Arab Journal of Gastroenterology 10 (2009) 10–13
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Arab Journal of Gastroenterology journal homepage: www.elsevier.com/locate/ajg
Serum HCV-RNA levels in patients with chronic hepatitis C: Correlation with histological features Luay E. Al-Khurri a, Khitam R. Al-Khafaji a, Suhair A. Al-Salihi b,*, Safa A.A. Alwaysi b, Raghad J. Al-Akayshi c a
Pathology Department, College of Medicine, Baghdad University, Baghdad, Iraq Laboratory Department, Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad, Iraq c Medical Department, Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad, Iraq b
a r t i c l e Keywords: HCV-RNA b-DNA Histological activity Hepatitis C Diagnosis
i n f o
a b s t r a c t Background and study aims: Liver disease in chronic hepatitis C virus (HCV) infection ranges from minimal lesion to liver cirrhosis and sometimes eventually evolving hepatocellular carcinoma. Whether and how HCV determines the different clinical and histological manifestation of the disease is not fully understood. It has not been clearly elucidated whether the extent of liver injury induced by HCV is influenced mainly by direct cytopathic damage or by an immune-mediated response against HCV-infected hepatocytes. The aim of this study is to verify whether the amount of virus in individual patient’s serum could be related to the severity of liver injury. Patients and methods: This study was carried out in the Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad. Serum levels of HCV-RNA were measured in 27 patients with chronic HCV using b-DNA assay. Core liver biopsies of the patients were evaluated according to Ishak histological activity index system. Results: The serum HCV RNA concentrations in the patients ranged from 3.2 103 to 1.2 107 copies/ml. In all patients no correlation was observed between the variable levels of viraemia and the age of the patients. Furthermore no correlations were observed between the serum HCV RNA concentrations and the biochemical liver function test levels: Total serum bilirubin, AST, ALT, and alkaline phosphatase. Histologically; patients were categorized into four subgroups: four patients (14.8%) had minimal activity, 17 patients (63%) had mild activity, and six patients (22.2%) had moderate activity. No significant correlation was found between viraemic levels and these histological findings or their individual components: Interface hepatitis, confluent necrosis, intralobular liver cell necrosis and portal inflammation. According to the stage of the fibrosis, the patients were categorized into seven subgroups: one patient (3.7%) with stage zero, seven patients with stage one (25.9%), four patients with stage two (14.9%), eight patients with stage three (29.6%), three patients with stage four (11.1%), two patients with stage five (7.4%), and two patients in cirrhotic stage six (7.4%). There was no correlation between the serum HCV RNA concentration and the stage of fibrosis. Hepatic steatosis was observed in 16/27 patients. It was mild in nine patients, moderate in five patients, and severe in two patients. Correlation has not been observed between the serum HCV RNA viraemic level and the severity of steatosis. Conclusion: Serum HCV-RNA level does not determine the degree of hepatic injury precisely and liver biopsy is necessary to accurately evaluate the extent of liver damage. Ó 2009 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
Introduction Chronic hepatitis C virus (HCV) remains a major health problem with around 200 million individuals affected worldwide [1]. Hepatitis C is a chronic progressive disease of the liver which is caused by infection with HCV [2]. Although 15 to 20% of individuals
* Corresponding author. Tel.: +9641 4141052; mobile: +9647901357290/ +963956425914; fax: +9641 4154642. E-mail addresses: [email protected], [email protected] (S.A. Al-Salihi).
infected with HCV spontaneously clear the virus in the acute phase, up to 85% develop persistent viraemia. Approximately 60% of those infected develop clinically overt chronic hepatitis, and in this group, the rate of liver disease progression is generally slow but variable [3]. Approximately 20% of patients with chronic hepatitis C develop cirrhosis within 20 years of infection, and those with cirrhosis are at risk of clinical decompensation and developing hepatocellular carcinoma [4]. The pathogenesis of HCV-induced liver injury has been attributed to either direct cytopathic damage [5–7] or an immune-mediated injury against HCV-encoded proteins [8,9]. Thus, different host and viral factors have been
1687-1979/$ - see front matter Ó 2009 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ajg.2009.03.003
11
L.E. Al-Khurri et al. / Arab Journal of Gastroenterology 10 (2009) 10–13
postulated to influence the clinical outcome of the disease. Discrete genomic variations may affect the expression of viral proteins and possibly the efficiency of replication [10,11]. Whether viral load in liver tissue and in the circulation has a role in determining the extent of liver disease is still controversial [12–15]. Most published studies have considered HCV RNA levels in serum, while less data are available on the amount of viral nucleic acid in the liver and on its relation with viraemia [16–18]. The prognostic and clinical management of still compensated chronic hepatitis C is largely based on the assessment of the activity grade and fibrosis stage. Therefore, several semi-quantitative scoring systems have been proposed and validated [12–15]. Liver biopsy is the gold standard for the evaluation of histological activity grade and fibrosis stage in chronic hepatitis C as in many other chronic liver diseases. To assess these issues, we have evaluated serum HCV RNA levels in relation to laboratory and histological findings in a series of patients with chronic hepatitis C.
