Takahashi, Satoshi MD*#; Shimizu, Toshiaki MD*#; Takeyama, Kou MD*#; Kunishima, Yasuharu MD*#; Hotta, Hiroshi MD*#; Koroku, Mikio MD†#; Tanda, Hitoshi MD†#; Saka, Taketoshi MD‡#; Nishimura, Masahiro MD§#; Iwasawa, Akihiko MD∥#; Furuya, Ryoji MD¶#; Hirose, Takaoki MD¶#; Kobayashi, Intetsu PhD**; Kumamoto, Yoshiaki MD*; Tsukamoto, Taiji MD*#
WHILE HUMAN PAPILLOMAVIRUS (HPV) INFECTION is closely linked with development of genital warts and cervical and penile cancers, depending on the DNA type, asymptomatic infection by the virus is more prevalent. Peyton et al. 1 reported that asymptomatic infection by HPV in cervical specimens was found in 39.2% of 3863 women and that the infection was strongly associated with age and the number of lifetime and recent sex partners. Richardson et al. 2 reported similar findings; they noted 21.8% overall HPV detection in asymptomatic female university students. The study also indicated that lifetime frequency of sexual intercourse and lifetime number of oral sex partners were associated with high-oncogenic-risk HPV infection.
However, partly because of the extremely low incidence of penile cancer in developed countries, there have been few studies of asymptomatic HPV infection in men. 3 Another factor hampering such studies is that we have not established how and what specimens we need to collect to determine the rate of asymptomatic HPV infection.
Since asymptomatic infection with the virus in men is partly responsible for asymptomatic infection in women, who more frequently develop cancer of genital lesions, it is crucial to determine the prevalence of such infection and the DNA types of the virus that are involved in it. Thus, we tried to detect HPV DNA in the external genitalia of healthy men and male patients with urethritis, all of whom were free of visible lesions caused by the virus. We also studied the natural clinical course of men who were positive for HPV DNA at the first examination. Although our study may not necessarily reveal a substantial rate of asymptomatic HPV infection in men, the results will enhance our understanding of the natural history of the infection.
Patients and Methods
The study included 75 healthy male volunteers who were recruited from among university students for 1 month by advertisements that explained the study design and its clinical relevance. For 3 months, consecutive patients with urethritis (130 men) who agreed to participate were included in the study. They visited a clinic or hospital with which one of the authors is affiliated, reporting symptoms related to urethritis. The response rate of the patients was >90%. Since the clinical significance of detection of HPV DNA in the absence of genital warts was not established, we informed participants of the result of the examination only when they wanted to know.
The healthy male volunteers were asked to respond to a self-administered questionnaire for information about age, marital status, history of sexually transmitted diseases (STDs), average frequency of sexual intercourse in the previous 3 months, and number of current sex partners. The average frequency of intercourse was assessed as 3 to 4 times per week, 1 to 2 times per week, 3 to 4 times per month, 1 to 2 times per month, less than 1 time per month, and none. The participants were also asked to mark on the questionnaire their preputial status according to four illustrated types: type A, in which the prepuce completely covers the glans; type B, in which the prepuce covers half of the glans; type C, in which the prepuce is beyond the sulcus but can be extended to cover half of the glans without compressing it; and type D, in which the prepuce is completely absent. 4
In addition, they were asked to confirm carefully by themselves that they did not have any visible genital warts on their external genitalia, including the glans, coronal sulcus, inner surface of the prepuce, or urethral meatus. The confirmation was made at the time of initiation of the study and then 3 and 6 months later, together with HPV DNA tests. The rationale for self-examination was based on the clinical experience of urologists that most patients with genital warts usually visit a clinic to report a visible tumor on the external genitalia that they find by themselves.
