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PVI-12 The hepatitis-A serology in patients infected with chronic hepatitis-B and -C
Posters, 12th ESCV, Istanbul / Journal of Clinical Virology 46 Suppl. 1 (2009) S15–S61 Materials and Methods: Blood samples were taken from all refugees who were captured while they were trying to run off from Izmir to European countries through sea route in June 2008. Hepatitis-B virus surface antigen (HBsAg), hepatitis-B virus surface antibody (Anti-HBs), hepatitis-B virus core antigen (Anti-HBc Total) and hepatitis-C virus antibody (Anti-HCV) were examined by means of ELISA method (Liason, Diasorin, Italy). Results: Among the total 222 refugees, 14 (6.36%) had positive HBsAg, 28 (12.6%) had positive Anti-HBs, 1 (0.4%) had isolated positive Anti-HBc Total and 10 (4.5%) had positive Anti-HCV. Discussion: Significantly higher HBV and HCV seroprevalences were found in illegal immigrants within Izmir than in normal population. In illegal immigrants HBV carriers should be identified, those who did not encounter yet should be vaccinated and those chronic infection was detected should be kept under control. In addition, we think that it is highly important to take general precautions to prevent HCV and HBV infected illegal immigrants from transmitting these infections to their environment. PVI-12 The hepatitis-A serology in patients infected with chronic hepatitis-B and -C S. Kose, M. Turken, G. Cavdar *. Izmir Tepecik Educational and Research Hospital, Infection Disease and Clinical Microbiology, Izmir, Turkey Aim: Hepatitis-A virus (HAV) infection is frequently seen during childhood in developing countries. HAV infection may demonstrate a fulminant progress in chronic hepatic patients and may have fatal consequences. In our study we evaluated the hepatitis-A serology in patients with viral chronic hepatitis-B and -C. Materials and Methods: In 401 viral chronic hepatitis patients who were followed by Hepatitis outpatient clinic of Izmir Tepecik Training and Research Hospital, Anti-HAV serology was investigated by Elisa (Liason, Diasorin, Italy) method retrospectively. Results: In 360 HBsAg-positive patients followed, the Anti-HAV IgG was investigated. In 5.5% of the patients Anti-HAV IgG was found negative whereas it was positive in 94.4%. Among 41 patients with positive Anti-HCV, Anti-HAV IgG was found negative in 17% and positive in 82.9%. Conclusion: In our country the prevalence of Anti-HAV varies depending on age and location. Age of onset of Hepatit-A has shifted to advanced ages with the improvement of hygienic conditions. In our study in patients with chronic hepatitis-B and C this rate was found as 5.5% and 17% respectively. Since the infection risk progressed to advanced ages will increase the risk in chronic liver patients, it is appropriate to vaccinate patients with viral chronic hepatitis who were not immunized against HAV infection. PVI-13 Concomitance of hepatitis-B and hepatitis-C viruses S. Kose, M. Turken, G. Cavdar *. Izmir Tepecik Educational and Research Hospital, Infection Diseases and Clinical Microbiology, Izmir, Turkey Aim: Hepatitis-B virus (HBV) and Hepatitis-C virus (HCV) infections are among the important chronic diseases that can progress to cirrhosis and hepatocellular carcinoma. Concomitance of chronic hepatitis-B and hepatitis-C is generally occurs through addition of another hepatitis viruses to preexisting chronic hepatitis carriage. In this study we evaluated those patients who had concomitant hepatitis-B and hepatitis-C viruses in our clinic. Materials and Methods: In HBsAg-positive patients and in anti-HCVpositive patients who were followed by Infectious Diseases and Clinic Microbiology Clinic of Izmir Tepecik Training and Research Hospital, antiHCV serology was investigated through microelisa method and HBsAg seroloji was investigated through macroelisa method, respectively. Results: A total of 1648 patients, 1552 with chronic hepatitis-B and 96 with chronic hepatitis-C were included in study. In 9 (0.54) patients with viral chronic hepatitis concomitant hepatitis-B and hepatitis-C were detected. Their mean age was 47.8 years; 44% were female and 55% were male. In 5 patients (55.5%) active hepatitis-B and in 4 patients (44.4%) active hepatitis-C viruses were found. Conclusion: Concomitance of hepatitis-B and C infections are very rarely seen due to viral interference. Their concomitance is more frequent in hemodialysis patients, IV drug addictive and immunocompromised patients. The patients in our study are not in the risk group. Concomitance of hepatitis-B and -C viruses has been found to be 0.54%.
