Role of Helicobacter pylori infection in pernicious anaemia

ALIMENTARYTRACT

DIGEST LIVER DIS 2000;32:756-62

Role of Helicobacter pylori in pernicious anaemia 6. E. C. G. F? C. R. G.

Annibale Lahner Bordil Martin0 Caruanal Grossi Negrini* Delle Fave

Flvm Gastroanterwiogy Department, University of Rome “La Swpienza”; ’ Anatomic fJatho\ogy, University of Pwrma, * Third Laboratory of Clinical Chemistry. Spedali Civili, Brescia, Italy. d by grant n. 02/12/01/10, 1996-1998 from the Itelian Ministry for the University. The e&hors era gratefi!l to Dr. Giancwrlo D’Ambra for his endoscopic support and Ms. Amelia Pwsquwli far technical assistance. Dr. B. Annibale, Dipartimento di Gastroentarologia, II Clinicw Medice. Policlinico Universitario Umberto I, Universitti dwgli Studi di Romw ‘La Sapienzw”% Vialw del Policlinica 5, 00761 Rome, Itwly. Fax: +39-064463737. E-mail: bruno. wnnibwle@unirome I. it Submitted July 17, 2000. Revised September 30, 2000. Acceptwd October 9, 2000.

Background. Pernicious anaemia is associated with atrophic body gastritis and considered an autoimmune disease. Whether Helicobacter pylori is involved in the induction of pernicious anaemia is uncertain. Aims. To investigate the prevalence of Helicobacter pylori infection in pernicious anaemia patients and to ascertain whether the Helicobacter pylori-positive patients had distinctive clinical and gastric morphofunctional characteristics. Patients and Methods. A series of 81 consecutive pernicious anaemia patients underwent serological, functional and endoscopit/histological investigations. Results. A total of 49 [60.5%] patients were Helicobacter pyloripositive [males 6 1.2% vs females 38.8%]. No difference was observed in clinical and morphofunctional characteristics between Helicobacter pylori-positive and negative patients, whereas distinctive functional/histological features between histologically Helicobacter pylori-positive [n=8] and serologically Helicobacter pyloripositive [n=41/ cases were detected. In the histologically Helicobacter pylori-positive group, Pepsinogen I was higher [I3 (O-58) vs 5 [026] rig/ml; p=O.O025]] and positivity for anti-parietal cell antibodies was lower [42.9% vs 76.9, p=O.O867]. Antral histological variables of the gastritis score were significantly higher in the histologically Helicobacter pylori-positive than in the serologically Helicobacter pylori-positive patients, but this latter group had a higher score of body atrophy (2.6320.12 vs 1.71_tO.29; p=O.O05 I]. Body inflammation was also significantly higher in the histologically Helicobacter pylori-positive group [chronic inflammation: I. 4320.2 vs I. 0520.06; p=O. 027 I; inflammation acitivity: 0.5720.3 vs 0.15~0.06, p=O.O220). Antral mucosa was normal Helicobacter pylori-positive in 24/4 I [58. ~Yo] of the serologically patients, but only in l/8 [12.5%] of the histologically Helicobacter pylori-positive patients [p=O.O232]. Conclusions. Almost two thirds of pernicious anaemia patients have evidence of Helicobacter pylori, but only those with an active Helicobacter pylori infection have distinctive functional and histological features. These findings support the hypothesis that Helicobacter pylori infection could play a triggering role in a subgroup of pernicious anaemia patients.

