Current available hpv molecular testing, detailed overview. Using tables wherever required.
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HPV molecular testing assays 2024 2025 FDA approved genotyping PCR overview
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HPV Molecular Testing: Detailed Overview
Human papillomavirus (HPV) molecular testing has become the cornerstone of cervical cancer screening and is increasingly important in head and neck oncology. Testing falls into two broad categories: tests for HPV nucleic acid (DNA or mRNA) and surrogate marker assays.
1. Background: Why Molecular Testing?
HPV is a double-stranded DNA virus. Its genome contains early genes (E1, E2, E4, E5, E6, E7) and late genes (L1, L2). The oncoproteins E6 and E7 drive malignant transformation - E6 degrades p53 via ubiquitin ligase E6AP, and E7 degrades retinoblastoma protein (pRb) via the cullin-2 complex. This pRb degradation releases E2F repression, causing overexpression of p16INK4a, which forms the basis of a key surrogate test.
Simply detecting HPV DNA is insufficient by itself - a positive result may reflect latent, bystander, or cleared infection. The goal of molecular testing is to detect biologically active HPV driving oncogenesis.
2. HPV Genotype Classification (Oncogenicity)
| Risk Category | HPV Genotypes |
|---|---|
| High-risk (HR) | 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 67, 68 |
| Probable high-risk | 26, 51, 53, 56, 66, 69, 82 |
| Low-risk (LR) | 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 74, 81, 83, 84 |
HPV-16 and HPV-18 together account for ~70% of cervical cancers worldwide. HPV-16 also drives the majority of HPV-positive oropharyngeal squamous cell carcinomas (OPSCC).
3. Categories of Molecular Testing
A. Signal Amplification Assays (Hybrid Capture)
| Feature | Detail |
|---|---|
| Assay name | Hybrid Capture 2 (HC2) - Digene/Qiagen |
| Nucleic acid target | HPV DNA |
| Mechanism | Solution hybridization: viral DNA + viral-specific RNA probes → RNA:DNA hybrids → captured by antibodies specific for RNA:DNA hybrids → signaling antibody conjugated to alkaline phosphatase → chemiluminescence measured as relative light units (RLUs) |
| Genotypes detected | 14 types (HR 16, 18, 31, 33, 35, 45, 51, 52, 56 + LR 6, 11, 42, 43, 44) |
| Genotyping | No (detects HR pool only) |
| FDA indications | ASC-US triage, Adjunct co-testing |
| Specimen types | SurePath, ThinPrep (PreservCyt) |
| Notes | First FDA-approved HPV test; no internal adequacy control - inadequate specimens can read as false negative; slightly lower sensitivity vs PCR-based assays |
B. Real-Time PCR-Based Assays
Cobas HPV Test (Roche Molecular Diagnostics)
| Feature | Detail |
|---|---|
| Nucleic acid target | HPV DNA (L1 gene region) |
| Mechanism | Automated sample prep + simultaneous real-time PCR amplification and detection; uses β-globin as internal adequacy control |
| Genotypes detected | 14 HR types: HPV 16 and 18 reported individually, plus 12 other HR types as a pool (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) |
| Genotyping | Partial - HPV 16 and 18 individually |
| FDA indications | ASC-US triage, Adjunct co-testing, Primary HPV screening (first approved 2014 for primary screening) |
| Specimen types | SurePath AND ThinPrep (PreservCyt) |
| Self-collection | FDA-approved May 2024 for self-collection in clinical settings |
| Key trial | ATHENA study - validated performance for primary screening |
BD Onclarity HPV Assay (Becton, Dickinson)
| Feature | Detail |
|---|---|
| Nucleic acid target | HPV DNA (E6/E7 gene region) |
| Mechanism | Real-time PCR |
| Genotypes detected | 14 HR types: HPV 16, 18, 45 each individually reported; 11 additional types in sub-pools (31/33/52/58; 51/56/66; 35/39/68) |
| Genotyping | Extended - HPV 16, 18, AND 45 individually |
| FDA indications | ASC-US triage, Primary HPV screening (approved 2018) |
| Specimen types | SurePath (and Specimen Transport Media - STM); also ThinPrep per expanded indications |
| Self-collection | FDA-approved May 2024 for self-collection in clinical settings |
Alinity m HR HPV Assay (Abbott Molecular Diagnostics)
| Feature | Detail |
|---|---|
| Nucleic acid target | HPV DNA |
| Mechanism | Real-time PCR on the Alinity m fully-automated platform |
| Genotypes detected | 14 HR types with HPV 16 and 18 individually identified |
| Genotyping | Partial - HPV 16 and 18 individually |
