LETTERS
TO THE EDITOR
405
HEPAT 00973
A significant number of patients with chronic hepatitis B virus (HBV) infection have no evidence of HBV replication, as determined by a negative serum HNV DNA, and yet have active liver disease. Some of them have hepatitis D virus (HDV) infection. In the remaining patients, it is important to ascertain whether hepatitis C virus (HCV) infection is the cause of active liver damage. Sera from 186 consecutive patients with chronic HBV infection seen over a l-year period were analysed for HBV e antigen (HBeAg), antibody to HBeAg (antiHBe) and total antibody to HDV, using commercially available radioimmunoassays (Abbott Diagnostics, Maidenhead, U.K.). Serum HBV DNA was measured by a semi-quantitative technique (1). Sera with elevated levels of serum aspartate transaminase (AST), seropositive for anti-HBe but with no detectable HBV DNA or total antibody to HDV, were tested for antibody to hepatitis C virus (HCV) using a peptide based ELISA (UBI, NY) (2) which we have found to be specific and reliable in
patients with chronic liver disease (Smith HM et al., personal observation). Anti-HBe was detected in 111 of the patients, 98 of whom were seronegative for HBV DNA. Thirty-five of the 98 patients with no serological evidence of active HBV replication had an elevated AST which could be attributed to HDV in 10 patients. Of the remaining 25 patients seropositive for HBsAg, seronegative for HBV DNA and total antibody to HDV but with an elevated AST. 10 were seropositive for antibody to HCV. This data indicates that testing for HCV in this group of patients is important since interferon-u has been shown to be effective in a proportion of patients with chronic HCV infection (3).
References
2 Hosein B. Fang CT, Popovsky MA, et al. Improved serodiagnosis
1 Fagan, EA, Guarner P. Pereira SDK, et al. Quantitation of hepatitis I3 virus DNA in serum using the spot hybridisation technique and scintillation counting. J Viral Methods 1985: 12: 251-62.
of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Nat1 Acad Sci USA 1991; in press. 3 Davis GL, Balart LA, Schiff ER. et al. Treatment of chronic hepatitis C with recombinant interferon alpha. A multlcenter randomized, controlled trial. N Engl J Med IYXY;321: 1501-6.
H.M. Smith, H.M. Daniels, C.J. Tibbs, J.Y.N. Lau and R. Williams Institute of Liver Shcdies, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, United Kingdom
HEPAT 00975
etection of erase We developed a PCR-based method to detect hepatitis C virus (HCV) RNA in liver needle biopsies. We used two sets of primers and the nested PCR technique and attempted to detect HCV RNA sequences in RNA isolated from tissue derived from liver needle biopsies (immediately frozen) from patients with HCV antibodies in their sera. HCV antibodies were detezted using an enzyme-linked immunoassay (Ortho Diagnostics, U.K.) following the instructions of the suppliers. The detailed characteristics of the patients are shown in Table 1. Histologically all patients had signs of chronic hepatitis. RNA was extracted from part of the material from the needle biopsy (1). The RNA was reverse transcribed to
sies by
liver nee ain reactio
using the reagents provided in the RNA PCR kit (Perkin Elmer). Five ~1 of cDNA were used for the first
CDNA
TABLE 1 Characteristics of the patients .__ ___ ~AST AgeALT No. /sex 1 2 3 4 5 6
60/F 52/M 62&l 34/F 26/F 61/M
434 250 110 103
357 120 224 49 *
25;
300
a-HCV + + + + + +
PCR set-l
PCR set-2
+ + +
+ + + +
-
F. female; M, male; ALT. ala&e amino transferase: aspartate amino trarsferase: *. reported as normal.
AST.
LETTERS
406
A
12345678
B
Fig. 1. PCR products of HCV cDNA using the d94, d95, Nl and N2 set of primers (A) and the NCR set of primers (B) on gel electrophoresis. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, sample from patient with proven hepatitis B infection: and lanes 5-8, samples from patients 1, 2, 3 and 4. respectively.
