Papers

Detection of Helicobacter pylori in stool specimens by non-invasive antigen enzyme immunoassay in children: multicentre Italian study

Giuseppina Oderda, researcher ^a,  Anna Rapa, head ^b,  Barbara Ronchi, research fellow ^a,  P Lerro, medical assistant ^c,  Maria Pastore, medical assistant ^d,  Annamaria Staiano, researcher ^e,  G L de'Angelis, researcher ^f,  P Strisciuglio, director of paediatric department ^g. 

^a Paediatric Endoscopy Units, Università del Piemonte Orientale, 28100 Novara, Italy, ^b Paediatric Research Laboratory, Università del Piemonte Orientale, ^c Paediatric Endoscopy Units, Turin University, Piazza Polonia 94, 10126 Turin, ^d Paediatric Endoscopy Units, Viale Cappuccini, 71013 San Giovanni Rotondo, ^e Paediatric Endoscopy Units, Naples University, Via Pansani 5, 80131 Naples, ^f Paediatric Endoscopy Units, Parma University, Via A Gramsci 14, 43100 Parma, ^g Paediatric Endoscopy Units, Via T Campanella, 88100 Catanzaro

Correspondence to: G Oderda oderda{at}med.unipmn.it

Helicobacter pylori infection is mainly acquired in childhood and may predispose to peptic ulcer or gastric cancer later in life.¹ Non-invasive diagnostic tools are particularly useful in children as screening tests and for epidemiological studies, but their accuracy has to be tested against that of invasive tests in symptomatic patients before they are used in any particular population.

Of the non-invasive tests now available, serological testing is not accurate in young patients and the ¹³C urea breath test is expensive. In 1998 an enzyme linked immunoassay (ELISA) (Premier-Platinum-HpSA, Meridian Diagnostics, Cincinnati, OH, USA) was approved by the Food and Drug Administration for both diagnosis in adult symptomatic patients, and monitoring the response to treatment. It is now commercially available, but its correlation with gastric infection has not been assessed in children. We evaluated its diagnostic accuracy against invasive tests in children undergoing endoscopy for clinical evaluation, and we determined the cut off values for the paediatric population.

	Patients, methods, and results

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Stool samples from 203 consecutive symptomatic children (97 male and 106 female; median age 7 (range 0.5-15) years) undergoing endoscopy in six Italian paediatric units were collected at home by parents, frozen at -20°C, and sent to the coordinating centre (Novara). There they were tested according to the manufacturer's instructions, except that a disposable 10 µl loop was used to improve reproducibility of sampling. Plates were read at 450 nm and 450/655 nm and were evaluated by direct visual reading without use of a plate reader (yellow microwell=positive, white microwell= negative). Stool testing was performed blind, without knowledge of the other test results. The cut off for children was determined with a receiver operating characteristic curve, confidence intervals were calculated by the exact method and likelihood ratios by CATmaker program (http://cebm.jr2.ox.ac.uk/docs/nomogram.html.), and values were expressed as optical densities.

At endoscopy, biopsy specimens from the antrum and gastric body were taken for histology, microscopic identification of H pylori (Giemsa staining), and urease testing. Patients were regarded as H pylori positive if H pylori was seen at histology and results of the urease test were positive, and as H pylori negative when both tests gave negative results. When histology and the urease test gave conflicting results the results of the urea breath test or culture were used if available (urea breath test and culture were routinely performed in only two centres).

Altogether 146 children were H pylori negative and 52 H pylori positive (49 children had positive results of both the urease test and histology; in two, histology was negative but results of the urea breath test and culture were positive; and in one, the result of the urease test was negative but histology and the result of the urea breath test were positive); and five cases were considered indeterminate because histology and the result of the urease test conflicted and no other test was available.

The sensitivity of the immunoassay was high, but the specificity and accuracy decreased to 93% and 95% respectively when the cut off suggested by the manufacturer for adults ^{2 3} was used and because a grey zone was used. With direct visual reading, sensitivity slightly decreased but specificity and accuracy significantly increased (P=0.01; table). When we used the cut off calculated from a receiver operating characteristic curve (0.185 at 450 nm and 0.135 at 450/655 nm) false positive results were obtained in only two cases and the sensitivity was 98%, specificity 99%, accuracy 98%, likelihood ratio positive 72, and likelihood ratio negative 0.02.

	Comment

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The enzyme linked immunoassay that we assessed is highly accurate in diagnosing childhood H pylori infection. The use of a grey zone may lower its accuracy, but the accuracy is satisfactory even with direct visual reading of the microwells without use of a plate reader; this makes it fairly cheap as a screening test (one determination cost is 22 euro (£14), half the average price of the urea breath test) and practical for epidemiological studies.

	Acknowledgments

This study was approved by and conducted within the guidelines of the gastric disease section of the Italian Society for Paediatric Gastroenterology and Hepatology.

Contributors: GO designed and coordinated the study and wrote the article. AR and BR collected the data from each centre, analysed the faecal samples and did the statistical analysis. PL, MP, AS, GLde'A, and PS all participated in the discussion about the design of the study and approved the study proposal and the final draft; they also recruited all cases and collected data from patients from each centre, did endoscopy in children, and collected faecal samples to be sent to the coordinator centre. GO will act as guarantor for the paper.

	Footnotes

Funding: Meridian Diagnostic Europe partially sponsored the study and provided free kits.

Competing interests: GO and AR have been reimbursed by Meridian Diagnostics, Europe (the manufacturer of HpSA) for attending a symposium on H pylori and gastroduodenal disease in Helsinki.

	References

Top Patients, methods, and results Comment References

1.	Blaser MJ, Chyou PH, Nomura A. Age at establishment of Helicobacter pylori infection and gastric carcinoma, gastric ulcer, and duodenal ulcer risk. Cancer Res 1995; 5: 562-565.
2.	Makristathis A, Pasching E, Schutze K, Wimmer M, Rotter ML, Hirschl AM. Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. J Clin Microbiol 1998; 36: 2772-2774[Abstract/Free Full Text].
3.	Vaira D, Malfertheiner P, Megraud F, Axon ATR, Deltenre M, Hirschl AM, et al, and European Helicobacter pylori HpSA Study Group. Diagnosis of Helicobacter pylori infection using a novel, non-invasive antigen based assay in a European multicentre study. Lancet 1999; 354: 30-33[CrossRef][Medline].

(Accepted 7 October 1999)