Detecting HPV

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Methods of detecting the Human Paplillomavirus

Over the years a wide range of methods have been developed for detecting human papillomavirus infection. The methods vary in their sensitivity and their ability to identify individual human papillomavirus types. Thus, some are appropriate for use in routine clinical practice (e.g.: colposcopy, cytology) while others (e.g.  immunocyto- histochemical methods, DNA hybridisation) have an important role in screening and research.

The methods and their uses and limitations are described below

Methods of detection of human papillomavirus infection

  1.  Examination of skin or other tissue with naked eye.
  2.  Magnified images of skin or mucous membrane  of the genital tract using the colposcope 
  3.  Morphological studies of histological sections or cervical smears
  4. Immunocytochemical staining of histological and cytological specimens
  5. Electron microscopy of warty lesions
  6. DNA hybridisation using PCR or Hybrid Capture 2

Classically, HPVs produce benign warty lesions of squamous epithelium which can be recognised macroscopically with the naked eye. The most common sites for these exophytic lesions are the skin (usually the hand) and the external genitalia. These lesions are highly infectious but are self limiting and usually regress spontaneously.

Colposcopy permits the detection of subclinical infections not normally visible to the naked eye. After the application of acetic acid the cervix assumes a shiny white appearance with an irregular outline and satellite lesions may be seen. Unless condylomatous changes are present the pattern may be difficult to distinguish from CIN  (Insert Fig 1 COLPSCOPY HPV CERVIX).

Histological specimens are characterised by the presence of koilocytosis, individual cell keratinisation, parakeratosis, acanthosis and multinucleation.

(Insert Fig 2 HISTOLOGY HPV CERVIX) These changes are reflected in the cytological specimens (Insert Fig3CYTOLOGY HPV CERVIX).  However neither colposcopy, histology, cytology nor immunological staining permit HPV typing or detection of virus integration and do not reveal latent infection.

(Insert Fig 4Fig 4 IMMUNOCYTOCHEMICAL STAINING OF CERVIX)

Electron microscopy can be used to demonstrate papillomavirus particles in scrapings of exophytic warts but the method is rarely used as complete virus particles are scanty. A satisfactory method of virus culture has not been developed.

DNA analysis has become the method of choice because of the limitations of other diagnostic method. The most satisfactory tests for HPV rely on the detection of viral DNA. Since all HPV types are closely related, assays can be   designed to target conserved regions of the genome to discriminate between different HPV types. Hybrid capture provides a viral profile of a number of different HPV types which can then be further identified using the polymerase chain reaction (PCR). PCR based method use a selection of consensus primers to identify specific  types of HPV. Hybrid Capture 2 (Digene Diagnostics, Gaithersburg, MD, USA) has recently been approved by the American Food and Drug Agency (FDA) as an adjunct to cytology screening of women aged 30 years and older. It is generally accepted that both PCR and HC2 reliably detect high –risk and other types of HPV in clinical samples

JOSHUA: Could you create a flow diagram to illustrate the key stage of Hybrid capture here. Also a flow diagram of PCR assay would be helpful. See text pp503

 (For further  reading see: Denny LA and Wright TC in Best Practice and Research in Clinical Obstetrics and  Gynaecology2005  vol 19, no4, pp501-505 Also available on line at www.sciencedirect.com ).