Hepatitis B - DNA Viral Load, Genotyping, and Drug Resistance Testing
Consistent with O. Reg. 671/92 of the French Language Services Act, laboratory testing information on this page is only available in English because it is scientific or technical in nature and is for use only by qualified health care providers and not by members of the public.
Background
This page provides testing information for hepatitis B virus (HBV) viral load, genotyping, and drug resistance testing at Public Health Ontario (PHO). HBV genotyping and drug resistance testing are referred to the Division of Sexually Transmitted and Bloodborne Infections at the National Microbiology Laboratory (NML) in Winnipeg, Manitoba.
For information regarding other testing options, refer to the following PHO webpages:
Updates
- Consolidated information from the HBV - Genotyping Test Information Sheet and added information on HBV drug resistance testing.
- Testing indications: added frequency of testing based on the testing indication.
Testing Indications
- HBV DNA viral load testing should not be used as a diagnostic test for HBV infection and should only be ordered in those with a confirmed HBV infection based on serologic testing. It is intended to aid in the management of HBV-infected individuals (i.e. HBsAg-reactive). HBV DNA viral load testing may be considered for the following:
- Treatment decisions and monitoring of treatment response. PHO will accept specimens for testing at the frequencies below.
- Minimal testing interval for treatment-naïve patients is every 6 months.
- Minimal testing interval for patients on treatment is every 3 months.
- Minimal testing interval for post-treatment monitoring is monthly.
- Testing outside of these intervals requires justification and approval. Call PHO customer service at 1-877-604-4567.
- Query viral breakthrough due to non-adherence to medication, drug resistance, or reactivation of HBV.
- Transplant recipient evaluation and post-transplant monitoring. HBV status of the donor must be indicated on the requisition, e.g., HBV serology and DNA, if applicable.
- Individuals at higher risk (e.g., injection drug use, HIV-positive GBMSM, high-risk occupational exposures) who are positive for HBsAg.
- Immunocompromised patients who are positive for HBsAg.
- Investigation of an occult HBV (e.g., isolated anti-HBc) infection.
- Management of HBsAg-positive pregnant individuals.
- Treatment decisions and monitoring of treatment response. PHO will accept specimens for testing at the frequencies below.
Note: HBV DNA viral load is not a diagnostic test and is not indicated for the screening of blood/blood products or to confirm HBV infection.
- HBV genotype testing may be indicated in the following:
- Pre-treatment evaluation, particularly in complex cases
- Therapeutic decision-making and prognostic assessment
- Monitoring of chronic HBV infection in patients with persistent viremia despite therapy
- HBV drug resistance testing may be indicated in the following:
- Baseline resistance for patients on treatment
- Viral breakthrough during antiviral therapy
- Observation of suboptimal response to therapy
- Before switching antiviral therapy
Acceptance/Rejection Criteria
- Dried Blood Spots (DBS) specimens are not accepted for HBV viral load, genotyping, and drug resistance testing.
- Unspun primary blood collection tubes will be rejected. Spun Serum separator tube (SSTs) are acceptable.
- Requests received other than those outlined in the testing indications will be rejected, unless approved by a PHO Microbiologist.
- Requests with incomplete information on the requisition will be rejected.
- Plasma is acceptable by the manufacturer for testing but has not been validated by PHO and will be reported with a disclaimer.
- HBV genotype and drug resistance testing on specimens with an HBV viral load that is detected below the limit of quantitation will be rejected.
Specimen Requirements
| Test Requested | Required Requisition(s) | Specimen Type | Minimum Volume | Collection Kit |
HBV DNA Viral Load |
Serum |
1.2 ml |
Serum separator tube (SST); vacutainer tubes without additives |
|
HBV Genotyping |
Serum or Previously submitted specimen for HBV DNA Viral Load |
1.0 ml |
Serum separator tube (SST); vacutainer tubes without additives |
|
HBV Drug Resistance |
Serum or Previously submitted specimen for HBV DNA Viral Load |
1.0 ml |
Serum separator tube (SST); vacutainer tubes without additives |
Submission and Collection Notes
Specimens may be stored at 2-25°C and must be centrifuged within 24 hours of collection. If using blood collection tubes without gel separators, serum must be separated and transferred to aliquot tubes immediately after spinning before sending to PHO. Spun SST can be sent to PHO, but a separated and aliquoted serum is preferred. Centrifugation should be performed according to the tube manufacturer’s instructions. Serum may be stored and/or transported at 2-8°C for up to 6 days or at ≤-18°C for up to 6 weeks. If extended storage is required, serum specimens can be frozen at ≤-60°C.
