Abstract

Eradication of Helicobacter pylori may lead to improvement of chronic immune thrombocytopenic purpura (ITP), although its efficacy over time is uncertain. We report the results of H pylori screening and eradication in 75 consecutive adult patients with ITP. We also used molecular methods to investigate lymphocyte clonality and H pylori genotypes in the gastric biopsies from 10 H pylori–positive patients with ITP and 19 H pylori–positive patients without ITP with chronic gastritis. Active H pylori infection was documented in 38 (51%) patients and successfully eradicated in 34 (89%) patients. After a median follow-up of 60 months, a persistent platelet response in 23 (68%) of patients with eradicated infection was observed; 1 relapse occurred. No differences in mucosal B- or T-cell clonalities were observed between patients with ITP and control participants. Of note, the frequency of the H pylori cagA gene (P = .02) and the frequency of concomitant H pylori cagA, vacAs1, and iceA genes (triple-positive strains; P = .015) resulted statistically higher in patients with ITP than in control participants. All asymptomatic H pylori–positive patients with ITP were suffering from chronic gastritis. Our data suggest a sustained platelet recovery in a proportion of patients with ITP by H pylori eradication alone. Overrepresentation of specific H pylori genotypes in ITP suggests a possible role for bacterium-related factors in the disease pathogenesis.

Introduction

The Gram-negative bacterium Helicobacter pylori is widely known as a causative agent of gastritis and peptic ulcers and as a high-risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma.1  The bacterium infection has also been implicated in the pathogenesis of extradigestive diseases, such as coronary heart disease, acne rosacea, idiopathic chronic urticaria, and also autoimmune diseases such as rheumatoid arthritis, autoimmune thyroiditis, Sjoegren syndrome, and Schoenlein-Henoch purpura.2  Moreover, in the last few years, several studies, primarily from Italy and Japan, found that the eradication of H pylori in infected patients with chronic immune thrombocytopenic purpura (ITP) leads to a substantial and persistent increase of platelet count in more than half of treated patients.3-17 

The pathogenetic mechanisms underlying the relationship between H pylori infection and ITP are still to be elucidated, but some data suggest that the effect of bacterium eradication may depend on either genetic factors of the host, country-related factors, or H pylori strains.18 

The real usefulness of bacterium eradication in ITP over time may be debatable, due to the limited duration of follow-up in most of the studies performed so far. To address this issue, we report the results of a 60-month follow-up of a study in 75 patients with ITP from a single center to conclude whether H pylori eradication may be an effective and long-term cure in chronic ITP. The second aim of the study is to give insights into the pathogenesis by investigating, in a selected group of patients, the possible association between either B- and T-cell clonality or specific H pylori strains and H pylori–associated ITP.

Patients and methods

Patients

Between January 1999 and December 2004, we enrolled 75 consecutive patients with adult chronic ITP: 33 men, 42 women with a median age of 56 years (range, 18-91 years). All but 2 patients were white, and came from Northern Italy; 93.3% were from Emilia-Romagna region, and 77.3% were from the province of Modena.

ITP was defined following the guidelines of the American Society of Hematology19 : idiopathic thrombocytopenia (platelets < 100 × 109/L) persistent for more than 6 months, with normal or increased megakaryocytosis in the bone marrow (patients more than 60 years old), and when other causes had been excluded, such as hepatits C virus (HCV) and HIV infections, drugs, lymphoproliferative disorders, other autoimmune diseases, and pseudothrombocytopenia.

Active H pylori infection was assessed by 13C urea breath test (UBT; Helicobacter test; Infai, Bochum, Germany) in all patients and, whenever possible, by histologic examination (Giemsa stain) of specimens obtained by an upper gastrointestinal endoscopy (22 patients; 29.3%). All immunosuppressive treatments and proton pump inhibitors were withdrawn at least 1 month before eradication. Bacterial eradication was performed with standard therapy: amoxicillin (1000 mg twice daily), clarithromycin (250 mg 3 times daily), and a proton-pump inhibitor (20 or 40 mg twice daily) for 1 week. Eradication was assessed by UBT 4 to 6 weeks after treatment withdrawal. Platelet counts were monitored every 2 weeks for the first 2 months, every month for the next 4 months and then every 6 months, after the end of treatment. A complete response (CR) was defined as a platelet count in the normal range for at least 3 months after eradication treatment. A partial response (PR) was defined as a platelet count not higher than 149 × 109/L or a doubling of initial value rising higher than 40 × 109/L. No response (NR) was defined as a platelet count that did not rise higher than 40 × 109/L, even if it was a doubling of initial value. The eradication treatment was also administered to 5 patients with ITP without H pylori infection, as control participants.

Biopsy and histology of the gastric mucosa

A total of 10 patients with ITP, with UBT positivity and without gastrointestinal symptoms, underwent upper gastrointestinal endoscopy; 2 distinct biopsy specimens were obtained from the corpus and antrum of the stomach. One fragment of each was placed in a sterile Eppendorf tube and stored at −80°C until DNA extraction; the second was fixed in 10% formalin and sent for routine histologic examination. Gastric biopsy specimens were also obtained from 35 patients without ITP with gastrointestinal symptoms who underwent the standard gastroscopy procedure and formed the control group. All paraffin-embedded tissue sections were stained with hematoxylin and eosin to detect gastric alterations and with Giemsa stain to detect H pylori.

For each patient of both groups, we evaluated the possible gastric mucosal B- and T-cell clonality and the presence of H pylori virulence factors: cagA (cytotoxin-associated gene A), vacA (vacuolating cytotoxin gene A) with s1, s2 (signal region) and m1, m2 (middle region) genotypes, iceA (induced by contact with epithelium gene; A1 and A2) and babA2 (blood group antigen-binding adhesin gene).

DNA extraction

Frozen gastric biopsy specimens were homogenized with a sterile micropestle, and the genomic DNA was extracted using Easy-DNA kit (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's instructions and stored at −20°C. Genomic DNA was also extracted from two 5-μm-thick paraffin wax–embedded sections, when frozen biopsy samples were not available, using the QIAamp DNA mini kit (tissue protocol; Qiagen, Valencia, CA) and stored at −20°C.

Primers and PCR design

The detection of the H pylori cagA (in the middle conservative region), signal regions s1 and s2 and middle regions m1 and m2 of the vacA and iceA variants iceA1 and iceA2 and babA2 genes was done by polymerase chain reaction (PCR) using established primers.20-24 

PCR reactions were performed in a reaction volume of 50 μL containing 1 × GeneAmp PCR Buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl2; Applied Biosystems, Foster City, CA), 225 μM dNTPs (Amersham Biosciences, Arlington Heights, IL), 0.5 μM of each primer, 2 U AmpliTaq Gold (Applied Biosystems), 10% glycerol for iceA, and 1 μg DNA. Negative and positive controls were assayed in each test.

A previous study25  revealed that the vacA midregion type was detected by PCR in only about 70% of the patients when the primers vac3R and vac4F were used. Therefore, these primers were substituted by primers vac3R* and vac4F*22  and additionally used in negative cases for vacA m1 or m2.

The PCR amplification was performed under the following conditions: initial denaturation at 95°C for 10 minutes, followed by 35 cycles (40 cycles for babA2) of denaturation at 95°C for 1 minute, annealing for 1 minute, extension for 1 minute at 72°C (1.5 minutes at 72°C for babA2), and a final extension at 72°C for 10 minutes. Annealing temperatures were set at 55°C for cagA primers, at 52°C for vacA (va1F-va1R, vac3F-vac3R, vac4F-vac4R), and at 50°C for vacA (vac3F-vac3R*, vac4F*-vac4R), iceA, and babA2 primer sets.

