448
JID 1992; 166 (August)
Correspondence
cases, the therapeutic options may be drug substitution or combination. Laurent Cotte, Martine Langlois, and Christian Trepo Hepatology and AIDS Unit. Hiipita! de I'Hotel-Dieu; Virology Laboratory. Universite Claude Bernard; and INSERM U 271. Lyon. France References I. Safrin S. Crumpacker C. Chatis P, et al. A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous
Serum Tumor Necrosis Factor in Acute and Fulminant Hepatitis B Colleagues-An increased concentration of tumor necrosis factor (TNF) serum levels has been demonstrated in infectious diseases, particularly in parasitic diseases such as malaria and leishmaniasis [I]. Some evidence exists of an enhanced TNF release during viral infections [2]. Sheron et a1. [3] recently demonstrated an increased plasma TNF concentration in patients with chronic hepatitis and seropositive for hepatitis B surface (HBsAg) and e antigens. We studied serum TNF levels in 30 patients with acute hepatitis Band 8 with fulminant hepatitis B; 15 healthy adults and 10 patients with acute non-A, non-B (NANB) hepatitis were tested as controls. Intravenous drug users were equally present among patients and controls (6/38 patients, 3/25 controls). A sandwich EIA (Biokine TNF test kit; T Cell Science, Cambridge, MA) was used. Two blood samples were obtained from each patient and each control with NANB hepatitis. The first sample coincided with the alanine aminotransferase (ALT) peak for each group (17 ± 2.4 days from the onset of symptoms). The second sample was collected during convalescence (95.4 ± 5 days from the onset of symptoms). Four patients died within 3 weeks of the onset of fulminant hepatitis. A blood sample had been collected from all 4 on the day of death. Data were analyzed using Student's t test and linear regression analysis. TNF was detectable in 26 of 30 patients with acute hepatitis B and in all 8 with fulminant hepatitis B. TNF values did not differ significantly between drug users and the others (18.3 ± 16.5 vs. 16.6 ± 7.3 pg/mL). Serum TNF was undetectable in 32 of 34 samples taken during convalescence. In the other 2 patients, TNF levels decreased from 20 and 16 pg/mL to 10 pg/rnl. (each); both of these subjects were drug users who continued to inject heroin intravenously during convalescence. TNF values were in correlation
herpes simplex in the acquired immunodeficiency syndrome. N Engl J Med 1991;325:551-5. 2. Langlois M, Allard JP, Nugier F. Aymard M. A rapid and automated colorimetric assay for evaluating the sensitivity of herpes simplex strains to antiviral drugs. J Bioi Stand 1986; 14:20 I-II. 3. Sacks SL. Rennie BA. Clinical resistance to acyclovir in herpes simplex virus type 2 infection in AIDS: false in vitro susceptibility associated with viral heterogeneity (abstract 1223). In: Programs and abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago). Washington, DC: American Society for Microbiology. 1991.
with ALT levels (P < .05). Only one of 25 controls had detectable serum TNF. TNF concentration in serum was significantly higher during fulminant hepatitis than during acute uncomplicated hepatitis B (24.5 ± 7.11 vs. 12.9 ± 10.70 pg/ml.; P < .01). On the other hand, TNF concentration did not differ significantly between patients who recovered and patients who died (16.8 ± 3.4 vs. 21.5 ± 6.12 pg/ml.). Although the immune-mediated process responsible for hepatocellular lysis induces contemporary TNF production, it can be hypothesized that TNF itself has a direct lytic action on infected liver cells, thus contributing to the elimination of hepatitis B virus. The evidence that only I of 10 patients with acute NANB hepatitis showed detectable serum TNF coinciding with the ALT peak confirms that our findings were not merely due to liver inflammation and necrosis. Two patients who became chronic HBsAg carriers had undetectable serum TNF during both acute and convalescent phases. This finding seems in accord with results of other studies, which relate the chronicity of HBV infection to a defect of cytokine production or to an impairment of monocyte function [4]. Excess TNF production is associated with fulminant viral hepatitis. Recently Zhang [5] observed an increase ofTNF levels in patients with virus-induced liver failure. Although mechanisms responsible for fulminant hepatitis are still not clear, a pathogenic role for TNF seems possible. Furthermore, some clinical features observed during fulminant hepatitis, such as fever, hypotension, hypoglycemia, clotting abnormalities, metabolic acidosis, and weight loss, might also be influenced by TNF. We failed to demonstrate a prognostic role of TNF in the evolution of fulminant hepatitis. However, it should be emphasized that in fulminant hepatitis other important factors may contribute to the clinical outcome, such as the regenerative capacity of liver cells or the duration of viral replication [6]. B. Cacopardo, F. Fatuzzo, R. Russo, B. M. Celesia, R. La Rosa, G. Lupo, S. Cosentino, and A. Nunnari Institute of Infectious Diseases, University ofCatania. Ital v
Reprints or correspondence: Dr. Bruno Cacopardo, Institute oflnfectious Diseases, Via Passo Gravina 187, 95125 Catania, Italy.