2 cm. for each biopsy two slides were stained with haematoxylin – eosin stain, and one slide was stained with Van Gieson stain for collagen. The slides were evaluated by three pathologists. Histological activity index grade (0–18) and fibrosis stage (0–6) were scored according to the protocol established by Ishak et al. [19]. Hepatic steatosis severity was evaluated by indicating the approximate amount of parenchyma involved (mild: less than one third; moderate: one third to two thirds; severe: more than two thirds) [20]. Statistical analysis Statistical analysis was done by using SPSS version 7.5 computer software (statistical package for social science) in association with Excel version 5. P-value < 0.05 was considered to be significant. Results
Patients and methods Thirty two patients; 22 males and 10 females, with an age range of 10–65 years, mean (SD) age: 37.5 (8.32) years, with chronic HCV infection were studied on referral to our Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad. All these patients were anti HCV positive by enzyme-linked immunosorbent assay (ELISA). Of these thirty two patients, five patients were excluded from the study because four patients were thalasaemic with secondary haemochromatosis, and one patient had a co-infection with hepatitis B virus. None of the patients included in this study had other potential causes of liver disease, such as alcoholism, autoimmune phenomena, or metabolic disorders. All the patients were not treated previously with antiviral drugs except for six patients (four males and two females) who received HCV treatment for last 3–6 months. Total serum bilrubin (TSB), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were collected in all cases within 30 days of liver biopsy and in the vast majority of cases within seven days of liver biopsy. HBs Ag, anti HDV, IgM anti HAV as well as anti HCV were tested by a third generation enzyme-linked immunosorbent assay test. HCV-RNA was detectable and quantifiable in serum samples from all 27 subjects. HCV-RNA levels were determined by a third generation branched –DNA assay (Bayer system, 340 b-DNA, 3.0 analyzer) with detection limit 3200-40000000 copies/ml. The versant HCV-RNA 3.0 assay (b-DNA) is a sandwich nucleic acid hybridization procedure for the direct quantitation of hepatitis (viral RNA in human serum and plasma). After HCV genomic RNA is released from the virions, the RNA is captured to a micro well by a set of specific, synthetic oligonucleotide capture probe. A set of target probes hybridizes to both the viral RNA and the pre-amplifier probes. The capture probes and the target probes bind to the 50 untranslated and core regions of HCV genome. The amplifier probe subsequently hybridizes to the pre-amplifier forming a branched DNA (b-DNA) complex. Multiple copies of an ALP labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the ALP-bound complex with a chemiluminescent substrate. Light emission is directly related to the amount of HCV-RNA present in each sample, and results are recorded as relative light units (RLUs) by the analyzer. A standard curve is defined by light emission from standards containing known concentrations of recombinant single standard phage DNA. Concentrations of HCV-RNA in specimens are determined from this standard curve. All biopsies were obtained by the standard Menghini procedure (needle diameter 1.6 mm) with a biopsy length of approximately
The age range of the 27 patients included in this study was 21– 65 years; the mean age was 43 years (SD 3.95). The serum HCV RNA concentrations in the patients ranged from 3.2 103 to 1.2 107 copies/ml, (Table 1). Low viral load level (< 3200 copies/ml) was demonstrated in six patients who had received antiviral drugs and in one who did not receive any antiviral treatment (patient no. 17, Table 1). In all patients no correlation was observed between the variable levels of viraemia and the age of the patients (p = 0.24). Furthermore, no correlations were observed between the serum HCV RNA concentrations and the biochemical liver profile: Total serum bilirubin (p = 0.2), AST (p = 0.89), ALT (p = 0.92) and ALP (p = 0.47). Core needle biopsies of the 27 chronic hepatitis C patients were evaluated according to Ishak modified histological activity index system. Depending on the grade of the disease activity, patients were categorized into four subgroups: four patients (14.8%) had
Table 1 Serum viraemia levels and histological activity index (HAI) in the patients included in this study. Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Serum viral load (copies/ml) 6
2.5 10 2.3 106 1 106 10 105 8.5 105 8.1 105 1 106 4.3 103 1.2 106 1.2 107 1.2 105 1.7 106 6.4 104 8.2 105 8.7 104 3.2 103 3.2 103 3.2 103 5.4 106 9.5 105 3.2 103 2 106 1.2 106 3.2 103 3.2 103 3.2 103 2.8 106
HAIa 1,0,2,1,3 1,0,2,1,3 1,0,2,1,1 1,0,1,1,3 1,1,2,2,2 1,2,1,1,2 0,0,1,1,1 1,0,1,1,1 3,0,2,2,3 2,0,2,2,2 1,0,1,1,6 1,0,2,1,1 2,4,2,2,5 2,4,2,2,3 1,2,2,1,1 1,2,2,2,3 1,2,1,1,5 2,1,2,2,3 3,2,2,3,3 1,0,2,1,1 1,1,2,2,3 2,1,2,2,4 3,1,2,3,4 2,2,2,3,2 1,0,2,2,0 3,2,2,3,4 1,0,1,1,6
a Numbers refer to scores of interface hepatitis, confluent necrosis, intralobular liver cell necrosis, portal inflammation and fibrosis, respectively.
