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.

Hepatitis C Virus RNA Strands Detection in Peripheral Blood Mononuclear Cells Legitimizes Virus Eradication in Negative Serum PCR Naive and Post-treatment Patients

Background and Aims:Hepatitis C virus(HCV)hepatotropism is associated with intra-peripheral blood mononuclear cell(PBMC)infection that causes post-treatment relapse in RNA seronegative patients.Our understanding of the association of non-viremic hepatic fibrosis with positive anti-HCV IgG antibodies and active hepatocellular damage might be increased by PBMCs screening for intracellular infection.Thus,the goals of this study included evaluation of PBMCs PCR for diagnosing HCV infection,addressing PBMCs plus serum real-time(SRT)PCR benefits over SRT-PCR alone,studying intraPBMCs distribution of RNA sense and antisense strands,and identifying treatment feasibility in solitary intracellular infection.Methods:Enzyme-linked immunosorbent assay,SRT-PCR and PBMCs PCR were used to evaluate HCV infection in 401 subjects.The patients were classified into groups of negative controls(n=30),positive controls(n=63),non-viremia post-treatment(experienced;n=166)and na(i)ve(n=49)cases,and non-viremia positive PBMCs PCR na(i)ve(n=35)and experienced(n=58)patients.Results:The diagnosis of true positive and negative by PBMCs PCR and SRT-PCR had 100%and 96.7%compatibility respectively.PBMCs PCR detected intracellular HCV infection in 49 out of 215 non-viremia patients;among them,na(i)ve cirrhotics had significantly higher number of intracellular infection than the na(i)ve non-cirrhotic(P<0.001)and experienced patients(P<0.0001).Antisense and sense strands were respectively recognized in na(i)ve and experienced cases(P=0.01218).Intracellular HCV strands were detected in 18.02%of experienced patients.Recognition of intracellular RNA strands showed significant decline in experienced compared to na(i)ve patients(P<0.05).Conclusion:PBMCs PCR is a valid diagnostic test that can diagnose intracellular HCV when SRT-PCR is negative.Antisense and sense strands are respectively recognized more often in na(i)ve and experienced patients.The expected overall relapsing rate in our cohort was 18.02%.IntraPBMC infections are associated with liver cirrhosis in na(i)ve non-viremia patients.Eradication of intracellular strands is recommended to avoid RNA seroconversion.