Development and Duration of Human Papillomavirus Lesions, after Initial Infection
Development and Duration of Human Papillomavirus Lesions, after Initial Infection
Rachel L. Winer 1 2
Nancy B. Kiviat 2
James P. Hughes 0 2
Diane E. Adam 1 2
Shu-Kuang Lee 0 2
Jane M. Kuypers 2
Laura A. Koutsky 1 2
0 Biostatistics, University of Washington , Seattle
2 Departments of
Background. To determine the potential value of human papillomavirus (HPV) vaccines, information concerning the incidence and duration of clinically important lesions is needed. Methods. A total of 603 female university students were followed for a mean duration of 38.8 months. Triannual gynecologic examinations included cervical and vulvovaginal specimen collection for Pap and HPV DNA testing. Women with cytologic evidence of a high-grade squamous intraepithelial lesion (SIL) were referred for colposcopically directed biopsy. Results. Among women with incident HPV infection, the 36-month cumulative incidence of cervical SILs found by cytologic testing (47.2%; 95% confidenc interval [CI], 38.9%-56.4%) was higher than that of vaginal SILs (28.8%; 95% CI, 21.3%-38.2%). The median time to clearance of cervical and vaginal SILs was 5.5 and 4.7 months, respectively. Among women with incident HPV-16 or HPV-18 infection, the 36-month cumulative incidence of cervical intraepithelial neoplasia (CIN) grade 2 was 20.0% (95% CI, 10.8%-35.1%), and that of CIN grade 3 was 6.7% (95% CI, 2.5%-17.0%). The 36-month cumulative incidence of clinically ascertained genital warts among women with incident HPV-6 or HPV-11 infection was 64.2% (95% CI, 50.7%-77.4%). Conclusions. Intraepithelial lesions are common early events among women with incident HPV infection, and the interval between incident HPV-16 or HPV-18 infection and biopsy-confi med CIN grade 2-3 appears to be relatively short.
Genital human papillomavirus (HPV) infections are
common among young women [
]. Although there
are abundant data describing the persistence of HPV
infection, few studies have examined the incidence and
duration of clinically important endpoints, including
squamous intraepithelial lesions (SILs) and genital warts.
Furthermore, there is an impression that SILs are
uncommon manifestations of HPV infection  and that
high-grade SILs (HSILs) usually take years to develop
after initial infection with HPV [
]. We hypothesized
that this may be a function of the screening interval and
that SILs would be detected more frequently and earlier
(but that the duration of lesions would be relatively
short) with more frequent screening. This prospective
study was designed to estimate the incidences and
induction periods of clinical endpoints among women with
incident HPV infection. With prophylactic HPV vaccines
showing promise [
], such information is needed to
determine the potential population-level impact of these
vaccines on rates of genital warts, abnormal Pap test
results, colposcopic examinations, biopsies, and
treatments for intraepithelial lesions.
PATIENTS, MATERIALS, AND METHODS
Study population. Female students 18–20 years of age
were recruited between July 1990 and September 1997
to participate in a longitudinal study of HPV infection.
Methods of recruitment and data collection have been
described elsewhere [
]. A total of 603 women (∼20%
of eligible women receiving letters of invitation) were
enrolled. Informed consent was obtained from all study
participants, and the human-experimentation
guidelines of the University of Washington Institutional
Review Board were followed.
Visits were scheduled at 4-month intervals. At every visit,
demographic, medical, and sexual history information was
obtained, and cervical and vulvovaginal Dacron-tipped swab
specimens were collected for HPV DNA testing. Cervical cytologic
specimens were obtained using a cytobrush to collect cellular
material from the endocervix and a plastic spatula to collect
cells from the squamocolumnar junction and ectocervix.
Vaginal cytologic specimens were obtained using a Dacron-tipped
swab to collect cells from the lateral vaginal walls. Colposcopic
examination of the cervix was performed at every visit. All
women with cytologic or colposcopic evidence of an HSIL were
referred for colposcopically directed biopsy. Women with
repeated cytologic test results showing a low-grade SIL (LSIL) or
equivocal finding were also referred.
