Helicobacter ISSN 1523-5378

doi: 10.1111/hel.12189
13
The Diagnostic Validity of Citric Acid-Free, High Dose C-Urea
Breath Test After Helicobacter pylori Eradication in Korea
Yong Hwan Kwon,* Nayoung Kim,*,† Ju Yup Lee,* Yoon Jin Choi,* Kichul Yoon,* Jae Jin Hwang,* Hyun
Joo Lee,* AeRa Lee,* Yeon Sang Jeong,* Sooyeon Oh,† Hyuk Yoon,* Cheol Min Shin,* Young Soo Park*
and Dong Ho Lee*,†
*Internal Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea, †Internal Medicine and Liver Research Institute, Seoul
National University College of Medicine, Seoul, South Korea

Keywords
Helicobacter pylori, 13C-urea breath test,
eradication, cutoff value, citric acid.
Reprint requests to: Nayoung Kim, Department
of Internal Medicine, Seoul National University
Bundang Hospital, 82, Gumi-ro 173 beon-gil,
Bundang-gu, Seongnam, Gyeonggi-do, 463-707,
South Korea. E-mail: nayoungkim49@empal.com
Telephone: + 82-31-787-7008
Fax: + 82-31-787-4051
Abstract
Background: The 13C-urea breath test (13C-UBT) is a noninvasive method
for diagnosing Helicobacter pylori (H. pylori) infection. The aims of this study
were to evaluate the diagnostic validity of the 13C-UBT cutoff value and to
identify influencing clinical factors responsible for aberrant results.
Methods: 13C-UBT (UBiTkit; Otsuka Pharmaceutical, cutoff value: 2.5&)
results in the range 2.0& to 10.0& after H. pylori eradication therapy were
compared with the results of endoscopic biopsy results of the antrum and
body. Factors considered to affect test results adversely were analyzed.
Results: Among patients with a positive 13C-UBT result (2.5& to 10.0&,
n = 223) or a negative 13C-UBT result (2.0& to < 2.5&, n = 66) after
H. pylori eradication, 73 patients (34.0%) were false positive, and one
(1.5%) was false negative as determined by endoscopic biopsy. The sensitiv-
ity, specificity, false-positive rate, and false-negative rate for a cutoff value of
2.5& were 99.3%, 47.1%, 52.9%, and 0.7%, respectively, and positive and
negative predictive values of the 13C-UBT were 67.3% and 98.5%, respec-
tively. Multivariate analysis showed that a history of two or more previous
H. pylori eradication therapies (OR = 2.455, 95%CI = 1.299–4.641) and
moderate to severe gastric intestinal metaplasia (OR = 3.359, 95%
CI = 1.572–7.178) were associated with a false-positive 13C-UBT result.
Conclusion: The 13C-UBT cutoff value currently used has poor specificity for
confirming H. pylori status after eradication, and this lack of specificity is exac-
erbated in patients that have undergone multiple prior eradication therapies
and in patients with moderate to severe gastric intestinal metaplasia. In addi-
tion, the citric-free 13C-UBT would increase a false-positive 13C-UBT result.

Helicobacter pylori (H. pylori) infection is the most impor-
tant cause of chronic atrophic gastritis, peptic ulcer dis-
ease, gastric cancer, and gastric lymphoma [1,2]. The
eradication of H. pylori infection reduces the incidence
of peptic ulcers, the risk of gastric cancer development,
and prevents recurrences of these diseases [3,4]. It was
proposed at the Kyoto Consensus Meeting on H. pylori
gastritis (held from January 30th to February 1st, 2014)
that “all H. pylori positive patients should be offered
H. pylori eradication” [5]. Several invasive and nonin-
vasive diagnostic methods have been developed to
detect H. pylori infections. Endoscopic biopsy is the gold
standard, but it is invasive and is prone to sampling
error because H. pylori tends to be heterogeneously
distributed in the stomach [6]. Serologic examinations
are noninvasive and convenient, but do not accurately
reflect infection status [7,8].
The urea breath test (UBT) using 13C-labeled urea is
a noninvasive test based on the potent urease activity of
H. pylori in gastric mucosa and was developed to over-
come the shortcomings of serologic testing. This test has
been widely used because it has been reported to have a
sensitivity and specificity greater than 90% for detecting
H. pylori infection, and to be more convenient to use
and safer for patients [9,10]. For these reasons, the
13
C-UBT is now routinely used to diagnosis of H. pylori
infections [11,12]. In Korea and Japan, it is generally
held that the optimum cutoff value of the 13C-UBT per-

