Percentage of Hepatitis C Virus-Infected Hepatocytes Is a Better Predictor of Response Than Serum Viremia Levels
2005; Elsevier BV; Volume: 7; Issue: 4 Linguagem: Inglês
10.1016/s1525-1578(10)60585-5
ISSN1943-7811
AutoresElena Rodríguez‐Iñigo, Juan Manuel López‐Alcorocho, Javier Bartolomé, Nuria Ortiz‐Movilla, Margarita Pardo, Vicente Carréño,
Tópico(s)Hepatitis B Virus Studies
ResumoPegylated α-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-α2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83 ± 4.50% versus 13.44 ± 10.05%; P = 0.00003) but not in serum HCV-RNA concentration [1.71 ± 2.70 (×106 IU/L) versus 1.32 ± 1.86 (×106 IU/L); P = 0.40694]. Other factors associated with response were age, γ-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065–1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109–0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated α-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample. Pegylated α-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-α2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83 ± 4.50% versus 13.44 ± 10.05%; P = 0.00003) but not in serum HCV-RNA concentration [1.71 ± 2.70 (×106 IU/L) versus 1.32 ± 1.86 (×106 IU/L); P = 0.40694]. Other factors associated with response were age, γ-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065–1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109–0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated α-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample. Hepatitis C virus (HCV) infection is a major health problem as it is estimated that 170 million people around the world are chronically infected by this virus.1World Health Organization Hepatitis C.Wkly Epidemiol Rec. 1997; 72: 65-69PubMed Google Scholar Pegylated α-interferon (PEG-IFN) plus ribavirin (a purine nucleoside analog) is the current therapy for the treatment of patients with chronic HCV infection. However, the sustained response rate (loss of serum HCV-RNA and normalization of alanine amino transferase (ALT) levels for more than 6 months after the end of the therapy) to these treatments is around 40% for HCV genotype 1-infected patients.2Manns MP McHutchison JG Gordon SC Rustgi VK Shiffman M Reindollar R Goodman ZD Koury K Ling M Albrecht JK Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial.Lancet. 2001; 358: 958-965Abstract Full Text Full Text PDF PubMed Scopus (5861) Google Scholar, 3Bruno S Camma C Di Marco V Rumi M Vinci M Camozzi M Rebucci C Di Bona D Colombo M Craxi A Mondelli MU Pinzello G Peginterferon alfa-2b plus ribavirin for naive patients with genotype 1 chronic hepatitis C: a randomized controlled trial.J Hepatol. 2004; 41: 474-481Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Shiffman ML Di Bisceglie AM Lindsay KL Morishima C Wright EC Everson GT Lok AS Morgan TR Bonkovsky HL Lee WM Dienstag JL Ghany MG Goodman ZD Everhart JE Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis Trial Group: Peginterferon alfa-2a and ribavirin in patients with chronic hepatitis C who have failed prior treatment.Gastroenterology. 2004; 126: 1015-1923Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 5Hadziyannis SJ Sette Jr, H Morgan TR Balan V Diago M Marcellin P Ramadori G Bodenheimer Jr, H Bernstein D Rizzetto M Zeuzem S Pockros PJ Lin A Ackrill AM PEGASYS International Study Group Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: a randomized study of treatment duration and ribavirin dose.Ann Intern Med. 2004; 140: 346-355Crossref PubMed Scopus (2733) Google Scholar Because these therapies have important side effects and high cost, it is important to identify which patients have the best chance to respond before the therapy. In this regard, several virological and clinical factors have been identified as associated with the likelihood of response. Among these factors, absence of fibrosis in the liver biopsy, viral genotype, and serum HCV-RNA concentration have been shown to be independent factors associated with a sustained response to the therapy.6Yuki N Hayashi N Kasahara A Hagiwara H Takehara T Oshita M Katayama K Fusamoto H Kamada T Pretreatment viral load and response to prolonged interferon-alpha course for chronic hepatitis C.J Hepatol. 