References
The Journal of Infectious Diseases 1992;166:448-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-0037$01.00
I. Scuderi P. Sterling KE, Lam KS, et al. Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 1986;2: 1364-5.
JID 1992; 166 (August)
449
Correspondence
2. Paya C, Kenmotsu N, Schoon RA, Leibson PJSO. Tumour necrosis factor and Iymphotoxin secretion by human natural killer celIs leads to antiviral cytotoxicity. J ImmunoI1988;141:1989-95. 3. Sheron N, Lou J, Daniels H, et at. Increased production of tumour necrosis factor alpha in chronic hepatitis B virus infection. J Hepatol 1991;12:241-5. 4. Nouri-Atia KT, Magrin S, Alexander GJM, Anderson MG, Williams R, Eddleston ALWF. Abnormal T cell activation in chronic hepatitis B
viral infection: a consequence of monocyte dysfunction. Immunology 1988;64:733-8. 5. Zhang D. Tumour necrotic factor in the pathogenesis ofliver necrosis in viral hepatitis and strategy for its prevention and treatment. Chung Hua I Hsueh Tsa Chih 1990;70:438-4 I. 6. Brahm J, Fagan EA, Budkowska A. et at. Prognostic significance of preS2antigen and antibody in fulminant hepatitis B: evidence for heterogeneous serological responses. J Hepatol 1991;13:49-55.
Indeterminate Second-Generation Hepatitis C Recombinant Immunoblot Test: Detection of Hepatitis C Virus Infection by Polymerase Chain Reaction
Table 1. ; Hepatitis C virus (HCV) RNA in sera with indeterminate secondgeneration recombinant immunoblot assay (RIBA-2) results according to immunodeficiency status.
Colleagues-The second-generation recombinant immunoblot assay (RIBA-2; Chiron, Emeryville, CA) apparently has resolved most difficulties in the serologic diagnosis of hepatitis C virus infection (HCV) [1). However, too many indeterminate results still remain. Detection of viral RNA using polymerase chain reaction (PCR) can then represent a useful way to discriminate between infected and uninfected patients. From January to July 1991,3942 sera were screened for HCV by a second-generation ELISA (Ortho Diagnostics, Raritan, NJ) and, when necessary, by RIBA-2. Thirty-three sera gave indeterminate results on RIBA-2 according to the manufacturer's instructions, reacting with only one of the four recombinant antigens. The same sera were then tested by HCV PCR. We used a nested PCR described by Garson and colleagues [2,3). The set of primers was from the fifth nonstructural gene (NS5) region and consisted of an outer primer pair (d94, d95) and an inner primer pair (Nl, N2). Among 15 human immunodeficiency virus (HIV)-positive sera, 8 were PCR-positive, whereas of 18 HIV-negative sera, 17 were PCR-positive. Moreover, this latter group included 4 transplant recipients with indeterminate RIBA-2 results who were all PCRpositive (table 1). This indicates that immunodeficient patients may not produce sufficient antibodies to the peptide of the nonstructural part of the viral genome [4]. In accordance with Garson et al. [2] and Van der Poe1et al. [5], we found no association between high alanine aminotransferase (ALT) levels and positive PCR results, as only 3 of our 33 patients had elevated ALT levels and 1 of them was PCR -positive. These data suggest that patients reactive for only one antigen by RIBA-2 may still be infectious. The manufacturer's criteria of indeterminate RIBA results are obviously inadequate, and detection of viremia by PCR remains definitive.
