Serum antibodies positivity to twelve Helicobacter pylori virulence antigens in patients with benign or malignant gastroduodenal diseases – cross-sectional study Authors



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Serum antibodies positivity to twelve Helicobacter pylori virulence antigens in patients with benign or malignant gastroduodenal diseases – cross-sectional study
Authors:

Tajana Filipec Kanizaj

Miroslava Katicic

Vladimir Presecki1

Slavko Gasparov2

Vesna Colic Cvrlje

Branko Kolaric3

Anna Mrzljak


Department of Internal medicine, University Hospital Merkur, Faculty of Medicine, Zajceva 19, 10 000 Zagreb, Croatia

1Department of Microbiology and Parasitology, Faculty of Medicine, Salata 3b, Zagreb, Croatia

2Department of Pathology, University Hospital Merkur, Faculty of Medicine, Zajceva 19, 10 000 Zagreb, Croatia

3Social medicine and gerontology service, Zagreb County Institute of Public Health, Matice hrvatske bb, 10410 Velika Gorica

Running head: Filipec et al. H. pylori Antigens in Gastroduodenal Diseases



Abstract

Aim: To investigate the association of gastric histological and endoscopic findings in patients with H. pylori, according to presence of seropositivity to 12 bacterial virulence antigens.

Methods: The study is a cross – sectional single center analysis of 360 consecutive outpatients referred in a single year to upper gastrointestinal endoscopy because of dyspeptic complaints. Patients sera were tested by Western blot method to determine the presence of serum antibodies to bacterial virulence antigens (p120 (CagA), p95 (VacA), p67 (FSH), p66 (UreB), p57 (HSP homolog), p54 (flagellin), p33, p30 (OMP), p29 (UreA), p26, p19, and p17). Upper gastrointestinal endoscopy was performed, endoscopic diagnosis recorded, and 4 mucosal biopsy samples obtained and assessed according to Updated Sydney protocol.

Results: The sera of 207 patients were analyzed. 30 patients had gastric adenocarcinoma, 126 peptic ulcers and 51 normal finding. P120 (CagA) seropositivity was significantly more often present in patients with higher activity grade in the antrum (P=0.025), p30 (OMP) with greater inflammation in the antrum (P=0.025) and the corpus (P=0.010), p33 with greater inflammation in the corpus (P=0.050) and p19 (OMP) with lower intestinal metaplasia grades in the corpus (P=0.025) of the stomach. Seroreactivity to all other bacterial proteins showed no association with the histological status of the stomach mucosa. Except for the seropositivity to protein p95 (VacA), which was more often present in patients with duodenal ulcer (P=0.006), there was no difference in seroreactivity to other bacterial proteins and upper gastrointestinal endoscopic findings.

Conclusions: P120 (CagA), p33, p 30 (OMP) and p19 (OMP) seropositivity were more often present in patients with higher grades of the histological parameters of gastritis and seropositivity to protein p95 (VacA) with endoscopic presence of duodenal ulcer. Histological parameters of gastritis are rather associated with bacterial virulence than endoscopic findings.
Keywords: H. pylori, gastritis, virulence antigens, CagA, VacA, p 19, p 30, p 33

Introduction

Helicobacter pylori (H. pylori) is recognized as a major gastric pathogen with worldwide distribution (1). Estimated prevalence of H. pylori infection in Croatia is 60.4% (2). The persistent colonization induces gastritis and in some patients peptic ulcer disease, atrophic gastritis or gastric carcinoma (3). However, most of patients infected with H. pylori never develop the cancer or ulcer disease and there is need to identify additional factors that may influence the risk for development of such diseases. The outcome of H. pylori infection is associated with specific (virulence associated) bacterial genotypes, environmental factors and host's immune status (4). A number of putative virulence factors for H. pylori infection have been identified including: CagA (cytotoxin - associated antigen), VacA (vacuolating cytotoxin), IceA (induced by epithelium antigen), BabA (blood-group antigen-binding adhesin), etc. It is likely that every other factor that results in increased inflammation will also consequently increase the risk of a disease occurrence.

