Carolyn A. Ragland August 2008

Hepatitis C *Paraenterally transmitted Contact with infected blood / blood products Blood transfusions – no longer the culprit * US – most likely reason, sharing of needles Sexual and perinatal transmission possible

Hepatitis C WHO - 3% have chronic HCV infections In US: 3+ million become infected yearly 8000 – 10,000 die annually from complications Cirrhosis Liver cancer

Hepatitis C Virus Positive sense, single stranded RNA Linear genome 9600 nucleotides Produces viral core and envelope structures Produces non-structural / operational products

Hepatitis C Virus The RNA 5’ end: highly conserved un-translated /non-coding region contains genotype specific arrangements *Analysis of 5’ end and the core region allows classification into one of six ‘clades’ and further sub-groupings.

Hepatitis C treatment *Knowledge of the genotype provides insight to which treatment and length of treatment will work best. Conventional - interferon alone (monotherapy) Combined conventional - interferon and ribavirin Pegylated interferon monotherapy Combined pegylated interferon and ribavirin

HCV Genotyping TRUGENE HCV 5' Genotyping Kit Abbott HCV Genotyping ASR Assay Invader HCV Genotyping Assay *VERSANT HCV Genotype 2.0 (LiPA)

Specimen & Handling Acceptable non-hemolyzed serum EDTA plasma Not acceptable Lithium anticoagulated plasma samples Hemolysis does have negative effects.

Specimen & Handling Store at RT for 24 hours, Refrigerate up to 5 days, or Freeze -20 C to -80 C.

Viral Nucleic Acid Extraction MagNA Pure LC Total Nucleic Acid Isolation Kit *a solid phase isolation methodology kit utilizing magnetic-bead technology

Viral Nucleic Acid Extraction *Roche MagnaPure robotic nucleic acid isolation system.

RT of the HCV RNA & cDNA Ampl. Reverse Transcription of the HCV RNA and cDNA Amplification VERSANT HCV ASR (analyte specific reagent) Amplification 2.0 Kit reverse transcription and subsequent amplification of the target cDNA obtained Produces biotinylated DNA product

RT of the HCV RNA & cDNA Ampl. Reaction tube has all reagents added reverse transcriptase Amplification polymerases UNG

RT of the HCV RNA & cDNA Ampl. GeneAmp PCR 2700

*Reverse Transcription Sensiscript enzyme RNA amounts less than 50 ng, Omniscript enzyme RNA amounts greater than 50 ng

Amplification Phase HotStar Taq polymerase *Two pairs of primers amplify portions of the 5'UTR and core regions producing biotinylated DNA fragments of 240 base pairs of 5’ UTR 270 base pairs of core

Electrophoresis Verification of an amplicon product 5uL sample a pre-formed polyacrylamide gel molecular weight ladder

Line Probe Assay (LiPA)

Tecan Auto-LiPA 48 Capable of processing 48 strips / samples All (proprietary) reagents kept at optimal temperatures and added in timed sequence

Interpretation of Results Evaluate control lines Position 1 – conjugate control Position2 – amplification control Position 23 - core products

Interpretation of Results Evaluate control samples Kit provides negative and positive controls Must perform according to manufacturer

Interpretation of Results Evaluate patient samples

Interpretation of Results Banding patterns