Importance of peniscopy , oncologic cytology and histopathology in the diagnosis of penile infection by human papillomavirus

Introduction: Male genital infection by human papillomavirus is of particular importance since it is often asymptomatic. The patient generally presents no clinicallesion. Therefore, men represent an important reservoir of virus, playing a special role in the transmission and perpetuation of the disease. Patients and methods: In the present prospective clinicai trial study, 190 sex partners of women with genital infection by human papillomavirus, associated or not with cervical intraepithelial neoplasia, were investigated. Ali patients were unaware of or denied the presence of a genitallesion. Results: Cytologic examination revealed koilocytosis in 9 cases (4.7%) in the urethra and in 3 cases (1.6%) in the corona of the glans and the distai prepuce. Peniscopy with the previous use of 5% acetic acid revealed white lesions in 97.9% of the patients. Toluidine blue stained most of the lesions. At least one fragment revealed koilocytosis in the histopathologic study of 97 cases (51.05%). Conclusion: The three methods complement one another; allowing a more precise diagnosis of this infection in men.


INTRODUCTION G
enital warts have been known since ancient titnes, being referred to by several Greek and Roman writers.They were considered to be a venereal disease transmitted among male homosexuals.They were then caIled condy loma, a term of Greek origin that is still used.
The role of papillomavirus in the etiology of condyloma is well defined.Molecular biology studies have led to a better understanding of the infection.Genital infection may be caused by more than 20 of the 70 types of papillomavirus known up to the present.Based on their properties and on the identification of the virus in both malignant and pretnalignant condylomatolls lesions, they are classified into low risk (6 and 11) and high risk (16 and 18) types as proposed by Syrjanen (1989).
After the studies of Meisels and Fortin (1976) and Meisels et aI.(1977), the interest in the infection was renewed.The authors described the cytological, colposcopic and histopathologic features of the fetnale genital infetion by the human papillomavirlls.
The real interest in papillomavirlls in the genesis of cervical cancer seems to have etnerged frolll the identification of morphological.aspects of viral cervical infecti0n compatible with intraepithelial neoplasia both in terms of oncologic cytology and histopathology (Meisels and Fortin, 1976).
The high incidence rates of the viral infection and the relation to cervical neoplasia, in addition to the fact that the men had no visible penile lesion, led the investigators to make use of other diagnostic methods for an earlier identification of the infection.(1984) were the first investigators to use magnification to examine the penis of the partners of the women with condyloma.
The aim of the present study was to ascertain the incidence ofthe infection and the value of magnified penile examination, cytology and histopathology in the diagnosis of papillomavirus lesions in the penis of men whose sex partners presented the same disease.All patients were submitted to careful anamnesis.Patients with genitallesions caused by human papillomavirus and visible to the naked eye were excluded from the study.

PATIENTS AND METHODS
The age of the patients ranged from 18 to 71 years, with a predominance of 20-to 39-year range (126 cases or 66.3%) (TabIe I).White patients were greater in number (144 or 75.80/0).Forty-two were black (22.1 %) and four were asian (2.1%).Sexual activity had started more frequently between 15 and 20 years (106 cases or 55.8%), as shown in Table 11.One hundred and fifteen (60.5%) patients were married and 82 (43.2%) had had sexual intercourse with more than one partner.One hundred and forty-four patients (75.8%) did not use condoms.
TabIe 111 shows the history of sexually transmitted diseases, which was positive in 87 (45.8%) patients.The most frequently reported disease was gonococcal urethritis (46 patients or 52.9%).In 7.4% of cases, the patients reported urogenitaI surgeries such as posthectolllY, CirCUlTIcisionor correction of hypospadias.

Table 111 Patient distribution by previous sexually transmissible disease
After anamnesis, the penis, distaI urethra and scrotum were examined with a coIposcope (D.F.Vasconcelos) under 4-and 6-fold magnification.The examination strictIy required the following steps: 1-Examination of the entire penis and scrotum with the patient in the sitting position.
11 -Examination of the 2 distaI centimeters of the urethra, using a urethraI speculum specially designed for this purpose.
111-Collecting of material for cytoIogic examination from the glans, from the distaI prepuce and from the distaI urethra.
IV -Wrapping of the penis and scrotum with gauzes soaked in 5% acetic acid.PIacement of a cotton tip soaked in the same solution into the distaI urethra.The acid was in contact with the structures for 5 minutes.v -Repeated examination of the penis and distaI urethra, after relTIoving the gauzes and cotton tipo The characteristics and sites of the images revealed were then identified.VI -Soaking of the penis and scrotum with 1% toluidine blue, removing the excess with 2% acetic acid after 3 11linutes.
In the present study, we define peniscopy performed before and after the use of acetic acid and toluidine blue as widened peniscopy.

