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- Clinical Experience -
Urine versus brushed samples in human papillomavirus screening: study in both genders
Kathleen D'Hauwers1, 2, Christophe
Depuydt3, John-Paul Bogers3,
4, Michel Stalpaert3, Annie
Vereecken3, Jean-Jacques
Wyndaele1, Wiebren Tjalma5
Departments of 1Urology,
4Pathology and 5Gynecology, University Hospital Antwerp, Wilrijkstraat 10, Antwerp B-2650,
Belgium
2Department of Urology, University Medical Centre Nijmegen, St Radboud, Geert Grooteplein 10, Internal Route 659,
6500 HB Nijmegen, the Netherlands
3AML, Algemeen Medisch Labo, Desguinlei 88, 2018 Antwerpen, Belgium
Abstract
Aim: To investigate whether urine is a good medium for screening and whether there is a correlation between the
amount of extracted DNA and human papillomavirus (HPV)-positivity.
Methods: In the present study, 30 first-voided
urine (FVU) specimens and 20 urethroglandular swabs using cervex-brushes from male partners of HPV-positive
patients, and 31 FVU specimens and 100 liquid-based cervix cytology leftovers sampled with cervix-brushes from
HPV-positive women were examined for the presence of
β-globin. Oncogenic HPV were detected using type-specific
PCR. Results: β-globin was found in all the brushed samples, whereas it was found in only 68.9% of the FVU
specimens. HPV-PCR was positive in 60.0% of the male brushes, in 29% of the female brushes and in 0% of the male
FVU specimens. DNA concentration was, respectively,
0.9998 ng/μL, 37.0598 ng/μL and
0.0207 ng/μL. Conclusion: Urine is not a good tool for HPV detection, probably because the low DNA concentration reflects a low amount of
collected cells. β-globin is measurable in FVU by real time quantitative PCR, but the DNA concentration is lower
compared to brush sampling for both genders. β-globin-positivity of urethral and cervical swabs is 100%, showing
a higher mean concentration of DNA, leading to a higher detection rate of HPV. This is the first article linking
DNA-concentration to the presence of HPV. (Asian J Androl 2007 Sep; 9: 705_710)
Keywords: DNA; human papillomavirus; men; polymerase chain reaction; screening; urine
Correspondence to: Dr Kathleen D'Hauwers, Department of Urology, University Medical Centre Nijmegen, St Radboud, Geert Grooteplein
10, Internal Route 659, 6500 HB Nijmegen, the Netherlands.
Tel: +31-24361-3735 Fax: +31-24354-1031
E-mail: K.D'Hauwers@uro.umcn.nl; kathleendhauwers@hotmail.com
Received 2006-06-12 Accepted 2007-04-04
DOI: 10.1111/j.1745-7262.2007.00287.x
1 Introduction
The human papillomavirus (HPV) causes the most common sexual transmitted infection (STI) of viral
aetiology. At least 70 types of HPV are known to infect
the anogenital region. Low-risk types (6, 11) are
responsible for rather benign conditions like genital warts,
whereas high-risk types (16, 18, 31, 33, 45) are related
with anogenital cancer [1]. The most common members of the low-risk group and the high-risk group are
HPV 6 and HPV 16, respectively. Cervical cytology can
detect (pre)malignant lesions in asymptomatic women.
Polymerase chain reaction (PCR) can be used to detect
oncogenic HPV types that are responsible for the
detected lesions. There is no standard screening method
for asymptomatic infected men. Detection of HPV DNA
in urine could be a screening possibility because
screening kits for other STI are available using PCR-testing of
first-voided urine (FVU) specimens [2]. To evaluate if
HPV-testing on urine is a good technique, β-globin
concentration and HPV positivity for oncogenic HPV types
are measured using real-time PCR in FVU specimens of
men and women and compared with the findings in swabs
of the glandular urethra and of the glans penis for men
and with swabs of the cervix for women. Samples are
always checked for β-globin first: the amount of β-globin
is important because it correlates with the amount of
DNA found in the sample. Furthermore, we looked for a
connection between the amount of DNA and HPV-positivity.
2 Patients and methods
2.1 HPV testing on FVU
After obtaining informed consent, FVU-specimens
of 30 men and 31 women were processed according to
the Roche Amplicor protocol [3]. All samples tested
negative for Chlamydia trachomatis and Neisseria
gonorrhoea (Roche Amplicor, San Diego, CA, USA). The rest of the
extracted material was used for HPV-testing by PCR. DNA
isolation from FVU specimen was performed according
the Roche Amplicor protocol. The DNA extracts were
stored at _20ºC until PCR was performed.
2.2 HPV on glandular scrape and urethra sampling
Urethro-glandular sampling for men (n = 20) and
cervical sampling for women (n = 100) were performed
using the Cervex-Brush (Rovers, Oss, the Netherlands).
