The Korean Journal of Internal Medicine: 23:134-139, 2008

Comparison of the 13C-urea breath test and the

endoscopic phenol red mucosal pH test in the
quantification of Helicobacter pylori infection loading
Young-Seok Cho, M.D.1, Hiun-Suk Chae, M.D.1, Se Na Jang, M.D.1,
1 1 1
Jin-Soo Kim, M.D. , Hye Suk Son, M.D. , Hyung-Keun Kim, M.D. ,
Byung-Wook Kim, M.D. , Sok-Won Han, M.D. , Kyu-Yong Choi, M.D.1,
1 1

Hae Kyung Lee, M.D.2 and Eun Deok Chang, M.D.2

Departments of Internal Medicine, Division of Gastroenterology1 and Clinical Pathology2

The Catholic University of Korea, College of Medicine Seoul, Korea2

Background/Aims: The 13C-urea breath test (UBT) is a semiquantitative test for measuring Helicobacter pylori
infection loading. H. pylori produces ammonia, which elevates the pH of the gastric mucosa and is detectable via
endoscopy using a phenol red indicator. We evaluated whether this test could be used to diagnose H. pylori infection
13
and whether phenol red staining was correlated with C-UBT results.
Methods: One hundred and twenty-three patients participated. The UBT was performed after ingestion of a capsule
containing urea. A change in 13C-UBT >2‰ was selected as the cutoff value for diagnosing infection. After spraying
evenly with a 0.1% phenol red solution, the pH of the gastric mucosal surface was measured using an antimony
electrode through the biopsy channel.
Results: The pH of stained mucosa (6.9±0.4) was significantly higher than that of unstained mucosa (1.9±0.8;
p<0.001), and the H. pylori detection rate confirmed via histology was higher in stained versus unstained mucosa
(p<0.01). Extensive mucosal staining resulted in a higher detection rate (p<0.001). The UBT produced results were very
similar to those obtained via histological detection in stained mucosa (p<0.001). The extent of staining, expressed as
a staining score, was positively correlated with the change in 13C-UBT (r=0.426, p < 0.001). A significant correlation was
13
also observed between the histologically determined H. pylori density and C-UBT results (r=0.674, p<0.001).
Conclusions: H. pylori infection elevates gastric mucosal surface pH, and endoscopic phenol red staining may be
an alternative method for the diagnosis of H. pylori infection.

Key Words: Helicobacter pylori; Mucosal surface pH; Phenol red; Urea breath test.

INTRODUCTION diagnosis of H. pylori infections, including direct culture,

histology, rapid urease testing, polymerase chain reaction (PCR)
Helicobacter pylori infection is associated with chronic active bacterial DNA amplification, the use of H. pylori-specific
13 14
gastritis, peptic ulcer disease, gastric adenocarcinoma, and antibodies, the urea breath test (UBT) using C- or C-labeled
4) 13
mucosa-associated lymphoid tissue lymphomas1-3). Many urea, and stool antigen detection . The C-UBT measures
noninvasive and invasive methods have been developed for the intragastric urease activity and is generally considered to be an

∙Received: February 15, 2008

∙Accepted: April 30, 2008
∙Correspondence to: Hiun-Suk Chae, M.D., The Catholic University of Korea, Department of Internal Medicine, Division of Gastroenterology, Seoul and
Uijeongbu St. Mary's Hospital, Uijongbu, Korea, 65-1, Geumo-dong, Uijongbu 480-717, Korea Tel: 82-31-820-3019,
Fax: 82-31-847-2719, E-mail: [email protected]
*This study has not been previously published or submitted for publication elsewhere. The authors have no conflict of interest to declare.
 Young-Seok Cho, et al: Comparison of UBT and endoscopic phenol red test 135