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Fig. 1. Chronic active hepatitis of mild grade according to the modified histological activity index grading system.
minimal activity, 17 patients (63%) had mild activity and six patients (22.2%) had moderate activity, while none of our patients had severe activity. No significant correlation was found between viraemic levels and these histological findings (p = 0.09) or their individual components: Interface hepatitis (p = 0.05), confluent necrosis (p = 0.43), intralobular degeneration (p = 0.56), and portal inflammation (p = 0.06) (Figs. 1 and 2). According to the stage of the fibrosis, the patients were categorized into seven subgroups: One patient (3.7%) with stage zero, seven patients with stage one (25.9%), four patients with stage two (14.9%), eight patients with stage three (29.6%), three patients with stage four (11.1%), two patients with stage five (7.4%), and
Fig. 2. Interface hepatitis and portal inflammation in chronic active hepatitis of moderate grade according to the modified histological activity index grading system.
Fig. 4. Chronic active hepatitis in a cirrhotic stage (Van Gieson stain).
two patients in cirrhotic stage six (7.4%) (Table 2). There was no correlation between the serum HCV RNA concentration and the stage of fibrosis (p = 0.86) (Figs. 3 and 4). Hepatic steatosis was observed in 16/27 patients (59.26%). It was mild in 9 patients (56.25%), moderate in five patients (31.25%), and severe in two patients (12.5%). No correlation between the serum HCV RNA viraemic level and the severity of steatosis (p = 0.29) was found. Discussion The polymorphic features of HCV infection are determined by both host and viral factors. These host-viral relationships driving chronic hepatitis C disease are presently unidentified. In the present study we have used b-DNA analysis method to determine the HCV viraemia level and we have correlated the results with individual components of the histological activity index in the liver biopsies of the patients with chronic HCV hepatitis. In our study, we could not find any correlation between the HCV viral load as measured by quantitative HCV RNA and serum liver function test biochemical levels (TSB, AST, ALT, and ALP), hepatic steatosis, necroinflammatory grade, or fibrosis stage. Several authors have tried to correlate the viral load in the serum or liver with the degree of liver injury with conflicting results. De Moliner et al. reported an association between the levels of viraemia and the amount of virus in the liver but they found no relationship between viral load and ALT level, genotype, or histological diagnosis [21]. Lagging et al. found no association between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necroinflammatory grade, steatosis, genotype, alanine transferase level, or alcohol consumption [22]. Furthermore, Saleem et al. concluded in their study that serum HCV RNA level does not determine the degree of hepatic injury [23]. Table 2 Histological activity grade and fibrosis stage of the chronic hepatitis C patients included in the study.
Fig. 3. Chronic active hepatitis in a cirrhotic stage (H & E stain).
Fibrosis stage
Minimal histological activity grade
Mild histological activity grade
Moderate histological activity grade
Zero One Two Three Four Five Six
0 3 0 0 0 0 1
1 4 3 6 1 1 1
0 0 1 2 2 1 0
1 7 4 8 3 2 2
Total
4
17
6
27
Total
L.E. Al-Khurri et al. / Arab Journal of Gastroenterology 10 (2009) 10–13
Likewise, Rodriguez et al. demonstrated a significant association between the proportion of infected hepatocytes and viral load but found no relationship with the histological activity index. Fanning et al., however, noted a weak association between viral load and degree of inflammation but no relationship between viral load and degree of fibrosis or ALT level [24]. On the other hand, Gretch et al. have reported a significant association between deterioration of the histological stage and amount of HCV viraemia [14]. Further controversial reports have been published on this point. Sampa et al. demonstrated a highly significant correlation between the percentage of hepatic cell with evidence of HCV replication (%R) and the three indices of liver disease activity and/or severity, namely, inflammation, fibrosis and serum ALT, while the percentage of liver cell harboring HCV infection (%G) was either marginally related or unrelated to these indices of liver injury [25]. It should be pointed out that we did not specify the HCV genotype infecting the patients included in this study. Genomic analysis and sequence comparison have allowed the classification of HCV into different genotypes [26] and a preferential association of genotype 1 with more severe forms of liver disease has been described [27], while genotype 2 has been found in the majority of asymptomatic HCV carriers [28]. However, in other studies in which large groups of anti-HCV positive patients with chronic liver disease have been studied, no clinical or histological differences could be identified among patients infected with different genotypes [29] indicating that the same mechanism of liver damage is provoked by different HCV genotypes [3]. It should be noted that the characteristics of the population studied are important variables for the interpretation of the results, since significant differences were only observed when extremely different clinical settings were considered, for