Improved Detection of Hepatitis C Virus Antibodies in High-risk Populations JOHN G. MCHUTCHISON,~ JOHN L. PERSON,^ SUGANTHA GOVINDARAJAN,~BOONTAR VALINLUCK, h A N POLIT0,3 DAVIDCHIEN,3 ROBERTDINELL0,3 TESSIE GORE,' STEVEN R. LEE,^ MITCHELLNELLES,~ STELLA Q U A N ,GEORGE ~ Kuo3 AND ALLANG. REDEKER~ 'Division of Gastrointestinal and Liver Diseases, University of Southern California School of Medicine, Los Angeles, California 90033; 'Ortho Diagnostics, Inc., Raritan, New Jersey 08869; and 'Chiron Corporation, Emeryville, California 98607
Sera from 483 patients at high (group 1, n = 313) and lower (group 2, n = 170) risk for exposure to hepatitis C were tested for antibodies to hepatitis C using first-generation (~100-3) and second-generation enzyme-linked immunosorbent assays and fourantigen recombinant immunoblot assay. The secondgeneration enzyme-linked immunosorbent assay and nitrocellulose-based immunoblot assay differ from c100-3-based systems in the addition of expression products from the NS3/NS4 (c33c, c200) and putative nucleocapsid (c22-3)region of the hepatitis C genome. In group 1, the sensitivity of detection of hepatitis C antibodies was 45%, 55% and 46% by the first- and second-generation enzyme-linked immunosorbent assays and recombinant immunoblot assay, respectively. In group 2, antibodies were detected by each test system in 26%, 32% and 7% of patients, respectively. Most sera (99%) reactive with the first-generation enzyme-linked immunosorbent assay were reactive with the second-generation enzyme-linked immunosorbent assay (in group 1, 89% of these specimens demonstrated reactivity to at least one antigen with the immunoblot assay, compared with only 31% in group 2). An additional 12% (group 1)and 6% (group2) of specimens demonstrated reactivity with the secondgeneration enzyme-linked immunosorbent assay only (ofthese, 75%[group 11 and 9% [group 21 demonstrated reactivity to at least one antigen with the immunoblot assay). Ninety-eight percent of specimens not reactive with both enzyme-linked immunosorbent assay test systems were also nonreactive by recombinant immunoblot assay. Antibodies to c22-3 and c33c were more frequently detected in chronic hepatitis C virus in-
Received May 9, 1991; accepted August 19, 1991. This work was supported in part by a grant provided by Ortho Diagnostic Systems, Inc., Raritan, New Jersey. Dr. Person is a naval physician currently working with the Division of Gastrointestinal and Liver Diseases at the University of Southern California. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of Defense or the US. Government. Address reprint requests to: John G. McHutchison, M.D., Scripps Clinic and Research Foundation, Division of Gastroenterology, 10666 North Torrey Pines Road, La Jolla, CA 92037. 31/1/33412
fection and appeared earlier in acute hepatitis C virus infection, compared with 5-1-1 and c100-3. These results indicate that the addition of structural and nonstructural hepatitis C virus antigens in the secondgeneration enzyme-linked immunosorbent assay improves the sensitivity of detection of hepatitis C antibodies. (HEPATOLOGY 1992;15:19-25.)
