Submit a Manuscript: http://www.wjgnet.

com/esps/ World J Gastroenterol 2015 November 28; 21(44): 12667-12675

Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx ISSN 1007-9327 (print) ISSN 2219-2840 (online)
DOI: 10.3748/wjg.v21.i44.12667 © 2015 Baishideng Publishing Group Inc. All rights reserved.

ORIGINAL ARTICLE

Prospective Study

Evaluation of a multiplex PCR assay for detection of

cytomegalovirus in stool samples from patients with
ulcerative colitis

Saifun Nahar, Atsushi Iraha, Akira Hokama, Ayako Uehara, Gretchen Parrott, Tetsuya Ohira, Masatoshi Kaida,
Tetsu Kinjo, Takeshi Kinjo, Tetsuo Hirata, Nagisa Kinjo, Jiro Fujita

Saifun Nahar, Ayako Uehara, Gretchen Parrott, Tetsu Kinjo, Peer-review started: June 3, 2015
Takeshi Kinjo, Tetsuo Hirata, Jiro Fujita, Department of First decision: July 10, 2015
Infectious, Respiratory, and Digestive Medicine, University of Revised: August 3, 2015
the Ryukyus, Okinawa 903-0215, Japan Accepted: September 28, 2015
Article in press: September 30, 2015
Atsushi Iraha, Akira Hokama, Tetsuya Ohira, Masatoshi Published online: November 28, 2015
Kaida, Nagisa Kinjo, Department of Endoscopy, University of
the Ryukyus, Okinawa 903-0215, Japan

Author contributions: Nahar S and Iraha A designed and

performed the research; Nahar S drafted the manuscript; Abstract
Hokama A and Parrott G reviewed the manuscript; Uehara A, AIM: To evaluate a multiplex PCR assay for the
Parrot G, Ohira T, Kaida M, Kinjo T, Kinjo T, Hirata T and detection of bacterial and viral enteropathogens in stool
Kinjo N provided technical support; and Hokama A and Fujita J samples from patients with ulcerative colitis (UC).
supervised the project.

Institutional review board statement: All stool samples were METHODS: We prospectively analyzed 300 individuals,
taken after informed consent and ethical permission was obtained including immunocompetent patients, immunocom­
from patients participating in the study. promised patients, and patients with UC. Stool samples
were collected from the recto-sigmoid region of the
Conflict-of-interest statement: To the best of our knowledge, colon by endoscopy. The samples were qualitatively
no conflict of interest exists. analyzed for bacterial and viral enteropathogens with
®
a multiplex PCR assay using a Seeplex Kit. Additional
Open-Access: This article is an open-access article which was clinical and laboratory data were collected from the
selected by an in-house editor and fully peer-reviewed by external
medical records.
reviewers. It is distributed in accordance with the Creative
Commons Attribution Non Commercial (CC BY-NC 4.0) license,
which permits others to distribute, remix, adapt, build upon this RESULTS: A multiplex PCR assay detected 397
work non-commercially, and license their derivative works on pathogens (191 bacteria and 206 viruses) in 215
different terms, provided the original work is properly cited and samples (71.7%). The most frequently detected
the use is non-commercial. See: http://creativecommons.org/ bacteria were Escherichia coli H7, 85 (28.3%); followed
licenses/by-nc/4.0/ by Aeromonas spp., 43 (14.3%); and Clostridium
perfringens , 36 (12.0%) samples. The most prevalent
Correspondence to: Akira Hokama, MD, PhD, Clinical viruses were Epstein-Barr virus (EBV), 90 (30.0%);
Professor, Department of Endoscopy, University of the Ryukyus,
followed by human herpes virus-6 (HHV-6), 53 (17.7%);
207 Uehara, Nishihara, Okinawa 903-0215,
Japan. hokama-a@med.u-ryukyu.ac.jp and cytomegalovirus (CMV), 37 (12.3%) samples.
Telephone: +81-98-8951144 The prevalence rate of CMV infection was significantly
Fax: +81-98-8951414 higher in the immunocompromised group than in the
immunocompetent group (p < 0.01). CMV infection
Received: June 1, 2015 was more common in patients with UC (26/71; 36.6%)

