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Accur ate Diagnosis of Helicobacter pylori infection by Stool Antigen Test and Six Other Cur r ently Available Tests in Children

Yen-Hsuan Ni, MD, PhD, Jaw-Town Lin*, MD, PhD, Shiu-Feng Huang#, MD, PhD,

Jyh-Chin Yang*, MD, and Mei-Hwei Chang, MD

Departments of Pediatrics, Internal Medicine*, and Pathology#

National Taiwan University Hospital,

College of Medicine, National Taiwan University,

Taipei, Taiwan

Corresponding author:

Mei-Hwei Chang, MD

Department of Pediatrics, National Taiwan University Hospital,

7, Chung-Shan South Road, Taipei, Taiwan 100

Tel: 886-2-23970800 ext 5131, 5509

Fax: 886-2-23938871

Email: mhchang@ha.mc.ntu.edu.tw

Key words: Helicobacter pylori, urease, 13

C-urea breath test, stool antigen test,

(2)

ABSTRACT

Helicobacter pylori (H. pylori) infection has been associated with gastritis,

peptic ulcer, and gastric malignancy. Invasive and noninvasive tests have been

developed for the diagnosis of H. pylori infection. Since H. pylori infection is mostly

acquired in childhood and adolescence, accurate diagnosis of the infection in the

pediatric population is important. The noninvasive diagnostic methods are particularly

feasible in children. We conducted a study to compare the invasive tests: culture,

biopsy urease test (BUT), histology, and polymerase chain reaction (PCR) on gastric

biopsy specimens, with noninvasive tests: serology, 13

C-urea breath test (13

C-UBT),

and a new diagnostic modality: stool antigen test to diagnose H. pylori infection in

children. A total of 53 symptomatic children were enrolled into this study and all had

completed the seven diagnostic tests for H. pylori. Our results showed all the

diagnostic tests except serology were excellent methods of diagnosing H. pylori

infection in children. The diagnostic accuracy of the seven tests were as follows: stool

antigen test 96.2%; BUT 96.2%; histology 98.1%; PCR 94.3%; culture 98.1%; 13

C-UBT 100%; and serology 84.9%. Stool antigen test, being highly sensitive and

specific as shown in our data, will be potentially very helpful in diagnosing H. pylori

(3)

List of Abbreviations

H. pylori Helicobacter pylori

UBT Urea breath test

PCR Polymerase chain reaction

(4)

INTRODUCTION

Helicobacter pylori (H. pylori) infection has been linked to gastritis, 1-3

duodenal

ulcer, 4,5

gastric cancer,6

and mucosa-associated lymphoid tissue lymphoma. 7

It is now

generally agreed that most H. pylori infections are acquired during childhood or

adolescence, both in developing and developed countries.8

H. pylori infection causes

similar disease patterns in children as adults except for the rarity of malignancies. H.

pylori infection was found in 90% of children with duodenal ulcers and in 25% of

children with gastric ulcers.5, 9

Acquisition of H. pylori at an early stage might

increase the risk of developing gastric cancer.10

The association of H. pylori infection

to many clinical conditions in childhood increases the demand for an accurate

diagnosis of H. pylori in childhood.

Two categories of diagnostic methods for H. pylori infection are defined:

invasive tests to detect the microorganisms in biopsies sample of the gastric mucosa

obtained through endoscopy, and noninvasive tests which obviate the need for

endoscopy. These diagnostic tests have been applied to diagnose H. pylori infection in

adults as well as in children. Deep sedation or even general anesthesia is sometimes

required for endoscopy in children, while this procedure remains valuable in pediatric

patients with symptoms suggesting peptic ulcer. Noninvasive tests, such as urea

(5)

following-up H. pylori infection in children.11

Although several studies have simultaneously compared the diagnostic accuracy

among different methods in adult patients,12-14

none have been conducted among

pediatric patients. In this study, we have analyzed the diagnostic values of seven

different tests for H. pylori infection, including culture, histology, biopsy urease test

(BUT), polymerase chain reaction (PCR), 13

C-UBT, serology, and particularly, a

newly developed stool antigen test.15

This comparison may help to validate the

diagnostic accuracy of the newly developed noninvasive stool antigen test. For those

who are unable to cooperate to inhale and exhale on command, stool antigen test will

be a good alternative for diagnosing H. pylori infection.

