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6 The NS3 protease gene of HCV is highly conserved between isolates of the same genotype
~l~stracts
May HCV VL or H C V genotype affect the immune response after starting HAART in HCV/HIV co-infected subjects? G Antonueci I A Cozzi-Lepri2, E Girardi1, MR Capobianchi ], M Solmone 1, F Carletti 1, M. Puoti3, A d ' ~ i o Monforte 4, G Ippollto, and the ICoNA Study Group INMI S p a l l ~ , Rome, I; Royal Free and Univ. College, London, LIK2; University of Brescia3; University o f Milan4, and the ICoNA Study Group Baekgreund and aim: Little is known about whether HCV viremia or HCV genotype alter the progression o f HIV infection in HAA T-treated co-infected patients. The identification of differeat categories o f coinfected subjects with different cell recoveries after HAART could be very useful to define the optimum time to start anti-HCV as well as antiHIV treatments. We assessed the CD4 response to HAART among coinfected subjects enrolled in the I.Co.N.A. cohort according to either HCV viremia or genotype. Methods: Prospective, observational study o f 319 HIV/HCV co-infected subjects with HCV RNA, and HCV genotype determined from plasma stored within 6 months prior to HAART initiation. Time to CD4 count response was defined as the time to achieve a rise o f 200 ceUs/ml. Results: O f the 319 patients included, 61.4% were IDUs and 77.0% males. Regarding HCV infection, subjects were grouped as follows: 40 (12.5%) had HCV RNA <5IU/ml,137 (49.1%) with HCV geaotype 1, 6 (2.2%) with genotype 2, 92 (33.0%) with genotype 3, and 44 (15.8%) with genotype 4. A multivariate Cox model showed no difference in the relative hazards of achieving the end-point according to different level o f HCV RNA [adjusted RH=l.06 per logl0 higher HCV RNA, 95% CI: 0.861.29; =0.56]. In contrast, among the subset o f 165 patients who responded virologically within the first 24 weeks o f HAART we found that higher levels ofHCV RNA load were significantly associated with a reduced probability o f achiaving an increase of >_200 cell/ml [adjusted RH=0.78 per lOgl0 higher HCV RNA, 95% CI: 0.63-0.97;/3=0.02]. Moreover, the CD4+ cell count response after HAART tended to he poorer in HCV genotype 3 compared to subjects with genotype 1 (adjusted RH o f CD4+ cell count response=0.71, 95% CI:0.48-1.06, p=0.09). Conelasion: Our data carried strong evidence that different levels of plasma HCV RNA are predictive o f different response o f CD4+ following initiation o f HAART in subjects who responded virologically to HAART within the first 24 weeks. This finding is compatible with the hypothesis e r a modification o f CD4+ cells kinetics driven by HCV.
THE NS3 PROTEASE GENE OF ]~CV IS HIGHLy CONSERVED BETWEEN ISOLATES OF THE SAME GENOTYPE 1 Lodfini S. 1 Bagaglio S., 2 Canducci F. 3 De IVfitriMS., l Lazzarin A., 2 Clementi M., 1 Morsica G. 1 Division of Infectious Diseases and 2 Laboratory of Microbiology, UniversitA Vita Salute, San Raffaele, Scientific Institute, Milan; 3 Department of Internal Medicine, Cardioangiology, Hepatology; University of Bologna BACKGROUND AND AIM Few data are available on the genetic diversity of the NS3 protease gnne that presents a potential target for antiviral therapy. We developed a nested PCR to obtain NS3 protease sequences from patients infected with HCV genoWpes la, lb, 3, respectively. METHODS The NS3 region of HCV was directly sequenced and alignment of nucleotide and amino acid sequences was performed by Clustal_X. Phylogenefic tree was constructed by Phylip package. RESULTS Nucleotides and amino acids alignment of the genotype 3 isolates showed 3.73% nucleotide divergence and 1.64% aa divergence, whereas isolates la exhibited 4.45 % nucleotide divergence and 4% aa divergence; finally, NS3 sequences from lb isolates showed 6.47% nuclsotide divergence and 3.5 % aa divergence Phylogenetic analysis showed distinct clades with three HCV isolates grouping corresponding to the HCV gesotype la, lti, and 3. Comparison