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ISSN 0956-4624 Volume 3 1992 international. TD AID f l1L S jjournal -~oS • For all clinicians with an interest in HIV, AIDS and the more "traditionaP' sexually transmissible diseases (STD) • In-depth editorial reviews provide a regular update • Special emphasis placed on papers dealing with the clinical aspects of HIV, AIDS and STD • AIDS Literature Index in each issue provides an up-to-date source listing of papers on AIDS and HIV published worldwide ished by Royal Society of Medicine Services Limited

Internatio fal TD & AID S o Journal S EDITOR W W Dinsmore (U) ASSOCIATE EDITORS J R W Harris (UKJ E Panconesi (/ta/y) A Lassus (Fin/and~ M A Waugh (LIA2 EDITORIAL BOARD D I Abrams (USA) S Adam (UKJ K Behbehani (/(uwait~ J S Bingham (I) A G Bird (LIA2 S Bygdeman (SwedenJ B Donovan (Austra/ia) D Freedman (Eire) B Gazzard (U) S K Hira (Zambia~ K King Holmes (Switzer/and~ D J Jeffries (U) J Jordan (LIA2 F N Judson (USA) G Kinghorn (LIA2 H C Korting (Geirnanj1) A Lewis (NewZea/andJ Low Bin Tick (Ma/aysia~ A Luger (Austricq) D McCance (USA) A McMillan (LIA2 J K Maniar (/ndia) J Mann (ZSA) RDMaw (U) A Meheus (Switzer/and~ F Mulcahy (Eire) A P Oranje (Nether/ands) J D Oriel (II) M E M Paalman (1vethe1/a17ds) C C Patterson (I) D Petzoldt (Getmanv) C R Philpot (Austra/ia) A J Pinching (I/A) T C Quinn (USA) G L Ridgway (Z) B Romanowski (CanadaJ A Stary (Austria) E Stolz (Nether/ands) J Stratigos (Greece) D Taylor Robinson (U) R N Thin (U) T Thiru Moorthy (Singapore) J N Weber (U) F Zacarias (USA) SUBSCRIPTION INFORMATION The International Journal of STD & AIDS is published bi-monthly by Royal Society of Medicine Services Limited, London, UK. Annual subscription rates for Volume 3, 1992 (six issues), including postage, are as follows: /nstitution.• £90.00/US$162.00. /ndividua/.• £65.00/US$120.00. Subscriptions are accepted only for the complete volume. Orders should be sent with payment to Royal Society of Medicine Services Limited, Publications Subscription Department, 'I Wimpole Street, London W1M 8AE, UK (Tel: 071-408 2119 ext 292; Fax: 071-355 3198). SOME RECENT PAPERS -9 Editorial Reviews Zidovudine in asymptomatic HIV infection: knowledge and uncertainty. A JPinching (UKJ Pelvic inflammatory disease: current approaches and ideas. JHare (UKJ The investigation of patients with HIV infection. CA Bowman, CJNLacey (L/KJ Condylomata acuminata in children. APOranje, FBde waard-van derSpek, VD Vuzevski fIA B Bi/o (Netherlands) Neurological manifestations of HIV infection. DRowen, CA Carne (U/4 Azithromycin in sexually transmitted diseases - an overview. MA Waugh (U/q Human papillomaviruses and genital neoplasia: the changing scene. JD Orie/ (ZIKJ HIV and its relationship to women. CSBradbeer(Ulq Sexual behaviour, AIDS and poverty in sub-Saharan Africa. A Prua/, S Chacko, DKoch-weser (N/gei & USA) Tuberculosis in HIV infection. PJHomer, FMMoss (ZA2 Clinical applications of the polymerase chain reaction. PA Ka'chin (Z//q Acquired immunodeficiency syndrome and the Epstein-Barr virus. fyKWLau (ZA2 Original Articles Evidence of HTLV-I infection in Singapore prostitutes. EHSing, TTh/rumoodhy, ALevin, SA/exander, /Sng, WB/atMei(Singapore, UK& USA) HIV-1 infection in HIV-1 enzyme-linked immunoassay seronegative patients in Kinshasa, Zaire. ACo%bunders, HFrancis, A•f-MDuma eta/. (Zaire) Anaerobic vaginosis: study of male sexual partners. J TArumainayagam, Yde Si/va, MShahmanesh (Z) Screening colposcopy in genitourinary medicine. PMNathan, TfI Moss (UKJ Management of intrameatal warts in men. JGMcKenna, AMcMi//an IUKI Rape and HIV. EC/aydon, SMurphy, EMOsbome, VKdchen, J H S m i t/ i, J R W Ha iri s(U/ Q A 2-year quantitative assessment of Ch/ainydia trachomatis in a sexually transmitted diseases clinic population by the MicroTrak direct smear immunofluorescence test. BJThomas, MFOsborn, PEMunday, H TEvans, D Tay/or-Hobinson (UKJ Sexually transmitted diseases in lesbians. AEoYvards, flNThin (UKf Prevalence of bacterial vaginosis and its association with genital infections, inflammation, and contraceptive methods in women attending sexually transmitted disease and primary health clinics. HMoi (Swede17) Evaluation of the significance of Mycop/asma hominis and Ureap/asma ureao/cum in female genital tract infection - a retrospective case study. 