1  

Original Article

Comparison of stool antigen immunoassay and serology for the screening of

Helicobacter pylori infection in intellectually disabled children

Masoumeh Douraghi, Ph.D,1,2* Mahmoud Nateghi Rostami, Ph.D,3 Hossein Goudarzi, MD, Ph.D,2 and Zohreh Ghalavand, Ph.D2

1 Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Department of Public Health, Faculty of Health, Qom University of Medical Sciences, Qom, Iran *Address correspondence to: Masoumeh Douraghi, PhD., Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, 14155-6446, Tel: +98 2188973901, Email: mdouraghi@tums.ac.ir Running title: Hp in intellectually disabled children Abbreviations: ELISA: enzyme linked immunosorbent assay; ID: Intellectual disabilities; SAT: stool antigen test; UBT: urea breath test

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, Typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/1348-0421.12099.

This article is protected by copyright. All rights reserved.

2  Abstract

Diagnosis of active Helicobacter pylori infection in intellectually disabled (ID) children is not easily

practicable since they are not enough conscientious to cooperate for performing invasive tests. In this

study, serum IgG antibody and stool antigen tests are used as noninvasive methods for screening of H.

pylori infection in ID children.

Eighty seven children with intellectual disabilities were included. The serum levels of IgG antibody

against H. pylori were measured using ELISA method. The stool samples were examined using the

Amplified IDEIA™ HpStAR™ kit. For categorical variables, Chi-square test, Fisher’s exact test, and

Kappa test were used.

The stool antigen test revealed that 93.1% of the children had H. pylori antigen; the serology test

showed that 85.1% of children were positive for H. pylori IgG antibodies. The agreement between the

results of H. pylori stool antigen (HpSA) test and IgG antibody serology test was 82.8%, but kappa

measure of agreement does not reach the statistical significant level (Value: 0.128; P=0.19).

Discordant results were observed for 15 children (17.2%): 11 (12.6%) who were positive in HpSA test

were negative by serology, and 4 (4.6%) who were IgG seropositive yielded negative results in HpSA

test.

This study showed a notably higher rate of H. pylori infection in ID children compared to other report

in non-ID children from the same area. The HpSA test is a valid method for the primary screening of

H. pylori infection in ID children, it detects the specific antigens shedding during active infections, and

it shows less cross-reactivity than serological tests which detect antibodies. HpSA is a sensitive non-

invasive method in detection of infection in ID children which might be used as an accurate alternative

for serology.

Keywords: H. pylori IgG antibody, H. pylori stool antigen test, Intellectual disability

3  Introduction

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 transmission

risk factors are poor sanitation, overcrowding, and low socioeconomic conditions (6-8). Preventive

measures during childhood might decrease the rate of H. pylori infection and consequently the risk for

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 noninvasive tests

are recommended for diagnosis of H. pylori infection. Diagnosis of active infection is based on rapid

urease test, histology, and culture as invasive tests as well as urea breath test (UBT) and H. pylori stool

antigen test (HpSA) as noninvasive tests (9-13). The measurement of serum anti-H. pylori antibodies

like other serological tests, cannot distinguish active from past infections and may remain positive for

several years after cure (14). Several consensus reports imply on the usefulness of noninvasive tests for

diagnosis of H. pylori infection in children (12, 15). UBT is considered as the gold standard

noninvasive test, but it demands patient’s consciousness, trained technician, and optimized analytical

instruments (16). The SATs are also technically simple and cost-effective methods for diagnosis of H.

pylori infection (15).

Intellectually disabled (ID) children are vulnerable population and may endure higher rates of

infections and morbidities (17). H. pylori infection as well as gastric cancer occur at higher rates in the

population with ID than in the general population (18, 19). Most of the ID children suffer from severe

neurologic impairments and are not enough conscientious to cooperate for performing of noninvasive

test such as UBT. In addition, such children have limitations in their intellectual and adaptive

functioning and are unable to complain the symptoms. Although life-long H. pylori associated

morbidities are well known but few studies addressed the status of H. pylori infection in ID children.

