Comparison of Hepatitis C Virus genotyping by sequencing of capsid and NS5B regions in Cameroonian patients

CaHReF 20116, Yaoundé Conges hall, 23 – 26 August 2016 , PL125

Faculty of Science,University of Yaoundé I, Yaoundé, Cameroon

Several methods have been developed for the determination of genotypes and subtypes of HCV. However, they have not yet been evaluated using isolates from Cameroonian patients. The objective of this study was to compare two methods of HCV genotyping: sequencing and phylogenetic analysis of nucleotidic sequences of the capsid and NS5B regions of HCV using Cameroonian samples.

Plasma from patients who came for a genotyping test of hepatitis C, and whose viremia was positive was used for the tests. Samples were analysed by RNA extraction using the "QIAamp ® Viral RNA Mini Kit" (Qiagen), a qualitative search for HCV RNA by the amplification of a portion of capsid (400nt) and NS5B (382nt) genes using specific primers, electrophoretic migration of the amplification products on a 1.5 % agarose gel, sequencing of amplicons using the Sanger technique, and phylogenetic analysis of the sequences obtained using the MEGA software.

Of 121 samples tested, 49 (41.2%) came from men and 70 (58.8%) from women. All samples were amplified with the capsid region compared to the NS5B region, in which the amplification ratio was 66.9 % (81).Phylogenetic analyses of the sequences were used to obtain genotypes 1, 2 and 4 for both regions.  For the capsid, 39 (32.2%) samples were of genotype 1, 22 (18.2%) of genotype 2, and 60 (49.5%) of genotype 4. For the NS5B region, 24 (29.6%) belonged to genotype 1, 17 (21%) to genotype 2, and 40 (49.4%) to genotype 4. A comparison of genotypes and subtypes for 81 samples amplified in both regions showed a level of inter-genotypic concordance of 100 % (81) and intra-genotypic discordance of 4.9% (4). Out of 81 samples amplified for both regions, the NS5B classified 51 (63%) samples into subtypes and, 25 (30.9%) for capsid.

These results confirmed the high genetic diversity of HCV in Cameroon, with three distinct genotypes in circulation. They showed that the capsid region could be recommended for the determination of genotypes in most patients. However, it is less discriminative than the NS5B region in determining subtypes. The low discordance ratio confirms the rarity of genetic recombination in HCV.

Genotyping, HCV, phylogenetic analysis, NS5B gene, capsid gene.