The global burden of infection with human papillomaviruses (HPVs) and associated diseases is profound. More than 99% of cervical cancers are HPV associated,1 with HPV16 and HPV18 accounting for approximately 70% of all invasive cervical cancers.2 Infections with HPV6 and HPV11 cause condylomas (genital warts), and case series of biopsies have found HPV6 or HPV11 in approximately 90% of condylomas.3–10
Currently, 2 prophylactic vaccines preventing HPV infections are available, a quadrivalent vaccine targeting HPV6, HPV11, HPV16, and HPV18 and a bivalent vaccine targeting HPV16 and HPV18.11,12 These vaccines are highly effective to prevent persistent infections and lesions.13 The quadrivalent vaccine prevents 82% of all condylomas.14
Condylomas are usually self-limiting and heal without problems, but a subgroup of patients may get chronic or recurrent disease. The incubation time is relatively short (1–6 months),15 and a decline in condyloma incidence was observed among young women in Australia shortly after the introduction of the quadrivalent vaccine.16,17
Although condyloma monitoring has been found to be useful for rapid follow-up of the effect of HPV vaccination programs, a monitoring system including HPV genotyping would enable surveillance of the incidence of disease caused both by vaccine HPV types and by nonvaccine HPV types. This would provide clearer answers on effectiveness and document any possible effects on cross-protection or type replacement. In addition, mapping the proportion of condylomas caused by vaccine types prevaccination enables prediction of the benefits of vaccination in terms of condyloma burden reduction.
In 2005 the World Health Organization initiated the establishment of a global HPV laboratory network (HPV LabNet) with the objective of further development and implementation of HPV vaccines by improving and standardizing the quality of HPV laboratory services used for HPV vaccination impact monitoring.18 In addition, the global reference laboratory in Sweden was given the task to design pilot projects to gain practical experience of how laboratory methods could be used to follow up the effects of HPV vaccination programs in a practical and (cost-)efficient manner.
The present study was designed as a pilot project of feasibility and technical requirements of adding HPV genotyping to routine condyloma reporting. We report on the results of comprehensive HPV genotyping in the, so far, largest case series of condyloma.
MATERIALS AND METHODS
Patients with condyloma visiting the Centre for Sexual Health (CSH) in Malmö, Sweden, were included in the surveillance program. Clinical diagnosis of condyloma19 was given by a doctor of medicine experienced in STI diagnostics.
The city of Malmö is the third largest city in Sweden with approximately 300,000 inhabitants. The CSH is the only specialized outpatient clinic treating mainly adult patients with suspected sexually transmitted infections in Malmö. Females younger than 20 years and males younger than 23 years are usually cared for by youth clinics. Approximately, 25% of all samples for diagnostics of Chlamydia trachomatis infection in the city of Malmö are obtained at CSH. The Centre for Sexual Health provides a walk-in service for patients but also handles referrals. Since 2006, all patients receiving a clinical diagnosis of condyloma were asked to participate in condyloma surveillance with HPV typing. The clinical diagnosis of condyloma is not specifically restricted to acuminate condylomas but includes all symptomatic condylomatous lesions (i.e., also papular or flat condylomas).19 Written and oral informed consent was obtained from all participants. Ethical permission for the project was obtained from the Ethical Review Board in Lund, Sweden. This report includes cases from 2006 to October 2009. Human papillomavirus detection was performed at the Malmö Clinical Microbiology Laboratory, which is 1 of the 2 World Health Organization global reference laboratories for HPV diagnostics.
A sterile cervical cytobrush (MedScand, Stockholm, Sweden) was dipped in sterile saline and brushed on the condyloma. There was no prior cleaning of the sampled skin area. The brush was then stirred in a tube with approximately 800 μL sterile saline, and the tube was sent to the Clinical Microbiology Laboratory for analysis.
