Prevalence of HPV infection by cervical cytologic status in Brazil.

Abstract

To assess the prevalence of human papillomavirus (HPV) infection according to cervical cytologic status in northeastern Brazil; identify other risk factors for low- and high-grade squamous intraepithelial lesions (LSILs and HSILs); and identify the most prevalent HPV genotypes associated with the lesions.
Two cervical smears were collected from 250 women referred for cancer screening, one for cytologic examination and the other to test for the presence of HPV by PCR with genotyping by dot blot hybridization.
There were 110 healthy cervices, 82 LSILs, and 58 HSILs. The overall HPV prevalence was 48%, with higher rates for HSILs, and HPV-16 was the most prevalent type. Age, multiple sexual partners, type of HPV present, smoking, and early onset of sexual activity were risk factors for cervical lesions.
Age, multiple sexual partners, and infection with HPV-16 increased the risk of having LSILs or HSILs. Early onset of sexual activity and smoking only increased the risk of having HSILs.

Full text

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CLINICAL ARTICLE
Prevalence of HPV infection by cervical cytologic status in Brazil
José Veríssimo Fernandesa,⁎, Rosely de Vasconcellos Meissnera, Maria Goretti Freire de Carvalhob,
Thales Allyrio Araújo de Medeiros Fernandesc, Paulo Roberto Medeiros de Azevedod, Luisa Lina Villae
aDepartment of Microbiology and Parasitology, Universidade Federal do Rio Grande do Norte, Natal - RN, Brazil
bDepartment of Pathology, Universidade Federal do Rio Grande do Norte, Natal - RN, Brazil
cDepartment of Biomedical Sciences, Universidade do Estado do Rio Grande do Norte, Mossoró - RN, Brazil
dDepartament of Statistics, Universidade Federal do Rio Grande do Norte, Natal - RN, Brazil
eLudwig Institute for Cancer Research, Hospital Alemão Oswaldo Cruz, São Paulo, SP, Brasil
a b s t r a c ta r t i c l ei n f o
Article history:
Received 9 September 2008
Received in revised form 12 November 2008
Accepted 1 December 2008
Keywords:
Cervical lesions
Human papillomavirus
Polymerase chain reaction
Risk factors
Objectives: To assess the prevalence of human papillomavirus (HPV) infection according to cervical cytologic
status in northeastern Brazil; identify other risk factors for low- and high-grade squamous intraepithelial
lesions (LSILs and HSILs); and identify the most prevalent HPV genotypes associated with the lesions.
Method: Two cervical smears were collected from 250 women referred for cancer screening, one for
cytologic examination and the other to test for the presence of HPV by PCR with genotyping by dot blot
hybridization. Result: There were 110 healthy cervices, 82 LSILs, and 58 HSILs. The overall HPV prevalence
was 48%, with higher rates for HSILs, and HPV-16 was the most prevalent type. Age, multiple sexual partners,
type of HPV present, smoking, and early onset of sexual activity were risk factors for cervical lesions.
Conclusion: Age, multiple sexual partners, and infection with HPV-16 increased the risk of having LSILs or
HSILs. Early onset of sexual activity and smoking only increased the risk of having HSILs.
© 2008 InternationalFederation ofGynecologyand Obstetrics.Publishedby Elsevier IrelandLtd. Allrights reserved.
1. Introduction
Human papillomavirus (HPV) is a large group of epitheliotropic
viruses of more than 120 different types. Of the 40 HPV types infecting
the human body, more than a dozen are associated with probable or
definite oncogenic risk [1,2]. Infection of the cervix is initiated when
infectious particles reach and then bind to the basal layer of the
cervical epithelium, where they enter cells through small tears. It has
been suggested that for maintenance of the infection, the virus has to
infect an epithelial stem cell [3].
