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.
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Abstract |
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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
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Introduction |
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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.
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Material and methods |
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Chiron RIBATM H. pylori strip immunoblot assay (SIA)
Chiron RIBATM H. pylori SIA system, as shown in Figure 1, consists of a nitrocellulose solid support on which are immobilized three bands of H. pylori antigens (bands 24) 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 1
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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 1). 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.
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Results |
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There was no difference between anti-H. pylori positive and negative patients on HD regarding gendermale frequency was 59% (76/127) vs 61% (62/101), NS and age64.06±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, 11.01.5) vs 4.2 (95% CI, 12.22.9) 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 1. 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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The pattern of reactivity by RIBATM H. pylori SIA in the subgroup of blood donors with H. pylori infection is shown in Table 1. 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.
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Discussion |
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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 racethis 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.
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Acknowledgments |
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References |
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