Presently, the only reliable way to identify the disease associated with H. pylori infection remains an endoscopic examination together with histological assessment of the gastric mucosa (5). The bacterial virulence antigens elicit specific antibodies during infection. But, the value of these antibodies as predictive factors for the severity of the disease remains controversial (6-15). To define the correlation so far several investigations were done, such as: detecting the level, the specificity or presence of isotypes of serum H. pylori antibodies (16-22). Because disease outcome depends on both, the strain characteristics and the host response, the serum antibody response to virulence antigens could provide clues in predicting the presence and severity of associated diseases (23, 24). On the other side, the fact that subjects without significant disease also have strains bearing this or other virulence antigens arise suspicion to assign disease to one virulence antigen alone. Thus other virulence antigens may be important. Exact role of other bacterial virulence antigens (p67 (FSH - flagellar sheath protein), p66 (UreB - urease enzyme heavy subunit), p57 (HSP homolog - heath shock protein homolog), p54 (flagellin), p33, p30 (OMP - outer membrane protein), p29 (UreA - urease enzyme light subunit), p26, p19 (OMP), p17) in pathogenesis of gastrointestinal diseases is still unclear.

In this study, we aimed to investigate the association of gastric histological and endoscopic findings in H. pylori positive patients according to presence of seropositivity to 12 bacterial virulence antigens. Since both bacterial virulence antigens and pattern of H. pylori gastritis may contribute to development of clinically relevant gastroduodenal disease, we sought to determine the antibodies which are best associated with higher grades of histology findings of gastritis, atrophy or intestinal metaplasia and different clinical diseases (peptic ulcer, gastric cancer, non - ulcer dyspepsia).

Methods
Patients

In year of 2000, 360 consecutive outpatients referred to upper gastrointestinal endoscopy because of dyspeptic complaints were screened. Before entering the study, all patients provided written informed consent. The research protocol was approved by the Clinical Research Ethical Committee of the University Hospital Merkur in Zagreb. All procedures were in accordance with the Declaration of Helsinki revision 2008 (25).

207 patients were eligible for the study. Inclusion criteria for the study were: age older than 18 years, positive to H. pylori (proven by histology and serology) and no data about previous eradication treatment for H. pylori infection. The exclusion criteria were: previous eradication treatment, any antimicrobial treatment 2 months preceding the study, concomitant medication with: bismuth preparations, proton pump inhibitors, H2-receptor antagonists or NASID (non-steroid anti-inflammatory drugs), other serious illnesses, and history of previous gastric surgery. H. pylori positive status was proven by histology and serology (Western blot).

At examination upper gastrointestinal endoscopy was performed and 4 gastric mucosa biopsy samples were obtained (2 from antrum and 2 from corpus). Additionally 1 vial of venous blood was obtained for serological examination. According to endoscopic findings patients were divided into five groups as follows: normal mucosa (N), duodenal ulcer (D), gastric ulcer (V), duodenal and gastric ulcer (VD), gastric adenocarcinoma (C).


Histology

For histological examination 4 biopsy samples were obtained: 2 from antrum and 2 from corpus of gastric mucosa. The biopsy samples were embedded in paraffin wax and stained with haematoxilyn and eosin, modified 2% Giemsa stain and alcian blue - PAS (periodic acid Schiff). All samples were analyzed for H pylori density, activity (intensity of polymorphonuclear cells infiltrates), inflammation (intensity of mononuclear cells infiltrates), atrophy, and intestinal metaplasia, as stipulated by Updated Sydney system classification (26). All parameters were graded as none (0), mild (1), moderate (2), or marked (3). Intestinal metaplasia was recognized morphologically with the presence of goblet cells, absorptive cells, and cells resembling colonocytes. Results from two antrum or corpus biopsy samples were combined and whenever differences were observed, the highest score was considered for statistical analysis.


Serology

Sera analysis was performed with commercially available Anti – Helicobacter pylori – Western blot test kit (Western blot, Euroimmun Medizinische Labordiagnostika AG, Lubeck). Procedure was performed according to manufacture instructions. Test is based on detection of anti H. pylori antigens IgG antibodies to twelve H. pylori specific and unspecific (cross – reacting or undefined) antigens. Antigen source for this commercially available anti – Helicobacter pylori - Western blot test is provided by the H. pylori type strain (27).

