- Email: [email protected]
Intrahepatic hepatitis C virus replication is increased in patients with regular alcohol consumption
H
lntrahepatic in patients
hepatitis C virus replication is increased with regular alcohol consumption
M. Romero-G6mez L. Grande M.C. Nogalesl M. Fern&ndez2 M. Chavez’ M. Castro
Aims.
To assess
clinical
significance
of liver
hepatitis
C virus
RNA
lev-
els and their relationship with epidemiological, biochemical and histological factors. Methods. A total of 50 patients [mean age 35.5*7 years] with biopsy-proven chronic hepatitis C infection were recruited. Risk factors were drug abuse (n=2 I), transfusion [n= IS], other parental routes (n=8; surgery=3, tattooing=5), and idiopathic [n=5). Duration of infection was 1629 years. All patients showed abnormal alanine aminotransferase levels and positive serum hepatitis C virus RNA. Hepatitis C virus genotype was assessed by Inno-Lipa. Liver biopsy was performed for histology and for hepatitis C virus RNA quantification by Amplicor-HCV-Monitor Daily alcohol consumption was recorded on two occasions by anamnesis. Inflammation grade was mild (n=31) or severe (n=l9]. Fibrosis was early stage [n=42) or advanced [n=8). Results. Mean hepatitis C virus RNA levels were 9.4~105 I. 5x1 iT copies/pg of total RNA in liver tissue, and 9. lx 105 I. 3xlP copies/ml in serum. Viral load in liver was positively correlated with that in serum [r=O. 51, p
burden
[r=O.53;
pcO.OOl].
Patients
infected
with
genotype
3a
showed lower intrahepatic hepatitis C virus load than patients infected with genotype I b; albeit without reaching statistical significance (0.49x I D%O. 89x I P vs I. 44x 10% I. 9x I @ copies/pg of total RNA; p=NS). No relationships were observed between liver viral burden and age, risk factor status, duration of infection, ferritin and alanine aminotransferase levels or with grading and staging. Conclusions. Hepatitis C virus load in serum is a mirror of intrahepatic hepatitis C virus levels. Chronic alcohol consumption enhances intrahepatic hepatitis C virus concentration.
Digest
Liver
Dis 2001;33:698-702
Key words: alcohol; amplicor-HCV-monitor; serum HCV RNA; Staging.
Fmm Units of Uepatology, I Microbiology and 2 Radiology, Hospital Universitariode Valme, Seville, Spain. Addmso tiw c~ndenc~ Dr. M. Romero-Gbmez, L1/Juan Antonio Cevestany l-4 G, 41003 Seville, Spain. fax: +34-954-681006. E-mail: [email protected] Submitted February 26, 2001. Accepted after revision June 27, 200 I.
699
grading;
hepatitis
C virus;
intrahepatic
HCV
levels;
Introduction The pathogenesis of hepatitis C virus (HCV)-induced hepatic injury remains unclear. There is little doubt that the immune response of the host plays a major role in HCV-induced liver disease I. However, genotype 3 has been implicated in the development of hepatocyte steatosis in chronic hepatitis C, and a cytopathic effect of this type of virus has been postulated2. A recent communication suggested that HCV liver injury could be induced by both mechanisms, consecutively or simultaneously 3. An assessment of intrahepatic HCV levels and their relationship with necro-inflammatory index and fibrosis could help to clarify the pathogenesis of chronic hepatitis C infection.
M. Aomero-GQmez et al.
Intrahepatic HCV replication has been detected using various methods such as in situ hybridization 4 5 or reverse transcription-polymerase chain reaction (RTPCR) 6-8. Commercial methods such as branched-DNA assay 9 or Amplicor-HCV-Monitor lo, which are available for measuring serum HCV replication, have been modified for use in the quantification of HCV RNA in liver. We assessed the intrahepatic HCV concentration in patients with chronic hepatitis C virus infection (increased alanine aminotransferase (ALT) and positive serum HCV RNA) and without previous interferon treatment. The aim of the study was to evaluate the relationship between liver HCV burden and serum HCV RNA level together with genotype, ALT and ferritin concentrations, liver histology, risk factor, duration of infection and daily alcohol consumption.
