria et al., (6) Soltani et al., (20) Mana et al., (19)
Portugal (Porto) Iran (Gorgan) Iran (Sanandaj) Belgium(Brussels) Brazil (Sa˜o Paulo) China (Beijing) Iran (Tehran)
Pacheco et al., (22) Yu et al., (18) This study
Table 4. Resistance rates of H. pylori in different countries Resistance rate N (%) Authors (reference)
Country (city)
Year
Sample size
Rafeey et al., (25) Fallahi et al., (23)
Iran (Tabriz) Iran (Tehran) Portugal (Lisboa) Iran (Tabriz) Brazil (Sa˜o Paulo) Europe South Korea (Jinju)
2007 2007
100 62
2011
1115
2012
China (Beijing)
Oleastro et al.,(29) Milani et al., (12) Ogata et al.,(26) Megraud et al.,(31) Seo et al.,(27) Su et al.,(16)
Mourad‐Baars et al., Netherlands (28) (Leiden) Maleknejad et al., Iran (Guilan) (24) Schwarzer et al.,(32) Europe This study
Iran (Tehran)
CLR
MTZ
AMX
TET
CIP
ERY
FUR
95 (95) 33 (54.16)
59 (59) 5 (8.33)
5 (5) 0 (0)
7 (7) -
1 (4.16)
9 (9) 0 (0)
155 (13.9)
0 (0)
0 (0)
51 (4.6)
-
-
112
16 (16) 1 (4.16) 387 (34.7) 2 (9.5)
17 (81)
5 (23.8)
1 (4.8)
6 (28.6)
3 (14.3)
-
2013
77
15 (19.5)
31(40)
8 (10.4)
0 (0)
-
-
0 (0)
2013
311
99 (31.8)
80 (25.7)
1 (0.3)
-
33
6 (18.2)
9 (27.3)
8 (24.2)
11 (33.3)
-
2013
0 (0) 4 (12.1)
2013
17731
3812 (21.5)
16915 (95.4)
18 (0.1)
-
-
-
18 (0.1)
2014
77
5 (6.5)
8 (10.4)
-
-
-
-
-
2015
169
24 (14.3)
96 (57.1)
8 (4.8)
-
-
2015
209
22 (73.3)
23 (71.9)
-
40 (23.8) -
-
-
2017
90
7 (22)
20 (62.5)
17 (53)
8 (25)
5 (15.2)
12 (37.5) 16 (50)
2 (6.1)
64 (38.1) 20 (62.5)
CLR; Clarithromycin; MTZ; Metronidazole, AMX; Amoxicillin, TET; Tetracycline; CIP; Ciprofloxacin; ERY; Erythromycin; FUR; Furazolidone.
S2213-7165(17)30092-9 http://dx.doi.org/doi:10.1016/j.jgar.2017.04.011 JGAR 416
To appear in: Received date: Revised date: Accepted date:
5-2-2017 5-4-2017 11-4-2017
Please cite this article as: Shima Mahmoudi, Setareh Mamishi, Maryam Banar, Sepideh Keshavarz Valian, Abbas Bahador, Mehri Najafi, Fatemeh Farahmand, Babak Pourakbari, Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: high frequency of A2143G point mutation associated with clarithromycin resistance (2010), http://dx.doi.org/10.1016/j.jgar.2017.04.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibiotic susceptibility of Helicobacter pylori strains isolated from Iranian children: high frequency of A2143G point mutation associated with clarithromycin resistance
Shima Mahmoudi1, Setareh Mamishi1,2, Maryam Banar1, Sepideh Keshavarz Valian3, Abbas Bahador4, Mehri Najafi5, Fatemeh Farahmand5, Babak Pourakbari1*
1- Pediatric Infectious Diseases Research Center, Tehran University of Medical Sciences, Tehran, Iran 2-Department of Pediatric Infectious Disease, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 3- School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 4- Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 5-Department of Pediatric Gastroenterology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Corresponding author Babak Pourakbari, PhD No. 62, Dr. Gharib St., Pediatric Infectious Disease Research Center, Children Medical Center Hospital, Tehran,
Iran Tel\fax: +98 21 66428996 E-mail : [email protected] Highlights
The Antibiotic resistance of H. pylori is rising in developing countries In our study, the prevalence of H. pylori infection among children was 35.5% (32 positive cultures out of 90 biopsy specimens). The resistance rate to CLR was 21.8%, which was higher than the resistance rate detected by other studies in Iran The A2143G point mutation was detected in 71% of CLR resistant isolates (5 out of 7 CLR resistant isolates).
