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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Background : Predicting factors for the outcome of conventional Helicobacter pylori triple therapy have been identified. Of these, the presence of the CagA gene is a strong predictor of successful treatment. Our preliminary data show that this factor becomes irrelevant when sequential therapy is used.

Aim : To identify predicting factors for the outcome of H. pylori eradication using two therapeutic schemes (triple and sequential) of equal duration (10 days).

Methods : Ninety-six patients with H. pylori infection were randomly assigned to receive one of the following therapeutic schemes: group A: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) for 5 days, followed by rabeprazole (20 mg b.d.) plus tinidazole (500 mg b.d.) and clarithromycin (500 mg b.d.) for a further 5 days; group B: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) and clarithromycin (500 mg b.d.) for 10 days. Age, sex, smoking, endoscopic and histological findings, and CagA and VacA status were considered as candidates for a model of multivariate analysis which used therapeutic outcome as the dependent variable. CagA and VacA status were assessed by polymerase chain reaction on DNA isolated from gastric antral specimens.

Results : The sequential scheme was significantly more effective than prolonged triple therapy (P < 0.05). Smoking (P < 0.001) and the absence of the CagA gene (P < 0.05) were significantly associated with the failure of triple therapy, but the effectiveness of sequential treatment was not predicted by these factors.

Conclusion : Our data suggest that sequential therapy is not affected by bacterial and host factors which have, until now, predicted the outcome of conventional eradication treatments.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Five years ago, a meta-analysis by Huang and Hunt indicated a better efficacy of current triple therapies for Helicobacter pylori infection in peptic ulcer disease than in non-ulcer dyspepsia.1 A significant difference (about 15%) in eradication rate was reported in this paper and this result was in agreement with that obtained in Mediterranean countries,2, 3 so demonstrating the world-wide nature of the phenomenon. The H. pylori genotype (predominantly CagA positive or cytotoxic in peptic ulcer disease and CagA negative or non-cytotoxic in non-ulcer dyspepsia) has been invoked to explain these differences in the outcome of therapy. After a preliminary report by Van Doorn et al.,4 Broutet et al. clearly stated that cytotoxic strains were more successfully treated than non-cytotoxic bacteria, with a difference of 14% in therapeutic outcome,5 and confirmed that peptic ulcer disease was more easily treated than non-ulcer dyspepsia in a sample of about 3000 patients.6 Recent experience from our geographical area (southern Italy) has corroborated the hypothesis that the CagA gene is a real predictor of H. pylori eradication, whereas other genetic traits of bacterial virulence, such as VacA and iceA, do not affect therapeutic outcome.7 Moreover, this study suggested that some bacteria-induced histological alterations, such as fibrosis, may contribute to the failure of therapy. Other identified risk factors for eradication failure have been reported, including clarithromycin resistance, smoking and low dose of antisecretory drugs.6

Two years ago, we investigated the efficacy of sequential therapy, a scheme which provides a rational use of conventional antibiotics and allows an eradication rate of close to 90% to be achieved,8 i.e. significantly higher than that of triple therapies.9 Moreover, our preliminary experience suggested that sequential therapy was equally effective in CagA-positive and CagA-negative bacteria.10 Recently, the efficacy of sequential therapy has been investigated in 1049 patients enrolled in a multi-centre study.11 In this report, sequential therapy was significantly more effective than 1-week classic triple therapy. It was suggested by de Boer and Tytgat,12 and confirmed by Broutet et al. in a large sample,6 that prolonging the duration of triple therapy improves the therapeutic outcome. Our experience confirms that it is possible to obtain a small increase in the eradication rate with 10-day triple therapy.13

For this reason, the present study was performed to compare the effectiveness of sequential therapy with a triple scheme of equal duration (10 days). Moreover, the study included the evaluation of bacterial and host aspects which have been shown to affect the outcome of eradication therapy.

Patient enrolment

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Patients complaining of symptoms in the upper gastrointestinal tract for at least 1 month, and referred to the endoscopic units located in three Italian towns (Foggia, Bari and Rome) by their primary care physicians for a diagnostic oesophago-gastro-duodenoscopy, were considered for recruitment in this study. Exclusion criteria were: (i) the consumption of proton pump inhibitors, H2-receptor antagonists, antibiotics, bismuth salts or non-steroidal anti-inflammatory drugs during the previous 8 weeks; (ii) a history of gastric surgery; (iii) systemic illness, such as liver cirrhosis, kidney failure or diabetes; (iv) alcohol abuse; (v) pregnancy; (vi) allergy to antibiotics; (vii) gastric cancer; and (viii) reflux oesophagitis. For all patients, written informed consent was obtained, and the protocol of the study was approved by the ethics committees in each centre.

