Highlighting a Potential Pitfall: Positive Treponema pallidum Immunohistochemical Stain in a Patient Without Syphilis.

The Treponema pallidum antibody immunohistochemical (IHC) stain has improved our ability to detect the organism histologically. We present a case of a man with genital condyloma acuminatum with a positive T. pallidum IHC stain but negative T. pallidum serologies and no syphilitic symptoms. It has been shown that the T. pallidum antibody IHC can cross-react, staining other spirochetes, including Borrelia burgdorferi and the Brachyspira family of intestinal spirochetes. Because of the proximity of our patient's lesions to the anus, and the persistently negative T. pallidum serologies, we believe nontreponemal spirochetes colonized the condyloma, giving a false-positive T. pallidum IHC. This cross-reactivity is a potential diagnostic pitfall and is important for the dermatopathologist to recognize, thereby avoiding false diagnosis of syphilis.

Patients With LR-HPV Infection Have a Distinct Vaginal Microbiota in Comparison With Healthy Controls.

Condyloma acuminatum (CA) is a benign epithelium hyperplasia mainly caused by human papillomavirus (HPV), which is now the second most common viral sexually transmitted infection (STI) in China. In total, 90% of CA patients are caused by the low-risk HPV 6 and 11. Aside from low-risk HPV infection there are likely other factors within the local microenvironment that contribute to CA and there has been related research before. In this study, 62 vaginal specimens were analyzed using 16S rRNA gene sequencing. The diversity of the vaginal microbiota was higher and the composition was different with LR-HPV infection. While the relative abundance of dominant Firmicutes was lower, Actinobacteria, Proteobacteria, and Fusobacteria phyla were significantly higher; at the genus level Gardnerella, Bifidobacterium, Sneathia, Hydrogenophilus, Burkholderia, and Atopobium were higher. This study firstly confirmed a more accurate and comprehensive understanding of the relationship between low-risk HPV infection and vaginal microbiota, in order to provide a theoretical basis for further research on the occurrence and development of CA.

Transmission of Chlamydia and genital warts during sleepwalking.

A boy aged 15 years infected a girl of 13 years with Chlamydia and genital warts. The boy has been engaged in sexual activities for the last four years. There was no dispute that the boy had had sex with the girl. He was prosecuted but acquitted by the Court on the ground of 'defence of sleepwalking'. This is the first case where sexually transmitted infections like Chlamydia and genital warts have been transmitted sexually through sleepwalking. This case also raises the issues of underage sex and issues of confidentiality.

Correlation between content of viral DNA and evidence of mature virus particles in HPV-1, HPV-4, and HPV-6 induced virus acanthomata.

In HPV-1 and HPV-4 induced warts as well as in HPV-6 positive condylomata acuminata the quantity of viral DNA encapsulated into virus particles was determined and compared to the total amount of viral DNA present in the papillomas. As shown by filter hybridization using 3H-labeled viral DNA molecularly cloned in Escherichia coli, the amount of total viral DNA found in HPV-1 or HPV-4 induced skin warts is similar. HPV-4 DNA, however, is encapsulated into virus particles with less efficiency. HPV-6 DNA can be detected only at minute amounts in condylomata acuminata and the percentage of DNA recovered from virions is extremely low.

Presence of human papillomavirus in genital tumors.

Human papillomavirus (HPV) types 16 and 18 have been identified in two different human cervical carcinomas. The viral DNAs were molecularly cloned and used as probes to screen a large number of genital tumors by Southern blot analysis. HPV-16 or HPV-18 sequences, respectively, were found in a high percentage of cervical carcinomas, but only in a small number of condylomata acuminata or flat condylomas. The majority of the latter lesions, however, contained HPV-6 or HPV-11 sequences, respectively, which in contrast were detected only rarely in carcinomas in situ or invasively growing carcinomas. A similar distribution of the different papillomaviruses was observed when cell swabs taken from the cervix were tested by in situ hybridization.

