Helicobacter pylori strains and histologically-related lesions affect the outcome of triple eradication therapy: a study from southern Italy
Correspondence to: Dr F. Russo, Laboratory of Biochemistry, IRCCS ‘Saverio de Bellis’, 70013 Castellana Grotte, Bari, Italy. E-mail: email@example.com
Background : Certain evidence suggests that Helicobacter pylori strains expressing genes for cytotoxin production show a higher sensitivity than non-cytotoxic organisms to eradication treatment. No data are available on the involvement of bacterium-related lesions in different therapeutic outcomes.
Aims : (i) To investigate whether differences in eradication rates may be related to the different expression of virulent strains (cagA, vacA, iceA) in patients undergoing proton pump inhibitor-based triple therapy, and (ii) to evaluate whether therapeutic outcome may be affected by bacterium-induced gastric lesions.
Methods : One hundred and ten H. pylori-positive subjects were enrolled. H. pylori was genotyped by polymerase chain reaction. Treatment consisted of lansoprazole–amoxicillin–clarithromycin, twice daily for 1 week. Eradication was checked by urea breath test.
Results : The eradication rate was 70%, and the absence of cagA was associated with unsuccessful treatment. No difference between the groups with successful and unsuccessful eradication was found with regard to vacA and iceA. Lympho-epithelial lesions and fibrosis were associated with unsuccessful treatment.
Conclusions : The present data confirm the importance of cagA (but not vacA and iceA) as a predictor of successful eradication. When fibrosis and lympho-epithelial lesions are present, therapy appears to be less effective. Therefore, these histological features may be involved in an unsuccessful therapeutic outcome.
Helicobacter pylori infection is associated with chronic gastritis and its sequelae. 1 Different genetic traits affect its virulence. Of these, the most important is the cagA gene, 2, 3 which codes for a hydrophilic surface exposed protein (CagA) expressed by 60–80% of strains. 4 This gene has been proposed as a marker of a ‘pathogenicity island’ associated with increased bacterial density, mucosal inflammation and cytokine release. 5 Another cytotoxin that injures epithelial cells is encoded by vacA , which is present in all H. pylori strains and contains two variable regions. The s -region (encoding the signal peptide) exists as s1 or s2 allelic types and the m -region (middle) as m1 or m2 . vacA type s1 has been demonstrated to be associated with cytotoxin activity in vitro , peptic ulcers and cagA expression. 6 In addition, H. pylori strains showing cagA positivity and vacA s1 provoke the most severe histological changes. 7 Recently, another gene, designated iceA (induced by contact with epithelium), 8 with two main allelic variants, iceA1 and iceA2 , has been reported to be independent of cagA and vacA status. Although some authors have suggested a significant association between iceA1 and peptic ulcers, 9 its exact role still remains controversial. 10, 11
Proton pump inhibitor-based triple therapy, one of the most commonly used therapeutic schemes, has been shown to produce H. pylori eradication rates that are not completely satisfactory,12 and compliance with therapy has been proposed as a significant variable affecting the outcome. Nonetheless, 1-week proton pump inhibitor-based triple therapy has been reported to show a uniformly high compliance,13 and therefore a more detailed analysis of other causes of treatment failure/success may be helpful. Recent evidence has suggested that different eradication rates are obtained according to the genetic characteristics of the H. pylori strains,14, 15 probably as a result of different interaction with the host.16 van Doorn et al. reported a higher cure rate in cagA/vacA s1 strains, and postulated that the s1 region produces more toxin, induces more inflammation and provides for an easier eradication of H. pylori as high concentrations of antibiotics are achieved in infected tissues.9 This hypothesis suggests an involvement of histological damage in therapeutic outcome, although no clear details are available on this topic. However, in some reports, no correlation has been found between cagA/vacA status and the eradication rate.17 Finally, no data are available on the therapeutic outcome in relation to iceA.
Differences in the predominant strain in different geographical regions may account for the different therapeutic results.18 In southern Italy, the prevalence of infection, as tested using immunoglobulin G antibodies against H. pylori, has been proven to be remarkably high,19 but the potentially different susceptibility to triple therapy in relation to particular virulence factors still needs to be investigated.
Thus, starting from this basis, the aims of the present study were: (i) to investigate whether differences in eradication rates are related to bacterial virulence genes (cagA, vacA and iceA) in H. pylori-infected patients in southern Italy undergoing standardized proton pump inhibitor-based triple therapy; and (ii) to evaluate whether therapeutic outcome is affected by bacterium-induced endoscopic and histological damage.
