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High Prevalence of Human Papillomavirus Infection in Mexican Males: Comparative Study of Penile-Urethral Swabs and Urine Samples

LAZCANO-PONCE, EDUARDO MD*; HERRERO, ROLANDO MD; MUÑOZ, NUBIA MD; HERNÁNDEZ-AVILA, MAURICIO MD*; SALMERÓN, JORGE MD; LEYVA, AHIDEE MSc*; MEIJER, CHRIS J. L. M. PhD§; WALBOOMERS, JAN M. M. PhD§

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Background Although extensive information has been gathered about the prevalence and determinants of human papillomavirus infection among women, little is known about the prevalence and natural history of this infection among males.

Goal To investigate the potential usefulness of urine specimens to assess the presence of genital human papillomavirus DNA infection.

Study Design The authors conducted a study of 120 healthy men from Cuernavaca, Mexico. A urine specimen and urethral and coronal sulcus swab samples were collected and tested for human papillomavirus using the GP5+/6+ polymerase chain reaction enzyme immunoassay method.

Results In 95% of the urethral-coronal sulcus samples, the β-globin gene was detectable, indicating adequacy of the specimen for DNA amplification; however, only 14% of the urine specimens had detectable β-globin. Removal of inhibitors by DNA purification in a sample of subjects produced β-globin amplification, but no increase in human papillomavirus DNA positivity was detected. Human papillomavirus DNA was not detectable in penile-urethral swab samples in any of the subjects who reported not having engaged in sexual activity but was present in 43% of men who reported sexual activity, a strong indication of the sexual transmission of human papillomavirus.

Conclusions Human papillomavirus is a common sexually transmitted infection among Mexican males, and urine sample specimens cannot adequately detect the presence of this infection in males.

From the *Center for Population Health Research-National Institute of Public Health of Cuernavaca Morelos, Mexico; the †International Agency for Research on Cancer, Lyon, France; the ‡Mexican Institute for Social Security, Mexico; and the §Free University Hospital, Amsterdam, the Netherlands

This study was made possible by the funding provided by the National Institute of Public Health of Mexico, the Mexican Institute for Social Security, the International Agency for Research on Cancer (FIS/97/09), and the Dutch Prevention Fund.

Reprint requests: Eduardo Lazcano, MD, Instituto Nacional de Salud Publica, Avenida Universidad 655, Colonia Sta. Maria Ahuacatitlan, Cuernavaca, Morelos, Mexico, C.P. 62508. E-mail: elazcano@insp3.insp.mx

Received for publication June 12, 2000, revised October 9, 2000, and accepted October 18, 2000.

HUMAN PAPILLOMAVIRUS (HPV) INFECTION is considered the cause of the majority of cervical cancers worldwide, and has been associated with cancer of the penis and other anogenital epithelia. 1 Although extensive information has been gathered about the prevalence and determinants of HPV infection among women, little is known about the prevalence and natural history of infection among men. Biologic behavior of male genital HPV infection is poorly understood, but this information will be useful for the planning of HPV vaccination strategies, which are likely to be targeted to both men and women. The collection of penile specimens from males without signs of HPV disease, which usually includes brushing of penile surfaces and urethra with different instruments, is not always feasible, and studies are often hampered by low response rates. Given the difficulty of obtaining adequate specimens for population-based studies, we were interested in exploring the viability of urine samples as a potentially easier method to obtain exfoliated cells representative of the genital epithelium. Previous reports in the literature have yielded inconclusive results, with some studies showing urine samples as promising 2–7 and others showing them as inadequate. 8,9 We present a brief report of a study in which sequential samples of urine and coronal sulcus-urethra were collected in a series of 120 asymptomatic males in Cuernavaca, Mexico, to compare the adequacy of these samples for detection of HPV DNA. The prevalence and correlates of HPV detection in this group of asymptomatic men are also presented.

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Methods

Study Population

The study was carried out in 1998 in a sample of 120 males aged 14 to 55 year living in Cuernavaca, Morelos State, Mexico. Eligible subjects were in good general physical and mental health, agreed to participate, and sign informed consent forms approved by the Ethical Committees of the participating institutions were obtained.

Samples were collected at the Urology Service of the Mexican Institute of Social Security (IMSS) in Cuernavaca. Two groups of participants were recruited; the first group (n = 43) included college students from one class at a public school, and the second group (n = 77) included industry workers from an automobile factory. A social worker visited the factory and invited the workers to participate in the study through the institution’s physician, and open appointments were given to all potential participants at the IMSS Urology Service, where the study procedures were carried out.

