Rapid Immunoassay Alone Is Insufficient for the Detection of Hepatitis C Virus Infection Among High-risk Population

R. Firdaus; K. Saha; P. C. Sadhukhan

Disclosures

J Viral Hepat. 2013;20(4):290-293. 

In This Article

Material and Methods

Clinical Samples

Blood samples were collected from different liver clinics, haemodialysis centres, HIV Apex Clinic and thalassaemic patients from Kolkata between March 2006 and February 2011. Among the 612 samples received during this period, 254 samples that were hepatitis B and HCV rapid immunoassay nonreactive but having increased levels of AST and ALT in patients with chronic liver diseases were included in this study. This was an IEC-approved study, and informed consent was obtained from the individuals during the collection of blood samples.

Detection of HCV Antibody by Rapid Immunoassay and ELISA

Rapid diagnosis of HCV was performed using fourth-generation HCV TRI-DOT (WHO-GMP certified, sensitivity 100% and specificity 98.9%; J. Mitra, New Delhi, India), as well as with Signal HCV Kit (sensitivity 98% and specificity 99.45%; Span Diagnostics Ltd, Surat, India).[4] The same sets of samples were tested for the presence of antibodies against HCV core, NS3 and NS5 regions using two commercially available ELISA kits Hepanostika anti-HCV Ultra Kit (Biomerieux, Boxtel, The Netherlands) and HCV Microlisa (J. Mitra) to verify the results. The fourth-generation HCV TRI-DOT utilizes a unique combination of modified HCV antigens conserved across all genotypes from the putative core, NS3, NS4 and NS5 regions of the virus to selectively identify all genotypes of HCV in human serum/plasma with a high degree of sensitivity and specificity.

Detection and Quantitative Estimation of HCV RNA

The viral RNA was extracted using QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. HCV viral RNA was detected by nested RT-PCR based on 5' noncoding region (5' NCR) of HCV genome according to Saha et al..[5] Briefly, the first-round RT-PCR was performed in 20-μL total reaction volume containing 2 μL of isolated RNA with outer forward primer (PS1) 5'-ACTGTCTTCACGCAGAAAGCGTCTAGCCAT-3' and outer reverse primer (PA1) 5'-CGAGACCTCCCGGGGCACTCGCAAGCACCC-3'. The second-round nested PCR was performed with inner forward primer (PS2) 5'-ACGCAGAAAGCGTCTAGCCATGGCGTTAGT-3' and inner reverse primer (PA2) 5'-TCCCGGGGCACTCGCAAGCACCCTATCAGG-3'. The primers were selected/used in this nested RT-PCR from highly conserved domains within the 5' NCR region of the HCV genome according to Bukh et al.,[6] which is well conserved across all the six HCV genotypes. A positive band at 256 bp in 1.5% agarose gel stained with ethidium bromide was observed in gel documentation system (Bio-Rad Laboratories, Hercules, CA, USA) for HCV RNA-positive samples. Quantitative hepatitis C viral RNA was determined using ABI real time RT-PCR kit (AgPath-IDTM One-Step RT-PCR kit, Applied Biosystems, Foster City, CA, USA). The HCV primer and probe sequences were directed against the 5' noncoding region of the HCV genome.

DNA Sequencing and Genotyping

Nested RT-PCR-amplified 256-bp amplicon of 5' NCR of HCV genome was gel-purified and directly used for DNA sequencing analysis in an automated DNA Sequencer, model 3130XL (Applied Biosystems) using Big Dye terminator 3.1 kit (Applied Biosystems). The genotypes of the sequences obtained were determined using the NCBI genotyping tool.[7]

Statistical Analysis

Mean and median values were calculated. The P-values ≤ 0.05 were considered statistically significant.

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