Hepatitis B Virus Dna Quantitative

What Is The Procedure For An Hbv Dna Quantitative Pcr Test

The procedure for an HBV DNA Quantitative PCR is quite simple, painless and non-invasive. Typically the test is performed at the patients residence. You will have to contact us to fix an appointment with the phlebotomist. The phlebotomist will arrive at your place with a pack of sterilized equipment.

To draw a blood sample from your vein in the hand, they will have to tie a rubber band on your arm above the elbow. A sterilized needle with an attached tube will be inserted in the vein and the sample will be collected. As soon as the collection of blood is done, the rubber band will be removed and a medical sticker will be applied at the puncture site to avoid infection.

The procedure takes 5-10 minutes as soon as the technician identifies the suitable vein to draw the blood. The collected sample will be labeled and send to a trusted lab to analyze it.

Discusses Physiology Pathophysiology And General Clinical Aspects As They Relate To A Laboratory Test

Diagnosis of acute or chronic hepatitis B virus infection is based on the presence of HBV serologic markers such as hepatitis B surface antigen and hepatitis B core IgM antibody , or the presence of HBV DNA detected by molecular assays. Although the diagnosis of acute and chronic HBV infection is usually made by serologic methods, detection and quantification of HBV DNA in serum are useful to:

-Diagnose some cases of early acute HBV infection

-Distinguish active from inactive HBV infection

-Monitor a patient’s response to anti-HBV therapy

The presence of HBV DNA in serum is a reliable marker of active HBV replication. HBV DNA levels are detectable by 30 days following infection, generally reach a peak at the time of acute hepatitis, and gradually decrease and disappear when the infection resolves spontaneously. In cases of acute viral hepatitis with equivocal HBsAg test results, testing for HBV DNA in serum may be a useful adjunct in the diagnosis of acute HBV infection, since HBV DNA can be detected approximately 21 days before HBsAg typically appears in the serum.

Factors Related To Liver Carcinogenesis

Comparison of background factors was conducted for the 9 patients who developed HCC during long-term NA therapy and for the 158 patients who did not develop liver cancer during the treatment period. Among the factors that were present before NA therapy, being male and the presence of hepatic cirrhosis were significant factors for the development of HCC. However, the ALT level normalization, low HBsAg level, and HBV-DNA negativation at the last observation at the end of the long-term NA therapy were not found to be significant factors for the development of HCC .

Table 3

Characteristics of patients with HCC and without HCC after long-term NA therapy

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Cobas Amplicor Hbv Monitor Assay

The Amplicor assay was performed at ARUP Laboratories . Briefly, the method involves PCR amplification of precipitated nucleic acids with one biotinylated primer. The biotinylated amplicon is serially diluted three times, and the four resulting solutions are captured onto magnetic particles coated with an oligonucleotide probe. The bound amplicon is detected with an avidin-horseradish peroxidase conjugate. A quantitation standard of plasmid DNA, modified for the probe binding sequence at a known concentration, is added to each sample. The HBV DNA level in each test sample is calculated by comparing the target signal with the quantitation standard signal. A sample volume of 100 l is processed, and DNA from the equivalent of a 50-l sample is used for amplification. Results from the Amplicor assay are reported as copies per milliliter, and the nominal range of quantitation is 200 to 200,000 copies/ml.

What Viral Load Can Tell You About Your Hepatitis B Infection

Hepatitis B is one of the most serious liver infections in the world. Untreated hepatitis B can cause fibrosis, cirrhosis, hepatocellular carcinoma, and end-stage liver disease. Doctors run several blood tests to see if the infection is harming your liver and to identify what stage of infection you are in. Heres why one of those blood tests the HBV DNA matters and what it can tell you about your hepatitis B infection.

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Conversion Of Copies To Iu Based On The Qcmd Panel

The conversion of proprietary units specific to each assay to copies resulted in a wide range of ratios .4). Because different standards and methods were used in developing the units of measure for each of these various assays, such a divergence in units of measure is not surprising . The means for target amplification assays were higher than those for signal amplification assays . The results from the QCMD panel indicated conversion factors of 2.6 copies per IU for genotype A and 7.0 copies per IU for genotype D. This high conversion factor for genotype D is unlikely to be a genotype difference but more likely to be an error of quantitation of the stock. Based on the results obtained during the collaborative study, a conversion factor of 2.6 copies per IU was proposed . The higher ratios obtained for the Amplicor and SuperQuant assays could be due to overquantitation by those assays. Despite attempts to unify conversion units, it is difficult to generate a consensus, and it is hoped that the WHO international standard will be adopted universally as a reference and that consistent reporting of results in IU per milliliter will emerge.

Lod Of The Duplex Real

The sensitivity test, which was performed using 20 replicates of different concentrations , showed that the lowest 100% positive concentration for the duplex real-time PCR assay was 20 IU/ml . These results were further subjected to probit regression analysis. The LODs of the S region, the C region, and the duplex real-time PCR assay were 15.2 IU/ml , 16.6 IU/ml , and 11.9 IU/ml , respectively.