Reprints or correspondence: Dr. Philippe Halfon, Centre Hospitalier Regional, Laboratoire de Biochimie, 147 Blvd. Bailie. 13385 Marseille Cedex 5, France. The Journal of Infectious Diseases 1992;166:449 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-0038$01.00
RIBA-2 recombinant antigen reacted to, HIV status (n) C22 Positive (13)* Negative (14)t C33 Positive (2}1: Negative (I)' C100-3 Negative (3)Total HIV positive (15) Total HIV negative (18)
HCV RNA on PCR Positive
Negative
6 13
7 I
2 I
0 0
3 8 17
0 7 I
NOTE. PCR, polymerase chain reaction; HIV, human immunodeficiency virus. * Centers for disease control (CDC) stages II and III (6 subjects), CDC stage IV (7). t Liver or renal transplantation (4), hemophilia A (2), hepatocellular carcinoma (3), intravenous drug users (5). t CDC stage II. I Drepanocytemia. - Mixed cryoglobulinemia type II (2). asymptomatic hospital employee with needlestick ( I ).
P. Halfon, S. Rousseau, C. Tamalet, M. Antoni, V. Gerolami, M. Levy, M. Bourliere, R. Planells, and G. Cartouzou Laboratoire de Biochimie and Service de Gastro-Enterologie, Hiipital de la Conception. Marseille. France References
I. Leon A. Canton R. Elia M. Mateos M. Second-generation RIBA to confirm diagnosis ofHCV infection. Lancet 1991;337:912. 2. Garson JA. Tedder RS. Briggs M. et at. 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;336:878-9. 4. Charnot E. Hirschel B, Wintsch J. Francois-Rebert C. Loss ofantibodies against hepatitis C virus in HIV-seropositive intravenous drug users. AIDS 1990;4: 1275-7. 5. Van der Poel CL, Cuypers HTM. Reesink HW. et al. Confirmation of hepatitis C virus infection by four antigen recombinant immunoblot assay. Lancet 1991 ;337:317-9.
JID 1992; 166 (August)
Correspondence
cases, the therapeutic options may be drug substitution or combination. Laurent Cotte, Martine Langlois, and Christian Trepo Hepatology and AIDS Unit. Hiipita! de I'Hotel-Dieu; Virology Laboratory. Universite Claude Bernard; and INSERM U 271. Lyon. France References I. Safrin S. Crumpacker C. Chatis P, et al. A controlled trial comparing foscarnet with vidarabine for acyclovir-resistant mucocutaneous
Serum Tumor Necrosis Factor in Acute and Fulminant Hepatitis B Colleagues-An increased concentration of tumor necrosis factor (TNF) serum levels has been demonstrated in infectious diseases, particularly in parasitic diseases such as malaria and leishmaniasis [I]. Some evidence exists of an enhanced TNF release during viral infections [2]. Sheron et a1. [3] recently demonstrated an increased plasma TNF concentration in patients with chronic hepatitis and seropositive for hepatitis B surface (HBsAg) and e antigens. We studied serum TNF levels in 30 patients with acute hepatitis Band 8 with fulminant hepatitis B; 15 healthy adults and 10 patients with acute non-A, non-B (NANB) hepatitis were tested as controls. Intravenous drug users were equally present among patients and controls (6/38 patients, 3/25 controls). A sandwich EIA (Biokine TNF test kit; T Cell Science, Cambridge, MA) was used. Two blood samples were obtained from each patient and each control with NANB hepatitis. The first sample coincided with the alanine aminotransferase (ALT) peak for each group (17 ± 2.4 days from the onset of symptoms). The second sample was collected during convalescence (95.4 ± 5 days from the onset of symptoms). Four patients died within 3 weeks of the onset of fulminant hepatitis. A blood sample had been collected from all 4 on the day of death. Data were analyzed using Student's t test and linear regression analysis. TNF was detectable in 26 of 30 patients with acute hepatitis B and in all 8 with fulminant hepatitis B. TNF values did not differ significantly between drug users and the others (18.3 ± 16.5 vs. 16.6 ± 7.3 pg/mL). Serum TNF was undetectable in 32 of 34 samples taken during convalescence. In the other 2 patients, TNF levels decreased from 20 and 16 pg/mL to 10 pg/rnl. (each); both of these subjects were drug users who continued to inject heroin intravenously during convalescence. TNF values were in correlation
herpes simplex in the acquired immunodeficiency syndrome. N Engl J Med 1991;325:551-5. 2. Langlois M, Allard JP, Nugier F. Aymard M. A rapid and automated colorimetric assay for evaluating the sensitivity of herpes simplex strains to antiviral drugs. J Bioi Stand 1986; 14:20 I-II. 3. Sacks SL. Rennie BA. Clinical resistance to acyclovir in herpes simplex virus type 2 infection in AIDS: false in vitro susceptibility associated with viral heterogeneity (abstract 1223). In: Programs and abstracts of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy (Chicago). Washington, DC: American Society for Microbiology. 1991.
