Evidence of a Higher Prevalence of Hpv Infection in Htlv-1-infected Women: a Cross-sectional Study

Introduction: HTLV-1 infection increases susceptibility to other infections. Few studies have addressed the co-infection between HPV and HTLV-1 and the immune response involved in this interaction. The aim of this study was to determine the prevalence of cervical HPV infection in HTLV-1-infected women and to establish the risk factors involved in this co-infection. Methods: A cross-sectional study was carried out in Salvador, Brazil, between September 2005 and December 2008, involving 50 HTLV-1-infected women from the HTLV Reference Center and 40 uninfected patients from gynecological clinic, both at the Bahiana School of Medicine. HPV infection was assessed using hybrid capture. HTLV-1 proviral load was quantified using real-time polymerase chain reaction (PCR). Results: The mean age of HTLV-1-infected women (38 ± 10 years) was similar to that of the control group (36 ± 13 years). The prevalence of HPV infection was 44% in the HTLV-1-infected group and 22.5% in uninfected women (p = 0.03). HTLV-1-infected women had lower mean age at onset of sexual life (17 ± 3 years versus 19 ± 3 years; p = 0.03) and greater number of lifetime partners compared with the control group (4 ± 3 versus 2 ± 1; p < 0.01). In the group of HTLV-1-infected patients, there was neither difference in HTLV-1 proviral load between HPV-infected women and the uninfected. Conclusions: The prevalence of HPV infection was higher in HTLV-1-infected women. Further studies should be performed to evaluate the progression of this co-infection.

Human papillomavirus (HPV) is the etiologic agent of one of the most prevalent sexually transmitted diseases in the world and is also directly related to cervical cancer 17 , the third most common cancer in Brazil.The HPV types 16 and 18 are the most frequent types found in cervical squamous cell carcinoma, accounting for more than 70% of cases 18 .A higher frequency of HPV infection and an increased risk of cervical cancer are found in immunosuppressed women, as can be observed in human immunodeficiency virus (HIV)-infected patients [19][20][21] .The studies that assessed the association of HPV infection in HTLV-1 patients have not reached a consensus on the epidemiological aspects of this co-infection [22][23][24][25] .The aim of this study was to determine the prevalence of HPV infection in HTLV-1-infected women in the City of Salvador, Bahia, Brazil, and to establish the risk factors involved in this co-infection.

METHODS
A cross-sectional pilot study with a control group was carried out in Salvador, Brazil, between September 2005 and December 2008, involving 50 HTLV-1-infected patients and 40 uninfected

RESULTS
Lôpo SS et al -HPV infection in HTLV-1-infected women women.All patients were followed at the Bahia School of Medicine and Public Health (EBMSP), Salvador, Brazil.HTLV-1-infected patients were selected from the HTLV Reference Center that has provided comprehensive care to a total of 1,050 patients since 2002.Of these, 50% are regularly seen at least twice a year (75% women).Uninfected controls were selected at the Gynecological Outpatient Clinic, located on the same campus.Patients were invited and sequentially included in the study upon their medical visit.Inclusion criteria were positive serology for HTLV-1 (HTLV-1 group), tested negative for HTLV-1 (control group), sexually active, and met criteria for sample collection for Pap smear.Patients with positive serology for HIV, pregnant women, transplant recipients, patients on chemotherapy or steroids, or those with diseases that compromise the immune system were excluded.
The patients answered a questionnaire that assessed demographics, medical, sexual, and gynecological data.After the interview, physical and gynecological examinations were performed.Specimens for Papanicolaou smears were collected from ectocervix and endocervix using an Ayres spatula and cytobrush, respectively.Squamous cell abnormalities seen in the Papanicolaou smears were classified as lowgrade or high-grade squamous intraepithelial lesions, in accordance with the Bethesda System 26 .The cervical samples were collected using a sterile swab and stored in universal collection medium (UCM) solution.The hybridization was performed using the commercial kit supplied by the Digene HPV (São Paulo, Brazil), according to the protocol specified by the manufacturer.The samples were processed in the laboratory of clinical analysis DNA (Salvador, Bahia).
The populations of CD4+ and CD8+ (cluster of differentiation) lymphocytes were analyzed using flow cytometry (FACScan), after labeling cells with monoclonal antibodies and CD3-FITC (fluorescein isothiocyanate) anti-CD4-PE (phycoerythrin) and anti-CD8-PerCP (peridinin chlorophyll protein).The HTLV-1 proviral load was measured using the technique of real-time polymerase chain reaction (PCR) after obtaining mononuclear cells from peripheral blood by density gradient and DNA extraction by the commercial kit (QIAGEN, Germany) previously described 27 .
Categorical variables were expressed as absolute values and percentage.Quantitative variables were described as mean and standard deviation or as median and interquartile interval.The Chisquare or the Fischer's exact tests were used to compare categorical variables between groups.The independent Student's t-test was used to compare continuous variables between groups.The association between HPV infection and the presence of HTLV-1 were verified, along with others known risk factors for HPV infection.Unadjusted odds ratio (OR) were calculated to screen for inclusion in an initial multivariate model.Variables that exhibited at least a moderate association with the outcome in the presence of these design variables were considered for inclusion in the final models.The analysis was performed using the SPSS software (Statistical Package for the Social Sciences), version 17.0.

