Candida colonization in these patients and its relationship
with H. pylori infection.
Materials and methods
Patients and sampling
A total of 125 stomach biopsies were collected from
December 2010 to April 2011 from outpatients with gas-
trointestinal diseases in BuuDien Hospital, Hanoi. These
included 27 antrum and 26 corpus biopsies from 27 patients
with gastric bleeding, 10 pairs of antrum and corpus
samples from 10 gastritis patients, and 52 antrum biopsies
from 52 patients with duodenal ulcers. Ten stomach biop-
sies were also collected from 10 healthy volunteers to serve
as controls. After collection, biopsies were immediately
snap-frozen in liquid nitrogen and stored at ? 80 ° C until
processed. The study protocol was approved by local insti-
tutional review boards and all subjects provided written
Culture isolation and H. pylori identifi cation
Frozen gastric biopsies were homogenized in 300 μ l sterile
1 ? PBS, from which an aliquot of 30 μ l of homogenate
was inoculated onto non-selective blood agar TSA, and
incubated under anaerobic condition (BBL GasPak ™
Received 8 October 2012 ; Received in fi nal revised form 1 April 2013 ;
Accepted 7 May 2013 .
Duong Thu Huong and Yanan Zhao contributed equally to this paper.
Correspondence: David S. Perlin, Public Health Research Institute,
UMDNJ-New Jersey Medical School, 225 Warren Street, Newark,
NJ 07103, USA. Tel.: ? 1 973 854 3200; fax: ? 1 973 854 3101;
Candida krusei colonization in patients with
DUONG THU HUONG ∗ , YANAN ZHAO † , NGUYEN THI NGUYET ∗ , TA THI LOAN ∗ ,
NGUYEN THI THANH BINH ∗ , NGUYEN VAN THINH ‡ , NGUYEN THI HONG HANH ∗ ,
GUILLERMO I. PEREZ-PEREZ § & DAVID S. PERLIN †
∗ Institute of Biotechnology , Vietnam Academy of Sciences and Technology , Viet Nam , † Public Health Research Institute,
UMDNJ, Newark, NJ, USA, ‡ Hospital Buu Dien, H à N ô. i, Viet Nam, and § New York University School of Medicine ,
New York , NY , USA
A total of 135 stomach samples from patients with gastrointestinal diseases and normal
controls were examined for Helicobacter pylori infection and Candida colonization.
Candida krusei was found in specimens from 20% bleeding, 52% ulcer, and 100%
gastritis patients, whereas H. pylori infection rates were 82%, 35% and 30%, respectively,
for the same groups of patients. C. krusei was not detected in stomach samples from
Keywords Candida krusei , colonization , Helicobacter pylori , gastrointestinal
At least 50% of the world population harbor Helicobacter
pylori in their upper gastrointestinal (GI) tract . The
incidence of its infection is mostly associated with child-
hood, socioeconomic and sanitary conditions and plays an
important role in the pathogenesis of upper GI tract disor-
ders. Meanwhile, Candida colonization of the gastric
mucosa has been considered to be involved with multiple
diseases of GI tract, including chronic gastritis and ulcer-
ous diseases . As such, Candida colonization may be of
clinical signifi cance, not only because it creates a vicious
cycle in which infl ammation and fungal colonization
are mutually promoted, but also because it raises the risk
of invasive candidiasis or candidemia, in which there may
be excessive morbidity and mortality [3,4]. This study
was initially carried out to evaluate the status of H. pylori
infection in Vietnamese patients with GI diseases. Molecu-
lar detection methods were performed later to explore
© 2013 ISHAM DOI: 10.3109/13693786.2013.804215
Medical Mycology November 2013, 51, 884–887
by guest on January 9, 2016
© 2013 ISHAM, Medical Mycology, 51, 884–887
C. krusei colonization in gastrointestinal tract 885
Anaerobic System) at 37 ° C for 3 – 5 days. Single colonies
were selected from the agar surface and purifi ed by sub-
culturing several times prior to DNA extraction and 16S
rRNA sequencing. DNA was extracted using genomic
DNA purifi cation Kit (Fermentas K0722).
H. pylori identifi cation methods included culture, rapid
urease test, PCRs to detect H. pylori biomarkers CagA
, HspA , 23S rRNA  and GlmM , detection by
serum ELISA , or PCR amplifi cation of VacA isotype
 or EPIYA motif of the cagA gene . Positive
H. pylori infection status was indicated by positive culture
or in the case of negative-culture samples, by positive
results from at least two of the tests indicated above.
Molecular detection of Candida colonization
Molecular beacon (MB) real-time PCR assays, including
pan- Candida PCR and species-specifi c PCRs for
Candida albicans , Candida glabrata , Candida krusei ,
Candida parapsilosis and Candida tropicalis , were used
for Candida detection . All probes targeted the 28S ribo-
somal RNA gene. The pan- Candida assay has been
described previously , with the species-specifi c probes
listed in Table 1. Probes were purchased from Biosearch
Technologies, Inc., (Novato, CA, USA) and labeled with
5-carboxyfl uorescein (FAM) at the 5 ′ end and a quencher,
benzoic acid succinimidyl ester (Dabcyl), at the 3 ′ end.
Real-time PCR amplifi cation was performed using the
Stratagene Mx3005P real-time PCR system (Agilent
Technologies, Santa Clara, CA, USA).