example, asymptomatic HCV carriers versus end stage liver disease [14]. In the present paper the study population included patients with disease severity ranging from mild chronic hepatitis to liver cirrhosis, however the majority of the patients had a mild activity grade, there were only a few cases with minimal features of hepatitis and none of our patients had a severe activity grade. It is possible that the wide range in viral load detected in individual patients does not allow identification of any difference, unless extreme situations are considered. On the other hand, the putative mechanism of liver injury is not yet fully clarified. While the contribution of a direct cytopathic effect of HCV to liver damage is still controversial, several lines of evidence, including the existence of chronic HCV infections without clinically overt disease [30] and the detection of diffuse viral antigens in the liver of immunosuppressed transplant patients [31], indicate that immune-mediated mechanisms, already described for hepatitis B virus infection [32], are likely to play an important role in the pathogenesis of hepatitis C. However, at variance with hepatitis B, where impairment of the virusspecific T cell response has been observed [33], in patients with hepatitis C a valid T cell response to HCV proteins has been detected both in the liver [34] and in the peripheral blood lymphocytes [35,36]. Whether the level of viral load is the result of immune surveillance or whether it acts as an independent variable awaits the development of reliable cell culture systems. References [1] Global surveillance and control of hepatitis C. Report of a WHO consultation organized in collaboration with the viral hepatitis prevention board, Antwerp, Belgium. J Viral Hepat 1999;6(1):35–47. [2] Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244(4902):359–62. [3] Seeff LB, Miller RN, Rabkin CS, et al. 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 2000;132(2):105–11. [4] Alberti A, Chemello L, Benvegnu L. Natural history of hepatitis C. J Hepatol 1999;31(Suppl. 1):17–24.
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[5] Shindo M, Di Bisceglie AM, Biswas R, et al. Hepatitis C virus replication during acute infection in the chimpanzee. J Infect Dis 1992;166(2):424–7. [6] Kato N, Yokosuka O, Hosoda K, et al. Quantification of hepatitis C virus by competitive reverse transcription-polymerase chain reaction: increase of the virus in advanced liver disease. Hepatology 1993;18(1):16–20. [7] Yuki N, Hayashi N, Kamada T. HCV viraemia and liver injury in symptom-free blood donors. Lancet 1993;342(8868):444. [8] Hu KQ, Yu CH, Vierling JM. Direct detection of circulating hepatitis C virus RNA using probes from the 50 untranslated region. J Clin Invest 1992;89(6):2040–5. [9] Di Bisceglie AM, Hoofnagle JH, Krawczynski K. Changes in hepatitis C virus antigen in liver with antiviral therapy. Gastroenterology 1993;105(3):858–62. [10] Tsukiyama Kohara K, Iizuka N, Kohara M, et al. Internal ribosome entry site within hepatitis C virus RNA. J Virol 1992;66(3):1476–83. [11] Wang C, Le SY, Ali N, et al. An RNA pseudoknot is an essential structural element of the internal ribosome entry site located within the hepatitis C virus 50 noncoding region. RNA 1995;1(5):526–37. [12] Lau JY, Davis GL, Kniffen J, et al. Significance of serum hepatitis C virus RNA levels in chronic hepatitis C. Lancet 1993;341(8859):1501–4. [13] Naito M, Hayashi N, Hagiwara H, et al. Serum hepatitis C virus RNA quantity and histological features of hepatitis C virus carriers with persistently normal ALT levels. Hepatology 1994;19(4):871–5. [14] Gretch D, Corey L, Wilson J, et al. Assessment of hepatitis C virus RNA levels by quantitative competitive RNA polymerase chain reaction: high-titer viremia correlates with advanced stage of disease. J Infect Dis 1994;169(6):1219–25. [15] Gretch DR, dela Rosa C, Carithers Jr RL, et al. Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications. Ann Intern Med 1995;123(5):321–9. [16] Coelho Little E, Jeffers LJ, Bartholomew M, et al. Correlation of HCV-RNA levels in serum and liver of patients with chronic hepatitis C. J Hepatol 1995;22(4):508. [17] Francesconi R, Manzin A, Giosta F, et al. Quantification of liver HCV-RNA: relation to number of HCV infected hepatocytes, HCV genotypes, HCV viremia and response to IFN treatment. Ital J Gastroenterol 1995;27:213–4. [18] Yathsuhashi H, Inoue O, Koga M, et al. Correlation between serum and liver tissue HCV-RNA levels in patients with chronic hepatitis C. Int Hepatol Commun 1995;3:35–40. [19] Ishak K, Baptista A, Bianchi L, et al. Histological grading and staging of chronic hepatitis. J Hepatol 1995;22(6):696–9. [20] Rosai J. Rosai and Ackerman’s surgical pathology. 