Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children

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1 ORIGINAL ARTICLE Microbiol Immunol 2013; 57: doi: / Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children Masoumeh Douraghi 1,2, Mahmoud Nateghi Rostami 3, Hossein Goudarzi 2 and Zohreh Ghalavand 2 1 Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, 2 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran and 3 Department of Public Health, Faculty of Health, Qom University of Medical Sciences, Qom, Iran ABSTRACT Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is problematic because they are unable to cooperate with performance of invasive tests. In this study, the non invasive methods of measuring serum IgG antibody concentrations and performing stool antigen tests were used to screen for H. pylori infection in ID children. Eighty seven children with intellectual disabilities were studied. The amount of serum IgG antibody against H. pylori was measured by the ELISA method. Stool samples were examined using an amplified IDEIA HpStAR kit. To assess categorical variables, X 2, Fisher's exact and Kappa tests were used. The stool antigen tests showed that 93.1% of the children had H. pylori antigen and the serology test that 85.1% of children were positive for H. pylori IgG antibodies. Agreement between results of H. pylori stool antigen (HpSA) testing and IgG antibody serology was 82.8%; however, according to the kappa measure of agreement this agreement is not statistically significant (value, 0.128; P ¼ 0.19). Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive on HpSA test but negative by serology and 4 (4.6%) who were IgG seropositive but had negative HpSA tests. This study showed a notably higher rate of H. pylori infection in ID children than has been reported by others for non ID children from the same geographical area. The HpSA test is a valid method for primary screening for H. pylori infection in ID children; it detects the specific antigens shed during active infections and has less cross reactivity than serological tests that detect antibodies. HpSA is a sensitive non invasive method for detecting infection in ID children and may serve as an accurate alternative to serology. Key words Helicobacter pylori IgG antibody, Helicobacter pylori stool antigen test, intellectual disability. Helicobacter pylori infection is primarily acquired in early childhood and chronic H. pylori infections may lead to severe gastroduodenal outcomes such as duodenal ulcer or gastric adenocarcinoma (1 4). Transmission of H. pylori occurs mainly through oral oral or fecal oral routes (5). Major risk factors for transmission are poor sanitation, overcrowding and poor socioeconomic conditions (6 8). Preventive measures during childhood might decrease the rate of H. pylori infection and consequently the risk of possible malignancies. Therefore, accurate and early diagnosis of H. pylori infection is of prime importance for management of H. pylori associated diseases. Various invasive and non invasive tests are recommended for diagnosis of H. pylori infection. Correspondence Masoumeh Douraghi, Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Tel: þ ; fax: þ ; mdouraghi@tums.ac.ir Received 5 July 2013; revised 25 August 2013; accepted 4 September List of Abbreviations: EIA, enzyme immunoassay; HpSA, H. pylori stool antigen; H. pylori, Helicobacter pylori; ID, intellectual disability; IgG, immunoglobulin G; OD, optical density; UBT, urea breath test The Societies and Wiley Publishing Asia Pty Ltd

2 Hp in intellectually disabled children Diagnosis of active infection is based on the invasive tests of rapid urease test, histology and culture and the noninvasive tests of UBTand HpSA (9 13). As is true of other serological tests, measurement of serum anti H. pylori antibodies cannot distinguish active from past infections: this test may remain positive for several years after cure (14). Several consensus reports have implied the usefulness of non invasive tests for diagnosis of H. pylori infection in children (12, 15). UBT is considered the gold standard non invasive test; however, it demands the patient's cooperation, trained technicians and optimized analytical instruments (16). Stool antigen tests are also technically simple and cost effective methods for diagnosis of H. pylori infection (15). Intellectually disabled children are a vulnerable subgroup and may experience higher rates of infections and morbidities (17). Both H. pylori infection and gastric cancer occur at higher rates in subjects with ID than in the general population (18, 19). Most ID children have severe neurologic impairments and are not able to cooperate with