The healthy volunteers underwent an HPV DNA detection test three times, at the initial examination and then 3 and 6 months later. They were carefully guided by one of us (S.T.) on how to obtain test specimens for the virus DNA. Using a body model, the urologist instructed them at each examination how and where they should wipe with a cotton swab for detection of HPV DNA. The glans, coronal sulcus, and inner surface of the prepuce were extensively wiped with a wet cotton swab by the volunteers themselves. Then the cotton swab was put in a storage bottle of a Digene Swab Specimen Collection Kit (Digene Corporation, Gaithersburg, MD) containing buffer solution provided by a manufacturer, and the bottles were kept at −20 °C until the detection test.
Hybrid Capture II (Digene Corporation) was used to detect HPV DNA and to determine the DNA type. The method identified two types of DNA: that of high-intermediate oncogenic risk and that of low oncogenic risk. The former included DNA types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and the latter included DNA types 6, 11, 42, 43 and 44. The viral DNA detection was done according to the instructions of the manufacturer (Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo).
Male patients with urethritis were asked to respond to a self-administered questionnaire as the healthy volunteers did. They underwent diagnostic examinations by a urologist, including urinalysis of the first-voided urine, Gram staining of urethral discharge, and detection of Chlamydia trachomatis and Neisseria gonorrhoeae in the first-voided urine, both of which were done with a commercially available PCR assay (Amplicor STD-I; Hoffmann-La Roche, Ltd., Basel, Switzerland). They also underwent standard physical examination by a urologist, including careful inspection of the glans, coronal sulcus, inner surface of the prepuce, and urethral meatus of the external genitalia, with confirmation of no visible genital warts and of preputial status, as described for healthy volunteers at the initial visit.
If patients visited again, at that visit a urologist confirmed by inspection that there were no visible warts on the external genitalia as well as the urethral meatus. HPV DNA detection tests were done at the first and following visits; the latter were not uniform but variable, depending on patients’ situations. The specimens for the viral DNA were obtained by urologists, who examined patients with the same methods as for healthy volunteers.
For 10 patients with urethritis who were positive for HPV DNA, including 8 with high-intermediate risk types and 2 with a low-risk one, the DNA types were confirmed by restriction fragment length polymorphism (RFLP) and a hybridization technique with type-specific probes, as reported previously, 5 at Mitsubishi Kagaku Bio-Clinical Laboratories, Inc., Tokyo.
Statistical analyses were done with the Fisher exact probability test, the Kruskal–Wallis test, and multiple regression, and StatView software for Windows, version 5 (SAS Institute, Cary, NC), was used for all analyses.
The protocol of this study was approved by the Institutional Review Board of Sapporo Medical University. Informed consent was obtained from all participants.
Detection of HPV DNA and DNA Types in Healthy Volunteers and Male Patients with Urethritis
Healthy male volunteers were clearly younger than patients with urethritis (Table 1). HPV DNA was detected on the genitalia of only one healthy volunteer (1.3%). The detection rate of HPV DNA was significantly higher among men with urethritis (18.5%) than among healthy volunteers (Table 1; P < 0.0001 by Fisher's exact probability test). Among the 130 men with urethritis, HPV DNA was detected in 6 (10%) of 60 patients with gonococcal infection, 11 (25%) of 44 with chlamydial infection, 3 (50%) of 6 with both gonococcal and chlamydial infections, and 4 (20%) of 20 with both nongonococcal and nonchlamydial infections.
The one positive healthy volunteer had HPV DNA of a high-intermediate-risk type (Table 1). Of 24 patients with urethritis who were positive for HPV DNA, the low-risk type was found in 4 (16.7%), the high-intermediate-risk type in 12 (50.0%), and both types in 8 (33.3%). Thus, of the 25 men positive for HPV DNA, 84% had HPV DNA of a high-intermediate risk type.