All brush-heads were placed in SurePath vials (Tripath
Imaging, Burlington, NC, USA). Enriched cell
suspensions were obtained by using the fully robotic AutoCyte
PREP System (AutoCyte, Tripath Imaging, Burlington,
NC, USA) [4]. Briefly, vials containing the brush-heads
were vigorously vortexed before transferring on top of a
density gradient. After centrifugation and washing, a 1-mL
cell suspension was obtained. From this cell suspension,
400 µL was used to extract DNA, as previously described [5].
2.3 PCR analysis of HPV DNA
First, each sample was subjected to a quantitative
real-time PCR amplification for the detection of β-globin.
This was done to confirm that the DNA quality was still
suitable for PCR analysis. Only samples that tested
positive for β-globin were tested with the MY 9/11
consensus PCR, and only samples that tested positive for the
consensus PCR were typed for oncogenic HPV types using type-specific PCR [5].
2.4 Real-time PCR and probe
Oligonucleotide primers were designed with the Primer
Express software (version 1.0; Applied Biosystems,
Foster City, CA, USA). The probe was designed to ensure a
higher Tm than that for the primers and was
manufactured by Applied Biosystems (Cheshire, UK). The size
of the β-globin PCR product was 167 bp (GenBank
Accession Number U01317). B-globine-143F 5'-TGCATTTGACTCCTGAGGAGAA-3', B-globine-223R
5'-GGGCCTCACCACCAACTTC-3', B-globine-167T 5'-CTGCCGTTACTGCCCT-3'.
β-globin probe was labeled with 6-carboxyfluorescein at the 5' end and minor groove
binding at the 3' end.
The PCR amplification was performed in a 25 µL
volume containing 2 × TaqMan Universal PCR master
mix (Applied Biosystems, Cheshire, UK), 200 nmol/L
concentration of each primer and probe and 10 µL of
extracted DNA. Amplification and detection were
performed using an ABI Prism 7000 sequence detection
system (Applied Biosystems, Cheshire, UK). The
amplification conditions were 2 min at 50ºC to activate the
uracil N'-glycosylase, followed by 10 min at 95ºC to
inactivate the uracil N'-glycosylase and release the
activity of the DNA polymerase, and by a two-step cycle
of 95ºC for 15 s and 60ºC for 60 s for a total of 45 cycles.
2.5 Positive and negative controls for PCR
A tube that contained all PCR components but no
template DNA was used in all runs to ensure that the
reagents were free of contamination. For the β-globin
real-time PCR a standard curve was obtained by
amplification of a dilution series of 150 ng to 0.015 ng of
female human DNA (Promega, San Luis Obispo, CA, USA).
2.6 Calculations
The threshold cycle number (Ct) was calculated with
Sequence Detection System software (Applied Biosystems, Cheshire, UK) and an automatic setting of
the baseline (10 standard deviations above the background
in the first 3_15 cycles). A standard curve was
generated by plotting the Ct values against the log of the amount
of β-globin DNA (in nanograms) and the amount of β-globin DNA for unknown samples inferred from the
regression line.
2.7 Statistical analysis
Comparisons of means were studied by analysis of
variance (ANOVA), followed by the Student_Newman_Keuls test for all pairwise comparisons. The
c2-statistics were used to verify the existence of a trend across
ordered groups, such as an increase in HPV positivity.
Statistical tests were considered significant at
P < 0.05. Statistical analysis was performed using the MedCalc
program (MedCalc Software, Mariakerke, Belgium) [6].
3 Results
An overview of β-globin concentrations and HPV positivity for the different groups is given in Table 1.
The percentage of FVU specimens with detectable β-globin concentration did not differ between men (66.7%)
and women (71.0%). In the group where the Cervex-brush was used to collect urethro-glandular or cervical
cells, β-globin was always detected. β-globin
concentration rises as follows: FVU specimens in men, FVU
specimens in women, Cervex-brush sampling in men, Cervex-brush sampling in women. Ultimately,
β-globin concentration in women was 100 times more elevated
than that in FVU specimens of men. Although the
β-globin is measurable in FVU specimens by real time
quantitative PCR, the DNA concentration is lower compared
to Cervex-brush sampling for both men (urethro-glandular, 100-fold) and women (cervical, 10 000-fold;
Figure 1). The DNA concentration (ng/µL) is
significantly higher in the brushes in both men (0.9998 ng/µL)
and women (37.0598 ng/µL) than that in FVU specimens
of men (0.0207 ng/µL) and of women (0.0916 ng/µL)
(ANOVA: P < 0.05). Oncogenic HPV were detected in
60.0% of the brushes from men and in 28.0% from women, and in only one sample (3.2%) of the FVU
specimens in women and was not detected in FVU specimens
of men. HPV prevalence is given in Table 2 for each of
the sampling methods. For men, HPV type 16 was the
most frequently detected HPV, whereas in women HPV
31 was more detected.
