Introduction:

Helicobacter pylori, a Gram-negative bacterium that

Colonizes human gastric mucosa, was first introduced in 1984 in biopsy of chronic antral gastritis and peptic ulcer disease.(1) It has been shown to be associated with serious diseases such as gastric cancer and gastric mucosa–associated lymphoid tissue lymphoma. Globally, it remains one of the most common infections, as an estimated 50% of the world’s population are carriers of this organism.(1,2).The mode of transmission for H pylori is not absolutely known; however, epidemiologic studies strongly support person-to-person transmission and fecal-oral or oral-oral routes are the most likely.(1)

It is known that the infection is mainly acquired during childhood, but the specific age of acquisition and the factors associated with its persistence are unknown.(3)infected mother and siblings is the most common familial source of this bacterium(4). Helicobacter pylori infection in children is usually acquired during the first 10 years of life in developing countries (3). In these countries, >80% of adults are colonized with H pylori, and >50% of children are infected at 10 years of age in comparison with 30% of adults and10% of children in developed countries.(3,5) comparison of  infection prevalence of various age in developing countries shows that infection is minimal in children aged    7 - 8 years (36.84%) and reaches maximum levels in students aged 14 years (66.67%).(6) Screening result shows that there are 2 waves of H pylori infection (the first peak was detected in 11 years, the second in 14 years). US data–as a developed nation - indicate that black children aged 5 to 9 years have an overall infection frequency of 30%. Around the world, infection among children ranges from approximately 35% in Russia, 20% in China and Poland, 12% in Korea and America to <10% in France, Belgium and Finland.(7)

In our review of literature there are two studies about prevalence of H pylori infection in pediatrics of Iran that showed high H pylori infection prevalence.(47% in center and 64.3% in west of Iran)(8,9)

there are invasive and noninvasive methods for diagnosis of infection. Invasive methods include endoscopy and biopsy for histology, culture and rapid urease test analysis.  noninvasive methods are serology, urea breath test and stool antigen analysis. The gold standard test for detection of H pylori remains histology from gastric biopsy.(1)

Serology-based evaluation of H pylori status is limited because of a 30% false-positive rate, as immunoglobulin G testing reveals both previous (treated)and present infections(1) To avoid detection of previous  H pylori infections, UBT (highly accurate for the diagnosis of H pylori infection in children older than 6 years)(3) and stool antigen testing are useful(1). These tests are more accurate during childhood (10,11,4,12)but urea breath test has a relatively high cost and requires trained staff and well equipped laboratory instruments despite of its high sensitivity and specificity. The new, noninvasive, low-cost H pylori antigen test in stool can replace UBT for detection of H pylori infection in children with comparable reliability and accuracy.(13) The sensitivity, specificity, and positive and negative predictive values of HpSA were found to be respectively 98%, 100%, 100%, and 96.5%.(14)there are 2 methods for detecting antigen: based on monoclonal and polyclonal antibody.in polyclonal antibody method, It is possible that cross reactivity of the HpSA with nonviable or coccoid forms of the Hp bacterium cause false-positive results.(15)

Comparisons between these two methods in children have shown that the monoclonal antibody has higher sensitivity than the polyclonal antibody(98% vs 93.8%)and replace it.(4,16)this test seems suitable to monitor the success of anti- H pylori therapy and screening of asymptomatic subjects.(17,18) also the test was approved by the United States Food and Drug Administration (FDA) as a pre-endoscopic diagnostic test for H pylori infection in adults . newly there is a new generation of rapid monoclonal antibody based HpSA test  that work with lateral flow immunochromatography technique in 5 minutes. It is a convenient office-based method for detection of        H pylori antigen in stool specimens.  the  diagnostic

accuracy of  this test was as high as that of HpSA ELISA in children (19)

some important risk factors for transmission of         H pylori include age , race , living in rural area , overcrowding , socioeconomic status(SES) , poor sanitary conditions , mothers with lower educational  level , poor diet and poor water supply (1,2,3,7,10,20).even in populations in the same country, low SES is associated with infection acquisition(2)there is no association between intestinal parasitosis and H pylori  infection but Giardia was closely associated with it.(21)some other associations are iron deficiency anemia, diarrheic disease, and impairment of growth , weight , and cognitive functions(4)

in attention of little evidence about prevalence of H pylori in children and importance of this issue due to its potential risk for severe gastrointestinal complications ,this study was designed. The aim of this study is to evaluate the prevalence of H pylori infection and its associated risk factors in school aged children in east of Iran, Birjand

material and  methods:

