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Published: June 2008Print Record of Viewing
Dr. Yao discusses Mayo’s preferred diagnostic approach and laboratory assessment of chronic viral Hepatitis B infection.
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 B and attributes for currently available tests.
Let’s look at a patient case of a 55 year-old Asian American woman who immigrated to the United States 15 years ago. She presented to her primary physician with fatigue and a history of jaundice 25 years ago. Physical examination of this patient did not reveal any abnormality, but she was found to have a serum alanine aminotransferase level of 85 U/L.
How could the care provider rule out the possibility of chronic viral hepatitis in this patient?
Mayo Medical Laboratories offers two test options for the testing of Hepatitis B. One is the Chronic Hepatitis Profile of Unknown Type, indicated as test number 9025. The other, to be used if chronic Hepatitis B is suspected, is test number 9023.
This particular provider chose the chronic hepatitis profile of unknown type. This patient was found to have a confirmed positive Hepatitis B surface antigen and anti-Hepatitis B core total antibodies with negative surface antibody and negative Hepatitis C antibodies.
Let’s review what we know about the typical serologic course of patients with acute Hepatitis B. In the majority of patients, i.e., 95% or more of adults infected with Hepatitis B, they present with this typical serologic profile in which the surface antigen becomes detectable first at about four weeks after exposure and at the same time there is detectable serum Hepatitis B DNA. This is followed by the appearance of anti-Hepatitis B core IgM indicated in the light orange line and of course anti-Hepatitis B core total antibodies which includes IgM is elevated at the same time.
Once the disease is resolved, the surface antigen disappear by about six months after exposure, and then there is a period of about eight weeks during which only Hepatitis B core IgM is detectable before the appearance of anti-Hepatitis B surface antibody. Note that the Hepatitis B E antigen is present during the acute phase of the infection and once their symptoms resolve then patients here will convert to anti-Hepatitis B E antibody.
For the minority of patients, namely adults who get Hepatitis B infection, they present with this serologic course in which the surface antigen is present and persists for years, and that’s the definition of chronic Hepatitis B or Hepatitis B carrier. The Hepatitis B E antigen is also present and persistent, but eventually, with immune tolerance and resolution of the infection they will convert to anti-Hepatitis B E antibody.
Note that Hepatitis B DNA (shown in the blue bar) is present and persists as long as the surface antigen is positive.
Next we will review the serologic markers. As I mentioned, the Hepatitis B surface antigen is a reflection of acute or chronic infection. The Hepatitis B surface antibody, which can be measured qualitatively or quantitatively, is a reflection of either recent or past infection, but is also present in successful immunity resulting from Hepatitis B immunization.
Hepatitis B core IgM antibody is only present during the acute infection, but the Hepatitis B core total antibodies are present in both acute and past infection.
Hepatitis B E antigen is present in chronic carriers with active viral replication whereas the Hepatitis B E antibody is present in either chronic carriers with minimal viral replication or those with past infection.
There are several laboratory testing methods for serologic testing of Hepatitis B. As you can see, the most commonly used is the enzyme immunoassay (EIA) and these are the methods used in the Abbott Commander, Bio-Rad, and Ortho ELISA systems. There is also the micro particle EIA, used by the Abbot AxSYm system.
The most recent advancement in immunoassays for hepatitis serologic testing is the application of chemiluminescence immunoassay (CIA) technology and here you see that the Ortho Vitros, the ADVIA Centaur, and the Immulite 2000 systems utilize this CIA.
The Roche Elecsys 2010 system used an electro-magnetic form of CIA in this assay. The latest system from Abbott, the Architect, uses chemiluminescent micro particle immunoassay.
Diagnosis of Hepatitis B should be based on the patient’s medical history, risk of exposure and system complex along with evidence of abnormality in physical examination and other chemistry laboratory tests.
Here is a table of the various combinations of serologic markers for those with acute, chronic carrier or previous infections due to Hepatitis B. Of note is that Hepatitis B core IgM antibody is present only during the acute infection phase and Hepatitis B E antigen can be present or absent during chronic Hepatitis B stage. In the case of the active viral replication of chronic Hepatitis B, the E antigen is positive whereas those with minimal or inactive viral replication but still has positive Hepatitis B surface antigen will have negative E antigen.
I want to mention briefly about the indications and use of qualitative versus quantitative Hepatitis B surface antibody. The qualitative Hepatitis B surface antigen is mainly indicated to stage Hepatitis B infection, to determine whether it’s past infection versus chronic. As in the case of chronic Hepatitis B, the surface antibody would be absent.
It is also useful to determine immunity status to Hepatitis B infection because when the result is positive, it is indicated that it’s greater than or equal to 10 miU/mL which is the World Health Organization standard for conferring protection from Hepatitis B infection.
For quantitative Hepatitis B surface antibody, this test is mainly indicated to determine adequacy of human Hepatitis B immunoglobulin therapy in post-operative liver transplant recipients with chronic Hepatitis B. This titer is helpful for clinicians and pharmacists to determine the optimal dose and dosing interval for HBIG therapy based on clearance of the Hepatitis B surface antibody in the recipient serum.
