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Human Papilloma Virus

Human papilloma virus (HPV) is a common sexually transmitted DNA virus comprised of more than 100 genotypes. HPV infections are very common and most women will clear HPV infections within 6 to 12 months. HPV is the etiological agent responsible for more than 99% of all cervical cancers.

The HPV viral genome is a double-stranded circular DNA approximately 7900 base pairs in length. 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.

Fourteen HPV genotypes are considered pathogenic or high-risk for cervical disease. Multiple studies have linked genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 to disease progression. Women with a persistent infection with one of these types have an increased risk for developing severe dysplasia or cervical carcinoma.

The Aptima HPV assay (Hologic) 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.

HPV testing is indicated: 

1. To screen women 21 years and older with atypical squamous cells of undetermined significance (ASC-US) cervical cytology results to determine the need for referral to colposcopy.

2. In women 30 years and older, Aptima HPV assay can be used with cervical cytology to adjunctively determine the presence or absence of high-risk HPV types.

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.

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