Endoscopy and H. pylori detection

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

On the basis of the endoscopic findings, H. pylori-related diseases were divided into peptic ulcer disease and non-ulcer dyspepsia. Patients with gastric hyperaemia and without macroscopic ulcerative or neoplastic lesions were placed in the non-ulcer dyspepsia group. Two gastric biopsies (body and antrum) were taken for rapid urease test (Cp-test Yamanouchi, Milan, Italy) and four (two from the body and two from the antrum) for histology, using Gram stain for H. pylori and haematoxylin–eosin for microscopic examination of the gastric mucosa, performed blind by two investigators (SM and EI). One antral biopsy was obtained from each patient to investigate the bacterial genetic traits by polymerase chain reaction (PCR) analysis. Precautions were taken to avoid contamination with bacterial DNA during biopsy collection. Instruments were cleaned with a detergent solution, disinfected with a solution that killed bacteria, viruses, fungal mycelia and spores, and rinsed with sterile water after each examination. Biopsy forceps were sterilized by autoclaving. Twenty-four hours after endoscopy, a 13C-urea breath test (Breathmat, Finniganmat, Bremen, Germany) was performed in all patients and breath samples were examined by a single operator (MFM). H. pylori was considered to be present when at least two of the three tests were positive, in agreement with the criteria advised by a panel of experts.14H. pylori eradication after re-treatment was tested by 13C-urea breath test within 6–8 weeks after the end of therapy.

Histology

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Histological features were classified using the updated Sydney system15 and a recent statement by a panel of experts.16 The degree of inflammatory activity (i.e. presence of polymorphonuclear cells in the lamina propria, epithelium and gastric pits) was graded using a semi-quantitative scale (0, absent; 1, low; 2, moderate; 3, severe). Histological features were defined in terms of gastritis activity, atrophy, lymphoid aggregates, fibrosis and intestinal metaplasia. Atrophy was classified as ‘true atrophy’ (with or without metaplasia) and ‘indefinite for atrophy’.

Therapeutic schemes

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Patients were randomized by a computer-generated list to receive one of the following treatments: group A: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) for 5 days, followed by rabeprazole (20 mg b.d.) plus tinidazole (500 mg b.d.) and clarithromycin (500 mg b.d.) for a further 5 days; group B: rabeprazole (20 mg b.d.) plus amoxicillin (1 g b.d.) and clarithromycin (500 mg b.d.) for 10 days.

Rabeprazole was prescribed before breakfast and dinner, whereas the antibiotics were administered after meals. At the end of therapy, side-effects were investigated by a personal interview. At the same time, therapeutic compliance was measured by returned pill count. A minimum pill intake of 90% was considered as acceptable.

DNA extraction and PCR analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Biopsies were placed in sterile vials and transported in dry ice to the collection centre (Bari) in order to avoid contamination and alteration. They were stored at − 80 °C until the extraction of DNA, performed using the method described by Lin et al.17 Briefly, gastric biopsy samples were incubated with lysis buffer and protease K for 2 h at 55 °C. Total DNA was extracted with phenol/chloroform, precipitated with acidic ethanol and dissolved in sterile water.

Amplification of ureA, CagA and VacA

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

The extracted DNA was subjected to PCR for the detection of the following H. pylori genes: (i) urease gene A (ureA), using previously described primers, which amplified a region of 411 base pairs (bp);18 (ii) CagA gene, using the primers described by Tummuru et al., which amplified a region of 400 bp;19 (iii) VacA gene, using primers described by Atherton et al., which evaluated the mid-region (m) and the region encoding for the signal peptide (s) of the gene;20 four different PCR products were obtained: s1 (259 bp) or s2 (286 bp) from the s-region, and m1 (290 bp) or m2 (352 bp) from the m-region.