© 2015 John Wiley & Sons Ltd, Helicobacter 1

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The Diagnostic Validity of the C-UBT
formed using the UBiTkit (Otsuka Pharmaceutical; d13C
values measured at 20 minutes after the administration
of 100 mg of urea) for the detection of H. pylori infec-
tion before and after eradication is 2.5& [13]. However,
many authors have suggested that 13C-UBT results
between 2.0& and 5.0& fall into a “gray zone”, because
in this region various factors can affect 13C-UBT results
[14,15]. Calvet X et al. [16] reported a high rate of
false-positive results for the UBiTkit in healthy Spanish
volunteers. In previous study, false-positive results had
delta values ranging from 8.5& to 12&. Somewhat sur-
prisingly, the optimal cutoff value of the 13C-UBT after
H. pylori eradication therapy has not been established,
and few have compared 13C-UBT and endoscopic biopsy
results [10,17].
Korea has one of the highest incidences of gastric
cancer [18]. A National Cancer Screening Program
(NCSP) offers barium meal studies (UGI series) or an
endoscopic examination for the initial screening of gas-
tric cancer. Furthermore, because the cost of an upper
endoscopic examination is relatively lower than that of
the 13C-UBT, H. pylori infection is usually diagnosed by
endoscopic biopsy (histology or rapid urease test), and
the 13C-UBT is usually used to confirm H. pylori status
after eradication. Given this background, the aims of
this study were to evaluate the diagnostic accuracy of
the 13C-UBT in the 2.0& to 10.0& range after H. pylori
eradication by comparing its results with that of endo-
scopic biopsy and to identify clinical factors that
adversely affect 13C-UBT results.
Methods
Patient Selection
Between April 2005 and March 2013, 14,972 patients
underwent the 13C-UBT in Seoul National University
Bundang hospital. We retrospectively screened 1,891
patients with a 13C-UBT result between 2.0& and
10.0& after H. pylori eradication and that subse-
quently underwent follow up endoscopic surveillance
to confirm final H. pylori status. Three endoscopic
biopsy methods [histology (the modified Giemsa test),
the rapid urease test (CLOtest, Delta West, Bentley,
Australia), and culture in antrum and body] were
used, and the final H. pylori infection was determined
by revealing H. pylori infection at least any two tests
of three. Endoscopic surveillances were performed at
least on two occasions during follow-up after
13
C-UBT without any further H. pylori treatment. The
exclusion criteria applied were as follows: 1) no
follow-up history of endoscopic surveillance after
13
C-UBT; 2) an age below 18 years; 3) the adminis-
2 © 2015 John Wiley & Sons Ltd, Helicobacter
Kwon et al.
tration of antibiotics or the consumption of bismuth
salts within 4 weeks or the administration of a
proton-pump inhibitor (PPI) within 2 weeks prior to
13
C-UBT; 4) H. pylori eradication failure because of
poor compliance; 5) H. pylori re-infection, where
H. pylori status became positive after more 1 year
after successful eradication [19]; and 6) a previous
history of gastrectomy. The study protocol was
approved by the Ethics Committee at Seoul National
University Bundang Hospital (SNUBH B-1406).
H. pylori Eradication
For the treatment of H. pylori infection, PPI-based tri-
ple therapy (standard dose of PPI b.i.d., clarithromycin
500 mg b.i.d., and amoxicillin 1 g b.i.d. for 1 week)
[20], and sequential therapy (initial 5-day therapy
with a combination of PPI b.i.d and amoxicillin 1 g
b.i.d, followed by 5 days of PPI b.i.d., clarithromycin
500 mg b.i.d., and metronidazole 500 mg t.i.d) [21]
were used as a first-line therapy in all study subjects.
When these first-line therapies failed, two types of
rescue therapies were used, that is, bismuth-contain-
ing quadruple therapy [PPI b.i.d., tripotassium dicitrate
bismuthate 300 mg q.i.d. (three tablets 30 min before
meals and one tablet 2 hours after dinner), metroni-
dazole 500 mg t.i.d., and tetracycline 500 mg q.i.d.]
for 1–2 weeks [22], or moxifloxacin-containing triple
therapy (moxifloxacin 400 mg q.d., amoxicillin 1 g
b.i.d., and PPI b.i.d.) for 1–2 weeks [23]. When sec-
ond-line therapy failed then the other rescue therapy
was used.
13
C-Urea Breath Test
Before the 13C-UBT, patients were instructed to stop
taking medications (such as, bismuth salts or antibiot-
ics for 4 weeks, and PPI for 2 weeks), and fasted for
a minimum of 4 hours. After washing the oral cavity
by gargling, a predose breath sample was obtained.
Then, 100 mg tablet of 13C-urea (UBiTkitTM; Otsuka
Pharmaceutical Co. Ltd., Tokyo, Japan) was adminis-
tered free of citric acid. Breath samples were collected
in the sitting position using special breath collection
bags before 13C-urea administration (baseline) and
20 minutes after administration. Collected breath sam-
ples were analyzed using an isotope-selective, nondis-
persive infrared spectrometer (UBiT-IR 300â; Otsuka
Pharmaceutical Co. Ltd, Tokyo, Japan). Despite the
lack of validation, a 13C-UBT cutoff value 2.5& was
used as recommended by the manufacturer, and thus,
a delta 13CO2 of ≥2.5& was considered positive for
H. pylori infection.
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Kwon et al. The Diagnostic Validity of the C-UBT

