Numerous risk factors for Helicobacter pylori antibiotic resistance revealed by extended anamnesis: a Bulgarian study

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1 Journal of Medical Microbiology (2012), 61, DOI /jmm Numerous risk factors for Helicobacter pylori antibiotic resistance revealed by extended anamnesis: a Bulgarian study Lyudmila Boyanova, 1 Juliana Ilieva, 2 Galina Gergova, 1 Lubomir Davidkov, 3 Zoya Spassova, 4 Victor Kamburov, 5 Nikolai Katsarov 6 and Ivan Mitov 1 Correspondence Lyudmila Boyanova l.boyanova@hotmail.com or l.boyanova@lycos.com 1 Department of Medical Microbiology, Medical University of Sofia, Zdrave Street 2, 1431 Sofia, Bulgaria 2 University Department of Social Medicine and Healthcare Management, Medical University of Sofia, Sofia, Bulgaria 3 Departments of Gastroenterology and General Surgery, University Hospital St Ekaterina, Sofia, Bulgaria 4 Department of Gastroenterology, University Hospital St Ivan Rilski, Sofia, Bulgaria 5 Urgent Endoscopy Unit, Emergency Hospital Pirogov, Sofia, Bulgaria 6 Second Surgery Department, University Alexander Hospital, Sofia, Bulgaria Received 21 June 2011 Accepted 19 August 2011 The aim of this study was to assess risk factors for primary Helicobacter pylori antibiotic resistance by an extended anamnesis. In total, 519 H. pylori strains from untreated symptomatic adults who answered a questionnaire were evaluated. Strain susceptibility was assessed by a breakpoint susceptibility test. Primary resistance rates were 29.5 % for metronidazole, 17.9 % for clarithromycin, 7.3 % for metronidazole+clarithromycin, 4.0 % for tetracycline and 10.8 % for ciprofloxacin. On multivariate analysis, younger ( 65 years) age was an independent predictor for metronidazole resistance. To our knowledge, for the first time, being a member of the health-care profession was revealed as a risk factor for H. pylori resistance to metronidazole and both metronidazole and clarithromycin. Respiratory and urinary tract infections were independent predictors of clarithromycin and ciprofloxacin resistance, respectively. The presence of co-infections was an independent risk factor for clarithromycin, metronidazole and ciprofloxacin resistance. Surprisingly, female sex was the only predictor for tetracycline resistance. The antibiotic resistance rates were not associated with disease type, place of residence, birthplace, educational level, non-steroidal anti-inflammatory drug or proton pump inhibitor use, smoking or dietary factors, such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey or meat. There was a trend for higher metronidazole resistance in strains from diabetic patients. In conclusion, the extended anamnesis of H. pylori-positive patients should include data on patient age, sex, whether they are in the health-care profession, co-infections and possibly diabetes to improve the choice of empiric therapy. Tailored treatment based on the extended anamnesis is suggested, and susceptibility testing of the strains is recommended for patients at risk for antibiotic resistance, especially to clarithromycin, fluoroquinolones or both metronidazole and clarithromycin. INTRODUCTION Resistance to antibiotics is the major reason for failure to eradicate Helicobacter pylori. The presence of clarithromycin-resistant Abbreviations: AUC, area under the curve; CI, confidence interval; NSAID, non-steroidal anti-inflammatory drug; OR, odds ratio; PPI, proton pump inhibitor; ROC, receiver operating characteristic. strains strongly diminishes (by %) the eradication success of clarithromycin-based triple regimens, and fluoroquinolone resistance can reduce the eradication rates of quinolone-based therapy by % compared with those against susceptible strains (Mégraud & Lehours, 2007; Perna et al., 2007; Nishizawa et al., 2009). The impact of metronidazole resistance on treatment success using triple nitroimidazole-based regimens is G 2012 SGM Printed in Great Britain 85

