HBV quantification
Related Terms
AFP, alkaline phosphatase, annealing, assay, chemiluminescent microparticle immunoassay, CT scan, denaturation, DNA dye, ELISA, extension, fluorescent
reporter probe, HBcAg, HBeAg, HBV DNA, HBsAg, hepadna, hepatitis B virus, hybridization, liver biopsy, liver ultrasound, mutation, quantification, PCR, real-time, RT-PCR, reverse transcription, Taq polymerase, signal amplification.
Background
Hepatitis B virus (HBV) quantification is a method of measuring the amount of viral nucleic acid in a blood sample collected from an individual infected with hepatitis B. Hepatitis B is a type of liver infection caused by the hepatitis B virus. The quantification tests, also called viral loads, are used to assess the ability of the virus to cause an infection and the disease status in an individual infected with HBV. Viral load is commonly divided into three categories: undetected (<1.6x103 viral DNA copies per milliliter); low titer (<105 copies per milliliter); and high titer (?105 copies per milliliter). The infection is considered severe or extensive if the viral load is high. The viral loads also help monitor treatment effects in infected individuals.
Genes are referred to as the building blocks of life because they provide instructions for making all of the proteins in the body. Genes are located inside of cells in a structure called the nucleus, and they control an organism's development and functions by instructing cells to make new molecules, usually proteins. Proteins are essential for the growth and maintenance of the body.
Deoxyribonucleic acid (DNA) is a long thread-like double-stranded molecule made up of large numbers of nucleotides. The sequence of nucleotide bases in DNA determines genetic information. Nucleotide bases are the foundation of DNA and are made of nitrogenous bases, sugars, and phosphate. Long strands of nucleotides form nucleic acids. RNA (ribonucleic acid) is a nucleic acid that plays a key role in protein synthesis. RNA is formed under the direction of DNA. Both RNA and DNA help in forming amino acids, which are the components of protein.
Virus: HBV is a hepadna (hepatitis DNA) virus that causes a serious liver infection known as hepatitis B. HBV infection can appear in two forms: acute, lasting less than six months, or chronic, lasting for longer than six months. During an acute infection, symptoms usually include liver inflammation, vomiting, jaundice (yellowing of the skin and eyes), and rarely, death. About 94-98% of adults infected with HBV recover completely and have no long-term effects. However, 2-6% of infected adults may develop chronic infections. When the condition is chronic, it may lead to liver failure; liver cancer; cirrhosis, a condition that causes permanent scarring and damage to the liver; or death.
The hepatitis B virus is transmitted through contact with bodily fluids such as blood and semen from an infected individual. It is categorized as a sexually transmitted disease (STD) because of its ease of transmission via semen and vaginal fluids. Even though HBV is transmitted the same way as the human immunodeficiency virus (HIV), the virus that causes AIDS, HBV is nearly 100 times more infectious than HIV. Individuals of any age, race, nationality, gender, or sexual orientation can become infected with HBV. Individuals who are more likely to become infected with HBV are those who use intravenous (IV) drugs, have unprotected sex, and are born in or travel to parts of the world where hepatitis B is prevalent (e.g., sub-Saharan Africa, Southeast Asia, the Amazon Basin, the Pacific Islands, and the Middle East). In addition, women who have HBV can transmit the infection to their babies during childbirth. When the infection is passed from mother to fetus, it is called vertical transmission.
Hepatitis B infection can be prevented with the hepatitis B vaccination. A hepatitis B vaccine (Engerix-B?) has been available since 1982. It is administered in a series of three injections and provides more than 90% protection for both adults and children, generally for at least 15 years. Almost anyone can receive the vaccine, including infants, older adults, and those with immune deficiencies. Infants usually receive the vaccine during the first year of life, with injections administered at two, four, and nine months of age.