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. ~ International Journal of STD & AIDS 1992; 3: 338-346 ORIGINAL ARTICLE Detection of human papillomavirus infections in the male sexual partners of women attending an STD clinic in Bologna Silvano Costa MD1, Stina Syrjanen 2, Calogero Vendral, Fuju Chang2, Gerardo Guidal, Arja Tervahauta2, Marita Hippelainen3 and Kari Syrjanen2-4 111 Department of Obstetrics and Gynecology, University of Bologna, Italy; 2Department of Pathology, University of Kuopio; 3Department of Gynecology & Obstetrics, University of Kuopio; 4Kuopio Cancer Research Centre, University of Kuopio, Finland Summary: A series of 65 male sexual partners of 65 women attending an STD clinic in Bologna, Italy for examination and treatment of genital human papillomavirus (HPV)-infections during 1990-1991, were examined using peniscopy and surgical biopsy, the latter being analysed by light microscopy, in situ hybridization'(ISH) and polymerase chain reaction (PCR) for HPV DNA. A detailed medical and sexual history was recorded from all men. Of the 65 men, 17 (26.2%) gave a history of a previous STD. The male partners with previous genital condylomata (14, 21.5% of men) were significantly associated with the detection of HPV DNA in the current lesions; 21.4% (3 of 14) and 10.2% (5 of 51) in those with and without previously treated condyloma, respectively. On colposcopy, 63 (96.9%) men presented with an abnormal pattern, the vast majority (49 of 65, 75.4%) showing an acetowhite lesion, and only 12 (18.5%) lesions being classified as condyloma acuminatum. HPV DNA was found, however, in only 4 of 12 (33.3%) condylomas by ISH and PCR, and in 4 of 49 (8.2%) and 6 of 49 (12.2%) acetowhite lesions by ISH and PCR, respectively. In a total of 41 (63%) patients, the biopsy was classified as non-HPV on light microscopy. HPV DNA detection rate was significantly higher in all morphologically HPV-suggestive lesions, compared with the non-HPV where ISH was invariably negative. PCR, however, disclosed HPV DNA in 4 of 41 (9.8%) cases. PIN (I or II) was present in 6 of 65 (9.2%) men. HPV DNA detection rate increased in parallel with the increasing grade of lesion, both HPV 16-positive cases containing a PIN lesion. Altogether, HPV DNA was found by ISH in 8 of 65 (12.3%) biopsies, and PCR amplification increased the detection rate by only two cases. HPV DNA was never present in men with only a single sexual partner, but increased significantly when the number of partners was increased, being highest (27.3%, 3 of 11) in those reporting 11-20 partners. HPV detection rate was lowest in those men whose partner had a flat condyloma, but significantly higher in those who presented with condyloma acuminatum (40%, 2 of 5), or HPV-CIN I and II lesions. Of interest was the finding that HPV DNA was never demonstrated in the men whose partner had only vaginal HPV lesions. Peniscopy is an applicable means of finding the abnormal patterns remaining undetectable by the naked eye, but because of its limited resolution, it is not a conclusive diagnostic tool capable of differentiating HPV- from non-HPV-lesions. Keywords: Male sexual partners, HPV infection, peniscopy, PIN, light microscopy INTRODUCTION exophytic condylomas) in the genital tract with precancer lesions (CIN, VIN, VAIN, PIN, AIN) and Current data strongly associates human papillo- invasive squamous cell carcinomas1-5. Almost 70 mavirus (HPV) lesions (ie, flat, inverted and different HPV types have been recognized during the past 10 years6, and a significant risk for the Correspondence to: Professor Kari Syrjanen, Department of development of an invasive cancer has been Pathology, University of Kuopio, POB 1627, SF-70211 Kuopio, ascribed to infections of the high risk HPV typesz-5. Finland Of special importance is the mode of transmission V 4