accurate, noninvasive, and semiquantitative method to collected 20 min later. Breath samples were then analyzed via
determine H. pylori loading5, 6). However, the UBT has limitations mass spectrometry (HeliView; MediChems, Seoul, Korea) to
13 12
inherent to noninvasive methods: it cannot pinpoint the position determine the C to C ratio, which was expressed per mil
13
of upper gastrointestinal disease or provide histological (‰). The change in C relative to the baseline value was
13
information. expressed as Δ C. A positive result was defined as a positive
H. pylori produces ammonia via the activity of a potent Δ13C >2‰.
bacterial urease, which is detectable via diagnostic assays such
7)
as the CLOtest . Urease activity can lead to mucosal damage Endoscopic Phenol Red Test
by degrading urea in human tissue8, 9), and the resultant The distribution of pH changes indicative of H. pylori infection
ammonia can elevate mucosal pH at the site of infection. in the gastric mucosa was examined via endoscopic phenol red
Therefore, we examined whether mucosal surface pH can be spraying. Patients were subjected to gastroscopy after first
determined endoscopically using phenol red as a pH indicator, receiving a local oral anesthetic (10% xylocaine) and an
and whether such a technique is useful in the detection of H. intramuscular injection of 20 mg of scopolamine butylbromide
pylori infection. To quantify H. pylori loading, we also examined (Buscopan; Boehringer Ingelheim Company Ltd., Seoul, Korea)
whether the extent of the affected region was correlated with to reduce gastric motility. A videoendoscope was inserted,
UBT values. In addition, we documented several noteworthy gastric juice was aspirated to improve visibility during
points in the phenol red spraying procedure and in the endoscopy, and the interior of the stomach was inspected. A
interpretation of results. 0.1% solution of phenol red was sprayed evenly over the entire
surface of the gastric mucosa using a spray catheter (PW-5L-1;
Olympus Co., Tokyo, Japan) passed through the biopsy channel
MATERIALS AND METHODS of the endoscope. A yellow to red color change occurred 2–3
min after applying the dye and persisted for at least 15 min. We
Patients classified the staining patterns into four types based on the
10)
From March to November 2006, 123 patients undergoing classification described by Kohli et al . The extent of staining
routine upper gastrointestinal endoscopy for dyspeptic symp- was scored on a scale of 0 to 3: 0, no staining; 1, patchy
toms were recruited into this study (46 men and 77 women staining in which red areas were observed in one part of the
aged 19–79 years). These patients suffered from nonulcer stomach, such as the antrum, body, or cardia; 2, regional
dyspepsia (n=109), duodenal ulcers (n=8), or gastric ulcers (n=6). staining in which red areas were observed in two parts of the
13
The C-UBT was performed 1 day after the patients had fasted stomach, such as the antrum and body or the body and cardia;
for at least 8 h. On the second day, the endoscopic phenol red 3, diffuse staining in which red areas were observed in all
test was performed after at least an 8-h fast. In addition, two to regions. An antimony pH electrode catheter (catalog
three gastric mucosal biopsies were taken at stained and #9012P3001; Medtronic A/S, Køpenhavn S, Denmark) connected
unstained sites in the antrum and the body histological analysis, to an ambulatory pH meter (Digitrapper Mk III; Synetics Medical
which remains the gold standard for the diagnosis of H. pylori AB, Stockholm, Sweden) was passed through the biopsy
infection. Exclusion criteria included previous eradication therapy channel of the endoscope. The tip was gently advanced under
or the use of bisthmus compounds, proton pump inhibitors, direct visualization until it abutted with the mucosa, at which
antibiotics, or anti-secretory drugs within the previous 2 months. point it was held steady to provide a stable mucosal reading in
Patients with hepatobiliary, pulmonary or metabolic diseases, or the red and yellow areas. In total, surface pH measurements
previous gastric surgery were also excluded. Written informed were obtained for 25 randomly selected stained areas (regional
consent was obtained from all patients. The protocol was or diffuse staining) and 20 randomly selected unstained sites.
approved by the institutional review board of the Catholic
University of Korea. Histology
To examine the relationship between positive phenol red
13
C-Urea Breath Test staining and the presence of H. pylori, endoscopic biopsies were
All patients were subjected to the 13C-UBT on the first day of taken at unstained and stained (patchy, regional, or diffuse)
the study after an overnight or 8-h fast. A baseline breath sites. Silver staining was used to histologically confirm the
sample (normal exhalation for 4 s) was collected into a presence of H. pylori bacilli, irrespective of color change,
13
collection tube. A capsule containing 38 mg C-urea in 50 mL staining patterns, or location (antrum or body). To quantify H.
of water (HeliFinder; Isodiagnostika, Edmonton, AB, Canada) pylori loading, we selected the densest H. pylori infection site for
was then administered orally. A second breath sample was comparison with UBT values. The specimens were fixed
136 The Korean Journal of Internal Medicine: Vol. 23, No. 3, September 2008

