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Published: April 2008Print Record of Viewing
Dr. Joseph Yao discusses the diagnosis and monitoring of patients with Hepatitis C, including the different screening tests available for detection and confirmation.
Hepatitis C, a blood-borne infectious disease caused by the Hepatitis C virus, is primarily diagnosed by the elevated serum levels of Alanine transaminase (ALT). Most patients are generally asymptomatic or experience mild symptoms. If the patients are untreated, the infection can later result in cirrhosis and liver cancer.
Presenter: Joseph Yao MD, from the Division of Clinical Microbiology at Mayo Clinic
Welcome to Mayo Medical Laboratories’ Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.
Our presenter for this program is Dr. Joseph Yao, from the Division of Clinical Microbiology at Mayo Clinic. Dr. Yao will be discussing Mayo’s approach to laboratory testing for Hepatitis C and attributes for currently available tests.
Today, we are going to discuss a patient case to illustrate optimal laboratory testing for Hepatitis C.
This is the case of a 45-year-old healthy-appearing man, a former injection drug user, presenting for a pre-employment health examination. There were no abnormalities found on physical examination, but laboratory tests revealed mildly elevated serum alanine aminotransferase (ALT) level of 115 U/L.
The care provider suspects possible Hepatitis C in this patient. How can this diagnosis be confirmed or ruled out?
Diagnosis of Hepatitis C begins by having a high index of suspicion, especially in those with high-risk behavior, such as injection drug use.
Laboratory tests can help us screen and confirm presence of Hepatitis C. Usually, it is advised to begin with screening assay for Hepatitis C Total Antibody. Currently in the United States, there are enzyme immunoassay (EIA, ELISA) and chemiluminescence immunoassay (CIA) that are FDA-approved for such screening test purposes.
Once the screening test results found to be positive, one can confirm the Hepatitis C by either using recombinant immunoblot assay (RIBA) for the Hepatitis C Total Antibody or by qualitative or quantitative molecular tests for presence or absence of Hepatitis C RNA.
For 25% of individuals infected with Hepatitis C, they follow this typical serologic course which is that of eventual recovery with no long-term sequelae. Here you notice that the symptoms peak at the time of the peak of the serum alanine transaminase levels at about 2-3 months from infection. This is also the time that Hepatitis C Total antibodies become detectable in the blood by the enzyme immunoassay and the chemiluminescence immunoassays that we talked about earlier. Note that the HCV RNA is present in the blood much earlier than the peak of ALT and the appearance of the HCV antibodies, but because the virus does not persist in those who recovered, the RNA disappears by 6 months after infection.
For the remaining three-quarters of individuals infected with Hepatitis C virus, they follow this typical serologic course which is one of chronic infection. The Hepatitis C RNA persists in the blood although at some time points, they may be undetectable because of low viral replication indicated by the interrupted red line. HCV antibodies appear around the same time as those who did recover, which is 2-3 months from infection. Symptoms appear again coinciding with the peak of serum ALT but here because of persistence of viral replication and continued inflammation of the liver, the serum ALT levels fluctuate over time indicated in the yellow line.
The serologic tests that I mentioned earlier have good sensitivity of greater than 95% but they lack specificity which is usually in the range of 90-95% bearing in mind that the HCV antibodies may not be detectable in the blood until 2-3 months after infection.
The commercially available assays defer in HCV viral antigen use in making these commercial tests and they provide qualitative results based on a signal to cutoff ratio in the given specimen.
Once the screening test results are positive, one can confirm the presence of HCV Antibody by requesting a recombinant immunoblot assay (RIBA) which use specific HCV viral antigens to detect specific bands and provide a qualitative result.
Here’s a schematic diagram of the HCV viral genome. The 5’ non-coding region is on the left and the 3’ non-coding region on the right. In between these two non-coding regions are genes that encode for specific viral proteins. It is these proteins that are used in the preparation of commercial assays for screening tests.
The early generation of enzyme immunoassays (Version 2) incorporates three viral antigens that are indicated in purple. The third generation of enzyme immunoassay and the current chemiluminescence immunoassay utilize four viral antigens, with the additions of the NS5V antigens.
RIBA also come in two versions. Version 2 utilizes four viral antigens, whereas the third generation utilizes the NS5 protein as well.
