US Pharm. 2011;36(6):HS-38-HS-40.
Hepatitis B virus (HBV), a blood-borne pathogen, originates from the hepadnavirus family.1 Over the years, the prevalence has increased--especially in Asia and Africa, where an estimated 350 million people are infected with chronic HBV. However, in western countries, the prevalence has decreased to less than 2%. In the United States, rates appear to be highest in males ranging from 25 to 44 years of age, and in African Americans, Hispanics, and those of Asian descent.2HBV infections may be transmitted by using infected needles or IV equipment, from mother to child, through sexual contact, or by the use of unscreened blood products. HBV is found in almost all bodily secretions; infectious secretions include blood, serum, semen, and vaginal fluid. The difference in the rate of transmission depends on the prevalence of the infection; mother-to-child or child-to-child transmission is higher when compared to IV drug use and high-risk sexual activity.1,3
Composed of three antigens, HBV is an encapsulated, circular, double-stranded DNA virus with a sequence of approximately 3,200 base molecules.4,5 HBV replication has a higher rate of mutation when compared to other DNA viruses. As such, eight genotypes--A through H--have been discovered. Therefore, it is important to determine the HBV DNA to predict the course of the infection and to monitor effects of antiviral drugs on replication.6,7 Earlier detection of resistant strains will enable physicians to determine the efficacy of treatment with antiviral drugs and to evaluate risk factors for cirrhosis and hepatocellular carcinoma.7
While some infected persons may be asymptomatic, the symptoms of HBV may include jaundice, anorexia, nausea, vomiting, fatigue, malaise, arthralgia, myalgias, headache, photophobia, pharyngitis, cough, coryza, low-grade fever, dark urine, clay-colored stool, tender hepatomegaly, and right upper-quadrant pain.6 In addition to monitoring the symptoms of HBV infection, some clinicians may choose to order a quantitative measurement of the HBV DNA to determine response to treatment.6,7 The Abbott RealTime HBV assay offers advanced technology that allows physicians to accurately measure viral loads and evaluate a patient's response to drug treatment.
Intended Use
The Abbott RealTime HBV assay is an in vitro polymerase chain reaction (PCR) assay that is used with Abbott RealTime m2000 SystemDNA reagents and Abbott m2000sp and m2000rt devices for the quantitation of HBV in human DNA (i.e., serum or plasma [EDTA]).6-9 This assay is not intended to diagnose; it is intended to aid in the management of chronically infected HBV patients by measuring HBV DNA levels at baseline and during treatment to assess patient response.6-9 Components of the RealTime HBV assay are listed in TABLE 1.
How It Works
The Abbott RealTime HBV assay utilizes PCR to produce amplified product from the DNA genome of HBV in clinical samples.9 The sample preparation is the first step in this process. The purpose of this step is to extract and concentrate target DNA to allow for easy amplification. This process also removes potential inhibitors of amplification from the specimen.8,9 The m2000sp is the instrument used in this portion of the testing. Employing magnetic microparticles to purify nucleic acids from the samples collected, this machine is designed to accept two sample sizes, 0.5 mL or 0.2 mL.6-9 The limit of detection (LOD) is different for each sample size. The LOD for the 0.5-mL and 0.2-mL sample sizes is 10 IU/mL and 15 IU/mL, respectively.10 The m2000sp reagents then lyse the viron and capture the nucleic acids. They also wash the particles to remove any unbound components of the specimen.8,9 Proteinase K is included to digest any proteins that are associated with the nucleic acids.8 The nucleic acids are subsequently transferred to a 96-well plate and are ready for amplification. At the beginning of the sample preparation, an internal control (IC) DNA sequence is introduced. IC is derived from the hydroxypyruvate reductase gene found in the pumpkin plant Cucurbita pepo.8 The control sequence is unrelated to the target DNA sequence of the HBV.