of consensus amino acid sequences derived from isolates la, ]b and 3, showed an higher amino acid sequence diversity between genotypes la, lb, and 3 (la and Ib subtypes showed 11% of amino acid divergence, whereas genotype 3 exhibited 22% of an divergence when compared with genotypes la and lb, respectively). However, the catalytic sites within the catalytic enzyme machinery were highly conserved within all the isolates analysed. CONCLIJSIONS Nuchiotides and amino acids analysis indicated that the NS3 protease enzyme is well conserved within the same gesotype, particularly within genotype 3. Furthermore, despite the higher amino acids divergence observed between different genotypes, the catalytic sites were found unchanged. This finding suggests that the NS3 protease enzyme of HCV is an important target for antiviral therapy
A4
CLINICAL EVENTS DURING LONG TERM LAMIVUDiNE TREATMENT IN ANTI-HBe/HBV-DNA POSITIVE CHRONIC LIVER DISEASES. V. Di Marco, A. Marzano, P, Lampertico, P. Andrecne, T. Santantonio, P. Almasio, M. R~letto, A. Crexi and the A.I,S.F. (Italian Association for the Study of Liver) Lamivudine Study Group, Italy, Background: The optimal length of lamivudine (I_AM) therapy for antiHBe/HBV-DNA positive chronic ~iverdisease is unknown. Frequent retapses are observed when stopping LAM and YMDD mutations emerge on long-term therapy. The rate of clinical events on virological remission and the impact of YMDD mutants in determining disease outcome are unclear. Aims: To analyse in a large cohort on long-term LAM the incidence of disease events in patients remaining in virological and biochemical remission and in patients with emergence of viral resistance. Study design: Data of all patients with histological or clinical diagnosis of antiHBe/HBV-DNA positive liver disease on continuous LAM observed in 22 Italian Centres were entered into a centralised database. HBV-DNA suppression (<10s genomes/ml by non-PCR assay), virological and biochemical breakthr~lghs, hepatitis flares (ALl" • 10 x UNL), development of HCC and liver decompensetion, OLT and death were recorded. YMDD mutations were detected at referral laboratories by direct sequencing or by a line probe assay (INNO-LIPA HBV DR, Innogenetics; Belgium) alter serum HBV-DNA breakthrough. Results: Of 656 patients (mean age 49.1 years, range 20-76, 84% males) 353 (53.8%) had chronic hepatitis, 198 (30.2%) Child A and 105 (16.0%) Child B-C cirrhosis. Mean duration on LAM was 23.3 months (range 1-66). Serum HBVDNA suppression (< 10s genomes/ml) was observed 616 (93.9%) patients. A biochemical and virological breakthrough due to YMDD mutants was seen in 209 cases (33.9%) with a mean time of 19.8 months. The virological remission rate was 88.1% at 1 year and 61.7 %, 46.2%, 42.5%, 37% at 2, 3, 4 and 5 years, in 371209 patients (17.7%) the emergence of mutants caused a hepatitic flare. After ALT flare no liver failure was observed in 17 patients with chronic hepatitis, tout 10 of 20 patients with cirrhosis developed liver decompensation and 5 patients died. Dudng treatment 47 subject (43 with 2:omplete virological suppression) had undergone OLT, HCC developed in 191209 (9.1%) patients with and 10/407 (2.5%) without YMDD mutants (p 0.02), liver deeampensetion was observed in 21/88 (23.8%) of cirrhotic with breakthrough and in 71194 (3.6%) cirrhotic on virological remission (p < 0.001). and 15/209 (7.2%) subjects with and 9/407(2.2%) without YMDD mutants died (p < 0.001) Diagnosis of cirrhosis and absence of sustained remission of serum HBV-DNA are independent variables predicting development of HCC and death. Conclusions: Lamivudine is highly effective in reducing viral load and ALT in anti-HBe/HBV-DNA chronic hepatitis. After 5 years of therapy 37% of patients have normal ALT and HBV-DNA still negative. After the emergence of YMDD mutants the clinical events are more frequent. Subjects with cirrhosis with YMDD mutants showed a significant deterioration of disease and may die after a hepatitic flare.