Endoscopic Surveillance for the Detection of

H. pylori Infection
Results

Subject Characteristics
Histologic evaluation
Of the 1,891 patients with a 13C-UBT result in the
Two biopsy specimens were obtained from the antrum
2.0& to 10.0& range, 289 (20.9% to 15.3%) fulfilled
and body, respectively [24]. The presence of H. pylori
our inclusion and exclusion criteria. Sixty-six patients
was assessed by modified Giemsa staining, and degrees
(22.8%) had a 13C-UBT result between ≥2.0 and <2.5,
of inflammatory cell infiltration, atrophy, and intestinal
and 223 patients (77.2%) had a value between ≥2.5
metaplasia were assessed by Hematoxylin and Eosin
and ≤10.0. Baseline patient characteristics are shown
staining (H&E staining). Histologic features of gastric
in Table 1. Mean patient age was 57.3 (21–82 years)
mucosae were graded using the updated Sydney scoring
years, and mean time from eradication treatment dis-
system, which has a four-point scale (i.e., 0 = none,
continuation to 13C-UBT was 5.7 weeks. Initial diagno-
1 = slight, 2 = moderate, and 3 = marked) [25].
ses were functional dyspepsia in 72 patients (24.9%),
atrophic gastritis in 83 (28.7%), benign peptic ulcer in
Mucosa Urease Test 72 (24.9%), gastric dysplasia in 21 (7.3%), gastric can-
cer in 17 (5.8%), gastric mucosa-associated lymphoid
Two biopsy specimens, one from the lesser curvature of
tissue (MALT) lymphoma in 11 (3.8%), and family
the antrum and body, were used for the rapid urease
history of gastric cancer in 13 patients (4.6%). 59
test (CLOtest, Delta West, Bentley, Australia). Antral
(20.4%) and 24 (8.3%) patients had hypertension and
and body biopsy specimens were evaluated separately,
diabetes mellitus, respectively. For initial eradication,
and all urease tests were monitored for color change
262 patients (90.6%) received PPI-based triple therapy,
for up to 24 hours.
and 27 patients (9.4%) received sequential therapy.
Before the 13C-UBT, 204 patients (70.6%) underwent
Microbiological Examination the first-line eradication therapy, 67 patients (23.2%)
underwent second-line eradication therapy, and 18
Two specimens from the antrum and body were sent
(6.2%) patients underwent third-line eradication ther-
for microbiological culture in brain heart infusion plates
apy (Table 1).
containing 7% horse blood. These plates were placed in
a glass tank in a 5% O2, 10% CO2, and 85% N2 atmo-
sphere at 37 °C for 3–5 days. Antral and body biopsy The Diagnostic Accuracy of 13
C-UBT in the 2.0& to
specimens were evaluated separately. Organisms were 10.0& Range
identified as H. pylori by Gram staining, colony mor-
A flowchart of the study is shown in Fig. 1. All 289
phology, and positive oxidase, catalase, and urease
patients were evaluated for H. pylori status by histology
reactions.
and using the CLOtest. H. pylori cultures were per-
formed on 25 patients during endoscopic surveillance
Statistical Analysis after the 13C-UBT. When H. pylori statuses was ana-
lyzed based on biopsy-based methods, 151 patients
Sensitivity, specificity, false-positive rate, false-negative
(52.2%) had a positive result for H. pylori infection, and
rate, positive predictive value (PPV), negative predic-
138 patients (47.8%) were H. pylori negative (Fig. 1).
tive value (NPV), likelihood ratio for a positive test
In patients with a 13C-UBT result from 2.0& to
result (LRp), and likelihood ratio for a negative test
<2.5&, histology and the CLOtest showed that 65
result (LRn) for the 13C-UBT in the 2.0& to 10.0&
patients (98.5%) were H. pylori negative and 1 patient
range were calculated. Statistical analysis was con-
(1.5%) was H. pylori positive (Fig. 1). However, in the
ducted using PASW Statistics ver. 18.0 (SPSS, Chicago,
group with a 13C-UBT result from 2.5& to 10.0&
IL). The Student’s t-test, Pearson’s chi-square test, and
(N = 223), histology, the CLOtest, and/or culture
Fisher’s exact test were used, as appropriate, for the
showed 150 patients (67.3%) were H. pylori positive
univariate analysis of factors affecting the accuracy of
and 73 patients (32.7%) were H. pylori negative.
the 13C-UBT, and a logistic regression model was used
When the diagnostic accuracy of the 13C-UBT in the
for the multivariate analysis. p-values of <0.05 were
2.0& to 10.0& range was calculated versus endoscopic
considered statistically significant.

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The Diagnostic Validity of the C-UBT Kwon et al.

13
Table 1 The Baseline Characteristics of Patients with a C-UBT value 5.0& range, 12.5% (2/16 patients) in the 5.0&
in the 2.0& to 10.0& Range to 6.0& range, 6.7% (1/15 patient) in the 6.0& to
7.0& range, 23.8% (5/21 patient) in the 7.0& to 8.0&
Number range, 12.5% (2/16 patients) in the 8.0& to
9.0& range, and 9.5% (4/42 patients) in the 9.0& to
Age (years) 57.3 (21–82)
Gender (Male: Female) 177 (61.2%): 112 (38.8%)
10.0& range had a false-positive 13C-UBT result.
The initial diagnosis at the time of 13C-UBT Table 3 shows the sensitivity and specificity of the
13
Functional dyspepsia 72 (24.9%) C-UBT when the cutoff value was changed. The spec-
Atrophic gastritis 83 (28.7%) ificity of the 13C-UBT was 47.1%, 72.5%, 84.9%,
Benign peptic ulcer 72 (24.9%) 89.6%, 91.3%, 92.0%, 95.7%, and 97.1% for cutoff
Gastric dysplasia 21 (7.3%) values of 2.5&, 3.0&, 4.0&, 5.0&, 6.0&, 7.0&, 8.0&,
Gastric cancer 17 (5.8%)
and 9.0&, respectively.
Gastric MALT lymphoma 11 (3.8%)
Family history of gastric 13 (4.6%)
cancer The Risk Factors for a False-Positive Result of the
Underlying disease 13
C-UBT after H. pylori Eradication
HTN 59 (20.4%)
DM 24 (8.3%) The mean delta value of the 13C-UBT after H. pylori
Total number of eradication therapies for H. pylori infection eradication was 4.1& in those with a false-positive
Single eradication therapy 204 (70.6%) 13
C-UBT result (p < .005). Among those patients with a
2 eradication therapies 67 (23.2%) 13
3 eradication therapies 18 (6.2%)
C-UBT value in the 2.5& to 10.0& range, 155
The 1st eradication regimen* patients (69.5%), 53 patients (23.8%), and 15 patients
PPI based triple therapy 262 (90.6%) (6.7%) underwent one, two, or three eradication thera-
Sequential therapy 27 (9.4%) pies for H. pylori infection before enrollment in this
The mean time from H. pylori eradication 5.7  5.5 weeks study. Mean time from eradication to 13C-UBT was not
to 13C-UBT (weeks) (4–24, median 5) significantly different for those with matching 13C-UBT
13 and biopsy results. However, univariate analysis of
C-UBT, 13C-urea breath test; MALT, mucosa-associated lymphoid tis-
sue; HTN, hypertension; DM, Diabetes mellitus; H. pylori, Helicobacter the risk factors that caused mismatched results showed
pylori. that total number of H. pylori eradication therapies
*
PPI-based triple therapy (PPI b.i.d., clarithromycin 500 mg b.i.d., and (p < .005) and degree of gastric intestinal metaplasia
amoxicillin 1 g b.i.d. for 1 week), bismuth-containing quadruple ther- (p < .005) significantly contributed to mismatching
apy (PPI b.i.d., tripotassium dicitrate bismuthate 300 mg q.i.d., metro- (Table 4). Logistic regression analysis confirmed that
nidazole 500 mg t.i.d., and tetracycline 500 mg q.i.d. for 1–2 weeks),
multiple prior eradication therapies (OR = 2.455, 95%
sequential therapy (initial 5-day therapy with a combination of PPI
CI = 1.299–4.641, p = .006) and moderate to severe
b.i.d and amoxicillin 1 g b.i.d, followed by 5 days of PPI b.i.d., clari-
thromycin 500 mg b.i.d., and metronidazole 500 mg t.i.d for 1– gastric intestinal metaplasia (OR = 3.359, 95%
2 weeks). CI = 1.572–7.178, p < .005) were associated with the
mismatching of 13C-UBT and biopsy results after
H. pylori eradication (Table 5).