WJG|www.wjgnet.com 12667 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

than in the immunocompetent patients excluding UC and 15.8%-34% of patients with inflammatory bowel
(6/188; 3.2%) (p < 0.01). CMV infection was more disease (IBD) who are treated with steroids and/or
prevalent in UC active patients (25/58; 43.1%) than [2]
other immunosuppressive drugs . The eyes, lungs,
in UC inactive patients (1/13; 7.7%) (p < 0.05). central nervous system, liver and intestine are the
Among 4 groups which defined by the UC activity and primary target organs for CMV infection. Typically,
immunosuppressive drugs, the prevalence rate of CMV individuals who are infected with CMV remain
infection was highest in the UC active patients with asymptomatic, but the infection may manifest with
immunosuppressive drugs (19/34; 55.8%). Epstein- mild mononucleosis-like symptoms. CMV, like other
Barr virus (EBV) infection was more common in the herpes viruses, persists in a life-long latency coupled
immunocompromised patients excluding UC (18/41; with a risk of intermittent reactivation in some
43.9%) than in the immunocompetent patients situations, such as the following: recipients of solid
excluding UC (47/188; 25.0%) (p < 0.05). The organ transplants, patients undergoing hemodialysis,
simultaneous presence of CMV and EBV and/or HHV6 patients with HIV, and patients who are treated with
in UC active patients (14/58; 24.1%) was greater than steroids and other immunosuppressive drugs.
in immunocompromised patients excluding UC (5/41; Typically, enteric infections are self-limiting and
12.2%) (p < 0.05). acute, but serious illness can occur in immuno­
compromised patients. The number of immuno­
CONCLUSION: The multiplex PCR assay that was used
compromised patients has been increasing dramatically
to analyze the stool samples in this study may serve as
in recent years due to the increased number of
a non-invasive approach that can be used to exclude
organ transplants, increased numbers of patients on
the possibility of CMV infection in patients with active
hemodialysis, or infected with HIV, and widespread
UC who are treated with immunosuppressive therapy.
use of immunosuppressive drugs and steroids. Due
Key words: Polymerase chain reaction; Ulcerative to defective or altered cellular and humoral immunity,
colitis; Cytomegalovirus; immunosuppressive drugs; immunocompromised patients are more susceptible to
Epstein-Barr virus infections. Any infection has the possibility to cause an
[3,4]
overwhelming disease in these populations .
© The Author(s) 2015. Published by Baishideng Publishing Patients with IBD such as ulcerative colitis (UC) and
Group Inc. All rights reserved. Crohn’s disease (CD) are often immunosuppressed.
Patients with severe, steroid-refractory or steroid-
Core tip: Infection with cytomegalovirus (CMV) dependent states, as well as those treated with other
can cause exacerbation of ulcerative colitis (UC). biologic therapies undergo even more intensive
Thus, early diagnosis of CMV infection is important. immunosuppression. Together with the disease activity,
Although endoscopic biopsy is the best approach these factors may contribute to the increased risk of
for the diagnosis of CMV infection, this procedure colonic reactivation of latent CMV or CMV reinfection
[5]
may be invasive for patients and damaging to the in patients with UC . CMV can cause the exacerbation
inflamed intestine. Our prospective study on the use of UC, particularly in those with steroid-dependent/
of the qualitative multiplex PCR assay in stool samples steroid- refractory diseases
[2,6-8]
. Histopathology and
revealed that CMV infection is significantly more the identification of CMV DNA in colonic tissue by
prevalent in UC active patients with immunosuppressive PCR or immunohistochemistry were recommended
drugs. The multiplex PCR assay for stool samples may as the gold standard diagnostic tool for the diagnosis
prove useful, non-invasive method to exclude the of CMV infection in immunosuppressed groups by
presence of CMV infection in patients with active UC the European Crohn’s and Colitis Organization in
who are treated with immunosuppressive drugs. [9]
2009 . Although histopathology may be the most
specific diagnostic approach, a biopsy is an invasive
procedure that requires an endoscopic examination. In
Nahar S, Iraha A, Hokama A, Uehara A, Parrott G, Ohira T, patients with UC, the inflamed colonic tissue is friable,
Kaida M, Kinjo T, Kinjo T, Hirata T, Kinjo N, Fujita J. Evaluation which leads to an increased risk of hemorrhage or
of a multiplex PCR assay for detection of cytomegalovirus [10]
perforation during invasive procedures . In addition,
in stool samples from patients with ulcerative colitis. World J
an extended length of time is required to obtain results
Gastroenterol 2015; 21(44): 12667-12675 Available from: URL:
of a histopathological analysis, and during this time, a
http://www.wjgnet.com/1007-9327/full/v21/i44/12667.htm DOI: [11]
given patient’s condition may deteriorate clinically .
http://dx.doi.org/10.3748/wjg.v21.i44.12667
Molecular diagnostic tools based on the PCR method,
have been developed to improve the detection of enter­
[12-14]
opathogens . Of late, additional advances have
been made to simultaneously identify enteropathogens
INTRODUCTION using multiplex real-time PCR
[15-18]
, but these methods
