Human Papillomavirus Persistence in Young Unscreened
Women, a Prospective Cohort Study
Channa E. Schmeink1*, Willem J. G. Melchers2, Albertus G. Siebers3, Wim G. V. Quint4, Leon F. A. G.
Massuger1, Ruud L. M. Bekkers1
1Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 2Department of Medical Microbiology,
Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, 3Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The
Netherlands, 4DDL Diagnostic Laboratory, Voorburg, The Netherlands
Objective: To evaluate hr-HPV persistence and associated risk factors in a prospective cohort of young unscreened women.
Additionally, the relation between hr-HPV status and cytology/histology results is examined.
Methods and Principal Findings: Two year follow-up of 235 out of 2065 young women (18–29 years), participating in a
large, one year epidemiological study, with questionnaires, self-collected cervico-vaginal samples (Vibabrush), and
SPF10LiPA for HPV detection. Only women hr-HPV positive at sample month 12 were invited for a second year of follow-up.
After study follow-up, available cytology/histology data were requested from PALGA (the national network and registry of
histo- and cytopathology in The Netherlands). These data were compared with available cytology/histology data of the
month 12 hr-HPV negative women from the same cohort. 44.1% of the hr-HPV types detected at study month 12, persisted
during follow-up. HPV types 45, 31, 16 and 18 were most likely to persist with percentages of 60.0%, 56.8%, 54.4%,and
50.0%, respectively. Compared to newly detected infections at month 12, infections present since 6 months or baseline had
an increased risk to persist (OR 3.09 [95% CI: 1.74–5.51] and OR 4.99 [95% CI: 2.67–9.32], respectively). Other co-factors
influencing persistence were, multiple HPV infections, smoking and multiple lifetime sexual partners. The percentage of
women with a HSIL/CIN2+ (12.1%) in the persistent HPV group, was not significantly different (p=0.107) from the 5.3% of
the women who cleared the hr-HPV infection, but was significantly (p 0.000) higher than to the 1.6% of women in the hr-
HPV negative control group.
Conclusion: We showed that HPV genotype, multiple infections, smoking, and multiple lifetime sexual partners are co-
factors that increase the risk of hr-HPV persistency. Most importantly, we showed that hr-HPV infections are more likely to
persist the longer they have been present and that women with a persistent hr-HPV infection have a high risk of HSIL/CIN2+
Citation: Schmeink CE, Melchers WJG, Siebers AG, Quint WGV, Massuger LFAG, et al. (2011) Human Papillomavirus Persistence in Young Unscreened Women, a
Prospective Cohort Study. PLoS ONE 6(11): e27937. doi:10.1371/journal.pone.0027937
Editor: Thomas F. Schulz, Hannover Medical School, Germany
Received June 26, 2011; Accepted October 28, 2011; Published November 23, 2011
Copyright: ? 2011 Schmeink et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: GlaxoSmithKline (GSK 2702-HPV-109932 to R.L.B., L.F.M.). The funder had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Within the past 24 months author C. E. Schmeink has been involved in research financially supported by GlaxoSmithKline; including
contract research. In 2009 R. L. M. Bekkers has been a consultant for GSK. Author W. G. V. Quint is Chief Executive Officer of DDL Diagnostic Laboratory. This does
not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
It has been well established that a human papillomavirus
(HPV) infection is a necessary cause in the development of
cervical intraepithelial neoplasia (CIN) and cervical cancer [1–3].
HPV appears to be the most common sexually transmitted
infection and about 80% of all sexually active women will
acquire an HPV infection during their life time . Fortunately,
only a small proportion of these infections lead to CIN and
cervical cancer. A persistent high risk (hr) HPV infection is
necessary for the development of cervical carcinoma. Therefore
the detection of a persistent hr-HPV infection represents an
important marker of an increased risk of CIN and cervical
carcinoma [4–6]. However, there is no consensus on the
definition of persistence.
Most investigators define a persistent HPV infection as
detection of the same HPV type, or group of types, on two
consecutive visits, but these could be from 2 months up to 72
months apart [7,8]. Several studies have shown that most
infections become undetectable within 1–2 years [5,9–11].
Additionally, infections lasting more than 1 year appear to be
associated with a lower clearance rate [5,9]. Therefore it is more
informative to monitor the duration of the infection rather than
the number of positive tests .
Several risk factors have been identified that are associated with
HPV persistence. Especially viral characteristics like viral load,
and HPV genotype are linked to persistence [13,14]. Given that
HPV16 and 18 are the most carcinogenic HPV genotypes, it
would be useful to know whether their risk of persistence differs
from other hr-HPV genotypes . Additionally, some authors
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have shown that multiple HPV infections are associated with an
increased duration of high-risk HPV infections [9,16], whereas
others have failed to show such an increase [11,17]. Other
cofactors like age, condom use, smoking, long-term use of oral
contraceptives, high parity, number of sex partners, and exposure
to other sexually transmitted diseases have also been associated
with HPV persistence [9,18,19].
