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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Background : Helicobacter pylori infection may persist after both first- and second-line current treatments.

Aim : To assess the efficacy of a third-line, culture-guided treatment approach for the eradication of H. pylori.

Methods : Patterns of resistance were analysed in H. pylori isolates from 94 consecutive patients in whom H. pylori infection had persisted after two eradication attempts. Using the epsilometer test, susceptibility analysis was performed for amoxicillin, clarithromycin, metronidazole, tetracycline and levofloxacin. Patients were then treated with a culture-guided, third-line regimen: 89 patients with a 1-week quadruple regimen including omeprazole, bismuth, doxycycline and amoxicillin, and five patients with a 1-week triple regimen containing omeprazole, amoxicillin and levofloxacin or clarithromycin.

Results : Ninety-four subjects (100%) were resistant to metronidazole, 89 (95%) to clarithromycin, 29 (31%) to levofloxacin and five (5%) to tetracycline. No resistance to amoxicillin was found in any patient. Overall, H. pylori eradication was obtained in 90% of subjects. The quadruple regimen was effective in 81 patients (92% by per protocol and 91% by intention-to-treat analysis). Four patients (80%, both per protocol and intention-to-treat analysis) were H. pylori-negative after the triple regimen.

Conclusions : A culture-guided, third-line therapeutic approach is effective for the eradication of H. pylori. Furthermore, the 1-week doxycycline- and amoxicillin-based quadruple regimen is a good third-line ‘rescue’ treatment option.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Recent decades have seen the standardization of treatment for Helicobacter pylori eradication, and all of the world-wide consensus conferences have recommended the use of triple therapy consisting of a proton pump inhibitor (double dose) and two antibiotics, mainly amoxicillin (1 g b.d.) and clarithromycin (500 mg b.d.) or metronidazole (500 mg b.d.), as first-line therapy. However, although this treatment has been shown to be effective in numerous clinical trials, the rate of success may vary widely, ranging from 70% to 95%.1–8 Treatment failures occur principally because of poor patient compliance or bacterial resistance.4, 6, 7 Patients for whom 1-week proton pump inhibitor-based triple therapy fails therefore require an effective ‘rescue’ regimen. The choice of second-line treatment depends on which treatment was used initially, because re-treatment with the same regimen is not recommended. Culture after a first eradication failure is not thought to be necessary, and the assessment of H. pylori sensitivity to antibiotics is suggested only after the failure of a second treatment attempt.

The question of second-line therapy has been considered in the Maastricht 2-2000 Consensus Report, which recommends the use of either a 7-day quadruple regimen or another course of triple therapy without resorting to bacterial culture assessment.9 Many versatile regimens of quadruple therapy, with eradication rates of 75–90%,10–12 and many other options, including combinations of amoxicillin–metronidazole, tetracycline–metronidazole or amoxicillin–rifabutin (or clarithromycin when H. pylori is still susceptible), with various rates of success,13 have been used. However, bacterial eradication may fail in up to 40% of cases after the suggested second-line regimens,14–18 and doctors who treat this infection are often faced with a certain number of patients for whom two treatment attempts have failed. Currently, a standard third-line therapy is lacking, and therapeutic options have only rarely been proposed.19–23 European guidelines recommend the use of bacterial culture in these patients to select a third-line treatment according to microbial sensitivity to antibiotics.9

In this study, from a single metropolitan hospital in Rome, Italy, we determined the pattern of resistance to antibiotics in H. pylori isolates obtained from 94 consecutive patients after the failure of two eradication attempts. Patients were treated with third-line, culture-guided, anti-H. pylori regimens.

Patients

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Ninety-four consecutive patients (43 males and 51 females; mean age, 47 ± 25 years), referred to our Gastroenterology Unit between January 2002 and July 2003 with persistent H. pylori infection after two treatment failures, were enrolled for this study. Patients underwent culture assessment for various reasons (history of duodenal or gastric ulcer, low-grade gastric mucosa-associated lymphoid tissue lymphomas, erosive gastritis; subjects who were first-degree relatives of patients with gastric cancer). Patients affected by serious concomitant illnesses and those with previous exposure to drugs capable of interfering with the susceptibility test, such as recent (up to 4 weeks before the first eradication attempt) or continuing use of antibiotics, were excluded. Good patient compliance with previous treatments was assessed by direct interview of all patients who underwent culture assessment. Therefore, 14 patients who had interrupted at least one of the previous eradication treatments because of severe adverse events or who had shown poor compliance with the prescribed regimens were not included in this study. Four eligible patients were also excluded because the H. pylori strains in the stomach did not survive culture processing. Table 1 shows the eradication regimens given to the 94 enrolled patients before antibiotic susceptibility testing.

