Epidemiology of Helicobacter pylori in chronic haemodialysis patients using the new RIBATM H. pylori SIA

Fabrizio Fabrizi1,3, Paul Martin1, Vivek Dixit1, Stella Quan2, Maria Brezina1, Heather Abbey1, Silvia Gerosa1, Ezra Kaufman2, Robert DiNello2, Alan Polito2 and Gary Gitnick1

1 Division of Digestive Diseases, UCLA School of Medicine, Los Angeles, 2 Chiron Corporation, CA, USA and 3 Division of Nephrology and Dialysis, Lecco Hospital, Lecco, Italy

Correspondence and offprint requests to: Fabrizio Fabrizi MD, Division of Nephrology and Dialysis, Lecco Hospital, via Ghislanzoni 22, I-23900 Lecco, Italy.



   Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background. There are few data concerning the epidemiology of H. pylori in patients on chronic haemodialysis (HD) treatment. These surveys concerned small populations and were made with ELISA technique. However, ELISA-based assays do not differentiate between strains of H. pylori that are associated with ulcers. Recent literature reports that formation of ulcers correlates strongly with the expression of cytotoxin-associated protein (CagA) and vacuolating cytotoxin (VacA) of H. pylori.

Methods. A novel serological test (RIBATM H. pylori strip immunoblot assay (SIA)) has been recently introduced, it uses the H. pylori lysate (Lys) along with two additional purified recombinant antigens derived from CagA and VacA of H. pylori.

Aim. To study the epidemiology of H. pylori using RIBATM H. pylori SIA among chronic HD patients and blood donors as a control group. In addition, the activity of H. pylori was analysed by immunoblot technique in a group of patients with documented ulcers and normal renal function.

Results. The prevalence of antibody towards H. pylori among HD patients, blood donors, and patients with documented ulcers was 56% (127/228), 53% (84/158), and 100% (21/21) respectively; the difference was significant (P=0.0001). The frequency of anti-H. pylori-positive individuals was significantly higher in patients with documented ulcers than HD patients and blood donors, 21/21 (100%) vs 211/386 (55%), P=0.0001. The frequency of antibody to H. pylori in the HD population was significantly associated with race (P=0.005); no relationship between anti-H. pylori antibody and numerous demographic, biochemical, and clinical features of patients was seen. The frequency of antibodies against virulent strains of H. pylori in HD patients and blood donors with H. pylori was 60% (76/127) and 61% (51/84) respectively; it was 86% (18/21) among individuals with documented ulcers. No significant difference among these three groups occurred.

Conclusions. The frequency of antibody towards H. pylori by RIBATM H. pylori SIA was high both in HD patients and blood donors; patients with documented ulcers and normal renal function had significantly higher frequency of anti-H. pylori antibody. The anti-H. pylori antibody rate among HD patients was strongly associated with race. The prevalence of antibody against virulent strains of H. pylori did not change among HD patients and control groups. Studies in large cohorts of HD patients with documented peptic ulcer disease are in progress.

Keywords: haemodialysis; Helicobacter pylori; immunoblot technique; peptic ulcer disease; virulent strains



   Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Helicobacter pylori has been identified as a major factor in the pathogenesis of peptic ulcer disease and gastritis in the general population [1,2]. Because peptic ulcer disease is frequent in patients with end-stage renal failure [3,4] preliminary reports have addressed the epidemiology of antibodies to H. pylori in patients on chronic haemodialysis [513], peritoneal dialysis [14], and renal transplant recipients [12,13]. However, these studies regarded small cohorts of patients, and an ELISA technique for detection of IgG antibodies to H. pylori has been used.

More recently the RIBATM H. pylori strip immunoblot assay has been developed [15,16] which improves the serological detection of H. pylori. RIBATM H. pylori SIA utilizes the H. pylori lysate along with two additional purified recombinant antigens derived from the cytotoxin-associated protein (CagA) and the vacuolating cytotoxin (VacA) of H. pylori. The reactivity of antibodies towards the lysate band is indicative of H. pylori infection. Additional band reactivity towards the CagA and/or VacA bands further indicates the presence of virulent strains of H. pylori. Gastric and duodenal ulcer formation has been recently reported to correlate strongly with the expression of CagA and VacA of H. pylori [17,18]. ELISA-based assays detect the presence of antibody to H. pylori, but do not differentiate between pathogenic and non-pathogenic strains.

