Helicobacter pylori Stool Antigen Test - MedIND

Original Article

Helicobacter pylori Stool Antigen Test E. Mahir Gulcan,1 Aydin Varol2, Tufan Kutlu,3 Fugen Cullu3, Tulay Erkan,3 Erdal Adal,2 Onder Ulucakli4 and Sibel Erdamar5 1

SSK Bakirkoy Maternity and Children’s Training Hospital, Department of Pediatric Gastroenterology, Department of Pediatrics, 4Department of Microbiology, 3Cerrahpasa , Faculty of Medicine, Department of Pediatric Gastroenterology, 5Department of Pathology, Istanbul, Turkey. 2

Abstract. Objective : Helicobacter pylori (H.pylori) infection is usually acquired in early childhood. Invasive techniques used for diagnosis of H.pylori infection require endoscopic examination which is expensive and inconvenient and may cause complications. The aim of this study was to evaluate the performance of a new noninvasive diagnostic method, stool antigen test for H.pylori in untreated children with recurrent abdominal pain. Methods : Eighty children (35 female, 45 male) who have undergone upper gastrointestinal endoscopy due to recurrent abdominal pain were included in the study. The H.pylori stool antigen test (HpSA) is based on a sandwich enzyme immunoassay with antigen detection. HpSA sensitivity, specificity, and positive and negative predictive values were determined with reference to the results of both histology and rapid urease test as a gold standard (H. pylori status). Results : While 49 of the 80 children (61%) tested were positive for H.pylori according to the results of both histology and rapid urease test, 28 children had negative H.pylori status. Among those 49 children, 48 were found to be positive by HpSA. Of 28 patients with negative H.pylori status, 28 were H.pylori-negative also in the stool test. The sensitivity, specificity, and positive and negative predictive values of HpSA were found to be 98%, 100%, 100%, and 96.5%, respectively. Conclusion : These findings have demonstrated that HpSA as a relatively simple, inexpensive and time saving noninvasive test is a reliable method for detection of H.pylori infections in children. [Indian J Pediatr 2005; 72 (8) : 675-678] E-mail: [email protected] Key words: Recurrent abdominal pain; Helicobacter pylori, diagnosis, Helicobacter pylori stool antigen test, HpSA.

Helicobacter pylori (H.pylori) infection has been linked to gastritis, duodenal ulcer, gastric cancer, and gastric lymphomas. It is now generally agreed that H. pylori infections are acquired during childhood or adolescence in developing as well as developed countries.1 Since H.pylori infection is associated with many clinical conditions in childhood, it requires to be diagnosed during this period. There are two categories of diagnostic methods used for detection of H.pylori: invasive tests such as histology, rapid urease test and culture, and noninvasive tests such as 13C-urea breath test (UBT) and serology.2 A noninvasive and practical diagnostic tool for detection of H.pylori infection is even more desirable in pediatrics, because upper gastrointestinal endoscopies in young children are usually performed in intubation anesthesia or conscious sedation.3 Serological tests and UBT have certain disadvantages. While serological tests lack in sufficient reliability in children and adolescence, UBT is expensive, causes administrative difficulties in young children and is not available in all countries although it is as reliable as invasive methods. 4 Because many studies support the hypothesis of a fecal-oral route of infection, and because H.pylori has been detected in the stool, interest has Correspondence and Reprint requests : E.Mahir Gulcan, Cami sok. Yesil Belgrat Evleri. F Blok. No:10. Göktürk-Kemerburgaz. 34077. Eyup/Istanbul-Turkey. Fax: 00902125714790.