2 L. Boyanova and others lower (by 25 %) (Mégraud, 2004a). The primary resistance rates of H. pylori to some antibiotics, e.g. to clarithromycin and quinolones, in many developed countries and those to metronidazole and tetracycline, mainly in some developing countries, increase over the years (Boyanova & Mitov, 2010). In some studies, primary H. pylori antibiotic resistance rates were shown to depend on countries or regions, national outpatient antibiotic use, prior use of the drug or similar agents for co-infections, patient age, sex, disease and co-morbidity, strain virulence (McMahon et al., 2003; Koivisto et al., 2004; Mégraud, 2004a; Boyanova & Mitov, 2010; Talebi Bezmin Abadi et al., 2010) and possibly other factors. Many of the reported factors are associated with previous use of the antibiotics. However, the role of dietary factors has not been elucidated, the impact of being in the health-care profession has not been determined and most studies have focused mainly on H. pylori resistance to clarithromycin and metronidazole. The aim of this study was to assess the risk factors for primary H. pylori antibiotic resistance by an extended anamnesis, involving data on sex, disease, age, place of residence, birthplace, educational level, membership of the health-care profession, co-infections, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) or proton pump inhibitors (PPIs) and the presence of diabetes and smoking, as well as dietary factors such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey and meat. METHODS Patients and specimens. In total, 519 consecutive H. pylori strains from untreated adults with upper gastrointestinal complaints who agreed to fill in a questionnaire were evaluated over the period Participation was voluntary and informed consent was obtained in all cases. The patients were men (318 subjects) and women (201) aged years (mean age, years), comprising 407 subjects aged years and 112 aged years. Categorization of patient disease was based on endoscopic and histological findings. The patients suffered from peptic ulcers, e.g. duodenal ulcer (143 cases), gastric ulcers (50) or non-ulcer diseases such as chronic gastritis (217), gastro-oesophageal reflux disease (104), gastric cancer (four) and mucosa-associated lymphoid tissue lymphoma (one). The presence of co-infections (urinary tract, respiratory tract or other infections), prior (during the last year) use of antibiotics, aspirin or other NSAIDs or PPIs, diabetes, place of residence and birthplace, membership of the health-care profession, educational level and smoking were also recorded (Table 1). Dietary habits such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey and meat were also taken into account. Two gastric biopsy specimens per patient were used. Specimens were transported in Stuart transport medium (Becton Dickinson). The study was approved by the Ethical Committee of the Medical University of Sofia. Microbiology. The specimens were used for direct Gram staining, an in-house rapid urease test and culture as described previously (Boyanova, 2007). Briefly, culture was carried out on Columbia agar (Becton Dickinson) with 10 % sheep blood (a non-selective agar) and the same medium supplemented with 1 % IsoVitaleX (Becton Dickinson) and the following agents: 10 mg vancomycin ml 21,5mg trimethoprim ml 21, 5 mg cefsulodin ml 21 and 5 mg amphotericin Bml 1 (a selective medium). Plates were incubated at 35 uc for up to 11 days in a microaerophilic atmosphere (CampyPak; Becton Dickinson). Identification of H. pylori was made by Gram staining of the colonies, lack of aerobic growth on blood agar plates and testing for the presence of urease, oxidase and catalase. Susceptibility testing. The susceptibility of the strains was evaluated by a breakpoint susceptibility test, as described previously (Boyanova et al., 2008). Briefly, ~30 60 ml H. pylori suspension, corresponding to a density of McFarland turbidity standard 2 3, was inoculated on Mueller Hinton blood agar plates (National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria) containing one of the following drug concentrations: 8, 16 or 32 mg metronidazole ml 1 ; 0.25, 0.5, 1 or 2 mg clarithromycin ml 1 ; 1 mg ciprofloxacin ml 1 ; and 4 mg tetracycline ml 1. Susceptibility testing for ciprofloxacin was carried out to reveal the strain susceptibility to levofloxacin (Mégraud & Lehours, 2007). The plates were incubated microaerophilically at 35 uc for 2 3 days. Non-selective Mueller Hinton blood agar plates were used as a control of the viability of the strains. Breakpoints for resistance were:.8 mg metronidazole ml 1, 1 mg clarithromycin ml 1,.1 mg ciprofloxacin ml 1 and.4 mg tetracycline ml 1 (Mégraud & Lehours, 2007; Boyanova et al., 2008; CLSI, 2010). Strain susceptibility was tested in duplicate. Statistical analysis. Statistical analysis was performed using SPSS 15.0 statistical software. A x 2 test and Fisher s exact test of independence were used to compare the following variables of interest: sex, disease (ulcer or non-ulcer), age, place of residence, birthplace, educational level, membership of the health-care profession, co-infections, use of NSAIDs