OCCULT HBV INFECTION IN I V D U s WITH CHRONIC HEPATITIS C : I M P A C T O N LIVER H I S T O L O G Y A N D O U T C O M E OF T H E R A P Y P. Fabris1 D. Brown 2 G. Tositti 2 L. Bozzoia 3, MT Giordani 2 p. Bevilacqua 3, F. de Lalta1 , G. Dusheiko2 1Dept of Infectious Diseases, S. Borto[o Hospital, Vicenza. 2 Centre for Hepatalogy and Gastroenterology, Royal Free Hospital, London, UK. 3 Dept of Pathology, S. Bertola Hospital Vicanza.
B a c k g r o u n d : T h e frequency and the impact of occult HBV infection in patients with chronic hepatitis C is still under discussion. Aims: To evaluate the prevalence of occult HBV infection and assess its impact on HCV viral titre, liver histology and on outcome of therapy in patients with chronic hepatitis C. Methods: Paired liver biopsies and serum samples collected from 51 patients with HBsAg negative chronic hepatitis C (M/F 37114, mean age 33.4, 84% IVDUs) were tested for HBV-DNA with nested PCR. Liver biopsies were further studied histologically, with morphometfic analyses and immunostaining techniques. After liver biopsy 25 were treated with alpha Interferon plus ribavifin and followed for at least 18 months. Results:HBV DNA was detected in 29.4% of liver tissue specimens and in only one (1.9%) serum sample. Three liver specimens were positive for Surface gene, 9 for Core gene, 3 for both and none for the X gone. No significant difference in mean transaminaees values, H C V viral titre, H C V genotype, or grading and staging and morphometdc analysis was observed in patients with or without HBV DNA. Moreover, all 51 liver specimens were negative f o r b o t h HBsAg and HBcAg, Sustained response to combination therapy was achieved in 40% of patients with and in 53% of patients without HBV DNA in the liver specimens (P=NS). Conclusions: HBV DNA is frequently found in the liver of patients with chronic hepatitis C. However, the lack of any significant impact on HCV viral titre, liver enzymes, histological parameters and response to therapy, suggests that in most cases HBV DNA detected in t h e liver by PCR may be either an integrated or low level replicative form
May HCV VL or H C V genotype affect the immune response after starting HAART in HCV/HIV co-infected subjects? G Antonueci I A Cozzi-Lepri2, E Girardi1, MR Capobianchi ], M Solmone 1, F Carletti 1, M. Puoti3, A d ' ~ i o Monforte 4, G Ippollto, and the ICoNA Study Group INMI S p a l l ~ , Rome, I; Royal Free and Univ. College, London, LIK2; University of Brescia3; University o f Milan4, and the ICoNA Study Group Baekgreund and aim: Little is known about whether HCV viremia or HCV genotype alter the progression o f HIV infection in HAA T-treated co-infected patients. The identification of differeat categories o f coinfected subjects with different cell recoveries after HAART could be very useful to define the optimum time to start anti-HCV as well as antiHIV treatments. We assessed the CD4 response to HAART among coinfected subjects enrolled in the I.Co.N.A. cohort according to either HCV viremia or genotype. Methods: Prospective, observational study o f 319 HIV/HCV co-infected subjects with HCV RNA, and HCV genotype determined from plasma stored within 6 months prior to HAART initiation. Time to CD4 count response was defined as the time to achieve a rise o f 200 ceUs/ml. Results: O f the 319 patients included, 61.4% were IDUs and 77.0% males. Regarding HCV infection, subjects were grouped as follows: 40 (12.5%) had HCV RNA <5IU/ml,137 (49.1%) with HCV geaotype 1, 6 (2.2%) with genotype 2, 92 (33.0%) with genotype 3, and 44 (15.8%) with genotype 4. A multivariate Cox model showed no difference in the relative hazards of achieving the end-point according to different level o f HCV RNA [adjusted RH=l.06 per logl0 higher HCV RNA, 95% CI: 0.861.29; =0.56]. In contrast, among the subset o f 165 patients who responded virologically within the first 24 weeks o f HAART we found that higher levels ofHCV RNA load were significantly associated with a reduced probability o f achiaving an increase of >_200 cell/ml [adjusted RH=0.78 per lOgl0 higher HCV RNA, 95% CI: 0.63-0.97;/3=0.02]. Moreover, the CD4+ cell count response after HAART tended to he poorer in HCV genotype 3 compared to subjects with genotype 1 (adjusted RH o f CD4+ cell count response=0.71, 95% CI:0.48-1.06, p=0.09). Conelasion: Our data carried strong evidence that different levels of plasma HCV RNA are predictive o f different response o f CD4+ following initiation o f HAART in subjects who responded virologically to HAART within the first 24 weeks. This finding is compatible with the hypothesis e r a modification o f CD4+ cells kinetics driven by HCV.
