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

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

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 adults²,³ 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

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.

Table.

Test performance calculated according to different reading techniques (at 450 nm or double reading at 450/655 nm or by direct visual reading without use of plate reader) and to different cut off values as suggested by manufacturer

Reading technique	Cut off according to manufacturer	Result				Sensitivity (%) (95% CI)	Specificity (%) (95% CI)	Accuracy (%) (95% CI)	Likelihood ratio (95% CI)		No of cases in grey zone*
		True positive	False negative	True negative	False positive
Positive	Negative
Reading at 450 nm	Negative <0.140, grey zone 0.140-0.160, positive >0.160	52	0	136	10	100 (93.2 to 100)	93 (87.8 to 96.7)	95 (90.9 to 97.6)	15 (7.6 to 30.3)	0 (0 to 0.08)	H pylori positive=0†H pylori negative=4
Reading at 450/655 nm	Negative <0.100, grey zone 0.100-0.120, positive >0.120	52	0	136	10	100 (93.2 to 100)	93 (87.8 to 96.7)	95 (90.9 to 97.6)	15 (7.6 to 30.3)	0 (0 to 0.08)	H pylori positive=0†H pylori negative=4
Direct visual reading	NA	49	3	144	2	94 (84.1 to 98.8)	99 (95.1 to 99.8)	97 (94.2 to 99.2)	69 (17.2 to 494)	0.06 (0.01 to 0.17)	NA

NA=Not applicable. 

^*According to manufacturer, these cases should be considered equivocal and second stool sample should be tested. The four H pylori negative results are included in the10 false positives. 

^†According to invasive tests (histology plus urease test or culture).