HCV infection in India HCV prevalence around the world

1
HCV infection in India

• HCV prevalence around the world 0.4-2
• 200 million people infected
• India
• 15 million infected individuals
• Seroprevalence 1.5-2
• Chronic liver disease 20-30
• Acute viral hepatitis 10
• Acute liver failure 10-15
• Post transfusion hepatitis 80

2
Natural history of HCV infection
3
Hepatitis C virus Genome organisation showing
different genes and proteins
4
Significance of qualitative RNA testing
Therapy Confirmation to prior to therapy Test
of responder at 3 or 6 months Assessment of
response at end of therapy (responder) Test of
cure (sustained response) 6 months after
cessation of therapy
5
Utility of qualitative and quantitative RNA
testing in HCV infection
Diagnosis Prognosis Tailoring Monitoring Test
of Rx Cure
Test Qualitative HCV RNA Test Quantitative HCV
RNA Test
6
Outline of quantitative assay of HCV RNA by a
competitive RT-PCR method (in house, AIIMS)
Mixture of HCV-RNA in Sample and Different
Amounts of Mutant HCV-RNA
Reverse- Transcription - PCR
HCV c-DNA
Mutant HCV c-DNA
Bam H I site
Bam H I digestion
251 bp.
161 bp 90 bp (Mutant HCV RNA)
Agarose Electrophoresis (ethidium-bromide)
251 bp. (HCV RNA)
160 bp.
90 bp.
Densitometry analysis
7
Competitive RT- PCR assay of target HCV RNA and
internal standard RNA
Patient 1
Patient 2
Patient 3
251bp
160bp
91bp
10 9
10 11
10 11
10 7
10 9
10 7
10 11
10 9
10 7
10 5
10 3
Log copies / ml. of serum
251bp
160bp
91bp
10 9
10 11
10 11
10 7
10 9
10 7
10 11
10 9
10 7
10 5
10 3
Log copies / ml. of serum
8
Validation of the quantitation assay (AIIMS, 2002)

• Viral load Competitive Amplicor
• (n 18) RT- PCR (In House) Monitor
• 105-106/ml
• (n8)
• gt107/ml
• (OD Value out of range)
• (n10)

9
Real-Time PCR using SYBR Green dye (n18)
10
Comparative analysis of HCV RNA titre by using
different quantitation assays
11
Problems in viral load estimation

• Criteria Quantiplex Amplicor Monitor Semiquant.
• b-DNA
• Methodology Signal PCRhybridization RT nested
  PCR
• Amplification Int. quality control Analysis of
  PCR
• product by
• densitometry Int.
• quality control
• Analytical sensitivity
• (Detection thresh hold) 2,00,000 geq/ml 1000
  copies/ml 100 copies/ml
• to 120 million to 107/ml to 5 x 106 /ml
• Clinical Sensitivity
• (Correlation coefficient) 1 0.677 0.806
• 0.677 1 0.632
• 0.806 0.632 1

Real time PCR is taken as gold standard.
12
Problems in viral load estimation

• Technical problems
• Carry over contamination, cross contamination,
  reproducibility
• Genotype specific
• I 2 (Indian, predominantly type 3)
• ? Threshold level
• To indicate replication in liver
• Reproducibility
• Variable
• Detection level
• Good correlation at lower level.
• At higher level poor correlation.
• Cost of assay
• Commercial quantitative assays are costly for
  Indian patients

13
Types of quantitative HCV RNA test and their costs
Name of the Test Sensitivity Cost per test 1.
Amplicor HCV monitor Low in detecting Rs.10,000
high copy no. 2. Branched DNA assay 2 x 105 2
x 107 Rs.6,000 copies/ml. 3. Competitive
RT-PCR 103 109 copies/ml. Rs.1,000 4.
Real-Time detection 50- 1012 copies/ml Rs.2,500

14
Methods of genotyping

• Molecular genotyping
• Direct sequencing of the amplified product from
  one structural and one nonstructural gene.
• RT-PCR with genotyping primers.
• RT-PCR followed by RFLP
• Hybridization of 5 UTR with genotype specific
  probe (InnoLipa).
• Serologic Genotyping
• (Based on genotype specific antibodies)
• RIBA SIA (Chiron Corp.)
• Murex HCV serotyping EIA

15
HCV genotypes

• 6 major genotypes and 65 sub types.
• HCV shows significant nucleotide sequence
  variation over the entire genome.
• Genotyping using envelope (E1), core and NS5
  sequence have been described.
• Significance in relation to geographic
  distribution, evolution and population migration,
  disease pattern and response to Interferon
  therapies.

16
Phylogenetic tree of HCV types
P Simmonds. Gut 1997 40291 Numbers indicate
types and alphabets denote subtypes
17
Worldwide geographic distribution of HCV
genotypes and subtypes
18
Unrooted tree of HCV core region (AIIMS) n15
4
1
3
19
Correlation between serum HCV titer in genotype 3
and non-genotype 3 by competitive RT-PCR method
HCV RNA (log copies per ml.) by comp. RT-PCR
Genotype 3
Non-genotype 3
20
Viral load/genotype - Influence on therapy

• How does it help?
• Doses of drug
• Duration of therapy
• Prediction in prognosis
• Indian Scenario
• RT-PCR for qualitative HCV RNA
• (Available in several centers)
• Quantitative PCR
• (expensive and limited availability)
  

21
Cost of Viral Genotype

• Commercial Labs
• Genotyping Rs 10,000
• 15 million anti HCV ve
• At least 1.5 million may need therapy
• Rs. 12500 Crores for quantitation genotyping

22
Viral Load/genotype (contd.)

• Is it necessary ? Answer No
• Western data No recommendation yet
• Indian data - Scant Needs evaluation
• Alternative methods to predict SVR and type of
  therapy ? Ansyes
• Can we treat patients without testing for
  quantitative PCR ? Ansyes

23
Genotype Influence on therapy (AIIMS)
Genotype (n51) (Direct sequencing of NS5 and
core)
SVR

• 3a - 20
• 3b - 12
• 3f - 1
• 3g - 2
• 1b - 7
• 1a - 3
• 2b/ 2a - 4
• 4 - 2

26/35 (74)
35 (69)
5/10 (50)
10 (19.6)
6 (12)
4/6 (66)
SVR Sustained virological response
24
Viral Load Influence on therapy (AIIMS)
Viral Load (n65) (competitive PCR)
SVR

• lt2 x 106/ml 29 (45) 22 (76)
• gt 2 x 106/ml 36 (55) 25 (69)
• gt 100x106/ml 30 (46) 20 (66)
• Total SVR 47/65 (72)

SVR Sustained virological response
25
Conclusion

• Genotype 3 is most predominant in our country.
  Most genotyping without sequencing gives
  erroneous result

• Despite high viral load the SVR is 73 amongst
  Indian patients.

• Qualitative assay is sufficient for therapy and
  is feasible due to high SVR in our population