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PVI-14 NS3 protease polymorphism and resistance profile to protease inhibitors in French patients infected with hepatitis C virus (HCV) genotypes 1 to 5 S. Vallet1,2 *, F. Viron1 , H. Le Guillou-Guillemette3 , C. Henquell4 , P. Trimoulet5 , F. Abravanel6 , P. Colson7 , J. Izopet6 , C. Payan1,2 , and the AC11-VHC Group (ANRS). 1 CHU de Brest, D´epartement de Microbiologie, Brest, France, 2 Universit´e de Brest, Facult´e de M´edecine et des sciences de la Sant´e, LUBEM EA3882, Brest, France, 3 CHU Angers, Laboratoire de Virologie, Angers, France, 4 CHRU de Clermont-Ferrand, Laboratoire de Virologie, Clermont-Ferrand, France, 5 Laboratoire de Virologie, Hˆopital Pellegrin Tripode, Bordeaux, France, 6 Laboratoire de Virologie, Institut F´ed´eratif de Biologie, CHU Toulouse Purpan, Toulouse, France, 7 Laboratoire de Virologie, F´ed´eration hospitali`ere de Bact´eriologie-Virologie Clinique et d’Hygi`ene Centre Hospitalier Universitaire Timone, Marseille, France NS3 protease is one of the most promising targets for specific anti-HCV therapy. Several NS3 protease inhibitors are being developed. Two agents, Telaprevir (TVR) and Boceprevir (BCV), are currently in phase 3 programs. Despite significant viral load reduction during TVR or BCV therapy, viral breakthrough may occur. It is associated with a number of well identified mutations. Few data on genotypes non-1 NS3 polymorphism are available. The aim of this ANRS-AC11 multicenter work was to define the NS3 protease polymorphism of HCV genotypes 1 to 5 circulating in France in the absence of any specific antiviral pressure. Genotype specific NS3 primers were designed to amplify and sequence the NS3 protease gene. Prevalence of dominant resistance mutations and polymorphism was then investigated in 113 HCV protease inhibitor-na¨ıve patients infected with HCV genotypes 1, 2, 3, 4 or 5. None of the analysed sequences contained TLV or BCV major resistance mutations (A156S/V/T, R155K/T and A156S/T, V170A respectively). The substitution V36L which confers a decreased sensibility to TVR was detected as a signature amino acid in 100% of genotypes 2, 3, 4 and 5 strains. None but one of the 113 analysed sequences showed the low-level associated resistance T54A/S substitutions. All analysed genotype 2, 3 and 5 sequences showed the conservative substitution V170I. We have developed an HCV protease NS3 inhibitor resistance genotyping tool suitable for HCV genotypes 1 to 5. Polymorphism data obtained will be valuable for interpretation of genotypic resistance profiles in cases of failure of any anti-HCV NS3 protease treatment. PVI-15 Developing a real-time PCR assay to determine the hepatitis B virus DNA in serum ¨ colpan. Dokuz Eylul University Faculty of Medicine A. Arzu Sayiner *, G. Oz¸ Microbiology and Clinical Microbiology Department, Turkey Aim: To develop a quantitative real-time PCR assay for HBV DNA that can be used for routine diagnostics. Material and Method: Amplification targets were a fragment of HBV preS and bovine herpes virus gC (internal control). Primers and hydrolysis probes were chosen from published studies. Standard curve was generated by dilutions of a plasmid carrying the HBV amplicon. DNA extracted by EZ1 virus mini kit (Qiagen). The assay was performed using Rotor Gene 6000 (Corbett) and “no-rox multipleks PCR” mix (Qiagen). The accuracy was evaluated by commercial quantification and proficiency panels. The assay was evaluated on 260 plasma samples, which also studied with a commercial assay (Qiagen). Results: Analytical sensitivity was 48 IU/ml and dynamic range was between 5×101 and 5×1010 IU/ml (PCR efficiency: 0.96). Intra- and interassay CV were between 0.3−4.1%. Results of the proficiency panel were correlated with expected results (r2 =0.92). The assay was able to detect all HBV genotypes. However, quantitative results of genotype B and C samples were ~2 log10 below the expected value. The correlation with the commercial assay was evaluated by 180 HBV DNA positive and 80 negative plasma samples. The sensitivity and specificity were 99% and 94%, respectively. Quantitative results of the samples detected by two assays showed a strong correlation (r = 0.86, p = 0.000, R2 =0.74). No false positivity was detected. The assay was 20 euros/sample cheaper than the commercial one. Conclusion: The developed assay could be an alternative for routine diagnostics.