Sequences were amplified following the PCR protocols described previously.16–18 The PCR products were analysed by electrophoresis on 2% agarose gel (Figure 1). Positive and negative controls were examined with each PCR batch. Positive controls consisted of DNA extracted from an H. pylori suspension (H. pylori strain NCTC 11637) and negative controls of DNA extracted from histological H. pylori-negative gastric specimens.

image

Figure 1. Ethidium bromide-stained agarose gel displaying the CagA fragment (396 bp) of Helicobacter pylori strains. Lanes 1, 2, 3, 4 and 5: CagA-positive samples; lane 6: CagA-negative sample; M: DNA markers.

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Statistical analysis

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Statistical analysis was performed by taking into consideration different variables related to the host [age, sex, smoking habit, endoscopic and histological findings (gastritis activity)] and the bacterium [CagA and VacA (s1-s2, m1-m2 subtypes) status]. A smoking habit was defined as the continuous consumption of more than five cigarettes daily.

Results were examined using the unpaired t-test for age and Fisher's exact test for categorical parameters (eradication rate, pathology, sex, smoking habit, gastritis activity and side-effect prevalence). Differences were considered to be significant at the 5% probability level. Therapeutic results were expressed using both intention-to-treat and per protocol analyses with 95% confidence intervals (95% CI).

Multivariate logistic regression analysis was performed using the therapeutic outcome as the dependent variable and including endoscopic and histological findings, age, sex, smoking habit and CagA/VacA status as candidates for the multivariate model. Variables were kept in the model only if their association with the eradication term improved the fit of the model. The odds ratio (OR) and 95% CI of unsuccessful eradication were calculated in relation to the different variables. For multivariate analysis, the age was considered as a categorical variable, pooling the patients into two distinct groups: < 45 years (young) and > 45 years (old). Moreover, the activity of gastritis was evaluated as a categorical variable, pooling the different grades as follows: grades 0–1, low activity; grades 2–3, high activity.

Statistical analysis was performed using statistical software (Statsoft 6.0 program for Windows 98; Microsoft, Redmond, WA, USA).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Ninety-seven patients were included in the study: 45 in group A (sequential therapy) and 52 in group B (10-day triple therapy). The baseline demographic and clinical characteristics of the enrolled patients are reported in Table 1. As shown, the groups were homogeneous with regard to sex, age, smoking habit and endoscopic findings (peptic ulcer disease/non-ulcer dyspepsia).

Table 1.  Baseline demographic and clinical characteristics of the enrolled patients
 Treatment group
AB
Number of patients4552
Age (years) (mean ± s.d.)44.2 ± 16.246.0 ± 15.3
Sex (male/female)20/2521/31
Smoking habit (yes/no)15/3017/35
Endoscopic findings
 Non-ulcer dyspepsia3635
 Normal findings1514
 Hyperaemia2220
 Antral chronic erosions911
 Peptic ulcer disease911
Histological findings
 Antral active chronic gastritis
  Low activity1928
  High activity2624
 Active chronic pangastritis45
 Atrophy53
 Intestinal metaplasia42
 Lymphoid aggregates13
 Fibrosis23
Helicobacter pylori genes
 CagA (positive/negative)36/939/13
 VacA
  s1/s225/2020/32
  m1/m220/2517/35

Endoscopic and histological findings

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

At endoscopy, 71 patients (73.1%) were diagnosed with non-ulcer dyspepsia (32 patients with gastric hyperaemia, 20 with gastric erosions, 19 without macroscopic abnormalities), and 26 patients (26.8%) were diagnosed with peptic ulcer disease. Histologically, all patients showed active antral gastritis. Of these, only nine (9.2%) showed active inflammation involving the gastric body (pangastritis). In eight patients (8.2%), ‘true atrophy’ was observed, six with associated intestinal metaplasia. Lymphoid aggregates were observed in four patients (4.1%), whilst mucosal fibrosis was evident in five patients (5.1%). The small number of cases showing pangastritis, lymphoid aggregates, atrophy, intestinal metaplasia and fibrosis did not enable us to include these findings in our statistical evaluation.

Treatment outcome

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

Ninety-five patients completed the study [two (2%) did not return after therapy for unreported reasons (one peptic ulcer disease in group A, one non-ulcer dyspepsia in group B)].

Overall, H. pylori eradication was obtained in 95.5% (95% CI, 89.53–101.58) and 97.7% (95% CI, 93.32–102.13) of patients in group A, and in 80.7% (95% CI, 70.06–91.48) and 82.3% (95% CI, 71.89–92.81) of patients in group B (intention-to-treat and per protocol analyses, respectively) (P < 0.05).