The target population in our study included H. pylori-infected subjects complaining of dyspeptic symptoms who had not been treated previously with proton pump inhibitor-based eradication therapy. One hundred and ten symptomatic patients (61 males and 49 females; median age, 49.5 years; range, 23–86 years), referred by their primary care physician for diagnostic upper endoscopy at our institute, participated in the study. Patients were considered to be eligible if they had not received non-steroidal anti-inflammatory drugs (NSAIDs), antibiotics, bismuth, antacids, H2-receptor antagonists, proton pump inhibitor, sucralfate or misoprostol in the 2 months before the examination, and had no previous history of gastric tumours or gastric surgery.
All patients underwent endoscopy and biopsy performed by two gastroenterologists who independently described the endoscopic appearance of the stomach. An Olympus GIF 100 endoscope (Olympus Corporation of America, Lake Success, NY, USA) was used in all cases. Precautions were taken to avoid contamination with bacterial DNA during biopsy collection. Instruments were cleaned with a detergent solution, disinfected with a solution against bacteria, viruses, fungal mycelia and spores, and rinsed with sterile water after each examination. Biopsy forceps were sterilized by autoclaving.
The endoscopic appearance was classified as ‘non-ulcer dyspepsia’ when no macroscopic mucosal abnormality, non-reflux oesophagitis or hiatus hernia was reported (non-ulcer dyspepsia group). If the endoscopic examination showed peptic ulcers or ulcer scars (as a sign of earlier ulcer presence), the patient was considered to have peptic ulcer disease (peptic ulcer disease group).20
Two antral (about 2 cm from the pylorus) and two body (small curve) biopsy specimens for histology, one antral specimen for intra-biopsy urease activity and one antral biopsy for polymerase chain reaction (PCR) analysis were obtained from each patient.
Histological features (haematoxylin–eosin stain) were classified according to the updated Sydney system,21 or a recent statement by a panel of experts.22 Additional Warthin–Starry stain was performed to confirm the presence of H. pylori on the mucosal stream of mucus and foveolar epithelium. Histological findings were assessed in terms of activity (polymorphonuclear cell infiltration), degradation of the mucus layer, lymphoid follicle formation, lympho-epithelial lesions, fibrosis and intestinal metaplasia.
Fibrosis was described as an ‘increase in the interglandular extracellular matrix’. In addition, a more specific stain (Azan–Mallory) was performed in cases of doubtful interpretation. Lympho-epithelial lesions were reported as ‘more than seven lymphocytes per 100 cells in gastric epithelium in the presence or absence of focal erosions’. The picture of atrophy was classified into ‘true’ atrophy (with and without intestinal metaplasia) and ‘indefinite for atrophy’.
The degrees of activity and lymphoid follicle formation were graded using a semi-quantitative scale (0, absent; 1, low; 2, moderate; 3, high). For further analysis, grades 0–1 and grades 2–3 were pooled together.
Assessment of H. pylori status
H. pylori status was determined by the rapid urease test, histology and PCR analysis. H. pylori infection was diagnosed in all patients if the results were positive with at least two methods.
DNA extraction and PCR analysis
The extraction of DNA from biopsy specimens was performed using the method described by Lin et al.23 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, vacA and iceA. 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);24 (ii) cagA gene, using the primers described by Tummuru et al., which amplified a region of 400 bp;25 (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;6 four different PCR products were obtained: s1 (259 bp) and s2 (286 bp) from the s-region, and m1 (290 bp) and m2 (352 bp) from the m-region; (iv) iceA gene; allele-specific PCR assays were used for iceA1 and iceA2; for the amplification of the iceA1 allele, forward primer iceA1-F and reverse primer iceA1-R yielded a fragment of 246 bp; for iceA2, primers iceA2-F and iceA2-R yielded a fragment of either 229 or 334 bp, because some iceA2 alleles contain a repeat sequence of 105 nucleotides encoding 35 amino acids.
Sequences were amplified following PCR protocols described previously.23–25 PCR products were analysed by electrophoresis on 2% agarose gel. Positive and negative controls were examined with each batch of PCR products. Positive controls consisted of DNA extracted from an H. pylori suspension (H. pylori strain NCTC 11637) and negative controls of DNA extracted from histologically H. pylori-negative gastric specimens.
Each subject received classic proton pump inhibitor-based triple therapy. The eradication regimen included lansoprazole, 30 mg twice daily, clarithromycin, 500 mg twice daily, and amoxicillin, 1000 mg twice daily, all for 1 week. Eradication was checked by urea breath test 4–6 weeks after therapy. H. pylori infection was considered to be present if the difference between the 13C/12C baseline value and the 30-min value exceeded 5.0‰.
