Poster Presentation

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Investigating HCV infectionDr P KarNew Delhi
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Investigating HCV infection

• Q. What is the list of tests recommended at
  registration of a new case and what is the market
  value of each test?
• Recommended tests at registration of a new
  case includes
• Test
  Cost
• 1.Liver function profile
  Rs 300/-
• 2.Prothrombin time
  Rs 50/-
• 3.HBsAg
  Rs 230/-
• 4.HIV
  Rs 300/-
• 5.Anti-HCV
  Rs 800/-
• 6.HCV-RNA
  Rs 2750

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Investigating HCV infection

• 7. Genotyping of Hepatitis C
  Rs 21,000/-
• 8. Auto antibodies
  Rs 4,680/-
• ANF
• Double stranded DNA
• Anti-smooth muscle antibody
• Anti-ALKM1 antibody
• 9. Endoscopy
  Rs 1500/-
• 10. Ultrasound abdomen
  Rs 600/-
• 11. Liver biopsy
  Rs 200/-

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Liver function tests

• The LFT is an important investigation at the
  entry point , as it gives information about the
  disease activity.
• The decision to treat anti-HCV positive patients
  is with persistently elevated ALT levels.
• ALTgt 60 IU/L along with anti-HCV positivity and
  detectable HCV-RNA forms the hallmark of starting
  the interferon therapy.
• Normalization of ALT levels is considered end
  point of therapy along with loss of HCV-RNA
• Almost 1/3 of anti-HCV patients may have normal
  ALT level. Most of these cases have histological
  damage ranging from mild hepatitis to cirrhosis.

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HBsAg and HIV

• HBsAg
• Dual infections of HBV and HCV is seen in 9 to
  25 of patients.
• The dual infection influences prognosis response
  rates to interferon therapy. (Hepatogastroenterolo
  gy 1994 41(5)438-41). JAPI 1991 (2)205-208
• HIV
• HCV and HIV co-infection is common in IV drug
  abuser.
• 1/3 of HIV cases globally are infected with HBV.
• 6 to 8 of HCV patients are HIV co-infected .
• Increases viral persistence.

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HBsAg and HIV

• Increases HCV-RNA levels.
• Increases risk of cirrhosis.
• Increases seronegative infection.
• Decreases response to Interferon- alpha.
• Increases drug interactions.

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The serologic window between HCV infection and
the detectability of specific antibodies.With
current assays, seroconversion occurs on average
at 7 to 8 weeks after onset of infection.Anti-HC
V is detectable in 50 to 70 of patients at the
onset of clinical symptoms and later in the
remaining patients.In patients with
spontaneously resolving infection, anti-HCV may
persist throughout life, or gradually disappear
after several years.
ANTI-HCV detection Immunoenzymatic
technique-indirect test
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ANTI-HCV detection Immunoenzymatic
technique-indirect test

• Anti-HCV is typically identified by using second
  or third generation EIAs. These assays detect a
  mixture of antibodies directed against various
  HCV epitopes located in the core, NS3, NS4, and (
  in case of third generation assays) NS5 proteins.
• Viral antigens can be coated onto microtiter
  plates, microbeads, or specific holders adapted
  to closed automated devices.
• The specificity of current EIAs for anti-HCV is
  greater than 99 of immunocompetent patients with
  detectable HCV-RNA.
• Anti-HCV persists indefinitely in patients who
  develop chronic infection, although antibodies
  may become undetectable i.e. negative (with
  ELISA) in hemodialysis patients, or in the case
  of profound immunodepression because of apparent
  sero-conversions and/ or seroconversions in whom
  the chronic nature of the infection is confirmed
  by the persistence of HCV-RNA

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ANTI-HCV detection Immunoenzymatic
technique-indirect test

• The test is unable to confirm viral infections
  during periods in the early phase of the
  infection before anti-HCV antibody has been
  produced.
• Antibody tests cannot distinguish between persons
  with anti-HCV antibodies who have recovered, and
  patients exhibiting an active infection, and they
  are not sufficient for the monitoring of therapy.
  Therefore a method that is able to detect HCV in
  samples is required.