with ALT levels (P < .05). Only one of 25 controls had detectable serum TNF. TNF concentration in serum was significantly higher during fulminant hepatitis than during acute uncomplicated hepatitis B (24.5 ± 7.11 vs. 12.9 ± 10.70 pg/ml.; P < .01). On the other hand, TNF concentration did not differ significantly between patients who recovered and patients who died (16.8 ± 3.4 vs. 21.5 ± 6.12 pg/ml.). Although the immune-mediated process responsible for hepatocellular lysis induces contemporary TNF production, it can be hypothesized that TNF itself has a direct lytic action on infected liver cells, thus contributing to the elimination of hepatitis B virus. The evidence that only I of 10 patients with acute NANB hepatitis showed detectable serum TNF coinciding with the ALT peak confirms that our findings were not merely due to liver inflammation and necrosis. Two patients who became chronic HBsAg carriers had undetectable serum TNF during both acute and convalescent phases. This finding seems in accord with results of other studies, which relate the chronicity of HBV infection to a defect of cytokine production or to an impairment of monocyte function [4]. Excess TNF production is associated with fulminant viral hepatitis. Recently Zhang [5] observed an increase ofTNF levels in patients with virus-induced liver failure. Although mechanisms responsible for fulminant hepatitis are still not clear, a pathogenic role for TNF seems possible. Furthermore, some clinical features observed during fulminant hepatitis, such as fever, hypotension, hypoglycemia, clotting abnormalities, metabolic acidosis, and weight loss, might also be influenced by TNF. We failed to demonstrate a prognostic role of TNF in the evolution of fulminant hepatitis. However, it should be emphasized that in fulminant hepatitis other important factors may contribute to the clinical outcome, such as the regenerative capacity of liver cells or the duration of viral replication [6]. B. Cacopardo, F. Fatuzzo, R. Russo, B. M. Celesia, R. La Rosa, G. Lupo, S. Cosentino, and A. Nunnari Institute of Infectious Diseases, University ofCatania. Ital v
Reprints or correspondence: Dr. Bruno Cacopardo, Institute oflnfectious Diseases, Via Passo Gravina 187, 95125 Catania, Italy.
References
The Journal of Infectious Diseases 1992;166:448-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-0037$01.00
I. Scuderi P. Sterling KE, Lam KS, et al. Raised serum levels of tumour necrosis factor in parasitic infections. Lancet 1986;2: 1364-5.
JID 1992; 166 (August)
449
Correspondence
2. Paya C, Kenmotsu N, Schoon RA, Leibson PJSO. Tumour necrosis factor and Iymphotoxin secretion by human natural killer celIs leads to antiviral cytotoxicity. J ImmunoI1988;141:1989-95. 3. Sheron N, Lou J, Daniels H, et at. Increased production of tumour necrosis factor alpha in chronic hepatitis B virus infection. J Hepatol 1991;12:241-5. 4. Nouri-Atia KT, Magrin S, Alexander GJM, Anderson MG, Williams R, Eddleston ALWF. Abnormal T cell activation in chronic hepatitis B
viral infection: a consequence of monocyte dysfunction. Immunology 1988;64:733-8. 5. Zhang D. Tumour necrotic factor in the pathogenesis ofliver necrosis in viral hepatitis and strategy for its prevention and treatment. Chung Hua I Hsueh Tsa Chih 1990;70:438-4 I. 6. Brahm J, Fagan EA, Budkowska A. et at. Prognostic significance of preS2antigen and antibody in fulminant hepatitis B: evidence for heterogeneous serological responses. J Hepatol 1991;13:49-55.
