This investigation was designed as a quasi-experimental study. The cases were collected among all admitted patients with acute ITP and platelet counts less than 30 × 10⁹/L during the sampling period in some general Hospitals in Iran.  The following variables were considered: age, gender, H. Pylori treatment, platelet count and chronic ITP. Criteria for the diagnosis of acute ITP included: 1) Having platelet count less than 30 × 10⁹/L with normal WBC, Hb and ESR, 2) Age of eighteen years and older of both sexes, 3) Having bone marrow aspiration/biopsy (BMA/B) compatible with ITP (if it was required), 4) Ruling out pseudo-thrombocytopenia, hereditary thrombocytopenia as well as secondary ITP due to human immunodeficiency virus (HIV) infection, autoimmune disorders and drug-induced thrombocytopenia, 5) No history of previous ITP, 6) No history of splenectomy.
From eighty seven cases of acute ITP who entered in this study, two patients were excluded due to the diagnosis of HIV and myelodysplastic syndrome (MDS) and eighty five patients with acute ITP and platelet counts below 30 × 10⁹/L were finally enrolled. Because of the low incidence of acute ITP in adults the number of patients was less than anticipated. After identifying patients with acute ITP eligible to participate in the study and also after disease stability regardless of response to treatment,   the cases were underwent UBT if they had no history of drug taking to interact with the mentioned test according to their medical histories. In the case of recent drug consumption, UBT was performed after one month of drug discontinuation. Patients were divided into 3 groups based on the UBT results: Group I) eradication of H. pylori in infected ITP cases using standard triple therapy by Amoxicillin 1000 mg twice daily, and Clarithromycin 500 mg twice daily and Pantoprazole 40 mg twice daily for 7 days, Group II) H. pylori uninfected cases with standard treatment of ITP) and Group III) patients infected with H. pylori which did not receive eradication therapy. All patients received standard therapy included corticosteroid with or without intravenous immunoglobulin (IVIG). UBT was carried out at the first and the six^th month after the onset of therapy.
 

Table 1. Comparisons of mean platelet counts in 3 groups during the study (mean±SD)

In the first group, the patients whose H. pylori infection were not eradicated (UBT was positive in the first month) or the patients who had recurrent infection (UBT was positive at the end of the six^th month) were transferred from group I to group III and data analysis was done with and without them. These groups received standard treatment for ITP. The platelet count was measured every month up to six months after the start of the treatment. All data required for this study included age, sex, time of entry into the study, tests requested, and date of treatment which were entered into the questionnaires.
Inclusion criteria were as follows: 1) Patients over eighteen years of age, 2) Patients who have not been elapsed from disease onset more than six months (acute ITP), 3) Other causes of thrombocytopenia were excluded. Exclusion criteria for the study were as follows: 1) A serious illness such as heart, liver and kidney diseases, and neoplastic disorders, 2) History of previous treatment for H. pylori, 3) Allergic reaction to therapies used for H. pylori, 4) Distinct gastrointestinal signs and symptoms for the pharmacologic intervention as well as treatment for H. pylori, 5) Secondary causes of ITP.
Chi-square (for qualitative variables), independent t-test (in the case of normally distributed quantitative variables) and Mann–Whitney U test (in the case of non-normal distribution of quantitative variables) were used for the statistical analysis.
In view of medical ethics, there was no compulsion to participate in the study. All patients were initially explained that the information was completely confidential and used solely for the scientific goals. Also, unknown effect of H. pylori treatment on patients with ITP was explained to the patients and a written informed consent was obtained from all the participants.
Ethically, there was no need to treat all patients with positive UBT, because all the patients with H. pylori did not have indications for treatment. Additionally, cases with indication for H. pylori treatment (like peptic ulcer histories) were excluded.

 

UBT was positive in sixty one patients (71.8%). Thirty seven of them (60.6%) were female and twenty four (39.4%) were male. UBT in twenty four patients (28.2%) was negative; among them fifteen (62.5%) were female and nine (37.5%) were male.
Thirty six patients (UBT positive group I cases) underwent eradication of infection. Twenty four patients in the second group (UBT negative cases) and twenty five patients in group III (UBT positive cases without eradication of infection) were enrolled. Three cases of group I and two cases of group II were excluded due to lack of H. Pylori eradication and infection recurrence at the end of the 6^th month, respectively. These cases were placed in group III and analysis was carried out with and without them. No significant difference was observed between gender and age of the patients in 3 groups.
The mean platelet count in different groups was not statistically significant at the beginning of the study (P=0.34, Table 1). At the end of the first month, mean differences of platelet counts between 3 groups of patients was statistically significant (P=0.001, Table 1). There was a significant difference between platelet mean in groups I and II (P=0.01) as well as groups II and III (P=0.002) at the end of the first month, but there was no significant difference between groups I and III (P=0.83). There were no significant statistical differences between mean platelet count in 3 groups after the end of the second month to six months (P=0.64, 0.55, 0.73, 0.86 and 0.75, respectively). It is noteworthy that the mean platelet count difference between the groups excluding 5 transmitted patients from group I to group III, showed no statistically significant difference during the second to six^th months either.
Platelet count higher than 100 × 10⁹/L was considered as a cutoff point for the assessment of chronicity at the end of the six^th month. There was no significant difference between groups I and II in terms of chronicity (P=0.22). However, chronicity of the disease in group I was statistically significant and much lower than group III (P=0.018).The disease chronicity in group I without excluded 5 transferred cases (3 refractory patients and 2 patients with relapsed H. pylori infection) was significantly lower than group III (P=0.016) at the end of the six^th month. The chronicity rate in the patients without H. pylori infection (Total Groups I and II) was significantly lower than in the patients with H. pylori infection (group III), (P=0.03). The mean platelet count elevation in groups I, II and III between the baseline and the end of the six^th month showed no significant difference (P=0.64). No significant difference was also observed between treatment groups at the end of the six^th month (P=0.67, Table 2).
As it is shown in Table 3, the likelihood of having platelet count higher than 100 × 10⁹/L was 0.14 in group III compared to group I (P=0.02) which showed significant effect of H. Pylori eradication on ITP chronicity despite age, gender, splenectomy and baseline platelet count.
 