Structure: HBV consists of a central core particle containing the viral genome in the form of a double-stranded DNA and an outer fat-soluble layer in which proteins are embedded. The genome is the total genetic material of an organism and carries information for its development and function. The central core of the viral genome also contains DNA polymerase, an enzyme that has reverse transcription activity because HBV, like HIV, is one of the few viruses that uses reverse transcription as part of its replication, or multiplication, process. Reverse transcription is a process of making DNA under the direction of RNA. In reverse transcription, the enzyme called reverse transcriptase has a high error rate in providing instructions to make the DNA, which allows mutations, or genetic errors, to accumulate at a high rate. Mutations may cause a wide range of diseases or infections in an individual. The reverse transcription mechanism has also helped researchers develop drugs that target this mechanism and suppress the virus' growth.
HBV contains a partially double-stranded circular DNA genome and three important antigens: HB surface antigen (HBsAg), HB core antigen (HBc), and HBe antigen. HBe antigen is a soluble component secreted by the virus core and expressed on the surface of liver cells. An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, which triggers an immune system response in the body, thereby helping to protect against any foreign invader.
Diagnosis: The hepatitis B infection is detected by conducting serum or blood tests called serological assays to identify the presence of the three viral antigens or the antibodies produced by the host in response to the antigens. Antibodies are proteins made by the body to fight off antigens, or foreign invaders, such as HBV. For HBV, which cannot be grown in a test tube, serological assays allow for an accurate diagnosis and follow-up of acute or chronic infection. Some of the blood tests conducted to detect the virus include the tests listed in this section.
Hepatitis B surface antigen (HBsAg): The outer surface of HBV has hepatitis B surface antigen (HbsAg) on it. This antigen is the earliest indicator of acute hepatitis B infection and frequently identifies infected people before symptoms appear, while the HBsAg disappears during the recovery period. In some cases, HBV may not produce new viruses that can infect others or it may produce them in such low quantities that they cannot be detected in the blood. Individuals with this form of infection are known as carriers. HBsAg is positive in both individuals with active, spreading infections and in carriers. A positive result indicates an active infection but does not indicate whether the virus can be passed to others. A negative result indicates that a person has never been exposed to the virus or has recovered from acute hepatitis and has rid themselves of the virus.
Antibody to hepatitis B surface antigens (anti-HBs): If an individual has antibodies to HBV, he or she has had previous exposure to HBV, but the virus is no longer present, and the person cannot transmit the virus to others. A positive result may indicate that the individual was previously infected with HBV or that the individual was vaccinated. If a patient has not been vaccinated but has anti-HBs, he or she was once infected with HBV, produced antibodies to the virus, and cannot be re-infected. This is because once antibodies to the virus are produced in an infected or vaccinated person, they protect against future infection.
Antibody to hepatitis B core antigen (anti-HBc): Anti-HBc is an antibody to the HBV core antigen (HBcAg). The core antigen is present on the central core of the virus particles and disappears early in the course of infection. Anti-HBc is produced during and after an acute HBV infection and is usually found in chronic HBV carriers as well as in those who have cleared the virus. The antibody usually persists for life. The blood test for antibodies to the hepatitis B core antigen identifies individuals who have a chronic infection. However, the results can sometimes be ambiguous. Individuals who test positive may have a chronic and contagious infection, may be recovering from an acute infection, or may have slight immunity to HBV that cannot otherwise be detected. The interpretation of this test depends on the results of other blood tests.
E-antigen test (HBeAg): HBeAg is a viral protein associated with HBV infection. The e-antigen is present in the blood only if the virus is present. Therefore, HBeAg is often used as a marker of an infected person's ability to spread the virus to other people. The HBeAg is also used to monitor the effectiveness of the treatment because it can determine whether the drug is effective in reducing the virus in the blood. A positive result means that the patient has high levels of the virus in the blood and that the virus is very contagious. If the test is negative, the patient has lower levels of HBV in the blood and the virus is less contagious.
Hepatitis B virus quantification is a more sensitive test than HBeAg for detecting and counting the number of viruses in the blood. In some cases, HBeAg is not sufficient to detect the ability of the virus to spread because some infected individuals may not show the presence of HBV antigens because of DNA mutations in the region encoding the 'e' antigen. Mutation is a change in the genetic constitution of an organism. Therefore, quantification detects and quantifies the HBV DNA although the antigen is not present, which prevents false-negative test results. It may also be used to monitor antiviral therapy in patients with chronic HBV infection.
