Multiplex PCR for HCV genotyping

IMPORTANCE OF HCV GENOTYPING AND

MULTIPLEX PCR FOR GENOTYPING

By, Ushanandini Mohanraj

FACTS:-

ONLY 20% OF PEOPLE INFECTED WITH HEPATITIS C VIRUS

WILL COMPLETELY RECOVER

HCV IS 10 TIMES MORE CONTAGIOUS THAN HIV WHEN

COMPARING DIRECT BLOOD-TO-BLOOD CONTACT

15 MILLION PEOPLE IN INDIA ARE AFFECTED BY HEPATITIS C

SILENT DISEASE

MORE THAN 3 LAKHS FOR CURRENT TREATMENT REGIMENT OF

24- 48 WEEKS WITH SIDE AFFECTS

WHY GENOTYPE HEPATITIS C VIRUS?

Genotype

HCV Treatment Recommendations by

AASLD

1 Harvoni for 12 weeks

2 Sovaldi + ribavirin for 12 weeks

3 Sovaldi + ribavirin for 24 weeks

4 Harvoni for 12 weeks / Sovaldi + ribavirin for

24 weeks

5 Sovaldi + ribavirin + PEG for 12 weeks

6 Harvoni for 12 weeks

Molecular methods:-

1] Innolipa

2] Restriction Fragment length

polymorphism

3] Phylogenetic analysis and sequencing

Serological method:-

1] Use of genotype specific antibodies

GENOTYPING METHODS

INNO LIPA

EXCEPT GENOTYPE 6 GOOD FOR GENOTYPING AT SUBTYPE LEVEL ONLY 1b AND 3b DISTINGUISHED ACCURATELY

PHYLOGENETIC ANALYSIS AND SEQUENCING:-

THREE STEPS:-

- IS GENOTYPING OF HEPATITIS C VIRUS BASED ON PHYLOGENETIC ANALYSIS OF 5’UTR REGION ACCURATE?

- IS THERE A NEED TO ANALYSE OTHER REGIONS OF HCV GENOME?

NS5B REGION:-

Sofosbuvir, an antiviral drug targeting NS5B, approved by the FDA in December 2013. 1.1 LAKHS FOR 24 WEEK TREATMENT Dasabuvir , Non-nucleoside NS5B polymerase inhibitor , Approved by FDA in December 2014

1000 BASES

CORE REGION:-

500 TO 600 BASES HCV core protein is a potential modulator of the host immune system

NS5B CORE

1]Specificity 2]Accurate results

1] sequencing is time consuming and a costly affair 2]sequencing of amplified DNA does not usually identify mixed infections in patients.

IDEAL GENOTYPING METHOD:-

1] ACCURATE AND RELIABLE

2] CHEAP

3] FASTER RESULTS

“GENOTYPING USING MULTIPLEX PCR”

Key features:-

1] results with just

electrophoresis

2] No need of sequencing

3] less cost and time

CHALLENGES:-

-- DECIDE WHICH REGION TO FOCUS -- TEST EFFICIENCY -- DESIGN SPECIFIC PRIMERS -- NEED FOR GENOTYPING STUDIES

IS GENOTYPING USEFUL FOR ONLY TREATMENT RECOMMEDATIONS?

-- The geographic distribution and diversity of HCV genotypes -- Tracing the source of an HCV outbreak in a given population. -- Potential Role of HCV heterogeneity and genotypes in mother-to-infant Transmission of HCV -- The role of HCV genotypes in the progression of liver disease is one of the most controversial areas of HCV research

WHATS’ MORE TO THE GENOTYPING RESEARCH?