or PPIs, the presence of diabetes and smoking, as well as dietary factors such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey and meat. Logistic regression was used to select significant predictor variables and to estimate odds ratios (ORs) of these variables and, if possible, to predict outcomes. Receiver operating characteristic (ROC) curves were performed to assess the ability of a test to distinguish between antibiotic-resistant and -susceptible strains in the patients. Prior antibiotic use was not included in the logistic regression because in most cases (76.5 %) no data for the antibiotic used were available. RESULTS Overall H. pylori primary resistance rates were 29.5 % for metronidazole, 17.9 % for clarithromycin, 7.3 % for both metronidazole and clarithromycin, 4.0 % for tetracycline and 10.8 % for ciprofloxacin (Table 1). On univariate analysis, significant risk factors (P,0.05) for H. pylori resistance were age for metronidazole, gender for tetracycline, place of residence for clarithromycin, presence of co-infections for metronidazole, clarithromycin and ciprofloxacin, and being a health-care professional for metronidazole and both metronidazole and clarithromycin. Conversely, the resistance rates to the antibiotics evaluated were not associated (P.0.05) with disease, place of residence, birthplace, educational level, use of NSAIDs or PPIs and smoking, or with dietary factors such as consumption of coffee, yogurt, green tea, raw garlic, raw onion, honey and meat, on univariate analysis. On univariate analysis, primary clarithromycin resistance was more common in patients living in Sofia (20.5 %) than in those living in villages (8.5 %, P50.049), in patients with 86 Journal of Medical Microbiology 61

3 Risk factors for H. pylori antibiotic resistance respiratory tract infections (31.4 %) compared with other patients (16.4 %, P50.008) and in patients with prior antibiotic use (24.5 %) compared with the remainder (15.9 %, P50.041). The primary clarithromycin resistance was 24.5 % in patients with co-infections and 16.3 % (P50.053) in other patients. Logistic regression analysis showed that the presence of co-infections was an independent risk factor for clarithromycin resistance [OR 1.79, 95 % confidence interval (CI) ]. The patients with respiratory tract infections were twice as likely (OR 2.05, 95 % CI ) as other patients to harbour clarithromycin-resistant strains (Table 2). On univariate analysis, primary metronidazole resistance was higher in younger (aged 65 years) patients compared with elderly subjects (31.9 vs 20.5 %, P50.019), in patients with co-infections compared with other patients (38.2 vs 27.3 %, P50.030), in subjects with prior antibiotic use compared with others (38.2 vs 27.5 %, P50.033) and in health-care staff compared with subjects with other professions (46.7 vs 28.4 %, P50.038). In diabetic patients, there was a trend for higher metronidazole resistance in H. pylori compared with that in non-diabetic patients (41.3 vs 28.3 %, P50.065); however, the lack of significance could be due to the low number (only 46) of diabetic patients evaluated. The logistic regression did not find any associations between metronidazole resistance and nonulcer diseases. On univariate analysis, more patients in the health-care profession than those in other professions harboured strains with double resistance to clarithromycin and metronidazole (16.7 vs 6.7 %, P50.043). The logistic regression analysis confirmed that the health-care workers had a 3.17-fold higher risk (OR 3.166, 95 % CI ) for double H. pylori resistance to clarithromycin and metronidazole (Table 2). Interestingly, on univariate analysis, women carried tetracycline-resistant strains more often than men (6.5 vs 2.5 %, P50.026). On the multivariate analysis, the female gender was confirmed as the only independent risk factor for tetracycline resistance. The woman were 2.7-fold more likely (OR 2.7, 95 % CI ) than the men to harbour tetracycline-resistant strains. On univariate analysis, the primary ciprofloxacin resistance rate was higher in patients with co-infections compared with other subjects (21.6 vs 8.2 %, P ), in patients with prior antibiotic use versus other patients (22.5 vs 7.9 %, P ) and in patients with urinary tract coinfections compared with other subjects (27.6 vs 9.8 %, P50.003). Logistic regression showed that the risk for primary ciprofloxacin resistance was associated with coinfections and urinary tract infections. Patients with urinary tract infections had a 2.7-fold higher risk (OR 2.73, 95 % CI ) for