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JOURNAL OF CANCER PREVENTION https://doi.org/10.15430/JCP.2019.24.4.240

Vol. 24, No. 4, December 2019 pISSN 2288-3649ㆍeISSN 2288-3657
www.jcpjournal.org

Short
Usefulness Analysis of Urine Samples for Early Communication
Screening of Human Papilloma Virus Infection

Yoon Sung Choi1, Hyunwoo Jin2, Kyung Eun Lee2

1
Department of Thoracic and Cardiovascular Surgery, Inje University Haeundae Paik Hospital, 2Department of Clinical Laboratory Science, College
of Health Sciences, Catholic University of Pusan, Busan, Korea

Human papilloma virus (HPV) is known to be a major cause of cervical cancer. In Korea, although the mortality of cervical cancer has
decreased, HPV infection rates are increasing rapidly in young women. One of the reasons for a high rate of human immunodeficiency
virus (HIV) infection appears to be associated with a low frequency to visit gynecology clinics because of the uncomfortable sampling
process for HPV testing. Therefore, it is necessary to develop a non-invasive method, such as urine testing to diagnose cervical cancer
rather than use of the existing invasive method. This study aimed to test validity of HPV DNA detection in urine specimens that can be
easily collected from women. Paired vaginal discharge and urine samples were collected prospectively from 203 women who visited the
local hospital between January and August 2018 in Busan, Korea. By using the Virocheck® assay kit (Optipharm), we found that 17.2%
(35/203) of vaginal discharge samples were HPV positive and 82.8% (168/203) were HPV negative. In urine samples, 15.8% (32/203) were
HPV positive and 84.2% (171/203) were HPV negative. The co-incident rate for HPV DNA detection was 84.8% in both vaginal discharge
and urine samples. These results suggest that the HPV DNA detection using urine samples might be an alternative way to diagnose HPV
infection in a non-invasive way. This analytical approach can be utilized as a screening test to identify HIV-infected patients who need
a follow-up process by using urine samples.
(J Cancer Prev 2019;24:240-244)

Key Words: Human papilloma virus, Vaginal discharge, Urine

INTRODUCTION human immunodeficiency virus infection which is caused by

specific types of high-risk HPV is persistent which can lead to
Cervical cancer is the third most commonly diagnosed cancer invasive cancer [6,7].
and the ninth leading cause of cancer death among women in In recent years, the prevalence of HPV in young women has
Korea [1]. The main cause of cervical cancer is human papilloma increased in many countries [8]. Other studies have shown that
virus (HPV) infection. The HPV family is comprised of a group of the rates of HPV infections that causes cervical cancer are highest
small (50-55 nm in diameter and –8 kb in length) double-stranded among young women, especially 18 to 29 years [9]. Therefore,
DNA viruses, including more than 200 genotypes that infect the screening tests are very important for early diagnosis and
keratinocytes of human skin and mucosa, causing a variety of management of HPV infection in young women. However,
lesions, such as skin warts, genital warts, papillomas, cervical screening test using cervical samples are limited because they are,
intraepithelial lesions, and also cancer [2,3]. Most HPV infections in general, invasive. According to a gynecological survey of 1,039
are asymptomatic, and the outcome of HPV infection is variable women in Korea, only 49.9% visited gynecology, and 56% of the
[4]. Approximately 90% of HPV infections are cleared by the host women in their 20s did not visit gynecology clinics [10]. One
immune system [5]. However, approximately 10% to 12% of reason for a low frequency to visit gynecology clinic is due to the

Received October 20, 2019, Revised December 17, 2019, Accepted December 17, 2019
Correspondence to: Kyung Eun Lee
E-mail: [email protected], ORCID: Kyung Eun Lee, https://orcid.org/0000-0001-9543-4159
Copyright © 2019 Korean Society of Cancer Prevention
cc This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