The demographic characteristics of patients are listed on
Table 2. H. pylori infection (Table 3) was detected in
81.5% (22/27) of patients with gastric bleeding, which is
signifi cantly higher than that noted in 34.6% (18/52;
P ? 0.001) of duodenal ulcer patients and 30% (3/10;
P ? 0.001) of gastritis patients. In contrast, all samples
from controls were negative for H. pylori infections-
positive. The most common species among 32 non-
H. pylori bacteria strains recovered from gastric bleeding
patients, were Streptococcus spp. ( S. australis , S. infantis )
and Bacillus spp. ( B. licheniformis , B. subtilis , B. cereus ,
B. megaterium ). The remaining non- H. pylori bacterial iso-
lates were identifi ed as Ochrobactrum intermedium , Rothia
mucilaginosa and R. dentocariosa . Only H. pylori isolates
were recovered from gastritis patients. Non- H. pylori
bacteria isolated from specimens collected from controls
included Acinetobacter spp., Mycobacterium spp. and
Using pan- Candida real-time PCR, 63% (17/27) antrum
and 50% (13/26) corpus samples from gastric bleeding
patients, 100% (10/10) antrum and 100% (10/10) corpus
samples from gastritis patients, and 77% (40/52) of
the antrum specimens from duodenal ulcer patients, were
found to be colonized by Candida spp. In contrast,
only 30% (3/10) of the samples from the control patients
were positive for Candida spp. Unexpectedly, C. krusei
was the only Candida species found in the specimens in
Table 1 Primers and probes used for species-specifi c Candida identifi cation.
SpeciesPrimer/probeSequences (5 ′ – 3 ′ )
C. albicans Forward primer
Table 2 Demographic characteristics of the patients.
( n ? 27)
( n ? 10)
( n ? 52)
( n ? 10)
Sex: male (%)
a In bleeding group, one patient had a stomach pH ? 4. b In gastric group,
three patients had a stomach pH ? 4. NT, not tested.
36.6 (25 – 57) 30.5 (21 – 36) 46.5 (18 – 71) 32.8 (20 – 49)
2 (20.0)23 (44.2)3 (30.0)
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© 2013 ISHAM, Medical Mycology, 51, 884–887
886 Huong et al.
was found in 22% (6/27) of gastric bleeding patients, 52%
(27/52) of patients with duodenal ulcers, and 100% (10/10)
of gastritis patients, with lower colonization rates for
corpus samples as compared to antrum specimens. The
level of C. krusei colonization was also lower in gastric
bleeding patients, who had the highest H. pylori infection
rate, compared to those with ulcer and gastritis (Figure 1).
The stomach microbiota is in a continual state of fl ux
determined by a variety of factors, such as age, diet,
hormonal state, health, and personal hygiene. Many
microorganisms are washed from the mouth into the stom-
ach, where most microorganisms are killed as a result of
the low pH (2 ∼ 3) environment. In adults with normal
acid secretion, the only organisms present in this micro-
biota are acid-tolerant bacteria, such as the lactic acid
producing bacteria Lactobacillus , Streptococcus and
H. pylori .
which 58% (52/90) of pan- Candida positive samples
from the three GI diseases groups were positive for this
species and the remaining samples were negative for all
species tested. The three pan- Candida positive samples
from control group did not elicit any detectable signal in
species-specifi c detection tests. The presence of C. krusei
Fig. 1 Candida krusei real-time PCR threshold cycle (Ct) value distribution by of disease category and anatomic position. Horizontal bars represent
Table 3 Helicobacter pylori infection status by patient group.
H. pylori infection status a
a Positive H. pylori infection status was determined by positive culture or
by at least two positive molecular detection results in the case of culture-
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© 2013 ISHAM, Medical Mycology, 51, 884–887 Download full-text
C. krusei colonization in gastrointestinal tract 887
Candida species colonizing the human GI tract are con-
sidered a component of the resident fl ora, and C. albicans
is the most common species recovered from such circum-
stances . However, C. albicans was not detected in the
present study, possibly because the levels of the yeast
in the stomach were too low to reach the detection limit
of the C. albicans -specifi c real-time PCR assay . Instead,
C. krusei seemed to be the best adapted to survive and
to establish itself as the predominant fungal colonizer in
the stomach microbiota of patients with three types of gas-
tric conditions. We cannot rule out the possibility that
other Candida species were present, but may not have
been detected by our assays due to the complexity of the
mixed organisms, as well as the limited spectrum of
the assays used in this study. However, the high (48%)
C. krusei colonization rates in patients with GI diseases is
unexpected and of clinical signifi cance given that C. krusei
is an emerging fungal nosocomial pathogen producing a
spectrum of clinical manifestations and intrinsically resis-
tant to fl uconazole .
H. pylori competes with other microbes in the stomach
microbiota. In this study, C. krusei colonization was
detected in 52% of ulcer and 100% of gastritis patients,
whose H. pylori infection rates were only 35% and 30%,
respectively. In contrast, 82% of gastric bleeding patients
had H. pylori infections but only 20% these individuals
were found to be C. krusei positive. Moreover, C. krusei
colonization in the absence of H. pylori infection was
found in 7/7 (100%) of gastritis and 14/34 (41%) of
ulcer patients, but was not seen in samples from normal
controls, indicating a possible causative role of C. krusei
in gastritis and peptic ulcer. In fact, the contributing role
of C. albicans in gastritis development in mice with altered
microbiota , and the etiologic role of C. tropicalis in
gastric ulcer in patients without H. pylori infection ,
has been reported recently. Nevertheless, more detailed
and proactive investigations are warranted to further study
the predominance of C. krusei colonization observed in the
In summary, C. krusei was found to be the predominant
fungal colonizer in Vietnamese patients with gastrointesti-
nal diseases. The exact role of this opportunistic Candida
pathogen in GI tract diseases and its interplay with other
GI microbiota components needs further exploration.
Declaration of interest: The authors report no confl icts of
interest. The authors alone are responsible for the content
and the writing of the paper.
This work was supported by research project no.
50/2010/HD-NDT from the Ministry of Sciences and
Technology, Vietnam, and research project RO1 DK090989
funded by the National Institutes of Health (NIH),
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