Methods
General: Hepatitis B virus (HBV) quantification is the method of measuring the amount of viral nucleic acid (DNA [deoxyribonucleic acid]) in a blood sample collected from an individual infected with hepatitis B. This is a sensitive method of detecting the infection when other methods, such as the hepatitis e-antigen test, are inconclusive. There are several methods for the quantification of HBV.
The number of HBV DNA can be detected, or quantified, using real-time polymerase chain reaction (RT-PCR) assay. This test is based on the conventional PCR method, which is an enzymatic method to make copies of the two strands of DNA in a particular gene sequence. It is widely used to produce multiple copies of minute quantities of DNA for further study. Several steps are included in the PCR technique, such as isolation, denaturation, annealing, and extension.
Isolation is the initial step in PCR. The genomic DNA from the blood of an infected individual is separated to remove interfering elements, such as other proteins, from the component of interest, the HBV DNA. Denaturation is a process in which the sample is heated to separate the double-stranded DNA into single strands to help in the next step, annealing. In annealing, the DNA strands are heated and cooled, which induces the pairing of primer strands with DNA strands that have complementary sequencing. This step initiates the synthesis of the HBV DNA. A primer is a short strand or sequence of nucleic acids that serves as the starting point for DNA or RNA replication and is designed to target the DNA region to be copied. A complementary strand is a nucleic acid sequence that can form a double-stranded structure by matching base pairs (adenine, A; guanine, G; cytosine C; and thymine, T). For example, the complementary strand for G-T-A-C is C-A-T-G. Extension or elongation is the process in which a new DNA strand is synthesized with the help of the enzyme DNA Taq polymerase, which results in the formation of a double-stranded DNA. By repeating the above steps in several cycles, there is a rapid increase in the specific target fragment of DNA.
Quantitative PCR or real-time PCR assay: The quantitative PCR test, based on PCR, amplifies and quantifies the HBV DNA. This test allows the detection and quantification of HBV DNA after each amplification cycle, thereby producing accurate and reliable results in quantifying the HBV DNA. The major advantage of this technique is that the amplified DNA can be quantified within a very short time, about 45 minutes, after each amplification cycle.
Quantification may be done in two ways: by using a fluorescent dye that binds by intercalation with the double-stranded DNA, or by using DNA oligonucleotide probes that pair with a complementary DNA. These probes are short segments of DNA, usually less than 20 nucleotides in length. Intercalation is the inclusion of a molecule between two other molecules, such as the dye attaching itself between the base pairs of the DNA. Base pairs include two nucleotide sequences located on opposite complementary DNA or RNA strands connected by hydrogen bonds, for example, A-T or C-G.
DNA dyes: In a PCR reaction, a DNA-binding dye (e.g., SYBR Green) binds to the double-stranded DNA, causing the dye to glow, or fluoresce. PCR leads to an increase in the copies of DNA, which causes the dye to glow brighter. With reference to a standard dilution, the viral DNA concentration in the PCR can be determined. Samples may be compared to the baseline and to one another, and by using these comparisons, variations may be determined. Viral DNA concentration is measured at the end of each cycle using a detector.
The levels of fluorescence are measured using a detector because the dye glows only when attached to the target sequence of HBV DNA. The viral DNA is present in larger quantities in the sample if the detected fluorescence is brighter. Hence, detection of the fluorescence intensity during the course of PCR enables verification as well as quantification of the product.
Fluorescent reporter probe method: The fluorescent reporter probe method uses a sequence-specific DNA-based probe to quantify only the DNA containing the probe sequence of HBV DNA. The DNA probes are single-stranded sequences with base pairs complementary to the target segment. This method is more specific and accurate as it quantifies HBV DNA even in the presence of DNA segments that do not belong to the viral genome. The labeled probe is added at the stage of annealing and when both the primers and probes are attached to the HBV DNA. Fluorescence is emitted once the polymerase, or enzyme, reaches the probe and breaks it down. The fluorescence is detected and measured in the RT-PCR machine, thus facilitating the quantification of the HBV DNA.