Detection of human papillomaviruses in paraffin-embedded condylomata acuminata--comparison of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

The polymerase chain reaction (PCR) is used for detection of human papillomavirus (HPV) DNA in paraffin-embedded tissue sections of condylomata acuminata. Incorporation of biotinylated nucleotides during the amplification process allows a highly sensitive, fast, and non-isotopic detection of viral DNA in a subsequent Southern dot blot. In 100% (13 of 13) of histologically confirmed condylomata, HPV-6 or -11 could be detected by polymerase chain reaction. By in situ hybridization 77% (10 of 13) and by immunohistochemistry (IHC) 69% (nine of 13) positive results were obtained. Because HPV genital infection is linked to penile and cervical dysplasia, polymerase chain reaction provides a powerful and highly sensitive tool for epidemiologic studies on sexual transmission of HPV.

Papillomavirus infection of the anogenital region: correlation between histology, clinical picture, and virus type. Proposal of a new nomenclature.

The clinical and histologic picture of 84 anogenital condylomatous and condyloma-like lesions of both sexes were analyzed in an effort to establish a correlation to the different papillomavirus (PV) types. The presence of human papillomavirus (HPV)-specific DNA sequences was confirmed through molecular hybridization and the presence of PV structure antigens was verified in thin sections by means of a group-specific anti-PV-antiserum using the peroxidase-antiperoxidase (PAP) technique. Three distinct clinical forms harboring distinct HPV types were distinguished: (1) Condylomata acuminata in which HPV-6 DNA was present in 37 of 59 samples and HPV-11 DNA in only 13 of 59 samples. HPV-16 DNA was not detected at all and 9 condylomatous lesions remained unclassified. (2) Flat condyloma-like lesions, where HPV-6 and HPV-11 were associated with lesions of low epidermal atypia in 8 and in 2 of 18 cases, respectively, and where HPV-16 was associated exclusively with 6 of 18 such lesions with severe atypia, called bowenoid papulosis. (3) Pigmented papules where HPV-16 was detected twice in lesions of bowenoid papulosis and HPV-11 in 2 of the benign pigmented lesions. The fourth clinical manifestation of genital papillomavirus infections--the so-called condylomata plana--was not available for virologic analysis. Histologically 5 different koilocytotic features were determined which could not be correlated either with one of the clinical pictures or with a specific PV type. HPV-16, however, was found frequently in non-koilocytotic lesions exhibiting the features of severe epithelial atypia known in bowenoid papulosis. The existence of PV structure antigens in these lesions could not be verified using the indirect immunoperoxidase--PAP-technique--in contrast to the koilocytotic lesions where clear evidence of the presence of HPV was proved in 36 of 56 (64.3%) of the cases.

A new type of human papillomavirus associated with oral focal epithelial hyperplasia.

Lesions from 10 patients suffering from focal epithelial hyperplasia (FEH) of the oral mucosa, including those of 4 Greenlandic Eskimos, were investigated for the presence of human papillomavirus (HPV) DNA sequences by blot hybridization experiments. Two distinct HPVs were detected in the DNA extracted from these lesions, and their genomes were molecularly cloned and characterized. One of these HPVs, detected in 4 patients, was found to be identical with HPV13, whose association with FEH was already known. The other one, detected in 6 patients, was only weakly related to HPV13 and to the other HPVs associated with lesions of the mucous membranes, and constituted a new HPV type, tentatively named HPV32. Lesions from other types of oral papillomas, obtained from 14 additional patients, were also analyzed. Human papillomavirus DNA sequences were detected in the DNA preparations extracted from 5 specimens: HPV6 DNA in a condyloma and in a papilloma, 2 as yet uncharacterized HPV DNAs in 2 papillomas, and HPV32 DNA in a papilloma which showed histologic similarities to FEH. Thus, it seems likely that FEH of the oral mucosa is a disease associated with 2 specific HPVs--HPV13 and HPV32.

Human papillomavirus DNA in cervical intraepithelial neoplasia detected by in situ hybridisation.

