Peripheral Blood Monocytes in Prediction of Response to Direct Acting Antiviral Drugs in Chronic HCV Patients

BACKGROUND: Eradication rate of hepatitis C virus (HCV) infection had dramatically increased in era of direct acting antivirals (DAAs). However, predicting relapse post-treatment had to be more speculated. AIM: To evaluate role of peripheral blood monocytes (PBMCs) HCV PCR as predictor of post-treatment relapse. Methods: For chronic HCV patients who achieved end of treatment response, HCV PCR at PBMNs was assessed in relation to SVR. RESULTS: 112 out of 118 cases had SVR 24 with only six relapsers ORIGINAL ARTICLE Peripheral Blood Monocytes in Prediction of Response to Direct Acting Antiviral Drugs in Chronic HCV Patients El-Sayed Ibrahim, Maha Elsabaawy, Ayman Alsebaey, Sabry Moawad Abdelmageed, Abd-ElAleem A.ElGendy, Eman Abdelsameea 3383 Journal of GHR 2020 December 21; 9(6): 3383-3388 ISSN 2224-3992 (print) ISSN 2224-6509 (online) Online Submissions: http: //www.ghrnet. org/index. /joghr/ doi: 10.17554/j.issn. 2224-3992.2020. 09.964 Journal of Gastroenterology and Hepatology Research Molecular testing HCV Real time PCR: Whole blood samples on EDTA have been collected to separate plasma by centrifugation. Then, used for extraction of viral RNA by using QIAamp® Viral RNA: Qiagen (Qiagen, Germany). Real Time PCR for HCV performed by using ArtusHCV RG RT-PCR Kit HandbookSeptember 2013. Triple PCR (1) PBMC isolation: PBMC were isolated by using Ficoll Technique. Washing steps have been skipped to keep part of plasma with the buffy coat for ability of detection of positive, negative strands in cells and positive strands in plasma. (2) RNA extraction: Cellular and plasma viral RNA were extracted from 200 μl of PBMCs& plasma sample using SV Total RNA Isolation System spin column-based technique to final 60 μl Elution volume. Protocol described originally by Promega in “SV Total RNA Isolation System manual 07/18 was followed (Promega Corporation, USA). (3) cDNA synthesis: RNA converted to cDNA by using GoScriptTM Reverse Transcription System: Promega as per manufacturer instructions (Promega Corporation, USA). Up to 5μg RNA extract + random primer (0.5μg) completed the volume to 5μl with nuclease free water were heated in a 70°C heat block for 5 minutes. Immediately, chilled in ice water for at least 5 minutes and then, centrifuged for 10 seconds in a micro centrifuge. The mix components were GoScriptTM 5X reaction buffer = 4. 0μl, MgCl2 (final concentration 1.5-5.0mM) = 1. 2μl, PCR nucleotide mix (final concentration 0.5mM each dNTP) = 1. 0μl, GoScriptTM reverse transcriptase = 1. 0μl and nuclease-free water = 7.8 μl to a final total volume of 15μl. RNA samples (5μL) were added and mixed with the previous prepared components then inserted in the thermal cycler: Veriti96: (Applied Biosystem, USA). Thermal profile was 25°C for 5 minutes (annealing), 42°C for up to one hour (extension), then, 70°C for 15 minutes (reverse transcriptase inactivation). (4) qPCR detection: First targets primer has been picked and blasted for the triple PCR HCV Target to target positive and negative HCV RNA strands. Primers were designed by the Oligo Primer D bioinformatics tool primer sequence (Gene Bank Accession number: D10749). And primers used were synthesized by (Metabion, Germany) as the following: Forward: 5’ GAGTGTCGTGCAGCCT 3’ Reverse: 5’ CACTCGCAAGCACCCTATCA 3’ Detection was done by using 5x HOT FIREPol® EvaGreen® PCR Supermix from (Solis BioDyne ,Estonia) on Rotor Gene Q-plex Real Time PCR Machine (Qiagen, Germany). Melting curve have been performed to eliminate nonspecific reaction (Figure1). Reaction components were as the following: 5x HOT FIREPol®EvaGreen® qPCR Super mix = 4 μl, primer forward (10 pmol/μl) = 0.3 μl, primer reverse (10 pmol/μl) = 0.3 μl, DNA template (0.1 ng/μl) = 2 μl and H2O PCR grade = 13.5 μl with total volume 20 μl. Thermal profile and cycling conditions were 95°C for 12 min as initial activation for one cycle, 95°C for 