primary ciprofloxacin resistance in H. pylori. Similarly, subjects with co-infections were about threefold more likely (OR 2.99, 95 % CI: ) than the other subjects to harbour ciprofloxacinresistant strains. In the logistic regression, a Hosmer Lemeshow goodnessof-fit test showed that the models adequately fitted the data, showing P for metronidazole, P50.72 for both metronidazole and clarithromycin, and P50.75 for tetracycline. The significance for clarithromycin was P50.83 when all co-infections were taken into account and P when only respiratory tract infections were considered. The significance for ciprofloxacin was P50.75 when all coinfections were taken into account and P when only urinary tract infections were considered. From the ROC curves, the area under the curve (AUC) for clarithromycin was 0.58 (SEM 0.034, P50.015, 95 % CI ) when all co-infections were taken into account and the result was similar (AUC 0.59, SEM50.033, P50.006, 95 % CI ) when only respiratory tract infections were considered. The AUC for metronidazole was 0.61 (SEM50.027, P50.00, 95 % CI ), that for both metronidazole and clarithromycin was 0.65 (SEM50.044, P50.002, 95 % CI ) and that for tetracycline was 0.64 (SEM50.063, P50.031, 95 % CI ). The AUC for ciprofloxacin was 0.62 (SEM50.042, P50.005, 95 % CI ) when all co-infections were considered and 0.56 (SEM50.043, P50.150, 95 % CI ) when only urinary tract infections were taken into account. DISCUSSION In some studies, H. pylori resistance to clarithromycin has been linked to years (recent vs previous) of study, prior use of macrolides for respiratory or dental infections, patient age, female sex, race, non-ulcer disease, smoking and geographical region (Koivisto et al., 2004; Boyanova & Mitov, 2010). In the present study, the multivariate analysis showed that clarithromycin resistance in H. pylori was strongly associated with the presence of co-infections and respiratory tract infections. The major factor was respiratory tract co-infections, as these are often treated with newer macrolides such as clarithromycin or azithromycin. Patients with respiratory tract infections were twice as likely as the other patients to harbour clarithromycin-resistant H. pylori. H. pylori resistance to metronidazole has been associated with previous use of nitroimidazoles for gynaecological diseases, e.g. trichomoniasis and bacterial vaginosis, leading to higher resistance rates in women than in men, and use for parasitic diseases in some developing countries, leading to elevated resistance rates in inhabitants of or immigrants from these countries (McMahon et al., 2003; Koivisto et al., 2004; Mégraud, 2004a Zullo et al., 2007; O Connor et al., 2010; Kostamo et al., 2011). In addition, resistance has been associated with birthplace and place of residence (Boyanova & Mitov, 2010). A recent study in Belgium showed patients aged years to be at risk for H. pylori resistance to metronidazole and ciprofloxacin (Miendje Deyi et al., 2011). 87

4 L. Boyanova and others Table 1. Influence of patient characteristics, prior antibiotic use and dietary habits on H. pylori resistance to antibiotics in the 519 patients evaluated Asterisks indicate statistical significance (P,0.05). Group No. of patients Resistance (%) to: Metronidazole Clarithromycin Metronidazole +clarithromycin Tetracycline Ciprofloxacin Age.65 years * years * Gender Female * 11.9 Male * 10.1 Disease Ulcer Non-ulcer NSAID used Yes No PPI use Yes No Diabetes Yes No Birthplace Sofia Town Village Place of residence Sofia * Town Village * Educational level High Lower Urinary infections Yes * No * Respiratory infections Yes * No * Co-infections Yesd * * No * * Prior antibiotic use Yes * 24.5* * No * 15.9* * Health-care worker Yes * * No * * Smoking Yes No Consumption of: Coffee Everyday Journal of Medical Microbiology 61

5 Risk factors for H. pylori antibiotic resistance Table 1. cont. Group No. of patients Resistance (%) to: Metronidazole Clarithromycin Metronidazole +clarithromycin Tetracycline Ciprofloxacin Less frequent No Yogurt Everyday Less frequent No Green tea Everyday Less frequent No Raw garlic Everyday Less frequent No Honey Everyday Less frequent No Meat.2 days weekly Less frequent No Raw onion Everyday Less frequent No All patients DMostly aspirin. drespiratory (n551), urinary tract (n529), gall bladder (n56), dental (n54), intestinal (n53) or unspecified infection (n516). Some patients reported more than one infection (n57). Clarithromycin (n51), azithromycin (n53), b-lactams (n510), quinolones (n59), metronidazole (n51) or