Occult HCV Infection (OCI) Diagnosis in Cirrhotic and Non-cirrhotic Naive Patients by Intra-PBMC Nested Viral RNA PCR

Background and Aims:Occult HCV infections(OCIs)include IgG antibody seronegative cryptogenic(COCIs),as well as seropositive secondary na(i)ve(SNOCIs)and experienced(SEOCIs)cases.We used peripheral-blood-mononuclear-cell(PBMC)-PCR to evaluate COCIs and SNOCIs prevalence,serum HCV spontaneous disappearance(SCSD)in na(i)ve cirrhotics and non-cirrhotics,intra-PBMC HCV-RNA strands in relation to cirrhosis density in na(i)ve non-viremia cases,and HCV-RNA seroconversion after 1 year of solitary naive intra-PBMC infection.Methods:The anti-HCV IgG antibodypositive na(i)ve-patients(n=785)were classified into viremic(n=673)and non-viremic[n=112,including non-cirrhotics(n=55)and cirrhotics(n=57)],and 62 controls without evidence of HCV-infection.Controls and post-HCV non-viremia cases(n=62+112=174)were submitted to hepatic FibroscanElastography evaluation.All subjects(n=847)were screened for intra-PBMC HCV-RNA sense and antisense strands by nested-PCR.Results:Na(i)ve-OCI cases(4.84%)that were diagnosed by PBMC-PCR significantly raised the total numbers of HCV-infection to 714(p=0.01).The percent positivity of SNOCIs(34.82%)was significantly higher than for asymptomatic-COCIs(3.125%,p=0.0001).Comparing PBMC-PCR with single-step-reverse-transcription(SRT)-PCR for identification of SCSD in na(i)ve IgG antibody-positive nonviremia patients(n=112)revealed a decline in SCSD prevalence by PBMC-PCR(from 14.27%to 9.3%),regardless of presence of hepatic cirrhosis(p=0.03).SCSD was found to be higher by PBMC-PCR in non-cirrhotics compared to cirrhotics(p=0.0001),with an insignificant difference when using SRT-PCR(p=0.45).Intra-PBMC HCV-RNA infection was significantly more frequent in cirrhotics compared to both non-cirrhotics and controls(p<0.0005).An increased hepatic fibrosis density was recognized in intra-PBMC HCV-RNA infection with sense(p=0.0001)or antisense strand(p=0.003).HCV-RNA seroconversion was associated with intra-PBMC infection when both sense and antisense strands were detected(p=0.047).Conclusions:Intracellular HCV-RNA evaluation is crucial for diagnosing OCIs and addressing relapse probability.

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

Background and Aims:Sustained virologic response is evaluated by single-step reverse transcription(SRT)PCR as-say,which assesses hepatitis C virus(HCV)clearance from plasma but not from tissues such as peripheral blood mono-nuclear cells(PBMCs).Persistence of HCV RNA in PBMCs beyond end of treatment(EOT)is associated with nonres-ponse.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:Compen-sated chronic HCV patients(n=278 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 2nd,6th,10th,14th,18th and 24th 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 inter-ruption to address cost effectiveness.Results:All patients tested negative by SRT PCR after therapy for 2 weeks.Appli-cation of the CTF2 protocol revealed:a)increasing HCV clear-ance rate from 75.9%at the end of 10th week to 90.3%at the end of 24th week(p<0.00001);b)faster clearance of HCV from plasma compared to PBMCs at each point of follow-up until the 18th week(p<0.05);c)higher viral elimination rates diagnosed by PBMC HCV RNA PCR(?)compared to PBMC HCV RNA PCR(+)from the 6th to 24th week of treatment(p<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 6th-18th week compared to the 24th week(p<0.01)—these results validated treatment continuation;and e)solitary evaluation of EOT sustained HCV infection and relapses by PBMC HCV RNA(p<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 pro-tocol application.