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Correlation of serum hepatitis C virus RNA titre with aminotransferases and liver histopathological findings in HCV-seropositive cases with end-stage chronic liver disease
Microbes and Infection, 1, 1999, 1091−1094 © 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
Correlation of serum hepatitis C virus RNA titre with aminotransferases and liver histopathological findings in HCV-seropositive cases with end-stage chronic liver disease Salah Aghaa*, Mohamed Al-Gendyb, Amgad El-Fikyc, Waffa El-Emshatya a
Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt b General Surgery Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt c Gastroenterology Centre, Faculty of Medicine, Mansoura University, Mansoura, Egypt (Received 22 March 1999; accepted 8 July 1999)
ABSTRACT – The quantity of circulating hepatitis C virus (HCV) RNA, aminotransferases and the degree of liver cell injury in relation to HCV serotype have not been fully studied. In this work, we estimated the HCV RNA titre in serum and correlated the findings with levels of aminotransferases, gamma glutamyltransferase (GGT), and liver histopathological changes and with HCV serotype. HCV RNA was found in 22 out of 30 HCV-seropositive cases included in this study (73.3%) and serotype 4 represented 90.9% (20/22). Levels of aminotransferases and GGT correlated with the levels of serum HCV RNA. Noticeably, GGT showed the highest positive correlation with the level of HCV RNA. Liver histopathological findings of 15 patients showed that eight had hepatocellular carcinoma and seven had cirrhosis. There was no significant difference between these two groups regarding levels of enzymes or serum HCV RNA titre. © 1999 Éditions scientifiques et médicales Elsevier SAS serum HCV RNA titre / liver enzymes / histopathology
1. Introduction Hepatitis C virus (HCV) is the predominant cause of posttransfusion and sporadic non-A, non-B hepatitis worldwide [1]. Chronic hepatitis C is an insidious disease and the progress is usually monitored by repeated measurement of aminotransferases and by polymerase chain reaction (PCR) [2]. Virological parameters of infection such as qualitative and quantitative determination of serum HCV RNA are significantly associated with underlying liver pathology irrespective of biochemical profile [3]. On the other hand, it has been reported that raised serum alanine aminotransferase (ALT) level is related to high circulating HCV RNA levels in individuals with anti-HCV [4]. However, the relationship of hepatocellular damage to the circulating HCV RNA level and aminotransferases has not been fully investigated [5]. The aim of this study was to find out the relation between HCV RNA level and liver enzymes (aminotransferases and * Correspondence and reprints Microbes and Infection 1999, 1091-1094
gamma glutamyltransferase) in HCV-seropositive (HCV+) individuals and to relate these findings to the degree of hepatocellular damage and HCV serotype.
2. Patients and methods This study was conducted on 30 HCV+ patients (17 males and 13 females), whose age ranged from 28 to 55 years. They were admitted to Mansoura University Hospital and Gastroenterology Centre. Serum samples from patients and the control group were divided into two parts, one part was subjected to liver function tests (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) bioMerieux kits, France) and to ELISA for detection of anti-HCV (Abbott HCV EIA 3.0), and this test was designed to detect antibodies to four recombinant HCV proteins: HC-34, HCr 43, c100-3, and NS5, hepatitis B virus surface antigen (HBs Ag), anti-HB core IgM and anti-HBc total.The other part was frozen immediately at -70 °C in RNase-free tubes for PCR (both qualitative and semiquantitative) and for serotyping. 1091
Original article
Agha et al.