Patients and Methods Patient population A total of 50 consecutive patients (age 39+ 10 years; 35 males, 15 females) with chronic HCV infection were enrolled. All patients showed positive anti-HCV (EIA3; Abbott Laboratories, North Chicago, IL, USA), increased ALT. and positive HCV RNA in serum. The duration of infection was 16*9 years and the related risk factors were drug abuse (n=21), transfusion (n=16), tattooing (n=5), surgery (n=3) and unknown (n=5). At the time of liver biopsy the patient’s history of alcohol use was solicited. Two members of the investigating team independently ascertained the quantity of alcohol consumed daily as well as the duration of consumption. The average alcohol consumption was calculated in terms of grams per day. No patient had had previous treatment with interferon or other antiviral drugs. Liver function status (ALT, aspartate aminotransferase (AST), alkaline phosphatase, gamma glutamyl transferase (yGT) and ferritin levels) were determined by routine methods. HbsAg, antiHBc, antiHIV were tested using commercially available kits. All patients were negative for HbsAg and anti-HIV.
Liver histology Percutaneous liver biopsies were performed under ultrasonographic control. A portion of the biopsy material was used for the histology diagnosis. The grading and staging assessments were as communicated by Scheuer ” as follows: Necro-inflammatory activity (Grade): a) Portal inflammation and interface hepatitis ranging in grade from absent (PO) to severe and wide-
spread interface hepatitis (P4); b) Lobular activity ranging from none (LO) to bridging confluent necrosis (L4); Fibrosis (stage) ranging from no fibrosis (FO) to cirrhosis (F4). Necro-inflammatory activity was further sub-classified as being mild (n=31) if Pl-P2 or Ll-L2 or severe (n=19) if P3-P4 or L3-L4. Stage of librosis was further sub-classified as being early (n=42) if F 1-F2 or advanced (n=8) if F3-F4. The other portion of the biopsy material was snap-frozen for subsequent batched analyses of intrahepatic HCV concentrations. Virology assessments HCV genotype was assessed using INNO-LIPA HCV II kits (Innogenetics, Zwijnaarden, Belgium) according to the manufacturer’s instructions. The genotypes identified were la in 14%, genotype lb in 52%, genotype 2 in 4% and genotype 3a in 30% of patients. Amplicor-HCV-Monitor (Perkin-Elmer, Norwalk, CT, USA) was used to quantify the HCV RNA level in serum. Semi-quantijkxxtion of HCV RNA in liver Total liver RNA (1 ug) was added to diluent buffer (50 ul) to which an internal standard (0.2 ~1) had been added. PCR master mix (50 ~1) was added and the rest of the procedure was identical to that of the serumbased assay. Statistical methods Spearman coefficient, U-Mann-Whitney, Fisher and Kruskal-Wallis tests were used, as appropriate. Values of p
All patients were viraemic and were intrahepatic HCV positive. Mean HCV RNA levels were 0.94x106+1.5x106 copies/mg of total liver RNA in hepatic tissue and 0.91x106~1.3x106 copies/ml in serum. There was a significant positive correlation between viral load in liver and serum (r=0.51; p
lntrahepatic
HCW replication
j*I 2
_
_
.-I
3
4
5
6
7
[m;;
Serum HCV RNA (Lg copies/ml) 0
Fig. 1. Correlation between intrahepatic HVC RNA and serum HCV 2NA using the same method of analysis. i-=0.51, p-&001.
Table I. Relationships between intrahepatic and serum HCV RNA levels with demographic [sex, risk factor status, age, duration of illness), biochemical [ALT, GGT, Farritinl and histological [grading and staging1 factors.
Sex Male Female Risk Factor IVOU Transfusion Tattoos Surgery Unknown
35 15 21 16
751ztl200 15OO~t2200 907+1200 56*46 682*918
; 5
Age [years1 Genotype la,b 3a 2a,b Others
1019i1552 510*505
39*10
25 13 2 10
r=0.29 940*1900 490*890 36391t4974 439zk.573
Duration
16.3*10
r=0.14
ALT
II&96
GGT Ferritin Grading Mild Severe
31 19
Staging Early Advanced
42 6
r=0.26 1100*1400 515*553 396*262 1050i2100
r=O. 14
r=0.07
r=0.22
55i65
r=0.21
r=-0.07
152~172
r=0.03
r=0.23
1000i1600 823i1400
730*1376 1090*1290
No statistkally significant differences serum HCV load and epidemiologica/,
700
991*1700 645&20 1800*2200 275~t.252 341*249
986i1600 732*772 were observed between liver or clinical or histological variables.