Detection and Typing of HPV
The sample was stored at +4°C and was processed within 5 working days. The sample was centrifugated (5 minutes, 3000g), and the cell pellet was resuspended in 500 μL of remaining saline. Two hundred microliters was used for DNA extraction with MagNA Pure LC using the Total Nucleic Acid Kit (Roche) and eluted in 100 μL. Five microliters was used for HPV DNA amplification using the GP5+/6+ primers (until April 15, 2008) or modified GP5+/6+ (MGP) primers (from April 17, 2008).20,21 Polymerase chain reaction (PCR) products were analyzed for specific HPV genotypes using a Luminex system that contains type-specific probes for mucosal HPV types (high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68a + 68b, 73, and 82; potential high-risk type 66; low-risk types 6, 11, 42, 43, and 70).22 The laboratory is accredited according to the ISO 15189 standard for 14 oncogenic types and for HPV6 and HPV11 in the used HPV test.
The Luminex assay also included 2 broadly reactive “universal” probes, and samples positive only for a universal probe were typed by sequencing. Nine additional HPV types (26, 30, 40, 54, 67, 81, 89, 90, and 91) were added to the large pool of Luminex probes from February 3, 2009.
Sample adequacy was assessed by assaying for the human β-globin gene by PCR and visualized by gel electrophoresis. After October 10, 2007, detection of the β-globin gene was performed with a real-time PCR in a 25-μL solution with 5 μL of sample, 0.2 μM of each primer (PCO3 14–39F 5′-ACACAACTGTGTTCACTAGCAACCTC-3′ and PCO4 123–103R 5′-CCAACTTCATCCACGTTCACCT-3′) and 0.2 μM of probe (55–82 5′-FAM-TGCACCTGACTCCTGAGGAGAAGTCTGC-TAMRA-3′), 1× Taq Man Universal PCR Master Mix (Applied Biosystems). The PCR was performed in 7500 Fast Real-Time PCR apparatus (Applied Biosystems) within 2 minutes at 50°C, followed by 10 minutes at 95°C and then 50 cycles of 15 seconds at 95°C and 1 minute at 60°C.
In a subset of 50 cases where no HPV was detected by the standard assay but were β-globin positive, we performed PCR using another general primer pair (FAP59/64),23 followed by cloning and sequencing. Samples still HPV negative were amplified by rolling circle amplification (RCA).24 Briefly, the RCA (Illustra Templiphi; GE Healthcare) used 1 μL of the extracted DNA sample mixed with 5 μL of sample buffer and incubated at 95°C for 3 minutes and then mixed with 5 μL of a reaction solution (5 μL of reaction buffer, 0.2 of μL enzyme, and 450 μM of dNTP [Roche]) and incubated at 30°C for approximately 16 hours, followed by 65°C for 10 minutes. The RCA was diluted 1:100 prior to analysis by FAP-PCR,23 GP5+/6+ PCR,20 MGP-PCR, and Luminex, as described previously.
Isolation and Amplification of a Novel HPV Type (HPV153)
Subsequent to RCA an amplicon generated by FAP-PCR23 was cloned (TOPO TA Cloning Kit; Invitrogen), and its sequence was used to design primers (HPV153.Fwd 5′-CCTCTTGGTATTGGTTCTACTGGC and HPV153.Rev 5′-ACCACCCCTATCTAATTCTAA ACC CTC) for amplification of the HPV genome. The 20-μL PCR mix included 1 μL of RCA-template, 0.3 μM of each primer, 0.5 mM of each dNTP, 1× buffer 2, and 0.89 U Expand Long Template enzyme mix (Roche). Polymerase chain reaction settings was 2 minutes at 94°C, 10 cycles of 94°C for 10 seconds, 68°C (decreased by 1°C in every cycle) for 30 seconds, and 68°C for 6 minutes, followed by 35 cycles of 94°C for 15 seconds, 57°C for 30 seconds, and 68°C for 6 minutes (increased by 10 seconds in every cycle) and finally 68°C for 15 minutes. The complete genome was sequenced after being obtained by E-Gel CloneWell 0.8% SYBR Safe gel (Invitrogen) and cloned (TOPO TA Cloning Kit; Invitrogen).
