Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation.

Distribution of human papillomavirus types in genital lesions from two temporally distinct populations determined by in situ hybridization.

We examined 341 paraffin-embedded cervical tissues for human papillomavirus (HPV) DNA by in situ hybridization. The genital lesions examined represented tissue biopsies from two temporally distinct populations (1964 to 1965 and 1988 to 1989). Biotinylated probes to 14 different HPV types were used in our analysis: HPV types 6, 11, 16, 18, 31, 33, 35, 42, 43, 44, 45, 51, 52, and 56. The number of HPV DNA-positive specimens and the distributions of HPV types were similar for these two populations. Human papillomavirus DNA sequences were detected in approximately 50% of the tissues from each time period. Of the low-grade lesions (condyloma/cervical intraepithelial neoplasia 1 [CIN 1]) 52% (1964 to 1965) and 35% (1988 to 1989) were positive for HPV DNA by in situ hybridization. Among the high-grade lesions (CIN 2/CIN 3), 41% (1964 to 1965) and 67% (1988 to 1989) had detectable HPV sequences. Approximately 15% of the tissues with minimal histopathologic changes also contained HPV DNA. Human papillomavirus types 16 and/or 18 were the most common viral types in lesions from both time periods, followed by types 31/33/35; 6/11, 51/52; and 42/43/44, 45/46. Types 16 and/or 18 were strongly associated with high-grade lesions. Five percent of the HPV-positive lesions demonstrated evidence of multiple infections. Our results indicate that HPV DNA sequences can be detected readily by in situ hybridization in archival materials, even those prepared more than 25 years ago. In addition, analysis of HPV type distributions demonstrates that recently isolated HPV types (42, 43, 44, 45, 51, 52, and 56) were equally represented in tissues from both time periods.

Comparative analysis of human papillomavirus detection by dot blot hybridisation and non-isotopic in situ hybridisation.

AIMS: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. METHODS: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. RESULTS: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). CONCLUSIONS: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.

Comparison of nucleic acid hybridization and cytologic examination for detection of human papillomavirus infection, with evaluation of two commercially available hybridization kits.

Human papillomavirus (HPV) infections were detected by analyzing exfoliated cervical cells for HPV DNA by use of nucleic acid hybridization; the results were correlated with cytologic findings on Papanicolaou smears. HPV infection was diagnosed in 154 women (20%) by either morphologic evidence on cervical smears or nucleic acid hybridization. Many of these women (38%; 58/154) exhibited Papanicolaou smears with no morphologic evidence of HPV infection. In those patients with cytologic evidence of HPV infection, only 28% were positive for HPV DNA. HPV 16 and (or) 18 were the most common types (27%) detected in women with cervical intraepithelial neoplasia, whereas all HPV groups tested were equally represented in patients with normal cervical smears. We also present an assessment of 17,000 clinical specimens submitted to this laboratory for analysis of HPV DNA.