Laboratory Methods for Detection of Human Papillomavirus Infection

Abstract

The diagnosis of human papillomavirus (HPV) infection can be inferred from morphological, serological, and clinical findings. In productive infections, such as warts, virus particles of about 50 nm diameter can be detected by electron microscopy and by immune detection of the virus capsid proteins (L1, L2). Immuno­logical detection of HPV in human cells or tissues has been hindered by three main factors: (a) the late, capsid proteins are only expressed in productive infections; (b) the early proteins are often expressed in low amounts in infected tissues; and (c) the lack of high-quality, sensitive, and specific antibodies against the viral proteins. Antibodies generated against bovine papillomavirus (BPV) late proteins have been widely used because of the observed cross-reaction with HPV late proteins; however, they have low sensitivity and fail to discriminate between HPV types, which is essential for disease risk determination. Detection of HPV early proteins is even more complicated due to the low expression levels generally observed in cells or tissues derived from HPV-positive lesions. Antibodies against E6 or E7 are available but their use is mostly restricted to in vitro assays including the direct visualization in cells or tissues (immunohistochemistry) or in protein extracts (Western blots and immune precipitation assays), not always with consistent results. HPV cannot be propagated in tissue culture and hence in most cases its accurate identification relies on molecular biology techniques. With a double-stranded DNA genome of about 8,000 base pairs (bp) in length and a well-known physical structure and gene organization, tests of choice for detecting HPV from clinical specimens are based on nucleic acid probe technology. Direct detection of HPV genomes and its transcripts can be achieved with hybridization procedures that include Southern and Northern blots, dot blots, in situ hybridization (ISH), Hybrid Capture (HC; formerly DIGENE Co., now QIAGEN, MA, USA), and DNA sequencing (reviewed in Iftner and Villa, 2003). A variety of signal-detection procedures are available that can further increase the sensitivity of these assays. The only procedure potentially capable of recognizing all HPV types and variants present in the biological specimen is DNA sequencing of the viral genome, either after cloning into plasmids or by direct sequencing of a polymerase chain reaction (PCR) fragment. This method, however, is presently labor-intensive and requires expensive equipment. Moreover, direct sequencing of specimens containing multiple HPVs awaits further developments.

Also contributed by Luisa Lina Villa, Ludwig Institute for Cancer Research, São Paulo, Brazil.