Comparison of human papillomavirus

(HPV) detection in urine and cervical

swab samples using the HPV GenoArray
Diagnostic assay
Pornjarim Nilyanimit1 , Jira Chansaenroj1 , Anant Karalak2 , Piyawat
Laowahutanont3 , Pairoj Junyangdikul4 and Yong Poovorawan1
1
Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
2
Department of Pathology, National Cancer Institute, Bangkok, Thailand
3
Department of Gynecology, National Cancer Institute, Bangkok, Thailand
4
Department of Pathology, Samitivej Srinakarin Hospital, Bangkok Hospital Group, Bangkok, Thailand

ABSTRACT
Human papillomavirus (HPV) is the leading cause of cervical cancer. Urine-based HPV
testing offers a simple and non-invasive method because of its increasing acceptance. A
total of 164 pairs of cervical swab and urine samples from Thai women who underwent
cervical cancer screening were used for HPV testing with HPV GenoArray Diagnostic
Kits. The overall concordance percentage for HPV detection in the cervical swab and
urine samples was 65.2%. The HPV genotypes most commonly detected were HPV16
and HPV18. An analysis of the urine samples and a second analysis of the cervical
swab samples showed that the differences in the overall HPV detection rate between
women with normal and abnormal cytology were not significant (p > 0.05). Urine
samples processed with the GenoArray assay is an alternative for women who decline
to undergo Pap smear even though it is not ideal as the first-line screening option.

Subjects Virology, Infectious Diseases, Oncology, Women’s Health

Submitted 13 June 2017 Keywords HPV, Urine, Genotyping, Cervical cancer
Accepted 19 September 2017
Published 9 October 2017
Corresponding author INTRODUCTION
Yong Poovorawan,
[email protected]
Human papillomavirus (HPV) causes cervical cancer (Koutsky, 1997). Approximately 170
genotypes have been identified (De Villiers et al., 2004) and at least 40 genotypes infect the
Academic editor
Salvatore Andrea Mastrolia human anogenital tract (De Villiers, 2013). The genital HPVs are classified into high-risk
Additional Information and and low-risk genotypes depending on their association with uterine cervical cancer (Muñoz
Declarations can be found on et al., 2003). The high-risk genotypes most commonly detected in uterine cervical cancer
page 8
are HPV16, 18, 31, 33, 35, 45, 52, 58, 39, 51, 56, and 59 (Bouvard et al., 2009).
DOI 10.7717/peerj.3910 The incidence of cervical cancer in young women is increasing in many countries
Copyright due to improved awareness and testing. HPV prevalence in Asia, Africa, Europe and
2017 Nilyanimit et al. South America varied significantly depending on the population and geographical regions
Distributed under (Clifford et al., 2005). In Europe, it ranges from 1.4% (Spain) to 9.2% (Italy). In South
Creative Commons CC-BY 4.0 America, the prevalence ranged from 11.9% (Chile) to 16.3% (Argentina). In Southeast
OPEN ACCESS Asia, the prevalence ranged from 1.6% (Vietnam) to 13.3% (Korea). Even within a country

