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Comparison of urine samples and penile swabs for detection of human papillomavirus in HIV-negative Dutch men
  1. Fleur Koene1,
  2. Petra Wolffs1,
  3. Antoinette Brink2,
  4. Nicole Dukers-Muijrers1,3,
  5. Wim Quint4,
  6. Cathrien Bruggeman1,
  7. Christian Hoebe1,3
  1. 1Department of Medical Microbiology, School of Public Health and Primary Care (CAPHRI), Maastricht University Medical Center (MUMC+), Maastricht, The Netherlands
  2. 2PathoFinder B.V., Maastricht, The Netherlands
  3. 3Department of Sexual Health, Infectious Diseases and Environment, South Limburg Public Health Service, Geleen, The Netherlands
  4. 4DDL Diagnostic Laboratory, Rijswijk, The Netherlands
  1. Correspondence to Dr Petra Wolffs, Department of Medical Microbiology, Maastricht University Medical Centre, PO Box 5800, Maastricht 6202 AZ, The Netherlands; p.wolffs{at}


Objectives Penile swab sampling is the method of choice when testing for human papillomavirus (HPV) in men. Urine sampling is already used in routine sexually transmitted infections (STI) diagnostics and could provide a less invasive sampling method in men to detect HPV. Therefore we compared detection of HPV types in urine samples and penile swabs by the highly sensitive SPF10-LiPA25 system.

Methods First void urine and self-obtained penile swab samples were collected from 120 men, with a mean age of 29.4 years, visiting a STI clinic in South Limburg, the Netherlands. In total 111 of 120 men were included in the analysis. Broad-spectrum HPV DNA amplification and mucosal HPV genotyping were performed using the SPF10 DEIA-LiPA25 system (SPF10 HPV LiPA, V.1).

Results In total 75 (68%) men were positive for HPV in the combined analysis. Sixty-six (59%) paired samples were concordant in being positive or negative. In 39% of the men HPV DNA was detected only in the penile swab. In 2% of the men HPV DNA was detected only in the urine sample. Considering penile swabs as the gold standard, a sensitivity of 41% (95% CI 30% to 53%) and a specificity of 95% (95% CI 81% to 99%) was found. In 6 (5%) urines high risk types were repeatedly found that were not detected in the matching swab.

Conclusions Urine samples are not comparable to penile swabs in the detection of HPV in men. However, the addition of urine samples to penile swabs could be of use in epidemiological or clearance studies.

  • HPV
  • MEN

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Human papillomavirus (HPV) infections are the most common sexually transmitted infections (STIs) and HPV related disease has a steadily increasing prevalence. In women, HPV is studied extensively for its role in the development of cervical cancer. At least 35 of over 100 HPV genotypes identified have a tropism for the genital tract.1 Although men in high-risk populations can develop HPV related oropharyngeal or anogenital cancer, the interest in genital HPV in men is increasing these past years mainly because of their role as a possible HPV reservoir, maintaining infection in their regular sexual partner.2 This leads consequently to a higher risk of development of cervical, anal or oropharyngeal cancer in their sexual partner. However, there are relatively few studies focusing on genital HPV in low-risk populations of men. To detect genital HPV in men from the supposed sites of infection, penile swabs (swabs from the glans penis and coronal sulcus) are the samples of choice. Penile swabs, as opposed to urine, are not part of standard of care in the detection of STIs and are perceived as relatively invasive. Urine would therefore be an easy-to-use and non-invasive sampling method for HPV in men. Earlier studies resulted in conflicting data considering the usage of urine and are difficult to compare because of the different populations studied and the usage of detection methods of variable analytical sensitivity.2–5 Studies comparing urine samples to penile swabs were not representative due to lesser sensitivity of the detection method used, missing data on genotype detection and false-negative results due to sampling bias.2 ,6 Therefore the use of urine samples versus penile swabs in HPV detection in men was assessed by comparing them for HPV positivity and detected genotypes. We chose the highly sensitive detection method SPF10-LiPA25 system for this study, because standard FDA approved clinical assays can only differentiate between the most relevant genotypes.7 Furthermore the PCR-based detection methods frequently used in research have a higher limit of detection. Moreover, we expected low viral loads3 as relatively few cells are recovered from the planned sampling techniques and we wanted to identify all HPV genotypes present.


Sampling and study population

Between March and May 2011, self-collected urine samples and penile swabs were obtained from 120 otherwise healthy male clients attending the STI outpatient clinic at the Public Health Service South Limburg (the Netherlands). Exclusion criterion was a HIV-positive status or use of immunosuppressive medication because of potential interference with HPV infection. Their age ranged between 18 years and 61 years, with a mean of 29.4 years. A questionnaire was used to obtain information about the reason for visiting the clinic, ethnicity, sexual behaviour, previous STIs and presence of symptoms. Clients were requested to catch their first void urine and subsequently perform a penile swab sampling the external urethral meatus, the glance, coronal sulcus and shaft.8 To ensure appropriate self-sampling, the presence of human DNA in swabs was evaluated via β-globin PCR.4 The samples were processed after delivery and kept at −20°C. The remaining urine was used for routine testing of STIs.

