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Alimentary Tract
Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients Zhiqiang Song a,1 , Jianzhong Zhang b,1 , Lihua He b , Minhu Chen c , Xiaohua Hou d , Zhaoshen Li e , Liya Zhou a,∗ a
Department of Gastroenterology, Peking University Third Hospital, Beijing, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China c Department of Gastroenterology, First Affiliated Hospital of SunYat-sen University, Guangzhou, China d Division of Gastroenterology, Union Hospital of Tongji Medical College, Huazhong University of Technology and Science, Wuhan, China e Department of Digestive Diseases, Changhai Hospital of Second Military Medical University, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Accepted 19 August 2014 Available online xxx Keywords: Antibiotic resistance China Helicobacter pylori
a b s t r a c t Background: Large-scale multi-region studies are urgently needed to provide comprehensive and up-todate information on the antibiotic resistance of Helicobacter pylori that is critical for selecting the most optimal eradication regimens. Aims: To determine the resistance patterns of Helicobacter pylori strains isolated from dyspeptic patients. Methods: This is a prospective, multicentre, cross-sectional, observational study. Helicobacter pylori cultures were successful in 600 patients (never receiving eradication therapy) from Northern, Eastern, Middle, and Southern regions between 2008 and 2012. Resistance to amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifampicin was determined by Epsilometer test. Results: The overall resistance rate was highest for metronidazole (403, 67.2%), followed by clarithromycin (225, 37.5%), levofloxacin (201, 33.5%), rifampicin (85, 14.2%), amoxicillin (41, 6.8%), and tetracycline (21, 3.5%). There were 16.3% isolates susceptible to all tested antibiotics, followed by mono-resistance (34.2%), double resistance (27.0%), triple resistance (16.8%), quadruple resistance (4.7%), quintuple resistance (0.7%) and sextuple resistance (0.3%). Independent factors influencing antibiotic resistance were gender (to levofloxacin), age (to levofloxacin), and endoscopic finding (to clarithromycin, metronidazole, and levofloxacin). Among the clarithromycin-resistant isolates, 75.6% and 48.0% were also resistant to metronidazole and levofloxacin, respectively. Conclusions: Helicobacter pylori resistance to commonly used antibiotics in China is a very serious issue, due to the high resistance rate and general multiple resistance. © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Although Helicobacter pylori (H. pylori) has been studied for about 30 years, eradication of H. pylori infection remains a challenge, mainly because of the increasing prevalence of H. pylori resistance to antibiotics [1,2]. Therefore, in order to achieve better long-term efficacy, a suitable therapeutic regimen for H. pylori infection should be based on the specific conditions of
∗ Corresponding author at: Department of Gastroenterology, Peking University Third Hospital, Beijing 100191, China. Tel.: +86 18910192576; fax: +86 10 62357303. E-mail address: [email protected] (L. Zhou). 1 These two authors contributed equally to this work.
patients, especially considering data on H. pylori antibiotic resistance in different geographical regions. In China, for example, there is a high prevalence of H. pylori infection and gastric cancer [3,4]. To date, the majority of the studies on H. pylori antibiotic resistance in Chinese patients have focused on patients from a single region, and few studies have been conducted in multiple regions. Moreover, most studies have investigated only few types of antibiotics, and more recent data on this topic are still lacking [5]. Therefore, further large-scale multi-region studies are urgently needed to provide comprehensive and up-to-date information on the antibiotic resistance of H. pylori that is critical to the selection of the most optimal therapeutic regimens for the eradication of H. pylori infection.
http://dx.doi.org/10.1016/j.dld.2014.08.038 1590-8658/© 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Song Z, et al. Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.08.038
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The aim of this study was to determine the resistance patterns of H. pylori strains isolated from dyspeptic patients in multiple regions of China.
oxidase activity. The organisms were identified as H. pylori if the isolates demonstrated curved Gram-negative rods that were similar to Helicobacter, along with positive urease, catalase, and oxidase reactions.
2. Methods 2.5. Antibiotic susceptibility testing This is a prospective, multicentre, cross-sectional, observational study. 2.1. Patients and selection criteria From March 2008 to February 2012, we recruited patients undergoing upper endoscopy due to dyspeptic symptoms in four clinical research centres from the Northern (Beijing), Eastern (Shanghai), Middle (Wuhan), and Southern (Guangzhou) regions of China. Patients were included if they were between 18 and 75 years old and had not received treatment for eradication of H. pylori infection prior to study entry. Patients with any of the following criteria were excluded from the study: (1) patients who had received proton pump inhibitors, H2 receptor antagonists, bismuth salts, or antibiotics during the four weeks prior to study entry; (2) patients with malignant tumours of the digestive system; (3) patients who received gastrointestinal tract surgery prior to inclusion; (4) patients who were complicated by severe heart, lung, blood, liver, or kidney dysfunction; and (5) patients who would not comply with study procedures. 2.2. Ethical consideration The study was approved by the independent Ethics Committee of the Peking University Health Science Centre (IRB000010520709), and was carried out in accordance with the ethical guideline of the 1975 Declaration of Helsinki (6th revision, 2008) and Good Clinical Practice. Written informed consent was obtained from each patient. 2.3. Study methods All patients were enrolled in the study by clinical gastroenterologists according to the established inclusion and exclusion criteria. After obtaining the patient’s general information, one gastric mucosal biopsy specimen from the antrum was collected during upper endoscopy for testing the presence of H. pylori by rapid urease test (HPUT-H102, San Qiang Biological and Chemical Co., Fujian, China). If the patient’s test was positive, two mucosal biopsy specimens (one from antrum and one from corpus) were collected and put into the same vial for culture of H. pylori. Two additional specimens (one from antrum and one from corpus) were also obtained for histological haematoxylin and eosin staining, and gastritis was evaluated using the updated Sydney system [6].
