How is human papillomavirus (HPV) testing performed?

How to Perform HPV Testing

HPV testing is performed using FDA-cleared molecular assays that detect viral DNA or messenger RNA from cervical specimens collected via clinician-obtained or self-collected samples, with specific test selection depending on the clinical indication (primary screening, cotesting, or triage). 1

Specimen Collection Methods

Cervical Specimen Collection

• Clinician-collected cervical samples remain the standard approach
• Specimens can be collected using:
  • The same swab for both Pap test and HPV DNA testing
  • A supplied swab at the time of the Pap test (for conventional cytology)
  • Reflex testing from liquid-based cytology vials if sufficient residual material exists
  • A separate follow-up appointment when Pap results are available 2

Self-Collection

• Self-collected cervicovaginal samples and first-void urine are emerging as credible alternatives, particularly for populations with low screening rates
• Important caveat: Self-collection for HPV testing is not FDA-cleared or recommended by U.S. medical organizations as of 2021, though this represents a potential future strategy 1

FDA-Cleared Testing Methods

Tests Approved for Primary Screening

Only two tests are FDA-cleared for primary cervical cancer screening 1:

1. Cobas 4800 HPV test (Roche Molecular Diagnostics)

   • Detects 14 oncogenic HPV types (16,18,31,33,35,39,45,51,52,56,58,59,66,68)
   • Provides individual genotyping for types 16 and 18

2. Onclarity HPV test (Becton Dickinson)

   • Same detection profile as Cobas 4800

Tests for Cotesting or Triage Only

These should NOT be used for primary HPV screening 1:

• Hybrid Capture 2 High-Risk HPV DNA test (Qiagen)
• Cervista HPV High-Risk DNA and HPV 16/18 DNA tests (Hologics)
• APTIMA HR HPV test (Gen Probe)

The distinction is critical—using non-approved tests for primary screening violates FDA clearance and may compromise patient care.

Technical Methodology

Detection Principles

• FDA-cleared tests detect viral DNA or messenger RNA through molecular techniques 1
• Methods include:
  • Non-amplified hybridization (less common clinically)
  • Signal amplification (Hybrid Capture 2 method—most widely used historically at 42.4% of laboratories) 3
  • Target amplification (PCR-based methods—increasingly common)
  • Real-time quantitative PCR for viral load determination 4

Laboratory Requirements

Testing must be performed in appropriately equipped settings with 5, 6:

• Standardized specimen handling protocols
• Rigorous internal and external quality control systems
• Clear documentation of HPV 16/18 status in positive cases
• Participation in proficiency testing programs (most use CAP Human Papillomavirus for Cytology Program) 3

Clinical Indications for HPV Testing

When to Use HPV Testing 1:

• Primary cervical cancer screening (ages ≥25-30 years, depending on guidelines)
• Cotesting with cytology (women aged ≥30 years)
• Triage of abnormal cytology (ASC-US, ASC-H, AGC, LSIL)
• Follow-up after abnormal screening results
• Post-colposcopy surveillance when no CIN 2/3 found
• Post-treatment monitoring of cervical precancers

When NOT to Use HPV Testing 1:

• Deciding whether to vaccinate against HPV
• Testing for low-risk (nononcogenic) HPV types (e.g., types 6 and 11)
• Managing persons with genital warts or their partners
• Routine screening in persons aged <25 years (exception: HIV-positive adolescents)
• Testing oral or anal specimens (not FDA-cleared for these sites)

Age-Specific Screening Algorithms

Ages 21-29 Years

• Cytology alone is recommended
• HPV testing should not be performed as part of routine screening 1
• HPV testing acceptable only for ASC-US triage

Ages ≥30 Years

Two acceptable strategies 1:

1. Primary HPV testing every 3-5 years (using FDA-cleared tests for primary screening)
2. Cotesting (cytology + HPV) every 3-5 years

Special Population: HIV-Positive Patients

• Use cytology, not primary HPV testing 1
• Cotesting acceptable for ages ≥30 years
• Annual screening initially; can extend to every 3 years after 3 consecutive normal results

Common Pitfalls to Avoid

1. Using non-approved tests for primary screening: Hybrid Capture 2, Cervista, and APTIMA are only cleared for cotesting/triage, not primary screening
2. Testing patients <25 years routinely: High HPV prevalence with high regression rates makes this inappropriate (except HIV-positive adolescents)
3. Testing for low-risk HPV types: Clinically not useful and not recommended
4. Attempting to use HPV tests on oral/anal specimens: Not FDA-cleared for these applications
5. Performing HPV testing to guide vaccination decisions: Inappropriate indication

Result Interpretation and Follow-Up

• Negative HPV test: Return to routine screening intervals (typically 3-5 years for primary HPV or cotesting)
• Positive HPV test: Management depends on specific genotype and cytology results
  • HPV 16/18 positive: Immediate colposcopy typically indicated
  • Other high-risk types positive: Follow 2019 ASCCP Risk-Based Management Guidelines 1
• Follow-up should be based on risk for CIN 3, not just specific test results 1

The shift toward molecular HPV testing represents a more sensitive and objective approach than cytology alone, with the critical requirement being proper test selection for the intended clinical application.