In this study, a prospective cohort of young unscreened women
(18–29 years) in the pre-vaccine era was followed for 2 years, to
examine the influence of viral factors (i.e. duration of infection,
HPV-type and co-infection), and co-factors (i.e. sexual behavior
and smoking) on hr-HPV persistence. Additionally we examined
the relation between hr-HPV status and follow-up cytology/
Materials and Methods
A large prospective cohort study on HPV prevalence, incidence
and clearance was performed in the Netherlands, in 2007–2010.
At study entry, 2065 unscreened women 18 to 29 years of age were
included . Exclusion criteria were, being pregnant, or not
fluent in Dutch. Of the 2065 women initially included, 1871
(90.6%) completed the first year of follow-up. Women, who were
hr-HPV positive at month 12 (n=257 (13.7%)), were invited to
participate in a second year of follow-up in order to study hr-HPV
persistence. In total 235 (91.4%) of the invited women, completed
the second year of follow-up and were included in this analysis of
persistence. The study was closed after the sample at month 24,
therefore all the hr-HPV positive women were referred to their
general practitioner or gynaecologist for additional follow-up or
treatment. Written informed consent was obtained from all
participants. This study was approved by the Local Medical
Specimen collection and HPV DNA detection and
The women provided five self-samples with a 3-month interval
(month 0, 3, 6, 9, and 12) during the first year of the study. In the
second year of follow-up, women provided two additional self-
samples with a 6 month interval (month 18 and 24). All women
received the self sample kit and the additional questionnaires by
The self sample kit contained an explanatory letter, a
questionnaire, an illustrated instruction form on how to perform
the cervico-vaginal self-sample, a small brush in a sterile cover
(Rovers VibabrushH, Rovers Medical Devices, Oss, the Nether-
lands), and a collection tube containing medium (SurePathtm,
Tripath ImagingH, Inc., Burlington NC, U.S.A.), as described
Broad-spectrum HPV DNA amplification was performed using
a short-PCR-fragment assay. Extracted DNA was used for PCR
amplification with the SPF10primer sets [22,23]. The samples were
run through an HPV DNA enzyme immunoassay (DEIA) to
obtain an OD reading, and categorized as HPV DNA negative,
positive, or borderline. The same SPF10amplimers were used on
SPF10-DEIA-positive samples to identify HPV genotype by reverse
hybridization on a line probe assay (LiPA) (SPF10HPVLiPA25,
version 1; Labo Bio-Medical Products B.V., Rijswijk, the Nether-
lands), which detects the following HPV genotypes: low-risk HPV
types: type 6, 11, 34, 40, 42, 43, 44, 53, 54, 55, 66, 70, 74, and
‘‘X’’ (DEIA positive and LiPA negative samples); and high-risk
HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73,
Type-specific hr-HPV; clearance, persistence, and history
An HPV infection was considered cleared when a woman had
two consecutive HPV negative samples [11,24,25]. Therefore, hr-
HPV positive women with one intermittent type-specific hr-HPV
negative sample were considered persistent for that hr-HPV type.
All women included in the second year of follow-up were hr-HPV
positive at sample month 12, thus only women who were negative
for hr-HPV at sample month 18 and 24 were considered to have
cleared their hr-HPV infection. (see table 1)
The sampling interval in the 2ndstudy year was 6 months and
persistence is generally defined as two consecutive HPV positive
samples. Thus, a types–specific hr-HPV infection was considered
persistent when it was detected for at least 6 months. Women who
were positive at sample month 18 and/or month 24 were therefore
considered to have a persistent type-specific hr-HPV infection in
the 2ndstudy year (see table 1). Four of the 235 women were
treated for abnormal cytology (and one cervical carcinoma) and
cleared hr-HPV before sample month 18, therefore the natural
course of clearance or persistence was disturbed. The data of these
women were excluded in the analysis of persistence.
To analyze whether the duration of the type-specific hr-HPV
infection (the history) prior to sample month 12 influences
persistence in the 2ndstudy year, we determined at which sample
moment the hr-HPV infection was first detected. In order to
Table 1. Definitions of duration of type-specific hr-HPV detection.
Study sample month
Type-specific hr-HPV history Month 0Month 6 Month 12*Month 18 Month 24
Type-specifc hr-HPV during 2ndstudy
Detected since baseline
Detected since study month 6
Newly detected at study month 12
*only women positive for hr-HPV at sample month 12 are included in the 2ndstudy year and in the analysis of HPV persistency.
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calculate with the same time interval between the samples in the
1stand the 2ndstudy year, the cut-off point for first detection of the
type-specific hr-HPV were made at baseline (month 0), sample
month 6 and sample month 12 (see table 1). This analysis was
performed for the total number of type-specific hr-HPV infections.
Hr-HPV status related to cytology and histology
The study protocol was based on self-sampling for HPV
detection. Gynecologic histo- and cytopathology data were thus
not available at the end of the study. To be able to evaluate the
relation between HPV status and cervical lesions, we requested
information from PALGA (the national network and registry of
histo- and cytopathology in The Netherlands) . To compare
data of the hr-HPV positive women included in the 2ndstudy year,
a control group was selected from the women who were hr-HPV
negative at sample month 12. These women were 27–29 years at
study baseline, and had received an invitation of the Dutch
national cervical cancer screening program. In order to relate
cytology/histology results to the hr-HPV status, information of all
sample points in the 1stand 2ndstudy year had to be available.
