Human papilloma virus (HPV) is a common sexually transmitted double-stranded DNA virus that is responsible for more than 90% of cervical cancers. Each year in the US, approximately 100 000 people are treated for cervical precancer, 1, 000 people are diagnosed with cervical cancer, and 4,000 die of cervical cancer.
The HPV viral genome is a double-stranded circular DNA that consists of approximately 7900 base pairs. The genome has eight overlapping open reading frames. There are six early (E) genes, two late (L) genes, and one untranslated long control region. The L1 and L2 genes encode the major and minor capsid proteins. Early genes regulate HPV viral replication. The E6 and E7 genes of high-risk HPV genotypes are known oncogenes. Proteins expressed from E6/E7 polycistronic mRNA alter cellular p53 and retinoblastoma protein functions, leading to disruption of cell-cycle check points and cell genome instability.
More than 450 genotypes of HPV have been identified but virtually all cervical cancers contain at least 1 of 13 carcinogenic HPV genotypes: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.
HPV infection causes more than 90% of cervical and anal cancer cases, 70% of cancers involving the vulva and vagina, 60% of cancers involving the penis, and 65% of cancers in the oropharynx.
HPV-16 is the most carcinogenic and is associated with more than 60% of cervical squamous cancers and adenocarcinomas and with oropharyngeal and other anogenital cancers. The other 12 carcinogenic HPV genotypes are each responsible for 1% to 4% of cancers.
Most sexually people become infected with HPV during their lifetimes but the majority of infections disappear within 12 to 24 months either due to lack of biological fitness or through suppression by the host cellular immune system. Cancers develop in people with persistence of an HPV infection that is not controlled by the immune system. The important factors in determining risk of cervical cancer are HPV positivity, HPV genotype, and cytological changes associated with HPV-related cell transformation.
Over a lifetime, cervical cancer develops in up to 5% of an unscreened population. Screening can reduce the lifetime risk to less than 0.5%. The risk of cervical cancer begins to increase around age 30 years and remains elevated for the remainder of the lifespan. Therefore, screening is recommended at least every 5 years for individuals aged 25 through 65 years who have a cervix (eg, women and transgender men who have not undergone hysterectomy).
The American Cancer Society recommends HPV testing alone at 5-year intervals for those aged 25 though 65 years. The sensitivity for detecting precancer is more than 90% for HPV testing and 50 to 70% for cytology. Therefore, cotesting provides limited additional information compared with HPV testing alone.
Up to 20% of individuals in the general population have a history of prior abnormal results, prior precancer or cancer, or immunosuppression. These individuals should undergo screening at 1 or 3-year intervals.
If a person tests HPV-positive, genotyping and cytology testing should be performed to assess the risk of cervical precancer and determine the need for colposcopy or treatment.
The Aptima HPV assay is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA (mRNA) from 14 high-risk types of human papillomavirus (HPV) in cervical specimens. The high-risk HPV types detected by the assay include: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. The Aptima HPV assay does not discriminate between the 14 high-risk types.
The Aptima HPV assay targets high-risk HPV mRNA instead of DNA. Identification of E6/E7 mRNA is indicative of those HPV infections destined to lead to disease. In contrast, HPV DNA from one of the 14 high-risk types indicates the presence, but not activity, of a high-risk HPV infection. Studies have shown that mRNA reflects the presence and activity of a high-risk HPV infection.
Because HPV DNA levels may decrease as infections progress toward cancer, some HPV DNA tests may provide false-negative results in more than 10% of the most severe cervical disease cases.
The Aptima HPV 16 18/45 genotype assay is an in vitro nucleic acid amplification test for the qualitative detection of E6/E7 viral messenger RNA (mRNA) of human papillomavirus (HPV) types 16, 18, and 45 in cervical specimens from women with Aptima HPV assay positive results. The Aptima HPV 16 18/45 genotype assay can differentiate HPV 16 from HPV 18 and/or HPV 45, but does not differentiate between HPV 18 and HPV 45.
The Aptima HPV assay involves three main steps, which take place in a single tube: target capture; target amplification by Transcription-Mediated Amplification (TMA®); and detection of the amplification products (amplicons) by the Hybridization Protection Assay (HPA).
Specimens are transferred to a tube containing specimen transport media (STM) that lyses the cells, releases mRNA, and prevents its degradation during storage. When the Aptima HPV assay is performed, target mRNA is isolated from the specimen by use of capture oligomers that are bound to magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of HPV mRNA as well as a string of deoxyadenosine residues. During the hybridization step, capture oligomers bind to the complementary regions of HPV mRNA. The capture oligomer:HPV mRNA complex is then captured out of solution by decreasing reaction temperature to room temperature. This temperature reduction allows hybridization to occur between the poly-deoxyadenosine molecules on the capture oligomer and the poly-deoxythymidine molecules that are covalently attached to the magnetic microparticles. The microparticles, including captured HPV mRNA, are pulled to the side of the reaction tube by magnetic force and the supernatant is aspirated. Particles are then washed to remove residual specimen matrix that may contain amplification inhibitors.
Following completion of target capture, HPV mRNA is amplified by a process called transcription mediated amplification (TMA), using two enzymes; MMLV reverse transcriptase and T7 RNA polymerase. MMLV reverse transcriptase is used to generate a DNA copy of the target mRNA sequence containing a promoter sequence for T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template.
Detection of the amplicon is achieved by the hybridization protection assay (HPA) using single-stranded nucleic acid probes with chemiluminescent labels that are complementary to the amplicon. The labeled nucleic acid probes hybridize specifically to the amplicon.
The Selection Reagent differentiates between hybridized and unhybridized probes by inactivating the label on the unhybridized probes. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals called Relative Light Units (RLU) in a luminometer. Final assay results are interpreted based on the analyte signal-to-cutoff (S/CO).
Internal control (IC) is added to each reaction via the Target Capture Reagent. The IC monitors the target capture, amplification, and detection steps of the assay. IC signal in each reaction is discriminated from the HPV signal by the differential kinetics of light emission from probes with different labels. IC-specific amplicon is detected using a probe with a rapid emission of light (flasher). Amplicon specific to HPV is detected using probes with relatively slower kinetics of light emission (glower). The Dual Kinetic Assay (DKA) is the method used to differentiate between the signals from the flasher and glower labels.
The following link contains a video that illustrates the entire assay.
Acceptable specimens for HPV testing include cervical specimens collected with broom-type or cytobrush/spatula collection devices that are submitted in ThinPrep Pap Test vials containing PreservCyt Solution.
Reference
Perkins RB et al. Cervical Cancer Screening: A Review. Published on line August 8, 2023.
How to resolve AdBlock issue?