PATIENTS AND METHODS

Patients. From July 1, 1998 to August 30, 1999, fifty-three consecutive symptomatic children who received endoscopic examination in Department of

Pediatrics, National Taiwan University Hospital, were enrolled into this study. There

were 31 females and 22 males with a median age of 12 years, ranging from 10 to 14

years. All patients had recurrent abdominal pain and epigastralgia for at least one

month. In addition, thirteen had hunger pain, five had family history of H.

pylori-associated duodenal ulcer, and two were evaluated for coexistent anemia. A booklet

(6)

procedures in this study was distributed to the patients and their parents. Informed

consents were obtained from all of their parents. Subjects who could not tolerate

endoscopy or who had been treated with antibiotics, H2-blockers, or proton pump

inhibitors in the recent six weeks were excluded from the study. If the subjects had

ever been documented to have H. pylori infection, they would also be excluded from

the study.

Endoscopy, biopsy, and biopsy urease test (BUT). All children received intramuscular injection of meperidine (2mg/kg, maximum 50 mg), and hyoscine

butylbromide (0.5 mg/kg) before endoscopic examination. None of them was deeply

sedated or underwent the endoscopic procedure under general anesthesia. Four

biopsied specimens were taken at the antrum, 1-3 cm from the pyloric canal, and

another three biopsied specimens were taken from the gastric corpus. These

specimens were processed for BUT, culture, histologic examinations, and PCR.

CLO test (Delta West, Bently, Australia) was adopted as BUT to detect the urease

activity in biopsied specimens. The chromogenic reaction was read after one hour and

24 hours after of incubation.

Histologic examinations. The specimens were snapped to phosphate-buffered formalin, subjected to paraffin-embedding, and stained with hematoxylin and eosin.

(7)

Polymerase chain reaction (PCR). The biopsied antrum tissues were minced and the DNA was extracted using the DNA extraction kit (Qiagen, Chatsworth, CA, USA).

Each biopsied specimen was finally adjusted to a DNA concentration of 0.1μg/μl.

The nucleotide sequences of primers (first and nested) are from H. pylori urease C

genome as previously described.17

If there was no product after the first run of PCR, 1 μl of first run PCR product was subjected to the nested PCR. A DNA fragment containing the sequences of urease C gene is cloned, expressed, and serially diluted.

This was used in the sensitivity assay as a positive control.

Bacterial culture. The procedures were described previously.18

Briefly, biopsied

specimens were immediately snapped into a brain-heart infusion broth. After grinding

and transformation, they were streaked in the blood agar plate and incubated at 35-37 ℃ under microaerophilic conditions (5% O2, 10%CO2, 85% N2) for 3-7 days. Organ-isms were identified as H. pylori on the basis of colony structure, results of Gram

stain, and the production of urease, oxidase, and catalase.

13

C-Urea breath test (13C-UBT). Infrared spectrophotometer (UBiT-IR200, Otsuka Electronics Co., Hirakata, Japan) was applied in this study.19,20

It had been validated

and correlated well with mass spectrometric analysis.21

The patient fasted overnight

before the test. The procedures were done following the manufacturer’s instruction.

(8)

ingestion of powder containing 100mg 13

C-urea in 100 cc drinking water. The two

bags were inserted into the inlets of infrared spectrophotometer and the increased

percentage of 13

CO2 urea could be shown in five minutes. The cut-off point was 4.5‰ .

Serology test. HEL-p II test kit (Amrad, Boronia, Victoria, Australia) for the determination of H. pylori IgG antibody was used in this study. This is an

enzyme-linked immunoadsorbent assay which has been validated in a previous adult

adenocarcinoma study.22

Because there was no data available to document the optimal

cut off point for H. pylori IgG antibody in children, we arbitrarily set it according to

the manufacturer’s instruction. All samples were tested in duplicate.

