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Poor Sensitivity of Polymerase Chain Reaction Assays of Genital Skin Swabs and Urine to Detect HPV 6 and 11 DNA in Men

FIFE, KENNETH H. MD, PhD*; COPLAN, PAUL M. MS, ScD; JANSEN, KATHRIN U. PhD; DiCELLO, ANTHONY C. BA; BROWN, DARRON R. MD*‡; ROJAS, CARLOS MD, PhD; SU, LING PhD

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Sexually Transmitted Diseases: March 2003 - Volume 30 - Issue 3 - p 246-248
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GENITAL TRACT HUMAN PAPILLOMAVIRUSES (HPVs) are probably the most prevalent of the sexually transmitted pathogens. 1,2 Numerous studies have shown a high frequency of detection of HPV in specimens from asymptomatic women. 3–6 Male sex partners are thought to be the major source of infection for women with symptomatic or asymptomatic genital tract HPV infection. 7 However, most attempts to demonstrate HPV DNA in male partners of women with symptomatic or asymptomatic HPV infection have detected infection in only a minority of male partners. 8–14

A possible reason for the failure to detect HPV DNA in asymptomatic men who are likely to be infected is the insufficient sensitivity of the detection methods. Another factor may be the location and type of samples obtained and the kind of skin sampled. The goal of this study was to identify a method for sampling the anogenital skin of men that was simple and well tolerated and that would permit the detection of asymptomatic or subclinical HPV infection, which is thought to occur commonly in sexually active men. Such a method would be extremely helpful in epidemiologic investigations of HPV transmission and in monitoring strategies for prevention of HPV infection, such as prophylactic vaccines.

We used a highly sensitive PCR-based assay for HPV 6 and 11 DNA to test the feasibility of using urine and genital skin swabs for detecting HPV infection in men, by comparing groups of men likely to be infected with HPV 6 or 11 (those with active genital warts), possibly infected with HPV 6 or 11 (those with no current warts but a history of genital warts), or of uncertain likelihood of infection with HPV 6 or 11 (those with no history of warts). We also took samples from several different anatomical locations to evaluate the adequacy of samples from specific locations for detecting HPV 6 or 11 infection.

Male subjects between the ages of 18 and 50 years attending a sexually transmitted diseases clinic were recruited for this study. Three groups of 20 subjects each were recruited on the basis of their clinical genital wart history. Group I consisted of men who had visible external genital warts present at the time of the clinic visit and no history of recent treatment of the warts. Group II comprised men who had a history of external genital warts that had been successfully treated between 30 and 365 days before the visit. Group III consisted of men who had no clinical history of genital warts at any time.

The local institutional review board approved the protocol and consent form, and all subjects provided written informed consent before any procedures were performed. Clinical and demographic information was collected, including a history of other sexually transmitted diseases and recent and lifetime numbers of sex partners.

Each subject had separate dry swab specimens collected from normal-appearing skin from the following sites: the glans penis (including the coronal sulcus), the penile shaft, the inguinal skin, the surface of the scrotum, the perineum, and the perianal area. For subjects who had genital warts present, a swab of the surface of one or more lesion was also obtained. All swabs were done vigorously in an effort to obtain skin cells. Each swab was placed in specimen transport medium (Digene Diagnostics, Gaithersburg, MD), frozen on dry ice, and transported to the laboratory. Each subject also provided a urine specimen (approximately 35 ml of urine collected at the beginning of the void). Subjects in group I were offered treatment of their external genital warts per clinic protocol (a choice of a prescription for a patient-applied treatment or in-clinic treatment with either liquid nitrogen or surgical excision). Those who elected to have their lesions treated by surgical excision had some of the excised wart tissue saved for HPV analysis (with subject consent). The tissue was placed in specimen transport medium, incubated at 50 °C for 8 to 12 hours, and then frozen. Subjects also completed a questionnaire regarding the acceptability of the specimen collection procedures.

Swabs were removed from the transport medium, and swab samples and wart tissue specimens were prepared for polymerase chain reaction (PCR) with a quick DNA purification method (Qiagen, Valencia, CA). Urine was spun and the cellular pellet was subjected to DNA extraction by the same method. DNA was analyzed by PCR with use of HPV type–specific primers based on the nucleotide sequence of the HPV L1, E6, and E7 genes of HPV types 6 and 11. PCR products were visualized by dot-blot hybridization with use of HPV-specific oligonucleotide probes. A positive result was defined as any hybridization signal above background (HPV-negative human DNA samples). Specimen adequacy was determined by amplifying a target sequence from the human β-globin gene. Specimens that did not yield a β-globin amplimer were labeled nonamplifiable and were considered inadequate for analysis.