| FDA indications | Primary HPV screening |
| Notes | Among the three currently FDA-approved assays for primary screening (Harrison's 22E, 2025) |
C. Transcription-Mediated Amplification (TMA) / RNA-Based Assays
Aptima HPV Assay (Hologic)
| Feature | Detail |
|---|---|
| Nucleic acid target | E6/E7 mRNA (oncogene transcripts) |
| Mechanism | Transcription-mediated amplification (TMA) of HR-HPV E6/E7 mRNA |
| Genotypes detected | 14 HR HPV types as pooled result - does not differentiate individual types |
| Genotyping | No (pool only) |
| FDA indications | ASC-US triage, Adjunct co-testing |
| Specimen types | ThinPrep (PreservCyt) only |
| Key advantage | Detects mRNA, not DNA - reflects active transcription, more specific for clinically significant infection; large amounts of mRNA are not present during viral latency, reducing over-detection of clinically irrelevant infections |
Aptima HPV 16 18/45 Genotype Assay (Hologic)
| Feature | Detail |
|---|---|
| Nucleic acid target | E6/E7 mRNA |
| Mechanism | TMA - reflex genotyping test |
| Genotypes | HPV 16 individually; HPV 18/45 combined |
| Use | Used adjunctively with Aptima HPV (on Aptima-positive samples) |
| FDA approval | 2012 |
D. Probe-Based Assays / In-Situ Hybridization (ISH)
| Assay Type | Target | Specimen | Notes |
|---|---|---|---|
| Chromogenic ISH (CISH) - High-risk HPV | HPV DNA (L1 gene sequences) | FFPE tissue | Commercially available kits for FFPE; blue dot signal under light microscopy; punctate pattern suggests integration; does not prove active oncogenic activity |
| Fluorescent ISH (FISH) | HPV DNA | FFPE tissue | Allows nuclear localization; less used clinically |
| RNA-ISH (RNAscope) | HPV E6/E7 mRNA | FFPE tissue | Overcomes dual-testing requirement; high sensitivity (97%) and specificity (93%) vs gold-standard qRT-PCR; commercial kits contain probes for E6/E7 mRNA of multiple HR genotypes; increasingly replacing PCR in routine FFPE analysis |
E. Cervista Assays (Hologic)
| Feature | Detail |
|---|---|
| Cervista HPV HR | Detects 14 HR types as pooled result; uses Invader chemistry (signal amplification); FDA-approved for ASC-US triage and co-testing |
| Cervista HPV 16/18 | Reflex genotyping for HPV 16 and 18 individually; used adjunctively with Cervista HPV HR |
| Specimen types | ThinPrep (PreservCyt) only |
4. Summary Table: FDA-Approved HR-HPV Assays
| Assay | Manufacturer | Technology | Nucleic Acid | Genotyping | ASC-US Triage | Adjunct (Co-test) | Primary Screening | Specimen |
|---|---|---|---|---|---|---|---|---|
| Digene HC2 | Qiagen | Signal amplification (hybrid capture) | DNA | None | Yes | Yes | No | PC, SP |
| Cervista HPV HR | Hologic | Invader signal amplification | DNA | None | Yes | Yes | No | PC only |
| Cervista HPV 16/18 | Hologic | Invader signal amplification | DNA | 16, 18 | Reflex only | Reflex only | No | PC only |
| Aptima HPV | Hologic | TMA | mRNA (E6/E7) | None | Yes | Yes | No | PC only |
| Aptima HPV 16 18/45 | Hologic | TMA | mRNA (E6/E7) | 16; 18/45 | Reflex only | Reflex only | No | PC only |
| Cobas HPV | Roche | Real-time PCR | DNA | 16, 18 separately | Yes | Yes | Yes | PC + SP |
| BD Onclarity | BD | Real-time PCR | DNA (E6/E7) | 16, 18, 45 individually | Yes | Yes | Yes | SP + STM |
| Alinity m HR HPV | Abbott | Real-time PCR | DNA | 16, 18 separately | Yes | Yes | Yes | PC |
PC = PreservCyt/ThinPrep; SP = SurePath; STM = Specimen Transport Media
5. Non-Commercial / Research-Use Molecular Methods
Consensus PCR (L1 Gene)
- Amplifies the highly conserved L1 capsid gene using degenerate primers
- Common primer systems:
- MY09/11 / PGMY - amplifies ~455 bp fragment
- GP5+/6+ - amplifies ~150 bp fragment
- SPF10 - amplifies <100 bp fragment (useful in degraded DNA)
- Results in HPV detection + genotyping by sequencing or reverse-line blot hybridization
- Most validated methodology for identifying and characterizing papillomaviruses
- Used widely in research and as the analytical comparator in FDA evaluations
qRT-PCR for E6/E7 mRNA
- Gold standard for demonstrating biological activity of HPV infection
- Quantifies E6 or E7 mRNA transcripts via cDNA synthesis followed by PCR amplification
- Historically restricted to frozen tissue; technically demanding and costly
- Key limitation: not readily applied to FFPE; labor-intensive; not suited for routine clinical use
Integration Detection Assays