Digest

Liver

Key words:

756

infection

gis 2000;32:756-62 atrophic

body

gastritis;

Helicobwcter pylori; pernicious

anaemia

8. Rnnibale et al.

Introduction Pernicious anaemia (PA) is associated with the presence of atrophic body gastritis (ABG) ‘. It is believed to be an autoimmune disease due to the presence of autoantibodies to the secretory elements of the gastric oxyntic mucosa. However, the way in which this form of gastritis initiates and evolves is unknown, even though it is believed that deep plasma cell and lymphocytic inflammation leads to the loss of oxyntic glands 2, with the progressive disappearance of parietal and chief cells and, therefore of the production of acid and the intrinsic factor 3. It is now apparent that Helicobacter pylori (H. pylori) is also involved in the induction of ABG 4-8.It is not certain, however, whether the infection is also involved in the induction of atrophic gastritis leading to the development of PA. Investigations on this hypothesis, based mainly on histological and/or serological findings, have given rise to contrasting results. Some Authors have, in fact, rejected the possibility that the infection may be involved in the process leading to PA 9-“, while others have provided convincing evidence arguing in favour of this hypothesis ‘2-‘4. Furthermore, the fact that H. pylori infection can induce gastric autoimmunity is well established, since the presence of the bacteria leads to the production of antibodies cross-reacting with human gastric mucosa I5 I6. It has been reported that the presence or absence of antibodies to H. pylori in PA patients cannot address aetiology alone “. H. pylori-related PA would, nevertheless, be expected to be associated with antral evidence of present or past infection, whereas in pure autoimmune PA, the antral mucosa would be normal “; however, so far, studies have failed to detect such differences 9 ‘“. We prospectively studied a consecutive series of newly diagnosed patients with PA with the aim of establishing the prevalence of H. pylori infection and to ascertain whether distinctive clinical and gastric morphofunctional characteristics differentiate the H. pyZori-positive PA patients.

Patients and Methods Patients From January 1992 to September 1999, we observed 8 1 consecutive patients referred from the Haematology Department of our University with a diagnosis of PA as previously described 6. The clinical and biochemical features of the study population are given in Table I. Criteria for the diagnosis of pernicious anaemia The diagnosis of PA was based on the following criteria: macrocytic anaemia [defined as a haemoglobin

Table I. Clinical and biochemical features of 81 pernicious anaemia patients. Data are expressed as median frangel. Males/Females Age, years History of anaemia”, months Fasting gastrin, pg/ml PAO, mEq/h+ Serum pepsinogen I, ns/ml Vitamin 8~ pg/ml

45136 62 BO-831 9 [O-1811 500 (55-27001 0.0 [O-l 2.641 5 [O-601 50 [I 3-1961

* History of anaemia is time interval fmm initial diagnosis of anaemia to first histological diagnosis of atrophic body gastritis. PAO: peak acid output; ‘Data auailsble in 51 patients. Normal values: See material and methods section.

concentration below 14 g/d1 for men (normal range 14 to 18 g/dl) and below 12 g/d1 for women (normal range, 12 to 16 g/dl), accompanied by a mean cellular volume >lOO fl], response to vitamin Blz therapy and/or low vitamin B12, histological confirmation of gastric body mucosa atrophy and pentagastrin-resistant hypo/achlorhydria, as described elsewhere 6. History of anaemia was defined as the time interval expressed in months between diagnosis of PA and endoscopy with appropriate biopsies (see below, Endoscopy, Biopsies, and Histological Procedures Section). A detailed anamnestic evaluation was performed, including a scrupulous search for associated autoimmune diseases in each patient and whenever possible, medical records were also checked. All patients gave informed consent to the study, which was approved by the local ethics committee. Blood assays Fasting gastrin levels were evaluated by means of a specific radioimmunoassay using antibody n. 4562 (Prof. J.F. Rehfeld), as previously described I8 19. Normal values: 440 pg/ml. Anti-intrinsic factor antibodies (IFA) were determined using a commercial kit (Anti-Intrinsic factor Abs, Cambridge Life Sciences, UK). Positive values >l U/ml. Pepsinogen I levels were measured using a commercial RIA kit (Pepsik, Sorin, Saluggia, Italy), as previously reported *O. Normal range: 20-80 rig/ml. Parietal cell antibodies (AbPC) were determined on serum using a solid phase immunosorbent assay commercial kit (AUTOSTAT, Cogent Diagnostic Ltd, Edinburgh, Scotland), as previously described 6. Positive values >125 U/ml. Anti-peroxidase antibodies (AbTPO) were measured by a radioligand assay (Radim Techland, Liege, Belgium). Positive values: >200 U/l. Circulating antibodies against H. pylori were detected