Stool antigen test. A fresh stool sample about the size of a peanut was collected and stored at –20°C for analysis as described previously.23

H. pylori stool antigens were

detected by a commercial kit (HpSA Microwell EIA, Meridian Diagnostic Inc,

Cincinnati, OH, USA) using an enzyme-linked immunoadsorbent assay. This is an

qualitative test utilizing a polyclonal rabbit anti-H. pylori antibody adsorbed to

microwells. Diluted stool samples and a peroxidase-conjugated secondary polyclonal

antibody were added to the microwells and incubated for one hour at room

temperature. Reading of the results was based on spectrophotometric analysis.

Reading of the results was based on spectrophotometric analysis: OD450< 0.14 is

(9)

should be repeated.

Standards for determination of H. pylori infection. All the tests were read

independently without knowing the results of other tests. The final results of H. pylori

infection status was defined as “infected” when the culture was positive or

concordance of at least two of the three conventional tests (histology, BUT, 13

C-UBT).

Patients with negative results on all the above four tests (culture, histology, BUT, and 13

C-UBT) were defined as “non-infected”. Those who could not be categorized into

the above criteria were classified as “indeterminant” and excluded from the analysis.

RESULTS

Patients. A total of 53 children (31 females and 22 males) were enrolled into this study. Endoscopic diagnosis revealed active duodenal ulcer in three, duodenal ulcer

scar with fold convergence in two, antral nodularity in 19, hyperemic mucosa at

antrum and corpus in 19, and negative findings in the remaining 10 patients. The

diagnostic results for culture, histology, BUT, and 13

C-UBT are described in Table 1.

H. pylori status was diagnosed as “infected” in 27 cases, “non-infected” in the other

26 cases, and 0 in the “indeterminant” group.

Diagnostic accuracy of seven tests. The diagnostic accuracy of the seven tests were as follows: stool antigen test 96.2%; BUT 96.2%; histology 98.1%; PCR 94.3%;

culture 98.1%; 13

(10)

values for a positive or negative test, and diagnostic accuracy were calculated and are

shown in Table 2. For the stool antigen, there was no false positive and two cases

were false negative. None of stool samples were read as “equivocal” in this study.

Stool antigen test had a 92.6% sensitivity, 100% specificity, positive predictive value

of 100%, and negative predictive value of 92.9%. BUT results showed two false

negative cases and zero false positive. For the 25 cases BUT positive, 24 cases were

read reactive within one hour (range 5-30 minutes) and only one case was read

reactive 80 minutes after the biopsy. For the PCR, there was one false positive and

two false negative result. For IgG anti-H. pylori, there were five false positive cases

and three false negative cases.

DISCUSSION

To the best of our knowledge, this is the first report to simultaneously compare

seven different diagnostic modalities for H. pylori in the pediatric population. In this

study, we have demonstrated that current modalities to diagnose H. pylori infection in

adults are similarly accurate in children, except for IgG antibody to H. pylori. Both

invasive and noninvasive tests were satisfactory, with an accuracy rate about 95%.

Duodenal ulcer in children is highly associated with H. pylori infection.5

The risk

of developing gastric cancer is also relatively high if H. pylori infection is acquired at

a young age.10

(11)

infection is important. To achieve this, accurate diagnosis of H. pylori infection in

children is essential. At present, diagnosis of H. pylori infection in children still

largely depends on the endoscopic biopsy of the gastric tissues for culture and urease

test.24

These methods were regarded as the gold standard. However, the invasive

nature limits its wide use in children. Noninvasive diagnostic tests, including 13

C-UBT and serology, were recently developed and shown to be promising in

establishing the diagnosis of H. pylori infection in children.

