Prevalence of Helicobacter pylori infection among children with primary nephrotic syndrome: a cross-sectional study

Background Limited data are available about the prevalence of helicobacter pylori (H.pylori) infection among primary NS children. Objectives To assess the frequency and risk factors of H.pylori infection among children with primary NS. Methods A cross-sectional study was carried out in Mansoura University Children's Hospital, Egypt during the period from 2017 to 2019 including 100 NS children (NS group) and 100 healthy controls. NS group included 88 steroid sensitive (SSNS) and 12 steroid resistant (SRNS) cases. All patients were assessed for H.pylori infection using H.pylori stool antigen (HpSA) test. Statistical analysis was done using chi-square, fisher exact and Mann-Whitney tests. Results With regard to HpSA test results, no significant differences were detected between control and NS groups (p = 0.193) and between SSNS and SRNS groups (p = 0.286). Concerning total biopsied cases and MCD (proven plus presumed) cases, no significant differences were found between those with positive and negative HpSA test (p = 0.648 and 0.126, respectively). The high dose of steroid therapy was associated with a higher risk of H.pylori infection among NS group (Odds ratio = 3.8; 95% confidence interval = 1.3–11.3). Conclusion The current study negates the increased risk of H.pylori infection in children with primary NS.


Introduction
Nephrotic syndrome (NS) is a relatively common pediatric disease 1 with an immunological background proved by the successful therapeutic effects of steroids and immune-modulating drugs 2,3 .
Helicobacter pylori (H.pylori) are gram-negative bacteria that selectively colonize the gastric mucosa. The organism is spiral shaped with three to five polar flagella, and characterized by being urease, oxidase and catalase positive 4 . A recent meta-analysis revealed that about four billion persons were colonized with H.pylori worldwide in 2015 5 . In developing countries, H.pylori infection is highly prevalent and represents a great challenge 6,7 . In Egypt, this infection is very common reaching up to 72% among school children 8 . Now, there is agreement that H.pylori is not only associated with gastric diseases but also with immune-mediated extra-gastrointestinal disorders 9 . It is associated with atopic dermatitis, Henoch-Schönlein purpura, idiopathic thrombocytopenic purpura, systemic sclerosis, recurrent aphthous stomatitis, alopecia areata and Sjogren's syndrome. In addition, H.pylori eradication could help in management of Behcet's disease. These effects could be attributed to the chronic systemic inflammatory state induced by the organism 10,11 . Limited trials were conducted in the pediatric age group to detect the association between H.pylori infection and NS with contradictory results 9,12,13 . The current study assessed the frequency of H.pylori infection among children with primary NS and the related risk factors.

Materials and methods Study design and participants
A cross-sectional comparative study was carried out from January 2017 to January 2019 including 100 primary NS children (NS group) and 100 healthy controls. NS patients were recruited from both inpatient wards and outpatient nephrology clinics of Mansoura Univer-sity Children's Hospital, Egypt. NS group included 88 steroid sensitive (SSNS) and 12 steroid resistant (SRNS) cases. Among SSNS cases, 43 patients were infrequent relapsers, 30 patients were steroid dependent, 12 patients were first attack and 3 patients were frequent relapsers. The controls were recruited from children attending the same hospital with minor complaints e.g. mild gastroenteritis and pharyngitis.
Our study was accepted by Institutional Research Board of Medical Faculty of Mansoura University, Egypt (Code No: MS/16.08.35) and followed the Helsinki Declaration of 1975, as revised in 2000. Informed written consents were obtained from legal guardians of all study participants.
All primary NS children with persistent dyspeptic symptoms were included. NS was defined as the presence of edema, serum albumin < 2.5 gm/dl, nephrotic range proteinuria (≥40 mg/m²/h or protein/creatinine ratio >2 mg/mg) and elevated serum cholesterol and triglycerides levels 14 . Patients were labeled as SSNS if in remission, infrequent relapse, frequent relapse or steroid dependent. Steroid resistance means no remission despite daily prednisolone therapy at a dose of 2 mg/ kg/d for four weeks. Patients were in remission if urinary albumin was trace or nil for three consecutive early morning specimens. Frequent relapse was defined as two or more relapses in the initial six months or more than three relapses in any 12 months of treatment. Steroid dependence means occurrence of two consecutive relapses within two weeks of steroid discontinuation or when on alternate day steroids 15 .
Patients were excluded if secondary NS, received H.pylori treatment within the last three months before participation in the study, received proton pump inhibitors within two weeks prior to sampling, or on an antibiotic treatment at the time of the testing procedure.

Sample size
It was calculated using G*Power 3.0.10 program. Based on 65.6% prevalence among NS patients and 44.4% among controls (Moriyama et al., 2006) 16 , and assuming α (type I) error = 0.05 and β (type II) error = 0.2 and power = 80%; a sample size of 88 children was assumed for each group with an effect size = 0.425. We added 15% to overcome drop out, and the presumed sample size was 100 children in each group.

History and clinical examination
Patients were subjected to detailed history taking with special emphasis on the response to steroids and the dose and duration of steroids therapy. Treatment of an initial episode of NS included oral prednisolone as a single daily dose starting at 2 mg/kg/d to a maximum 60 mg/d for 4 weeks followed by alternate-day dosing as a single daily dose starting at 1.5 mg/kg (maximum 40 mg/d) and continued for 2-5 months with gradual dose tapering. The steroid therapy should be given for at least 12 weeks 17 . Low-dose steroid was initiated for relapse of SSNS using oral prednisolone 1 mg/kg/d (maximum 40 mg/d) for a minimum of 7 days. Once the patient was in remission and/or had completed 7 days of steroid therapy, gradual tapering of prednisolone was done over a month. If the patient developed progressive edema or failed to get remission within 7 days of therapy initiation, the prednisolone dose was increased to the standard high-dose regime (2 mg/ kg/d) 18 . All patients were assessed for gastritis symptoms as dyspepsia, vomiting, recurrent abdominal pain and epigastric tenderness.