Serum samples were assayed for 12 antibodies to H. pylori antigens: p120 (CagA; cytotoxin - associated antigen), p95 (VacA; vacuolating cytotoxin), p67 (FSH; flagellar sheath protein), p66 (UreB; urease enzyme heavy subunit), p57 (HSP homolog; heath shock protein homolog), p54 (flagellin), p33, p30 (OMP; outer membrane protein), p29 (UreA; urease enzyme light subunit), p26, p19 (OMP) and p17 by Western blot method. Proteins p 120 (CagA), p 95 (VacA), p33, p30 (OMP), p29 (UreA), p26, p19 (OMP), p17 are H. pylori strain specific antigens and p67 (FSH), p66 (UreB), p57 (HSP homolog), p54 (flagellin) are cross-reacting or undefined antigens.
Statistics

The associations between H. pylori individual virulence antigens and presence of mucosal histological changes or clinical disease were analyzed using 2 – test. Odds ratios (ORs) with confidence intervals were assessed by bivariate logistic regression. The t- test was used to analyze the age distribution between the different patients groups. STATISTICA for Windows, Release 5.5 H* and SPSS for Windows Release 17.01 were used in statistical analysis.



Results
Study population

All 207 patients included in the study were H. pylori positive, confirmed by histology and serology (Western blot). Among them 113 were males (mean age 57.96±14.10 SD) and 94 were females (mean age 57.86±14.69 SD) (P= 0.960). In Table 1. demographic, endoscopic diagnosis, serological and patohistological findings of all 207 patients are presented.

In corpus mucosa inflammation was present in 91.1 % patients, activity 50.1%, atrophy 7.2%, and intestinal metaplasia 11.1 % respectively. Figure 1. represent distribution of different grades of patohistological parameters of gastritis in corpus of patients with benign and malignant gastroduodenal diseases.

Patohistological analysis of the antrum mucosa revealed inflammation in 51.2% patients, activity 74.9%, atrophy 10.6% and intestinal metaplasia 23.2% respectively. Figure 2 represent distribution of different grades of patohistological parameters of gastritis in antrum mucosa of patients with benign and malignant gastroduodenal diseases.

Out of 30 patients with gastric adenocarcinoma, 23 had intestinal and 7 diffuse respectively. Of all patients 18 had tumor in antrum and 12 in corpus part of the stomach.
Correlation of H. pylori virulence antigens and gastric histology

The correlation of histological features and seropositivity status revealed statistically significant results for proteins p120 (CagA), p30 (OMP), p33 and p19 as followed: p120 (CagA) seropositivity was associated with higher activity grades (P=0.025) in antrum, p30 (OMP) seropositivity with higher inflammation grades in both, corpus (P=0.010) and antrum (P=0.025) and antigen p33 higher inflammation grades in corpus (P=0.050). Contrary, seropositivity to p19 (OMP) antigen was associated with presence of lower grades of intestinal metaplasia in corpus (P=0.025) of gastric mucosa. Complete data of histological finding of gastric mucosa according to seropositivity to p 120 (CagA), p19, p30 (OMP) and p33 proteins are provided in Table 2. There was no statistically significant difference in the distribution of histological parameters of gastritis with respect to serological status to other tested bacterial virulence antigens.


Correlation of H. pylori virulence antigens and endoscopic diagnosis

Seropositivity to antigen p95 (VacA) was statistically significantly more often observed in patients with duodenal ulcer and less in those with normal mucosa and gastric carcinoma (P=0.006). Sensitivity and specificity of positive serology finding to p95 (VacA), in order to detect patients with duodenal ulcer, were 91.18% and 31.65%. Analogously, to gastric carcinoma 60.00% and 21.47%, gastric ulcer 71.74% and 22.98%, simultaneous gastric and duodenal ulcer 66.67% and 23.59%, and normal mucosa finding 70.59% and 22.44% respectively. There was no association between seropositivity to any other of eleven H. pylori virulence proteins and endoscopic finding. The distributions of different endoscopy findings and antibodies to H. pylori virulence antigens are summarized in Table 3.