151

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by competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody against a 64kD H. pylori antigen, as elsewhere described 21. Briefly, 100 pl of undiluted serum sample and calibrators were added to microwells coated with a H. pylori sonicated preparation. 100 ul of peroxidase conjugated HpN4.5 were then added to all wells. After 1 hour of incubation, the plate was washed, colour was developed with a TMB solution and absorbances read at 450. Antibody concentration was expressed as units/ml. Negative values are considered ~450 U/ml *l. Sensitivity was 100% and specificity was 89.6% 21. Vitamin B12 levels were determined by a commercial kit (SimulTRAC-SNB, radioassay kit Vitamin BQ, Becton Dickinson Italia SpA, Milano, Italy). Normal values: 160-970 pg/ml. Gastric acid secretion studies Pentagastrin-stimulated acid output (PAO) (6 g subcutaneously, Peptavlon, ICI, England) was determined as previously described I9 22. The median reference value for PAO, in our laboratory, is 31.1 mEq/h (range 1344.4) 6. Endoscopy, biopsies and histological procedures Patients underwent upper GI endoscopy with collection of antral (n=3) and gastric body (n=4) biopsies according to a previously published protocol 6. Specimens were fixed immediately in Bouin’s solution for 4-8 hrs at room temperature, rinsed in 0.1 phosphatebuffered saline, pH 7.4, and routinely processed to wax. Serial paraffin sections (5 urn) were stained with haematoxylin-eosin for conventional histological evaluation and with Giemsa stain for H. pylori determination. Assessment of the degree of gastritis was performed according to the Updated Sydney system 23. Atrophy of the gastric body mucosa was defined as focal or complete oxyntic gland loss and/or replacement by metaplastic pyloric or intestinal glands. To each graded variable the following scores were assigned: 0 for absence and 1, 2, 3 for mild, moderate or severe presentation, respectively. The biopsies were examined independently by two experienced histopathologists (PC, CB), unaware of the clinical data of the patients as previously described 24. The antral mucosa was defined as completely spared if all the graded variables scored 0. H. pylori status H. pylori status was considered to be positive either when the organism was detected at histology and/or on the basis of the presence of a positive H. pylori IgG titre, since it is generally held that in the absence of evidence of colonization, the presence of antibodies to H. pylori reflect previous or hidden infection 2526. 758

Statistical evaluation Analysis was performed using the t-test test for unpaired non parametric data and data are expressed as median (range). Graded histological variables of the gastric body mucosa have been expressed as mean+SEM and analysis performed using the t-test for unpaired data. Differences between groups have been assessed by Fisher’s exact test. A p value of less than 0.05 has been considered statistically significant. Results

Prevalence of H. pylori infection A total of 49 patients [60.5%, median age 62 years (range 30-78)] were classified as H. pylori-positive. Of these, 41 (50.6%) patients had only a positive IgG titre to H. pylori, while in 8 (9.9%) patients with positive serology, H. pylori organisms were found also at histology. In two cases, the bacteria were present both in the body and in the antrum, in another two cases they were detected only in the body, while in four cases they were found only in the annum. The remaining 32 pa-

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A. Annibale et al.

