Prevalence and genotyping of human papillomavirus infection in women with vulvodynia

Transcription

1 Acta Obstetricia et Gynecologica. 2007; 86: ORIGINAL ARTICLE Prevalence and genotyping of human papillomavirus infection in women with vulvodynia AUGUSTO ORLANDI 1, ARIANNA FRANCESCONI 1, CATERINA ANGELONI 1, GIAMPIERO PALMIERI 1, GLORIA FULVIA 2, MARCO CIOTTI 1, ANNA CRISCUOLO 3, FRANCESCO SESTI 3 & LUIGI GIUSTO SPAGNOLI 1 1 Anatomic Pathology Institute, Department of Biopathology and Image Diagnostics, 2 Division of Preventive and Social Pediatrics, and 3 Section of Obstetrics and Gynecology, Tor Vergata University of Rome, Rome, Italy Abstract Background. Evidence of vulvar human papillomavirus infection varies and the frequency of the different genotypes has not been adequately assessed. Methods. Fifty consecutive sexually active healthy patients with vulvodynia and suspected of human papillomavirus infection underwent a vulvoscopy and biopsy. Ten normal vulvar samples were also enrolled as control. Histological and vulvoscopic findings were compared in relation to human papillomavirus-dna presence and genotyping by a broad-spectrum polymerase chain reaction and reverse hybridization line probe assay. Results. Although the clinical and histological diagnoses did not always coincide, a good association was found (p B0.0001). Human papillomavirus-dna was detected in 42% of all biopsies and in none of the controls, and less frequently in acetowhitepositive patients (33.3%, p B0.03). Squamous papillomatosis (74%) was the most frequent histological diagnosis, followed by condyloma (20%). Condyloma (90%) but not squamous papillomatosis (29.7%) was significantly associated with human papillomavirus-dna presence. Out of the vulvoscopically normal patients, one (33%) was human papillomavirus-dna positive. Out of the recorded microscopic features, only koilocytosis was associated with human papillomavirus-dna presence. Eight different human papillomavirus genotypes were detected: high-risk 16 (43%), 31 (19%), 52 (14.3%), 68, and 59 (4.8% each), and low-risk types 6 (71.4%), 11, and 40 (4.8% each); 33.3% of infections were multiple, ranging from 2 to 4 genotypes. Out of the human papillomavirus-dna positive squamous papillomatosis, 72.7% showed a high-risk type but the infection remained episomal. Conclusions. Our data confirm human papillomavirus as a frequent cause of vulvodynia and its frequent association with squamous papillomatosis or condyloma. The high-risk human papillomavirus in squamous papillomatosis suggests screening for possible undiagnosed cervical infection. Key words: HPV genotype, squamous papillomatosis, vulvar condyloma, reverse hybridization Abbreviations: HPV: human papillomavirus, NE: normal epithelium, VP: vestibular papillomatosis, VC: vestibular condyloma, CG: control group, NT: normal tissue, SP: squamous papillomatosis, HC: condyloma, PCR: polymerase chain reaction, OR: odds ratio, CI: confidence interval The importance of human papillomavirus (HPV) infections is related to its capacity to cause genital infections, its transmissibility, and its role in the development of vulvocervical neoplasms, while recognizing the high-risk and low-risk carcinogenic types (1 6). Vulvar HPV infections classically referred to the presence of wart-like lesions (7), but there is now a general agreement that clinical and vulvoscopic evidence may vary (810). When clinically evident, chronic vulvar pain or vulvodynia may represent the only recognizable sign of HPV infection (1115). This complex clinical pathological view emphasizes how important confirming the presence and possibly defining the type of vulvar HPV infection are. Although the link between HPV infection and condyloma (HC) is widely accepted Correspondence: Augusto Orlandi, Anatomic Pathology, Department of Biopathology and Image Diagnostics, Tor Vergata University, Rome 00133, Italy. orlandi@uniroma2.it (Received 24 August 2006; accepted 3 April 2007) ISSN print/issn online # 2007 Taylor & Francis DOI: /