Cytomegalovirus (CMV), a double-stranded enveloped can be low-throughput and expensive. Therefore, the
DNA virus and a member of β-herpesviridae family, implementation of a rapid, sensitive, powerful, and
[1]
commonly infects 40%-100% of adult populations non-invasive molecular tool is necessary to determine

WJG|www.wjgnet.com 12668 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

the etiology of diarrhea in UC patients. Only then will it contained 5 μl of the Meningitis ACE NC and PC,
be possible to provide early and specific interventions respectively. DNA amplification was performed in an
for the prevention and control of infections in both Eppendorf Mastercycler under the following conditions:
individuals and the community. an initial denaturation at 94 ℃ for 15 min, 94 ℃ for 30
The aim of this prospective study was to evaluate s followed by 40 cycles at 63 ℃ for 1.5 min, 72 ℃ for
the feasibility of a qualitative multiplex PCR assay of 1.5 min with a final cycle at 72 ℃ for 10 min and then
stool for the simultaneous detection of bacterial and a hold at 4 ℃.
viral enteropathogens, focusing on CMV infection In regards to the Diarrhea B1/B2 ACE Detection
for adult patients, including patients with UC, immu­ assay, the multiplex PCR was performed in a 20-μL
nocompetent patients, and immunocompromised volume that contained 4 μL of 5 × DB1 PM, 3 μL of
patients. 8-MOP solution, 10 μL of 2 × master mix and 3 μL of
nucleic acid template. Negative and positive controls
samples were included in each PCR reaction and
MATERIALS AND METHODS contained 3 μL of DB1 NC and DB1 PC, respectively,
Study population and specimens instead of nucleic acid.
We prospectively analyzed 300 patients who underwent All multiplex PCR mixtures underwent the same
®
colonoscopy at the Department of Endoscopy at amplification conditions shown above. The Seeplex
the University of the Ryukyus Hospital from August Meningitis V1 kit is able to detect five pathogens in
2014 to January 2015. Stool samples were collected a single reaction tube including CMV, human herpes
endoscopically from the recto-sigmoid region of virus-6 (HHV-6), Epstein-Barr virus (EBV), herpes
the colon and were transported to the laboratory. simplex virus type 1 (HSV-1), HSV-2, and varicella
®
Stool samples were immediately processed for the zoster virus (VZV). The Seeplex Diarrhea B1 & B2 ACE
multiplex PCR. Additional clinical and laboratory data detection assay permits the simultaneous amplification
were collected from medical records. UC activity was of the target DNA of the following: Salmonella
[19]
assessed using the Mayo scoring system . spp. (S. bongori and S. enterica), Shigella spp. (S.
flexneri, S boydii, S. sonnei, and S. dysenteriae),
DNA extraction Vibrio spp. (V. cholerae, V. parahaemolyticus, and V.
DNA was extracted using Ribospin™ vRD kit (GeneAll vulnificus), Clostridium difficile toxin B, Campylobacter
Biotechnology, Seoul, South Korea) according to spp. (C. jejuni and C. coli), Clostridium perfringens
manufacturer’s instructions. Briefly, 300 µL of endo­ toxin, Yersinia enterocolitica, Aeromonas spp.
scopically collected stool was transferred into a (A. salmonicida, A. sobria, A. bivalvium, and A.
1.5-ml microcentrifuge tube followed by the addition hydrophila), Escherichia coli O157: H7, and Verotoxin-
of 500 µl of buffer to lyse the fecal matter. After an producing E. coli (VTEC). In total, we were able to test
incubation of 10-15 min at room temperature, 700 µl for 25 pathogens simultaneously.
of nucleic binding buffer was added; the solution was
then vortexed and mixed. The resultant solution was Analysis of PCR product
transferred to a mini spin column and was centrifuged PCR products were analyzed by microchip electro­
at 10000 g for 30-60 s. Total DNA was bound to the phoresis system using the DNA-1000 Reagent kit in
glass fiber membrane while the remaining impurities the MCE-202 MultiNA machine (Shimadzu, Japan).
on the membrane were removed by two successive The data was analyzed using MultiNA viewer software
wash buffers. Pure DNA was eluted to a final volume (Shimadzu, Japan).
of 50 µL of nuclease-free water. All procedures were
performed at room temperature. The extracted DNA Ethical considerations
was stored at 4 ℃ for immediate use or at -20 ℃ for This prospective study was approved by the
long-term use. institutional review board of the University of the
Ryukyus, and written informed consent was obtained
PCR amplification from all patients prior to their inclusion in the study.
The multiplex PCR was performed according to
the manufacturer’s instructions using a Seeplex Statistical analysis
®
Meningitis V1, Diarrhea B1 and Diarrhea B2 ACE Statistical comparisons were conducted by two-tailed
detection kits (Seegene, Seoul, South Korea). In χ 2 test with Yates’ correction using SPSS (version 21.0,
regards to the Meningitis V1 ACE Detection assay, SPSS Inc., Chicago, IL, United States). A P value < 0.05
multiplex PCR was performed in a 20-μL total volume, was considered statistically significant.
which included 2 μL of each 10 × MV1 PM and 8-MOP
solution, 1 μL of the Meningitis ACE internal control,
10 μL of the 2 × multiplex master mix and 5 μL of RESULTS
nucleic acid template. Negative and positive control Study population
samples were included in every PCR procedure and The clinical characteristics of the included patients

