Hepatitis C Rna Quantitative Pcr

The Quantitative Hcv Rna Test Is Checked Before A Patient Starts Treatment

For each patient, the result can be described as either a “high” viral load, which is usually > 800,000 IU/L, or a “low” viral load, which is usually < 800,000 IU/L. It’s not uncommon to have a viral load in the millions. Today’s hepatitis C treatments are very effective with both high and low viral loads. An undetectable HCV viral load 10-12 weeks after hepatitis C is completed is associated with a cure.

Requisitions And Kit Ordering

1Based on internal validation studies performed at PHO Laboratory, HCV RNA testing conducted on DBS is less sensitive than venous-collected samples. The lower limit of detection of HCV RNA using two DBS per test is approximately 1.6 to 2.0 logs higher than a concomitantly tested EDTA plasma or serum sample thus, DBS samples should NOT be used to rule out active HCV infection or to determine whether a patient on treatment has achieved an undetectable HCV RNA level.

No additional sample is usually required for HCV genotyping, provided there is sufficient volume. The first pre-treatment sample submitted for HCV RNA viral load testing will be used to automatically perform HCV genotyping if the HCV viral load is 500 IU/mL. Below this level, HCV genotyping cannot be performed.

Results From The Qualitative Test

Doctors use the qualitative HCV RNA PCR test to determine whether or not the hepatitis C virus is present in the blood.

If the virus is present, the test will be positive. If the test does not detect the virus, the result will be negative.

If the result is positive, a person will then need a quantitative HCV RNA PCR test. For this reason, many doctors now prefer to skip the first test and use the quantitative test straight away.

The quantitative test results show how much HCV is in the body. However, whether low or high, the viral load does not reflect levels of damage to the liver.

Other blood tests, ultrasounds, and, rarely, a liver biopsy will help a doctor determine overall liver health.

After using an HCV RNA PCR test to confirm the presence of HCV, doctors will work out which strain of the virus is active in the body. This helps a doctor plan the course of treatment.

The primary goal of treatment is to bring down the viral load in the body until it is entirely free of the virus. Doctors know this as a sustained virologic response .

SVR occurs when the virus is undetectable for 12 weeks or longer after treatment.

Achieving SVR is the best outcome of treatment, as it often means the person is free from hepatitis C, or that treatment has cured hepatitis C.

Doctors will also combine treatments with other tests that monitor for complications of HCV, including cirrhosis and liver cancer.

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Reproducibility And Specificity Of The Real

In order to determine the reproducibility of intra- and inter-measurements of the qPCR, serial dilutions were tested on 2 separated days with triplicates of each dilution in each run . shows the mean CV of Ct values and the input copy number of HCV within a run the CV calculated for these repetitions was less than 1.06%. Interassay CV was low for standard curve and samples , a reasonable target for the percentage of CV in routine testing is 10%15%. On the other hand, the reproducibility of the RT-PCR was determined using 4 samples from patients chronically infected with HCV genotypes 1, 2, and 3 the most frequent genotypes in Northeast Mexico.20,23 shows detection of all HCV subtypes tested. The CV of these assays was less than 0.45%, and each sample was tested in triplicate.

The specificity of the RT-PCR we developed was assessed by testing serum/plasma samples from 5 healthy blood donors. All of these samples were negative for HCV-RNA.

Hepatitis C Qualitative Pcr Tissue

Hepatitis C Qualitative PCR, Tissue

Lab Code

Hepatitis C Qualitative PCR, Tissue

Description

The Abbott real-time FDA approved HCV assay detects and quantifies Hepatitis C virus by real-time PCR amplification. The Abbott assay utilizes three master mixes, with each mix containing three primer/probe sets, with 1a and 1b subtyping in the NS5b region, and the remaining primer sets are in the 5UTR region. The RNA concentration detected with a probability of 95% or greater is 12 IU/mL .

For HCV core sequencing, see HCV Genotype by Sequencing .