The recent isolation and cloning of the genome of hepatitis C virus (HCV) has resulted in numerous reports of the prevalence, sensitivity and significance of detectable antibodies to this virus in non-A, non-B (NANB) hepatitis and other liver diseases. Worldwide, 60% to 80% of patients with chronic NANB hepatitis appear to have detectable antibodies to the recombinant HCV protein c100-3 (1-8).A high prevalence of antibody to HCV (anti-HCV)has generally been reported in other groups of individuals at risk for exposure to this virus (7, 9-11). The role of HCV in other liver diseases has not been so clearly established (12-17). Varying prevalence rates and false-positive results have been reported in some of these groups (18-21). Some of the remaining 20% to 30% of patients with NANB hepatitis without detectable c100-3antibodies do appear to have antibodies to other structural and nonstructural HCV proteins (22, 23). Whether another agent is responsible for some of these presumed infections or how many have an undetectable antibody response to these recombinant viral proteins is yet unknown. These problems in HCV infection highlight the need for improved screening test systems and a specific confirmatory test. In this report, we evaluated a second-generation ELISA and a recombinant immunoblot assay that detect antibodies to both structural and nonstructural HCV proteins in patients at high and lower risk for HCV infection. PATIENTS AND METHODS Group I : Patients at High Risk for HCV Exposure. Specimens from 56 patients with chronic NANB hepatitis were studied. All patients had at least two samples with elevated ALT values (at least two times normal, at least 6 mo apart) with no other apparent explanation for these abnormal liver 19
20
McHUTCHISON ET AL.
HEPATOLOGY
test results. Also, 106 serum samples from 49 patients with 0.600 + negative control for the first- and second-generation acute NANB hepatitis were included. Criteria for this diag- ELISAs, respectively). Sera were also tested using the newly developed fournosis included a clinical illness compatible with acute viral hepatitis, a peak ALT value at least 10 times the normal and antigen recombinant immunoblot assay (RIBA) (Chiron RIBA negative results of tests for HBsAg, HBc IgM antibody and HCV Test System; Chiron Corp., Emeryville, CAI. This antibody to hepatitis A virus IgM. Multiple samples (at least nitrocellulose stripbased assay is capable of detecting antitwo) were assayed in 37 of these 49 patients from the period of bodies to the recombinant HCV antigens 5-1-1, c100-3, c33c 6 to 25 wk after the peak ALT value. The earliest specimen and c22-3. During incubation of each strip with serum or tested was always from within 4 wk of the peak ALT value. We controls, HCV antibodies will react to the corresponding specifically included only patients with no identifiable risk nitrocellulose strip. After removal of nonspecific antibodies, factors for their presumed acute or chronic infection (i.e., all each strip is reacted with conjugated goat antihuman IgGpatients denied illicit intravenous drug use, prior blood horseradish peroxidase. Colored band patterns develop after transfusions or other percutaneous modes of transmission, as the addition of a solution containing hydrogen peroxide and well as multiple sexual contacts). Patients were excluded if any 4-chloro-1-naphthol. The intensity of the color is proportional other cause of hepatocellular injury could be identified (al- to the amount of specific antibody bound (graded as 1+ to 4 + 1. cohol, medications, hepatotoxins or other systemic diseases). Results are reported as reactive (reactivity with at least two These patients were selected from a hepatitis clinic at our antigen bands), indeterminate (reactivity with a single antigen band) or nonreactive (no reactive bands). tertiary referral center. Statistical Analysis. Student's t test and Fisher's exact test Specimens from 112 intravenous drug abusers attending a walk-in health-care clinic in the Los Angeles metropolitan area were used to compare the differences among groups and the (100 men, 12 women) were studied. Ten patients were known relative frequencies within groups. A p value 1:80) andlor ELISA only. Twenty-six were reactive when tested with antinuclear