A minimum of 3 mL of serum is required to perform HBV DNA viral, genotyping and resistance testing together.
Complete all fields on the dedicated requisition. Select a testing indication (diagnostic, pre-treatment, or post-treatment). An indication for testing must be provided to avoid testing delays and/or cancellation. The date of collection must be recorded on the test requisition.
HBV genotyping and drug resistance testing require a quantifiable viral load. Specimens with viral loads below the limit of quantification (<10 IU/mL) will be rejected.
Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or health card number. For additional information, see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay. Patient information on the specimen must match the requisition, or the specimen will be rejected.
Limitations
HBV DNA viral load test is not a diagnostic test and is not indicated for screening of blood/blood products or to confirm HBV infection.
Grossly haemolysed, icteric, lipemic or microbially contaminated serum is not recommended for testing.
Storage and Transport
Place the specimen tube in the biohazard bag and seal it. Then, place the completed HCV/HBV Viral Load, Genotyping, and Drug Resistance Testing Requisition in the pouch at the front of the biohazard bag.
Ship specimens to PHO immediately after collection or processing to avoid delays in testing. Whole blood that has not been centrifuged must be received at PHO by 2:00 p.m. on the same day it is collected, Monday through Friday. Serum may be stored and/or transported at 2-8°C for up to 6 days or at ≤-18°C for up to 6 weeks. If extended storage is required, serum specimens can be frozen at ≤-60°C. Frozen serum specimens must be shipped on dry ice.
Shipping of specimens should be done by Transportation of Dangerous Goods (TDG) certified individuals in accordance with TDG regulations.
Test Frequency and Turnaround Time (TAT)
- HBV DNA viral load testing is performed daily Monday to Friday. Turnaround time is up to 8 business days from receipt by PHO’s laboratory.
- HBV genotyping and drug resistance requests are forwarded to the NML weekly on Thursdays from PHO’s laboratory, Toronto location. Turnaround time is up to 19 business days from receipt by NML.
- HBV DNA Viral Load: tested using the Roche cobas® HBV Quantitative Assay on the cobas® 8800 system. The cobas® HBV assay is an in vitro nucleic acid amplification test for the quantitation of HBV DNA in serum of HBV-infected individuals. The cobas® HBV DNA viral load test has a limit of detection of ~2.4 IU/mL and a lower limit of quantitation of 10 IU/mL.
- HBV Genotyping: tested by conventional PCR followed by sequencing and phylogenetic analysis compared to HBV genotype reference sequences.
- HBV Drug Resistance: tested using conventional PCR followed by sequencing and analysis compared to HBV reference sequences.
Algorithm
Specimens submitted for HBV viral load testing will be reviewed for a history of HBsAg reactivity. If there is no history, the specimen will be tested for HBsAg first, and only HBsAg-reactive specimens will be tested for HBV viral load. An exception is testing for occult HBV infection.
Interpretation
Results should be interpreted in the context of the patient’s clinical presentation and history, risk factors, and exposure history. The table below is a guide for the interpretation of HBV DNA viral load results.
Result |
Interpretation |
Comments |
|---|---|---|
Not Detected |
HBV DNA not detected. |
|
<1.00E+01 IU/mL |
HBV DNA detected below the lower limit of quantitation. Unable to quantify. |
An exact value for HBV viral load cannot be calculated as it is below the lower limit of quantitation of 10 IU/mL. |
1.00E+01 to 1.00E+09 IU/mL |
Viral load will be reported in IU/mL. |
|
>1.00E+09 IU/mL |
HBV DNA detected above the upper limit of quantitation. Unable to quantify. |
An exact value for HBV viral load cannot be calculated as it is above the upper limit of quantitation of 1.0E+09 IU/mL. |
MyPHO
Don’t have a MyPHO account? Register Now