The antibiotic resistance of H. pylori isolates was determined with the Epsilometer test (E-test) [7]. According to the clinical breakpoints for H. pylori proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [8], the resistance breakpoints for amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifampicin were >0.12 mg/L, >0.5 mg/L, >8 mg/L, >1 mg/L, >1 mg/L, and >1 mg/L, respectively. Susceptibility testing for amoxicillin was performed again after the H. pylori isolates had been stored at −80 ◦ C for two weeks. All the cultures and tests were conducted at the National Institute for Communicable Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. 2.6. Statistical analysis All statistical analyses were performed using the statistical software SPSS, version 18.0 (SPSS Inc., Chicago, IL, USA). Frequencies and percentages were used to describe the antimicrobial resistance of H. pylori isolates. Univariate analyses were performed to identify the factors that could influence the antibiotic resistance of H. pylori isolates. The Chi-square test was used to compare the resistance rates of patients of different genders (males vs. females), age groups (18–35 vs. 36–50 vs. 51–75 years), regions (Northern vs. Eastern vs. Middle vs. Southern), and endoscopic findings (peptic ulcer disease vs. non-ulcer disease). Patients with duodenal and/or gastric ulcer were considered as having peptic ulcer disease; those without ulcers were considered as non-ulcer dyspeptic patients. Thereafter, binary logistic regression was used to analyse the relationships between independent variables (gender, age group, region, and endoscopic finding) and dependent variables (antibiotic susceptibility or resistance) with backward likelihood ratio analysis, and thus to determine the independent factors influencing the antibiotic resistance of H. pylori. The odds ratio (OR) and 95% confidence interval (CI) of different variables related to antibiotic resistance were calculated. Differences with P-values of less than 0.05 from a 2-tailed test were considered statistically significant. 3. Results Nine hundred and twenty-eight patients were H. pylori-positive at rapid urease test, and bacterial cultures were successfully obtained for 600 of these patients (64.7%). The general characteristics as well as the endoscopic and histological findings of the study population are shown in Table 1.
2.4. Isolation and identification of H. pylori 3.1. Determination of antibiotic resistance Briefly, gastric mucosal biopsy specimens were stored in brain heart infusion broth (Oxoid, Basingstoke, UK) with 5% glycerin at −80 ◦ C and brought in dry ice to the laboratory at the National Institute for Communicable Disease Control and Prevention, China. Frozen samples were thawed at room temperature for 30 min, then ground and cultivated on Columbia Agar plates (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated sheep blood, 3 g/mL trimethoprim, 2.5 g/mL vancomycin, 2 g/mL amphotericin B, and 2 g/mL polymyxin B. The cultured plates were incubated in a micro-aerobic atmosphere (5% O2 , 10% CO2 , and 85% N2 ). After culture, the translucent colonies of about 0.5–2 mm from selective agar plates were Gram-stained and tested for urease, catalase, and
The overall resistance rate of 600 H. pylori isolates to all tested antibiotics was highest for metronidazole (403, 67.2%), followed by clarithromycin (225, 37.5%), levofloxacin (201, 33.5%), rifampicin (85, 14.2%), amoxicillin (41, 6.8%), and tetracycline (21, 3.5%). 3.2. Multiple antibiotic resistance In the 600 H. pylori isolates investigated, 16.3% were susceptible to all tested antibiotics. The resistance patterns were recorded as follows: mono-resistance (34.2%), double resistance (27.0%), triple resistance (16.8%), quadruple resistance (4.7%), quintuple
Please cite this article in press as: Song Z, et al. Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.08.038
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Values
Male gender Mean age (years) Regions, n (%a ) Northern (Beijing) Eastern (Shanghai) Middle (Wuhan) Southern (Guangzhou) Endoscopic findings, n Peptic ulcer Gastric ulcer Duodenal ulcer Gastric and duodenal ulcer Gastric erosions No macroscopic abnormality Histological findings, n Non-atrophic gastritis Atrophic gastritis Intestinal metaplasia
290 (48.3%) 42.5 ± 13.2
a
283 (77.1%) 102 (52.8%) 105 (56.1%) 110 (60.8%) 95 18 62 15 125 380 408 192 178
The rate of positive culture.
resistance (0.7%), and sextuple resistance (0.3%). The multiple resistance patterns are shown in Table 2. In the clarithromycin-resistant isolates, 75.6% and 48.0% of the isolates were also resistant to metronidazole and levofloxacin, respectively. Among the metronidazole-resistant isolates, levofloxacin-resistant isolates were detected in 37.5% of cases.
