Detection of HPV Infection
Replication of HPV is tightly linked to squamous epithelial cell differentiation with capsids being produced only in terminally differentiated squamous cells. For this reason, traditional techniques for culturing viruses cannot be employed for HPV. Further, due to their strict species specificity, it is not possible to easily propagate HPVs in normal laboratory animals. Clinical and colposcopic examination, as well as microscopic examination of exfoliated cell samples (pap smears) or tissue biopsies for koilocytes (cells whose nuclei demonstrate perinuclear clearing, with an increase in the density of the surrounding rim of cytoplasm) were used in the past for identification of HPV infections. However, these methods are both insensitive and non-specific. Recently, the production of "viral like particles" has allowed the development of serologic assays, but since it is believed that development of such antibodies occurs within 12 to 15 months after incident infection of HPV, detection of serum antibodies to HPV are of little interest for detection of acute infection.
Molecular detection of HPV DNA or RNA is currently the gold standard for identification of HPV. Three categories of molecular assays are available for detection of HPV infection in tissue and exfoliated cell samples. All are based on detection of HPV DNA and include: (1) non-amplified hybridization assays (Southern transfer hybridization, (STH), dot blot hybridization (DB) and in situ hybridization (ISH)); (2) Signal amplified hybridization assays such as hybrid capture assays; (3) Target amplification assays, such as PCR and in situ PCR. Southern blot hybridization (STH) and in situ hybridization (ISH) can be been used, but have serious shortcomings. STH requires large amounts of DNA, is laborious, and is not reproducible, while ISH has only moderate sensitivity for HPV. PCR based detection of HPV is both extremely sensitive and specific. Using this approach, the viral DNA is amplified in vitro by DNA polymerase to generate adequate amount of target, which is then either directly visualized on gels, or (the more specific approach) detected by specific probe using traditional hybridization methods. In theory, PCR is able to detect one copy of a target sequence in a given sample. In practice, the sensitivity of PCR based method is about 10-100 HPV viral genomes in a background of 100 ng cellular DNA. Since PCR can be performed on very small amounts of DNA (10-100 ng), it is ideal for use on specimens with low DNA content. Currently, the only available FDA approved HPV detection method is the Hybrid capture II assay. In this assay HPV DNAs are hybridized to RNA probes, and RNA-DNA hybrids are captured and detected by a chemiluminescent system. The sensitivity of this assay is similar to that of PCR based assays, with high sensitivity being achieved by signal, rather than target amplification. The current HC II has the sensitivity to detect 1pg HPV (about 50,000 copies) per ml sample. Proper sample collection is essential to achieve maximal sensitivity, and a brush-sampling device has been shown to be optimal. Although the assay allows one to differentiate between high and low risk types of HPV, individual HPV types cannot be identified without the purchase of additional special reagents.