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Detection of anti-hepatitis C virus antibodies in patients undergoing dialysis by utilizing a hepatitis C virus 3.0 assay: Correlation with hepatitis C virus RNA
Detection of anti-hepatitis C virus antibodies in patients undergoing dialysis by utilizing a hepatitis C virus 3,0 assay: Correlation with hepatitis C virus RNA M. DE MEDINA, M. HILL, H. O. SULLIVAN, B. LECLERQ, J. P. PENNELL, L. JEFFERS, K. R. REDDY, E. R. SCHIFF, and G. O. PEREZ MIAMI, FLORIDA
Hepatitis C virus (HCV) infection is endemic in long-term dialysis units. We assessed the performance of a recently developed HCV 3.0 assay for the detection of HCV antibodies in patients undergoing dialysis. The study evaluated 128 patients undergoing long-term maintenance hemodialysis. Anti-HCV was detected by 2.0 and 3.0 enzyme immunoassay (EIA). Results were confirmed with recombinant immunoblot assays (RIBATM 2.0 and RIBATM 3.0). HCV RNA was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR).Thirty-two patients (25%) were HCV EIA 2.0 positive. Of these, I was RIBATM 2.0 negative (PCR positive), 3 were indeterminate (3 PCR positive), and 28 were positive (23 PCR positive). Thirtyfive (27%) were HCV EIA 3.0 positive. One was RIBATM 3.0 negative (PCR positive), I was indeterminate (c33c, PCR positive), and 33 were positive (27 PCR positive) by RIBATM 3.0. Thus only I PCR-positive patient was negative with RIBATM 2.0 and 3.0 assays. Two of the 3 RIBATM 2.0 indeterminate samples were positive with RIBATM 3.0. One remained indeterminate but was HCV RNA positive. In summary, HCV 3.0 EiA detected 4 additional viremic patients but was positive in 6 PCR-negative subjects. A high correlation of the presence of antibody to c33c with HCV RNA (28 of 34, 82%) was found, and it was found in all anti-HCV positive samples and in I indeterminate sample. We conclude that the HCV EIA 3.0 test with the supplemental confirmatory RIBATM 3.0 test may improve the sensitivity for the detection of antiHCV. Nevertheless, in potentially immunocompromised patients undergoing dialysis, PCR continues to be the only reliable test for detecting viremia. (J Lab Clin Med 1998;132:73-5)
Abbreviations: EIA : enzyme immunoassay; HCV = hepatitis C virus; RIBATM= recombinant immunoblot assay; RT-PCR : reverse transcriptase-polymerase chain reaction; SIA = strip immunoblot assay
From the Division of Hepatology and Nephrology, University of Miami School of Medicine, and the Veterans Administration Medical Center. Submitted for publication Dec. 9, 1997; revision submitted March 7, 1998; accepted March 11, 1998. Reprint requests: Guido O. Perez, MD, Chief, Dialysis Unit (11 I-C), Veterans Administration Medical Center, Room a-1005, 1201 N.W. 16th St., Miami, FL 33125. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5lllQ0337
H
epatitis C virus infection is endemic in longterm dialysis units. Detection o f a n t i - H C V with E I A was substantially i m p r o v e d by the introduction of second-generation assays. 1 A secondgeneration R I B A T M (RIBA T M 2.0) is routinely utilized to confirm the E I A results.2, 3 Nevertheless, many R I B A T M 2.0 results are indeterminate, 4,5 and both tests underestimate the prevalence of viremia.6, 7 The present study was d e s i g n e d to assess the p e r f o r m a n c e o f a
73
74
J Lab Clin Med July 1998
de Medina et al.
T a b l e I. Results of HCV a n t i b o d y test a c c o r d i n g to RT-PCR
HCV RNA (+) HCV RNA (-) Totals
EIA2.0 (+)
(+)
RIBATM 2.0 IND
27 5 32
23 5 28
3 0 3
(-)
1 0 1
EIA3.0 (+)
(+)
RIBATM 3.0 IND*
29
27
1
1
6
6
0
0
35
33
1
1
(-)
IND, Indeterminate.