The four women who were treated for abnormal cytology before
sample month 18, were also excluded in the analysis of persistence
related to cytology. This resulted in 224 women hr-HPV positive
at sample month 12 and 336 women hr-HPV negative at sample
month 12 (control group). PALGA data on gynecologic histo- and
cytopathology, test date, and age of the woman at time of the test,
were available for 166 (74.1%) hr-HPV positive women and 246
(73.2%) women of the control group. Due to privacy legislation,
identification data were made anonymous by a Third Trusted
Party (ZorgTTP, Houten, The Netherlands). To relate these
anonymous gynecologic histo- and cytopathology test results with
the hr-HPV status, women were grouped.
1)Month 12 hr-HPV positive women:
a. type-specific hr-HPV type persistent in the 2ndstudy year
Cleared type-specific hr-HPV type in the 2ndstudy year
2) Control group: Month 12 hr-HPV negative women
The baseline characteristics of the study population were
analyzed and presented in frequencies, mean and standard
deviation (SD). The percentage and 95% confidence interval
(95% CI) of the hr-HPV prevalence at sample month 12 and
persistence in the second year of follow-up were calculated using
the number of infections instead of the number of women. Data
from the questionnaires were used to determine, with simple
logistic regression analysis, whether co-factors like OCP use,
smoking and sexual behavior influenced the persistence in the 2nd
study year. To analyze the relation between hr-HPV status and
histo- and cytopathology test results significance was calculated
with the Fischer’s exact test. All statistical analyses were performed
using SPSS version 16.0 (SPSS Inc., Chicago, IL).
The 235 hr-HPV positive women included in this analysis had a
mean follow-up time of 25.3 months (SD 1.5) and were all born in
the Netherlands. At study baseline these women were 18-29 years
of age with a mean age of 24 years (SD 3.2), and 233 (99.1%)
women reported to ever have had sexual intercourse. Sixty six
(28.1%) women reported that they were current smokers, and
there were 165 (70.2%) current oral contraceptive pill (OCP)
users. These baseline data are shown in table 2.
In order to be included in the 2ndstudy year, women had to be
hr-HPV positive at sample month 12. Of the 235 women, 163 had
one hr-HPV type, 54 had two hr-HPV types, 15 had 3 hr-HPV
types, two had 4 hr-HPV types and one woman had 6 hr-HPV
types at sample month 12. This resulted in a total of 330 type-
specific hr-HPV infections detected at sample month 12. HPV 16
had the highest prevalence of 30.2%, followed by HPV 51
(19.1%), HPV 31 (16.6%), and HPV 52 (14.9%). HPV 18 was
prevalent in 11.1% of the women. (see table 3)
The percentage of the type-specific hr-HPV infections, present
at sample month 12, persisting in the 2ndstudy year are shown in
table 3. For 315 of the 330 type-specific hr-HPV infections data
were available on persistence. Of these 315 type-specific infections,
139 (44.1%) infections were persistent in the 2ndstudy year. HPV
45 was the most persistent type, with a persistence rate of 60.0%,
followed by HPV 31 (56.8%), HPV 16 (54.4%), and HPV type 18
(50.0%). Notably, HPV type 45 was only number 10 in prevalence
with 11 (4.7%) type-specific infections at month 12, whereas HPV
type 51 (second in prevalence with 45 infections) was one of the
least persistent infections with a persistence rate of 25.0%.
Not only the hr-HPV type, but also the duration of the infection
prior to sample month 12, seemed to influence persistence in the
Table 2. Baseline Demographics.
Sample size N (%)
19 8 (3.4)
21 19 (8.1)
22 16 (6.8)
24 15 (6.4)
25 25 (10.6)
27 18 (7.7)
28 33 (14.0)
Education Lower secondary/Lower vocational
Higher secondary/Vocational training46 (19.6)
Higher vocational training/University 184 (78.3)
Current SmokingYes 66 (28.1)
Using OCP Yes 165 (70.2)
No 70 (29.8)
Ever had sexual
No 2 (0.9)
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2ndstudy year. Overall, a type-specific infection detected since
baseline had an odds ratio (OR) of 4.99 (95% CI: 2.67-9.32) to
persist in the 2ndstudy year, compared to infections newly detected
at month 12. Infections first detected at sample month 6 also had
an increased risk, OR of 3.09 (95% CI: 1.74-5.51), to persist in the
2ndstudy year, compared to newly detected infections. (see table 4)
Having multiple HPV infections at sample month 12, irrespec-
tive whether these infections were only hr-HPV or also lr-HPV
types, increased a woman’s risk to have a type-specific hr-HPV
persistent infection in the 2ndstudy year. This was almost a
twofold increased risk, OR 1.93 (95% CI: 1.14–3.26). (See table 5)
Furthermore, table 5 shows that smoking and number of life-
time sexual partners are co-factors influencing hr-HPV persis-
tence. Smokers have an almost twofold increased risk of persistence
than non-smokers (OR: 1.87 [95% CI: 1.03–3.39]) and women
with multiple lifetime sexual partners at baseline have and in-
creased risk of persistence compared to women with 1 lifetime
sexual partner. The OR’s for 2-5, 6-10 and more than 10 lifetime
sexual partners were 4.29 [95% CI: 1.13–16.26, 6.82 [95% CI:
1.76–26.33] and 4.15 [95% CI: 1.05–16.34], respectively. Of the
15 women with 1 lifetime sexual partner at baseline, only three
had a type-specific persistent infection during follow-up. Of the
three persistent infections in women with 1 life time sexual
partner, one (HPV 16) was newly detected at month 12, and two
(HPV 16 and HPV 56) were detected since month 6.