Table 1.  First and second failed treatments for the eradication of Helicobacter pylori infection in 94 patients, and the antibiotic resistance of the H. pylori isolates after the second treatment failure. Drugs which have not been used in the previous two failed eradication attempts are highlighted with an asterisk
First failed treatment (number of patients treated)Second failed treatment (number of patients treated)Multi-drug resistance (number) after the second treatment failure
  1. Amo, amoxicillin; Bis, bismuth salts; Cla, clarithromycin; Lev, levofloxacin; Met, metronidazole; Ome, omeprazole; PPI, proton pump inhibitor; Tet, tetracycline; Tin, tinidazole; b.d., twice daily; q.d.s., four times daily.

PPI (double dose per day)/ Amo (1 g b.d.)/ Met (250 mg q.d.) or Tin (500 mg b.d.) for 7 days (n = 37)PPI (double dose per day)/ Amo (1 g b.d.)/ Cla (250 or 500 mg b.d.) for 7 days (n = 27)Met/Cla/*Lev (2) Cla/Met (24) Met/*Lev (1)
PPI (double dose per day)/ Amo (1 g b.d.)/ Lev (500 mg b.d.) for 7 days (n = 7)Met/*Cla/Lev (2) *Cla/Met (4) Met/Lev (1)
Ome (20 mg b.d.)/ Bis (120 mg q.d.)/ Amo (1 g b.d.)/ Tet (500 mg q.d.) for 7 days (n = 3)Met/*Lev/Tet (1) *Cla/Met (2)
PPI (double dose per day)/ Amo (1 g b.d.)/ Cla (250 or 500 mg b.d.) for 7 days (n = 34)PPI (double dose per day)/ Bis (120 mg q.d.)/ Met (250 mg q.d.) or Tin (500 mg b.d.)/ Tet (500 mg q.d.) for 7 days (n = 28)Met/Cla/*Lev (1) Met/Cla/Tet (3) Cla/Met (23) Met/*Lev (1)
PPI (double dose per day)/ Amo (1 g b.d.)/ Lev (500 mg b.d.) for 7 days (n = 6)*Met/Cla/Lev (6)
PPI (double dose per day)/ Amo (1 g b.d.)/ Lev (500 mg b.d.) for 7 days (n = 23)PPI (double dose per day)/ Amo (1 g b.d.)/ Cla (250 or 500 mg b.d.) for 7 days (n = 11)*Met/Cla/Lev (7) Cla/*Met (3) *Met/Lev (1)
Ome (20 mg b.d.)/ Bis (120 mg q.d.)/ Amo (1 g b.d.)/ Tet (500 mg q.d.) for 7 days (n = 2)*Met/*Cla/Lev (1) *Met/*Cla/Tet (1)
PPI (double dose per day)/ Bis (120 mg q.d.)/ Met (250 mg q.d.) or Tin (500 mg b.d.)/ Tet (500 mg q.d.) for 7 days (n = 10)Met/*Cla/Lev (5) *Cla/Met (5)