PCR was carried out on frozen samples and on two known PCR-negative (PCR–) and two known PCR-positive (PCR+) samples as a test for the validity of the kit. HCV RNA was extracted from serum samples using a solution of guanidine salts and urea which acts as a denaturating reagent. A specific RNA-binding resin was then added to purify total RNA, and diethylpyrocarbonate treated water was used to elute RNA from the resin pellet (Ultraspec kit, Biotec Laboratories Inc., Houston, USA). Two microlitres of the extracted RNA was added to 18 µL of the master mix: 2 µL of first-strand buffer, 1 µL of Moloney murine leukaemia virus reverse transcriptase, 11.2 µL RNase-free water, 2 µL 20 A primer, 0.8 µL deoxynucleoside triphosphate and 1 µL RNase block (Stratagen Diagnostic Inc., CA, USA). Tubes were placed in TECHNE thermal cycler (TECHNE Cambridge Ltd., UK) at 37 °C for one hour and 99 °C for 5 min.The resulting cDNA from this step was subjected to amplification. For each sample, 80 µL of the master mix was prepared (10 µL of 10x buffer, 2 µL HCV 20 SB primer, 0.5 µL Taq polymerase and 67.5 µL sterile distilled water) and added to each tube resulting from the previous reverse transcription step. Amplification was carried out in a TECHNE thermal cycler at 94 °C for 1 min, 40 cycles at 94 °C for 30 sec, 59 °C for 30 sec, 72 °C for 1 min, and at 72 °C for 7 min.
Figure 1. Correlation between serum HCV RNA titre (copies/100 µL) and ALT values (U/mL) (No. of patients = 22).
2.1. Standard
A positive control DNA containing 105 copies/µL of plasmid DNA in Tris-EDTA buffer with 10 µg/mL of herring sperm DNA, containing the region amplified by the corresponding SHARP signal probe/primer set was used. Tenfold dilutions of this positive control was prepared in Tris-EDTA buffer. To each 2 µL of the prepared 10-fold dilutions, 98 µL of master mix was added (10 µL of 10× buffer, 0.8 µL of dNTP, 2 µL of HCV 20 A primer, 2 µL HCV 20 SB primer, 0.5 µL Taq polymerase and 82.7 µL of sterile distilled water). Amplification was performed according to the above protocol. The amplified PCR products and amplified standards were subjected to both qualitative and semiquantitative detection. Qualitative determination was carried out using agarose gel electrophoresis with DNA marker from 100–1 500 bp. Bands detected at 150 bp were considered positive. Semiquantitative determination was carried out using the Digene diagnostic kit (Digene Diagnostic Inc., Beltsville, MD), which is a sandwich capture molecular hybridization assay that utilizes colorimetric detection. The absorbance
Table I. Mean (± SD) of ALT, AST, and GGT in HCV+/ PCR+ patients versus HCV+/PCR– patients. Variable
HCV+/PCR+ patients (n = 22) Mean ± SD
HCV+/PCR– patients (n = 8) Mean ± SD
ALT * AST ** GGT***
72.53 ± 42 77.14 ± 50.29 53.09 ± 31.81
27.13 ± 8.7 27.38 ± 6.46 13.13 ± 3.48
T
P
3.39 < 0.01 4.54 < 0.0001 5.8 < 0.0001
* Normal range is up to 45 U/mL. ** Normal range is up to 40 U/mL. *** Normal range is males 11–43 U/L and females 9–37 U/L
1092
Figure 2. Correlation between serum HCV RNA titre (copies/100 µL) and AST values (U/mL) (No. of patients = 22). was read using ELISA reader (at wavelength 405 nm) and the curve was drawn on log/log paper using absorbance value at 405 nm versus concentrations of the standards. Serotyping of HCV was carried out on serum samples (Murex HCV serotyping 1–6 assay, Murex Diagnostics Ltd., England). In this test, synthetic peptides representing the variable antigenic region from NS4 of HCV types 1, 2, 3, 4, 5, and 6 were used. One strip of eight wells was used for each sample or control. An appropriate competing solution (1–6 or ‘all’) was added to the relevant wells. Readings were obtained at 450 nm and results were calculated.
3. Results This work included 30 HCV+ patients. They were negative for HBsAg, anti-HBc ‘IgM’ and anti-HBc ‘total’. Microbes and Infection 1999, 1091-1094
Serum HCV RNA titre, liver enzymes, and histopathology
Original article
Table II. Mean (± SD) of ALT, AST, and GGT and HCV RNA level/100 µL in patients with cirrhosis versus patients with HCC.