2 1004 A. Orlandi et al. (9,16), there is less agreement regarding the association of vulvar HPV infection with other lesions, including vulvodynia and papillomatosis (10,12,13, 17,18). In addition, the possible coexistence of these different clinical conditions in HPV-positive patients is debated (10,12,19). More than 80 HPV genotypes have been identified and about 40 can infect the genital tract (5). Nevertheless, most studies concerning the vulva and HPV infection derive from the examination of a limited number of HPV genotypes (2022). Recently developed methodologies allow an increased number of HPV genotypes to be routinely detected (5,23). In this study, we investigated 50 consecutive women undergoing clinical examination for vulvodynia lasting at least 3 months and suspected of HPV infection. Vulvoscopic and histological findings were compared using statistical criteria and correlated with the presence and genotyping of HPV-DNA detected by reverse hybridization line probe assay. Materials and methods Patients Fifty consecutive sexually active healthy women (mean age 33, range years) with marked vulvar pain or vulvodynia, with variable symptoms such as burning and pruritus, were considered. Vulvodynia was referred as provoked and lasted for at least 3 months. Vulvodynia was defined according to the recent published guideline of the International Society for the Study of Vulvovaginal Disease (14). Patients had no previous history of genital condylomas. Vulvovaginal microbic infections, including candida, group B streptococcus, Trichomonas, herpes, Gardnerella, and chlamydia, were excluded by performing cultural exams. Clinical investigation for inflammatory skin diseases such as lichen planus, cicatricial pemphigoid, or pemphigus vulgaris was also negative. Consequently, the patients were screened as suspected of HPV infection and directed biopsies obtained. Three percent acetic acid was applied for 5 min and a positive or negative reaction evaluated. Vulvoscopy was also performed and the women were assigned to one of the following groups: i.e. normal epithelium (NE), vestibular papillomatosis (VP), or vestibular condyloma (VC) (24,25). Histological examination Vulvar biopsies were immediately formalin-fixed for 24 h and embedded in paraffin. Intact vulvar tissue from the free edges of 10 consecutive surgical samples of benign neoplastic diseases, including seborrheic keratosis (n 4), fibroepithelial polyp (n 3), and lentigo simplex (n 3), was obtained from the paraffin block archive and enrolled as control group (CG; mean age 35 years, range 2262). Four-micrometer thin sections were stained with hematoxylin-eosin and examined by two experienced pathologists. HPV-related microscopic features such as inflammation, hyperkeratosis, koilocytosis, epithelial acanthosis, and basal hyperplasia (16,26) were also recorded. The presence of vulvar intraepithelial neoplasia (VIN) (27) was also excluded in all cases. The blinded histological diagnoses were normal tissue (NT), squamous papillomatosis (SP), and HC, according to the most widely accepted criteria (26,28). Interobserver variability was less than 5%. DNA extraction and b-globin amplification To isolate DNA from vulvar biopsies following histological examination, 5 10-mm thick serial paraffin tissue sections were cut and accurately collected in sterile 1.5-ml microtubes and dewaxed with two xylene washes followed by rehydration with a decreasing ethanol concentration addition. The DNA extraction was carried out using the commercial QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer s instructions. Recovered DNA was quantified using a spectrophotometer (Ultrospec 3000, Pharmacia). To ensure adequate DNA quality, polymerase chain reaction (PCR) of the b-globin gene was performed in a separate reaction using the GH20 and PC04 primers (29). PCR products (10 ml) were examined with electrophoresis on 2.5% agarose gel (Invitrogen, Carlsbad, CA, USA) and viewed under UV light following ethidium bromide staining. All PCRs were carried out using appropriate precautions to avoid cross-contamination with appropriate positive and negative controls. Human papillomavirus detection and typing Broad-spectrum HPV DNA amplification was performed using the short PCR fragment (SPF 10 ) primer set (Innogenetics, Belgium), containing a mixture of 10 sequences, and amplifying a 65-bp fragment from the L1 region of the HPV genome (23). Initial denaturation was at 958C (9 min) followed by 40 cycles of denaturation at 948C (30 min), annealing at 528C (45 min), and extension at 728C (45 min) with additional 7 min. PCR amplicons were analyzed by the Inno-line probe assay (LiPA; Innogenetics) using reverse hybridization (23). The assay simultaneously