Stool antigen test is a new noninvasive test and was reported to have a

comparable diagnostic value to any other diagnostic test in adults.23,25

However, there

have been no reports about the application of stool antigen test in children. Our data

proves that this test works well in children. It gave 100% specificity and 92.6%

sensitivity and the diagnostic accuracy is 96.2%. All the values were comparable to

other tests (Table 2). Its advantage over 13

C-UBT for children is no need to exhale air,

making it much easier for children who still cannot cooperate to inhale and exhale on

command. Further investigation of stool antigen test in infants are needed to validate

its use.

Non-dispersive infrared spectrometry was used to analyze 13

CO2 in this study.

Although it requires more exhaled gas to be collected than the conventional 13 CO2

(12)

sensitive and specific in adult patients.26

Rowland et al. reported that the sensitivity

and specificity of 13

C-UBT could be as high as 100%, and 97.6% if the subjects were

fasting.27

Our data also supported that 13

C-UBT is of good diagnostic accuracy in

school-aged children. It offers a feasible way for diagnosing H. pylori infection, and

monitoring the therapeutic effects in children who can successfully follow the

procedure of 13

C-UBT.

Most commercially available IgG antibody to H. pylori kits performed equally.28

They have some pitfalls for diagnosis and therapeutic monitoring in children.29,30

Probably due to the duration of infection and the difference in immunity and bacterial

load, the antibody levels in children differ from the adults.31

Moreover, spontaneous

clearance of H. pylori may occur in some children with persistent antibody, thus

resulting in false positive serological tests.32,33

The serological tests are not good to

detect previous infections since the titers decrease below the cut off value within

months after eradication of H. pylori infection. Our data also demonstrated this test is

of the least diagnostic value among the seven tests.

Noninvasive tests are not able to completely replace invasive tests such as

endoscopic examinations. The majority of pediatricians will prefer to eradicate H.

pylori infection according to the endoscopic and histologic examinations, rather than

(13)

severity of H. pylori infection still depends upon these invasive tests. Endoscopic

examination is especially indispensable since the current guidelines for eradication

therapy do not advocate treatment for all H. pylori-infected children.34

At present, we

suggest an endoscopic examination should be done before H. pylori eradication to

define the gastroduodenal pathology both endoscopically and histologically.

In conclusion, this study demonstrated the currently available invasive (culture,

BUT, PCR, histology) and noninvasive (UBT, and stool antigen test) diagnostic

methods for H. pylori infection are excellent in children. Serology is the least valuable

diagnostic method in children. Particularly, stool antigen test is potentially valuable in

diagnosing and following up H. pylori infection in children because of its accessibility

(14)

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7. Delchier JC, Ebert M, Malfertheiner P. Helicobacter pylori in gastric lymphoma

(15)

8. Jones NL, Sherman PM. Helicobacter pylori infection in children. Curr Opin

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13. Thijs JC, van Zwet AA, Thijs WJ, Oey HB, Karrenbeld A, Stellaard F, et al.

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(16)

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reaction. J Infect 1998;36:105-10.

18. Yang JC, Wang TH, Wang HJ, Kuo CH, Wang JT, Wang WC. Genetic analysis of the cytotoxin-associated gene and the vacuolating toxin gene in Helicobacter

pylori strains isolated from Taiwanese patients. Am J Gastroenterol

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1996;31(suppl 9): 37-40..

20. Ohara H, Suzuki T, Nakagawa T, Yoneshima M, Yamamoto M, Tsujino D, et al. 13

(17)

and for monitoring the effects of lansoprazole. J Clin Gastroenterol 1995;20 (supple

2): S115-7.

21. Ohara S, Kato M, Asaka M, Toyota T. The UBiT-100 13

CO2 infrared analyzer:

comparison between infrared spectrometric analysis and mass spectrometric

analysis. Helicobacter 1998;3:49-53.

22. Lin JT, Wang JT, Wu MS, Huang TS, How SW, Wang HP, et al. Serological,

histological, and polymerase chain reaction studies of Helicobacter pylori infection

in patients with gastric adenocarcinoma. J Formos Med Assoc 1994;93:15-9.

23. Chang MC, Wu MS, Wang HH, Wang HP, Lin JT. Helicobacter pylori stool

antigen (HpSA) test – A simple, accurate and noninvasive test for detection of

Helicobacter pylori infection. Hepato-Gastroenterology 1999;46:299-302.