round. Using the d94, d95, Nl, N2 set of primers (2) we performed the first round of PCR consisting of 35 cycles of denaturation for 80 s at 95 “C, annealing for 40 s at 58 “C and extension for 60 s at 72 “C. Ten ~1 of amplified material were subjected to the second round of PCR consisting of 25 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 46 “C and extension for 60 s
TO THE EDITOR
72 “C. Using the NCR 1,2,3 and 4 set of primers (3) we performed the first PCR round consisting of 35 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 50 “C and extension for 60 s at 72 “C. The second PCR round consisted of 30 cycles of denaturation for 60 s at 94 “C, annealing for 60 s at 46 “C and extension for 60 s at 72 “C. The last cycle of each PCR round was followed by an extension of 7 min at 72 “C. Reagents without template and RNA isolated from a liver with proven hepatitis B were used as negative controls. As a positive control we used RNA isolated from the liver of a patient who had a partial hepatectomy because of a hepatocellular carcinoma and a nonalcoholic cirrhosis. The patient’s serum had HCV antibodies, tested by an enzyme immunoassay (Abbott Wiesbaden). By using the first set of primers we amplified HCV cDNA sequences in three of six cases (Fig. lA), while by using the second set of primers we amplified HCV cDNA in four of six cases (Fig. 1B). The use of this method to detect HCV RNA in material from liver needle biopsies may help in the definite diagnosis of HCV. Since only part of the material is enough for the detection of HCV RNA, this could complement the diagnosis of HCV based upon morphological criteria. at
PCR
References 1 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9. 2 Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, et al.
Ioannis D. Diamantis’, Christine E. McGandy’, Irmgard Pult’, Jean-Jacques Widmann2, Fred Gudat’ and Leonardo Bianchi’ ‘Institute of Pathology, University Hospital, Schdnbeinstrasse 40, CH-4003, Base1 and ‘Institute of Pathology, University Hospital, CMU, CH-1200 Geneva, Switzerland
Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 3 Garson JA, Ring C, Tuke P, Tedder RS. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990; 336:878
HEPAT 00976
Hormonal treatment of hepatocellular In response to the letter of Farinati et al. (1) about hormonal manipulation of hepatocellular carcinoma (HCC), we would like to correct one small omission. In their discussion of anti-androgen therapy, they stated that they were aware of three studies. We would like to draw their attention to a fourth, in which the progestogenie anti-androgen cyproterone acetate was administered to 25 cirrhotic patients with HCC (23 male) (1).
carcinoma
There was an objective response to therapy in five patients, and furthermore, the response appeared to be correlated with a fall in free levels of the potent androgen Sa-dihydrotestosterone. In this study, while the compounding variables or ‘biases’ referred to by Farinati et al. were not entirely eliminated, the aetiology of cirrhosis, Gkuda grade and, in most patients, the pre- and post-treatment hormonal
TO THE EDITOR
405
HEPAT 00973
A significant number of patients with chronic hepatitis B virus (HBV) infection have no evidence of HBV replication, as determined by a negative serum HNV DNA, and yet have active liver disease. Some of them have hepatitis D virus (HDV) infection. In the remaining patients, it is important to ascertain whether hepatitis C virus (HCV) infection is the cause of active liver damage. Sera from 186 consecutive patients with chronic HBV infection seen over a l-year period were analysed for HBV e antigen (HBeAg), antibody to HBeAg (antiHBe) and total antibody to HDV, using commercially available radioimmunoassays (Abbott Diagnostics, Maidenhead, U.K.). Serum HBV DNA was measured by a semi-quantitative technique (1). Sera with elevated levels of serum aspartate transaminase (AST), seropositive for anti-HBe but with no detectable HBV DNA or total antibody to HDV, were tested for antibody to hepatitis C virus (HCV) using a peptide based ELISA (UBI, NY) (2) which we have found to be specific and reliable in
patients with chronic liver disease (Smith HM et al., personal observation). Anti-HBe was detected in 111 of the patients, 98 of whom were seronegative for HBV DNA. Thirty-five of the 98 patients with no serological evidence of active HBV replication had an elevated AST which could be attributed to HDV in 10 patients. Of the remaining 25 patients seropositive for HBsAg, seronegative for HBV DNA and total antibody to HDV but with an elevated AST. 10 were seropositive for antibody to HCV. This data indicates that testing for HCV in this group of patients is important since interferon-u has been shown to be effective in a proportion of patients with chronic HCV infection (3).
References
2 Hosein B. Fang CT, Popovsky MA, et al. Improved serodiagnosis
1 Fagan, EA, Guarner P. Pereira SDK, et al. Quantitation of hepatitis I3 virus DNA in serum using the spot hybridisation technique and scintillation counting. J Viral Methods 1985: 12: 251-62.
of hepatitis C virus infection with synthetic peptide antigen from capsid protein. Proc Nat1 Acad Sci USA 1991; in press. 3 Davis GL, Balart LA, Schiff ER. et al. Treatment of chronic hepatitis C with recombinant interferon alpha. A multlcenter randomized, controlled trial. N Engl J Med IYXY;321: 1501-6.