IgH gene rearrangement

Analysis of IgH gene rearrangement was carried out as a seminested PCR according to 2 different previously described protocols.26 

TCRγ gene rearrangement

Analysis of T-cell receptor γ (TCRγ) gene rearrangement was performed using 2 mixtures of primers for the various V and J segments by a single round of amplification as previously published,27  but with some modifications in PCR reaction conditions. Briefly, PCRs were performed using 2.5 U AmpliTaq Gold DNA polymerase in a 50-μL reaction that contained 1 × GeneAmp PCR Buffer, 1.5 mM MgCl2, 200 μM dNTPs, 300 ng of each primer, and 0.5 μg DNA under the following conditions: 10 minutes at 95°C, 45 cycles of 95°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, and final extension step for 10 minutes at 72°C.

A total of 20 μL of each PCR product was visualized by electrophoresis on 2% to 3% agarose gel stained with ethidium bromide under UV light and photographed (Gel Doc 2000; Bio-Rad, Hercules, CA).

In statistical analysis, data were expressed as the mean (SD) or median (range) as appropriate and were analyzed by using the t test. Percentages were compared by chi-square test (Fisher exact test for values < 5). A P value less than .05 was considered statistically significant. Disease-free survival was estimated using the Kaplan-Meier method. The statistical software used for the analyses was STATA/SE version 8.0 for Windows (Stata Corp, College Station, TX).

The study protocol was approved by the Provincial Ethical Committee of Modena, and an informed consent from all patients was obtained in accordance with the Declaration of Helsinki.

A second, separate consent was obtained from the patients with UBT positivity to allow gastric biopsy. The research aim of the procedure has been clarified. In the 5 patients with platelet counts less than 30 × 109/L, a 4-day course of intravenous immunoglobulin (IVIg) was administered. In these patients, gastric biopsy was performed when platelet counts reached 50 × 109/L, while eradication treatment was initiated when platelet counts decreased to lower than 30 × 109/L, which usually occurred after 1 month. No bleeding was noted in any patient.

Results

The clinical data concerning the 75 patients are shown in Tables 1,Table 23. Demographics of the first enrolled 37 patients, already reported elsewhere,4,5  as well as of the last enrolled 38 patients are shown in Table 2. On the whole, 22 (30%) patients entered the study at diagnosis of chronic ITP (7-9 months of persistent thrombocytopenia), while the remaining 53 (70%) have been previously diagnosed. A total of 52 percent of the patients have had previous treatment for ITP: all but 2 treated patients received steroids (often intermittently); 3 patients also received vincristine, 4 received cyclophosphamide, 7 received IVIg, 2 received cyclosporine, and 4 underwent splenectomy; 24 (61%) of 39 previously treated patients were refractory or dependent on steroid administration; 1 patient was also refractory to splenectomy.

Table 1

H pylori infection and ITP: our series

TimeNo. of patientsInfected, no. (%)Eradicated, no. (%)Responders, no. (%)CR/PR/NR, no.Median follow-up, mo (range)Reference
2001 30 13 (43) 12 (92) 6 (50)* 4/2/6 81 Emilia et al4  
2002 3 (43) 3 (100) 2 (66) 2/0/1 67 Emilia et al5  
2002-2004 38 22 (58) 19 (86) 15 (79) 10/5/5 32 (18-46) Present report 
Total 75 38 (51) 34 (89) 23 (68) 16/7/12 60 (18-90) — 
TimeNo. of patientsInfected, no. (%)Eradicated, no. (%)Responders, no. (%)CR/PR/NR, no.Median follow-up, mo (range)Reference
2001 30 13 (43) 12 (92) 6 (50)* 4/2/6 81 Emilia et al4  
2002 3 (43) 3 (100) 2 (66) 2/0/1 67 Emilia et al5  
2002-2004 38 22 (58) 19 (86) 15 (79) 10/5/5 32 (18-46) Present report 
Total 75 38 (51) 34 (89) 23 (68) 16/7/12 60 (18-90) — 

Number of men, 33; women, 42. Median age, 56 y (range, 18-91 y). A total of 14 patients had platelet counts lower than 30 × 109/L. A total of 10 (71%) showed response (1 relapse at 7 months). A total of 2 patients died from nonhematologic diseases. CR indicates complete response; PR, partial response; NR, no response; and —, not applicable.

*

1 relapse 7 months after eradication.

1 patient resistant to eradication, achieved response during follow-up.