20
40
60
Daily alcohol consumption
60
lrm
120
140
(g/d)
Fig. 2. Correlation between liver HCV RNA load and daily alcohol consumption. r=0.53; p
and staging (r=0.14; p=ns). Also, serum HCV RNA levels were not related to the demographic, biochemical or histological factors assessed. Daily alcohol consumption correlated significantly with degree of fibrosis (r=0.35; ~~0.05). A positive correlation was observed between daily alcohol consumption a&l intrahepatic HCV load (r=0.53; p
Discussion In the present study, chronic alcohol consumption was associated with increased intrahepatic HCV levels, Chronic hepatitis C is a slow, progressive disease. Regular alcohol intake has been associated with an increased risk of liver cirrhosis development 12-14,a higher serum viral load I5 16, and a lower response to interferon therapy I7 in patients with chronic hepatitis C. The mechanism of interaction between alcohol and HCV remains to be defined. An experimental study in mice indicated that chronic alcohol intake could have a direct effect on the host immune response directed towards the viral protein. Upon alcohol withdrawal, the immune response is reversible but the cytotoxic lymphocyte (CTL)-response is not IS. Further, in patients with chronic hepatitis C virus infection, neopterin levels in serum (a marker for activation of CTL-mediated immunity) were observed to be lower in alcoholic than
M. Romero-Cimez
non-alcoholic patients I9. Intrahepatic viral load depends on two processes: viral replication and/or viral excretion from the cell and, since intrahepatic viral RNA concentration measurements are standardized relative to total hepatic RNA, changes in overall liver RNA could lead to difficulties with interpretation of the results ?O.It is possible that chronic alcohol consumption could affect both the cellular excretion and cellular RNA content. Haydon et al. did not relate alcohol consumption to intrahepatic HCV concentration in their study 2’ but it should be pointed out that the study had included a group of patients with normal ALT concentrations and negative serum HCV RNA. Furthermore, liver viral load was related to alcohol abuse without the daily alcohol consumption being described. The mean daily alcohol intake is very difficult to calculate from an interview or questionnaire since it is a very subjective parameter 16. To-date, no other study has focused on this point. In the present study, although alcohol consumption was associated with higher intrahepatic levels, this relationship was weak (R’=0.25) and this variable alone could not explain the intrahepatic HCV burden and, as such, others factor need to be invoked. Thus, factors associated with more severe fibrosis progression (such as HIV-HCV co-infection or alcohol consumption) have also been related to higher liver HCV RNA levels E. There is a lack of data on HCV replication in patients with chronic hepatitis due to the clinical and ethical constraints of performing serial liver biopsies in these patients. It has recently been reported that the amount of hepatic viral RNA was similar in wedge and needle biopsies and that the former was representative of HCV levels at other sites in the liver and that HCV could be detected in very small specimens of liver tissue :?. As in previous studies ?’ 2J25, we observed a correlation between intrahepatic viral load and serum HCV burden. Using a PCR-based assay that is currently available for semi-quantification in serum and liver, we observed that serum HCV RNA reflected intrahepatic HCV concentrations and that this was consistent with the results of the study of Martin et al. I6 in which the same method was used. HCV RNA has been detected in the liver of patients with HCV infection but with normal ALT concentrations and negative serum HCV RNA. This favours the hypothesis that the liver is the last of the sites of HCV replication. However, a relationship between liver HCV burden and ALT or ferritin concentrations was not observed as previously reported 24. As with previous studies 21 25 there were no correlation between HCV RNA levels in liver and demographic factors such as age, sex, risk factor status and duration of infection. Alcohol consumption has been associated with higher serum HCV RNA I5 Ih, however, in the current study this relation-
et al.