T a b l e II. HCV a n t i g e n p a t t e r n distribution for RIBATM
SIA 3.0 RIBATM SIA 3.0 pattern
cl 00(p)+c33c+c22(p)+NS5 cl00(p) + c33c+c22(p) cl00(p) + c33c c33c+c22(p)+NS5 c33c+c22(p) c33c
Number
HCV RNA PCR(+) Number(%)
14 8 7 3 1 1
12(86) 6(75) 5(71 ) 3(100) 1(100) 1(100)
recently developed HCV 3.0 EIA assay. Positive results were confirmed by RIBA T M 3.0, and testing for HCV RNA was performed by RT-PCR in all patients. The results suggest that 3.0 tests improve the detection of HCV antibodies in patients undergoing dialysis. METHODS Patients. One hundred and twenty-eight patients undergoing maintenance hemodialysis for at least 3 months at the University of Miami Affiliated Dialysis Unit were consecutively screened. All serum samples were coded, aliquoted, and stored at -70 ° C until analysis. Routine hemodialysis techniques were used, with 3 to 5 hours of treatment performed three times a week with synthetic membranes and bicarbonate solutions of standard composition. Artificial kidneys were reused an average of five times. Universal blood precautions were instituted, and anti-HCV positive patients were not isolated.
Serologic testing. Anti-HCV was detected by EIA (HCV 2.0 and HCV 3.0; Abbott Laboratories, North Chicago, Ill.). HCV 3.0 is designed to detect antibodies to four recombinant HCV proteins: HC-34, HCr43, c100-3 and NS5. Positive results were confirmed by a four-antigen SIA (RIBATM HCV 2.0; Chiton, Emeryville, Calif.) that uses four recombinant HCV-encoded antigens (5-1-1, c100-3, c33c, and c22-3) and by HCV 3.0 SIA with two recombinant antigens (c33c and NS5) and two synthetic peptides (c22p and cl00p). PCR procedure. HCV RNA was detected directly in human serum by using RT-PCR (AMPLICORTM HCV Test; Roche Diagnostic Systems Inc., Branchburg, N.J) followed by a hybridization reaction of the amplified product to a specific oligonucleotide probe and detection of the probe-bound
amplified product by color formation. This test uses the primers KY78 and KY80 to define a 244-nucleotide sequence within the highly conserved 5"-untranslated region. The sensitivity of the test is 1000 copies/ml. RESULTS
Included in the study were 75 men and 53 women ranging in age from 21 to 76 years. Sixty-seven of the patients were black. Duration of maintenance hemodialysis was between 3 months and 9 years. Fortyseven patients had a history of blood transfusion. Twelve patients gave a history of intravenous drug abuse and 13 had a diagnosis of HIV infection. Thirty-two patients (25%) were HCV EIA 2.0 positive (Table I). Of these, one was RIBA T M 2.0 negative (PCR positive), three were indeterminate (3 PCR positive), and 28 positive (23 PCR positive). Thirty-five (27%) were HCV EIA 3.0 positive. One was RIBA T M 3.0 negative (PCR positive), 1 was indeterminate by c33c (PCR positive), and 33 were positive (27 PCR positive) by RIBAT M 3.0. Only 1 PCR positive patient was negative with RIBAT M 2.0 and 3.0 assays (Table I). Two of the 3 RIBAT M 2.0 indeterminate samples were positive with RIBAT M 3.0. One remained indeterminate but was HCV RNA positive. The results of the RT-PCR in the different reactive patterns of the four common antigen components of RIBA T M 3.0 are shown in Table II. HCV RNA was detected in 86% of patients reactive with the four antigens. The HCV antigen band c33c exhibited the strongest correlation with HCV RNA (82%), followed by c100(p)(68%), c22(p) (65%), and NS5 (44%). DISCUSSION
HCV infection is endemic in long-term dialysis units. Detection of HCV antibodies in patients undergoing dialysis was substantially improved with the introduction of HCV 2.0. 8 In the present study the introduction of HCV EIA 3.0 and RIBA TM 3.0 further increased the sensitivity of the HCV assay by detecting as positive 3 additional patients and resolving 2 of 3 indeterminate results by RIBAT M 2.0. HCV RNA was detected in 2 of the 3 additional patients detected by HCV EIA 3.0. Both EIA tests are not specific, however, because