of this virus by sexual contact, thus conferring a potential risk for the development of genital precancer (and eventually cancer) lesions to both sexual partners7-1z. Before appropriate prevention can be attempted, it is essential to identify the reservoirs of the virus2-4.7-z°. During the past few years, several reports have been published, in which HPV DNA has been disclosed by different hybridization techniques and PCR in normal squamous epithelium of the genital tract in both sexes, suggesting that these sites of latent HPV infections might act as such a reservoir13.14.z1-25. The role of male sexual partners in transmission of HPV infections is still highly controversial4•19.26,27This is partly because of our limited understanding of the true prevalence of genital HPV infections in men. Although the prevalence of clinically manifest infections (detectable by PAP smear) in a non- selected female population has been established as 2-3%18-20, and the estimated life-time risk at least one infection by HPV has been calculated to approach 80%18, no such figures are available for men28. It has been estimated that the prevalence of genital HPV lesions in asymptomatic sexually active men is probably no higher than 10% in the general population26.27, whereas the rate of infected male partners of women with overt condyloma or abnormal PAP smears is suspected to be considerably higher, figures from 65% to 85% having been reported7- 34. However, substantially lower figures (between 10% and 40%) have been found more recently in the male sexual partners of HPV-infected women16•35-37 To clarify some of these controversies, the present study was conducted to assess the prevalence of genital HPV infections in a high risk male population, i.e., in the current male sexual partners of women attending an STD clinic in Bologna, and reporting a high number of female partners as well. The techniques used included peniscopy, punch biopsy, in situ hybridization (ISH) and PCR. Despite the use of these highly sensitive techniques, the detection rate of HPV infections remained sur- prisingly low in this series. MATERIALS AND METHODS Patients A series of 65 male sexual partners of 65 women attending an STD clinic in Bologna for their genital HPV infections were invited to take part in the present study. They were examined at the 2nd Department of Gynecology & Obstetrics, University of Bologna between February 1990 and January 1991 using peniscopy and surgical biopsy. A personal record was filled out and the questions included: socio-economic status, age, age at first intercourse, duration of sexual intercourse (at least six months to be included in the study), number of partners in total and in the last year, number of episodes of Costa et al. HPV infection in male sexual partners 339 sexual intercourse a week, symptoms, drug or alcohol addiction, cigarette smoking, immuno- suppression as well as previous diagnosis and treat- ment for condyloma and other venereal infections. Couples using barrier contraceptive methods (con- dom, diaphragm), were not included in the study. Peniscopy Examination of the male genitalia was performed with the patient in a lithotomy position on a standard gynaecology table. The external genitalia were inspected grossly for evidence of visible lesions, followed by soaking for 5 min in 5% acetic acid. The urethral meatus and the entire external surface of the penis, scrotum, and anus were systematically inspected by a colposcope with a magnification of x 7-12. Small surface irregularities, colour differences and changes in vascularization can be visualized by this magnification. Additional acetic acid was applied during the examination by covering the external genitalia with cotton gauze pads soaked in acetic acid. The distal urethra was inspected by a paediatric nasal speculum. If anal condylomata were present, the anal canal was inspected by a proctoscope. A small surgical biopsy was obtained from one or two representative lesions after subepidermal injection of 1% lidocaine without epinephrine, using a tuberculin syringe and a 26-gauge needle. Topical lidocaine jelly was often sufficient for taking a biopsy of the urethral lesions. Cultures were taken from the urethra for chlamydia. The peniscopic abnormalities were classified into one of the following categories: (1) exophytic condyloma acuminata, (2) acetowhite lesion or (3) papular lesions35. Histopathology The biopsies were fixed in 10% neutral formalin, embedded in paraffin and processed for light microscopy according to standard procedures. All biopsies were evaluated on light microscopy by a single pathologist (KS), who was not aware of the colposcopical findings at that stage of the study. The histological criteria used to classify the lesions into papillary, flat or endoph tic condylomas have been previously described2•3.1~20. In addition, papulosis, pigmented papulosis and Bowenoid papulosis were recorded using the criteria outlined previously3s,39 Koilocytosis and superficial cell parakeratosis were the two most reliable signs of HPV. The lesion was graded HPV-suggestive if acanthosis or hyper- keratosis were present. In all the above lesions, the grade of concomitant penile intraepithelial neoplasia (PIN) was graded as PIN I, II or III, according to criteria in common use2. HPV-DNA in situ hybridization (ISH) In situ DNA hybridization on formalin-fixed, paraffin-embedded tissues using biotin-labelled

340 International )ournal of STD & AIDS Volume 3 September/October 1992 Table 1. Characteristics of 65 male patients and their current female sexual partner Age (mean±sD) 31.6±7.6 Number of sexual partners (mean+_so) 17.5 +_ 23.0 Previously treated for genital HPV 14 (21.5%) Smoker 26 (40.0%) Previously treated for STD: Chlamydia trachomatis 3 (4.6%) Candida albicans 9 (13.8%) Neisseria gonorrhoeae 2 (3.1%) HSV 1 (1.5%) Trichomonas vaginalis and condyloma 2 (3.1%) Clinical or colposcopic findings in female partner: Flat condyloma 44 (67.7%) Condyloma acuminatum 5 (7.7%) HPV-CIN I 5 (7.7%) HPV-CIN II 1 (1.5%) HPV-CIN III 4 (6.2%) Flat condyloma & condyloma acuminatum 2 (3.1%) HPV-CIN I & flat condyloma 1 (1.5%) No data 3 (4.6%) Site of lesions in female partner: Cervix 29 (44.6%) Vagina 5(7.7%) Vulva 17 (26.2%) Cervix and vulva 11 (16.9%) No data 3(4.6%) Table 2. Peniscopic pattern related to detection whole ~enomic HPV DNA probes was per- formed 41. Briefly, 4 µm sections were cut from each biopsy and mounted on microscopic slides pre- treated with 1% gamma-amino propyl-triethoxy- silane (Sigma, St. Louis, MO, USA). The specimens were hybridized in a mixture of 50% formamide, 10% dextran sulfate, 2 x SSC, 0.4 mg/mi salmon sperm DNA and biotinylated probe 1.0 µg/ml (HPV 6/11, 16, 18, 31/33) for 16 h at high stringency conditions (Tm-17). Following the hybridization, the slides were washed with 2 x SSC for 20 min at room temperature, 0.2 x SSC at 60°C, followed by 2 x SSC for 5 min at room temperature. Specimens were incubated with streptavidin-alkaline phosphatase complex and successively developed with nitroblue tetrazolium and bromo-chloro-indoxyl phosphate. Polymerase chain reaction 11LC VJ I.QDCD Vl 1V111L0.111L-11ACU, 1Ja10.1111L-C11LVClAUCIA biopsies were further analysed by the PCR 42. One or more 7 µm sections with an average surface area > 1 cmz were cut from each block. For PCR, 15 µl of supernatant was used. The primers were the consensus primers from L1 (MY 11 and MY09) described by Manos et al.43. A total of 40 cycles of of HPV DNA by ISH and PCR Peniscopic pattern No. of cases HPV type present 6/11 16 18 Acetowhite 49 0 2 0 Condyloma acuminatum 12 3 0 0 Papules 1 0 0 0 Acetowhite and condyloma 1 0 0 0 No data 2 0 0 0 Total series 65 3 2 0 31 33 42 Table 3. Lesion site related to detection of HPV DNA by ISH and PCR Site of lesion No. of cases HPV type present 6/11 16 18 31 Preputium 50 1 1 0 0 Glans 1 0 0 0 0 Shaft 6 2 0 0 0 Preputium and glans 5 0 1 0 0 Preputium and anus 1 0 0 0 0 No data 2 0 0 0 0 Total series 65 3 2 0 0 33 42 HPV DNA in PCR Negative Positive 43(87.8%) 6(12.2%) 8(66.7%) 4(33.3%) 1(100%) 0 1(100%) 0 2(100%) 0 55(84.6%) 10(15.4%) HPV DNA in PCR Negative Positive 44(88.0%) 6(12.0%) 1(100%) 0 3(50.0%) 3(50.0%) 4(80.0%) 1(20.0%) 1(100%) 0 2(100%) 0 55(84.6%) 10(15.4%) Table 4. Lesion morphology related to detection of HPV DNA by ISH and PCR Lesion morphology No. of cases HPV type present HPV DNA in PCR 6/11 16 18 31 33 42 Negative Positive Non-HPV 41 0 0 0 0 0 0 37(90.2%) 4(9.8%) Papillary condyloma 1 1 0 0 0 0 0 1(100%) 0 Flat condyloma 14 1 1 0 0 0 0 11(78.6%) 3(21.4%) Pigmented papulosis 8 1 0 0 0 0 3 6(75.0%) 2(25.0%) Bowenoid papulosis 1 0 1 0 0 0 0 0 1(100%) Total series 65 3 2 0 0 0 3 55(84.6%) 10(15.4%) R 0 ~ w

Costa et al. HPV infection in male sexual partners amplification of HPV target sequences were carried out in 50 µl of the reaction mixture with the Gene Ampli Taq kit and the Perkin Elmer-Cetus automatic thermal cyder (Perkin Elmer-Cetus, Emeryville, CA, USA). After initial denaturation, each cycle involved heating at 95°C for 30 s (DNA denaturation), followed by cooling at 55°C for 30 S (primer- annealing), and finally heating at 72°C for 1 min (chain elongation). Ten microlitres of the amplified product was electrophoresed in a 3% NuSieve agarose gell, and the DNAs were visualized in the gel by ethidium bromide staining. The specific HPV DNA sequences amplified by the PCR were subsequently confirmed by Southern blot hybridi- zation and dot