antibody with high serum globulin and high ALT RIBA, including all 22 sera reactive with both ELISA level before treatment. All patients showed a dramatic re- systems and the four patients reactive with the secondsponse to immunosuppressive treatment. Pre- and post- generation ELISA alone. A single specimen was reactive treatment sera were available for comparison in 13 cases. only with the first-generation ELISA (and was unreAnti-HCV Testing. Serum was stored at - 20" C until each active with RIBA). assay was performed. Retrospectively and prospectively HCC. Twenty-two specimens (48%) had anti-HCV gathered specimens were used in this study. Specimens were tested for anti-HCV using the first-generation (~100-3) detected by both ELISA systems, 8 demonstrated reacand second-generation (c200/c22-3) Ortho HCV ELISA test tivity with the second-generation ELISA only and 1 systems (Ortho Diagnostics, Inc., Raritan, NJ). The second- specimen was reactive with the first-generation ELISA generation assay differs from the available c100-3 based assay only. Reactivity with RIBA was demonstrated in 24 in that it detects antibodies to proteins derived from three patients, including all 21 that were reactive with both distinct regions of the HCV genome. The c200 recombinant ELISA systems. The three remaining sera reactive with antigen is an expression product of the putative NS3/NS4 RIBA were reactive with the second-generation ELISA region of the HCV genome and is derived from that region of only (another specimen reactive with the secondthe genome encoding both the c33c and c100-3 recombinant generation ELISA was indeterminately reactive with antigens. The c22-3 recombinant antigen is an expression product of the putative nucleocapsid region of the HCV RIBA). Intravenous Drug Users. Anti-HCV was detected by genome. All initially reactive specimens were retested in duplicate. For either assay, a reactive result was recorded if at the first- and second-generation ELISAs in 74 (66%) least two of the three optical-density values were equal to or patients. Another eight patients' sera were reactive with greater than the cutoff value (0.400 + negative control and the second-generation ELISA only. Of the 70 specimens
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
21
TABLE1. Prevalence of anti-HCV by three test systems ELISA
Diagnosis
Group 1 (high riskIb Acute HCV Chronic HCV HCC IV drug use Dialysis Group 2 (lower risky Hepatitis A or B PBC Al CAH
First-generation
Second-generation
RIBAa
49 56 46 112 50
20 23 23 74 2
29 26 30 a2 6
20 26 24 70 4
107 32 31
24 11 10
25 14 14
9 2 1
IV = intravenous; A1 CAH = autoimmune CAH. “RIBA reactivity implies at least two reactive antigen bands. Specimens classified as indeterminate (a single reactive antigen band) are not shown. b313 patients; 370 specimens. “170 patients; 184 specimens.
reactive with RIBA, 65 were reactive with both ELISA systems and 5 were reactive with the second-generation ELISA only. Two other patients with a reactive secondgeneration ELISA only were indeterminately reactive with RIBA. Hemodialysis. Two specimens (4%) were reactive with both ELISA systems and also with RIBA. An additional four specimens were reactive with only the second-generation ELISA (two were RIBA reactive and two were indeterminate). The remaining 44 sera were negative by all three tests. Viral Hepatitis A and B. Twenty-three patients were reactive with both ELISA formats, and two others were reactive with the second-generation ELISA only. Of these 25 patients, 9 were reactive, two were indeterminate and 12 were unreactive with RIBA. The specimens reactive with the second-generation ELISA only were unreactive with RIBA. Eighty-one patients were negative with both ELISA formats; 77 of these were RIBA unreactive (four were indeterminate). PBC. Eighteen (56%) of 32 patients tested were negative by all three tests. Eleven patients were reactive with both ELISA test systems, but only 2 of these 11 were RIBA reactive. Another three cases were reactive with the second-generation ELISA only (two were RIBA unreactive; one was indeterminate). Autoimmune CAH. Sera from 13patients were tested before and after successful treatment with prednisone. All were RIBA unreactive. Six patients had reactive firstand second-generation ELISA results before treatment; these later became negative. Three other patients had reactive second-generation ELISA results before treatment that remained reactive in one patient and disappeared in two patients after treatment. The remaining four patients were negative by all three assays, irrespective of treatment. In 18 additional patients with sera tested only after treatment, 12 were negative by all three assays, 4 were reactive by the second-generation ELISA (but unreactive with RIBA) and 2 were reactive with both ELISA systems (one was RIBA reactive and