Abstract Objective: Increasing antibiotic resistance in Helicobacter pylori is a global concern and is associated with treatment failure. The aim of this study was to evaluate antibiotic resistance of H. pylori to different antibiotics including amoxicillin, erythromycin, ciprofloxacin, furazolidone, tetracycline, metronidazole and clarithromycin and to detection of A2143G mutation in clarithromycin resistant isolates by real-time PCR technique. Methods: Ninety patients with upper gastrointestinal symptoms were enrolled in this study. H. pylori were isolated from 32 specimens and the resistance rate of these strains to amoxicillin, erythromycin, ciprofloxacin, furazolidone, and tetracycline was tested by disc agar diffusion method. The resistance level to metronidazole and clarithromycin was determined by agar dilution method. The presence of A2143G point mutation in clarithromycin resistant isolates was determined using real-time PCR technique.
Results: The resistance rates to amoxicillin, erythromycin, ciprofloxacin, furazolidone, tetracycline, metronidazole and clarithromycin were 53%, 50%, 37.5%, 62.5%, 25%, 62.5% and 22%, respectively. The A2143G point mutation was detected in 71% of clarithromycin resistant strains (5 out of 7). Conclusions: The prevalence of H. pylori resistance to metronidazole, ciprofloxacin, erythromycin, amoxicillin and furazolidone in Iran is high. Determination of antibiotic susceptibility plays an important role in selecting of the appropriate anti H. pylori regimen.
Keywords: Helicobacter pylori, antibiotic resistance, A2143G mutation, Iran. Introduction Helicobacter pylori (H. pylori) is a Gram-negative curved microaerophilic bacterium that can survive and colonize in the acidic condition of gastric mucosa (1, 2). It is one of the most common human bacterial infections (3). It is the main cause of chronic gastritis and is responsible for peptic ulcer disease (PUD), mucosa-associated lymphoid tissue (MALT) lymphoma and gastric adenocarcinoma (4, 5). The prevalence of H. pylori infection in developing countries is more than in developed countries, and studies show that its prevalence in Iran is about 54% (6, 7). Infection of this bacterium usually occurs in childhood (8) and plays a decisive role in infection rates in adults; therefore, evaluation of H. pylori infection in children is important (4) and can help to implement appropriate preventive and therapeutic programs (6). The current therapy regimen for eradication of H. pylori infections consists of one or two antibiotics such as clarithromycin, amoxicillin, or metronidazole in combination with a Proton Pomp Inhibitor (PPI) (2, 9). One of the most important antibiotics in this regimen is clarithromycin and occurrence of point mutations such as the transition of Adenine to Guanine at
position 2143 (A2143G) in the bacterial 23SrRNA gene is responsible for the emergence of clarithromycin resistant isolates (10). The aim of this study was to evaluate antibiotic resistance of H. pylori isolates, and determine A2143G mutation in clarithromycin resistant strains by real-time PCR. Materials and Methods Patients and specimens Between April 2014 and March 2015, one gastric biopsy specimens was taken from 90 patients with upper gastrointestinal symptoms who were referred to children’s medical center hospital, Tehran, Iran. Ethical approval was obtained by Tehran University of Medical Sciences. The patients’ demographic data including name, sex, age, symptoms and treatment history were collected and sampling was accomplished with their consent. One part of the specimen was used for Rapid urease test (RUT) (11) and another section was added into BHI broth (Merck, Germany) and was transferred to the microbiology laboratory within 3 hours. Culture The isolation of H. pylori strains from biopsy specimens were performed according to the method of Milani et al. with some modifications (12). At first, specimens were crushed between two sterile glass slides, then the resulting extracts was cultured on Brucella agar (Merck, Germany) supplemented with 7% defibrinated sheep blood (Jihad daneshgahi of Tehran University, Tehran, Iran), 5% fetal bovine serum (FBS) (Jihad daneshgahi of Tehran University, Tehran, Iran), vancomycin (6mg/L, Sigma), trimethoprim (5 mg/L, Sigma) and amphotricin B (2mg/L, Sigma). Then plates were incubated in the microaerophilic conditions (6% O2, 10% CO2 and 84% N2) using the gas pack incubation method (Merck, Germany) at 37°C for 3-5 days.