THE NS3 PROTEASE GENE OF ]~CV IS HIGHLy CONSERVED BETWEEN ISOLATES OF THE SAME GENOTYPE 1 Lodfini S. 1 Bagaglio S., 2 Canducci F. 3 De IVfitriMS., l Lazzarin A., 2 Clementi M., 1 Morsica G. 1 Division of Infectious Diseases and 2 Laboratory of Microbiology, UniversitA Vita Salute, San Raffaele, Scientific Institute, Milan; 3 Department of Internal Medicine, Cardioangiology, Hepatology; University of Bologna BACKGROUND AND AIM Few data are available on the genetic diversity of the NS3 protease gnne that presents a potential target for antiviral therapy. We developed a nested PCR to obtain NS3 protease sequences from patients infected with HCV genoWpes la, lb, 3, respectively. METHODS The NS3 region of HCV was directly sequenced and alignment of nucleotide and amino acid sequences was performed by Clustal_X. Phylogenefic tree was constructed by Phylip package. RESULTS Nucleotides and amino acids alignment of the genotype 3 isolates showed 3.73% nucleotide divergence and 1.64% aa divergence, whereas isolates la exhibited 4.45 % nucleotide divergence and 4% aa divergence; finally, NS3 sequences from lb isolates showed 6.47% nuclsotide divergence and 3.5 % aa divergence Phylogenetic analysis showed distinct clades with three HCV isolates grouping corresponding to the HCV gesotype la, lti, and 3. Comparison of consensus amino acid sequences derived from isolates la, ]b and 3, showed an higher amino acid sequence diversity between genotypes la, lb, and 3 (la and Ib subtypes showed 11% of amino acid divergence, whereas genotype 3 exhibited 22% of an divergence when compared with genotypes la and lb, respectively). However, the catalytic sites within the catalytic enzyme machinery were highly conserved within all the isolates analysed. CONCLIJSIONS Nuchiotides and amino acids analysis indicated that the NS3 protease enzyme is well conserved within the same gesotype, particularly within genotype 3. Furthermore, despite the higher amino acids divergence observed between different genotypes, the catalytic sites were found unchanged. This finding suggests that the NS3 protease enzyme of HCV is an important target for antiviral therapy
A4
CLINICAL EVENTS DURING LONG TERM LAMIVUDiNE TREATMENT IN ANTI-HBe/HBV-DNA POSITIVE CHRONIC LIVER DISEASES. V. Di Marco, A. Marzano, P, Lampertico, P. Andrecne, T. Santantonio, P. Almasio, M. R~letto, A. Crexi and the A.I,S.F. (Italian Association for the Study of Liver) Lamivudine Study Group, Italy, Background: The optimal length of lamivudine (I_AM) therapy for antiHBe/HBV-DNA positive chronic ~iverdisease is unknown. Frequent retapses are observed when stopping LAM and YMDD mutations emerge on long-term therapy. The rate of clinical events on virological remission and the impact of YMDD mutants in determining disease outcome are unclear. Aims: To analyse in a large cohort on long-term LAM the incidence of disease events in patients remaining in virological and biochemical remission and in patients with emergence of viral resistance. Study design: Data of all patients with histological or clinical diagnosis of antiHBe/HBV-DNA positive liver disease on continuous LAM observed in 22 Italian Centres were entered into a centralised database. HBV-DNA suppression (<10s genomes/ml by non-PCR assay), virological and biochemical breakthr~lghs, hepatitis flares (ALl" • 10 x UNL), development of HCC and liver decompensetion, OLT and death were recorded. YMDD mutations were detected at referral laboratories by direct sequencing or by a line probe assay (INNO-LIPA HBV DR, Innogenetics; Belgium) alter serum HBV-DNA breakthrough. Results: Of 656 patients (mean age 49.1 years, range 20-76, 84% males) 353 (53.8%) had chronic hepatitis, 198 (30.2%) Child A and 105 (16.0%) Child B-C cirrhosis. Mean duration on LAM was 23.3 months (range 1-66). Serum HBVDNA suppression (< 10s genomes/ml) was observed 616 (93.9%) patients. A biochemical and virological breakthrough due to YMDD mutants was seen in 209 cases (33.9%) with a mean time of 19.8 months. The virological remission rate was 88.1% at 1 year and 61.7 %, 46.2%, 42.5%, 37% at 2, 3, 4 and 5 years, in 371209 patients (17.7%) the emergence of mutants caused a hepatitic flare. After ALT flare no liver failure was observed in 17 patients with chronic hepatitis, tout 10 of 20 patients with cirrhosis developed liver decompensation and 5 patients died. Dudng treatment 47 subject (43 with 2:omplete virological suppression) had undergone OLT, HCC developed in 191209 (9.1%) patients with and 10/407 (2.5%) without YMDD mutants (p 0.02), liver deeampensetion was observed in 21/88 (23.8%) of cirrhotic with breakthrough and in 71194 (3.6%) cirrhotic on virological remission (p < 0.001). and 15/209 (7.2%) subjects with and 9/407(2.2%) without YMDD mutants died (p < 0.001) Diagnosis of cirrhosis and absence of sustained remission of serum HBV-DNA are independent variables predicting development of HCC and death. Conclusions: Lamivudine is highly effective in reducing viral load and ALT in anti-HBe/HBV-DNA chronic hepatitis. After 5 years of therapy 37% of patients have normal ALT and HBV-DNA still negative. After the emergence of YMDD mutants the clinical events are more frequent. Subjects with cirrhosis with YMDD mutants showed a significant deterioration of disease and may die after a hepatitic flare.
OCCULT HBV INFECTION IN I V D U s WITH CHRONIC HEPATITIS C : I M P A C T O N LIVER H I S T O L O G Y A N D O U T C O M E OF T H E R A P Y P. Fabris1 D. Brown 2 G. Tositti 2 L. Bozzoia 3, MT Giordani 2 p. Bevilacqua 3, F. de Lalta1 , G. Dusheiko2 1Dept of Infectious Diseases, S. Borto[o Hospital, Vicenza. 2 Centre for Hepatalogy and Gastroenterology, Royal Free Hospital, London, UK. 3 Dept of Pathology, S. Bertola Hospital Vicanza.
B a c k g r o u n d : T h e frequency and the impact of occult HBV infection in patients with chronic hepatitis C is still under discussion. Aims: To evaluate the prevalence of occult HBV infection and assess its impact on HCV viral titre, liver histology and on outcome of therapy in patients with chronic hepatitis C. Methods: Paired liver biopsies and serum samples collected from 51 patients with HBsAg negative chronic hepatitis C (M/F 37114, mean age 33.4, 84% IVDUs) were tested for HBV-DNA with nested PCR. Liver biopsies were further studied histologically, with morphometfic analyses and immunostaining techniques. After liver biopsy 25 were treated with alpha Interferon plus ribavifin and followed for at least 18 months. Results:HBV DNA was detected in 29.4% of liver tissue specimens and in only one (1.9%) serum sample. Three liver specimens were positive for Surface gene, 9 for Core gene, 3 for both and none for the X gone. No significant difference in mean transaminaees values, H C V viral titre, H C V genotype, or grading and staging and morphometdc analysis was observed in patients with or without HBV DNA. Moreover, all 51 liver specimens were negative f o r b o t h HBsAg and HBcAg, Sustained response to combination therapy was achieved in 40% of patients with and in 53% of patients without HBV DNA in the liver specimens (P=NS). Conclusions: HBV DNA is frequently found in the liver of patients with chronic hepatitis C. However, the lack of any significant impact on HCV viral titre, liver enzymes, histological parameters and response to therapy, suggests that in most cases HBV DNA detected in t h e liver by PCR may be either an integrated or low level replicative form