Annals of Clinical and Laboratory Research

Keywords

Helicobacter pylori; Children; Serology; Muong; Ethnic; Seroprevalence

Introduction

Helicobacter pylori (H. pylori) is now recognized as a worldwide problem, with an estimated of about half of the world’s population being infected [1]. It is the most common cause of chronic gastritis, peptic ulcer and is strongly linked to gastric cancer [1,2]. The sources of H. pylori and the mechanisms of acquisition remain poorly understood [1-3]. Childhood has been identified as the critical time for acquisition of H. pylori infection [3-5]. The prevalence of H. pylori infection varies by country from 20% to 90%, with rates of over 60% in developing countries [1-6]. Person-to-person transmission within the family have been considered as major modes of transmission by epidemiological and microbiological studies, both in developed and developing countries [1,6]. Differences in prevalence among racial and ethnic groups have been described worldwide, but it is unclear to what extent such differences can be ascribed to socioeconomic factors and other possible risk factors [4,7]. Vietnam is the easternmost country in Southeast Asia with an estimated 90.5 million inhabitants. Vietnam is also home to 54 ethnic groups with different cultures, of which 75% to 80% are living in rural or remote areas [8,9]. High rates of H. pylori infection were reported in both hospital-based and community-based studies from Vietnam [10-13]. Different risk factors for H. pylori infection have been identified in the Kinh ethnic majority [10-12] and among ethnic minorities [13-15]. As the socioeconomic level and the lifestyle vary considerably among ethnic groups in the country, the prevalence of H. pylori infection and particularly the risk factors for this infection differed in previous studies [10-15]. According to the General Statistics Office of the Government of Vietnam 2009 [9], the Muong is the thirst largest ethnic group in Vietnam after the majority Kinh and Tay ethnic groups with an estimated more than one million people. No data is available about prevalence of H. pylori infection and risk factors for this infection in Muong people. Further knowledge about the mode of transmission could be valuable for primary prevention of the H. pylori infection in this subpopulation. The aim of the present study was to determine the prevalence of H. pylori infection and to identify factors associated with the infection in Muong children living in Vietnam.

Population and Methods

Study population

Muong people inhabit the mountainous slopes of north central Vietnam, from the lower reaches of the Da river to the upper reaches of the Ma river. Each village generally consists of 10 to 50 households. This native ethnic group shares some distant roots with the Kinh (majority people) after the split into two groups (in the 10th century) but has all the elements of an ethnic group, having preserved and developed their own habits, customs, cultural practices and economic condition that differ from those of the Kinh people [9]. The Muong people live in traditional stilt houses where they also worship their ancestors and are involved in tasks like weaving and embroidery. A traditional stilt house is made from wood, bamboo, cane, or rattan and set one or two steps below the main level. The area under the house is either unused or used as a pen for livestock [9].

Hoabinh is a mountainous province in the North of Vietnam that covers an area of 4,684.2 square kilometers and has a population of 793,500 people. The Muong people comprises 63.4% of the population [9]. Kyson is a rural district of Hoabinh with population of 34.800 people over a total area of 202 square kilometers where we implemented the study, enrolling 918 family members of all generations living together in the same home from 219 households. All family members were included in order to investigate whether the size and composition of the families and H. pylori infection rates in family members would represent a risk factor for the children under study.

To avoid a selection bias, children aged less than 6 months were excluded due to the likelihood of residual maternal H. pylori antibody, as well as those with severe diseases or immunocompromised status due to the possibility of an altered immune response. Data were collected after obtaining written consents from local administration and health authorities. Informed consent was obtained from each household member. The study was granted ethical clearance in Vietnam by the ethics committee of the Hanoi Medical University

Data collection

In this cross-sectional study, door-to-door sampling method was adopted. We used a structured questionnaire, as in previous studies [12-14] for data collection on sociodemographic, health status and potential exposure. The information was collected from the head of the households while the study investigators completed the questionnaires. Blood samples (5 mL) were obtained from each subject by peripheral venipuncture under aseptic conditions and were immediately centrifuged. Sera were separated and preserved in vaccine thermos, then sent to the reference laboratory (Microbiology Division of Digestive Diseases, National Institute of Epidemiology and Hygiene) on the same day where sera were stored at -20°C and processed as previously described [16].