3 HPV, vulvodynia, and vulvar lesions 1005 recognizes 24 individual HPV genotypes (high-risk HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and low-risk HPV6, 11, 40, 4244, 53, 54, 70, 74). Human papillomavirus physical status The physical status of HPV16 positive cases was studied using a multiplex PCR amplification of the E2 and E6 gene open reading frames (30). The primers for each sequence were 5?-CTTGGGCAC- CGAAGAAACAC-3? (nucleotides ) and 5?-TTGGTCACGTTGCCATTCAC-3? (nucleotides ) for E2 gene; 5?-AAGGGCGTAA- CCGAAATCGGT-3? (nucleotides 26 46) and 5?-CATATACCTCACGTCGCAG-3? (nucleotides ) for E6 gene. PCR products were electrophoresed, viewed as reported above, and quantized with an image scanner (Biorad Fluor-S MAX). The relative ratio of HPV E2 to E6 PCR products was calculated. The detection of equivalent E2 and E6 products was interpreted as an indicator of HPV- DNA in episomal form. Statistical analysis The Statistical Package for Social Science software package (SPSS Inc., Chicago, IL) was used. HPV association with acetowhitening of the vulva and aging, clinical, and histological classification, and HPV-related microscopic features were assessed comparing data with x 2 test. The odds ratio (OR) was estimated with a 95% confidence interval (CI); p values B0.05 were considered statistically significant. Results Clinical and pathological findings Examples of vulvoscopic and histological findings are reported in Figure 1. The vulvoscopy showed vestibular acetowhite changes in 78% of all cases, whereas the remaining ones did not. Vulvoscopic diagnosis was NE in 12% (mean age 42.3, range 2851 years), VP in 64% (mean age 31.3, range years), and VC in 24% of all cases (mean age 33.0, range 2350 years). A histological examination of the biopsies from the same patients revealed NT in 6% (mean age 33, range 2546 years), SP in 74% (mean age 34, range 2154 years), and HC in 20% of all cases (mean age 29.5, range years). Distributions according to clinical and histological criteria are summarized in Table I. Although the clinical and histological diagnoses did not coincide in all cases, a good association between the overall clinical and histological subtyping was found (x , p B0.0001). Human papillomavirus DNA detection Amplifiable DNA was isolated from all vulvar samples with a similar positive detection of the b- globin gene (not shown). HPV-DNA was detected in 42% of all biopsies and was absent in CG vulvar skin. HPV-DNA was present in 72.7% and 33.3% of negative and positive cases to acetic acid reaction, respectively, and this difference was significant (x , p B0.03). HPV-DNA detection frequency in different groups according to the clinical and pathological diagnosis is reported in Figure 2. HPV-positive cases included 90% of all HC, 29.7% of all SP, and 33.3% of NT. Statistical analysis revealed a significant association between the presence of HPV-DNA and the histological grouping (x , p B0.004), whereas no association between HPV detection and clinical definition was found (x 2 1.8, p0.4). The strong possibility of detecting HPV-DNA in HC was emphasized by the OR (21.27, CI , p B0.002). Correlation between human papillomavirus-dna detection and age To verify a possible correlation between age and HPV-prevalence, patients were divided into three subgroups: aged less than 30 (n24), 3040 (n13), and more than 40 years (n24). Although HPV prevalence was reduced with increased age, being 50% in the younger, 46.2% in the middle, and 23.1% in the older groups respectively, differences were not statistically significant. Correlation between human papillomavirus-dna detection and microscopic features We compared microscopic features with the presence of HPV-DNA. As reported in Figure 3, an association was found between HPV-DNA and koilocytosis (x , p B0.007) but not with inflammation, hyperkeratosis, or acanthosis. Conversely, an inverse relationship between HPV- DNA and basal hyperplasia presence was found (x , p B0.02). Human papillomavirus DNA genotyping As reported in Figure 4, we detected 8 different HPV genotypes, 3 of which were low-risk (6, 11, and 40) and 5 high-risk types (16, 31, 52, 59, and 68) (5).