Isolates were identified as H. pylori based on colony morphology, gram staining and positive reactions of urease, catalase and oxidase tests. Antibiotic Susceptibility Test Resistance of strains to amoxicillin (AMX), erythromycin (ERY), ciprofloxacin (CIP), furazolidone (FUR) and tetracycline (TET) antibiotics was assessed by using the disc agar diffusion method. Bacterial suspension equivalent to McFarland turbidity standard no. 3 (approximately 1.0×109 CFU/ml) were prepared in sterile normal saline and spread on brucella blood agar plates (without antibiotics) which had been enriched with 5% FBS. After plates dried, antibiotic disks were placed and plates were incubated in microaerophilic atmosphere at 37°C for 72 h, then the zones of inhibition were measured and the sensitive and resistant isolates were determined based on CLSI criteria (13). The minimum inhibitory concentrations (MIC) of the clarithromycin (CLR) and metronidazole (MTZ) antibiotics were determined using agar dilution method (13). Various concentrations of metronidazole (ranging from 2 to 32 mg/ml) and clarithromycin (ranging from 0.125 to 4 mg/ml) were added to brucella agar supplemented with 5% defibrinated sheep blood and 5% FBS. Bacterial suspension with turbidity equivalent to McFarland standard no. 2 was prepared in sterile normal saline and 3 μl of them was inoculated on the plates and MICs were determined after 3 days incubation. A strain was considered as resistant to metronidazole if the MIC value was > 8 mg/ml and MIC > 1 was indicative of resistance to clarithromycin. H. pylori ATCC43504 was used as quality control of the two tests. TaqMan real-time PCR A TaqMan real-time PCR assay was used to detect A2143G point mutation in CLR resistant H. pylori isolates. DNA was extracted from fresh isolates using boiling method (14). An 86-bp
fragment of the 23S rRNA gene of H. pylori was amplified and coupled with detection of the product by probe hybridization (15). The assay was accomplished in a final volume of 20 μl containing: 2 μl of PCR buffer, 0.2 unit of Taq DNA polymerase, 0.5 μl of dNTP , 0.5 μl of MgCl2 , 0.5 μl of each primer, 0.5 μl of probe, 1 μl of template DNA and 14.3 μl of dH 2O. The thermal cycler conditions were a first denaturation cycle at 95°C for 10 min, followed by 40 cycles of 20 s at 95 °C, 20 s at 61°C and extension at 72°C for 20 s. An ABI step one real-time PCR system (Applied Biosystems, Foster City, California, USA) was used and acquired results were evaluated on the basis of the melting curves. Statistical analysis Descriptive statistics were used to summarize all demographic characteristics of the patients. Categorical data were tested using the χ2 test for dependence and a P-value <0.05 considered significant. All statistical analyses were done using statistical packages SPSS 16.0 (SPSS Inc. Chicago,
IL).
