Rationale
Testing for hepatitis C virus (HCV) infection by using anti-HCV is performed for 1) clinical diagnosis of patients with signs or symptoms of liver disease; 2) management of occupational and perinatal exposures; and 3) screening asymptomatic persons to identify HCV-infected persons who should receive counseling and medical evaluation. Anti-HCV test results also are used for public health surveillance to monitor incidence and prevalence and to target and evaluate HCV prevention efforts.
Anti-HCV testing is performed in multiple settings, including hospitals and other health-care facilities, physicians' offices, health department clinics, HIV or other freestanding counseling and testing sites, employment sites, and health fairs. The interpretation of anti-HCV screening-test-positive results in these settings can be problematic. Clinical information related to the persons tested often is lacking, and even persons with risk factors for HCV infection might be at sufficiently low enough risk for infection that their screening test results could be falsely positive (e.g., health-care professionals are at occupational risk for HCV infection, but their overall prevalence of infection is low). (Alter, 2002) Without knowledge of the origin of the test sample or clinical information related to the person being tested, the accuracy of a screening-test-positive result for any given specimen cannot be determined.
However, despite previous recommendations for reflex supplemental testing of all anti-HCV screening-test-positive results (CDC, 1998), the majority of laboratories report positive anti-HCV results based only on a positive screening assay. To facilitate and improve the practice of reflex supplemental testing, the recommended anti-HCV testing algorithm has been expanded to include an option for more specific testing based on the signal to cut-off (s/co) ratios of screening-test-positive results that can be implemented without substantial increases in testing costs.
Implementation of these recommendations will provide more reliable results for physicians and their patients, so that further counseling and clinical evaluation are limited to those confirmed to have been infected with HCV. This is critical for persons being tested for HCV infection for the first time, for persons being tested in nonclinical settings, and for those being tested to determine the need for postexposure follow-up. Implementation of these recommendations also will improve public health surveillance systems for monitoring the effect of HCV prevention and control activities.
Laboratory Algorithm for Anti-HCV Testing and Result Reporting
All laboratories that provide anti-HCV testing should perform initial screening with a Food and Drug Administration (FDA)-licensed or approved anti-HCV test according to the manufacturer's labeling.
- Screening-test-negative (i.e., nonreactive) samples require no further testing and can be reported as anti-HCV-negative.
- Screening-test-positive samples require reflex serologic or nucleic acid supplemental testing according to the testing algorithm. Laboratorians can choose to perform reflex supplemental testing 1) based on screening-test-positive s/co ratios, or 2) on all specimens with screening-test-positive results.
- For screening-test-positive samples that require reflex supplemental testing (according to the testing option chosen), the anti-HCV result should not be reported until the results from the additional tests are available.
Reflex Supplemental Testing Based on Screening-Test-Positive Signal to Cut-Off (S/Co) Ratios
- Laboratories should use only screening tests that have been evaluated for this purpose* and for which high s/co ratios have been demonstrated to predict a supplemental-test-positive >95% of the time among all populations tested.
- Screening-test-positive samples with high s/co ratios can be reported as anti-HCV-positive without supplemental testing.
- A comment should accompany the report indicating that supplemental serologic testing was not performed, and it should include a statement that samples with high s/co ratios usually (>95%) confirm positive, but <5 of every 100 samples with these results might be false-positives. The ordering physician also should be informed that more specific testing can be requested, if indicated.
- Screening-test-positive samples with low s/co ratios should have reflex supplemental testing performed, preferably recombinant immunoblot assay (RIBA) (see Figure 4 in the original guideline document).
*Note: Data are available from three screening assays. For the two enzyme immunoassays (HCV EIA 2.0 or HCV Version 3.0 ELISA), high s/co ratios are defined as screening-test-positive results with average s/co ratios >3.8, and low s/co ratios as screening-test-positive results with average s/co ratios <3.8. For chemiluminescence immunoassay (VITROS Anti-HCV), high s/co ratios are defined as screening-test-positive results with s/co ratios >8, and low s/co ratios as screening-test-positive results with s/co ratios <8.
Reflex Supplemental Testing on All Specimens with Screening-Test-Positive Results
- RIBA only
- Nucleic acid test (NAT), followed by RIBA for specimens with NAT-negative results
Considerations When Choosing a Reflex Supplemental Testing Option
Serologic Supplemental Testing
- RIBA can be performed on the same sample collected for the screening test.
- RIBA is the most cost-effective supplemental test for verifying anti-HCV status for screening-test-positive samples with low s/co ratios.
- The RIBA result is used to report the anti-HCV result.
Nucleic Acid Supplemental Testing
- NATs can be performed in laboratories that have facilities specifically designed for that purpose.
- Serum or plasma samples must be collected, processed, and stored in a manner suitable for NATs to minimize false-negative results (Davis et al., 1994).
- Blood should be collected in sterile collection tubes with no additives or in sterile tubes by using ethylenediaminetetraacetic acid (EDTA).
- Serum or EDTA plasma must be separated from cellular components within 2-6 hours after collection.
- Storage of serum or EDTA plasma at 2 degrees C to 5 degrees C is limited to 72 hours; for longer storage, freezing at -20 degrees C or -70 degrees C is recommended. If shipping is required, frozen samples should be protected from thawing.
- Samples collected for serologic testing can be used only if the previous conditions are met.
- Because of assay variability, rigorous quality assurance and control should be standards of practice in clinical laboratories performing this assay; proficiency testing is recommended, including monitoring for false-positive results.
- Technician proficiency can vary and increases in direct relation to experience.
- Intra-assay contamination can occur, including aerosolization, splashing, and carry-over.
- If the HCV ribonucleic acid (RNA) result is positive, the presence of active HCV infection can be reported as well as a positive anti-HCV result.
- An HCV RNA-negative result requires that RIBA be performed and the RIBA result used to report the anti-HCV result.
Other Reflex Supplemental Testing Options
Certain laboratories might choose to modify the recommended supplemental testing options to provide additional information before reporting results. One such modification might include reflex NAT of screening-test-positive results with high s/co ratios, which might be of interest to hospital-based laboratories that usually test specimens from patients being evaluated for liver disease. If the NAT result is positive, the presence of active HCV infection can be reported as well as a positive anti-HCV result. However, if the NAT result is negative, reflex RIBA testing still is required before reporting the results to verify the anti-HCV status. Certain specimens will test RIBA-positive, indicating that the person should receive further evaluation, including repeat testing for HCV RNA (see Interpretation of Anti-HCV Test Results).