Twelve of the 15 women with 1 lifetime sexual partner at
baseline reported to have a new sexual partner during the study
follow-up. Together they had 16 type-specific hr-HPV infections
detected at sample month 12, of whom 10 were newly detected,
four were first detected at month 6, and two at baseline. Because
most hr-HPV types were newly detected at month 12 and newly
detected hr-HPV infections are less likely to persist, this might be a
confounding factor. However, confounding could not be ruled out
with multiple logistic regression analysis because ‘duration of hr-
HPV detection’ and ‘number of lifetime partners’ are based on
different dependent variables, ‘number of infections’ and ‘number
of women’, respectively.
The following variables; age, OCP use, age of first sexual
intercourse, sexual age, having a new sexual partner during study
follow-up, current type of relationship, current number of sexual
partners, current frequency of sexual intercourse, current condom
use and having a STI during study follow-up, did not influence
persistence of a type-specific hr-HPV infection in the 2ndstudy
year. (see table 5).
Hr-HPV persistence related to cytology and histology
The results from PALGA on cytology and histology related to
the hr-HPV status of the women are presented in table 6. The
mean age of the women with available cytology results was 28,4
(SD: 2.8). It was not possible to measure the exact time interval
between sample month 12 and first cytology because the cases
were made anonymous. However, all dates of self-sampling and
cytology were available. We calculated the time interval between
the date when 50% of the women had taken their self-sample en
Table 3. Ranking of type-specific hr-HPV prevalence at month 12 and type-specific hr-HPV persistence in 2ndstudy year.
Prevalence at month 12
persistence in 2ndstudy year *
N % (95% CI)N % (95% CI)
1 1671 30.2 (24.4–36.5)45 6/1060.0 (26.2–87.8)
2 5145 19.1 (14.3–24.8)31 21/37 56.8 (39.5–72.9)
3 3139 16.6 (12.1–22.0)16 37/68 54.4 (41.9–66.5)
4 52 35 14.9 (10.6–20.1)18 13/26 50.0 (29.9–70.1)
5 56 28 11.9 (8.1–16.8)33 5/10 50.0 (18.7–81.3)
6 18 2611.1 (7.4–15.8) 5612/2646.6 (26.6–66.6)
7 3925 10.6 (7.0–15.3) 39 10/2540.0 (21.1–61.3)
8 68 218.9 (5.6–13.3) 687/19 36.8 (16.3–61.6)
9 3312 5.1 (2.7–8.7) 5212/34 35.3 (19.7–53.5)
1045 114.7 (2.4–8.2) 352/728.6 (3.7–71.0)
11 599 3.8 (1.8–7.1)5111/4425.0 (13.2–40.3)
12 358 3.4 (1.5–6.6) 592/9 22.2 (2.8–60.0)
Total330 140.4% Total139/31544.1 (38.6–49.8)
*Four of the 235 women were treated for abnormal cytology before sample month 18, therefore the natural fluctuation of clearance and persistence was disturbed. The
data of these women were excluded in the analysis of persistence in the 2ndstudy year.
Table 4. Influence of the duration of type-specific hr-HPV detection on persistence in 2ndstudy year.
Type-specific hr-HPV historyN persistent N (%)OR (95% CI)P-value
Detected since baseline63 43 (68.3)4.99 (2.67-9.32)0.000
Detected since study month 670 40 (57.1) 3.09 (1.74-5.51)0.000
Newly detected at month 12166 50 (30.1) 1 (ref)
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when 50% of the women had their cytology performed. For the
study group women (1a and 1b) this interval was 13 months and for
the control group women (2a and 2b) the interval was 12 months.
In the study group 1a; 11 (12.1%) of the 91 women with a
persistent hr-HPV infection were identified with HSIL/CIN2+.
Four of the 75 women (5.3%) in group 1b, who cleared their type-
Table 5. Woman’s risk of having a persistent type-specific hr-HPV type in the 2ndstudy year.