4 1006 A. Orlandi et al. A B C D E F Figure 1. (A, C, E) Vulvoscopic and (B, D, F) histological findings in 50 consecutive patients with suspected HPV infection: (A) acetowhite change of vestibular epithelium after staining with 3% acetic acid for 5 min, (B) histological section demonstrating normal vestibular epithelium and a mild chronic inflammatory infiltrate of the underlying lamina propria (magnification; 200), (C) vulvoscopic appearance of diffuse vestibular papillomatosis, (D) histological features of squamous papillomatosis, with multiple thin papillae covered from hyperkeratotic epithelium, (magnification; 100), (E) vulvoscopic and (F) histological appearance of condyloma with frond-like hyperkeratotic epithelium (magnification, 100). HPV6 was the most frequent, followed by 16, 31, and 52 genotypes. Low-risk HPV types prevailed in HC (p B0.01); the frequency of the high-risk HPV types in SP (72.7%) was higher than what was observed in HC (22.2%, p B0.01). Nevertheless, multiplex E2/E6 PCR showed that in these cases HPV-DNA remained in the episomal form (data not shown).

5 HPV, vulvodynia, and vulvar lesions 1007 Table I. Correlation between vulvoscopic (NE, VP, VC) and histological diagnosis (NT, SP, HC) in a group of 50 patients with vulvodynia and suspected for HPV infection. Histological diagnosis NT SP HC Total Vulvoscopic diagnosis NE VP VC Total NE, vulvoscopic normal epithelium; HC, condyloma by histological examination. Single and multiple human papillomavirus infections In Table II, single/multiple and low/high-risk HPV infections in different groups are listed. Single and multiple genotype infections were detected in 61.9 and 33% of HPV-DNA-positive cases, respectively. Multiple infections ranged from 2 to 4 genotypes (242.9%, %, and 4 genotypes 14.2%). Reverse hybridization prevented genotyping in 1 case (HPVX). Out of all multiple infections, multiple low-risk and high-risk was observed in 1 case of HC and SP each, and mixed low/high-risk genotypes in the remaining cases. In the SP group, multiple low/high-risk HPV-DNA genotype infections were the most frequent (45.5%), whereas in HC, single low-risk infections prevailed (66.7%). Figure 3. Bar graph showing the different percentages of microscopic features in HPV-negative and -positive groups; a positive association of HPV infection is found only with koilocytosis. Discussion Our results show that in a series of 50 consecutive patients with provoked vulvodynia negative for microbic infections and inflammatory vulvar skin diseases, viral DNA was present in 42% of all the biopsies and none in the control vulvar skin. Our data confirm HPV as a relevant causal factor of vulvodynia (11 13,15,17,18). HPV-DNA detection was significantly associated with absence of whitening change of the vestibular epithelium after 3% acid acetic staining (10,31). Although a good correlation was found between the overall clinical and microscopic classification of the vulvar lesions, the histological diagnosis of HC was the only one significantly Figure 2. Frequency of HPV-DNA detection in clinical and histological groups; (NE) negative, (VP) vestibular papillomatosis, (VC) vestibular condyloma, (NT) normal tissue, (SP) squamous papillomatosis and (HC) condyloma. Figure 4. HPV genotypes detected by reverse hybridization line probe assay in different groups with histological diagnosis of (NT) normal tissue, (SP) squamous papillomatosis, and (HC) condyloma; HPVX, HPV-DNA positive detection with SPF 10 -PCR but not enclosed among 24 tested genotypes (see Materials and Methods).