Table 2

Patient demographics

Patient no.Age in y, sexDisease duration, mo.Previous therapyH pylori infectionPlatelets, × 109/L
Follow-up, moPresent condition
BeforeAfter (at 6 mo)
Previously reported patients 
52, M 16 None Yes 80 67 90 NR-stable 
58, F 15 None Yes 80 111 87 NR-stable 
63, M 14 None Yes 52 68 86 NR-stable 
60, F 16 Yes 30 31 86 NR-P 
67, M 46 None Yes 50 126* 85 PR 
79, F 70 P, V Yes 33 220* 83 CR 
60, M 15 Yes 25 176* 80 CR 
61, M 39 Yes 80 76 80 NR-stable 
75, M 20 None Yes 80 84 80 NR-stable 
10 77, F 21 Yes 27 172* 79 CR 
11 33, M 35 P,Sple Yes 80 371* 79 CR 
12 89, M 13 Yes 15 35 34 NR-lost 
13 48, F 53 Yes 50 125* 78 PR 
14 70, F 110 Ig,Cy No 37 80 88 Stable 
15 79, M 65 No 41 130 87 Stable 
16 36, M 52 P,V No 30 110 87 Stable 
17 19, F 19 None No 62 58 86 Stable 
18 18, F 18 None No 58 70 85 Stable 
19 20, F 29 No 38 120 83 Stable 
20 53, F 64 P,Ig,C No 35 80 83 Stable 
21 55, F 84 P,C,V No 32 90 83 Stable 
22 39, F 34 No 43 100 82 Stable 
23 26, F 15 No 65 150 80 Stable 
24 64, M 79 Ig,P No 45 130 80 Stable 
25 64, M 31 P,C,Sple No 120 81 Stable 
26 66, M 23 No 40 140 79 Stable 
27 49, F 32 Ig,Sple No 35 130 79 Stable 
28 26, F 21 No 40 140 79 Stable 
29 35, F 18 No 42 130 79 Stable 
30 68, F 120 P,Sple,Cy No 40 115 79 Stable 
31 66, M 11 P,C Yes 38 50 70 NR-stable 
32 41, F 12 Yes 38 160* 68 CR 
33 18, F 10 None Yes 46 180* 66 CR 
34 62, M 12 None No 48 50 72 Stable 
35 38, M 10 None No 46 145 65 Stable 
36 46, F 12 None No 42 190 64 Stable 
37 58, M 10 None No 43 185 64 Stable 
Last enrolled patients 
38 69, M Yes 29 190* 46 CR 
39 83, M 10 None Yes 43 170* 45 CR 
40 24, F None Yes 50 180* 45 CR 
41 78, M Yes 10 20 44 NR-P 
42 30, F 12 Yes 34 38 43 NR-sple 
43 52, M 16 None Yes 45 100* 43 PR 
44 85, F Yes 12 195* 23 CR 
45 69, F Yes 50 163* 43 CR 
46 18, F Ig/P Yes 22 41 NR-P 
47 47, M 186 Ig/P Yes 173* 39 CR 
48 27, M None Yes 80 120* 39 PR 
49 37, M 18 None Yes§ 25 99* 32 PR 
50 37, M 14 Yes 27 246* 31 CR 
51 73, F 18 None Yes 60 75 30 NR-P 
52 77, M None Yes 70 120* 28 PR 
53 26, F Yes 485* 28 CR 
54 52, F Ig/P Yes 20 235* 26 CR 
55 77, F 21 Yes 18 187* 26 CR 
56 77, F 12 Yes 65 120* 25 PR 
57 70, F Yes 31 40 21 NR-P 
58 45, F 10 None Yes§ 50 60 21 NR-stable 
59 34, M Yes§ 12 18 NR-P 
60 57, M None No 113 41 
61 68, M None No 15 95 39 
62 81, F 12 None No 248 34 Stable 
63 46, M None No 50 60 33 Stable 
64 82, F 19 None No 78 70 32 Stable 
65 36, F None No 25 100 32 P-stable 
66 26, M None No 24 115 31 
67 38, F 12 None No 50 48 31 Stable 
68 85, F None No 20 80 29 Dead 
69 42, M 16 None No 60 55 28 Stable 
70 35, M 14 None No 70 60 28 Stable 
71 91, F 10 None No 25 90 27 P-stable 
72 24, M None No 12 85 25 
73 47, M None No 25 90 19 P-stable 
74 48, M None No 45 50 18 Stable 
75 26, F None No 40 48 18 Stable 
Patient no.Age in y, sexDisease duration, mo.Previous therapyH pylori infectionPlatelets, × 109/L
Follow-up, moPresent condition
BeforeAfter (at 6 mo)
Previously reported patients 
52, M 16 None Yes 80 67 90 NR-stable 
58, F 15 None Yes 80 111 87 NR-stable 
63, M 14 None Yes 52 68 86 NR-stable 
60, F 16 Yes 30 31 86 NR-P 
67, M 46 None Yes 50 126* 85 PR 
79, F 70 P, V Yes 33 220* 83 CR 
60, M 15 Yes 25 176* 80 CR 
61, M 39 Yes 80 76 80 NR-stable 
75, M 20 None Yes 80 84 80 NR-stable 
10 77, F 21 Yes 27 172* 79 CR 
11 33, M 35 P,Sple Yes 80 371* 79 CR 
12 89, M 13 Yes 15 35 34 NR-lost 
13 48, F 53 Yes 50 125* 78 PR 
14 70, F 110 Ig,Cy No 37 80 88 Stable 
15 79, M 65 No 41 130 87 Stable 
16 36, M 52 P,V No 30 110 87 Stable 
17 19, F 19 None No 62 58 86 Stable 
18 18, F 18 None No 58 70 85 Stable 
19 20, F 29 No 38 120 83 Stable 
20 53, F 64 P,Ig,C No 35 80 83 Stable 
21 55, F 84 P,C,V No 32 90 83 Stable 
22 39, F 34 No 43 100 82 Stable 
23 26, F 15 No 65 150 80 Stable 
24 64, M 79 Ig,P No 45 130 80 Stable 
25 64, M 31 P,C,Sple No 120 81 Stable 
26 66, M 23 No 40 140 79 Stable 
27 49, F 32 Ig,Sple No 35 130 79 Stable 
28 26, F 21 No 40 140 79 Stable 
29 35, F 18 No 42 130 79 Stable 
30 68, F 120 P,Sple,Cy No 40 115 79 Stable 
31 66, M 11 P,C Yes 38 50 70 NR-stable 
32 41, F 12 Yes 38 160* 68 CR 
33 18, F 10 None Yes 46 180* 66 CR 
34 62, M 12 None No 48 50 72 Stable 
35 38, M 10 None No 46 145 65 Stable 
36 46, F 12 None No 42 190 64 Stable 
37 58, M 10 None No 43 185 64 Stable 
Last enrolled patients 
38 69, M Yes 29 190* 46 CR 
39 83, M 10 None Yes 43 170* 45 CR 
40 24, F None Yes 50 180* 45 CR 
41 78, M Yes 10 20 44 NR-P 
42 30, F 12 Yes 34 38 43 NR-sple 
43 52, M 16 None Yes 45 100* 43 PR 
44 85, F Yes 12 195* 23 CR 
45 69, F Yes 50 163* 43 CR 
46 18, F Ig/P Yes 22 41 NR-P 
47 47, M 186 Ig/P Yes 173* 39 CR 
48 27, M None Yes 80 120* 39 PR 
49 37, M 18 None Yes§ 25 99* 32 PR 
50 37, M 14 Yes 27 246* 31 CR 
51 73, F 18 None Yes 60 75 30 NR-P 
52 77, M None Yes 70 120* 28 PR 
53 26, F Yes 485* 28 CR 
54 52, F Ig/P Yes 20 235* 26 CR 
55 77, F 21 Yes 18 187* 26 CR 
56 77, F 12 Yes 65 120* 25 PR 
57 70, F Yes 31 40 21 NR-P 
58 45, F 10 None Yes§ 50 60 21 NR-stable 
59 34, M Yes§ 12 18 NR-P 
60 57, M None No 113 41 
61 68, M None No 15 95 39 
62 81, F 12 None No 248 34 Stable 
63 46, M None No 50 60 33 Stable 
64 82, F 19 None No 78 70 32 Stable 
65 36, F None No 25 100 32 P-stable 
66 26, M None No 24 115 31 
67 38, F 12 None No 50 48 31 Stable 
68 85, F None No 20 80 29 Dead 
69 42, M 16 None No 60 55 28 Stable 
70 35, M 14 None No 70 60 28 Stable 
71 91, F 10 None No 25 90 27 P-stable 
72 24, M None No 12 85 25 
73 47, M None No 25 90 19 P-stable 
74 48, M None No 45 50 18 Stable 
75 26, F None No 40 48 18 Stable 

Of the previously reported patients, nos. 1-30 are from Emilia et al;4  nos. 31-37 are from Emilia et al.5  For uninfected patients, platelet values are before and after immunosuppressive therapy.

P indicates prednisone; C, cyclosphosphamide; Cy, cyclosporine; V, vincristine; Ig, intravenous immunoglobulin; sple, splenectomy; CR, complete response; PR, partial response; and NR, no response.

*

Eradication-responsive patients.

Patient relapsed 7 months after eradication; at present, the patient is in CR with intermittent prednisone.

Died from heart failure.

§

Eradication failed after at least 2 regimens, including metronidazole.

Patient achieved response during follow-up.

Table 3

Response to H pylori eradication treatment in patients with platelets less then 30 × 109/L

Patient no.Age in y, sexDuration, moH pylori infectionPlatelets, × 109/L
Follow-up, mo.Present condition
BeforeAfter (at 6 mo)
69, M Yes 29 190* 46 CR 
10 77, F 21 Yes 27 172* 79 CR 
50 37, M 14 Yes 27 246* 31 CR 
60, M 15 Yes 25 176* 80 CR 
49 37, M 18 Yes 25 99* 32 PR 
54 52, F Yes 20 235* 26 CR 
55 77, F 21 Yes 18 187* 26 CR 
12 89, M 13 Yes 15 35 34 NR 
44 85, F Yes 12 195* 23 Died in CR 
41 78, M Yes 10 20 44 NR-P 
46 18, F Yes 22 41 NR-P 
47 47, M 186 Yes 173* 39 CR 
53 26, F Yes 485* 28 CR 
59 34, M Yes 12 18 NR-P 
65 36, F No 25 100 32 P-stable 
71 91, F No 25 90 27 P-stable 
73 47, M No 25 90 19 P-stable 
66 26, M No 24 115 31 
68 85, F No 20 80 29 Dead 
61 68, M No 15 95 39 
72 24, M No 12 85 25 P-stable 
62 81, F 12 No 248 34 Stable 
25 64, M 31 No 120 81 Stable 
60 57, M No 113 41 
Patient no.Age in y, sexDuration, moH pylori infectionPlatelets, × 109/L
Follow-up, mo.Present condition
BeforeAfter (at 6 mo)
69, M Yes 29 190* 46 CR 
10 77, F 21 Yes 27 172* 79 CR 
50 37, M 14 Yes 27 246* 31 CR 
60, M 15 Yes 25 176* 80 CR 
49 37, M 18 Yes 25 99* 32 PR 
54 52, F Yes 20 235* 26 CR 
55 77, F 21 Yes 18 187* 26 CR 
12 89, M 13 Yes 15 35 34 NR 
44 85, F Yes 12 195* 23 Died in CR 
41 78, M Yes 10 20 44 NR-P 
46 18, F Yes 22 41 NR-P 
47 47, M 186 Yes 173* 39 CR 
53 26, F Yes 485* 28 CR 
59 34, M Yes 12 18 NR-P 
65 36, F No 25 100 32 P-stable 
71 91, F No 25 90 27 P-stable 
73 47, M No 25 90 19 P-stable 
66 26, M No 24 115 31 
68 85, F No 20 80 29 Dead 
61 68, M No 15 95 39 
72 24, M No 12 85 25 P-stable 
62 81, F 12 No 248 34 Stable 
25 64, M 31 No 120 81 Stable 
60 57, M No 113 41 

Data represent patients with ITP with platelet counts less than 30 × 109/L. Disease duration data from Table 2. For uninfected patients, platelet values are before and after immunosuppressive therapy.