tients (39.5%, 15 males and 17 females, median age 64 years, range 33-83) negative at histology for the presence of H. pylori organisms and with a median IgG titre to H. pylori below the cut-off value of 450 U/ml were consequently classified as H. pylori-negative. As shown in Figure 1, the median IgG titres to H. pylori were significantly different in the histologically and serologically H. pylori-positive group [ 17.138 U/ml (range 1.06535.040)] with respect to the only serologically H. pylori-positive [1410 U/ml (range 46520.595)] and the H. pylori-negative [ 180 U/ml (range O-435)] group. Among the H. pylori-positive patients, a male prevalence, albeit not statistically significant, was observed [30 males (61.2%) vs 19 females (38.8%)] with respect to H. pylori-negative PA patients [15 males (46.9%) vs 17 females (53.1%)]. No differences in clinical and morphofunctional characteristics, between H. pylori-positive and H. pylori-negative patients, were observed (data not shown). Distinct functional and histological features between histologically and seroZogicaZZy II. pylori-positive PA patients As shown in Table II, histologically and serologically H. pylori-positive patients had some distinctive functional and histological features with respect only to serologically H. pylori-positive patients. Median age, sex distribution, and median values of fasting serum gastrin did not vary between the two groups. Serum

Pepsinogen I levels were higher in the histologically H. pylori-positive with respect to the serologically H. pyZori-positive group [13 (O-58) vs 5 (O-26) rig/ml; p=O.O025)]; in fact, serum Pepsinogen I values within the normal range were found in 2 patients in this group (41 and 58 pg/ml, respectively). Furthermore, two of the patients in the histologically H. pylori-positive group showed a response to pentagastrin-stimulated acid secretion, with PA0 values of 2.38 and 11.64 mEq/h, respectively. The comparative analysis of the graded histological variables in the antral and body mucosa of the histologically and serologically H. pylori-positive PA patients is shown in Table II. Score of body atrophy was higher in the serologically H. pylori-positive as compared to the histologically H. pylori-positive group (2.63kO. 12 vs 1.7 lkO.29; p=O.O05 1; t-test). Chronic inflammation and inflammation activity of the body was significantly higher in the histologically H. pyloripositive group (chronic inflammation: 1.43kO.2 vs 1.05+0.06; p=O.O27 1; inflammation acitivity: 0.57kO.3 vs 0.15kO.06, p=O.O220). The score of body intestinal metaplasia did not differ between the two groups. In the histologically H. pylori-positive group, all antral histological variables of the gastritis score were significantly higher than those of the serologically H. pyloripositive patients. In fact, only 1 out of the 8 histologically H. pylori-positive patients (12.5%) had a completely spared antrum compared to 24 out of the 41

Table II. Clinical, serological and histological features of the 49 H. pylori-positive pernicious anaemia patients. Data are expressed as number positive (%I, median [rangel, end mean&SEM H. ~&fbpemee

at It&elegy and aa?-

N. #ffar&laaltlae

&Zeedegg e&y

ClinicolunU~~ No. patients Males/Females Age, years Fasting gastrin, pg/ml Serum pepsinogen I, q/ml

Him

5/i 63 (48-78) 322 [70-5501 13 CO-581

254 62 t30-741 475 [55-2.700) 5 0261

ns ns ns p=o.o025

sari&h

Bellr

Chronic inflammation Inflammation activity Atrophy Intestinal metaplasia

1.43*0.2 0.57*0.3 1.71*0.3 1.43zt0.5

Chronic inflammation Inflammation activity Atrophy Intestinal mataplasie Antrum spared

l.OTtO.22 0.43*0.3 1.14*0.34 1.29zt0.42 1 (12.51

1.05*0.06 0.15ztO.06 2.63kO.12 1.68kO.15

p=O.O271 p=o.o220 p=o.0051 ns

0.32*0.07 0.07*0.04 0.22ztO.08 0.24*0.08 24 (58.51

p=o.o013 p=o.o253 p=o.o004 p=o.o003 p=O.O232

fls: not significant

759

Il. pylariand

pernicious anaemia

serologically H. pylori-positive Fisher’s test) patients.