1995; 22: 457-463Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 7Garson JA Brillanti S Whitby K Foli M Deaville R Masci C Miglioli M Barbara L Analysis of clinical and virological factors associated with response to alpha interferon therapy in chronic hepatitis C.J Med Virol. 1995; 45: 348-353Crossref PubMed Scopus (56) Google Scholar, 8Lindsay KL Davis GL Schiff ER Bodenheimer HC Balart LA Dienstag JL Perrillo RP Tamburro CH Goff JS Everson GT Silva M Katkov WN Goodman Z Lau JY Maertens G Gogate J Sanghvi B Albrecht J Response to higher doses of interferon alfa-2b in patients with chronic hepatitis C: a randomized multicenter trial. Hepatitis Interventional Therapy Group.Hepatology. 1996; 24: 1034-1040PubMed Google Scholar Regarding serum viremia levels, the threshold for a favorable response to 24 weeks of therapy has been established at 800,000 IU/ml.9Pawlotsky JM Bouvier-Alias M Hezode C Darthuy F Remire J Dhumeaux D Standardization of hepatitis C virus RNA quantification.Hepatology. 2000; 32: 654-659Crossref PubMed Scopus (168) Google Scholar However, measurement of serum HCV-RNA concentration may not be accurate enough because it depends on several factors such as storage conditions of serum samples, efficiency of HCV-RNA extraction procedures or presence of inhibitors of thermo-stable polymerase chain reaction (PCR) enzymes in serum samples.10Davis GL Lau JY Urdea MS Neuwald PD Wilber JC Lindsay K Perrillo RP Albrecht J Quantitative detection of hepatitis C virus RNA with a solid-phase signal amplification method: definition of optimal conditions for specimen collection and clinical application in interferon-treated patients.Hepatology. 1994; 19: 1337-1341Crossref PubMed Scopus (209) Google Scholar, 11Lau JY Simmonds P Urdea MS Implications of variations of “conserved” regions of hepatitis C virus genome.Lancet. 1995; 346: 425-426PubMed Scopus (91) Google Scholar In situ hybridization is a technique that allows the localization of a target nucleic acid within individual cells in a tissue section12McNicol AM Farquharson MA In situ hybridization and its diagnostic applications in pathology.J Pathol. 1997; 182: 250-261Crossref PubMed Scopus (73) Google Scholar with a sensitivity of 10–20 copies of a given RNA per cell.13Höfler H Childers H Montminy MR Lecham RM Goodmann RH Wolfe HJ In situ hybridization methods for the detection of somatostain mRNA in tissue sections using antisense RNA probes.Histochem J. 1986; 18: 597-604Crossref PubMed Scopus (157) Google Scholar Because it has been estimated that the number of HCV genomes per infected cell ranges from 7 to 64 molecules,14Chang M Williams O Mittler J Quintanilla A Carithers Jr, RL Perkins J Corey L Gretch DR Dynamics of hepatitis C virus replication in human liver.Am J Pathol. 2003; 163: 433-444Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar in situ hybridization is a technique sensitive enough to detect HCV-infected hepatocytes in liver biopsies. In fact, using this technique, we have detected HCV-infected hepatocytes in liver biopsies from all chronic hepatitis C patients with detectable HCV-RNA in serum analyzed so far,15Rodriguez-Inigo E Bartolome J de Lucas S Manzarbeitia F Pardo M Arocena C Gosalvez J Oliva H Carreno V Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.Am J Pathol. 1999; 154: 1877-1881Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16de Lucas S Bartolome J Rodriguez-Inigo E Casqueiro M Millan A Ruiz-Moreno M Oliva H Carreno V Distribution of hepatitis C virus infection in liver biopsies from children and adults with chronic hepatitis C.J Med Virol. 