performance of non invasive test such as UBT. In addition, because of limitations in their intellectual and adaptive functioning, such children are unable to report their symptoms. Although life long H. pylori associated morbidities are well known, few studies have addressed the status of H. pylori infection in ID children. In this study, the non invasive methods of serum IgG antibody and stool antigen tests were used to screen for H. pylori infection in children with IDs. MATERIALS AND METHODS Participants and ethical considerations In all, 87 children aged less than 18 years from three centers were studied, comprising 17 from center I, 13 from center II and 57 from center III. All subjects were permanent residents of long stay centers located in the urban area of Tehran. The number of randomly chosen cases from each center was proportional to the total number of children with IDs in each center. The centers provide several services including medical, dietary and physical therapy for children with mild to severe IDs. Relevant clinical characteristics and medical histories were obtained from medical records. The study was approved by the local Ethical Committees. Informed consents were given by welfare guardianships as well as by the children's legal guardians. Sampling Inclusion criteria were the absence of diarrhea at the time of sampling and no treatment with antibiotics or proton pump inhibitors for the last 2 weeks. Peripheral blood samples were collected aseptically from all children under the supervision of physicians. The resultant serum samples were stored at 20 C until performance of IgG ELISA. Because some of the ID children had fecal incontinence, their caregivers collected stool specimens from them. Stool samples were frozen at 70 C until use for detection of H. pylori antigens. Stool antigen test The amplified IDEIA HpStAR (Oxoid, Cambridge, UK) test, a sandwich type EIA, was used to detect H. pylori antigens in stools, according to the manufacturer's instructions. Briefly, approximately 0.1 g of stool sample was mixed with the sample diluent. Fifty microliters of stool supernatant and 50 ml of conjugate were added to microwells and incubated for 60 min at room temperature. Following five wash cycles, 100 ml of substrate solution was added to each microwell and incubated for 10 min at room temperature. The reactions were stopped by adding 100 ml of stop solution. The results were read by an EIA plate reader at 450 nm. OD values of 0.20 were considered positive; ODs between 0.2 and 0.5 weakly positive, and those > 0.5 strongly positive. Values < 0.20 were considered negative. Helicobacter pylori immunoglobulin G serum enzyme linked immunosorbent assay The amounts of IgG antibody to H. pylori were measured using ELISA according to the manufacturer's instructions (IMMUNOLAB GmbH, Frickenhausen, Germany). Briefly, the diluted sera (1:101), standards and controls were added to the wells. Anti human IgG peroxidase conjugate was added to each well and the reactions developed using 3,3,5,5 0 tetramethyl benzidine substrate. ODs were read at a wavelength of 450 nm using an EIA plate reader. Sera with ODs 0.05 were considered positive. Values between 0.05 and 0.49 were considered weakly positive and those 0.5 strongly positive. Statistical analysis Statistical analysis was performed using SPSS version For comparison of categorical variables, x 2 and Fisher's exact tests were used. For assessment of the consistency of two diagnostic tests, the Kappa statistic was used. Continuous variables are presented as means and standard deviations. Using HpSA as the reference test, the sensitivity, specificity and positive and negative predictive values of the serology test were calculated with 95% confidence intervals (CIs). P value of 0.05 was considered statistically significant The Societies and Wiley Publishing Asia Pty Ltd 773

3 M. Douraghi et al. RESULTS Relevant clinical characteristics of participants Among the 87 ID children, 43 (49.4%) were girls and 44 (50.6%) boys with an age range of 8 months to 18 years (mean SD; ). Nine (10.3%) children were younger than 4 years, 24 (27.6%) were aged from 5 9 years, 34 (39.1%) from years and 20 (23%) from years. The mean duration of institutionalization was months. These characteristics according to center are as follows: the distribution of girls to boys was 11:6, 2:11 and 30:27 in centers I, II and III, respectively. The mean age was higher in center III (years, mean SD; ) than in the other two centers (center I, ; center II, ). The duration of institutionalization was also longer in center III (months, ) compared to the other two centers (center I, ; center II, ). The cause of ID was developmental