How to cite this article Nilyanimit et al. (2017), Comparison of human papillomavirus (HPV) detection in urine and cervical swab sam-
ples using the HPV GenoArray Diagnostic assay. PeerJ 5:e3910; DOI 10.7717/peerj.3910
 such as Thailand, HPV prevalence can vary from 7.2% in northern region to 15.1% in
central region (Kantathavorn et al., 2015).
The Papanicolaou (Pap) test is a cost-effective way to screen for cervical cancer. Test
results help physicians detect precancerous lesions and determine the course of treatment.
Pap test has been shown to reduce the incidence of mortality from cervical cancer (Mählck,
Jonsson & Lenner, 1994). However, it is primarily used for detecting invasive cervical
cancer and cannot identify asymptomatic HPV infection (Safaeian, Solomon & Castle, 2007;
Leyden et al., 2005). In addition, barriers to testing include the lack of information, personal
preference, fear, embarrassment and lack of trust in healthcare under certain circumstances.
False information regarding the procedure, the lack of spousal support towards screening,
cultural taboos, and stigmatization of women with cervical cancer further contribute to
the limitations of the Pap test. Therefore, alternative and supplementary HPV DNA assays
are often required in combination with the traditional Pap smear test (Cox et al., 1995).
HPV DNA detection in urine samples presents a feasible alternative to HPV DNA
detection in cervical specimens. Urine testing provides an especially simple, non-invasive
method for screening (Prusty et al., 2005). The benefits of using urine for HPV DNA
detection have been evaluated in disease surveillance and screening for cervical cancers
involving specific genotypes. HPV DNA urine testing can be used to identify abnormal
cells in adolescent girls and young women who do not wish to have a vaginal examination
(Vorsters et al., 2014; Enerly, Olofsson & Nygård, 2013). Some studies have reported a high
HPV detection sensitivity for urine-based assays (Forslund et al., 1993; Hagihara et al.,
2016; Bernal et al., 2014), while other studies have reported a low HPV detection sensitivity
from urine-based assays (D’Hauwers et al., 2007; Nilyanimit et al., 2013).
Molecular methods for HPV testing have been explored, such as PCR/sequencing (De
Roda Husman et al., 1995), the INNO-LiPA HPV Assay (Van Hamont et al., 2006), and the
Hybrid Capture 2 test (HCII) assay (Kubota et al., 1998). In addition, the HPV GenoArray
Diagnostic Kit (Hybribio Ltd., Sheung Wan, Hong Kong) is a recently developed PCR-based
HPV genotyping assay, which uses L1 consensus primers to amplify 21 HPV genotypes. It is
then followed by flow-through hybridization with immobilized genotype-specific probes.
This test is currently used in several hospitals in China (Liu et al., 2010). A previous study
showed that the sensitivity of the HPV GenoArray assay was 97.8% and the specificity
was 100% (Du et al., 2013). If its use expanded to other parts of Asia where individuals
share similar cultural beliefs, more women would benefit from increased cervical cancer
screening. This study aimed to evaluate the use of a urine-based assay as a non-invasive
method for HPV detection and to genotype the samples using the HPV GenoArray assay
in a Thai population.

MATERIALS & METHODS

The research protocol was approved by the Ethics Committee of the National Cancer
Institute, Thailand (number EC COA 037/2012), and the Institutional Review Board of the

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 2/13

 Faculty of Medicine at Chulalongkorn University (number 389/2555). The objective of the
study was explained to the patients, and written consent was obtained from all participants.
Each specimen was sent for testing anonymously, which only included participant-specific
numerical code and age information.

Clinical specimens
All patients underwent the Pap smears test and were subsequently asked to participate in
this study. In all, 164 women consented and were willing to provide paired specimens (a Pap
smear sample from the cervix and a first-void urine sample). All specimen were recruited
between March to December 2014. Specimens were classified into three groups: 95 samples
indicating normal cytology, 50 samples indicating low-grade squamous intraepithelial
lesions (LSIL), and 19 samples indicating high-grade squamous intraepithelial lesions
(HSIL). The ages of the patients enrolled in this study were between 19 and 69 years.

Sample preparation
Each Pap smear sample (which are the standard samples for HPV genotyping) was
evaluated by a specialized cytotechnologist and the results were confirmed by a pathologist.
Samples were suspended in the liquid-based cytology (LBC) buffer (ThinPrep, Hologic,
Marlborough, MA, USA). Collection of the first-void urine (FVU) samples was performed
in a sterile Cell PrepPlus (Biodyne, Gyeonggi-do, Korea) urine bottle, stored at 4 ◦ C, and
processed within 3 days. For each sample, approximately 15 mL of LBC and FVU were
centrifuged at 3,000 rpm for 5 min, and the supernatant was removed. The residual 800
µL of the sample suspension containing cell debris was washed and centrifuged at 8,000
rpm for 5 min. The DNA was extracted from the pellets using a DNA Prep Kit (Chaozhou
Hybribio Biochemistry Ltd., Guangdong, China) and stored at −20 ◦ C until testing.

HPV GenoArray Diagnostic assay

The extracted DNA from the cervical swab and urine samples was subjected to an HPV
genotyping assay using HPV GenoArray Diagnostic Kits (Hybribio Ltd., Sheung Wan,
Hong Kong) according to the manufacturer’s instructions. This PCR-based assay enables
the amplification of 21 HPV genotypes including 13 high-risk types (HPV 16, 18, 31, 33,
35, 39, 45, 51, 52, 56, 58, 59, and 68), two probable high-risk types (HPV 53 and 66),
and six low-risk or unknown risk types (HPV 6, 11, 42, 43, 44, and CP8304 [HPV-81]).
This assay uses an L1 consensus primer-based PCR and is different from the Linear Array
HPV Genotyping Test, which uses MY09 and MY11 primers (Liu et al., 2010). After PCR
amplification, the amplicons were subjected to flow-through hybridization on a nylon
membrane covered in immobilized HPV genotype-specific oligonucleotide probes. The
hybrids were detected by the addition of streptavidin-horseradish peroxidase conjugate
and substrate (NBT/BCIP). The presence of a positive result for the internal control
and the biotin dots within the membrane serves to validate DNA quality, good enzyme
conjugate, and successful hybridization process. Results were manually interpreted using
the manufacturer’s guidelines. The normal detection limit is ∼500 copies/µL of target
HPV DNA. There were no cross-reactivities from the amplification/detection of the 21

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 3/13

 Table 1 Detection of HPV genotypes and concordance between cervical swab and urine samples.