Ethical considerations
The project was approved by the Ethics Research Center from Gonçalo Muniz Center, Fundação Oswaldo Cruz, Bahia.All patients read and signed an informed consent before being included in the study.
The mean age of the HTLV-1-infected group was similar to that of the control group.HTLV-1-infected patients had a lower age of first sexual intercourse (17 years versus 18.6 years; p = 0.03), a higher number of sexual partners (4.1 versus 1.9; p < 0.01), and a higher number of abortions (1 versus 0.5; p = 0.02) compared with the control group (Table 1).
The HPV prevalence was higher among HTLV-1-infected women than in the control group (44% versus 22.5%; p = 0.03) (Table 2).There was no difference in the frequency of HPV type among the groups.One HTLV-1-infected patient showed a highgrade squamous intraepithelial lesion (HSIL), whereas women of the control group had cytological findings classified as normal or inflammatory.There was no difference in the prevalence of HPV   Categorical variables were expressed as n (%).Quantitative variables were described as mean and standard deviation.
The OR for HPV infection in HTLV-1-infected patients was 2.7 (95% confidence interval, CI: 0.8-8.2;p = 0.07) after adjusting the variables.The OR of age for HPV infection was 0.9 (95% CI: 0.8-0.9;p = 0.03), and the number of sexual partners showed an OR of 1.1% (95% CI: 0.9-1.4,p = 0.09) (Table 3).The HTLV-1infected patients with HPV co-infection showed a higher number of sexual partners compared with the HTLV-1infected patients not infected with HPV (5.3 versus 2.6; p = 0.01).There were no observed differences regarding other possible risk factors associated with this co-infection (Table 4).

DISCUSSION
The present study indicated a higher prevalence of cervical HPV infection in HTLV-1-infected women.The HTLV-1 infection and the number of sexual partners appeared to be associated with increased risk for HPV infection, despite the absence of statistical difference in this study.Only the age of patients had a significant association with HPV infection: younger patients had an increased risk for HPV infection.In spite of the higher frequency of cervical HPV infection in HTLV-1-infected women, there was no occurrence of cervical cancer in these patients.
A few studies have assessed the association between HTLV-1 and cervical HPV infection.In Japan, a cross-sectional study 22 observed higher prevalence of HTLV-1 infection in patients with cervical cancer, assigning a threefold higher risk for this malignancy.In Jamaica, a case-control study suggested that HTLV-1 infection was a risk factor for progression of cervical intraepithelial neoplasia 23 .However, another study performed in that same area did not confirm this association 24 .Moreover, a study conducted in Mexico failed to find a relation between HTLV-1 infection and risk for cervical cancer 25 .
As this is a pilot cross-sectional study, involving a small sample size, it was not possible to estimate the prevalence of cervical cancer or premalignant lesions.The absence of cervical cancer in this population could also be explained by the young age of patients, although cervical cancer may occur from the third decade of life; the peak age-specific cervical cancer is around the sixth decade of life 28 .Also, it was not possible to identify the HPV types involved in the infections or to determine whether this population has lower tissue viral clearance.
The HTLV-1 infection induces a spontaneous proliferation of lymphocyte subsets.As a result, HTLV-1-infected individuals have an increased population of activated lymphocytes, an increased expression of proinflammatory cytokines 29 , and lower proliferative response to recall antigens 30 , which may induce an immunosuppression.These immunological abnormalities and the highest exposure to risk factors common to HTLV-1 and HPV could explain the high prevalence of HPV infection 31 .Other immunosuppressive diseases are also associated with a high prevalence of HPV infection, such as HIV infection 20 and systemic lupus erythematosus 32 .However, no difference was found in the proportion of CD4-T lymphocytes and in the HTLV-1 proviral load of HTLV-1-infected patients with HPV, compared with HTLV-1infected women.