Discussion

The results of our study demonstrate the association of seropositivity to p120 (CagA), p33 and p 30 (OMP) with higher grades of the histological parameters of gastritis and seropositivity to p19 (OMP) with lower grades of intestinal metaplasia. Only protein p95 (VacA) seropositivity was strongly associated with endoscopic presence of duodenal ulcer.

So far, many attempts have been made before to find serological markers of H. pylori virulence which could be correlated with the severity of the H. pylori-associated diseases. Data from these studies indicate that seropositivity to some H. pylori antigens could be used as serological markers for bacterial virulence (19, 28-33).

Comparing serology findings to twelve H. pylori virulence antigens with gastric mucosa histopathological findings we noticed that p120 (CagA) seropositivity was associated with the higher activity grades in antrum, p30 (OMP) seropositivity with higher inflammation grades in both antrum and corpus, p33 seropositivity with higher inflammation grades in corpus and p19 (OMP) seropositivity with lower grades of intestinal metaplasia in corpus of gastric mucosa. Protein CagA is the most investigated H. pylori virulence factor but, its connection with presence of various mucosal histological changes is still controversial and there are many studies with contradictory results (34-37). Our results are consistent with previous observations where CagA elicit high neutrophil accumulation measured as inflammation activity in gastric mucosa (33). According to the results of previously published studies, this can be related to strong stimulation of IL-8 production by CagA positive strains (38, 39). There are no previously published data on association of seropositivity to proteins p30 (OMP), p33 and p19 (OMP) and different patterns of gastritis. First step in all H. pylori related diseases is emergence of gastritis. Possible pathogenetic mechanisms involved in association of these proteins with higher grades of gastritis should be additionally investigated regarding implications in occurrence of gastroduodenal diseases.

Protein p95 (VacA), p67 (FSH), p66 (UreB), p57 (HSP homolog), p54 (flagellin), p29 (UreA), p26 and p17 seropositivity were not associated with the histological patterns of gastritis. Several studies consistently showed that different vacA genotypes are associated with disease appearance in patients from various countries. On the other hand, there are different VacA isoforms with distinct antigenic properties and consequently multiple forms of VacA could elicit the different antibody responses in H. pylori positive humans (40). Based on our findings, we can conclude that present serology testing to antigen VacA is not sensitive enough to predict toxic and histolopathological effects of vacuolisating toxin. Obviously distinct serological response to VacA toxin cannot distinct between more and less toxic isoforms of VacA related with different grades of histopathological findings. Better results in this field of research could be provided with investigation of different vacA genotypes in relation with presence of various histopathological findings.

Also, none of the tested serologic markers have been associated with appearance of atrophic gastritis. It may be necessary to look for other antigens, or the atrophy could be unrelated to serologic status to H. pylori virulence antigens.

In the present work, we found that only antibodies to the protein p95 (VacA) were more frequently present in patients suffering from duodenal ulcers than other endoscopic findings. Thus the anti-VacA antibody is a more powerful marker of ulcers than anti-CagA. This is not surprising, because the vacuolisating toxin has been suspected to be involved in the development of mucosal ulcerous lesions (41-43). It has been reported in earlier studies that the toxin causes epithelial damage, but appears to have little effect on inflammatory cell infiltration. These results are in agreement with previous works representing that anti-CagA, anti-VacA and anti-p35 appears to be the best markers of ulcers (44). However, the clinical relevance of this association remains controversial (45). Contrary to this, we found no association of anti-CagA antibodies to endoscopic finding. Explanation of results discrepancy could be due the fact that in some areas there is high prevalence of CagA and VacA positive patients. In our study 91.3% of patients were CagA and 75.8% VacA positive and larger group of patients is needed to validate possible difference. Another explanation could be the fact that peptic ulcers may be intermittently present and certain patients may be non – ulcerous at the time of endoscopic examination but may later evolve to an ulcerous state. Other authors have attempted to establish correlations between certain antibody patterns and gastric cancer (31). We also found anti- p95 (VacA) to be less frequently detected in patients with endoscopic finding of gastric carcinoma or normal mucosa than peptic ulcer. Possible clinical implications of observed phenomena could be used in future in order to distinguish patients infected with more virulent strains of bacteria especially in those patients infected with therapy resistant strains. Also proteins which elicit strong serological response in higher number of patients could be possibly used as a part of anti H. pylori vaccine. But, in order to confirm its real prognostic significance and clinical usefulness of these observations, a larger study population is necessary.

