Viral Kinetics of Hepatitis C Virus genotype 5 in South African patients treated with Pegylated-Interferon-alfa and Ribavirin Martin Nieuwoudt , Schalk van der Merwe: Hepatology and GI-research Unit, Dept Immunology and Internal Medicine, University of Pretoria, South Africa Arnon Arazi, Avidan U Neumann: Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel Ernie Song: Liver Unit, University of Witwatersrand, Johannesburg, South Africa

Introduction and Aims Viral kinetics of HCV-RNA is different between HCV genotypes 1 and 4 vs 2 and 3. Sustained viral response (SVR) correlates with early viral kinetics (day 1 to 14 of therapy). A rapid viral response (RVR) allows individualization of treatment duration by genotype. Genotypes 2,3 more likely to show RVR than genotypes 1,4. Genotype 5 is common in South Africa but kinetics not yet studied. Challenges of study: Sampling schedule, Not remunerated, Non-linear mathematics. First and most complete viral kinetics study of genotype 5. Also compares with historical controls of genotypes 1 and 2-3 The challenges of such a study are not immediately apparent: Very thorough sampling schedule is required, especially on the first day of treatment. The recruited patients do not pay for this study, research funding must be found, and in this case was kindly provided by Roche. The mathematical methods are challenging. The universally accepted Bi-phasic model of viral kinetics was first designed by Avidan Neumann. It requires non-linear regression and very thorough subsequent verification of the results. While this is possible locally, we thought it best to associate with him.

Methods 9 chronically infected HCV genotype 5-positive Caucasian patients. All naïve to treatment with abnormal liver functions. Cirrhosis excluded by histology/sonography. Serum sampling and analysis: Baseline, prior to Peg-Interferon (PEG-α2a) injection and Ribavirin (RIB). Day 1: Hours 1, 2, 3, 5, and 8, Days 2, 3, 4, 7, 14, 21, 28, End of treatment at 48 weeks, Follow-up at 24 weeks. HCV-RNA measured with Roche COBAS TaqMan kit. Non-linear curve fitting to bi-phasic model of Neumann et al (Science 1998, J Inf Dis 2000). Results compared to historical controls of HCV genotypes 1, 2 and 3 from Zeuzem et al, (DITTO study 2003). Significant differences: Non-parametric Mann-Whitney, Fisher exact tests.

The Bi-phasic Model (Neumann et al Science 1998;282:103-7) For the sake of illustration. Make note of the importance of the fraction “E” and the RVR that takes place within the first day, thus, comprehensive short term sampling required. Summary: A system of 3 differential equations (1-3) with exact solutions (4,5) when particular variables are assumed constant (e.g. ‘T’). Variables: T = number of target cells (‘patient mass’), I = number of infected cells, V = viral load. Constants: s = production rate of target cells, d = death rate of target cells, β = cell infection rate, p = virion production rate, c = virion clearance rate. Fractional parameters: (1 - ε) = reduction of virion production rate by therapy, (1-η) = reduction of new infections to cells.

Results Baseline viral load was (6.3 log IU/ml), not different to other genotypes. Viral decline was bi-phasic in all except one (non responder) patient. First phase decline for genotype 5 significantly (P < 0.03) faster than genotype 1. No transient rebound in HCV-RNA at end of week prior to next PEG injection, as often seen in genotype 1. Second phase decline also significantly (P < 0.01) faster than genotype 1. Similar to genotypes 2,3. RVR observed in 75% of genotype 5 patients, significantly more (P<0.003) than genotype 1 (14%), and similar to genotypes 2,3 (86%).

Viral load kinetics for patients of genotypes 1, 2/3 and 5. Genotype 5 responds similarly to genotypes 2,3. Notes: The values are the medians of each group. Error bars represent the first and third quartiles. Similar baseline viral loads

Discussion and Conclusions Similarity of early viral kinetics of genotype 5 to 2-3 suggests genotype 5 may respond to shorter treatment duration (24 vs 48 weeks). However, when comparing genotype 5 data from France, Belgium and South Africa we have observed variations in response to treatment. The strains have evolved independently dating from western trading with central and southern africa in the late 1800’s. Thus, intra-genotype virulence differences may have developed. (Verbeeck et al Jnl Virol 2005, and Nieuwoudt, van der Merwe et al ‘Multinational study of HCV genotype 5’ in progress at our institution). Clinical studies are warranted to determine ideal treatment duration for genotype 5. Until then, longer durations (48 weeks) are suggested to ensure a SVR.

Phylogram of HCV genotype 5 variants by nationality, Ask for questions while this is displayed Phylogram of HCV genotype 5 variants by nationality, based on a 470 bp fragment of the NS3-NS4B region of the genome. The degrees of divergence between the clusters indicate independent evolution for more than 100 years. Thus, viral genetic differences may explain the differences in response to treatment. (Verbeeck et al and our Multinational study in progress).