Main

Helicobacter pylori is a common human gastric pathogen with worldwide prevalence in both children and adults(13). Infection with H. pylori is associated with significant gastroduodenal disease, including chronic-active gastritis and duodenal ulcers(1, 2). Most persons infected with H. pylori acquire this infection during childhood(46). It is postulated that individuals with more severe H. pylori-related gastroduodenal disease acquire infection before the age of 10 y(4, 5). Recent epidemiologic evidence suggests that chronic H. pylori infection, acquired during very early childhood, can result in gastric carcinoma and primary gastric lymphoma both in adults and in children(68).

Many features of H. pylori infection remain poorly characterized; these include evolution of the host humoral immune response to gastric mucosal colonization(911) and the protective role of vertical passive maternal antibody transfer to the infant(12, 13). In addition, studies of susceptibility to H. pylori infection in infants are lacking(14).

We obtained serum specimens from a cohort of Taiwanese mothers and their infants to examine the natural history of H. pylori acquisition and the host immune response. Maternal-fetal passive transfer of anti-H. pylori IgG antibodies to their infants and the duration of these antibodies before their disappearance was assessed. We used an ELISA to determine the cross-sectional prevalence of H. pylori antibodies in the mothers(15). The incidence of H. pylori infection, determined by the reappearance of IgG antibodies, was evaluated in their infants over the first 14 mo of life. Finally, we characterized the specificity of the IgM response to acute H. pylori infection, the length of the IgM response, and period for antibody switching (IgM to IgG).

METHODS

Subject characteristics. Study subjects had been enrolled voluntarily from a group of Taiwanese women and infants entered in a hepatitis B vaccine efficacy study at the National Taiwan University, Taipei, Taiwan. Informed consent for se rum collection had been obtained from all maternal subjects and by the mothers for their infants, as well as appropriate approval from the Human Investigations Committees at the participating institute. We analyzed sera obtained from 80 mothers at delivery (single specimen) and serum specimens from their infants at birth and at 1, 2, 3, 6, 12, and 14 mo of age. Demographic characteristics of 62 of the 80 subjects were obtained by telephone interview, and the following characteristics were ascertained: parental age, occupation and educational level at the child's birth, family income, house size, number of individuals and children in the family, the infant's primary caretaker, infant diet, whether or not the child was breast-fed, a history of premastication of food before administration to the infant, and previous endoscopic evidence of upper gastrointestinal disease in the mothers.

Determination of maternal anti- H. pyloriIgG, maternal-fetal IgG transfer, and infant H. pylori infection. IgG assay development. The prevalence of H. pylori infection in the mothers was defined on the basis of IgG seropositivity. A highly sensitive (96%) and specific (95%) microtiter ELISA for the detection of anti-H. pylori IgG was used to evaluate the sera of the mothers and their infants(15). Briefly, standardization and validation of our ELISA for the detection of anti-H. pylori total IgG was accomplished using sera from 203 children undergoing diagnostic upper endoscopy and biopsy from the United States, Canada, China, and South Africa. Each serum specimen was analyzed without access to the subject's clinical status. Definitive H. pylori infection, for the purpose of ELISA validation, was established by demonstration of the organism in endoscopically obtained tissue through histology and staining of organisms by Geimsa or Warthin Starry stain, primary culture, and/or a positive CLO®-test (Trimed Specialties, Overland Park, Kansas)(1618). For the initial development of our ELISA, a patient was considered infected by H. pylori(i.e. H. pylori positive) if any one of the above criteria was met. A patient was considered H. pylori-negative if all the three diagnostic modalities employed were negative.

Bacterial proteins from 48 different H. pylori isolates including the ATCC type strain (American Type Culture Collection, Gaithersburg, MD) were evaluated for use as the solid phase antigen. ELISA sensitivity was determined using five well characterized H. pylori-positive and five H. pylori-negative pediatric and adult sera. Antigen evaluation was undertaken using whole cell and purified outer membrane protein preparations from both single strains and a combined pool of H. pylori isolates(15,18). Using a standard competitive inhibition assay, cross-reactivity and specificity of the antigen was evaluated by examining 15 heterologous, genetically related bacteria (see Table 1)(1921). Cross-reactivity of the solid phase antigen was further evaluated with sera from H. pylori-negative, “sick controls.” Controls were patients with gastroenteritis and one of the following pathogenic species isolated from stool culture: Escherichia coli, Campylobacter, Salmonella, or Shigella. The cross-reactivity of these sera against the ELISA solid-phase antigens was determined using serial dilutions(undiluted to 1:64 000) while holding the solid-phase antigen and secondary antibody concentrations constant.