Statistical analysis used multivariate logistic regression analyses adjusting for age using STATA/SE 11.0.
Altogether, 703 patients with condyloma were reported. Among these, 11.6% (82/703) had inadequate samples and were excluded from further analysis. Inadequate samples were more common among males (15.6%; 70/449) than among females (4.6%; 12/259) (P < 0.001). In total, 621 samples from 376 males and 245 females were included in the analysis. Ages ranged from 15 to 69, with a mean of 28.4 years and median of 28 years. Approximately 90% of the samples were from patients between 20 and 40 years of age. The men were older than the women: mean age, 29.5 and 26.7 years, respectively. Age and sex of patients with regard to HPV detection and the occurrence of multiple infections are presented in Table 1.
Of 621 samples, 96.3% were positive for HPV DNA. Genital HPV types were found in 93.9% of the patients with 35 different HPV types from the alpha genus detected (Table 2). A single infection was detected in 62.3% of the samples, and more than 1 HPV type was detected in 31.6%, with up to 8 types in 2 samples (Table 3). On average, 1.5 HPV types were detected in each sample. Detection of multiple HPV types was more common among women (42.0%) than among men (24.7%) (odds ratio [OR], 2.0; 95% confidence interval [CI],1.4–2.9).
HPV Types Associated With a Low Risk for Cervical Cancer
Genital HPV types known to be associated with low risk for malignancy2 were identified in 76.3% of the condylomas. The low-risk HPV types detected were as follows: HPV types 6, 11, 40, 42, 43, 54, 70, and 81. Human papillomavirus type 6 was identified in 61.7%, HPV11 in 10.3%, and either of these HPV types in 71.0% of the condylomas (Table 2). Simultaneous detection of HPV6 and HPV11 was found in 0.01% (6 condylomas). Human papillomavirus type 6 or 11 were more common among condylomas in males (Table 4). Other HPV types associated with a low risk for malignancy were detected in 13.2% (82/621), but 60% (49/82) of these samples also contained HPV6 or HPV11. Overall, low-risk types were detected in 76.3% of the condylomas and were less common in women (71.8%) than in men (79.3%) (OR, 0.62; 95% CI, 0.42–0.93).
HPV Types Associated With a High Risk for Cervical Cancer
Overall, high-risk HPV types were detected in 34.9% of the samples. High-risk HPV types were distinctly more common among women (45.3%) than among men (28.2%) (OR, 1.9; 95% CI, 1.3–2.7) (Table 4). Samples with high-risk HPV types only were more common among women (17.6%) than among men (11.4%) (OR, 1.6; 95% CI, 1.0–2.7) (Table 4). Human papillomavirus type 16 was detected in 12.6% of the samples (Table 2). Human papillomavirus type 18 was identified in 4.8% of the patients and was more common among women (8.2%) than among men (2.7%) (OR, 2.7; 95% CI, 1.2–6.2) (Table 4). Single infection of HPV16 (31 cases) and of HPV18 (4 cases) and 1 double infection with HPV16/18 were found in 5.8% (36/621). Other high-risk HPV types (not including probable high-risk HPV types2) were found in 23.2% (144/621) of the samples. In 20.8% (30/144) of these, either HPV16 or HPV18 were also detected.
The following 15 high-risk types were detected: HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 (a and b), 73, and 82. Four probable high-risk types were also found (HPV types 26, 53, 66, and 67) (Table 2).
Additional HPV Types
The following HPV types with unknown risk were also detected: 7, 10, 30, 84, 86, 89, 90, and 91 (Table 2).
Of the 50 initially HPV DNA–negative samples, 12 did contain genital HPV types after extended analysis: HPV6 (7 samples) and single infection of HPV types 16, 39, 53, 89, and 90. Twenty-one patients had cutaneous HPV with single infection of HPV types 36, 80, 49, 75, 112, 134, and 153. Human papillomavirus type 153 (GenBank accession no. JN171845) is a novel type and was isolated from a condyloma in a 27-year-old man, belongs to the Gammapapillomavirus genus, and has a genome size of 7240 bas pairs. The closest nucleotide identity is with HPV128 (71.2% similarity in the L1 open reading frame). Thirteen putative HPV types in the beta and gamma genera were also detected (Table 2).