Indian Journal of Pediatrics, Volume 72—August, 2005

focused on the diagnostic detection of H.pylori antigens in stool samples. 3 H.pylori stool antigen test (HpSA) is an enzyme immunoassay (EIA) to detect H.pylori antigen in stool specimen.5 This test was approved in USA in 1998 for both diagnosis and monitoring the response to treatment of H.pylori infection in adult patients.4 HpSA was found to have very reliable results particularly in adult patients as a noninvasive method.2,6-8 Similar results have also been obtained in recent studies conducted in children.1,3,4,9 The aim of this study was to compare invasive methods with a noninvasive method which detects H.pylori antigen in stool specimens for the diagnosis of H.pylori infection in untreated Turkish children with recurrent abdominal pain. MATERIAL AND METHODS Eighty children admitted to the Department of Pediatric Gastroenterology due to recurrent abdominal pain and underwent upper gastrointestinal endoscopy were included in the study. The mean age of the children (mean ± SD) was 8.9 ± 3.6 yr (age range: 2 to 15 year). Thirty-five of them were girls and 45 were boys. Recurrent abdominal pain was defined as occurrence of abdominal pain limiting school or other activities at regular intervals for more than 3 months in at least 1 year. Those who 675

E. Mahir Gulcan et al received antibiotic treatment, H2-blockers or proton pump inhibitors due to any reason for six weeks before undergoing endoscopic examination were excluded from the study. The study was performed according to the Declaration of Helsinki, and all parents gave informed consent for the participation of their child in the study. Biopsy-based methods Gastroscopy was performed in all patients. During endoscopy, three biopsy specimens were collected in the antrum of the stomach of each patient: two for histology and one for rapid urease test. The rapid urease test (CLO test; Delta West, Perth, WA, Australia) was considered as positive for H.pylori if a color change from orange to pinkish was seen within 24 hours. In the histological test, the presence of H.pylori was based on the identification of curved rods in the hematoxylin and eosin, or modified Giemsa-stained sections.

infected, 28 were not infected, and 3 were of indeterminate status. In 48 of the 49 patients, H.pylori antigen could be detected in stool. On the other hand, 28 children were negative by HpSA. There was only one false-negative result, and none of the cases were falsepositive. None of the stool samples was read as equivocal in the present study. The sensitivity, specificity, and positive and negative predictive values of HpSA were found to be 98%, 100%, 100%, and 96.5%, respectively (Table 1). TABLE 1. Results of HpSA Compared with H. pylori Status of Children Infected positive (49)

Noninfected negative (28)

positive

48

0**

Helicobacter pylori status

negative

1*

28

A patient was considered as H.pylori-infected if both histology and rapid urease tests were positive, and as H.pylori-negative when both tests gave negative results. Patients who had only one positive test on rapid urease test or histology were considered to be of indeterminate status.

sensitivity (%) specificity (%) positive predictive value (%) negative predictive value (%)

HpSA

HpSA

98 100 100 96.5

(*) false-negative test result (**) false-positive test result

DISCUSSION

A fresh stool sample with approximately the size of a peanut was collected and stored at -20oC for analysis as described previously. HpSA (Premier Platinum HpSA, Meridian Diagnostics, Cincinnati, OH) was performed according to the manufacturer’s recommendations without knowledge of the H.pylori status. The test is based on a sandwich EIA with antigen detection. This is a qualitative test with a polyclonal rabbit anti-H.pylori antibody adsorbed to microwells as capture antibody. First, 100 µl of a diluted stool sample (10 µl stool in 0.5 ml sample diluent) and thereafter, peroxidase-conjugated polyclonal antibody solution were added to the wells and incubated for 1 hour at room temperature. Unbound material was removed by washing. After addition of a substrate solution, H.pylori antigen could be detected by a color change. A stop solution was added and the absorbance was read at 450 nm by a spectrophotometer. The results were interpreted as follows: OD4500.160 was positive, and the value in between was equivocal. Equivocal results should be repeated. Statistical analysis The sensitivity, specificity, and positive and negative predictive values of HpSA were calculated against the H.pylori status for the results of HpSA. RESULTS Based on the proposed H.pylori status, 49 patients were 676