The aim of this study was to assess the epidemiology of H. pylori by RIBATM H. pylori SIA in a large cohort of HD patients compared to blood donors. Moreover, an additional control group of patients with normal renal function and documented peptic ulcers was studied.



   Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Patients
Three distinct populations were analysed by RIBATM H. pylori SIA. The first cohort was a large (n=228) population of patients undergoing chronic HD treatment in three units in the greater Los Angeles area. There were 138 (61%) males and 90 (39%) females, the mean age was 61.8±13.8 years. There were 18 (8%) Asian, 91 (40%) African-American, 34 (15%) Hispanic, and 85 (37%) Caucasian individuals. The prevalence of HBsAg infection was 2.6% (6/228), the anti-HCV positivity was 16.5% (35/212). The mean ALT levels were 9.09 U/l (95% CI, 42.9–1.9). The causes of end-stage renal disease (ESRD) were as follows: chronic glomerulonephritis (n=36), nephroangiosclerosis (n=7), diabetic nephropathy (n=65), chronic pyelonephritis (n=4), polycystic kidney disease (n=8), unknown causes and others (n=42). The median time on HD was 29 months (range, 6–331); the mean pre-dialysis level of creatinine and azotaemia was 10.3±3.5 and 69.3±21.1 mg%, respectively; the mean Kt/V value was 1.47±0.28. There were 20 (9%) of 228 patients using antibiotics and 47 (21%) antacids, 187 (82%) of 228 received phosphate binders and 10 (4%) corticosteroids. Bicarbonate dialysis was used for all the patients. Eighty-nine (39%) of 228 underwent HD at unit 1, 97 (43%) and 42 (18%) were on HD at units 2 and 3, respectively. The patients were dialysed with standard HD technique 3–4 h three times a week. The second group was a population of blood donors (n=158) in the same region matched by sex and race; 91 (58%) were males and 67 (42%) females; there was no significant difference between HD patients and blood donors with regard to age, 61.8±13.8 vs 58.9±13.9 years (NS). In the third group there were patients (n=21) evaluated for peptic ulcer disease at UCLA/Medical Center and the UCLA/VA Cure Ulcer Clinic. They had endoscopically confirmed duodenal (n=12) and gastric (n=9) ulcers with normal renal function.

Chiron RIBATM H. pylori strip immunoblot assay (SIA)
Chiron RIBATM H. pylori SIA system, as shown in Figure 1Go, consists of a nitrocellulose solid support on which are immobilized three bands of H. pylori antigens (bands 2–4) and two bands of high- and low-level immunoglobulin G (IgG) controls (bands 1 and 5 respectively). The IgG controls are used to assess the level of RIBATM SIA reactivity. The remaining bands are coated with H. pylori antigens as follows: band 2 contains modified bacterial antigen lysate; band 3 contains recombinant VacA from the vacuolating cytotoxin (VacA) of H. pylori; band 4 contains recombinant CagA from the cytotoxin-associated protein (CagA) of H. pylori. The identity of the antibodies is defined by the specified location of the antigen band as shown in Figure 1Go.



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Fig. 1. Chiron RIBA H. pylori SIA strip diagram.

 
The procedure for performing manual RIBA H. pylori SIA is as follows: 30 µl of each sample is added to a tube containing a RIBA H. pylori 1.0 strip and 1 ml of RIBA H. pylori SIA specimen diluent. The next step is a 2–h incubation of the specimen at room temperature, a 30-min incubation in specimen diluent, two washes in wash buffer, a 10-min incubation in conjugate (peroxidase-labelled goat anti-human IgG (heavy and light chains)) followed by three washes in wash buffer, and a 15-min incubation in substrate (4-chloro-1 naphthol) followed by two washes with deionized water.

Algorithm for RIBATM H. pylori SIA
The band reactivity pattern on the strip is interpreted by comparing the intensity of each specific H. pylori antigen band with the intensities of the human IgG (level I and level II) internal control bands on each strip (Figure 1Go). The intensity of the H. pylori antigen bands is scored in relation to the intensities of the internal IgG controls. Band intensity equal to the intensity of the level I IgG control band is scored as 1+. Band intensity greater than that of level I IgG but less than that of level II IgG is scored as 2+. Band intensity equal to that of level II IgG is scored as 3+. Band intensity greater than that of level II IgG is scored as 4+. Band intensity less than that of the level I IgG control band is scored as ±, and the absence of any band reactivity is scored as-.