Comparison of categorical data (H. pylori genotype, endoscopic and histological findings) between groups of patients with successful and unsuccessful eradication treatment was performed using the chi-squared test. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated to evaluate the OR of unsuccessful eradication in relation to different variables. To evaluate whether infection with particular H. pylori genotypes increased the risk of unsuccessful treatment, multiple logistic regression was performed. All variables (age and sex of patients, endoscopic findings, histological results, cagA gene expression, vacA and iceA alleles) were considered as candidates for the multivariate models. Variables were kept in the model only if their association with the eradication term improved the fit of the model. Age was considered as a continuous variable, whereas sex (male or female), endoscopic appearance of the gastric mucosa (non-ulcer dyspepsia, peptic ulcer disease), histological findings (degree of activity, degradation of mucus layer, fibrosis, atrophy and lympho-epithelial lesions) and H. pylori genotyping [cagA (positive and negative), vacA (s1 and s2 segment), iceA (A1 and A2 alleles)] were scored as categorical variables. Statistical analysis was performed with the statistical software package STATA 6.
Endoscopic and histological findings
At endoscopy, 45 (40.9%) patients were diagnosed with non-ulcer dyspepsia (eight patients showed no macroscopic mucosal abnormality and 37 showed mucosal hyperaemia, oedema or a nodular appearance) and 65 (59.1%) patients were diagnosed with peptic ulcer disease (seven showed endoscopic signs of gastric ulcer and 58 of duodenal ulcer).
Histologically, two patients had minimal non-specific changes, whereas 108 had evidence of chronic gastritis. This feature was confined to the antrum in most patients (82/110; 74.5%), and only twenty-eight (25.5%) suffered from histological pan-gastritis. Seventy-nine patients showed atrophy. In detail, 38 (34.5%) patients showed a picture of atrophy, which was associated with intestinal metaplasia in thirty-six cases. In the remaining 41 patients (37.3%), histology was indefinite for atrophy. Prominent lymphoid follicle formation (scores 2–3) was detected in 30 patients (27.3%), lympho-epithelial lesions in 23 (20.9%), degradation of the mucus layer in 72 (65.4%), high-grade activity (scores 2–3) in 52 (47.3%) and fibrosis in 60 (54.5%). Fibrosis was present in 34 of the 38 patients with atrophy (four patients, all with intestinal metaplasia, showed atrophy without fibrosis). Therefore, fibrosis without atrophy was observed in 26 patients. The degree of fibrosis was associated with a predominant chronic inflammation (in all patients, an increase in the extracellular matrix was confirmed by Azan–Mallory stain). Atrophy, fibrosis and intestinal metaplasia were exclusively found in the antrum.
H. pylori genotyping
cagA was present in 91 of the 110 (82.7%) patients, whereas vacA alleles were distributed as follows: vacA s1 in 87 of 110 (79.1%), vacA s2 in 21 of 110 (19.1%) and vacA mixed forms in two of 110 (1.8%). The predominant vacA genotype was s1 and it was always associated with the presence of the cagA gene (82/110 patients; 74.5%). iceA1 was detected in 40 of 110 (36.4%) isolates examined. iceA2 was found in 51 isolates (46.4%). Twelve isolates (10.9%) were positive for both iceA1 and iceA2 , and seven (6.4%) did not yield any product for iceA .
With regard to the relationship with the endoscopic findings, both cagA expression and the vacA s1 allele were significantly associated with the presence of peptic ulcer disease (P = 0.0098 and P = 0.0086, respectively; chi-squared test). No significant association resulted for the iceA genotypes (Table 1).
Table 1. Relationship between Helicobacter pylori genotyping and antral endoscopic findings in 110 dyspeptic patients
|cagA (+) ( n = 91) ||5 (5.5)||54 (59.3)||32 (35.2)|
|cagA (–) ( n = 19) ||2 (10.5)||4 (21.0)||13 (68.5) |
P = 0.0098‡
|vacA s1 ( n = 87) ||5 (5.7)||51 (58.6)||31 (35.7)|
|vacA s2 ( n = 21) ||2 (9.5)||5 (23.8)||14 (66.7)|
|vacA mixed forms ( n = 2) ||0 (0.0)||2 (100.0)|| 0 (0.0) |
P = 0.0086‡
|iceA1 ( n = 40) ||3 (7.5)||20 (50.0)||17 (42.5)|
|iceA2 ( n = 51) ||3 (5.9)||25 (49.0)||23 (45.1)|
|iceA mixed forms ( n = 12) ||1 (8.3)||10 (83.4)|| 1 (8.3)|
|iceA not detected ( n = 7) ||0 (0.0)||3 (42.9)||4 (57.1) |
P = 0.335‡
Histologically, the presence of atrophy was not significantly associated with particularH. pylori alleles. In contrast, a high grade of inflammation, prominent lymphoid follicle formation (scores 2–3) and the degradation of the mucus layer were significantly associated with cagA positivity (P < 0.05; chi-squared test), but not with particularvacA or iceA alleles (Table 2).