(58.5%;

p=O.O232,

Autoimmunity

The pattern of associated autoimmune phenomena in PA patients (Table III) shows that 56 patients (31 males, 25 females) had positive antibodies to parietal cells and 34 (15 males, 19 females) had positive antibodies against the intrinsic factor; 33 patients (10 males, 22 females) had positive anti-thyroid peroxidase antibodies, but chronic autoimmune thyroiditis was present in 30 (9 males, 21 females) of these patients. Other autoimmune diseases were present in 12 PA patients (11 males, 1 female), as summarized in Table III. No differences in autoimmune phenomena between H. pylori-positive and H. pylori-negative patients were observed (data not shown). However, in the histologically H. pylori-positive group the positivity for anti-parietal cell antibodies was lower, but did not [42.9% vs 76.9, reach statistical significance p=O.O867] with respect to the serologically H. pyloripositive group. Furthermore, 5 1.2% of the only serologically H. pylori-positive group had associated autoimmune diseases with respect to only 25% of the hispatologically and serologically H. pylori-positive tients, although this difference did not reach statistical significance. It is of interest that the two histologically H. pylori-positive patients with a response to pentagastrin-stimulated acid secretion were negative to antiparietal cell and anti-thyroid peroxidase antibodies and had no associated autoimmune diseases. TMe III. Associated autoimmune phenomena in 81 patients with pernicious anaemie”. &rum atibodii Anti-parietal cell Anti-intrinsic factor Anti-thyroid peroxidase

56 t76.71 34 (421 33 (40.7)

AmmcW mWmmmte diruw HI Chronic autoimmune thyroid disease Vitiligo Alopecia Psoriasis Type I diabetes mellitus

30 t371 5 (6.21 3 t3.71 2 (2.5%1 2 t2.51

“Data are expressed than one associated

as number autoimmune

positive (%I. +” 5 patients disease.

with more

Discussion The main finding of this study was the high prevalence of H. pylori infection in a consecutive population of patients with pernicious anaemia. In fact, histological and/or serological evidence of H. pylori infection was 760

revealed in 60.5% of the PA patients investigated. Although this study lacks a control group, our finding is in agreement with the prevalence of H. pylori infection in the general age-related population, a well-documented figure in Western countries. In fact, considering that the median age, at diagnosis, in our PA series was 62 years, the prevalence of infection in this age class of the general population is about 50-60% 2728. In contrast, the prevalence of H. pylori infection in PA has been reported to be lower than that in age- and sexmatched controls (0%-21.4%) I*. The majority of our PA patients (50.6%) had only serological evidence of H. pylori infection. This finding is in agreement with a previous study in which a relatively high prevalence of H. pylori antibodies and low H. pylori staining in atrophic body gastritis were observed 2529. This phenomenon may reflect a previous or hidden H. pylori infection in these patients. This hypothesis is supported by the findings of Perez Perez et al. 12, who in their prospective study on PA patients, initially positive for the presence of antibodies to H. pylori, observed a seroreversion rate of more than 6% year. It has been amply demonstrated that serology determination should be considered a valid tool in the diagnosis of H. pyEori infection in PA patients. It has been shown that anti-H. pylori antibody levels are elevated in patients with atrophic body gastritis without histological evidence of infection 263o3’ suggesting that gastric biopsies might give a false negative result especially if H. pylori infection is patchy or if the bacteria colonization is low. Furthermore, it has recently been documented that positive serology indicates active H. pylori infection in ABG patients 26 and eradication therapy is able to reduce the serological levels 26‘I. Some Authors have suggested the possibility that H. pyZori infection may play a role in the development of pernicious anaemia, the end-stage consequence of ABG I3 32. Recently, it has been reported that H. pylori infection is involved in the induction of ABG 4-72s. Furthermore, the fact that H. pylori infection can induce gastric autoimmunity is well established, since the presence of the bacteria leads to the production of antibodies cross-reacting with human gastric mucosa I5 16. Moreover, if PA were related to H. pylori infection, pathological involvement of arm-al mucosa due to present or past infection, could be expected, whereas in pure autoimmune PA the antral mucosa would be normal 17. Contrasting data concerning the presence of antral gastritis in PA patients have been reported, however, previous studies have failed to detect differences between H. pyZori-related and non H. pylori-related PA 9 I”. In the present study, we observed that, considering the whole group of H. pylori-positive PA patients vs those H. pylori-negative, no clinical or gastric morpho-functional features were found. However, analysing the