Table 2 Multiple resistance patterns in the 600 Helicobacter pylori isolates. Multiple resistance patterns Double resistance CLA + MTZ MTZ + LEV CLA + LEV MTZ + RIF AMX + MTZ CLA + RIF MTZ + TET AMX + CLA AMX + RIF CLA + TET Triple resistance CLA + MTZ + LEV CLA + MTZ + RIF MTZ + LEV + RIF AMX + CLA + MTZ CLA + MTZ + TET AMX + MTZ + LEV AMX + MTZ + RIF CLA + LEV + RIF AMX + CLA + LEV CLA + LEV + TET MTZ + LEV + TET Quadruple resistance CLA + MTZ + LEV + RIF AMX + CLA + MTZ + LEV CLA + MTZ + LEV + TET AMX + CLA + MTZ + RIF AMX + MTZ + LEV + TET AMX + MTZ + LEV + RIF Quintuple resistance AMX + CLA + MTZ + LEV + TET AMX + CLA + MTZ + LEV + RIF AMX + MTZ + LEV + TET + RIF CLA + MTZ + LEV + TET + RIF Sextuple resistance
No. of isolates
Resistance rate (%)
60 49 19 19 5 3 3 2 1 1
10.0 8.2 3.2 3.2 0.8 0.5 0.5 0.3 0.2 0.2
55 13 8 6 5 5 4 2 1 1 1
9.2 2.2 1.3 1.0 0.8 0.8 0.7 0.3 0.2 0.2 0.2
16 6 3 1 1 1
2.7 1.0 0.5 0.2 0.2 0.2
1 1 1 1 2
0.2 0.2 0.2 0.2 0.3
AMX, amoxicillin; CLA, clarithromycin; MTZ, metronidazole; LEV, levofloxacin; TET tetracycline; RIF, rifampicin.
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3.3. Factors influencing antibiotic resistance Univariate analysis showed that there were significant differences in the resistance rates to clarithromycin, metronidazole, and levofloxacin between patients with peptic ulcer disease and nonulcer disease. Moreover, significant differences in the levofloxacin resistance rates were also observed between men and women, as well as among different age groups. Multivariate analysis demonstrated that the above-mentioned factors were independent factors influencing the antibiotic resistance of H. pylori (Table 3).
4. Discussion The results from the present study show that the treatment of H. pylori infection remains a challenge in China due to the high rates of H. pylori resistance to commonly used antibiotics. Furthermore, multiple antibiotic resistance patterns were also commonly observed in the H. pylori isolates investigated. In the present study, resistance to clarithromycin was identified in 37.5% of H. pylori isolates. According to the Maastricht IV/Florence consensus report [2], clarithromycin-based standard triple therapy is unsuitable for eradication of H. pylori in China, unless susceptibility testing for H. pylori is performed before treatment to confirm the lack of resistance to this specific antibiotic. Some studies reported that sequential and concomitant therapies are effective treatment options for patients who show clarithromycin resistance. Thus, these therapies can be recommended for patients from those regions with high clarithromycin resistance rates [2,9–12]. Unfortunately, meta-analyses also showed that dual resistance to clarithromycin and metronidazole could reduce the ability of sequential and concomitant therapies to eradicate H. pylori [13,14], indicating that these therapies could only be used in regions with high rates of isolated clarithromycin resistance. Our data showed a high rate of resistance to metronidazole (75.6%) in clarithromycin-resistant isolates. Based on these results, it is reasonable to speculate that sequential and concomitant therapies are not suitable for the management of H. pylori infection in China. In recent years, due to the high frequency of resistance to clarithromycin and metronidazole, levofloxacin has been extensively studied in patients with H. pylori infection. Moreover, levofloxacin has been proven to be effective in eradicating H. pylori in some trials [15–17]. Nevertheless, these studies were mainly conducted in regions in which levofloxacin resistance was low. In our study, we found that the resistance rate of H. pylori isolates to levofloxacin was as high as 33.5%, with a high level of combined resistance to clarithromycin and metronidazole, indicating that a levofloxacincontaining therapy may not be a good choice for the initial empiric treatment of H. pylori in China. In the current study, the H. pylori isolates were found to be susceptible to tetracycline with an overall resistance rate of 3.5%, which suggested that tetracycline could be used in the initial treatment of H. pylori infections in China. Additionally, it is generally thought that the increased dosage, and dosing frequency, of metronidazole can reduce high-level resistance to metronidazole [1,18]. Currently bismuth salts are widely used in clinical practice since they are easily obtained in China. Therefore, the classic quadruple therapy, which contains metronidazole, tetracycline, bismuth, and proton pump inhibitor, can be suggested as the best empirical first-line regimen for H. pylori eradication in China, a concept that is consistent with the recommendations of the Maastricht IV/Florence consensus report [2]. Unfortunately, tetracycline is not generally available in China, and this has affected its clinical application. Although semi-synthetic tetracycline derivatives, including minocycline, are easily obtainable in the clinic, it is still uncertain
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Table 3 The influence factors of antibiotic resistance of Helicobacter pylori isolates. Resistance rate (%)
Region Northern (n = 283), Eastern (n = 102), Middle (n = 105), Southern (n = 110)
Gender Male (n = 290), Female (n = 310)
Age (years) 18–35 (n = 189), 36–50 (n = 227), 51–75 (n = 184)
Endoscopic finding PUD (n = 95), NUD (n = 505)
Amoxicillin Clarithromycin
6.4, 7.8, 5.7, 8.2 38.2, 39.2, 36.2, 35.5
7.9, 5.8 34.8, 40.0
7.9, 5.7, 7.1 38.6, 40.1, 33.2
Metronidazole
69.3, 64.7, 66.7, 64.5
63.4, 70.6
3.2, 7.5 22.1, 40.4* (OR 2.316; 95%CI 1.374–3.904) 56.8, 69.1* (1.624; 1.031–2.558) 17.9, 36.4* (2.516; 1.431–4.424) 2.1, 3.8 10.5, 14.9
Levofloxacin
33.9, 35.3, 31.4, 32.7
Tetracycline Rifampicin
3.5, 3.9, 2.9, 3.6 13.1, 14.7, 16.2, 14.5
64.0, 70.5, 66.3 *
24.3, 37.9, 37.5* (1.378; 1.103–1.722) 3.2, 1.8, 6.0 13.8, 14.1, 16.7
27.9, 38.7 (1.462; 1.028–2.080) 3.8, 3.2 13.8, 14.5
PUD, peptic ulcer disease; NUD, non-ulcer disease; OR, odds ratio; CI, confidence interval. * P < 0.05.