Sixty percent of the subjects were white and 30% were black. Most subjects had one or more previous sexually transmitted diseases, and the majority had 10 or more lifetime sex partners, although most had 0 or 1 partner in the previous 30 days. All but 1 of the 60 subjects were circumcised, and only 8 subjects were consistent condom users. The three groups were generally similar, although group III subjects were less likely to have a history of a sexually transmitted disease (45%) than were group I subjects (85%) or group II subjects (95%;P < 0.05, Fisher exact test).

The PCR results for each anatomical site are summarized in Table 1. Fifteen subjects in group I had at least one positive PCR result for HPV 6 or 11, while only one in group II and two in group III had positive results. In group I, 12 subjects had HPV 6 detected and three subjects had HPV 11 detected. All of the group II and group III subjects who had HPV detected were positive for HPV 6. No subject had both HPV types detected. Fourteen of the 18 subjects in whom HPV DNA was detected had HPV DNA at two or more sites. The four subjects who had HPV DNA detected in only one site were all in group I, and the single positive site was the wart surface in all cases. Many of the skin sites contained no amplifiable DNA. Urine contained amplifiable DNA in most cases, but only two urine specimens were positive in group I.

TABLE 1
TABLE 1:
Results of HPV PCR on Urine and Genital Skin Swabs for Men, by Subject Group and Specimen Site

Wart tissue was available from seven subjects in group I who elected to have their warts treated by surgical excision. All seven were positive for HPV DNA (five for HPV 6 and two for HPV 11). In all cases, one or more surface swab specimen was positive for the same HPV type. The lesions were not examined histologically.

Nearly all of the subjects felt that the specimen collection procedures were quite acceptable. Only one of the 60 subjects found the perianal swabbing difficult to tolerate. All other site samplings were rated as easy to tolerate by all subjects.

Although our method of using dry swabs of several anatomical areas was simple and well tolerated, it lacked the sensitivity to make it a useful approach for screening. Much of the poor sensitivity was due to specimen inadequacy. Half or more of the swabs obtained at several anatomical sites contained no amplifiable human DNA.

While no attempt was made to quantitate the amount of DNA recovered on the swabs, the large number of specimens that contained no DNA suggests that many of the other specimens probably contained very little DNA. Thus, many of the specimens that were considered adequate because we were able to detect human β-globin gene sequences were probably marginal for detecting HPV sequences from subclinical lesions.

Urine was the specimen source that was most likely to contain amplifiable DNA. However, only 10% of group I subjects (10.5%, excluding inadequate specimens) and only one of the 40 subjects in groups II and III had HPV DNA in their urine. This suggests that urethral carriage of HPV 6 and 11 is unusual (or that HPV is present is quantities too small to be detected by our PCR assay). Thus, screening urine for HPV 6 and 11 sequences is unlikely to be helpful in epidemiological and vaccine studies.

The only site for which this sampling method was efficacious was the surface of the lesions in subjects with active external genital warts. Because HPV DNA is amplified and virions are produced in the upper layers of an infected epithelium 15,16 and because cells of the upper stratum corneum are easily removed from skin, it is not surprising that these specimens would yield detectable HPV DNA in many cases. Shedding of such HPV-infected cells has been postulated to be an important mechanism for transmission of HPV among sex partners. 17 It is not clear if this type of noninvasive sampling of genital warts will be of any practical value in clinical or research settings.

Most subjects who had HPV DNA detected had two or more anatomical sites that were positive. The only exceptions were four subjects whose only positive specimen was that taken from the surface of a wart. This finding of multifocal HPV infection of even normal-appearing skin is consistent with findings in previous studies 11 and suggests that in many patients the visible warts represent only a fraction of the HPV-infected epithelium.

Noninvasive sampling of genital skin to identify individuals with subclinical HPV infection remains a challenge. The method must be disruptive enough to remove epithelial cells from the surface of the skin but gentle enough to be tolerated by subjects who are being screened. One previous study used cyanoacrylate glue to accomplish this task 11 and reported good research-subject acceptance. However, an informal survey of our subject population found a general reluctance to permit removal of genital epithelial cells with cyanoacrylate glue. Future studies should use more abrasive sampling devices (such as cytobrushes), perhaps combined with the use of some type of soap to try to dislodge more epithelial cells.

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