Indeterminate Second-Generation Hepatitis C Recombinant Immunoblot Test: Detection of Hepatitis C Virus Infection by Polymerase Chain Reaction
Table 1. ; Hepatitis C virus (HCV) RNA in sera with indeterminate secondgeneration recombinant immunoblot assay (RIBA-2) results according to immunodeficiency status.
Colleagues-The second-generation recombinant immunoblot assay (RIBA-2; Chiron, Emeryville, CA) apparently has resolved most difficulties in the serologic diagnosis of hepatitis C virus infection (HCV) [1). However, too many indeterminate results still remain. Detection of viral RNA using polymerase chain reaction (PCR) can then represent a useful way to discriminate between infected and uninfected patients. From January to July 1991,3942 sera were screened for HCV by a second-generation ELISA (Ortho Diagnostics, Raritan, NJ) and, when necessary, by RIBA-2. Thirty-three sera gave indeterminate results on RIBA-2 according to the manufacturer's instructions, reacting with only one of the four recombinant antigens. The same sera were then tested by HCV PCR. We used a nested PCR described by Garson and colleagues [2,3). The set of primers was from the fifth nonstructural gene (NS5) region and consisted of an outer primer pair (d94, d95) and an inner primer pair (Nl, N2). Among 15 human immunodeficiency virus (HIV)-positive sera, 8 were PCR-positive, whereas of 18 HIV-negative sera, 17 were PCR-positive. Moreover, this latter group included 4 transplant recipients with indeterminate RIBA-2 results who were all PCRpositive (table 1). This indicates that immunodeficient patients may not produce sufficient antibodies to the peptide of the nonstructural part of the viral genome [4]. In accordance with Garson et al. [2] and Van der Poe1et al. [5], we found no association between high alanine aminotransferase (ALT) levels and positive PCR results, as only 3 of our 33 patients had elevated ALT levels and 1 of them was PCR -positive. These data suggest that patients reactive for only one antigen by RIBA-2 may still be infectious. The manufacturer's criteria of indeterminate RIBA results are obviously inadequate, and detection of viremia by PCR remains definitive.
Reprints or correspondence: Dr. Philippe Halfon, Centre Hospitalier Regional, Laboratoire de Biochimie, 147 Blvd. Bailie. 13385 Marseille Cedex 5, France. The Journal of Infectious Diseases 1992;166:449 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6602-0038$01.00
RIBA-2 recombinant antigen reacted to, HIV status (n) C22 Positive (13)* Negative (14)t C33 Positive (2}1: Negative (I)' C100-3 Negative (3)Total HIV positive (15) Total HIV negative (18)
HCV RNA on PCR Positive
Negative
6 13
7 I
2 I
0 0
3 8 17
0 7 I
NOTE. PCR, polymerase chain reaction; HIV, human immunodeficiency virus. * Centers for disease control (CDC) stages II and III (6 subjects), CDC stage IV (7). t Liver or renal transplantation (4), hemophilia A (2), hepatocellular carcinoma (3), intravenous drug users (5). t CDC stage II. I Drepanocytemia. - Mixed cryoglobulinemia type II (2). asymptomatic hospital employee with needlestick ( I ).
P. Halfon, S. Rousseau, C. Tamalet, M. Antoni, V. Gerolami, M. Levy, M. Bourliere, R. Planells, and G. Cartouzou Laboratoire de Biochimie and Service de Gastro-Enterologie, Hiipital de la Conception. Marseille. France References
I. Leon A. Canton R. Elia M. Mateos M. Second-generation RIBA to confirm diagnosis ofHCV infection. Lancet 1991;337:912. 2. Garson JA. Tedder RS. Briggs M. et at. 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;336:878-9. 4. Charnot E. Hirschel B, Wintsch J. Francois-Rebert C. Loss ofantibodies against hepatitis C virus in HIV-seropositive intravenous drug users. AIDS 1990;4: 1275-7. 5. Van der Poel CL, Cuypers HTM. Reesink HW. et al. Confirmation of hepatitis C virus infection by four antigen recombinant immunoblot assay. Lancet 1991 ;337:317-9.
