Hepatitis C Antibody with Reflex to HCV RNA, PCR with Reflex to Genotype

The reflex algorithm allows 3 important HCV tests to be completed, if necessary, with a single blood draw. Each of the 3 tests provides information that is important when a patient enters care.¹

• HCV antibody testing is the initial screening test. A positive result is consistent with prior resolved infection, active infection, or a false-positive antibody, but a supplemental test is needed to confirm active infection.
• The reflex to quantitative HCV RNA testing serves 2 important functions: 1) as a supplemental test to confirm active infection in patients with a positive HCV Ab result; and 2) to establish the baseline viral load (concentration of HCV virus in blood). Knowing the viral load at baseline helps physicians monitor response to therapy.
• HCV genotype testing is used to inform treatment decisions and duration of therapy.

The tests included in this reflex panel are recommended to guide the selection (and, for some patients, duration) of antiviral treatment.¹ However, only about half of patients have had confirmatory HCV testing by the time of their first visit to an HCV treatment provider, and an even lower proportion have had quantitative RNA and genotype results.² The reflex approach should improve the proportion of patients who have this complete series of important HCV assay results.

Results are reported in international units per milliliter (IU/mL) to facilitate comparisons between results generated by different test methods. This is important because the various methods used by different laboratories are not standardized against each other. Use of IU/mL reporting units helps to make the comparison of viral load results across different methods more reliable.

This makes it easier to understand whether a change in viral load is clinically meaningful.

Replicate PCR test results using the same specimen can vary analytically by as much as 0.5 log IU/mL; thus, only changes greater than 0.5 log IU/mL from one measurement to the next (or across several measurements) are considered to represent true changes in viral load.³ Reporting the viral load results in log IU/mL units helps the healthcare provider accurately interpret changes in viral load and better assess a patient's response to antiviral treatment.

A “<15 Detected” viral load result means the assay detected HCV RNA in the patient’s specimen at a very low level (<15 IU/mL), but could not measure the precise level. A “<15 Not Detected” viral load result means the assay did not detect HCV RNA in the patient’s specimen.

This test is performed using a Taqman^® assay. The lowest viral load this assay can accurately quantify is 15 IU/mL, but the qualitative limit of detection is in the 10 to 13 IU/mL range. Therefore, even when the viral load is below 15 IU/mL, we can still report qualitative detection of HCV RNA consistent with active infection in some cases.

The LiPA genotype assay can identify all 6 major HCV genotypes (1–6). In many cases, it can also differentiate among HCV subtypes, including 1a, 1b, 2a-c, 3a, 3b, 3c, 3k, 4a/c/d, 4f, 4h, 5a, 6a/b, and 6c-l. However, if the LiPA banding pattern for a patient specimen does not sufficiently differentiate between subtypes, only the genotype may be reported (for example, "genotype 1" or "genotype 2").

The LiPA usually requires a minimum viral load of 300 IU/mL to successfully obtain a genotype. Since the viral load assay used in this reflex test has a much lower limit of quantitation, it is possible for the patient to have a detectable viral load (below 300 IU/mL) and not have a reportable genotype result. Therefore, this test code does not reflex to HCV genotype if the patient’s viral load is <300 IU/mL.

Not necessarily. The HCV genotype assay is performed by analyzing LiPA banding patterns that are indicative of the genotype. Two regions of the genome are assessed: the 5' UTR and the core region. Genotype 6 subtypes c-l have the same LiPA banding pattern as genotype 1 when this technique is performed. Therefore, the core region is used to differentiate type 1 from type 6. If the core region LiPA banding pattern is not conclusive, we report a result of “HCV genotype 1 but cannot rule out type 6 subtypes c-l.”

This result means that the specimen produced a LiPA banding pattern indicating that HCV subtypes 1a and 2b were both present. This test result is consistent with HCV coinfection by both subtypes.

The LiPA genotype test is designed to identify all 6 major HCV genotypes. In contrast, the NS3, NS5a, and NS5b drug resistance tests detect mutations associated with drug resistance for a particular HCV genotype. They are not intended for determining HCV genotype and subtype. Separate test codes for drug resistance testing are available, depending on the HCV genotype and the gene of interest:

• Hepatitis C Viral RNA Genotype 1 NS3 Drug Resistance (test code 90924)
• Hepatitis C Viral RNA Genotype 1 NS5a Drug Resistance(test code 92447)
• Hepatitis C Viral RNA Genotype 1 NS5b Drug Resistance(test code 92204)
• Hepatitis C Viral RNA Genotype 3 NS5a Drug Resistance(test code 93325)

The HCV genotype (LiPa) test should be performed before ordering an applicable HCV drug resistance test.