9th ed. Mosby; 2004. [21] De Moliner L, Pontisso P, De Salvo GL, et al. Serum and liver HCV RNA levels in patients with chronic hepatitis C: correlation with clinical and histological features. Gut 1998;42(6):856–60. [22] Lagging LM, Garcia CE, Westin J, et al. Comparison of serum hepatitis C virus RNA and core antigen concentrations and determination of whether levels are associated with liver histology or affected by specimen storage time. J Clin Microbiol 2002;40(11):4224–9. [23] Saleem N, Mubarik A, Qureshi AH, et al. Is there a correlation between degree of viremia and liver histology in chronic hepatitis C? J Pak Med Assoc 2004;54(9):476–9. [24] Fanning L, Kenny E, Sheehan M, et al. Viral load and clinicopathological features of chronic hepatitis C (1b) in a homogeneous patient population. Hepatology 1999;29(3):904–7. [25] Pal S, Shuhart MC, Thomassen L, et al. Intrahepatic hepatitis C virus replication correlates with chronic hepatitis C disease severity in vivo. J Virol 2006;80(5):2280–90. [26] Simmonds P. Variability of hepatitis C virus. Hepatology 1995;21(2):570–83. [27] Kobayashi M, Tanaka E, Sodeyama T, et al. The natural course of chronic hepatitis C: a comparison between patients with genotypes 1 and 2 hepatitis C viruses. Hepatology 1996;23(4):695–9. [28] Prati D, Capelli C, Zanella A, et al. Influence of different hepatitis C virus genotypes on the course of asymptomatic hepatitis C virus infection. Gastroenterology 1996;110(1):178–83. [29] Yamada M, Kakumu S, Yoshioka K, et al. Hepatitis C virus genotypes are not responsible for development of serious liver disease. Dig Dis Sci 1994;39(2):234–9. [30] Brillanti S, Foli M, Gaiani S, et al. Persistent hepatitis C viraemia without liver disease. Lancet 1993;341(8843):464–5. [31] Krawczynski K, Beach MJ, Bradley DW, et al. Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification. Gastroenterology 1992;103(2):622–9. [32] Thomas HC, Jacyna M, Waters J, et al. Virus-host interaction in chronic hepatitis B virus infection. Semin Liver Dis 1988;8(4):342–9. [33] Barnaba V, Balsano F. Immunologic and molecular basis of viral persistence. The hepatitis B virus model. J Hepatol 1992;14(2–3):391–400. [34] Koziel MJ, Dudley D, Wong JT, et al. Intrahepatic cytotoxic T lymphocytes specific for hepatitis C virus in persons with chronic hepatitis. J Immunol 1992;149(10):3339–44. [35] Ferrari C, Valli A, Galati L, et al. T-cell response to structural and nonstructural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections. Hepatology 1994;19(2):286–95. [36] Cerny A, McHutchison JG, Pasquinelli C, et al. Hepatitis C virus specific cytotoxic T cell response: identification of multiple HLA-2 restricted epitopes. J Clin Invest 1995;95:521–30.
Contents lists available at ScienceDirect
Arab Journal of Gastroenterology journal homepage: www.elsevier.com/locate/ajg
Serum HCV-RNA levels in patients with chronic hepatitis C: Correlation with histological features Luay E. Al-Khurri a, Khitam R. Al-Khafaji a, Suhair A. Al-Salihi b,*, Safa A.A. Alwaysi b, Raghad J. Al-Akayshi c a
Pathology Department, College of Medicine, Baghdad University, Baghdad, Iraq Laboratory Department, Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad, Iraq c Medical Department, Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad, Iraq b
a r t i c l e Keywords: HCV-RNA b-DNA Histological activity Hepatitis C Diagnosis
i n f o
a b s t r a c t Background and study aims: Liver disease in chronic hepatitis C virus (HCV) infection ranges from minimal lesion to liver cirrhosis and sometimes eventually evolving hepatocellular carcinoma. Whether and how HCV determines the different clinical and histological manifestation of the disease is not fully understood. It has not been clearly elucidated whether the extent of liver injury induced by HCV is influenced mainly by direct cytopathic damage or by an immune-mediated response against HCV-infected hepatocytes. The aim of this study is to verify whether the amount of virus in individual patient’s serum could be related to the severity of liver injury. Patients and methods: This study was carried out in the Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad. Serum levels of HCV-RNA were measured in 27 patients with chronic HCV using b-DNA assay. Core liver biopsies of the patients were evaluated according to Ishak histological activity index system. Results: The serum HCV RNA concentrations in the patients ranged from 3.2 103 to 1.2 107 copies/ml. In all patients no correlation was observed between the variable levels of viraemia and the age of the patients. Furthermore no correlations were observed between the serum HCV RNA concentrations and the biochemical liver function test levels: Total serum bilirubin, AST, ALT, and alkaline phosphatase. Histologically; patients were categorized into four subgroups: four patients (14.8%) had minimal activity, 17 patients (63%) had mild activity, and six patients (22.2%) had moderate activity. No significant correlation was found between viraemic levels and these histological findings or their individual components: Interface hepatitis, confluent necrosis, intralobular liver cell necrosis and portal inflammation. According to the stage of the fibrosis, the patients were categorized into seven subgroups: one patient (3.7%) with stage zero, seven patients with stage one (25.9%), four patients with stage two (14.9%), eight patients with stage three (29.6%), three patients with stage four (11.1%), two patients with stage five (7.4%), and two patients in cirrhotic stage six (7.4%). There was no correlation between the serum HCV RNA concentration and the stage of fibrosis. Hepatic steatosis was observed in 16/27 patients. It was mild in nine patients, moderate in five patients, and severe in two patients. Correlation has not been observed between the serum HCV RNA viraemic level and the severity of steatosis. Conclusion: Serum HCV-RNA level does not determine the degree of hepatic injury precisely and liver biopsy is necessary to accurately evaluate the extent of liver damage. Ó 2009 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved.