Variable definitions

H. pylori infection was determined by in-house ELISA for dosage of H. pylori IgG antibody against specific H. pylori antigen. The ELISA was carried out in the reference laboratory (Microbiology Division of Digestive Diseases, National Institute of Epidemiology and Hygiene) using sonicated Swedish and Vietnamese H. pylori strains as antigens prepared and validated in the Microbiology Department, Karolinska Institute, Stockholm for use in Vietnamese adults and children. The method had been established and evaluated previously, giving with a cut-off level of OD 0.22 a sensitivity of 93.9% to 99.6% and a specificity of 90.7%. [16]. Since this type of assay can have ±10% variability around the established cut-off level, we used the lower cut-off of 0.2 in order to ensure a high sensitivity in the assay.

Socio-demographic variables consisted of age and sex of all children and adults in every household, household monthly income per person (calculated by taking the total gross household monthly income divided by the total number of family members living together) classified into two categories (i.e. ≤ one million VND (<50 US/capita) and >one million VND (≥50 US/capita)), household space (in 2 categories, i.e. up to 20 m2/capita and ≥20 m2/capita), parents’ occupation and parents’ education level [9].

Potential exposure variables were divided into three major subgroups

Environmental, individual hygiene and life-style variables including water sources were classified in 2 main sources (family well and others, e.g. streams, collected rain or ponds), regular latrine in 2 main types (existence or not), pet ownership (dog, cat or pig) in the house in 2 categories (yes or no), behavior of children on hand washing before meal and after toilet in 2 categories (not regular, i.e. those who practiced hand washing only once or twice, and regular, i.e. those who practiced hand washing every time or most of the time), mouth-to-mouth feeding from mothers or caregivers to child in 2 categories (regular or not), breast feeding duration in 2 categories (shorter or longer than until 12 months of age).

Living conditions were evaluated by dividing the size of the household in 2 categories (≤4 or ≥5 persons), number of sibling in 2 categories (≤2 or ≥3), sharing a bed in 2 categories (with ≤2 and with ≥3 persons), collective life initiation (age for a child to start daycare) in 2 categories (<3 years and ≥3 years of age).

Health status variables consisted of H. pylori infection status in parents and sibling (H. pylori seropositivity or seronegativity), gastro-duodenal history of child and parents (previous gastro-duodenal disease, diagnosed and treated by health-givers from district health center or higher levels) in 2 categories (presence or absence).

Statistical analysis

First, study population characteristics were compared according to their H. pylori infection status using the Chi square (χ2) test. As one of the major aims of our study was to detect the factors potentially influencing H. pylori infection in this subpopulation, the appropriate strategy of analysis must be able to control for mutual confounding, point out the risk factors and avoid overlooking important associated variables. We then analyzed separately the associations between H. pylori infection status with the demographic and socioeconomic variables and with variables related to potential exposure. Analysis was performed firstly by univariate technique, by adjusting in each group on every variable, and finally by using backward stepwise conditional logistic regression to select variables importantly associated with H. pylori infection within each group, including all variables significantly associated with H. pylori seropositivity after adjustment and those with p values less than 0.05 by Chi square test. Associations were expressed as odds ratio (OR) and their confidence intervals (95% CI). Finally, backward stepwise conditional procedures were used again to include in the final model not only variables independently associated with H. pylori serological status in each group, but also those known to be important for transmission pathways. Statistical significance was set up at the 0.05 level. All p values were 2- tailed. Data were analyzed using SPSS so ware (SPSS® for WindowsTM version 16.0 Copyright SPSS Inc.).

Results

Overall 918 healthy individuals of all generations living together in the same home from 219 families were enrolled in the study. The proportion of H. pylori infected adults and children ≤18 years old was 52.1% (307/589) and 42.8% (141/329), (p=0.05) respectively (data not shown). Sociodemographic variables associated with the prevalence of H. pylori infection in Muong children aged 6 months to 18 years are presented in Table 1. There was no significant difference in H. pylori seropositivity based on gender (p>0.05). The prevalence of H. pylori infection was 32.7% in children under 3 years of age, rising to 45.9% for those older than 15 years. Monthly income was significant determination for seropositive of children in the multivariate models. The adjusted OR for infection in children was 0.62 (95%CI: 0.39-0.99, p=0.004) among family having monthly income more than 50 US/capita compared to family having monthly income less than 50 US/ capita. A significant relationship was found between H. pylori seropositivity and some variables related to socioeconomic status of the household such as father's occupation and mother’s education (Table 1).

OR: Odds Ratio; CI: Confidence Interval; NS: Non-significant Difference. *Adjusted for age, sex, house space, number of people in the same household, education level of mother and father, water source, latrine and hygienic status and children lifestyle

Table 1: Socio-demographic variables associated with H. pylori seropositivity in Muong children aged 6 months to 18 years.