ship did not achieve statistical significance. Fluctuations in serum HCV RNA have been reported and peaks in viral load would have been missed if quantification had been performed only on one occasion 27. We did not find a correlation between intrahepatic or serum HCV concentration and grading or staging of liver injury. This is a controversial issue since some reports have indicated that a higher liver viral load correlates with more severe liver disease 3 while, in other studies, no correlation was found 2’ 25. Nevertheless, following orthotopic liver transplantation, chronic active hepatitis showed lower intrahepatic HCV levels than lobular or minimal hepatitis 2s. Those studies that suggested a relationship between hepatic viral load and liver injury had various aspects of possible bias such as recruitment of relatively small numbers of cases and the inclusion of patients with normal ALT concentrations 2’. In conclusion, daily alcohol intake increases intrahepatic HCV replication and, probably, induces impairment of the cellular immune response that could exacerbate the liver disease. Hence, it is to be recommended that patients with chronic hepatitis C infection be advised to eliminate their alcohol consumption.
List of abbreviations ALT: alanine aminotransferase; AST: aspartate aminotransferase; yGT: gamma glutamyl transferase; CTL: cytotoxic lymphocyte; YGT: gamma glutamyl transferase; HCV: hepatitis C virus; HIV: human immuno-deficiency virus; ns: not significant; RT-PCR: reverse transcription-polymerase chain reaction,
References ’ Di Martin0 V, Brenot C, Samuel D, Saurini F, Paradis V, Reynes M, et al. Influence of liver hepatitis C virus RNA and hepatitis C virus genotype on FAS-mediated apoptosis after liver transplantation for hepatitis C. Transplantation 2000;70: 1390-6. 2 Rubbia-Brandt L, Quadri R, Abid K, Giostra E, Male PJ, Mentha G, et al. Hepatocyte steatosis is a cytopathic effect of hepatitis C virus genotype 3. J Hepatol 2000;33:106-15. ’ Nakagawa H, Shimomura H, Hasui T, Tsuji T. Quantitative detection of hepatitis C virus genome in liver tissue and circulation by competitive reverse transcription-polymerase chain reaction. Dig Dis Sci 1994;39:225-33. -) Lamas E, Baccarini P, Housset C, Kremsdorf D, Brechot C. Detection of hepatitis C virus (HCV) RNA sequences in liver tissue by in situ hybridization. J Hepatol 1992; 16:219-23. 5 Gastaldi M, Massacrier A, Planells R, Robaglia-Schlupp A, Portal-Bartolome I. Bourliere M, et al. Detection by in situ hybridization of hepatitis C virus positive and negative RNA strands using digoxigenin-labeled cRNA probes in human liver cells. J Hepatol 1995;23:509-18. h Sakamoto N, Enomoto N, Kurosaki M, Marumo F, Sato C. Detection and quantification of hepatitis C virus RNA replication in the liver. J Hepatol 1994;20:593-97.
701
lntrahepatic
HCV replication
’ Grassi G, Pozzato G, Moretti M, Giacca M. Quantitative analysis of hepatitis C virus RNA in liver biopsies by competitive reverse transcription and polymerase chain reaction. J Hepatol 1995;23:403-11. * Negro F, Giostra E, Krawczynski K, Quadri R, Rubbia-Brandt L, Mentha G, et al. Detection of intrahepatic hepatitis C virus replication by strand-specific semi-quantitative RT-PCR: Preliminary application to the liver transplantation model. J Hepatol 1998;29:1-11. ’ Pessoa MG, Terrault NA, Detmer J, Kolberg J, Collins M, Hassoba HM, et al. Quantitation of hepatitis G and C viruses in the liver: Evidence that hepatitis G virus is not hepatotropic. Hepatology 1998;27:877-80. I” McGuinness, Bishop GA, Painter DM, Chan R, McCaughan GW. lntrahepatic hepatitis C RNA levels do not correlate with degree of liver injury in patients with chronic hepatitis C. Hepatology 1996;23:676-87. ” Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991; 13:372-3. ‘* Corrao G, Arico S. Independent and combined action of hepatitis C virus infection and alcohol consumption on the risk of symptomatic liver cirrhosis. Hepatology 1998;27:914-9. ” Verbaan H, Wide11 A, Bondeson L, Andersson K, Erikson S. Factors associated with cirrhosis development in chronic hepatitis C patients from an area of low prevalence. J Viral