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Data and Specimen Collection

Two specially trained male interviewers administered a questionnaire regarding sociodemographic characteristics, smoking history, and sexual behavior (regular and casual partners, visits to commercial sex workers, condom use, circumcision, and anal, oral, and homosexual sex).

Subjects were instructed not to wash their genitals the night or morning before the examination and were asked to bring to their appointment 50 ml first urine (taken in the morning from the first part of a void). The specimens were collected by the subjects directly in a 50-ml conic tube provided by the investigators. At the clinic, a physical examination of the genital area was performed and exfoliated cells were collected with an Accellon Multi biosampler swab (Medscand, Hollywood, FL) that was inserted 2 cm in the urethra and rotated 360 degrees. Immediately after retraction of the prepuce, the same device was swabbed along the circumference of the coronal sulcus, was introduced, and was vigorously shaken in a 50-ml conic tube containing 20 ml phosphate-buffered saline. Both the urine and urethral cells were centrifuged, and the cell pellets were suspended in phosphate-buffered saline and frozen at −70 °C until testing was performed.

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HPV Testing

All specimens were numbered randomly and tested blindly at the laboratory. To assess the quality of the samples for DNA amplification, they were prescreened by a 209-bp amplifying β-globin polymerase chain reaction (PCR), as previously described. 10 Testing for HPV was done by PCR-based enzyme immunoassay, which used HPV general primer-GP5+/6+ mediated PCR. 10,11 One assay was used to test for 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and six low-risk HPV types (6, 11, 40, 42, 43, 44). In addition, the PCR amplification products were analyzed for individual high-risk HPV types 16, 18, 31, and 33, and the low-risk types 6 and 11. The same PCR products were also tested with radioactive Southern blot hybridization using a general probe. 12 Samples positive after the radioactive Southern blotting but negative for high-risk and low-risk HPV in the PCR enzyme immunoassay were labeled HPV-X. Special precautions to minimize false-positive results in the PCR have been described in detail elsewhere. 13

In an effort to investigate the effect of inhibitors in the lack of amplification of the β-globin gene, we further tested urine specimens from a sample of 14 participants who were HPV negative by urine testing (12 were β-globin negative, 2 were β-globin positive). However, based on results of urethra-coronal sulcus specimen testing, 7 of these 14 participants HPV positive. The DNA was isolated from these samples using the High Pure PCR template (Boehringer, Mannheim, Germany) assay, which uses guanine thiocyanate in combination with silica gel columns, according to the manufacturers instructions. 14 Detection for HPV was carried out on the extracted DNA as described previously.

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Statistical Methods

Univariate statistics were calculated for all variables. Variables measured on a continuous scale were explored in terms of their original distribution and in categories. A three-level index of socioeconomic status was developed by combining five variables (number of persons living in the house, number of rooms, availability of drinking water, sanitary conditions, and education). Bivariate analysis of selected characteristics was conducted using standard chi-square and t tests.

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Results

Characteristics of the Population

A total of 120 male participants with a mean age of 29.3 years (range, 14–55 years) were included in the study. The subjects included 43 students (mean age, 18.1 years; range, 14–20 years) and 77 car industry workers (mean age, 31.3 years; range, 20–55 years).

A total of 18 subjects reported no previous sexual intercourse (mean age, 18.2; range, 15–20 years). Most of these nonsexually active subjects (89%) were students and had a higher education and socioeconomic level than factory workers of similar age. Workers included in the study had limited education and middle socioeconomic status. None of the males included in the sample had evident external genital lesions at clinical examination.

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β-Globin Assay Results

After initial processing and amplification independent of sexual activity history, β-globin sequences were detected in 114 (95%) of the 120 specimens of exfoliated cells from the urethra and coronal sulcus, indicating sufficient DNA for gene amplification and the absence of PCR inhibitors in these samples. However, only 17 (14.1%) of the urine specimens were β-globin positive. When the procedure to remove inhibitors was completed in the sample of these subjects, all the urine samples were β-globin positive, but none were positive for HPV DNA.

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Prevalence of HPV

After exclusion of β-globin–negative samples, 114 subjects were included for the following analysis, for which only results from exfoliated cells from the urethra-coronal sulcus are considered. The HPV DNA was not detected in any of the 18 subjects who reported no previous sexual intercourse. Among 96 previously sexually active subjects, HPV positivity was 42.7% (Table 1).