H. pylori seropositivity in relation to environment, individual hygienic status and the children’s lifestyle are presented in Table 2. No significant differences were found in rates of H. pylori infection based on water source, regular latrine presence, regular hand washing before meal, eating with fingers and having pets. Prevalence of H. pylori infection in children who do not regularly wash their hands after defecation was significant higher than in those regularly washing their hands (adjusted OR: 38.6, 95%CI: 11.8-126.3, p=0.021). Children who did not regularly receive chewed food were less likely to be H. pylori seropositive (adjusted OR: 0.58, 95%CI 0.31-0.80, p=0.04). Breastfeeding more than 12 months was negatively and independently associated with H. pylori seropositivity (adjusted OR (95% CI): 0.58 (0.35-0.94), p=0.036).

OR: Odds Ratio; CI: Confidence Interval; NS: Non-Significant difference. *Adjusted for age, sex, house space, number of people in the same household, education level of mother and father, water source, latrine and hygienic status and children lifestyle

Table 2: H. pylori seropositivity in relation to environment, individual hygienic status and children lifestyle.

H. pylori seropositivity in Muong children in relation to variables related to living conditions and health status of study population as well as antibiotic use are presented in Table 3. An association was found between H. pylori seropositivity and the size of the household (adjusted OR: 1.46; 95% CI, 1.08-2.68, p=0.043). A family clustering of H. pylori infection was found. The adjusted OR for the risk of infection in the children when the mothers were infected was 1.34 (95% CI 1.01-2.32, p=0.001); when the siblings were infected it was 1.37 (95% CI 0.99-1.98, p=0.045) and it was 1.73 (95% CI 1.01-4.43, p=0.003) when grandfathers were infected compared to children without any infected family members. We did not find any relationship between H. pylori seropositivity in children and variables related to other living conditions (i.e. number of sibling, H. pylori infection status of the father, regularly sharing bed, collective life initiation.

OR: Odds Ratio; CI: Confidence Interval; NS: Non-Significant difference; GI: Gastrointestinal
*Adjusted for age, sex, house space, number of people in the same household, education level of mother and father, water source, latrine and hygienic status and children lifestyle

Table 3: H. pylori seropositivity in Muong children in relation to variables related to promiscuity and health status of study population.

Discussion

This is the first study describing the prevalence of H. pylori infection in Muong people in Vietnam. Our study showed an overall prevalence of H. pylori infection in Muong people was 48.8%. H. pylori seroprevalence was 52.1% in adults and 42.8% in children ≤18 years old. Our findings are comparable to those reported in other community-based studies in Vietnam [10-15]. In one community-based study carried out in Kinh majority ethnic, using the same serological test, Hoang et al. found an overall seroprevalence of H. pylori of 58.2% with 47.3% in children [10]. Data from one community-based study carried out in a population of Kinh and four other minority ethnic groups in the central highland previously showed an overall rate of 45.2% with 40.0% in children under 15 years [15]. Nguyen et al. conducted another community-based study in a Tay population in a rural area of the Langson province, found an overall rate of H. pylori infection of 46.8% and 41.4% in children [13].

It is believed that H. pylori is mainly acquired during childhood and little is known about its age of onset, rate, or mode of infection [3-5]. We found an increasing prevalence with age. H. pylori infection rate was 32.7% for children under 3 years of age, rising to 45.9% for those older than 15 years. The odd of infection was 1.74-fold for children aged over 15 years compared with those under 3 years old (OR, 1.74; 95% CI, 1.01-3.76) confirming that children from developing countries are at greater risk of infection. Several studies supported the observation that early childhood is the main period of acquisition of H. pylori infection in high prevalence population. One study conducted in Cameroon, using an enzyme-linked immunosorbent assay, the H. pylori stool antigen (HpSA) test reported a similar prevalence of 37.5% in children younger than 3 years [17]. Ceylan et al. [18] reported that the prevalence of H. pylori ranged from 4.62% in children less than 5 years old to 70.76% among group aged 11-15 years, in Turkey. In another study conducted in China, Zhang et al. showed a significant relationship between the H. pylori seropositivity and age group. A higher rate of H. pylori infection in children 13 to 16-year-old compared to those aged 3 to 7-year old (54.4% versus 39.5%, p<0.05) [19]. Nguyen et al. reported an increase of H. pylori seroprevalence from 30.9% in children under 3% to 53.1% in 15-18 year group (OR: 1.59; 95% CI: 1.1-2.32) in a study population of 476 children [13]. Our results are comparable with some other findings [13,16,20,21].