Table 3 Limit of detection for the S and C regions using the duplex real-time PCR assay

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Quantitative Measurement Of The Extent Of Dna Methylation Of The Hbv Genome In Various Liver Disease Tissues

To evaluate whether the extent of DNA methylation was associated with the progression of liver diseases, we developed quantitative MSP assays for each CpG island to measure the DNA methylation in a larger sample size. As some of the HBV infected tissues may not contain any detectable HBV DNA, each DNA sample was first subjected to the quantitative HBV DNA PCR assays for CG1 and CG3, as described in the Materials and Methods. Only samples positive for at least one HBV DNA assay were subjected to methylation analysis. The clinicopathological characteristics of the study subjects are listed in Supplementary Table S2.

Figure 4

Quantitative analysis of HBV DNA methylation in infected diseased livers.

Scatter plots of the quantity of methylated HBV DNA detected by qMSP assays for each CpG island. Each data point represents one sample and is the average of two duplicate assays per input of 3000 copies of BS-actin DNA. p values indicate the statistical comparison between HCC and non-HCC samples for each CpG island by Mann-Whitney U test.

Questions For Your Doctor About Test Results

Patients may find it helpful to ask questions about their hepatitis B test results. Questions that may be helpful include:

• What was my test result?
• Do I have an acute or chronic hepatitis B infection?
• Does the test result suggest that I have immunity for hepatitis B?
• Would I benefit from hepatitis B vaccination?
• Do I need any follow-up tests based on my hepatitis B test results?

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Submission And Collection Notes

Freshly drawn whole blood specimens may be stored and/or transported at 2°C to 25°C for up to 24 hours before centrifugation. Following centrifugation, remove serum from cells immediately. Serum specimens may be stored and/or transported at 2°C to 8°C for up to 6 days or at -18°C for up to 12 weeks. If more extended storage of serum specimens is required, it must be frozen at -60°C.

Sensitivity Of Detection Related To Amplification

The target-capture PCR, Amplicor, and SuperQuant assays utilize PCR for amplification of the target, with significant differences in the three detection mechanisms. Both the Amplicor and SuperQuant assays require processing of the amplicons and elaborate technology for quantitation they estimate the initial amount of DNA by measuring the amount of amplified PCR product at the end of the amplification reaction. This end-point analysis is prone to error at high target concentrations, because the concentrations of the reaction components can become limiting, not resulting in the expected logarithmic increase in the amplicon for each cycle of amplification. The Amplicor assay involves serial dilution of the amplicon, followed by hybridization, washing, and enzyme-associated color development, and requires at least 4 h following amplification to obtain results. The SuperQuant assay involves gel separation, transfer to a membrane, hybridization, washing, and scanning and takes more than 24 h to generate results. Processing of amplicons in an open system, such as that required in the SuperQuant assay, can result in contamination and is generally considered undesirable.

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Superquant Hbv Dna Assay

The SuperQuant assay was performed at National Genetics Institute. The method involves PCR amplification of extracted nucleic acids followed by Southern hybridization. Samples are centrifuged, and nucleic acids are isolated by proteinase K extraction. Following PCR amplification of the unknowns and the standard, the amplicons are electrophoresed and transferred to a membrane. Southern blotting is performed with a nonradioactive digoxigenin-labeled DNA probe. The profile of each Southern blot membrane is scanned into a computer by using an automated scanner-densitometer. The resulting electronic images are measured for band area and mean band density. A sample volume of 2 ml is used, and the results are reported as copies per milliliter. The nominal range of the SuperQuant assay is 100 to 108 copies/ml, requiring dilution of high-titer samples before analysis to obtain measurements in the linear range.

Available Hbv Dna Tests

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Rates Of Vr And Sr Among Patients With Favorable Baseline Qanti

The sum of sensitivity and specificity was maximal in predicting VR and SR at year 10 when the cut-off value was 3.1 log10 IU/mL. Patients were stratified into two groups according to the cut-off value. Eighty percent and 100% of patients with qAnti-HBc3.1 log10 IU/mL achieved VR and SR, respectively, after 10 years of antiviral therapy. However, only 36.4% and 45.5% of patients in the group with qAnti-HBc< 3.1 log10 IU/mL achieved VR and SR, respectively, at year 10 .

Performance Of The Target

As part of the 2003 QCMD BBV program, an HBV proficiency panel consisting of eight coded members was tested by the target-capture PCR assay. The results are shown in Table . The results indicated no detection of HBV DNA in the HBV-negative sample. The assay detected and quantitated the genotype A sample with the lowest copy number, 200 copies/ml, demonstrating high sensitivity. All of the genotype A samples with higher copy numbers as well as genotype D samples were detected. For all of the quantitated genotype A samples, the conversion factor was 1 IU equals 2.6 copies.