ALT AST GGT HCV RNA level
Patients with cirrhosis (n = 7) Mean ± SD
Patients with HCC (n = 8) Mean ± SD
64 ± 44.06 82.8 ± 47.65 51.57 ± 33.62 Range (1 × 102–8.0 × 105) Median (2 × 103)
107.63 ± 79.36 99 ± 60.37 72.13 ± 27.4 Range (9.50 × 102–8.0 × 105) Median (1 × 105)
Test of significance
P
T = 1.29 > 0.05 T = 0.57 > 0.05 T = 1.3 > 0.05 Z* = -1.39 (Mann Whitney test > 0.05)
* Value of Mann-Whitney test.
liver enzyme, determining the highest positive correlation with the level of HCV RNA. Liver needle biopsy revealed cirrhosis in seven patients, and hepatocellular carcinoma (HCC) in eight, while the remaining seven cases were not amenable for pathological interpretation due to tissue crushing with less than three portal areas. It was of interest that we found no significant difference in levels of HCV RNA and liver enzymes between the cirrhotic and HCC patients (P > 0.05, table II). The results of HCV serotyping (table III) confirmed that serotype 4 was the most prevalent (90.9, 20/22).
4. Discussion
Figure 3. Correlation between serum HCV RNA titre (copis/100 µL) and GGT values (U/L) (No. of patients = 22). The results obtained from this study showed that 73% (22/30) of the HCV+ patients were PCR+ . In patients who were HCV+/PCR+, a significant difference was observed in levels of ALT (P < 0.01), and a highly significant difference (P < 0.0001) was found in levels of AST and GGT compared to HCV+/PCR– patients (table I). Regarding the relationship between level of HCV RNA and liver enzymes in the 22 HCV+/PCR+ patients, there was a significant positive correlation between the quantity of HCV RNA and values of ALT (r + 0.92, P < 0.001), AST (r + 0.83, P < 0.001) and GGT (r + 0.97, P < 0.0001) (figures 1, 2, and 3), respectively. It is obvious that GGT was the most sensitive
Table III. HCV serotyping data. Serotype I II III IV V VI Microbes and Infection 1999, 1091-1094
No. of patients
% out of total
2/22 0/22 0/22 20/22 0/22 0/22
9.1% 0% 0% 90.9% 0% 0%
HCV infection is a major cause of morbidity and mortality worldwide [6]. In spite of the asymptomatic clinical course of chronic hepatitis C, cirrhosis develops in approximately 20% of patients after ten years [7] and HCC superimposes in a subset of them [8]. Qualitative PCR is a sensitive and reliable means for detection of HCV RNA, but it gives no information about the quantity of HCV RNA. HCV quantitation will become more and more important, since it can have significant clinical implications [9]. Circulating viral titres may be useful to evaluate the clinical evolution of infection, to gain information on infectivity of biological samples and to monitor the efficacy of antiviral therapy [10]. Therefore, this study aimed at finding the relation between HCV RNA level, aminotransferases and GGT in HCV+ individuals and to show the correlation of these findings with both the degree of hepatocellular damage and HCV serotype.The study was conducted on 30 HCV+ cases and 22 of them were PCR+ (73.3%). Serotype 4 was the most common (20/22; 90.9%) and this finding supports our previously reported data [12]. The remaining two patients had serotype 1 (table III). Shistosomal infestation was a frequent finding in the 30 studied patients. However, the eight HCV+/PCR– cases had normal liver enzymes, indicating that the elevated enzymes in the 22 HCV+/PCR+ patients were due to viraemia. This raises the hypophesis that shistosomiasis worsens the outcome in patients with a high titre of circulating HCV RNA. There were significantly higher levels of ALT (P < 0.01), AST (P < 0.0001) and GGT (P < 0.0001) in serum of HCV+/PCR+ patients compared with HCV+/ PCR– 1093
Original article