Low socioeconomic status and poor sanitary standards have been considered as risk factors for the acquisition and transmission of H. pylori [21,22]. Our findings showed a significant relationship between H. pylori seropositivity and some variables related to socioeconomic status of their household such as monthly income, father's occupation and mother’s education. The H. pylori infection rate decreasing along with socioeconomic development was shown in a multicenter study from Czech Republic [23]. The seroprevalence of H. pylori was significantly decreased from 41.7% in 2001 to 23.5% in 2011. Decrease in the prevalence of H. pylori infection in children was also shown in Estonia [24] and Russia [25]. It has been explained by improving socio-economic conditions and standards of living together. Hasosah et al. found that an income of <5000 Saudi riyal was positively associated with the presence of H. pylori in multivariable analyses (OR=2.06, 95% CI=1.07-3.95) [21]. The finding that it was the mother’s educational levels rather than the father’s that were associated with the rate of H. pylori infection in children has been reported in Turkish study [26]. One Nigerian study showed that the prevalence of H. pylori infection was lower in children from families of higher socioeconomic status [27]. The poor socioeconomic status of people in the south of Iran was correlated with a higher incidence of infection (p<0.05) [28]. Our data is thus in line with these findings of others previous studies [26-28].

Epidemiological studies of H. pylori infection have always looked to find potential exposure variables in relation to individual hygiene as well as habits and life-style [22,23,27]. Our data showed no significant differences in H. pylori infection rates based on water source, regular latrine presence, regular hand washing before meal and having pets. Prevalence of H. pylori infection in children without regular hand washing after defecation was significantly higher than those doing regular hand washing (OR: 38.6, 95% CI 11.8-126.3, p=0.021). In some previous studies, Nguyen et al. has shown that owing latrine in the family and regular washing hands before meal is significantly associated to lower H. pylori seropositivity [29]. Regular washing hand after defecation and no taking food by hand appeared as protective factors for H. pylori infection in ethnic people in Vietnam [12,29]. Abebaw et al. also reported higher rate of H. pylori infection in people who used unprotected surface water and irregular washing hand before meal [30]. Children who did not regularly receive chewed food were less likely to be H. pylori seropositive (adjusted OR: 0.58, 95% CI 0.31-0.80, p=0.04). In our previous study, we found no association between a rate of H. pylori infection and behavior of regularly chewed food in Tay ethnic people [13]. Using multivariate logistic regression analysis, mouth-to-mouth feeding from mothers or caregivers to a child was a risk factor for H. pylori infection in Thai and Kinh population in Vietnam (OR: 1.81, 95%CI 1.08-3.03) [14].

Some studies have demonstrated that breastfeeding is protective against the acquisition of H. pylori infection [31-33], while other studies have reported no protective effect of breastfeeding [3,5,34]. Prevalence of H. pylori infection in children breastfed longer than 12 months was lower than those breastfed for a shorter duration [OR (95% CI): 0.58 (0.35-0.94)] (Table 2). In one study conducted in Germany, Rothenbacher et al. [3] found that breastfeeding longer than 6 months after birth may increase the rate of H. pylori infection [OR (95% CI): 2.57 (1.19-5.55)]. Results from a previous study in Tay children showed that children breastfed longer than 12 months were likely to be more infected by H. pylori than those breastfed for a shorter duration [OR (95% CI): 1.42 (1.07-2.68)] [13]. A systematic review conducted by Chak showed that breast-feeding is protective against H. pylori infection (OR 0.78 (95% CI, 0.61-0.99; p=0.02) [32]. OR was 0.55 (95% CI, 0.33-0.93; p=0.01) in studies in which the subjects resided in developing countries compared with 0.93 (95% CI, 0.73-1.19; p=0.28) in those of developed nations. Our data is thus in line with findings from this systematic review [32].

