GASTROENTEROLOGY
1992;102:692-694
Detection of Hepatitis C Virus RNA in Chronic Non-A, Non-B Liver Disease HIDEKI HAGIWARA, NOR10 HAYASHI, EIJI MITA, NAOKI KEIJI UEDA, TETSUO TAKEHARA, NOBUKAZU YUKI, AKINORI
KASAHARA,
HIDEYUKI
HIRAMATSU,
FUSAMOTO,
and TAKENOBU KAMADA First Department of Medicine, Osaka University Medical School, Osaka, Japan
Serum samples were tested for detection of hepatitis C virus (HCV) RNA from 156 patients with chronic non-A, non-B liver disease. HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-ClOO-3 but was also found in 15 (55.6%) of 27 patients negative for anti-ClOO-3. The rate of positivity for HCV RNA was not significantly different among various stages of liver diseases. These results showed that HCV continues to replicate even in advanced liver disease and that it seems to be related to half of the cases of chronic non-A, non-B liver disease negative for anti-C169-3. n assay for anti-C100-3 is now available for diagnosis of hepatitis C virus (HCV) infection.lm5 However, in some cases, HCV can be transmitted from plasma negative for anti-C100-3, which indicates a limitation to the precise diagnosis of HCV carriers using anti-ClOO-3. In contrast, detecting HCV RNA in serum can be useful for diagnosing infectious HCV carriers.6*7 To determine the interrelationship between replication of HCV and various stages of chronic non-A, non-B (NANB) liver disease, we tested serum samples for the presence of HCV RNA using a combination assay of reverse transcription and polymerase chain reaction (RT-PCR).
A
Materials and Methods The subjects were 156 patients with chronic NANB liver disease. Hepatitis B virus infection was excluded by commercial serological tests (absence of detectable hepatitis B surface antigen and high-titer anti-hepatitis B core antigen). Liver biopsy was performed on 117 patients, and the histology showed the features of chronic persistent hepatitis (CPH) in 20 patients, chronic active hepatitis (CAH) in 69 patients, and liver cirrhosis (LC) in 28 patients. The remaining 39 patients had a diagnosis of hepatocellular carcinoma (HCC) by diagnostic imaging. Anti-ClOO-3 was assayed by enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Tokyo, Japan). The primer sequences for RT-PCR were derived from the 5’-terminal sequence of a complementary DNA (cDNA) clone of HCV [5’-CATGGTGCACGGTCTACGAG-3’, anti-
sense; 5’-ACTCCACCATAGATCACTCC-3’, sense).’ For extraction of RNA, 100 pL of 21% polyethylene glycol6000 and 1.5 mol/L NaCl were added to 200 pL of serum sample. This was left standing for 2 hours at 4’C. After centrifugation, the pellet was mixed with 600 pL of homogenization buffer, containing 4 mol/L guanidinium thiocyanate, 0.1 mol/L Tris-HCl, pH 7.5, 0.5% sodium lauryl sarcosinate, and 1% j3-mercaptoethanol. After two extractions with phenol chloroform, 10 pg of transfer RNA (tRNA), one-tenth volume of 3 mol/L sodium acetate (pH 6.0), and 2.5 volumes of absolute ethanol were added to the aqueous phase. This mixture was left at -8O’C for 1 hour, and then the RNA was pelleted by centrifugation. The pellet was washed with 70% ethanol, dried, and dissolved in 7 pL of distilled water. RNA solution was made up to 20 FL with reaction solution containing 20 pm01 of anti-sense primer, 1 mmol/L of each deoxynucleotide triphosphate, 20 units of ribonuclease inhibitor (Promega Corp., Madison, WI), and 200 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD). The reverse transcriptase reaction was performed according to the manufacturer’s instructions. The cDNA preparation was added to 80 yL of PCR solution containing 20 pmol of sense primer, 1.2 mmol/L of MgCl,, 45.2 mmol/L of KCl, and 2.5 units of Taq polymerase (Perkin Elmer Cetus Corp., Norwalk, CT). The mixtures were amplified by 40 cycles of PCR (94°C for 30 seconds, 55’C for 1 minute, and 72’C for 1 minute), followed by a lo-minute extension at 72°C. A lo-pL sample of PCR products was analyzed on 2% agarose gels, blotted onto a nylon membrane, hybridized to a 32P-end-labeled HCV cDNA (-1.0 X 10~ cpm/pg), which lay between the RT-PCR primers. The membrane was exposed to a preflashed film at -7O’C for 6 hours using an intensifying screen. We could detect a single copy of HCV cDNA in this condition. All samples were assayed at least two times with reproducible results. The x2 test or Fisher’s Exact Test and Student’s t test were used for statistical analysis. Results higher