N* (n=231) Persistent N (%)OR (95% CI)P-value
Age Mean: 24.0 years231 122 (52.1) 1.07 (0.99–1.16)0.106
Education Lower secondary/Lower
5 3 (60.0) 1.31 (0.21–8.04)0.769
45 23 (51.1) 0.92 (0.48–1.76)0.898
180 96 (53.3)1 (ref)
Current SmokingNo 166 81 (48.8)1 (ref)
Yes6441 (64.1) 1.87 (1.03–3.39)0.039
OAC at T12 No7536 (48.0) 0.72 (0.41–1.25) 0.240
Yes151 85 (56.3)1 (ref)
Age at first sexual intercourse
# 138 6 (75.0)2.57 (0.37–17.83) 0.339
14–16 119 61 (51.3)0.90 (0.29–2.84)0.859
17–199048 (53.3) 0.98 (0.31–3.15) 0.972
$ 20137 (53.8) 1 (ref)
Sexual Age ˆ Mean: 7.5 228122 (52.6) 1.07 (0.99–1.16)0.197
Lifetime sexual partners1 153 (20.0) 1 (ref)
2–5 87 45 (51.7) 4.29 (1.13–16.26)0.032
6–10 7346 (63.0) 6.82 (1.76–26.33) 0.005
.10 5528 (50.9) 4.15 (1.05–16.34)0.042
New sexual partner during study period?No 5535 (63.6)1.75 (0.90–3.43) 0.103
Yes102 51 (50.0)1 (ref)
Type of relationship at study month 12Married or Living together4728 (59.6) 1.47 (0.73–2.98)
Couple, living apart 8344 (53.0)1.13 (0.63–2.03) 0.281
Single98 49 (50.0)1 (ref) 0.686
Number of sexual partners past 3 months at
study month 12
0 32 14 (43.8)1.36 (0.33–5.59) 669
1 16186 (53.4)2.01 (0.57–7.12)0.281
2 2618 (69.2)3.94 (0.89–17.37) 0.070
$3 11 4 (36.4) 1 (ref)
Sexual frequency past 3 months at T121–3 4226 (61.9)1.49 (0.68–3.26)0.318
4–12 2510 (40.0)0.61 (0.24–1.55)0.299
13–27 61 35 (57.4) 1.23 (0.62–2.47)0.552
.27 69 36 (52.2)1 (ref)
Condom use at study month 12Never 101 60 (59.4)1.13 (0.45–2.81)0.800
Sometimes 39 18 (46.2)0.66 (0.23–1.86) 0.431
Most of times34 16 (47.1) 0.68 (0.24–1.98)0.484
Always23 13 (56.5) 1 (ref)
Having multiple infections at study month 12?#
Yes 10766 (61.7) 1.93 (1.14–3.26)0.015
No123 56 (45.5)1 (ref)
Other STI during study period? No 209112 (53.6)1.27 (0.52–3.12) 0.602
Yes 2110 (47.6)1 (ref)
SD: standard deviation.
*Number = based on available data, as not all 231 women completely filled-in the questionnaire.
ˆSexual age = years of sexual activity (current age minus age of first sexual intercourse).
#Multiple infections = irrespective whether these infections were only hr-HPV or also lr-HPV.
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specific hr-HPV infection in the 2ndstudy year had HSIL/CIN2+.
This difference between both groups was, not significant (p 0.107).
In the control group, 4 of the 246 women (1.6%) developed HSIL/
CIN2+. Women with a persistent hr-HPV type were significantly
more often diagnosed with HSIL/CIN2+ compared to women in
the control group (p 0.000).
In this 2-year prospective cohort study among young unscreen-
ed women, we showed that an already persistent hr-HPV infection
has an increased risk to persist during follow-up compared to
newly detected infections. Hr-HPV types persistent for 6 and 12
months or longer had a respective, threefold or fivefold, increased
risk of persistence. Other studies also reported that HPV infections
are more likely to persist the longer they have been present
[4,5,11,14,27]. Of the newly detected infections 70% were cleared
before the next sample (6 month interval). This is a slightly higher
clearance rate than reported in some other studies, who found a
mean time to clearance of 6-8 months for newly acquired HPV
Persistence was also influenced by the genotype. The overall
persistence of type-specific hr-HPV in this study is 44.1%, HPV
types 45, 31, 16, and 18, had the highest rate of persistence and
the highly prevalent HPV type 51 had a low tendency to persist.
In agreement with our findings, other studies also showed that
HPV 16, 18, 31, and 45 have an increased risk to persist
compared to other HPV types [11,17,29–32]. Additionally, the
IARC multicentre case-control study showed that HPV 16, 18,
45 and 31 were the most common HPV types in cervical
Having multiple HPV infections is a risk factor for hr-HPV
persistence. The risk to have a persistent type-specific hr-HPV
infection during follow-up increased almost twofold in women
with multiple (high-risk and/or low-risk HPV) HPV types at
month 12. These results are in agreement with other studies
showing that women with multiple infections have a higher risk to
have a persistent type-specific HPV infection during follow-up
[9,16,18]. A possible explanation may be that the multiple
infections increase the overall viral load of the infection which
by sheer volume overcomes immune control. Whether this only
happens in women with a lower immunity or also in the general
population is not well known. Another explanation may be that
there are specific interactions between different HPV genotypes.
The impact of having multiple infections on persistence, however,
probably ceases in time. Cuschieri et al., showed that over a period
of two to three years multiple hr-HPV infections did not constitute
a higher risk factor for the development of cervical neoplasia
compared with single hr-HPV infections .
Only two co-factors, smoking and number of lifetime sexual
partners, were found to correlate with the likelihood that a hr-
HPV type would persist. Smokers had a two-fold increased risk of
hr-HPV persistence. Smoking is a known immune suppressor and
a risk factor for cervical carcinoma . However, some studies
did not find a difference in HPV persistence between current
and non-current smokers [27,36]. In our study only smoking at
baseline was registered, therefore we could not identify differences
in the risks between current or past smokers, nor for the duration
of smoking, or number of cigarettes a day.
