Human Papillomavirus DNA Probes Research Articles

Evaluation of two commercialised in situ hybridisation assays for detecting HPV-DNA in formalin-fixed, paraffin-embedded tissue

The role of human papilloma virus (HPV) in the pathogenesis of anogenital dysplasia is now conclusive. However, HPV detection in formalin-fixed and paraffin-embedded tissues remains controversial. Therefore, the aim of this study was to evaluate morphological changes directly in tissue specimens using a HPV-DNA detection system involving HPV in situ hybridisation. Samples from patients with cervical carcinoma were analysed using the GenPoint HPV DNA Probe Cocktail (Dako, Glostrup, Denmark) and the ZytoFast HPV Screening CISH-Kit (Zytomed, Berlin, Germany). Three cervical carcinoma cell lines with a well-defined HPV copy number per cell (SiHa, HeLa, and CaSki) served as positive controls for sensitivity testing, while two HPV-negative cell lines (AC-1M32, MCF-7) and brain tissue samples served as negative controls. Moreover, to assess the validity of the in situ hybridisation, the expression of HPV-16 DNA in cell lines was demonstrated by HPV-16 E6-specific PCR. Both HPV-screening assays revealed strong signals of episomal and integrated HPV-DNA at a HPV copy number of more than 50 copies/cell. All cervical carcinoma samples were positive in the Dako assay, which identifies 13 high-risk HPV genotypes, whereas HPV-DNA could be detected in 9/10 cervical carcinoma samples using the Zytofast assay, identifying HPV 16, 18, 31, 33, and 35. HPV in situ hybridisation is a convenient and powerful tool for detecting HPV-DNA in formalin-fixed and paraffin-embedded tissue samples. Therefore, this technique is suitable for analysis of a potential HPV infection using archival pathological slides.

Human Immunodeficiency Virus and Carcinoma Cervix

Problem statement: Carcinoma of the cervix is the second leading cause of death in women in the world. Human Papilloma Virus (HPV) which plays a major role in the etiology of cervical cancer is said to have the same mode of transmission as the Human Immunodeficiency Virus (HIV). Hence the curiosity to learn the prevalence, incidence and association of cervical cancer in HIV positive patients paves the way for the present study. Approach: Forty patients who were positive for HIV were scrutinized for other diseases at our hospital. The patients underwent gynaecological examination at the department of obstetrics and gynaecology. The blood samples of the patients were tested by both the Enzyme Linked Immunosorbent Assay (ELISA) and confirmed by Western Blot in the Serology Department. The cervical biopsies were examined microscopically in the Department of Pathology. Results: Two of the patients had invasive cervical carcinoma and were undergoing radiotherapy for the same. One patient was detected HIV positive when his spouse was diagnosed with cervical invasive carcinoma. A total of three out of forty patients had cervical cancer. Therefore the prevalence of invasive cervical cancer in HIV infected patients is 7.5%. Conclusion: A percentage of 7.5 is nearly close to the prevalence of 6 and 5% reported by other studies. This study therefore emphasized the association of cervical cancer and possibly the human papilloma virus with HIV infection. Further studies using HPV DNA probes in concurrence with HIV testing in patients with carcinoma cervix and intraepithelial lesions would contribute much and confirm the association of HPV and HIV co-infection.

Effect of human papilloma virus expression on clinical course of laryngeal papilloma

Conclusion. Our observations suggest that human papilloma virus (HPV) 6/11 is the main causative agent of laryngeal papilloma and that detection of active HPV DNA expression may be helpful in identifying patients with aggressive recurrent laryngeal papilloma. Objectives. HPV is assumed to be the main causative agent of this disease. We investigated the expression of the entire genotype of HPV in cases of laryngeal papilloma and correlated their expression with the clinical course of the disease. Subjects and methods. Seventy cases of laryngeal papilloma were evaluated for the presence of the HPV genome by in situ hybridization (ISH) using wide-spectrum HPV DNA probe. Specific types of HPV infection were determined by DNA ISH using type-specific HPV DNA probes (HPV 6, 11, 16, 18, 31, 33). Separate analyses were conducted comparing viral types, frequency of recurrences and duration of disease-free periods. Results. We detected HPV DNA in 40 of the 70 laryngeal papilloma cases (57%). In particular, HPV DNA was detected in 75% of the juvenile types. There were significant associations between HPV and laryngeal papilloma (p<0.01). Among the HPV-positive cases, major specific types were HPV 6/11 (97%). Significant associations were also noted between viral expression and clinical course.