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PVI-14 NS3 protease polymorphism and resistance profile to protease inhibitors in French patients infected with hepatitis C virus (HCV) genotypes 1 to 5 S. Vallet1,2 *, F. Viron1 , H. Le Guillou-Guillemette3 , C. Henquell4 , P. Trimoulet5 , F. Abravanel6 , P. Colson7 , J. Izopet6 , C. Payan1,2 , and the AC11-VHC Group (ANRS). 1 CHU de Brest, D´epartement de Microbiologie, Brest, France, 2 Universit´e de Brest, Facult´e de M´edecine et des sciences de la Sant´e, LUBEM EA3882, Brest, France, 3 CHU Angers, Laboratoire de Virologie, Angers, France, 4 CHRU de Clermont-Ferrand, Laboratoire de Virologie, Clermont-Ferrand, France, 5 Laboratoire de Virologie, Hˆopital Pellegrin Tripode, Bordeaux, France, 6 Laboratoire de Virologie, Institut F´ed´eratif de Biologie, CHU Toulouse Purpan, Toulouse, France, 7 Laboratoire de Virologie, F´ed´eration hospitali`ere de Bact´eriologie-Virologie Clinique et d’Hygi`ene Centre Hospitalier Universitaire Timone, Marseille, France NS3 protease is one of the most promising targets for specific anti-HCV therapy. Several NS3 protease inhibitors are being developed. Two agents, Telaprevir (TVR) and Boceprevir (BCV), are currently in phase 3 programs. Despite significant viral load reduction during TVR or BCV therapy, viral breakthrough may occur. It is associated with a number of well identified mutations. Few data on genotypes non-1 NS3 polymorphism are available. The aim of this ANRS-AC11 multicenter work was to define the NS3 protease polymorphism of HCV genotypes 1 to 5 circulating in France in the absence of any specific antiviral pressure. Genotype specific NS3 primers were designed to amplify and sequence the NS3 protease gene. Prevalence of dominant resistance mutations and polymorphism was then investigated in 113 HCV protease inhibitor-na¨ıve patients infected with HCV genotypes 1, 2, 3, 4 or 5. None of the analysed sequences contained TLV or BCV major resistance mutations (A156S/V/T, R155K/T and A156S/T, V170A respectively). The substitution V36L which confers a decreased sensibility to TVR was detected as a signature amino acid in 100% of genotypes 2, 3, 4 and 5 strains. None but one of the 113 analysed sequences showed the low-level associated resistance T54A/S substitutions. All analysed genotype 2, 3 and 5 sequences showed the conservative substitution V170I. We have developed an HCV protease NS3 inhibitor resistance genotyping tool suitable for HCV genotypes 1 to 5. Polymorphism data obtained will be valuable for interpretation of genotypic resistance profiles in cases of failure of any anti-HCV NS3 protease treatment. PVI-15 Developing a real-time PCR assay to determine the hepatitis B virus DNA in serum ¨ colpan. Dokuz Eylul University Faculty of Medicine A. Arzu Sayiner *, G. Oz¸ Microbiology and Clinical Microbiology Department, Turkey Aim: To develop a quantitative real-time PCR assay for HBV DNA that can be used for routine diagnostics. Material and Method: Amplification targets were a fragment of HBV preS and bovine herpes virus gC (internal control). Primers and hydrolysis probes were chosen from published studies. Standard curve was generated by dilutions of a plasmid carrying the HBV amplicon. DNA extracted by EZ1 virus mini kit (Qiagen). The assay was performed using Rotor Gene 6000 (Corbett) and “no-rox multipleks PCR” mix (Qiagen). The accuracy was evaluated by commercial quantification and proficiency panels. The assay was evaluated on 260 plasma samples, which also studied with a commercial assay (Qiagen). Results: Analytical sensitivity was 48 IU/ml and dynamic range was between 5×101 and 5×1010 IU/ml (PCR efficiency: 0.96). Intra- and interassay CV were between 0.3−4.1%. Results of the proficiency panel were correlated with expected results (r2 =0.92). The assay was able to detect all HBV genotypes. However, quantitative results of genotype B and C samples were ~2 log10 below the expected value. The correlation with the commercial assay was evaluated by 180 HBV DNA positive and 80 negative plasma samples. The sensitivity and specificity were 99% and 94%, respectively. Quantitative results of the samples detected by two assays showed a strong correlation (r = 0.86, p = 0.000, R2 =0.74). No false positivity was detected. The assay was 20 euros/sample cheaper than the commercial one. Conclusion: The developed assay could be an alternative for routine diagnostics.