Table 1 Specificity of the IgG ELISA solid-phase H. pylori antigens

The well characterized pediatric and adult control serum samples (five each) from H. pylori-infected and uninfected patients were then used to determine cutoff values for the IgG ELISA. A relatively narrow window separated the limits of the H. pylori-positive and -negative values, which was then designated the borderline range. When sera were evaluated for IgG antibodies to H. pylori, any value falling within the borderline range was retested in triplicate, and, if it was still in the borderline range, the patient's serum was considered indeterminate.

Detection of maternal H. pylori IgG and infant H. pylori infection. In the present study, all sera were run in triplicate on each plate and H. pylori-seropositivity determined as above(15,18,22). Each serum specimen was analyzed without access to the subject's demographic data. H. pylori IgG antibodies present in infant's blood at birth were assumed to be maternal antibodies against H. pylori that had crossed the placental barrier during gestation and, thus, not indicative of active infection(12,23). Detection of the reappearance of H. pylori IgG in the sera of infants after the disappearance of maternal IgG antibodies in those with maternal-fetal transplacental transfer was designated as infection acquisition.

Detection of IgM response and antibody switching. Sera from infants who acquired H. pylori infection were used for IgM antibody characterization and determination of ELISA conditions. Serial dilutions of infant sera were assessed by optical density measurements for the determination of IgM antibody response to H. pylori infection by using a standard 96-well microtiter plate ELISA spectrophotometer at wave-length (λ) of 492 nm (Fisher Scientific, Pittsburgh, PA). The optimum concentration of the secondary antibody, a mouse monoclonal anti-human IgM antibody conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, MO), was then determined.

All infant sera were then evaluated in triplicate, for H. pylori IgM-specific antibodies. The mean OD reading for IgM antibodies to H. pylori, for all children positive for H. pylori infection by IgG, was further analyzed by running serial dilutions of individual patient sera against a constant concentration of the solid-phase antigen whole-cell proteins obtained from a single clinical isolate of H. pylori (10μg/mL). In this manner the appropriate concentrations of the primary(serum) and secondary antibodies (anti-human IgM) were determined. As with the anti-H. pylori IgG assay(15), a relatively narrow window separated the H. pylori IgM-positive and -negative OD reading: sera in this range were designated indeterminate. The ELISA was carried out as described with total IgM from all 80 infants at a dilution of 1:100 and secondary antibody at a dilution of 1:10 000.

Anti- H. pylori IgM verification/competitive inhibition assay. Infant sera were incubated with goat anti-human IgG and anti-human IgA agarose-coated beads (Sigma Chemical Co.) for 2 h at room temperature to adsorb total IgG and total IgA. Adsorbed total IgG and total IgA were then separated from the sera by centrifugation at 10 000 rpm (Beckman Instruments, Palo Alto, CA) for 5 min(19). Adsorption efficiency for this procedure was verified by Western blot analysis of supernatants using anti-human IgG, IgA and IgM antibodies(19). Supernatants containing only total IgM were collected and used immediately (within 30 min) to prevent antibody degradation. Isolated IgM samples were run in triplicate by ELISA, and the time course of the IgM response over the 14 mo was determined for 49/80 infants due to limited sera available from each infant.

Subsequently, a competitive inhibition assay to determine specificity of IgM antibodies against H. pylori antigens was performed(19,21). Cross-reactivity and specificity of isolated total IgM antibodies against H. pylori whole-cell proteins, the solid phase antigen in our microtiter ELISA, were tested against whole-cell proteins obtained from two species of genetically related bacteria(Campylobacter and E. coli) that are commonly found as diarrheal pathogens in infants (see (Table 2)(15,20). These bacteria have been shown to have cross-reactivity with the H. pylori antigens used in many commercially available serological preparations(16,20,22). The competitive inhibition assay was performed using up to 40 μg/ml of heterologous bacterial whole cell proteins (preincubated with isolated total IgM from patient sera).

Table 2 Specificity of IgM antibodies to H. pylori solid-phase antigens

Statistical analysis. Associations between maternal H. pylori seropositivity, infant seroconversion (i.e. infection acquisition), and potential risk factors obtained from the questionnaire were analyzed using either χ2 or Fisher's exact test as appropriate.χ2 test for trend was used to examine the relationship between paternal education and H. pylori status. The relationship between H. pylori status and age was determined by the Kruskal-Wallis test.

RESULTS

Maternal and infant sera. The demographic characteristics of the 62 infants 35 (57%) were male, 17 (28%) were breastfed and two (3%) had food premasticated before eating. Median family income was $8,200.00 (US), mean maternal age at delivery, 28 y, mean paternal age at delivery, 30 y, and family sizes ranged from 3 to 12 members.

Fifty of the 80 mothers (62.5%; 95% CI 51-73%) were seropositive. Forty-eight of the 80 infants were H. pylori-positive at birth(i.e. blood drawn at 3 d of life); all of those had seropositive mothers. Ninety four percent of the infants with evidence of passive transfer of maternal IgG antibodies showed no detectable antibodies by 3 mo of age and 98% of these infants by 6 mo of age (Fig. 1).

