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Journal of Clinical Microbiology, October 2000, p. 3710-3714, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Two Enzyme Immunoassays and PCR for Detection of Helicobacter pylori in Stool Specimens from Pediatric Patients before and after Eradication Therapy

Athanasios Makristathis,1,* Wolfgang Barousch,1 Eva Pasching,1 Christa Binder,2 Christa Kuderna,3 Petra Apfalter,1 Manfred L. Rotter,1 and Alexander M. Hirschl1

Department of Clinical Microbiology, Hygiene Institute of the University of Vienna,1 St. Anna Pediatric Hospital,2 and Department of Pediatric Infectious Diseases, Wilhelminen Hospital,3 Vienna, Austria

Received 21 April 2000/Returned for modification 2 June 2000/Accepted 10 August 2000


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

This study of pediatric patients was intended to determine the suitability of stool PCR and two antigen enzyme immunoassays (EIAs; Premier Platinum HpSA and the novel FemtoLab H. pylori), which detect Helicobacter pylori antigens in feces, as pretreatment diagnostic tools and especially as posttreatment control. Forty-nine H. pylori-infected children with dyspepsia received eradication therapy. Successful treatment was determined by a negative [13C]urea breath test 4 and 12 weeks after discontinuation of therapy. Fecal specimens were collected prior to eradication therapy as well as 4 weeks after the end of treatment. Successfully treated children delivered stool samples at 6, 8, and 12 weeks posttreatment also. Specimens were examined by seminested PCR and Premier Platinum HpSA and were reexamined by both EIAs as soon as FemtoLab H. pylori was available. In the first test series, the overall sensitivities of PCR and Premier Platinum HpSA were 93.0 and 91.1%, respectively. With specimens collected at 4 weeks after treatment, the respective specificities were 68.8 and 79.3%. After longer follow-up periods, however, they gradually increased to 100 and 96.9%, respectively. In the new test series, Premier Platinum HpSA delivered a considerably lower number of false-positive results (4 versus 18), indicating intertest variations. The overall test sensitivity was 94.6%, and the overall specificity was 97.5%. FemtoLab H. pylori showed an excellent performance with an overall sensitivity and specificity of 98.2 and 98.1%, respectively. Thus, in contrast to PCR, both EIAs were shown to be suitable for early posttreatment control.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Helicobacter pylori causes gastritis and peptic ulcer disease and has been associated with gastric malignancies (2, 16, 21). During recent years, noninvasive diagnostic tests have gained in significance. This is of particular importance for children, especially in posttreatment control, which---according to the Canadian and European consensus conference reports (7, 17)---should preferentially be performed by noninvasive tests.

Well-established noninvasive tests are serology and the [13C]urea breath test ([13C]UBT). Serology, however, is not suitable for an early follow-up examination due to the slow reduction of the anti-H. pylori antibody titer after successful treatment (5). The [13C]UBT---delivering accurate results both in the pretreatment examination of infected individuals and in the early posttreatment control---fulfills the demands for such a test (6). However, expensive instrumentation and a specialized technician are required. In addition, the performance of the test has been associated with some disadvantages with infants and very young children as well as patients with certain neurological disorders. Since infected individuals excrete H. pylori in stool specimens (9, 11, 13, 18), a sufficiently accurate test using feces would be an important alternative to [13C]UBT.

In previous studies, fecal detection of H. pylori DNA by PCR or of H. pylori antigen by a commercially available antigen enzyme immunoassay (EIA; Premier Platinum HpSA; Meridian Diagnostics, Inc., Cincinnati, Ohio) delivered accurate results, suggesting the usefulness of these methods as pretreatment diagnostic tools (8, 10, 20). However, follow-up examination of stool specimens revealed a high percentage of false-positive results by PCR, and reports of the suitability of Premier Platinum HpSA in follow-up tests were controversial (10, 19, 20).