P indicates intermittent prednisone.

*

Eradication-responsive patients.

Relapse 7 months after eradication.

Lost follow-up.

H pylori infection was found in 38 (51%) patients. No differences in the prevalence of infection were noted between infected men (19 infected vs 17 uninfected) and women (19 infected vs 20 uninfected). The median age of infected patients was 58 years (± 19 years), while the median age for uninfected patients was 49.9 years (± 20.7 years; P = .082). The baseline platelet count was similar in infected and uninfected patients (40.6 × 109/L [± 25.0 × 109/L] vs 37.2 × 109/L [± 18.6 × 109/L]; P = .507). A total of 14 (37%) of 38 patients infected with H pylori showed, at diagnosis, a severe form of ITP (platelet count less than 30 × 109/L). Eradication of the bacterium has been obtained in 34 (89%) infected patients, of which 18 (53%) were younger than 65 years, while 16 (47%) were older than 65 years (P = .387).

The median platelet count follow-up time (calculated from the date of registration to either June 31, 2006, or the date of death or loss of follow-up) was 60 months (range, 18-81 months). Only 2 non-ITP–related deaths occurred, caused by heart failure. At the end of follow-up, 23 (68%) of the 34 patients with eradicated infection had a significant and persistent increase in platelet count (95% confidence interval [CI] = 48%-81%); 1 patient refractory to eradication treatment showed response during follow-up (Tables 1,2); and 16 (46%) patients achieved CR (95% CI = 29%-73%), while 7 (20%) patients achieved PR (95% CI = 8%-37%) and 12 (34%) were nonresponders (95% CI = 19%-52%). The response rate was 40% (4 of 10 patients; 1 CR and 3 PR) in patients with baseline platelet counts greater than 50 × 109/L; 64% (9 of 14 patients; 6 CR and 3 PR) in patients with platelet counts between 50 and 30 × 109/L, and 71% (10 of 14 patients; 9 CR and 1 PR) in patients with platelet counts less than 30 × 109/L. Further details of disease duration and response in this latter patient group are shown in Table 3. All of the 6 patients with platelet counts between 29 and 20 × 109/L were responders (CR = 83%); 50% of the 4 patients with platelet counts between 19 and 10 × 109/L and 50% of 4 patients with platelet counts between 9 and 1 × 109/L were responders, and achieved CR. All of the 10 uninfected patients with platelet counts less than 30 × 109/L were responsive to immunosuppressive treatment (Table 3). The initial platelet count improvement was usually observed 2 weeks after completion of eradication therapy, with assessment at 6 months (Figure 1A,B). Disease-free survival analysis of H pylori–positive patients with ITP (responders and nonresponders to eradication treatment) showed that 55% of patients are disease free both at 7 and 60 months (95% CI = 38.3-69.3), with no late relapses (Figure 1C). No significant differences in the platelet response were noted in respect to sex and age: 11 of 17 men and 12 of 18 women were responders; 12 (52%) patients aged 65 years and younger and 11 (48%) patients older than 65 years showed a response (CR, 8 and 8, respectively; PR, 4 and 3, respectively; P = .505). Responders were not younger than nonresponders (59 ± 19 years vs 61 ± 19 years; P = .497), and had neither a higher baseline platelet count (39 ± 23 × 109/L vs 46 ± 24 × 109/L; P = .402) nor a shorter disease duration (21 ± 39 months vs 10 ± 11 months; P = .368). Among 23 responder patients, 15 (65%) had received prior therapy (including prednisone, immunoglobulin [2 patients], vincristine [1patient], and splenectomy [1 patient]) versus 9 (60%) of 15 nonresponders (P = .307).

Figure 1

Comparison of platelet counts between patient groups; disease-free survival in infected patients. (A) Platelet (PLT) count of H pylori–positive patients with ITP, both responders and nonresponders to eradication, and H pylori–negative patients with ITP, at baseline. Black lines represent the median platelet value. ● represents single patient platelet values. (B) Platelet (PLT) count of H pylori–positive patients with ITP, both responders and nonresponders to eradication, and H pylori–negative patients with ITP, at 6 months. Black lines represent the median platelet value. ● represents single patient platelet values. (C) Disease-free survival analysis of H pylori–positive patients measured by the Kaplan-Meier method. A total of 55 percent of patients are disease free both at 7 and 60 months (95% CI = 38.3-69.3).

Figure 1

Comparison of platelet counts between patient groups; disease-free survival in infected patients. (A) Platelet (PLT) count of H pylori–positive patients with ITP, both responders and nonresponders to eradication, and H pylori–negative patients with ITP, at baseline. Black lines represent the median platelet value. ● represents single patient platelet values. (B) Platelet (PLT) count of H pylori–positive patients with ITP, both responders and nonresponders to eradication, and H pylori–negative patients with ITP, at 6 months. Black lines represent the median platelet value. ● represents single patient platelet values. (C) Disease-free survival analysis of H pylori–positive patients measured by the Kaplan-Meier method. A total of 55 percent of patients are disease free both at 7 and 60 months (95% CI = 38.3-69.3).

The ITP recurred in only 1 patient 7 months after eradication, although the UBT remained negative. In 3 of 4 patients in whom eradication failed, the platelet count remained unchanged during follow-up; 1 patient (no. 49) achieved response during the follow-up (about 10 months after the third-line regimen, including metronidazole) without any further treatment. At the end of follow-up, all living patients were in good condition. No side effects after eradication treatment were noted in any patient. No platelet response was observed in any H pylori–negative patients in whom eradication therapy had been administered.

Among the 37 H pylori–negative patients, the platelet count remained substantially unchanged during follow-up in the 10 patients not requiring therapy; immunosuppressive treatment (prednisone and IVIg, or cyclophosphamide or vincristine) was often intermittently administered in the remaining 27 patients, including 10 patients with platelet counts less than 30 × 109/L, allowing CR in 4 patients and a PR in 23 patients (Table 2).

At histologic examination, all 10 clinically asymptomatic patients with ITP were confirmed to be infected with H pylori and were found to be suffering from chronic and/or atrophic gastritis. Of 35 clinically symptomatic patients without ITP, 23 were found to be infected with H pylori: 4 had peptic ulcer and 19 had chronic and/or atrophic gastritis; these latter patients were selected as controls, to be compared with patients with ITP who had the same gastric pathology. Regarding the mucosal B-cell clonality, no significant differences were noted between patients with ITP and control patients: 6 (60%) of 10 patients with ITP and 11 (58%) of 19 control patientss were mono- or oligoclonal (P = .61), while the remaining patients were polyclonal. Similarly, no differences were noted in respect to T-cell clonality: 3 (33%) of 9 patients with ITP were monoclonal, and 5 (29%) of 17 control patients were mono- or oligoclonal (P = .587; Table 4). As far as the responsiveness to eradication treatment, B-cell oligo-monoclonality was detected in 5 (83%) of 6 responsive patients and 1 (25%) of 4 nonresponsive patients (P = .065). Likewise, 2 (40%) of 5 responsive patients showed T-cell monoclonality versus 1 (25%) of 4 nonresponsive patients (P = .635; Table 4).

