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| Helicobacter Pylori |
Diagnostics: a 14C-urea breath
test
Therapy: less than 7 days; a systematic overview. |
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Author: Eveline van Hattum
Student number: 9628347
Supervisors:
Dr. W. de Boer Internist / Gastroenterologist, Bernhoven Hospital,
Oss.
Dr. M. Janssen / Prof. Dr. J. Jansen Stomach, Intestines and
Liver diseases UMCN. |
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| Summary |
The 14C-urea breath test
is a non-invasive method for the detection of H. pylori. In
this research this test is compared with the biopsies taken
during gastroscopy. A second group of patients is also subjected
to the 14C-urea breath test in addition to the 13C-urea
breath test, after informed consent. It appears from the results
that the 14C-urea breath test is a good non-invasive
alternative test, which can be applied easily.
Two extra biopsies were taken from the antrum during a gastroscopy,
in addition to the Golden standard. These two extra biopsies
have been used for the GUT-test a new, fast urease test. The
GUT-test is compared with the CLO-test, the most used urease
test, and separately with the culture and the histology of the
antrum.
The results from the GUT-test and the CLO-test are the same,
with a sensitivity of 100% and a specificity of 100%. In comparison
to the histology and the culture, the sensitivity and specificity
are respectively 94.1% and 96.6%. The difference is particularly
related to the costs and the fact that the GUT-test already
provides a reliable result after 60 minutes.
Presently the treatment of Helicobacter pylori consists of a
combination of medicines: a PPI with two or three antibiotics,
being prescribed for 7 days. The therapies achieve good results
in clinical trials, but in practice it appears that the success
percentage is lower. This has got to do with the compliance.
The object of this study is to determine the effect of a treatment
duration of less than 7 days on the effectiveness of therapies
for H. pylori eradication, by means of a systematic literature
review. It is shown from the results that a quadruple therapy
already has sufficient effect within 2 to 4 days. A modern triple
therapy only has a sufficient effect after 5 days. These results
show that it is possible to stop at an earlier stage. However,
this is dependent on which therapy a patient has obtained. A
quadruple and a modern PPI triple therapy does make this possible
but the old bismuth triple therapy has to be applied for at
least 7 days. |
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| Preface |
When choosing a subject for my research
training period I immediately thought of one of the subjects
regarding Helicobacter pylori. I have always been interested
in this bacterium. Previously I had given a presentation about
it. Thus I considered this training period to connect well with
my interests. I have chosen a combined training period, which
means a part consisting of patient directed research and a part
consisting of literature searching. It was my intention to look
at both aspects of research.
The practice directed part I have accomplished in the Bernhoven
hospital in Oss. I have asked patients if they were willing
to voluntarily do a breath test to show the existence of the
bacterium. Most patients are very curious as to what it is and
how it works. Therefore most of them were willing to co-operate
with the research. I have enjoyed doing the literature searching,
although it is not always easy, because it takes a lot of time.
But the results obtained make most of it very satisfying. I
have enjoyed doing both training periods and have learned a
lot from them. Furthermore I would like to express my gratitude
to my supervisors for the support and time they have given me
after the passing away of my mother. |
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| Part 1: Validation of the Heliprobe
and the GUT-test |
| Section 1: General Introduction |
Helicobacter pylori is a Gram negative
spirally bacterium that exclusively exists in the mucosa of
the stomach, particularly in the antrum. But in addition to
this it also exists in the corpus and fundus.1,2
H. pylori causes gastritis in anyone who is infected by the
bacterium. The prevalence of H. pylori in the entire population
is 30-50% in the western world; in developing countries it is
up to 90%. Eradication of H. pylori results in the cure of gastritis.3
5-20% of the persons with a H. pylori-infection get a peptic
ulcer.
The correlation the other ways around between a duodenal ulcer
and H. pylori is 95-100%. The correlation between an ventricular
ulcer and H. pylori is 70-90%.3 This infection also
gives an increased risk of a stomach carcinoma.4 The risk of
getting a stomach carcinoma concerns persons with a H pylori-associated
gastritis and is approx. 6 times higher than for persons without
H. pylori gastritis.
An infection with H. pylori can be shown in different ways.
