MOLECULAR (DNA) DIAGNOSIS OF ANOGENITAL HPV INFECTION

hrHPV testing is currently only used in the management and follow-up of women with squamous lesions because glandular lesions have a lower hrHPV positivity rate than squamous lesions.

Introduction

Molecular diagnosis of HPV relies on detection of viral DNA. A variety of DNA detection methods are available.

Polymerase chain reaction amplification (PCR) of a short region of specific HPV DNA is probably the most sensitive method.(88)

hrHPV testing was introduced in New Zealand to the NCSP management pathway in October 2009 as an adjunct to cervical cytology in specific clinical situations (key points above). Two internationally validated PCR-based commercial tests are currently being used by National Cervical Screening Programme (NCSP) approved laboratories.

hrHPV testing has been repeatedly found to have a high negative predictive value (~99%). This means that women with a negative hrHPV test are most unlikely to have any of previously listed 14 hrHPV types and are most unlikely to progress to a pre-cancerous abnormality. It is the strong negative predictive value of hrHPV testing that has the most effective clinical use in conjunction with cervical cytology.

hrHPV testing is more sensitive for detecting high-grade cervical abnormalities than LBC cytology. Cytology is more specific because it identifies actual cell abnormalities whereas HPV testing identifies the presence of infection, not cell changes. A positive hrHPV test indicates increased risk of developing an abnormality. hrHPV testing is performed in conjunction with cytology using liquid-based cytology (LBC). This allows testing for both cytology and hrHPV on one cervical sample.

HPV testing in the National Cervical Screening Programme (NCSP)

(The full NCSP Guidelines can be accessed at www.nsu.govt.nz/health-professionals/national-cervical-screening-programme/ cervical-screening-guidelines).

The testing laboratory relies on the sample taker discussing and gaining consent from the woman for possible hrHPV testing.

The areas where the management of women with abnormal cervical samples within the NCSP may benefit from hrHPV testing include the following:

1. The triage of women 30 years and over with ASC-US or LSIL cytology (without an abnormal cytology sample in the last 5 years). 
(See Chart 1 below.)

• ‘Reflex’ testing is ordered by the laboratory for women with ASC-US/LSIL and eliminates the need for repeat cytology testing for those women who are hrHPV detected.
• A negative hrHPV test can reassure a woman that she is very unlikely to have a significant lesion and can be safely followed by a repeat cytology test in 12 months. If the repeat cytology result at 12 months is normal, she can return to regular 3-yearly screening.
  
• Women with ASC-US/LSIL smears who test positive for hrHPV should be referred for colposcopy.

2. The follow-up of women who have been treated for a histologically confirmed high-grade squamous lesion (CIN2/3)

– Test of Cure. (See Chart 2 below.)

• Women who have been previously treated for histologically confirmed CIN-2/3 are at a small increased risk of further disease and cervical cancer. Recurrence may be due to limitations of colposcopy, inadequate treatment/persistent disease or new infection.
  Cotesting using cytology and hrHPV testing as a combined test allows better identification of women at risk of persistent or recurrent lesions,      while enabling many women to return to regular 3-yearly screening.
  
• Women treated for CIN-2/3 currently undergo follow-up colposcopy and cytology within 6-12 months after treatment. It should be noted that hrHPV testing should not be carried out sooner than 12 months after treatment of high-grade lesions, as viral clearance may take more than 12 months to occur.
  
• hrHPV testing and cytology should be carried out 12 months after treatment and annually thereafter until a woman has tested negative by both tests on two consecutive occasions, 12 months apart. A woman can then return to regular three-yearly screening.
  
• Women who test hrHPV positive, despite having cytology assessed as negative post-colposcopy, should be referred back to colposcopy. The reason for this is the risk of residual disease requiring assessment and further treatment, despite the apparently negative cytology
  
• It is the sample taker’s responsibility to order hrHPV testing in this situation.

As indicated in flowchart HPV Testing Guidance 2 (pages 51 and 52 of Guidelines for Cervical Screening in New Zealand). (See Chart 2 below.)

3. Women with high-grade lesions (cytology HSIL/ASC-H; histology CIN2/3) more than 3 years previously, treated or untreated and currently managed by cytology alone (Test of Cure – historical testing). (See Chart 3 below.)

It is the smear taker’s responsibility to order hrHPV testing in this situation.

• This group of women, currently managed by annual cytology for more than 3 years with all tests assessed as negative, can be managed with cotesting (cytology and hrHPV) to ascertain if they can safely return to regular 3-yearly screening.
  
• hrHPV testing and cytology should be carried out and annually thereafter until a woman has tested negative by both tests on two consecutive occasions, 12 months apart. A woman can then return to regular 3-yearly screening.
• It is the sample taker’s responsibility to gain consent and order hrHPV testing in this situation.
  
• Those women who test positive for hrHPV despite repeated negative cytology are likely to have a low risk of CIN-2/3 and can be retested with cytology and hrHPV annually thereafter, until a woman has tested negative by both tests on two consecutive occasions, 12 months apart. Persistent hrHPV test positivity can be discussed with a colposcopist and referral can occur if deemed appropriate.
  
• Refer to colposcopy for any clinical concern or an abnormal cytology result.

4. Post-colposcopy management of women with discordant results e.g. high-grade cytology and negative, satisfactory colposcopy.

It is the colposcopist/specialist responsibility to order hrHPV testing in this situation.

• A single colposcopic examination can miss significant lesions.
  
• Where findings on colposcopy/histology are negative or show low-grade changes only and the discordance persists following case review, hrHPV testing can be a useful adjunct to further management.
  
• The NCSP recommends a woman return to 3-yearly screening only after two negative sets of hrHPV plus cytology tests 12 months apart.
  
• Failure to detect CIN-2/3 lesions in a woman with high-grade cytology (following review of the smear) should lead to consideration of a diagnostic excisional procedure, or observation for 1 year with colposcopy, cytology and HPV testing.
  
• Specialist colposcopists are responsible for ordering hrHPV testing in this situation.

Note:

• Women with a history of genital warts do not need to begin screening at an earlier age or have screening more frequently.
  
• hrHPV testing should be used for squamous lesions (as indicated in the HPV flowcharts) but is not currently recommended for the follow-up of glandular abnormalities. For historical testing situations, it is therefore very important that the sample taker identifies whether a previous abnormality was squamous or glandular. This is particularly important for women who were investigated or treated overseas, as their results may not be held on the New Zealand NCSP Register. 

Samples required for hrHPV DNA detection and typing

1. Specimens are collected using a broom-type collection device and either:

• vigorously rinsed in the sample vial fluid if using ThinPrep (do not leave the broom head in ThinPrep vial), or
  
• the head of the device must be detached and placed into the vial fluid if using SurePath.

2. Liquid-based cytology sample (ThinPrep or SurePath) are sent directly to the laboratory by the sample taker. The sample needs to be taken and sent in accordance with your local laboratory protocol.

3. If lubrication is necessary, NCSP recommends use of warm water where possible or lubricant sparingly away from the opening end/tip of the speculum (use waterbased gel ) because lubricant can mask cells limiting or making LBC sample unsatisfactory for testing. This is particularly important when using ThinPrep LBC.

NB: The following charts are taken from NCSP Guidelines, see www.nsu.govt.nz/health-professionals/nationalcervical-screening-programme/cervical-screening-guidelines.