Introduction Chronic hepatitis C virus (HCV) remains a major health problem with around 200 million individuals affected worldwide [1]. Hepatitis C is a chronic progressive disease of the liver which is caused by infection with HCV [2]. Although 15 to 20% of individuals
* Corresponding author. Tel.: +9641 4141052; mobile: +9647901357290/ +963956425914; fax: +9641 4154642. E-mail addresses: [email protected], [email protected] (S.A. Al-Salihi).
infected with HCV spontaneously clear the virus in the acute phase, up to 85% develop persistent viraemia. Approximately 60% of those infected develop clinically overt chronic hepatitis, and in this group, the rate of liver disease progression is generally slow but variable [3]. Approximately 20% of patients with chronic hepatitis C develop cirrhosis within 20 years of infection, and those with cirrhosis are at risk of clinical decompensation and developing hepatocellular carcinoma [4]. The pathogenesis of HCV-induced liver injury has been attributed to either direct cytopathic damage [5–7] or an immune-mediated injury against HCV-encoded proteins [8,9]. Thus, different host and viral factors have been
1687-1979/$ - see front matter Ó 2009 Arab Journal of Gastroenterology. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ajg.2009.03.003
11
L.E. Al-Khurri et al. / Arab Journal of Gastroenterology 10 (2009) 10–13
postulated to influence the clinical outcome of the disease. Discrete genomic variations may affect the expression of viral proteins and possibly the efficiency of replication [10,11]. Whether viral load in liver tissue and in the circulation has a role in determining the extent of liver disease is still controversial [12–15]. Most published studies have considered HCV RNA levels in serum, while less data are available on the amount of viral nucleic acid in the liver and on its relation with viraemia [16–18]. The prognostic and clinical management of still compensated chronic hepatitis C is largely based on the assessment of the activity grade and fibrosis stage. Therefore, several semi-quantitative scoring systems have been proposed and validated [12–15]. Liver biopsy is the gold standard for the evaluation of histological activity grade and fibrosis stage in chronic hepatitis C as in many other chronic liver diseases. To assess these issues, we have evaluated serum HCV RNA levels in relation to laboratory and histological findings in a series of patients with chronic hepatitis C.
2 cm. for each biopsy two slides were stained with haematoxylin – eosin stain, and one slide was stained with Van Gieson stain for collagen. The slides were evaluated by three pathologists. Histological activity index grade (0–18) and fibrosis stage (0–6) were scored according to the protocol established by Ishak et al. [19]. Hepatic steatosis severity was evaluated by indicating the approximate amount of parenchyma involved (mild: less than one third; moderate: one third to two thirds; severe: more than two thirds) [20]. Statistical analysis Statistical analysis was done by using SPSS version 7.5 computer software (statistical package for social science) in association with Excel version 5. P-value < 0.05 was considered to be significant. Results
Patients and methods Thirty two patients; 22 males and 10 females, with an age range of 10–65 years, mean (SD) age: 37.5 (8.32) years, with chronic HCV infection were studied on referral to our Gastroenterology and Hepatology Teaching Hospital, Medical City, Baghdad. All these patients were anti HCV positive by enzyme-linked immunosorbent assay (ELISA). Of these thirty two patients, five patients were excluded from the study because four patients were thalasaemic with secondary haemochromatosis, and one patient had a co-infection with hepatitis B virus. None of the patients included in this study had other potential causes of liver disease, such as alcoholism, autoimmune phenomena, or metabolic disorders. All the patients were not treated previously with antiviral drugs except for six patients (four males and two females) who received HCV treatment for last 3–6 months. Total serum bilrubin (TSB), Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were collected in all cases within 30 days of liver biopsy and in the vast majority of cases within seven days of liver biopsy. HBs Ag, anti HDV, IgM anti HAV as well as anti HCV were tested by a third generation enzyme-linked immunosorbent assay