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Immunoenzymatic technique-indirect test. total
HCV core antigen

• The icosahedral HCV capsid is formed by the
  polymerization of HCV core protein, a 21 Kd
  structural phospoprotein composed of the first
  191 amino acids of the viral polyprotein.
• Total HCV core antigen levels correlate with HCV
  RNA levels. During the preseroconversion period,
  the core antigen is detected on an average 1 to 2
  days later than HCV-RNA. Therefore, core antigen
  kinetics run closely parrallel to HCV RNA
  kinetics.The HCV core antigen titer can thus also
  be used as a marker of HCV replication.
• Total HCV core antigen can be detected and
  quantified by means of an EIA assay.

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Immunoenzymatic Technique-Indirect Test. Total
HCV Core Antigen

• The HCV core antigen titer (in pg/ml) correlates
  closely with the HCV-RNA level and can thus be
  used as a surrogate marker of viral replication.
• It has been estimated that 1 pg of total HCV core
  antigen per milliliter is equivalent to
  approximately 8,000 IU HCV RNA, but there are
  slight between patient differences.
• The current version of the assay does not detect
  HCV core antigen when the HCV RNA level is below
  approximately 20,000IU/ml, limiting its use in
  clinical setting.

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Direct testsHepatitis C virus-RNA

• The presence of HCV RNA in peripheral blood is a
  reliable marker of active HCV replication, which
  takes place principally in liver.
• HCV-RNA is detectable in serum within 1 to 2
  weeks after infection.
• In most patients progressing to chronic
  infection, the decrease in HCV-RNA gradually
  slows then stabilizes occasionally, however
  HCV-RNA may become undetectable for a few days or
  weeks before reappearing and reaching a plateau.
• HCV-RNA levels are stable over time in patients
  with chronic infection. The HCV-RNA level may
  increase slightly after several years of chronic
  infection.

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Direct testsHepatitis C virus-RNA

• The HCV RNA level is not affected by the
  severity of liver disease, except in patients
  with end stage liver disease, who generally have
  low or even undetectable HCV-RNA levels. The
  decrease in viral level with end-stage liver
  disease is probably due to hepatocyte depletion
  and extensive fibrosis.
• QUALITATIVE DETECTION OF HCV-RNA
• Qualitative (i.e. non-quantitative) HCV-RNA
  detection tests are still useful, as they are
  significantly more sensitive than most available
  quantitative assays.
• Qualitative assays are based on the principle of
  target amplification using either PCR or TMA.
• The specificity of the 2 assays is 98 to
  99.Qualitative assays can detect the presence of
  HCV-RNA, but they cannot measure the viral load.

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Quantitative detection of HCV RNA

• Hepatitis C virus viral load testing is one of
  the most common procedures done in many molecular
  biology laboratories.
• Recently the REAL TIME PCR techniques have been
  developed. The principle is to detect the
  amplicon synthesis and to deduce the amount of
  viral genomes in the starting clinical sample
  during rather than at the end of the PCR
  reaction.
• These methods are theoretically more sensitive
  than classical target amplification techniques
  and are not prone to carry over contamination.
  Their dynamic range of quantification is
  consistently wider, making them particularly
  useful for quantifying the full range of viral
  loads observed in untreated and treated patients
  with HCV-infection.

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Quantitative detection of HCV RNA

• While HCV viral load does not correlate with the
  severity of the hepatitis or with a poor
  prognosis, viral load does correlate with the
  likelihood of response to antiviral therapy.
• The lower the virus level at the initiation of
  therapy, the higher the response rate to therapy
  and better the chance of a long-term response.
• HCV-REAL TIME RT-PCR assays have a sensitivity of
  1000 RNA copies per reaction, with a dynamic
  range of detection between 103 and 107 RNA
  copies.
• Real Time PCR has the potential for clinical
  diagnosis, and is used to examine the HCV-RNA
  levels in plasma from sero-positive and negative
  subjects this shows that the assay is highly
  sensitive and has specificity of 100

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HCV-genotypeSerological determination of the
HCV-genotype
Indirect tests

• The HCV-Genotype is an intrinsic characteristic
  of the transmitted HCV strain(s) and does not
  change during the course of the infection.
• HCV-genotypes form 6 clads or types (numbered 1
  to 6) and themselves subdivided into a large
  number of subclads or subtypes identified by
  lower-case letters (1a,1b, 1c etc).
• Phylogenetic analysis can distinguish HCV types,
  subtypes, and isolates on the basis of average
  sequence divergence rates of approximately 30,
  20 and 10 respectively.