6 1008 A. Orlandi et al. Table II. Single and multiple HPV infections detected by reverse hybridization line probe assay. HPV-DNA was checked in vulvar biopsies from 50 patients with vulvodynia and suspected for HPV infection, grouped according to histological diagnosis. HPV Single Single Multiple Multiple Multiple low/ Cases presence low-risk high-risk low-risk high-risk high-risk HPVX n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Normal tissue 3 (6) 1 (33.3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (100) Squamous papillomatosis 37 (74) 11 (29.7) 3 (27.3) 3 (27.3) 0 (0) 1 (9) 4 (36.4) 0 (0) Condyloma 10 (20) 9 (90.0) 6 (66.7) 1 (11.1) 1 (11.1) 0 (0) 1 (11.1) 0 (0) HPVX, HPV-DNA-positive detection with SPF 10 -PCR but not enclosed among 24 tested genotypes (see Materials and Methods). associated with HPV-DNA, in particular with a single low-risk HPV genotype infection. This coincides with what was previously reported (9,26). In fact, contradictory opinions characterize the capacity of vulvoscopy to discriminate among vulvar lesions (24,32). In addition, the histological diagnosis of SP and HC was associated to the presence of vulvar symptoms in 74% and 20% of all cases, respectively. The term papillomatosis is used clinically to describe the presence of multiple vestibular papillae, which may cover the entire surface of the labia minora (19). Unlike isolated papillae, SP has also been described in patients with vulvodynia and sometimes along with HPV infection (10,12,33,34). Unlike HC, the association between HPV infection and papillomatosis is still questioned (10,35,36). We found that 29.7% of SP was HPV-positive. Although this low frequency does not support a causal relationship (18), we cannot exclude that transient HPV infections are responsible for the clinical outset of vulvar SP. This is also indirectly supported by the clinical documentation of frequent spontaneous regression (10,19). In addition, the variability in the reported percentages of HPV-DNA detection in SP (10,18 20) is likely due, at least partly, to the criteria in selecting patients and to methodological differences. DNA amplification by PCR is considered a sensitive method (37,38), but single PCR assays are not easily performed because of an increasing number of HPV genotypes (5). The PCR technique performed using the SPF 10 primer set improves HPV detection sensitivity in DNA extracted from formalin-fixed, paraffin-embedded tissue by amplifying a 65-bp fragment (23), an amplification product shorter than that obtained with MY09/11 or GP5/6 primer sets (37,38). The presence of vulvodynia (14,15) is a common condition. A recent prevalence study indicates that 15.7% of routine patients suffered from chronic vulvar discomfort (39). The terminology and classification of vulvodynia continue to evolve, and much remains to be understood about the prevalence, pathogenesis, natural history, and management of this condition (40). Some authors subdivide vulvodynia according to its clinical presentation (15,41). Actually, vulvodynia recognizes different causal agents (14,15). Although in a limited series, we observed HPV-DNA in 42% of all cases, supporting viral infection among possible etiopathogenetic agents of vulvodynia. The frequent association with HC or papillomatosis, in accordance with other authors (10,12,19,33), strengthens the causal role of HPV infection in vulvodynia. Concerning HPV genotyping, unlike HC, a high frequency of high-risk types was observed among the HPV-positive SP. In the latter, our results also indicated the episomal status of high-risk HPV- DNA, excluding a possible link with vulvar cancer progression (10). Nevertheless, the vestibular presence of high-risk HPV infections suggests the need of screening for a possible co-infection of the cervix (19). In fact, we documented a cervical co-infection in all patients with high-risk HPV-positive SP, sometimes associated with squamous intraepithelial lesions (Orlandi et al., unpublished results). In an attempt to narrow diagnostic criteria, we also tried to identify which morphological features are more frequently associated with HPV-DNA presence. We found a positive association only between HPV-DNA and koilocytosis. The diagnostic criteria of koilocytosis as a marker of HPV infection are questioned (18). Difficulties in the morphological diagnosis of koilocytosis derive from the young age of patients and glycogen production, which results in cytoplasm clearing (18,26). Our results coincide with other reported frequencies of koilocytosis and HPV-DNA association (10,19). In conclusion, we documented that HPV-DNA is present in 42% of all biopsies obtained from patients with vulvodynia. Among HPV-DNA-positive cases, SP and HC were diagnosed in 29.7 and 90%, respectively. Histological diagnosis was more predictive of HPV-DNA infection than vulvoscopy. Although not causal, the finding of high-risk HPV genotypes in SP suggests screening for cervical coinfection.

