evaluation of stool antigen detection for diagnosis of helicobacter ...

EVALUATION OF STOOL ANTIGEN DETECTION FOR DIAGNOSIS OF HELICOBACTER PYLORI INFECTION IN ADULTS.

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Original articles

EVALUATION OF STOOL ANTIGEN DETECTION FOR DIAGNOSIS OF HELICOBACTER PYLORI INFECTION IN ADULTS B Aguemon1, M. Struelens1, J. Deviere2, O. Denis1, P. Golstein2, N. Nagy3 I. Salmon3 Key words: Helicobacter pylori, diagnosis, antigen detection test, ulcer disease

ABSTRACT Objective: The aims of this study were to evaluate the performance of Helicobacter pylori stool antigen test in the diagnosis of H. pylori infection. Method: The study included 63 out patients attending the ULB-Lothier Clinic between January 1 and July 31, 2002. They underwent an upper endoscopy, as well as biopsies for histological examination and for culture of H. pylori. Stool samples of these patients were collected either the day of the endoscopy or within 24 hours and tested for H. pylori antigen (HpSA Test) Results: The mean age of study patients included 29 men and 34 women was 51(± :16) years. H. pylori infection was detected in 29 cases (46 %) by culture and histology, and in 31 cases (49.2 %) by

––––––––––––––– 1 Unit of Epidemiology of Infectious Diseases, School of Public Health, Université Libre de Bruxelles; Department of Microbiology. 2 Department of Gastroenterology and Hepato-Pancreatology. 3 Department of anatomo-pathology, Erasme Hospital, Brussels – Belgium Address for correspondence : Dr B. AGUEMON Department of Infectious Diseases Epidemiology Unit, School of Public Health -ULB CP 594, 808 route de Lennik, 1070 Brussels –Belgium E-mail: [email protected]

Acta Clinica Belgica, 2004; 59-5

detection of the antigen in the feces. In 27 patients, all methods were positive whereas 5 in they provided discrepant results. Compared to the reference methods (culture and histology), the HpSA test had a sensitivity of 96.5% and a specificity of 91.2%. PPV of 90.3% and NPV of 96.8%. Conclusion: The good correlation found between the results of the HpSA test and the methods based on endoscopy supports its use as an alternative to invasive methods of diagnosis of H. pylori infection and therapeutic follow-up.

INTRODUCTION The major role of H. pylori in the etiopathogenicity of various gastroduodenal affections (B type gastritis, gastric and duodenal ulcers, gastric lymphomas) is well settled today. Infection with H. pylori occurs worldwide but the prevalence varies greatly. As many as 30 - 50 % of the adult population are infected in industrialised countries versus 80 - 90 % in developing countries. The usual methods allowing the diagnosis of the gastric infection by H. pylori are either invasive, requiring a gastric endoscopy and biopsies (fast urease test, anatomopathological examination, culture and PCR), or non-invasive (breath test with 13C or 14C marked urea, serology and stool antigen detection (1-5). Each method has its advantages and its disadvantages. Whereas histology and culture are indicated for diagnosis, stool antigen detection test is recommended in the eradication follow-up in adults and the diagnosis in young children (3). We evaluated the performance of a stool antigen detection test for the diagnosis of


H. pylori infection in adults (before treatment) compared with reference methods (culture and histology).

PATIENTS AND METHODS The prospective study was carried out from January 1 to July 31, 2002 in Lothier Clinic, in collaboration with the departments of digestive endoscopy, bacteriology and anatomopathology of the University hospital Erasme. A total of 63 out patients presenting with upper gastro- intestinal symptoms (epigastralgia, abdominal pain, pyrosis, acid regurgitation, nausea or vomiting) were included. They underwent an upper gastro-intestinal endoscopy with gastric biopsy sampling for histologic examination and for culture (two samples from the antrum and two from the corpus). The samples were collected in a formalin solution for histologic examination (one from the antrum and one from the corpus). Microscopic detection of curved bacilli of H. pylori morphology was done after purple cresyl staining and the associated digestive lesions were identified. In case of doubt about the presence of bacteria, a immunohistochemistry staining was carried out to provide confirmation (polyclonal anti-H.pylori antibody, Dako, Glostrup, Denmark). Each sample intended for bacteriological analysis was conveyed to the laboratory in a transport medium (Cultiplast). Outside working days, the samples for the bacteriology were frozen at – 70°C to ensure bacterial viability. The biopsies were crushed and homogenized in 1 ml of distilled water. Two media were inoculated: (i) - a selective medium (Pylori Agar, BioMérieux SA, Marcy l’Etoile, France) compound of an agar base, growth supplements (horse plasma) and selective antibiotics (vancomycin, trimethoprim, colistin and amphotericin B). (ii)-a non-selective medium (Columbia medium) compound of peptones and a growth supplement (horse blood). The inoculated media were incubated at 37° C in a microaerophilic atmosphere. After 3 days of incubation, plates were inspected daily during 10 days. H. pylori was identified by colony morphology, curved bacilli morphology after Gram staining and positive biochemical reaction for catalase, oxydase and urease. Antibiogram was carried out by the disk diffusion method (NeoSensitabs, Rosco, Denmark) with amoxicillin, tetracycline, metronidazole and clarithromycin. Stool specimens of these same patients were collected within 24 hours after the endoscopy. Antigen detection