Cytology Specimen (% positive) Concordance

(percentage)
Any HPV positive HPV16&18
Cervical swab Urine Cervical swab Urine
Normal (N = 95) 11 (11.6) 10 (10.5) 1 (1.1) 4 (4.2) 68 (71.6)
LSIL (N = 50) 35 (10.0) 35 (10.0) 11 (22.0) 13 (26.0) 31 (62.0)
HSIL (N = 19) 19 (100.0) 8 (42.1) 8 (42.1) 3 (15.8) 8 (70.5)
Total (N = 164) 65 (39.6) 53 (32.3) 20 (12.2) 20 (12.2) 107 (65.2)

HPV genotypes. The provided positive control and two negative controls (including
HPV-negative C33-A cells) were included in each set of PCR to assess the performance of
the test.

Statistical methods
A statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA).
Pearson’s chi-square test for matched pairs was used to compare the performance of the
two types of samples regarding the detection of HPV genotypes. Statistical significance was
defined as p < 0.05.

RESULTS
The mean age of the 164 participants was 45.8 years. Among the women with normal
cytology, the mean age was 50.5 years, while among those with abnormal cytology (LSIL
or HSIL), the mean age was 41.1 years. Results from the cervical swab samples showed
that 39.6% (65/164) were HPV DNA-positive (Table 1). In total, 11% (18/164) contained
multiple HPV genotypes. The most common HPV genotypes detected were HPV16 (12
samples) and HPV18 (8 samples). Thus, 12.2% (20/164) contained either HPV16 or HPV18.
In the normal cytology group, 11 of the 95 samples (11.6%) were HPV DNA-positive. In
contrast, the LSIL and HSIL groups had 35 (10.0%) and 19 (100.0%) HPV DNA-positive
samples, respectively.
For the urine samples, 32.3% (53/164) were HPV DNA-positive (Table 1), of which
7.9% (13/164) had multiple HPV genotypes. The most commonly detected HPV genotypes
were HPV18 (17 samples) and HPV16 (four samples). In total, 12.2% (20/164) contained
HPV16 or HPV18. In the normal cytology group, 10 of the 95 samples (10.5%) were HPV
DNA-positive. In contrast, the LSIL and HSIL groups had 35 (10.0%) and 8 (42.1%) HPV
DNA-positive samples, respectively.
We next compared HPV detection efficacy between the standard Pap smear samples and
the urine samples. Comparison between the cervical swab and urine specimen resulted in
the overall concordance of 65.2% (107/164) (Table 1). In the normal cytology group, the
concordance was 71.6% (68/95). In the abnormal cytology group, the concordance was
56.5% (39/69). Using the Pap smear results as reference, the sensitivity and specificity of the
urine-based HPV GenoArray Detection Kit were 56.5% and 70.6%, respectively. However,

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 4/13

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910

Table 2 Studies of human papillomavirus DNA detected in paired urine and cervical samples from females of all ages.

Author Country HPV detection assay Age, years Total sample Lesion/HPV Sensitivity Specificity Concordance
range size types (%) (%) (%)
Strauss et al. (1999) UK PCR with MY and GP 16–57 144 All/any type 76.4 73.3 75.7
primers
Daponte et al. (2006) Greece In house type-specific N/A 77 All/HPV16/18 70.3 100.0 85.7
primers and commercial
Gupta et al. (2006) India In house L1 consensus N/A 30 All/any type 100.0 100.0 100.0
primers
Cuschieri et al. (2011) UK HPV INNO–LiPA 16–25 90 All/any type 90.5 67.6 59.8
Nilyanimit et al. (2013) Thailand Electrochemical DNA 27–61 116 All/HR-HPV 64.3 100.0 75
chip
Bernal et al. (2014) Spain Cobas 4800HPV test 21–65 125 All/any type 90.5 85 88
Hagihara et al. (2016) Japan Anyplex II HPV28 19–58 240 All/any type 68.4 99.9 98.4
This study Thailand Hybribio GenoArray 19–69 164 All/any type 56.5 70.6 65.2
Notes.
N/A, not applicable.
5/13
 Table 3 Number of HPV genotypes detected using the HPV GenoArray assay.