HPV DNA testing. As described elsewhere, specimens were
analyzed for the presence of HPV DNA by use of polymerase
chain reaction (PCR) amplificatio and dot-blot hybridization
]. One-fiftiet of each sample was amplifie in
duplicate with the consensus primers MY09, MY11, and HMB01
and with human b-globin control primers. The products of
these amplification were then probed with a biotin-labeled
generic probe designed to detect most genital HPV types.
Specimens found to be positive by generic probe were tested with
individual and mixtures of biotin-labeled, type-specifi
oligonucleotide probes, to determine the presence of HPV types 6,
11, 16, 18, 31, 45, and 56 and the following type mixtures: 31/
33/35/39, 40/42/53/54, and 51/52/55/58. Samples hybridizing
with the generic probe but not with one of the type-specifi
probes were classifie as positive for uncharacterized genital
HPV types. Samples that were found to be negative for both
HPV and b-globin DNA by PCR were considered inadequate
for analysis. Unless noted otherwise, cervical and vulvovaginal
HPV DNA test results were combined.
Cytologic and histologic testing. Pap smears were reviewed
by a cytotechnologist; all smears showing abnormalities and all
biopsies were reviewed by the study pathologist. The study
pathologist has participated in blinded studies of interpathologist
agreement, and her cytologic and histologic interpretations were
found to be similar to those of other expert gynecologic
]. Pap smear finding were classified according to the
Bethesda system [
], as normal, atypical squamous cells of
undetermined significanc (ASCUS), LSIL, or HSIL. Biopsy tissue
was diagnosed as showing cervical intraepithelial neoplasia (CIN)
grade 1, 2, or 3. None of the women developed cytologic or
histologic evidence of invasive cervical cancer.
Statistical analysis. An incident HPV infection was define
as the firs positive HPV DNA test result among women who
tested negative at enrollment. In analyses restricted to
typespecifi HPV infections, an incident infection was define as
the firs positive type-specifi result among women who tested
negative for that type at enrollment.
The cumulative probabilities of developing HPV-related
lesions were estimated using the Kaplan-Meier method. In analyses
restricted to women with incident HPV infection, at-risk time
was calculated from the date of firs incident HPV DNA positivity
to the date of detection of firs abnormality. To assess
development of HPV-related lesions among women without HPV
infection, at-risk time was calculated from enrollment to the date
of lesion detection (including the enrollment visit), and women
were censored at the date of firs positive HPV DNA test result.
Women could contribute at-risk time to both HPV-negative and
incident HPV analyses, provided they did not develop an
abnormality prior to detection of incident HPV infection. For the
genital-warts analysis, separate cumulative incidences were
estimated among women with incident HPV-6 or HPV-11 infection
and among women with incident infections other than HPV-6
or HPV-11. The same method was used to estimate the
cumulative incidence of CIN grade 2–3 among women with and
without incident HPV-16 or HPV-18 infection.
In analyses distinguishing between cervical and vaginal SILs,
date of firs detection was define as the firs date at which a
cervical or vaginal lesion was detected, regardless of whether a
lesion had been detected at the other site at an earlier date.
Date of CIN grade 2–3 was define as the study visit date
immediately preceding the date at which a biopsy-confi med
diagnosis was made.
The Kaplan-Meier method was also used to estimate median
time to clearance of LSILs (based on cytologic testing) and genital
warts. Date of development was define as the date at which the
abnormality was firs detected, and date of clearance was define
as the firs date at which the abnormality was not detected.
Cox proportional-hazards methods were used to compare
durations of firs and second episodes of SILs and to make
comparisons between cervical and vaginal SILs with respect to
both duration and time to development. Robust variances [
were used when multiple observations from the same women
were included in the analysis.