J Lab Clin Med Volume 132, Number 1
antibodies were found in 6 patients with undetectable HCV RNA, which m a y represent past infection or intermittent viremia. Only one viremic patient was negative b y both H C V R I B A TM 2.0 and H C V 3.0, suggesting that the patient acquired the infection without the ability to mount a detectable antibody response. The R I B A TM S I A 3.0 results demonstrated that c33c is the most sensitive antigen for the accurate diagnosis o f viremia. C33c exhibited the strongest correlation with H C V RNA, followed by cl00(p), c22(p), and NS5. Previous studies have d e m o n s t r a t e d the increased sensitivity of H C V 3.0 in the detection of H C V antib o d i e s in patients with post-transfusion hepatitis and normal renal function. 9 A comparison o f H C V 2.0 and H C V 3.0 in the evaluation o f H C V antibodies in patients undergoing dialysis has been recently reported from Europe and Saudi Arabia. 10-12 In these studies, H C V 3.0 also was shown to increase the sensitivity of H C V antibody detection when c o m p a r e d with H C V 2.0. In conclusion, the H C V E I A 3.0 with the supplemental confirmatory R I B A TM 3.0 tests increased the detection of H C V antibodies in our patients undergoing longterm dialysis. Nevertheless, RT-PCR remains the only reliable test to establish the presence of viremia in this patient population. REFERENCES
1. Alter HJ. New kit on the block: evaluation of second-generation assays for detection of antibody to hepatitis C virus. Hepatology 1992;15:350-3. 2. Vanderpeol CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, Dinello R, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991;337:317-9.
de Medina et al.
75
3. Evans CS, Tobler L, Polito A, Stewart J, Chien D, Wilber J, et al. Comparative evaluation of supplemental hepatitis C virus antibody test systems. Transfusion 1991;32:408-14. 4. Nordenfelt E, Lofgren B, Widell A, Hansson BG, Zhang YY, Hagstam KG, et al. Hepatitis C virus infection in hemodialysis patients in Southern Sweden: epidemiological, clinical and diagnostic aspects. J Med Virol 1993;40:266-70. 5. Pawlotsky JM, Fleury A, Deforges L, Choukroun V, Aumont P, Duval J, et al. Highly positive c22-3 "indeterminate" second generation RIBA: resolution by third generation RIBATM and HCV RNA PCR [abstract]. Hepatology 1993;18:86A. 6. Pereira BJG, Milford EL, Kirkman RL, Quan S, Sayre KR, Johnson PJ, et al. Prevalence of hepatitis C virus RNA in organ donor positive for hepatitis C antibody and in the recipients on their organs. N Engl J Med 1992;327:910-5. 7. Lau JYN, Davis GL, Brunson ME, Qian KP, Lin HJ, Quan HJ, et al. Hepatitis C virus infection in kidney transplant recipients. Hepatology 1993;18:1027-31. 8. de Medina M, Ortiz C, Krenc C, Leete J, Vallafi D, Hill M, et al. Improved detection of antibodies to hepatitis C virus in dialysis patients using a second-generation enzyme immunoassay. Am J Kidney Dis 1992;20:589-91. 9. Barrera JM, Francis B, Ercilla G, Nelles M, Achord D, Darner J, et al. Improved detection of anti-HCV in post transfusion hepatitis by a third-generation ELISA. Vox Sang 1995;68:15-8. 10. Courouce AM, Bouchardeau F, Chauveau P, Le Marrec N, Girault A, Zins B, et al. Hepatitis C virus (HCV) infection in haemodialysed patients: HCV-RNA and anti-HCV antibodies (third generation assay). Nephrol Dial Transplant 1995;10:234-9. 11. Denfico P, Volpe A, Buongioruo R, Manno C, Carabellese S, Monno L, et al. HCV third generation test in hemodialysis patients. Ital J Gastroenterol 1995;27:300-2. 12. A1 Meshari K, A1 Ahdal M, Alfurarh O, Ali A, DeVol E, Kessie G. New insights into hepatitis C virus infection of hemodialysis patients: the implications. Am J Kidney Dis 1995;25:572-8.