Human papillomavirus (HPV) infection was investigated by in situ hybridisation in histological sections from 38 women with abnormal Papanicolaou smears. 13 patients had condylomatous lesions without atypia, 15 cervical intraepithelial neoplasia (CIN) I, 4 CIN II, 3 CIN III and 2 carcinoma in situ (CIS). HPV DNA was detected in 29 cases (78%) (1 specimen was technically inadequate). HPV 16 and 18, and 31, 33 and 35 were both present (67%) in CIN III. HPV 6 and 11 were more frequent in CIN I (56%) and in condylomatous lesions (38%). 31% of the condylomatous lesions without atypia contained HPV 31, 33, and 35 and 31% of those with CIN I were infected with HPV 16 and 18. These data confirm the frequent association of HPV infection with cervical cancer and CIN, and indicate that in situ hybridisation can identify patients with specific types of HPV infection at risk for cervical cancer.

Correlation of histology and human papillomavirus DNA detection in condyloma acuminatum and condyloma-like vulvar lesions.

The diagnosis of a vulvar condyloma is made when perinuclear halos are seen with nuclear atypia and binucleate forms (koilocytotic atypia). These changes are most prominent in the granular layer and are associated with the presence of human papillomavirus (HPV). However, these changes may be absent or minimal in patients with papillary vulvar lesions; this situation can thus present diagnostic difficulties. We analyzed the histologic features of 53 biopsies from 48 patients who had vulvar lesions suggestive of condylomata. Of the 26 biopsy specimens with koilocytotic atypia, 20 (77%) had sequences homologous to HPV DNA as detected by Southern blot hybridization analysis using a probe of HPV s 6/11, 16, 18, 31, 35, and 51. In cases where the histologic features were suggestive but not diagnostic of condylomata, because unequivocal koilocytotic atypia was not noted, five of 27 (19%) had detectable HPV DNA. In this latter group, we found no histologic feature to distinguish the cases that had detectable HPV DNA from those that did not. Analysis for HPV DNA by in situ hybridization in the cases that were histologically equivocal for condyloma was uniformly negative. We conclude that there is a marked decrease in the detection rate of the HPV types associated with genital tract neoplasms in vulvar lesions that lack koilocytotic atypia. Southern blot hybridization analysis was the only reliable way to distinguish the "equivocal for condyloma" cases that had HPV from those where HPV DNA was not detected.

Human papillomavirus (HPV) in condylomatous lesions of cervix.

Ninety-seven cervical condylomata classified histologically as flat condyloma (planum), papillary condyloma (acuminatum), and endophytic condyloma were studied by transmission electron microscopy (TEM) and by immunoperoxidase technique (IPT) for the presence of human papillomavirus (HPV) particles and antigen, respectively. Both techniques localized HPV chiefly in nuclei of koilocytotic cells. HPV particles were found in 25% of the cases by TEM and HPV antigen was detected in 48% of the cases by IPT. All cases positive by TEM were also positive by IPT, thus confirming the specificity of the immunological staining. The viral antigen was detected in 56% of 68 flat condylomata, 35% of 26 papillary condylomata, and in none of 3 cases of endophytic condylomata. However, when histiotypes of virus-positive condylomata were controlled for the intraepithelial extent of koilcytotic cells, the prevalence of HPV correlated with the extent of koilocytosis rather than with the histiotype. The immunologic technique will be of value for the further characterization of cervical condylomata and of the relationship between HPV infection and cervical intraepithelial neoplasia.

Histological distribution of polymerase chain reaction--amplified human papillomavirus 6 and 11 DNA in penile lesions.

