Investigating HCV infection Dr P Kar New Delhi

3. 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

4. 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/-

5. 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. ALT> 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.

6. 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. (Hepatogastroenterology 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.

7. Increases HCV-RNA levels. Increases risk of cirrhosis. Increases seronegative infection. Decreases response to Interferon- alpha. Increases drug interactions. HBsAg and HIV

8. 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-HCV 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.

9. 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

10. 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.

11. 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. Immunoenzymatic technique-indirect test. total HCV core antigen

12. 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. Immunoenzymatic Technique-Indirect Test. Total HCV Core Antigen

13. 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.

14. 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. Direct testsHepatitis C virus-RNA

15. 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.

16. 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% Quantitative detection of HCV RNA

17. HCV-genotypeSerological determination of the HCV-genotype 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.

18. 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. HCV-genotypeSerological determination of the HCV-genotype

19. 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.

20. 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. Molecular determination of the HCV genotype- direct tests

21. 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 1994;19:841-8) Auto-immune markers were seen in18/25 patients of chronic HCV infection. (IJMR 2001;113: 170-174)

22. ENDOSCOPY

23. ABDOMINAL ULTRASOUND

24. 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

25. 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

26. 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

27. Algorithm for evaluation of anti-HCV positive patients