Microbiol Immunol 2013; 57: 772–777doi: 10.1111/1348-0421.12099 O R I GIN A L A R T I C L E Comparison of stool antigen immunoassay and serology for screening for Helicobacter pylori infection in intellectually disabled children Masoumeh Douraghi1,2, Mahmoud Nateghi Rostami3, Hossein Goudarzi2 and Zohreh Ghalavand2 1 Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, 2Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran and 3Department 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, X2, 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 ditions (6–8). Preventive measures during childhood childhood and chronic H. pylori infections may lead to might decrease the rate of H. pylori infection and severe gastroduodenal outcomes such as duodenal ulcer consequently the risk of possible malignancies. There- or gastric adenocarcinoma (1–4). Transmission of H. fore, accurate and early diagnosis of H. pylori infection is of pylori occurs mainly through oral–oral or fecal–oral prime importance for management of H. pylori‐associated routes (5). Major risk factors for transmission are poor diseases. Various invasive and non‐invasive tests are sanitation, overcrowding and poor socioeconomic con- 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, 14155‐ 6446 Tehran, Iran. Tel: þ98 2188973901; fax: þ98 2188954913; email:
[email protected] Received 5 July 2013; revised 25 August 2013; accepted 4 September 2013. 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. 772 © 2013 The Societies and Wiley Publishing Asia Pty Ltd Hp in intellectually disabled children Diagnosis of active infection is based on the invasive tests samples were collected aseptically from all children under of rapid urease test, histology and culture and the non‐ the supervision of physicians. The resultant serum invasive tests of UBTand HpSA (9–13). As is true of other samples were stored at ‐ 20°C until performance of IgG serological tests, measurement of serum anti‐H. pylori ELISA. Because some of the ID children had fecal antibodies cannot distinguish active from past infections: incontinence, their caregivers collected stool specimens this test may remain positive for several years after from them. Stool samples were frozen at ‐ 70°C until use cure (14). Several consensus reports have implied the for detection of H. pylori antigens. usefulness of non‐invasive tests for diagnosis of H. pylori infection in children (12, 15). UBT is considered the gold Stool antigen test standard non‐invasive test; however, it demands the patient's cooperation, trained technicians and optimized The amplified IDEIA HpStAR (Oxoid, Cambridge, UK) analytical instruments (16). Stool antigen tests are also test, a sandwich‐type EIA, was used to detect H. pylori technically simple and cost‐effective methods for diag- antigens in stools, according to the manufacturer's nosis of H. pylori infection (15). instructions. Briefly, approximately 0.1 g of stool sample Intellectually disabled children are a vulnerable was mixed with the sample diluent. Fifty microliters of subgroup and may experience higher rates of infections stool supernatant and 50 mL of conjugate were added to and morbidities (17). Both H. pylori infection and gastric microwells and incubated for 60 min at room tempera- cancer occur at higher rates in subjects with ID than in the ture. Following five wash cycles, 100 mL of substrate general population (18, 19). Most ID children have severe solution was added to each microwell and incubated for neurologic impairments and are not able to cooperate 10 min at room temperature. The reactions were stopped with performance of non‐invasive test such as UBT. In by adding 100 mL of stop solution. The results were read addition, because of limitations in their intellectual and by an EIA plate reader at 450 nm. OD values of 0.20 adaptive functioning, such children are unable to report were considered positive; ODs between 0.2 and 0.5 their symptoms. Although life‐long H. pylori associated weakly positive, and those > 0.5 strongly positive. Values morbidities are well known, few studies have addressed < 0.20 were considered negative. the status of H. pylori infection in ID children. In this study, the non‐invasive methods of serum IgG antibody Helicobacter pylori immunoglobulin G and stool antigen tests were used to screen for H. pylori serum enzyme‐linked immunosorbent infection in children with IDs. assay The amounts of IgG antibody to H. pylori were measured MATERIALS AND METHODS using ELISA according to the manufacturer's instructions (IMMUNOLAB GmbH, Frickenhausen, Germany). Participants and ethical considerations