Recently, a novel antigen EIA (FemtoLab H. pylori; Connex, Martinsried, Germany) using monoclonal antibodies directed against H. pylori antigens was developed. At present, no published data are available with respect to the performance of this new test. This study was intended to evaluate the usefulness of FemtoLab H. pylori in the pretreatment diagnosis of H. pylori infection in pediatric patients. Moreover, it was of particular interest to determine in a long-term follow-up whether the two antigen EIAs and PCR are appropriate for posttreatment examination of stool specimens.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Forty-nine H. pylori-infected children with recurrent abdominal pain were recruited for this study in two pediatric gastroenterology centers. The patients were aged between 3 and 15 years (mean age, 11.1 years), and none of them had received antibiotic or proton pump inhibitor therapy during the last 2 months. A positive H. pylori status was assumed if both [13C]UBT and serology were positive, which was the case for all 49 patients. All children received a 7-day regimen of amoxicillin combined with clarithromycin and omeprazole. Eradication control was performed by [13C]UBT 4 weeks after therapy was discontinued, and if yielding a negative test result, [13C]UBT was repeated 12 weeks after the end of treatment. Fecal specimens were collected prior to eradication therapy and 4 weeks after the end of treatment. Patients with a negative [13C]UBT result at this time delivered additional stool specimens 6, 8, and 12 weeks after discontinuation of therapy. The specimens were stored at -70°C. In a first test series, the specimens were examined by PCR and Premier Platinum HpSA. As soon as the new FemtoLab H. pylori test was available, all specimens were reexamined by the two antigen EIAs (second test series).

[13C]UBT. The test was performed after an overnight fast. Breath samples were collected in duplicate before and 30 min after ingestion of 200 ml of orange juice and 75 mg of [13C]urea dissolved in 30 ml of tap water. Breath samples were analyzed by a mass spectrometer (Breath Mat; Finnigan, Bremen, Germany). A delta-over-baseline value of above 3.5 per mil was considered to be a positive result (4).

Serology. Helori-test IgG (Eurospital SpA, Trieste, Italy) was used for the quantitative determination of specific anti-H. pylori immunoglobulin G antibodies. This fluorescence EIA was performed according to the manufacturer's instructions.

Stool specimen PCR. DNA extraction and purification as well as target DNA amplification by seminested PCR were performed as described elsewhere (10).

Premier Platinum HpSA. This commercially available antigen EIA using polyclonal antibodies to H. pylori was performed as indicated by the manufacturer, and the results were read by spectrophotometry. Specimens with absorbance values (A450/630) of >= 0.120 were positive, those with values of >= 0.100 and <0.120 were equivocal, and those with values of <0.100 were negative.

FemtoLab H. pylori. This novel antigen EIA was provided by Connex for research use only. Microwells are coated with monoclonal antibodies to H. pylori antigens. A stool suspension with sample diluent was centrifuged for 5 min at a minimum of 7,000 × g. A small aliquot of the supernatant and peroxidase-conjugated monoclonal antibodies were pipetted into the wells and incubated for 1 h on a shaker. After four washes followed by a 10-min incubation with the substrate, the reaction was stopped and the results were read by spectrophotometry. According to the manufacturer's instructions, specimens with absorbance values (A450/630) of >= 0.180 were positive, those with values of >0.130 and <0.180 were equivocal, and those with values of <= 0.130 were negative.

Statistical analysis of data. Sensitivity, specificity, and positive and negative predictive values (PPVs and NPVs, respectively) were calculated according to standard methods. The coefficient of correlation between the two EIAs was calculated by the Spearman rank correlation test. Statistical significance was set at P = 0.01, and testing was two-sided.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

After eradication therapy, 9 of the 49 patients either refused to undergo follow-up investigations or discontinued the study protocol. Four weeks after the end of therapy, 32 (80%) of the remaining 40 patients were negative by [13C]UBT, suggesting that they had been treated successfully. The eight patients still positive underwent no further monitoring during follow-up. Twelve weeks after the end of treatment, all of the 32 patients remaining in follow-up delivered a negative [13C]UBT result, which was a further confirmation of their negative H. pylori status.

First test series. Stool samples were examined by PCR and Premier Platinum HpSA. Forty-five of the 49 fecal specimens collected before treatment were positive by PCR (91.8%). By Premier Platinum HpSA, 43 specimens yielded positive results. A further specimen delivered an equivocal result; it was not considered for the calculation of the sensitivity value, which was 89.6%.

Stool samples obtained 4 weeks after the end of therapy from the eight children having a positive [13C]UBT were all positive by both tests. For the fecal specimens from the 32 successfully treated patients collected at that time as well as 6, 8, and 12 weeks after the end of therapy, PCR still delivered positive results in 10, 8, 4, and 0 specimens, respectively (Table 1). The corresponding test specificities were 68.8, 75.0, 87.5, and 100%. Premier Platinum HpSA still delivered positive results in six, four, one, and two of the specimens (Table 1), suggesting specificities of 79.3, 86.7, 96.9, and 93.8%, respectively. For three specimens collected 4 weeks and two collected 6 weeks after the end of treatment, Premier Platinum HpSA delivered equivocal results (Table 1), which were not taken into account for the calculation of the respective specificity values. Upon closer evaluation, the data in Table 1 suggest persistence of H. pylori DNA and/or antigen for certain time periods after successful eradication therapy in all cases of false-positive results with PCR and in most of the cases of false-positive or equivocal results with the EIA.