Table 1. Gastric mucosa staining patterns after phenol red application mucosal sites. The data were analyzed using SPSS (version
10.0; SPSS Inc., Chicago, IL, USA).
Staining pattern No. of patients (%)
Diffuse 24 (19.5)
Regional 34 (27.6)
Patchy 38 (30.9)
RESULTS
Unstained 27 (22.0)
Phenol Red Staining Pattern and Mucosal Surface pH
Total 123 (100)
Phenol staining was classified into four types as described in
the Materials and Methods (Figure 1). Table 1 shows the
separately in 10% formalin, embedded in paraffin wax, and 5 μm distribution of staining patterns, of which patchy staining (score
sections were stained with hematoxylin and eosin and the of 1) was the most common. The surface pH of stained mucosa
Warthin–Starry silver technique. Two pathologists, who were (6.9±0.4) was significantly higher than that of unstained (yellow)
13
unaware of the clinical, endoscopic, and Δ C-UBT results, gastric mucosa (1.9±0.8, p<0.001) (Figure 2).
evaluated the sections. The H. pylori bacterial load (H. pylori
score) was classified according to the Updated Sydney Phenol Red Staining and UBT Compared with Histo-
10)
System . pathology
Seventy-eight of 96 patients showing patchy, regional, or
Statistical Analysis diffuse staining were also histologically positive for H. pylori
Spearman’s rank correlation test was used to detect any (sensitivity, 81.3%), whereas 22 of 27 people with unstained
correlation between Δ13C-UBT values and H. pylori bacterial mucosa were histologically negative for H. pylori (specificity,
loading, gastric histological variables, and endoscopic phenol 81.5%; Table 2). In comparison to UBT results, phenol red
red scoring. Student’s t-test was used to compare gastric staining also showed fair sensitivity (81/96 or 84.4%), but
surface pH values between stained and unstained mucosal relatively low specificity (20/27 or 74.1%; Table 2). Among
sites, and chi-square tests were applied to compare the positive staining patterns, the H. pylori detection rate was higher at sites
rate of H. pylori infection between stained and unstained showing broad staining (regional and diffuse pattern) compared to

Figure 1. After spraying the gastric mucosa with phenol red, mucosal staining
patterns were classified into four types: diffuse (A), regional (B), patchy (C), or
unstained (D).
 Young-Seok Cho, et al: Comparison of UBT and endoscopic phenol red test 137

13
Table 2. Comparison of H. pylori detection via phenol red staining versus histological and Δ C-UBT results

Phenol red staining

Histological staining cases (%) Δ13C-UBT results cases (%)
Positive Negative ≥2 (‰) <2 (‰)
Stained (n=96) 78 (81.3) 18 (18.7) 81 (84.4) 15 (15.6)
Unstained (n=27) 5 (18.5) 22 (81.5) 7 (25.9) 20 (74.1)

13
Table 3. Comparison of phenol red staining patterns with histological and Δ C-UBT results

13
Histological staining cases (%) Δ C-UBT results cases (%)
Staining patterns
Positive Negative ≥2 (‰) <2 (‰)
Diffuse (n=24) 20 (83.3) 4 (16.7) 23 (95.8) 1 (4.2)
Regional (n=34) 33 (97.1) 1 (2.9) 33 (97.1) 1 (2.9)
Patchy (n=38) 25 (65.8) 13 (34.2) 25 (65.8) 13 (34.2)
Unstained (n=27) 5 (18.5) 22 (81.5) 7 (25.9) 20 (74.1)

Figure 2. Gastric mucosa surface pH values in stained versus Figure 3. Correlation of Δ13C-UBT values with phenol red scores
unstained areas after spraying with a 0.1% phenol red solution (r=0.426, *p<0.001).
(*p<0.001).

patchy or no staining (Table 3), and H. pylori detection was most noninvasive procedures, such as the UBT and serologic
sensitive in sites showing regional staining (Table 3). The UBT and examination, are as accurate in predicting H. pylori status as
histological analysis showed very similar results (Table 3). invasive tests in untreated patients12). Previous studies have
indicated that the high incidence of gastric cancer is closely
Correlation of UBT with Phenol Red Staining Patterns associated with the high rate of H. pylori infection, particularly in
13-17)
or Histology Results Korea . Therefore, it is important to use simultaneous
Phenol red staining scores were correlated with Δ13C-UBT invasive endoscopic observation and direct histological evalua-
results (r=0.426, p<0.001; Figure 3). However, a stronger correla- tion of H. pylori status to detect gastric cancer and develop a
13
tion was observed between Δ C-UBT and the histologically treatment strategy.
determined H. pylori density (r=0.674, p<0.001) (Figure 4). Currently, gastric biopsies for the purpose of histological H.
pylori detection are generally taken from within the lesser
curvature of the stomach, approximately 2～3 cm from the
DISCUSSION pylorus; this technique yields a relatively high rate of detection
(>90%) under the correct orientation11). However, because H.
Numerous tests are available for H. pylori detection4), and pylori infection is usually unevenly distributed across the gastric
138 The Korean Journal of Internal Medicine: Vol. 23, No. 3, September 2008