WJG|www.wjgnet.com 12669 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

100
Table 1 Demographic and clinical characteristics of the
patients n (%) CMV positive
80
Characteristics Number of patients 300 (100)

Prevalence rate (%)

Gender 60
Male 177 (59) P < 0.01
Female 123 (41)
Age in years 59.04 (16-86 ) 40
25/80
Underlying disease
Immunocompromised 41 (13.7)
Ulcerative colitis 71 (23.7) 20
One or more bacteria/viruses detected 215 (71.7) 12/220
One or more bacteria detected 146 (48.7)
Number of bacteria detected 0
Immuno-competent Immuno-compromised
1 108 (36.0)
patients patients
2 33 (11.0)
3 5 (1.67)
One or more viruses detected 148 (49.3) Figure 1 Comparison of prevalence rate of cytomegalovirus infection. The
Number of viruses detected prevalence rate was significantly higher in the immunocompromised patients
1 96 (32.0) than in the immunocompetent patients. CMV: Cytomegalovirus.
2 45 (15.0)
3 7 (2.3)
Bacteria detected detected. The most prevalent viruses were EBV, 91
Clostridium difficile toxin B 15 (5.0) (30.3%); followed by HHV-6, 53 (17.7%); and CMV,
Salmonella spp. 4 (1.3) 37 (12.3%). The least prevalent viruses were HSV2,
Campylobacter spp. 3 (1.0)
Vibrio spp. 1 (0.3)
6 (2.0%) and VZV, 6 (2.0%). Internal control was
Clostridium perfringens 36 (12.0) positive for all samples.
E. coli H7 85 (28.3) Patients who were treated with immunosuppressive
E. coli O157 1 (0.3) drugs (e.g., anticancer drugs, T-cell inhibitors,
VTEC 1 (0.3)
steroids), HIV patients, and patients on hemodialysis
Aeromonas spp. 43 (14.3)
Virus detected for chronic kidney disease were included in the
CMV 37 (12.3) immunocompromised group (80; 26.7%). Patients
HHV6 53 (17.7) who were not treated with immunosuppressive drugs
EBV 91 (30.3) were incorporated into the immunocompetent group
HSV1 14 (4.7)
HSV2 6 (2.0)
(220; 73.3%). The prevalence rate of CMV infection
VZV 6 (2.0) was significantly higher in the immunocompromised
group than in the immunocompetent group (p <
E. coli: Escherichia coli; VTEC: Verotoxin-producing E. coli; CMV: 0.01) (Figure 1). CMV infection was more common
Cytomegalovirus; EBV: Epstein-Barr virus; HHV6: Human herpes virus-6; in patients with UC (26/71; 36.6%) than in the
VZV: Varicella zoster virus.
immunocompetent patients excluding UC (6/188;
3.2%) (p < 0.01) (Figure 2).
are summarized in Table 1. Three hundred patients In UC patients, CMV infection was more prevalent
were enrolled, including 41 immunocompromised in UC active patients (25/58; 43.1%) than in UC
patients and 71 patients with UC. The average age inactive patients (1/13; 7.7%) (p < 0.05) (Figure 3).
of the patients was 59.04 years (range 16-86 years), UC patients were further categorized into 4 groups
and there were more males (59%) than females. All based on the UC activity and the administration
of the UC active patients had diarrhea. A total of 300 of immunosuppressive drugs. The prevalence rate
samples collected by colonoscopy were examined. of CMV infection was highest among individuals
Among the 300 samples, multiplex PCR showed a in the active UC group who were treated with
positive reaction in 215 samples (71.7%) (Table immunosuppressive drugs compared with individuals
1). Among the 300 samples, one or more bacteria in the other 3 groups (Figure 4). Among the 58
and viruses were identified in 146 (48.7%) and 148 patients with active UC, immunosuppressive drugs
(49.3%) samples, respectively. One bacterium was were prescribed for 34 patients. The CMV infection
detected in 108 (36.0%) samples. Two and three rate was significantly higher in those who were treated
other types of bacteria were detected in 33 (11.0%) with immunosuppressive drugs (19/34; 55.8%)
and 5 (1.67%) samples, respectively. Ninety six compared with those who were not treated with
(32.0%) had a single viral infection while 45 (15.0%) immunosuppressive drugs (6/24; 25.0%) (p < 0.05).
and 7 (2.3%) had 2 and 3 viruses, respectively. The As for EBV infection, EBV infection was more
most frequently detected bacteria were E. coli H7, 85 common in the immunocompromised patients excluding
(28.3%); followed by Aeromonas spp., 43 (14.3%); UC (18/41; 43.9%) than in the immunocompetent
C. perfringens, 36 (12.0%); and C. difficile Toxin B, 15 patients excluding UC (47/188; 25.0%) (p < 0.05)
(5.0%). Shigella spp. and Y. enterocolitica were not (Figure 5). Figure 6 shows no significant differences