H. pylori status Histology and rapid urease test

Detection of H. pylori infection in childhood today mostly depends on the endoscopic biopsy of the gastric tissue which is an invasive method performed for rapid urease test, histology, and culture.10 These methods have been recognized as gold standard. In pediatric patients, however, invasive procedures have major disadvantages such as risk of anesthesia, discomfort, and terrifying the patients and their parents. For this reason, common use of those procedures in children is limited.1,3 Therefore, a noninvasive, practical, and sensitive diagnostic test for the detection of H.pylori infection is desirable.3 The most common noninvasive tests are serology and UBT.1,4 Serology is based on the detection of specific IgG and IgA antibodies by using the ELISA method in patients infected by H. pylori.11 It is not solely used in diagnosis of the infection but particularly used for epidemiological or screening studies. 12 Serological methods are not reliable in children and fail in the diagnosis and in monitoring the success of anti-H.pylori therapy.13 Up to the present, UBT is the favorite diagnostic tool in children for the diagnosis of H.pylori infection because it avoids upper gastrointestinal endoscopy.14-16 The accuracy of noninvasive UBT in diagnosing H.pylori infection has also been evaluated in children with reference to histology and culture.17-21 UBT is based on the stable isotope technique and combines the advantages of noninvasiveness, excellent sensitivity and specificity.14-16 However, there are some disadvantages of the test Indian Journal of Pediatrics, Volume 72—August, 2005

Helicobacter pylori Stool Antigen Test in Children including its requirement of a mass spectrometer device which is very expensive and its wrong results due to lack of cooperation of young children for performing the inhalation and exhalation exercises.1,20 Besides, this test is available only in a very few centers in Turkey. HpSA is a newly developed noninvasive EIA. It is not time-consuming and is cheaper than the UBT. The analytical technique of the immunoassay in stool samples can be performed easily in any laboratory. Feces can be obtained easily, even in newborn children. Spot samples of the stool are sufficient; homogenization of the stool is not required.3 This new test utilizes a polyclonal antiH.pylori capture antibody that is adsorbed to microwells and shows good performance characteristics.5 HpSA has been found to have comparable diagnostic value for detection of H. pylori infection and for monitoring the response to treatment in adult patients.22-25 In the present study, results of HpSA were compared with H.pylori status. Positive H.pylori status and positive HpSA results were consistent in 48 patients, while negative H.pylori status and negative HpSA results were consistent in all patients. We observed one false-negative stool test result, but there was no false-positive test result. Therefore, the sensitivity, specificity, and positive and negative predictive values of HpSA in untreated children were 98%, 100%, 100%, and 96,5%, respectively. Other investigators have reported similar high sensitivities (86.9-100%) and specificities (92-100%) of HpSA in untreated children. Positive and negative predictive values have been found similarly high in these studies.1,3,9,26-30 Besides, it has been claimed that HpSA can be used for monitoring treatment success and that successful eradication of H.pylori can be confirmed after treatment.26 The accuracy of HpSA has been confirmed in children, and this may be the optimal test to confirm the success of eradication therapy.1,4,27 HpSA has been found to be a useful method for post-treatment eradication testing of H.pylori infection in children. 27 However, some investigators have reported that results of HpSA reveal an unsatisfactory sensitivity after treatment.26,31 In conclusion, HpSA as a noninvasive test, does not have the disadvantages seen in UBT and is as reliable as invasive tests. HpSA is suitable to use particularly in developing countries like ours. Detection of H.pylori antigens using HpSA shows a high sensitivity and specificity and might be useful for noninvasive diagnosis of H.pylori infection in children. HpSA may be useful particularly in selection of the cases requiring endoscopic examination, in monitoring the response to treatment and in epidemiological studies. REFERENCES 1. Ni YH, Lin JT, Huang SF, Yang JC, Chang MH. Accurate diagnosis of Helicobacter pylori infection by stool antigen test and 6 other currently available tests in children. J Pediatr 2000;


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