Cytology
Itllmediately after the first examination, material for cytology was colIected "frOlll the distaI urethra, the distaI prepuce and the corona of the gIans with a brush commonly used for endocervicaI smears (Terreiro et aI., 1990).

His topa thology
Direct biopsies were coIlected in order to obtain representati ve fraglllents of each type of lesion, identified by peniscopy with and without acetic acid and toluidine blue.The biopsies were obtained under anesthesia, using a 2-3-11lm wide denllatome.Hemostasis was obtained with a cotton tip soaked in concentrated methacresolsulfonic acid applied onto the raw area.
In cases in which urethral lesion could be easily reached, biopsies were eventually obtained using the forceps for the uterine cervix of the Gaylor type, modified by Medina (Medina et aI., 1977).this alteration "koilocytotic atypia", a tenll originating frOlll the Greek koilos, which means hole OI'cavity.
The initial examination with the peniscope revealecl suspect lesions in 72 patients (37.9%).
Toluidine blue stained 11l0Stof the lesions describecl above, mainly the central area of the 11licropapules and of the annular lesions (Fig. 2).In SOllle instances, it stainecl areas in which no leucoacetic lesions had been previously found.SimilarIy, some leucoacetic lesions were not stainecl by toluidine blue.Papillomatous urethral lesions were observed in 4 cases (2.10/0); koilocytosis was not detected in only one of these cases.
Cytology revealed koilocytosis in 9 cases (4.7%) in the urethra, and in 3 cases (1.6%) in the corona of the .gIans and distaI prepuce.Koilocytosis was detected in the biopsy specimens in these 3 cases.Only two (22.20/0) of the nine cases of urethral cytology with koilocytosis presented a papilIomatous lesion upon exalllination of the distaI urethra.These Iesions were SUblllitted to biopsy and koilocytosis was detected by histopathologic examination.
Biopsies were colIected frOlll alI patients.In the 4 patients in WhOlTI the acetic acid test proved negati ve, SOllle epithelial areas were stained with toulidine blue anel were biopsied.In one patient, smaII lesions were observed after the initial examination with magnification.

RESULTS
The diagnosis of human papillomavirus infection was based on the cytologic and histopathologic alterations compatible with kolocytosis, i.e., presence of Iarge, round and vacuolized squamous ceIls, which may be Iocated at any leveI of the stratified epithelium, though harelly in the basaI or parabasal layers.
The ceIl shows a Iight perinuclear halo anel a hyperchromatic anel retracted nucleus which seems to be suspended in empty space.Binucleated forms are COlTImon.Koss and Durfee (I 956) ~al1ed Koilocytosis was detected in at least one fragment in 97 (51.05%) of the 190 patients.
Histopathologic exalnination also detected Inollusculn contagiosuln in 3 patients, and genital herpes in another.