Invasive and non-invasive tests5,6 exist. All tests
have advantages as well as disadvantages, which is why in effect
a screening has never been set up for Helicobacter pylori. The
Golden standard for the diagnostics consists of: a gastroscopy
with biopsies for a direct urease test (for example the CLO
test), a biopsy for culture and biopsies for histology7.
These biopsies are taken both from the antrum and the corpus.
Thus this is an invasive test. The non-invasive tests are a
13C-urea breath test, a 14C-urea breath
test, specific H. pylori serology or a faecal antigen test.8,9
The urea breath tests are considered presently as reference
methods for the non-invasive diagnosis of H. pylori infection
and undergoing the therapy.10,11 IgG-antibodies are
shown during serology with the aid of ELISA. The sensitivity
varies between 80 and 95%, the same applies for the specificity.
This test can only be carried out with untreated patients, because
after eradication therapy the titres are only decreasing slowly,
and a sero-conversion mostly is not forthcoming. If one would
like to do a follow up with serology then 2 sera have to be
collected, one from before the treatment and one from at least
6 months after the treatment. A decrease of titre of more than
40% proves eradication.9 The faeces antigen test
has a specificity of 90% and a sensitivity of 80-90%. H. pylori
antigens are shown in the stools with this test. This can be
both before and after eradication therapy. |
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| Section 2; Validation of Heliprobe |
In this study I have reviewed a 14C-urea
breath test, which makes use of the HeliCap®
capsule as a source for the labeled urea, and the breath samples
are analysed in the HeliprobeTM analyser. 12,13
The HeliprobeTM method is based on the fact that
the bacterium, H. pylori, produces an enzyme. This enzyme, urease,
catalyses the conversion of urea into ammonium and CO2.
H. pylori needs this ammonium in order to create an optimal
local mucous environment in the acid stomach, thus increasing
the pH. The urea can be labeled with a 13C or 14C-atom.
The Heliprobe makes use of the isotope 14C, which
has a very low amount of ß-radiation. The radioactive
amount used here is 1 µCurie. This is many thousands of
times less than the radiation load of a Stomach x-Ray or a Thoracic
x-Ray. The test can be used both for the primary diagnosis and
for the follow-up after eradication therapy, minimum 4 weeks
after the end of the therapy. Thus this 14C-urea
breath test is a new non-invasive method to prove H. pylori.
In this research we have compared this new breath test with
the Golden standard and in addition to it also with a validated,
commercial 13C-urea breath test, the HELICO STATETM
with the BreathID analyzer. We would like to know if this test
is reliable and safe and if the test would be a good alternative
in daily practice for the tests presently in use. |
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| Section 2.1: Method |
Patients, who have undergone a gastroscopy
and from whom biopsies were taken, were asked to also undergo
the 14C-urea breath test on a voluntary basis. The
indications for a gastroscopy are mostly ulcer complaints not
responding to acid inhibition within 4-8 weeks, primary recrudescent
ulcer complaints, secondary recrudescent reflux complaints and
secondary recrudescent non-specific stomach complaints within
1 year.
Following gastroscopy informed consent was obtained from most
patients and we made a new appointment for them. Before the
gastroscopy and the breath test they had stopped swallowing
a proton pump inhibitor and antibiotics for a minimum of 1 week.
Before commencement of the test the patients were asked again
if they were taken a stomach acid inhibitor and if they had
indeed stopped using it. Furthermore the patients had fasted
since the previous midnight.
The Helicap®, a small capsule containing 1µCi
labeled 14C-urea and critic acid, is ingested with
50 ml water. If an infection with H. pylori exists, then the
bacteria produce urease, which convert labeled urea. The 14CO2
is absorbed in the blood via the mucosa and transported to the
lungs where it is exhaled. By exhaling into a BreathCard®,
the quantity of 14CO2 present in the exhaled air can be measured
with the aid of the HeliprobeTM apparatus. The BreathCard®
consists of 2 pads containing a concentrated quantity of alkaline.
The pads become saturated with exhaled air, which is to stay
with a certain amount of CO2. After approx. 1 to
2 minutes the BreathCard® is saturated. This
can be seen because an indicator on the BreathCard®
changes colour from yellow to orange.
