Indeterminate Prediction of Hepatitis C Virus Genotype by Commercial Real-Time Polymerase Chain Reaction Assay Resolving by Sequencing to Avoid the Consequence of Inaccurate Typing.

Abstract

Pakistan is ranked second highest after Egypt in hepatitis C virus (HCV) infection. Accurate typing is mandatory to be compliant with the World Health Organization strategy to eliminate HCV infection in 2030. We characterized the HCV genotypes using Abbott real-time polymerase chain reaction assay and indeterminate samples were sequenced. We also investigated the distribution of HCV genotype among different age groups and gender in chronic HCV patients. One thousand thirteen samples were tested for HCV genotyping using Abbott real-time HCV genotyping assay. RNA extraction from plasma was done using the m2000sp platform. The amplification and detection of genotypes was done on m2000rt instrument. The lower limit of detection assay is 500 IU/mL. The indeterminate genotypes were analyzed by sequencing of the NS5B region. We found genotype 1 in 1.68%, genotype 1b in 0.89%, genotype 1a in 0.79%, genotype 2 in 0.6, genotype 3 in 94.37%, genotype 4 in 0.4%, genotype 5 in 0.09%, and indeterminate genotype result were found in 1.18%. Abbott assay could not identify 12 samples of genotype 3 (1.18%) and gave the indeterminate result. It also fails to assign some of the samples of genotype 1 into 1a and 1b. The indeterminate genotypes were resolved by sequencing followed by phylogenetic analysis. Genotype 3 is the predominant genotype and significantly higher in females as compared with males. Genotype 1a is more common in males than in females. Indeterminate HCV genotypes on sequencing analysis identify as genotype 3a and likewise subtype of genotype1 as 1a.