7 HPV, vulvodynia, and vulvar lesions 1009 Acknowledgements We thank Prof. B. Czernobilsky (Ness Ziona, Israel) for his advice during the preparation of the manuscript. We also thank S. Cappelli, A. Colantoni, and L. Santangelo for their technical work. References 1. Munoz N, Bosch FX, de Sanjose S, Tafur L, Izarzugaza I, Gili M, et al. The causal link between human papillomavirus and invasive cervical cancer: a population-based case-control study in Colombia and Spain. Int J Cancer. 1992;/52:/ van Beurden M, ten Kate FJ, Smits HL, Berkhout RJ, de Craen AJ, van der Vange N, et al. Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus. Cancer. 1995;/75:/ Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol. 1999;/189:/ zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer. 2002;/2: / Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med. 2003;/348:/ Hillemanns P, Wang X. Integration of HPV-16 and HPV-18 DNA in vulvar intraepithelial neoplasia. Gynecol Oncol. 2006;/100:/ Bafverstedt B. Condylomata acuminata past and present. Acta Derm Venereol. 1967;/47:/ Boden E, Eriksson A, Rylander E, von Schoultz B. Clinical characteristics of papillomavirus-vulvovaginitis. A new entity with oncogenic potential. Acta Obstet Gynecol Scand. 1988;/ 67:/ Dias EP, Gouvea AL, Eyer CC. Condyloma acuminatum: its histopathological pattern. Sao Paulo Med J. 1997;/115:/ Costa S, Rotola A, Terzano P, Secchiero P, Di Luca D, Poggi MG, et al. Is vestibular papillomatosis associated with human papillomavirus? J Med Virol. 1991;/35:/ di Paola GR, Rueda NG. Deceptive vulvar papillomavirus infection. A possible explanation for certain cases of vulvodynia. J Reprod Med. 1986;/31:/ Turner ML, Marinoff SC. Association of human papillomavirus with vulvodynia and the vulvar vestibulitis syndrome. J Reprod Med. 1988;/33:/ McKay M, Frankman O, Horowitz BJ, Lecart C, Micheletti L, Ridley CM, et al. Vulvar vestibulitis and vestibular papillomatosis. Report of the ISSVD Committee on Vulvodynia. J Reprod Med. 1991;/36:/ Haefner HK, Collins ME, Davis GD, Edwards L, Foster DC, Hartmann ED, et al. The vulvodynia guideline. J Low Genit Tract Dis. 2005;/9:/ Fischer G. Management of vulvar pain. Dermatol Ther. 2004;/17:/ Kurman R. Benign disease of the vulva. In: Kurman RJ, editor. Blaustein s Pathology of the Female Genital Tract. New York: Springer; p Growdon WA, Fu YS, Lebherz TB, Rapkin A, Mason GD, Parks G. Pruritic vulvar squamous papillomatosis: evidence for human papillomavirus etiology. Obstet Gynecol. 1985;/66:/ Origoni M, Rossi M, Ferrari D, Lillo F, Ferrari AG. Human papillomavirus with co-existing vulvar vestibulitis syndrome and vestibular papillomatosis. Int J Gynaecol Obstet. 1999;/ 64:/ Welch JM, Nayagam M, Parry G, Das R, Campbell M, Whatley J, et al. What is vestibular papillomatosis? A study of its prevalence, aetiology and natural history. Br J Obstet Gynaecol. 1993;/100:/ Bergeron C, Moyal-Barracco M, Pelisse M, Lewin P. Vulvar vestibulitis. Lack of evidence for a human papillomavirus etiology. J Reprod Med. 1994;/39:/ Stellato G, Paavonen J. Vulvar and vaginal colposcopic pictures of human papillomavirus infection. Eur J Gynaecol Oncol. 