Our condyloma monitoring sentinel system has provided the largest, so far, reported condyloma series with comprehensive HPV typing. Notable findings include the following: (i) the large spectrum of HPV types detected, with 35 different genital HPV types being identified overall, implying that condyloma monitoring systems will require methods capable to detect and type a broad range of HPV types; (ii) the HPV types that are the dominating cause of condylomas (HPV6/11) were detected in most (71%) condylomas; (iii) high-risk HPV types were commonly found and were particularly common in condylomas of females; and (iv) in addition to genital HPV types, cutaneous types/FA isolates from the beta and gamma genera were found in a proportion of the cases (21/50). The significance of these cutaneous HPV types including the novel HPV153 is unknown, but they may represent HPV in superficial layers or HPV from healthy adjacent skin not necessarily present throughout the condylomas.
The diagnosis of condyloma is based on clinical examination. Verification by histopathology is rarely used, and some misclassification is therefore possible. However, estimates of the disease burden of condylomas have been instrumental for evaluating the cost-effectiveness of HPV vaccination programs.25 Such estimates are uniformly based on estimates obtained using clinical diagnosis. Therefore, the most accurate study design for determining the HPV type–specific disease burden is not to study specially selected and verified cases but to study the HPV types present in the same type of clinically diagnosed case series as used for the condyloma statistics that form the basis of the health economic evidence base for condyloma prevention. Morphologic appearance of the condylomas was registered only on a consecutive subset of 52 patients. We observed flat condylomas only in 4 cases (3 cases without concurrent acuminate condylomas). In 3 of the 4 flat condylomas, either HPV6 or HPV11 was detected. Thus, that the clinically diagnosed condylomas also included a minority of flat condylomas is not likely to have substantially affected the HPV type distribution.
We detected HPV6/11 in 71% of condylomas. This proportion is similar to the rates reported in other large-scale studies26,27 but lower than the estimate in most of the smaller studies3–10 (Table 5). One reason for the lower HPV6/11 rate could be that we, to avoid selection bias, used consecutive enrollment of patients with condyloma diagnosed at a primary patient contact center rather than more selective inclusion of “typical” patients with acuminate condyloma seen at special referral centers. There is also a difference in sampling methodology, with smaller studies typically using biopsies, whereas the large-scale studies have used cytobrush samples (Table 5). In addition, differences in diagnostics practices or real differences between populations are also conceivable explanations for our relatively low detection rate of HPV6/11 in condylomas.
Although the results of this study and of the other large condyloma series are at some variance with the smaller condyloma biopsy series, it should be noted that our results that HPV6/11/16/18 were present in 77% of condylomas are essentially in line with the results of primary HPV vaccination trials, where near-complete protection against HPV6/11/16/18 infection reduced the overall condyloma burden by 83%.14 A sentinel condyloma surveillance system without HPV typing has been used in Australia. After launching of an HPV vaccination program, the condyloma incidence was found to decrease by 59% among females in the target age group (11–26 years),17 the first ever report of population efficacy of HPV vaccination. In one Sexual Health Centre, genital warts declined by almost 90%.28
The proportion of high-risk HPV types detected (35%) is in agreement with other studies that found 33% to 48% high-risk HPVs in condylomas from nonimmunocomprimised patients.3,4,26,27 Interestingly, we found a significant proportion (6%) of condylomas that contained HPV16/18 without HPV6/11, which is similar to the 3% reported from a large French condyloma case series by Aubin et al.26 This suggests that also vaccination against only HPV16/18 could have some effect on condyloma incidence, albeit only quite. Our finding that high-risk HPV is more common among condylomas in females than in males (45% vs. 27%) is almost identical to the French series (41% vs. 25%)26 and to a multicenter study including 8 countries (47% vs. 35%).27 Conversely, low-risk HPV types were slightly more common among males (79% vs. 72%), a tendency also reported by Aubin et al. (94% vs. 86%).26 Possible explanations for these sex differences include differences in sexual behavior (a proportion of homosexual men were estimated to be <5% in our study), possible preferred virus tropism for cornified epithelium, or an effect of female sex hormones. Other possible reasons could be more efficient sampling by cytobrush and increased risk of contamination from other anogenital sites among women.