WJG|www.wjgnet.com 12670 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

100 100

CMV positive CMV positive

80 80
Prevalence rate (%)

Prevalence rate (%)

P < 0.01 P < 0.05
60 60

25/58
40 26/71 40
P < 0.05

20 20
5/41
1/13
6/188
0 0
Immuno- Immuno- Ulcerative colitis Ulcerative colitis Ulcerative colitis
competent compromised patients inactive patients active patients
patients excluding patients excluding
ulcerative colitis ulcerative colitis Figure 3 Comparison of prevalence rate of cytomegalovirus infection. The
prevalence rate was significantly higher in ulcerative colitis active patients than
Figure 2 Comparison of prevalence rate of cytomegalovirus infection in in ulcerative colitis inactive patients. CMV: Cytomegalovirus.
3 groups, including the immunocompetent patients excluding ulcerative
colitis, the immunocompromised patients excluding ulcerative colitis, and
ulcerative colitis patients. CMV: Cytomegalovirus. infection in patients with active UC.
In addition, CMV infection is significantly correlated
with the administration of immunosuppressive drugs
of the prevalence rate of HHV6 infection among the
among the patients with active UC (p < 0.05). UC
groups. The overlapping prevalence patterns for CMV,
patients who were treated with immunosuppressive
EBV, and HHV6 are shown for UC active patients (Figure
drugs such as corticosteroids, tacrolimus, azathioprine,
7) and the immunocompromised patients excluding UC
6-mercaptopurine, or cyclosporine A, and those
(Figure 8). The simultaneous presence of CMV and EBV
with existing inflammation are considered to be
and/or HHV6 in UC active patients (14/58; 24.1%)
at an increased risk for the development of CMV
was greater than in immunocompromised patients [22-24]
infection . Although some studies have suggested
excluding UC (5/41; 12.2%) (p < 0.05). No patients in
that CMV infection may be an occasional finding or
our cohort were simultaneously infected with all three [22,25,26]
that CMV may be an inactive participant in UC ,
viruses. CMV itself can be a major cause of exacerbation of
UC. As a result, CMV subsequently leads to the worst
[27]
DISCUSSION clinical conditions seen among patients with UC .
Therefore, an early diagnosis of CMV infection in UC is
Multiplex PCR has been widely applied to the crucial, and several modalities have been developed
[18]
diagnosis of gastrointestinal infectious diseases . for the diagnosis of CMV infection.
®
Notably, the Seeplex Diarrhea ACE Detection assay Currently, the guidelines of the European Colitis and
has shown itself to be a rapid, sensitive, specific, Crohn’s Organization recommend the combined use of
and reliable diagnostic tool for the direct detection hematoxylin-eosin (HE) staining, immunohistochemical
of the most common enteropathogens in stool (IHC) staining with a CMV-specific monoclonal
[20]
samples . Other multiplex PCR assays (e.g., the antibody and PCR for CMV in colonic tissue for the
FilmArray gastrointestinal panel and Luminex xTag detection of colonic CMV infection in patients with
gastrointestinal pathogen panel) have also yielded an [9]
UC . IHC staining has a higher sensitivity than
increased percent positive rate compared with routine conventional histology (78%-93%) for the detection of
[21]
testing . Based on these recent developments, CMV in colonic tissue .
[28]

we have conducted this prospective assay with the Several studies have reported that the quantitative
®
Seeplex Diarrhea and Meningitis Detection method. real-time PCR technique is a highly sensitive method
We have analyzed (1) the results of a multiplex PCR for the diagnosis of CMV infection in inflamed colonic
assay as a rapid and non-invasive molecular diagnostic tissue compared with non-inflamed colonic tissue in the
tool for the early diagnosis of CMV infection in patients [29]
setting of UC . The detection of CMV DNA in mucosal
with UC; (2) the prevalence rate of CMV infection in biopsies by PCR analysis has been regarded as the
UC in comparison with control populations; and (3) the most sensitive assay for the diagnosis of CMV infection
relationship of CMV infection with immunosuppressive [30]
of the intestinal tract . However, this technique
drugs and disease activity. We have found that CMV requires invasive procedures such as sigmoidoscopy
infection is significantly more prevalent in the active or colonoscopy to collect biopsy material and in some
UC group compared with the immunocompetent group instances a physician may not be able to perform a
and the immunocompromised group (p < 0.05), colonoscopy. Furthermore, inflamed colonic tissue in
which further indicates the strong association of CMV cases of UC is mostly friable, edematous, and eroded

WJG|www.wjgnet.com 12671 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

100 CMV positive

80
P < 0.05
Prevalence rate (%)

60 19/34

40

6/24
1/5
20

0/8
0
Ulcerative colitis Ulcerative colitis inactive Ulcerative colitis Ulcerative colitis active
inactive patients with patients without active patients with patients without
immunosuppressive drugs immunosuppressive drugs immunosuppressive drugs immunosuppressive drugs

Figure 4 Comparison of prevalence rate of cytomegalovirus infection in 4 groups, including, ulcerative colitis inactive patients with immunosuppressive
drugs, ulcerative colitis inactive patients without immunosuppressive drugs, ulcerative colitis active patients with immunosuppressive drugs, and
ulcerative colitis active patients without immunosuppressive drugs. CMV: Cytomegalovirus.

