A highly specific and sensitive hepatitis C virus antigen enzyme immunoassay for One‐step diagnosis of viremic hepatitis C virus infection

The current standard in diagnosing hepatitis C virus (HCV) infection requires two sequential steps: anti‐HCV test to screen, followed by HCV RNA reverse‐transcription polymerase chain reaction to confirm viremic HCV (V‐HCV) infection. HCV core antigen tests provided potential for possible one‐step diagnosis. However, low sensitivity and specificity limit their clinical utility. The present study developed a novel HCV antigens enzyme immunoassay (HCV‐Ags EIA) and assessed its sensitivity, specificity, and utility for one‐step diagnosis of V‐HCV infection using 365 serum specimens, including 176 without and 189 with V‐HCV infection. First, we confirmed the presence of HCV nonstructural proteins 3, 4b, and 5a besides HCV core antigen during HCV infection and developed a novel HCV‐Ags EIA through simultaneous detection of all four HCV proteins. For the first time, the present study demonstrated that serum sample denaturation decreases the test specificity due to release of HCV‐Ags sequestered in HCV immune complexes and should not be used in any HCV‐Ags, including all the current HCV core antigen assays. On the other hand, using sample nondenaturation, the HCV‐Ags EIA results showed 98.9% specificity and 100% sensitivity compared to serum anti‐HCV and HCV RNA reverse‐transcription polymerase chain reaction results. Using serum sample dilution, and nondenaturation, the lowest limits of detection of the HCV‐Ags EIA were equivalent to serum HCV RNA levels of approximate 150‐250 IU/mL. Conclusions: The highly specific and sensitive HCV‐Ags EIA developed in the present study has the lowest limit of detection equivalent to serum HCV RNA levels of 150‐250 IU/mL; using nondenaturation of serum samples, our HCV‐Ags EIA reliably differentiated V‐HCV infection from resolved HCV infection, accomplishing screening and diagnosis of V‐HCV infection in one step. (Hepatology 2016;64:415‐424)