test. HCV-RNA was detectable and quantifiable in serum samples from all 27 subjects. HCV-RNA levels were determined by a third generation branched –DNA assay (Bayer system, 340 b-DNA, 3.0 analyzer) with detection limit 3200-40000000 copies/ml. The versant HCV-RNA 3.0 assay (b-DNA) is a sandwich nucleic acid hybridization procedure for the direct quantitation of hepatitis (viral RNA in human serum and plasma). After HCV genomic RNA is released from the virions, the RNA is captured to a micro well by a set of specific, synthetic oligonucleotide capture probe. A set of target probes hybridizes to both the viral RNA and the pre-amplifier probes. The capture probes and the target probes bind to the 50 untranslated and core regions of HCV genome. The amplifier probe subsequently hybridizes to the pre-amplifier forming a branched DNA (b-DNA) complex. Multiple copies of an ALP labeled probe are then hybridized to this immobilized complex. Detection is achieved by incubating the ALP-bound complex with a chemiluminescent substrate. Light emission is directly related to the amount of HCV-RNA present in each sample, and results are recorded as relative light units (RLUs) by the analyzer. A standard curve is defined by light emission from standards containing known concentrations of recombinant single standard phage DNA. Concentrations of HCV-RNA in specimens are determined from this standard curve. All biopsies were obtained by the standard Menghini procedure (needle diameter 1.6 mm) with a biopsy length of approximately
The age range of the 27 patients included in this study was 21– 65 years; the mean age was 43 years (SD 3.95). The serum HCV RNA concentrations in the patients ranged from 3.2 103 to 1.2 107 copies/ml, (Table 1). Low viral load level (< 3200 copies/ml) was demonstrated in six patients who had received antiviral drugs and in one who did not receive any antiviral treatment (patient no. 17, Table 1). In all patients no correlation was observed between the variable levels of viraemia and the age of the patients (p = 0.24). Furthermore, no correlations were observed between the serum HCV RNA concentrations and the biochemical liver profile: Total serum bilirubin (p = 0.2), AST (p = 0.89), ALT (p = 0.92) and ALP (p = 0.47). Core needle biopsies of the 27 chronic hepatitis C patients were evaluated according to Ishak modified histological activity index system. Depending on the grade of the disease activity, patients were categorized into four subgroups: four patients (14.8%) had
Table 1 Serum viraemia levels and histological activity index (HAI) in the patients included in this study. Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Serum viral load (copies/ml) 6
2.5 10 2.3 106 1 106 10 105 8.5 105 8.1 105 1 106 4.3 103 1.2 106 1.2 107 1.2 105 1.7 106 6.4 104 8.2 105 8.7 104 3.2 103 3.2 103 3.2 103 5.4 106 9.5 105 3.2 103 2 106 1.2 106 3.2 103 3.2 103 3.2 103 2.8 106
HAIa 1,0,2,1,3 1,0,2,1,3 1,0,2,1,1 1,0,1,1,3 1,1,2,2,2 1,2,1,1,2 0,0,1,1,1 1,0,1,1,1 3,0,2,2,3 2,0,2,2,2 1,0,1,1,6 1,0,2,1,1 2,4,2,2,5 2,4,2,2,3 1,2,2,1,1 1,2,2,2,3 1,2,1,1,5 2,1,2,2,3 3,2,2,3,3 1,0,2,1,1 1,1,2,2,3 2,1,2,2,4 3,1,2,3,4 2,2,2,3,2 1,0,2,2,0 3,2,2,3,4 1,0,1,1,6
a Numbers refer to scores of interface hepatitis, confluent necrosis, intralobular liver cell necrosis, portal inflammation and fibrosis, respectively.
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Fig. 1. Chronic active hepatitis of mild grade according to the modified histological activity index grading system.
minimal activity, 17 patients (63%) had mild activity and six patients (22.2%) had moderate activity, while none of our patients had severe activity. No significant correlation was found between viraemic levels and these histological findings (p = 0.09) or their individual components: Interface hepatitis (p = 0.05), confluent necrosis (p = 0.43), intralobular degeneration (p = 0.56), and portal inflammation (p = 0.06) (Figs. 1 and 2). According to the stage of the fibrosis, the patients were categorized into seven subgroups: One patient (3.7%) with stage zero, seven patients with stage one (25.9%), four patients with stage two (14.9%), eight patients with stage three (29.6%), three patients with stage four (11.1%), two patients with stage five (7.4%), and
Fig. 2. Interface hepatitis and portal inflammation in chronic active hepatitis of moderate grade according to the modified histological activity index grading system.