100 100

EBV positive HHV6 positive

80 80
Prevalence rate (%)

Prevalence rate (%)

P < 0.05
60 60

18/41
40 26/71 40
47/188 10/41
35/188
20 20
8/71

0 0
Immuno- Immuno- Ulcerative colitis Immuno- Immuno- Ulcerative colitis
competent compromised patients competent compromised patients
patients excluding patients excluding patients excluding patients excluding
ulcerative colitis ulcerative colitis ulcerative colitis ulcerative colitis

Figure 5 Comparison of prevalence rate of Epstein-Barr virus infection in Figure 6 Comparison of prevalence rate of human herpes virus-6
3 groups, including the immunocompetent patients excluding ulcerative infection in 3 groups, including the immunocompetent patients excluding
colitis, the immunocompromised patients excluding ulcerative colitis, and ulcerative colitis, the immunocompromised patients excluding ulcerative
ulcerative colitis patients. EBV: Epstein-Barr virus. colitis, and ulcerative colitis patients. HHV6: Human herpes virus-6.

and bleeds when touched. As a result, there is a blood for the detection of CMV in patients with UC
high risk of bleeding during the collection of tissues. and showed lower sensitivities for the diagnosis of
[10]
Additionally, endoscopic biopsy may lead to false- CMV infection compared with a previous study . The
negative results due to sampling bias. To reduce presence of a CMV infection in the intestine cannot be
sampling error, tissues in different locations along ruled out in the case of a negative CMV antigenemia
the colon are needed. Therefore, serology, a CMV assay. In many cases, additional testing will be
antigenemia assay and PCR of blood samples for CMV required to reach the diagnosis of CMV infection. The
[11,31]
are more convenient than invasive procedures . CMV antigenemia assay also has limited clinical value
Serological tests, such as the detection of CMV- in the prediction of the reactivation of CMV infection in
[33]
specific IgM antibodies, have been shown to have a the gastrointestinal tract and is therefore ineffective
[32]
100% sensitivity and a 99% specificity . However, at preventing the development of CMV colitis.
the presence of IgM antibodies can only reveal acute The CMV antigenemia assay is also relatively time
infection, and as such, cases of reactivation are often consuming and demands expert pathologists to reduce
undiagnosed. Therefore, serology also has limited subjective bias during interpretation of the slides.
65
value in the diagnosis of CMV infection in patients with As the pp antigen for the CMV antigenemia assay
UC. is examined in blood leukocytes, it may also reveal
[11] [27]
A recent study evaluated the diagnostic per­ false-negative results in leucopenic patients . The
formance of a CMV antigenemia assay and PCR of diagnostic accuracy of CMV antigenemia may depend

WJG|www.wjgnet.com 12672 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

HHV6 HHV6

37.9% negative 41.5% negative

for all viruses for all viruses
5.2% 9.7%

1.7% 6.9% 4.9% 9.7%

0% 0%
CMV EBV CMV EBV

19% 22.4% 6.9% 0% 7.3% 26.8%

n = 58 n = 41

Figure 7 Overlapping prevalence patterns for cytomegalovirus, Epstein- Figure 8 Overlapping prevalence patterns for cytomegalovirus,
Barr virus, and human herpes virus-6 infection in active ulcerative colitis Epstein-Barr virus, and human herpes virus-6 infection in the immuno­
patients. CMV: Cytomegalovirus; EBV: Epstein-Barr virus; HHV6: Human compromised patients excluding ulcerative colitis. CMV: Cytomegalovirus;
herpes virus-6. EBV: Epstein-Barr virus; HHV6: Human herpes virus-6.

[34]
on site of organ/tissue involvement . Under these prospective study that assesses the multiplex PCR
circumstances, the testing of stool samples by PCR assay as a diagnostic tool for the diagnosis of CMV in
was chosen as an alternative to these techniques. patients with active UC, and in both immunocompetent
We have found advantages in the detection of CMV and immunocompromised patients. As for EBV and
DNA in stool samples by the multiplex PCR technique. HHV6, this study has showed a possible synergistic
Although this prospective study was designed so that role for these viruses in the pathogenesis in active UC
[38,39]
fresh samples would be prepared from stool samples activation, as suggested by the prior reports .
obtained by endoscopy, stool analysis is traditionally a Our study has some limitations. First, we did
non-invasive procedure. The procurement of tissues by not quantify the CMV DNA. However, in patients
biopsy may have potential risks when the intestine is with UC, any positive result with respect to CMV is
inflamed. In addition, the stool may better reflect the clinically significant without quantification of the viral
CMV enterocolitis that is located in the proximal colon load. Quantification of CMV in stool specimens is not
and small intestine. We are planning to analyse stool feasible during specimen processing (e.g., diluted
[40]
samples which are collected by the patients at home. versus non-diluted) , and positive results should be
Several studies have evaluated CMV DNA in carefully considered, especially in patients with UC
[26,35,36] [37] [37]
stool samples . Ganzenmueller et al have who are treated with immunosuppressive drugs .
retrospectively evaluated quantitative real-time PCR Furthermore, this prospective study was not designed
in 66 fecal samples and intestinal biopsies for the to compare the different modalities for the detection
diagnosis of CMV intestinal disease. Their study also of CMV, including histopathology, CMV antigenemia
[36]
compared CMV DNA in stool and colonic biopsies with assay, serology, and tissue CMV PCR. Herfarth et al
the results of histopathology and the CMV antigenemia have shown that the sensitivity and specificity of the
assay. In their study, the sensitivity and specificity stool PCR analysis compared with PCR in mucosal
of stool CMV DNA for the diagnosis of CMV intestinal biopsies were 83% and 93%, respectively. They have
disease are 67% and 96%, respectively. Therefore, noted that it is not clear whether CMV DNA detected
CMV DNA in the stool is more sensitive and specific in the stool samples was due to leakage from the
than the gold standard method (histopathology and blood compartment into the intestinal tract, or if it was
IHC) for the diagnosis of CMV in inflamed colonic derived from intestinal CMV infection.
tissue. In conclusion, this prospective study proposes
Additional studies have evaluated the sensitivity, multiplex PCR as a successful, non-invasive diagnostic
specificity and reliability of the multiplex PCR assay as technique for rapid detection of CMV infection among
a rapid molecular diagnostic tool for the investigation UC patients in clinical in-patient and out-patient
of the etiology of enteric infections in pediatric settings. Additionally, we present a new protocol for
[20]
patients . However, the diagnostic significance of the the broad analysis of the enteropathogens in stool
multiplex PCR assay in stool samples for the diagnosis samples in adult populations. This method will also
of CMV infection in patients with UC has not yet been help predict CMV infection prior to the development
evaluated. In our prospective study, we evaluated of intestinal symptoms, which is important for the
the ability of a commercially available multiplex PCR prevention of exacerbation of UC by CMV reactivation.
assay to diagnose CMV infection in UC using stool Positive PCR results may help to rapidly diagnose
samples. To our knowledge, this is the first large-scale patients at a high risk for CMV infection. Further