whether these derivatives can be used as alternatives to tetracycline for the treatment of H. pylori infection. In this study, the rate of resistance to amoxicillin was relatively low, suggesting that amoxicillin should be fully used in the eradication of H. pylori infection. With the general increase in the rate of resistance to commonly used antibiotics, dual therapy (proton pump inhibitor plus amoxicillin) has drawn attention again, but the study results are not consistent and conclusive [19,20]. Shirai et al. have shown a very good eradication efficacy in their study, but rabeprazole and amoxicillin should be administered four times a day [19]. So far no studies on this regimen have been conducted in China. Rifampicin is rarely used for the treatment of H. pylori infection In China. In the present study, the rate of resistance to this drug (14.2%) in the H. pylori isolates analysed may be due to its frequent use in the treatment of tuberculosis in China, where a high prevalence of tuberculosis infection is observed. Our results show that the resistance rates of H. pylori isolates were significantly higher in the patients with non-ulcer disease than with peptic ulcer disease, consistently with the results of previous studies [21,22]. Additionally, levofloxacin resistance was more frequent in women than men, and in middle-aged and elderly patients than younger patients. These findings may be related to the more frequent use of levofloxacin in these groups of patients, especially in women with gynaecological diseases, as antimicrobial resistance is dependent on antimicrobial use. In Western countries the commercialization of quinolones such as levofloxacin is more recent than that of clarithromycin, and levofloxacin has a more limited use as an antibiotic. Therefore, in those areas, the resistance rate of H. pylori to levofloxacin is relatively low [2]. However, in China, over the past 30 years, quinolones have been widely used as non-prescription drugs even for animal husbandry and aquaculture, thus resulting in such a high resistance rate (33.5%). Recently, restrictions in the use of levofloxacin have been demanded. In the present study, the resistance rate to metronidazole in females was higher than in males (70.6% vs. 63.4%), despite not being statistically significant. This finding is in disagreement with the literature [23], which reports higher rates in females due to a large use of imidazole for gynaecological diseases. The similar resistance rates obtained in our study may be related to the general use of metronidazole in China, in males as well as females, for a variety of oral and gastrointestinal diseases, a feature that probably reduces the significance of this difference. In our study, the antimicrobial susceptibility of the H. pylori isolates was determined with the E-test, primarily because this method can be easily performed, especially in a multicentre clinical study such as ours. Despite some previous studies suggested that the rate of metronidazole resistance may be overestimated by the E-test [24], these results were not consistent or conclusive. A high resistance rate to metronidazole was also found in a recent
study, conducted in the Southeast coastal region of China, with the reference agar dilution method [25]. The requirements for the culture of H. pylori are relatively high. The success rate of a culture is usually between 60% and 90% [23]. The overall success rate of culture in this study was relatively low (64.7%), and may be mainly related to the need of transferring the gastric specimens. In order to unify and standardise the culture, we sent all the specimens to the National Institute for Communicable Disease Control and Prevention, in Beijing, for carrying out the culture and sensitivity testing. Because the Chinese territory covers a vast area, the distances from Beijing to Shanghai, Wuhan, and Guangzhou are of about 1200 km, 1200 km, and 2200 km, respectively. Although we used air transport in dry ice and with a multi-layer insulation box, at least 24–48 h were needed for specimen transfer, and these conditions may be detrimental to the survival of some fragile strains of H. pylori. This also explains why the positive culture rate of the centre located in Beijing (only two hours by car needed for specimen transfer) was relatively higher (77.1%) than those of the other three centres outside Beijing. In this study, two gastric mucosal biopsy specimens (one from antrum and one from corpus) were collected and put into the same vial for the culture of H. pylori. Due to restrictions in research funding and study conditions, we did not carry out the culture of H. pylori in the mucosal specimens of antrum and corpus separately. Thus, we could not analyse the differences in the antimicrobial resistance profiles of H. pylori according to the localisation; thus, the antimicrobial resistance rates may be underestimated, representing a limitation of this study. In conclusion, we carried out a prospective, multi-region study with a large sample size to determine the resistance patterns of H. pylori isolates collected from dyspeptic patients in China. Our results showed that H. pylori resistance to commonly used antibiotics in China is a very serious issue due to the high resistance rate and universal multiple resistance. This comprehensive and up-todate information on H. pylori resistance will be very helpful to select the most optimal eradication regimens in China as well as in other regions with a high prevalence of antibiotic resistance. With the current rates of resistance, the priority would be obtaining new drugs and/or improved methods for the detection of resistance. The use of a more accessible and easier method to obtain H. pylori has already been suggested [26], and progresses in the molecular detection of resistance from faecal samples [27] also represent a very promising line of research. Conflict of interest None declared. References [1] Graham DY, Fischbach L. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut 2010;59:1143–53.