1995;/16:/ Gentile G, Formelli G, Pelusi G, Flamigni C. Is vestibular micropapillomatosis associated with human papillomavirus infection? Eur J Gynaecol Oncol. 1997;/18:/ Kleter B, van Doorn LJ, Schrauwen L, Molijn A, Sastrowijoto S, ter Schegget J, et al. Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J Clin Microbiol. 1999;/37:/ Reid R, Greenberg MD, Daoud Y, Husain M, Selvaggi S, Wilkinson E. Colposcopic findings in women with vulvar pain syndromes. A preliminary report. J Reprod Med. 1988;/33:/ Byrne MA, Walker MM, Leonard J, Pryce D, Taylor- Robinson D. Recognising covert disease in women with chronic vulval symptoms attending an STD clinic: value of detailed examination including colposcopy. Genitourin Med. 1989;/65:/ Fu YS. Infectious and inflammatory diseases of the lower female genital tract. In: Livolsi VA, editor. Pathology of the Uterine Cervix, Vagina and Vulva. Philadelphia: Saunders (Elsevier Science); p Scurry J, Wilkinson EJ. Review of terminology of precursors of vulvar squamous cell carcinoma. J Low Genit Tract Dis. 2006;/10:/ Kurman R, Wilkinson E. Tumors of the vulva. In: Rosai MJ, editor. Tumors of the Cervix, Vagina, and Vulva. Washington, DC: Armed Forces Institute of Pathology; p Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985;/230:/ Yoshinouchi M, Hongo A, Nakamura K, Kodama J, Itoh S, Sakai H, et al. Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers. J Clin Microbiol. 1999;/37:/ Pao CC, Hor JJ, Fu YL. Genital human papillomavirus infections in young women with vulvar and vestibular papillomatosis. Eur J Clin Microbiol Infect Dis. 1994;/13:/ van Beurden M, van der Vange N, de Craen AJ, Tjong AHSP, ten Kate FJ, ter Schegget J, et al. Normal findings in vulvar examination and vulvoscopy. Br J Obstet Gynaecol. 1997;/ 104:/ Wang AC, Hsu JJ, Hsueh S, Sun CF, Tsao KC. Evidence of human papillomavirus deoxyribonucleic acid in vulvar squamous papillomatosis. Int J Gynecol Pathol. 1991;/10:/ Boden E, Rylander E, Evander M, Wadell G, von Schoultz B. Papilloma virus infection of the vulva. Acta Obstet Gynecol Scand. 1989;/68:/17984.

8 1010 A. Orlandi et al. 35. Morin C, Bouchard C, Brisson J, Fortier M, Blanchette C, Meisels A. Human papillomaviruses and vulvar vestibulitis. Obstet Gynecol. 2000;/95:/ de Deus JM, Focchi J, Stavale JN, de Lima GR. Histologic and biomolecular aspects of papillomatosis of the vulvar vestibule in relation to human papillomavirus. Obstet Gynecol. 1995;/86:/ Manos MM, Ting Y, Wright DK, Lewis AJ, Broker TR, Wolinsky SM. The use of polymerase chain reaction amplification for the detection of anogenital human papillomaviruses. Cancer Cells. 1989;/7:/ de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. 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:/ Harlow BL, Stewart EG. A population-based assessment of chronic unexplained vulvar pain: have we underestimated the prevalence of vulvodynia? J Am Med Womens Assoc. 2003;/ 58:/ Lotery HE, McClure N, Galask RP. Vulvodynia. Lancet. 2004;/363:/ Edwards L. New concepts in vulvodynia. Am J Obstet Gynecol. 2003;/189:/S2430.