In the final step, Abbott RealTime HBV amplification components are combined. Amplification components include HBV oligonucleotide reagent, Amplitaq Gold enzyme, and activation reagent. This mixture is added to the Abbott 96-well optical reaction plate with the aliquots of nucleic acids that were previously prepared. Samples are then ready to be transferred to the m2000rt device.8,9 During amplification, the target DNA sequence is amplified by Amplitaq Gold enzyme, which requires magnesium as a cofactor and is subjected to several rounds of thermal cycling in which the amplification products dissociate to single strands at a high temperature.8,9 The amount of HBV target sequence present at each amplification cycle is quantified by measuring the fluorescence of the HBV probe that binds to the target during the previous step.8,9 The Abbott RealTime HBV assay is standardized against the World Health Organization (WHO) International Standard for Hepatitis B Virus DNA (NIBSC Code 97/746).8 See TABLE 2 for a summary of the above steps.
Abbott RealTime HBV Assay Versus Cobas TaqMan HBV Test
The Abbott RealTime HBV assay and the Cobas TaqMan HBV (Roche) test are both real-time PCRs used in the quantitation of HBV DNA. In all comparisons between HBV DNA PCRs, these two real-time assays provide the best correlation in terms of results. Both meet the requirements for the use of monitoring viral response to antiviral treatment. However, a few differences were noted between the two products.8 The Abbott RealTime HBV assay reports a higher sensitivity for detecting viral loads than that of the Cobas TaqMan (HBV test 10 IU/mL [for 0.5-mL sample size] and 12 IU/mL, respectively). Additionally, the variability of sample size with the Abbott device allows clinicians to use either 0.5-mL or 0.2-mL sample sizes while the Roche device affords only a 0.85-mL sample size. The Abbott device tests both serum and plasma while the Roche instrument only tests plasma.10 This device targets the N-terminal third of the Surface gene, which is required for assembly and secretion of subviral particles and is not highly impacted by mutations, therefore affording better accuracy in determining viral loads.10 A comparison of the Abbott RealTime HBV assay and the Cobas TaqMan HBV test can be found in TABLE 3.
Conclusion
Monitoring the HBV viral count of patients with chronic HBV has been a challenge for many clinicians. The treatment goals are to keep the viral count low and to prevent complications of the disease. The Abbott RealTime HBV offers a tactical advantage when compared to traditional PCR exams. Advantages of the device include greater sensitivity, which allows physicians to accurately monitor and make proper changes in treatment as needed, and location of gene testing, since the selected location is not easily affected by mutation or drug resistance. For more information please visit Abbott Molecular, Inc. at www.abbottmolecular.com.
REFERENCES
1. Nettleman M, El Mortada M, Marks JW. Hepatitis B (HBV). MedicineNet.com. 2009. www.medicinenet.com/scrpit/
2. Hepatitis B FAQs for health professionals. Centers for Disease Control and Prevention. 2009. www.cdc.gov/hepatitis/HBV/
3. Lexi-Comp Online, Harrison's Practice Online. Hepatitis B, chronic. Hudson, Ohio: Lexi-Comp, Inc.; 2010. Accessed February 10, 2011.
4. Ciotti M, Marcuccilli F, Guenci T, et al. Evaluation of the Abbott RealTime HBV DNA Assay and comparison to the Cobas AmpliPrep/Cobas TaqMan 48 Assay in monitoring patients with chronic cases of Hepatitis B. J Clin Microbiol 2008;46(4):1517-1519.
5. Cobas AmpliScreen HBV Test. Clifton, NJ: Roche; 2007.
6. Lexi-Comp Online, Infectious Diseases Online, Hepatitis B virus. Hudson, Ohio: Lexi-Comp, Inc.; 2010. Accessed February 10, 2011.
7. Pol J, Pendeven C, Beby-Defaux A, et al.
Prospective comparison of Abbott RealTime HBV DNA and Versant HBV DNA 3.0 assays for hepatitis B DNA quantitation: impact on HBV genotype monitoring. J Virol Methods. 2008;154:1-6.
8. Thibault V, Pichoud C, Mullen C, et al. Characterization of a new sensitive PCR assay for quantification of viral DNA isolated from patients with hepatitis B virus infections. J Clin Microbiol. 2007;45(12):3948-3953.
9. Abbott RealTime HBV. Assay [package insert]. Abbott Park: IL. 1-26.
10. Abbott RealTime HBV assay [product brochure]. Abbott Park, IL: Abbott Molecular. 1-3.
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