No sample size calculation or power analysis was performed that is a possible limitation of the study. Marginal results of statistical significance were observed in difference of distributions of various grades corpus inflammation and intestinal metaplasia according to anti- p 120 (CagA) finding (P=0.087 and P=0.114 respectively, data not shown). Additional investigation with higher number of patients could answer the question of true association of protein CagA and histopathological patterns of gastritis. Moreover, presence of the bacterial virulence antigens was evaluated indirectly by host serological response only. Many of tested bacterial virulence antigens are highly immunogenic and can induce antibody response in almost all patients (46). But, it is generally known that serological response depends on antigen type and immunological reactivity of the host. Some patients are infected with more than one type of bacteria possessing a variety of different (more or less virulent) proteins encoded by different genotypes of virulence genes. All of this explains why negative serological response does not always implicate that H. pylori negativity to tested virulence antigen. Seropositivity to specific antigen does not absolutely indicate the relationship with pathogenesis of specific disease. Furthermore, these observations lead to the direction for future investigations in pathogenesis of gastroduodenal diseases.



In conclusion, histological parameters of gastritis show better association with bacterial virulence than an endoscopic finding. But, no single entity or array of antigens has yet been identified that can serve as a serological marker(s) for predicting the development of specific gastric disease. This could be attributed to the role of other factors that govern disease expression for example, other undetected virulence antigens, changes in gastric physiology or host immune-inflammatory response (47).

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Table 1. Demographic, endoscopic, serological and patohistological findings of all 207 Croatian patients with benign and malignant gastroduodenal diseases.

Parameters

N (%)

Age

(average ± SD)

54.89 ± 14.3

Gender

males

113 (54.6)




females

94 (45.4)




Endoscopic finding

Gastric ulcer (V)

46 (22.2)

Duodenal ulcer (D)

68 (32.8)

Gastric carcinoma (C)

30 (14.5)

Normal mucosa (N)

51 (24.6)

Gastric and duodenal ulcer (VD)

12 (5.8)




Virulence antigens

positive

negative

P 120 (CagA)

189 (91.3)

18 (8.7)

P 95 (VacA)

157 (75.8)

50 (24.2)

P 67 (flagellin)

83 (40.1)

124 (59.9)

P 66 (ureB)

130 (62.8)

77 (37.2)

P 57 (heath shock protein homolog)

149 (71.9)

58 (28.1)

P 54 (flagellin)

50 (24.1)

157 (75.9)

P 33

52 (25.1)

155 (74.9)

P 30 (outer membrane protein)

57 (27.5)

150 (72.5)

P 29 (ureA)

93 (44.9)

114 (55.1)

P 26

87 (42.1)

120 (57.9)

P 19 (outer membrane protein)

111 (53.6)

96 (46.4)

P 17

76 (36.7)

131 (63.3)




Patohistological finding

positive

negative

Corpus

Inflammation

201 (97.1)

6 (2.9)

Activity

104 (50.2)

103 (49.8)

Atrophy

15 (7.2)

192 (92.8)

Intestinal metaplasia

23 (11.1)

184 (88.9)

Antrum

Inflammation

203 (98.1)

4 (1.9)

Activity

153 (73.9)

54 (26.1)

Atrophy

22 (10.6)

185 (89.4)

Intestinal metaplasia

48 (23.2)

159 (76.8)

Figure 1. Distribution of different grades (0- no change, 1- mild, 2- moderate, 3- marked) of histological parameters (infammation, activity, atrophy and intestinal metaplasia) of gastritis in corpus mucosa of 207 Croatian patients with benign and malignant gastroduodenal diseases.