Results Ninety children with chronic abdominal pain were included in this study (Table1). Among them, H. pylori were isolated from 32 individuals (35.5%) with average age of 9.4 years. The distribution of female and male within H. pylori positive patients were 17 female (53%) and 15 male (47%) and there was no relationship between H. pylori infection and gender (p>0.5). Thirty- four samples were RUT positive (38%). The antimicrobial resistance of 32 H. pylori isolates to TET, CIP, AMX, ERY and FUR were determined by disc agar diffusion method and results are listed in Table 2. The highest resistance rate was observed against FUR (62.5%) and the lowest resistance rate was seen against TET (25%). The MIC values of MTZ and CLR were determined by agar dilution method. Resistance to CLR was found in 7 isolates (22%) and 20 isolates (62.5%) were resistant to MTZ. seven strains were resistant to both CLR and MTZ (double resistant) . The presence of A2143G point mutation was assessed in CLR resistant isolates by TaqMan Real-time PCR. This mutation was detected in 5 (71%) out of 7 resistant strains. Discussion The Antibiotic resistance of H. pylori is rising in developing countries (9), which can affect the effectiveness of current therapeutic regimens and decrease their eradication rates (16). Given the role of H. pylori infections in developing gastric malignancies, controlling of its infections and monitoring of antibiotic resistance is important (16, 17). In this study, we investigated the resistance of H. pylori isolates to AMX, TET, CIP, ERY, FUR, MTZ and CLR by disk agar diffusion and agar dilution methods.
In our study, the prevalence of H. pylori infection among children was 35.5% , which was similar to the rate reported from China (18). However, it was higher than the prevalence rate in Belgium (19) and lower than those reported in Iran (6, 20), Portugal (21) and Brazil (22) (Table 3). As is evident, the prevalence of H. pylori infection in developing countries is higher than developed countries, which can be due to the use of effective combination therapies, higher level of hygiene and restricted use of antibiotics in these communities (9). The resistance rate to CLR was 22%, which was higher than the resistance rate detected by other studies in Iran (12, 23-25), Brazil (26), South Korea (27) and the Netherlands (28) and was lower than the rate reported from Portugal (29) (Table 4). Several factors may be involved in the emergence of resistance to CLR including extensive use of CLR for the treatment of respiratory infections in children (26), applying monotherapy regimen and cross-reactivity between CLR and ERY (members of macrolides) (30). The overall resistance to MTZ was 62.5% and was lower than the results of previous studies in Iran (12, 25) and China (16). The resistance rate to MTZ was higher than the rates reported by other studies in Iran(23, 24), South Korea (27), Europe (28, 29, 31, 32) and Brazil (26) (Table 4).. It seems that the extensive use of MTZ for the treatment of genital and parasitic infections led to the emergence of MTZ resistance in H. pylori strains (17). Resistance rate to AMX in this study was 53%. This rate was lower than the rates reported by Rafeey et al., in Iran (25). However, the resistance rate to AMX in our study was much higher than the results of some studies conducted in Iran (12, 23, 24), Europe (29, 31), Brazil (26), South Korea (27) and China (16) (Table 4).Frequent use of AMX for the treatment of sick children and consumption of antibiotics without a prescription can be the reason for the high level resistance to AMX observed in our study (26).
The rate of resistance to TET in this study was 25%, which was higher than the results of some reports in Iran (12, 23, 25), Brazil (26) and Europe (31) and lower than the results of one study in Iran (24) and South Korea (27) (Table 4).. TET is not prescribed to Iranian children, especially because of its adverse effects on color and calcification of children tooth (26). Probably emergence of resistance to this antibiotic is related to acquisition of genetic elements containing multidrug resistance genes.