Enzyme-linked immunosorbent assay (ELISA): ELISA is a blood test that looks for an antigen specific to HBV. ELISA is most widely used as an initial blood test for screening large numbers of samples on a daily basis. It is based on the principle of antigen-antibody interaction, which is similar to the lock and key fit of enzyme-substrate reaction. The reaction produces a color variation, which allows researchers to easily see the results. An antigen is any substance, such as a virus, bacterium, toxin, or foreign protein, that triggers an immune system response in the body. Antibodies are proteins made by the body to fight off antigens, or foreign invaders such as HBV. It also facilitates the quantification of HBsAg (surface antigen) or HBeAg (e-antigen), thereby assisting in the detection of viral load.
Radioimmunoassay: Radioimmunoassay is a procedure meant for detection and quantification of antigens using radioactive antigens (e.g., radioactive isotope of iodine). Radioactive versions of antigens are mixed with antibodies and inserted into a sample of the patient's blood. The same nonradioactive antigen in the blood takes the place of the isotope in the antibodies, leaving the radioactive substance free. The amount of free antigen is then measured to see how much of the original substance was in the blood, thus enabling detection and quantification.
Quantitative HBsAg: The quantitative HBsAg test is a reliable and early marker that predicts the outcome and severity of the infection in acute hepatitis B infection. Several studies found a significant association between the HBsAg (surface antigen) concentration and the HBV DNA level, suggesting that concentration of HBsAg may be used to analyze the viral load accurately. The hepatitis B surface antigen (HBsAg) concentrations are measured using a fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT, Abbott). The chemiluminescence method uses a chemical dye that binds with the target molecule. The chemical reaction emits light (luminescence), which is detected by a luminescent imager. In this technique, microparticles of glass between 0.1 and 100 micrometers, various metals, or magnetic materials are coated with a labeled molecule specific to the HBsAg assay. They are used along with a chemiluminescent tracer or substrate, such as N-sulfonylacridinium-9-carboxamide, to quantify the viral surface antigen. The analyzer measures the intensity of chemiluminescence, thereby quantifying the hepatitis B virus.
bDNA (branched DNA) signal amplification assays: bDNA signal amplification assay is a simple and nonradioisotopic method in which radioactive substances are not used for detecting HBV DNA, making it a safe method of quantification. The assay is commercially available as QuantiplexT HBV DNA Assay, Chiron, USA. It is a nucleic acid hybridization procedure for the quantification of HBV DNA in human serum, the liquid part of blood that does not contain clotting factors. Hybridization is the process of combining complementary single-stranded nucleic acids (DNA) to form a double-stranded structure, which makes the detection of target nucleic acid sequences possible. In this hybridization method, a probe bound to an enzyme, such as alkaline phosphatase, is hybridized to a target probe containing viral DNA with the use of chemiluminescent substrate. Detection is made possible by measuring the amount of light emitted, which is proportional to the amount of HBV DNA present in each sample. This method is expensive and is recommended for those already diagnosed with the infection to assist in monitoring treatment strategies, but not for use as a screening tool.
Research
General: Several studies have been conducted to develop methods that could improve the existing hepatitis B virus (HBV) quantification methods. Some of the studies described below.
Real-time fluorogenic method: Researchers have used a new method of quantifying HBV by using loop-mediated isothermal amplification (LAMP) to develop a real-time fluorogenic method of HBV quantification. LAMP is an alternative to polymerase chain reaction (PCR) for making many copies of DNA. It uses single temperature incubation, thus eliminating the need for thermocyclers, an expensive laboratory apparatus used to amplify DNA in the PCR process. Incubation is the act of maintaining controlled environmental conditions to enhance growth or development of microbial or tissue cultures or to maintain optimal conditions for a chemical or immunologic reaction. This method is precise, accurate, rapid, and comparable to PCR. LAMP has the potential to be a valuable detection tool for HBV in large clinical and epidemiological studies that look at factors influencing the health of populations to develop strategies to prevent illness.