No. of HPV Normal (N = 95) Abnormal (N = 69)

genotypes detected
Cervical swab Urine Cervical swab Urine
a
0 84 (88.4) 85 (89.5) 15 (21.7) 26 (37.7)
1 11 (11.5) 9 (9.4) 36 (52.2) 31 (44.9)
2 – 1 (1.1) 14 (20.3) 7 (10.1)
≥3 – – 4 (5.8) 5 (7.3)
Notes.
a
Samples were HPV DNA-negative.

these results were lower than other studies (Table 2). The positive and negative predictive
values were 53.8% (95% CI [41.9–65.4]) and 72.7% (95% CI [63.2–80.5]), respectively.
For multiple HPV infections, the cervical swab-based assays were able to detect more
HPV genotypes in each sample. However, in the normal cytology group, for each pair of
biospecimens, the most common number of genotypes per sample was one. Similarly, in
the abnormal cytology group, 36 of the 69 cervical swab samples (52.2%) and 31 of the 69
urine samples (44.9%) had a single genotype (Table 3).
An analysis of the urine samples and a second analysis of the cervical swab samples
showed that the differences in the overall HPV detection rate between the normal and
abnormal cytology groups were not significant (p > 0.05).

DISCUSSION
Despite the benefits offered by the Pap test, screening attendance remains low (Gakidou,
Nordhagen & Obermeyer, 2008), while the estimated incidence of invasive cervical cancer
remains high (Leyden et al., 2005). In Thailand, 25–38% of women aged 30–65 years have
had only one Pap test (Sriamporn, Khuhaprema & Parkin, 2006). When cervical testing for
HPV is required, these results suggest that urine sample collection provides an alternative
non-invasive sampling method for monitoring HPV infection in women. In a previous
study, the overall percentage agreement between HPV detection in urine and cervical
samples was 88% using the Cobas 4800 HPV test (Bernal et al., 2014), 75% using an
electrochemical DNA chip (Nilyanimit et al., 2013) and, in this study, the percentage was
65.2%. The undeniable advantage in testing urine sample is its acceptance and convenience
for the patients, although better results are obtained with first urine in the morning
(Vorsters et al., 2014). However, the results must be interpreted with caution owing to
variations among the studies in terms of the participant characteristics, surrogate nature of
using cervical HPV detection to screen for cervical disease, and lack of standardized urine
testing methods.
Urine sample assays cannot be used to detect all of the genital HPV infections, but these
assays provide an alternative for use in epidemiological surveys in which invasive sampling
is difficult to perform. Under these circumstances, testing urine for HPV DNA offers a
distinct advantage (Prusty et al., 2005). Previous studies have compared HPV detection
rates between cervical and urine samples in order to evaluate the ability of urine-based