Synonyms

HCV RNA Qualitative, Hepatitis C by PCR, PCR-Hepatitis C

Components

Tissue Biopsy, Bone Marrow, Swab or Cellular fluid

Collection

Tissue Biopsy: Pea Size in sterile tube/universal transport media.

Bone Marrow: 5 mL blood in EDTA tube, No Heparin aspirate in syringe.

Swab: In sterile tube/universal transport media .

Cellular fluid: 1 mL in sterile tube

Unacceptable samples: Buffy coat collected with heparin syringe. Paraffin samples.

Handling Instructions

Bone Marrow: Send LAVENDER TOP to Laboratory at room temperature within 24 hour of collection.

Note: Separate specimens MUST be submitted when both Viral Culture and PCR testing are ordered.

Quantity

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Data Acquisition And Analysis

A PCR amplification curve for each amplicon was generated by plotting the peak height versus the cycle number based on the growth of the target peak as the PCR progressed. The quantitative cycle values were determined from the amplification curves using a threshold peak height of 5. Cq values were then converted to IU/mL based on a predetermined calibration curve traceable to the 5th WHO International Standard for HCV. The analysis were conducted using a proprietary software developed for the TASWako g1 instrument.

What Is The Role Of Quantitative Hepatitis C Virus Rna Assays In The Diagnosis Of Hepatitis C Infection

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Quantitative assays ascertain HCV RNA quantity in blood, using signal amplification or target amplification techniques . RT-PCR is more sensitive than bDNA testing. The HCV RNA level in blood helps predict the likelihood of a response to treatment, and the change in HCV RNA level can also be used to monitor the therapeutic response.

The same quantitative test should be used throughout therapy to avoid confusion, and results should be reported in international units to standardize data. The Versant HCV RNA Assay, version 3.0, is based on bDNA technology and has a dynamic range of 615-7,700,000 IU/mL. Another FDA-approved HCV quantitative test is the Aptima HCV Quant Dx Assay its limit of detection is 3.9 IU/mL in plasma and 3.4 IU/mL in serum.

The following are the best laboratory evidence of acute HCV infection:

• A positive HCV RNA test in the setting of a negative HCV antibody test
• A positive HCV antibody test after a prior negative HCV antibody test

It should be noted that impaired antibody production in immunosuppressed individuals may result in misleading information.

References

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Hepatitis C Viral Load / Hcv Rna Quantitative Testing

Hepatitis C

The viral load of hepatitis C refers to the amount of virus present in the bloodstream. The quantitative HCV RNA tests measure the amount of hepatitis C virus in the blood. The result will be an exact number, such as “1,215,422 IU/L.” Many people refer to the quantitative measurement as the hepatitis C “viral load.”

Viral load tests are used to confirm active hepatitis C infection and are used during treatment to help determine response. If you have lower levels of virus in your blood when you start treatment, you may have a better chance of getting rid of the virus.

Hepatitis C Rna Quantitative

Hepatitis C RNA, Quantitative

Description

The Abbott real-time FDA approved HCV assay detects and quantifies Hepatitis C virus by real-time PCR amplification. The Abbott assay utilizes three master mixes, with each mix containing three primer/probe sets, with 1a and 1b subtyping in the NS5b region, and the remaining primer sets are in the 5UTR region. The RNA concentration detected with a probability of 95% or greater is 12 IU/mL .

For Tissue Biopsy, Bone Marrow, Swab, or Cellular fluid , see Hepatitis C Qualitative PCR, Tissue .

For HCV core sequencing, see HCV Genotype by Sequencing .