Figure 1
figure 1

Percentage of infants born to H. pylori-positive mothers with maternal anti-H. pylori IgG antibodies at birth. The solid bars represents the percentage of infants at each age with presence of transplacental maternal IgG antibodies against H. pylori infection detectable in their sera. Maternal IgG antibodies to H. pylori declined by 6 mo of age in nearly all of the infants seropositive at birth.

Six infants (7.5%; 95% CI 3-16%) acquired H. pylori infection. Two of these infants who seroconverted at 6 and 14 mo were born to H. pylori-negative mothers and were H. pylori-seronegative at birth. Three additional infants had IgG antibody levels in the indeterminate range at 14 mo of age.

There was no statistical association between maternal H. pylori seropositivity and maternal or paternal age, maternal or paternal education level, or family income. In addition, no statistical associations were obtained between maternal H. pylori seropositivity and the size of the dwelling, gastrointestinal disease in the infant, number of individuals or number of children occupying the same domicile, or breast-feeding. There was no association between infant seroconversion and maternal seropositivity, maternal or paternal age, maternal or paternal education level, family income, size of the house, number of individuals or children living in the household, primary caretaker of the child, or infant gender.

Breast-feeding was, however, associated with seroconversion of the infant(odd ratio = 13.2; 95% CI; 1.2-347; p = 0.02). Of the six infants who acquired H. pylori infection, demographic data were collected on five. Of these five infants, four (80%) were breast-fed. Of the 72 infants who failed to seroconvert, demographic data were collected on 56. Of these 56, 13(23%) were breast-fed. This association remained when analysis was limited to those infants whose mothers were the primary care givers. Both infants whose food was premasticated before feeding were cared for primarily by their grandparents, and neither infant seroconverted.

Detection of IgM response and antibody switching. Specificity of the solid-phase antigen for the detection of anti-H. pylori total IgG antibodies is shown in Tables 1 and 2. All six infants acquiring IgG antibodies to H. pylori infection had evidence of anti-H. pylori IgM, and five of the six had IgM antibodies which appeared before the rise in IgG antibodies (time course for the antibody response in these infants is shown in Fig. 2,A-F). Three of these six infants had IgM antibodies above the positive cutoff and three had IgM values in the indeterminate range. The IgM ELISA performed best at a solid-phase antigen concentration of 10 μg/ml, with sera at 1:100 dilution and secondary antibody (anti-human IgM-horseradish perotidal) at a dilution of 1:10 000. An IgM response was determined by antibodies above the positive and indeterminate cutoff values. The positive cutoff OD for H. pylori IgM was 1.08; with an indeterminate range of 0.78-1.07. An IgM response was observed in four infants who did not acquire H. pylori infection. Three of these infants had IgG antibodies in the indeterminate range at 14 mo of age. Antibody switching in the infected infants appeared to take place within 2 mo of demonstrating IgM antibodies against H. pylori.

Figure 2
figure 2

(A-F) The systemic IgM and IgG antibody response to H. pylori infection in the six infants who acquired H. pylori infection after the decline in maternal anti-H. pylori IgG antibodies. The open bars represents IgG antibodies against H. pylori at each time point at which sera were obtained. Black bars represents whole serum IgM response to H. pylori infection at each time point. The solid horizontal line represents the ELISA OD cutoff value for H. pylori IgG seropositivity (1.3). The dashed line represents the ELISA OD cutoff value for H. pylori IgM seropositivity (0.8).(A) A graph of one of the infants who acquired H. pylori infection shown to demonstrate similarities between the anti-H. pylori IgM antibody response over time, pre- and postadsorption of IgA and IgG. The gray bars represents this infant's IgM response at each time point after adsorption of IgG and IgA from the serum specimen. (B-F) The other five infants who acquired H. pylori infection and the IgG and IgM antibody response over time (the isolated IgM antibody response,i.e. gray bars in A, have not been included in these five graphs).

Anti- H. pylori IgM verification/competitive inhibition assay. There was a negligible difference in the two IgM curves over the 14 mo for all infants with an anti-H. pylori IgG and IgM response (Fig. 2A). As had been previously shown with the use of the ELISA for the detection of H. pylori IgG antibodies(Table 1), the competitive inhibition assay demonstrated no cross-reactivity with any of the enteric pathogens' whole-cell proteins and the solid-phase H. pylori antigens (Table 2).

DISCUSSION

Our study demonstrated that children appear to acquire H. pylori in infancy. Data suggest that these children may be at a greater risk of peptic ulcer disease and possibly gastric cancer(46). In this cohort, 8% of children demonstrated seroconversion with IgG antibodies against H. pylori. At least three additional infants had IgG antibodies at 14 mo of age that fell in the ELISA indeterminate range.