Table 2. Comparison of different treatment procedures performed during the study

*Others: Rituximab, Cellcept, Cyclophosphamide, Cyclosporine, Azathioprine
 

Table 3. The logistic regression model results of H. Pylori eradication on ITP chronicity in association with and without age, gender, splenectomy and baseline platelet count

Discussion

A link between ITP and H. pylori infection was proposed by Gasbarrini et al., who showed a significant increase in platelet count after H. pylori eradication in ITP patients infected with these bacteria (19).
Eradication of H. pylori infection has been variably associated with a platelet response in the patients with ITP. The response occurs in some ITP patients infected with this bacterium but not in all patients. Eradication therapy seems to be more effective in the patients with less severe ITP (platelet count >30 × 10⁹/L) and a shorter duration of disease. Most of the studies regarding H. pylori infection and ITP have been carried out on the patients with chronic ITP, but patients with chronic ITP have already experienced various treatments that have affected their platelet count. Therefore, the present survey on acute ITP in adults is one of the few existing studies. According to our results, no significant difference was observed for ITP chronicity at the end of the six^th month between the groups with and without H. pylori infection. The first report about ITP patients whom H. pylori infection were eradicated was provided by Gasbarrini et al., (19) and some studies were conducted by other investigators (20-23). Gan et al., found that the overall platelet response rate was 30% which was similar to those reported in America but was lower compared to the studies done in Japan and Europe. They also showed that there was no platelet recovery in the patients who demonstrated initial response (24). Baseline platelet count over 30 × 10⁹/L and previous treatments for ITP, such as prednisone, are factors that affect the response to eradication of H. pylori infection. In our study, the patients had no history of treatment with corticosteroids. However, our study differs from other studies due to the inclusion of patients with a platelet count less than 30 × 10⁹/L. Veneri et al., showed that there was no significant benefit of H. pylori infection eradication in the patients with marked thrombocytopenia, however, they showed that early eradication therapy was more effective in their patients as the thrombocytopenia was moderate (25). Our study suggests that H. pylori infection may be a cause of ITP chronicity which is hoped to be controlled by eradicating infection. The mechanism by which H. pylori may cause ITP has not been established, but a chronic immunological stimulation or immunological mimicry between H. pylori antigens and platelet has been proposed as a cause of ITP (16). The protein Cag-A (cytotoxin associate gene-A) is one of the molecules which has molecular cross-reaction with platelets. This protein is more prevalent in Asian countries (such as Iran, Korea and Japan) than in Western countries (18). As a result of H. pylori eradication, Cag-A antibody is disappeared and the platelet count is increased in ITP patients. Difference in H. pylori antigen genotype (prevalence of H. pylori with positive Cag-A) may be increased in the patients with ITP and platelet which influence the treatment outcome. Although, we have not tested for the presence or absence of this protein in our patients, the significant differences seen in ITP chronicity in our groups may be due to the differences in H. pylori Cag-A protein. In most studies, cases with a slight decrease in platelet count have shown a better response to treatment (4). Suzuki et al., found that the eradication of H. pylori infection in the patients with ITP was associated with a platelet response of 46.2% in the eradication group and 0% in non-eradication group. They also showed that the platelet response was significantly more common in the patients with infection sustained by CagA-positive strains of H. pylori (22).
Prevalence of ITP at older age in H. pylori-positive patients compared to H. pylori-negative patients may be related to the long-term underlying susceptibility to H. pylori infection (3,22). However, in our study there was no significant difference between the groups in terms of age. The results of our study indicated that eradication of H. pylori infection in the patients with acute ITP will prevent the chronicity of the disease because the platelet count higher than 30 × 10⁹/L in groups I and II (which were negative for H. pylori infection at the end of the six^th month) was more common than in group III (H. pylori infection at the end of the six^th month). These results showed an association between H. pylori infection and ITP, thus, eradication of H. pylori can diminish the chronicity of ITP. At least in part, these findings suggest that screening of the patients with acute ITP  in order to eradicate H. pylori infection can prevent ITP chronicity in a number of patients with H. pylori infection. Finally, we should mention that the results of the present study cannot be compared with the studies that have been conducted on the patients with chronic ITP infected with H. pylori, because the patients with chronic ITP have already a milder or moderate thrombocytopenia due to use of corticosteroids. Rather, patients with acute ITP and severe thrombocytopenia were entered in our study and received corticosteroids afterwards.

Conclusion

Our study confirm some of the previous results and suggest that H. pylori infection may be a cause of ITP. Therefore, eradication of H. pylori infection in patients with acute ITP may prevent the chronicity of ITP. However, further studies on larger number of infected and non-infected patients with acute ITP are recommended to confirm these results.

Acknowledgements

We are grateful to all patients who participated in this study. Dr Moghimi designed the project and contributed to the drafting. Dr Mortazavi, Dr Keyhanian, Dr Mansouri and Dr Asadi-Khiavi contributed to the design of research study and also drafting the manuscript. Dr Mazloomzadeh analyzed the data and Dr Razavi-Dizaji, Miss Ghadimi and Miss Baba Ali contributed to the reagents, Lab works and experiments. This work was fully supported by the grant from Zanjan University of Medical Sciences, Zanjan, Iran.

Conflicts of Interest

Authors declared no conflict of interests.