The interpretation of results is based on the pattern of band reactivity on the strip. Reactivity of 1+ or greater to the modified lysate band, and/or the CagA band, and/or the VacA band is interpreted as a positive result for antibody to H. pylori. No band having 1+ or greater reactivity is interpreted as a negative result.

Statistical analysis
Means between groups were compared with Student t-test. Percentages were compared by means of Fisher's exact test or X2 test. The statistical program Primer (by Stanton A. Glantz, 1992) was used.



   Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The frequency of anti-H. pylori antibody among HD patients was 56% (127/228). The frequency of antibody towards H. pylori among blood donors and individuals with documented ulcers was 53% (84/158) and 100% (21/21) respectively. The difference among the three groups was highly significant (P=0.0001); the anti-H. pylori antibody rate was higher in patients with documented ulcers (21/21=100%) than individuals on HD and blood donors (211/386=55%), P=0.0001. No difference was apparent with regard to the frequency of antibody against H. pylori by RIBATM H. pylori SIA between HD patients and blood donors, 56% (127/228) and 53% (84/158), NS.

There was no difference between anti-H. pylori positive and negative patients on HD regarding gender—male frequency was 59% (76/127) vs 61% (62/101), NS and age—64.06±12.4 vs 64.08±15.4 years (NS). The rate of H. pylori seropositivity in African-American and Hispanic patients was higher in H. pylori-positive than -negative patients on HD, 65% (83/127) vs 41% (42/101), P=0.0005.

No difference was present between anti-H. pylori-positive and -negative patients on HD concerning pre-dialysis urea and creatinine levels, 67.9±21 vs 70.9±21 mg% (NS) and 10.5±3.6 vs 10.2±3.3 mg% respectively, and ESRD aetiology or patient location among the HD units (NS). The depurative adequacy of dialysis treatment (Kt/V) did not significantly change in anti-H. pylori-positive vs -negative individuals on HD, 1.49±0.3 vs 1.45±0.2 (NS). No difference was present in anti-H. pylori-positive vs -negative patients on HD with regard to the mean time on HD, 4.1 (95% CI, 11.0–1.5) vs 4.2 (95% CI, 12.2–2.9) months (NS) and HBsAg rate or anti-HCV positivity (data not shown).

The frequency of HD patients using phosphate binders (aluminium hydroxide, calcium carbonate, or calcium acetate) did not significantly change between anti-H. pylori-positive and -negative individuals, 84% (107/127) vs 79% (80/101), NS. The rate of HD patients using antacids (H2 antagonists or proton prompt inhibitors) and antibiotics did not change between anti-H. pylori-positive and -negative patients, 29% (37/127) vs 29% (30/101) and 11% (14/127) vs 6% (6/101) respectively. The frequency of individuals receiving corticosteroids was slightly lower among H. pylori-positive than -negative individuals, 1.5% (2/127) vs 8% (8/101), P=0.046.

The pattern of reactivity by RIBATM H. pylori SIA in the subset of HD patients with anti-H. pylori antibody is shown in Table 1Go. Fifty-one (40%) patients of 127 tested lysate positive, 72 (56%) were Lys+CagA and/or VacA positive. Three patients (2%) were CagA positive and one (0.7%) was VacA positive.


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Table 1. Patterns of reactivity by RIBATM H. pylori SIA in HD patients and blood donors with H. pylori infection
 
There was an increased prevalence of anti-H. pylori antibody with age in both the patients on HD treatment and the healthy controls, as shown in Figure 2Go.



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Fig. 2. Prevalence of antibody towards H. pylori according to age.

 
There was significant difference between anti-H. pylori-positive vs negative blood donors with regard to age, 61.5±13.1 and 54.3±13.7 years, P=0.0005. There was no difference between anti-H. pylori-positive and -negative blood donors with regard to gender, male frequency 61% (51/84) vs 54% (40/74), P=0.491. There was no difference between anti-H. pylori-positive vs -negative blood donors with regard to race; however, H. pylori seropositivity was more frequent in Hispanic and African-American individuals 61% (51/84) vs 45% (33/74), P=0.062.

The pattern of reactivity by RIBATM H. pylori SIA in the subgroup of blood donors with H. pylori infection is shown in Table 1Go. Thirty-three (39%) of 84 were lysate positive, 48 (57%) were positive by Lys+CagA and/or VacA. Two (2%) patients were CagA positive and one (1%) was VacA positive.

The prevalence of antibodies against virulent strains of H. pylori among HD patients and blood donors with H. pylori was 60% (76/127) and 61% (51/84) respectively; the frequency of antibody against pathogenic strains of H. pylori was 86% (18/21) among patients with documented ulcers and normal renal function. The difference among the three groups was not significant.



   Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The presence of H. pylori in the gastric mucosa of humans is associated with peptic ulcers and gastric carcinoma. However, a clear discrepancy between the number of infected individuals and patients with clinical symptoms exists. Although host and environmental factors are considered important, there is also evidence for a role of specific H. pylori genotypes. Infection with some H. pylori genotypes (i.e. producing vacuolating cytotoxins or CagA-positive) is related to more severe morbidity, whereas other variants appear less pathogenic.

In our HD patients, the prevalence of antibody to H. pylori was high (56%). The frequency of anti-H. pylori antibody among chronic uraemic patients reported in the literature ranges between 21 and 64% [514]. These conflicting results may be related to various factors including: the methods of detecting H. pylori infection, the size of the population studied, the local prevalence of H. pylori in the health population, the clinical or demographic features of the study group, and other unknown factors.

In this study the prevalence of antibody towards H. pylori was high both in HD patients and blood donors. Earlier studies [19,20] reported a higher prevalence of H. pylori among dialysis patients and renal transplant recipients than in patients with normal renal function. A potential explanation is H. pylori's urease activity which may produce ammonia and so neutralize the bactericidal effect of gastric acid. As gastric juice urea is elevated in renal failure there is abundant substrate for H. pylori [11]. Also, the low gastric motility [21] and hypochlorhydria frequent in uraemic patients could be synergistic risk factors for gastric colonization with H. pylori. However, our results do not support these theoretical assumptions and indeed some investigators [6,9,10] have found a lower prevalence of H. pylori among dialysis patients than in individuals with normal renal function and concluded that patients with renal dysfunction appear to be partially protected against H. pylori. Possible protective mechanisms might include antibiotic use [9] or aluminium-containing antacids [22], prescribed for HD patients during the course of their illness. Also, increased uraemia could change bacterial colonization of the upper gastrointestinal tract with overgrowth of other bacteria and reduced H. pylori colonization [23].

We did not find difference in blood urea levels between seropositive and seronegative patients on HD. It indicates that high levels of urea are not a risk factor for acquiring H. pylori in this population, as previously mentioned by various investigators [6].

The prevalence of antibody against H. pylori in our HD patients was not significantly related to age; a significant association between H. pylori and increasing age in HD population has been found by some investigators [59]. A relationship between anti-H. pylori antibody and age was present in our blood donors. On the other hand, it is generally accepted that an increasing prevalence of H. pylori with rising age occurs in the general population [24].

In the current study we found an important link between antibody towards H. pylori and race—this may be related to socioeconomic status. The prevalence of H. pylori infection in the general population is higher in developing countries [24]. On the contrary, numerous demographic, biochemical and clinical features did not show association with H. pylori seropositivity.

No important relationship between anti-H. pylori antibody and several drugs commmonly prescribed to HD patients was apparent, this is in contrast with some suggestions present in the literature [9,22].

The analysis by immunoblot technique showed a high frequency of antibody to virulent strains of H. pylori among patients with documented ulcers; however, the prevalence of additional band reactivity toward CagA and/or VacA bands showed no changes in HD population seropositive for H. pylori in comparison with the controls with H. pylori. On the other hand, it is possible that the relatively small group of patients with documented peptic ulcers prevented the finding of a significant difference among the three groups. Further studies on larger HD cohorts with documented peptic ulcer disease are warranted to clarify this issue.

In conclusion, the frequency of antibody to H. pylori by RIBATM H. pylori SIA was high both in HD patients and in blood donors; patients with documented ulcers and normal renal function had a higher frequency of anti-H. pylori activity than among HD population. The anti-H. pylori antibody rate was strongly associated with race in our HD population; the rate of antibody against virulent strains of H. pylori did not change in HD patients vs control patients with H. pylori. Studies in large cohorts of HD patients with documented peptic ulcer disease are in progress.



   Acknowledgments
 
Fabrizio Fabrizi MD, is a staff nephrologist at the Nephrology and Dialysis Division, Hospital, Lecco, Italy. This study was in part supported by a Research Fellowship Award from the Society of Italian-American Nephrologists (to Dr F. Fabrizi). This work has been conducted at the Department of Medicine, Division of Digestive Diseases, University of California at Los Angeles, Los Angeles, California, USA.



   References
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received for publication: 9. 6.98
Accepted in revised form: 21. 4.99





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