blot hybridization with 32P-labelled specific HPV probes as described earlier44. RESULTS The characteristics and clinical data of the 65 male patients and their current female sexual partners are given in Table 1. The age of the 65 males examined ranged from 20 to 59 years (median 30 years). Altogether 17 (25.4%) gave a history of a previous STD. Prior to enrolment in the study, 14 men had received treatment for genital condylomata. Previous genital condylomata were significantly associated with the current detection of HPV DNA; 3 of 14 (21.4%) and 5 of 49 (10.2°!0) in those with and without previous HPV, respectively. Socioeconomic status, age, age at first intercourse, duration of sexual intercourse (at least 6 months to be included in the study), number of episodes of sexual intercourse a week, symptoms, drug or alcohol addiction, cigarette smoking, or immunosuppression did not correlate the detection rate of HPV DNA. On colposcopy, 63 (96.9%) men presented with an abnormal pattern, in 12 (18.5%) the lesion being classified as condyloma acuminatum (Table 2). The 341 vast majority (49, 75.4%) showed an acetowhite lesion in the genital tract. HPV DNA was found in only 4 of 12 (33.3%) condylomas by ISH and PCR, and in 4 of 49 (8.2%) and 6 of 49 (12.2%) acetowhite lesions by ISH and PCR, respectively. The localization of the peniscopic lesions as well as the detection of HPV DNA are summarized in Table 3. Most lesions (76.9%) were found on the preputium, followed by the penile shaft (6 cases). Involvement of multiple sites was seen in another 6 cases. The highest detection rate of HPV DNA was encountered in the shaft lesions, where PCR was positive in 3 of 6 (50%) cases. The histological findings of the biopsies are shown in Table 4. In a total of 41 (63%) patients, the biopsy was classified as non-HPV on light microscopy. A characteristic flat and exophytic condyloma was disclosed in 14 (21.5%) cases and 1(1.5°l0) case, respectively. HPV DNA detection rate was significantly higher in all morphologically HPV-suggestive lesions, compared with the non- HPV, where ISH was invariably negative. PCR, however, disclosed HPV DNA in 9.8% of these cases. PIN was present in 6 (9.2%) men, being graded as PIN I or II. HPV DNA detection rates increased in parallel with the increasing grade of lesion (Table 5). Both HPV 16-positive cases contained a PIN lesion. Altogether, HPV DNA was found by ISH in 12.3% (35.7%) of the biopsies. PCR amplification increased the detection of HPV DNA by only two cases. Table 6 summarizes the relationship between the number of sexual partners and the detection of HPV DNA in the men studied. HPV DNA was never present in men with only a single sexual partner, but increased significantly when the number of partners was increased, being highest (27.3%) in those reporting 11-20 partners. Table 5. Grade of lesions related to detection of HPV DNA by ISH and PCR Lesion grade No. of cases HPV'type present 6/11 16 18 31 33 Non-HPV 41 0 0 0 0 0 HPV-NPIN 18 2 0 0 0 0 HPV-PIN 1 5 1 1 0 0 0 HPV-PIN 11 1 0 1 0 0 0 Total series 65 3 2 0 0 0 HPV DNA in PCR 42 Negative Positive 0 37(90.2%) 4(9.8%) 2 15(83.3%) 3(16.7%) 1 3(60.0%) 2(40.0%) 0 0 1(100%) 3 55(84.6%) 10(15.4%) Table 6. Number of sexual partners related to detection of HPV DNA by ISH and PCR Number of partners No. of cases HPV type present HPV DNA in PCR 6/11 16 18 31 33 42 Negative Positive 1 6 0 0 0 0 0 0 6(100%) 0 2-5 14 1 0 0 0 0 0 11(78.6%) 3(21.4%) 6-10 16 0 0 0 0 0 1 15(93.8%) 1(6.3%) 11-20 11 0 1 0 0 0 1 8(72.7%) 3(27.3%) >20 16 2 1 0 0 0 1 13(81.3%) 3(18.8%) No data 2 0 0 0 0 0 0 2(100%) 0 Total series 65 3 2 0 0 0 3 55(84.6%) 10(15.4%)

~ 342 International Journal of STD & AIDS Volume 3 September/October 1992 ~ Table 7. Partner lesions related to detection of HPV DNA by ISH and PCR Male lesions Type of lesion No. of cases HPV type present HPV DNA in PCR in female partner 6/11 16 18 31 33 42 Negative Positive Flat condyloma 44 0 0 0 0 0 3 41(93.2%) 3(6.8%) Condyloma acuminatum 5 2 0 0 0 0 0 3(60.0%) 2(40.0%) HPV-CIN I 5 1 0 0 0 0 0 3(60.0%) 2(40.0%) HPV-CIN II 1 0 1 0 0 0 0 0 1(100%) HPV-CIN III 4 0 0 0 0 0 0 4(100%) 0 Flat condyloma & CA 2 0 1 0 0 0 0 0 2(100%) HPV-CIN I & Flat 1 0 0 0 0 0 0 1(100%) 0 No data 3 0 0 0 0 0 0 3(100%) 0 Total series 65 3 2 0 0 0 3 55(84.6%) 10(15.4%) Table 8. Partner lesion site related to detection of HPV DNA by ISH and PCR Male lesions Site of lesion