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Alimentary Tract
Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients Zhiqiang Song a,1 , Jianzhong Zhang b,1 , Lihua He b , Minhu Chen c , Xiaohua Hou d , Zhaoshen Li e , Liya Zhou a,∗ a
Department of Gastroenterology, Peking University Third Hospital, Beijing, China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China c Department of Gastroenterology, First Affiliated Hospital of SunYat-sen University, Guangzhou, China d Division of Gastroenterology, Union Hospital of Tongji Medical College, Huazhong University of Technology and Science, Wuhan, China e Department of Digestive Diseases, Changhai Hospital of Second Military Medical University, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 4 June 2014 Accepted 19 August 2014 Available online xxx Keywords: Antibiotic resistance China Helicobacter pylori
a b s t r a c t Background: Large-scale multi-region studies are urgently needed to provide comprehensive and up-todate information on the antibiotic resistance of Helicobacter pylori that is critical for selecting the most optimal eradication regimens. Aims: To determine the resistance patterns of Helicobacter pylori strains isolated from dyspeptic patients. Methods: This is a prospective, multicentre, cross-sectional, observational study. Helicobacter pylori cultures were successful in 600 patients (never receiving eradication therapy) from Northern, Eastern, Middle, and Southern regions between 2008 and 2012. Resistance to amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifampicin was determined by Epsilometer test. Results: The overall resistance rate was highest for metronidazole (403, 67.2%), followed by clarithromycin (225, 37.5%), levofloxacin (201, 33.5%), rifampicin (85, 14.2%), amoxicillin (41, 6.8%), and tetracycline (21, 3.5%). There were 16.3% isolates susceptible to all tested antibiotics, followed by mono-resistance (34.2%), double resistance (27.0%), triple resistance (16.8%), quadruple resistance (4.7%), quintuple resistance (0.7%) and sextuple resistance (0.3%). Independent factors influencing antibiotic resistance were gender (to levofloxacin), age (to levofloxacin), and endoscopic finding (to clarithromycin, metronidazole, and levofloxacin). Among the clarithromycin-resistant isolates, 75.6% and 48.0% were also resistant to metronidazole and levofloxacin, respectively. Conclusions: Helicobacter pylori resistance to commonly used antibiotics in China is a very serious issue, due to the high resistance rate and general multiple resistance. © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Although Helicobacter pylori (H. pylori) has been studied for about 30 years, eradication of H. pylori infection remains a challenge, mainly because of the increasing prevalence of H. pylori resistance to antibiotics [1,2]. Therefore, in order to achieve better long-term efficacy, a suitable therapeutic regimen for H. pylori infection should be based on the specific conditions of
∗ Corresponding author at: Department of Gastroenterology, Peking University Third Hospital, Beijing 100191, China. Tel.: +86 18910192576; fax: +86 10 62357303. E-mail address: [email protected] (L. Zhou). 1 These two authors contributed equally to this work.
patients, especially considering data on H. pylori antibiotic resistance in different geographical regions. In China, for example, there is a high prevalence of H. pylori infection and gastric cancer [3,4]. To date, the majority of the studies on H. pylori antibiotic resistance in Chinese patients have focused on patients from a single region, and few studies have been conducted in multiple regions. Moreover, most studies have investigated only few types of antibiotics, and more recent data on this topic are still lacking [5]. Therefore, further large-scale multi-region studies are urgently needed to provide comprehensive and up-to-date information on the antibiotic resistance of H. pylori that is critical to the selection of the most optimal therapeutic regimens for the eradication of H. pylori infection.
http://dx.doi.org/10.1016/j.dld.2014.08.038 1590-8658/© 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Song Z, et al. Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.08.038
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The aim of this study was to determine the resistance patterns of H. pylori strains isolated from dyspeptic patients in multiple regions of China.
oxidase activity. The organisms were identified as H. pylori if the isolates demonstrated curved Gram-negative rods that were similar to Helicobacter, along with positive urease, catalase, and oxidase reactions.
2. Methods 2.5. Antibiotic susceptibility testing This is a prospective, multicentre, cross-sectional, observational study. 2.1. Patients and selection criteria From March 2008 to February 2012, we recruited patients undergoing upper endoscopy due to dyspeptic symptoms in four clinical research centres from the Northern (Beijing), Eastern (Shanghai), Middle (Wuhan), and Southern (Guangzhou) regions of China. Patients were included if they were between 18 and 75 years old and had not received treatment for eradication of H. pylori infection prior to study entry. Patients with any of the following criteria were excluded from the study: (1) patients who had received proton pump inhibitors, H2 receptor antagonists, bismuth salts, or antibiotics during the four weeks prior to study entry; (2) patients with malignant tumours of the digestive system; (3) patients who received gastrointestinal tract surgery prior to inclusion; (4) patients who were complicated by severe heart, lung, blood, liver, or kidney dysfunction; and (5) patients who would not comply with study procedures. 2.2. Ethical consideration The study was approved by the independent Ethics Committee of the Peking University Health Science Centre (IRB000010520709), and was carried out in accordance with the ethical guideline of the 1975 Declaration of Helsinki (6th revision, 2008) and Good Clinical Practice. Written informed consent was obtained from each patient. 2.3. Study methods All patients were enrolled in the study by clinical gastroenterologists according to the established inclusion and exclusion criteria. After obtaining the patient’s general information, one gastric mucosal biopsy specimen from the antrum was collected during upper endoscopy for testing the presence of H. pylori by rapid urease test (HPUT-H102, San Qiang Biological and Chemical Co., Fujian, China). If the patient’s test was positive, two mucosal biopsy specimens (one from antrum and one from corpus) were collected and put into the same vial for culture of H. pylori. Two additional specimens (one from antrum and one from corpus) were also obtained for histological haematoxylin and eosin staining, and gastritis was evaluated using the updated Sydney system [6].
