Human Papillomavirus Typing by Polymerase Chain Reaction-Reverse Dot Blot Hybridization and Comparison with Hybrid Capture SystemTM

Yoon, Young Ho; Choi, Sun Ju; Yoo, Ki Sook; Ko, Chang Won; Kim, Young Tak

Abstract

BACKGROUND: Infection with certain Human Papillomavirus (HPV) type is strongly associated with the development of dysplasia and cancer of the cervix uteri. About 70 HPV types have been identified and some 25 of these have been found in the genital tract. HPV typing has diagnostic and prognostic importance to discriminate between 'low', 'intermediate', 'high' risk types. A Polymerase Chain Reaction-Reverse Dot Blot Hybridization (PCR-RDBH) method was developed for typing of HPV with consensus biotinylated primer generated PCR products in a single test. We attempted to know the clinical usefulness of PCR-RDBH and also compared PCR-RDBH with Hybrid captureTM system (HCS) method in same specimens.
METHODS
HPV typing was performed on cervical swab samples obtained from 100 women with abnormal cervical cytology: 37 with Atypical Squamous Cells of Undetermined Significance (ASCUS), 14 with Low Grade Squamous Intraepithelial Lesions (LGSIL), 44 with High Grade Squamous Intraepithelial Lesions (HGSIL) and 5 with invasive carcinoma of the uterine cervix. HPV PCR screening was tested with consensus biotinylated primer. If HPV PCR screening was positive, RDBH was done for the typing of HPV. In RDBH, biotinylated PCR product was used in hybridization with a membrane on which 12 different oligonucleotide probes (type 6/11/16/18/ 31/33/35/39/45/51/52/56) of genital HPV types had been immobilized. Hybrid captureTM system (HCS, Digene Diagnostics) was used for screening of HPV.
RESULTS
Of 100 abnormal cervical cytology specimens, the positivity of HPV PCR screening was 67%. In 52 specimens, HPV could be typed by RDBH. Type 16 was the most frequent and mixed infection was found in 6 cases, all combined with type 16. Among the 13 cervical cancer specimens confirmed by biopsy, 12 specimens was found to be infected high and intermediate risk types of HPV. In cervical swab, there was signifincant discrepancy in positivity of HPV infection between PCR-RDBH and HCS method. In 51 cases, negative for PCR-RDBH, 16 cases (31%) were positive by HCS. In 49 cases, positive for PCR-RDBH, 15 cases (31%) were negative by HCS.
CONCLUSIONS
PCR-RDBH method can do HPV typing fast and easy with non-radioactive biotinylated primer in cervical swab specimens. It is shown to be useful method for HPV typing and have a high clinical applicability. The results between PCR-RDBH and HCS methods show a significant discrepancy, so further investigation is needed.