Our patient was found to have a positive HCV Total Antibody by a chemiluminescence immunoassay, which is the method used here at Mayo Medical Laboratories. With a signal-to-cutoff ratio of 15.1 for enzyme immunoassay and, any signal-to-cutoff greater than 1 is considered positive. The value of the ratios do not necessarily correlate with the amount of antibodies present, but in general, the higher the signal-to-cutoff ratio value, the more antibodies are usually detected in the specimen.
The natural questions to this provider then are “What supplementary test(s) should be done to confirm HCV infection and determine infection status?”
The CDC has issued recommendations to laboratories on how to report and test subsequently to confirm the pres of Hepatitis C infection. This is a figure taken from that publication which appeared in the Morbidity and Mortality Weekly Report in 2003.
This algorithm illustrates that one should begin with screening test for anti-Hepatitis C Total Antibody. At Mayo Medical Laboratories, the test name is Hepatitis C Virus Antibody (Anti-HCV), in serum, by the chemiluminescence immunoassay. If the result is negative, then it’s reported out to the provider. If the test is positive, then laboratories and providers have the choice of either determining subsequent testing based on the signal-to-cutoff ratio or by testing all positive regardless of the signal-to-cutoff ratio, to reflex to one of two confirmatory tests.
In selection 2, one can choose an automatic reflex to confirmation of HCV Antibody by RIBA, or by choosing selection 3, with automatic reflex to reverse transcription PCR which is a molecular test to confirm the presence of HCV RNA.
One can also request Anti-HCV Confirmation (selection 4) alone after a screening test is positive, if an automatic reflex test was a not an issue. One can also request HCV RNA Detection and Quantification by RT-PCR (selection 5) here at Mayo Medical Laboratories if the HCV Anitbody reflex testing was not requested initially.
Note that with the recombinant immunoblot assay being positive, one cannot distinguish between past infections versus chronic Hepatitis C. With a positive screening test and a positive confirmation test by radio immunoblot assay, it is still necessary to do the nucleic acid test to determine if there is chronic Hepatitis C which is indicated by the presence of HCV RNA. In our Mayo Medical Laboratories practice, it is our preference to reflex after a positive HCV Antibody to the RT-PCR test since a positive HCV RNA is sufficient to confirm the presence of chronic or acute Hepatitis C without need to confirm HCV Anitbody by RIBA.
I want to spend a few minutes describing the molecular methods that are currently used commercially for qualitative and quantitative molecular tests. In the first column in the left, you see a polymerase chain reaction (PCR), which amplifies the viral nucleic acid target. In this case, it’s HCV RNA but because PCR is a DNA-target amplification process and the virus is an RNA virus, it is necessary to have the first step of reverse transcription by making a complimentary DNA copy of the viral RNA target. For HCV RNA amplification by PCR, it’s really a reverse transcription PCR (RT-PCR). From the complimentary DNA copy, one can generate multiple copies of subsequent DNA.
With transcription mediated amplification (TMA) for HCV RNA the viral target is copied by reverse transcription to make complimentary DNA, and then using transcription the complimentary DNA is used to make multiple copies of RNA. Both of these methods are amplifying the viral genome, so it’s a target amplification method.
The last method which is used commercially is the branch DNA (bDNA) method. Here, the viral RNA target is not amplified, but it is captured by oligonucleotide probes that are specific for the region of the HCV RNA gene. The probe contains multiple attachment sites for signal reagents to be attached, so a single viral target could be captured and the signal is amplified to indicate the presence or absence of the target. So here, you see this is a signal amplification method as opposed to a target amplification method.
Now commercially, molecular assays for laboratory testing of Hepatitis C come in qualitative as well as quantitative tests. Keep in mind that HCV RNA is the first viral marker that is detectable in the infected individual, usually by 2 weeks after infection.
These qualitative tests are useful for confirming the presence of acute or chronic Hepatitis C. However it is not useful for distinguishing between these two states of Hepatitis C. It is helpful for distinguishing chronic from past Hepatitis C infection, because in the latter, HCV RNA should not be present. Finally, it is useful to determine sustained virologic response which is defined as absence of Hepatitis C RNA to antiviral treatment.