biopsy results, its sensitivity, specificity, false-positive

rate, and false-negative rate were 99.3%, 47.1%,
52.9%, and 0.7%, respectively. Its positive and negative
predictive values were 67.3% and 98.5%, respectively
(Table 2), and its LRp and LRn values were 1.87 and
0.01, respectively.
13
False-Positive Results of the C-UBT in the 2.5&
to 10.0& Range
Figure 2 shows discordant H. pylori status results for
the 13C-UBT and endoscopic biopsy in the 2.5& to
10.0& range; 46.1% (35/76 patients) in the 2.5& to
3.0& range, 77.3% (17/22 patients) in the 3.0&
to 4.0& range, 46.7% (7/15 patients) in the 4.0& to
Discussion
The most important finding of this study was that a
high proportion of mismatch was found between the
13
C-UBT and endoscopic biopsy-based results in the
2.5& to 10.0& 13C-UBT range after H. pylori eradica-
tion. This discordance resulted in a low specificity
(47.1%) and LRp (1.87) for the 13C-UBT, despite its
high sensitivity (99.3%) and LRn (0.01).
Generally, the 13C-UBT has been reported to be one
of the most accurate diagnostic tools for assessing
H. pylori status [26,27], and due to its speed, cost-
effectiveness, and convenience, this test has been widely
adopted in clinical practice [28]. However, the cutoff
value of the 13C-UBT has not been validated and it

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Kwon et al. The Diagnostic Validity of the C-UBT

Figure 1 Flow chart of 13C-UBT and endoscopic biopsy-based results of Helicobacter pylori status after eradication. 13
C-UBT, 13
C-urea breath test;
H. pylori, Helicobacter pylori

recommended that the cutoff value be lowered from

Table 2 The sensitivity, specificity, and predictive values of the
13
C-UBT compared with endoscopic biopsy methods for detecting the
3.5& to 3.0& [14]. Others suggested a cutoff value of
Helicobacter pylori status 2.5& with a smaller dose of urea, and subsequently,
this value was accepted as the best cutoff point [34–36].
Using the biopsy-based methods for However, in a recent Spanish study, it was reported the
13
H. pylori status C-UBT (UBit 100 mg, Otsuka Pharmaceutical Europe)
without citric acid had low specificity (60%) when a
Positive Negative cutoff value of 2.5& is used [16] and that a higher cut-
13 off value (8.5&) had more reliable a sensitivity and
C-UBT value
2.5&–10.0& 150 73 PPV 67.3%
specificity of > 90% in healthy volunteers.
2.0&–2.5& 1 65 NPV 98.5% The appropriate 13C-UBT cutoff value after H. pylori
Sensitivity 99.3% Specificity 47.1% eradication is a controversial issue. In several studies,
cutoff points between 1.3& and 7.4& resulted in high
13
C-UBT, 13C-urea breath test; H. pylori, Helicobacter pylori; PPV, posi- sensitivity and specificity after H. pylori eradication
tive predictive value, NPV; negative predictive value.
[17,34,36–40]. In the present study, the false-negative
rate for 13C-UBT in the 2.0& to 2.5& range was very
could depend on several factors, such as, the dose of low (1.5%). However, the false-positive rates of
13
urea administered, the indication for 13C-UBT, the mea- C-UBT at cutoff values between 2.5& and 10.0&
suring equipment used to determine delta13CO2, the ranged from 6.7% to 77.3% (Fig. 2), and as the cutoff
type of test meal, and the time elapsed from urease value was increased, specificity tended to increase
ingestion to the collection of gas samples [29–32]. Previ- (Table 3), which concurs with the above-mentioned
ously, Logan et al. [33] suggested a cutoff value for the Spanish study [16]. This observation means that at a
13
C-UBT of 5.0& based on the normal distribution of low cutoff value of 2.5&, the 13C-UBT has difficulty
excess delta 13CO2 values for H. pylori negative subjects determining H. pylori infection status before and after
that had never been infected. A later study eradication. In previous study, it was supposed the

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The Diagnostic Validity of the C-UBT Kwon et al.

Figure 2 False-positive 13C-UBT result rates in the 2.5& to 10.0& range after Helicobacter pylori eradication. 13
C-UBT, 13
C-urea breath test;
H. pylori, Helicobacter pylori