Table 4

Virulence factor distribution: gastric B- and T-cell clonality

Patient no.Sex, age in ycagAvacA s1vacA s2vacA m1vacA m2ice A1ice A2bab A2IgHTCRγ
Patients with ITP 
    39 M, 83 − − − − Oligoclonal ND 
    41* M, 78 − − − − − Polyclonal Polyclonal 
    42* F, 30 − − − − − Polyclonal Polyclonal 
    47 M, 47 − − Polyclonal Monoclonal 
    48 F, 27 − − − − monoclonal Polyclonal 
    49 M, 37 − − − − − Oligoclonal Monoclonal 
    50 M, 37 − − − − monoclonal Polyclonal 
    40 F, 24 − − ND Oligoclonal Polyclonal 
    59* M, 34 − − − monoclonal Polyclonal 
    58* F, 45 − − − Polyclonal Monoclonal 
Control patients without ITP, with gastritis 
    1 M, 34 − − Oligoclonal Monoclonal 
    2 M, 89 − − − − − monoclonal Polyclonal 
    3 F, 36 − − − − − Polyclonal Polyclonal 
    4 M, 65 − − − − − Oligoclonal Monoclonal 
    5 M, 60 − − − − monoclonal Polyclonal 
    6 M, 67 − − − − Polyclonal Polyclonal 
    7 F, 34 − − − − ND Polyclonal Polyclonal 
    8 M, 74 − − − − − monoclonal ND 
    9 M, 40 − − − Oligoclonal Oligoclonal 
    10 F, 53 − − − − Polyclonal Polyclonal 
    11 M, 78 − − − − ND Polyclonal Polyclonal 
    12 M, 36 − − − monoclonal Polyclonal 
    13 F, 40 − − − − − Polyclonal Polyclonal 
    14 F, 51 − − − − − − Polyclonal Oligoclonal 
    15 F, 71 − − − monoclonal Monoclonal 
    16 F, 73 − − − − − monoclonal Polyclonal 
    17 F, 77 − − − − − Oligoclonal Polyclonal 
    18 F, 51 − − − − Polyclonal ND 
    19 F, 58 − − − − Oligoclonal Polyclonal 
Patient no.Sex, age in ycagAvacA s1vacA s2vacA m1vacA m2ice A1ice A2bab A2IgHTCRγ
Patients with ITP 
    39 M, 83 − − − − Oligoclonal ND 
    41* M, 78 − − − − − Polyclonal Polyclonal 
    42* F, 30 − − − − − Polyclonal Polyclonal 
    47 M, 47 − − Polyclonal Monoclonal 
    48 F, 27 − − − − monoclonal Polyclonal 
    49 M, 37 − − − − − Oligoclonal Monoclonal 
    50 M, 37 − − − − monoclonal Polyclonal 
    40 F, 24 − − ND Oligoclonal Polyclonal 
    59* M, 34 − − − monoclonal Polyclonal 
    58* F, 45 − − − Polyclonal Monoclonal 
Control patients without ITP, with gastritis 
    1 M, 34 − − Oligoclonal Monoclonal 
    2 M, 89 − − − − − monoclonal Polyclonal 
    3 F, 36 − − − − − Polyclonal Polyclonal 
    4 M, 65 − − − − − Oligoclonal Monoclonal 
    5 M, 60 − − − − monoclonal Polyclonal 
    6 M, 67 − − − − Polyclonal Polyclonal 
    7 F, 34 − − − − ND Polyclonal Polyclonal 
    8 M, 74 − − − − − monoclonal ND 
    9 M, 40 − − − Oligoclonal Oligoclonal 
    10 F, 53 − − − − Polyclonal Polyclonal 
    11 M, 78 − − − − ND Polyclonal Polyclonal 
    12 M, 36 − − − monoclonal Polyclonal 
    13 F, 40 − − − − − Polyclonal Polyclonal 
    14 F, 51 − − − − − − Polyclonal Oligoclonal 
    15 F, 71 − − − monoclonal Monoclonal 
    16 F, 73 − − − − − monoclonal Polyclonal 
    17 F, 77 − − − − − Oligoclonal Polyclonal 
    18 F, 51 − − − − Polyclonal ND 
    19 F, 58 − − − − Oligoclonal Polyclonal 

ND indicates not done.

*

Nonresponders to eradication treatment.

A different frequency distribution of the various H pylori virulence genes in patients with ITP compared with the controls was noted: the cagA, vacAs1, and iceA1 genes are each independently overrepresented in patients with ITP compared with the control patients, while conversely, the vacAs2 and the babA2 genes are overrepresented in the control group. Nevertheless, only the higher frequency of the cagA gene in patients with ITP was statistically significant (P = .02; Table 5). The expression of the babA2 gene was detected in 3 (30%) of 10 ITP patients and in 9 (47%) of 19 control patients (P = .309). In patients with ITP, the expression of the babA2 gene is apparently associated with the absence of response to eradication treatment. Of 4 ITP patients not responsive to eradication, 2 showed cagA negativity, and 2 showed cagA positivity (Table 4). The patient (no. 49) with late response was cagA negative. If a concomitant expression of at least 3 genes—the so-called “triple-positive strains”—is considered, the frequency of concomitant cagA, vacAs1, and iceA genes was significantly higher in patients with ITP than in control patients (7 [70%] of 10 vs 4 [22%] of 19; P = .015; Table 6).

Table 5

Virulence factor distribution between patients with ITP and control patients

cagA, no. (%)vacAs1, no. (%)s2, no. (%)m1, no. (%)m2, no. (%)iceA1, no. (%)A2, no. (%)babA2, no. (%)
Patients with ITP 7/10 (70) 7/10 (70) 3/10 (30) 2/10 (20) 8/10 (80) 7/10 (70) 4/10 (40) 3/9 (33) 
Control patients 5/19 (26) 9/19 (47) 10/19 (53) 4/19 (21) 14/19 (74) 11/19 (57) 8/19 (42) 9/17 (53) 
P .02 .24 .24 .9 .7 .5 .9 .3 
cagA, no. (%)vacAs1, no. (%)s2, no. (%)m1, no. (%)m2, no. (%)iceA1, no. (%)A2, no. (%)babA2, no. (%)
Patients with ITP 7/10 (70) 7/10 (70) 3/10 (30) 2/10 (20) 8/10 (80) 7/10 (70) 4/10 (40) 3/9 (33) 
Control patients 5/19 (26) 9/19 (47) 10/19 (53) 4/19 (21) 14/19 (74) 11/19 (57) 8/19 (42) 9/17 (53) 
P .02 .24 .24 .9 .7 .5 .9 .3 
Table 6

Distribution of ″triple-positive strains″ in patients with ITP and control patients

Patients with ITP, no. (%) 7/10 (70) 
Control patients, no. (%) 4/19 (22) 
P .015 
Patients with ITP, no. (%) 7/10 (70) 
Control patients, no. (%) 4/19 (22) 
P .015 

Concomitant expression of cagA, vacAs1, and iceA.

Discussion

The results of our study demonstrate the remarkable therapeutic efficacies (ie, bacterium eradication rate, platelet response rate, and durability of response) of antibiotics plus proton-pump inhibitors for an homogeneous ethnic group of adult patients with chronic ITP and active H pylori infection.

These findings are similar to those reported by several groups with 3 exceptions.3-17,28-35  The prevalence (51%) of bacterium infection in our series is not higher than that (58%) in the general population of the area of Modena province,36  or that (67%) in healthy Italian blood donors aged 50 years or older,37  in keeping with the data from the other countries, where the prevalence of infection in patients with ITP is similar or lower than that of the general population of the same area,6,10,13,38,39  with rare exceptions.15,40  In our study, we observed a platelet improvement in 68% of patients with eradicated infection (60.5% of patients infected; 30.6% of total patients with ITP); in about 1500 patients so far reported in the literature, including the 3 US and Spanish cohorts with the lowest responses, the rate of response was about 53%.17 

The highest response rate was reported in cohorts from Italy, Japan, and the United Kingdom. A better response in younger Italian and British patients with high baseline platelet counts and minor duration of the disease,10  and better results in Japanese patients older than 60 years40  or in Italian patients not previously treated7  have been reported. In our series, no differences between responders and nonresponders were noted in sex ratio, mean age, disease duration, previous treatments, and baseline platelet counts. Platelet recovery was found in patients previously refractory to immunosuppression or splenectomy, as reported by others.7,40  We also observed a response rate of 71% in patients with a severe form of ITP (platelets < 30 × 109/L), similar to that of other reports,7,40  whereas minor41  or nearly negative10  results were also reported.