The assays differ commercially in the limit of detection. Currently there are two FDA-approved assays in the United States: the COBAS AMPLICOR HCV Test Version 2 from Roche Diagnostics utilizes the RT-PCR method and it has a lower limit of detection at 50 IU/mL whereas the transcription mediated amplification method from Siemens Healthcare Diagnostics known as the VERSANT HCV RNA Qualitative Test, which has a lower limit of detection at 10 IU/mL.
Now quantitative molecular tests are used for similar indications as those for qualitative tests. However, they have the added advantage of monitoring precise response to antiviral therapy using a quantitative result.
The desirable responses to treatment are indicated here for treatment of Hepatitis C chronic infection, the current state of mgmt requires determining rapid virologic response which is measured at 4 weeks into initiation of treatment. The desirable response is at least 100-fold or 2 log decrease in the serum HCV RNA from the baseline.
Subsequent responses are measured at 12 weeks, which is known as early virologic response. End of treatment response occurs at 24 or 48 weeks depending on the duration of treatment decided by the provider. Finally, six months after the end of treatment which is known as sustained virologic response. The desirable responses at these three points are undetectable HCV RNA levels.
These assays differ in their limits of quantification. There is currently one FDA-approved assay in the US, and that is the VERSANT (name) form Siemens Healthcare Diagnostics which has a lower limit of quantification at 615IU. The other three assays indicated here are either analyte specific reagent (ASR) or are research-use only assays. Note that the real-time RT-PCR assays, such as Taq Man HCV RNA and the Abbott Molecular ASR, have quite good lower limits of quant detection at 10-25 IU/mL.
Interpretation of laboratory test results depends on the profile that is indicated here. Those with chronic Hepatitis C infection, all three markers -HCV Antibody screen, confirmation with RIBA and HCV RNA -should all be positive. For those with resolved infection, the difference from chronic infection is there should be an absence of HCV RNA.
Now, in acute HCV infection, depending on when the patient presents for medical attention and laboratory testing, the Hepatitis C Antibody may or may not be detectable by screening tests and by confirmatory antibody tests indicated by the plus and minus signs for these two tests. However, HCV RNA should be detectable in these individuals.
As the infection progresses, and if the patient presents later in the course of infection during the acute phase, then all three markers should also be detectable. So, the difference between recent HCV infection versus chronic HCV infection is the absence or presence of symptoms because in the former, symptoms of liver inflammation should be present, whereas in chronic infection, symptoms may be minimal or absent.
It is also important to recognize there may be false-positive HCV Antibody results. In this case, both the HCV Antibody confirmation by RIBA and HCV RNA would be negative. Or the recombinant immunoblot assay could be indeterminate as well. It’s also possible to have an indeterminate or false-negative Hepatitis C Antibody by RIBA, in which case the HCV RNA was positive.
In our patient, he was found to have an HCV RNA of 795,000 IU/mL. Because of the absence of clinical signs and symptoms, with the exception of an elevated serum, an alanine-transaminase level, we can conclude that he has chronic Hepatitis C.
The provider needs to know which additional tests should be requested to help guide specific anti-HCV therapy.
The final series of tests to present are the genotyping tests for Hepatitis C. They’re important to determine and guide the duration of anti-HCV treatment because HCV genotypes 1 and 4 requires prolonged treatment duration, namely 48 weeks, versus a shorter duration necessary to achieve desirable responses for those infected with genotypes 2, 3, 5, and 6. These assays are important to provide a genotype that will predict virologic response to anti HCV treatment.
Commercially there are three assays that are available in the US. They differ in the assay method and limit of detection. A method of reverse hybridization of the line probe assay (LiPA) from Siemens Healthcare Diagnostics, which market this assay manufactured by Innogenetics, the lower limit of detection is around 3,700 IU/mL.
Another assay from Siemens Diagnostics which utilizes gene sequencing assay known as to TRUGENE HCV 5’NC, has a lower limit of detection of 5,000 IU/mL.
Finally, an analyte specific reagent assay from third wave technologies, using a proprietary cleavase enzyme, with an oligonucleotide probe assay, known as the Invader HCV Genotyping Assay has a lower limit of detection at 1,000 IU/mL.
For our patient, he was found to have HCV genotype 1b, which is the most frequent HCV genotype in North America for those individuals infected with chronic Hepatitis C. In fact, around 75% of chron Hepatitis C patients are infected with genotype 1.