H. pylori culture and antibiotic susceptibility test

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Antral biopsies were collected during upper endoscopy and immediately transported to the laboratory for culture and antibiotic susceptibility testing. Biopsy materials were placed directly into a transport medium and then immediately plated onto Helicobacter-selective agar at 36.5 °C in a micro-aerophilic atmosphere (O2, 50%; CO2, 10%; Campygen, Oxoid Ltd., Basingstoke, UK) for 5–10 days. Biochemical identification of H. pylori was made using catalase tests, oxidase tests and miniaturized enzymatic and conventional tests (apiCampy, bioMerieux, Marcy I'Etoile, France). For each strain, minimal inhibitory concentrations (MICs) were obtained by the epsilometer test method (AB Biodisk, Dalvagen, Sweden)24 for the following agents: amoxicillin, clarithromycin, metronidazole, tetracycline and levofloxacin. The epsilometer test was performed in accordance with the manufacturer's instructions using aged sheep in a Mueller–Hinton Z agar (MH2-D; bioMerieux) with aged sheep blood. Bacterial suspensions were prepared in Brucella broth and adjusted to an opacity equivalent to a MacFarland turbidity of two. All plates were read after 3–5 days. MICs were measured as the point of intersection of the inhibition ellipse with the epsilometer test strip edge.25 Clarithromycin resistance was defined according to the criteria of the National Committee for Clinical Laboratory Standards (NCCLS), whereas the breakpoint for levofloxacin was selected according to the NCCLS breakpoint used for other Gram-negative isolates.26 Resistance criteria for amoxicillin, metronidazole and tetracycline were defined according to parameters used by other authors.27 Any test in which the quality control MIC fell outside the accepted range was repeated. MIC was defined as the lowest concentration of antibiotics that completely inhibited the growth of the inoculum. The resistant breakpoints were: ≥ 2 µg/mL for amoxicillin; ≥ 2 µg/mL for clarithromycin; ≥ 8 µg/mL for metronidazole; ≥ 8 µg/mL for levofloxacin; > 2 µg/mL for tetracycline. Strains of H. pylori that were resistant to at least two antibiotics were defined as multidrug-resistant H. pylori isolates.

Third-line therapies and follow-up

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Following the evaluation of resistance patterns in the H. pylori isolates, patients were treated with third-line, culture-guided, anti-H. pylori‘rescue’ regimens. These regimens did not include drugs demonstrated to be ineffective by culture. In particular, 89 of the 94 patients, in whom H. pylori isolates were found to be susceptible to tetracycline and amoxicillin, were treated with a 1-week quadruple drug combination including bismuth salts (120 mg; two tablets twice per day, with patients fasted for at least 2 h before the consumption of the bismuth-containing tablets), omeprazole (20 mg; one capsule 30 min before breakfast and dinner), amoxicillin (1 g; one tablet after breakfast and dinner) and doxycycline (100 mg; one tablet after breakfast and dinner). Four patients in whom H. pylori isolates were found to be susceptible to levofloxacin and amoxicillin, but were resistant to tetracycline, metronidazole and clarithromycin, received a 1-week third-line regimen containing omeprazole (20 mg; one capsule 30 min before breakfast and dinner), amoxicillin (1 g; one tablet after breakfast and dinner) and levofloxacin (500 mg; one tablet after breakfast and dinner). Another patient, who harboured H. pylori strains resistant to tetracycline, metronidazole and levofloxacin, was treated with a 1-week regimen including omeprazole (20 mg; one capsule 30 min before breakfast and dinner), amoxicillin (1 g; one tablet after breakfast and dinner) and clarithromycin (500 mg; one tablet after breakfast and dinner). All patients were given a diary to record the side-effects and symptoms during therapy. Side-effects were also documented by a direct questioning approach by an investigator 1 week after treatment. Compliance was measured by counting the tablets returned after the 7-day treatment, together with patient interview. At least 2 months after the end of the third-line treatment, patients underwent 13C-urea breath testing for the assessment of H. pylori status.

Antibiotic resistance data

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Table 2 shows the MIC50 and MIC90 values of the antibiotics tested. The first- and second-line regimens previously given to the 94 enrolled subjects are shown in Table 1. Amoxicillin was included in at least one of the failed eradication regimens in all subjects (100%), metronidazole in 75 (80%), clarithromycin in 72 (77%), levofloxacin in 36 (38%) and tetracycline in 33 (35%). Amongst the H. pylori isolates obtained from the enrolled subjects, 94 (100%) were resistant to metronidazole, 89 (95%) to clarithromycin, 29 (31%) to levofloxacin and five (5%) to tetracycline; none of the H. pylori strains showed resistance to amoxicillin. Overall, in 29 cases (31%), we found a triple resistance: to metronidazole, clarithromycin and levofloxacin in 24 subjects (26%); to metronidazole, clarithromycin and tetracycline in four subjects (4%); and to metronidazole, levofloxacin and tetracycline in one subject (1%). In the remaining 65 subjects (69%), double resistance was found: to clarithromycin and metronidazole in 61 subjects (65%) and to metronidazole and levofloxacin in four subjects (4%). In no case was there resistance to a single antibiotic. Furthermore, isolates were resistant to clarithromycin in 19 of 22 cases (86%), to metronidazole in 19 of 19 cases (100%) and to levofloxacin in five of 55 cases (9%) in which these drugs had not been included in any of the previously failed eradication regimens (primary resistance) (see Table 1).