The purpose of this study was to ascertain the histological pattern of distribution of human papillomavirus (HPV) 6 and 11 DNA in penile lesions by in situ hybridization after amplification by the polymerase chain reaction (PCR). HPV DNA was routinely detected by in situ hybridization with or without PCR amplification in granular layer cells that showed perinuclear halos and nuclear atypia. Cells that lack these histological features rarely exhibited HPV DNA with conventional in situ hybridization. However, after PCR amplification, in situ analysis showed that many of the cells that lacked halos and atypia contained HPV DNA. The hybridization signal often localized to crevices in the epithelium where there was relative hyperkeratosis and a thickened granular layer. HPV DNA was not noted in the basal cells and was rarely identified in other parts of the lesion. It is concluded that penile tissues may contain HPV DNA when lacking the diagnostic features of a condyloma/low-grade intraepithelial lesions and that such tissues usually demonstrate specific histological changes characterized by a focally thickened granular layer often associated with epithelial crevices.

Condylomata acuminata of the urinary bladder. Natural history, viral typing, and DNA content.

Three patients with condylomata acuminata of the urinary bladder are reported. Two of the patients were immunosuppressed, and one had longstanding extensive condylomata acuminata of the external genitalia and adjacent areas. All lesions recurred at least once and were difficult to treat. The diagnosis was confirmed by in situ hybridization on archival material with human papillomavirus (HPV) DNA probes under stringent conditions. In two of the patients, probes for HPV types 6 and 11 were positive; HPV 11 only was identified in one patient. Probes for HPV types 16 and 18 and pBR322 vector controls were negative. In one patient with a strong hybridization signal, the lesion was also positive for common papillomavirus antigen. DNA content measured by cytophotometry of Feulgen-stained whole nuclei isolated from lesions in two patients revealed a markedly aneuploid DNA pattern. Whether this is a factor in the behavior of the lesions is not known at this time. Although rare, HPV infection of the urinary bladder may result in widespread condylomatosis and may mimic giant condylomas of Buschke-Löwenstein or even verrucous carcinomas, sometimes necessitating radical treatment. Nevertheless, until there is proof to the contrary, the lesions must be considered benign and should not be confused with squamous cancer of the bladder.

Basaloid and warty carcinomas of the vulva. Distinctive types of squamous cell carcinoma frequently associated with human papillomaviruses.

In a previous study, we described an elevated prevalence of human papillomavirus (HPV) in two specific types of squamous cell carcinoma of the vulva designated basaloid carcinoma (BC) and warty carcinoma (WC) compared with the conventional type of keratinizing squamous cell carcinoma (KSC). To determine whether there were other differences in their clinical presentation or behavior, we examined 100 cases of squamous cell carcinoma of the vulva classified as BC (28 cases), WC (seven cases), and KSC (65 cases). We included only cases in which tissue adjacent to the tumor was present so that the presence of intraepithelial lesions (squamous hyperplasia, lichen sclerosus, and vulvar intraepithelial neoplasia [VIN]) could be correlated with the different types of invasive carcinomas. Microscopically, BC was characterized by a relatively uniform population of small, ovoid cells with a high nuclear-cytoplasmic ratio resembling VIN 3. Although WC was similar to typical squamous cell carcinoma, it contained many squamous cells that displayed marked nuclear pleomorphism, enlargement, atypia, and multinucleation in conjunction with cytoplasmic cavitation resembling koilocytotic atypia in intraepithelial lesions. The majority of the women with BC and WC were less than 60 years of age, and the proportion of black women was higher as compared with the women with KSC, the majority of whom were white and over 65 years of age. On crude comparison, women with BC appeared to have a survival advantage compared with women with KSC; however, through multivariate modelling, when all possible confounding variables were taken into account, there was little residual impression of a survival advantage of women with BC compared with those having KSC. Substantial differences were found among the three types of carcinoma with regard to the prevalence of adjacent intraepithelial lesions. Squamous hyperplasia was found adjacent to KSC in 54 (83%) of the 65 cases, whereas 27 (77%) of 35 cases of BC and WC had adjacent basaloid or warty VIN. These findings suggest that VIN is a precursor of BC and WC. In view of the high frequency of HPV-DNA detected in VIN and in BC and WC, the findings support the view that HPV has a role in the development of these tumors. In addition, a difference was found in the distribution of associated cervical and vaginal tumors with the three types of vulvar carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Human papillomaviruses associated with cervical intraepithelial neoplasia. Great diversity and distinct distribution in low- and high-grade lesions.