Synonyms

HCV RNA Quant, Hepatitis C by PCR, PCR-Hepatitis C, Viral Load-Hepatitis C

Components

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Hepatitis C Rna Pcr Qualitative Blood Test

The Hepatitis C RNA PCR Qualitative test is used to look for infections with the Hepatitis C virus. This test looks for the genetic material of the virus. Because viral genetic material may be detectable earlier than antibodies which develop in response to an infection, PCR testing can be used to screen for a recent exposure. This test is also useful as a confirmation for people who have had a positive result from a HCV Abs test. Results for this test are qualitative meaning they will come back as positive or negative.Hepatitis C is a virus spread through contact with infected blood. Nearly 80% of Hepatitis C infections develop into chronic Hepatitis. The number of people worldwide with chronic Hepatitis C infections is around 150 million. Chronic Hepatitis C infections can lead to serious health complication such as Cirrhosis and Liver Cancer. Many HCV infections display no symptoms. When symptoms do occur, some of the most common include:

Fever Grey feces Jaundice

Note: Result turn around times are an estimate and are not guaranteed. Our reference lab may need additional time due to weather, holidays, confirmation/repeat testing, or equipment maintenance.

Detection Period:

The Hepatitis C PCR test can typically detect the virus about 3 weeks from a suspected contact or exposure or anytime after. Some people may be detectable earlier.

Requirements:

Other Things To Know:

• The viral load measurement does not tell us anything about the severity of a patient’s liver disease or the degree of fibrosis . For that information, the patient would need additional testing.
• It is not necessary to check the viral load repeatedly during treatment.
• If a quantitative HCV RNA result is reported as “< 15 IU/L,” this means that the quantitative test cannot measure the hepatitis C virus. It may mean that there is no detectable HCV RNA at all, but it may mean that the level of virus is just too low for the test to pick it up.

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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 6 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 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.

Refer to for collection criteria and instructions for RNA testing using DBS. DBS samples, when prepared appropriately, will be stable at room temperature for up to 30 days.

Roche Taqman Hcv Assay

The Cobas TaqMan HCV test is a real-time nucleic acid amplification assay for quantitative detection of HCV RNA in human serum or plasma. Like the TaqMan HCV analyte-specific reagent , this assay was developed for use with the recently introduced Cobas TaqMan 48 analyzer . Amplification and detection were performed according to the manufacturer’s instructions for TaqMan HCV with a CTM 48 analyzer and Amlilink software, v. 3.0.1 . The lower limit of detection is 15 IU/ml, with 95% probability, using 1 ml of serum .

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Hcv Rna Hepatitis C Viral Rna Quantitative

5,000

This test is generally useful in monitoring Hepatitis C Virus.

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Synthesis Of Cdna And Qualitative Detection Of Hcv

The isolated RNA was subjected to reverse transcription using a high-capacity cDNA Archive Kit according to the manufacturers specifications. The resultant cDNAs were subsequently used for conventional PCR and qPCR assay. For the qualitative detection of HCV-RNA by nested PCR, the primers were designed to amplify a portion of 5 untranslated region of HCV genome. The primer pair for the first round of PCR was: sense-primer HCV-U1 5-CTGTGAGGAACTACTGTCTTC-3 and antisense primer HCV-D2 5-CAACACTACTCGGCTAG-CAGT-3. For the second round a set of sense-primer HCV1–N3 5-ACGCAGAAAGCGTCTAGCCAT-3 and antisense primer HCV-N4 5-ACTCGGCTAGCAGTCTTGCGG-3 were used. The PCR reagents and thermal cycling conditions were used according to the protocol previously described.

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Preparation Prior To Transport

Label the specimen container with the patients full name, date of collection and one other unique identifier such as the patients date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.Place specimen in a biohazard bag and seal. It is recommended to ship specimens for testing to PHO Laboratory immediately after collection or processing to avoid delays in testing. Whole blood that has not been centrifuged must be received at PHO Laboratory within 6 hours of collection, before 2:00 p.m. Monday – Friday. Serum stored at 2°C – 8°C must be shipped with ice packs within 6 days of separation. Frozen serum must be shipped on dry ice.Filter cards containing DBS specimens should be shipped to the PHO Laboratory in individual resealable bags containing a desiccant sachet. When the sample is appropriately prepared, it will be stable at room temperature for 30 days. Do not refrigerate samples. Do not ship on weekends.Shipping of specimens shall be done by TDG certified individuals in accordance with TDG regulations.