Women with 1 lifetime sexual partner at baseline were at lower
risk for a persistent infection than women with multiple lifetime
sexual partners. The highest risk was found for women with 6–10
Table 6. Histo- and cytopathology follow-up results.
classificationn (%) Normal CIN 1 CIN 2 CIN 3+ +
1. Study group*
a. Persisting type specific hr-HPV
No cytology1 (1.1)1
Normal55 (60.4) 55
HSIL 9 (9.9)351
b. Cleared type-specific hr-HPV
Normal 52 (69.3) 52
ASCUS 15 (20.0)31 11
2. Control group#
ASCUS 14 (5.7)1 13
LSIL 2 (0.8)11
HSIL 3 (1.2)21
*Study group: study month 12 hr-HPV positive women. Mean interval between study month 12 and cytology=13 months.
#Control group: study month 12 hr-HPV negative women. Mean interval between study month 12 and cytology=12 months.
Human Papillomavirus Persistency in Young Women
PLoS ONE | www.plosone.org6 November 2011 | Volume 6 | Issue 11 | e27937
lifetime sexual partners at baseline. It should be noted, however,
that the majority of the women with 1 lifetime sexual partner at
baseline, had a new sexual partner during study follow-up and
newly identified hr-HPV types. Newly detected hr-HPV types are
less likely to persist, so this might be a confounding factor. In
literature, the number of lifetime sexual partners is not a consistent
risk factor for hr-HPV persistency.[36,37] Therefore, the potential
influence of the number of lifetime sexual partners on hr-HPV
persistency still needs to be elucidated.
The use of OCP’s is also a potential risk factor for the deve-
lopmentofcervicalcarcinoma.However,results fromour study
and others did not find a relation between OCP use and persistence
[21,27,36]. This increased risk is reported to be strongly related to
the duration of use and the effect proved reversible after cessation.
This influence of OCP use on HPV persistence, however, is still
Previously it has been shown that women with a persistent type-
specific hr-HPV infection are significantly more likely to have or
develop CIN than those who were sequentially infected by
different hr-HPV types or who cleared their infection [34,39,40].
We also found that having a persistent type-specific hr-HPV
infection is associated with a higher (not significant) rate of HSIL/
CIN2+ development (12.1%) compared to women who cleared the
type-specific hr-HPV infection (5.3%), and significantly higher (p
0.000) compared to women who were hr-HPV negative at study
month 12 (1.6%).
Due to the anonymous histo- and cytopathology data, we were
not able to correlate the histo- and cytopathology results to the
type-specific hr-HPV types, nor if the woman had multiple HPV
types. Therefore we could not confirm whether the infections with
HPV types 45, 31, 16, and 18 were more likely to cause HSIL/
CIN2+ and whether the number of infections was correlated to the
severity of the lesion as described previously .
There is considerable interest in the possibility of using HPV
testing as a primary cervical cancer screening tool [42,43]. Our
data support that following prevalently detected hr-HPV types for
persistence is useful to identify women with an increased risk of
CIN in the following years . In order to do so, a clear
definition of a clinically relevant persistent HPV infection should
be determined. This definition should be based on the duration of
the type-specific hr-HPV presence that is predictive of CIN deve-
lopment rather than based on two consecutive hr-HPV positive
Based on our results in this population of young women, we
suggest that this interval should be a minimal of 6 months, because
a 6 month persistent hr-HPV type had a threefold increased risk to
persist during follow-up compared to a newly detected hr-HPV
infection. However, this interval may be extended to 12 months,
because 68.3% of the hr-HPV types already persistent for 12
months or longer continued to persist during follow-up. Therefore
a 12 month interval for detection of type-specific hr-HPV per-
sistency will select a group of women that needs close surveillance
for HSIL/CIN2+ development in the following year(s).
In conclusion, we showed that co-factors increasing the risk of
hr-HPV persistency are, genotype specific (45, 31,16, and 18, are
most likely to persist), multiple infections, smoking, and multiple
sexual lifetime partners. Most importantly, we showed that hr-
HPV infections are more likely to persist the longer they have been
detected and that women with a persistent hr-HPV infection have
a higher rate of HSIL/CIN2+ detection in the following year.
Thus, women with a persistent hr-HPV infection should be
monitored for HSIL/CIN2+ development.
We are grateful for the work of C.H. Lenselink in the study design and data
collection. We thank the women that participated in this longitudinal study
on HPV epidemiology.
Conceived and designed the experiments: CES WJGM LFAGM RLMB.
Performed the experiments: WGVQ WJGM AGS. Analyzed the data:
CES AGS. Contributed reagents/materials/analysis tools: WJGM
WGVQ. Wrote the paper: CES WJGM LFAGM AGS RLMB.
1. Schiffman MH, Bauer HM, Hoover RN, Glass AG, Cadell DM, et al. (1993)
Epidemiologic evidence showing that human papillomavirus infection
causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 85:
2. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, et al. (1995)
Prevalence of human papillomavirus in cervical cancer: a worldwide perspective.
International biological study on cervical cancer (IBSCC) Study Group. J Natl
Cancer Inst 87: 796–802.
3. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, et al. (1999)
Human papillomavirus is a necessary cause of invasive cervical cancer
worldwide. J Pathol 189: 12–19.
4. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S (2007)
Human papillomavirus and cervical cancer. Lancet 370: 890–907.
5. Plummer M, Schiffman M, Castle PE, Maucort-Boulch D, Wheeler CM (2007)
A 2-year prospective study of human papillomavirus persistence among women
with a cytological diagnosis of atypical squamous cells of undetermined
significance or low-grade squamous intraepithelial lesion. J Infect Dis 195:
6. Syrjanen K, Kulmala SM, Shabalova I, Petrovichev N, Kozachenko V, et al.
(2008) Epidemiological, clinical and viral determinants of the increased
prevalence of high-risk human papillomavirus (HPV) infections in elderly
women. Eur J Gynaecol Oncol 29: 114–122.
7. Schiffman M, Kjaer SK (2003) Chapter 2: Natural history of anogenital
human papillomavirus infection and neoplasia. J Natl Cancer Inst Monogr
8. Koshiol J, Lindsay L, Pimenta JM, Poole C, Jenkins D, et al. (2008) Persistent
human papillomavirus infection and cervical neoplasia: a systematic review and
meta-analysis. Am J Epidemiol 168: 123–137.
9. Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD (1998) Natural history of
cervicovaginal papillomavirus infection in young women. N Engl J Med 338:
10. Moscicki AB, Shiboski S, Broering J, Powell K, Clayton L, et al. (1998) The
natural history of human papillomavirus infection as measured by repeated
DNA testing in adolescent and young women. J Pediatr 132: 277–284.
11. Richardson H, Kelsall G, Tellier P, Voyer H, Abrahamowicz M, et al. (2003)
The natural history of type-specific human papillomavirus infections in female
university students. Cancer Epidemiol Biomarkers Prev 12: 485–490.
12. Woodman CB, Collins SI, Young LS (2007) The natural history of cervical HPV
infection: unresolved issues. Nat Rev Cancer 7: 11–22.
13. van Duin M, Snijders PJ, Schrijnemakers HF, Voorhorst FJ, Rozendaal L, et al.
(2002) Human papillomavirus 16 load in normal and abnormal cervical scrapes:
an indicator of CIN II/III and viral clearance. Int J Cancer 98: 590–595.
14. Munoz N, Hernandez-Suarez G, Mendez F, Molano M, Posso H, et al. (2009)
Persistence of HPV infection and risk of high-grade cervical intraepithelial
neoplasia in a cohort of Colombian women. Br J Cancer.
15. Castle PE (2008) Invited commentary: is monitoring of human papillomavirus
infection for viral persistence ready for use in cervical cancer screening?
Am J Epidemiol 168: 138–144.
16. Trottier H, Mahmud S, Prado JC, Sobrinho JS, Costa MC, et al. (2008) Type-
specific duration of human papillomavirus infection: implications for human
papillomavirus screening and vaccination. J Infect Dis 197: 1436–1447.
17. Kulmala SM, Shabalova IP, Petrovitchev N, Syrjanen KJ, Gyllensten UB, et al.
(2007) Type-specific persistence of high-risk human papillomavirus infections in
the New Independent States of the former Soviet Union Cohort Study. Cancer
Epidemiol Biomarkers Prev 16: 17–22.
18. Nielsen A, Kjaer SK, Munk C, Osler M, Iftner T (2010) Persistence of high-risk
human papillomavirus infection in a population-based cohort of Danish women.
J Med Virol 82: 616–623.
19. Louvanto K, Rintala MA, Syrjanen KJ, Grenman SE, Syrjanen SM (2010)
Genotype-specific persistence of genital human papillomavirus (HPV) infections
in women followed for 6 years in the Finnish Family HPV Study. J Infect Dis
Human Papillomavirus Persistency in Young Women
PLoS ONE | www.plosone.org7 November 2011 | Volume 6 | Issue 11 | e27937
20. Lenselink CH, Melchers WJ, Quint WG, Hoebers AM, Hendriks JC, et al. Download full-text
(2008) Sexual behaviour and HPV infections in 18 to 29 year old women in the
pre-vaccine era in the Netherlands. PLoS ONE 3: e3743.
21. Schmeink CE, Massuger LF, Lenselink CH, Quint WG, Melchers WJ, et al.
(2010) Effect of the menstrual cycle and hormonal contraceptives on human
papillomavirus detection in young, unscreened women. Obstet Gynecol 116:
22. Melchers WJ, Bakkers JM, Wang J, de Wilde PC, Boonstra H, et al. (1999) Short
fragment polymerase chain reaction reverse hybridization line probe assay to
detect and genotype a broad spectrum of human papillomavirus types. Clinical
evaluation and follow-up. Am J Pathol 155: 1473–1478.