The mean age of LC patients was significantly than that of CPH or CAH patients: that of HCC
0 1992by
the American Gastroenterological 0016-5065/92/$3.00
Association
HCV
February 1992
RNA IN NANB LIVERDISEASE 693
Table 1. Positive Rate for HCV RNA in Chronic NANB Liver Disease Positive rate for HCVRNA (%) Disease CPH CAH LC HCC Total (%)
(yr, mean + SD] 44.1 47.6 56.0 61.6 51.1
f + + + f
11.0 9.8 8.4’ 6.8b 11.1
Positive for anti-ClOO-3
Negative for anti-ClOO-3 l/2 6/9 3/6 5/10 15/27 (55.6)
17/18 56/60 21/22 27/29 121/129 (93.8)
Total (%) 18/20 62/69 24/28 32/39 136/156
(90.0) (89.9) (85.7) (82.1) (87.2)
“P < 0.01 vs. CPH, CAH, and HCC. bP < 0.01 vs. CPH, CAH, and LC.
patients was significantly higher than that of CPH, CAH, or LC patients (Table 1). Of the 156 patients, 129 (82.7%) were positive for anti-ClOO-3. The rate was 90.0% for CPH,87.0% for CAH, 78.6% for LC, and 74.4% for HCC patients. HCV RNA in serum was detected in 136 patients (87.2%). The rates of positive results for HCV RNA in advanced chronic liver disease including LC (85.7%) and HCC (82.1%) were as high as those in the early stage of chronic liver disease including CPH (90.0%) and CAH (89.9%). The positive HCV RNA rates did not significantly differ among diseases. In comparison with anti-ClOO-3 and HCV RNA, HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-ClOO-3 and in 15 (55.6%) of 27 patients negative for anti-ClOO-3 (Table 1).
reverse transcriptase reaction step. We could not confirm whether serum negative for HCV RNA contained HCV particles, because this was below the threshold of sensitivity of the RT-PCR assay. Furthermore, Ulrich et ala7 reported that a low concentration of HCV RNA could be detected from 2.4 mL of plasma, but not from 0.6 mL. HCV RNA may be detected if >2OO pL of serum can be analyzed for these eight patients. Twelve patients were both negative for anti-ClOO-3 and HCV RNA in chronic NANB liver disease. These patients might account for hepatitis induced by other unknown viruses, although a low concentration of HCV RNA should be excluded. References 1.
Discussion Recent reports have documented the detection of HCV RNA by RT-PCR assay.6*7.g*‘oUlrich et a1.7 found HCV RNA in 2.4 mL of plasma from 11 (100%) of 11 volunteer blood donors positive for anti-ClOO-3, and Kato et al.” detected it in 1 mL of serum from 13 (81%) of 16 patients positive for antiCIOO-3 and 7 (50%) of 14 patients negative for antiClOO-3 with chronic NANB liver disease. In the present study, HCV RNA was detected in 200 yL of serum from 136 patients (87.2%) with chronic NANB liver disease. Although the mean ages of the patients with HCC and LC were significantly higher than those with chronic hepatitis, there was no significant difference in the positive rates for HCV RNA between the different chronic NANB liver diseases. These results indicated that replication of HCV occurs even in the advanced liver disease. Furthermore, HCV RNA was detected in 15 (55.6%) of 27 patients negative for anti-ClOO-3. This showed that HCV seems to be related to half of the chronic NANB viral liver diseases negative for anti-ClOO-3. Eight patients were negative for HCV RNA but positive for anti-ClOO-3 in the present study. Although the detection limit of this PCR assay is a single copy of cDNA, there is likely to be some loss of generating cDNA fragments in the extraction, precipitation, and
2.
3.
4.
5.
6.
7.
Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-364. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989:321:14941500. Hopf U, Moller B, Kuther D, Stemerowicz R, Lobeck H, Ludtke-Handjery A, Walter E, Blum HE, Roggendorf M, Deinhardt F. Long-term follow-up of posttransfusion and sporadic chronic hepatitis non-A, non-B and frequency of circulating antibodies to hepatitis C virus (HCV). J Hepatol1990;10:69-76. Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, Alter HJ. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990;12:671-675. Esteban JI, Gonzalez A, Hernandez JM, Viladomiu L, Sanchez C, Lopez-Talavera JC, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia J. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990;323:1107-1112. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JAJ, Contreras M, Aloysius S. Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 1990;335:1419-1422. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel LJ, Vyas GN. Detection, semiquantitation, and genetic variation in hepatitis C virus sequence amplified from the plasma of blood do-
694
HAGIWARA ET AL.
nom with elevated alanine aminotransferase. J Clin Invest 1990:86:1609-1614. 8. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, Miyakawa Y, Mayumi M. The 5’-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990;60:167-177. 9. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo QL, Houghton M. Detection of hepatitis C viral sequence in non-A, non-B hepatitis. Lancet 1990; 335:1-3.
GASTROENTEROLOGY Vol. 102, No. 2
10. Kato N, Yokosuka 0, Omata M, Hosoda K, Ohto M. Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J Clin Invest 1990;86:1764-1767.
Received March 29, 1991. Accepted July 17, 1991. Address requests for reprints to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima l-l-59, Fukushima, Osaka 553, Japan.
1992;102:692-694
Detection of Hepatitis C Virus RNA in Chronic Non-A, Non-B Liver Disease HIDEKI HAGIWARA, NOR10 HAYASHI, EIJI MITA, NAOKI KEIJI UEDA, TETSUO TAKEHARA, NOBUKAZU YUKI, AKINORI
KASAHARA,
HIDEYUKI
HIRAMATSU,
FUSAMOTO,
and TAKENOBU KAMADA First Department of Medicine, Osaka University Medical School, Osaka, Japan
Serum samples were tested for detection of hepatitis C virus (HCV) RNA from 156 patients with chronic non-A, non-B liver disease. HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-ClOO-3 but was also found in 15 (55.6%) of 27 patients negative for anti-ClOO-3. The rate of positivity for HCV RNA was not significantly different among various stages of liver diseases. These results showed that HCV continues to replicate even in advanced liver disease and that it seems to be related to half of the cases of chronic non-A, non-B liver disease negative for anti-C169-3. n assay for anti-C100-3 is now available for diagnosis of hepatitis C virus (HCV) infection.lm5 However, in some cases, HCV can be transmitted from plasma negative for anti-C100-3, which indicates a limitation to the precise diagnosis of HCV carriers using anti-ClOO-3. In contrast, detecting HCV RNA in serum can be useful for diagnosing infectious HCV carriers.6*7 To determine the interrelationship between replication of HCV and various stages of chronic non-A, non-B (NANB) liver disease, we tested serum samples for the presence of HCV RNA using a combination assay of reverse transcription and polymerase chain reaction (RT-PCR).