Detection of Cervical Human Papillomavirus Infection by In Situ Hybridization in Fetuses from Women with Squamous Intraepithelial Lesions

To evaluate for human papillomavirus (HPV) infection in the cervix of fetuses from mothers with documented squamous intraepithelial lesions. Fetal cervical epithelium was obtained from the Human Fetal Tissue Repository as per Institutional Review Board protocol. Fetal cervical epithelium was dissected, fixed in formalin, and embedded in paraffin. Sections were tested by in situ hybridization using a wide-spectrum HPV DNA probe. Cases were specimens from mothers with low- and high-grade squamous intraepithelial lesions, and controls were specimens from women with no documented squamous intraepithelial lesions. A total of 14 controls and 10 cases were evaluated for HPV DNA. No reactivity was detected in the controls. Two cases showed focal intracellular reactivity with the HPV DNA probe. To our knowledge, this is the first study demonstrate fetal cervical HPV infection due to intrauterine exposure. These findings have important implications in understanding the pathogenesis of cervical neoplasia and its management.

Detection of Human Papillomavirus in Laryngeal Squamous Dysplasia and Carcinoma. An In Situ Hybridization and Signal Amplification Study

We examined the presence of human papillomavirus (HPV) DNA in 65 cases of laryngeal squamous dysplasia and carcinomas using in situ hybridization with signal amplification in paraffin sections. Hybridization was performed with biotynilated DNA probes for HPV 6/11, 16/18, 31/33 and wide-spectrum HPV (6, 11, 16, 30, 31, 45, 51 and 52). HPV DNA was found in 7 cases of the total sample (10.7%); it was also found in 4 out of 45 (8.8%) cases of invasive carcinoma and in 5 out of 33 (15.5%) cases of squamous dysplasia. Morphological signs suggestive of HPV infection were observed in 35.5% of our sample but they were not related to HPV DNA positivity. In conclusion, HPV probably plays little, if any, role in laryngeal carcinogenesis among the population studied.

Human Papillomavirus Involvement in Esophageal Carcinogenesis in the High-Incidence Area of China: A Study of 700 Cases by Screening and Type-Specific in Situ Hybridization

Background: Human papillomavirus (HPV) DNA has been identified in esophageal precancerous lesions and carcinomas. However, there are marked variations in the prevalence of HPV infection reported in different studies. Most previous studies on HPV and esophageal carcinomas have been based on a limited number of biopsy samples studied by different HPV detection methods with highly variable sensitivity and specificy, making systematic studies of larger series clearly warranted. Methods: A series of 1876 surgical specimens (primary tumor, adjacent epithelium, regional lymph nodes, resection margins) from 700 patients surgically resected for an invasive squamous cell carcinoma of the esophagus in the high-incidence area of China was analyzed for the presence of HPV DNA with screening in situ hybridization (ISH) using biotinylated HPV DNA probes and followed by type-specific ISH for HPV 6, 11, 16, 18, 30, and 53. Results: Of the 700 esophageal carcinomas, 118 (16.9%) were shown to contain HPV DNA sequences by screening ISH. Positive signals were most frequent in the cancer cells (16.6%), more rare in the surrounding hyperplastic and dysplastic epithelia (5.6%), and infrequently present in the resection margins (0.2%). HPV signals were also detected in cancer cells in 6.9% of the lymph node metastases. HPV types 6, 11, 16, 18, and 30 account for 39.8% of the HPV-positive lesions, of which the high-risk types HPV 16 and 18 were present in 27.1% (32 of 118). Notably, 60.2% of the HPV-positive lesions contained DNA sequences other than HPV types 6, 11, 16, 18, 30, and 53. Conclusions: This study reports the largest series of esophageal cancers ever analyzed for the presence of HPV DNA. Our results confirm the presence of common mucosal HPV types in esophageal carcinomas but also suggest the involvement of other (novel?) HPV types that are unusually detected in genital cancers in a significant proportion of these lesions. The results further indicate that HVP has an etiologic role in esophageal carcinogenesis, at least in the high-incidence area of northern China.