Hepat 1998;5:43-5 1. I4 Pol S, Fontaine H, Carnot F, Zylberberg H, Berthelot P, Brechot C, et al. Predictive factors for development of cirrhosis in parenterally acquired chronic hepatitis C: a comparison between immunocompetent and immunocompromised patients. J Hepatol 1998;29: 12-9. I5 Cromie SL, Jenkins PJ, Bowden DS, Dudley FJ. Chronic hepatitis C: Effect of alcohol on hepatic activity and viral titre. J Hepato1 1996;25:821-6. I6 Pessione F, Degos F, Marcellin P, Duchatelle V, Njapoum C, Martinot-Peignoux M, et al. Effect of alcohol consumption on serum hepatitis C virus RNA and histological lesions in chronic hepatitis C. Hepatology 1998;27:1717-22. ” Loguercio C, Di Pierro M, Di Marino MP, Federico A, Disalvo D, Crafa E, et al. Drinking habits of subjects with hepatitis C virus-
702
related chronic liver disease: Prevalence and effect on clinical, viand pathological aspects. Alcohol Alcohol rological 2000;35:296-301. I8 Encke J, Wands JR. Ethanol inhibition: the humoral and cellular immune response to hepatitis C virus NS5 protein after genetic immunization. Alcohol Clin Exp Res 2000;24: 1063-9. I9 Oshita M, Hayashi N, Karahara A, Hagiwara H, Mita E, Naito M, et al. Increased serum hepatitis C virus RNA levels among alcoholic patients with chronic hepatitis C. Hepatology 1994;20: 1115-20. XJNegro F. Detection of hepatitis C virus RNA in liver tissue: an overview. ltal J Gastroenterol Hepatol 1998;30:205-10. ” Haydon GH, Jarvis LM, Blair CS, Simmonds P, Harrison DJ, Simpson KJ, et al. Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection. Gut 1998;42:570-5. ** Bonacini M, Govindarajan S, Blatt LM, Schmid P, Conrad A, Lindsay KL. Patients co-infected with human immunodeficiency virus and hepatitis C virus demonstrate higher levels of hepatic HCV RNA. J Viral Hepat 1999;6:203-8. ” Terrault NA, Dailey PJ, Ferrell L, Collins ML, Wilber JC, Urdea MS, et al. Hepatitis C virus: Quantitation and distribution in liver. J Med Virol 1997:51:217-24. 24 Di Martin0 V, Saurini F, Samuel D, Gigou M, Dussaix E, Reynes M, et al. Long-term longitudinal study of intrahepatic hepatitis C virus replication after liver transplantation. Hepatology 1997;26: 1343-50. 25 De Moliner L, Pontisso P, De Salvo GL, Cavalletto L, Chemello L, Alberti A. Serum and liver HCV RNA levels in oatients with chronic hepatitis C: Correlation with clinical and histological features. Gut 1998;42:856-60. *’ Martin J, Navas S, Quiroga JA, Colucci G, Pardo M, Carreiio V. Quantitation of hepatitis C virus in liver and peripheral blood mononuclear cells from patients with chronic hepatitis C virus infection. J Med Virol 1998;54:265-70. *’ Pontisso P, Bellati G, Brunetto M, Chemello L, Di Stefano R, Nicoletti M, et al. Hepatitis C virus RNA profiles in chronically infected individuals: Do they relate to disease activity? Hepatology 1999;29:585-9.
lntrahepatic in patients
hepatitis C virus replication is increased with regular alcohol consumption
M. Romero-G6mez L. Grande M.C. Nogalesl M. Fern&ndez2 M. Chavez’ M. Castro
Aims.
To assess
clinical
significance
of liver
hepatitis
C virus
RNA
lev-
els and their relationship with epidemiological, biochemical and histological factors. Methods. A total of 50 patients [mean age 35.5*7 years] with biopsy-proven chronic hepatitis C infection were recruited. Risk factors were drug abuse (n=2 I), transfusion [n= IS], other parental routes (n=8; surgery=3, tattooing=5), and idiopathic [n=5). Duration of infection was 1629 years. All patients showed abnormal alanine aminotransferase levels and positive serum hepatitis C virus RNA. Hepatitis C virus genotype was assessed by Inno-Lipa. Liver biopsy was performed for histology and for hepatitis C virus RNA quantification by Amplicor-HCV-Monitor Daily alcohol consumption was recorded on two occasions by anamnesis. Inflammation grade was mild (n=31) or severe (n=l9]. Fibrosis was early stage [n=42) or advanced [n=8). Results. Mean hepatitis C virus RNA levels were 9.4~105 I. 5x1 iT copies/pg of total RNA in liver tissue, and 9. lx 105 I. 3xlP copies/ml in serum. Viral load in liver was positively correlated with that in serum [r=O. 51, p
burden
[r=O.53;
pcO.OOl].