Fig. 4. Chronic active hepatitis in a cirrhotic stage (Van Gieson stain).
two patients in cirrhotic stage six (7.4%) (Table 2). There was no correlation between the serum HCV RNA concentration and the stage of fibrosis (p = 0.86) (Figs. 3 and 4). Hepatic steatosis was observed in 16/27 patients (59.26%). It was mild in 9 patients (56.25%), moderate in five patients (31.25%), and severe in two patients (12.5%). No correlation between the serum HCV RNA viraemic level and the severity of steatosis (p = 0.29) was found. Discussion The polymorphic features of HCV infection are determined by both host and viral factors. These host-viral relationships driving chronic hepatitis C disease are presently unidentified. In the present study we have used b-DNA analysis method to determine the HCV viraemia level and we have correlated the results with individual components of the histological activity index in the liver biopsies of the patients with chronic HCV hepatitis. In our study, we could not find any correlation between the HCV viral load as measured by quantitative HCV RNA and serum liver function test biochemical levels (TSB, AST, ALT, and ALP), hepatic steatosis, necroinflammatory grade, or fibrosis stage. Several authors have tried to correlate the viral load in the serum or liver with the degree of liver injury with conflicting results. De Moliner et al. reported an association between the levels of viraemia and the amount of virus in the liver but they found no relationship between viral load and ALT level, genotype, or histological diagnosis [21]. Lagging et al. found no association between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necroinflammatory grade, steatosis, genotype, alanine transferase level, or alcohol consumption [22]. Furthermore, Saleem et al. concluded in their study that serum HCV RNA level does not determine the degree of hepatic injury [23]. Table 2 Histological activity grade and fibrosis stage of the chronic hepatitis C patients included in the study.
Fig. 3. Chronic active hepatitis in a cirrhotic stage (H & E stain).
Fibrosis stage
Minimal histological activity grade
Mild histological activity grade
Moderate histological activity grade
Zero One Two Three Four Five Six
0 3 0 0 0 0 1
1 4 3 6 1 1 1
0 0 1 2 2 1 0
1 7 4 8 3 2 2
Total
4
17
6
27
Total
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Likewise, Rodriguez et al. demonstrated a significant association between the proportion of infected hepatocytes and viral load but found no relationship with the histological activity index. Fanning et al., however, noted a weak association between viral load and degree of inflammation but no relationship between viral load and degree of fibrosis or ALT level [24]. On the other hand, Gretch et al. have reported a significant association between deterioration of the histological stage and amount of HCV viraemia [14]. Further controversial reports have been published on this point. Sampa et al. demonstrated a highly significant correlation between the percentage of hepatic cell with evidence of HCV replication (%R) and the three indices of liver disease activity and/or severity, namely, inflammation, fibrosis and serum ALT, while the percentage of liver cell harboring HCV infection (%G) was either marginally related or unrelated to these indices of liver injury [25]. It should be pointed out that we did not specify the HCV genotype infecting the patients included in this study. Genomic analysis and sequence comparison have allowed the classification of HCV into different genotypes [26] and a preferential association of genotype 1 with more severe forms of liver disease has been described [27], while genotype 2 has been found in the majority of asymptomatic HCV carriers [28]. However, in other studies in which large groups of anti-HCV positive patients with chronic liver disease have been studied, no clinical or histological differences could be identified among patients infected with different genotypes [29] indicating that the same mechanism of liver damage is provoked by different HCV genotypes [3]. It should be noted that the characteristics of the population studied are important variables for the interpretation of the results, since significant differences were only observed when extremely different clinical settings were considered, for example, asymptomatic HCV carriers versus end stage liver disease [14]. In the present paper the study population included patients with disease severity ranging from mild chronic hepatitis to liver cirrhosis, however the majority of the patients had a mild activity grade, there were only a few cases with minimal features of hepatitis and none of our patients had a severe activity grade. It is possible that the wide range in viral load detected in individual patients does not allow identification of any difference, unless extreme situations are considered. On the other hand, the putative mechanism of liver injury is not yet fully clarified. While the contribution of a direct cytopathic effect of HCV to liver damage is still controversial, several lines of evidence, including the existence of chronic HCV infections without clinically overt disease [30] and the detection of diffuse viral antigens in the liver of immunosuppressed transplant patients [31], indicate that immune-mediated mechanisms, already described for hepatitis B virus infection [32], are likely to play an important role in the pathogenesis of hepatitis C. However, at variance with hepatitis B, where impairment of the virusspecific T cell response has been observed [33], in patients with hepatitis C a valid T cell response to HCV proteins has been detected both in the liver [34] and in the peripheral blood lymphocytes [35,36]. Whether the level of viral load is the result of immune surveillance or whether it acts as an independent variable awaits the development of reliable cell culture systems. References [1] Global surveillance and control of hepatitis C. Report of a WHO consultation organized in collaboration with the viral hepatitis prevention board, Antwerp, Belgium. J Viral Hepat 1999;6(1):35–47. [2] Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989;244(4902):359–62. [3] Seeff LB, Miller RN, Rabkin CS, et al. 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 2000;132(2):105–11. [4] Alberti A, Chemello L, Benvegnu L. Natural history of hepatitis C. J Hepatol 1999;31(Suppl. 1):17–24.
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