HPV detection and genotyping

DNA was extracted with the QIAamp DNA mini kit (QIAGEN, Netherlands) according to the manufacturer's instructions. The SPF10 DEIA-LiPA25 system (V.1, manufactured by LBP Rijswijk, the Netherlands) was used as previously described and a quantified processing control was added.9 In brief, the SPF10-LiPA25 assay is based on the SPF10 PCR primer set, which amplifies a fragment of 65 bp within the L1 region. The SPF10 amplimers are tested for HPV positivity in a DNA enzyme immune assay (DEIA), able to detect more than 50 anogenital HPV types. The HPV-positive amplimers are subsequently analysed by SPF10 LiPA25, permitting the identification of 25 HPV genotypes (high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, possible high-risk HPV types: 34, 53, 66, 68/73, 70 and low-risk HPV types: 6, 11, 40, 42, 43, 44, 54 and 74). In our analyses the possible carcinogenic HPV types are considered to be high-risk types. Between HPV types 68 and 73 no differentiation was made because of non-unique interprimer sequences. Samples that showed a positive DEIA signal but had no positive result in the LiPA typing were characterised as HPV type X. This indicates the presence of (sub)types and/or variants that were not available on the typing blots and are considered to be low-risk HPV types.10


For statistical analysis Pearson's χ2 test and Fischer's exact test were used. p<0.05 was considered statistically significant.


Of the 120 penile swabs tested, 4 did not contain sufficient human DNA as shown by a negative β-globin PCR, suggesting inadequate sampling. These samples were excluded from further analysis. Five other men were excluded because of positive HIV status at time of sampling. No samples were inhibited in their amplification before further processing via the SPF10-LiPA25 system.

In total HPV DNA was found in 75 (68%) of 111 men, using the SPF10 LiPA25 system. In 43 (39%) of the men HPV DNA was detected only in the penile swab. In two (2%) of the men HPV DNA was detected only in the urine sample. Sixty-six paired samples were concordant in being HPV-positive or HPV-negative. Of these samples 30 (27%) pairs were positive and 36 (32%) pairs were negative for HPV (see table 1).

Table 1

Cross table HPV outcome in urine and penile swab samples of 111 men

Eleven of these 30 positive matching samples were concordant in genotype and 14 pairs were comparable (≥1 genotype identical). The remaining five pairs were discordant in their genotypes, that is, there were no identical type(s) in the corresponding samples. Surprisingly, in one of the comparable pairs and in three of the five discordant pairs, independent repeated testing confirmed that the urine samples contained high-risk types (types 16, 39 and 66) which were not found in the corresponding penile swabs.

Furthermore, the two positive urines with a negative swab contained high-risk types as well (types 53 and 66, respectively). To ensure true discrepancy between these genotypes and HPV positivity in discordant samples, the procedure was repeated and resulted in the same outcome for all samples. The results are summarised in online supplementary tables S2 and S3. There were no significant associations between HPV positivity in urine or swab and patient characteristics or sexual behaviour.


In this study we compared urine samples with penile swabs for detection of HPV in HIV-negative men and found that urine samples cannot replace penile swabs. Considering the penile swab to be the gold standard, an HPV prevalence of 66% (95% CI 56% to 74%), a sensitivity of 41% (95% CI 30% to 53%) and a specificity of 95% (95% CI 81% to 99%) was found for the detection of HPV in urine, which is comparable to what Payan et al3 found in women. Although the usage of urine samples in men seemed promising for the detection of HPV, these positive test results were seen in men with HPV-related penile lesions or female partners with proven HPV infection2 and HIV-positive men. As seen in a recent review of literature on HPV prevalence in urine of men by Enerly et al,11 only few studies focused on healthy men. Among these, only Cuschieri et al5 found a higher prevalence of HPV in urines compared with penile swabs (37% and 29%, respectively). The relatively high HPV prevalence in urine samples in the study of Cuschieri might be explained by the use of the InnoLiPA detection method, which detects more low-risk HPV types then the SPF10-LiPA25 system or might be due to sampling errors of the penile swabs leading to false-negative results, which was not controlled for in this study. In our study we ensured the absence of sampling errors by using positive β-globin results as an inclusion criterion.

Notably in our study, by repeated testing (possible) high-risk types were found in the urine samples of six men that could not be detected in the corresponding penile swab. Forslund et al2 also found HPV-negative swabs with HPV-positive urines in 2% of men (n=3). In two of these men consensus primers were used (HPV-X) and in one man high-risk type 35 specific primers (n=1) were used because a HPV-type 35 positive lesion was present on the posterior margin of the glans penis, confirming the possible additional value of a urine sample. Our study was limited however by the lack of sample volume because of which we were not able to further confirm our results by sequencing or quantification of viral loads.

Despite the high analytical sensitivity of the method used in our study, we are not able to recommend urine as a substitute sampling method instead of penile swabs in testing for HPV in men. To our knowledge this is the first report showing such a high prevalence of additional HPV detection in urine in men. These findings suggest that urine does have an additional value in epidemiology and clearance research.

The clinical relevance of the addition of urine sampling needs to be further assessed, but these results could be an indication of increased value in the detection of (high-risk) HPV in men.


The authors thank Dr A T Hesselink from the VU medical centre, Amsterdam for his helpful comments concerning the usage of β-globin PCR.



  • Handling editor Jackie A Cassell

  • Contributors PW and CH had the idea for the study and reviewed the manuscript. FK processed and analysed the samples and wrote the article. AB helped with the molecular methods and interpretation of the samples and reviewed the manuscript. CB reviewed the manuscript. ND-M helped with statistical analyses and reviewed the manuscript.

  • Competing interests None declared.

  • Ethics approval Medical Ethical Committee of the Maastricht University Medical Centre (no. METC 10-4-085).

  • Provenance and peer review Not commissioned; externally peer reviewed.