No. of cases HPV type present HPV DNA in PCR in female partner 6/11 16 18 31 33 42 Negative Positive Cervix 29 1 1 0 0 0 1 23(79.3%) 6(20.7%) Vagina 5 0 0 0 0 0 0 5(100%) 0 Vulva 17 2 0 0 0 0 1 15(88.2%) 2(11.8%) Cervix and vulva 11 0 1 0 0 0 1 9(81.8%) 2(18.2%) No data 3 0 0 0 0 0 0 3(100%) 0 Total series 65 3 2 0 0 0 3 55(84.6%) 10(15.4%) Table 7 depicts the female lesions as related to HPV DNA detection in their male partners. HPV detection rate was lowest in those males whose partner had flat condyloma, but significantly higher in those who presented with condyloma acuminatum (40%), or HPV CIN I and II lesions (40% and 100%, respectively). The site of the female lesion as related to HPV detection in the male is shown in Table 8. There are no significant differences between the cervical and vulvar (or those combined) sites in their association with male HPV detection rate. Of interest are the women with only vaginal HPV. lesions in whose partners HPV DNA was never demonstrated. DISCUSSION It seems obvious that the highly divergent pre- valence of male genital HPV infections reported are critically dependent on the detection techniques used, i.e., whether peniscopy, PAP smear, biopsy, hybridization or PCR, as recently emphasized for female HPV lesions as we113.4,19,44 The first three are capable of disclosing only the clinically manifest lesions, whereas hybridization and especially PCR can detect the minute quantities of HPV DNA present in the subclinical and latent infections as we113•4.19,4¢ It seems that the majority of the male sexual partners of women with condyloma are rarely aware of their penile lesions, most of them remaining subclinical or latent45. It is equally clear that much of the present controversy in reporting the prevalence of male HPV infections can be also ascribed to the in- consistent diagnostic criteria, especially concerning the subclinical and latent infections, currently defined as different entities by different wor- kers3,4,7,i3,14,i8,i9,2i-zs,2s,29,3i-34. Unfortunately, no consistently reliable diagnostic methods exist for routine diagnosis of male genital HPV lesions as yet. During recent years, however, examination of the male genitalia by colposcopic equipment after application of 5% acetic acid has been claimed to be the most reliable method for identification of subclinical HPV infections29•3o•46 However, no uniformly accepted colposcopic criteria for the male lesions have been outlined so far. The aim of the present study was to establish the frequency of HPV infections in the male sexual partners of 65 women attending an STD clinic for genital HPV infections, and to compare the sensitivity and specificity of peniscopic examination with (a) histopathological assessment of the biopsy and (b) HPV-DNA detection techniques (ISH, and PCR). While viewing the sexual history of the present patients, only 17 (25.4%) gave a history of a previous STD (HPV not included). Prior to enrolment in the present study, only 14 men had received treatment for genital condylomata. Interestingly, this previously treated condyloma seemed to be strongly associated with the current detection of HPV DNA in their genital tract; 21.4% and 10.2%, respectively for those with and those without previous genital condylomata. This is in full agreement with the recent findings of Hippelainen et al.35 who found that previously experienced STD 2063654641

(including HPV) was the most reliable predicting factor for current HPV infection. This has been previously suggested by other workers as well45,47The most feasible explanation is to be found in the complex natural history of this disease, where fluctuation between manifest and subclinical or latent infection during the prolonged disease course frequently occurs. Thus, in males with previous genital warts, either reactivation of a regressed (latent or subclinical) infection or a reinfection from the sexual partners can be considered as a course of the currently detectable genital HPV lesionz-4,1$-z°. Several studies have confirmed the important role of peniscopic examination in diagnosis of HPV lesions on the male external genitalia8-3o,4s This view is substantiated by the present results. On peniscopy, 63 (96.9%) men presented with an abnormal pattern, the vast majority (75.4%) showing an acetowhite lesion in the genital tract. This is in agreement with the recent reports where acetowhite lesions were