This is a population based cross sectional study in 9-12 year old children live in Birjand . just being a 9-12 year old child who study in Birjand  is the inclusion criteria, and exclusion criterias are dissatisfaction in cooperation ,significant gastrointestinal sign and symptom ,antibiotic or antacid usage(PPI or H2 blocker) and diarrhea at the time of sampling .the study  was reviewed and authorized by the ethics and investigation committees of the Birjand University of Medical Science. The parents signed an informed consent form authorizing their children’s participation.

Sample size of this study is 282 persons that calculate with comparison of proportions formula  according to the prevalence that achieve from Dr Soltani and et al. study (Prevalence of Helicobacter Pylori infection in  children) (9). sampling was done in a randomized cluster manner from 12 primary schools (4 male and 8 female primary schools). schools was selected from 4 economic regions in Birjand . after explaining of study, completion of a written consent by student`s parents and verbal consent by students, and dismissing of exclusions, the participants fill a questionnaire of some demographic data and  stool samples was obtained and analysed with H pylori Antigen EIA Test Kit (ACON company). This is a solid phase enzyme qualitative and quantitative detection of H pylori antigen in human stool.The microwell plate is coated with anti-H. pylori antibodies. During testing, the antigens are extracted out with extraction solution and added to the antibodies coated microwell plate with the enzyme conjugated antibodies to H. pylori and then incubated. If the specimens contain H. pylori antigens, it will bind to the antibodies coated on the microwell plate and simultaneously bind to the conjugate to form immobilized antibody-H. pylori antigen-conjugate complexes. If the specimens do not contain H. pylori antigens, the complexes will not be formed. After initial incubation, the microwell plate is washed to remove unbound materials. Substrate A and substrate B are added and then incubated to produce a blue color indicating the amount of H. pylori antigens present in the specimens. Sulfuric acid solution is added to the microwell plate to stop the reaction producing a color change from blue to yellow. The color intensity, which corresponds to the amount of H. pylori antigens present in the specimens, is measured with a microplate reader at 450/630-700 nm or 450 nm. The sensitivity and specificity of this kit have been obtained 98.6%and  95.4% in a previous survey(according to the data in brochure of kit).In each run of testing 65-70 sample, 2 control and 4 calibrator  was analysed . to increase the accuracy of test results in each run 3 samples was tested twice and the results compare with each other. in this study only quantitative results was achieved and interpreted. values below 0.045 µg/ml, between 0.045-0.055µg/ml ,and above 0.055µg/ml considered as negative, equivocal and positive, respectively. finally data was analysed with IBM SPSS 21.0   and STATISTICA 10.0 and the level of significance considered below 5%.

results:

The mean age (± SD) of the participants for girls was 10.54years and for boys 10.42 years. 191 (67.7%%) of participants are girls and 91(32.2%) are boys. The overall prevalence of Helicobacter pylori colonization in 282 students was 13.1%( table-1).the prevalence of infection in female population of our sample is 16.2% and in male population is 6.6%, so the relationship between H pylori infection and gender  was statistically significant.(p-value:0.025)(table-1).the prevalence of infection among 9, 10, 11 and 12year old children is respectively 12.9%,13.4%,16.4% and 10.5% and there is no significant relationship between age of children and H pylori infection(table-1) (p-value:0.789) . surprisingly there is strong negative relationship between duration of breast feeding and prevalence of H pylori infection (p:0.002) (table-1).

number of case`s siblings (family crowding) and H pylori infection has a significant positive relationship too(p:0.004)(table-1).