To determine what the cumulative probabilities of incident
cervical SILs would be if women were screened yearly or every
2 years rather than every 4 months, analyses were restricted to
women who were sexually experienced at the time of
enrollment. At-risk time was calculated from the enrollment date,
and follow-up data were restricted to visits closest to 12, 24,
36, and 48 months after enrollment for yearly screening and
24 and 48 months after enrollment for biennial screening.
A total of 602 women provided at least 1 set of adequate
samples for HPV DNA testing and cytologic evaluation. Altogether,
these women completed 5500 visits. Their mean follow-up time
was 38.8 months (SD, 19.5 months), the mean number of visits
per person was 9.1 (SD, 4.1), and the median time between
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visits was 4.3 months. The mean age of these women at
enrollment was 19.2 years (SD, 0.5 years). At enrollment, 170
women were virgins, and the mean lifetime number of partners
of the 432 women who were sexually active was 2.5 (SD, 2.8).
HPV DNA was detected in the genital-tract specimens from
119 (20.3%) of 585 women providing adequate samples for
testing at enrollment, and SILs were detected in 24 (3.5%) of
589 women. Of these 24 women, 23 (95.8%) also tested positive
for HPV DNA at the enrollment visit. Four virgins did not
provide samples for cytologic evaluation at enrollment.
During the course of the study, there were 129 incident cases
of SIL and 23 incident cases of biopsy-confi med CIN grade
2–3. Of the 129 incident cases of SIL, 26 (20.2%) were detected
on the same day that the woman was firs found to be positive
for HPV DNA. Eighty-fiv cases of SIL were firs detected in
the cervix, 17 in the vagina, and 27 in both sites. Vaginal SILs
were detected at subsequent visits in 12 (14.1%) of the 85
women whose firs SIL was detected in the cervix, and cervical
SILs were detected at subsequent visits in 6 (35.3%) of the 17
women whose firs SIL was detected in the vagina.
Seventeen of the 23 cases of histologically confi med CIN
grade 2–3 were diagnosed among women with incident HPV
infection. Sixteen cases were referred for biopsy on the basis
of abnormal cytologic test results, and 1 case had colposcopic
signs of an HSIL.
Cumulative incidence of lesions. Among women with
incident HPV infection, the cumulative incidence of cervical SILs
(47.2% at 36 months; 95% confidenc interval [CI], 38.9%–
56.4%) was higher than that of vaginal SILs (28.8% at 36 months;
95% CI, 21.3%–38.2%; P ! .01) (figu e 1A and 1B). Among
women who developed lesions within 36 months of incident
infection (95%), the median time from firs incident HPV
infection to detection of lesions was 4.0 months (interquartile range
[IQR], 0–14.3 months) for cervical SILs and 8.0 months (IQR,
3.3–20.7 months) for vaginal SILs. The distribution of HPV types
detected at the same visit that SILs were detected was similar for
cervical and vaginal SILs.
Among women with incident HPV infection of any type, the
36-month cumulative incidence of CIN grade 2–3 was 11.1%
(95% CI, 6.5%–18.5%) (figu e 1C), and, among women with
incident HPV-16 or HPV-18 infection, it was 27.2% (95% CI,
16.3%–43.3%) (figu e 2). Among women who received
diagnoses within 40 months of incident infection (94%), the median
time from firs HPV detection to CIN grade 2–3 was 14.1
months (IQR, 6.7–31.2 months). The median time from
detection of incident HPV infection to diagnosis was similar for
CIN grade 2 and CIN grade 3. Among women with CIN grade
1 (n p 16), CIN grade 2 (n p 13), and CIN grade 3 (n p 4),
the median time from detection of incident HPV infection to
detection of firs cervical cytologic abnormality was ∼4 months
and did not differ by lesion grade. Nine (52.9%) of 17 cases
of CIN grade 2–3 (including all 4 cases of CIN grade 3) among
women with incident HPV infection were positive for HPV-16
or HPV-18 DNA at the visit prior to biopsy (figu e 3). Six of
the other 8 cases of CIN grade 2 were found to be positive for
other high-risk types. There were no cases of CIN grade 2–3
among HPV-negative women within 1048 person-years of
Although the 36-month cumulative incidence of cervical
SILs among HPV-negative women was only 1.6% (95% CI,
0.7%–4.0%), the 36-month cumulative incidence of ASCUS
or a more severe abnormality was 25.2% (95% CI, 20.3%–
31.1%) (figu e 1D).