The infection occurs mainly inyoung, sexuallyactive women in the
first years following their first sexual encounters [4,5]. The highest
HPV prevalence is among women younger than 25 years. It decreases
from age group to age group and is lowest among women aged
between 45 and 50 years, but increases in the postmenopausal years
[6]. Most HPV infections clear spontaneously, but cervical lesions can
develop in women who have persistent infection with high-risk types
of HPV [7]. A current epidemiologic challenge is to identify etiologic
cofactors that lead to HPV persistence and progression to neoplastic
lesions, and clarify how these cofactors contribute to carcinogenesis
[8]. The high-risk types of HPV have a higher tendency to cause
persistent infection and, in the presence of other environmental and
host factors, are associated with an increased risk of cervical lesions
that may progress to cervical cancer [8,9].
Various studies of screening testing for cervical cancer were
recently realized in the south and southeast regions of Brazil as part of
the Latin America Screening Study. These studies have analyzed the
relations between the cytologic and histologic abnormalities present
in cervical smears, and between HPV infection and the predictive
factors of these anomalies [10–12], but so far have not included the
Northeast region of Brazil. Moreover, the method used for HPV de-
tection in these studies was Hybrid Capture 2, which does not identify
HPV type.
The present study was conducted to analyze prevalence of HPV
infection in women with healthy and abnormal uterine cervices in the
Northeast region of Brazil, and to identify the HPV types prevalent in
the local population, as well as risk factors for HPV infection.
2. Materials and methods
The cervical samples used were obtained between April 2001 and
June 2002 at Luis Antonio Hospital in Natal, Rio Grande do Norte State,
Northeastern Brazil, from 258 women referred for cancer screening
because of a history of cytologic alterations. The women's age ranged
from 15 to 65 years. All women were informed about the methods and
objectives of the research. All signed an informed consent form and
International Journal of Gynecology and Obstetrics 105 (2009) 21–24
⁎ Corresponding author. Departamento de Microbiologia e Parasitologia, Centro de
Biociências, Universidade Federal do Rio Grande do Norte, Av. Sen. Salgado Filho, S/N,
Campus Universitário, Lagoa Nova, CEP: 59072-970, Natal, RN, Brazil. Fax: +55 84
32119210.
E-mail address: veris@cb.ufrn.br (J.V. Fernandes).
0020-7292/$ – see front matter © 2008 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ijgo.2008.12.004
Contents lists available at ScienceDirect
International Journal of Gynecology and Obstetrics
journal homepage: www.elsevier.com/locate/ijgo

Page 2

answered a standardized questionnaire about their marital status,
ethnicity,sexualbehaviorandreproductiveactivities,oralcontraceptive
use,smokinghabits,and family historyof cancer.The patients'ethnicity
was defined based on self-reports according to the criterion of the
Instituto Brasileiro de Geografia e Estatística (IBGE) that classifies
ethnicity into 5 categories: white, black, mulatto, yellow, and native. In
this study, the black, mulatto, yellow, and native categories were
combined into a nonwhite category. The study was approved by the
Ethical Committee in Research of Federal University of Rio Grande do
Norte.
The cervical smears were obtained using a cytobrush for optimal
collection of exfoliated cells. A trained cytopathologist from the
Department of Pathology of Federal University of Rio Grande do
Norte stained the smears and analyzed them according to the
Bethesda classification (available at: http://bethesda2001.cancer.
gov/terminology.html). All abnormal smears were re-examined for
result confirmation. Samples with scant squamous cellularity were
considered unsatisfactory for evaluation and excluded from analysis.
The women with abnormal cytologic results were followed up
according to the guidelines of the National Cancer Program of Brazil.
Based on the results of the cytologic evaluation, the 250 retained
participants were allocated to 1 of 3 groups: a normal cytology group
(n=110); a low-grade squamous intraepithelial lesions (LSIL) group
(n=82); and a high-grade squamous intraepithelial lesions (HSIL)
group (n=58).