A. Annibale at al.

subgroup of histologically H. pylori-positive PA patients, we observed that they showed a significantly greater involvement of the antral mucosa with respect to the serologically H. pylori-positive group, not unlike our previous finding on histologically proven H. pyloripositive ABG patients 6. In addition, the histologically H. pylori-positive group showed a lower score of body mucosal atrophy and a higher median concentration of serum Pepsinogen I, indicating less oxyntic damage since this enzyme is a reliable marker of secretory function in the oxyntic mucosa 33.In addition, these patients had a higher score of chronic inflammation and inflammation activity, both in the antrum and in the body mucosa, a finding considered as a very sensitive indicator of the presence of H. pylori infection 34. Since it has been described that in PA the antral mucosa shows a tendency to progressive healing 35,our findings suggest that this group of histologically H. pylori-positive PA patients could be in an earlier phase of the pathological process leading to the full picture of PA. Possible explanations for the differences between our results and those of other earlier studies regarding H. pylori infection and PA are manifold. First, our study was conducted on a group of consecutive PA patients, with a homogeneous ethnic background. Thus, it was not retrospective in nature and not based on data obtained from filed pathology records as in studies by other authors ‘I 36. Moreover, in the present study, we employed both serology and histology to establish the status of H. pylori infection rather than a single technique I1 36-38.Although this approach has previously been used by other Authors 9 ‘O, we examined a larger number of patients, adopted a more extensive biopsy protocol and a different histological scoring system, all aspects that can account for some apparently contrasting results. PA was considered, until now, an exclusively autoimmune disease. Even if the autoimmune processes operating in the full-blown picture of this disease have been extensively studied 39, its initiating events have not been clearly defined. In fact, another interesting finding in our study is the significantly higher prevalence of males among our PA patients, in contrast with the general observation that ABG with an autoimmune component is more frequently seen in women than in men 39-4’. The high male prevalence of PA patients is accounted for by the H. pylori positive patients since this infection is reported to be more frequent in the male sex 1 27. Conversely, autoimmune thyroid phenomena were prevalent among female patients. In fact, when analysing the associated autoimmune phenomena with respect to H. pylori infection we failed to observe a higher prevalence in females. Furthermore, in the histologically H. pylori-positive group of PA patients, autoimmune phenomena were less frequent,

since only one patient had positive anti-thyroid peroxidase antibodies and two patients (1 male and 1 female) had associated autoimmune diseases. This group of patients presented less extensive damage in the oxyntic mucosa indicated by higher Pepsinogen I levels and presence of response to pentagastrin-stimulated acid secretion in two patients. Moreover, in the histologically H. pylori-positive patients, the presence of anti-parietal cell antibodies was lower with respect to serologically H. pylori-positive patients, but even present in more than 40% of patients. This finding is in agreement with the evidence that H. pylori infection is able to trigger gastric autoimmunity and that anti-parieta1 cell antibodies can be detected in patients with H. pylori infection without ABG or PA I5 I6 42-44.Thus, it may be hypothesized that, at least in histologically H. pylori-positive patients, PA may be triggered by H. pyZori infection. In conclusion, our results indicate that in a consecutive population of PA patients, documented H. pylori infection is associated with 60% of PA cases. Thus, it is feasible to suggest that in a subgroup of PA patients, oxyntic atrophy is related to H. pylori and cure of infection should be considered also in this condition.

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