Thereafter the BreathCard® is put into the Heliprobe®
apparatus. This apparatus consists of 2 Geiger-M?ller counters
that can count the two pads at the same time. The results are
known after 4 minutes.
Heliprobe 0 means that less than 25 counts per measurement (cpm)
are observed on both pads. Thus the bacterium is not present
there.
Heliprobe 2 means that more than 50 cpm have been detected.
Thus the patient is infected with H. pylori.
Heliprobe 1 means that between 25 and 50 cpm have been detected.
This implies that it is not certain if the patient is infected
or not. This is the grey area of this test, these results are
only found with 0-2% of the examined patients.
The number of counts per measurement (cpm) is called the d-value.
This is the number of cpm of both pads together. This d-value
is not known for all patients. This is because the printer connected
to the Heliprobe apparatus was out of order for some time and
it took quite a while before a new one arrived. Also no d-values
obtained by the Canisius Wilhelmina hospital in Nijmegen are
known.
After a week the results from the Golden standard are known,
which are the results from the culture, PA and CLO-test. These
results are compared with the results from the Heliprobe. |
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| Table1: Definition of Golden
Standard |
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Histology (PA) |
Culture |
CLO-test |
| Patient H. pylori + |
+ |
+ |
+ |
| Patient H. pylori + |
+ |
+ |
- |
| Patient H. pylori + |
+ |
- |
+ |
| Patient H. pylori + |
- |
+ |
+ |
| Patient H. pylori + |
- |
- |
+ |
| Patient H. pylori + |
- |
+ |
- |
| Patient H. pylori + |
+ |
- |
- |
| Patient H. pylori - |
- |
- |
- |
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A second patient group had undergone
a regular 13C-urea breath test, the HelicoSTATETM
with the BreathID analyzer, and those people were asked to voluntarily
participate in the 14C-urea breath test as well.
This as a comparison between both breath tests. The patients
who are participating in a 13C-urea breath test are
always told that they have to fast starting from the previous
evening. They also should not have taken, for at least the previous
week, any stomach acid inhibitors or antibiotics. For the version
of the urea breath test used in Oss, patients are connected
via a flexible nose tube with the BreathId analyzer. Then the
basic measurements starts, that is to say that the baseline
is determined for the quantity of 13CO2
present in the exhaled air. Then the patient is asked to drink
water with 75 mg 13C-urea and citric acid dissolved in it. The
quantity of 13CO2 in the exhaled air is
determined again and the apparatus measures if a change has
occurred. A graph will appear on the screen showing whether
a patient has been infected with the bacterium or not. The test
takes a minimum of 20 minutes. The cut-off value of the graph
is 5 DOB. That means that all patients with a value above the
5 DOB are Helicobacter pylori positive.
If informed consent was obtained, first the 14C-urea
breath test was carried out because the quantity of urea, 1µCi
labeled 14C-urea, which is used in this test is many
times less than the 75 mg labeled 13C-urea breath
test. Conversely, there is a strong possibility that the bacteria
are already saturated with the labeled 13C-urea and
thus will not absorb 14C-urea anymore. Thus there
is a larger theoretical possibility of false-negative results. |
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| Section 2.2: Results |
| 2.2.1; Comparison of 14C-urea
breath test with the gastroscopic biopsies. |
In total 77 patients participated in
the first part of the research in which the breath test was
compared with the biopsies. Twenty two patients from the Canisius
Wilhelmina hospital in Nijmegen and 55 patients from the Bernhoven
hospital in Oss were examined.
The research sample consisted of 52 men with an age between
25 and 83 years and 25 women with an age between 54 and 89 years.
Thirteen patients used a proton pump inhibitor at the moment
they were examined and 64 patients did not use a stomach acid
inhibitor.
The d-values varied between -21 and 516 cpm. These values originated
only from the patients in Oss. In the positive group the d-values
were between 94 and 516 cpm. In the negative group they were
between -21 and 19 cpm. For one patient (1.3%), a man of 76
years of age, the result from the 14C-urea breath test was a
1, doubtful positive, and both the CLO-test and the culture
and histology were positive. Thus the patient had received treatment.