2001; 64: 1-5Crossref PubMed Scopus (7) Google Scholar and we have found that the serum HCV-RNA concentration is related with the percentage of HCV-infected hepatocytes determined by in situ hybridization.17Gosalvez J Rodriguez-Inigo E Ramiro-Diaz JL Bartolome J Tomas JF Oliva H Carreno V Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis.Hepatology. 1998; 27: 1428-1434Crossref PubMed Scopus (36) Google Scholar However, there are no studies comparing serum HCV viremia and the percentage of HCV-infected hepatocytes as predictors of response to 48 weeks of therapy. Thus, in the present study, we have compared the percentage of HCV-infected hepatocytes, determined by in situ hybridization in the pretreatment liver biopsy, with the HCV-RNA concentration in serum samples obtained the same day as the liver biopsy, as predictors of response to 48 weeks of therapy with pegylated α-IFN plus ribavirin. In the present study, individual data from 97 patients (62 males and 37 females) with chronic HCV infection (abnormal ALT values and anti-HCV and serum HCV-RNA positive for at least 6 months) were analyzed. All patients were infected by the HCV genotype 1 as determined with a reverse hybridization assay (INNO LIPA HCV-II; Innogenetics, Gent, Belgium) and were hepatitis B surface antigen negative, and none of them had antibodies against human immunodeficiency virus 1 and 2. Patients were treated with pegylated-IFN-α2b (Peg-Intron; Schering Corporation, Kenilworth, NJ) at doses of 1.5 μg/kg body weight once weekly plus ribavirin (Rebetol; Schering Corporation) at doses of 1000 to 1200 mg/day for 12 months. Forty-nine of the 97 patients (50.5%) were nonresponders to previous antiviral therapies (44 were treated with 3 MU/thrice weekly (tiw) IFN-α for 6 to 12 months and 5 with 3 MU/tiw IFN-α plus 1000 to 1200 mg ribavirin for 6 to 12 months), but none of them was under antiviral or immunosuppressive therapy for at least 12 months before entry in the present study. The study was performed following the guidelines of the 1975 Declaration of Helsinki, and a written informed consent was obtained from each patient. Virological response was determined with the Amplicor HCV Monitor 2.0 test (Roche Diagnostics System, Basel, Switzerland) as described below and confirmed by an in-house reverse transcriptase (RT)-PCR with primers derived from the 5′ noncoding region of the viral genome, with a sensitivity of 10 IU/ml.18Castillo I Pardo M Bartolome J Ortiz-Movilla N Rodriguez-Iñigo E de Lucas S Salas C Jimenez-Heffernan JA Perez-Mota A Graus J Lopez-Alcorocho JM Carreño V Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.J Infect Dis. 2004; 189: 7-14Crossref PubMed Scopus (227) Google Scholar Patients were defined as sustained responders when they presented normal ALT values and did not have detectable serum HCV-RNA for at least 6 months after the end of the therapy. The remaining patients were considered as nonresponders. A baseline liver biopsy was obtained from each patient in the 1-month period before the therapy. Liver biopsies were immersed in 4% paraformaldehyde-phosphate-buffered saline in less than 30 seconds after they were obtained and fixed overnight in this buffer. The next day, tissue samples were dehydrated through successive baths of ethanol and embedded in paraffin, and the paraffin blocks were stored at 4°C until the histological diagnosis and in situ hybridization were performed. Hepatic necroinflammation and fibrosis were assessed according to the METAVIR score system.19Bedossa P Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C: METAVIR Cooperative Study Group.Hepatology. 1994; 20: 15-20Crossref PubMed Scopus (1797) Google Scholar, 20Bedossa P Poynard T An algorithm for the grading of activity in chronic hepatitis C: The METAVIR Cooperative Study Group.Hepatology. 