Another sample of exfoliated cervical cells was collected from each
participant and the collection brush was placed in a tube containing
PBS, vancomycin, and nystatin. The tube was kept at 4 °C until
processing for DNA extraction. The content was centrifuged at 3000 g
for 10 minutes following vigorous tube agitation and brush removal;
the supernatant was removed; and the remaining pellet was
processed for DNA extraction, using rapid isolation of DNA from
mammals, with proteinase K [13].
To check the qualityof theobtained DNA, aliquotswithabout 30 ng
of DNA were submitted to polymerase chain reaction (PCR) using
the primers PCO3+/PCO4+, specific for the amplification of a 110 bp
fragmentof theβ-globingene [14].Onlythe 250DNA samplespositive
to β-globin were checked for HPV DNA.
Of the 250 good-quality DNA samples 110 were from participants
in the normal cytology group, 82 from participants in the LSIL group,
and 58 fromparticipantsin the HSIL group. The presence orabsence of
HPV DNA was established using the degenerate primers MY09/M11,
specific for the L1 open reading frame (ORF) of the HPV genome [15].
The products of PCR were submitted to electrophoresis on 7%
polyacrilamide gel, followed by silver staining [16]. The specimens
were considered positive for HPV DNA if they presented a band of
450 bp. The amplicons were submitted to HPV genotyping by Dot blot
hybridization as described [17].
To check for associations between sociodemographic character-
istics and cervical lesions, and for differences in the prevalences of
HPV infection in the 3 groups, we used the Pearson χ2test for
comparison of proportions. To identify variables that could be
considered risk factors for cervical lesions we used all the considered
variables in a multivariate logistic regression analysis, with the
dependent variable assigned a value of 1 in the presence of low- or
high-grade intraepithelial cervical lesions and a value of 0 in the
absence of lesions. To check for associations between HPV type and
health status we used univariate logistic regression. All analyses were
performed using the software SPSS, version 13.0 (SPSS, Chicago,
Illinois, USA). P≤0.05 was considered statistically significant.
3. Results
Statistical analysis by the χ2test showed an association between
age and presence of HSIL (P=0.000) and between ethnicity and
presence of HSIL (P=0.002). Considering separately married and
single women, we did not observe an association between marital
status and the presence of LSIL or HSIL (P=0.09 and P=0.62),
respectively (Table 1).
We observed that most (81.5%) of the women with both a normal
cytology result and HPV infectionwere younger than 34 years, with a
lesser prevalence of HPV infection in the older age groups. Among
women with LSILs, the prevalence of HPV infection increased as they
reached the age of 34 years, decreased as they aged from 35 to
44 years, and increased again after they were 45 years old. Most
(77.8%) of the women with HSILs were between 25 and 54 years old
(Table 1). Differences in HPV prevalence between the normal
cytology group (24.5% of the participants) and the LSIL (58.5%) and
HSIL groups (77.6%) were statistically significant by comparison of
the proportions, revealing that HPV infection increased the risk of
developing both low-grade (P=0.000) and high-grade (P=0.000)
premalignant lesions. Age was associated with the occurrence of
HSIL (P=0.000), as was nonwhite status (P=0.002). We observed
no associations between marital status and the presence of cervical
lesions (Table 1).
The women older than 34 years were at greater risk for LSILs and
those older than 45 years were at greater risk for HSILs. The women
who had their first sexual intercourse between the ages of 14 and
17 years were at greater risk only for HSILs. Having had multiple
sexual partners and being a smoker also increased the risk of having
HSILs. There was no evidence that age at first pregnancy, number of
pregnancies, use of oral contraceptives, and a family history of cancer
increased the risk of either LSIL or HSIL (Table 2).
Cervical HPV infection increased the risk of both LSIL and HSIL.