successively less as the three hydrogens were replaced

Table 3 The Assessment of the appropriate cutoff value of the with sodium [42,46]. Graham et al. showed citric acid
13
C-UBT for discriminating the false-positive results after H. pylori dose-dependently increased urease activity and breath
eradication 13
CO2 activity, and reported that urease activity was
13
markedly enhanced 10 min after administering urea in
C-UBT value
citric acid [31], which indicates that citric acid delays
(&) <2.5 <3.0 <4.0 <5.0 <6.0 <7.0 <8.0 <9.0
gastric emptying and enhances the intragastric distribu-
Sensitivity (%) 99.3 72.2 68.9 63.6 54.3 45.0 34.4 25.2 tion of urea. In a randomized trial, it was reported that
13
Specificity (%) 47.1 72.5 84.9 89.6 91.3 92.0 95.7 97.1 C-UBT with a low dose of urea (75 mg) and a citric
acid test meal had excellent diagnostic accuracy with-
13 13
C-UBT, C-urea breath test. out prior fasting [32]. These results suggest that the use
of citric acid increases delta 13CO2 and the discrimina-
main causes of the observed high false-positive rate of tive ability of the 13C-UBT.
the 13C-UBT were an insufficiency of data to confirm In the present study, we were unable to define a
13
the validity of the test and the nonuse of citric acid [16]. C-UBT cutoff value suitable for discriminating
Using citric acid as the test meal could increase the H. pylori status after eradication. Based on our figures, a
amount of 13CO2 in the breath after the administration 13
C-UBT cutoff of 6.0& would be most appropriate in
of 13C-urea via several mechanisms. In an in vitro terms of test specificity (>90%), but this cutoff value
study [41], the urease activities of H. pylori-containing caused a high false-negative rate. Thus, according to
antral mucosal biopsies were found to be markedly our results, 13C-UBT cutoff values between 2.5& and
increased at acid pH values. This finding suggests that 6.0& would be a “gray zone”.
citric acid has the ability to reduce pH at locations In the present study, we also sought to identify clin-
where H. pylori resides in the stomach [31]. Further- ical factors that caused false-positive 13C-UBT results
more, previous studies [42–46] showed that citric acid after H. pylori eradication. It is known that some factors
retards gastric emptying; maximum slowing of gastric can alter gastric H. pylori bacterial loading and cause
emptying was seen with citric acid and slowing became false-negative results. A low intragastric bacterial load,

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Kwon et al. The Diagnostic Validity of the C-UBT

Table 4 Risk Factors for the mismatched 13

C-UBT result in the 2.5& to 10.0& range compared with biopsy-based methods after H. pylori eradi-
cation

13
C-UBT result compared with biopsy based methods

Matched group (n = 150) Mismatched group (n = 73) Univariate p-value Multivariate p-value

Gender (Male:Female) 86(57.3%)/64(42.7%) 42(57.5%)/31(42.5%) NA

Age (≥ 60 years old) 68 (45.3%) 33 (45.2%) NA
Underlying disease
DM 11 (7.3%) 4 (5.6%) NA
HTN 32 (21.3%) 10 (13.9%) NA
The initial diagnosis at the time of 13C-UBT
Functional dyspepsia 40 (26.7%) 20 (27.4%) NA
Atrophic gastritis 53 (35.3%) 18 (24.7%)
Benign peptic ulcer 39 (26.0%) 18 (24.7%)
Gastric dysplasia 7 (4.7%) 9 (12.3%)
Gastric cancer 4 (2.7%) 5 (7.0%).
Gastric MALT lymphoma 3 (1.9%) 2 (2.7%)
Family history of gastric cancer 4 (2.7%) 1 (1.2%)
Time to 13C-UBT after eradication (weeks) 5.21 5.45 NA
The mean delta value of the 13C-UBT (&) 6.3  1.5 4.1  2.0 <.005
The total number of H. pylori eradication therapies
Single 116 (77.3%) 39 (53.4%) <.005 .006
Two 27 (18.0%) 26 (35.6%)
Three 7 (4.7%) 8 (11.0%)
The degree of gastric mucosal atrophy
None 106 (70.7%) 46 (63.0%) NA
Mild 27 (18.0%) 13 (17.8%)
Moderate 14 (9.3%) 11 (15.1%)
Marked 3 (2.0%) 3 (4.1%)
The degree of gastric mucosal intestinal metaplasia
None 93 (62.0%) 33 (45.2%) <.005 <.005
Mild 36 (24.0%) 11 (15.1%)
Moderate 15 (10.0%) 17 (23.3%)
Severe 6 (4.0%) 12 (16.4%)

13
C-UBT, C-urea breath test; NA, not available; DM, diabetes mellitus; HTN, hypertension; H. pylori, Helicobacter pylori.
13

Table 5 Logistic regression model for risk factors of the mismatching inhibiting drugs, such as, the recent use of antibiot-
result between 13C-UBT and endoscopic biopsy-based methods in the ics, bismuth, or PPI, are known to be risk factors of
diagnosis of H. pylori infection
false-negative results [47,48]. In contrast, urease-pro-
ducing bacteria (Streptococcus, Staphylococcus, Gardnerella,
p-
Lactococcus, and Enterococcus) could cause false-positive
Risk factors b SE value OR 95% CI
results [49–52]. Furthermore, if gastric atrophy is
Multiple eradication 0.898 0.325 .006 2.455 1.299–4.641 severe, the stomach environment becomes hypochlo-
therapies for H. pylori rhydric, which accelerates colonization and the over-
infection growth of non- H. pylori urease-positive bacteria. In the
Moderate to severe 1.212 0.387 <.005 3.359 1.572–7.178 present study, degrees of gastric atrophy were similar in
gastric intestinal patients with matched and mismatched 13C-UBT and
metaplasia
endoscopic biopsy results. However, it is rather tricky to
13
C-UBT, 13C-urea breath test; H. pylori, Helicobacter pylori; b, coeffi- assess the degree of gastric atrophy based on histologic
cient; S.E, standard error; OR, odds ratio; CI, confidence interval. and gross findings and pepsinogen test results [53].
Instead, moderate to severe gastric intestinal metaplasia
fast gastric emptying (shorter contact time between determination by histology, which developed 10 years
ingested urea and infected mucosa), previous gastric after developing gastric atrophy [54] was found to be
surgery, and the concomitant administration of urease an important risk factor of false-positive 13C-UBT