For molecular testing, Hepatitis B treatment requires baseline viral load monitoring prior to starting treatment. However, the current molecular tests are not indicated or helpful for diagnostic purposes. Diagnosis is still based on serologic findings and presence of Hepatitis B surface antigens will provide a diagnosis for chronic or acute Hepatitis B.
Currently, there are various test methods, commercially as well as laboratory-developed methods for determining Hepatitis B DNA in serum and plasma. The commercially-available COBAS Amplicor HBV Monitor from Roche Diagnostics is a PCR-based assay with a range of 58-38,462 IU/mL.
The Digene Hybrid Capture II HBV DNA Test comes in two forms, standard and ultra-sensitive. The range of results is different for these two. The Digene Hybrid Capture is now part of QIAGEN Incorporated.
The Versant HBV DNA 3.0 is a branch DNA signal-amplification method from Siemens HC Diag. It has a range from 357-17.8 million IU/mL.
Finally, the TaqMan HBV assay is a real-time PCR method available in the United States as an analyte specific reagent, has the widest result range of all these tests with a lower limit of quantitative as low as 10 IU/mL.
Here is a graphical representation of the various commercially-available as well as laboratory developed HBV DNA quantification assays. The blue line indicates the range of results according to IU/mL, whereas the orange line indicates the values in copies per mL.
Let’s return to the patient’s case. This patient subsequently was found to have a Hepatitis B DNA level of 8,500,000 IU/mL in serum. This is quantified by the Mayo Medical Laboratories test number 88634. The provider also has requested an HBV genotype. She was found to be infected with the genotype b strain of Hepatitis B virus.
This patient was started with Entecavir, which is one of the current FDA-approved antiviral agents for treating chronic Hepatitis B.
In the United States, currently the therapeutic options that are FDA-approved are shown on this slide. There are two forms of interferon preparations for treating chronic Hepatitis B, and there are three nucleoside analogs, (top 3). The other two agents are actually approved by the FDA for treating HIV infection, but they have been used by hepatologists for treating drug resistant chronic Hepatitis B.
Finally the last class of FDA-approved anti-Hepatitis B viral agent is Adefovir which is a nucleotide analog.
Our patient was monitored closely every three months with her serum HBV DNA levels. As you can see form the slide, she had a good response three months into therapy with a decline in her serum DNA from 8,500,000 to 5,100 IU/mL, and her serum DNA HBV units were undetectable at six and nine months. Interestingly, by 12 months into mono therapy with Entecavir, there was a reemergence of viral DNA to 1,050 IU.
So the question posed by her treating physician is “What could be the cause(s) of the detectable HBV DNA level in serum at 12 months of therapy?”
We know from natural history of anti-HBV treatment that there is emergence of resistance in the HBV virus after antiviral treatment is started and this emergence of resistance can be detected using molecular methods looking at genotypic resistance in the target gene of these drugs. This is followed by a virologic breakthrough which in this case meaning that the serum HBV DNA will rise and when there is full virologic breakthrough, then we will see the HBV DNA level returns to baseline, even when it’s being treated.
This is in contrast to the Wild-type HBV virus indicated in the green line.
Currently there are several genotypic methods for determining HBV genotype. Meaning determining HBV genotype A, B, C, D, E, F, G and H as well as determining genotypic mutations in the drug target of the viral genome.
The INNO-LiPA assay from Innogenetics uses differential probe hybridization to the RT polymerase gene region of the virus as well as determining core/precore mutations in the core/precore gene regions of the virus. This assay requires a minimum viral load of 150 IU/mL present in the patient’s serum.
A DNA sequencing method from Siemens HC Diagnostics known as the Trugene HBV Genotyping Assay also detects mutations in the polymerase and surface gene regions of the HBV virus and that requires a min HBV DNA level of 200 IU/mL to yield consistent results.
Finally there are laboratory-developed assays that use the DNA sequencing method mainly looking at the polymerase gene region as well as the core/precore gene regions.
The literature has shown that individuals with HBV virus showing different genotypic resistance mutations at these sites confer resistance to these drugs.
Here in the pink color, the four resistance mutations codons confer resistance to Lamivudine; the green show resistance to Adefovir; the blue resistance to Entecavir; and the light purple are resistance mutation codons to Telbivudine. Lastly, the black colored mutation confers resistance to Telbivudine.
Our patient, by gene sequencing method testing, was found to have resistance mutation codons (listed). These codons confer resistance to Lamivudine, Entecavir, Telbivudine and Telbivudine.
The patient’s therapy was then switched to Adefovir.
In summary, diagnosis of Hepatitis B infection should be based on a high index of suspicion based on the patient’s presentation, med history, epidemiologic exposure, and laboratory findings.
Immunocompetent patients can be screened effectively and appropriately first with serologic marker tests.
I wish to disclose that I receive research grants currently and in the past from Roche Diagnostics, Siemens Healthcare Diagnostics, and Third Wave Technologies. I have also served on the advisory board for Roche Diagnostics and Siemens Healthcare Diagnostics.