We also observed that 32% of the samples contained multiple HPV types, which is similar to that of other large studies (34%–40%) that used brush samples and PCR for HPV detection (Table 5). To determine which HPV type(s) that is truly causal among multiple HPV types in brush samples, we suggest that paired brush and biopsy samples should be analyzed for the presence of HPV DNA and messenger RNA, respectively. Our sampling method using a cytobrush is similar to the method used in the large French condyloma case series, which, in many respects, had results in line with our study.26 However, concerning sample adequacy, we excluded only samples that were negative for both β-globin and HPV, which may explain the relatively low frequency of nonadequate samples of 12% (4.7% females and 15.7% males) in our study. The French study classified all β-globin–negative brush samples as nonadequate (i.e., β-globin–negative and HPV-positive cases were excluded) and excluded 17% of the samples,26 and another study with the same criteria excluded 25% of brush samples from males.29 Although biopsies would probably be the most sensitive method for analysis of HPV DNA in condylomas, sampling using cytobrush is acceptable by most patients and realistic to use also in large-scale studies of condylomas.26,27 Organized HPV vaccination will commence in 2012 in Sweden. The vaccine has been commercially available, but few women older than 17 years of age have taken the vaccine. For the age group 20 to 40 years, which comprises approximately 90% of our data set, only approximately 0.5% of women have been vaccinated, and our data should therefore be considered as reflecting the HPV type distribution before the launch of the vaccination program.
Condyloma surveillance is an attractive option to monitor the efficacy of quadrivalent HPV vaccination programs because it is a readily identifiable clinical condition with short incubation time, providing rapid feedback of effectiveness of the programs. Condyloma surveillance with HPV typing is essential to obtain a thorough knowledge of the HPV type–specific disease burden before vaccination. This is needed to make estimations of the cost-effectiveness of vaccination and to provide a baseline to obtain clear answers on the control of specific HPV types after vaccination program launch. This report, which we believe to be the, so far, largest investigation of HPV types in condylomas, demonstrates the feasibility to include monitoring of HPV types in condylomas in a sentinel clinic-based reporting system.
1. Walboomers JM, Jacobs MV, Manos MM, et al.. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189: 12–19.
2. Munoz N, Bosch FX, de Sanjose S, et al.. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: 518–527.
3. Ball SL, Winder DM, Vaughan K, et al.. Analyses of human papillomavirus genotypes and viral loads in anogenital warts. J Med Virol 2011; 83: 1345–1350.
4. Brown DR, Schroeder JM, Bryan JT, et al.. Detection of multiple human papillomavirus types in condylomata acuminata lesions from otherwise healthy and immunosuppressed patients. J Clin Microbiol 1999; 37: 3316–3322.
5. Gissmann L, deVilliers EM, zur Hausen H. Analysis of human genital warts (condylomata acuminata) and other genital tumors for human papillomavirus type 6 DNA. Int J Cancer 1982; 29: 143–146.
6. Gissmann L, Wolnik L, Ikenberg H, et al.. Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci U S A 1983; 80: 560–563.
7. Gross G, Ikenberg H, Gissmann L, et al.. Papillomavirus infection of the anogenital region: Correlation between histology, clinical picture, and virus type. Proposal of a new nomenclature. J Invest Dermatol 1985; 85: 147–152.
8. Hagiwara M, Sasaki H, Matsuo K, et al.. Loop-mediated isothermal amplification method for detection of human papillomavirus type 6, 11, 16, and 18. J Med Virol 2007; 79: 605–615.
9. Lowhagen GB, Bolmstedt A, Ryd W, et al.. The prevalence of “high-risk” HPV types in penile condyloma–like lesions: Correlation between HPV type and morphology. Genitourin Med 1993; 69: 87–90.
