Open Jo urnal of Obstetr ics and Gynecology, 2011, 1, 104-108 OJOG
doi:10.4236 /ojog.2011.13018 Publ ished O nline S ept ember 2011 (http ://
Published Online September 2011 in SciRes.
Age-specific distribution of Human Papilloma Vir us (HPV)
mucosal infection among young females
Annalisa Pieralli*, Maria Graz ia Fallani, Virginia Lozza, Serena Corioni, Manuela Longinotti,
Massimiliano Fambrini, Carlo Penna
Department of the Sci ences for Woman’s and Child’s Health, University of Floren ce, Italy.
E-mai l : *
Received 30 May 2011; re v i s e d 5 July 2011; accepted 13 July 2011.
The goal of our study was to describe the age-specif ic
distribution of HPV genotypes and related disease
among females under the age of 25 years. A prospec-
tive cohort study was carried out. We enrolled 85
young females aged 16 - 25 years (30 aged 16 - 19 and
55 aged 20 - 25 years) ref erred to our colposcopic unit
after a repeated abnormal Pap smear result . Every
patient unde rw ent an H PV DN A test ing, a co lposcopy
and eventually a cervical biopsy. Participants were
proposed to follow-up or trea t ment on request. Treat-
ment was performed by destructive or excisional laser
CO2 therapy. Data were analyzed by Fisher’s Exact
test. The overall preva lence of low -ri sk HPV amounted
to 80% among 16 - 19-year-old girls, while the o ver all
prevalence of high-risk HPV was 85.5% among 20 -
25-year-old patients. The univariate analysis of cho-
sen characteristics of HPV-disease demonstrates the
statistically significative difference of this infection
between the two groups of age (P < 0.005). We ob-
served a particular age-specific stratification of HPV
genotypes and related disease, which appeared to be
characterized by a cut-off at the age of 20 years. Ac-
cording to our data, cervical screening program in
Italy seems to start later than the beginning of
HPV-related pathology.
Keywords: HPV; Epidemiology; Adolescents; Genital
According to available data, genital infection with the
Human Papillomavirus is highly prevalent in young fe-
male population from all countries and it is the most
common sexually transmitted infection among sexually
active girls [1-6]. Its prevalence in young women ranges
from about 20 to 46% in various countries [7-9].
Sexually active adolescents show the highest HPV
prevalence and incidence and over 50% - 80% of them
acquire the infection within two-three years of initiating
sexual intercourse.
Data about the re al di strib utio n of HPV mucosal i nfec-
tion and re lated histological l esions in young wo men are
scarce in literature. The actual knowledge of HPV-linked
genital pathology and its natural history in adolescence
principally derives from screening test results and not
from clinical and histological findings, because care pro-
viders are sure that cell abnormalities are usually tran-
sient in adolescent population, and they advise observa-
tion by repeating cytology or HPV DNA testing and not
second level evaluation [10].
Since a quadrivalent (HPV 6, 11, 16 and 18) and a bi-
valent (HPV16 and 18) prophylactic HPV vaccine have
been licensed, since the vaccination does not affect the
course of an existing vaccine-type infection and the ef-
fectiveness of immunization for subsequent infections is
uncer t ain, pre-vaccination data on the occurrence of HPV
infection and its nat ural history in the adolescence popu-
lation would be essential to mo nito r the impact of vacci-
nation itself.
There is only one study in literature describing the
age-specific distribution of HPV types within a popula-
tion o f girls aged 11 - 26 and it was conducted determin-
ing the specific sero p r evalence [11].
It has been already demonstrated that only 50% HPV
infected women have detectable levels of antibodies to
the HPV type with which they were infected and that
there are specific risk factors which spoilt seropositivity
as marker of HPV infection and linked pathology [12].
The aim of the present study was to describe the age-
specific distribution of HPV infecting genotypes and
their related lesions among females under the age of 25,
and to provide a first knowledge on Italian adolescents’
HPV infection occurre nce.
2.1. Study Population
From September 2009 to April 2010 a prospective cohort
A. Pieralli et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 104-108
Copyright © 2011 Sc iRes. OJOG
study was carried out at the Colposcopy and Laser The-
rapy Office of Careggi U niversity Hospital in Florence.
Recruitment included young females aged 16 - 25
years referred to our colposcopy unit after a repeated
abnormal Pap smear result performed on a voluntary
basis. Ever y patie nt was submitted to HPV DN A test ing,
colposcopic evaluation of the entire genital area and
eventual bioptical sampling of unclear lesions. A brief
personal medical history was collected to be informed
about recurrent HPV genital lesions. Informed consent
on procedures to be performed was obtained from each
patient or, when under 18 of age, from her legal tutor.