The antibiotic resistance of H. pylori isolates was determined with the Epsilometer test (E-test) [7]. According to the clinical breakpoints for H. pylori proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [8], the resistance breakpoints for amoxicillin, clarithromycin, metronidazole, levofloxacin, tetracycline, and rifampicin were >0.12 mg/L, >0.5 mg/L, >8 mg/L, >1 mg/L, >1 mg/L, and >1 mg/L, respectively. Susceptibility testing for amoxicillin was performed again after the H. pylori isolates had been stored at −80 ◦ C for two weeks. All the cultures and tests were conducted at the National Institute for Communicable Disease Control and Prevention of the Chinese Center for Disease Control and Prevention. 2.6. Statistical analysis All statistical analyses were performed using the statistical software SPSS, version 18.0 (SPSS Inc., Chicago, IL, USA). Frequencies and percentages were used to describe the antimicrobial resistance of H. pylori isolates. Univariate analyses were performed to identify the factors that could influence the antibiotic resistance of H. pylori isolates. The Chi-square test was used to compare the resistance rates of patients of different genders (males vs. females), age groups (18–35 vs. 36–50 vs. 51–75 years), regions (Northern vs. Eastern vs. Middle vs. Southern), and endoscopic findings (peptic ulcer disease vs. non-ulcer disease). Patients with duodenal and/or gastric ulcer were considered as having peptic ulcer disease; those without ulcers were considered as non-ulcer dyspeptic patients. Thereafter, binary logistic regression was used to analyse the relationships between independent variables (gender, age group, region, and endoscopic finding) and dependent variables (antibiotic susceptibility or resistance) with backward likelihood ratio analysis, and thus to determine the independent factors influencing the antibiotic resistance of H. pylori. The odds ratio (OR) and 95% confidence interval (CI) of different variables related to antibiotic resistance were calculated. Differences with P-values of less than 0.05 from a 2-tailed test were considered statistically significant. 3. Results Nine hundred and twenty-eight patients were H. pylori-positive at rapid urease test, and bacterial cultures were successfully obtained for 600 of these patients (64.7%). The general characteristics as well as the endoscopic and histological findings of the study population are shown in Table 1.
2.4. Isolation and identification of H. pylori 3.1. Determination of antibiotic resistance Briefly, gastric mucosal biopsy specimens were stored in brain heart infusion broth (Oxoid, Basingstoke, UK) with 5% glycerin at −80 ◦ C and brought in dry ice to the laboratory at the National Institute for Communicable Disease Control and Prevention, China. Frozen samples were thawed at room temperature for 30 min, then ground and cultivated on Columbia Agar plates (Oxoid, Basingstoke, UK) supplemented with 5% defibrinated sheep blood, 3 g/mL trimethoprim, 2.5 g/mL vancomycin, 2 g/mL amphotericin B, and 2 g/mL polymyxin B. The cultured plates were incubated in a micro-aerobic atmosphere (5% O2 , 10% CO2 , and 85% N2 ). After culture, the translucent colonies of about 0.5–2 mm from selective agar plates were Gram-stained and tested for urease, catalase, and
The overall resistance rate of 600 H. pylori isolates to all tested antibiotics was highest for metronidazole (403, 67.2%), followed by clarithromycin (225, 37.5%), levofloxacin (201, 33.5%), rifampicin (85, 14.2%), amoxicillin (41, 6.8%), and tetracycline (21, 3.5%). 3.2. Multiple antibiotic resistance In the 600 H. pylori isolates investigated, 16.3% were susceptible to all tested antibiotics. The resistance patterns were recorded as follows: mono-resistance (34.2%), double resistance (27.0%), triple resistance (16.8%), quadruple resistance (4.7%), quintuple
Please cite this article in press as: Song Z, et al. Prospective multi-region study on primary antibiotic resistance of Helicobacter pylori strains isolated from Chinese patients. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.08.038
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Values
Male gender Mean age (years) Regions, n (%a ) Northern (Beijing) Eastern (Shanghai) Middle (Wuhan) Southern (Guangzhou) Endoscopic findings, n Peptic ulcer Gastric ulcer Duodenal ulcer Gastric and duodenal ulcer Gastric erosions No macroscopic abnormality Histological findings, n Non-atrophic gastritis Atrophic gastritis Intestinal metaplasia
290 (48.3%) 42.5 ± 13.2
a
283 (77.1%) 102 (52.8%) 105 (56.1%) 110 (60.8%) 95 18 62 15 125 380 408 192 178
The rate of positive culture.
resistance (0.7%), and sextuple resistance (0.3%). The multiple resistance patterns are shown in Table 2. In the clarithromycin-resistant isolates, 75.6% and 48.0% of the isolates were also resistant to metronidazole and levofloxacin, respectively. Among the metronidazole-resistant isolates, levofloxacin-resistant isolates were detected in 37.5% of cases.