trauma. In addition, interobserver variation may have influenced

our results, especially in patients showing patchy staining, which
was associated with relatively low sensitivity and specificity. For
these reasons, we excluded ten cases that may be been
affected by the above uncertainties. However, the remaining
three staining patterns presented no difficulty in terms of
endoscopic interpretation.
To our knowledge, we are the first to demonstrate that
changes in gastric mucosal pH, as determined via the
application of a urea-free phenol red solution, can be used to
assess H. pylori infection. In addition, phenol red staining scores
were positively correlated with Δ13C-UBT values. As such, the
development of a device to measure the area of color change
may one day allow for the direct quantification of H. pylori
loading from phenol red staining. Previous studies have
13
indicated that Δ C-UBT values are correlated with bacterial
13
Figure 4. Correlation of Δ C-UBT values with H. pylori coloni- density19, 20), although conflicting data have also been reported5, 21).
zation density (r=0.674, *p<0.001). However, Rauws et al.22) reported a positive correlation between
UBT values and quantitative antral culture results. In the present
13
mucosa18), sampling errors leading to false negatives are study, bacterial density was more strongly correlated with Δ C-
unavoidable. Therefore, it is important to consider the UBT values than phenol red staining scores.
13
intragastric distribution of H. pylori infection. In this study, we In the clinical setting, Δ C-UBT values are useful to
used phenol red staining to detect local pH changes due to determine the success of bacterial eradication therapy and to
ammonium production by urease, which revealed the intragastric evaluate the efficacy of new antibiotics. Moreover, bacterial load
distribution of H. pylori. We found that phenol red staining may play an important role in determining the outcome of H.
shows high detection sensitivity in stained mucosa (histologically pylori eradication. A high bacterial load appears to be
positive rate, 81.3%; UBT, 84.4%) and high specificity in associated with an increased risk of gastroduodenal endoscopic
unstained mucosa (histologically negative rate 81.5%; UBT lesions, such as peptic ulcer disease, and with the reduced
23-25)
74.1%). These results demonstrate a higher rate of H. pylori efficacy of eradication therapy . Based on the correlation
13
detection in mucosal regions with a high pH (stained mucosa) between Δ C-UBT values and phenol red scores, H. pylori
than in mucosal regions with low pH (unstained mucosa). In eradication may be more challenging in patients with regional or
addition, phenol red staining may help to select infected biopsy diffuse staining compared to patchy staining. Thus, endoscopists
sites, thus decreasing the rate of false negatives for H. pylori may be able to estimate the efficacy of eradication therapy
infection compared to the current blind biopsy approach. directly based on endoscopic phenol red spraying, without
According to staining pattern, phenol red staining showed a waiting for UBT results.
significantly higher detection sensitivity in areas of broad staining Although endoscopic phenol red application and biopsy have
(regional, 97.1%; diffuse, 83.3%) compared to patchy (65.8%) or some disadvantages in terms of time requirements, misinterpre-
unstained areas (18.5%). tation of color change or staining patterns, and biopsy-
The rate of H. pylori detection was lower in patients showing associated complications, this technique offers several
diffuse staining compared to those showing regional staining, advantages in terms of direct detection of the infection site,
which may reflect poor acid secretion due to severe gastric histological evaluation, potential area-based quantification of H.
mucosal atrophy. Patchy staining was the most commonly pylori loading, and little mucosal injury. Moreover, this
observed pattern, but this may also reflect false positives endoscopic procedure is a relatively easy method of detecting
10)
resulting from localized injury caused by the catheter or by H. pylori infection and is not harmful to humans . No
high-pressure spray. Alternatively, patchy staining may result complication was observed in the course of this study.
from the leakage of extracellular fluid due to injuries before
endoscopy, or the patient may experience bile reflux before or
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