© 2015 John Wiley & Sons Ltd, Helicobacter 7

 13
The Diagnostic Validity of the C-UBT
results after H. pylori eradication (OR = 3.359, 95%
CI=1.572–7.178, p < .005).
It is also possible that the presence of dying
H. pylori, but with urease activity, could increase false-
positive 13C-UBT results after H. pylori eradication,
which is in line with the drop in delta 13C values
observed at 3 and 6 months after treatment. This
implies an alternative strategy of retesting would be
needed in patients with 13C-UBT results in the 2.5& to
5.0& range at 6 months later 13C-UBT [55]. In the
present study, a previous history of multiple H. pylori
eradication therapies was found to be correlated with
false-positive 13C-UBT results (OR = 2.455, 95%CI =
1.299–4.641, p = .006). Of the 15 patients that under-
went third-line eradication therapy for H. pylori infec-
tion in the 13C-UBT range 2.5& to 10.0&, eight had a
false-positive 13C-UBT result, and of these eight, six
had a 13C-UBT result in the 2.5& to 6.0& range after
2nd eradication therapy. Because delta 13C-UBT results
were lower (although positive) at 4 weeks after the 3rd
eradication therapy, we postponed further H. pylori
eradication in these patients and performed endoscopic
biopsies after 6 months. Finally, histology and the CLO-
test demonstrated no evidence of H. pylori infection in
gastric mucosa, which could be explained by “delayed
response”.
The observed false-positive 13C-UBT results raise the
question as to whether endoscopic biopsy-based meth-
ods are reliable for determining the final status of
H. pylori. A previous study showed that the 13C-UBT
occasionally has poor diagnostic ability as compared
with endoscopic biopsy-based methods [10]. However,
endoscopic biopsy-based methods are susceptible to
sampling errors because of discontinuous H. pylori colo-
nization of the stomach [56]. In the present study, gas-
tric biopsies were performed and the samples were
obtained from the antrum and from the body for histo-
logic analysis (modified Giemsa staining), and a gastric
sample was obtained from both antrum and body for
rapid urease testing. H. pylori culture was performed
using samples from the gastric biopsies, that is, two
samples from the antrum and from the body. Further-
more, all patients received at least two or more follow-
up endoscopic surveillances, which reconfirmed final
H. pylori statuses. Thus, we believe the possibility of a
gastric biopsy sampling error was slight.
The present study has several strengths. First, many
studies have sought to determine an appropriate cutoff
value for the 13C-UBT, but few studies have compared
13
C-UBT results in the indeterminate zone (2& to
10&) with those of endoscopic biopsy-based methods
[13,26]. Furthermore, the numbers of patients enrolled
in these previous studies were less than in the present
8 © 2015 John Wiley & Sons Ltd, Helicobacter
Kwon et al.
study, in which we included 289 patients (15.3%) with
a 13C-UBT value in the 2.0& to 10.0& range that were
assessed by histology, the mucosa urease test, and cul-
ture. In addition, these results were obtained after the
eradication of H. pylori. This is an important point, as it
should encourage clinicians to consider the inaccuracy
of the 13C-UBT after H. pylori eradication, especially
when it is performed instead of endoscopic surveillance
after H. pylori eradication. Second, the findings of the
present study suggest that clinicians should undertake
additional H. pylori eradication more cautiously when
13
C-UBT results fall into the gray zone after H. pylori
eradication, because this would reduce antibiotic usage
and prevent unnecessary medical spending.
However, the study also has limitations. First, we
could not compare 13C-UBT and endoscopic biopsy
results at the same time. According to the Korean
Health Insurance service, these two tests should not be
performed simultaneously after H. pylori eradication.
Therefore, in this study, we evaluated H. pylori status
endoscopically at least 6 months after the 13C-UBT.
With respect to the gray zone of the 13C-UBT after
H. pylori eradication and the delayed response of
H. pylori to eradication, this approach is probably better
aligned to decision making. Second, although the sever-
ity of gastric intestinal metaplasia was found to be an
independent risk factor of a false-positive 13C-UBT
result, we did not evaluate the colonization or over-
growth of non- H. pylori urease-positive bacteria. The
nature of relation between degree of gastric intestinal
metaplasia and overgrowth of non- H. pylori urease-
positive bacteria could be evaluated by pyrosequencing,
and this is currently in progress. Third, the results of
our study could be applied to post- H. pylori eradication
status rather than initial diagnosis of H. pylori infection.
Under the Korean National Medical Insurance system,
it is difficult to obtain 13C-UBT values before H. pylori
eradication. Furthermore, a well-designed, randomized,
controlled study is needed to confirm the diagnostic
validity of the 13C-UBT before H. pylori eradication.
Forth, this study is intrinsically limited by its retrospec-
tive design. In particular, only 15% of patients who
showed 13C-UBT result between 2.0& and 10.0& ini-
tially were enrolled, which introduces the possibility of
sampling bias. However, our study had both prospective
and retrospective features. Patients with a 13C-UBT
result in the range 2.5& to 3.0& were prospectively
enrolled after providing informed consent, whereas the
other patients were retrospectively enrolled. Finally,
we did not investigate the effect of citric acid use on
13
C-UBT results. Further investigation is needed to
determine the effect of citric acid inclusion on diagnos-
tic accuracy.