H.M. Smith, H.M. Daniels, C.J. Tibbs, J.Y.N. Lau and R. Williams Institute of Liver Shcdies, King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, United Kingdom
HEPAT 00975
etection of erase We developed a PCR-based method to detect hepatitis C virus (HCV) RNA in liver needle biopsies. We used two sets of primers and the nested PCR technique and attempted to detect HCV RNA sequences in RNA isolated from tissue derived from liver needle biopsies (immediately frozen) from patients with HCV antibodies in their sera. HCV antibodies were detezted using an enzyme-linked immunoassay (Ortho Diagnostics, U.K.) following the instructions of the suppliers. The detailed characteristics of the patients are shown in Table 1. Histologically all patients had signs of chronic hepatitis. RNA was extracted from part of the material from the needle biopsy (1). The RNA was reverse transcribed to
sies by
liver nee ain reactio
using the reagents provided in the RNA PCR kit (Perkin Elmer). Five ~1 of cDNA were used for the first
CDNA
TABLE 1 Characteristics of the patients .__ ___ ~AST AgeALT No. /sex 1 2 3 4 5 6
60/F 52/M 62&l 34/F 26/F 61/M
434 250 110 103
357 120 224 49 *
25;
300
a-HCV + + + + + +
PCR set-l
PCR set-2
+ + +
+ + + +
-
F. female; M, male; ALT. ala&e amino transferase: aspartate amino trarsferase: *. reported as normal.
AST.
LETTERS
406
A
12345678
B
Fig. 1. PCR products of HCV cDNA using the d94, d95, Nl and N2 set of primers (A) and the NCR set of primers (B) on gel electrophoresis. Lane 1, molecular weight marker; lane 2, positive control; lane 3, negative control; lane 4, sample from patient with proven hepatitis B infection: and lanes 5-8, samples from patients 1, 2, 3 and 4. respectively.
round. Using the d94, d95, Nl, N2 set of primers (2) we performed the first round of PCR consisting of 35 cycles of denaturation for 80 s at 95 “C, annealing for 40 s at 58 “C and extension for 60 s at 72 “C. Ten ~1 of amplified material were subjected to the second round of PCR consisting of 25 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 46 “C and extension for 60 s
TO THE EDITOR
72 “C. Using the NCR 1,2,3 and 4 set of primers (3) we performed the first PCR round consisting of 35 cycles of denaturation for 60 s at 95 “C, annealing for 60 s at 50 “C and extension for 60 s at 72 “C. The second PCR round consisted of 30 cycles of denaturation for 60 s at 94 “C, annealing for 60 s at 46 “C and extension for 60 s at 72 “C. The last cycle of each PCR round was followed by an extension of 7 min at 72 “C. Reagents without template and RNA isolated from a liver with proven hepatitis B were used as negative controls. As a positive control we used RNA isolated from the liver of a patient who had a partial hepatectomy because of a hepatocellular carcinoma and a nonalcoholic cirrhosis. The patient’s serum had HCV antibodies, tested by an enzyme immunoassay (Abbott Wiesbaden). By using the first set of primers we amplified HCV cDNA sequences in three of six cases (Fig. lA), while by using the second set of primers we amplified HCV cDNA in four of six cases (Fig. 1B). The use of this method to detect HCV RNA in material from liver needle biopsies may help in the definite diagnosis of HCV. Since only part of the material is enough for the detection of HCV RNA, this could complement the diagnosis of HCV based upon morphological criteria. at
PCR
References 1 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium-thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9. 2 Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, et al.
Ioannis D. Diamantis’, Christine E. McGandy’, Irmgard Pult’, Jean-Jacques Widmann2, Fred Gudat’ and Leonardo Bianchi’ ‘Institute of Pathology, University Hospital, Schdnbeinstrasse 40, CH-4003, Base1 and ‘Institute of Pathology, University Hospital, CMU, CH-1200 Geneva, Switzerland
Detection of hepatitis C viral sequences in blood donations by ‘nested’ polymerase chain reaction and prediction of infectivity. Lancet 1990; 335: 1419-22. 3 Garson JA, Ring C, Tuke P, Tedder RS. Enhanced detection by PCR of hepatitis C virus RNA. Lancet 1990; 336:878
HEPAT 00976
Hormonal treatment of hepatocellular In response to the letter of Farinati et al. (1) about hormonal manipulation of hepatocellular carcinoma (HCC), we would like to correct one small omission. In their discussion of anti-androgen therapy, they stated that they were aware of three studies. We would like to draw their attention to a fourth, in which the progestogenie anti-androgen cyproterone acetate was administered to 25 cirrhotic patients with HCC (23 male) (1).
carcinoma
There was an objective response to therapy in five patients, and furthermore, the response appeared to be correlated with a fall in free levels of the potent androgen Sa-dihydrotestosterone. In this study, while the compounding variables or ‘biases’ referred to by Farinati et al. were not entirely eliminated, the aetiology of cirrhosis, Gkuda grade and, in most patients, the pre- and post-treatment hormonal