240
 Yoon Sung Choi, et al: Usefulness of Urine Samples for Screening HPV Infection 241

uncomfortable sampling process for HPV testing [11,12]. protein degraded with 200 of PBS buffer and 20 L of proteinase
Considering these problems, it is necessary to use a K and added 200 L of gel binding buffer to the sample. Samples
non-invasive method, such as urine testing, to diagnose HPV were mixed immediately by vortex mixing, followed by
o
infection rather than the conventional invasive method [13]. HPV incubation at 60 C for 10 minutes. Thereafter, 400 L of absolute
detection in urine samples has been reported as one of the most ethanol was added to samples and mixed well, and transferred to
effective non-invasive methods for early screening of cervical the lysate in the upper reservoir of the binding column tube,
cancer in young women [14,15]. In a recent meta-analytical study, followed by centrifugation at 9,000 ×g for 1 minute. The
the detection of high-risk HPV in urine samples was found to have solution from the collection tube was discarded and the column
high accuracy when compared with the detection in cervical was then washed twice with WA1 and WA2 from the kit. Finally,
samples, with a sensitivity of 77% and specificity of 88% [15]. DNA was eluted with 50 L of elution buffer at room temperature
However, few studies have evaluated the clinical performance of for at least 1 minute and centrifuged at 9,000 ×g for 1 minute to
urine-based HPV detection in the prediction of cervical elute.
precancerous lesions and cancer in Korea. Therefore, this study
3. Quantitative PCR TaqMan assay targeting human
aimed to evaluate the validity of urinary HPV DNA testing.
papilloma virus E6/E7 genomic DNA

MATERIALS AND METHODS Detection of HPV E6/E7 DNA in vaginal discharge and urine
samples was performed by the qPCR TaqMan assay using the HPV
1. Participants and clinical samples ®
ViroCheck assay kit (Optipharm, Osong, Korea). This was based
To evaluate the performance of molecular diagnostic methods on HPV E6/E7 oncogene detection using ABI 7,500 Fast (Applied
for HPV detection, paired vaginal discharge and urine samples Biosystems, Foster City, CA, USA) real-time PCR systems for
were collected prospectively from 203 women who visited the thermalcycling and fluorescence detection.
local hospital between January and August 2018 in Busan, Korea. The PCR primers and the corresponding TaqMan probes for
Their ages ranged from 18 to 81 years (median age: 45.81 years). detection of HPV genotypes were designed for three different sets
This study was approved by the Institutional Ethics Committee of of HPVs (set 1: FAM-HPV genotype 16, HEX-HPV genotype 31, 33,
Catholic University of Pusan (approval number CUP IRB 35, 52, and 58, Cy5-IC; set 2: FAM-HPV genotype 18, HEX-HPV
18-01-003). Study participants provided approximately 15 mL of genotype 39, 45, 59, and 68, Cy5-IC; set 3: HEX-HPV genotype 53,
urine samples. Each urine sample was washed with normal 56, 66, 51, and 69; and glyceraldehyde-3-phosphate dehydro-
saline. Briefly, 15 mL urine sample was centrifuged at 1,500 ×g genase [GAPDH]: FAM).
for 10 minutes. After removing the supernatant, the pellet was Real-time PCR amplification for HPV E6/E7 DNA was
resuspended in 1 mL of normal saline and transfer to 1.5 mL tube. performed in a total volume of 20 L containing 10 L 2 ×
The vaginal discharge samples were collected by a clinician using Thunderbird probe qPCR mix (Toyobo, Osaka, Japan), 5 L
liquid based cytology bottle. Briefly, vaginal discharge samples primer, 3 L template DNA, and distilled water (D.W.) to hive a
were transferred to 1.5 mL tube and centrifuged at 11,000 ×g for final volume of 20 L for each sample. The multiplex qPCR assay
5 minutes. After removing the supernatant, the pellet was detected HPV E6/E7 genes simultaneously in a single tube by
resuspended in 1 mL of normal saline and used for the HPV test. incorporating three targets specific TaqMan probes, which were
labeled with different fluorophores (FAM, HEX, and Cy5).
2. Genomic DNA extraction from vaginal discharge
Positive and negative controls were included throughout the
and urine samples
procedure. No-template controls with sterile D.W. instead of
Genomic DNA was extracted from vaginal discharge and urine template DNA were incorporated into each run. Cycling
® o o
samples using the Accuprep Genomic DNA Extraction Kit conditions were 95 C for 3 minutes, followed by 40 cycle of 95 C
(Bioneer, Daejeon, Korea) according to manufacturer for 3 seconds and 55oC for 30 seconds. The HPV DNA was
instructions. The vaginal discharge and urine samples were identified by determining the cycle threshold which is the
washed by addition of 1 mL saline, followed by incubation for 3 number of PCR cycle required for the fluorescence to exceed a
minutes at room temperature, and centrifugation for 2 minutes at value significantly higher than the background fluorescence. To
12,000 ×g. The supernatant was discarded, and the pellet was avoid false negative due to degradation of DNA, GAPDH was used
washed twice with 1 mL absolute ethanol for re-hydration. The as a control.
242 Journal of Cancer Prevention Vol. 24, No. 4, 2019