Sexual Activity, Preputial Status, and Significant Risk Factor(s) for HPV DNA Detection in Healthy Volunteers and Patients with Urethritis
Marital status differed for volunteers and patients with urethritis (Table 1). Patients with urethritis had sexual intercourse more frequently and more sex partners than healthy volunteers. Patients had a history of STD more frequently than did volunteers (Table 1). The history was more prominent for patients positive for HPV DNA than for those who were negative (Table 2). Almost 50% of patients negative for HPV DNA and more than 70% of those positive for HPV DNA had sexual intercourse one or two times per week or more frequently, which was in contrast to the finding that only 30% of volunteers had intercourse at such a frequency (Table 1).
As for sex partners, 20% of patients with urethritis positive for HPV DNA and 10% of those negative for HPV DNA had three or more current sex partners, while no volunteers had such a number (Tables 1 and 2). Status of the prepuce did not affect detection of HPV DNA. The four prepuce types were almost equally distributed among healthy volunteers and patients with urethritis who were positive or negative for HPV DNA.
Risk factors for positive detection of HPV DNA, including age, marital status, history of STD, average frequency of sexual intercourse, number of current sex partners, and type of prepuce, showed statistically significant differences among volunteers, patients with urethritis who were negative for HPV DNA, and patients with urethritis who were positive for HPV DNA (except for preputial status;Table 3). However, multiple regression analysis of these factors revealed a history of STD to be a significant, independent risk factor for HPV DNA positivity (P = 0.0122).
In follow-up of the 75 healthy volunteers, all were examined for HPV DNA, with repeated instruction on obtaining specimens and confirmation that there were no visible warts at 3 and 6 months after the initial examination. The one volunteer positive for HPV DNA at the initial examination showed persistent infection with HPV of the same DNA type 3 months later. However, no HPV DNA was found 6 months later. No volunteers were found to become positive for HPV DNA during the 6-month follow-up. Development of genital warts was not noticed in any volunteers during this period.
Of the 130 patients with urethritis, 69 (including the 18 positive for HPV DNA at the first visit) underwent a detection test and were confirmed not to have genital warts by inspection again by a urologist at the second or third visit. The same type of HPV DNA was detected again at the second visit (median follow-up, 11 days; range, 3–30) in 16 of 18 who were positive at the first visit; two patients were negative at this second follow-up visit. Only 2 of 16 patients who were positive for HPV DNA at both the first and second visits had a third visit (at 35 and 145 days, respectively, after the diagnosis). They were persistently positive for the same type of HPV DNA. The remaining 51 patients negative for HPV DNA at the initial visit had second visits, with a median follow-up period of 10 days (range, 2–62) and one of these had a third visit as well (follow-up: 29 days). None of these 51 patients became positive for HPV DNA at the second or third visit.
Confirmation of HPV DNA Types with the PCR Method
We confirmed HPV DNA types for eight patients who were positive for high-intermediate-risk DNA and two of those positive for low-risk DNA with use of Hybrid Capture II. Their DNA types were analyzed by the RFPL and hybridization methods, 5 which revealed that specimens from eight patients who were positive for HPV DNA high-intermediate-risk types contained DNA types 16, 31, and 58. The method also revealed that swab specimens from the two patients positive for a low-risk type contained DNA type 6.
The prevalence of asymptomatic HPV infection in men who were positive for HPV DNA on their external genitalia but had no visible lesion by pneoscopy was reported to be 7.2% in a study of voluntary participants of the Finnish Army. 3 In addition, the study indicated that having many sex partners, a history of STD, and nonuse of condoms were significant risk factors for HPV infection of the genitalia. In a study of asymptomatic Mexican men, a higher HPV DNA detection rate, 43%, was noted, 6 although the backgrounds of controls and method of detection were not the same as those in our study. Unfortunately, the precise prevalence of asymptomatic infection in men has not been determined because the backgrounds of subjects recruited in studies and study designs are not always similar.