Patients
infected
with
genotype
3a
showed lower intrahepatic hepatitis C virus load than patients infected with genotype I b; albeit without reaching statistical significance (0.49x I D%O. 89x I P vs I. 44x 10% I. 9x I @ copies/pg of total RNA; p=NS). No relationships were observed between liver viral burden and age, risk factor status, duration of infection, ferritin and alanine aminotransferase levels or with grading and staging. Conclusions. Hepatitis C virus load in serum is a mirror of intrahepatic hepatitis C virus levels. Chronic alcohol consumption enhances intrahepatic hepatitis C virus concentration.
Digest
Liver
Dis 2001;33:698-702
Key words: alcohol; amplicor-HCV-monitor; serum HCV RNA; Staging.
Fmm Units of Uepatology, I Microbiology and 2 Radiology, Hospital Universitariode Valme, Seville, Spain. Addmso tiw c~ndenc~ Dr. M. Romero-Gbmez, L1/Juan Antonio Cevestany l-4 G, 41003 Seville, Spain. fax: +34-954-681006. E-mail: [email protected] Submitted February 26, 2001. Accepted after revision June 27, 200 I.
699
grading;
hepatitis
C virus;
intrahepatic
HCV
levels;
Introduction The pathogenesis of hepatitis C virus (HCV)-induced hepatic injury remains unclear. There is little doubt that the immune response of the host plays a major role in HCV-induced liver disease I. However, genotype 3 has been implicated in the development of hepatocyte steatosis in chronic hepatitis C, and a cytopathic effect of this type of virus has been postulated2. A recent communication suggested that HCV liver injury could be induced by both mechanisms, consecutively or simultaneously 3. An assessment of intrahepatic HCV levels and their relationship with necro-inflammatory index and fibrosis could help to clarify the pathogenesis of chronic hepatitis C infection.
M. Aomero-GQmez et al.
Intrahepatic HCV replication has been detected using various methods such as in situ hybridization 4 5 or reverse transcription-polymerase chain reaction (RTPCR) 6-8. Commercial methods such as branched-DNA assay 9 or Amplicor-HCV-Monitor lo, which are available for measuring serum HCV replication, have been modified for use in the quantification of HCV RNA in liver. We assessed the intrahepatic HCV concentration in patients with chronic hepatitis C virus infection (increased alanine aminotransferase (ALT) and positive serum HCV RNA) and without previous interferon treatment. The aim of the study was to evaluate the relationship between liver HCV burden and serum HCV RNA level together with genotype, ALT and ferritin concentrations, liver histology, risk factor, duration of infection and daily alcohol consumption.
Patients and Methods Patient population A total of 50 consecutive patients (age 39+ 10 years; 35 males, 15 females) with chronic HCV infection were enrolled. All patients showed positive anti-HCV (EIA3; Abbott Laboratories, North Chicago, IL, USA), increased ALT. and positive HCV RNA in serum. The duration of infection was 16*9 years and the related risk factors were drug abuse (n=21), transfusion (n=16), tattooing (n=5), surgery (n=3) and unknown (n=5). At the time of liver biopsy the patient’s history of alcohol use was solicited. Two members of the investigating team independently ascertained the quantity of alcohol consumed daily as well as the duration of consumption. The average alcohol consumption was calculated in terms of grams per day. No patient had had previous treatment with interferon or other antiviral drugs. Liver function status (ALT, aspartate aminotransferase (AST), alkaline phosphatase, gamma glutamyl transferase (yGT) and ferritin levels) were determined by routine methods. HbsAg, antiHBc, antiHIV were tested using commercially available kits. All patients were negative for HbsAg and anti-HIV.