sequence-based assay. Among the 35 HPV E6/E7 gene positive

4. Statistical analysis
cases in vaginal discharge samples, 9 (25.7%), 19 (54.2%), 2 (5.7%),
Statistical analysis was performed using IBM Statistical 10 (28.5%), and 8 cases (22.8%) were Group I FAM, Group I HEX,
Package for the Social Sciences (SPSS) for Windows Standard ver. Group II FAM, Group II HEX, and Group III HEX, respectively (Fig.
19.0 (IBM Corp., Armonk, NY, USA). Fisher exact test was 1).
performed to assess the relationship between the HPV positive Among the 32 HPV E6/E7 gene positive cases in urine samples,
prevalence of vaginal discharge and urine samples. A P -value less 7 (21.8%), 12 (37.5%), 2 (6.2%), 8 (25.0%), and 10 cases (31.2%) were
than 0.05 was considered to be statistically significant. Group I FAM, Group I HEX, Group II FAM, Group II HEX, and
Group III HEX, respectively (Fig. 1).
RESULTS 3. Comparison of the positivity of human papilloma
1. Detection of human papilloma virus E6/E7 virus E6/E7 gene according to the age between
genomic DNA by the quantitative PCR TaqMan vaginal discharge and urine samples
assay
The positivity of HPV E6/E7 genomic DNA in vaginal discharge
Analysis of vaginal discharge samples using the qPCR TaqMan samples was 18.4% (7/38), 18.6% (8/43), 17.5% (7/40), 18.4% (7/38),
assay showed that 17.2% (35/203) were HPV positive and 82.8% and 13.6% (6/44) in under 20, 30, 40, 50, and over 60 years of age,
(168/203) were HPV negative. In urine samples, 15.8% (32/203) respectively. The positivity of HPV E6/E7 gene DNA in urine
were HPV positive and 84.2% (171/203) were HPV negative. In samples was 21.1% (8/38), 16.3% (7/43), 22.5% (9/40), 7.8% (3/38),
both clinical samples, 12.3% (25/203) of all cases were and 11.3% (5/44) in under 20, 30, 40, 50, and over 60 years of age,
concurrently HPV positive and 79.3% (161/203) of all cases were respectively (Fig. 2). The age-specific HPV infection rates by
HPV negative. Among the 17.2% (35/203) of HPV positive cases in detection of HPV E6/E7 gene DNA were different in both vaginal
vaginal discharge samples, 4.9% (10/203) were HPV negative in discharge and urine samples, but younger women especially
urine samples. Among the 15.8% of HPV positive cases (32/203) in under 40s showed higher HPV infection rate (Fig. 2).
urine samples, 3.4% (7/203) were HPV negative in vaginal
discharge samples. The co-incident rate for HPV E6/E7 genomic
DNA detection in both vaginal discharge and urine samples was
84.8%. These results indicate that there is a correlation between the
vaginal discharge and urine samples for HPV E6/E7 genomic DNA
detection (P ＜ 0.001; Table 1).