one was unreactive). Thus only 1 of 31 patients evaluated was reactive with all three assays. Interrelationship Between the Three Test Systems. As shown in Figure 1, more sera were reactive with either or both ELISA systems in group 1patients at high risk for exposure to HCV. In these patients, another 12% of individuals were only reactive with the secondgeneration ELISA. In groups 1 and 2, most sera unreactive with both ELISA formats were also unreactive when tested with RIBA. Most specimens reactive with the first-generation ELISA were also reactive with RIBA in group 1 patients, but significantly fewer were confirmed as reactive with RIBA in group 2 patients (p < 0,0001, group 1 vs. 2). Note that in group 1 patients, the second-generation ELISA detected all remaining individuals who demonstrated reactivity with RIBA and most specimens that had been classified as indeterminate. Irrespective of the risk of exposure to HCV, most specimens classified as indeterminate RIBA were reactive with one or both ELISA systems. Figure 2 illustrates the results of RIBAin sera reactive by either or both the first- and second-generation ELISA. In group 1, 78% of ELISA-reactive sera were confirmed as reactive with RIBA, another 8% had a single reactive antigen band. The differences in the prevalence of RIBA-reactive results in these ELISAreactive sera in the different high-risk groups were not significant. In comparison, group 2 patients reactive with ELISA were much less likely to demonstrate reactivity with RIBA. Fifty-five specimens were found to be discordant when tested with the ELISA systems in that they were only reactive with the second-generation ELISA (Table 2). Most of these were patients at high risk for HCV exposure; of them, 50% were reactive with RIBA and another 25%were classified as indeterminate. In those cases displaying some reactivity with RIBA, the reactivity was directed toward the c33c and c22-3 antigen bands, the two HCV proteins present in the secondgeneration ELISA but not in the first-generation
22
McHUTCHISON ET AL.
HEPATOLOGY
GROUP 1 (HIGH RISK) 151 GEN
ELISA (c-100-3)
No. 01
ELSA REACTIVIN
2nd GEN ELISA
RlEA RESULT
SPECIMENS
POS
lsland 2nd GEN.
155
2nd GEN. ONLY
44
111 GEN. ONLY
NEGATIVE
Reactivity
IN0
NEG
135
5
15
22
11
11
2
0
0
2
169
0
1
168
GROUP 2 (LOWER RISK) 1st GEN ELISA (c-100-3)
No. of
ELSA REACTlVlW
+
2nd GEN. ELSA
-
Reactivity
RlEA RESULTS
SPECIMENS
POS
IN0
NEG
1sl and 2nd GEN
44
12
2
30
2nd GEN. ONLY
11
0
1
10
1st GEN. ONLY
1
0
0
1
NEGATIVE
128
0
4
124
Reactivily
FIG.1. Comparison between the three test systems in patients at high risk (group 1) and lower risk (group 2) for exposure to HCV. Boxed numbers represent the relationship between the first- and second-generation ELISAs. RIBA results are shown as positive or reactive (POS), indeterminate (IND) and negative or unreactive (NEG). More sera were reactive with the second-generation than the first-generation ELISA (254 vs. 202; p < 0.005).
1
croup 1
I
7Croup 2 1
100%
v)
[I
i
._
80%
V W
a v)
W
.-2
60%
V
0
? I Q
40%
J
w
r
0
20%
? a 0% Acute NANB Chronic NANB IV drug use
RIBA non--rmctive RIBA indeterminate
Dialysis (n=6)
PBC (n=14)
Hap A & 8 (n=26)
(n=16)
19%
0%
79x
57x
94%
3%
33%
77.
8%
78%
67%
14Z
35%
OX 6%
(n=55)
(n=Z7)
__
(n=82)
Liver Cancer (n=31)
25% 15% 60%
4%
9%
0% 96%
6% 85%
1
RIBA rmctive
RIBA indelerminale
0RIBA non-raoctive
A1 CAH
I
FIG.2. Results of the four-antigen recombinant immunoblot assay in specimens reactive by one or both ELISA test systems (n = 257). Data are shown as the percentage in each subgroup that was reactive, indeterminate or unreactive with the four-antigen RIBA. More group 1 sera were reactive with RIBA than were group 2 sera (78% vs. 12%;p < 0.0001).