Hepatitis C virus (HCV) infection is endemic in long-term dialysis units. We assessed the performance of a recently developed HCV 3.0 assay for the detection of HCV antibodies in patients undergoing dialysis. The study evaluated 128 patients undergoing long-term maintenance hemodialysis. Anti-HCV was detected by 2.0 and 3.0 enzyme immunoassay (EIA). Results were confirmed with recombinant immunoblot assays (RIBATM 2.0 and RIBATM 3.0). HCV RNA was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR).Thirty-two patients (25%) were HCV EIA 2.0 positive. Of these, I was RIBATM 2.0 negative (PCR positive), 3 were indeterminate (3 PCR positive), and 28 were positive (23 PCR positive). Thirtyfive (27%) were HCV EIA 3.0 positive. One was RIBATM 3.0 negative (PCR positive), I was indeterminate (c33c, PCR positive), and 33 were positive (27 PCR positive) by RIBATM 3.0. Thus only I PCR-positive patient was negative with RIBATM 2.0 and 3.0 assays. Two of the 3 RIBATM 2.0 indeterminate samples were positive with RIBATM 3.0. One remained indeterminate but was HCV RNA positive. In summary, HCV 3.0 EiA detected 4 additional viremic patients but was positive in 6 PCR-negative subjects. A high correlation of the presence of antibody to c33c with HCV RNA (28 of 34, 82%) was found, and it was found in all anti-HCV positive samples and in I indeterminate sample. We conclude that the HCV EIA 3.0 test with the supplemental confirmatory RIBATM 3.0 test may improve the sensitivity for the detection of antiHCV. Nevertheless, in potentially immunocompromised patients undergoing dialysis, PCR continues to be the only reliable test for detecting viremia. (J Lab Clin Med 1998;132:73-5)
Abbreviations: EIA : enzyme immunoassay; HCV = hepatitis C virus; RIBATM= recombinant immunoblot assay; RT-PCR : reverse transcriptase-polymerase chain reaction; SIA = strip immunoblot assay
From the Division of Hepatology and Nephrology, University of Miami School of Medicine, and the Veterans Administration Medical Center. Submitted for publication Dec. 9, 1997; revision submitted March 7, 1998; accepted March 11, 1998. Reprint requests: Guido O. Perez, MD, Chief, Dialysis Unit (11 I-C), Veterans Administration Medical Center, Room a-1005, 1201 N.W. 16th St., Miami, FL 33125. Copyright © 1998 by Mosby, Inc. 0022-2143/98 $5.00 + 0 5lllQ0337
H
epatitis C virus infection is endemic in longterm dialysis units. Detection o f a n t i - H C V with E I A was substantially i m p r o v e d by the introduction of second-generation assays. 1 A secondgeneration R I B A T M (RIBA T M 2.0) is routinely utilized to confirm the E I A results.2, 3 Nevertheless, many R I B A T M 2.0 results are indeterminate, 4,5 and both tests underestimate the prevalence of viremia.6, 7 The present study was d e s i g n e d to assess the p e r f o r m a n c e o f a
73
74
J Lab Clin Med July 1998
de Medina et al.
T a b l e I. Results of HCV a n t i b o d y test a c c o r d i n g to RT-PCR
HCV RNA (+) HCV RNA (-) Totals
EIA2.0 (+)
(+)
RIBATM 2.0 IND
27 5 32
23 5 28
3 0 3
(-)
1 0 1
EIA3.0 (+)
(+)
RIBATM 3.0 IND*
29
27
1
1
6
6
0
0
35
33
1
1
(-)
IND, Indeterminate.