Liver histology Percutaneous liver biopsies were performed under ultrasonographic control. A portion of the biopsy material was used for the histology diagnosis. The grading and staging assessments were as communicated by Scheuer ” as follows: Necro-inflammatory activity (Grade): a) Portal inflammation and interface hepatitis ranging in grade from absent (PO) to severe and wide-
spread interface hepatitis (P4); b) Lobular activity ranging from none (LO) to bridging confluent necrosis (L4); Fibrosis (stage) ranging from no fibrosis (FO) to cirrhosis (F4). Necro-inflammatory activity was further sub-classified as being mild (n=31) if Pl-P2 or Ll-L2 or severe (n=19) if P3-P4 or L3-L4. Stage of librosis was further sub-classified as being early (n=42) if F 1-F2 or advanced (n=8) if F3-F4. The other portion of the biopsy material was snap-frozen for subsequent batched analyses of intrahepatic HCV concentrations. Virology assessments HCV genotype was assessed using INNO-LIPA HCV II kits (Innogenetics, Zwijnaarden, Belgium) according to the manufacturer’s instructions. The genotypes identified were la in 14%, genotype lb in 52%, genotype 2 in 4% and genotype 3a in 30% of patients. Amplicor-HCV-Monitor (Perkin-Elmer, Norwalk, CT, USA) was used to quantify the HCV RNA level in serum. Semi-quantijkxxtion of HCV RNA in liver Total liver RNA (1 ug) was added to diluent buffer (50 ul) to which an internal standard (0.2 ~1) had been added. PCR master mix (50 ~1) was added and the rest of the procedure was identical to that of the serumbased assay. Statistical methods Spearman coefficient, U-Mann-Whitney, Fisher and Kruskal-Wallis tests were used, as appropriate. Values of p
All patients were viraemic and were intrahepatic HCV positive. Mean HCV RNA levels were 0.94x106+1.5x106 copies/mg of total liver RNA in hepatic tissue and 0.91x106~1.3x106 copies/ml in serum. There was a significant positive correlation between viral load in liver and serum (r=0.51; p
lntrahepatic
HCW replication
j*I 2
_
_
.-I
3
4
5
6
7
[m;;
Serum HCV RNA (Lg copies/ml) 0
Fig. 1. Correlation between intrahepatic HVC RNA and serum HCV 2NA using the same method of analysis. i-=0.51, p-&001.
Table I. Relationships between intrahepatic and serum HCV RNA levels with demographic [sex, risk factor status, age, duration of illness), biochemical [ALT, GGT, Farritinl and histological [grading and staging1 factors.
Sex Male Female Risk Factor IVOU Transfusion Tattoos Surgery Unknown
35 15 21 16
751ztl200 15OO~t2200 907+1200 56*46 682*918
; 5
Age [years1 Genotype la,b 3a 2a,b Others
1019i1552 510*505
39*10
25 13 2 10
r=0.29 940*1900 490*890 36391t4974 439zk.573
Duration
16.3*10
r=0.14
ALT
II&96
GGT Ferritin Grading Mild Severe
31 19
Staging Early Advanced
42 6
r=0.26 1100*1400 515*553 396*262 1050i2100
r=O. 14
r=0.07
r=0.22
55i65
r=0.21
r=-0.07
152~172
r=0.03
r=0.23
1000i1600 823i1400
730*1376 1090*1290
No statistkally significant differences serum HCV load and epidemiologica/,
700
991*1700 645&20 1800*2200 275~t.252 341*249
986i1600 732*772 were observed between liver or clinical or histological variables.