A total of 9 (64%) of our 14 patients with severe, refractory ITP who failed to maintain a platelet count higher than 30 × 109/L after 6 to 12 months of standard therapy maintained a response for a median of 34 months (range, 26-46 months); 1 patient maintained a response for 73 months. This finding seems to be of interest if compared, for example, with the results of treatment of ITP with rituximab. A recent systematic review on more than 300 patients treated with rituximab, many of whom had a severe form of ITP, showed an overall response of 62.5% with a median response duration of 10.5 months, while relapse was found to occur in 10.5% of the patients. Of note, 21.6% of the patients experienced mild or moderate adverse events, and 3.7% experienced severe events, while 2.9% of deaths (even if not necessarily attributed to rituximab) were recorded.42  Conversely, we never observed toxicity related to H pylori eradication treatment.

The administration of eradication therapy to H pylori–negative patients with ITP failed to show any platelet increase in all treated patients, as observed by others,8,15,32,41,43  suggesting the crucial role of bacterium eradication rather than an effect of antibiotics by itself.

Of interest, all of our patients with ITP who underwent gastric biopsy showed at histologic examination chronic and/or atrophic gastritis, although gastric symptoms have never been disclosed. A similar finding, but in dyspeptic patients with ITP, has only been reported by another group.40 

Finally, at the end of follow-up, our living patients are all in remission, without any treatment; only 1 patient relapsed, and 2 died from non-ITP–related disease. The follow-up of 60 months in our study is the longest reported so far, whereas in the literature, it varies from 4 to 24 months, except for 1 series followed for 31.2 months.14 

The variability in the clinical outcome of eradicating the bacterium in patients from different countries and some differences in clinical features of responsive patients, even from the same area, raise the issue of whether and how H pylori infection plays a role in the development or persistence of a proportion of ITP. The pathogenetic mechanisms of ITP have still to be fully elucidated, and include platelet destruction due to phagocytosis by macrophages, antibody-dependent cell-mediated cytotoxicity,44  and T-cell–mediated destruction.45  The disease has also been associated with impaired megakaryocyte production.46  Several hypotheses have been raised to explain the possible interaction between H pylori and thrombocytopenia.10,41,46-49  Some H pylori strains bind von Willebrand factor and interact with glycoprotein Ib to induce platelet aggregation, but its contribution to immune thrombocytopenia remains to be demonstrated.50  Nevertheless, these hypotheses have yet to be confirmed and cannot be easily reconciled with the observation that improvement in the platelet count was generally seen 2 weeks after completion of eradication therapy, as reported also by others.10 

To address these issues and to give further insights into the role of H pylori in chronic ITP, we investigated the possible clonality of B and T cells in the gastric mucosa and assessed the H pylori genotypes in patients with ITP compared with those without ITP with the same gastric pathology. Gastric B-cell and T-cell monoclonality or oligoclonality have been observed in certain instances of H pylori chronic gastritis, without ITP.51,52  Peripheral blood B-cell clonality has elsewhere been reported in a low percentage of infected patients with ITP.53  No differences in the pattern of B-cell and T-cell clonality have been found between our infected patients with ITP and control participants. A prevalence of B-cell clonality was noted in 83% of our responsive patients versus the 25% of nonresponsive patients; a prevalence of T-cell clonality was present in 40% of responsive patients versus 25% of nonresponsive patients. Peripheral blood T-cell clonality has been observed in patients with ITP and H pylori infection, prevailing in responder patients.54  Peripheral blood B- and/or T-cell clonality has been noted in noninfected patients with ITP, with better response to therapy.54,55  On the whole, the findings from our limited series do not suggest a clear role of B- and T-cell clonality in the pathogenesis of ITP associated with H pylori infection.

The analysis of frequency distribution of the various H pylori virulence genes showed a marked difference between patients with ITP and control participants: the cagA, vacAs1, and iceA genes are each independently overrepresented in patients with ITP compared with control participants, while conversely, the vacAs2 and babA2 genes are overrepresented in the control group. Nevertheless, the main finding appears to be the higher frequency of the cagA gene in patients with ITP, even statistically significant (P = .02). In addition, the concomitant expression of at least 3 genes, cagA, vacAs1, and iceA, “triple -positive strains,” is significantly more frequent in patients with ITP than in control participants (P = .015). Conversely, the frequency of babA2 gene expression is significantly lower in patients with ITP than in controls, but of note, in 2 of 3 patients with ITP it is apparently associated with the absence of platelet response to eradication treatment. No differences in the virulence profile of cagA, vacA, and iceA strains were found in Japanese patients with ITP compared with control patients with ulcer or gastritis.40  On the other hand, other Japanese investigators found no differences in H pylori virulence factors between responder and nonresponder patients with ITP.13  The association of some specific H pylori genotypes with ITP is reminiscent of the association between certain H pylori genotypes and other human diseases, such as gastric ulcer and gastric adenocarcinoma, which correlate with “triple-positive strains” babA2, vacAs1, and cagA,24  or intestinal metaplasia, correlating with cagA, vacAs1m1, and babA2,56  while the iceA1 gene was suggested to be a marker of peptic ulcer.57 

The existence of some specific genotypes of H pylori associated with ITP might explain the different platelet response rates to eradication treatment in ITP from different geographic areas, which may have different genotype distribution. The existence of some specific genotypes of H pylori, with a different pathogenic potential in relation to ITP, might overcome the issue raised by the finding of similar seroprevalence rates of H pylori infection in patients with ITP compared with control participants. Although our small series may not adequately represent the whole ITP population, it may provide some useful insights into the pathogenesis of H pylori–related ITP. In summary, whichever is the mechanism by which H pylori might contribute to ITP, the eradication of the bacterium has proved to be of significant clinical value in a considerable number of patients with ITP. The long follow-up and the extremely low percentage of relapse in our series suggest a sustained recovery of disease for most patients with ITP responsive to H pylori eradication. Thus, the screening of all patients with ITP for H pylori at diagnosis and the eradication of the bacterium in H pylori–positive patients should precede other specific treatments for ITP whenever possible. This strategy is a useful and inexpensive way to avoid the discomfort and side effects of either immunosuppressive treatments or splenectomy in a subset of patients with ITP.

Further investigations are warranted to address how some of the H pylori virulence factors, identified in our study, may affect disease pathogenesis in ITP.

An Inside Blood analysis of this article appears at the front of this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

We are indebted to Dr Alberto Merighi, Dr Anna Maria Primerano, and Dr Antonella Scancelli (Gastroenterology Unit, Modena); Prof Roberto Marasca, Prof Alessandro Liberati, Dr Katia Cagossi, Dr Leonardo Ferrara, Dr Annalisa Imovilli, Dr Giovanni Riva, and Dr Roberto D'Amico (Department of Oncology and Hematology, Modena); and Dr Daniele Vallisa (Department of Oncology and Hematology, Azienda Unità Sanitaria Locale (AUSL) Piacenza).

This work was supported in part by the Italian Ministry for University and Scientific Research (MIUR), Rome, Italy; Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy; and the European Commission FP6 Life-Sciences-Health Program (INCA Project: LSHC-CT-2005–018704).

Authorship

Contribution: G.E., M.L., and G.T. designed the research; G.E. and M.L. analyzed data and wrote the paper; P.Z. performed molecular studies; G.J. attended to statistical analysis; M.M., L.P., F.F., F.V., G.L., and A.D. performed the clinical studies. G.E. and M.L. contributed equally to the study.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Giovanni Emilia, Department of Oncology and Hematology, Policlinico, via del Pozzo 71, 41100 Modena, Italy; e-mail: emilia.giovanni@unimore.it.