On the other hand, 37.5% of our isolates were resistant to CIP, which was higher than the results of previous studies conducted by Rafeey et al., and Milan et al., from Iran(12, 25). The rate of resistance to CIP in our study was also higher than the rate of resistance in Portuguese and South Korean children (27, 29) (Table 4). These results represent a rising trend of resistance to CIP in Iran. Possibly the widespread use of fluoroquinolones in the treatment of H. pylori infections, is the main cause of increasing levels of resistance to this group. Frequency of ERY resistance in our study was 50%, which was higher than the results of previous studies performed in Iran (12, 23) and South Korea (27). ERY is a macrolide antibiotic and high level resistance to this drug may be related to cross-reactivity with other macrolides. At the present work, the resistance level to FUR was 62.5%, which was higher than those reported by Maleknejad et al., (24), Rafeey et al., (25) and Fallahi et al., (23) in Iran. The resistance level to FUR in other countries such as Brazil, China and South Korae was lower than our study (16, 26, 27) (Table 4). Due to the genotoxicity and carcinogenic effects of FUR, its consumption in Europe and
USA is banned and it is available in some developing countries such as Iran (26) and it is probable that the high resistance observed in our study is due to the excessive intake of this medicine in Iran. According to the results, it can be stated that the antibiotic resistance pattern of H. pylori varies between different geographic areas and depends on the different factors such as population density, age, race and availability of antibiotics (33). CLR belongs to the macrolide family and inhibits protein synthesis in bacteria through binding to the peptidyl transferase of 23SrRNA (10). Occurrence of some point mutations such as A2143G, A2142G and A2142C in bacterial 23SrRNA gene leads to the emergence of CLR resistance in H. pylori (34). In the present study, the A2143G point mutation was detected in 71% of CLR resistant isolates (5 out of 7 CLR resistant isolates). Our report is in consistent with previous studies in which A2143G mutation was detected in 73.68% (35) and 71.4% [34] of CLR resistant isolates. However, in the study conducted by Eghbal et al., (10) and Vala et al., (36) A2143G mutation was detected in 100% of the CLR resistant isolates. These data demonstrated the importance of A2143G point mutation in the development of CLR resistance in H. pylori. Because the presence of other point mutations (A2142G and A2142C) and other resistance mechanisms such as RNA methylation and efflux pumps were not evaluated in this study, the observed difference between phenotypic and genotypic resistance of CLR can be explained. Based on the guidelines proposed in the Conference of Maastricht III (37), the first-line of treatment against H. pylori infections that is used in our hospital is AMX, omeprazole, and CLR for 7 days.
This study has some limitations. First, its sample size (n=90) was small compared to other studies and biopsy specimens were obtained from the admitted patients of one hospital (i.e. CMC hospital). Therefore, the results should be analyzed cautiously when compared with other studies and are not generalizable to the entire city of Tehran. Second, the presence of other point mutations in H. pylori 23srRNA gene (i.e. A2142G and A2142C) that are associated with CLR resistance was not evaluated in this study. The use of Realtime PCR method which is a highly sensitive technique can be the strength point of our survey. Conclusion According to the results of our study, resistance of H. pylori isolates to MTZ, CIP, ERY, FUR and AMX in our study was higher than other countries, which could be the reason for treatment failure in Iran. These results indicate the necessity of determination the antibiotic resistance patterns of H. pylori before prescribing drugs, which can play an important role in preventing the spread of drug resistance in this bacterium and leads to selection of appropriate treatment regimen. A majority of the CLR resistant isolates showed A2143G mutation in 23S rRNA gene that indicates the importance of this mutation.
Declarations Funding: This study was supported by a grant (grant number: 92-01-88-19590) from Tehran University of Medical Sciences to Dr. Babak Pourakbari. Competing Interests: None Ethical Approval: Ethical approval was obtained by Tehran University of Medical Sciences.
References 1. Khademi F, Poursina F, Hosseini E, Akbari M, Safaei HG. Helicobacter pylori in Iran: A systematic review on the antibiotic resistance. Iranian journal of basic medical sciences. 2015;18(1):2. 2. Khodadad A, Farahmand F, Najafi M, Shoaran M. Probiotics for the treatment of pediatric helicobacter pylori infection: a randomized double blind clinical trial. 2013. 3. Pourakbari B, Mirsalehian A, Maleknejad P, Mamishi S, Azhdarkosh H, Daryani NE, et al. Evaluation of a new antigen for diagnosis of Helicobacter pylori infection in stool of adult and children. Helicobacter. 2011;16(1):42-6. 4. Kim N. Prevalence and Transmission Routes of H. pylori. Helicobacter pylori: Springer; 2016. p. 3-19. 5. Sayehmiri F, Kiani F, Sayehmiri K, Soroush S, Asadollahi K, Alikhani MY, et al. Prevalence of cagA and vacA among Helicobacter pyloriinfected patients in Iran: a systematic review and meta-analysis. The Journal of Infection in Developing Countries. 2015;9(07):686-96. 6. Ghasemi-Kebria F, Ghaemi E, Azadfar S, Roshandel G. Epidemiology of Helicobacter pylori infection among Iranian children. Arab Journal of Gastroenterology. 2013;14(4):169-72. 7. Moosazadeh M, Lankarani KB, Afshari M. Meta-analysis of the prevalence of helicobacter pylori infection among children and adults of Iran. International journal of preventive medicine. 2016;7. 8. Mamishi S, Eshaghi H, Mahmoudi S, Bahador A, Hosseinpour Sadeghi R, Najafi M, et al. Intrafamilial transmission of Helicobacter pylori: genotyping of faecal samples. British journal of biomedical science. 2016;73(1):38-43. 9. Shokrzadeh L, Alebouyeh M, Mirzaei T, Farzi N, Zali MR. Prevalence of multiple drug-resistant Helicobacter pylori strains among patients with different gastric disorders in Iran. Microbial Drug Resistance. 2015;21(1):105-10. 10. Eghbali Z, Mojtahedi A, Ansar MM, Asl SF, Aminian K. Detection of 23SrRNA Mutations Strongly Related to Clarithromycin Resistance in Helicobacter pylori Strains Isolated From Patients in the North of Iran. Jundishapur journal of microbiology. 2016;9(2). 11. Pourakbari B, Ghazi M, Mahmoudi S, Mamishi S, Azhdarkosh H, Najafi M, et al. Diagnosis of Helicobacter pylori infection by invasive and noninvasive tests. Brazilian Journal of Microbiology. 2013;44(3):795-8. 12. Milani M, Ghotaslou R, Somi MH, Rafeey M, Akhi MT, Nahaei MR, et al. The status of antimicrobial resistance of Helicobacter pylori in Eastern Azerbaijan, Iran: comparative study according to demographics. Journal of Infection and Chemotherapy. 2012;18(6):848-52. 13. CLSI MfDASTfBTGAasp-. 14. Queipo-Ortuño MI, Colmenero JDD, Macias M, Bravo MJ, Morata P. Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clinical and Vaccine Immunology. 2008;15(2):293-6. 15. De Francesco V, Zullo A, Ierardi E, Giorgio F, Perna F, Hassan C, et al. Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits. Journal of antimicrobial chemotherapy. 2010;65(2):327-32. 16. Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, et al. Antibiotic resistance of Helicobacter pylori isolated in the Southeast Coastal Region of China. Helicobacter. 2013;18(4):274-9.
17. Khademi F, Faghri J, Poursina F, Esfahani BN, Moghim S, Fazeli H, et al. Resistance pattern of Helicobacter pylori strains to clarithromycin, metronidazole, and amoxicillin in Isfahan, Iran. Journal of Research in Medical Sciences. 2013;18(12). 18. Yu Y, Su L, Wang X, Wang X, Xu C. Association between Helicobacter pylori infection and pathological changes in the gastric mucosa in Chinese children. Internal Medicine. 2014;53(2):83-8. 19. Mana F, Vandebosch S, Deyi VM, Haentjens P, Urbain D. Prevalence of and risk factors for H. pylori infection in healthy children and young adults in Belgium anno 2010/2011. Acta Gastro Enterol Belg. 2013;76:381-5. 20. Soltani J, Amirzadeh J, Nahedi S, Shahsavari S. Prevalence of helicobacter pylori infection in children, a population-based cross-sectional study in west iran. 2013. 21. Bastos J, Peleteiro B, Pinto H, Marinho A, Guimarães JT, Ramos E, et al. Prevalence, incidence and risk factors for Helicobacter pylori infection in a cohort of Portuguese adolescents (EpiTeen). Digestive and Liver Disease. 2013;45(4):290-5. 22. Pacheco SL, Ogata SK, Machado RS, da Silva Patrício FR, Pardo ML, Kawakami E. Diagnosis of Helicobacter pylori Infection by Means of Reduced-Dose 13C-Urea Breath Test and Early Sampling of Exhaled Breath. Journal of pediatric gastroenterology and nutrition. 2013;57(5):60711. 23. Fallahi G-H, Maleknejad S. Helicobacter pylori culture and antimicrobial resistance in Iran. The Indian Journal of Pediatrics. 2007;74(2):127. 24. Maleknejad S, Mojtahedi A, Safaei-Asl A, Taghavi Z, Kazemnejad E. Primary Antibiotic Resistance to Helicobacter pylori Strains Isolated From Children in Northern Iran: A Single Center Study. Iranian journal of pediatrics. 2015;25(6):e2661-e. 25. Rafeey M, Ghotaslou R, Nikvash S, Hafez AA. Primary resistance in Helicobacter pylori isolated in children from Iran. Journal of Infection and Chemotherapy. 2007;13(5):291-5. 26. Ogata SK, Godoy APO, da Silva Patricio FR, Kawakami E. High Helicobacter pylori resistance to metronidazole and clarithromycin in Brazilian children and adolescents. Journal of pediatric gastroenterology and nutrition. 2013;56(6):645-8. 27. Seo JH, Jun JS, Yeom JS, Park JS, Youn HS, Ko GH, et al. Changing pattern of antibiotic resistance of Helicobacter pylori in children during 20 years in Jinju, South Korea. Pediatrics International. 2013;55(3):332-6. 28. Mourad‐Baars PE, Wunderink HF, Luisa Mearin M, Veenendaal RA, Wit JM, Veldkamp KE. Low Antibiotic Resistance of Helicobacter pylori in The Netherlands. Helicobacter. 2015;20(1):69-70. 29. Oleastro M, Cabral J, Ramalho PM, Lemos PS, Paixão E, Benoliel J, et al. Primary antibiotic resistance of Helicobacter pylori strains isolated from Portuguese children: a prospective multicentre study over a 10 year period. Journal of antimicrobial chemotherapy. 2011;66(10):2308-11. 30. Golrokhi MM, Fakheri H, Haghshenas MR, Ahanjan M. The Determination of Antibiotic Resistance of Helicobacter Pylori Isolated from Patients Living in North of Iran (Sari). 2016. 31. Megraud F, Coenen S, Versporten A, Kist M, Lopez-Brea M, Hirschl A, et al. 853 Helicobacter pylori Resistance to Antibiotics in Europe and Its Relationship to Antibiotic Consumption. Gastroenterology. 2012;142(5):S-146. 32. Schwarzer A, Bontems P, Urruzuno P, Kalach N, Iwanczak B, Roma‐Giannikou E, et al. Sequential Therapy for Helicobacter pylori Infection in Treatment‐naïve Children. Helicobacter. 2015.
33. Gahbauer AM, Gonzales ML, Guglielmo BJ. Patterns of antibacterial use and impact of age, race/ethnicity, and geographic region on antibacterial use in an outpatient medicaid cohort. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2014;34(7):67785. 34. Liu Q, Qi D, Kang J, Jin Y, Liu W, Gao W, et al. Efficacy of real-time PCR-based detection of Helicobacter pylori infection and genotypic resistance-guided quadruple therapy as the first-line treatment for functional dyspepsia with Helicobacter pylori infection. European journal of gastroenterology & hepatology. 2015;27(3):221-5. 35. Mohammadi M, Doroud D, Mohajerani N, Massarrat S. Helicobacter pylori antibiotic resistance in Iran. World journal of gastroenterology. 2005;11(38):6009. 36. Vala MH, Eyvazi S, Goudarzi H, Sarie HR, Gholami M. Evaluation of Clarithromycin Resistance Among Iranian Helicobacter pylori Isolates by E-Test and Real-Time Polymerase Chain Reaction Methods. Jundishapur journal of microbiology. 2016;9(5). 37. 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(6):772-81.