Real Time Detection Of Hcv Target Amplicon By Capillary Electrophoresis

RT-qPCR assay of the 5th WHO International Standard for HCV spanning the range of 1510000 IU/mL in EDTA-plasma was performed on the µTASWako g1 Analyzer. Figure shows the overlay of electropherograms of the PCR cycles for 1000 IU/mL of HCV RNA. The graph depicts the growth of the amplicons of HCV RNA target and the non-competitive internal control , MS2 IC as the thermal cycle progressed. Peak alignment was performed using the 300-bp and 500-bp DNA markers. The fluorescent signal was normalized by the peak height of the 300-bp DNA marker. A growth curve for each HCV concentration was generated by plotting the amplicon peak height against the PCR cycle number, and the Cq value was determined by using a threshold value of 5. The composite PCR amplification curves and the linearity plot from 15 to 10000 IU/mL of the 5th WHO International Standard for HCV are shown in Fig. . The linearity plot, shown with serial dilution of the WHO standard, revealed a linear response over the range of HCV concentration tested. The amplification efficiency was calculated to be 96% 1).

Figure 1

Real time detection of HCV RNA by RT-qPCR-CE. Overlay of CE electropherograms for 1000 IU/mL of the 5th WHO International Standard for HCV. HCV amplification growth curves. , PCR linearity plot. PCR efficiency = 101=96%. RFU stands for Relative Fluorescence Unit.

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Preparation Of The Standard Curve

To perform an absolute quantitative qPCR, a plasmid construct harboring genotype 1b HCV subgenomic replicon was used to prepare the standard curve. We first calculated the mass of a single plasmid molecule based on the pFKI389-NS3-3 plasmid size, the mass of plasmid containing the copy number of interest that was 20,000,000 to 10 copies, and the concentration of plasmid needed to achieve the copy number of interest. This was followed by the preparation of 10-fold serial dilutions of plasmid template to create the standard curve. Finally, with the threshold cycle values, a linear regression model was calculated.

Absolute Quantitative Pcr Assay

cDNAs were subjected to qPCR for HCV-RNA quantification. Amplifications were conducted in triplicate using the following primers: HCV-Forward 5-GCGTCTAGCCATGGCGTTA-3 and HCV-Reverse 5-GGTTCCGCAGACCACTATGG-3 and the TaqMan probe 5-FAM-CTGCACGA-CACTCATAC-NFQ-3. The Perkin-Elmer ABI Prism 7000 Sequence Detection System was used for real-time analysis and thermal cycling conditions were: initial setup at 50°C for 2 minutes, then 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Fluorescence was monitored during each annealing step and amplification plots were generated. For each PCR reaction, 12.5 L of TaqMan PCR Master Mix, 1.25 L of 20x Assay Mix, and 11.25 L of cDNA diluted were added.

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Hepatitis B Pcr Quantitative Blood Test

This test is used to look for the presence of Hepatitis B viral genetic material in the blood. The results for this test are quantitative, meaning they provide a numerical result rather than a simple positive or negative. Because Viral DNA is often detectable sooner than antibodies developed in response to an infection, Hep B PCR testing can be used as a screening for people who have had a recent exposure. This test can also be used as a confirmatory test for a previous positive result or to assist in monitoring the effects of antiviral therapy.

Hepatitis B is a viral liver infection that is spread through exposure to infected blood or bodily fluids. It is the most common cause of acute viral Hepatitis. Hepatitis B infections often show no symptoms but when symptoms do occur they are often described as flu-like. Common symptoms include abdominal pain, fever, loss of appetite, nausea, joint pain, fatigue, jaundice, and dark-colored urine. Chronic Hep B infections can cause serious health complications like Cirrhosis and Liver Cancer.

This test is typically ordered by people:

• Who require screening for Hepatitis B but may not yet be at a point after exposure where a Hep B surface Antigen test will be accurate.
• Are receiving treatment for Hep B and wish to monitor their viral levels to see how effective their course of treatment is.
• Require a highly sensitive test to confirm the results of a previous Hepatitis B test.

Detection Period:

Requirements:

Description: