This case discusses a 75-year-old man with multiple blood transfusions who had an initially reactive anti-HCV test but undetectable HCV RNA. This could indicate either a past resolved HCV infection (true positivity) or a false positive anti-HCV result. Supplemental testing is recommended to distinguish between these possibilities, as single negative HCV RNA results cannot rule out current infection. Repeat anti-HCV testing using a different assay platform can help determine if the initial result was a biological false positive or indicative of past infection. Interpretation of HCV status requires considering results from immunoassays for anti-HCV, nucleic acid tests for HCV RNA, and supplemental tests.

1. Anti HCV reactive-
true or false positivity?

2. February 2020
• 75 years old man
• DM/ESRD on regular HD since 2018
• Anti HCV Non reactive (Medi-Vance lab
7/2/2020)
• Admitted in Feb 2020 for TURP and TURBT
• 2 pint PC transfused

3. April 2020
• Readmitted for haematuria
• 4 pint PC transfused
• Rapid test for Hepatitis C negative
• Anti HCV reactive (Roche Cobas analyser –
HPP)
• HCV RNA by PCR : Not detected (Limit of
detection 12 IU/ml)
• Memo to HD centre given

4. Aduan (May 2020): Contraction of Hepatits C
during blood transfusion in HPP
• Repeat Anti HCV reactive (Roche Cobas analyser
Serology Lab)
• Repeat HCV RNA by PCR : Not detected
• Blood bank investigated all donors – all non reactive
• Anti HCV (Abbott analyser at Blood Bank) Non
reactive
• Repeat Anti HCV Non reactive (Medi-Vance lab
5/6/2020)

5. Anti HCV
Detectable average 8 weeks after an exposure.
2 main methods to detect antibodies against
recombinant HCV poylpeptides
• Enzyme immunoassay (EIA)
• Chemiluminescence immunoassay (CIA)
**HCV has not been cultured, natural viral proteins
were unlikely obtained.
**No structural antigens and proteins have been
derived from HCV up to now.

6. 3 generations of HCV enzyme
immunoassay
• Although third-generation HCV are more sensitive and specific than
older generation assays, they still have a high percentage of false
positive reactions
• CDC has recommended the confirmation of all anti-HCV results by
either RIBA or HCV-RNA assay

7. Among immunocompromised populations (e.g., hemodialysis
patients) average rate of false-positive results approximately: 15 %.

8. HCV RNA
• The earliest marker of infection- appears 1 to
2 weeks after infection before any alterations
in liver enzyme levels and appearance of anti-
HCV antibodies can be detected.
• A direct indicator of ongoing viral replication.
– If the nucleic acid testing (NAT) result is positive,
active HCV infection is confirmed.
– If NAT result is negative, the HCV antibody or
infection status can not be determined.

9. Situations HCV RNA result can be
negative in persons with HCV infection
• As the titer of anti-HCV increases during acute
infection, the level of circulating HCV-RNA
declines
• Intermittent HCV RNA positivity has been
observed among persons with chronic HCV
infection.
• A negative HCV RNA result also can indicate
resolved infection.

10. Serum HCV core Antigen
• A surrogate marker of HCV replication
• HCV core antigen can be detected on average
1 to 2 days after HCV RNA during the pre-
seroconversion period.
• HCV Ag test presented as a ‘’Direct marker for
diagnosis of HCV infection’’. Sensitivity of HCV
core antigen test is lower than HCV-RNA assay

11. Immunoassay
for Anti-HCV
Nucleic acid test
for HCV RNA
Supplemental
test for anti-HCV
Interpretation of
HCV status
Negative - - Never infected
with HCV
Positive Not done Not done Unknown;
Screening test
positive needs
confirmation
Positive Positive Positive/ Not
done
Active HCV
infection
Positive Negative Not done Unknown; Single
negative HCV RNA
result cannot
determine
infection status
Perform RIBA TRO
false positive

12. Anti-HCV positive
but HCV PCR RNA negative
• No evidence of current (active) HCV infection,
– with no further testing necessary,
– unless there are ongoing risk factors for and suspicion of
recent infection.
– In this case, repeat HCV RNA test is recommended.
• To determine if the HCV antibody test represents
– a remote HCV infection that has resolved (true positivity)
– or a false-positive result
 CDC recommends a second FDA-approved HCV
antibody test that is different from the test used for
initial antibody testing. A biological false result is not
likely to occur with 2 different tests (Vermeersch 2008).

13. Supplemental test for anti-HCV
• Recombinant Immunoblot Assay (RIBA)
• Also a kind of serologic assay
• A positive RIBA result interpreted as anti-HCV
positive.
• Not enough to confirm current HCV infection
• A negative RIBA is indicative of unspecific
reactivity in the anti-HCV screening test.

14. Disadvantages of RIBA
• Difficulties of performing the test ( lengthy and technically complex
laboratory procedures).
• High percentage of the indeterminate results and high cost-
effectiveness
– causing the less use or preventing the common use in
many developing countries, in routine diagnostic
laboratory procedures
• In patients with a high pretest probability of infection, a
positive serological screening test should be ‘’confirmed’’ by
performing a qualitative test for HCV RNA rather than the
supplemental recombinant immunoblot assay. This strategy is
cost-effective and more informative than using the RIBA to
confirm positive antibody screening tests in a diagnostic
setting

16. Immunoassay
for Anti-HCV
Nucleic acid test
for HCV RNA
Supplemental
test for anti-HCV
Interpretation of
HCV status
Positive Negative Positive Has been infected
with HCV; Repeat
HCV RNA TRO
active infection as
HCV levels may
fluctuate
Positive Not done Positive Has been infected
with HCV; HCV
RNA , liver
enzymes to
determine current
infection status
Positive Negative/Not
done
Negative Never infected
with HCV

18. HCV antibody
reactive
HCV RNA not
detected
If distinction between true positivity and
biologic false positivity for HCV infection is
desired,and if sample is repeatedly reactive in
the initial test, test with another HCV antibody
assay