15 seconds as denaturation step, 57°C for 20 seconds as annealing step, 72°C for 30 seconds as elongation step. Denaturation, annealing and elongation steps were repeated for 45 cycles. Then, final step was 70°C95°C continuous as melting step (Table 1). Statistical analysis Data were tabulated, computerized and shown in the form of rate (%) and the standard deviation (SD). Chi-square (χ2) test and Fischer’s exact test were used where appropriate. Predictors of SVR and peripheral blood mononuclear cells (PBMCs) confirms the role of these cells as HCV reservoir both during ongoing antiviral treatment and most importantly after its completion[5]. The discordance between serum and PBMCs HCV RNA was reported by Cavalheiro et al. 2007 as an early sign of HCV persistence or reinfection[6]. HCV relapse might be a consequence of continuous or recurrent intracellular PBMCs viral replication. Occult hepatitis C (negative viremia with positive HCV PCR RNA in PBMCs and/ or liver tissue) prevalence had been investigated among SVR achievers post DAAs and was assigned to be present in 25% chronic HCV patients treated with the combination of sofosbuvir and daclatasvir for 3 months[7-8]. HCV relapse might be a consequence of continuous or recurrent intracellular PBMCs viral replication[9]. PATIENTS AND METHODS Study population This study was conducted on 118 chronically HCV infected patients (males represented 51.7%, n = 61) who were eligible for treatment with DAAs and they were recruited from hepatology and gastroenterology department, National Liver Institute, Menoufia University and Al-Hussein University hospital in the period from March 2017 to June 2018. Establishment of diagnosis and evaluation were based on clinical examination, laboratory investigations and abdominal ultrasound examination (US). The study was approved by the local Ethics Committee, National Liver Institute, Menoufia University. Enrolment of the individuals to the study was conditioned by an obtained written informed consent. One hundred and eighteen patients were consecutively selected and submitted to either dual or triple therapy treatment protocols (sofosbuvir 400 mg, daclatasvir 60 mg with or without ribavirin) and achieved end of treatment response. At 24 weeks post treatment; 112 cases achieved sustained virological response (SVR24) (57 males / 55 females; with mean age 46.9 ± 10.2 years old), with only six relapsers. Non-responders (HCV/RNA positive in serum after completion of treatment), patients co-infected with HBV, patients with previous treatment with DAAs and/or immunosuppressive agents and chronic hepatitis C patients complicated with hepatocellular carcinoma all were excluded from the study. All cases were subjected to thorough history taking, clinical examination, and routine laboratory examinations including complete blood counts, liver tests [aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum total and direct bilirubin, albumin, prothrombin concentration and international normalized ratio (INR)]. HBsAg was assessed by third generation ELISA, detection of HCV RNA level by real time PCR with lower detection limit (15 Iu/mL), serum creatinine, HCV RNA in the PBMCs and serum were investigated at the end treatment &12 weeks later. Model of End-Stage Liver Disease (MELD) was calculated[10]. Cirrhosis was diagnosed based on clinical findings, imaging studies (abdominal ultrasound) and laboratory results. Laboratory investigations Ten ml venous blood samples collected from all patients were divided into three aliquots. Two aliquots used for routine laboratory investigations using fully automated auto analyzer SYNCHRON CX9ALX (Beckman Coulter Inc., CA, USA) for liver and renal tests, Sysmex K-21, (Sysmex Corporation, Kobe, Japan) for CBC and immunoassay (Abbott Laboratories, Abbott Park, IL, USA) for serum HBsAg and Anti-HCV. The third one was collected in EDTA containing tube for molecular and Triple PCR. 3384 El-Sayed I et al . Peripheral blood monocytes and DAAs