brain abnormalities in 61 (70.1%) cases:, whereas eight subjects (9.2%) had genetic disorders and four (4.6%) birth injuries. Other causes such as meningitis, autism, convulsions, seizures, hyperbilirubinemia, metabolic causes and head trauma each accounted for less than 3% of participants. The cause of the disability was unknown in five children (5.7%). Helicobacter pylori stool antigen test Helicobacter pylori infection was detected in 93.1% (81/87) of children (40 boys and 41 girls, mean age years). The total OD values for the HpSA test ranged from 0.06 to 0.94 (mean SD, ) and OD values in infected children ranged from 0.21 to 0.94 (mean, ). The distribution of HpSA test values in H. pylori infected and non infected children is shown in Figure 1a. Among the infected cases, 57 subjects (70.4%) had strong HpSA test results and 24 (29.6%) weak results. The mean age of infected participants ( years) was not significantly different from that of non infected participants ( years). Infection rate did not correlate with age group (P ¼ 0.901). The proportions of infected to non infected children in the three centers were as follow: in center I, 16 1 (94.1%); in center II, 13 0 (100%); and in center III, 52 5 (91.2%). Anti Helicobacter pylori immunoglobulin G antibody Serum IgG antibody to H. pylori antigens was detected in 85.1% (74/87) of children (39 boys and 35 girls, mean age years). H. pylori seropositive cases were Fig. 1. The distribution of test values in H. pylori infected and non infected children for (a) stool antigen and (b) serum antibody tests. Ab, antibody; Ag, antigen. classified as weakly positive (n ¼ 38; 51.4%) and strongly positive (n ¼ 36; 48.6%). The total OD values for anti H. pylori IgG ranged from (mean SD, ) and OD values in H. pylori seropositive children varied from (mean SD, ). The distribution of anti H. pylori IgG values is shown in Figure 1b. The mean age of infected cases ( years) was not significantly different from that of non infected cases ( years). The seropositivity rate did not correlate with age group (P ¼ 0.544). The proportions of seropositive to seronegative children in the three centers were as follow: in center I, 14 3 (82.4%); in center II, 10 3 (76.9%); and in center III, 50 7 (87.7%) The Societies and Wiley Publishing Asia Pty Ltd

4 Hp in intellectually disabled children Table 1. Positivity rate of H. pylori stool antigen versus IgG antibody tests according to age group Age group (years) H. pylori stool antigen, n (%) Test H. pylori IgG antibody, n (%) (88.9) 7 (77.8) (95.8) 20 (83.3) (94.1) 29 (85.3) (90) 18 (90) Stool antigen testing versus serology According to stool antigen assay and serology, 93.1 and 85.1% of children, respectively, were positive for H. pylori infection. This difference was not statistically significant (P ¼ 0.218). The positivity rate of HpSA versus IgG antibody tests according to age group is shown in Table 1. There were no significant differences between concordant results in different age groups. Correlations of ODs between results of stool antigen and serum antibody tests in children with ID are shown in Figure 2. Discordance was found between results of HpSA and anti H. pylori IgG tests in 15/87 (17.2%) children. Eleven children (12.6%) who tested negative by serology had positive results by HpSA testing. In addition, four children (4.6%) who were IgG seropositive had negative results by HpSA. With HpSA as the reference test, the sensitivity of serology was 86.4% and its specificity 33.33%. The positive and negative predictive values were 94.6 and 15.4%, respectively. Fig. 2. The correlation of ODs between stool antigen and serum antibody tests in children with ID. Broken lines show cut off values. Ab, antibody; Ag, antigen. DISCUSSION Diagnosis of H. pylori infection can be achieved by invasive and non invasive tests. The children in this study had intellectual and developmental disabilities: there are various barriers that can influence the access of such children to health services and resources. Most invasive methods, such as endoscopy, histology and culture, are considered the gold standard for diagnosis of H. pylori infection. However, these methods require expensive procedures and are not tolerated by ID children. For instance, endoscopy is not recommended for asymptomatic children and is not feasible for children with various deformities or cerebral palsy. Although UBT is a noninvasive test, it is used mainly for follow up of treatment and assessment of eradication therapy (13). Collection of breath for UBT requires the subject's cooperation and as reported previously, few adults with IDs will provide breath specimens (20). Because of their cognitive abnormalities and