Laboratory evaluation
All participants were subjected to urine analysis, 24-h urine protein assessment/protein-creatinine ratio and serum levels of creatinine, albumin, cholesterol and triglycerides. Data about renal histopathology were retrieved from patients' files. Renal biopsy was done for only 38 cases (29 SSNS and 9 SRNS cases). Four SSNS cases (steroid dependent) and three SRNS cases were not biopsied due to patient guardians' refusal or current contraindications for renal biopsy (bleeding tendency).
H.pylori infection was assessed using H.pylori stool antigen test (HpSA test; ImmunoCard STAT! ®; Meridian Bioscience Europe, European Union; Catalog Number: 750220). HpSA test is simple, rapid, non-invasive, and can detect the active current infection. It is considered as a 2 nd generation rapid lateral flow immunoassay test using monoclonal anti-H.pylori antibodies. The specificity and sensitivity of the test are 99.2% and 94.3%, respectively as reported in the manufacturer's product information. The test value for H.pylori diagnosis was well recognized in previous reports 19 .
Stool samples were collected from all study participants, transported in airtight containers and tested as soon as possible but might have been held at 2°-8°C for 72 hours before testing. If testing could not be done with-in this time frame, specimens were frozen immediately upon receipt and stored at -20° to -80°C until tested. Each stool sample (5-6 ml) was mixed thoroughly with 1 ml diluent in a test tube. Then, we dipped the reaction strip in the test tube for 10 seconds, and read the results after 5 minutes. We considered the test negative if only a blue colored band (the control line) appeared across the white central area of the reaction strip, and positive if an additional pink red band (the test line) also appeared. Any pink red line, even very weak, was considered positive. The band intensity varied according to the concentration of the antigen in the specimen. Any color or line appearing after 10 minutes had no diagnostic value. The test was considered invalid if the control band was absent.
Statistical analysis SPSS version 21 was used to analyze data. Kolmogorov-Smirnov test was used to assess the data for normality. Qualitative data were described using numbers and percent. Chi-square and Fisher exact tests were used to describe the associations between the categorical variables. Non-parametric continuous variables were expressed as median (min-max), and analyzed using Mann-Whitney test. Logistic regression analysis was done for the risk factors associated with positive HpSA test among NS group. P ≤ 0.05 was considered significant.

Results
Both NS and control groups were matched for the gender and age. The median age of NS group was 9 (1-17) years, while that of the control group was 8.5 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) years (p = 0.083). Males constituted the majority of the study participants (67 children in NS group and 57 children in control group, p = 0.145). With regard to HpSA test results, no significant difference was found between control and NS groups (       (Table 5). The only report that agrees with our results was that of Nutpho and Ukarapol 13 ( Table  5). This discrepancy could be explained by variability of the screening methods for H.pylori, different sample size and study design. This observation may negate the increased risk of H.pylori infection in children with primary NS. On reviewing the literature, some authors reported presence of H.pylori antigens in glomeruli of renal biopsy specimens, but failed to confirm the link between the organism and NS pathogenesis 20 . They depended upon the presumed hypothesis that H.pylori could increase the risk of NS through immune-mediated reactions induced by direct antigenic effects 20 or auto-antibodies formation 22,23 . The effects of H.pylori eradication on the treatment response was also tested in few cohort studies. Some authors found insignificant improvement in proteinuria after H.pylori eradication 24 , while others failed to prove that the improvement in proteinuria was due to H.pylori treatment only and not due to spontaneous remission 19 . Even for studies that proved improvement in proteinuria due to eradication of H.pylori, the small sample size limits generalization of their results {three out of studied 32 cases in Moriyama et al 16  Moreover, the current study was the first to describe an insignificant difference between SSNS and SRNS chil- dren with regard to H.pylori infection. However, the high dose of steroid therapy was associated with a higher H.pylori risk among NS group, possibly due to the immuno-suppressant effect of the high-dose steroid 9 .
In the current study, the age of patients was not considered a risk factor for H.pylori among NS group. This observation copes with a previous national report 9 .
Other international studies with larger sample sizes proved that H.pylori infection rates increase with advancing age, mostly due to cumulative frequencies [25][26][27] . Also, the gender of patients was not a risk factor for H.pylori infection that agrees with many international reports 25,26,28,29 . However, other studies reported male predominance 27, 30 . This discrepancy could be explained by differences in study design and sample size.
Unexpectedly, the total duration of steroids therapy did not increase the risk for H.pylori, possibly due to interrupted courses of therapy with variable intervals that increased the therapy duration among patients. This finding was in contrast to Zajaczkowska et al 12 .
Concerning total biopsied NS cases and MCD (proven plus presumed cases), the insignificant differences found between those with positive and negative HpSA tests suggest lack of association between H.pylori infection and renal histopathology in NS group. Our findings were in variance with Nagashima et al 20

Conclusion
The current study provides useful information about H.pylori prevalence among primary NS children in Egypt. Our study negates the increased risk of H.pylori infection in children with primary NS. The high dose of steroid therapy was associated with a higher H.pylori risk among those patients. Large scale RCTs multicenter studies are still needed.

Limitations of the study
A single center association study with limited studied H.pylori risk factors, lack of long term follow-up and a potential selection bias of patients.