References

References
1
Suerbaum
 
S
Michetti
 
P
Helicobacter pylori infection.
N Engl J Med
2002
, vol. 
347
 (pg. 
1175
-
1186
)
2
Gasbarrini
 
A
Franceschi
 
F
Autoimmune diseases and Helicobacter pylori infection.
Biom Pharmacother
1999
, vol. 
53
 (pg. 
223
-
226
)
3
Gasbarrini
 
A
Franceschi
 
F
Tartaglione
 
R
Landolfi
 
R
Pola
 
P
Gasbarrini
 
G
Regression of autoimmune thrombocytopenia after eradication of Helicobacter pylori.
Lancet
1998
, vol. 
352
 pg. 
878
 
4
Emilia
 
G
Longo
 
G
Luppi
 
M
, et al. 
Helicobacter pylori eradication can induce platelet recovery in idiopathic thrombocytopenic purpura.
Blood
2001
, vol. 
97
 (pg. 
812
-
814
)
5
Emilia
 
G
Luppi
 
M
Morselli
 
M
Potenza
 
L
D'Apollo
 
N
Torelli
 
G
Helicobacter pylori infection and idiopathic thrombocytopenic purpura.
Br J Haematol
2002
, vol. 
118
 (pg. 
1198
-
1199
)
6
Kohda
 
K
Kuga
 
T
Kogawa
 
K
, et al. 
Effect of Helicobacter pylori eradication on platelet recovery in Japanese patients with chronic idiopathic thrombocytopenic purpura and secondary autoimmune thrombocytopenic purpura.
Br J Haematol
2002
, vol. 
118
 (pg. 
584
-
588
)
7
Veneri
 
D
Franchini
 
M
Gottardi
 
M
, et al. 
Efficacy of Helicobacter pylori eradication in raising platelet count in adult patients with idiopathic thrombocytopenic purpura.
Haematologica
2002
, vol. 
87
 (pg. 
1177
-
1179
)
8
Hino
 
M
Yamane
 
T
Park
 
K
, et al. 
Platelet recovery after eradication of Helicobacter pylori in patients with idiopathic thrombocytopenic purpura.
Ann Hematol
2003
, vol. 
82
 (pg. 
30
-
32
)
9
Ando
 
K
Shimamoto
 
T
Tauchi
 
T
, et al. 
Can eradication therapy for Helicobacter pylori really improve the thrombocytopenia in idiopathic thrombocytopenic purpura? Our experience and a literature review.
Int J Hematol
2003
, vol. 
77
 (pg. 
239
-
244
)
10
Stasi
 
R
Rossi
 
Z
Stipa
 
E
Amadori
 
S
Newland
 
AC
Provan
 
D
Helicobacter pylori eradication in the management of patients with idiopathic thrombocytopenic purpura.
Am J Med
2005
, vol. 
118
 (pg. 
414
-
419
)
11
Inaba
 
T
Mizuno
 
M
Take
 
S
, et al. 
Eradication of Helicobacter pylori increases platelet count in patients with idiopathic thrombocytopenic purpura in Japan.
Eur J Clin Invest
2005
, vol. 
35
 (pg. 
214
-
219
)
12
Fujimura
 
K
Kuwana
 
M
Kurata
 
Y
, et al. 
Is eradication therapy useful as first line treatment in Helicobacter pylori-positive idiopathic thrombocytopenic purpura? Analysis of 207 eradicated chronic ITP cases in Japan.
Int J Hematol
2005
, vol. 
81
 (pg. 
162
-
168
)
13
Suzuki
 
T
Matsushima
 
M
Masui
 
A
, et al. 
Effect of Helicobacter pylori eradication in patients with chronic idiopathic thrombocytopenic purpura: a randomized controlled trial.
Am J Gastroenterol
2005
, vol. 
100
 (pg. 
1265
-
1270
)
14
Veneri
 
D
Krampera
 
M
Franchini
 
M
High prevalence of sustained remission of idiopathic thrombocytopenic purpura after Helicobacter pylori eradication: a long-term follow-up study.
Platelets
2005
, vol. 
16
 (pg. 
117
-
119
)
15
Suvajdzic
 
N
Stankovic
 
B
Artiko
 
V
, et al. 
Helicobacter pylori eradication can induce platelet recovery in chronic idiopathic thrombocytopenic purpura.
Platelets
2006
, vol. 
17
 (pg. 
227
-
230
)
16
Sayan
 
O
Akyol
 
E
Ozturk
 
A
The efficacy of Helicobacter pylori eradication in the treatment of idiopathic thrombocytopenic purpura: the first study in Turkey.
Acta Haematol
2006
, vol. 
116
 (pg. 
146
-
149
)
17
Campuzano-Maya
 
G
Proof of an association between Helicobacter pylori and idiopathic thrombocytopenic purpura in Latin America.
Helicobacter
2007
, vol. 
12
 (pg. 
265
-
273
)
18
Emilia
 
G
Luppi
 
M
Torelli
 
G
Infectious agents and human immune diseases: lessons from Helicobacter pylori.
Am J Med
2005
, vol. 
118
 (pg. 
420
-
421
)
19
George
 
JN
Woolf
 
SH
Raskob
 
GE
, et al. 
Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology.
Blood
1996
, vol. 
88
 (pg. 
3
-
40
)
20
Tummuru
 
MKR
Cover
 
TL
Blaser
 
MJ
Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.
Infect Immun
1993
, vol. 
61
 (pg. 
1799
-
1809
)
21
Atherton
 
JC
Cao
 
P
Peck
 
RM
, et al. 
Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori: association of specific vacA types with cytotoxin production and peptic ulceration.
J Biol Chem
1995
, vol. 
270
 (pg. 
17771
-
17777
)
22
Rudi
 
J
Rudy
 
A
Maiwald
 
M
, et al. 
Direct determination of Helicobacter pylori vacA genotypes and cagA gene in gastric biopsies and relationship to gastrointestinal diseases.
Am J Gastroenterol
1999
, vol. 
94
 (pg. 
1525
-
1531
)
23
van Doorn
 
LJ
Figueiredo
 
C
Sanna
 
R
, et al. 
Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori.
Gastroenterology
1998
, vol. 
115
 (pg. 
58
-
66
)
24
Gerhard
 
M
Lehn
 
N
Neumayer
 
N
, et al. 
Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin.
Proc Natl Acad Sci U S A
1999
, vol. 
96
 (pg. 
12778
-
12783
)
25
Rudi
 
J
Kolb
 
C
Maiwald
 
M
, et al. 
Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated diseases.
J Clin Microbiol
1998
, vol. 
36
 (pg. 
944
-
948
)
26
De Re
 
V
De Vita
 
S
Carbone
 
A
, et al. 
The relevance of VDJ PCR protocols in detecting B-cell clonal expansion in lymphoma and other lymphoproliferative disorders.
Tumori
1995
, vol. 
81
 (pg. 
405
-
409
)
27
Trainor
 
KJ
Brisco
 
MJ
Wan
 
JH
Neoh
 
S
Grist
 
S
Morley
 
AA
Gene rearrangement in B- and T-lymphoproliferative disease detected by polymerase chain reaction.
Blood
1991
, vol. 
78
 (pg. 
192
-
196
)
28
Nam
 
S-H
Kim
 
B-S
Lee
 
JH
, et al. 
The clinical efficacy of Helicobacter pylori eradication in patients with Helicobacter pylori–positive chronic thrombocytopenic purpura [abstract].
Blood
2005
, vol. 
106
 pg. 
80b
  
Abstract no. 4002
29
Jang
 
JH
Kim
 
HI
Lee
 
HW
, et al. 
Correlation between Helicobacter pylori eradication and platelet count in immune thrombocytopenic purpura (ITP) [abstract].
Blood
2005
, vol. 
106
 pg. 
80b
  
Abstract no. 4003
30
Jaing
 
TH
Yang
 
CP
Hung
 
IJ
Chin
 
CH
Chang
 
KW
Efficacy of Helicobacter pylori eradication on platelet recovery in children with chronic idiopathic thrombocytopenic purpura.
Acta Paediatr
2003
, vol. 
92
 (pg. 
1153
-
1157
)
31
Neefjes
 