patients (table I). PCR remained negative in the eight HCV+/PCR– cases, and this finding suggests that HCV+ individuals with normal liver enzymes are most likely to be PCR–. Similarly, Banschoten et al. [11] found that none of 39 HCV+/PCR– patients had serum ALT level above 50 U/mL. There was a significant positive correlation between HCV RNA level and values of ALT (P < 0.001, figure 1), AST (P < 0.001, figure 2) and GGT (P < 0.0001, figure 3) in the 22 HCV+/PCR+ patients. GGT showed the highest positive correlation with HCV RNA quantity, and it might be considered as a good indicator for the underlying liver injury in hepatitis C. Donald [13] stated that GGT is the most sensitive chemical test for the presence of hepatobiliary disease and reported that the isolated elevation of GGT could be observed in chronic and minimal liver disease because of its great sensitivity. High ALT levels in patients with high titres of circulating HCV RNA might reflect viral replication which leads to hepatocellular damage with subsequent elevation of ALT. Similar observations were reported by Hagiwara et al. [4]. In Italy, Ballardini et al. [14] concluded that in genotype Ib, liver parameters of HCV infection were clinically more significant than viraemia levels. There was insignificant difference (Mann-Whitney test > 0.05) between the seven cirrhotic patients and the eight patients with HCC as regards level of HCV RNA (table II). This is in agreement with results obtained by Nousbaum et al. [15], who did not observe any difference in the level of HCV RNA in patients with chronic hepatitis, cirrhosis or with HCC. They stated that sustained HCV multiplication persists during the course of HCV infection when cirrhosis and HCC develop. Similarly, Shindo et al. [16] concluded that HCV RNA titers in serum and liver in chronic hepatitis C did not differ significantly with different histological activity index. Also, there was no significant difference between cirrhotic patients and patients with HCC as regards levels of ALT, AST and GGT (table II). In conclusion, in patients with HCV (serotype 4) a significant positive correlation was found between the level of viraemia and values of ALT, AST, and GGT. GGT showed the highest positive correlation with the level of HCV RNA, and thus it is the most sensitive liver enzyme for the detection of liver injury. No significant difference was observed between cirrhotic patients and patients with HCC as regards viraemia, ALT, AST, or GGT levels.
Acknowledgments We acknowledge Dr Yasser El-Sherbiny for the assistance in manuscript preparation. Dr Salah Agha wishes to thank Dr Amina Sameh for invaluable help. This study was supported by a grant from Mansoura University, Egypt.
References [1] Kuo G., Choo O.I., Alter H.J., Gitnick G.I., Redeker A.G., Purcell R.H., Miyamur T., An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis in the Netherlands, Science 244 (1989) 362–364. 1094
Agha et al.
[2] Alberti A., Recurrent HCV infection after liver transplantation, Lancet 341 (1993) 378–379. [3] Nalto M., Hoyashi N., Hagiwara H., Hiramatsu N., Serum hepatitis C virus RNA quantity and histological features of hepatitis C virus carriers with persistently normal ALT levels, Hepatology 19 (1994) 871–875. [4] Hagiwara H., Hayashi N., Mita E., Naito M., Kasahara A., Fusamoto H., Kamado T., Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease, Hepatology 17 (1993) 545–550. [5] Masayuki K., Nobuyukie Chifumi S., Naoya S., Yujji H., Hideaki H., Fumiaki M., Correlation of plasma hepatitis C virus RNA levels with serum alanine aminotransferase in Non-A, non-B chronic liver disease, J. Med. Virol. 39 (1993) 246–250. [6] Schreiber G.B., Busch M.P., Kleinman S.H., Korelitz J.J., The risk of transfusion-transmitted viral infections, N. Engl. Med. 334 (1996) 1685–1690. [7] DiBisceglie A.M., Goodman Z.D., Ishok K.G., Hoofinagl J.H., Melopolder J.J., 17 Alter H. J., Long term clinical and histo pathological follow up of chronic posttransfusion hepatitis, Hepatology 14 (1991) 969–974. [8] Colombo M., De Franchis R., Delninno E., Songiowanni A., DeFazio C., Tommasini M., Donato M.F., Hepatocellular carcinoma in Italian patients with cirrhosis N. Engl. J. Med. 325 (1991) 675–680. [9] Lau J.Y.N., Davis G.L., Kniffen J., Significance of serum hepatitis C virus RNA levels in chronic hepatitis, Lancet 41 (1993) 1501–1504. [10] Ravaggi A., Zonara A., Mazza C., Albertini A., Cariai E., Quantification of hepatits C virus RNA by competitive amplification of RNA from denatured serum and hybridization on microtiter plates, J. Clin. Microbiol. 