T a b l e II. HCV a n t i g e n p a t t e r n distribution for RIBATM
SIA 3.0 RIBATM SIA 3.0 pattern
cl 00(p)+c33c+c22(p)+NS5 cl00(p) + c33c+c22(p) cl00(p) + c33c c33c+c22(p)+NS5 c33c+c22(p) c33c
Number
HCV RNA PCR(+) Number(%)
14 8 7 3 1 1
12(86) 6(75) 5(71 ) 3(100) 1(100) 1(100)
recently developed HCV 3.0 EIA assay. Positive results were confirmed by RIBA T M 3.0, and testing for HCV RNA was performed by RT-PCR in all patients. The results suggest that 3.0 tests improve the detection of HCV antibodies in patients undergoing dialysis. METHODS Patients. One hundred and twenty-eight patients undergoing maintenance hemodialysis for at least 3 months at the University of Miami Affiliated Dialysis Unit were consecutively screened. All serum samples were coded, aliquoted, and stored at -70 ° C until analysis. Routine hemodialysis techniques were used, with 3 to 5 hours of treatment performed three times a week with synthetic membranes and bicarbonate solutions of standard composition. Artificial kidneys were reused an average of five times. Universal blood precautions were instituted, and anti-HCV positive patients were not isolated.
Serologic testing. Anti-HCV was detected by EIA (HCV 2.0 and HCV 3.0; Abbott Laboratories, North Chicago, Ill.). HCV 3.0 is designed to detect antibodies to four recombinant HCV proteins: HC-34, HCr43, c100-3 and NS5. Positive results were confirmed by a four-antigen SIA (RIBATM HCV 2.0; Chiton, Emeryville, Calif.) that uses four recombinant HCV-encoded antigens (5-1-1, c100-3, c33c, and c22-3) and by HCV 3.0 SIA with two recombinant antigens (c33c and NS5) and two synthetic peptides (c22p and cl00p). PCR procedure. HCV RNA was detected directly in human serum by using RT-PCR (AMPLICORTM HCV Test; Roche Diagnostic Systems Inc., Branchburg, N.J) followed by a hybridization reaction of the amplified product to a specific oligonucleotide probe and detection of the probe-bound
amplified product by color formation. This test uses the primers KY78 and KY80 to define a 244-nucleotide sequence within the highly conserved 5"-untranslated region. The sensitivity of the test is 1000 copies/ml. RESULTS
Included in the study were 75 men and 53 women ranging in age from 21 to 76 years. Sixty-seven of the patients were black. Duration of maintenance hemodialysis was between 3 months and 9 years. Fortyseven patients had a history of blood transfusion. Twelve patients gave a history of intravenous drug abuse and 13 had a diagnosis of HIV infection. Thirty-two patients (25%) were HCV EIA 2.0 positive (Table I). Of these, one was RIBA T M 2.0 negative (PCR positive), three were indeterminate (3 PCR positive), and 28 positive (23 PCR positive). Thirty-five (27%) were HCV EIA 3.0 positive. One was RIBA T M 3.0 negative (PCR positive), 1 was indeterminate by c33c (PCR positive), and 33 were positive (27 PCR positive) by RIBAT M 3.0. Only 1 PCR positive patient was negative with RIBAT M 2.0 and 3.0 assays (Table I). Two of the 3 RIBAT M 2.0 indeterminate samples were positive with RIBAT M 3.0. One remained indeterminate but was HCV RNA positive. The results of the RT-PCR in the different reactive patterns of the four common antigen components of RIBA T M 3.0 are shown in Table II. HCV RNA was detected in 86% of patients reactive with the four antigens. The HCV antigen band c33c exhibited the strongest correlation with HCV RNA (82%), followed by c100(p)(68%), c22(p) (65%), and NS5 (44%). DISCUSSION
HCV infection is endemic in long-term dialysis units. Detection of HCV antibodies in patients undergoing dialysis was substantially improved with the introduction of HCV 2.0. 8 In the present study the introduction of HCV EIA 3.0 and RIBA TM 3.0 further increased the sensitivity of the HCV assay by detecting as positive 3 additional patients and resolving 2 of 3 indeterminate results by RIBAT M 2.0. HCV RNA was detected in 2 of the 3 additional patients detected by HCV EIA 3.0. Both EIA tests are not specific, however, because