Table 2 Multiple resistance patterns in the 600 Helicobacter pylori isolates. Multiple resistance patterns Double resistance CLA + MTZ MTZ + LEV CLA + LEV MTZ + RIF AMX + MTZ CLA + RIF MTZ + TET AMX + CLA AMX + RIF CLA + TET Triple resistance CLA + MTZ + LEV CLA + MTZ + RIF MTZ + LEV + RIF AMX + CLA + MTZ CLA + MTZ + TET AMX + MTZ + LEV AMX + MTZ + RIF CLA + LEV + RIF AMX + CLA + LEV CLA + LEV + TET MTZ + LEV + TET Quadruple resistance CLA + MTZ + LEV + RIF AMX + CLA + MTZ + LEV CLA + MTZ + LEV + TET AMX + CLA + MTZ + RIF AMX + MTZ + LEV + TET AMX + MTZ + LEV + RIF Quintuple resistance AMX + CLA + MTZ + LEV + TET AMX + CLA + MTZ + LEV + RIF AMX + MTZ + LEV + TET + RIF CLA + MTZ + LEV + TET + RIF Sextuple resistance
No. of isolates
Resistance rate (%)
60 49 19 19 5 3 3 2 1 1
10.0 8.2 3.2 3.2 0.8 0.5 0.5 0.3 0.2 0.2
55 13 8 6 5 5 4 2 1 1 1
9.2 2.2 1.3 1.0 0.8 0.8 0.7 0.3 0.2 0.2 0.2
16 6 3 1 1 1
2.7 1.0 0.5 0.2 0.2 0.2
1 1 1 1 2
0.2 0.2 0.2 0.2 0.3
AMX, amoxicillin; CLA, clarithromycin; MTZ, metronidazole; LEV, levofloxacin; TET tetracycline; RIF, rifampicin.
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3.3. Factors influencing antibiotic resistance Univariate analysis showed that there were significant differences in the resistance rates to clarithromycin, metronidazole, and levofloxacin between patients with peptic ulcer disease and nonulcer disease. Moreover, significant differences in the levofloxacin resistance rates were also observed between men and women, as well as among different age groups. Multivariate analysis demonstrated that the above-mentioned factors were independent factors influencing the antibiotic resistance of H. pylori (Table 3).
4. Discussion The results from the present study show that the treatment of H. pylori infection remains a challenge in China due to the high rates of H. pylori resistance to commonly used antibiotics. Furthermore, multiple antibiotic resistance patterns were also commonly observed in the H. pylori isolates investigated. In the present study, resistance to clarithromycin was identified in 37.5% of H. pylori isolates. According to the Maastricht IV/Florence consensus report [2], clarithromycin-based standard triple therapy is unsuitable for eradication of H. pylori in China, unless susceptibility testing for H. pylori is performed before treatment to confirm the lack of resistance to this specific antibiotic. Some studies reported that sequential and concomitant therapies are effective treatment options for patients who show clarithromycin resistance. Thus, these therapies can be recommended for patients from those regions with high clarithromycin resistance rates [2,9–12]. Unfortunately, meta-analyses also showed that dual resistance to clarithromycin and metronidazole could reduce the ability of sequential and concomitant therapies to eradicate H. pylori [13,14], indicating that these therapies could only be used in regions with high rates of isolated clarithromycin resistance. Our data showed a high rate of resistance to metronidazole (75.6%) in clarithromycin-resistant isolates. Based on these results, it is reasonable to speculate that sequential and concomitant therapies are not suitable for the management of H. pylori infection in China. In recent years, due to the high frequency of resistance to clarithromycin and metronidazole, levofloxacin has been extensively studied in patients with H. pylori infection. Moreover, levofloxacin has been proven to be effective in eradicating H. pylori in some trials [15–17]. Nevertheless, these studies were mainly conducted in regions in which levofloxacin resistance was low. In our study, we found that the resistance rate of H. pylori isolates to levofloxacin was as high as 33.5%, with a high level of combined resistance to clarithromycin and metronidazole, indicating that a levofloxacincontaining therapy may not be a good choice for the initial empiric treatment of H. pylori in China. In the current study, the H. pylori isolates were found to be susceptible to tetracycline with an overall resistance rate of 3.5%, which suggested that tetracycline could be used in the initial treatment of H. pylori infections in China. Additionally, it is generally thought that the increased dosage, and dosing frequency, of metronidazole can reduce high-level resistance to metronidazole [1,18]. Currently bismuth salts are widely used in clinical practice since they are easily obtained in China. Therefore, the classic quadruple therapy, which contains metronidazole, tetracycline, bismuth, and proton pump inhibitor, can be suggested as the best empirical first-line regimen for H. pylori eradication in China, a concept that is consistent with the recommendations of the Maastricht IV/Florence consensus report [2]. Unfortunately, tetracycline is not generally available in China, and this has affected its clinical application. Although semi-synthetic tetracycline derivatives, including minocycline, are easily obtainable in the clinic, it is still uncertain
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Table 3 The influence factors of antibiotic resistance of Helicobacter pylori isolates. Resistance rate (%)
Region Northern (n = 283), Eastern (n = 102), Middle (n = 105), Southern (n = 110)
Gender Male (n = 290), Female (n = 310)
Age (years) 18–35 (n = 189), 36–50 (n = 227), 51–75 (n = 184)
Endoscopic finding PUD (n = 95), NUD (n = 505)
Amoxicillin Clarithromycin
6.4, 7.8, 5.7, 8.2 38.2, 39.2, 36.2, 35.5
7.9, 5.8 34.8, 40.0
7.9, 5.7, 7.1 38.6, 40.1, 33.2
Metronidazole
69.3, 64.7, 66.7, 64.5
63.4, 70.6
3.2, 7.5 22.1, 40.4* (OR 2.316; 95%CI 1.374–3.904) 56.8, 69.1* (1.624; 1.031–2.558) 17.9, 36.4* (2.516; 1.431–4.424) 2.1, 3.8 10.5, 14.9
Levofloxacin
33.9, 35.3, 31.4, 32.7
Tetracycline Rifampicin
3.5, 3.9, 2.9, 3.6 13.1, 14.7, 16.2, 14.5
64.0, 70.5, 66.3 *
24.3, 37.9, 37.5* (1.378; 1.103–1.722) 3.2, 1.8, 6.0 13.8, 14.1, 16.7
27.9, 38.7 (1.462; 1.028–2.080) 3.8, 3.2 13.8, 14.5
PUD, peptic ulcer disease; NUD, non-ulcer disease; OR, odds ratio; CI, confidence interval. * P < 0.05.