33 (1995) 265–269. [11] Banschoten E.P., Bresters D., van Drimmelen A.A., Rodsenders G., Cuypers H.T., Reesink H.W., van derPoel C.L., van Denberg H.M., Lelie P.N., Hepatic C infection and viraemia in Dutch hemophilia patients, J. Med. Virol. 45 (1995) 24–26. [12] Agha S., Sherif L., Allam M.A., Fawzy M., Transplacental transmission of hepatitis C virus (HCV) in human immuno defeciency virus (HIV) negative mothers, Res. Virol. 149 (1998) 229–234. [13] Donald M., Liver function tests, Med. Int. 22 (1994) 425–431. [14] Ballardini G., Manzin A., Giostra F., Francesconi R., Groff P., Grassi A., Solforosi L., Ghetti S., Zauli D., Clementi M., Bianchi F.B., Quantitative liver parameters of HCV infection: relation to HCV genotypes, viraemia and response to interferon treatment, J. Hepatology 26 (1997) 779–86. [15] Nousbaum J.B., Pol S., Nalpas B., Landais P., Berthelot P., Brechot C., Hepatitis C virus type (Ib) II infection in France and Italy, Ann. Intern. Med. 122 (1995) 161–168. [16] Shindo M., Arail K., Sokawa Y., Tadao A., The virological and histological states of anti-HCV positive subjects with normal liver biochemical values, Hepatology 22 (1995) 418–425. Microbes and Infection 1999, 1091-1094
Correlation of serum hepatitis C virus RNA titre with aminotransferases and liver histopathological findings in HCV-seropositive cases with end-stage chronic liver disease Salah Aghaa*, Mohamed Al-Gendyb, Amgad El-Fikyc, Waffa El-Emshatya a
Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt b General Surgery Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt c Gastroenterology Centre, Faculty of Medicine, Mansoura University, Mansoura, Egypt (Received 22 March 1999; accepted 8 July 1999)
ABSTRACT – The quantity of circulating hepatitis C virus (HCV) RNA, aminotransferases and the degree of liver cell injury in relation to HCV serotype have not been fully studied. In this work, we estimated the HCV RNA titre in serum and correlated the findings with levels of aminotransferases, gamma glutamyltransferase (GGT), and liver histopathological changes and with HCV serotype. HCV RNA was found in 22 out of 30 HCV-seropositive cases included in this study (73.3%) and serotype 4 represented 90.9% (20/22). Levels of aminotransferases and GGT correlated with the levels of serum HCV RNA. Noticeably, GGT showed the highest positive correlation with the level of HCV RNA. Liver histopathological findings of 15 patients showed that eight had hepatocellular carcinoma and seven had cirrhosis. There was no significant difference between these two groups regarding levels of enzymes or serum HCV RNA titre. © 1999 Éditions scientifiques et médicales Elsevier SAS serum HCV RNA titre / liver enzymes / histopathology
1. Introduction Hepatitis C virus (HCV) is the predominant cause of posttransfusion and sporadic non-A, non-B hepatitis worldwide [1]. Chronic hepatitis C is an insidious disease and the progress is usually monitored by repeated measurement of aminotransferases and by polymerase chain reaction (PCR) [2]. Virological parameters of infection such as qualitative and quantitative determination of serum HCV RNA are significantly associated with underlying liver pathology irrespective of biochemical profile [3]. On the other hand, it has been reported that raised serum alanine aminotransferase (ALT) level is related to high circulating HCV RNA levels in individuals with anti-HCV [4]. However, the relationship of hepatocellular damage to the circulating HCV RNA level and aminotransferases has not been fully investigated [5]. The aim of this study was to find out the relation between HCV RNA level and liver enzymes (aminotransferases and * Correspondence and reprints Microbes and Infection 1999, 1091-1094
gamma glutamyltransferase) in HCV-seropositive (HCV+) individuals and to relate these findings to the degree of hepatocellular damage and HCV serotype.