Fourteen different types of HPV were identified in the study
population, and most had a high oncogenic potential. Infection was
causedby1 typeof virus in 19.0%of thewomen in thenormalcytology
group, 40.2% of the women in the LSIL group, and 67.2% of the women
in the HSIL group, and HPV-16 was the most prevalent type in all 3
groups. The second most common type was HPV-58 in the first 2
groups and HPV-45in the HSILgroup. Infectionwith2 HPV typeswere
detected in the 3 groups, the most frequent coinfection being with
HPV-56andHPV-57inthefirst2groups,andwithHPV-31andHPV-33
or HPV-45 and HPV-59 in the HSIL group. With the probes we used, it
was not possible to identify the HPV types carried by 2 participants in
the normal cytology group, 1 participant in the LSIL group, and 1
participantin theHSIL group.SingleinfectionbyHPV-16 increased the
Table 1
Distribution of socio-demographic characteristics, according to health status
VariableNormal cytology group LSIL group HSIL group
No. of
patients
No. of
patients
with HPV (%)
No. of
patients
No. of
patients
with HPV (%)
No. of
patients
No. of
patients
with HPV (%)
Age at study entrya
b25
25-34
35-44
45-54
≥55
Total
Ethnicityb
White
Nonwhite
Marital statusc
Single
Married
Widowed
37
44
23
9 (24.3)
13 (29.5)
5 (21.7)
0
0
27 (24.5)
19
30
24
13 (68.4)
17 (56.7)
11 (45.8)
3 (60.0)
4 (100.0)
48 (58.5)
4 4 (100.0)
11 (78.6)
13 (72.2)
11 (84.6)
6 (66.7)
45 (77.6)
14
18
13
9
58
4
2
5
4
110 82
87
23
19 (21.8)
8 (34.8)
64
18
40 (62.5)
8 (44.4)
33
25
24 (72.7)
21 (84.0)
32
78
0
11 (34.4)
16 (20.5)
0
15
67
0
12 (80.0)
36 (53.7)
0
14
41
3
10 (71.4)
32 (78.0)
3 (100.00)
aStatistical analysis for age among the groups: Group I×Group II: χ2=5.262,=0.261;
Group I×Group III: χ2=39.606 P=0.000.
bStatistical analysis for ethnicity among the groups: Group I×Group II: χ2=0.030,
P=0.862; Group I×Group III: χ2=9.166, P=0.002.
cStatistical analysis for marital status among the groups: Group I×Group II:
χ2=2.963, P=0.085; Group I×Group III: χ2=0.241, P=0.623.
22
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Page 3

risk of both LSIL and HSIL, whereas single infection by HPV-58 and
coinfection with HPV-56 and HPV-58 only increased the risk of LSIL
(Table 3).
4. Discussion
The prevalence of HPV infection in women with normal cytology
resultswashighinthisstudy.Higherthaninanotherstudyconductedin
Recife, also in Northeast Brazil [18], it was similar to that estimated for
AfricaandhigherthanthatestimatedforSouthAmericain2largestudies
of meta-analyses [19,20]. This high prevalence of HPV infection in
womenwithnormalcytologyresultscouldbeduetothehighproportion
of women whose sexual activity began before they were 18 years old,
when they were biologically the most vulnerable to this virus. It could
also be attributed to false-negative results, which commonly occur in
cytologic studies [2,21]. Besides, most infections were caused by high-
risk HPVtypes,mainlybyHPV-16,whichhasa greatercapacitythanthe
other types to maintain infection without cytologic alterations.
The overall HPV prevalence among the participants with normal
cytologyresultswas24.5%.Itwashighestinthe25to34yearsagegroup
and less in the 35 to 44 years age group, a finding similar to those
reported for other World Regions [20]. Among women with LSILs, the
Table 3
Distribution of HPV types according to health status and associated odds ratio obtained by univariate logistic regression model
VariableNormal cytology group(n=110) LSIL group(n=82)HSIL group(n=56)
No. of patients No. of patients OR95% CI No. of patientsOR 95% CI
Only type
HPV 16
HPV 18
HPV 58
HPV X
Other
Double Infection
HPV 16+18
HPV 16+58
HPV 55+58
HPV 56+58
Other
HPV status
Positive
Negative
14
1
3
2
1
15
2
7
1
8
2.62
4.88
5.70
1.22
19.53
[1.14–6.00]a
[0.43–55.65]
[1.39–23.33]a
[0.11–13.91]
[2.35–162.18]a
2913.68
6.38
4.26
3.19
31.92
[5.77–32.42]a
[0.38–108.49]
[0.65–27.96]
[0.7–37.76]
[3.45–295.46]a
2
2
1
5
1
1
1
2
1
0
1
1
8
5
NA
2.44
2.44
9.76
12.21
NA
[0.15–40.16]
[0.15–40.16]
[1.97–48.37]a
[1.38–108.39]a
0
1
0
0
5
NA
6.38
NA
NA
31.92
NA
[0.38–108.49]
NA
NA
[3.45–29.546]a
27
83
48
34
4.34
1
[2.34–8.05]a
[Reference]
45
13
10.64
1
[5.00–22.63]a
[Reference]
Abbreviations and note: See Table 2.
Table 2
Distribution of patients by cytological status according to the considered variables, and associated odds ratio (OR) obtained by multivariate logistic regression model
VariableNormal cytology group(n=110) LSIL group(n=82)HSIL group(n=56)
No. of patientsNo. of patientsOR 95% CI No. of patientsOR95% CI
Age at study entry, y
b25
25–34
35–44
45–54
N54
Age at 1st sexual intercourse, y
14–17
18–21
≥22
Age at 1st pregnancy, y
Never pregnant
14–17
18–21
≥22
No. of pregnancies
0
1–3
4–6
N6
Oral contraceptive use, y
0
1–3
4–6
7–9
≥9
Multiple sexual partners
Yes
No
Smoking
Yes
No
Family history of cancer
Yes
No
37
44
23
19
30
24
1
2.27
3.44
9.66
30.18
[Reference]
[0.91–5.64]
[1.17–10.14]a
[1.30–71.56]a
[2.01–453.90]a
41
1.72
5.49
28.04
332.80
[Reference]
[0.32–9.25]
[0.94–32.04]
[2.24–350.29]a
[7.58–14621.26]a
14
18
13
9
4
2
5
4
49
38
23
52
20
10
2.34
1.19
1
[0.71–7.76]
[0.37–3.83]
[Reference]
4926.09
9.68
1
[1.93–352.05]a
[0.80–117.00]
[Reference]
8
1
17
14
43
36
11
18
36
17
NA NA
[0.70–8.43]
[0.64–4.41]
[Reference]
10.90
0.61
0.50
1
[0.03–26.53]
[0.11–3.50]
[0.12–2.19]
[Reference]
2.43
1.69
1
27
21
9
17
76
11
6
10
54
12
1[Reference]
[0.0–∝]
[0.0–∝]
[0.0–∝]
21
2.52
6.32
[Reference]
[0.23–28.11]
[0.47–84.25]
NA
101.44
53.42
27.09
22
22
126 NA
37
43
20
5
5
32
27
13
6
4
1
0.65
0.56
1.01
0.75
[Reference]
[0.29–1.47]
[0.20–1.53]
[0.23–4.48]
[0.14–4.09]
281
0.56
0.68
0.47
1.74
[Reference]
[0.12–2.58]
[0.15–3.06]
[0.03–8.31]
[0.23–13.41]
9
15
1
5
29
81
46
36
3.56
1
[1.80–7.04]a
[Reference]
45
13
10.50
1
[3.04–36.35]a
[Reference]
12
98
13
69
1.44
1
[0.52–4.02]
[Reference]
32
26
4.80
1
[1.36–16.94]a
[Reference]
21
89
26
56
1.33
1
[0.58–3.05]
[Reference]
18
40
0.74
1
[0.21–2.66]
[Reference]
Abbreviations: CI, confidence interval; HSIL, High-grade lesion; LSIL, low-grade lesion; NA, not applicable; OR, odds ratio.
aDenotes statistical significance.
23
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prevalence was higher in this study than that reported for Porto Alegre,
in Southern Brazil [22]; it was similar to that estimated for Africa [23];
and it was less than that reported for Recife [18], Brasilia [24], or
estimated for Central and South America [23]. It was highest among
womenyounger than 34 years, less in the 35 to 44 years age group, and
higher again among women 45 years or older. Most of the womenwith
HSILswerebetween25and54yearsold,andtherewasnolinearrelation
between age group and HPV prevalence. The overall HPV prevalence in
thisstudyforwomenwithHSILswashigherthanthatreportedforPorto
Alegre [22], but less than that reported for Recife or Brasilia [18,24].
These differences can be explained not only by variations usually
observed between populations of different geographic regions, but also
bydifferencesinageandlesiongradesamongstudypopulationsandthe
sensitivity of the HPV DNA assays used in different studies [2,25].
An association between ethnicity and cervical lesion was observed
only for HSILs, suggesting a trend for lesions to progress faster in
nonwhitewomen. Mostof thewomen in the3 groups were marriedor
had only 1 sexual partner. No associations were observed between
HPV prevalence or development of cervical lesions and marital status.
Compared with the participants with normal cytology results, HPV
prevalence was significantly higher in participants with either lesion
grade. The overall prevalence of HPV-16 in this study was similar to
that estimated for Africa, but higher than that reported for Recife [18]
or estimated for South America [19,20]. We found an association
between HPV infection and the presence of LSILs among women
infected with HPV-16 and HPV-58 as single infections as well as
among those infected with both HPV-56 and HPV-58. We also found
an association between HPV-16 infection and presence of HSILs.
We found both LSILs and HSILs to be associated with age and
multiple sexual partners. On the other hand, we found only HSIL to be
associated with early age at first sexual intercourse or with smoking,
even when all variables were considered simultaneously in a logistic
regressionmodel.Andwefoundnoassociationbetweenthepresenceof
premalignantlesionsofeithergradeandoralcontraceptiveuseorfamily
history of cancer. These results are concordant with those reported in
studies conducted in São Paulo, Porto Alegre, and Buenos Aires [12,22].
Inthe3 studygroups,themostof thewomentestingpositivefor HPV
were infected with 1 or 2 high-risk types. Among women with normal
cytologyresults,theHPV-16prevalencewasverysimilartothatestimated
forSouthAmerica[19],buthigherthanthatestimatedforAfricaandtwice
than that reported for Recife [18]. These results show that HPV-16 is
highlyprevalentinthefemalepopulationofNatal,Brazil.Andsinceother
high-risk HPV types alsoinfect the same population, thesewomen inour
region are at high risk for HPV infection and, consequently, for cervical
cancer. The distribution of the HPV types in this study was similar to that
described for women in Africa and in Central and South America.
The results of this studylead us toconcludethat high-grade lesions
are associated with age and ethnicity. Multiple lifetime sexual
partners,singleinfectionwithHPV-16orHPV-58,anddoubleinfection
withHPV-56andHPV-58increasedtheriskofhavingLSILs.Earlyageat
first sexual intercourse and multiple sexual partners, smoking, and
infection with HPV-16 all increased the risk of having HSILs. The most
prevalent virus type was HPV-16 in the 3 groups, followed by HPV-58
in the normal cytologyand LSIL groups, and HPV-45 in the HSIL group.
Despite similarities in the order of prevalence of some HPV types
foundforNatalinthisstudyandotherregionsofBrazilindifferentstudies,
therewereimportantdifferencesintheprevalenceofHPVtypes31,33,45,
56, 57, and 59. By virtue of the existence of multiple HPV types and
variations in their distributions, evenwithin the same geographic region,
more local studies should be conducted to evaluate the cost-benefit and
expected efficacy of a HPV vaccination campaign, if found justified.
Acknowledgment
This study was supported by funds from the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (National Council of
Scientific and Technological Development [CNPq]) and the Coorde-
nação de Aperfeiçoamento de Pessoal de Nível Superior (Coordination
of Improvement of Higher Education [CAPES]).
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