HCV treatment outcome depends on SNPs of IFNL3-Gene polymorphisms (rs12979860) and cirrhotic changes in liver parenchyma.

The allelic discrimination of IFNL3-(rs12979860 C > T) polymorphism reveals ambiguous associations with the effectiveness of oral HCV treatment. Solitary intra peripheral-blood-mononuclear-cells (PBMCs) HCV-RNA antisense-strands are independently detected in naïve and experienced cases regardless of viremia or hepatic-parenchymal alterations. We examined the frequencies of IFNL3-genetic variants with chronic-HCV-induced liver changes during the sustained virologic response (SVR) by evaluating the PBMCs- HCV-PCR after oral antiviral therapy. Methods: Twelve weeks after finishing oral antiviral therapy, the effects of IFNL3-genetic variants were evaluated in three groups of patients: Group-I (n = 25) showed HCV-RNA negativity in both serum and PBMCs-, group II (n = 52) showed positivity of HCV-RNA in PBMCs, and group-III (n = 25) had positive HCV-RNA in serum. The genetic variants of the IFNL3-gene were estimated for all the enrolled cases and correlated with their hepatic image changes. Results: IFNL3-(rs12979860) genotyping in post-direct acting antivirals (DAAs) SVR and HCV-relapse revealed: a) high frequency of CC-genotype and C-allele in group I compared to group II (P < 0.005) and group III(P ≤ 0.05) when hepatic-parenchyma looks normal by ultrasound b) frequent CT-genotype and T-allele in group II compared with I(P < 0.01) and III(P < 0.05) when liver tissues are bright (early cirrhotic-changes) c) frequent TT-genotype and T-allele in group III relative to I (P < 0.05) and II (P ≤ 0.08) when liver-tissues appear coarse by ultrasound. Conclusion: Outcomes of HCV treatment depend on host IFNL3-gene polymorphism and hepatic-parenchymal changes. A high frequency of wild-CC-genotype and C-allele is observed in patients with normal hepatic parenchyma and that achieved SVR. Solitary relapse in PBMCs occurs on increasing CT-genotype frequency when liver tissues are bright. Serologic relapse is detected when TT-genotype and T-allele are dominant in association with the cirrhotic liver. Therefore, IFNL3-gene-SNP analysis as a genetic predictor in relation to ultra-sonographic hepatic-parenchymal changes could be valuable for selecting the patients with the highest priority for treatment.

Treatment of hepatitis C virus infection with direct-acting antivirals plus ribavirin eliminates viral RNA from peripheral blood mononuclear cells and reduces virologic relapse in diverse hepatic parenchymal changes.

Elimination of hepatitis C virus (HCV) may fail, leading to a non-response outcome because of inappropriate testing for viral RNA in peripheral blood mononuclear cells (PBMCs). Sequelae of HCV genotype 4 therapy with sofosbuvir and daclatasvir ± ribavirin were assessed in our study at the 12th week after end of treatment (EOT) by screening for viral genomic RNA in serum and PBMCs with correlation to hepatic parenchymal changes. We recruited 102 out of 2165 patients who had received sofosbuvir/daclatasvir, either alone (n = 1573) or together with ribavirin (n = 592). Subjects were classified into three groups based on testing by single-step reverse transcription PCR: group I, HCV negative in both serum and PBMCs (n = 25); group II, HCV positive in PBMCs only (n = 52); and group III, HCV positive in both serum and PBMCs (n = 25). Groups I and II (n = 77) were selected out of 2102 (every 27th subject), while group III (n = 25) were selected from every second or third serologic relapse (n = 63). The pre-sampling population (n = 2165) showed sustained virologic response (SVR) in 33.21%; serologic relapse in 2.91%; HCV RNA only in PBMCs (66.79%) compared to serologic relapses and potential cure (P < 0.0001); higher serologic (38 out of 63, P = 0.03210) and cellular (36 out of 52, P = 0.0002) relapses in dual therapy than in triple therapy. The post-sampling population (n = 102) showed more HCV relapses in dual (50 out of 60) than in triple (27 out of 42) therapy (P = 0.0351); increased HCV antisense RNA strand in relapses compared to positive-sense strands alone (P < 0.001); and significant SVR events in undetectable (15 out of 31) compared to early (10 out of 55, P = 0.0058) and cirrhotic liver tissue changes (0 out of 16, P = 0.0006). In summary, HCV treatment with sofosbuvir/daclatasvir is followed by higher rates of serologic and intracellular viral RNA relapse than treatment with sofosbuvir/daclatasvir plus ribavirin. Cellular and serum viral RNA relapses are accompanied by HCV-induced hepatic pathology. An increased SVR with no detectable liver tissue changes was observed after triple therapy due to elimination of HCV RNA from PBMCs.