2. Patients and methods This study was conducted on 30 HCV+ patients (17 males and 13 females), whose age ranged from 28 to 55 years. They were admitted to Mansoura University Hospital and Gastroenterology Centre. Serum samples from patients and the control group were divided into two parts, one part was subjected to liver function tests (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma glutamyltransferase (GGT) bioMerieux kits, France) and to ELISA for detection of anti-HCV (Abbott HCV EIA 3.0), and this test was designed to detect antibodies to four recombinant HCV proteins: HC-34, HCr 43, c100-3, and NS5, hepatitis B virus surface antigen (HBs Ag), anti-HB core IgM and anti-HBc total.The other part was frozen immediately at -70 °C in RNase-free tubes for PCR (both qualitative and semiquantitative) and for serotyping. 1091
Original article
Agha et al.
PCR was carried out on frozen samples and on two known PCR-negative (PCR–) and two known PCR-positive (PCR+) samples as a test for the validity of the kit. HCV RNA was extracted from serum samples using a solution of guanidine salts and urea which acts as a denaturating reagent. A specific RNA-binding resin was then added to purify total RNA, and diethylpyrocarbonate treated water was used to elute RNA from the resin pellet (Ultraspec kit, Biotec Laboratories Inc., Houston, USA). Two microlitres of the extracted RNA was added to 18 µL of the master mix: 2 µL of first-strand buffer, 1 µL of Moloney murine leukaemia virus reverse transcriptase, 11.2 µL RNase-free water, 2 µL 20 A primer, 0.8 µL deoxynucleoside triphosphate and 1 µL RNase block (Stratagen Diagnostic Inc., CA, USA). Tubes were placed in TECHNE thermal cycler (TECHNE Cambridge Ltd., UK) at 37 °C for one hour and 99 °C for 5 min.The resulting cDNA from this step was subjected to amplification. For each sample, 80 µL of the master mix was prepared (10 µL of 10x buffer, 2 µL HCV 20 SB primer, 0.5 µL Taq polymerase and 67.5 µL sterile distilled water) and added to each tube resulting from the previous reverse transcription step. Amplification was carried out in a TECHNE thermal cycler at 94 °C for 1 min, 40 cycles at 94 °C for 30 sec, 59 °C for 30 sec, 72 °C for 1 min, and at 72 °C for 7 min.
Figure 1. Correlation between serum HCV RNA titre (copies/100 µL) and ALT values (U/mL) (No. of patients = 22).
2.1. Standard
A positive control DNA containing 105 copies/µL of plasmid DNA in Tris-EDTA buffer with 10 µg/mL of herring sperm DNA, containing the region amplified by the corresponding SHARP signal probe/primer set was used. Tenfold dilutions of this positive control was prepared in Tris-EDTA buffer. To each 2 µL of the prepared 10-fold dilutions, 98 µL of master mix was added (10 µL of 10× buffer, 0.8 µL of dNTP, 2 µL of HCV 20 A primer, 2 µL HCV 20 SB primer, 0.5 µL Taq polymerase and 82.7 µL of sterile distilled water). Amplification was performed according to the above protocol. The amplified PCR products and amplified standards were subjected to both qualitative and semiquantitative detection. Qualitative determination was carried out using agarose gel electrophoresis with DNA marker from 100–1 500 bp. Bands detected at 150 bp were considered positive. Semiquantitative determination was carried out using the Digene diagnostic kit (Digene Diagnostic Inc., Beltsville, MD), which is a sandwich capture molecular hybridization assay that utilizes colorimetric detection. The absorbance
Table I. Mean (± SD) of ALT, AST, and GGT in HCV+/ PCR+ patients versus HCV+/PCR– patients. Variable
HCV+/PCR+ patients (n = 22) Mean ± SD
HCV+/PCR– patients (n = 8) Mean ± SD
ALT * AST ** GGT***
72.53 ± 42 77.14 ± 50.29 53.09 ± 31.81
27.13 ± 8.7 27.38 ± 6.46 13.13 ± 3.48
T
P
3.39 < 0.01 4.54 < 0.0001 5.8 < 0.0001
* Normal range is up to 45 U/mL. ** Normal range is up to 40 U/mL. *** Normal range is males 11–43 U/L and females 9–37 U/L
1092
Figure 2. Correlation between serum HCV RNA titre (copies/100 µL) and AST values (U/mL) (No. of patients = 22). was read using ELISA reader (at wavelength 405 nm) and the curve was drawn on log/log paper using absorbance value at 405 nm versus concentrations of the standards. Serotyping of HCV was carried out on serum samples (Murex HCV serotyping 1–6 assay, Murex Diagnostics Ltd., England). In this test, synthetic peptides representing the variable antigenic region from NS4 of HCV types 1, 2, 3, 4, 5, and 6 were used. One strip of eight wells was used for each sample or control. An appropriate competing solution (1–6 or ‘all’) was added to the relevant wells. Readings were obtained at 450 nm and results were calculated.