4  In this study, serum IgG antibody and stool antigen tests are used as noninvasive methods for

screening of H. pylori infection in children with intellectual disabilities.

Materials and Methods

Participants and ethical consideration

In total, 87 children younger than 18 years from three centers were included. All cases were permanent

resident of long-stay centers located in urban area of Tehran. The number of randomly included cases

from each center was proportional to the total number of ID population in each center. Seventeen cases

from center I, 13 from center II, and 57 from center III were included. The centers provide several

services including medical, dietary, and physical therapy for children with mild to severe intellectual

disabilities. Demographic characteristics and medical histories were collected from medical records.

The study was approved by the local ethical committees. The informed consents were given by welfare

guardianships. In addition, informed consents were signed by the legal guardians of children.

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 from all

children aseptically under supervision of physicians. Serum samples were obtained from blood and

were stored at -20 °C until IgG ELISA was performed. Because of fecal incontinence in ID children,

caregivers collected stool specimens from children. Stool samples were frozen at −70 °C until use for

detection of H. pylori antigens.

5  Stool antigen test

Amplified IDEIA™ HpStAR™ (OXOID, UK) test is a sandwich-type enzyme immunoassay (EIA)

which was used for the detection of H. pylori antigens in stool, according to manufacturer's instruction.

Briefly, approximately 0.1 gram of stool samples was mixed with the sample diluent. Fifty microliter

of stool supernatant and 50 μl of conjugate were added to the microwells and incubated for 60 min at

room temperature. Following five wash cycles, 100 μl of substrate solution was added to each

microwell and incubated for 10 min at room temperature. The reaction was stopped by adding 100 μl

of stop solution. The results were read by EIA plate reader at 450 nm. An optical density (OD) value of

≥ 0.20 was considered positive; ODs between 0.2 and 0.5 were considered weak positive, and those >

0.5 were considered strong positive. The values < 0.20 were considered negative.

H. pylori IgG serum ELISA

The levels of IgG antibody to H. pylori were measured using enzyme-linked immunosorbent assay

(ELISA) method according to manufacturer’s instructions (IMMUNOLAB GmbH, Germany). Briefly,

the diluted sera (1:101), standards and controls were added into the wells. Anti-human IgG peroxidase

conjugate was added to each well and the reaction was developed using 3,3 ,5,5´-tetramethyl benzidine

(TMB) substrate. ODs were read at the wavelength of 450 nm using an EIA plate reader. The serum

with ODs ≥ 0.05 was considered positive. The values between 0.05 and 0.49 were considered weak

positive, and those ≥ 0.5 were considered strong positive.

Statistical analysis

The analysis was performed using SPSS version 11.5. For comparison of the categorical variables,

Chi-square test and Fisher’s exact test were used. For assessment of the consistency of two diagnostic

tests, Kappa statistic was used. Continuous variables are shown by mean and standard deviations.

6  Using HpSA as reference test, sensitivity, specificity, positive predictive value (PPV), and negative

predictive value (NPV) of serology test were calculated with 95% confidence intervals (CI). A P value

of ≤ 0.05 was considered statistically significant.

Results

Demographic characteristics of participants

Among 87 ID children, 43 (49.4%) were females and 44 (50.6%) were males with age range of 8

months to 18 years (mean ± SD; 10.89 ± 4.83). Nine (10.3%) children were younger than 4 years old,

24 (27.6%) children aged 5 to 9 years, 34 (39.1%) aged 10 to 14 years, and 20 (23%) aged 15 to 18

years. The mean duration of institutionalization was 54.14 ± 36.92 months. Demographic

characteristics of children in three centers are as following: The distribution of female to male was

11:6, 2:11, and 30:27 in center I, II, and III respectively. The mean of age was higher in center III

(years, mean ± SD; 13.01± 3.50) compared to the two other centers (center I: 3.57± 1.75; center II:

11.15± 2.96). The duration of institutionalization was also higher in center III (months, 66.60± 37.39)

compared to others (center I: 27.65± 19.54; center II: 34.15± 23.92).