WJG|www.wjgnet.com 12673 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

studies are needed to confirm these results. Future 519-524 [PMID: 12588074]
studies that involve colonic biopsies will be needed to 5 McCurdy JD, Jones A, Enders FT, Killian JM, Loftus EV, Smyrk
TC, Bruining DH. A model for identifying cytomegalovirus in
confirm the reliability of the multiplex PCR test results.
patients with inflammatory bowel disease. Clin Gastroenterol
Hepatol 2015; 13: 131-137; quiz e7 [PMID: 24993369 DOI:
10.1016/j.cgh.2014.05.026]
ACKNOWLEDGMENTS 6 Cottone M, Pietrosi G, Martorana G, Casà A, Pecoraro G, Oliva
The authors are grateful to clinical staff in the L, Orlando A, Rosselli M, Rizzo A, Pagliaro L. Prevalence of
cytomegalovirus infection in severe refractory ulcerative and
Department of Infectious, Respiratory, and Digestive
Crohn’s colitis. Am J Gastroenterol 2001; 96: 773-775 [PMID:
Medicine and the Department of Endoscopy at the 11280549]
University of the Ryukyus. 7 Domènech E, Vega R, Ojanguren I, Hernández A, Garcia-
Planella E, Bernal I, Rosinach M, Boix J, Cabré E, Gassull MA.
Cytomegalovirus infection in ulcerative colitis: a prospective,
COMMENTS
COMMENTS comparative study on prevalence and diagnostic strategy. Inflamm
Bowel Dis 2008; 14: 1373-1379 [PMID: 18452205 DOI: 10.1002/
Background ibd.20498]
Infection with cytomegalovirus (CMV) can cause exacerbation of ulcerative 8 Maher MM, Nassar MI. Acute cytomegalovirus infection is a risk
colitis (UC). Thus, early diagnosis of CMV infection is important. Although factor in refractory and complicated inflammatory bowel disease.
endoscopic biopsy is the best approach for the diagnosis of CMV infection, this Dig Dis Sci 2009; 54: 2456-2462 [PMID: 19093204 DOI: 10.1007/
procedure may be invasive for patients and damaging to the inflamed intestine. s10620-008-0639-6]
9 Rahier JF, Ben-Horin S, Chowers Y, Conlon C, De Munter P, D’
Research frontiers Haens G, Domènech E, Eliakim R, Eser A, Frater J, Gassull M,
Molecular diagnostic advances have been made to simultaneously identify Giladi M, Kaser A, Lémann M, Moreels T, Moschen A, Pollok R,
enteropathogens using multiplex polymerase chain reaction (PCR). The Reinisch W, Schunter M, Stange EF, Tilg H, Van Assche G, Viget
aim of this prospective study was to evaluate the feasibility of a qualitative N, Vucelic B, Walsh A, Weiss G, Yazdanpanah Y, Zabana Y, Travis
multiplex PCR assay of stool for the simultaneous detection of bacterial and SP, Colombel JF. European evidence-based Consensus on the
viral enteropathogens, focusing on CMV infection for adult patients, including prevention, diagnosis and management of opportunistic infections
patients with UC, immunocompetent patients, and immunocompromised in inflammatory bowel disease. J Crohns Colitis 2009; 3: 47-91
patients. [PMID: 21172250 DOI: 10.1016/j.crohns.2009.02.010]
10 Nagata N, Kobayakawa M, Shimbo T, Hoshimoto K, Yada T,
Gotoda T, Akiyama J, Oka S, Uemura N. Diagnostic value of
Innovations and breakthroughs antigenemia assay for cytomegalovirus gastrointestinal disease in
This is the first large-scale prospective study that assesses the multiplex PCR immunocompromised patients. World J Gastroenterol 2011; 17:
assay in stool samples as a diagnostic tool for the diagnosis of CMV in patients 1185-1191 [PMID: 21448424 DOI: 10.3748/wjg.v17.i9.1185]
with UC. This assay may prove useful, non-invasive method to exclude the 11 Kim JW, Boo SJ, Ye BD, Kim CL, Yang SK, Kim J, Kim SA,
presence of CMV infection in patients with active UC who are treated with Park SH, Park SK, Yang DH, Jung KW, Kim KJ, Byeon JS, Myung
immunosuppressive drugs. SJ, Kim JH. Clinical utility of cytomegalovirus antigenemia
assay and blood cytomegalovirus DNA PCR for cytomegaloviral
Applications colitis patients with moderate to severe ulcerative colitis. J Crohns
This multiplex PCR as a successful, non-invasive diagnostic technique will help Colitis 2014; 8: 693-701 [PMID: 24405983 DOI: 10.1016/
predict CMV infection prior to the development of intestinal symptoms, which is j.crohns.2013.12.014]
important for the prevention of exacerbation of UC by CMV reactivation. 12 de Boer RF, Ott A, Kesztyüs B, Kooistra-Smid AM. Improved
detection of five major gastrointestinal pathogens by use of
a molecular screening approach. J Clin Microbiol 2010; 48:
Terminology 4140-4146 [PMID: 20861334 DOI: 10.1128/JCM.01124-10]
Multiplex PCR assay: a rapid, sensitive, specific, and reliable diagnostic tool for 13 Wolffs PF, Bruggeman CA, van Well GT, van Loo IH. Replacing
the simultaneous detection of bacterial and viral enteropathogens by PCR. traditional diagnostics of fecal viral pathogens by a comprehensive
panel of real-time PCRs. J Clin Microbiol 2011; 49: 1926-1931
Peer-review [PMID: 21430103 DOI: 10.1128/JCM.01925-10]
In this study, the authors detected CMV and other enteric pathogens using 14 Cunningham SA, Sloan LM, Nyre LM, Vetter EA, Mandrekar
multiplex PCR in stool samples collected from 300 patients who underwent J, Patel R. Three-hour molecular detection of Campylobacter,
colonoscopy including patients with UC. Salmonella, Yersinia, and Shigella species in feces with accuracy
as high as that of culture. J Clin Microbiol 2010; 48: 2929-2933
[PMID: 20519461 DOI: 10.1128/JCM.00339-10]
15 Barletta F, Mercado EH, Lluque A, Ruiz J, Cleary TG, Ochoa
REFERENCES TJ. Multiplex real-time PCR for detection of Campylobacter,
1 Staras SA, Dollard SC, Radford KW, Flanders WD, Pass RF, Salmonella, and Shigella. J Clin Microbiol 2013; 51: 2822-2829
Cannon MJ. Seroprevalence of cytomegalovirus infection in the [PMID: 23761159 DOI: 10.1128/JCM.01397-13]
United States, 1988-1994. Clin Infect Dis 2006; 43: 1143-1151 16 Barbut F, Monot M, Rousseau A, Cavelot S, Simon T, Burghoffer
[PMID: 17029132] B, Lalande V, Tankovic J, Petit JC, Dupuy B, Eckert C. Rapid
2 Papadakis KA, Tung JK, Binder SW, Kam LY, Abreu MT, Targan diagnosis of Clostridium difficile infection by multiplex real-time
SR, Vasiliauskas EA. Outcome of cytomegalovirus infections in PCR. Eur J Clin Microbiol Infect Dis 2011; 30: 1279-1285 [PMID:
patients with inflammatory bowel disease. Am J Gastroenterol 21487764 DOI: 10.1007/s10096-011-1224-z]
2001; 96: 2137-2142 [PMID: 11467645] 17 Enserink R, Scholts R, Bruijning-Verhagen P, Duizer E, Vennema
3 Rozenblyum EV, Allen UD, Silverman ED, Levy DM. H, de Boer R, Kortbeek T, Roelfsema J, Smit H, Kooistra-
Cytomegalovirus infection in childhood-onset systemic lupus Smid M, van Pelt W. High detection rates of enteropathogens in
erythematosus. Int J Clin Rheumtol 2013; 8: 137-146 [PMID: asymptomatic children attending day care. PLoS One 2014; 9:
24527062 DOI: 10.2217/ijr.12.82] e89496 [PMID: 24586825 DOI: 10.1371/journal.pone.0089496]
4 Taylor GH. Cytomegalovirus. Am Fam Physician 2003; 67: 18 McAuliffe GN, Anderson TP, Stevens M, Adams J, Coleman