Indeed, there are no widely accepted standards for healthy volunteers. In our study, although we recruited university students, they might not have been ideal controls because their lifestyle, including sexual behavior, may have differed somewhat from that of ordinary workers. In addition, the most appropriate specimens for sampling have not been established for screening asymptomatic HPV infection. Iwasawa et al. 7 reported that urine samples could be used to detect HPV DNA in patients with genital warts and suggested that such samples might be applicable for screening asymptomatic subjects. However, a recent publication indicated that urine samples had very low sensitivity for such detection, in comparison with samples obtained by wiping the external genitalia. 6
Obtaining urethral samples from the meatus by wiping with a swab is not more useful for subjects in the screening setting because of its painful and uncomfortable nature. Thus, our finding that the detection rate of HPV DNA for healthy volunteers was extremely low may stem from their background. We believe that the sampling method did not contribute to the rate, although a false-negative result might have been possible in the volunteers because they obtained specimens by themselves.
Nevertheless, what is important in the results of our study is that nearly 1 of 5 men with urethritis had asymptomatic HPV infection of their external genitalia that was revealed by HPV DNA detection with Hybrid Capture II, indicating that the asymptomatic infection might have been associated with other STDs such as urethritis.
High-intermediate-risk HPV DNA was detected more frequently than the low-risk type in the current study. Because there are reports of similar findings in female studies, 2,8 the HPV DNA types detected may have the same tendency in men and women. However, there is controversy about whether sex partners have the same type of HPV DNA. 9 In addition, while the efficacy of the Hybrid Capture method has been clinically established for the diagnosis of HPV infection, 10,11 undetermined HPV DNA types might not be detected by this method, since there are currently more than 80 types and the number is still growing. The detection of more types of HPV DNA is thus necessary to achieve greater accuracy.
A history of STD was determined by multiple regression analysis to be the only significant risk factor for HPV DNA positivity in our study. In addition, patients with urethritis who were positive for HPV DNA reported more sexual activity than those who were negative and volunteers. Thus, our results indicated that HPV DNA detection in men was closely linked with sexual behavior that was characterized by a history of STD and sexual frequency, which was consistent with the results of previous studies.
As for the association of preputial status in men with HPV infection and cervical or penile cancer, circumcision was reported to reduce the risk of penile HPV infection, and the absence of neonatal circumcision and potential resulting complications were associated with development of penile cancer. 12,13 Although phimosis is one of the risk factors of penile cancer, other factors such as sexual activity, smoking, 12 and inadequate hygiene may be involved.
In this study, no significant difference was found between the HPV infection rate and the status of the prepuce. Rather, multiple regression analysis showed that a history STD was the only significant risk factor for asymptomatic HPV infection. Because HPV prevalence differs among countries, 9 various environmental factors may be involved in genital HPV infection.
While the natural history of cervical cancer associated with HPV infection has been partly clarified, 14 the natural history of asymptomatic HPV infection has not been determined, particularly for men. A population-based cohort study that recruited young Swedish women who were asymptomatic revealed that HPV infection was transient, and a new HPV-type-specific infection was associated with a new sex partner or an abnormal smear after enrollment. 15 In our study, HPV infection persisted for the first 3 months in the one volunteer who was positive for HPV DNA at the initial evaluation. Patients with urethritis who were positive for HPV DNA at the initial evaluation were followed-up, although for a short interval, and most of them were positive for HPV at the second visit.
Because the number of patients enrolled in the follow-up study was small and their follow-up period was short and variable, depending on personal reasons, we could not fully elucidate the natural history of asymptomatic male HPV infection. However, HPV infection may persist, although the infection is transient, and reinfection is not excluded. What is important is that men positive for HPV DNA can transmit it to female partners during its persistent period. The natural history of asymptomatic infection by HPV will be clarified only with studies that provide longer follow-up and precise determination of HPV DNA types.
In conclusion, HPV DNA was detected in patients with urethritis more frequently than in healthy male volunteers. The high-intermediate-risk type was detected predominantly in men who were positive for HPV DNA. The risk factor for such detection was a history of STD, a circumstance suggesting association with the sexual activity of men.
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