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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
1. Herrero R, Muñoz N. HPV and cancer. Cancer Surv 1999; 33: 75–98.
2. Melchers WJ, Schift R, Stolz E, Lindeman J, Quint WGV. Human papillomavirus detection in urine samples from male patients by the polymerase chain reaction. J Clin Microbiol 1989; 27: 1711–1714.
3. Nakazawa A, Inoue M, Fujita M, Tanizawa O, Hakura A. Detection of human papillomavirus type 16 in sexual partners of patients having cervical cancer by polymerase chain reaction. Jpn J Cancer Res 1991; 82: 1187–1190.
4. Hillman RJ, Ryait BK, Botcherby M, Walker MN, Taylor-Robinson D. Human papillomavirus DNA in the urogenital tracts of men with genital dermatoses: evidence for multifocal infection. Int J STD AIDS 1993; 4: 147–154.
5. Hillman RJ, Botcherby M, Ryait BK, Hanna N, Taylor-Robinson D. Detection of human papillomavirus DNA in the urogenital tracts of men with anogenital warts. Sex Transm Dis 1993; 20: 21–27.
6. Forslund O, Goran B, Rymark P, Bjerre B. Human papillomavirus DNA in urine samples compared with that in simultaneously collected urethra and cervix samples. J Clin Microbiol 1993; 31: 1975–1979.
7. Iwasawa A, Hiltunen E, Reunala T, Nieminen P, Paavonen J. Human papillomavirus DNA in urine specimens of men with condyloma acuminatum. Sex Transm Dis 1997; 24: 165–168.
8. Geddy PM, Wells M, Lacey CJN. Lack of detection of human papillomavirus DNA in male urine samples. Genitourin Med 1993; 69: 276–279.
9. Astori G, Pipan C, Muffato G, Botta GA. Detection of HPV-DNA in semen, urine, and urethral samples by dot blot and PCR. Microbiologia 1995; 18: 143–149.
10. De Roda Husman AM, Walboomers JMM, Van den Brule AJC, Meijer CJLM, Snijders PJF. The use of general primers GP5 and GP6 elongated at their 3′ ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995; 76: 1057–1062.
11. Jacobs MV, Snijders PJF, Van den Brule AJC, Helmerhorst THJM, Meijer CJLM, Walboomers JMM. A general primer GP5+/6+ mediated PCR-enzyme immunoassay method for rapid detection of 14 high risk and 6 low risk human papillomavirus genotypes in cervical scrapings. J Clin Microbiol 1997; 35: 791–795.
12. Brule AJC van den, Meijer CJLM, Bakels V, Kenemans P, Walboomers JMM. Rapid detection of human papillomavirus in cervical scrapes by combined general primer mediated and type specific polymerase chain reaction. J Clin Microbiol 1990; 28: 2739–2743.
13. Walboomers JMM, Melkert PWJ, Van den Brule AJC, Snijders PJF, Meijer CJLM. The polymerase chain reaction for human papillomavirus screening in diagnostic cytopathology of the cervix. In: Herrington CS, McGee OD, eds. Diagnostic Molecular Pathology: A Practical Approach. Oxford: Oxford University Press, 1992: 152–172.
14. Mahony J, Chong S, Jang D, et al. Urine specimens from pregnant and non pregnant women inhibitory to amplification of chlamydia trachomatisnucleic acid by PCR,LCR and TMA: identification of urine substances associated with inhibition and removal of inhibitory activity. J Clin Microbiol. 1998; 36: 3122–3126.
15. Bosch FX, Castellsagué X, Muñoz N, et al. Male sexual behavior and human papillomavirus DNA: key risk factors for cervical cancer in Spain. J Natl Cancer Inst 1996; 88: 1060–1067.
16. Muñoz N, Castellsagué X, Bosch FX, et al. Difficulty in elucidating the male role in cervical cancer in Colombia, a high risk area for the disease. J Natl Cancer Inst 1996; 88: 1068–1075.
17. Morre SA, van Valkengoed IG, de Jong A, et al. Mailed, home-obtained urine specimens: a reliable screening approach for detecting asymptomatic Chlamydia trachomatis
infections. J Clin Microbiol 1999; 37: 976–980.
18. Wikstrom A, Popescu C, Forslund O. Asymptomatic penile HPV infection: a prospective study. Int J STD AIDS 2000; 11: 80–84.