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test (HpSA test, Meridian Diagnosis, Cincinnati, Ohio, USA) was performed on the fresh stool specimens either within 12 hours or on samples frozen at -20° C until testing according to manufacturer’s instructions. A little portion of stool (5 mm diameter) was diluted in 200µl of diluant and 50 µl of these dilutions were added to the wells of a microplate coated with anti-H. pylori antibodies. After emulsifying and vortexing, a peroxidase-conjugated polyclonal antibody was added. After a one hour incubation at room temperature (2227° C), the wells were washed with a buffer solution in order to remove unbound material. Substrate (urea peroxide + tetramethyl benzidin) was added and incubated for 10 minutes, a stop solution was added and absorbance measured with a spectrophotometer at a double wavelength (450/630 nm). The results were classified according to the manufacturer’s criteria as: positive > 0.120; negative < 0.100 and equivocal > 0.100 and < 0.120. Positive and negative controls were included in each test. Infected patients were defined as infected if at least one of the two standard test (histology and/or culture) were positive. Statistical analyses were carried out using EPI-INFO software version 6.04. The threshold of significance was 95% (α = 5 %). Statistical tests included: χ 2 test of Pearson or exact Fisher test for proportion comparison and Student t test for comparing the mean values.

RESULTS Between January 1 and July 31, 2002, 63 patients (29 men and 34 women; mean age 51± S.D of 16.3 years) were included in this study. Of these, 91 % did not have a past history of H. pylori infection. The previously infected patients had had no relapse for the past 6 month to 2 years. Table1 gives the distribution of the endoscopic lesions found in the patients. A positive result for H. pylori was observed in 29(46 %) patients by culture and histology and in 31(49.2 %) patients by stool antigen detection test. Among these cases, 27 (42.9 %) were positive by the three methods. Compared to the two usual methods, (culture and histology) the HpSA test sensitivity was 96.5% and a specificity was 91.2%. In the patient population, the positive predicting value (PPV) was 90.3% and the negative predicting value (PNV) was 96.8%. The HpSA antigen test with both result (culture and histology) showed 27 cases of concordant positive, 31 cases of negative correlation with cell tests and 5 cases of discrepancies including 3 Acta Clinica Belgica, 2004; 59-5


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Table 1: Distribution of endoscopic lesions in infected and non infected patients. Endoscopic lesion (N= 63)

N(%) of patient

: Number of patients detected by

Culture Hp +

Hp-

Histology Hp +

Antigen

Hp-

Hp +

Hp-

Normal mucosa(n= 4)

3(10.3)

1( 2.9)

3(10.3)

1( 2.9)

3(9.7)

1( 3.1)

Gastric ulcer(n= 5)

4(13.8)

1( 2.9)

3(10.3)

2( 5.9)

4(12.9)

1( 3.1)

Duodenal ulcer(n= 6)

5(17.2)

1( 2.9)

5(17.2)

1( 2.9)

5(16.1)

1( 3.1)

UGD*(n= 2)

0( 0.0)

2( 5.9)

1( 3.8)

1( 2.9)

0( 0.0)

2( 6.3)

Esophagitis(n=24)

4(13.8)

20(58.8)

4(13.8)

20(58.8)

5(16.1)

19(59.4)

13(44.8)

9(26.5)

13(44.8)

9(26.5)

14(45.2)

8(25.0)

Other gastropathy** (n=22)

* UGD : Gastroduodenal ulcer; ** erythematous/atrophic/nodular/Gastropathy

patients with only HPSA test positive and one patient each which only culture or histology positive.