1996; 24: 289-293Crossref PubMed Google Scholar After histological diagnosis, the remaining tissue was used for in situ hybridization. HCV-RNA concentration in the baseline serum sample taken the same day as the liver biopsy was measured with the Amplicor HCV Monitor 2.0 test (Roche Diagnostics System). Serum samples were aliquoted and stored at −80°C until used. When the viral RNA concentration of a given sample was above the upper dynamic range of quantitation of the assay (500,000 IU/ml), the serum sample was retested diluted (1/10 and 1/100) to obtain an accurate quantitation. Genomic HCV-RNA was detected with a complementary RNA probe labeled with digoxigenin 11-UTP obtained by in vitro transcription of the pC5′NCR, which contains the complete 5′NC region of the HCV genome. Hybridization conditions for the in situ detection of the HCV-RNA were as described previously.15Rodriguez-Inigo E Bartolome J de Lucas S Manzarbeitia F Pardo M Arocena C Gosalvez J Oliva H Carreno V Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.Am J Pathol. 1999; 154: 1877-1881Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16de Lucas S Bartolome J Rodriguez-Inigo E Casqueiro M Millan A Ruiz-Moreno M Oliva H Carreno V Distribution of hepatitis C virus infection in liver biopsies from children and adults with chronic hepatitis C.J Med Virol. 2001; 64: 1-5Crossref PubMed Scopus (7) Google Scholar, 17Gosalvez J Rodriguez-Inigo E Ramiro-Diaz JL Bartolome J Tomas JF Oliva H Carreno V Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis.Hepatology. 1998; 27: 1428-1434Crossref PubMed Scopus (36) Google Scholar Specificity of the in situ hybridization was assessed by: 1) digestions of the sections with RNase A (0.2 mg/ml) or DNase I (20 U/ml) for 2 hours at 37°C before hybridization; 2) hybridization with an unrelated RNA probe (a 360 base fragment of the chloramphenicol acetyl transferase gene); and 3) omission of the probe in the hybridization mixture. To further demonstrate the specificity of the HCV-RNA detection by in situ hybridization, liver biopsies from 10 patients with chronic hepatitis B virus infection, 10 patients with alcoholic hepatitis, and 5 patients with chronic autoimmune hepatitis (all of them without HCV markers) were hybridized with the same probe and under the same conditions used for the detection of the HCV-RNA. The percentage of infected cells was determined by visual inspection of at least 20 microscopic fields, counting at least 2000 cells from each liver section. To test the reproducibility of our in situ hybridization technique, the percentage of infected hepatocytes was determined in serial sections of two liver biopsies (from which enough material was available) hybridized in different runs (four sections from each biopsy in three different runs carried out on three different days). All statistical tests described below were carried out with SPSS package release 9.0 (SPSS, Chicago, IL). All tests performed were two-sided, and statistical significance was established at P < 0.05. In each biopsy, the mean percentage of the infected hepatocytes observed in each run and its 95% confidence interval (CI) were estimated and compared by a one-way analysis of variance, once the equality between the variances of the variables was checked with the Levene's test. Several parameters were compared between responder and nonresponder patients. Continuous variables included in the analysis were as follows: serum HCV-RNA concentration (IU/ml); percentage of infected hepatocytes; age; body mass index; ALT level (IU/L); aspartate amino transferase level (IU/ml); γ-glutamyl transpeptidase (GGTP) level (IU/L); ferritin level (ng/ml); iron level (μg/dl); necroinflammatory activity; and fibrosis score. The categorical variables analyzed were as follows: gender (0, male; 1, female); and previous antiviral therapy (0, yes; 1, no). After exploring the continuous variables for normality using the Kolmogorov-Smirnov test, the mean was compared with the Student's t-test in those with normal distribution. In these variables, equality