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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 |
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 |
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 |
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 |
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.
View this table:
[in this window]
[in a new window]
|
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 |
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.
 |
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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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