3. Results This work included 30 HCV+ patients. They were negative for HBsAg, anti-HBc ‘IgM’ and anti-HBc ‘total’. Microbes and Infection 1999, 1091-1094
Serum HCV RNA titre, liver enzymes, and histopathology
Original article
Table II. Mean (± SD) of ALT, AST, and GGT and HCV RNA level/100 µL in patients with cirrhosis versus patients with HCC.
ALT AST GGT HCV RNA level
Patients with cirrhosis (n = 7) Mean ± SD
Patients with HCC (n = 8) Mean ± SD
64 ± 44.06 82.8 ± 47.65 51.57 ± 33.62 Range (1 × 102–8.0 × 105) Median (2 × 103)
107.63 ± 79.36 99 ± 60.37 72.13 ± 27.4 Range (9.50 × 102–8.0 × 105) Median (1 × 105)
Test of significance
P
T = 1.29 > 0.05 T = 0.57 > 0.05 T = 1.3 > 0.05 Z* = -1.39 (Mann Whitney test > 0.05)
* Value of Mann-Whitney test.
liver enzyme, determining the highest positive correlation with the level of HCV RNA. Liver needle biopsy revealed cirrhosis in seven patients, and hepatocellular carcinoma (HCC) in eight, while the remaining seven cases were not amenable for pathological interpretation due to tissue crushing with less than three portal areas. It was of interest that we found no significant difference in levels of HCV RNA and liver enzymes between the cirrhotic and HCC patients (P > 0.05, table II). The results of HCV serotyping (table III) confirmed that serotype 4 was the most prevalent (90.9, 20/22).
4. Discussion
Figure 3. Correlation between serum HCV RNA titre (copis/100 µL) and GGT values (U/L) (No. of patients = 22). The results obtained from this study showed that 73% (22/30) of the HCV+ patients were PCR+ . In patients who were HCV+/PCR+, a significant difference was observed in levels of ALT (P < 0.01), and a highly significant difference (P < 0.0001) was found in levels of AST and GGT compared to HCV+/PCR– patients (table I). Regarding the relationship between level of HCV RNA and liver enzymes in the 22 HCV+/PCR+ patients, there was a significant positive correlation between the quantity of HCV RNA and values of ALT (r + 0.92, P < 0.001), AST (r + 0.83, P < 0.001) and GGT (r + 0.97, P < 0.0001) (figures 1, 2, and 3), respectively. It is obvious that GGT was the most sensitive
Table III. HCV serotyping data. Serotype I II III IV V VI Microbes and Infection 1999, 1091-1094
No. of patients
% out of total
2/22 0/22 0/22 20/22 0/22 0/22
9.1% 0% 0% 90.9% 0% 0%
HCV infection is a major cause of morbidity and mortality worldwide [6]. In spite of the asymptomatic clinical course of chronic hepatitis C, cirrhosis develops in approximately 20% of patients after ten years [7] and HCC superimposes in a subset of them [8]. Qualitative PCR is a sensitive and reliable means for detection of HCV RNA, but it gives no information about the quantity of HCV RNA. HCV quantitation will become more and more important, since it can have significant clinical implications [9]. Circulating viral titres may be useful to evaluate the clinical evolution of infection, to gain information on infectivity of biological samples and to monitor the efficacy of antiviral therapy [10]. Therefore, this study aimed at finding the relation between HCV RNA level, aminotransferases and GGT in HCV+ individuals and to show the correlation of these findings with both the degree of hepatocellular damage and HCV serotype.The study was conducted on 30 HCV+ cases and 22 of them were PCR+ (73.3%). Serotype 4 was the most common (20/22; 90.9%) and this finding supports our previously reported data [12]. The remaining two patients had serotype 1 (table III). Shistosomal infestation was a frequent finding in the 30 studied patients. However, the eight HCV+/PCR– cases had normal liver enzymes, indicating that the elevated enzymes in the 22 HCV+/PCR+ patients were due to viraemia. This raises the hypophesis that shistosomiasis worsens the outcome in patients with a high titre of circulating HCV RNA. There were significantly higher levels of ALT (P < 0.01), AST (P < 0.0001) and GGT (P < 0.0001) in serum of HCV+/PCR+ patients compared with HCV+/ PCR– 1093
