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Typing and distribution of HCV strains in South Italy (Apulia region) by a cost-effective direct sequencing test

Abstract number: 1733_100

Tagliaferro L., Menegazzi P., Reho E., Varnier O.

Objectives: Standardisation of a rapid, direct and cost-effective test for typing of HCV strains, by sequencing from real time PCR amplicons. Evaluation of epidemiological distribution of HCV isolates in the population of a southern region of Italy (Apulia).

Methods: HCV strains typing is performed by direct sequencing of purified amplicons, using the OpenGene System (Visible Genetics, Bayer diagn.) after removal of FRET probes.

We amplified the HCV 5' UTR region by an ``in house'' rapid, single tube LightCycler Real Time PCR with FRET technology. The accuracy and the reproducibility of this test have been confirmed by the very low crossing point coefficient of variation and standard deviation values (3.55 and 1.03, respectively) of the used international WHO HCV human reference standard plasma (HCV Accurun series, BBI Inc.), calculated on more than 4,500 samples tested in 359 runs.

Results: A total of 490 HCV LightCycler amplicons were sequenced using OpenGene System from October 2002 to November 2006. We observed 5 of the 6 wide-world known principal HCV genotypes, identifying 16 types and subtypes, with a homology average of 99.9%. Along with genotypes 1–4, HCV 5 has been found in Italy as well. The sequencer has been able to distinguish 60 different HCV isolates; 186 of 490 samples (38%) have been typed but not subtyped, because of the presence of some undistinguishable sequences, common to several strains.

Conclusions: This HCV sequencing test revealed an interesting cost effectiveness, saving a lot of time and money, and was able to subtype the 62% of observed HCV genotype, with a very high percentage of sequence homology (99.9%). In Apulia region (southern Italy) the most frequently observed genotypes have been 1b (35%) and 2 (28%), with a prevalence of the K0014, HCJ5 and S83 isolates. A different subtypable region and an External Quality Assessment (EQA) programme for HCV typing would be a needed addition.

Session Details

Date: 31/03/2007
Time: 00:00-00:00
Session name: European Society of Clinical Microbiology and Infectious Diseases
Subject:
Location: ICC, Munich, Germany
Presentation type:
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