Passive transplacental transfer of maternal anti-H. pylori IgG occurred in 96% of the infants studied. The remaining two infants born to H. pylori- positive mothers had IgG levels that fell in the indeterminate range at birth, and at 1 mo of age, were seropositive. These results suggest that probably all infants born to H. pylori-positive mothers in this cohort had passive transplacental antibody transfer. We, like others(3032), have shown that these antibodies decrease in the infant between 3 and 6 mo of age.

A cross-sectional study from Peru showed a high prevalence of H. pylori infection among infants (71.4%), as determined by 13C-labeled urea breath testing, but a lower prevalence between the ages 1 and 4 y (49.4%)(25). A retrospective serologic study by Blecker et al.(12) of a population from a developed country showed a low level (<1%) of H. pylori infection acquisition in the 1 st y of life. These authors speculated that the lack of infection acquisition in the infants studied may have been due to protective maternal anti-H. pylori antibodies(12). The demographic characteristics of our subjects were more comparable with the Peruvian children evaluated by Klein et al.(25).

Increased risk factors for H. pylori infection in infants and children include 1) living in developing countries, 2) lower socioeconomic circumstances, 3) living in overcrowded conditions, and 4) being of African-American or Hispanic decent(5,23,24). Although demographic and clinical data were not collected on the entire study group, the majority of the families were in a low income bracket. Overcrowding also appeared to be a factor of subjects evaluated in this study, as many of the family sizes were greater than six persons. The above demographic profile of our cohort may explain the differences in the findings of this study and the findings of the cohort described by Blecker et al.(12).

Saliva and/or dental plaque have been postulated as both a reservoir for H. pylori and a vehicle for transmission of this organism(26,27). The data concerning feeding practices were insufficient in this cohort to address the question of the role of feeding practice on H. pylori transmission. Similarly, some water sources have been previously reported as reservoirs for H. pylori infection transmission(28,29). Information about the water sources available to this cohort was also not available.

Because only six infants seroconverted and demographic data were available on five, the power for detecting associations between seroconversions and potential risk factors was extremely low and the lack of associations seen should be interpreted with caution. Despite this, a positive association with breast-feeding and H. pylori infection acquisition was documented. These findings contrast with other studies that have shown that H. pylori infection is less frequent in breast-fed infants(33,34). Possibly our results could be explained by the more intimate contact between infants and caretakers who breast-fed, thus leading to an increased number of close contacts between a breast-fed infant and an H. pylori-infected mother, and thereby to and increased risk for infection transmission. The duration and pattern of breast-feeding were not ascertained. These and other data(23,3335) suggest that further studies on the relationship between breast-feeding and acquisition of H. pylori infection are required.

In most infectious diseases, evolution of host immune response proceeds along a predictable pattern(3032). After initial infection, there is a primary, initial host IgM response followed by a persistent IgG response(36,37). Previous reports evaluating the immune response to H. pylori infection have demonstrated a rise in IgM within 2 wk after gastric colonization of H. pylori, followed by a rise in IgG and a decline in IgM antibodies(3640). In five of the six infants who acquired H. pylori infection in our study, IgG seroconversion was proceeded by a rise and fall of IgM antibodies. Unfortunately, no infant sera were available from 6 through 12 mo of age, and this appeared to be the period when most infants acquired H. pylori IgM antibodies. In addition, of the four infants who had detectable IgM antibodies that did not seroconvert, three had a rise in H. pylori IgG antibodies that were in the indeterminate range at 14 mo. We were unable to determine whether seroconversion subsequently occurred in these four patients as the cohorts were followed for only 14 mo. Understanding the duration of the IgM response and the timing of antibody switching requires further study.

Of the many serologic assays available for the detection of H. pylori infection, none is sensitive and specific for the evaluation of IgM(36,37,41). The majority of the commercial IgG assays were standardized and validated in adult populations, where most individuals are chronically infected(41,42). Attempts to develop IgM assays have been complicated by cross-reactivity with other common enteric pathogens(38,43) and failure to eliminate IgG in the serum preparations due to epitope similarities between the two immunoglobulin classes(37,38,42). In this study, we used two methods to show a highly specific IgM response to H. pylori infection. No cross-reactivity was observed between the isolated anti-H. pylori IgM antibodies and antigens from other common enteric pathogens. Further validation of this assay with upper endoscopy performed on children with acute infection may prove to be an important tool for studying the natural history of H. pylori infection.

In summary, maternal-fetal transplacental transfer of anti-H. pylori IgG antibodies occurs, and these antibodies decline by 3-6 mo of age. In this cohort, H. pylori infection is acquired in early infancy. A specific, systemic IgM response to H. pylori infection appears to be short-lived and precedes development of a systemic IgG response. These findings should be further evaluated in a prospective study and are critical for future vaccine strategies.