Using triple therapy, by per protocol analysis, eradication was achieved in all 17 (five smokers and 12 non-smokers) peptic ulcer disease patients and 25 (four smokers and 21 non-smokers) of 34 non-ulcer dyspepsia patients (five smokers and 29 non-smokers) (100% vs. 73.5%, P < 0.05); in 34 of 38 CagA-positive and eight of 13 CagA-negative strains (89.4% vs. 61.5%, P < 0.05); and in 33 (12 peptic ulcer disease and 21 non-ulcer dyspepsia) of 34 non-smokers (12 peptic ulcer disease and 22 non-ulcer dyspepsia) and nine (five peptic ulcer disease and four non-ulcer dyspepsia) of 17 smokers (five peptic ulcer disease and 12 non-ulcer dyspepsia) (97.0% vs. 52.9%, P < 0.001).

These differences were not observed using the sequential regimen, which allowed H. pylori eradication to be obtained in all eight (three smokers and five non-smokers) peptic ulcer disease patients and 35 (11 smokers and 24 non-smokers) of 36 non-ulcer dyspepsia patients (12 smokers and 24 non-smokers) (100.0% vs. 97.2%, P = N.S.); in 34 of 35 CagA-positive and all nine CagA-negative strains (97.1% vs. 100.0%, P = N.S.); and in all 29 (five peptic ulcer disease and 24 non-ulcer dyspepsia) non-smokers and 14 (three peptic ulcer disease and 11 non-ulcer dyspepsia) of 15 smokers (three peptic ulcer disease and 12 non-ulcer dyspepsia) (100.0% vs. 93.3%, P = N.S.).

In detail, the sequential regimen was more effective than triple therapy in non-ulcer dyspepsia (97.2% vs. 73.5%, P < 0.01), in smokers (93.3% vs. 52.9%, P < 0.05) and in the presence of histological findings of gastritis with mild activity (100.0% vs. 77.7%, P < 0.05). Age and sex did not affect the eradication efficacy of the two regimens.

Overall, the eradication and failure rates for each considered variable are reported in Table 2. Multivariate logistic regression revealed that only smoking showed a negative and significant correlation with the success of eradication (P < 0.01; r = − 0.3; OR = 6.0; 95% CI, 1.4–26.5).

Table 2.  Multivariate analysis: age, sex, smoking habit, endoscopic and histological findings, and CagA and VacA status are considered as candidates for a model which uses therapeutic outcome as the dependent variable
 Eradication (89.4%)Failure (10.6%)P
Treatment
 Sequential therapy (44)43 (97.7%)1 (2.3%) 
 Triple therapy (51)42 (82.3%)9 (17.7%)< 0.05
Age
 < 45 years (44)39 (88.6%)5 (11.4%) 
 > 45 years (51)46 (90.1%)5 (9.9%)N.S.
Sex
 Male (40)36 (88.6%)4 (10.0%) 
 Female (55)49 (89.0%)6 (11.0%)N.S.
Smoking habit
 Smokers (32)23 (71.8%)9 (28.2%) 
 Non-smokers (63)62 (98.4%)1 (1.6%)< 0.001
Endoscopic finding
 Non-ulcer dyspepsia (70)60 (85.7%)10 (14.3%) 
 Peptic ulcer disease (25)25 (100%)0 (0%)N.S.
Histological finding
 Degree of activity of gastritis
  Low (46)40 (86.9%)6 (13.9%) 
  High (49)45 (91.8%)4 (8.2%)N.S.
CagA status
 Positive (73)68 (93.1%)5 (6.9%) 
 Negative (22)17 (77.2%)5 (22.2%)< 0.05
VacA status
 s1 (44)40 (90.9%)4 (9.1%) 
 s2 (51)46 (90.1%)5 (9.9%)N.S.
 m1 (37)33 (89.1%)4 (10.9%) 
 m2 (58)52 (89.6%)6 (10.4%)N.S.