encountered in the vast majority of male partners of HPV-infected women14,ss•34,49-51 In 12 (18.5%) cases, the lesion was classified as condyloma acuminatum on peniscopy. The detection rate of HPV DNA was significantly associated with the peniscopic pattern; 4 of 12 (33.3%) condylomas by ISH and PCR, and 4 of 49 (8.2%) and 6 of 49 (12.2%) acetowhite lesions by ISH and PCR, respectively. This clearly indicates that the accuracy of peniscopy is critically dependent on the morphology of the findings. Difficulties arose especially in the correct assessment of flat lesions, because of the variety of factors responsible for acetowhite staining; nonspecific infections, healing areas after treatment, etc. The majority of the flat lesions were invisible before acetic acid. Acetowhite areas varied in number, size, surface characters, and time of reaction. In particular, this feature was frequently found on the prepuce, less frequently on the glans, and on the penile skin as has been reported by other authors48. HPV DNA detection rate was highest in the shaft lesions (50%), ~however, indicating that colposcopic abnormality in that area is much more frequently associated with HPV as compared with those in preputium, where HPV DNA was found in 12% of the lesions only (Table 3). This also suggests that the accuracy of peniscopic diagnosis is dependent on the localization of the lesion, as recently discussed by Hippelainen et al.35, being high in the meatus, less so in the glans, and lowest in the preputium. The above data clearly indicate that acetowhite lesions in the preputium frequently develop due to causes other than HPV. This impression was confirmed by the histological examination of the biopsies (Table 4). On light microscopy, the biopsy was classified as non-HPV in 41 (63%) patients. A characteristic flat and exophytic condyloma was disclosed in 14 (21.5%) and 1 (1.5%) cases, res- pectively. HPV DNA detection rate was significantly higher in all morphologically HPV-suggestive lesions, compared with the non-HPV, where ISH Costa et al. HPV infection in male sexual partners 343 was invariably negative. The latter observation is fully consistent with the previous experience in the female genital tract where HPV DNA was never found in a histologically normal epithelium52. Similarly, in a recent study where biopsies were taken from peniscopically normal male genitalia, 1 cm adjacent to the suspected HPV lesion, and HPV DNA was never found by ISH35. In the present series, however, PCR disclosed HPV DNA in 9.8% of such cases. Similarly, Nuovo et a1.16 found HPV DNA in 20% of normal penile biopsies negative with ISH. In a recent report of Kataoka et al. 12, HPV DNA was found in 6% of normal epithelium by Southern blot, and in 12% by PCR. It is reasonable that the frequency of HPV infections increases when DNA techniques are applied23-29,53 With the highly sensitive DNA techniques (ISH, PCR), minute amounts of viral DNA can be detected that are most probably not capable of causing cytopathic effects in epithelial cells. With these techniques, subclinical and latent HPV infections could be detected in the epithelium that appears normal on gross, colposcopic, cytologic and histologic examination4-44. The correlation between colposcopy, histology and ISH (or PCR) was not particularly good in the present series. HPV DNA was found in only 33.3% even of the peniscopically typical condylomas and much less frequently in acetowhite lesions (12.2%). The correlation between histology and DNA hybrid- ization was of the same order, only 8 lesions containing HPV DNA by ISH (Table 4). The use of PCR increased the detection rate by two cases only. Overall, the detection rate of HPV DNA seems to be significantly lower than that obtained in cervical or vaginal biopsies of clinically manifest HPV lesions54. In fact, the DNA detection rate was of similar magnitude to that of subclinical and latent HPV infections in the female genital tract". As discussed above, HPV DNA detection rates sig- nificantly higher than those of the present series have been previously reported7-29-34. On the other hand, there are well conducted studies where the prevalence of male genital HPV lesions seem to be close to that of the present series11,16,36•37The reasons for our relatively low figures might be either one or all of the following: (1) the penile lesions contained HPV types other than those included