althogh there are some evidence that advocate potential anti H pylori activity of probiotics, we don`t find any significant association or trend between the number of days that family consume yogurt (as a probable source of probiotics) and H pylori infection(p:0.487).presence of gastric complaint(such as flatulence, heart burn, regurgitation, as a general term:  dyspepsia) in households has a visual positive trend with H pylori infection but the relationship is not statistically significant.(p:0.096)there is not any relationship between H pylori infection and living regions that economically stratified across the city (0.726)

Discussion:

The prevalence of infection in our study is surprisingly lower than in other similar studies.many predisposing and inhibitory factors involves in prevalence of this infection in different populations .some of these factors are socio-economic level, crowding, level of sanitation, care taker `s literature, and positive family history of gastro-intestinal complaint . in attention to the difference of prevalence between two related studies in west and center of Iran(8,9) and our study it seems that may be such regional factors like climate and food habits influence it but it  needs to be further studied .moreover in Falsafi and et al. study in Tehran(8)  ,the study population is both symptomatic and asymptomatic children and adolescent that explain the larger prevalence. our study is designed in asymptomatic primary school student population, most of families are young and the mean number of siblings is 2.5.we try to sample homogeneously in socio-economic status from Birjand`s student population . for this reason we subgroup the population into four socio-economic level by living region and randomly select the primary schools in each region because we want to reach the total prevalence of infection in Birjand .these reasons  may be some causes for lower prevalence in our study.

There is statistically significant relationship between gender and infection in our study (16.2% in female and 6.6% in male). two studies in our literature review (4,30) have significant relationship between gender and infection but in those infection in male is higher.so it seems that this is an accidental relationship.

Our population`s age range is narrow (9-12 year).may be this is the reason for that There is not a significant relationship between increasing age and prevalence of infection in present study .despite us in six studies these have significant relationship (3,8,9,10,21,24)

We found that number of siblings have a significant relationship with prevalence of infection (p value: 0.004)(5.6% in 1child vs 25% in 6 children family).it can be explained by the root of transmission of         H pylori infection(person to person by fecal-oral and oral-oral)because of poor sanitation that usually is in larger families . two studies (3,9)agree and two studies (10,32) disagree us in this issue.

In order of family member`s dyspepsia and infection in the case we find  just a positive trend but it is not statistically significant(p value:0.096).in two studies of related articles this relationship surveyed and is significant(7,8).

After searching for risk factors we try to find some probable protective factors against acquiring infection. there was very significant negative relationship between duration of breast feeding and  H pylori infection( p value:0.002)that support the protective effect of breast feeding against this infection like a lot of other protective and immunologic effect of breast milk .there are two studies that advocate this result(30,33) and three studies in opposition of it(9,10,31).

 there are some evidence that probiotics - new generation of therapy in a wide range of infectious and non-infectious diseases – have good effects in treatment of H pylori infection(36) and some that assert dairy product based probiotics have a better effect in this issue(37). We consider the number of days that family consume yogurt as a variable and its relationship with prevalence of infection but we diden`t find a significant relationship between these two factors (p value:0.487) .however we could not assert about role of probiotics in control of infection because we did not sure that the dairy products inevitably have probiotic strains. this issue is not studied in any other research.

The last factor that we investigate is relationship between economic region of living and  infection acquiring risk. We divide the area of city into four  economic region by the aid of  office of education in Birjand and from each region we select one male and one female primary school. after completion of this stage because the sample size was insufficient we have to continue sampling from four randomly selected female primary school from each region again.so finally we have samples from two female and one male primary school from each region. we don`t find any relation between these two issues .Miranda and et al.(8)- that investigate H pylori by serology – didn`t find such relationship too but two studies-that investigate H pylori by detection of stool antigen- find positive relationship between these two variables(31,33).this positive relationship seems that provide by logic but it is difficult to determine absolute factors that indicate the economic state of family.it seems that living region in the city may be not enough  indicator  of economic level of family in our study and should be modify simultaneously by other indicators.

Conclusion:

The prevalence of Helicobacter Pylori is different between areas of a country and health care providers should plan strategies for exploring and modifying its risk factors.

Table-1: relationship between some associated factors and H pylori colonization