Correlation between HPV types associated with oral squamous cell carcinoma and cervicovaginal cytology: An in situ hybridization study

Epidemiologic and clinical evidence suggests a possible relationship between the human papillomavirus types found in oral squamous cell carcinoma and cervicovaginal cancer. To try to address this question, we analyzed by in situ hybridization 50 paraffin-embedded biopsies of oral squamous cell carcinomas and their corresponding Papanicolaou smears using nonradioactive DNA probes for human papillomavirus types 6 11 and 16 18 . This analysis revealed that 70% (35 of 50) of oral squamous cell carcinomas and 56% (28 of 50) of Papanicolaou smears were positive for one or both of these human papillomavirus DNA probes. In 23 of the 28 patients who were positive for human papillomavirus sequences in the cervicovaginal tract, we found the same human papillomavirus type in the oral cavity. Five of the positive in situ hybridization Papanicolaou smears were associated with negative in situ hybridization oral squamous cell carcinomas. Our data suggest that after a primary infection by human papillomavirus, other mucosal sites may eventually become infected. Local environmental factors may contribute to the pathogenesis of lesions such as oral squamous cell carcinoma.

HPV Typing by in Situ Hybridization on Cervical Cytologic Smears with ASCUS

To assess the prognostic significance of atypical squamous cells of undetermined significance (ASCUS) using an in situ hybridization (ISH) method for destined cervical cytologic smears and a cocktail of biotinylated DNA probes for human papillomavirus (HPV) 6, 11, 16, 18, 31 and 33. Two HPV DNA probe mixtures were applied to the same smear for the simultaneous detection of high-risk HPV types 16, 18, 31 and 33 and low-risk HPV 6 and 11. ISH was carried out on 192 smears. Among them, 59 showed koilocytosis, 91 ASCUS and 42 normal features. Low-risk HPV types were rarely found and associated mainly with koilocytosis (17%). High rates of potentially oncogenic HPV were detected in ASCUS (41%) and condyloma (73%). In addition, similar levels of positivity were found to be associated with ASCUS when using two probe mixtures specific to high-risk HPV: one included HPV genotypes 16 and 18 and the other, genotypes 31 and 33. HPV DNA typing by ISH on cervical cytologic smears might improve the identification of women at high risk of developing precancerous and cancerous cervical lesions.

Human Papillomavirus 16 and 18 Infection is Absent in Urinary Bladder Carcinomas

The role of human papillomavirus (HPV) in the pathogenesis of bladder neoplasia remains controversial. Studies to date have mainly utilized polymerase chain reaction and DNA blot techniques to detect HPV DNA. The detection rates have varied from 0 to 81%. One of the major limitations of these techniques is that they lack topographic information. We have used a highly sensitive in situ hybridization (ISH) technique which detects single copies of viral genome and provides topographic information for investigation of HPV infection in bladder carcinomas.Thirty-one samples of formalin-fixed paraffin-embedded bladder carcinomas (4 adenocarcinomas, 5 squamous cell and 22 transitional cell carcinomas) were examined using non-isotopic ISH with biotin-labelled DNA probes of HPV 16 and 18 subtypes. HPV-positive skin and anal carcinoma samples were used as controls.No positive signals for HPV 16 or 18 were detected in any of the bladder carcinoma samples. Given the sensitivity of the technique, the result indicates that HPV 16 and 18 are not implicated in the development of bladder neoplasia compared to certain other epithelial malignancies. The possibility that other subtypes of HPV contribute to bladder epithelial carcinogenesis remains to be clarified.

Human papillomavirus 16 and 18 infection is absent in urinary bladder carcinomas.