Table 2.  MIC50 and MIC90 values of the different antibiotics tested
Antimicrobial agentRangeMIC50 (µg/mL)MIC90 (µg/mL)
  1. MIC, minimal inhibitory concentration.

Amoxicillin0.016–0.0320.0160.032
Clarithromycin12–256256256
Metronidazole200–256256256
Tetracycline0.064–10.1250.5
Levofloxacin0.064–320.1258

Eradication of H. pylori and side-effects

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

Drug compliance was excellent: 93 patients (99%) (one patient defaulted follow-up) completed the study medications. Of the subjects treated with the doxycycline- and amoxicillin-based quadruple regimen, one patient interrupted therapy after 3 days of treatment due to the onset of severe side-effects. Eighty-five of the 94 subjects (90%) were H. pylori-negative at follow-up. The quadruple regimen eradicated H. pylori in 81 of the 89 patients treated (92% by per protocol and 91% by intention-to-treat analysis). Four of the five patients (80%, by both per protocol and intention-to-treat analysis) who received a triple regimen (three of the four patients who received the levofloxacin-based regimen and one patient treated with a clarithromycin-based drug combination) were H. pylori-negative at follow-up. Overall, 54 of 94 subjects (57%) reported one or multiple side-effects. Of these, 52 individuals (58%) received the quadruple regimen. In one patient, severe side-effects (diarrhoea, vomiting, malaise) caused the interruption (after only 3 days) of treatment with the quadruple regimen. The number and mean duration of side-effects are shown in Table 3.

Table 3.  Side-effects in each treatment group
Side-effectQuadruple regimen (mean duration in days)Triple regimens (mean duration in days)
Diarrhoea13 (3) 
Headache7 (3) 
Nausea23 (4)1 (3)
Vomiting3 (2) 
Taste disturbance31 (6)2 (4)
Dizzines6 (4) 
Malaise22 (5)1 (3)
Glossitis4 (6) 
Abdominal pain10 (2) 

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References

The increasing problem of failed eradication of H. pylori infection due to the development of resistance is of importance for all physicians treating dyspeptic patients. The results obtained in this study confirm the following: (i) the low estimated rate of resistance to tetracycline (5%) and (ii) the absence of resistance to amoxicillin after two failed treatments; (iii) the effectiveness of a third-line, culture-guided treatment strategy; and (iv) that a doxycycline- and amoxicillin-based quadruple regimen, including omeprazole and bismuth salts, may constitute an effective third-line option for treatment.

Quadruple regimens, including amoxicillin and tetracycline plus a proton pump inhibitor and bismuth salts, have already been demonstrated to be effective as second-line therapy, being capable of overcoming possible metronidazole resistance.28 Although already used in one of the two previous unsuccessful eradication regimens, our findings show that both amoxicillin and tetracycline may also be taken into consideration for a third-line ‘rescue’ treatment of H. pylori infection. In contrast, our data show that 100% and 95% of H. pylori isolates were resistant to metronidazole and clarithromycin, respectively. Therefore, these two drugs cannot be recommended for use in third-line treatment, and clinicians should expect a strong negative impact on the outcome of ‘rescue’ regimens including metronidazole and clarithromycin. Indeed, it has been demonstrated that resistance to these two drugs reduces the probability of eradication when using a drug combination including either of them.19, 29 Furthermore, our data showed high resistance rates to metronidazole and clarithromycin when the previously used regimens did not include either of these two drugs (for clarithromycin, in 86% of cases; for metronidazole, in 100% of cases). These latter findings suggest that a ‘primary resistance’ to clarithromycin and metronidazole may negatively affect a second-line treatment attempt.