in the present test panel; (2) biopsies were not from the most representative areas of HPV lesions; or (3) the lesions disclosed are not related to HPV infection. The previously suggested explanation that DNA content was below the detection limits of ISH can be excluded in this case, because of the fact that PCR increased the ISH detection rate only by two cases. Altogether, these data fully confirm the discrepancy still existing in the prevalence figures of male genital HPV lesions, depending on (a) the patient population studied, and (b) the technique used. Concerning the latter, also the importance of standardization of the PCR techniques (e.g. primer selection) should be emphasized.

344 International Journal of STD & AIDS Volume 3 September/October 1992 In the present series, six PIN lesions were discovered, four of which contained HPV DNA either by ISH or PCR (Table 5). Three of these positive cases represented the high risk HPV types, i.e. HPV 16 or 42. In general, HPV DNA detection rate increased in parallel with the increasing grade of lesion, which is also in agreement with a number of recent studies7. A single case of Bowenoid papulosis contained HPV 16 DNA, which is consistent with the previous concepts on this particular entity3$•39. These results indicate that among the male genital lesions, PIN lesions are quite rare occurrences which supports the data from other recent series35. As related to the number of sexual partners, the detection of HPV DNA increased significantly when the number of partners was increased, being highest (27.3%) in those reporting 11-20 female partners. Noteworthy was the observation that HPV DNA was never found in those males with only a single sexual partner (Table 6). Even the existence of 2-5 partners increased the detection rate to 21.4%, whereas the history of more than 20 partners did not further increase these figures. These observations support the concept of the important role of direct sexual contact as a mode of transmission of male genital HPV infections, as emphasized before7-12. It may well be that there are significant differences in infectivity between the clinical, subclinical and latent HPV infections, as well as between HPV lesions with different morphology, and between those at different localization. The latter concept is also supported by the observations in the present study, where HPV detection rate was lowest in the males whose partner had a flat condyloma, but significantly higher in those who presented with condyloma acuminatum (40%), or HPV CIN I and II lesions (40% and 100%, respectively). Similarly, of interest was the finding that HPV DNA was never demonstrated in the males whose partners had only vaginal HPV lesions, although no significant differences were observed between the cervical and vulvar (or those combined) sites in this respect. Barrasso et al.7 suggested that the acetic acid test can be used as a screening method by any physician with help of a hand lens, because most of the macular and papular lesions that become white upon application of acetic acid could be detected by a careful clinical examination. In our practice, peniscopic examination was carried out entirely analogous to the technique of vulvar and perianal colposcopy in women. According to the present results, acetic acid test and peniscopy were not specific enough to discriminate between HPV- lesions and other non-HPV-related epithelial changes. It seems justified to conclude that peniscopic examination of the male genitalia seems to be helpful in the diagnosis of HPV infection, but it should not be used as the only conclusive diagnostic tool. Peniscopic overdiagnosis is the rule when biopsies are evaluated on light microscopy or by HPV DNA detection with ISH or PCR. To summarize, more data are needed to elucidate fully the significance of the male sexual partners in patho- genesis of the squamous cell tumours of the female genital tract. Acknowledgements: This study was supported by a research grant from the Finnish Cancer Society, a research contract (No. 1041051) from the Medical Research Council of the Academy of Finland, a joint research grant from Fabriques de Tabac Reunies S.A., and British <American Tobacco Company Ltd, and CNR Grant No. 8800783, Progetto finalizzato oncologia e finanziamento Regione Emilia-Romana (Progetto di ricerca Oncologia). 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