Comparison study between biochemical and molecular assays in chronic hepatitis C virus patients in Egypt

Mohamed M.S. Farag. Biochemical parameters and Molecular assays are used in monitoring patients with chronic hepatitis C (CHC). The aim of the present study was to investigate the relationship between biochemical markers and HCV RNA titers in patients with CHC. The study was conducted on 50 known HCVinfected patients, recruitment of patients was random. All samples were collected from the medical department of Al-Azhar University, Cairo, Egypt during the period from January 2013 to June 2014. For the HCV-RNA positive patients, blood samples were collected for different biochemical analysis at the time of routine clinic attendance. All serum samples were assayed for anti-HCV by ELISA. Regarding to molecular assay HCV RNA was detected by RT-PCR. Our results indicated that ALT and AST biochemical markers were increased in CHC in relation with HCV RNA titre on the other hand WBCs and Platelets (PLT) were decreased. In Conclusion the results introduced in this work confirmed that biochemical analysis was correlated to HCV RNA titre and this indicated that Biochemical and Molecular assays are essential for monitoring patients with CHC.

Biochemical parameters and Molecular assays are used in monitoring patients with chronic hepatitis C (CHC). The aim of the present study was to investigate the relationship between biochemical markers and HCV RNA titers in patients with CHC. The study was conducted on 50 known HCVinfected patients, recruitment of patients was random. All samples were collected from the medical department of Al-Azhar University, Cairo, Egypt during the period from January 2013 to June 2014. For the HCV-RNA positive patients, blood samples were collected for different biochemical analysis at the time of routine clinic attendance. All serum samples were assayed for anti-HCV by ELISA. Regarding to molecular assay HCV RNA was detected by RT-PCR. Our results indicated that ALT and AST biochemical markers were increased in CHC in relation with HCV RNA titre on the other hand WBCs and Platelets (PLT) were decreased. In Conclusion the results introduced in this work confirmed that biochemical analysis was correlated to HCV RNA titre and this indicated that Biochemical and Molecular assays are essential for monitoring patients with CHC. HCV infection is a significant public health issue. Currently, it is estimated that worldwide there are 175 million chronic hepatitis infection cases and 350.000 patients die every year due to complications of HCV such as cirrhosis and hepatic-cellular carcinoma (HCC) (Zaltron et al., 2012). HCV infection is an insidious disease with slow progression. HCV infection can be manifested as an acute infection and in around 20% of the patients, the disease spontaneously resolves but becomes chronic in 80% of cases (Saadeh et  The main objective of this study was to evaluate and determine the potential correlation between HCV viral load and different biochemical parameters in chronic hepatitis C.

Material and methods:-Blood Sampling:-
The study was conducted on 50 known HCV-infected patients, recruitment of patients was random. The median age of CHC patients was around 22. All samples were collected from the medical department of Al-Azhar University, Cairo, Egypt during the period from January 2013 to June 2014. For the HCV-RNA positive patients, blood samples were collected for different analysis at the time of routine clinic attendance.

Serological Assay (ELISA):-
All serum samples were assayed for anti-HCV positive by ELISA (Third-generation enzyme-linked immunosorbent assay, murex anti-HCV version), following the manufacturer's instructions. All the reagents were allowed to reach room temperature before running the assay. Liquid reagents were mixed before use. Concentrate washing solution was diluted 1/10 with distilled water. The concentrated conjugate was diluted 1/51 with the conjugate diluents. Diluted samples or controls were loaded into a 96-well plate pre-coated with a recombinant HCV-specific antigen. The plate was then incubated for one hour at 37°C to allow for the formation of the Ag-B complex. The plate was washed, the conjugate was added, and the plate was incubated for 30 minutes at 37°C. After incubation, the washing step was carried out and a substrate solution (TMB) was added for detection. Finally, the reaction was stopped using H2SO4 and the colorimetric signal was measured by absorbance at 450 nm using a spectrophotometer.

Molecular Assay:-
Extraction:-RNA was extracted using RTP® DNA/ RNA Virus Mini Kit. Briefly 200 μl of sample was transferred into the provided Extraction Tubes, 200 μl dd H2O was added. For samples which have a smaller volume than 200 μl were filled up to a total volume of 400 μl with ddH2O and incubated for 15 minutes at 65°C in a thermo-mixer after that incubated for 10 minutes at 95°C in a thermomixer (optional).For optimal binding conditions 400 μl Binding Solution was added and mixed completely by pipetting up and down. The sample was transferred on the RTA Spin Filter, incubated for 1 min then centrifuged for 2 mins at 11.000 x g (11.000 rpm), the flow-through with the RTA Receiver Tube was discarded and the RTA Spin Filter was put in a new RTA Receiver Tube. 500 μl Wash Buffer R1 was pipetted onto the RTA Spin Filter, centrifuged for 1 min at 11.000 x g (11.000 rpm) then the flow-through and the RTA Receiver Tube were discarded. the RTA Spin Filter was transferred into a new RTA Receiver Tube. 700 μl Wash Buffer R2 was pipette onto the RTA Spin Filter, centrifuged for 1 min at 11.000 x g (11.000 rpm), then the flow-through and the RTA Receiver Tube were discarded, after that, the RTA Spin Filter was transferred into a new RTA Receiver Tube. To eliminate any traces of ethanol, we centrifuged again for 4 min at maximum speed, discarded the RTA Receiver Tube. The RTA Spin Filter was transferred into an RNase-free 1.5 ml Elution Tube pipetted 60 μl of Elution Buffer R (preheated to 65°C) directly onto the membrane of the RTA Spin Filter, incubated for 3 min, centrifuged for 1 min at 11.000 x g (11.000 rpm) finally the RTA Spin Filter was discarded and the eluted viral DNA/ RNA was placed on ice.