H. pylorigenotyping

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

CagA positivity was found in 75 of the 97 patients (77.3%); VacA alleles were divided in our patients as follows: s1 in 45 of 97 (46.3%), s2 in 52 of 97 (53.6%); m1 in 37 of 97 (38.1%) and m2 in 60 of 97 (61.8%). The VacA s1 allele was associated with CagA status in 42 of 45 cases (93.3%). Twenty-five of the 26 patients with peptic ulcer disease were infected with a CagA-positive strain, whereas this association involved only 39 of 71 subjects with non-ulcer dyspepsia (96.1% vs. 54.9%, P < 0.0001). Similarly, the s1 allele was observed in 23 of 26 patients with peptic ulcer disease but in only 22 of 71 patients with non-ulcer dyspepsia (88.4% vs. 30.9%, P < 0.0001). Finally, the distribution of m1/m2 alleles was not significantly different between patients with peptic ulcer disease and non-ulcer dyspepsia. A high degree of gastritis severity was observed in 59 of 75 (78.6%) CagA-positive and in 35 of 45 (77.7%) VacA s1-positive patients; this was statistically significant when compared with CagA-negative (P < 0.0001) and s2-positive (P < 0.0001) patients, respectively. No significant association was found between gastritis activity and m1/m2 alleles.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References

In this paper, we compared the effectiveness of two therapeutic schemes for H. pylori eradication (triple and sequential), with equal duration (10 days), taking into account bacterial and host features which have been shown to affect the results of eradication therapy.

Our main finding was that the CagA status, which has been reported by our group7 and others4, 5 to be an important predictor of successful outcome for 7-day triple therapy, has an effect on the efficacy of this therapeutic scheme, even when prolonged to 10 days, despite the possible increase in eradication rate. However, the CagA status is irrelevant when sequential treatment is used. In a preliminary study, we reported that sequential therapy was equally effective for CagA-positive and CagA-negative strains in patients with non-ulcer dyspepsia,10 but this study was limited by the fact that the H. pylori cytotoxic status had been assessed by serology, which does not provide information on the actual infection in the stomach and therefore cannot be considered as a reliable technique. In the present study, however, the CagA gene was detected by polymerase chain reaction directly on biopsy specimens. This method has a sensitivity similar to that of colony hybridization,21 and therefore allows very reliable information to be obtained.

From a biological point of view, the relationship between the outcome of eradication therapy and CagA status has been explained by the severity of gastritis due to cytotoxic strains; in this case, mucosal hyperaemia may favour antibiotic diffusion through an increased blood flow.22 Another mechanism that has been invoked is the increased antibiotic activity on dividing cells; this capacity may be increased in the case of CagA strains by their rapid growth.4, 23 Our experience agrees with both of these hypotheses. Indeed, a good correlation between CagA positivity and high histological scores of gastritis severity was confirmed by the results of this study. Moreover, we have previously observed that semi-quantitative measurements of bacterial load show significantly higher values in cytotoxic than non-cytotoxic strains, thus suggesting faster replication.24

Nevertheless, our study clearly showed that the eradication rate of the sequential scheme was unaffected by the bacterial strain or gastritis severity. The reason why a short-period double therapy followed by a short-period triple therapy is equally effective in both CagA-positive and CagA-negative strains is unclear, although some hypotheses may be proposed.

A possible explanation may be based on the key role of amoxicillin in the first phase of treatment. Indeed, the relevance of this step to the therapeutic outcome is confirmed by evidence indicating that a sequence consisting of triple followed by double therapy shows a poor performance when compared with the inverse sequence.25 Moreover, it is well known that short-term double therapy with a proton pump inhibitor and amoxicillin can eradicate H. pylori in about 50% of infected patients and reduce its load in the remaining cases.26 This reduction in bacterial amount with double therapy may improve the response to the subsequent short course of triple therapy; indeed, amoxicillin can destroy the wall of bacterial cells, increasing the intracellular diffusion of the macrolide, with a consequent improved outcome.

Furthermore, resistance to amoxicillin is extremely rare and has not yet been demonstrated in vivo, as it requires more than one mutation in the bacterial genome;27, 28 it has been found that regimens containing this antibiotic may prevent the selection of clarithromycin-resistant strains.28 On the other hand, clarithromycin resistance has been reported as a very strong predictor of the failure of triple therapy, independent of CagA status.5 Although this aspect was not investigated in the present study, previous experience has shown that sequential treatment is much more effective than standard triple therapy (77.8% vs. 16.7%) in the eradication of clarithromycin-resistant strains.11 Therefore, the capacity of sequential therapy to eradicate even clarithromycin-resistant strains could help to explain our results.