Original article
patients (table I). PCR remained negative in the eight HCV+/PCR– cases, and this finding suggests that HCV+ individuals with normal liver enzymes are most likely to be PCR–. Similarly, Banschoten et al. [11] found that none of 39 HCV+/PCR– patients had serum ALT level above 50 U/mL. There was a significant positive correlation between HCV RNA level and values of ALT (P < 0.001, figure 1), AST (P < 0.001, figure 2) and GGT (P < 0.0001, figure 3) in the 22 HCV+/PCR+ patients. GGT showed the highest positive correlation with HCV RNA quantity, and it might be considered as a good indicator for the underlying liver injury in hepatitis C. Donald [13] stated that GGT is the most sensitive chemical test for the presence of hepatobiliary disease and reported that the isolated elevation of GGT could be observed in chronic and minimal liver disease because of its great sensitivity. High ALT levels in patients with high titres of circulating HCV RNA might reflect viral replication which leads to hepatocellular damage with subsequent elevation of ALT. Similar observations were reported by Hagiwara et al. [4]. In Italy, Ballardini et al. [14] concluded that in genotype Ib, liver parameters of HCV infection were clinically more significant than viraemia levels. There was insignificant difference (Mann-Whitney test > 0.05) between the seven cirrhotic patients and the eight patients with HCC as regards level of HCV RNA (table II). This is in agreement with results obtained by Nousbaum et al. [15], who did not observe any difference in the level of HCV RNA in patients with chronic hepatitis, cirrhosis or with HCC. They stated that sustained HCV multiplication persists during the course of HCV infection when cirrhosis and HCC develop. Similarly, Shindo et al. [16] concluded that HCV RNA titers in serum and liver in chronic hepatitis C did not differ significantly with different histological activity index. Also, there was no significant difference between cirrhotic patients and patients with HCC as regards levels of ALT, AST and GGT (table II). In conclusion, in patients with HCV (serotype 4) a significant positive correlation was found between the level of viraemia and values of ALT, AST, and GGT. GGT showed the highest positive correlation with the level of HCV RNA, and thus it is the most sensitive liver enzyme for the detection of liver injury. No significant difference was observed between cirrhotic patients and patients with HCC as regards viraemia, ALT, AST, or GGT levels.
Acknowledgments We acknowledge Dr Yasser El-Sherbiny for the assistance in manuscript preparation. Dr Salah Agha wishes to thank Dr Amina Sameh for invaluable help. This study was supported by a grant from Mansoura University, Egypt.
References [1] Kuo G., Choo O.I., Alter H.J., Gitnick G.I., Redeker A.G., Purcell R.H., Miyamur T., An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis in the Netherlands, Science 244 (1989) 362–364. 1094
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