whether these derivatives can be used as alternatives to tetracycline for the treatment of H. pylori infection. In this study, the rate of resistance to amoxicillin was relatively low, suggesting that amoxicillin should be fully used in the eradication of H. pylori infection. With the general increase in the rate of resistance to commonly used antibiotics, dual therapy (proton pump inhibitor plus amoxicillin) has drawn attention again, but the study results are not consistent and conclusive [19,20]. Shirai et al. have shown a very good eradication efficacy in their study, but rabeprazole and amoxicillin should be administered four times a day [19]. So far no studies on this regimen have been conducted in China. Rifampicin is rarely used for the treatment of H. pylori infection In China. In the present study, the rate of resistance to this drug (14.2%) in the H. pylori isolates analysed may be due to its frequent use in the treatment of tuberculosis in China, where a high prevalence of tuberculosis infection is observed. Our results show that the resistance rates of H. pylori isolates were significantly higher in the patients with non-ulcer disease than with peptic ulcer disease, consistently with the results of previous studies [21,22]. Additionally, levofloxacin resistance was more frequent in women than men, and in middle-aged and elderly patients than younger patients. These findings may be related to the more frequent use of levofloxacin in these groups of patients, especially in women with gynaecological diseases, as antimicrobial resistance is dependent on antimicrobial use. In Western countries the commercialization of quinolones such as levofloxacin is more recent than that of clarithromycin, and levofloxacin has a more limited use as an antibiotic. Therefore, in those areas, the resistance rate of H. pylori to levofloxacin is relatively low [2]. However, in China, over the past 30 years, quinolones have been widely used as non-prescription drugs even for animal husbandry and aquaculture, thus resulting in such a high resistance rate (33.5%). Recently, restrictions in the use of levofloxacin have been demanded. In the present study, the resistance rate to metronidazole in females was higher than in males (70.6% vs. 63.4%), despite not being statistically significant. This finding is in disagreement with the literature [23], which reports higher rates in females due to a large use of imidazole for gynaecological diseases. The similar resistance rates obtained in our study may be related to the general use of metronidazole in China, in males as well as females, for a variety of oral and gastrointestinal diseases, a feature that probably reduces the significance of this difference. In our study, the antimicrobial susceptibility of the H. pylori isolates was determined with the E-test, primarily because this method can be easily performed, especially in a multicentre clinical study such as ours. Despite some previous studies suggested that the rate of metronidazole resistance may be overestimated by the E-test [24], these results were not consistent or conclusive. A high resistance rate to metronidazole was also found in a recent
study, conducted in the Southeast coastal region of China, with the reference agar dilution method [25]. The requirements for the culture of H. pylori are relatively high. The success rate of a culture is usually between 60% and 90% [23]. The overall success rate of culture in this study was relatively low (64.7%), and may be mainly related to the need of transferring the gastric specimens. In order to unify and standardise the culture, we sent all the specimens to the National Institute for Communicable Disease Control and Prevention, in Beijing, for carrying out the culture and sensitivity testing. Because the Chinese territory covers a vast area, the distances from Beijing to Shanghai, Wuhan, and Guangzhou are of about 1200 km, 1200 km, and 2200 km, respectively. Although we used air transport in dry ice and with a multi-layer insulation box, at least 24–48 h were needed for specimen transfer, and these conditions may be detrimental to the survival of some fragile strains of H. pylori. This also explains why the positive culture rate of the centre located in Beijing (only two hours by car needed for specimen transfer) was relatively higher (77.1%) than those of the other three centres outside Beijing. In this study, two gastric mucosal biopsy specimens (one from antrum and one from corpus) were collected and put into the same vial for the culture of H. pylori. Due to restrictions in research funding and study conditions, we did not carry out the culture of H. pylori in the mucosal specimens of antrum and corpus separately. Thus, we could not analyse the differences in the antimicrobial resistance profiles of H. pylori according to the localisation; thus, the antimicrobial resistance rates may be underestimated, representing a limitation of this study. In conclusion, we carried out a prospective, multi-region study with a large sample size to determine the resistance patterns of H. pylori isolates collected from dyspeptic patients in China. Our results showed that H. pylori resistance to commonly used antibiotics in China is a very serious issue due to the high resistance rate and universal multiple resistance. This comprehensive and up-todate information on H. pylori resistance will be very helpful to select the most optimal eradication regimens in China as well as in other regions with a high prevalence of antibiotic resistance. With the current rates of resistance, the priority would be obtaining new drugs and/or improved methods for the detection of resistance. The use of a more accessible and easier method to obtain H. pylori has already been suggested [26], and progresses in the molecular detection of resistance from faecal samples [27] also represent a very promising line of research. Conflict of interest None declared. References [1] Graham DY, Fischbach L. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut 2010;59:1143–53.
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