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 6/13

 assays to detect the prevalence of HPV independently of cervical cytology assays (Daponte
et al., 2006; Munoz et al., 2013). Evidence suggests that the sensitivity of urine testing
for HPV 16 and 18 was higher for participants with cervical cancer (88.8%) than for
those with high- and low-grade lesions (Daponte et al., 2006). However, since our samples
comprised more of normal than abnormal cytology, proportion of HPV16-positive samples
are thus admittedly lower in this study. The larger-scale HPV genotyping for cervical
cancer screening in China showed the most common high risk HPV genotypes in women
population worldwide were HPV16, 18, 31, 58, 52, 51 and 33, however frequencies varied
by region (De Sanjosé et al., 2007). In contrast, some regions showed that HPV52 is higher
detection than HPV16 because of the geographical and biological interaction between
HPV genotypes and host immunogenic factors (De Sanjosé et al., 2007; Ye et al., 2010).
Alternatively, it is possible that certain urine samples yield low-efficiency amplification due
to the presence of inhibitory substances in the urine or HPV DNA loss during processing
(Brinkman et al., 2004).
The HPV DNA analysis of urine samples needs to be developed further before a urine-
based assay can replace the Pap smear test. It is possible that a greater amount of urethral
cells in the urine samples helped to increase the sensitivity of the test. An analysis of the
urine samples and a second analysis of the cervical swab samples showed that the differences
in the overall HPV detection rate between women with normal and abnormal cytology
were not significant (p > 0.05). This result suggests that urine represents a viable substitute
for cervical swabs. However, the urine samples should be optimized by preventing DNA
degradation during extraction and storage, recovering cell-free HPV DNA in addition
to cell-associated DNA, processing a sufficient volume of urine, and collecting the first
portion of the urine stream in the morning (Vorsters et al., 2014).
Using traditional cytological analysis, it is difficult to determine accurate screening
results for HPV-associated anogenital tumors. Therefore, HPV genotyping is an alternative
screening method to be used in combination with traditional cytology for identifying
patients at high risk of developing squamous cell carcinoma (Saslow et al., 2012; WHO,
2013). Nowadays, there are many HPV genotyping techniques for detecting HPV DNA, such
as PCR, real-time PCR, restriction fragment length polymorphism (RFLP), Hybrid Capture,
and Linear Array (Bernard et al., 1994; Cox et al., 1995; Castle et al., 2008). However, PCR
and real-time PCR need specific expensive equipment (such as a thermal cycler), and these
methods have not yet become common procedures in hospital laboratories (Hagiwara
et al., 2007). This study used the HPV GenoArray Diagnostic Kit for HPV genotyping,
which is a commercial kit that has recently been started to be used, especially in China
(Liu et al., 2010). The results from the HPV GenoArray assay used in this study were a
percentage-point (39.6%) higher compared to the results from a previous survey of Thai
women (7.6%) (Chansaenroj et al., 2010) and one of Japanese women (22.5%) (Onuki et
al., 2009). The higher percentage may be due to the small number of participants in our
study sample.
The Linear Array HPV Genotyping Test has been widely used as a standard reference
method for evaluating new methods. However, the HPV GenoArray Diagnostic Kit is an
alternative technique for studies conducted in resource-limited laboratories because the

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 7/13

 cost of the HPV GenoArray Diagnostic Kit is lower than that of the Linear Array HPV
Genotyping Test and the hybridization time is also lower (Li et al., 2012; Liu et al., 2010).
Moreover, the HPV GenoArray assay can distinguish and identify HPV 52, which is one
of the most common high-risk HPV genotypes in women in eastern and southeastern Asia
(Sukvirach et al., 2003; Takehara et al., 2011).
In conclusion, the HPV GenoArray assay is an alternative for HPV genotyping using both
cervical swab and urine samples, the latter of which is an alternative for women declining
to undergo Pap smears. Although it is not the gold standard, utilization of this method is
expected to will be increase the number of women who undergo cervical cancer screening.

ACKNOWLEDGEMENTS
We thank the staff of the Center of Excellence in Clinical Virology for their generous
assistance.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
This work was supported by the National Research Council of Thailand, the Research
Chair Grant from NSTDA, the Center of Excellence in Clinical Virology (GCE 59-009-
30-005) and King Chulalongkorn University, the Centenary Academic Development
Project (CU56-HR01), the Ratchadaphiseksomphot Endowment Fund of Chulalongkorn
University (RES560530093), Office of Higher Education Commission (NRU 59-002-HR)
and Dutsadi Piphat Scholarship. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
National Research Council of Thailand.
Research Chair Grant from NSTDA.
Center of Excellence in Clinical Virology: GCE 59-009-30-005.
King Chulalongkorn University.
Centenary Academic Development Project: CU56-HR01.
Ratchadaphiseksomphot Endowment Fund of Chulalongkorn University: RES560530093.
Office of Higher Education Commission: NRU 59-002-HR.
Dutsadi Piphat Scholarship.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Pornjarim Nilyanimit conceived and designed the experiments, performed the
experiments, analyzed the data, wrote the paper, prepared figures and/or tables.
• Jira Chansaenroj analyzed the data, prepared figures and/or tables, reviewed drafts of
the paper.

Nilyanimit et al. (2017), PeerJ, DOI 10.7717/peerj.3910 8/13

 • Anant Karalak and Piyawat Laowahutanont contributed reagents/materials/analysis
tools.
• Pairoj Junyangdikul contributed reagents/materials/analysis tools, reviewed drafts of the
paper.
• Yong Poovorawan conceived and designed the experiments, contributed reagents/ma-
terials/analysis tools, reviewed drafts of the paper.

Human Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
The research protocol was approved by the Ethics Committee of the National Cancer
Institute, Thailand (number EC COA 037/2012), and the Institutional Review Board of the
Faculty of Medicine at Chulalongkorn University (number 389/2555).

Data Availability
The following information was supplied regarding data availability:
The genotyping data comparison between urine and cervical swab samples has been
provided as a Data S1.

Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.3910#supplemental-information.

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