Table 1

Table 1

Considering HPV types, 19 subjects (19.8%) had high-risk HPV types as detected with the combined probe (including HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) or as individual types (HPV 16, 18, or 31). An additional 17 subjects (17.7%, 41.5% of positives) had low-risk HPV types, including those detected with the six combined probes (HPV 6, 11, 40, 42, 43, and 44) and those in which HPV 6 and 11 were detected as individual types. In addition, five subjects (5.2%) had uncharacterized types, which are probably low-risk HPV types. Multiple infections were detected in 11 subjects (11.4%, 26.8% of the positives), 9 of which included high-risk and low-risk types (and were included as high-risk types in Table 1).

Originally, 17 β-globin–positive urine samples were tested for HPV from subjects with a history of sexual activity. Only two (11.8%) of these samples were HPV positive. Extraction of inhibitors, which constitutes a laborious process that can lead to contamination, was carried out on a sample of 14 subjects. After extraction, an additional 12 urine specimens (14 minus two overlapping samples) scored β-globin positive and were tested for HPV. A combined total of 29 urine samples were tested for HPV, of which only two were positive.

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Prevalence of HPV by Age and Other Risk Factors

Human papillomavirus was detected in 23% of subjects younger than 20 years, 50% among those 20 to 29 years, and 43% among older subjects, a difference that was not statistically significant. There were no clear differences in HPV prevalence by socioeconomic status, but a somewhat higher HPV prevalence was observed among subjects with limited education. No differences in HPV prevalence were observed in occupation, marital status, age at first intercourse, number of sexual partners, or history of visits to commercial sex workers. A slightly higher prevalence was observed among subjects reporting using condoms (49% versus 38%), history of anal sex (50% versus 40%) and smoking (48.2% versus 34.2%), but these differences were not statistically significant.

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Discussion

Human papillomavirus DNA was detectable in nearly half of sexually active males in Cuernavaca. This prevalence is higher than that of HPV DNA reported among husbands of healthy women included as controls in a cervical cancer case–control study conducted Spain (3.5%) 15 and Colombia (18.9%). 16 The complete lack of detection of HPV DNA among men reporting no previous sexual activity provides strong evidence of the sexual acquisition and transmission of HPV. Although the majority of nonsexually active men were students, HPV infection among sexually active students was similar to that of factory workers. Interestingly, none of these men had clinically apparent lesions or complaints, which together with the rarity of cancer of the penis indicates that infection has a generally benign course in males.

Urine specimens do not appear to be adequate for the detection of genital HPV infection compared with the collection of exfoliated cells from the urethra and coronal sulcus in asymptomatic males. In this study, there was limited amplification of the β-globin gene in urine specimens, a finding that could be explained by the presence of inhibitors in urine. 14 However, after removing those inhibitors by DNA purification, which resulted in a β-globin–positive PCR, we did not obtain additional positivity in urine specimens.

Urine specimens have been shown to detect HPV in cases of intrameatal warts or urethral infection, 2,7 but not in men with genital dermatoses 5 or asymptomatic HPV infection located in the penis outside of the urethra. 3,6,7 Urine specimens could be of value in the assessment of genital HPV infection in males if infection generally affected large portions of the entire genital epithelium, including the urethra to the extent of being detectable in urine. However, this does not seem to be the case in this study. We are unable to differentiate between infection of the glans and infection of the urethra because both samples were collected simultaneously to represent the usual method of collection.

The few studies that have used β-globin gene amplification as a marker of the adequacy of DNA in urine specimens 8,9 reported successful amplification of β-globin in approximately 50% to 80% of subjects, but in our study β-globin was rarely detectable. Whether this reflects differences in the laboratory methods used or in the collection procedures in unclear.

One of the potential limitations of the current study is that urine specimens were collected at home by the participants and were not immediately processed (processing started within a few hours of collection). However, previous studies have tested the quality of DNA in urine specimens for PCR purposes after different storage times. 17 It appeared that a 209-bp β-globin fragment could be amplified without an apparent reduction in efficiency after a storage of 1 week at room temperature.

Finally , we observed an increasing trend in HPV positivity with increasing age that could be related to the possibility of acquiring infection with repeated exposure, though we did not observe an association with increasing number of sexual partners. Although we did not explore the full age range, this finding is in contrast with the observed tendency among women of decreasing HPV prevalence with age. Wikstrom et al 18 reported that among sexually active males, subclinical latent HPV infection is common and repeated sampling increases its prevalence. Additional research on the prevalence and natural history of HPV infection in males is warranted.

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References

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