unspecified antibiotics (n578). Fifteen were physicians and 15 nurses. In the present study, a younger age, being a member of the health-care profession and the presence of co-infections were important independent predictors of primary metronidazole resistance in H. pylori. The risk for metronidazole resistance in younger patients may be related to the use of metronidazole to treat urogenital and gynaecological infections. The logistic regression did not confirm the associations between metronidazole resistance and nonulcer disease or birthplace in towns that were found in our previous study (Boyanova et al., 2009). However, in 607 adults evaluated in , primary H. pylori resistance to metronidazole was also less common in adults with peptic ulcers (19.2 %) than in non-ulcer subjects (28.1 %) (Boyanova et al., 2008). Similarly, in Ireland, Taneike et al. (2009) related higher overall metronidazole resistance to caga-negative strains, which are more common in non-ulcer patients, compared with virulent strains. The difference between the results of our present and previous studies can be explained by the larger (7 years, ) period of the present study compared with that (,5 years, 2004 March 2008) of the earlier study (Boyanova et al., 2009). Moreover, in the present study, more (34.2 %, 78/228) patients evaluated since March 2008 had coinfections, mainly respiratory infections, compared with 8.2 % (24/291, P ) of those studied previously. This emphasizes again the role of co-infections in H. pylori resistance to antibiotics. The effect of birthplace or place of residence could be evaluated further by taking into account regional and smaller towns separately. Double H. pylori resistance to clarithromycin and metronidazole has been associated with female sex, older age, ethnicity and non-ulcer diseases (Kist & Glocker, 2004; Boyanova & Mitov, 2010). To our knowledge, the present study provides the first data about the health-care profession as a risk factor for H. 89

6 L. Boyanova and others Table 2. Risk factors for primary H. pylori resistance to antibiotics among 519 Bulgarian symptomatic patients confirmed by logistic regression analysis Outcome variable: resistance to Exposure (independent) variable B* SED Waldd P OR 95 % CI Metronidazole Age (.65 vs 65 years) Metronidazole Profession (health care vs other) Metronidazole Co-infections (presence vs absence) Clarithromycin Co-infections (presence vs absence) Clarithromycin Respiratory tract infections (presence vs absence) Metronidazole+ Profession (health-care worker vs other) clarithromycin Tetracycline Gender (female vs male) Ciprofloxacin Co-infections (presence vs absence) Ciprofloxacin Urinary infections (presence vs absence) *B, Regression coefficient of the logistic regression. DSE, Standard error of the regression coefficients. dwald statistics (logistic regression). 95 % CI for Exp(B), i.e. 95 % CI for the OR. pylori resistance to metronidazole and both metronidazole and clarithromycin. This can be explained, at least partly, by the direct contact of health-care workers with patients infected by H. pylori strains resistant to antibiotics, by intravenous antibiotic handling, by exposure to hospital waste or by a higher antibiotic consumption in this group compared with patients with other professions. In support of this suggestion, other data from the literature can be considered. Although reports regarding the prevalence of H. pylori infection in medical staff are still contradictory, infection was considered a professional risk in health-care workers in Belgian institutions for people with intellectual disability (De Schryver et al., 2008). Therefore, some medical workers can be infected, co-infected or reinfected by resistant H. pylori strains more easily than subjects in other professions. Moreover, Arfons et al. (2005) reported that, in a US medical centre, 45 % of health-care workers had received antibiotics within the past year, suggesting frequent antibiotic treatments in some groups of medical staff. Additionally, Lindberg et al. (2007) reported relatively high metronidazole and ciprofloxacin concentrations (100 mg l 21 ) in a Swedish hospital effluent and hence an additional possible exposure risk to metronidazole in health-care personnel. Primary tetracycline resistance has been associated with national outpatient antibiotic use (Boyanova & Mitov, 2010). A study in Belgium, where tetracycline resistance is rare, showed the female sex to be associated with H. pylori resistance to clarithromycin, metronidazole and ciprofloxacin (Miendje Deyi