The developmental brain abnormalities were diagnosed in 61 (70.1%) cases as etiology of disability,

whereas 8 (9.2%) had genetic disorders, 4 (4.6%) had injures at birth, and other etiologies such as

meningitis, autism, convulsion, seizure, hyperbilirubinemia, metabolic cause, and head trauma were

reordered less than 3% each. The etiologies of disability were unknown in 5 children (5.7%).

H. pylori stool antigen (HpSA) test

H. pylori infection was detected in 93.1% (81/87) of children (40 males and 41 females, mean age

10.87±4.77 years). The total OD values of 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 (Mean= 0.59±0.2). The

7  distribution of HpSA test values in H. pylori infected and non-infected children is illustrated in figure

1a. Among infected cases, 57 (70.4%) had strong HpSA test results and 24 cases (29.6%) showed

weak results. The mean of age of infected cases (10.87±4.77 years) was not significantly different from

that of non-infected cases (11.13±6.11 years). The infection rate was not associated with age group

(P=0.901). The proportions of infected to non-infected children in the three centers were as follow: In

center I 16 to 1 (94.1%), in center II, 13 to 0 (100%), and in center III 52 to 5 (91.2%).

Anti-H. pylori IgG antibody

Serum IgG antibody to H. pylori antigens was detected in 85.1% (74/87) of children (39 males and 35

females, mean age 11.02±4.78 years). H. pylori seropositive cases classified as weak positive (n=38;

51.4%) and strong positive (n=36; 48.6%). The total OD values of anti-H. pylori IgG ranged from 0.02

to 1.195 (Mean±SD =0.39±0.3) and OD values in H. pylori seropositive children varied from 0.05 to

1.195 (Mean±SD = 0.46±0.28). The distribution of anti-H.pylori IgG values is shown in figure 1b. The

mean of age of infected cases (11.02± 4.78 years) was not significantly different with non-infected

cases (10.13 ± 5.25 years). The seropositivity rate was not associated with age group (P=0.544). The

proportions of seropositive to seronegative children in the three centers were as follow: In center I 14

to 3 (82.4%), in center II 10 to 3 (76.9%), and in center III 50 to 7 (87.7%).

Stool antigen test vs. serology

The tests results revealed that 93.1% and 85.1% of children were positive for H. pylori infection using

stool antigen assay and serology, respectively. This difference was not statistically significant

(P=0.218). Positivity rate of H. pylori stool antigen vs. IgG antibody tests in different age groups is

shown in table 1. No significant differences were found between the concordant results in different age

groups.

8  The correlation of ODs between stool antigen and serum antibody tests in children with ID is shown in

figure 2. The discordant results between HpSA test and anti-H. pylori IgG test were found in 15 of 87

(17.2%) children. Eleven (12.6%) children who tested negative using serology, showed positive results

by HpSA test. In addition, 4 children (4.6%) who were IgG seropositive, showed negative results using

HpSA. Given HpSA as reference test, the sensitivity of serology was 86.4% whereas the specificity

was 33.33%. The positive predictive (PPV) and negative predictive (NPV) values were 94.6% and

15.4%, respectively.

Discussion

Diagnosis of H. pylori infection could be done using invasive and non-invasive tests. Children who

included in this study suffer from intellectual and developmental disabilities and various barriers may

influence the access of such children to health services and resources. Most of the invasive methods

such as endoscopy, histology, and culture are indicated as 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 children with no alarming symptoms and it is not

feasible for children with body deformities or children with cerebral palsy. Although UBT is a non-

invasive test, it is used mainly for follow-up of treatment and assessment of eradication therapy (13).

UBT requires patient’s cooperation for collecting breath and as reported previously, a few adults with

intellectual disabilities provided breath specimens (20). Due to cognitive abnormalities and

dependency of ID children, breath test collection is difficult and UBT is not a convenient 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 active infections

among children. Several characteristics of HpSA test prompted its application for children. HpSA

detects the viable or dead H. pylori antigens shedding in gastric lumen (21), is cost-effective with

9  simple sampling particularly in ID children who were incontinent of feces. To avoid variations of

polyclonal antibodies, we assessed the presence of H. pylori using a kit which includes monoclonal

antibodies specific for H. pylori antigens.

