Hepatitis C Virus HCV Quantitative NAAT

The Aptima^® HCV Quant Dx assay is a real-time transcription-mediated amplification (TMA) test used for both detection and quantitation of hepatitis C virus (HCV) RNA in fresh and frozen human serum and plasma from HCV-infected individuals. Plasma may be prepared in ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose (ACD) solution, and plasma preparation tubes (PPT). Serum may be prepared in serum tubes and serum separator tubes (SST). Specimens are tested using the Panther® system for automated specimen processing, amplification, detection, and quantitation. Specimens containing HCV genotypes 1 to 6 are validated for detection and quantitation in the assay.

The Aptima HCV Quant Dx assay is indicated for use as an aid in the diagnosis of active HCV infection in the following populations: individuals with antibody evidence of HCV infection with evidence of liver disease, individuals suspected to be actively infected with HCV antibody evidence, and individuals at risk for HCV infection with antibodies to HCV. Detection of HCV RNA indicates that the virus is replicating and, therefore, is evidence of active infection. Detection of HCV RNA does not discriminate between acute and chronic states of infection.

The Aptima HCV Quant Dx assay is also indicated for use as an aid in the management of HCV infected patients undergoing HCV antiviral drug therapy. The assay can be used to measure HCV RNA levels periodically prior to, during, and after treatment to determine sustained virological response (SVR) or nonsustained virological response (NSVR). Assay performance characteristics have been established for individuals infected with HCV and treated with certain direct-acting antiviral agents (DAA) regimens. No information is available on the assay’s predictive value when other therapies are used. The results from the Aptima HCV Quant Dx assay must be interpreted within the context of all relevant clinical and laboratory findings.

The Aptima HCV Quant Dx assay is not approved for use as a screening test for the presence of HCV RNA in blood or blood products.

About HCV

HCV is a blood-borne pathogen and a worldwide public health burden with up to 170 million people infected globally and 350,000 annual deaths due to HCV related conditions, including cirrhosis and liver cancer.^1,2 Transmission of HCV is through exposure to blood, blood products, or activities with potential for percutaneous exposure.^3,4 Genetically, HCV contains a positive-strand RNA genome of approximately 9500 nucleotides encoding structural proteins (core, E1 and E2 glycoproteins, p7 ion channel protein) and non-structural proteins (NS2, NS3, NS4A/B, NS5A/B), the latter being key viral replicative proteins and targets of direct acting antivirals.^4,5 Two untranslated regions (UTR) of the genome, 5’UTR and 3’UTR, function in genome translation and replication/packaging roles, respectively.⁵ The 5'-UTR is the most highly conserved genomic region across the six major HCV genotypes.⁶

Clinically, there is a high prevalence of asymptomatic HCV infection, and, chronic HCV infection occurs in up to 75% of patients.² HCV laboratory testing algorithms require diagnosis of active HCV infections in antibody positive individuals through detection of HCV RNA in plasma or serum to allow appropriate link to care.^7,8,9

In the era of direct-acting antivirals (DAAs), quantitation of HCV RNA (viral load) has played a pivotal role in defining and monitoring successful HCV treatment. Sustained virological response (SVR) is defined as undetectable HCV RNA (with an assay that has a limit of detection of <25 IU/mL) after therapy.10,11 Recent guidelines from the AASLD suggest testing HCV RNA not only at baseline, but also periodically during treatment (i.e., 4 weeks) and at 12 weeks following completion of treatment.^10,12,13

The Aptima^® HCV Quant Dx assay is a nucleic acid amplification test that uses real-time TMA technology to detect and quantitate HCV RNA for aiding diagnosis or to establish baseline viral load, as well as to measure on-treatment and post-treatment responses. The assay targets a conserved region of the HCV genome, detecting and quantitating genotypes 1, 2, 3, 4, 5, and 6. The assay is standardized against the 2nd WHO International Standard for Hepatitis C Virus (NIBSC Code 96/798).¹²

The Aptima HCV Quant Dx assay involves three main steps, which all take place in a single tube on the Panther system*: target capture, target amplification by TMA, and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches).

During target capture, viral RNA is isolated from specimens. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic RNA. Capture oligonucleotides hybridize to highly conserved regions of HCV RNA, if present, in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube.

Target amplification occurs via TMA, which is a transcription-mediated nucleic acid amplification method that utilizes two enzymes, Moloney murine leukemia virus (MMLV) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. The Aptima HCV Quant Dx assay utilizes the TMA method to amplify a portion of the 5’ UTR of the HCV genome. Amplification of this region is achieved using specific primers which are designed to amplify HCV genotypes 1, 2, 3, 4, 5, and 6.

Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and that hybridize specifically to the amplicon in real time. Each torch has a fluorophore and a quencher. When the torch is not hybridized to the amplicon, the quencher is in close proximity of the fluorophore and suppresses the fluorescence. When the torch binds to the amplicon, the quencher is moved farther away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. As more torches hybridize to amplicon a higher fluorescent signal is generated. The time taken for the fluorescent signal to reach a specified threshold is proportional to the starting HCV concentration. Each reaction has an internal calibrator/internal control (IC) that controls for variations in specimen processing, amplification, and detection. The concentration of a sample is determined by the Panther system software using the HCV and IC signals for each reaction and comparing them to calibration information.

Aptima^® HCV Quant is a product of Hologic^® and is FDA approved for in vitro diagnostic use. See package insert for more information.

*All references to the Panther system in this document are applicable to the Panther system and the Panther Fusion system. There are no changes to the indications for use, labeling, and principles of operation for the Aptima HCV Quant Dx Assay on the Panther system as a result of the add-on Panther Fusion Module.

Potential cross-reactivity to the pathogens listed in Table 13 of the package insert. was evaluated in HCV negative human plasma in the presence or absence of approximately 15 IU/mL (1.2 log10 IU/mL) or 1995 IU/mL (3.3 log10 IU/mL) HCV. No cross-reactivity was observed. No interference was observed in the presence of the pathogens.