A
Materials and Methods The subjects were 156 patients with chronic NANB liver disease. Hepatitis B virus infection was excluded by commercial serological tests (absence of detectable hepatitis B surface antigen and high-titer anti-hepatitis B core antigen). Liver biopsy was performed on 117 patients, and the histology showed the features of chronic persistent hepatitis (CPH) in 20 patients, chronic active hepatitis (CAH) in 69 patients, and liver cirrhosis (LC) in 28 patients. The remaining 39 patients had a diagnosis of hepatocellular carcinoma (HCC) by diagnostic imaging. Anti-ClOO-3 was assayed by enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Tokyo, Japan). The primer sequences for RT-PCR were derived from the 5’-terminal sequence of a complementary DNA (cDNA) clone of HCV [5’-CATGGTGCACGGTCTACGAG-3’, anti-
sense; 5’-ACTCCACCATAGATCACTCC-3’, sense).’ For extraction of RNA, 100 pL of 21% polyethylene glycol6000 and 1.5 mol/L NaCl were added to 200 pL of serum sample. This was left standing for 2 hours at 4’C. After centrifugation, the pellet was mixed with 600 pL of homogenization buffer, containing 4 mol/L guanidinium thiocyanate, 0.1 mol/L Tris-HCl, pH 7.5, 0.5% sodium lauryl sarcosinate, and 1% j3-mercaptoethanol. After two extractions with phenol chloroform, 10 pg of transfer RNA (tRNA), one-tenth volume of 3 mol/L sodium acetate (pH 6.0), and 2.5 volumes of absolute ethanol were added to the aqueous phase. This mixture was left at -8O’C for 1 hour, and then the RNA was pelleted by centrifugation. The pellet was washed with 70% ethanol, dried, and dissolved in 7 pL of distilled water. RNA solution was made up to 20 FL with reaction solution containing 20 pm01 of anti-sense primer, 1 mmol/L of each deoxynucleotide triphosphate, 20 units of ribonuclease inhibitor (Promega Corp., Madison, WI), and 200 units of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD). The reverse transcriptase reaction was performed according to the manufacturer’s instructions. The cDNA preparation was added to 80 yL of PCR solution containing 20 pmol of sense primer, 1.2 mmol/L of MgCl,, 45.2 mmol/L of KCl, and 2.5 units of Taq polymerase (Perkin Elmer Cetus Corp., Norwalk, CT). The mixtures were amplified by 40 cycles of PCR (94°C for 30 seconds, 55’C for 1 minute, and 72’C for 1 minute), followed by a lo-minute extension at 72°C. A lo-pL sample of PCR products was analyzed on 2% agarose gels, blotted onto a nylon membrane, hybridized to a 32P-end-labeled HCV cDNA (-1.0 X 10~ cpm/pg), which lay between the RT-PCR primers. The membrane was exposed to a preflashed film at -7O’C for 6 hours using an intensifying screen. We could detect a single copy of HCV cDNA in this condition. All samples were assayed at least two times with reproducible results. The x2 test or Fisher’s Exact Test and Student’s t test were used for statistical analysis. Results higher
The mean age of LC patients was significantly than that of CPH or CAH patients: that of HCC
0 1992by
the American Gastroenterological 0016-5065/92/$3.00
Association
HCV
February 1992
RNA IN NANB LIVERDISEASE 693
Table 1. Positive Rate for HCV RNA in Chronic NANB Liver Disease Positive rate for HCVRNA (%) Disease CPH CAH LC HCC Total (%)
(yr, mean + SD] 44.1 47.6 56.0 61.6 51.1
f + + + f
11.0 9.8 8.4’ 6.8b 11.1
Positive for anti-ClOO-3
Negative for anti-ClOO-3 l/2 6/9 3/6 5/10 15/27 (55.6)
17/18 56/60 21/22 27/29 121/129 (93.8)
Total (%) 18/20 62/69 24/28 32/39 136/156
(90.0) (89.9) (85.7) (82.1) (87.2)
“P < 0.01 vs. CPH, CAH, and HCC. bP < 0.01 vs. CPH, CAH, and LC.
patients was significantly higher than that of CPH, CAH, or LC patients (Table 1). Of the 156 patients, 129 (82.7%) were positive for anti-ClOO-3. The rate was 90.0% for CPH,87.0% for CAH, 78.6% for LC, and 74.4% for HCC patients. HCV RNA in serum was detected in 136 patients (87.2%). The rates of positive results for HCV RNA in advanced chronic liver disease including LC (85.7%) and HCC (82.1%) were as high as those in the early stage of chronic liver disease including CPH (90.0%) and CAH (89.9%). The positive HCV RNA rates did not significantly differ among diseases. In comparison with anti-ClOO-3 and HCV RNA, HCV RNA was detected in 121 (93.8%) of 129 patients positive for anti-ClOO-3 and in 15 (55.6%) of 27 patients negative for anti-ClOO-3 (Table 1).
reverse transcriptase reaction step. We could not confirm whether serum negative for HCV RNA contained HCV particles, because this was below the threshold of sensitivity of the RT-PCR assay. Furthermore, Ulrich et ala7 reported that a low concentration of HCV RNA could be detected from 2.4 mL of plasma, but not from 0.6 mL. HCV RNA may be detected if >2OO pL of serum can be analyzed for these eight patients. Twelve patients were both negative for anti-ClOO-3 and HCV RNA in chronic NANB liver disease. These patients might account for hepatitis induced by other unknown viruses, although a low concentration of HCV RNA should be excluded. References 1.