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HCV-genotypeSerological determination of the
HCV-genotype

• The HCV genotype can be determined by testing for
  type-specific antibodies with a competitive EIA
  (so called serotyping). The available assay
  provides interpretable results in approximately
  90 of immunocompetent patients with chronic
  hepatitis C.
• Its sensitivity is lower in hemodialysis and
  immunodepressed patients.
• The assay identifies the type (1 to 6), but does
  not discriminate between subtypes of HCV ( 1a vs.
  1b). Concordance with molecular assay is of the
  order 95 and is better for genotype 1 than for
  other genotype.
• Mixed serologic reactivity is sometimes observed,
  and this test cannot distinguish between true
  mixed infection and cross- reactivity or recovery
  from one genotype infection and persistence of
  viremia with another.

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Molecular determination of the HCV genotype-
direct tests

• The gold standard for genotyping is direct
  sequencing of the NS5B or E1 region, followed by
  sequence alignment with reference sequences and
  phylogenetic analysis.
• In clinical practice, HCV can be genotyped by
  direct sequence analysis, by reverse
  hybridization to genotype-specific
  oligonucleotide probes, or by restriction
  fragment length polymorphisms analysis.
• Two standardized kits based on PCR amplification
  of the 5 non-coding region are commercially
  available. The true gene HCV 5 noncoding
  genotyping kit is based on direct sequencing of
  PCR amplicons and sequence comparision with a
  reference sequence database.

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Molecular determination of the HCV genotype-
direct tests

• The line-probe assay is based on reverse
  hybridization of PCR amplicons to a
  nitrocellulose strip coated with genotype
  specific-oligonucleotide probes followed by
  colorimetric revelation probes.
• Both assays can identify the 6 HCV types and a
  large number of subtypes.
• Knowledge of the HCV genotype has been shown to
  be helpful in making recommendations regarding
  HCV therapy. Patients with genotype 2 and 3 are
  almost 3 times more likely to respond to therapy
  with alpha interferon and ribavirin.
• For patients with genotypes 2 and 3, a 24 week
  course of combination therapy is usually
  adequate, whereas for patients with genotype 1, a
  48 week course is recommended.

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Autoantibodies

• The presence of auto-antibodies in hepatitis C is
  an expression of generalized immune expression by
  cytokines.
• Cryoglobulins 11-36
• Rheumatoid factor 44-76
• Anti-LKM1 antibodies 2.4-5
• ASMA 5-635
• AMA 1-4
• ACL-antibodies 0-22
• 
  (Hepatology 199419841-8)
• Auto-immune markers were seen in18/25 patients of
  chronic HCV infection.
• (IJMR 2001113 170-174)

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ENDOSCOPY
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ABDOMINAL ULTRASOUND
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Liver biopsy

• The Liver biopsy is considered an accurate means
  to assess necronflammatory activity.
• The level of aminotransferases l does not
  adequately reflect the severity of disease and
  correlate with histological grading.
• It is the easiest way to determine the stage of
  the disease by assessing type and extent of
  fibrosis together with recognition of
  architectural disturbance.
• Liver biopsy helps us to look for
• Periportal or periseptal interface
  hepatitis (piecemeal necrosis)
• Confluent necrosis
• Focal lytic necrosis
• Focal inflammation
• Portal inflammation
• Presence of cirrhosis

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Investigating HCV infection

• Q. What is the minimum numbers of tests
  necessary in reference to the above question in
  clinical practice?
• ANTI-HCV
• HCV-RNA
• HCV-GENOTYPING
• LIVER BIOPSY

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Investigating HCV infection

• Q. Which tests can be exclusively reserved for
  research and may not be done in clinical
  practice?
• QUANTIFICATION OF HCV-RNA
• ANALYSIS OF HCV QUASISPECIES
• Molecular cloning of RT-PCR
• Amplicons and sequencing
• Single strand conformational polymorphism
• Temperature gradient gel electrophoresis
• Heteroduplex mobility assay

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Algorithm for evaluation of anti-HCV positive
patients
Low risk group
High risk group
HCV RNA by PCR
Negative false ve EIA
Positive
High ALT
Normal ALT
Candidate for Rx?
Yes
No
Liver biopsy Genotype viral load
Follow clinically serial labs every 6-12 months
biopsy every 5-7 years?
Liver biopsy? Consider therapy under research
protocol