J Lab Clin Med Volume 132, Number 1
antibodies were found in 6 patients with undetectable HCV RNA, which m a y represent past infection or intermittent viremia. Only one viremic patient was negative b y both H C V R I B A TM 2.0 and H C V 3.0, suggesting that the patient acquired the infection without the ability to mount a detectable antibody response. The R I B A TM S I A 3.0 results demonstrated that c33c is the most sensitive antigen for the accurate diagnosis o f viremia. C33c exhibited the strongest correlation with H C V RNA, followed by cl00(p), c22(p), and NS5. Previous studies have d e m o n s t r a t e d the increased sensitivity of H C V 3.0 in the detection of H C V antib o d i e s in patients with post-transfusion hepatitis and normal renal function. 9 A comparison o f H C V 2.0 and H C V 3.0 in the evaluation o f H C V antibodies in patients undergoing dialysis has been recently reported from Europe and Saudi Arabia. 10-12 In these studies, H C V 3.0 also was shown to increase the sensitivity of H C V antibody detection when c o m p a r e d with H C V 2.0. In conclusion, the H C V E I A 3.0 with the supplemental confirmatory R I B A TM 3.0 tests increased the detection of H C V antibodies in our patients undergoing longterm dialysis. Nevertheless, RT-PCR remains the only reliable test to establish the presence of viremia in this patient population. REFERENCES
1. Alter HJ. New kit on the block: evaluation of second-generation assays for detection of antibody to hepatitis C virus. Hepatology 1992;15:350-3. 2. Vanderpeol CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, Dinello R, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lancet 1991;337:317-9.
de Medina et al.
75
3. Evans CS, Tobler L, Polito A, Stewart J, Chien D, Wilber J, et al. Comparative evaluation of supplemental hepatitis C virus antibody test systems. Transfusion 1991;32:408-14. 4. Nordenfelt E, Lofgren B, Widell A, Hansson BG, Zhang YY, Hagstam KG, et al. Hepatitis C virus infection in hemodialysis patients in Southern Sweden: epidemiological, clinical and diagnostic aspects. J Med Virol 1993;40:266-70. 5. Pawlotsky JM, Fleury A, Deforges L, Choukroun V, Aumont P, Duval J, et al. Highly positive c22-3 "indeterminate" second generation RIBA: resolution by third generation RIBATM and HCV RNA PCR [abstract]. Hepatology 1993;18:86A. 6. Pereira BJG, Milford EL, Kirkman RL, Quan S, Sayre KR, Johnson PJ, et al. Prevalence of hepatitis C virus RNA in organ donor positive for hepatitis C antibody and in the recipients on their organs. N Engl J Med 1992;327:910-5. 7. Lau JYN, Davis GL, Brunson ME, Qian KP, Lin HJ, Quan HJ, et al. Hepatitis C virus infection in kidney transplant recipients. Hepatology 1993;18:1027-31. 8. de Medina M, Ortiz C, Krenc C, Leete J, Vallafi D, Hill M, et al. Improved detection of antibodies to hepatitis C virus in dialysis patients using a second-generation enzyme immunoassay. Am J Kidney Dis 1992;20:589-91. 9. Barrera JM, Francis B, Ercilla G, Nelles M, Achord D, Darner J, et al. Improved detection of anti-HCV in post transfusion hepatitis by a third-generation ELISA. Vox Sang 1995;68:15-8. 10. Courouce AM, Bouchardeau F, Chauveau P, Le Marrec N, Girault A, Zins B, et al. Hepatitis C virus (HCV) infection in haemodialysed patients: HCV-RNA and anti-HCV antibodies (third generation assay). Nephrol Dial Transplant 1995;10:234-9. 11. Denfico P, Volpe A, Buongioruo R, Manno C, Carabellese S, Monno L, et al. HCV third generation test in hemodialysis patients. Ital J Gastroenterol 1995;27:300-2. 12. A1 Meshari K, A1 Ahdal M, Alfurarh O, Ali A, DeVol E, Kessie G. New insights into hepatitis C virus infection of hemodialysis patients: the implications. Am J Kidney Dis 1995;25:572-8.