All together, 30 genital human papillomavirus (HPV) types have been characterized so far. To evaluate the importance of HPV diversity in associated cervical diseases, we analyzed 188 biopsy specimens obtained from patients with a recent diagnosis of cervical HPV infection or intraepithelial neoplasia (CIN). Of these 188 specimens, 116 were classified as low-grade CIN (48 cases), high-grade CIN (53 cases), condylomata acuminata (10 cases), flat condylomas (five cases). Seventy-two specimens were considered nondiagnostic. Using probes specific for 18 genital HPV types, HPV DNA sequences were detected by Southern blot hybridization in 100 lesions and 21 nondiagnostic specimens. When further analyzed by the polymerase chain reaction, eight HPV-negative biopsy specimens, four CIN, and four nondiagnostic specimens were positive. Of the 129 positive biopsy specimens, 92 contained at least one of 18 known HPV types and 37 HPV that have not yet been identified. Nine specimens had more than one type. Thirteen HPV types were identified in CIN. The detection rate of HPV 16 increased from 21% in low-grade CIN to 57% in high-grade CIN. HPV 18 was detected in only 3% of CIN; HPV 31, 33, and 35 were found in 8%. HPV 30, 39, 45, 51, 52, 56, 58, and 61 were detected in 44% of low-grade CIN but in only 8% of high-grade CIN. Unidentified HPV were detected in about 25% of low-grade and high-grade CIN. Fifty-seven CIN positive for at least one HPV type were further analyzed by in situ hybridization. Thirty-five (65%) biopsy specimens were positive, including 21 of 24 low-grade CIN and 14 of 33 high-grade CIN. Ten of the 13 previously identified HPV types were detected. Thus, CIN represents an heterogeneous disease from a virologic viewpoint. This fact could explain their variable clinical evolution.

Differential expression of HPV types 6 and 11 in condylomas and cervical preneoplastic lesions.

Tumor biopsies from exophytic and flat condylomas at different locations on the genital epithelium (10 cases) and in cervical intraepithelial neoplasia (CIN) grades 1-2 (6 cases) were analysed for HPV types 6 and 11 DNA and RNA. The presence of mRNA species which could encode the E6, E7, E1M, E2, E2C, E4, E5 and L1 proteins was determined using the RNA polymerase chain reaction (PCR) technique with primers that flank previously mapped or predicted splice sites. The state of the viral DNA in the tumor biopsies was established by Southern blot analysis. We could detect the various mRNA species in biopsies from condylomas associated with both HPV types. The size of the RNA PCR products were in agreement with the previously mapped splice sites of mRNAs recovered from an experimental condyloma induced by HPV11. The major viral transcript encoding the E1i--E4 protein was detected in all the tumor biopsies. From the rare transcripts the rate of detection of mRNA species encoding the E1M, E2C proteins was the highest. In 2 of 6 CIN biopsies analysed only the major viral transcript was detected. The overall results of this study suggest that early gene products of HPV types 6 and 11 may be important in the induction of cellular proliferation and condylamatous differentiation but these possibly may not be required for the development of the HPV-associated cervical neoplasia.

Detection of multiple types of human papillomavirus in a giant condyloma from a grafted patient. Analysis by immunohistochemistry, in situ hybridisation, Southern blot and polymerase chain reaction.