2. Human papilloma virus genotype distribution

based on the human papilloma virus E6/E7
genomic DNA sequence

A total of 203 samples were analyzed using the HPV E6/E7 gene

Table 1. Detection rate of HPV E6/E7 gene DNA by qPCR TaqMan

assay

Urine
Variable Total Figure 1. Human papilloma virus (HPV) genotype distribution based
HPV positive HPV negative
on the HPV E6/E7 gene DNA sequence. HPV positive was detected
Vaginal discharge in 35 vaginal discharges and 32 urine samples; 9 (25.7%), 19 (54.2%),
HPV positive 25 (12.3) 10 (4.9) 35/203 (17.2) 2 (5.7%), 10 (28.5%), and 8 cases (22.8%) in vaginal discharge sam-
HPV negative 7 (3.4) 161 (79.3) 168/203 (82.8) ples, 7 (21.8%), 12 (37.5%), 2 (6.2%), 8 (25.0%), and 10 cases (31.2%)
Total 32/203 (15.8) 171/203 (84.2) in urine samples cases were Group I FAM, Group I HEX, Group II
Co-incident rate (%) 84.8 P ＜ 0.001 FAM, Group II HEX, and Group III HEX, respectively (Group I FAM:
HPV 16; Group I HEX: HPV 31, 33, 35, 52, 58; Group II FAM: HPV
Values are presented as number (%) or percent only. HPV, human 18; Group II HEX: 39, 45, 59, 68; Group III HEX: HPV 51, 53, 56,
papilloma virus. 66, 69).
 Yoon Sung Choi, et al: Usefulness of Urine Samples for Screening HPV Infection 243

statistically significant, use of urine samples for HPV DNA

detection has a practical merit. Using urine samples for HPV DNA
testing has a number of advantages. Urine testing is a
non-invasive self-sampling method, which would enable frequent
samplings as well as the sampling of a large number of
populations participating in HPV vaccination programs.
Moreover, urine sampling, unlike cervical sampling, is preferable
to and better accepted by women, which may lead to better
population coverage in screening programs.
While there are some differences in the distribution of HPV
genotypes according to countries and regions, HPV 16 accounts
for 50% of all cervical neoplasia, and HPV 18 accounts for about
Figure 2. Comparison of positive rate of human papilloma virus 10% to 20% [18]. The prevalence of HPV types 52 and 58 have been
(HPV) using ViroCheck® by age between vaginal discharge and urine reported to be high in Korea [19]. In the present study, Group I
samples; The average standard deviations of each positive rate be-
tween the duplication data for HPV detection.
HEX was the most frequently positive group, including HPV 31,
33, 35, 52, and 58 genotypes, whereas the HPV 16 genotype was
the most frequently identified single infection genotype.
DISCUSSION Therefore, continuous monitoring of high-risk HPV infection is
considered to be necessary.
HPV is one of the most common sexually transmitted In conclusion, HPV detection was examined as a potential
infections worldwide. Although the estimated risk of HPV is screening tool for the prevention of cervical cancer, and this study
notably high over a woman’s lifetime (over 80%), most women found that easy-to-collect urine samples can detect high-risk HPV
who acquire HPV infection do not develop the more serious types. Urine samples are more easily collected which is
high-grade cervical neoplasia or invasive cancer because most non-invasive than current screening test collection methods.
infections are transient [5,6]. However, HPV persistent infection Therefore, the results from this study can be applied to an
causes cervical cancer [7]. Other studies have reported a high non-invasive disgnostic method to identify patients who need
incidence of HPV infection among young women under 30 years follow-up process. In addition, for the early screening of young
of age [9], raising concerns about the development of cervical women who refuse to see a gynecologist, the evaluation of the
cancer as a consequence of persistent HPV infection. In the usefulness of urine samples through cytological diagnosis or
present study, we analyzed the rate of HPV infection among the sequencing should continue to be expanded.
general population by age and found the highest positive rates in
women in their 40s, and similar infection rates among young ACKNOWLEDGMENTS
women in their 20s and 30s. Therefore, early detection of HPV is
important for preventing cervical cancer. However, young This work was supported by RESEARCH FUND offered from
women have a low frequency of visiting gynecology clinics due to Catholic University of Pusan (No. 2016-1-044).
the uncomfortable sampling process for HPV testing.
Several studies have reported the usefulness of HPV DNA CONFLICTS OF INTEREST
testing with urine specimens for cervical cancer prevention
[13,16,17]. However, its validity as compared with that assessed No potential conflicts of interest were disclosed.
with cervical sample and urine sample from the same person not
been well studied. In the present study, the HPV infection rates ORCID
were compared in vaginal discharge and urine samples from the
®
same population. Using the Virocheck assay kit (Optipharm), Yoon Sung Choi,
17.2% of vaginal discharge samples (35/203) were HPV positive https://orcid.org/0000-0003-1338-4579
and 15.8% of urine samples (32/203) were HPV positive. Since the Hyunwoo Jin,
results with urine and vaginal discharge samples were https://orcid.org/0000-0003-1773-023X
244 Journal of Cancer Prevention Vol. 24, No. 4, 2019

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