The role of human papillomavirus (HPV) in the pathogenesis of bladder neoplasia remains controversial. Studies to date have mainly utilized polymerase chain reaction and DNA blot techniques to detect HPV DNA. The detection rates have varied from 0 to 81%. One of the major limitations of these techniques is that they lack topographic information. We have used a highly sensitive in situ hybridization (ISH) technique which detects single copies of viral genome and provides topographic information for investigation of HPV infection in bladder carcinomas. Thirty-one samples of formalin-fixed paraffin-embedded bladder carcinomas (4 adenocarcinomas, 5 squamous cell and 22 transitional cell carcinomas) were examined using non-isotopic ISH with biotin-labelled DNA probes of HPV 16 and 18 subtypes. HPV-positive skin and anal carcinoma samples were used as controls. No positive signals for HPV 16 or 18 were detected in any of the bladder carcinoma samples. Given the sensitivity of the technique, the result indicates that HPV 16 and 18 are not implicated in the development of bladder neoplasia compared to certain other epithelial malignancies. The possibility that other subtypes of HPV contribute to bladder epithelial carcinogenesis remains to be clarified.

Presence of human papillomavirus type 6f in tonsillar condyloma acuminatum and clinically normal tonsillar mucosa.

An attempt was made to detect human papillomavirus (HPV) in 8 cases of oral papilloma by Southern blot hybridization. HPV type 6 was identified in a case of tonsillar condyloma acuminatum, but neither HPV type 6 nor 11 was found in the 7 other cases of squamous papilloma. The hybridization pattern of the HPV type 6 DNA after digestion with restriction enzymes revealed that the HPV was of the 6f subtype. DNA samples isolated from the condyloma acuminatum, 3 peripheral specimens of clinically normal tonsillar mucosa, a swab sample and saliva from the male patient with this tonsillar condyloma acuminatum were examined further by polymerase chain reaction (PCR). HPV type 6 DNA was detected in the tonsillar condyloma acuminatum and the 3 specimens of clinically normal tonsillar mucosa, but no HPV type 6 DNA was detectable in the normal tonsillar mucosa by Southern blot hybridization using a whole HPV DNA probe. The presence of a small amount of HPV in clinically normal tissue detectable only by PCR suggests latent infection of peripheral tissues with HPV produced from the condyloma acuminatum. Although HPV type 6 DNA was not detectable in the swab and saliva samples of this single case, further study is necessary for the clinical application of preoperative virological diagnosis by PCR using these samples.

Detection of human papillomavirus DNA in nongenital seborrhoeic keratoses.

The histological similarities of seborrhoeic keratoses and common warts led to the investigation of the possible occurrence of human papillomavirus DNA (HPV-DNA) in a large number of nongenital seborrhoeic keratoses using the in situ hybridization technique. All specimens derived from normal skin (n = 173) were negative for the applied HPV-DNA probe, whereas the HPV genome was detected in 34 of 173 seborrhoeic keratosis specimens (19.65%). Of 34 HPV-positive specimens, 15 contained types 6/11 and 14 types 31/33/35, and 5 showed no positive reaction to the applied types. These results suggest that a considerable percentage of nongenital seborrhoeic keratoses may be related to an HPV infection.

Human papillomavirus DNA in oesophageal carcinomas in South Africa

Using morphological criteria, the presence of human papillomavirus (HPV) in oesophageal carcinomas has been inferred in patients from Finland and South Africa. However, studies to demonstrate the viral antigen in tissue sections of these tumours have proved disappointing. This study investigates 48 archival oesophageal carcinoma biopsies from South Africa for the presence of HPV DNA using non-isotopic in situ hybridization (NISH) with HPV DNA probes to HPV 6, 11, 16, 18, 31, and 33. HPV DNA sequences were detected in 25/48 (52 per cent) oesophageal cancers. HPV 16 was present in 84 per cent of the HPV-positive cancers. A NISH type 2 signal pattern (punctate/dot) was present in all HPV-positive tumours. This signal pattern was previously shown to represent integrated HPV DNA within host chromosome. Integrated HPV DNA in oesophageal cancers has also been demonstrated in patients from China and Japan. In addition, the prevalence of HPV DNA in oesophageal cancers from high-risk countries like South Africa (52 per cent) and China (49 per cent) would appear to be consistent.

Frequent mutations of p53 gene in oesophageal squamous cell carcinomas with and without human papillomavirus (HPV) involvement suggest the dominant role of environmental carcinogens in oesophageal carcinogenesis.