The study population was selected as not to be influ-
enced to a considerable extent by demographic charac-
teristics such as educational level, co-morbid itie s, smoke,
oral contraceptives use, condom use. The distribution of
these characteristics in our study population had to be
similar to that in the general I talian female p op ulation for
this group of age. Moreover the entire sampled popula-
tion consisted of indigenous Italian ethnic origi n.
2.2. HPV DNA Testing
Two cellular samples were collected from the infected
genital area by scrab.
Both specimens were stored overnight at 4˚C grades
until proc essing.
Up to five mL of cervical samples were centrifugated
at 2000 g for 15 minutes at room temperature; 200 ųl of
the obtained cell pellets were diluted in 190 ul of diges-
tion buffer and incubated with proteinase K at 56˚C fo r 2
- 5 hours (EZ1 DNA Tissue kit, QIAGEN, Germany).
An automated DNA purification was performed (Bio-
Robot EZ1, QIAGEN, Germany).
PCR (Polymerase Chain Reaction) amplification of
HPV-DNA sequences was carried out by a thermocycler
(MJ Research) with a two-step commercial PCR kit
First step consisted in screening the presence of HPV-
DNA by L1 consensus primers (6, 11, 16, 18, 26, 31, 33,
35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 55, 56, 57, 58,
59, 66 and 68) while in a second time positive screens
wer e t ype d b y E6 -E7 primers (6, 11 for low risk; 16, 18,
31, 33, 35, 45, 52 and 58 for high risk). The amplified
products were identified by agarose gel at 2% electro-
phoresis and visualized by ultraviolet light. The quality
of the DNA samples was validated by detection of the
housekeeping gene beta-globin, as internal control.
2.3. Management Protocol
All cohort participants underwent a colposcopy using a
3% acetic acid solution followed b y Lugol test. The col-
poscopic aspect was i nterpreted according to the Inter na-
tional No menclature [13].
A cervical biopsy specimen was taken from the HPV
lesion in all girls with colposcopic diagnosis of ANTZ
(abnormal transformation zone) and it was analyzed in
order to obtain the histological diagnosis of the lesion.
Treatment of genital warts was performed in accor-
dance with patient’s request.
Histologically proven CIN (cervical intraepithelial
neoplasia) were treated by destructi ve o r exci sio nal laser
CO2 therapy.
The choice of treatment method was based on the type
of diagnosed disease. A laser vaporization therapy (de-
structive method) was performed for florid and flat warts,
every site. Most cases of CIN 2/3 were treated by laser
vaporization, in respect of patients’ young age and ferti-
lity status; laser conization was reserved only to lesions
extended into the end o cer vix (excisional method).
Laser procedures were performed by a SmartXide 50
HS (Deka Inc. Italia) CO2 laser with maximum power
output o f 50 Watt, used in super-pulsed mode at 25 Watt
and connected to a Zeiss OPMI colposcope (Carl Zeiss,
Oberkochen, Germany). The beam spot diameter ranged
from 0.5 to 1 mm with an irradiance ranging from 3500
to 4000 W/cm 2, guided by a micromanipulator.
All treated women were checked three months after
the procedure. Follow-up visits were scheduled after six
mont hs from diagno sis.
All the procedures involved in the study were per-
formed in accordance with our Institutional guidelines
and in respect of the principles of the Declaration of Hel-
Ethic approval for this study was obtained by the
University of Florence and the AOUC teaching Hospital
review board.
2.4. Statistical Analysis
The distribution of five characteristics of HPV-related
disease (presence of HR HPV, co-infection, histologi-
call y proven high grade CIN, relapsing disease and mul-
tiple site lesions) in different age groups was evaluated
analyzing variables by Fishers Exact test as discrete
The mea n age of st udy popul ation ( n = 85) was 21.36 (±
3.54) years; 30 patients belonged to the age group 1 6-19
and 55 to the age group 20 - 25 years.
Of the participants, 24.7% (n = 21) had already had
previous experience o f HPV-related disease and had been
treated by laser therapy; 61.9% (n = 13) of them were
younger than 20 years at the time of recurrence and
38.1% (n = 8) were older than 20; 80.9% (n = 17) of
them were at the first recurrence, 14.3% (n = 3) at the
second one and 4.8% (n = 1) at the third one. No patient
A. Pieralli et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 104-108
Copyright © 2011 Sc iRes. OJOG
had a previous history of CIN 2/3: all previous treat-
ments had been performed for flat (38.4%), mixed
(15.4%) or florid (46.2%) warts on genital areas. Among
recurrent episodes no case of CIN 2/3 occurred.
