Hepatitis B Virus (HBV) Quantitative NAAT

The Aptima^® HBV Quant assay is an in vitro nucleic acid amplification test for the quantitation of hepatitis B virus (HBV) DNA in human plasma and serum on the fully automated Panther^® system.

Plasma may be prepared in ethylenediaminetetraacetic acid (EDTA), anticoagulant citrate dextrose (ACD) solution, and plasma preparation tubes (PPTs). Serum may be prepared in serum tubes and serum separator tubes (SSTs). Specimens are tested using the fully automated Panther system for sample processing, amplification, and quantitation. Specimens containing HBV genotypes A, B, C, D, E, F, G, and H are validated for quantitation in the assay.

The Aptima HBV Quant assay is intended for use as an aid in the management of patients with chronic HBV infections undergoing HBV antiviral drug therapy. The assay can be used to measure HBV DNA levels at baseline and during treatment to aid in assessing viral response to treatment. The results from the Aptima HBV Quant assay must be interpreted within the context of all relevant clinical and laboratory findings. Assay performance for determining the clinical stage of HBV infection has not been established. Clinical performance characteristics have been established for individuals treated with tenofovir disoproxil fumarate or entecavir.

The Aptima HBV Quant assay is not approved for use as a screening test for the presence of HBV DNA in blood or blood products or as a diagnostic test to confirm the presence of HBV infection.

About HBV

Hepatitis B virus, one of several viruses known to cause hepatitis, has been attributed to lifelong HBV infection, cirrhosis of the liver, liver cancer, liver failure, and potentially, death. The World Health Organization (WHO) lists HBV as one of the world’s most common infectious diseases. The prevalence of HBV infection and method of transmission varies greatly around the world. About one third of the world’s population has serological evidence of past or present HBV infection with chronic HBV infection occurring in an estimated 240 million people worldwide.^1,2,3 HBV infection results in increased risk of hepatic decompensation, cirrhosis, and hepatocellular carcinoma (HCC) with a mortality of 0.5 to 1.2 million deaths and 5-10% of cases of liver transplantation worldwide annually.^4,5 Without appropriate treatment, intervention, and monitoring after diagnosis, the 5 year cumulative incidence of cirrhosis ranges from 8-20%. Once cirrhosis has developed, the annual risk of hepatocellular carcinoma is 2-5%.⁶

HBV contains a circular, partially double-stranded DNA genome of approximately 3200 base pairs, which encode four partially overlapping open reading frames (ORF) expressing the polymerase, surface, precore/core, and X proteins. The polymerase ORF overlaps the other three ORFs and encodes a key viral replication protein, polymerase. The surface ORF expresses three proteins, which are essential for viral morphogenesis, viral entry into hepatocytes, and provoking the host’s immune response.⁷ There are 8 HBV genotypes (A-H), and these are typically found in distinct geographical locations.

Due to the dynamic nature of chronic hepatitis B infection, continuous monitoring of HBV DNA and alanine aminotransferase (ALT) levels is important.¹ For the majority of HBV infected individuals who are undergoing antiviral treatment, the goal is HBV DNA suppression. Quantitative nucleic acid tests with a broad linear range are effective tools to monitor HBV DNA viral load during the course of treatment.

The Aptima HBV Quant assay is an in vitro nucleic acid amplification test that uses real time transcription-mediated amplification (TMA) technology on the Panther system to quantitate HBV DNA, genotypes A, B, C, D, E, F, G, and H. The Aptima HBV Quant assay targets two highly conserved regions in the polymerase and surface genes (for increased tolerance to potential mutations). The assay is standardized to the 3rd WHO International Standard for Hepatitis B Virus (NIBSC code: 10/264).

The Aptima HBV Quant 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 DNA is isolated from specimens. The specimen is treated with a detergent to solubilize the viral envelope, denature proteins, and release viral genomic DNA. Capture oligonucleotides hybridize to highly conserved regions of HBV DNA, 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 HBV Quant assay utilizes the TMA method to amplify two regions of the HBV genome (polymerase gene and surface gene). Amplification of those regions is achieved using specific primers designed to amplify HBV genotypes A, B, C, D, E, F, G, and H. The dual target region approach with primer design targeting the highly conserved regions ensures accurate quantitation of the HBV DNA.

Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target, which 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 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 HBV 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 HBV and IC signals for each reaction and comparing them to calibration information.

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

Potential cross-reactivity to the pathogens listed in Table 12 of the package insert was evaluated in HBV negative human plasma in the presence or absence of 30 IU/mL (1.5 log10 IU/mL) and 20,000 (4.3 log10 IU/mL) HBV DNA. No cross-reactivity was observed. No interference was observed in the presence of the pathogens.