between the variances of the variables was checked with the Levene's test. In the continuous variables without normal distribution, the median between responder and nonresponder patients was compared using the nonparametric Mann-Whitney U test. The variables with normal distribution were expressed as the mean ± SD, and those without normal distribution were expressed as the median (range). Categorical variables were compared between responders and nonresponders using the χ2 or Fisher's exact tests. The same univariate analysis was also performed in the subgroup of previously treated and untreated patients, comparing responders and nonresponders. Binary logistic regression analysis was performed to explore the influence of the above described variables on the response. Dependent variable was defined as “Response” (0, responder patient; 1, nonresponder patient). First, a global model with all of the variables included in the univariate analysis was considered. Nonsignificant variables were excluded from the model one by one. Overall significance was assessed by the log of likelihood ratio with the χ2 test, and goodness-of-fit was studied by the Hosmer-Lemeshow test. Statistical significance of the coefficients in the regression equation was contrasted with the Wald test. Odds ratios (OR) and their respective 95% CI were also estimated. To study the ability of the definitive model in the discrimination between responder and nonresponder patients, receiver operating characteristic (ROC) curve was constructed using the predicted probability values estimated with this model as the test variable and “Response = 1” as the value of the state variable. A ROC curve is a graphic representation of the trade-off between the false-negative and false-positive rates for every possible cutoff. The accuracy of the model depends on how well it separates the group of patients being tested into responders and nonresponders. Accuracy is measured by the area under the ROC curve (AUC), being an area of 1, a perfect model. The cutoff probability value to discriminate between responder and nonresponder patients was estimated by examining the coordinates of the ROC curve. This cutoff probability value was established at the maximum specificity and sensitivity. Finally, specificity, sensitivity, false-positive and false-negative rates, positive and negative predictive values, and overall accuracy or diagnostic efficiency of the model were also estimated, according to the coordinates of the ROC curve. Of the 97 patients analyzed in this study, 38 (39%) were sustained responders, whereas the remaining 59 (61%) patients were nonresponders. Positive hybridization signals were observed in the liver biopsies from the 97 patients with chronic hepatitis C analyzed in this study, whereas no signals were detected in the liver samples from the 10 patients with chronic hepatitis B, the 10 patients with alcoholic hepatitis, or the 5 patients with autoimmune chronic hepatitis. Furthermore, when the liver biopsies from the patients with chronic hepatitis C were digested with RNase before the hybridization, no signals were observed, whereas no changes in the hybridization pattern were seen when the liver biopsies were predigested with DNase. Finally, no hybridization signals were observed when the liver biopsies were hybridized with an unrelated probe or when the specific probe was omitted in the hybridization mixture. All of these results demonstrated the specificity and the accuracy of the detection of HCV-RNA in liver biopsies by the in situ hybridization technique. To demonstrate the reproducibility of the in situ hybridization for the detection of HCV-RNA, serial sections of two liver biopsies from two different patients were tested in the same run and in different runs. As shown in Table 1, in the two liver biopsies analyzed, no statistical differences in the mean percentage of infected hepatocytes in the sections