By modeling the screening interval (that is, by assuming
screening every year, every 2 years, or every 4 months), we
showed that the frequency of screening greatly impacted
estimates of the frequency of detecting cervical cytologic
abnormalities (table 1). Whether a Pap test findin of ASCUS or a
more severe abnormality or of SIL was used as the threshold,
more-frequent screening yielded higher cumulative incidence
estimates at both 24 and 48 months.
Lesion duration. Of 112 women who developed incident
LSILs before their last follow-up visit, 96 (85.7%) cleared their
firs episode of SILs while enrolled in the study, and 10 (8.9%)
subsequently developed CIN grade 2–3, were referred for
treatment, and were removed from the duration analysis of
cervical SILs. The median time to clearance was slightly longer for
cervical SILs (5.5 months; IQR, 4.2–7.9 months) than for
vaginal SILs (4.7 months; IQR, 3.9–6.8 months) (P p .08). The
duration of histologically confi med HSILs could not be
determined, because all women were referred for treatment.
Fourteen (15.7%) of 89 women had a recurrent episode of cervical
LSILs. Durations were similar for firs (median, 5.5 months;
IQR, 4.1–8.0 months) and second (median, 5.9 months; IQR,
4.2–7.1 months) episodes (P p .55).
Genital warts. The cumulative incidence of genital warts
was highest among women with incident HPV-6 or HPV-11
infection (66.2% at 36 months; 95% CI, 52.8%–79.2%) (figu e
4). Given the rarity of incident HPV-11 infections, compared
with incident HPV-6 infections, we did not have sufficien
power to determine whether the cumulative incidences of
genital warts were similar for each type. It is worth noting, however,
that during the course of the study period, clinically visible
genital warts were detected in 2 of 4 women with incident
HPV-11 infection (and no HPV-6 infection) and in 28 of 41
women with incident HPV-6 infection (and no HPV-11
infection). Of 31 women developing clinically ascertained genital
warts after incident HPV infection (2 women had cervical warts,
1 woman had vaginal warts, and 28 women had vulvar, perineal,
or perianal warts), 83.9% were positive for HPV-6 or HPV-11
DNA prior to or during the time that warts were present. The
median time between detection of incident HPV-6 or HPV-11
infection and detection of genital warts was 2.9 months (IQR,
0–5.7 months). All women with probable or definit genital warts
were referred for treatment; the median time to clearance with
treatment was 5.9 months (IQR, 3.9–8.0 months).
Contrary to the theory that prolonged infection is necessary
for progression to high-grade neoplasia [
], our results
support the hypothesis that HSILs are often an early
manifestation of HPV infection in young women. Half of the incident
cases of biopsy-confi med CIN grade 2–3 in our study occurred
within 14 months of an incident HPV infection. This findin
is consistent with previous studies that reported HSILs within
2 years of detection of HPV infection [
]. Our data also
suggest that the time between firs detection of HPV and firs
detection of cytologic abnormalities is similar for all grades of
CIN. Many of the HSILs we detected were CIN grade 2.
Although CIN grade 2 is less reproducible than CIN grade 3 ,
it is reassuring that 100% of women with diagnoses of CIN
grade 2 were positive for HPV DNA at the visit prior to biopsy
and that 88.2% were positive for high-risk types.
Approximately 50% of women in our cohort developed
cervical LSILs within 3 years of an incident HPV infection. Two
other cohort studies reported significantl lower proportions
of cervical abnormalities in women with incident HPV
infection. Moscicki et al. [
] followed a cohort of young women in
San Francisco every 6 months until firs HPV detection and
every 4 months thereafter and found that only 15% developed
LSILs within 3 years of initial HPV infection. Woodman et al.