Hepatitis C virus serologic relapse after treatment with direct-acting antivirals is dependent on viral RNA levels in peripheral blood mononuclear cells and the grade of liver cirrhosis.

The disappearance of hepatitis C virus (HCV) from serum and tissues for 12 weeks after the end of treatment (EOT) with direct-acting antivirals (DAAs) is known as a "sustained virologic response" (SVR) and occurs more frequently in non-cirrhotic patients than in cirrhotic patients. In this study, we evaluated the outcome of HCV treatment with sofosbuvir (SOF) plus ledipasvir (LDV) at both EOT and 12 weeks after EOT in patients with and without hepatic cirrhosis to address the relationship of serologic relapse to persistent infection of PBMCs and the frequency of hepatic encephalopathy and hepatocellular carcinoma (HCC) after treatment. Seventy-five patients with post-HCV liver cirrhosis were assigned to one of three groups (A, B, and C), each of which included 25 patients and corresponded to the patients' Child-Turcotte-Pugh (CTP) classification. All of the patients received a daily dose of SOF (400 mg) plus LDV (90 mg) for 24 weeks and were tested using HCV single-strand reverse transcription (SRT) and PCR analysis of PBMCs at both EOT and 12 weeks after EOT. Fourteen (18.7%) out of 75 patients (all study populations) had intra-PBMC HCV RNA, but only nine of them (64.3%) developed HCV RNA serum relapse (seroconversion) 12 weeks after EOT (P < 0.001). Encephalopathy was significantly higher in group C at EOT and 12 weeks after EOT (P < 0.05). Development of HCC was observed in decompensated patients of group C (2 out of 5 = 40.0%) 12 weeks post-EOT (P = 0.03). In conclusion, detection of HCV RNA within PBMCs at the EOT provides an indication of potential relapse after 12 weeks. Moreover, development of encephalopathy and HCC after HCV eradication by SOF plus LDV therapy is perhaps a future warning for post-treatment hepatic decompensation in cirrhotic patients.

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 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 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 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 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 protocol application.

Occult HCV Infection (OCI) Diagnosis in Cirrhotic and Non-cirrhotic Naïve 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 naïve (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 naïve cirrhotics and non-cirrhotics, intra-PBMC HCV-RNA strands in relation to cirrhosis density in naïve non-viremia cases, and HCV-RNA seroconversion after 1 year of solitary naïve intra-PBMC infection. Methods: The anti-HCV IgG antibody-positive naïve-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 Fibroscan-Elastography evaluation. All subjects (n = 847) were screened for intra-PBMC HCV-RNA sense and antisense strands by nested-PCR. Results: Naïve-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 naïve IgG antibody-positive non-viremia 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.

Hepatitis C Virus RNA Strands Detection in Peripheral Blood Mononuclear Cells Legitimizes Virus Eradication in Negative Serum PCR Naïve 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 intra-PBMCs 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 naïve (n = 49) cases, and non-viremia positive PBMCs PCR naïve (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, naïve cirrhotics had significantly higher number of intracellular infection than the naïve non-cirrhotic (p < 0.001) and experienced patients (p < 0.0001). Antisense and sense strands were respectively recognized in naïve 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 naïve 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 naïve and experienced patients. The expected overall relapsing rate in our cohort was 18.02%. Intra-PBMC infections are associated with liver cirrhosis in naïve non-viremia patients. Eradication of intracellular strands is recommended to avoid RNA seroconversion. Ethical approval certificate: Registration number 10231.