Table 2. Relationship between Helicobacter pylori genotyping and histology in 110 dyspeptic patients
|cagA (+) ( n = 91) ||31||36||24||43/48*||62/29*||15/76*||68/23*||50/41||28/63|
|cagA (–) ( n = 19) || 7|| 5|| 7||15/4||18/1|| 8/11|| 4/15||10/9|| 8/11|
|vacA s1 ( n = 87) ||31||35||21||47/40||62/25||16/71||60/27||47/40||30/57|
|vacA s2 ( n = 21) || 7|| 6|| 8||10/11||16/5|| 6/15||12/9||12/9|| 6/15|
|Mixed forms (n = 2)|| 0|| 0|| 2|| 1/1|| 2/0|| 1/1|| 0/2|| 1/1|| 0/2|
|iceA1 ( n = 40) ||18||14|| 8||27/13||27/13|| 9/31||30/10||21/19|| 8/32|
|iceA2 ( n = 51) ||14||19||18||23/28||40/11||11/40||31/20||30/21||23/28|
|Mixed forms (n = 12)|| 5|| 5|| 2|| 4/8|| 7/5|| 2/10|| 7/5|| 7/5|| 3/9|
|Not detected (n = 7)|| 1|| 3|| 3|| 4/3|| 6/1|| 1/6|| 4/3|| 2/5|| 2/5|
No patient in the study dropped out before the determination of the H. pylori eradication status. Successful eradication was achieved in 77 of 110 patients (70.0%). There were no differences in age (52.27 ± 14.07years vs. 48.39 ± 11.20 years; mean ± s.d.) or sex (36 females/41 males vs. 13 females/20 males) between the successful and unsuccessful eradication groups.
Table 3 reports the endoscopic and histological findings categorized according to the treatment outcome. With regard to the relationship between endoscopic lesions and rates of eradication, a higher, but not significantly higher ( P = 0.29; chi-squared test), percentage of patients with peptic ulcer disease were present in the successful eradication group than in the unsuccessful eradication group.
Table 3. Endoscopic and histological findings in patients with successful or unsuccessful eradication of Helicobacter pylori
| Non-ulcer dyspepsia||29 (64.4)||16 (35.6)|| |
| Peptic ulcer disease||48 (73.8)||17 (26.2)||0.29|
| No||51 (68.9)||23 (31.1)|| |
| Yes||26 (72.2)||10 (27.8)||0.723|
| Absent||20 (64.5)||11 (35.5)|| |
| Indefinite||27 (65.8)||14 (34.2)|| |
| True||30 (78.9)||8 (21.1)||0.320|
| Low grade (score 0–1)||40 (69.0)||18 (31.0)|| |
| High grade (score 2–3)||37 (71.1)||15 (28.9)||0.803|
|Lymphoid follicle formation|
| Low grade (score 0–1)||56 (70.0)||24 (30.0)|| |
| High grade (score 2–3)||21 (70.0)||9 (30.0)||1.00|
| No||69 (79.3)||18 (20.7)|| |
| Yes||8 (34.8)||15 (65.2)||< 0.001|
|Degradation of the mucus|
| No||26 (68.4)||12 (31.6)|| |
| Yes||51 (70.8)||21 (29.2)||0.793|
| No||42 (84.0)||8 (16.0)|| |
| Yes||35 (58.3)||25 (41.7)||0.003|
Of the considered histological variables, lympho-epithelial lesions and fibrosis were significantly (P < 0.005; chi-squared test) associated with unsuccessful eradication. Differences in the distribution of H. pylori strains in patients with successful and unsuccessful eradication are reported in Table 4. cagA expression and the vacA s1 segment were significantly associated with successful eradication treatment (P = 0.004; chi-squared test). No association with eradication was found for iceA alleles.