dependency, breath test collection from ID children is so difficult that UBT is not a practicable test. Given the numerous limitations of ID children, there is a need for a simple and reliable test for screening of H. pylori infection in such children. We selected the non invasive HpSA test to study the prevalence of active infections among these children. Several characteristics of the HpSA test prompted this choice. HpSA, which detects viable or dead H. pylori antigens that have been shed into the gastric lumen (21), is cost effective and sampling is simple, particularly from ID children who are incontinent of feces. To avoid variations in polyclonal antibodies, we used a kit that includes monoclonal antibodies specific for H. pylori antigens to assess the presence of H. pylori. We found that the majority of children were infected with H. pylori according to HpSA findings. The infection rates were similar in all age groups and all centers. To our knowledge, there are no previous reports of assessing H. pylori infection rates by stool antigen testing of ID children. Wallace et al. used stool antigen testing and found that 75% of ID adults living in a residential center were infected with H. pylori (20). A study of non ID children living with their parents in various districts of Tehran showed that 47% of children have positive stool antigen tests (22). Thus, the current study identified a higher rate of H. pylori infection than that reported for non ID, non institutionalized children in Tehran. The high rate of infection may be in part attributable to sharing objects, utensils and services in an overcrowded environment. Most ID children are dependent on caregivers for feeding, toileting, hygiene and so on. Hence, common caregivers may play a role in transmission of various infections among these children. Another 2013 The Societies and Wiley Publishing Asia Pty Ltd 775

5 M. Douraghi et al. possible explanation for the high rate of H. pylori infection is common food and water sources. Fecal incontinence may also facilitate the transmission of infection and result in circulation of bacteria among institutionalized children with IDs. According to the most recent European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition joint recommendations, a combination of two or more invasive tests is necessary as the reference diagnostic tool for H. pylori infection in comparative studies (23). In ID children, neither invasive tests (endoscopy, culture, histology) nor UBT are practicable for diagnosis of H. pylori.thus, this study was potentially limited by lack of the traditional gold standard tests. H. pylori IgG assay is classified as a cost effective method for assessing H. pylori infection in symptomatic or high risk asymptomatic cases (14). However, because IgG antibody titers persist for up to several months after H. pylori infections, positive serological tests may indicate previous exposure to or infection with H. pylori. In contrast, HpSA tests detect specific antigensthatare shed during active infections and have less crossreactivity than serological tests that detect antibodies. Hence, in this study, we used HpSA as a proxy reference test for screening for H. pylori infection and compared the results with those of serology tests. We found that IgG serology had a sensitivity of 86.4% for detection of H. pylori antibodies. The discrepancy between the results of HpSA and serologic tests was not statistically significant. In four children, HpSA tests were negative and serology tests positive. Given the high sensitivity of HpSA, the positive serology result may have been attributable to persistence of IgG antibodies after clearing of the infection, rather than to active infection. In 11 children, serology tests were negative and HpSA tests positive. One possible explanation for this finding is that some ID children have incompetent immune systems. Altered immune responses in such children may have resulted in lack of reactivity against purified natural H. pylori antigens, including CagA, VacA and urease, which are used in IgG ELISA methods (24). Another possible explanation is that the infections were at an early stage, prior to increases in IgG antibody. The HpSA test is reportedly a reliable method for detection of Hp antigen in stool samples of children (25 28). This study showed that stool antigen tests can be used as accurate non invasive tools for screening for H. pylori infection in ID children. This procedure eliminates the need for invasive methods and needle stick procedures for serologic tests, hence HpSA is a good candidate method for replacing serological tests in ID children. 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