et al., 2011). Tetracyclines can be used to treat urogenital infections caused by Chlamydia trachomatis, Mycoplasma species and Neisseria gonorrhoeae (Aupee et al., 2009). In the present work, the higher risk in women than in men for harbouring tetracycline-resistant strains can be explained, at least partly, by the more frequent use of tetracyclines in women to treat urogenital infections (Aupee et al., 2009). In a recent Bulgarian study, in specimens from patients presenting for diagnosis of chlamydial infections, those from women were fourfold more common compared with samples from men (Raikova, 2010). Carothers et al. (2007) found only a 2 % levofloxacin resistance rate in patients without prior fluoroquinolone use versus.50 % in six patients with three or more previous prescriptions of the antibiotics. H. pylori resistance to fluoroquinolones has been also associated with years (recent vs previous) of study, older age (.45 years) and high-level resistance to either clarithromycin or metronidazole (Zullo et al., 2007; Boyanova & Mitov, 2010). Other risk factors for H. pylori resistance to fluoroquinolones have been reported to be hospital centres and place of residence (Boyanova & Mitov, 2010). In the present study, patients with co-infections and those with urinary tract infections were at risk for primary ciprofloxacin resistance in H. pylori. In these patients, prior use of quinolones to treat urogenital or respiratory tract infections can increase the risk for primary H. pylori resistance. AUCs were calculated to predict the ability of each model to correctly classify the patients having antibiotic-resistant and -susceptible strains. The AUCs for the antibiotics in the present study showed that the ability to predict patients who harboured a resistant H. pylori strain failed for ciprofloxacin when only urinary tract infections were considered, as well as for clarithromycin (AUCs,0.6), and was slightly higher but poor (AUCs ) for ciprofloxacin when all co-infections were considered, as well as for metronidazole, tetracycline, and both metronidazole and clarithromycin. 90 Journal of Medical Microbiology 61

7 Risk factors for H. pylori antibiotic resistance Dietary factors such as yogurt consumption may influence theprevalence ofh. pylori infection (Ornelas et al., 2007). Therefore, it was interesting to evaluate the role of dietary habits in H. pylori antibiotic resistance. However, in the present study, the resistance rates to the antibiotics evaluated were not linked to disease type, place of residence, birthplace, educational level, NSAID or PPI use, smoking or dietary factors. Diabetes mellitus can be a risk factor for H. pylori resistance. In diabetic patients, alterations in gastric microvasculature and drug absorption, as well as possible gastroparesis and common antibiotic treatments for other infections, can be associated with suboptimal eradication and high resistance levels in H. pylori (Ojetti et al., 2005). In Turkey, H. pylori clarithromycin resistance was 1.8- fold higher in diabetic patients than in other subjects (Demir et al., 2009). The trend for higher metronidazole resistance in strains from diabetic patients in the present study should be evaluated further in larger patient groups. The limitations of this study involve the fact that the smoking and dietary data were self-reported by the patients. However, the proportion of smokers in the present work (33.7 %) was similar to data from the World Health Organization for adult smoker prevalence in Bulgaria in 2007 (38.1 %; annex8_prevalence_data_2007.pdf). In addition, many patients were unable to provide the exact names of the antibiotics used and hence the presence of co-infections instead of the antibiotics used was included in the multivariate analysis. Tailored treatment based on the extended anamnesis can be suggested. Generally, standard triple regimens such as a PPI, amoxicillin (or metronidazole) and clarithromycin therapy give suboptimal results in patients at risk for antibiotic-resistant H. pylori strains (Rimbara et al., 2011). No therapy has been highly active against strains resistant to both clarithromycin and metronidazole (Graham & Shiotani, 2008). However, in patients at risk for double resistance, such as the health-care workers in the present study, a 2-week empiric bismuth quadruple therapy (PPI, bismuth subsalicylate/subcitrate, tetracycline and metronidazole) or a high-dose esomeprazole/amoxicillin/metronidazole regimen for 2 weeks (Graham & Shiotani, 2008; Schwarzer et al., 2011) can be successful. Fluoroquinolone-based