Table 4. Differences in the distribution of Helicobacter pylori strains between patients with successful and unsuccessful eradication
|cagA (–) ||8 (42.1)||11 (57.9)|| |
|cagA (+) ||69 (75.8)||22 (24.2)||0.004|
|s1||67 (77.0)||20 (23.0)|| |
|s2||9 (42.9)||12 (57.1)|| |
|Mixed forms||1 (50.0)||1 (50.0)||0.004|
|iceA1||27 (67.5)||13 (32.5)|| |
|iceA2||33 (64.7)||18 (35.3)|| |
|Mixed||11 (91.7)||1 (8.3)|| |
|Not detected||6 (85.7)||1 (14.3)||0.23|
The final model of the multivariate logistic regression analysis with eradication outcome as the dependent variable included sex, age, cagA expression and the presence of atrophy, fibrosis and lympho-epithelial lesions. Table 5 reports the model used to evaluate the associations between the variables. The absence of cagA and the presence of fibrosis and lympho-epithelial lesions were significantly associated with a failure of eradication treatment.
Table 5. Multiple linear regression analysis of eradication rates, clinicopathological variables and Helicobacter pylori genotype
|cagA (–/+) ||0.15||0.10||0.005||0.04–0.56|
|Atrophy (absent/true + indefinite)||3.19||1.92||0.059||0.98–10.39|
|Lympho-epithelial lesions (no/yes)||9.73||6.31||< 0.001||2.73–34.69|
Differences in H. pylori strains have been reported to be important in determining the clinical outcome of infection, and a number of putative virulence factors for H. pylori have been identified (cagA, vacA and iceA). Previous studies have indicated that cagA expression and the vacA s1 segment are predictive for the success of eradication treatment.26 Our data confirm that the absence of cagA is strongly associated with treatment failure, and this association persists in the final model of logistic regression. In addition, cagA expression is associated with a low prevalence of lympho-epithelial lesions, together with a high inflammatory activity and a marked reduction in the mucus layer in the gastric antrum. This finding is in agreement with the well-stated cagA association with bacterial growth and a severe inflammatory response.27, 28 Moreover, polymorphonuclear infiltration in the antrum of patients with a high grade of inflammation (scores 2–3) is associated with a significantly higher eradication rate when compared with that in patients with inflammation of grades 0–1,29 and, consequently, it has been hypothesized that the related increased blood flow may help in the diffusion of antibiotics.13 Our results seem to support this hypothesis.
With regard to the vacA status, the s1 segment is significantly associated with particular endoscopic features and successful eradication treatment, but this association does not persist when the final model of multivariate logistic regression analysis is performed. Thus, the claim that the vacA genotype (in particular the s1 segment) might provide clinically useful information on the treatment outcome or predict the presentation of major diseases9 does not seem to be confirmed in our study. Conversely, our data seem to be in agreement with reports suggesting a role for vacA genotype s1 as a surrogate for the cagA pathogenicity island.30
Finally, with regard to the iceA status, no statistically or clinically relevant difference could be found between the groups with or without successful eradication. Therefore, our results are in agreement with those that do not assign a critical role to the expression of iceA alleles in the induction of mucosal damage.10, 11 They indicate a lack of influence of iceA expression in the eradication outcome.
All patients in this study received the same eradication regimen, which achieved an eradication rate of 70%. The protocol was well tolerated, without clinically important adverse sequelae during short-term administration.31 Therefore, compliance cannot be invoked as a possible cause of treatment failure. Multivariate logistic regression analysis showed that significant treatment failure occurred in the absence of cagA and in the presence of fibrosis and lympho-epithelial lesions. Therefore, these histological features could be actively involved in determining the eradication outcome. Both provide evidence of a long-standing inflammatory process, and fibrosis can act as an inhibitory factor in the local diffusion of antibiotics. More interesting, but less explicable, is the association between cagA-negative status and lympho-epithelial lesions. The main picture of H. pylori infection is chronic active gastritis, characterized by predominant neutrophil polymorphs in both the lamina propria and epithelium.21 However, the polymorphonuclear density is high in the presence of cagA-positive strains, but often minimal when cagA-negative organisms colonize the gastric mucosa.32 In this case, a background of chronic inflammation, i.e. increased amount of mononuclear leucocytes (especially cytotoxic T lymphocytes), in both the epithelium and lamina propria may be unmasked,33 and this phenomenon may account for our result.
In conclusion, the results of this study are substantially in agreement with current reports, and demonstrate that vacA and iceA are not important in the determination of the eradication outcome, but that cagA expression is significantly associated with successful eradication. In addition, the results provide evidence that histological changes occurring in the infected gastric mucosa may contribute to the success of treatment.
The authors would like to thank Mr Vito Guerra for his invaluable help with the statistical analysis.