The baseline cytologic diagnosis was viral cytopathic
effect s in 20 cases (23.5%), ASC-US (atypical squamous
cells of undetermined significance) in 14 cases (16.5%),
LSIL (low grade squamous intraepithelial lesion) in 43
cases (50.6%) and HSIL (high grade squamous intraepi-
thelial lesion) in 8 cases (9.4%). No case of ASC-H
(atypical squamous cells cannot exclude HSIL) occurred
duri ng the study and in the cho s en category.
By colposcopy, we diagnosed florid warts affecting a
single genital area in 13 patients (15.3%) and florid warts
affecting multiple genital areas in 6 patients (7.1%); a
diagnosis of ANTZ (abnormal transformation zone)
grade 1 and ANT Z grade 2 was mad e in 62 cases (72.9%)
and in 12 cases (14.1%), respectively.
A cervical biopsy was performed in all cases (n = 74)
of ANTZ i n order to obtain the final histological diagno-
sis of the lesion and to plan the correct treatment; the
biopsy showed cervical flat warts/CIN 1 in 54 cases
(73%) and CIN 2/3 in 20 cases (27%).
Viral genotyping showed 42 (49.4%) single HR (high
risk) infections, 31 (36.5%) single LR (low risk) infec-
tions and 12 (14.1%) co-infections. In 11 cases (91.7%)
of co-infection both infecting viruses were high risk
types, in one case there was a co-infection with two low
risk types. There was no reported case of co-infection
with a LR genotype and a HR one.
Seventeen HPV types were detected in the study co-
hort. The most common HPV type was HPV-6 (overall
prevalence 15.3%), followed by HPV-16 (14.3%), HPV-
11 (12.24%), HPV-56 (9.18%), HPV-31 (7.14%), HPV-
18 and HPV-52 (6.12%), HPV-33 (5.1%), HPV-51
(4.1%), HPV-54 and HPV-45 (8.4%), HPV-58 and HPV-
68 (3.06%), HPV-35 and HPV-72 (2.04%), HPV-53 and
HPV-73 (1.02%).
We observed a predominance of low risk infections
among 16-19-year-o ld girls: t he overa ll pre valence o f L R
HPV amounted to 80% in this age group (83.3% and
79.2% among 16 - 17 and 18-19-year- old girls, respec-
tively). On the contrary, we found a predominance of
high risk infections among 20-25-year-old females: the
overall prevalence of HR HPV was 85.5% in this age
group (72.7%, 89.5% and 88% among 20 - 21, 22 - 23
and 24 - 25-year-old girls, respectiv e l y) .
The univariate analysis of chosen characteristics of
HPV-disease demonstrates the statistically significative
difference of this infection between the two groups of
age (Table 1).
All participa nts demanded for trea tment by destr uctive
Table 1. Age-stratified characteri stics of HPV-related disease.
Age 16 - 19
(30 pt s) Age 20 - 25
(55 pt s) P < 0.0 05
HR HPV 6 47 <0.0001
INFE CT IO N 1 11 0.04
CIN 2/3 0 20 < 0.0001
RELAPSE 17 9 <0.0001
DISE AS E 13 2 < 0.000 1
or excisional physica l therap y in our clinic. A laser vapo-
rization was perfor med in 81 patients: 23.4% (n = 19) of
them had perineal florid or mixed warts, 56.8% (n = 46)
had cervical flat warts and 19.8% (n = 16) had a diagno-
sis o f CIN 2 /3 . I n fo ur c ase s of high gra de CIN (two C I N
2 and two CIN 3) with not visible squamo-columnar
junction (SCJ) a laser conization was preferred.
All patients were negative at the cyto-colposcopic
examination performed three months after treatment.
To our pr ese nt k nowledge thi s is the f ir st s t ud y p r oviding
the age-specific distribution of HPV types within muco-
sal sa mples of suc h a young population.
The common tho ught o f H PV in t his age gr oup is t hat
the infections result in frequent abnormal cytology not
correlated to histological proven lesions, most of them
are transient, with frequent clearance of HPV and of the
cytological alteration itself. Thus current strategies for
adolescents with abnormal cytology include conservati ve
mana gement, avoiding invasive procedures [ 14].
All patients included in our observational study were
young sexually active females who started to make cer-
vical screening by their own will. This is a common ac-
tual practice, thanks to screening campaigns, women are
sensitive to cervical cancer prevention and often send
their s exually active daughters to such examinations.
The present study relevance, whether influenced by a
small sample size, is linked to show the real burden that
HPV-li nked pa tho logies may have on yout h.