regimens or a prolonged PPI/amoxicillin therapy for 4 weeks (Wüppenhorst et al., 2011) may be also of benefit. Sequential (PPI and amoxicillin for 5 days and PPI, clarithromycin and metronidazole/tinidazole for another 5 days) therapy and concomitant therapy (PPI, amoxicillin, clarithromycin and metronidazole/tinidazole) are currently highly effective regimens but have not been recommended for eradication of strains with double resistance (Graham & Shiotani, 2008). However, it should be stressed that metronidazole resistance has a lower effect on the eradication success compared with that to clarithromycin or fluoroquinolones, and the sequential therapy may overcome clarithromycin resistance in most H. pylori strains according to Francavilla et al. (2010). The benefit of the sequential therapy to treat patients at risk for clarithromycin-resistant strains, such as subjects with co-infections and especially respiratory tract infections in the present study, should be evaluated further, as the data from the literature are controversial (Francavilla et al., 2010; Rimbara et al., 2011). These patients can be treated empirically by bismuth quadruple therapy, fluoroquinolone-based regimens or by triple combinations of PPI (ranitidine bismuth), amoxicillin (or tetracycline) and metronidazole (Mégraud, 2004b). However, fluoroquinolones should be used mainly as a third-line therapy because of concern about the increase in resistance with growing use (Graham & Shiotani, 2008). For patients aged 65 years, health-care staff and subjects with co-infections, a triple nitroimidazole-based regimen should be avoided because of the risk for metronidazoleresistant strains, whilst a bismuth quadruple regimen could be administered for more than 1 week (Rimbara et al., 2011). For patients with urinary tract infections, fluoroquinolone-based regimens should be avoided unless susceptibility testing of the strains is performed. The need to avoid metronidazole in eradication regimens for diabetic patients should be evaluated further. Most H. pylori-positive patients are treated empirically, although susceptibility testing of the strains is preferable for revealing the antibiotic resistance compared with the extended anamnesis. Therefore, susceptibility testing should be recommended for patients at risk for antibiotic resistance, especially to clarithromycin, fluoroquinolones or both clarithromycin and metronidazole. Finally, probiotics, which reduce side effects from standard regimens and may increase patient compliance as well as the eradication success by 10 % (Boyanova & Mitov, 2010), should be administered for all patients at risk for antibiotic resistance. In conclusion, independent risk factors for H. pylori resistance identified by logistic regression were urinary tract infections and all co-infections for quinolones, respiratory tract infections for clarithromycin, a younger age and being a health-care professional for metronidazole, being a health-care professional for double resistance to metronidazole and clarithromycin, and female gender for tetracycline. The presence of co-infections was an independent risk factor for clarithromycin, metronidazole and ciprofloxacin resistance. The extended anamnesis of H. pylori-positive patients should include data on patient age, sex, whether they are in the health-care profession, present/ past co-infections and possibly diabetes to improve the choice of empiric therapy for chronic infection in this era of increasing antibiotic resistance. 91

8 L. Boyanova and others ACKNOWLEDGEMENTS This study was supported by grants from the Medical University of Sofia (Council of Medical Science), project no. 2/2010, grant no. 37/ REFERENCES Arfons, L., Ray, A. J. & Donskey, C. J. (2005). Clostridium difficile infection among health care workers receiving antibiotic therapy. Clin Infect Dis 40, Aupee, O., Almeras, D., Le Garlantezec, P. & Bohand, X. (2009). [Doxycycline]. Med Trop (Mars) 69, (in French). Boyanova, L. (2007). Detection of Helicobacter pylori infection in symptomatic Bulgarian adults. Clin Microbiol Infect 13, Boyanova, L. & Mitov, I. (2010). Geographic map and evolution of primary Helicobacter pylori resistance to antibacterial agents. Expert Rev Anti Infect Ther 8, Boyanova, L., Gergova, G., Nikolov, R., Davidkov, L., Kamburov, V., Jelev, C. & Mitov, I. (2008). Prevalence and evolution of Helicobacter pylori resistance to 6 antibacterial agents over 12 years and correlation between susceptibility testing methods. 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