The d-value was 48, just below the cut-off value of the test.
Thus he is probably infected with the bacterium.
He uses a PPI, possibly he did not stop using it in time. It
is already known from previous research that the d-value for
patients with a proton pump inhibitor decreases. And thus the
test can yield false negative results. This patient was excluded
by us on the basis of the doubtful breath test results, and
so the definitive patient sample consisted of 76 patients. Biopsies
were taken from these patients in order to prove H. pylori,
by means of the Golden standard. The 14C-urea breath
test was positive for 21 patients, negative for 55 patients.
The number of patients having a positive Golden Standard was
in total 23. Moreover, 17 patients had both a positive CLO-test
and a positive culture and positive histology. Four patients
had a positive CLO-test and a positive histology, but the culture
was negative. For 2 patients only the CLO-test was positive.
53 patients had a negative Golden Standard, that is to say all
3 test were negative. The results from the Golden standard and
the 14C-urea breath test are listed in table 2. |
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| Table2: 14C-urea
breath test in comparison with the Golden standard |
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Golden
Standard + |
Golden
Standard - |
| 14C-urea
breath test + |
21 |
0 |
| 14C-urea
breath test - |
2 |
53 |
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| Sensitivity 91%, Specificity 100%,
Accuracy 97.3% |
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| PPI+ |
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Golden
Standard + |
Golden
Standard - |
| 14C-urea
breath test + |
1 |
0 |
| 14C-urea
breath test - |
1 |
10 |
|
| Sensitivity 50%, Specificity 100%,
Accuracy 91.7% |
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| PPI- |
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Golden
Standard + |
Golden
Standard - |
| 14C-urea
breath test + |
20 |
0 |
| 14C-urea
breath test - |
1 |
43 |
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| Sensitivity 95%, Specificity 100%,
Accuracy 98.4% |
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For 21 out of the 76 patients both the
Golden Standard and the 14C-urea breath test were
positive. The sensitivity is 91% and the specificity 100%.
If a distinction is made between patients who do use PPI and
those who don’t use PPI, then the results are as follows:
With PPI: sensitivity 50%, specificity 100%
Without PPI: sensitivity 95%, specificity 100%
For a patient, a man of 61 years of age, the results from the
Golden standard (both the culture and the histology were positive)
did not agree with the results from the 14C-urea
breath test. The d-value is not known. This patient does not
use PPI. Maybe he had not fasted, or his bacteria count is so
slow that it was false-negative in this test. For another man,
78 years of age, the breath test was also negative while the
histology and the CLO-test were positive. The d-value was 0
cpm. He uses a PPI, but stopped using it in time for the examination. |
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| 2.2.2: Comparison of the 14C-urea
breath test with the 13C-urea breath test |
In a second patient group both the
14C-urea breath test and the 13C-urea
breath test were carried out. In total 69 patients participated
it, all originating from the Bernhoven hospital in Oss.
There were 29 women with an age between 21 and 80, and 40 men
with an age between 18 and 83.
Twenty six out of the 69 patients had a positive 14C-urea
breath test and 43 a negative 14C-urea breath test.
Twenty seven patients had a positive 13C-urea breath
test and 42 a negative 13C-urea breath test.
All patients who use stomach acid inhibitors stopped them at
least 1 week before the examination. At the time of the examination
the patients did not use antibiotics.
The results are shown in table 3. |
Table 3: 14C-urea breath
test in comparison with 13C-urea breath test
Total |
| |
13C-urea
breath test + |
13C-urea
breath test - |
| 14C-urea breath
test + |
26 |
0 |
| 14C-urea breath
test - |
1 |
42 |
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| Sensitivity 96%, Specificity 100% |
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For 26 out of the 69 patients both the
14C-urea breath test and the13C-urea breath
test were positive and for 42 out of the 69 both tests were
negative. This results in a sensitivity of 96% and a specificity
of 100%.
For one patient, a man of 77 years of age, the 13C-urea breath
test was positive and the 14C-urea breath test negative.
The d-value of the 14C-urea breath test was 0 cpm
and the value of the 13C-urea breath test was 7 DOB.