24. Bourke B, Jones NL, Sherman PM. Helicobacter pylori infection and peptic

ulcer disease in children. Pediatr Infect Dis J 1996;15:1-13.

25. Vaira D, Malfertheiner P, Mégraud F, Axon ATR, Deltenre M, Hirschi AM, et al.

Diagnosis of Helicobacter pylori infection with a new non-invasive antigen-based

assay. Lancet 1999;354:30-33.

26. Koletzko S, Haissch M, Seeboth I, Braden B, Hengels K, Koletzko B.

Isotope-selective nondispersive infrared spectrometry for detection of Helicobacter pylori

infection with 13

(18)

27. Rowland M, Lambert I, Gormally S, Daly LE, Thomas JE, Hetherington C, et al.

Carbon 13-labeled urea breath test for the diagnosis of Helicobacter pylori

infection in children. J Pediatr 1997;131:815-20.

28. Glupczynski Y. Microbiological and serological diagnostic tests for Helicobacter

pylori: an overview. Br Med Bull 1998;54:175-86.

29. Khanna B, Cutler A, Israel NR, Perry M, Lastovica A, Fields P, et al. Use

Caution with serologic testing for Helicobacter pylori in children. J Infect Dis

1998;178:460-5.

30. Thomas JE, Dale A, Harding M, Coward A, Cole TJ, Weaver LT. Helicobacter

pylori colonization in early life. Pediatr Res 1999;45:218-23.

31. Casswall TH, Nilsson HO, Bergstrom M, Aleljung P, Wadstrom T, Dahlstrom

AK, et al. Evaluation of serology, 13

C-urea breath test and polymerase chain

reaction of stool samples to detect Helicobacter pylori in Bangladeshi chldren. J

Pediatr Gastroenterol Nutr 1999;28:31-6.

32. Perri F, Pastore M, Clemente R, Festa V, Quitadamo M, Niro G, et al.

Helicobacter pylori infection may undergo spontaneous eradication in children: A

2-year follow-up study. J Pediatr Gastroenterol Nutr 1998;27:181-3.

33. Kumagai T, Malaty HM, Graham DY, Hosogaya S, Misawa K, Furihata K, et al.

(19)

8-year birth cohort study. J Infect Dis 1998;178:717-21.

34. Sherman PM, Hunt RH. Why guidelines are required for the treatment of

(20)

Table 1. Concordance of diagnosis of H. pylori infection in 43 children by four diagnostic tests as gold standard*

Culture Histology BUT 13C-UBT H. pylori status No. of patients negative negative negative negative noninfected 26

positive positive positive positive infected 24 negative positive positive positive infected 1

positive negative negative positive infected 1 positive positive negative positive infected 1

gold standard: either culture is positive or concordance positive with at least two of

the other three tests (Histology, BUT, and 13

C-UBT).

BUT: biopsy urease test 13

C-UBT: 13

(21)

Table 2. Sensitivity, specificity, and diagnostic accuracy of seven diagnostic tests for H. pylori infection in children

BUT Histology PCR Culture 13C-UBT IgG SA Sensitivity 92.6% 96.3% 92.6% 96.3% 100% 88.9% 92.6% Specificity 100% 100% 96.2% 100% 100% 80.8% 100%

PPV 100% 100% 96.2% 100% 100% 82.8% 100%

NPV 92.9% 96.1% 92.6% 96.1% 100% 87.5% 92.9%

Accuracy 96.2% 98.1% 94.3% 98.1% 100% 84.9% 96.2%

PPV: positive predictive value, NPV: negative predictive value

BUT: biopsy urease test by CLO test

PCR: polymerase chain reaction 13

C-UBT: urea breath test by infrared spectrometer

IgG: serum IgG antibody to H. pylori

數據

Table 1. Concordance of diagnosis of H. pylori infection in 43 children by four
Table 2. Sensitivity, specificity, and diagnostic accuracy of seven diagnostic tests

參考文獻

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