Clinical and laboratorial study of HPV infection in men infected with HIV

ARTIcLe InfO _______________________________________________________________ _____________________


InTRODUcTIOn
In the literature, several studies point out that 10% to 20% of sexually active adults have HPV infection, although only 1% presents classic condyloma and 2% visible lesions after acetic acid application (1).According to world literature, it is rational to expect the existence of 3 to 6 million males infected with HPV (2).
The relationship between HPV infection and cervical cancer is well established and there are strong evidences that it may also be implicated in the etiology of anal and genital cancer (3).
The studies related to the determination of prevalence of HPV infection in males are very important, since they can present a subclinical and asymptomatic infection and become potential source of infection of HPV to their male or female sexual partners (4).
In view of all these facts, our study was designed to determine the prevalence of precursor lesions of penile cancer, to establish the concordance among different diagnostic techniques (PCR, Hybrid Capture (HC) and Peniscopy with acetic acid 5%) in the diagnosis of infection with the Human Papilloma Virus (HPV) of the penis of Objectives: To determine the prevalence of precursor lesions of penile cancer, to establish the concordance of diagnostic techniques (PCR, Hybrid Capture (HC) and peniscopy with acetic acid 5%) in the diagnosis of Human Papilloma Virus (HPV) of the penis of men infected with HIV and to evaluate the infl uence of the immune status.Patients, Methods and Results: 276 men were studied, with a median age of 34.6 years.Prevalence of High Risk HPV, Low Risk HPV and infection with both, according to HC, was 43%, 32% and 22%, respectively.PCR showed 50% of positivity for HPV DNA.Peniscopy was positive in 27% of individuals.Peniscopy showed good specifi city and low sensitivity for the detection of penile HPV, and low concordance with PCR.Men with white lesions had a 3.6 higher relative risk of positivity for HPV.The most common clinical lesion observed was vegetation, identifi ed in 29% of patients.PCR and HC techniques showed high sensitivity for HPV DNA and there was an excellent correlation between them.Immunosuppressed individuals with CD4< 200 cells/mm3 had the highest prevalence of premalignant lesions that were observed in 10% of the studied individuals.Conclusions: Peniscopy was important for identifi cation and treatment of subclinical lesions.PCR and HC techniques were sensitive methods for the detection of HPV DNA with high concordance.Severely immunosuppressed individuals showed a higher prevalence of pre-malignant lesions of the penis.
HIV positive males and to evaluate the influence of the immunologic status on the occurrence of the lesions.

MATeRIAIS AnD MeTHODS
This is a cross-sectional descriptive study of men affected with HIV attended at the Fundação de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD).Data including demographic, epidemiologic and clinical characteristics of patients were collected.Physical examination (urological inspection and peniscopy with acetic acid 5%), molecular biological tests (PCR in house and Hybrid Capture II, Digene & Co®) and conventional histopathology study were also performed.Criteria for inclusion were: HIV positive males, with ≥ 18 years old, who provided written informed consent to join the study.Criteria for exclusion were: HIV negative males, Indians, psychiatric patients and those that didn't complete all steps of the study.Data were collected at Epi Info® version 6.04 platform and the statistical analysis was made through Statistical Package for Social Sciences® (SPSS) version 16.0 for Windows.
Patients lied in supine position in order to be submitted to the urological and scrotal inspection.A surgical brush with saline was rubbed against the foreskin, balanopreputial sulcus, glans and navicular fossa of the penis.The brush was immersed in an Eppendorf vial containing 1 mL of T1 buffer (commercial kit for nuclear extraction Spin Tissue-Macherey-Nagel®).The vial was tightly closed and sent to the laboratory, where was maintained at -70 o C until PCR analysis.
Another brush was used to Hybrid Capture (HC) for High and Low Risk HPV, and it was stored in an appropriate kit.
After the cytological collection, we proceeded with peniscopy and penile and scrotal inspection.A gauze soaked with acetic acid 5% was placed around the penis for 10 minutes.Positive lesions (white lesions) were biopsied, except in patients with previous histopathological or laboratorial diagnosis or that didn't allow the procedure.The samples were fixed in buffered formalin 10% and were sent to histopathological studies.
For statistical analysis, it was used Pearson's Chi-Square test with Yates correction whenever necessary; Fisher's exact test was used to categorical variables for values under 5 and significance analysis including Odds Ratio (OR) and 95% confidence intervals was performed.Significance was established for p < 0.05 (5%).
For concordance analysis, it was used the kappa (k) associative test.