- APOT (Amplification of Papillomavirus Oncogene Transcripts) - distinguishes mRNA from integrated vs episomal viral genomes based on structural differences at the 3' end of viral transcripts
- DIPS (Detection of Integrated Papillomavirus Sequences) - PCR-based detection of viral-cellular junctions
- Southern blotting - original method; radioactive probe hybridization; high quality data but technically demanding, low throughput
6. Surrogate Marker Assay: p16INK4a Immunohistochemistry (IHC)
| Feature | Detail |
|---|---|
| Principle | E7 degrades pRb → releases pRb-mediated repression of p16INK4a → overexpression of p16INK4a |
| Interpretation | Strong, diffuse nuclear AND cytoplasmic staining in >70% of malignant cells = positive |
| Sensitivity | High for transcriptionally active HR-HPV |
| Specificity | Limited - p16 can be overexpressed by non-HPV mechanisms (e.g., RB mutation) in 10-15% of cases |
| Advantages | Cheap, widely available, easy to perform on FFPE, well-established protocols |
| Clinical use | Primarily for oropharyngeal SCC (OPSCC) - not considered valid surrogate outside the oropharynx |
| AJC C 8th edition | Separate staging systems for p16-positive and p16-negative OPSCC |
7. Diagnostic Algorithm (Head and Neck / OPSCC)
The Johns Hopkins algorithm (Westra) is the most widely accepted approach for OPSCC:
FFPE tissue
↓
p16 IHC (screening)
↓
p16 POSITIVE
↓
HPV-16-specific ISH
↓ ↓
HPV-16+/p16+ HPV-16–/p16+ (minority)
→ HPV-16+ve → Broad HR-HPV ISH (other genotypes)
↓
+ or – → assign HPV status
This two-step algorithm is necessary because of the variable sensitivity and specificity of individual tests and the requirement to demonstrate biologically active HPV infection.
8. Cervical Cancer Screening Algorithms (Clinical Use)
| Age Group | Recommended Strategy |
|---|---|
| 21-29 years | Cytology (Pap) every 3 years; no HPV co-testing (young women often HPV DNA+ but at very low cancer risk) |
| 25-29 years | HPV DNA co-testing if cytology shows ASCUS |
| 30-65 years | Co-testing (Pap + HPV DNA) every 5 years (preferred), OR cytology alone every 3 years, OR primary HPV testing every 5 years |
| >65 years | Cessation of screening if adequate prior negative screening history |
| HIV-positive women <30 | Cytology preferred; HPV co-testing not recommended |
| HIV-positive women ≥30 | Cytology + HPV DNA co-testing acceptable |
Management of positive HPV results:
- HPV 16 or 18 positive → direct colposcopy (high positive predictive value for CIN 2+; risk ~11.4% for CIN 2+ vs 6.1% for other HR types per ATHENA trial)
- Other HR HPV types positive + normal cytology → retest in 1 year
9. Self-Collection (FDA-Approved, May 2024)
Two self-collection options gained FDA approval in May 2024 for use in clinical settings (in-clinic self-collection, not home testing):
- cobas HPV Test (Roche) - vaginal self-swab by patient; specimen processed in lab same as provider-collected sample
- BD Onclarity HPV Assay (BD) - vaginal self-swab; similar workflow
This expands access to screening in primary care, urgent care, pharmacies, and mobile clinics.
10. Key Performance Considerations
| Parameter | DNA Assays | mRNA Assays (Aptima) |
|---|---|---|
| Sensitivity for CIN 2+ | Very high (~94-97%) | Slightly lower sensitivity, higher specificity |
| Specificity | Lower (detects latent/cleared DNA) | Higher (detects active transcription only) |
| Risk of over-referral | Higher | Lower |
| Use in FFPE | Some assays (PCR-based) | RNA-ISH preferred for FFPE |
| Genotyping capability | Variable by platform | Aptima HPV 16 18/45 available as reflex |
11. Non-Cervical Applications
| Site | Testing Approach |
|---|---|
| Oropharynx (OPSCC) | p16 IHC (surrogate) + confirmatory HR-HPV ISH or PCR; RNA-ISH (RNAscope) increasingly used |
| Anal canal | DNA-based HPV testing; anal cytology + HPV accepted in high-risk groups (MSM, HIV+) |
| Vulva/Vagina | Cytology + HPV DNA testing as per cervical protocols |
| Penile | No established molecular screening guideline; biopsy + p16 IHC used diagnostically |
Sources: Tietz Textbook of Laboratory Medicine 7th Ed. | Harrison's Principles of Internal Medicine 22E (2025) | Scott-Brown's Otorhinolaryngology Head & Neck Surgery | Henry's Clinical Diagnosis and Management by Laboratory Methods | Cummings Otolaryngology | FDA HPV Device Summary | PMC review of HPV molecular tests
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