Figure 2. Distribution of different grades (0- no change, 1- mild, 2- moderate, 3- marked) of histological parameters (infammation, activity, atrophy and intestinal metaplasia) of gastritis in antrum mucosa of 207 Croatian patients with benign and malignant gastroduodenal diseases.



Table 2. Presence of different grades of histological parameters of gastritis in patients serologicaly positive and negative to H. pylori virulence proteins p120 (CagA), p19, p30 (OMP) and p33 (n=207).



Histology

Antibodies

c2 test

Bivariate logistic

regression






grade

negative

positive

sensitivity

specificity

P

P

OR

(95% CI)





p 19

Intestinal

metaplasia

corpus


0

79

105







0.025

0.005

0.441

(0.248-0.785)



1

4

3

2.70

95.83

2

11

3

2.70

88.54

3

2

0

0.00

97.92

sum

96

111










p 30 (outer membrane protein)

Inflammation

corpus


0

6

0







0.010

0.003

1.956

(1.252-3.055)



1

88

27

47.37

41.33

2

49

20

35.09

67.55

3

7

10

17.54

95.33

sum

150

57










p 30 (outer membrane protein)

Inflammation

antrum


0

1

3







0.025

0.367

1.224

(0.789-1.900)



1

54

16

28.07

64.00

2

80

26

45.61

46.67

3

15

12

21.05

90.00

sum

150

57










p 33

Inflammation

corpus


0

4

2







0.050

0.026

1.666

(1.061-2.616)



1

95

20

38.46

38.71

2

45

24

46.15

70.97

3

11

6

11.54

92.90

sum

155

52










p 120 (CagA)

Activity

antrum


0

4

50







0.025

0.095

2.295

(0.911-3.175)



1

14

77

40.74

22.22

2

0

47

24.87

100.00

3

0

15

7.94

100.00

sum

18

189







Table 3. Serological findings to twelve H. pylori virulence antigens in various benign and malignant gastroduodenal diseases (n=207).



Antigen

Endoscopic finding*

c2 test

Bivariate logistic regression

D

C

N

V

VD

P

OR

(95% CI)


P

p120

pos.

65

28

43

42

11

0.301

0.794

(0.511-1.233)



0.304

neg.

3

2

8

4

1

p95

pos.

62

18

36

33

8

0.006

1.147

(0.862-1.524)



0.347

neg.

6

12

15

13

4

p67

pos.

24

11

27

15

6

0.209

1.264

(0.981-1.628)



0.070

neg.

44

19

24

31

6

p66

pos.

42

23

32

27

6

0.455

0.854

(0.663-1.101)



0.233

neg.

24

7

19

19

6

p57

pos.

48

22

40

30

9

0.691

1.128

(0.860-1.479)



0.384

neg.

20

8

11

16

3

p54

pos.

14

10

13

11

2

0.680

0.872

(0.656-1.159)



0.347

neg.

54

20

37

35

10

p33

pos.

14

5

19

12

2

0.174

1.199

(0.902-1.593)



0.211

neg.

54

25

32

34

10

p30

pos.

20

10

17

7

3

0.291

1.092

(0.831-1.434)



0.529

neg.

48

20

34

39

8

p29

pos.

28

16

24

19

6

0.790

0.983

(0.770-1.256)



0.894

neg.

40

14

27

27

6

p26

pos.

28

13

28

12

6

0.391

1.272

(0.989-1.635)



0.061

neg.

40

17

23

34

6

p19

pos.

41

11

31

20

8

0.076

1.409

(1.094-1.814)



0.008

neg.

27

19

20

26

4

p17

pos.

28

12

19

13

4

0.699

1.023

(0.795-1.316)



0.859

neg.

40

18

32

33

8

sum

68

30

51

46

12




*C- carcinoma. D- duodenal ulcer. N- normal mucosa. V- gastric ulcer. VD- gastric and duodenal ulcer

Corresponding author:

Tajana Filipec Kanižaj

Department of Internal medicine, University Hospital Merkur, Faculty of Medicine Zajčeva 19, 10 000 Zagreb, Croatia

Tel. 0038598623903

Fax. 0038512431393

e-mail: tajana_filipec@yahoo.com






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