Identification of mutation: A mutation is a change in a gene, specifically a change in the sequence of base pairs in the DNA that makes up a gene. Studies are being conducted to develop methods of detection and quantification of genetically altered strains of HBV, which have become resistant to lamivudine (Epivir?), an antiviral drug. This research is helping researchers understand the underlying mechanism of viral mutation to develop preventive strategies against the mutated organism and helps in selecting an appropriate drug for the affected patient because lamivudine should be avoided in patients with lamivudine-resistant strains of HBV.
Implications
According to the U.S. Centers for Disease Control and Prevention (CDC), an estimated 1.25 million Americans have chronic hepatitis B infection. The CDC speculates that vaccinating one in 1,000,000 high-risk adults would save up to $100 million in future direct medical costs by preventing hepatitis B infection and its associated complications. Hence, accurate diagnosis and monitoring of treatment in infected individuals using hepatitis B virus (HBV) quantification may help reduce the disease burden.
Accurate quantification of viral load helps in screening HBV infection and thereby in the diagnosis of the infection and analysis of its severity. It is a useful method of monitoring HBV replication, or multiplication. Quantification helps clinicians understand the infective status of an affected individual because it helps to calculate the number of hepatitis B viruses in the blood sample. Quantification also helps in determining the most appropriate treatment for an affected individual.
HBV quantification can help in monitoring the effectiveness of the treatment prescribed in an individual infected with HBV by determining the viral load. If the viral load is decreasing, it indicates that the treatment is effective for that patient. However, if the viral load is not decreasing because of drug resistance, it indicates that an alternative treatment strategy is needed. Drug resistance refers to the ability of an organism such as HBV to overcome the effects of a drug.
Limitations
In real-time polymerase chain reaction (RT-PCR), dyes such as SYBR Green bind to all PCR products, including nonspecific PCR products such as DNA segments that do not belong to the viral genome. This non-specific binding can interfere with the accurate quantification of the hepatitis B virus (HBV) DNA, and may falsely indicate a high viral load. PCR is an enzymatic method for repeated copying of the two strands of DNA in a particular gene sequence. It is widely used to amplify minute quantities of biologic material to provide adequate specimens, i.e., PCR products, for laboratory study.
Standardization was a major problem in early tests of HBV quantification. However, with the development of techniques such as PCR, standardization has allowed comparison of the results of different tests, thereby ensuring accurate and reliable results.
Future research
Quantification of viruses: Researchers have developed a new approach using microarray to identify and quantify hepatitis C, hepatitis B virus (HBV), and HIV (human immunodeficiency virus) simultaneously. Microarray is a technology that consists of tiny spots of DNA oligonucleotides, or short segments of DNA, fixed to a supporting material in a regular pattern for genetic analysis. Genetic analysis is the study and analysis of genes that may be normal or associated with certain diseases.
This technique is based on real-time reverse transcription-polymerase chain reaction (RT-PCR) performed within microarray hydrogel pads. Reverse transcription is a process of DNA synthesis under the direction of RNA. Polymerase chain reaction (PCR) is an enzymatic method for repeated copying of the two strands of DNA in a particular gene sequence. It is widely used to amplify minute quantities of biologic material to provide adequate specimens (i.e., PCR products, for laboratory study).
RT-PCR is used to measure the change in expression of a gene or to detect any variation in a DNA sequence. Further study is required to evaluate the accuracy of the method to detect and quantify different genetic targets.
Detection of reference genes: Quantitative RT-PCR requires reference genes to normalize sample variations while quantifying a particular target (e.g., HBV DNA) to get accurate results. Samples may be compared with the baseline and with one another, and by using these comparisons, variations may be determined. Quantitative RT-PCR makes possible the measurement of differences between samples and enables accurate identification of gene expression. Two software tools, geNorm and Normfinder, were used to check for the reference gene stability. These tools determine the most stable genes from a set of tested genes in a given DNA sample. Based on the findings, scientists have identified GUS and PMM1 as suitable reference genes for normalization in gene expression studies of liver tissue from patients with chronic hepatitis B.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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