Epidemiological evidence suggests that alcohol intake, use of tobacco, ingestion of mycotoxins and nitrosamines and nutritional deficiencies are high-risk factors for the development of oesophageal cancer. Similarly, viral infections have been postulated to play a role in some tumours. However, the molecular events underlying the development of oesophageal carcinoma are poorly understood as yet. Loss of p53 tumour-suppressor gene function has been found in different human malignancies, and it can occur in a variety of ways, including gene mutation and interaction with the E6 protein of oncogenic human papillomaviruses (HPVs). Because the oesophageal mucosa is potentially exposed to mutagens and HPVs, we studied DNA samples derived from nine HPV-positive squamous cell carcinomas and 12 HPV-negative tumours. Exons 5-9 of the p53 gene containing phylogenetically conserved domains were examined using the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique. HPV detection was done using DNA in situ hybridisation with biotin-labelled HPV DNA probes. Mutations were detected in eight (38%) out of the 21 cases. Three mutations were found in exons 5/6, three in exon 7 and two in exon 8/9. Six (50%) of the 12 HPV-negative carcinomas showed p53 mutations. Two (22.2%) of the nine HPV-positive carcinomas were found to contain p53 mutations as well; one contained HPV 16 DNA sequences and showed p53 mutation in exon 8/9, and the other was HPV 6/11 positive with the mutation in exon 5/6. Although mutations were more common in HPV-negative tumours (50.0% vs 22.2%), the difference in p53 mutations in HPV-positive and -negative tumours did not reach statistical significance (P = 0.1946). These data indicate that inactivation of the p53 gene is a frequent event in oesophageal squamous cell carcinomas and such an inactivation might be an important molecular pathway for the development of oesophageal cancer. The findings of p53 mutations in HPV-positive oesophageal carcinomas suggest that HPV and p53 mutation were not mutually exclusive events. The presence of frequent mutations of p53 gene in both HPV-positive and -negative oesophageal carcinomas suggests a dominant role of environmental carcinogens in oesophageal carcinogenesis.

HPV typing of cervical squamous lesions by in situ HPV DNA hybridization: influence of HPV type and therapy on the follow-up of low-grade squamous cervical disease.

Papanicolaou (Pap)-stained cervical specimens from 160 squamous lesions were processed for the detection of human papillomavirus (HPV) DNA by an in situ hybridization (ISH) assay. Three biotinylated HPV DNA probes were employed, each containing HPV genotypes 6/11, HPV genotypes 16/18, or HPV genotypes 31/35/51. The HPV etiology of 86 lesions was ascertained (53.8%). In 74 out of 135 (58.8%) HPV-typed low-grade squamous intraepithelial lesions (SILs), HPV 6/11 was found in nine (6.6%), HPV 16/18 in 46 (34.2%), and HPV 31/35/51 in 19 lesions (14.1%); in 11 out of 18 HPV-typed high-grade SILs (61.1%), seven lesions (38.9%) were typed for HPV 16/18 and four (22.2%) for HPV 31/35/51. Of seven invasive carcinomas, only one (14.3%) reacted with the HPV 16/18 DNA probe. A cohort of 124 low-grade SILs were followed cytologically for a year. The results of this study are discussed in light of HPV type association and therapy.

Screening for human papillomavirus infections in esophageal squamous cell carcinomas by in situ hybridization.

Infections with specific types of human papillomavirus (HPV) have been closely linked with human squamous cell carcinomas, those of the anogenital tract in particular. Increasing number of reports also suggest that HPV infection could be a risk factor for esophageal cancer. However, most of the previous studies on HPV involvement in esophageal carcinomas have included only small numbers of biopsy specimens, thus necessitating additional studies based on extensive series of esophageal samples. A series of 776 biopsy specimens derived from 363 patients who had undergone esophagectomy for squamous cell carcinoma in the high-incidence area of China were analyzed for the presence of HPV DNA by screening and specific typing in situ hybridization with biotinylated HPV DNA probes. Under low-stringency conditions, 85 (23.4%) tumors were demonstrated to contain HPV DNA: Positive signals were found on the nuclei of cancer cells in 71 (19.6%), in the surrounding epithelial cells with hyperplastic or dysplastic changes in 13 (3.6%), in the cancer cells and the surrounding epithelial cells in 10 (2.8%), and in the resected margins in 1 (0.3%). Thirty-four (40%) of the 85 HPV-positive tumors were shown to contain at least one type of HPV 6, 11, 16, 18, or 30 DNA sequences. HPV 16 was the type found most frequently, occurring in 18.8% of the 85 HPV-positive specimens. In addition to the primary tumors, HPV DNA sequences were found in 12.3% (7 of 57) of the lymph node metastases. The results confirm the previously reported HPV involvement in esophageal squamous cell lesions and implicate HPV as a potential etiologic agent in the multifactorial pathogenesis of esophageal carcinoma.