analyzed in three different runs were found, which indicates that the technique is highly reproducible.Table 1Reproducibility of the in Situ Hybridization TechniqueInfected hepatocytes (%)Biopsy ABiopsy BRun 1 Section 14.96.0 Section 24.45.5 Section 35.45.0 Section 44.06.2 Mean (95% CI)4.7 (3.7–5.6)5.7 (4.8–6.5)Run 2 Section 14.25.4 Section 25.05.2 Section 33.95.9 Section 45.26.5 Mean (95% CI)4.6 (3.6–5.6)5.8 (4.8–6.7)Run 3 Section 14.96.1 Section 25.34.9 Section 34.16.5 Section 44.87.1 Mean (95% CI)4.8 (4.0–5.6)6.2 (4.7–7.6) P value0.889090.60837 Open table in a new tab Regarding HCV-RNA concentration in the basal serum sample, there were no statistical differences between sustained responder (1.71 × 106± 2.70 × 106 IU/ml) and nonresponder patients (1.32 × 106± 1.86 × 106 IU/ml) (Figure 1). In contrast, sustained responders had a significantly lower percentage of HCV-infected hepatocytes in the pretreatment liver biopsy than the nonresponder patients (6.83 ± 4.50% versus 13.44 ± 10.05%; P = 0.00003) (Figure 1). Other baseline characteristics significantly associated with a sustained response were age (responder patients were younger), GGTP levels (responder patients had lower levels of this enzyme), and absence of a previous antiviral treatment. The remaining variables analyzed were not significantly associated with a sustained response to the therapy (Table 2).Table 2Results of Univariate Analysis Performed in All of the PatientsVariableResponders (n = 38)Nonresponders (n = 59)P ValueAge (years)*Expressed as the mean ± SD.43.92 ± 11.0148.92 ± 8.940.01611Gender Male28/38 (73.7%)34/59 (57.6%)}0.10795 Female10/38 (26.3%)25/59 (42.4%)Body mass index*Expressed as the mean ± SD.24.11 ± 2.7325.57 ± 3.450.07827Infected hepatocytes (%)*Expressed as the mean ± SD.6.83 ± 4.5013.44 ± 10.050.00003Serum HCV-RNA [(IU/ml)× 106]*Expressed as the mean ± SD.1.71 ± 2.701.32 ± 1.860.40694ALT (IU/L)*Expressed as the mean ± SD.114.66 ± 107.37105.31 ± 58.610.48027AST (IU/L)*Expressed as the mean ± SD.70.29 ± 70.9872.97 ± 37.760.80948GGTP (IU/L)†Expressed as the median (range).30 (11–239)43 (9–326)0.01856Ferritin (ng/ml)†Expressed as the median (range).216 (41–1250)132 (6–1122)0.34071Iron (μg/dl)*Expressed as the mean ± SD.128.27 ± 57.45133.18 ± 56.980.72552Necroinflammatory activity†Expressed as the median (range).4 (1–6)4 (1–7)0.60170Fibrosis score†Expressed as the median (range).2 (0–4)2 (0–4)0.53835Previous treatment Yes14/38 (36.8%)35/59 (59.3%)}0.03065 No24/38 (63.2%)24/59 (40.7%)Statistically significant P values are highlighted in boldface.AST, aspartate aminotransferase.* Expressed as the mean ± SD.† Expressed as the median (range). Open table in a new tab Statistically significant P values are highlighted in boldface. AST, aspartate aminotransferase. When previously untreated patients were analyzed alone, there were statistical differences between responder and nonresponder patients in the percentage of infected hepatocytes in the basal liver biopsy (6.28 ± 4.29% versus 17.74 ± 11.39%; P = 0.00007) and in the GGTP levels [30 IU/L (range, 11–239 IU/L) versus 43 IU/L (range, 9–326 IU/L); P = 0.01856], whereas no differences were found in the remaining variables, including the baseline serum HCV-RNA concentration (Table 3). The same analysis was performed in previously treated patients, and no significant differences in the variables (including the percentage of hepatocytes) were found (Table 4).Table 3Results of Univariate Analysis Performed in Previously Untreated PatientsVariableResponders (n = 24)Nonresponders (n = 24)P valueAge (years)*Expressed as the mean ± SD.43.67 ± 12.2246.42 ± 9.720.39270Gender Male16/24 (66.7%)11/24 (45.8%)}0.14573 Female8/24 (33.7%)13/24 (54.2%)Body mass index*Expressed as the mean ± SD.24.10 ± 2.8225.78 ± 3.900.10149Infected hepatocytes(%)*Expressed as the mean ± SD.6.28 ± 4.2917.74 ± 11.390.00007Serum HCV-RNA [(IU/ml)× 106]*Expressed as the mean ± SD.1.46 ± 1.821.86 ± 1.740.24816ALT(IU/L)*Expressed as the mean ± SD.110.17 ± 84.19117.29 ± 69.870.50932AST(IU/L)*Expressed as the mean ± SD.64.71 ± 41.7284.92 ± 47.250.12312GGTP(IU/L)†Expressed as the median (range).32 (11–239)54 (13–263)0.01332Ferritin(ng/ml)†Expressed as the median (range).236 (41–1250)128 (6–884)0.57109Iron(μg/dl)†Expressed as the median (range).134 (38–214)116 (42–250)1.00000Necroinflammatory activity*Expressed as the mean ± SD.4.08 ± 1.353.96 ± 1.270.74226Fibrosis score*Expressed as the mean ± SD.1.71 ± 1.331.67 ± 1.170.90884Statistically significant P values are highlighted in boldface.AST, aspartate aminotransferase.* Expressed as the mean ± SD.† Expressed as the median (range). Open table in a new tab Table 4Results of Univariate Analysis Performed in Previously Treated PatientsVariableResponders (n = 14)Nonresponders (n = 35)P valueAge (years)*Expressed as the mean ± SD.44.36 ± 8.9850.63 ± 8.070.02134Gender Male12/14 (85.7%)23/35 (65.7%)}0.29361 Female2/14 (14.3%)12/35 (34.3%)Body mass index*Expressed as the mean ± SD.24.13 ± 2.3025.19 ± 2.590.63773Infected hepatocytes(%)*Expressed as the mean ± SD.7.79 ± 4.8410.49 ± 7.910.40603Serum HCV-RNA [(IU/ml)× 106]*Expressed as the mean ± SD.2.12 ± 3.830.96 ± 1.860.73154ALT(IU/L)*Expressed as the mean ± SD.122.36 ± 142.3697.06 ± 48.850.45837AST(IU/L)*Expressed as the mean ± SD.79.86 ± 105.3864.78 ± 27.430.14698GGTP(IU/L)†Expressed as the median (range).28.5 (15–175)38 (9–326)0.28791Ferritin(ng/ml)†Expressed as the median (range).197 (83–451)160 (33–1122)0.36006Iron(μg/dl)*Expressed as the mean ± SD.117.43 ± 67.24136.03 ± 62.520.50088Necroinflammatory activity†Expressed as the median (range).3 (1–5)4 (1–7)0.08681Fibrosis score†Expressed as the median (range).1 (0–3)2 (0–4)0.32197Statistically significant P values are highlighted in boldface.AST, aspartate aminotransferase.* Expressed as the mean ± SD.† Expressed as the median (range). Open table in a new tab Statistically significant P values are highlighted in boldface. AST, aspartate aminotransferase. Statistically significant P values are highlighted in boldface. AST, aspartate aminotransferase. When a viremia level of 800,000 IU/ml and 7% of infected hepatocytes (the mean of infected hepatocytes in responder patients) were chosen as the threshold for sustained response, it was found that 54 patients had a basal serum HCV-RNA concentration lower than 800,000 IU/ml, and 21 (38.9%) of them were sustained responders (Table 5). In contrast, 40 patients had 7% or fewer infected hepatocytes, and 21 (52.5%) of them were sustained responders. On the other hand, of the 43 patients with viremia levels higher than 800,000 IU/ml, 17 (39.5%) were sustained responders; whereas 57 patients had more than 7% infected hepatocytes in the basal liver biopsy, and 17 (29.8%) of them were responders (Table 5).Table 5Percentage of Responder and Nonresponder Patients, according to the HCV-RNA Concentration or the Percentage of Infected HepatocytesRespondersNonrespondersP valueHCV-RNA concentration 7% (n = 57)17/57 (29.8%)40/57 (70.2%) Open table in a new tab A global binary logistic analysis model was constructed to study the effect of all of the variables analyzed in the univariate analysis on the “Response” as dependent variable (0, responder patients; 1, nonresponder patients). Table 6 shows the best fitted model obtained after the exclusion of the nonsignificant variables (one by one). Log of likelihood ratio contrasted by the χ2 test demonstrated that the model was highly significant [χ2 = 31.212; degrees of freedom (df) = 4; P = 0.0000028]. The Hosmer-Lemeshow test, which evaluates the differences between the probabilities predicted by the model and those observed, showed that the goodness-of-fit of the model was acceptable (χ2 = 10.605; df = 8; P = 0.225). As shown in Table 6, only the percentage of infected hepatocytes and previous an
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