Table 1. Demographic data of the patients Parameters
N
%
3 to 10 years
50
55.5
10 to 16 years
40
44.5
Female
53
59
male
37
41
90
100
Previous anti-H.pylori treatment (Recurrence)
10
11
Consumption of antibiotics before endoscopy
37
41
H. pylori positive
32
35.5
Age
Gender
Symptoms Abdominal pain History
Table2. Antibiotic susceptibility of the H.pylori isolates by disk diffusion method Isolates, N (%) Antimicrobial agent Tetracycline (TET)
Susceptible 24 (75)
Resistant 8 (25)
Ciprofloxacin (CIP)
20 (62.5)
12 (37.5)
Amoxicillin (AMX)
15 (46.9)
17 (53)
Erythromycin (ERY)
16 (50)
16 (50)
Furazolidone (FUR)
12 (37.5)
20 (62.5)
Table 3. Prevalence rate of H. pylori infection in different countries
2012 2013 2013 2013
Sample size 21160 194 294 516
Infection rate (%) 66.20 50.50 64.20 11
2013
129
41.10
2014 2017
17731 90
32.10 35.5
Authors (reference)
Country (city)
Year
Bastos et al., )12( Ghasemi-Kebria et al., (6) Soltani et al., (20) Mana et al., (19)
Portugal (Porto) Iran (Gorgan) Iran (Sanandaj) Belgium(Brussels) Brazil (Sa˜o Paulo) China (Beijing) Iran (Tehran)
Pacheco et al., (22) Yu et al., (18) This study
Table 4. Resistance rates of H. pylori in different countries Resistance rate N (%) Authors (reference)
Country (city)
Year
Sample size
Rafeey et al., (25) Fallahi et al., (23)
Iran (Tabriz) Iran (Tehran) Portugal (Lisboa) Iran (Tabriz) Brazil (Sa˜o Paulo) Europe South Korea (Jinju)
2007 2007
100 62
2011
1115
2012
China (Beijing)
Oleastro et al.,(29) Milani et al., (12) Ogata et al.,(26) Megraud et al.,(31) Seo et al.,(27) Su et al.,(16)
Mourad‐Baars et al., Netherlands (28) (Leiden) Maleknejad et al., Iran (Guilan) (24) Schwarzer et al.,(32) Europe This study
Iran (Tehran)
CLR
MTZ
AMX
TET
CIP
ERY
FUR
95 (95) 33 (54.16)
59 (59) 5 (8.33)
5 (5) 0 (0)
7 (7) -
1 (4.16)
9 (9) 0 (0)
155 (13.9)
0 (0)
0 (0)
51 (4.6)
-
-
112
16 (16) 1 (4.16) 387 (34.7) 2 (9.5)
17 (81)
5 (23.8)
1 (4.8)
6 (28.6)
3 (14.3)
-
2013
77
15 (19.5)
31(40)
8 (10.4)
0 (0)
-
-
0 (0)
2013
311
99 (31.8)
80 (25.7)
1 (0.3)
-
33
6 (18.2)
9 (27.3)
8 (24.2)
11 (33.3)
-
2013
0 (0) 4 (12.1)
2013
17731
3812 (21.5)
16915 (95.4)
18 (0.1)
-
-
-
18 (0.1)
2014
77
5 (6.5)
8 (10.4)
-
-
-
-
-
2015
169
24 (14.3)
96 (57.1)
8 (4.8)
-
-
2015
209
22 (73.3)
23 (71.9)
-
40 (23.8) -
-
-
2017
90
7 (22)
20 (62.5)
17 (53)
8 (25)
5 (15.2)
12 (37.5) 16 (50)
2 (6.1)
64 (38.1) 20 (62.5)
CLR; Clarithromycin; MTZ; Metronidazole, AMX; Amoxicillin, TET; Tetracycline; CIP; Ciprofloxacin; ERY; Erythromycin; FUR; Furazolidone.