Sequential treatment was equally effective in patients with peptic ulcer disease and non-ulcer dyspepsia, suggesting that this scheme, unlike triple therapy, does not discriminate between CagA-positive and CagA-negative H. pylori strains.

In a previous report, we observed that histologically proven fibrosis may predict the failure of triple therapy probably as a result of a decrease in antibiotic diffusion.7 In the present study, the prevalence of this finding was very low (< 10%) and therefore it was not considered in our analysis.

Finally, our results confirmed that smoking may predict the failure of triple therapy, as multivariate logistic regression showed a significantly negative correlation of this factor with the success of eradication. To explain the better performance of treatment in non-smokers vs. smokers, it has been hypothesized that smoking may induce a decrease in gastric blood flow and mucus secretion, and therefore reduce the efficacy of treatment by reducing the delivery of antibiotics to the gastric mucosa.29, 30 In particular, triple therapy seems to fail in non-ulcer dyspepsia patients who are smokers. A possible explanation for this observation may be that non-ulcer dyspepsia is associated with less marked inflammation and therefore with a further reduction in mucosal blood flow. Nevertheless, even smoking did not change the outcome of sequential treatment.

In conclusion, our study shows that sequential therapy is more effective than triple therapy, even when the latter is prolonged. Moreover, the high eradication rate of sequential therapy is not affected by the factors known to affect the results of conventional triple therapy. Therefore, it may be argued that the marked increase in eradication obtained by this new modality of treatment may cancel out the statistical relevance of predicting factors. On the basis of our results, further studies should be encouraged in order to confirm whether, finally, we have at our disposal a treatment resulting in a very satisfactory outcome that is not affected by the bacterial and host factors known to affect the results of conventional triple therapy.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Patient enrolment
  6. Endoscopy and H. pylori detection
  7. Histology
  8. Therapeutic schemes
  9. DNA extraction and PCR analysis
  10. Amplification of ureA, CagA and VacA
  11. Statistical analysis
  12. Results
  13. Endoscopic and histological findings
  14. Smoking habit
  15. Treatment outcome
  16. H. pylorigenotyping
  17. Discussion
  18. References
  • 1
    Huang JQ, Hunt RH. Are one-week anti-Helicobacter pylori treatments more effective in patients with peptic ulcer disease than in those with non ulcer dyspepsia? A meta-analysis. Am J Gastroenterol 1998; 93: 1639.
  • 2
    De Francesco V, Sgarro C, Cela E, et al. Helicobacter pylori eradication rates in non-ulcer dyspepsia (NUD) and duodenal ulcer (DU) patients. Gut 2001; 48(Suppl. 11): A94(Abstract).
  • 3
    Gisbert JP, Marcos S, Gisbert JL, Pajares JM. Helicobacter pylori eradication therapy is more effective in peptic ulcer than in non-ulcer dyspepsia. Eur J Gastroenterol Hepatol 2001; 13(11): 13037.
  • 4
    Van Doorn LJ, Schneeberger PM, Nohuan N, Plaisier AP, Quint WGV, De Boer WA. Importance of Helicobacter pylori cagA and vacA status for the efficacy of antibiotic treatment. Gut 2000; 46: 3216.
  • 5
    Broutet N, Marais A, Lamouliatte H, et al. cag A status and eradication treatment outcome of anti-Helicobacter pylori triple therapies in patients with non-ulcer dyspepsia. J Clin Microbiol 2001; 39(4): 131922.
  • 6
    Broutet N, Tchamgoué S, Pereira E, Lamouliatte H, Salamon R, Mègraud F. Risk factors for failure of Helicobacter pylori therapy — results of an individual data analysis of 2751 patients. Aliment Pharmacol Ther 2003; 17: 99109.
  • 7
    Russo F, Berloco P, Cuomo R, et al. Helicobacter pylori strains and histologically related lesions affect the outcome of triple eradication therapy: a study from southern Italy. Aliment Pharmacol Ther 2003; 17: 18.
  • 8
    Zullo A, Rinaldi V, Winn S, et al. A new highly effective short-term therapy schedule for Helicobacter pylori eradication. Aliment Pharmacol Ther 2000; 14: 7158.