Kwon et al.
Summarizing, this study shows that false-positive
rate of the 13C-UBT without citric acid is high in the
2.5& to 6.0& range after the eradication of H. pylori
infection, and therefore, we suggest that 13C-UBT
results in this range be referred to as “probably positive
result” and not “positive”. Accordingly, when a “proba-
bly positive 13C-UBT result” is obtained after H. pylori
eradication, additional endoscopic surveillance using
biopsy-based methods should be considered to help
avoid unnecessary additional treatment, especially in
patients that have undergone multiple prior eradication
therapies and in patients with moderate to severe gas-
tric intestinal metaplasia.
Acknowledgements and Disclosures
The authors thank the Division of Statistics of the Medical
Research Collaborating Center at Seoul National University
Bundang Hospital for their assistance with the statistical analy-
sis. This work was supported by a Korean National Research
Foundation (NRF) grant to the Global Core Research Center
(GCRC) funded by the Korea government (MSIP) (No. 2011-
0030001).
Competing interests: The authors declare that they have no
proprietary, commercial, or financial interests that could be
construed to have inappropriately influenced this study.
References
1 Forbes GM, Warren JR, Glaser ME, et al. Long-term follow-up
of gastric histology after Helicobacter pylori eradication. J Gastro-
enterol Hepatol 1996;11:670–3.
2 Asaka M, Kato M, Takahashi S, et al. Guidelines for the man-
agement of Helicobacter pylori infection in Japan: 2009 revised
edition. Helicobacter 2010;15:1–20.
3 Hopkins RJ, Girardi LS, Turney EA. Relationship between
Helicobacter pylori eradication and reduced duodenal and
gastric ulcer recurrence: a review. Gastroenterology
1996;110:1244–52.
4 Kwon YH, Heo J, Lee HS, et al. Failure of Helicobacter pylori
eradication and age are independent risk factors for recurrent
neoplasia after endoscopic resection of early gastric cancer in
283 patients. Aliment Pharmacol Ther 2014;39:609–18.
5 Lee JY, Kim N. Future trends of Helicobacter pylori eradication
therapy in Korea. Korean J Gastroenterol 2014;63:158–70.
6 Morris A, Ali MR, Brown P, et al. Campylobacter pylori
infection in biopsy specimens of gastric antrum: laboratory
diagnosis and estimation of sampling error. J Clin Pathol
1989;42:727–32.
7 Newell DG, Hawtin PR, Stacey AR, et al. Estimation of preva-
lence of Helicobacter pylori infection in an asymptomatic elderly
population comparing [14C] urea breath test and serology. J
Clin Pathol 1991;44:385–7.
8 Lee JH, Kim N, Chung JI, et al. Long-term follow-up of Helicob-
acter pylori IgG serology after eradication and reinfection rate of
H. pylori in South Korea. Helicobacter 2008;13:288–94.
9 Cutler AF, Havstad S, Ma CK, et al. Accuracy of invasive and
noninvasive tests to diagnose Helicobacter pylori infection. Gastro-
enterology 1995;109:136–41.
© 2015 John Wiley & Sons Ltd, Helicobacter
13
The Diagnostic Validity of the C-UBT
10 Gisbert JP, Pajares JM. Review article: 13C-urea breath test in
the diagnosis of Helicobacter pylori infection - a critical review.
Aliment Pharmacol Ther 2004;20:1001–17.
11 Malfertheiner P, Megraud F, O’Morain C, et al. European Heli-
cobacter Pylori Study Group. Current concepts in the management
of Helicobacter pylori infection – The Maastricht 2-2000 Consensus
Report. Aliment Pharmacol Ther 2002;16:167–80.
12 Hunt RH, Fallone CA, Thomson AB; Canadian Helicobacter
Study Group. Canadian Helicobacter pylori Consensus Confer-
ence update: infections in adults. Can J Gastroenterol
1999;13:213–7.
13 Ohara S, Kato M, Asaka M, et al. Studies of 13C-urea breath
test for diagnosis of Helicobacter pylori infection in Japan. J Gas-
troenterol 1998;33:6–13.
14 Mion F, Rosner G, Rousseau M, et al. 13C-urea breath test for
Helicobacter pylori: cut-off point determination by cluster analy-
sis. Clin Sci (Lond) 1997;93:3–6.
15 Graham DY, Klein PD. Accurate diagnosis of Helicobacter pylori.
13
C-urea breath test. Gastroenterol Clin North Am 2000;29:885–
93.
16 Calvet X, Sanchez-Delgado J, Montserrat A, et al. Accuracy of
diagnostics test for Helicobacter pylori: a reappraisal. Clin Infect
Dis 2009;48:1385–91.
17 Kato C, Sugiyama T, Sato K, et al. Appropriate cut-off value of
13C-urea breath test after eradication of Helicobacter pylori infec-
tion in Japan. J Gastroenterol Hepatol 2003;18:1379–83.
18 Parkin DM, Bray F, Ferlay T, Pisani P. Global cancer statistics,
2002. CA Cancer J Clin 2005;55:74–108.
19 Kim MS, Kim N, Kim SE, et al. Long-term follow-up Helicobact-
er pylori reinfection rate and its associated factors in Korea. Heli-
cobacter 2013;18:135–42.
20 Lee JY, Kim N, Kim MS, et al. Factors affecting first-line triple
therapy of Helicobacter pylori including CYP2C19 genotype and
antibiotic resistance. Dig Dis Sci 2014;59:1235–43.
21 Lee JW, Kim N, Kim JM, et al. A comparison between 15-day
sequential, 10-day sequential and PPI-based triple therapy for
Helicobacter pylori infection in Korea. Scand J Gastroenterol.
2014;49:917–24.
22 Lee BH, Kim N, Hwang TJ, et al. Bismuth-containing quadru-
ple therapy as second-line treatment for Helicobacter pylori
Infection: effect of treatment duration and antibiotic resistance
on the eradication rate in Korea. Helicobacter 2010;15:38–45.
23 Yoon H, Kim N, Lee BH, et al. Moxifloxacin-containing triple
therapy as second-line treatment for Helicobacter pylori infection:
effect of treatment duration and antibiotic resistance on the
eradication rate. Helicobacter 2009;14:77–85.
24 Kim SE, Park YS, Kim N, et al. The effect of Helicobacter pylori
eradication on functional dyspepsia. J Neurogastroenterol Motil
