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Background and Aims:  Sustained virologic response is evaluated by single-step reverse transcription (SRT) PCR assay, which assesses hepatitis C virus (HCV) clearance from plasma but not from tissues such as peripheral blood mononuclear cells (PBMCs). Persistence of HCV RNA in PBMCs beyond end of treatment (EOT) is associated with nonresponse. Our goal was to measure intra-PBMC HCV RNA levels during oral antiviral therapy according to the HCV therapy follow-up fractionation (CTF2) protocol.  Methods:  Compensated chronic HCV patients ( n = 2 78 SRT-PCR positive) were scheduled to receive oral antiviral therapy. Subjects were followed-up by SRT and intra-PBMCs HCV RNA PCR at the end of the 2 nd , 6 th , 10 th , 14 th , 18 th and 24 th weeks to evaluate virus clearance from plasma and PBMCs, respectively. The CTF2 protocol evaluated SRT and PBMC PCR status at each follow-up point for determining therapy continuation or interruption to address cost effectiveness.  Results:  All patients tested negative by SRT PCR after therapy for 2 weeks. Application of the CTF2 protocol revealed: a) increasing HCV clearance rate from 75.9% at the end of 10 th week to 90.3% at the end of 24 th week ( p &lt; 0.00001); b) faster clearance of HCV from plasma compared to PBMCs at each point of follow-up until the 18 th week ( p &lt; 0.05); c) higher viral elimination rates diagnosed by PBMC HCV RNA PCR(-) compared to PBMC HCV RNA PCR(+) from the 6 th to 24 th week of treatment ( p &lt; 0.0001); d) higher over-time increase curve of combined plasma and PBMC HCV RNA determined negativity compared to the decline in positivity curves by PBMC PCR at the 6 th -18 th week compared to the 24 th week ( p &lt; 0.01)-these results validated treatment continuation; and e) solitary evaluation of EOT sustained HCV infection and relapses by PBMC HCV RNA ( p &lt; 0.001).  Conclusions:  Early elimination of serum and tissue (PBMC) HCV infection by oral antiviral therapy can be achieved and evaluated during a cost-effective CTF2 protocol application.

HCV Therapy Follow-up Fractionation (CTF2) by Intra-PBMC Nested RNA PCR Recognizes Early Virologic Response and Relapse