20
40
60
Daily alcohol consumption
60
lrm
120
140
(g/d)
Fig. 2. Correlation between liver HCV RNA load and daily alcohol consumption. r=0.53; p
and staging (r=0.14; p=ns). Also, serum HCV RNA levels were not related to the demographic, biochemical or histological factors assessed. Daily alcohol consumption correlated significantly with degree of fibrosis (r=0.35; ~~0.05). A positive correlation was observed between daily alcohol consumption a&l intrahepatic HCV load (r=0.53; p
Discussion In the present study, chronic alcohol consumption was associated with increased intrahepatic HCV levels, Chronic hepatitis C is a slow, progressive disease. Regular alcohol intake has been associated with an increased risk of liver cirrhosis development 12-14,a higher serum viral load I5 16, and a lower response to interferon therapy I7 in patients with chronic hepatitis C. The mechanism of interaction between alcohol and HCV remains to be defined. An experimental study in mice indicated that chronic alcohol intake could have a direct effect on the host immune response directed towards the viral protein. Upon alcohol withdrawal, the immune response is reversible but the cytotoxic lymphocyte (CTL)-response is not IS. Further, in patients with chronic hepatitis C virus infection, neopterin levels in serum (a marker for activation of CTL-mediated immunity) were observed to be lower in alcoholic than
M. Romero-Cimez
non-alcoholic patients I9. Intrahepatic viral load depends on two processes: viral replication and/or viral excretion from the cell and, since intrahepatic viral RNA concentration measurements are standardized relative to total hepatic RNA, changes in overall liver RNA could lead to difficulties with interpretation of the results ?O.It is possible that chronic alcohol consumption could affect both the cellular excretion and cellular RNA content. Haydon et al. did not relate alcohol consumption to intrahepatic HCV concentration in their study 2’ but it should be pointed out that the study had included a group of patients with normal ALT concentrations and negative serum HCV RNA. Furthermore, liver viral load was related to alcohol abuse without the daily alcohol consumption being described. The mean daily alcohol intake is very difficult to calculate from an interview or questionnaire since it is a very subjective parameter 16. To-date, no other study has focused on this point. In the present study, although alcohol consumption was associated with higher intrahepatic levels, this relationship was weak (R’=0.25) and this variable alone could not explain the intrahepatic HCV burden and, as such, others factor need to be invoked. Thus, factors associated with more severe fibrosis progression (such as HIV-HCV co-infection or alcohol consumption) have also been related to higher liver HCV RNA levels E. There is a lack of data on HCV replication in patients with chronic hepatitis due to the clinical and ethical constraints of performing serial liver biopsies in these patients. It has recently been reported that the amount of hepatic viral RNA was similar in wedge and needle biopsies and that the former was representative of HCV levels at other sites in the liver and that HCV could be detected in very small specimens of liver tissue :?. As in previous studies ?’ 2J25, we observed a correlation between intrahepatic viral load and serum HCV burden. Using a PCR-based assay that is currently available for semi-quantification in serum and liver, we observed that serum HCV RNA reflected intrahepatic HCV concentrations and that this was consistent with the results of the study of Martin et al. I6 in which the same method was used. HCV RNA has been detected in the liver of patients with HCV infection but with normal ALT concentrations and negative serum HCV RNA. This favours the hypothesis that the liver is the last of the sites of HCV replication. However, a relationship between liver HCV burden and ALT or ferritin concentrations was not observed as previously reported 24. As with previous studies 21 25 there were no correlation between HCV RNA levels in liver and demographic factors such as age, sex, risk factor status and duration of infection. Alcohol consumption has been associated with higher serum HCV RNA I5 Ih, however, in the current study this relation-
et al.
ship did not achieve statistical significance. Fluctuations in serum HCV RNA have been reported and peaks in viral load would have been missed if quantification had been performed only on one occasion 27. We did not find a correlation between intrahepatic or serum HCV concentration and grading or staging of liver injury. This is a controversial issue since some reports have indicated that a higher liver viral load correlates with more severe liver disease 3 while, in other studies, no correlation was found 2’ 25. Nevertheless, following orthotopic liver transplantation, chronic active hepatitis showed lower intrahepatic HCV levels than lobular or minimal hepatitis 2s. Those studies that suggested a relationship between hepatic viral load and liver injury had various aspects of possible bias such as recruitment of relatively small numbers of cases and the inclusion of patients with normal ALT concentrations 2’. In conclusion, daily alcohol intake increases intrahepatic HCV replication and, probably, induces impairment of the cellular immune response that could exacerbate the liver disease. Hence, it is to be recommended that patients with chronic hepatitis C infection be advised to eliminate their alcohol consumption.
List of abbreviations ALT: alanine aminotransferase; AST: aspartate aminotransferase; yGT: gamma glutamyl transferase; CTL: cytotoxic lymphocyte; YGT: gamma glutamyl transferase; HCV: hepatitis C virus; HIV: human immuno-deficiency virus; ns: not significant; RT-PCR: reverse transcription-polymerase chain reaction,
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