4 Discussion
Persistent HPV-infection is a risk factor for anogenital
dysplasia and carcinoma. Normally, the body clears the
infection in 6_12 months [7], but persistent HPV-load is
maintained in patients with a weakened immunity
system and by the presence of HPV in the partners' anogenital
region. In vitro, the role of sperm as a vector for HPV
has clearly been suggested [8]. HPV prevalence in a
younger population (18_29 years) varies around 33% [9]:
it rises with age and with promiscuity. Cytological
examination of endocervical smears can detect abnormal
cellular changes in asymptomatic women. PCR on
cervical scrapes is the golden standard to confirm the
presence of HPV. A satisfactory counterpart does not exist
for asymptomatic men. Cytological screening of urine
for abnormal cells is not a good diagnostic tool; it cannot
confirm or rule out the presence of visible intraurethral
disease: there is no correlation between cytological
abnormalities and the presence of HPV-DNA [10]. Urinary
HPV-DNA reflects the presence of infected cells shed
from the epithelium of the urethral meatus. The amount
of β-globin is an indirect method for measuring these
cells. A low amount of β-globin could result in a lower
HPV detection rate, although approximately 6% is
positive for HPV-DNA while negative for β-Globin [11]. In
these cases HPV-DNA is transferred by desquamated,
anucleated cells, packed with HPV virions. In the present
study, one of the β-globin-negative-FVU specimens in
the female group was HPV positive. The number of HPV copies per infected cell can be as low as one, so a
minimum of infected cells is required to obtain a positive
PCR signal. The first part of voided urine contains most
of the exfoliated cells; delayed catching of the FVU
specimens or collecting a larger amount of urine will adversely
affect the amount of collected cells. Urine samples can
be stored for 1 week at room temperature without
reduction of β-globin. Urinary HPV-detection can be
impaired by white urea at a concentration of higher than
50 mmol/L, by proteins, or by iron and heparin, all present
in the urine sample. These inhibitory components can
be removed by boiling the specimen, by
phenol-chloroform extraction, by diluting or by dialysis, or by
pre-treating the samples with glass beads using GeneClean
(Bio 101, Inc., La Jolla, CA, USA) [12]: all are time
consuming and generate extra costs. Logically, the chance
of detecting HPV in urine is higher in cases of intrameatal
warts and lower in patients with genital dermatoses or
warts located outside the urethra [12_14]. Some authors
found additional value in urine testing: Weaver
et al. [11] identified an additional 7% of HPV-DNA positive men.
Others have failed to find HPV in urine. Laczano
et al. [15] compared urethral swabs and urine samples of 120
sexual active Mexican men, finding detectable β-globin
in respectively 95% and 14% of the samples. In 43% of
the sample, HPV-DNA was found in penile-urethral swabs,
never in urine. For women, the results are somewhat
better, probably because of the close proximity of the
urinary tract to the vagina, vulva and cervix [16_21].
We found only one positive HPV 18 sample in FVU of
women. The women in our study do not have invasive
cervical cancer, so we are unlikely to find many positive
cases. In Zimbabwe, cervical and urine samples of 43
women with invasive cervical cancer were investigated
for the presence of HPV DNA [18]. HPV was identified
and typed in 98% of cervical swabs and 72% of urine
samples. Type-specific concordance between cervical
and urine samples was only 79%. The distance from the
cervix to the sampling spot has an impact on the
detection rate: Sellors et al. [21] noted that the concordance
between cervical specimens and the vaginal, vulva and
urine specimens for the presence of HPV was 0.76, 0.55
and 0.41, respectively.
We would like to evaluate which medium was appropriate for HPV screening in men. We know from the
published literature that urine is not suitable for
cytological examination. Therefore, urine (of both genders) and
male urethral scrapes were tested for
β-globin-concentration and HPV-positivity. We found
β-globin in 69% of the urine samples and in 100% of the brushed samples,
whereas HPV-PCR was positive in 3.2% of the female
FVU specimens, in 0% of the male FVU specimens and
in 60% of the male brushes. Our results show that for
both men and women, the percentages as well as the
absolute concentration of DNA is significantly lower in
urine (68%) then in swabs taken with the Cervex-brush
(100%). Despite the encouraging amount of β-globin in
urine, the amount of HPV-DNA is disappointingly low.
We conclude that urine is not the first choice for HPV
screening. Testing on urine might only be beneficial in
high-risk populations with a high HPV viral load (e.g.
cervical cancer and HIV positive women). It is not only
crucial to be able to amplify β-globin but also the
absolute amount of DNA present is important. We have shown
for the first time that the amount of human DNA
measured by real-time quantitative PCR is important for HPV
detection in the same sample.
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