ReSULTS
Two hundred and seventy six HIV-positive male patients older than 18 years old were included.Median age was 34.6 years.Peniscopy was positive for white lesions in 27% of patients.The most frequent lesion was vegetation (29%) (Table-4).Biopsy was obtained in 22% of participants; some of them had more than one lesion and a total of 75 fragments of skin were collected for conventional histopathological study .Pre-malignant lesions were observed in 10% of patients , and most of them (59%) had CD4 < 200 cells/mm3.
According to HC, the prevalence for High Risk, Low Risk and both High and Low Risk HPV infection were 43%, 32% and 22%, respectively.PCR had 50% of positivity for HPV DNA.
The concordance between peniscopy and PCR was observed in 62% of samples, revealing a "weak concordance" according to kappa associative test (k = 0.2317).Patients with white lesions observed at peniscopy had a 3.6 higher risk of HPV infection.
When PCR and HC for High Risk and Low Risk HPV results were compared, 88% of samples showed similar results.There was an "excellent concordance" between the different techniques according to kappa associative test (k = 0.7522).

DIScUSSIOn
The prevalence of HPV in our study was higher than of the study of Goldstone et al.They evaluated 602 HIV negative males who were engaged in sex with other males and observed a prevalence of 18.2% of HPV infection of the penis using also PCR (6).
Peniscopy showed high specificity and low sensitivity.However, most studies showed a weak specificity of the exam and also a good sensitivity (7)(8)(9).We believe that our results were biased due to the high prevalence of HPV in the studied population (around 50%), explaining the good specificity, and that most of the patients had subclinical or latent infection with HPV, that impaired the identification through peniscopy, only with biomolecular techniques, explaining the low sensitivity.
Some risk characteristics for HPV infection of penis of HIV positive males were identified in our study, including heterosexual behavior (higher rate of penile HPV infection compared to bisexuals and homosexuals, probably due to the high rate of female infection in our population, demonstrated in several studies done in Manaus (10)(11)(12).
PCR and HC techniques had high concordance and sensitivity for the detection of HPV.Rodrigues et al. (13) demonstrated that HC and PCR techniques for the detection of HPV in clinical samples had a fair concordance, including conventional and real time techniques (k = 0.338).When they compared conventional PCR with real time PCR they observed an almost perfect concordance (k = 0.818).
There are very few studies related to intraepithelial neoplasms or penile cancer of HIV positive men.Kreuter et al. studied 263 HIV-positive homosexual men and found penile intraepithelial neoplasms of penis in 11 (4.2%) and of anus in 156 (59.3%) (14).
The limitations of our study included the small size of sample that prevented a strong association among the techniques.The study was conducted in an AIDS ambulatory.However, most HIV+/AIDS patients from Amazonas are attended at FMT-HVD, which we believed allowed the study of a significant sample of patients.We believe that the present results can be used to delineate preventive programs for early detection of penile cancer, in individuals with higher risk, including immunosuppressed patients.Diagnosis and treatment of male partners infected with HPV would also allow a reduction of sexually transmitted diseases.cOncLUSIOnS Prevalence of DNA HPV was approximately 50%.
Peniscopy proved to be a high specific and low sensitive exam.
Concordance of peniscopy and PCR for the detection of HPV was low.
Concordance of PCR and HC for HPV detection was excellent.
We observed a prevalence of 10% of patients with pre-malignant lesions determined by histopathological studies and that most of them were severely immunosuppressed (TCD4 < 200 cells/mm 3 ).

cOnfLIcT Of InTeReST
None declared.
Table-1 depicts socio-demographics characteristics of patients.Table-2 shows the variables related to sexual behavior, use of condoms and previous STDs and Table-3 those related to HIV virus.

Table 2 -Distribution of sexual behavior variables, use of condoms and previous STDs of 276 HIV+/AIDS males.
*sexually transmitted diseases.