] followed young women in the United Kingdom at 6-month
intervals and reported that 33% developed any type of cervical
cytologic abnormality within 3 years of infection. More
frequent screening and the fact that the median duration of lesions
was !6 months could explain the higher cumulative incidence
of lesions in our cohort. It is possible that Woodman et al. [
and Moscicki et al. [
] missed detection of lesions that
developed and cleared within 6 months. Irrespective of HPV status,
detection rates tend to increase with more frequent screening,
as is demonstrated in our projections of the number of cervical
abnormalities that would have been detected if the sexually
active women in our cohort had been tested yearly or every
other year (intervals consistent with current screening
]) rather than every 4 months. Furthermore, by testing
every 4 months, we were able to more finel estimate the
median duration of LSILs. Our estimate of !6 months was lower
than Woodman et al.’s [
] estimate of 9 months for the median
duration of any type of cervical abnormality and was lower
than estimates in earlier studies reporting that half of lesions
regress within several years [
2, 17, 18
]. None of these earlier
studies included active follow-up; rather, they relied on women
attending screening visits according to their own schedules.
Although less common than cervical SILs, vaginal SILs were
not rare among women with HPV infection (almost 30% of
women developed vaginal SILs within 3 years of incident HPV
infection). The present study is, to our knowledge, the firs study
to assess the incidence of vaginal SILs. Although the high
incidence of vaginal SILs is somewhat surprising, given the rarity of
vaginal cancers [
], all lesions cleared within 16 months,
suggesting that early vaginal lesions are associated with a low risk
for progression. Vaginal SILs were more likely to precede cervical
SILs than vice versa, an observation that is consistent with the
findin that HPV DNA is more frequently detected in the vulva
or vagina before it is detected in the cervix [
One limitation of our study is that we did not test for
individual HPV types other than types 6, 11, 16, 18, 31, 45, and
56. Although this decreased our ability to assess the associations
between certain HPV types and risk of lesions, we were able
to evaluate risk associated with the 2 types most
frequently detected in cervical cancers (HPV-16 and HPV-18) and the
2 types most often associated with genital warts (HPV-6 and
HPV-11). However, without tissue dissection, we cannot know
with certainty which HPV types were present in the cells of all
lesions. Furthermore, our projections of the 1- and 2-year
cumulative incidences of abnormal Pap smear results are
conservative, since women with confi med HSILs were treated and
removed from the analysis. Also, although specimens for
cervical and vaginal cytologic testing were obtained by
independent sampling of the endo-/ectocervix and lateral vaginal walls,
respectively, it is possible that cervical cells could have been
picked up in vaginal cytologic specimens, or vice versa. Finally,
as with any test, there is always the inherent possibility of
falsepositive or false-negative results in HPV, cytologic, colposcopic,
and histologic testing.
The present study is one of the firs to examine the incidence
and duration of clinically important endpoints of incident HPV
infection. Although the incidence of LSILs in our study was
higher than those previously reported, these lesions tended to
regress quickly. Although the recommendations to refer women
directly to colposcopically directed biopsy after detection of
] may be appropriate for older women, our results
suggest that incident LSILs detected in young women (i.e., those
18–24 years of age) behave differently from prevalent LSILs
detected in older women with persistent HPV infection.
Applying such recommendations to young women would result
in a largely unnecessary expense.
When HSILs developed, they did so relatively early after
incident HPV infection, primarily HPV-16 infection. Genital
warts also developed quickly after infection with HPV-6 or
HPV-11. These results have important implications for the
implementation of a prophylactic HPV vaccine. Our results
suggest that, within 4 years of achieving widespread vaccine
coverage, a quadrivalent vaccine for HPV types 6, 11, 16, and 18
could reduce a substantial proportion of cases of CIN grade
2–3 and genital warts in young women.
We thank Sandra O’Reilly for her contributions as research coordinator
and Connie Nelson for her assistance in creating the figu es for the article.
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