                              
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TABLE 1.   False-positive and equivocal results obtained by PCR and Premier Platinum HpSA in the first test series with specimens collected after H. pylori eradication therapy

Second test series. All stool specimens were reexamined by both Premier Platinum HpSA and the novel EIA FemtoLab H. pylori as soon as the latter was available. Stool specimens from an additional 30 [13C]UBT- and serology-negative individuals were also examined, mainly in order to evaluate the pretreatment specificity of the new test.

Of the original 49 stool specimens obtained from infected patients before treatment, 48 were positive by FemtoLab H. pylori (Fig. 1). The reexamination of the specimens by Premier Platinum HpSA gave three false-negative results (Fig. 1). Another specimen yielded an equivocal result and was not considered for statistical evaluations. Of the 30 new specimens from noninfected individuals, only one was positive by both tests.


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FIG. 1.   Results of [13C]UBT and FemtoLab H. pylori and second test series results of Premier Platinum HpSA with specimens collected from pediatric patients before and 4 weeks after discontinuation of therapy. Before treatment, a positive H. pylori status was assumed if both [13C]UBT and serology were positive. A posttreatment negative H. pylori status was assumed if [13C]UBT was negative at both 4 and 12 weeks after the end of treatment. Positive and negative patient H. pylori status is indicated by solid and open dots, respectively.

Of the 32 specimens obtained from successfully treated patients 4 weeks after the end of therapy, FemtoLab H. pylori delivered one false-positive result while Premier Platinum HpSA yielded two false-positive results and another one that was equivocal (Fig. 1). The false-positive result of FemtoLab H. pylori was obtained for a specimen also false positive in the Premier Platinum HpSA test in both test series (patient 7, Table 1). All stool specimens from the eight children still infected after treatment were positive by both tests (Fig. 1).

Stool specimens obtained 6 and 8 weeks after the end of treatment gave no false-positive results with FemtoLab H. pylori. However, one equivocal and one false-positive result were found in specimens obtained 6 and 12 weeks after treatment, respectively. With Premier Platinum HpSA, one of the specimens obtained 6 weeks after treatment was false positive. All specimens obtained 8 and 12 weeks after treatment were negative by this test. Values for sensitivities and specificities as well as PPVs and NPVs for both tests before and after eradication therapy are shown in Table 2.

                              
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TABLE 2.   Performance of Premier Platinum HpSA and FemtoLab H. pylori in the second test series with specimens collected before and after eradication therapya

The comparison of all values delivered by the two EIAs revealed a coefficient of correlation of 0.749 (P < 0.01). This correlation could have been higher if a considerable number of positive specimens had not been above the measuring range with FemtoLab H. pylori (Fig. 1).


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

During recent years, great efforts were made to develop accurate noninvasive diagnostic tests for both pre- and posttreatment examination of H. pylori-infected individuals. In our previous study (10), a sensitive and highly specific DNA extraction and amplification protocol by seminested PCR for H. pylori DNA detection in feces was developed. In the first test series of the present study, using stool PCR and Premier Platinum HpSA, the examination of specimens from successfully treated pediatric patients collected 4 weeks after the end of treatment revealed a large number of false-positive results with both tests. This was in accordance with the findings of our previous report (10). Furthermore, during recent years, the lower posttreatment specificity of Premier Platinum HpSA has been demonstrated in a number of publications (15, 19) and abstracts (M. Forné, J. Dominguez, M. Esteve, S. Quintana, F. Fernández, J. C. Espinós, N. Gali, V. Ausina, and J. M. Viver, Abstr. XIth Int. Workshop Gastroduodenal Pathol. Helicobacter pylori, abstr. 06/168, Gut 43[Suppl. 2]:A50, 1998).