The detection of human papillomaviruses in cervical biopsies by immunohistochemistry and in situ hybridization.

The presence of human papillomavirus (HPV) types 6, 16 and 18 in cervical biopsies can be detected by an immunoperoxidase technique using type-restricted monoclonal antibodies raised against fusion proteins representing the L1 major capsid proteins of these three HPV types. In a retrospective study (n = 54) we have used these antibodies and biotinylated DNA probes of HPV 6, 16 and 18 to detect and type HPV in formalin-fixed material from the cervix. The biopsies were classified histologically into normals, wart infections without dysplasia, cervical intraepithelial neoplasia (CIN) and squamous cell carcinomas. Antibody staining showed that 22% of all CIN was positive for HPV 16 and 40% of cervical warts were positive for HPV 6, 16 and 18. There was no HPV capsid protein detected in the normals and squamous cell carcinomas using these antibodies, whereas 25% of the tumours were positive for HPV 16 by in situ hybridization. Sections of cervical warts and CIN positive for HPV types by in situ hybridization were also positive by antibody staining which suggests that both techniques are detecting replicating virus. We feel these two techniques complement each other in detection and typing of HPV in cervical biopsies from patients with active disease.

Southern blot hybridization and PCR in detection of oral human papillomavirus (HPV) infections in women with genital HPV infections.

The presence of human papillomavirus (HPV) in biopsies taken from clinically normal buccal mucosa (n = 212) and clinical lesions (n = 60) was examined by Southern blot hybridization (SBH) using 32P-labelled HPV DNA probes. Furthermore, one hundred formalin-fixed, paraffin-embedded biopsies were analyzed by using polymerase chain reaction (PCR), combined with dot blot hybridization and biotinylated HPV DNA probes. With SBH and PCR, 15.4% and 29.4% of the biopsies, respectively, contained HPV DNA. In clinically normal epithelium, 15.6% and 23.1% of the samples were HPV-positive with SBH and PCR, respectively. The HPV types detected in the genital and oral mucosa of index patients differed in all except two cases. Histology could not be relied on distinguishing HPV DNA positive and HPV DNA negative samples. Hand warts were encountered significantly more frequently in patients with a concomitant oral HPV infection. To conclude, oral HPV infections as detected by SBH and PCR are surprisingly common, but similar to the genital tract, the virus seems to exist in a latent form in the vast majority of cases. The frequent concomitant finding of skin warts and oral HPV infection may suggest some kind of HPV-specific immunosuppression.

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminata by in situ hybridization with biotinylated DNA probes.

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with biotinylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Dot blot hybridization in detection of human papillomavirus (HPV) infections in the oral cavity of women with genital HPV infections.

The presence of human papillomavirus (HPV) types 2, 6, 7, 11, 13 and 16 DNA in cytologic scrapings of oral mucosa was studied in 309 women with genital HPV infections. The objective was to test the usefulness of oral mucosal scrapings (3 sequential swabs) in HPV DNA detection by dot blot hybridization. Based on hybridization with the 32P-labelled Alu-repeat probe, most samples contained more than 10(5) cells, which is an adequate number of cells for dot blot hybridization. Hybridization with 32P-labelled HPV DNA probes showed that 3.8% of the 309 women had an oral HPV infection. Of these, only 2 had clinical lesions indicative of HPV. All other oral HPV positive subjects had clinically healthy mucosa. HPV 6 was the most common (3.1%) type, followed by HPV 11 and 16 (1.1%). In 3 cases the genital mucosa harboured the same HPV type as found in the oral cavity. The results indicate that oral mucosal scraping results in adequate number of cells for dot blot hybridization with HPV DNA. Although the method is likely to result in an underestimation of latent and subclinical HPV infections, it is useful for studying the clinical HPV infections as well as other viral infections known to be present in exfoliated cells.