The bacteria count for this patient is probably too low. This
man used a PPI, so it is possible that he did not stop using
it in time. It has been shown in previous research that the
13C-urea breath test used by us is less sensitive
for the effect of a PPI.14 For patients who use a
PPI, false negative breath tests results are often obtained.
For that reason, we advise the patient to stop using a PPI one
week before the examination. However, some researchers advise
a period of 14 days. In that way it can be explained why the
14C-urea breath test is false-negative and the 13C-urea
breath test is (true) positive. |
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| Section 2.3: Discussion |
For patients with stomach complaints
the general practitioners can choose from multiple strategies.
Recently a new NHG-Standard for Stomach Complaints has been
published in which advice is provided for the general practitioner
regarding the policy to be followed.2,15 Elderly patients, above
55 years of age are advised to undergo a endoscopy, in connection
with the risk of malignancy. Patients with typical reflux complaints
first undergo a test treatment with a PPI. For all other patients
the “test & treat” strategy is advised.16
This “test & treat” strategy, by means of a
non-invasive diagnostic test, determines if the bacterium, H.
pylori, is present. Patients with positive results can obtain
immediately an eradication therapy. Stomach ulcer disease is
excluded in patients with negative results. An endoscopy is
medically not necessary for Helicobacter-negative patients who
are younger than 55 years of age, and without alarm symptoms.
Only a small number of patients are still advised to undergo
gastroscopy after the “test & treat” protocol.17
In the directives from the CBO and NHG about stomach complaints2,
the Urea Breath Test is indicated as the non-invasive test method
with the best test characteristics. Therefore this method is
preferred to other non-invasive methods. For that reason I have
analysed further, in the Bernhoven Hospital in Oss, the value
of the Urea Breath Tests.
My results show that the 14C-urea breath test, executed with
the Heliprobe TM system, is a good and reliable non-invasive
method to show H. pylori, in comparison with the Golden standard
(endoscopic biopsies). It is a method that can easily be carried
out and moreover is not expensive. This test does not have to
be carried out in a hospital environment, but can also be carried
out in the general practitioner’s office. Here the test
can be used optimally within the framework of the “test
& treat” strategy. When comparing the 14C-urea breath
test with the previously evaluated 13C-urea breath test, in
fact the same results are obtained. Both tests are equally effective
in tracing H. pylori infection. But patients do have to stop
using acid inhibitors and antibiotics in time.
The Heliprobe TM system is less expensive than the 13C-urea
Breath Test and easier to use. It is only a small portable apparatus.
The patients do not find it inconvenient to exhale into a BreathCard,
they don’t have to blow hard. The tablets and the BreathCards
have a long shelf life. The only, particularly emotional disadvantage
is that an extremely small quantity of radioactive ß-radiation
is used. Working with the capsule does not pose a problem either,
because it concerns a very low amount of ß-radiation,
which cannot get outside the capsule because of its low penetrating
capacity.
The test is much less radioactive than an X-ray photo. But the
word radioactivity deters patients, because they are still afraid
that it can pose a danger to their health. The radiation load
is equal to drinking 3-4 glasses of orange juice; therefore
in America the test does not come under the stipulations of
the law regarding working with radioactivity.
An analyzer, which is an expensive purchase, is necessary for
the 13C-urea breath test. In this case many different tests
have also been marketed, but in Oss use is made of the BreathID
analyzer from Oridion. This test can only be applied in a cost-effective
manner if many tests per month are being carried out. In a general
practitioner’s practice that will not be feasible, and
patients will still be referred to the hospital or to the general
practitioner’s surgery.
Thus the 14C-urea breath test appears to be a good
alternative for the 13C-urea breath test. It can
be used as a reliable non-invasive Golden standard for the detection
of H. pylori. The 14C-urea breath test is a valid
possible choice for the “test & treat” strategy,
which can be applied well in a general practitioner’s
practice. This test can be carried out completely autonomously
by the general practitioner in his own practice. A disadvantage
is that the 14C-urea capsule is not yet registered in the Netherlands
and the 13C-urea capsule with 75 mg urea is. |
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