Briefly, the diluted sera (1:101), standards and controls In all, 87 children aged less than 18 years from three were added to the wells. Anti‐human IgG peroxidase centers were studied, comprising 17 from center I, 13 conjugate was added to each well and the reactions from center II and 57 from center III. All subjects were developed using 3,3,5,50 ‐tetramethyl benzidine substrate. permanent residents of long‐stay centers located in the ODs were read at a wavelength of 450 nm using an EIA urban area of Tehran. The number of randomly chosen plate reader. Sera with ODs 0.05 were considered cases from each center was proportional to the total positive. Values between 0.05 and 0.49 were considered number of children with IDs in each center. The centers weakly positive and those 0.5 strongly positive. provide several services including medical, dietary and physical therapy for children with mild to severe IDs. Statistical analysis Relevant clinical characteristics and medical histories Statistical analysis was performed using SPSS version were obtained from medical records. The study was 11.5. For comparison of categorical variables, x2 and approved by the local Ethical Committees. Informed Fisher's exact tests were used. For assessment of the consents were given by welfare guardianships as well as by consistency of two diagnostic tests, the Kappa statistic was the children's legal guardians. used. Continuous variables are presented as means and standard deviations. Using HpSA as the reference test, the Sampling sensitivity, specificity and positive and negative predictive Inclusion criteria were the absence of diarrhea at the time values of the serology test were calculated with 95% of sampling and no treatment with antibiotics or proton confidence intervals (CIs). P‐value of 0.05 was pump inhibitors for the last 2 weeks. Peripheral blood considered statistically significant. © 2013 The Societies and Wiley Publishing Asia Pty Ltd 773 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; 10.89 4.83). Nine (10.3%) children were younger than 4 years, 24 (27.6%) were aged from 5– 9 years, 34 (39.1%) from 10–14 years and 20 (23%) from 15–18 years. The mean duration of institutionalization was 54.14 36.92 months. These characteristics accord- ing 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; 13.01 3.50) than in the other two centers (center I, 3.57 1.75; center II, 11.15 2.96). The duration of institutionalization was also longer in center III (months, 66.60 37.39) compared to the other two centers (center I, 27.65 19.54; center II, 34.15 23.92). The cause of ID was developmental brain abnormali- ties 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 10.87 4.77 years). The total OD values for the HpSA test ranged from 0.06 to 0.94 (mean SD, 0.56 0.22) and OD values in infected children ranged from 0.21 to 0.94 Fig. 1. The distribution of test values in H. pylori infected and (mean, 0.59 0.2). The distribution of HpSA test values non‐infected children for (a) stool antigen and (b) serum in H. pylori infected and non‐infected children is shown antibody tests. Ab, antibody; Ag, antigen. 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 (10.87 4.77 years) was not significantly different from classified as weakly positive (n ¼ 38; 51.4%) and strongly that of non‐infected participants (11.13 6.11 years). positive (n ¼ 36; 48.6%). The total OD values for anti‐H. Infection rate did not correlate with age group pylori IgG ranged from 0.02–1.195 (mean SD, (P ¼ 0.901). The proportions of infected to non‐infected 0.39 0.3) and OD values in H. pylori seropositive children in the three centers were as follow: in center I, children varied from 0.05–1.195 (mean SD, 16–1 (94.1%); in center II, 13–0 (100%); and in center III, 0.46 0.28). The distribution of anti‐H. pylori IgG values 52–5 (91.2%). is shown in Figure 1b. The mean age of infected cases (11.02 4.78 years) was not significantly different from that of non‐infected cases (10.13 5.25 years). The Anti‐Helicobacter pylori immunoglobulin G seropositivity rate did not correlate with age group antibody (P ¼ 0.544). The proportions of seropositive to seroneg- Serum IgG antibody to H. pylori antigens was detected in ative children in the three centers were as follow: in center 85.1% (74/87) of children (39 boys and 35 girls, mean age I, 14–3 (82.4%); in center II, 10–3 (76.9%); and in center 11.02 4.78 years). H. pylori seropositive cases were III, 50–7 (87.7%). 