10. Meyer T, Arndt R, Christophers E, et al.. Association of rare human papillomavirus types with genital premalignant and malignant lesions. J Infect Dis 1998; 178: 252–255.
11. Future II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007; 356: 1915–1927.
12. Paavonen J, Jenkins D, Bosch FX, et al.. Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: An interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007; 369: 2161–2170.
13. Pathirana D, Hillemanns P, Petry KU, et al.. Short version of the German evidence-based guidelines for prophylactic vaccination against HPV-associated neoplasia. Vaccine 2009; 27: 4551–4559.
14. Dillner J, Kjaer SK, Wheeler CM, et al.. Four year efficacy of prophylactic human papillomavirus quadrivalent vaccine against low grade cervical, vulvar, and vaginal intraepithelial neoplasia and anogenital warts: Randomised controlled trial. BMJ 2010; 341: c3493.
15. Winer RL, Kiviat NB, Hughes JP, et al.. Development and duration of human papillomavirus lesions, after initial infection. J Infect Dis 2005; 191: 731–738.
16. Fairley CK, Hocking JS, Gurrin LC, et al.. Rapid decline in presentations of genital warts after the implementation of a national quadrivalent human papillomavirus vaccination programme for young women. Sex Transm Infect 2009; 85: 499–502.
17. Donovan B, Franklin N, Guy R, et al.. Quadrivalent human papillomavirus vaccination and trends in genital warts in Australia: Analysis of national sentinel surveillance data. Lancet Infect Dis 2011; 11: 39–44.
18. Ferguson M, Wilkinson DE, Zhou T. WHO meeting on the standardization of HPV assays and the role of the WHO HPV Laboratory Network in supporting vaccine introduction held on 24–25 January 2008, Geneva, Switzerland. Vaccine 2009; 27: 337–347.
19. Fazel N, Wilczynski S, Lowe L, et al.. Clinical, histopathologic, and molecular aspects of cutaneous human papillomavirus infections. Dermatol Clin 1999; 17: 521–536, viii.
20. de Roda Husman AM, Walboomers JM, van den Brule AJ, et al.. 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.
21. Soderlund-Strand A, Carlson J, Dillner J. Modified general primer PCR system for sensitive detection of multiple types of oncogenic human papillomavirus. J Clin Microbiol 2009; 47: 541–546.
22. Schmitt M, Bravo IG, Snijders PJ, et al.. Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol 2006; 44: 504–512.
23. Forslund O, Antonsson A, Nordin P, et al.. A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin. J Gen Virol 1999; 80: 2437–2443.
24. Rector A, Tachezy R, Van Ranst M. A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification. J Virol 2004; 78: 4993–4998.
25. Jit M, Choi YH, Edmunds WJ. Economic evaluation of human papillomavirus vaccination in the United Kingdom. BMJ 2008; 337: a769.
26. Aubin F, Pretet JL, Jacquard AC, et al.. Human papillomavirus genotype distribution in external acuminata condylomata: A large French national study (EDiTH IV). Clin Infect Dis 2008; 47: 610–615.
27. Vandepapeliere P, Barrasso R, Meijer CJ, et al.. Randomized controlled trial of an adjuvanted human papillomavirus (HPV) type 6 L2E7 vaccine: Infection of external anogenital warts with multiple HPV types and failure of therapeutic vaccination. J Infect Dis 2005; 192: 2099–2107.
28. Read TR, Hocking JS, Chen MY, et al.. The near disappearance of genital warts in young women 4 years after commencing a national human papillomavirus (HPV) vaccination programme. Sex Transm Infect 2011; 87: 544–547.
29. Chan PK, Luk AC, Luk TN, et al.. Distribution of human papillomavirus types in anogenital warts of men. J Clin Virol 2009; 44: 111–114.
30. Greer CE, Wheeler CM, Ladner MB, et al.. Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts. J Clin Microbiol 1995; 33: 2058–2063.