We found that the majority of children were infected with H. pylori according to HpSA results. The

infection rates were similar in all age groups and all of the centers. To our knowledge, there is no

report about the H. pylori infection rates using stool antigen test in ID children. Wallace et al. found

that 75% of ID adults living in a residential center were infected with H. pylori using stool antigen test

(20). A study conducted on non-ID children living with parents in various districts of Tehran showed

that 47% of children have a positive result in stool antigen test (22). The current study showed higher

rate of H. pylori infection than that of non-ID, non-institutionalized children in Tehran. The high rate

of infection may attribute in part to sharing subjects, utensils, and services in an overcrowded

population. Most of ID children are dependent on caregivers for feeding, toilet, hygiene, etc. Hence,

common caregivers may play a role in transmission of various infections among children. Another

possible explanation for 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 intellectual disabilities.

According to the last European Society for Pediatric Gastroenterology, Hepatology, and Nutrition

(ESPGHAN) and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition

(NASPGHAN) joint recommendations, combination of two or more invasive tests are necessary as

reference diagnostic tool of H. pylori infection in comparative studies (23). In ID children, none of

endoscopy, culture, histology, and UBT is applicable for H. pylori diagnosis and this study was

potentially limited by lack of the traditional gold standard tests. H. pylori IgG assay is classified as a

cost-effective method to assess H. pylori infection in symptomatic or high risk asymptomatic cases

(14). However, because IgG antibody titers persist up to several months after H. pylori infections,

10  positive serological tests may show the previous exposure or infection to H. pylori. In contrast, HpSA

tests detect the specific antigens which are shed during active infections and it shows less cross-

reactivity than serological tests which detect antibodies. Hence, in this study HpSA is used as a proxy

reference test for screening of H. pylori infection and the results of serology tests were compared with

results of HpSA. This study showed that IgG serology had a sensitivity of 86.4% in detection of H.

pylori antibodies. In the current study, the discrepancy between the results of HpSA and serologic tests

was not statistically significant. For four children, HpSA test showed negative and serology test

showed positive results. Given the high sensitivity of HpSA, the positive serology result may occur

due to the persistence of IgG antibodies even after clearing of the infection not active infection.

For eleven children, the serology test was negative whereas HpSA test showed positive results. A

possible reason of this difference is the incompetency of immune system response in children suffering

from a kind of intellectual disabilities. The altered immune response in such children may result 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 might be that the infection is in its

early phases and consequently there is not enough time for IgG antibody to rise. There are reports

showing the HpSA test as a reliable method for detection of Hp antigen in stool samples of children

(25-28). This study showed that the stool antigen test can be used as an accurate noninvasive tool for

screening of H. pylori infection in ID children. This procedure eliminates the need for invasive

methods and the needle stick procedure in serologic tests, hence HpSA is a good candidate method to

replace serological tests in ID children.

Acknowledgements

The authors would like to thank the healthcare staff at welfare organization of Tehran.

11  Disclosure

The authors have no financial relationships relevant to this article to disclose. The authors declare no

conflict of interest.

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References

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14   Figure legends Figure 1. The distribution of test values in H. pylori infected and non-infected children for stool

antigen (a) and serum antibody (b) tests.

Figure 2. The correlation of ODs between stool antigen and serum antibody tests in children with ID. Bricked lines show cut off values.

15  

Table 1. Positivity rate of H. pylori stool antigen vs. IgG antibody tests in different age groups

Test

Age group (year)

H. pylori stool antigen

n (%)

H. pylori IgG antibody

n (%)

0-4 8 (88.9) 7 (77.8)

5-9 23 (95.8) 20 (83.3)

10-14 32 (94.1) 29 (85.3)

15-18 18 (90) 18 (90)

16  

Figure 1

17  

Figure 2

0.0 0.5 1.0 1.50.0

0.2

0.4

0.6

0.8

1.0

H. pylori serum Ab

H. p

ylor

i st

ool A

g