23. van Hamont D, van Ham MA, Bakkers JM, Massuger LF, Melchers WJ (2006)
Evaluation of the SPF10-INNO LiPA human papillomavirus (HPV) genotyping
test and the roche linear array HPV genotyping test. J Clin Microbiol 44:
24. Winer RL, Lee SK, Hughes JP, Adam DE, Kiviat NB, et al. (2003) Genital
human papillomavirus infection: incidence and risk factors in a cohort of female
university students. Am J Epidemiol 157: 218–226.
25. Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, et al. (2002) Incidence,
prevalence, and clearance of type-specific human papillomavirus infections: The
Young Women’s Health Study. J Infect Dis 186: 462–469.
26. Casparie M, Tiebosch AT, Burger G, Blauwgeers H, van Krieken JH, et al.
(2007) Pathology databanking and biobanking in The Netherlands, a central role
for PALGA, the nationwide histopathology and cytopathology data network and
archive. Cell Oncol 19-24.
27. Maucort-Boulch D, Plummer M, Castle PE, Demuth F, Safaeian M, et al. (2009)
Predictors of human papillomavirus persistence among women with equivocal or
mildly abnormal cytology. Int J Cancer.
28. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, Rozendaal L, Remmink AJ,
et al. (1999) Relation of human papillomavirus status to cervical lesions and
consequences for cervical-cancer screening: a prospective study. Lancet 354:
29. Schiffman M, Herrero R, Desalle R, Hildesheim A, Wacholder S, et al. (2005)
The carcinogenicity of human papillomavirus types reflects viral evolution.
Virology 337: 76–84.
30. Schiffman M, Rodriguez AC, Chen Z, Wacholder S, Herrero R, et al. (2010) A
population-based prospective study of carcinogenic human papillomavirus
variant lineages, viral persistence, and cervical neoplasia. Cancer Res 70:
31. Syrjanen K, Shabalova I, Naud P, Kozachenko V, Derchain S, et al. (2009)
Persistent High-Risk Human Papillomavirus Infections and Other End-Point
Markers of Progressive Cervical Disease Among Women Prospectively Followed
up in the New Independent States of the Former Soviet Union and the Latin
American Screening Study Cohorts. Int J Gynecol Cancer 19: 934–942.
32. Iftner T, Eberle S, Iftner A, Holz B, Banik N, et al. (2010) Prevalence of low-risk
and high-risk types of human papillomavirus and other risk factors for HPV
infection in Germany within different age groups in women up to 30 years of
age: an epidemiological observational study. J Med Virol 82: 1928–1939.
33. Munoz N (2000) Human papillomavirus and cancer: the epidemiological
evidence. J Clin Virol 19: 1–5.
34. Cuschieri KS, Cubie HA, Whitley MW, Gilkison G, Arends MJ, et al. (2005)
Persistent high risk HPV infection associated with development of cervical
neoplasia in a prospective population study. J Clin Pathol 58: 946–950.
35. Castellsague X, Munoz N (2003) Chapter 3: Cofactors in human papillomavirus
carcinogenesis--role of parity, oral contraceptives, and tobacco smoking. J Natl
Cancer Inst Monogr 20-28.
36. Bae J, Seo SS, Park YS, Dong SM, Kang S, et al. (2009) Natural history of
persistent high-risk human papillomavirus infections in Korean women. Gynecol
37. Fukuchi E, Sawaya GF, Chirenje M, Magure T, Tuveson J, et al. (2009) Cervical
Human Papillomavirus Incidence and Persistence in a Cohort of HIV-Negative
Women in Zimbabwe. Sex Transm Dis.
38. Appleby P, Beral V, Berrington de GA, Colin D, Franceschi S, et al. (2007)
Cervical cancer and hormonal contraceptives: collaborative reanalysis of
individual data for 16,573 women with cervical cancer and 35,509 women
without cervical cancer from 24 epidemiological studies. Lancet 370:
39. Sundstrom K, Eloranta S, Sparen P, Arnheim DL, Gunnell A, et al. (2010)
Prospective study of HPV types, HPV persistence and risk of squamous cell
carcinoma of the cervix. Cancer Epidemiol Biomarkers Prev.
40. Lai CH, Chao A, Chang CJ, Chao FY, Huang HJ, et al. (2008) Host and viral
factors in relation to clearance of human papillomavirus infection: A cohort
study in Taiwan. Int J Cancer 123: 1685–1692.
41. Bello BD, Spinillo A, Alberizzi P, Cesari S, Gardella B, et al. (2009) Cervical
infections by multiple human papillomavirus (HPV) genotypes: Prevalence and
impact on the risk of precancerous epithelial lesions. J Med Virol 81: 703–712.
42. Brink AA, Snijders PJ, Meijer CJ, Berkhof J, Verheijen RH (2006) HPV testing
in cervical screening. Best Pract Res Clin Obstet Gynaecol 20: 253–266.
43. Franco EL, Mahmud SM, Tota J, Ferenczy A, Coutlee F (2009) The expected
impact of HPV vaccination on the accuracy of cervical cancer screening: the
need for a paradigm change. Arch Med Res 40: 478–485.
44. Castle PE, Rodriguez AC, Burk RD, Herrero R, Wacholder S, et al. (2009)
Short term persistence of human papillomavirus and risk of cervical precancer
and cancer: population based cohort study. BMJ 339: b2569.
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