Immunosuppressed patients such as transplant recipients are known to develop multiple lesions suggestive of human papillomavirus (HPV) infection. A giant anal condyloma was obtained from a transplant patient; several fragments taken from different areas were examined for the presence of HPV DNA using in situ hybridisation, polymerase chain reaction (PCR) and Southern blot. Typical koilocytes were seen in routinely stained tissue sections, suggesting an HPV infection; furthermore, group specific HPV antigen was detected in one of four frozen fragments. Different results were obtained by in situ hybridisation according to the fragment tested. HPV types 6/11 were detected in each of the five fragments, frozen or fixed in Bouin's or formalin solutions. However, the number of HPV DNA positive cells and the intensity of the reaction greatly varied with the specimen. HPV 16 and 18 probes also reacted positively with the sample fixed in formalin; a stronger signal was observed with HPV 18 in one large focus than with HPV 16. HPV type 5 was detected in a few isolated cells of two frozen fragments. With the Southern blot technique, the profile of an HPV 6/11 was seen only in one of two frozen fragments; in this case, the bands were intense. A slight positive reaction was also obtained in one frozen fragment with HPV 16 probe. Four frozen fragments were analyzed with PCR: HPV 6/11 was detected in each fragment; HPV 18 was detected in the four samples but with different intensities; HPV types 5 and 16 did not show any positive signal. In conclusion, the lesion is an example of infection with several HPV types, demonstrated by three different techniques. This suggests the need for careful dermatological or colposcopic follow-up of transplant recipients, in order to prevent possible malignant transformation of anogenital lesions.

Identification of human papillomavirus types in male urethral condylomata acuminata by in situ hybridization.

An in situ hybridization technique was applied under stringent conditions to paraffin sections of urethral condylomata from male patients to determine the presence of DNA sequences of human papillomavirus (HPV) types 6, 11, 16, and 18. The material consisted of 15 classical condylomata acuminata, two flat condylomata, and five recurrent lesions. HPV DNA sequences could be identified in all 15 condylomata acuminata; in 13 lesions, two types of viral DNA were observed (types 6 and 11 in 12, types 6 and 18 in one). In the remaining two condylomata acuminata, only HPV type 11 was present. One of the two flat condylomata was negative with all the probes, and one was borderline-positive for HPV 6. Four of five recurrent lesions contained the same types of viral DNA as the primary lesions, albeit with slight differences in the intensity of viral expression. One lesion was negative with all probes. We conclude that urethral condylomata in males contain the same types of HPV as seen in other anogenital lesions of both sexes and that infection with two viral types is common. In situ hybridization with HPV DNA probes is applicable to archival material and therefore may prove to be of value in future epidemiologic studies comparing lesions in sexual partners. The determination of viral type may have therapeutic implications.

In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma.

Routinely processed formalin-fixed paraffin-embedded sections from anogenital condyloma acuminatum and an invasive squamous cell carcinoma of the cervix were examined by in situ hybridization for the detection of human papillomavirus (HPV) DNAs and messenger RNAs. Asymmetric, single-stranded, tritium-labeled RNA probes for both the coding and the nonsense strands of HPVs 6, 11, 16, 18, and 31 were hybridized and washed under stringent conditions and detected by autoradiography. Type-specific HPV DNAs were detected with specific nuclear localization, while HPV messenger RNAs gave much higher signals and had clear-cut cytoplasmic localization. Cross-hybridization was observed only with closely related viruses. The level of signal obtained seemed to be linked to the degree of cellular differentiation, with koilocytotic cells labeling the most heavily. However, messenger RNA could be detected in even relatively undifferentiated cells within areas of dysplasia and invasive carcinoma. In situ hybridization is a sensitive and specific method for investigation of the dynamic interplay of papillomavirus replication and gene expression, cellular differentiation, and neoplastic transformation.

Detection of human papillomavirus infection in tissue blocks by in situ hybridization as compared with a polymerase chain reaction procedure.

Tissue blocks from 25 cases of condyloma and/or dysplasia were used for human papillomavirus typing with DNA in situ hybridization, compared with the very sensitive polymerase chain reaction. Only one of these cases was negative with both methods: a case of vaginal "koilocytosis." Polymerase chain reaction, as expected, was the more sensitive method, positive in 24 cases, with seven double infections. In situ hybridization was positive in 18 cases, with only two detected double infections. There was excellent agreement between the two methods in typing results. In all cases in situ hybridization showed a positive reaction in areas of koilocytosis and/or dysplasia.