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Clinical Characteristics Of Study Subjects

Nine Hundred and seventy six HBsAg-positive patients were categorised into four phases of persistent HBV-infection. The demographics and baseline characteristics of all the subjects were presented in . Their median age was 32 years, with a range between 3 and 82. Male predominance was seen with 668 in the study subjects. The median value of the HBsAg titres was 3.01 log IU/ml, and the median value of serum HBV DNA was 3.41 log IU/ml.

Correlation Between Serum Hbsag Titres And Hbv Dna Quantitative Levels

In the entire cohort of persistent HBV-infections, serum HBsAg titres showed a significant positive correlation . The correlation between HBsAg levels and HBV DNA in various phases was shown in . There was a strong correlation in the IT and IC phase . LR phase shows moderate correlation and weak correlation was seen ENH phase .

Correlation between serum HBsAg titres log and HBV-DNA log in HBV infections in overall population as well as sequential phases of infection: IT, immune tolerance phase, IC, immune clearance phase LR, low-replicative phase ENH, HBeAg hepatitis CI, confidence interval.

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Quantitation Of Serum Hbv Dna

Viral DNA from serum samples was extracted by using High Pure System Viral Nucleic Acid kit provided by as per the manufacturer’s instructions. Extracted HBV DNA was subsequently amplified by Genome Diagnostic HBV quantification kit for Real-Time PCR by using which has a lower limit of detection of 6 IU of HBV DNA/mL.

Primers And Probes Selection For The Duplex Real

One thousand HBV complete sequences from Chinese patients were downloaded from the NCBI database, which include genotypes A, B, C, D, and G and were aligned by the DNAman software. Primers and probes were designed by Primer 5 to target the conserved sequences of the S and C regions of the HBV genome. Primers were selected by testing using EvaGreen, and primers with significant dimerization were removed. Probes with lower count values were chosen for further experiments . The primers and probes are listed in Table .

Table 1 The primers and probe sequences for the S and C regions

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What Does Hbv Quant Measure

The Hepatitis B Virus – Viral Load, Quantitative test measures the actual amount of hepatitis B present in a blood sample. This test helps determine whether Hepatitis B Virus is getting reproduced in the liver. If HBV viral load is greater than 20,000 international units per milliliter in a person with detectable Hepatitis B envelope antigen then it indicates that the virus is active and has the greatest potential to cause damage to the liver. Similarly, if HBV viral load is greater than 2,000 IU/mL in HBeAg-negative chronic hepatitis B patients then it indicates that the virus is active and has the potential to cause damage to the liver. If the HBV viral load is above these numbers, treatment is considered necessary.

Can I Take The Test At Home

Samples for hepatitis B testing can be collected at home. At-home hepatitis B testing requires a patient to collect a blood sample, typically from a fingerstick using a very small needle provided in the test kit. Once a blood sample is collected, it is prepared according to the instructions contained in the test kit and mailed to a laboratory for testing.

Because there are numerous types of tests for HBV, it is important to look closely at the specific components of any at-home test kit. Many at-home test kits only look for hepatitis B surface antigen .

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Conversion Of Assay Units To Iu

The WHO international standard uses IU as the definition for HBV DNA. Therefore, results reported as copies per milliliter by other assays were correlated with the IU obtained from the target-capture PCR assay .4). Because the Amplicor, SuperQuant, and target-capture PCR assays use PCR-based target amplification, several points of overlap could be established to obtain a ratio of copies per milliliter to IU. For the Amplicor and SuperQuant assays, the values in the early phase of seroconversion with low viral titers, resulting in an exponential phase of amplification, were used to calculate this ratio. For the Hybrid Capture and Quantiplex signal amplification assays, few such overlapping points were noted. Consequently, the values in the later phase of seroconversion with high titers were used to calculate the ratio of copies per milliliter to IU for these two assays. The mean ratios obtained from this experiment were as follows: target-capture PCR assay, 2.6 Amplicor, 7.3 SuperQuant, 6.2 Quantiplex, 4.5 and Hybrid Capture, 2.3. Similar observations of a failure to obtain unique ratios with different NAT assays have been reported by Pas et al. and Pawlotsky et al. .

Quantiplex Hbv Version 20 Bdna Assay

The Quantiplex assay was performed at Bayer Reference and Testing Laboratories. The method uses bDNA technology with signal amplification. The target is isolated by hybridization to specific complementary oligonucleotides. The bDNA assay achieves sensitivity by using target probes that bind to selective regions of the full viral nucleic acid sequence, which in turn hybridizes to the bDNA amplifiers. Alkaline phosphatase-labeled probes then hybridize to the bDNA amplifiers, and signal detection is accomplished by using chemiluminescence technology. Reference to an external standard curve with known concentrations of HBV DNA permits quantitation of the unknowns. A sample volume of 0.2 ml is used, and the results are reported as milliequivalents per milliliter, with 1 mEq/ml 106 copies/ml. The limits of quantitation of the assay are from 7 × 105 to 4.8 × 109 copies/ml.

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