As we observed, HPV-related disease heavily mani-
fests also under the age of 25, which is the initial term
for Italian cervical screening.
Girls under the age of 20 years have a low frequency
of HR HPV infections, a low incidence of co-infections
and not-existent incidence of high grade cervical intra-
epithelial neoplasia, but they experiment a disease af-
fecting multiple genital areas simultaneously (43.3%)
and tending to relapse frequently (43.3%), thus the vo-
lunteer request of treatment.
Moreover 85.5% of girls over 20 years of age experi-
ment the grade of disease that is generally coupled to
A. Pieralli et al. / Open Journal of Obstetrics and Gynecology 1 (2011) 104-108
Copyright © 2011 Sc iRes. OJOG
older women, because principally infected by HR HPV.
On the basis of these data we are able to observe that
the initial age of the actual cervical cytological screening
pro gr am i n It al y se e ms to b e much old er than t he star ting
of the HPV linked pathology within the young popula-
tion, determi ning a loss rate of CIN, which are part of the
discussed failure of national cervical cancer screening
programs together with never reached women and pa-
tients loss at follow-up.
Within the study group , a mon g the gi rl s ag i ng b e t wee n
16 and 19 years old, we observed that 70% of diagnosed
CIN 1 were associated with florid genital warts. This
association makes the HPV-linked pathology of the
youngest a clinically manifested disease in the majority
of the cases, thus not requiring institutionalized screen-
ing or diagnostic programs. Otherwise, in our study
group, girls of 20 - 25 years of age were affected by cer-
vical isolated HPV-linked lesions in 96.4% of the cases,
witho ut external clinical manifestations of the infection.
Moreover, as previously said, these girls are the ones
prevalently infected by HR HPV, suffering by CIN 2/3.
This observation may suggest the inclusion in the Ita-
lian cervical cancer screening programs of gir ls a ging 20
- 24, as other worldwide countries already did [15].
The particular stratification of HPV genotypes in dif-
ferent age gro ups t hat thi s st ud y sho ws a nd the cut -off of
incidence of LR HPV in the girls older than 20 suggest
further cons iderations.
Contemporary literature already explained the role of
humoral and cellular immune response towards HPV in-
fections. There are many observations that demonstrate
how antibodies are failing in the protection against HR
HPV and their consequent disease. One of this is the ob-
servation that only 50% of HR genotypes infected wo-
men have circulating antibodies against HPV and more-
over that the circulating HPV-antibod y level is as higher
as more severe the cervical le sion is [1 2]. Further more is
already well known how low CD4 count HIV positive
patients are at major risk for cervical dysplasia than
normal CD4 count ones, demonstrating how the cellular
immune response is fundamental as protection against
HR HPV linked disease [1 6 ] .
After the introduction of HPV quadrivalent vaccine in
Australia, a relevant reduction of HPV linked pathology
rate among wo me n you nger than 28 years was observed,
while the prevalence of HPV-linked disease rema i n ed
stable among older wome n. Benign lesions (cer vical, vu-
lvar or other genital warts) were reduced most with sub-
stantial stability of CIN 2/3 prevalence in the popula-
tion [17].
Pr esent da ta support this observation in suggesting that
antibody protection, natural or vaccine inducted, best
expressed towards low risk HPV linked pathology, could
be one of the hypothetical explanation of such a stratifi-
cation of HPV genotypes within different groups of age.
Another hypothesis bears from a recent study on bio-
logical differences in mucosal immune function which
were evacuated to play a fundamental role in the increate
vulnerability of adolescents compared to adults to sex-
ually transmitte d infections [18 ].
This study evaluated in healthy young women the as-
sociation between their type of cervical epithelium and
the levels of inflammatory and regulatory cytokines in
their mucus. The results of this study show that inflam-
matory cytokines are si gnific antl y higher in the immature
group compared to the mature one.
The hypothesis is that a higher cytokine profile at the
columnar epithelium mounts to protect the columnar
epithelium which is only single layered and more prone
to proliferative infections such as LR HPV, until a
prompt immune response is evocated and able to clear
them. At t he same ti me t hi s hi gher c yt o ki ne p r ofi le could
foreshadow a tendency towards a more severe inflam-
matory response that leads to harmful tissue damage
such as multisite H PV le sions.
These data on reactive inflammatory response of ado-
lescent cervical epithelium are produced only on health y
non-infected women and implications for infections de-
rive only from speculative association between them and
the present data of an existent particular age-specific
distribution of differ ent HPV genotypes.
Perspectives for Authors are to plan further studies for
understanding the role of cervical epithelial type in ac-
quisition of HPV infection and the consequent immune
response during active infection.
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