DISCUSSION
Several investigators have recomlnendeel exalnination of the penis with the aid of an optical instrulnent, the colposcope, with the aim of eliagnosing genital infections, specially when these are subclinical or not visible (Levine et  Although sOlne authors use the term colposcopy of the penis (Barrasso et aI., 1986; Krebs and Schneider, 1987), we prefer to call it peniscopy.Other words such as androscopy OI'genitoscopy are also useel.
The criterion used in the present study for the cytologic and histopathologic eliagnosis of this viral infection was the presence of koilocytosis.
Considering the criteria described in the cytology, with 1000/0 results proving positive in the presence of cytoplaslnic anel nuclear alterations, we reached a cytologic diagnosis of the penile and urethral infection in 1.6% and 4.7% of the cases, respectivelly.
In spite of the few positive cases detected, urethral cytology was Inore effective than cytology of the elistal prepuce and the corona ofthe glans.Only 2 patients presenteel papilIolnata in the distaI urethra anel the others elielnot have aIteration of the aspects when uneler Inagnfication.Cytologic finelings were itnportant for the eliagnosis.
In the distaI part of the penis, cytology was positive in cases where histopathology showed koilocytosis.The s p e c i m e n s h a el In a n y u n n u c 1e a te el keratinized cells, thus being inappropriate for the identification of koi 10cytosis.
The ideal ti nle for collecti ng Inaterial for cytology with a brush shoulel no t b e t h e s a In e as fo r p e rf o nn in g peniscopy.The brush injures the gIans anel prepuce epitheliuln and the urethral Inucosa, possibIy leading to false-positive peniscopic findings.Peniscopy detecteel lesions Úl alI cases, anel was considered to be positive.In 97.9% it showeel alterations with acetic acid and in the others only with tolllieline blue.
We used a 5% acetic acid concentration.The Inain action of the acid is to coagulate cytoplasnlic anel nuclear proteins of the epithelial cells, turning theln white.It is a progressive, superficial, and reversibIe processo In wonlen, it occurs between 10 and 30 seconels anel elisappears after 30-40 seconds (Cartier, 1986).In Inen, not only the latency tilne but also its effect lasts longer.
The urethra shouId be equally exalnineel with acetic acid, for sOlnetimes it is possible to eletect snlall lesions not visible under silnple Inagnification.In fact, Wosnitzer (1988) recommends exalnining the urethra with a coIposcope, both before and after the use of acetic aciel in order to eletect small lesions not visible to the nakeel eye.
The effect of 1% toluidine bllle in aqueous solution is noteel in the nuclei.Since this is a cell elye that behaves as a base, it has affinity for, and binels to, aciel structures sllch as DNA and RNA, silnilarly to Inethylene blue (Junqueira anel Carneiro, 1977).
Nicolau et aI.(1991) cOlnpareel the etfects of the 2 solutions (acetic aciel and toluidine) in 863 biopsies anel concluded that acetic acid reacteel slightly Inore frequently with lesions with koilocytotic cells.
In the present stuely, we concluded that the two tests cOlnplelnent each other, since sOlne lesions showed reaction with only one of the two solutions.Therefore, both tests should be applied.The action of the dye has the advantage of lasting much longer, which permits to decide about the area to be biopsied and to anesthetize it before the lesion disappears.Many lesions revealed by peniscopy do not present koilocytosis.A large part of them were detected because of inflammatory or infectious processes causing balanoposthitis.UsualIy patients are not circumcised (in the present study, only 7.4% were).This hampers appropriate hygiene and facilitates the growth of infectious .agents such as Candida albicans, Gardnerella vaginalis, herpes siI11plex, Tricholnonas vaginalis, bacteroids and other anaerobic organisms (Peutherer et aI., 1979;Cree et aI., 1982;Kinghorn et aI.,1982).
We preferred to examine the patient while seated because this position is more cOI11fortable for both examiner and patient.
In medicaI literature there are reports of macroscopic urethraI lesions in 0.5-5% of cases (Oriel, 1971;Cetti, 1984).In the present study, distaI urethroscopy revealed lesions in 2.1 % but there were cytoIogic alterations in 4.7%.This is the reason for performing both procedures.
HistopathoIogic examination was performed in alI patients, several lesions being biopsied in each case.The great majority of the lesions were flat.We could note no correlation between the clinicaI aspect of the lesion and the anatomopathologic alterations; thus, it is necessary to biopsy alI lesions, especialIy the flat ones.
In the present study, intraepitheIial neopIasia was found to be associated with the viraI infection in 1.58% of cases.These Iesions were unremarkable when examined by peniscopy.
The biopsies were well tolerated.The most difficuIt step was anesthesia, which is sOI11ewhatuncol11fortabIe. The specil11en is small, which allows performing of l110rethan one biopsy, with a better representation of the Iesions.In addition, due to the small size of the biopsy, the heaIing period is short, approximately 2 weeks.About 50% of the cases did not present koi Iocytosis.According to the most recent reports, it is possible that these patients are carriers of a virus in the penis, urethra and semen, especialIy viruses of the types associated with premaIignant and malignant lesions (Grussendorf- Based upon these facts, it becomes clear that, frol11a clinicaI point of view, the examination of the penis with magnification is insufficient; performing ofpeniscopy after applying acetic acid and toluidine bIue is necessary.

I. Peniscopy alIowed the diagnosis of papilIol11avirus
infection, especialIy with the use of acetic acid and toluidine blue; the white Iesions were present in 97.9% of the cases and in the others the Iesions were stai ned with toluidine bIue.2. AIthough a papillomatous lesion was rare (2.1 %), the examination of the distaI urethra, preferentially with a specuIum, was important for the diagnosis.

A
total of 190 sex partners of women with genital infection by human papillomavirus, associated or not with intraepithelial cervical neoplasia, diagnosed by colposcopic, histopathologic and cervicovaginal cytologic examinations, were studied between April 1988 and March 1991.