Discussion Recent reports have documented the detection of HCV RNA by RT-PCR assay.6*7.g*‘oUlrich et a1.7 found HCV RNA in 2.4 mL of plasma from 11 (100%) of 11 volunteer blood donors positive for anti-ClOO-3, and Kato et al.” detected it in 1 mL of serum from 13 (81%) of 16 patients positive for antiCIOO-3 and 7 (50%) of 14 patients negative for antiClOO-3 with chronic NANB liver disease. In the present study, HCV RNA was detected in 200 yL of serum from 136 patients (87.2%) with chronic NANB liver disease. Although the mean ages of the patients with HCC and LC were significantly higher than those with chronic hepatitis, there was no significant difference in the positive rates for HCV RNA between the different chronic NANB liver diseases. These results indicated that replication of HCV occurs even in the advanced liver disease. Furthermore, HCV RNA was detected in 15 (55.6%) of 27 patients negative for anti-ClOO-3. This showed that HCV seems to be related to half of the chronic NANB viral liver diseases negative for anti-ClOO-3. Eight patients were negative for HCV RNA but positive for anti-ClOO-3 in the present study. Although the detection limit of this PCR assay is a single copy of cDNA, there is likely to be some loss of generating cDNA fragments in the extraction, precipitation, and
2.
3.
4.
5.
6.
7.
Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M. An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 1989;244:362-364. Alter HJ, Purcell RH, Shih JW, Melpolder JC, Houghton M, Choo QL, Kuo G. Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989:321:14941500. Hopf U, Moller B, Kuther D, Stemerowicz R, Lobeck H, Ludtke-Handjery A, Walter E, Blum HE, Roggendorf M, Deinhardt F. Long-term follow-up of posttransfusion and sporadic chronic hepatitis non-A, non-B and frequency of circulating antibodies to hepatitis C virus (HCV). J Hepatol1990;10:69-76. Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furuta S, Akahane Y, Nishioka K, Purcell RH, Alter HJ. Interrelationship of blood transfusion, non-A, non-B hepatitis and hepatocellular carcinoma: analysis by detection of antibody to hepatitis C virus. Hepatology 1990;12:671-675. Esteban JI, Gonzalez A, Hernandez JM, Viladomiu L, Sanchez C, Lopez-Talavera JC, Lucea D, Martin-Vega C, Vidal X, Esteban R, Guardia J. Evaluation of antibodies to hepatitis C virus in a study of transfusion-associated hepatitis. N Engl J Med 1990;323:1107-1112. Garson JA, Tedder RS, Briggs M, Tuke P, Glazebrook JA, Trute A, Parker D, Barbara JAJ, Contreras M, Aloysius S. Detection of hepatitis C viral sequences in blood donations by “nested” polymerase chain reaction and prediction of infectivity. Lancet 1990;335:1419-1422. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel LJ, Vyas GN. Detection, semiquantitation, and genetic variation in hepatitis C virus sequence amplified from the plasma of blood do-
694
HAGIWARA ET AL.
nom with elevated alanine aminotransferase. J Clin Invest 1990:86:1609-1614. 8. Okamoto H, Okada S, Sugiyama Y, Yotsumoto S, Tanaka T, Yoshizawa H, Tsuda F, Miyakawa Y, Mayumi M. The 5’-terminal sequence of the hepatitis C virus genome. Jpn J Exp Med 1990;60:167-177. 9. Weiner AJ, Kuo G, Bradley DW, Bonino F, Saracco G, Lee C, Rosenblatt J, Choo QL, Houghton M. Detection of hepatitis C viral sequence in non-A, non-B hepatitis. Lancet 1990; 335:1-3.
GASTROENTEROLOGY Vol. 102, No. 2
10. Kato N, Yokosuka 0, Omata M, Hosoda K, Ohto M. Detection of hepatitis C virus ribonucleic acid in the serum by amplification with polymerase chain reaction. J Clin Invest 1990;86:1764-1767.
Received March 29, 1991. Accepted July 17, 1991. Address requests for reprints to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima l-l-59, Fukushima, Osaka 553, Japan.