Having infected family members was a risk factor for H. pylori infection in children in the present study. The prevalence of H. pylori was higher among children with infected mothers as compared to children whose mothers were uninfected [OR (95% CI): 1.34 (1.01-2.32)]. Our data found that also the presence of infected siblings and grandfather were risk factors for H. pylori infection in children. The adjusted OR for infection was [OR (95% CI): 1.37 (0.99-1.98)] and [OR (95% CI): 1.73 (1.01-4.34)], respectively. The prevalence of H. pylori infection in children living with more than 5 people in the household was higher than in those having less than 4 people (OR: 1.46, 95% CI: 1.08-2.68, p=0.043). These results are similar to findings in other studies [27,33,34]. We did not find any relationship between H. pylori seropositivity in children and some other variables such as the number of siblings, H. pylori infection in fathers, regularly sharing bed, collective life initiation and history antibiotic use within 6 or 12 months in children. Infection clusters in families and familial spread were considered as the mode of H. pylori transmission [5,20,35,36]. Data from those studies provided an evidence of having infected family members is highly associated with the infection in children. Kivi et al. showed that having an infected mother [OR 11.6 (95% CI: 2.0-67.9)] or at least one infected sibling [OR 8.1, (95%CI: 1.8-37.3)] was a major risk factor for H. pylori infection in Swedish children [37]. Urita et al. reported a significantly higher seroprevalence of H. pylori in children who had infected siblings compared to the control group [38]. Malaty et al. also provided data demonstrating an increased prevalence of infected children to infected parents [4,39]. The higher prevalence of infection due to H. pylori in parents of infected children suggests person-to-person transmission within the family [5,40]. The H. pylori status of the mother was found to be a strong determinant for childhood infection and more predictive than the status of the father [13,14,36]. The importance of both infected siblings and mothers was recently corroborated in a Brazilian high-prevalence community [41]. The findings are substantiated by a previous report of H. pylori strain concordance between mothers and offspring and amongst siblings, demonstrated by using bacterial molecular typing in a subset of the currently studied families [40]. Aguemon et al. also reported the higher infection rate in children whose parents were both infected or only infected mother. By using logistic regression analysis, sharing bed (OR, 95% CI: 3.85, 1.53-9.67, p 0.003) and infected mothers (OR, 95% CI: 9.82, 4.13-23.31, p<0.001) were independent predictors for H. pylori infection [42]. Family contact with infected persons and crowded living conditions were associated with increased risk of infection [5,37,40]. In the present study, we found higher rates of seropositivity among children who had H. pylori positive mothers, siblings or grandfather compared to those whose family members were negative. Results from our study are similar to those from other studies in Vietnam [13,14,29]. These findings might be explained by longer and closer contact between siblings, mothers and grandfathers in Muong people.

Mouth secretions of mother or grandfather could be contaminated with H. pylori and may be transmitted to the infant and child. Transmission may occur also for sharing common cups, spoons, chopsticks, teats of feeding bottles, or for chewing or tasting children’s food. Vomitus has been suggested as an important vehicle for H. pylori transmission as this organism had been successfully cultured from gastric juice and vomitus [43]. The faecal-oral route is another potential route of transmission. Evidence for a faecal-oral transmission route of H. pylori has been reported in several studies using DNA to detect H. pylori in stool of infected patients [44] although it is difficult to detect H. pylori in faecal samples by DNA methods because of potential inhibitors. In addition, this bacterium has also been isolated by culture of faecal samples in several studies [43,44]. This mode of transmission has been proposed to commonly occur in developing countries because of limitations in hygienic conditions and high risk of diarrheal disease [1,2].

Strength of the present study is that all members of all generations living together in the same household recruited in this cross-sectional community based-study. This method of sampling facilitated the data analysis and interpretation, provided a more comprehensive understanding about the interaction or interrelation between studied variables and H. pylori infection. Therefore, it rendered more feasible and more reliable for risk factors to be identified among studied sociodemographic and potential exposure variables. The possible limitations of our study emanate from the limitation of structured questionnaire reluctantly adopted according to human and financial conditions of the study. Since we had to rely on self-reported from householder, the first limitation resides in recall biases inevitably committed by respondents during interview. Another limitation may be the difficulty to calculate exact income per capita per month given the diversity of homemade and self-serving products, very popular in this population living in a remote area of a mountain province.

Conclusion

The present first community-based study in a Muong population found a moderate prevalence of H. pylori infection among Muong children at an early age and an increase with age. The findings showed a familial clustering in these multigeneration familial structures and supported the hypothesis of person-to-person transmission in H. pylori infection.

Acknowledgement

We sincerely thank,

National Fund of Scientific and Technological Development (NAFOSTED) for sponsoring this study.

Hanoi Medical University and the National Institute of Epidemiology and Hygiene for material and technical supports indispensable for the study.The personnel of the Provincial Department Health of Kyson District, Hoabinh province whose invaluable and primordial assistance and involvement in organization and participation make these funding possible.

And all household members participating to the study for their preciously collaborative spirit.

Funding

This study was funded by National Fund of Scientific and Technological Development (NAFOSTED) of Vietnam.

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