774 © 2013 The Societies and Wiley Publishing Asia Pty Ltd Hp in intellectually disabled children Table 1. Positivity rate of H. pylori stool antigen versus IgG antibody DISCUSSION tests according to age group Diagnosis of H. pylori infection can be achieved by invasive Test and non‐invasive tests. The children in this study had Age group H. pylori stool H. pylori IgG intellectual and developmental disabilities: there are (years) antigen, n (%) antibody, n (%) various barriers that can influence the access of such children to health services and resources. Most invasive 0–4 8 (88.9) 7 (77.8) 5–9 23 (95.8) 20 (83.3) methods, such as endoscopy, histology and culture, are 10–14 32 (94.1) 29 (85.3) considered the gold standard for diagnosis of H. pylori 15–18 18 (90) 18 (90) infection. However, these methods require expensive procedures and are not tolerated by ID children. For instance, endoscopy is not recommended for asymptom- atic children and is not feasible for children with various Stool antigen testing versus serology deformities or cerebral palsy. Although UBT is a non‐ invasive test, it is used mainly for follow‐up of treatment According to stool antigen assay and serology, 93.1 and and assessment of eradication therapy (13). Collection of 85.1% of children, respectively, were positive for H. pylori breath for UBT requires the subject's cooperation and as infection. This difference was not statistically significant reported previously, few adults with IDs will provide (P ¼ 0.218). The positivity rate of HpSA versus IgG breath specimens (20). Because of their cognitive antibody tests according to age group is shown in Table 1. abnormalities and dependency, breath test collection There were no significant differences between concordant from ID children is so difficult that UBT is not a results in different age groups. practicable test. Given the numerous limitations of ID Correlations of ODs between results of stool antigen children, there is a need for a simple and reliable test for and serum antibody tests in children with ID are shown in screening of H. pylori infection in such children. We Figure 2. Discordance was found between results of HpSA selected the non‐invasive HpSA test to study the and anti‐H. pylori IgG tests in 15/87 (17.2%) children. prevalence of active infections among these children. Eleven children (12.6%) who tested negative by serology Several characteristics of the HpSA test prompted this had positive results by HpSA testing. In addition, four choice. HpSA, which detects viable or dead H. pylori children (4.6%) who were IgG seropositive had negative antigens that have been shed into the gastric lumen (21), results by HpSA. With HpSA as the reference test, the is cost‐effective and sampling is simple, particularly from sensitivity of serology was 86.4% and its specificity ID children who are incontinent of feces. To avoid 33.33%. The positive and negative predictive values were variations in polyclonal antibodies, we used a kit that 94.6 and 15.4%, respectively. 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 Fig. 2. The correlation of ODs between stool antigen and serum caregivers for feeding, toileting, hygiene and so on. antibody tests in children with ID. Broken lines show cut off values. Hence, common caregivers may play a role in transmis- Ab, antibody; Ag, antigen. sion of various infections among these children. Another © 2013 The Societies and Wiley Publishing Asia Pty Ltd 775 M. Douraghi et al. possible explanation for the high rate of H. pylori infection ACKNOWLEDGMENTS is common food and water sources. Fecal incontinence The authors would like to thank the healthcare staff in the may also facilitate the transmission of infection and result welfare organization of Tehran. in circulation of bacteria among institutionalized children with IDs. According to the most recent European Society for DISCLOSURE Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenter- The authors have no financial relationships relevant to ology, Hepatology, and Nutrition joint recommenda- this article to disclose. The authors declare no conflict of tions, a combination of two or more invasive tests is interest. necessary as the reference diagnostic tool for H. pylori infection in comparative studies (23). In ID children, REFERENCES neither invasive tests (endoscopy, culture, histology) nor UBT are practicable for diagnosis of H. pylori. Thus, 1. Kindermann A., Lopes A.I. (2009) Helicobacter pylori infection in pediatrics. Helicobacter 14 (Suppl 1): 52–7. this study was potentially limited by lack of the 2. Mourad‐Baars P., Hussey S., Jones N.L. (2010) Helicobacter pylori traditional gold standard tests. H. pylori IgG assay is infection and childhood. 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