VME
Heijboer
 
H
Tamminga
 
RYJ
H. pylori infection in childhood chronic immune thrombocytopenic purpura.
Haematologica
2007
, vol. 
92
 pg. 
576
 
32
Michel
 
M
Cooper
 
N
Jean
 
C
Frissora
 
C
Bussel
 
JB
Does Helicobacter pylori initiate or perpetuate immune thrombocytopenic purpura?
Blood
2004
, vol. 
103
 (pg. 
890
-
896
)
33
Ahn
 
ER
Tiede
 
MP
Jy
 
W
Bidot
 
CJ
Fontana
 
V
Ahn
 
YS
Platelet activation in Helicobacter pylori-associated idiopathic thrombocytopenic purpura: eradication reduces platelet activation but seldom improves platelet counts.
Acta Haematol
2006
, vol. 
116
 (pg. 
19
-
24
)
34
Jarque
 
I
Andreu
 
R
Llopis
 
I
, et al. 
Absence of platelet response after eradication of Helicobacter pylori infection in patients with chronic idiopathic thrombocytopenic purpura.
Br J Haematol
2001
, vol. 
115
 (pg. 
1002
-
1003
)
35
Garcia-Perez
 
A
Valverde de la Osa
 
J
Gimenez Samper
 
M
Alonso Garcia
 
I
Resolution of an autoimmune thrombocytopenic purpura after eradicating treatment of Helicobacter pylori.
Sangre (Barcelona)
1999
, vol. 
44
 (pg. 
387
-
388
)
36
Dominici
 
P
Bellentani
 
S
Di Biase
 
AR
, et al. 
Familial clustering of Helicobacter pylori infection: population based study.
BMJ
1999
, vol. 
319
 (pg. 
537
-
540
)
37
Russo
 
A
Eboli
 
M
Pizzetti
 
P
, et al. 
Determinants of Helicobacter pylori seroprevalence among Italian blood donors.
Eur J Gastroenterol Hepatol
1999
, vol. 
11
 (pg. 
867
-
873
)
38
Franchini
 
M
Veneri
 
D
Helicobacter pylori infection and immune thrombocytopenic purpura: an update.
Helicobacter
2004
, vol. 
9
 (pg. 
342
-
346
)
39
Michel
 
M
Khellfar
 
M
Desforges
 
L
, et al. 
Autoimmune thrombocytopenic purpura and Helicobacter pylori infection.
Arch Intern Med
2002
, vol. 
162
 (pg. 
1033
-
1036
)
40
Ando
 
T
Tsuzuki
 
T
Mizuno
 
T
, et al. 
Characteristics of Helicobacter pylori-induced gastritis and the effect of H. pylori eradication in patients with chronic idiopathic thrombocytopenic purpura.
Helicobacter
2004
, vol. 
9
 (pg. 
443
-
452
)
41
Takahashi
 
T
Yujiri
 
T
Shinohara
 
K
, et al. 
Molecular mimicry by Helicobacter pylori CagA protein may be involved in the pathogenesis of H pylori-associated chronic idiopathic thrombocytopenic purpura.
Br J Haematol
2004
, vol. 
124
 (pg. 
91
-
96
)
42
Arnold
 
DM
Dentali
 
F
Crowther
 
MA
, et al. 
Systematic review: efficacy and safety of rituximab for adults with idiopathic thrombocytopenic purpura.
Ann Intern Med
2007
, vol. 
146
 (pg. 
25
-
33
)
43
Ohguchi
 
H
Kameoka
 
J
Harigae
 
H
, et al. 
Can the Helicobacter pylori eradication regimen induce platelet recovery in H. pylori-negative patients with idiopathic thrombocytopenic purpura?
Am J Hematol
2005
, vol. 
78
 (pg. 
164
-
165
)
44
Kuwana
 
M
Okazaki
 
Y
Kaburaki
 
J
Kawakami
 
Y
Ikeda
 
Y
Spleen is a primary site for activation of platelet-reactive T and B cells in patients with immune thrombocytopenic purpura.
J Immunol
2002
, vol. 
168
 (pg. 
3675
-
3682
)
45
Olsson
 
B
Andersson
 
PO
Jernas
 
M
, et al. 
T-cell-mediated cytotoxicity toward platelets in chronic idiopathic thrombocytopenic purpura.
Nat Med
2003
, vol. 
9
 (pg. 
1123
-
1124
)
46
McMillan
 
R
Wang
 
L
Tomer
 
A
Nichol
 
J
Pistillo
 
J
Suppression of in vitro megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP.
Blood
2004
, vol. 
103
 (pg. 
1364
-
1369
)
47
Shimoyama
 
T
Crabtree
 
JE
Bacterial factors and immune pathogenesis in Helicobacter pylori infection.
Gut
1998
, vol. 
43
 (pg. 
52
-
55
)
48
Veneri
 
D
Gottardi
 
M
Guizzardi
 
E
Zanuso
 
C
Krampera
 
M
Franchini
 
D
Idiopathic thrombocytopenic purpura, Helicobacter pylori infection, and HLA class II alleles.
Blood
2002
, vol. 
100
 (pg. 
1925
-
1926
)
49
Takahashi
 
T
Yujiri
 
T
Tanizawa
 
T
Helicobacter pylori and chronic ITP: the discrepancy in the clinical responses to eradication therapy might be due to differences in the bacterial strains.
Blood
2004
, vol. 
104
 pg. 
594
 
50
Byrne
 
MF
Kerrigan
 
SW
Corcoran
 
PA
, et al. 
Helicobacter pylori binds von Willebrand factor and interacts with GPIb to induce platelet aggregation.
Gastroenterology
2003
, vol. 
124
 (pg. 
1846
-
1854
)
51
Kraft
 
M
Tiemann
 
M
Riedel
 
S
, et al. 
Compartment analysis of T cell receptor gamma-and immunoglobulin V (H) rearrangements by microdissection in patients with malt lymphoma and chronic gastritis with lymphatic hyperplasia.
Cell Mol Biol (Noisy-le-grand)
2002
, vol. 
48
 (pg. 
253
-
263
)
52
Georgopoulos
 
SD
Triantafyllou
 
K
Fameli
 
M
, et al. 
Molecular analysis of B-cell clonality in Helicobacter pylori gastritis.
Digest Dis Sci
2005
, vol. 
50
 (pg. 
1616
-
1620
)
53
Veneri
 
D
De Matteis
 
G
Solero
 
P
, et al. 
Analysis of B- and T-cell clonality and HLA class II alleles in patients with idiopathic thrombocytopenic purpura: correlation with Helicobacter pylori infection and response to eradication treatment.
Platelets
2005
, vol. 
16
 (pg. 
307
-
311
)
54
Ishiyama
 
M
Teramura
 
M
Iwabe
 
K
Kato
 
T
Motoji
 
T
Clonally expanded T-cells in the peripheral blood of patients with idiopathic thrombocytopenic purpura and Helicobacter pylori infection.
Int J Haematol
2006
, vol. 
83
 (pg. 
147
-
151
)
55
Kim
 
J
Park
 
CJ
Chi
 
HS
, et al. 
Idiopathic thrombocytopenic purpura: better therapeutic responses of patients with B- or T-cell clonality than patients without clonality.
Int J Haematol
2003
, vol. 
78
 (pg. 
461
-
466
)
56
Zambon
 
CF
Navaglia
 
F
Basso
 
D
Rugge
 
M
Plebani
 
M
Helicobacter pylori babA2, cagA, and S1 vacA genes work synergistically in causing intestinal metaplasia.
J Clin Pathol
2003
, vol. 
56
 (pg. 
287
-
291
)
57
Yamaoka
 
Y
Kodama
 
T
Gutierrez
 
O
, et al. 
Relationship between Helicobacter pylori iceA, cagA, and vacA status and clinical outcome: studies in four different countries.
J Clin Microbiol
1999
, vol. 
37
 (pg. 
2274
-
2279
)