2013;19:233–43.
25 Dixon MF, Genta RM, Yardley JH, et al. Classification and
grading of gastritis: the updated Sydney system. Am J Surg
Pathol 1996;20:1161–81.
26 Bazzoli F, Zagari M, Fossi S, et al. Urea breath tests for the
detection of Helicobacter pylori infection. Helicobacter 1997;2:
34–7.
27 Savarino V, Vigneri S, Celle G. The 13C urea breath test in the
diagnosis of Helicobacter pylori infection. Gut 1999;45:18–22.
28 Savarino V, Mela GS, Zentilin P, et al. Comparison of isotope
ratio mass spectrometry and nondispersive isotope-selective
infrared spectroscopy for 13C-urea breath test. Am J Gastroenter-
ol 1999;94:1203–8.
29 Klein PD, Malaty HM, Martin RF, Graham KS, Genta RM, Gra-
ham DY. Noninvasive detection of Helicobacter pylori infection
9
 13
The Diagnostic Validity of the C-UBT
in clinical practice: the 13C urea breath test. Am J Gastroenterol
1996;91:690–4.
30 Wong WM, Wong BC, Li TM, et al. Twenty-minute 50 mg
13
C-urea breath test without test meal for the diagnosis of Heli-
cobacter pylori infection in Chinese. Aliment Pharmacol Ther
2001;15:1499–504.
31 Graham DY, Runke D, Anderson SY, Anderson SY, Malaty
HM, Klein PD. Citric acid as the test meal for the 13C-urea
breath test. Am J Gastroenterol 1999;94:1214–7.
32 Graham DY, Malaty HM, Cole RA, Martin RF, Klein PD. Sim-
plified 13C-urea breath test for detection of Helicobacter pylori
infection. Am J Gastroenterol 2001;96:1741–5.
33 Logan RP, Dill S, Bauer FE, et al. The European 13C-urea
breath test for the detection of Helicobacter pylori. Eur J Gastroen-
terol Hepatol 1991;3:915–21.
34 Malaty HM, el-Zimaity HM, Genta RM, Klein PD, Graham DY.
Twenty-minute fasting version of the US 13C-urea breath test
for the diagnosis of H. pylori infection. Helicobacter, 1996;1:165–7.
35 Lotterer E, Ramaker J, L€ udtke F, et al. The simplified 13C-urea
breath test-one point analysis for detection of Helicobacter pylori
infection. J Gastroenterol 1991;29:590–4.
36 Epple HJ, Kirstein FW, Bojarski C, et al. 13C-urea breath test in
Helicobacter pylori diagnosis and eradication. Correlation to his-
tology, origin of ‘False’ results, and influence of food intake.
Scand J Gastroenterol 1997;32:308–14.
37 Chen TS, Chang FY, Chen PC, et al. Simplified 13C-urea breath
test with a new infrared spectrometer for diagnosis of Helicob-
acter pylori infection. J Gastroenterol Hepatol 2003;18:1237–43.
38 Kopacova M, Bures J, Vorisek V, et al. Comparison of different
protocols for 13C-urea breath test for the diagnosis of Helicobact-
er pylori infection in healthy volunteer. Scand J Clin Lab Invest
2005;65:491–8.
39 Gatta L, Vakil N, Ricci C, et al. A rapid, low-dose, 13C-urea
tablet for the detection of Helicobacter pylori infection before
and after treatment. Aliment Pharmacol Ther 2003;17:793–8.
40 Leodolter A, Dominguez-Munoz JE, von Arnim U, et al. Valid-
ity of a modified 13C-urea breath test for pre- and posttreat-
ment diagnosis of Helicobacter pylori infection in the routine
clinical setting. Am J Gastroenterol 1999;94:2100–4.
41 Miederer SE, Grubel P. Profound increase of Helicobacter pylori
urease activity in gastric antral mucosa at low pH. Dig Dis Sci
1996;41:944–9.
42 Hunt JN, Knox MT. The effect of citric acid and its sodium salts
in test meals on the gastric outputs of acid and of chloride. J
Physiol 1973;230:171–84.
10
Kwon et al.
43 Hunt JN, Knox MT. The slowing of gastric emptying by
four strong acids and three weak acids. J Physiol 1972;222:
187–208.
44 Hunt JN, Knox MT. The slowing of gastric emptying by nine
acids. J Physiol 1969;201:161–79.
45 Hunt JN, Knox MT. Control of gastric emptying. Am J Dig Dis
1968;13:372–5.
46 Hunt JN, Knox MT. The regulation of gastric emptying of meals
containing citric acid and salts of citric acid. J Physiol
1962;163:34–45.
47 Graham DY, Opekun AR, Hammoud F, et al. Studies regard-
ing the mechanism of false negative urea breath tests
with proton pump inhibitors. Am J Gastroenterol 2003;98:
1005–9.
48 Di Rienzo TA, D’Angelo G, Ojetti V, et al. 13C-Urea breath test
for the diagnosis of Helicobacter pylori infection. Eur Rev Med
Pharmacol Sci 2013;17:51–8.
49 Brandi G, Biavati B, Calabrese C, et al. Urease-positive bacteria
other than Helicobacter pylori in human gastric juice and
mucosa. Am J Gastroenterol 2006;101:1756–61.
50 Gurbuz AK, Ozel AM, Narin Y, et al. Is the remarkable contra-
diction between histology and 14C urea breath test in the
detection of Helicobacter pylori due to false-negative histology or
false-positive 14C urea breath test? J Int Med Res 2005;33:
632–40.
51 Osaki T, Mabe K, Hanawa T, et al. Urease-positive bacteria in
the stomach induce a false-positive reaction in a urea breath
test for diagnosis of Helicobacter pylori infection. J Med Microbiol
2008;57:814–9.
52 Slomianski A, Schubert T, Cutler AF. [13C] urea breath test to
confirm eradication of Helicobacter pylori. Am J Gastroenterol
1995;90:224–6.
53 Lee JY, Kim N, Lee HS, et al. Correlations among endoscopic,
histologic and serologic diagnoses for the assessment of atrophic
gastritis. J Cancer Prev 2014;19:47–55.
54 Kim N, Park YS, Cho SI, et al. Prevalence and risk factors of
atrophic gastritis and intestinal metaplasia in a Korean popula-
tion without significant gastroduodenal disease. Helicobacter
2008;13:245–55.
55 Miwa H, Ohkura R, Nagahara A, et al. [13C]-urea breath test
for assessment of cure of Helicobacter pylori infection at 1 month
after treatment. J Clin Gastroenterol 1998;27:150–3.
56 Perri F, Festa V, Clemente R, Quitadamo M, Andriulli A. Meth-
odological problems and pitfalls of urea breath test. Ital J Gas-
troenterol Hepatol 1998;30:315–9.
© 2015 John Wiley & Sons Ltd, Helicobacter