  • 9
    De Francesco VA, Zullo A, Hassan C, et al. Two new treatment regimens for Helicobacter pylori eradication: a randomised study. Digest Liver Dis 2001; 33(8): 6769.
  • 10
    De Francesco V, Panella C, Margiotta M, Faleo D, Ierardi E. Sequential eradicating therapy: a treatment that does not discriminate Helicobacter pylori strains in patients with non-ulcer dyspepsia? Am J Gastroenterol 2002; 97: 910.
  • 11
    Zullo A, Vaira D, Vakil N, et al. High rates of Helicobacter pylori with new sequential treatment. Aliment Pharmacol Ther 2003; 17: 71926.
  • 12
    De Boer WA, Tytgat GN. Should anti Helicobacter therapy be different in patients with dyspepsia compared with patients with peptic ulcer diathesis? Eur J Gastroenterol Hepatol 2001; 13: 12814.
  • 13
    De Francesco V, Ierardi E, Zullo A, et al. Triple therapy prolongation is of doubtful advantage for Helicobacter pylori eradication. Digest Liver Dis 2003; 35(4S): S104.
  • 14
    Working Party of the European Helicobacter pylori Study Group. Technical annex: tests used to assess Helicobacter pylori infection. In: Guidelines for clinical trials in Helicobacter pylori infection. Gut 1997; 41(2S): S108.
  • 15
    Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston, 1994. Am J Surg Pathol 1996; 20: 116181.
  • 16
    Rugge M, Correa P, Dixon MF, et al. Gastric mucosal atrophy: interobserver consistency using new criteria for classification and grading. Aliment Pharmacol Ther 2002; 16: 124959.
  • 17
    Lin TT, Yeh CT, Yang E, Chen PC. Detection of Helicobacter pylori by polymerase chain reaction assay using gastric biopsy specimens taken for CLO-test. J Gastroenterol 1996; 31: 32932.
  • 18
    Clayton CL, Kleanthous H, Coates PJ, Morgan DD, Tabaqchali S. Sensitive detection of Helicobacter pylori by using polymerase chain reaction. J Clin Microbiol 1992; 30: 192200.
  • 19
    Tummuru MKR, Cover TL, Blaser MJ. Cloning and expression of a high molecular mass major antigen of Helicobacter pylori. Infect Immun 1993; 61: 1799809.
  • 20
    Atherton JC, Cao P, Peek RM, Tummuru MKR, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori: association of specific vacA types with cytotoxin production and peptic ulcer secretion protein. J Biol Chem 1995; 270: 17 771–7.
  • 21
    Labigne A, Lamouliatte H, Birac C. Distribution of the CagA gene among Helicobacter pylori strains associated with peptic ulcer. Am J Gastroenterol 1994; 89: 1326.
  • 22
    Maeda S, Yoshida T, Ikenoue T, et al. Structure of cag pathogenicity island in Japanese Helicobacter pylori isolates. Gut 1999; 44: 33641.
  • 23
    Censini S, Lange Z, Xiang JE, et al. Cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA 1996; 93: 14 648–53.
  • 24
    Ierardi E, Margiotta M, Monno R, et al. A new semiquantitive method of quantifying Helicobacter pylori. J Clin Gastroenterol 2002; 35: 3758.
  • 25
    Rinaldi V, Zullo A, Pugliano F, Valente C, Diana F, Attili AF. The management of failed dual or triple therapy for Helicobacter pylori eradication. Aliment Pharmacol Ther 1997; 11: 92933.
  • 26
    Moschkowitz FM, Peled Y, Santo M, et al. Helicobacter pylori numbers are associated with low eradication rate after triple therapy. Gut 1995; 36: 8457.
  • 27
    Mégraud F, Lamouliatte H, Delchier JC., et al. Helicobacter pylori resistance to antimicrobial agents after failure of an initial eradication treatment and impact on results of second-line treatment strategies: a multicenter prospective study. Gut 2001; 49(Suppl. II): A83(Abstract).
  • 28
    Paul R, Postius S, Melchers K, Schafer KP. Mutations of Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin. Antimicrob Chemother 2001; 45: 9625.
  • 29
    Iwao T, Toyonaga A, Ikegami M, et al. Gastric mucosal blood flow after smoking in healthy human beings assessed by laser Doppler flowmetry. Gastrointest Endosc 1993; 39: 4003.
  • 30
    Kamada T, Haruma K, Miyoshi E, et al. Cetraxate, a mucosal protective agent, combined with omeprazole, amoxycillin and clarithromycin increases the eradication rate of Helicobacter pylori in smokers. Aliment Pharmacol Ther 2000; 14: 108994.