However, the number of false-positive results with both PCR and Premier Platinum HpSA gradually diminished with specimens collected after longer follow-up periods (Table 1). Specimens collected from successfully treated patients 12 weeks after discontinuation of therapy were all negative with the exception of two, which were false positive with Premier Platinum HpSA, exhibiting values barely above the cutoff (patient 3, 12 weeks, 0.129, and patient 10, 0.133 [Table 1]). Moreover, even if the results were false positive, there was a considerable reduction of the Premier Platinum HpSA values with posttreatment specimens from successfully treated children (data not shown).

Thus, the findings of the first test series indicate that, in some cases and under unknown circumstances, H. pylori DNA and at least some antigens persist for longer periods so that they can be detected by PCR and Premier Platinum HpSA, which utilizes polyclonal anti-H. pylori antibodies. A recent report suggested that the persistence of H. pylori coccoid forms may be responsible for posttreatment discrepancies between Premier Platinum HpSA and [13C]UBT results (G. Masoero, L. Lombardo, P. Della Monica, L. Andrini, S. Vicari, F. Sallio, and A. Pera, Abstr. XIIth Int. Workshop Gastroduodenal Pathol. Helicobacter pylori, abstr. 15/33, Gut 45[Suppl. 3]:A131, 1999). This could also be an explanation for the observed discrepancies between the EIA and invasive tests used as reference methods to assess treatment efficacy (10, 19).

In the second---and more recent---test series, the number of false-positive or equivocal results delivered by Premier Platinum HpSA with specimens obtained after the end of treatment was considerably lower than that of the first test series (4 versus 18). Moreover, the improvement of specificity was not accompanied by a loss of sensitivity. Thus, the second test series showed an overall sensitivity of 94.6% and an overall specificity of 97.5%. The comparison of the results obtained with Premier Platinum HpSA in both test series, using the same specimens, indicates intertest variations. In this context, differences between batches cannot be excluded, which could be at least partly an explanation for the controversial reports in the literature (10, 19, 20). However, the results of this study together with the latest reports in this field (3, 12) suggest that Premier Platinum HpSA has been improved and now delivers sufficiently accurate results in early posttreatment control as well.

Considering the results delivered by Premier Platinum HpSA in the second test series, an adequate cutoff value was evaluated on the basis of a receiver-operator characteristic curve. This analysis revealed 0.110 as the appropriate cutoff value. This value is in accordance with that suggested by the manufacturer but considerably lower than that of 0.300 reported by Ohkura and colleagues (15). However, if the values delivered by the antigen EIA correspond to the bacterial density in the gastric mucosa, this would explain the higher values reported in this Japanese study, since infection with cagA+ strains, which account for >90% of H. pylori isolates in Japan, is characterized by higher densities of the pathogen (1, 22). Furthermore, the calculated cutoff value in the present study is also lower than that of 0.135 proposed recently for an Italian pediatric population (14). Thus, a local test validation seems to be of importance.

In the present study, the novel antigen EIA FemtoLab H. pylori was evaluated for the first time in a clinical trial and showed excellent performance both as a diagnostic tool and as an early follow-up method after eradication therapy. With only a few exceptions, positive samples gave values far above and negative samples gave values clearly below the cutoff value (Fig. 1). Therefore, monoclonal antibodies utilized by this test may recognize water-soluble H. pylori proteins with a high turnover. The overall sensitivity of FemtoLab H. pylori was 98.2%. The overall test specificity was 98.1%, thus being similar to that obtained with samples from noninfected individuals (96.7%) or to that shown with specimens collected as early as 4 weeks after the end of therapy (96.9%). For this study, a cutoff value of between 0.300 and 0.500 would increase the specificity of the test to 100% without affecting sensitivity.

Thus, for our pediatric study population both Premier Platinum HpSA and, especially, the novel FemtoLab H. pylori delivered sufficiently accurate results and showed themselves to be a suitable alternative to [13C]UBT, which is of particular importance for the early posttreatment control. In contrast, PCR delivering accurate results only at 12 weeks after discontinuation of therapy did not qualify for early monitoring of treatment efficacy.


    ACKNOWLEDGMENTS

We thank the European Helicobacter pylori Study Group (EHPSG) for supporting this study.


    FOOTNOTES

* Corresponding author. Mailing address: Department of Clinical Microbiology, University Hospital of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria. Phone: 43 (1) 40400-5157. Fax: 43 (1) 40400-5162. E-mail: Athanasios.Makristathis{at}akh-wien.ac.at.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Clinical Microbiology, October 2000, p. 3710-3714, Vol. 38, No. 10
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.



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