DISCUSSION Our results confirm the good correlation between the H. pylori stool antigen test (HpSA test) and detection by culture and histology of gastric biopsies. Our results on in agreement with the observations reported by Mustafa and al.(2) in a comparative study (of the histology detection and the fast test with urea), showing a sensitivity of 97.8 % and a specificity of 94.9 %. Several studies concluded to similar rates of sensitivity of 85 to 92 % and a specificity of 82 to 98 % in children (13-14). In another pediatric study (15), the HpSA test was compared to six other diagnostic tests (UBT, culture, fast urease test on biopsy, histology, serology and PCR). The accuracy of the diagnosis obtained by the detection of the antigen in the feces was identical to the other tests (sensitivity 92.6%; specificity 100%). A large European prospective multicentric study (22) evaluated the performance of HpSA compared to the endoscopic invasive tests and concluded to a sensitivity of 93.8 % and a specificity of 96.9 %. Makristathis et al.(17) showed that HpSA test is as sensitive as PCR for detecting H. pylori in the stools and these two methods were almost as sensitive as the methods based on biopsies. Our results displayed a specificity of 91.2% for the HpSA test. It is important to emphasise that if used Acta Clinica Belgica, 2004; 59-5

in a population with a lower prevalence of the infection (e g, 20%), the PPV of the test will decrease and one positive result out of three will be a false positive. This should be kept in mind when applying this test as a screening method. The HpSA test is generally proposed for diagnosis in children, whereas in adults, it is recommended for eradication follow-up. We carried out an eradication control study comparing results by the HpSA test and the Breath test which showed a sensitivity of 100 % and a specificity of 91% (unpublished data).

Table 2 : Results of H.pylori detection tests by method Result of test Culture

No of patients

Histology Antigen

+

+

+

27

-

+

-

1

+

-

-

1

-

-

+

3

-

-

-

31

+ : Positive ; - : negative; 27 cases of positive correlation and 31 cases of negative correlation


The occurrence of antigenic cross reactions with other Helicobacter spp strains in the stomach, or the presence of non cultivable coccoïd cells of H. pylori in the stools could be responsible for false positives of HpSA test result observed in the present study. Inadequate transport conditions of the stools are probably responsible for the false negative results observed with HpSA. The good correlation between the results of the HpSA test and the methods based on endoscopy found in this study confirm previous studies. Taken together, these data plead for its broad use as an alternative to invasive methods of diagnosis of H. pylori infection and follow-up of therapy. This could be proposed to adult patients with dyspepsia who want to escape endoscopy or to patients for whom biopsy is contra-indicated such as patient treated by anticoagulants. The HpSA test is also appropriate for epidemiologic studies. Its lower cost compared to the radiolabeled urea breath test is one more argument for its use in eradication follow-up of therapy. Furthermore, its point-of care version (a five minutes immunochromatographic antigen detection test) could become a widespread used bed-side test providing that its accuracy is validated in such conditions.

RESUME UTILITE DE LA DETECTION DE L’ANTIGENE DANS LES SELLES CHEZ L’ADULTE DANS LE DIAGNOSTIC DE L’INFECTION A H. PYLORI Objectif : Le but de cette étude est d’évaluer la performance de la recherche de l’antigène d’Helicobacter pylori dans les selles, dans le diagnostic de l’infection. Méthode : 63 patients ambulatoires ont bénéficié à la polyclinique Lothier entre le 1er janvier et le 31juillet 2002 d’une endoscopie digestive haute avec biopsies (pour examen histologique et pour culture à la recherche de H. pylori). Les selles de ces patients prélevées dans les 24 heures de l’endoscopie ont été testés pour la détection de l’antigène (Test HpSA : Méridian diagnostic). Résultats : L’âge moyen des patients était de 51 ± DS : 16 ans dont 29 hommes et 34 femmes. H. pylori a été détecté chez 29(46 %) par la culture et l’histologie, et 31(49.2 %) par la détection de l’antigène fécal. Parmi ces cas, 27 étaient positifs par les trois méthodes et 5

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patients ont présenté des discordances. La sensibilité du test HpSA par rapport aux deux méthodes usuelles (culture et histologie) est de 96.5% pour une spécificité de 91.2%. La valeur prédictive positive (VPP) est de 90.3% et la valeur prédictive négative (VPN) est de 96.8%. Conclusion : La bonne corrélation entre les résultats du test HpSA et les méthodes basées sur l’endoscopie confirme l’intérêt de son utilisation en tant qu’alternative aux méthodes invasives de diagnostic et de suivi thérapeutique. Au vu de nos observations, il convient bien au diagnostic pré-traitement, aux études épidémiologiques, et d’un confort agréable pour le patient ce qui justifie son remboursement par la sécurité sociale.

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