WJG|www.wjgnet.com 12674 November 28, 2015|Volume 21|Issue 44|

 Nahar S et al . Multiplex PCR for CMV in UC

R, Mahagamasekera P, Young S, Henderson T, Hofmann M, Matsuura M, Ohmori K, Sakurai T, Nagayama S, Hasegawa S,

Jennings LC, Murdoch DR. Systematic application of multiplex Sakai Y, Chiba T. Usefulness of quantitative real-time PCR assay
PCR enhances the detection of bacteria, parasites, and viruses in for early detection of cytomegalovirus infection in patients with
stool samples. J Infect 2013; 67: 122-129 [PMID: 23603249 DOI: ulcerative colitis refractory to immunosuppressive therapies.
10.1016/j.jinf.2013.04.009] Inflamm Bowel Dis 2007; 13: 1516-1521 [PMID: 17828781 DOI:
19 Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 10.1002/ibd.20253]
5-aminosalicylic acid therapy for mildly to moderately active 30 Kandiel A, Lashner B. Cytomegalovirus colitis complicating
ulcerative colitis. A randomized study. N Engl J Med 1987; 317: inflammatory bowel disease. Am J Gastroenterol 2006; 101:
1625-1629 [PMID: 3317057] 2857-2865 [PMID: 17026558 DOI: 10.1111/j.1572-0241.2006.00869.
20 Onori M, Coltella L, Mancinelli L, Argentieri M, Menichella D, x]
Villani A, Grandin A, Valentini D, Raponi M, Russo C. Evaluation 31 Chun J, Lee C, Kwon JE, Hwang SW, Kim SG, Kim JS, Jung HC,
of a multiplex PCR assay for simultaneous detection of bacterial Im JP. Usefulness of the cytomegalovirus antigenemia assay in
and viral enteropathogens in stool samples of paediatric patients. patients with ulcerative colitis. Intest Res 2015; 13: 50-59 [PMID:
Diagn Microbiol Infect Dis 2014; 79: 149-154 [PMID: 24656922 25691843 DOI: 10.5217/ir.2015.13.1.50]
DOI: 10.1016/j.diagmicrobio.2014.02.004] 32 Revello MG, Gerna G. Diagnosis and management of human
21 Khare R, Espy MJ, Cebelinski E, Boxrud D, Sloan LM, cytomegalovirus infection in the mother, fetus, and newborn infant.
Cunningham SA, Pritt BS, Patel R, Binnicker MJ. Comparative Clin Microbiol Rev 2002; 15: 680-715 [PMID: 12364375 DOI:
evaluation of two commercial multiplex panels for detection of 10.1128/CMR.15.4.680-715,]
gastrointestinal pathogens by use of clinical stool specimens. J Clin 33 Ganzenmueller T, Henke-Gendo C, Schlué J, Wedemeyer J,
Microbiol 2014; 52: 3667-3673 [PMID: 25100818 DOI: 10.1128/ Huebner S, Heim A. Quantification of cytomegalovirus DNA
JCM.01637-14] levels in intestinal biopsies as a diagnostic tool for CMV intestinal
22 Lawlor G, Moss AC. Cytomegalovirus in inflammatory bowel disease. J Clin Virol 2009; 46: 254-258 [PMID: 19748823 DOI:
disease: pathogen or innocent bystander? Inflamm Bowel Dis 2010; 10.1016/jcv.2009.08.008]
16: 1620-1627 [PMID: 20232408 DOI: 10.1002/ibd.21275] 34 Hamada Y, Nagata N, Shimbo T, Igari T, Nakashima R, Asayama N,
23 Nakase H, Honzawa Y, Toyonaga T, Yamada S, Minami N, Nishimura S, Yazaki H, Teruya K, Gatanaga H, Kikuchi Y, Akiyama
Yoshino T, Matsuura M. Diagnosis and treatment of ulcerative J, Ohmagari N, Uemura N, Oka S. Assessment of antigenemia assay
colitis with cytomegalovirus infection: importance of controlling for the diagnosis of cytomegalovirus gastrointestinal diseases in
mucosal inflammation to prevent cytomegalovirus reactivation. HIV-infected patients. AIDS Patient Care STDS 2013; 27: 387-391
Intest Res 2014; 12: 5-11 [PMID: 25349558 DOI: 10.5217/ [PMID: 23799239 DOI: 10.1089/apc.2013.0115]
ir.2014.12.1.5] 35 Chan KS, Yang CC, Chen CM, Yang HH, Lee CC, Chuang YC,
24 Goodman AL, Murray CD, Watkins J, Griffiths PD, Webster Yu WL. Cytomegalovirus colitis in intensive care unit patients:
DP. CMV in the gut: a critical review of CMV detection in the difficulties in clinical diagnosis. J Crit Care 2014; 29: 474.e1-474.
immunocompetent host with colitis. Eur J Clin Microbiol Infect e6 [PMID: 24556151 DOI: 10.1016/j.jcrc.2014.01.003]
Dis 2015; 34: 13-18 [PMID: 25097085 DOI: 10.1007/s10096-014- 36 Herfarth HH, Long MD, Rubinas TC, Sandridge M, Miller MB.
2212-x] Evaluation of a non-invasive method to detect cytomegalovirus
25 Matsuoka K, Iwao Y, Mori T, Sakuraba A, Yajima T, Hisamatsu T, (CMV)-DNA in stool samples of patients with inflammatory bowel
Okamoto S, Morohoshi Y, Izumiya M, Ichikawa H, Sato T, Inoue disease (IBD): a pilot study. Dig Dis Sci 2010; 55: 1053-1058
N, Ogata H, Hibi T. Cytomegalovirus is frequently reactivated and [PMID: 20165976 DOI: 10.1007/s10620-010-1146-0]
disappears without antiviral agents in ulcerative colitis patients. 37 Ganzenmueller T, Kluba J, Becker JU, Bachmann O, Heim A.
Am J Gastroenterol 2007; 102: 331-337 [PMID: 17156136 DOI: Detection of cytomegalovirus (CMV) by real-time PCR in fecal
10.1111/j.1572-0241.2006.00989.x] samples for the non-invasive diagnosis of CMV intestinal disease.
26 do Carmo AM, Santos FM, Ortiz-Agostinho CL, Nishitokukado J Clin Virol 2014; 61: 517-522 [PMID: 25453330 DOI: 10.1016/
I, Frota CS, Gomes FU, Leite AZ, Pannuti CS, Boas LS, Teixeira j.jcv.2014.10.009]
MG, Sipahi AM. Cytomegalovirus infection in inflammatory bowel 38 Wakefield AJ, Fox JD, Sawyerr AM, Taylor JE, Sweenie CH,
disease is not associated with worsening of intestinal inflammatory Smith M, Emery VC, Hudson M, Tedder RS, Pounder RE.
activity. PLoS One 2014; 9: e111574 [PMID: 25387236 DOI: Detection of herpesvirus DNA in the large intestine of patients with
10.1371/journal.pone.0111574] ulcerative colitis and Crohn’s disease using the nested polymerase
27 Nakase H, Matsumura K, Yoshino T, Chiba T. Systematic review: chain reaction. J Med Virol 1992; 38: 183-190 [PMID: 1287131]
cytomegalovirus infection in inflammatory bowel disease. J 39 Bertalot G, Villanacci V, Gramegna M, Orvieto E, Negrini R,
Gastroenterol 2008; 43: 735-740 [PMID: 18958541 DOI: 10.1007/ Saleri A, Terraroli C, Ravelli P, Cestari R, Viale G. Evidence of
s00535-008-2246-x] Epstein-Barr virus infection in ulcerative colitis. Dig Liver Dis
28 Garrido E, Carrera E, Manzano R, Lopez-Sanroman A. Clinical 2001; 33: 551-558 [PMID: 11816543]
significance of cytomegalovirus infection in patients with 40 Binnicker MJ, Espy ME. Comparison of six real-time PCR assays
inflammatory bowel disease. World J Gastroenterol 2013; 19: for qualitative detection of cytomegalovirus in clinical specimens.
17-25 [PMID: 23326158 DOI: 10.3748/wjg.v19.i1.17] J Clin Microbiol 2013; 51: 3749-3752 [PMID: 24006002 DOI:
29 Yoshino T, Nakase H, Ueno S, Uza N, Inoue S, Mikami S, 10.1128/JCM.02005-13]

P- Reviewer: Annese V, Zhang L S- Editor: Ma YJ L- Editor: A

E- Editor: Ma S

WJG|www.wjgnet.com 12675 November 28, 2015|Volume 21|Issue 44|

 Published by Baishideng Publishing Group Inc
8226 Regency Drive, Pleasanton, CA 94588, USA
Telephone: +1-925-223-8242
Fax: +1-925-223-8243
E-mail: bpgoffice@wjgnet.com
Help Desk: http://www.wjgnet.com/esps/helpdesk.aspx
http://www.wjgnet.com

I S S N 1 0  0 7  -   9  3 2  7

4   4

9   7 7 10  0 7   9 3 2 0 45

© 2015 Baishideng Publishing Group Inc. All rights reserved.