Real Time PCR (RT-PCR):-
A RT-PCR test was done using RT-PCR reagents that constitute a ready-to-use system for the detection of HCV RNA by PCR in a Stratagene' Mx3000P quantitative RT-PCR system. The HCV RT-PCR kit included reagents and enzymes for the reverse transcription and specific amplification of a specific region of the HCV genome in a fluorescence detector FAM (reporter dye). The kit has a second heterologous amplification system to identify possible PCR inhibition. HCV PCR Master Mix (Applied Biosystems) was added including an optimized RT-PCR buffer, MgCl2, Taq DNA polymerase, and Reverse transcriptase, and stabilizers. HCV-RNA was amplified by RT-PCR using primers KY80 (5′GCAGAAAGCGTCTAGCCATGGCGT) and KY78 (5′CTCGCAAGCACCCTATCAGGCAGT) targeting the 244-base region located within the highly conserved 5′ noncoding region of the HCV genome. The reaction took place under stander thermal profile: incubation at 40°C for 60 minutes to transcribe viral RNA to cDNA by RT. This was followed by AmpliTaq gold activation at 95°C for 3 minutes. Denaturation was performed at 95°C for 15 seconds, followed by annealing at 94°C for 5 second and extension at 62°C for 10 second with end point fluorescence detection. 34 The fluorescence intensity increases proportionally with each amplification cycle in response to the increased amplicon concentration. This allows quantification of the template to be based on the fluorescent signal during the exponential phase of amplification, before limiting reagents, accumulation of inhibitors, or inactivation of the polymerase has started to have an effect on the efficiency of amplification. Software provided in the computer system should connect to the apparatus allowing real-time amplification plots to be viewed and to be analyzed during the PCR run.
The biochemical assessment included:-AST, ALT, and Albumin (Alb.) which were measured using Spin React Kit respectively and Bilirubin (BIL.) which was measured using Diamond kit.

Aspartate aminotransferase (ALT):-
Briefly 1ml of AST working reagent(one tablet of R2 (NADH+1200 U/L Lactate Dehydrogenase+α-ketoglutarate) dissolved in 15 mL of R1(TRIS pH 7.8 + L-Alanine))were pipetted and mixed with 100 µl sample into a cuvette, then incubated for 1 min at 37°C. After that initial absorbance (A) of the sample was read, also absorbances at 1minute intervals thereafter for 3 minutes were read at 340 nm. Finally, the difference between absorbances and the average absorbance differences per minute (A/min) were calculated.

Albumin (ALB):-
One ml of R (Bromocresol green PH 4.2) was pipetted and mixed with 5 µl of sample. Then incubated for 5 min at 37ºC. The absorbance (A) of the Blank (1 ml R). The colour is stable for 1 hour at room temperature.

Blood count:-
A complete blood count of haemoglobin (HB), white blood cells (WBC), and platelets (PLT), were counted using Beckman Coulter Machine. All obtained from the same automated blood sample at the time of admission to the study.

Results:-
Virological findings:-Serological assay: All patients included in our study were anti-HCV positive by third-generation enzyme-linked immunosorbent assay (ELISA) ( Table 1).

Molecular assay:-
The presence of the viral genome in serum was detected by qualitative polymerase chain reaction (PCR). All patients included in our study were positive for HCV RNA ( Table 2). Biochemical assays:-Our results showed increasing in ALT, AST, and Bilirubin and decreased in WBCS and Platelets with a relation to HCV RNA titres (Table 3). Table 3:-Biochemical analysis for chronic HCV patients

Correlation between biochemical and molecular assays:-
The present study was aimed to investigate the relation between biochemical and molecular assays in CHC patients. Our results indicated that some biochemical analysis including ALT and AST were increased in relation with HCV RNA titre (Viral load) on the other hand WBCs and Platelets (PLT) were decreased. These results have implied that biochemical parameters may contribute to monitoring patients with CHC (Table 4). In recent years, various studies investigated the association between the grade of liver injury and serum ALT levels, HCV RNA titers in CHC patients and HCV genotype were performed, but the results were inconsistent.
Fanning et al have found that serum HCV RNA viral load and ALT level were significantly correlated with the grade of liver inflammation but no such correlation was found between these parameters and liver fibrosis (Fanning  et al., 1999). Al Swaff have found an association between grade 1 and grade 4 liver fibrosis and higher ALT levels in patients with CHC (genotype 4) infection and have detected higher HCV RNA levels in grade 3 liver fibrosis (Al  Swaffet al., 2012).
Zechini et al have found a significant correlation between HCV RNA and ALT. in CHC patients and have also found a correlation between histological activity index (HAI) and HCV RNA levels aswell as between HAI and AST and ALT levels. They have reported in their study that particularly AST might be associated with liver injury (Zechinietal., 2004). Shahid 2014) that have been showed there was no association between HCV RNA level and grade of liver injury in chronic HCV carriers but serum ALT level was associated with portal inflammation and periportal necrosis. (Lee et al .,2014)In some studies, no clinically feasible association was found between ALT level and liver injury or liver fibrosis (Liu et al., 2009).
In Conclusion The present study have implied that non-invasive biochemical parameters may contribute to the monitoring of CHC disease and evaluation of its grade. However, further studies including larger patient population and measuring biochemical parameters and HCV RNA titers simultaneously with histopathological evaluation are needed.