Quadruple regimens represent the most widely used ‘rescue’ therapy. They include bismuth salts, which have a synergistic effect on antibiotics, possibly by decreasing the bacterial load, a proton pump inhibitor, which facilitates antibiotic activity by increasing the gastric pH, and tetracycline, an antibiotic for which resistance is not yet a great problem (as also confirmed by our data). Nevertheless, classical quadruple regimens also include metronidazole, an antibiotic for which we found, after two failure attempts, 100% resistance. Therefore, in the quadruple ‘package’, metronidazole has been successfully replaced in some trials by other effective drugs, such as furazolidone23, 30 or rifabutin. In particular, a 2-week rifabutin-based ‘rescue’ therapy (including omeprazole and amoxicillin) has been found to be an encouraging option for treatment after two eradication failures, with key antibiotics such as amoxicillin, clarithromycin, metronidazole and tetracycline (achieving an eradication rate of 79%).20 Other 2-week quadruple regimens have been successfully tested as salvage therapies without culture after multiple failures.31, 32 However, by performing culture after two treatment failures and therefore following the experts' recommendations,9 in the present study, we demonstrated that a culture-guided, third-line, 1-week therapeutic approach is safe and effective against H. pylori. Nevertheless, in the quadruple regimen, we included doxycycline, a tetracycline antibiotic widely used in out-patients, particularly for the treatment of pelvic inflammatory disease, acne and rickettsial infections. In some previous trials, doxycycline-containing therapies have been variously used, always in triple drug combinations. In particular, Perri et al. found that tetracycline (including doxycycline)–amoxicillin combinations were inadequate as therapy, when used with a proton pump inhibitor, for the eradication of H. pylori. In the study by Perri et al., however, doxycycline showed a slightly superior eradication capability with respect to tetracycline (36% vs. 35% by intention-to-treat analysis after 2 weeks of treatment).33 In contrast, Borody et al. showed that tetracycline was superior to doxycycline in triple therapy containing bismuth subcitrate and metronidazole (92% vs. 65%).34 Finally, Realdi et al. found that doxycycline was superior to amoxicillin and clarithromycin when included in a triple regimen containing metronidazole and omeprazole,35 while Heep et al., in a study to determine secondary resistance in H. pylori isolates from patients in whom one or more therapies for the eradication of H. pylori had failed, found 0% resistance to doxycycline.36 To our knowledge, our study is the first to use doxycycline in association with amoxicillin in a quadruple regimen as third-line treatment for H. pylori infection. Quadruple therapy is usually suggested in this situation, but there is some reluctance to use it in clinical practice due to the large number of tablets that need to be taken and concern about side-effects. Recently, a study by Chi et al. has demonstrated that tetracycline and amoxicillin may effectively co-exist in a quadruple, second-line regimen against H. pylori.28 An advantage of doxycycline with respect to tetracycline is that it requires the administration of only two tablets per day, therefore leading to better compliance of patients undergoing third-line ‘rescue’ treatment against H. pylori infection. Interestingly, in the present investigation, all patients completed the study, returning empty medicine boxes, and in no case did the number of tablets per day pose a problem. In addition to achieving a high eradication rate as third-line treatment (91% by intention-to-treat analysis), the quadruple regimen used in this study obtained excellent patient compliance: 99% of patients who received this regimen completed the study.

In contrast, our data indicate a resistance rate to levofloxacin of 31%, with a primary resistance to this drug after two failed treatments of 9%. Levofloxacin has been demonstrated to be effective when included in second-line regimens (in association with a proton pump inhibitor, rifabutin, amoxicillin and tinidazole),37–39 achieving in some studies a better rate of efficacy than standard quadruple regimens.38 In a single pilot study, it has been demonstrated that a 10-day levofloxacin- and amoxicillin-based triple therapy may constitute a promising third-line therapeutic approach for H. pylori eradication.21 Our results confirm that this drug may be taken into consideration for use in a third-line ‘rescue’ regimen, as three of four patients were successfully treated with a levofloxacin-based triple combination.

In conclusion, our data show that a culture-guided, third-line approach allows a high rate of eradication of multidrug-resistant H. pylori isolates to be achieved. Therefore, although some discrepancies between antibiotic susceptibility in vitro and H. pylori eradication in vivo may occur (due, for example, to the possibility of co-infection with different H. pylori strains),40 we emphasize the role of culture guidance to avoid the use of drugs which are likely to be ineffective in a ‘rescue’ therapeutic approach. Furthermore, a doxycycline- and amoxicillin-based quadruple regimen, including omeprazole and bismuth salts, may constitute an attractive option for third-line treatment.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patients
  6. H. pylori culture and antibiotic susceptibility test
  7. Third-line therapies and follow-up
  8. Results
  9. Antibiotic resistance data
  10. Eradication of H. pylori and side-effects
  11. Discussion
  12. References
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