ELISA. In comparison, most group 2 patients who were discordant with ELISA testing demonstrated no reactivity with RIBA. In both groups, none of the specimens with an optical density value of less than 2.0 in the second-generation ELISA were reactive or indeterminate when tested with RIBA. Three other patients were discordant in that they were reactive with only the first-generation ELISA (one had chronic NANB hepatitis; one had HCC and one had viral hepatitis B). All three were unreactive with RIBA.
Patterns of anti-HCV Reactivity. One hundred sixty-nine specimens were reactive when tested with RIBA (at least two reactive antigen bands), and 24 were interpreted as indeterminate (one reactive band). In the 124 group 1patients with reactive immunoblots whom we presume had chronic HCV infection (not including the acute NANB hepatitis group), 100% had detectable antibodies to c33c, 94% had detectable antibodies to c22-3, 69%had detectable antibodies to 5-1-1and 66% had detectable antibodies to the c100-3 HCV protein
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
(Fig. 3). Twelve group 2 patients were also reactive with RIBA, and the prevalence of antibodies detected to these four recombinant HCV antigens was similar to that in group 1 cases (c33c, 100%; c22-3, 92%; 5-1-1, 67%; c100-3, 67%). Most (53%) of the 136 patients with reactive immunoblots had antibodies detected to all four recombinant HCV antigens. There was no consistent pattern of antibody observed in cases with three detectable antibodies; when antibody was detected to only two of these HCV proteins, they were usually c33c and ~22-3. In evaluating the first specimen that displayed a reactive antigen band with RIBA in acute NANB hepatitis, development of antibodies to c33c and c22-3 preceded the development of antibodies to 5-1-1 and c100-3 in 11 of the 22 cases that displayed reactive or indeterminate RIBA results. In the other 11patients, a variable pattern of antibodies to these structural and nonstructural regions of the HCV genome was observed first. In only one case were antibodies to 5-1-1 and c100-3 detected first. There was no pattern evident in assessing the first detectable antibody in acute HCV infection (i.e., c22-3, c33c or both). Although most patients in this group who were serially followed appeared to have constant or increasing amounts of detectable antibody to each HCV antigen, antibody reactivity occasionally decreased or disappeared with time. Twenty-four patients had a single reactive antigen band on RIBA; they were classified as indeterminate. As shown in Table 3, most of these patients were at high risk for HCV infection and displayed reactivity to either the c33c or c22-3 recombinant antigen. On subsequent testing of the eight patients with acute NANB hepatitis indeterminate on RIBA, reactivity developed in four (50%)to two or more HCV antigens. DISCUSSION
The prevalence of anti-HCV detected by the firstgeneration ELISA in our subgroups of patients at high and lower risk for HCV infection is comparable to that in previous reports. With the newer test systems, we were able to detect anti-HCV in approximately 50% of patients with chronic sporadic NANB hepatitis -a lower prevalence than those seen in other reports (1,2, 6,241. Whether some of these individuals lack an immune response detectable by these assays, are infected with another NANB agent or have some other viral or nonviral cause for the observed liver injury is unknown. Despite the fact that we excluded patients with known percutaneous risk factors for HCV infection, we found a high percentage of patients with other types of liver diseases that were reactive with ELISA only and that presumably represent false-positive results. In particular, in autoimmune CAH, most sera reactive with ELISA were unreactive when tested with RIBA. Reactivity in these cases disappeared after successful immunosuppressive therapy. The results of this study demonstrate a high rate of concordance between the first- and second-generation
23
100%
80% U z D ._
6
L
60%
1
u
OI
1 L
.Z
40%
L
(D C
2 a
20%
0%
(n=26)
(n=24)
_
~
_
_
Dialysis (n=4)
75:
c 100-2
7551
5-1-1 ~22-3
,
92%
93%
100%
100%
c100-3
5-1-1
1005 100% _____
0c22-3
100%
c33c
1
FIG. 3. Pattern of reactivity to each of the recombinant HCV antigens in group 1 patients. Bars = percentage of patients in each group with each antibody detected. Only patients displaying reactivity to a t least two HCV antigens are included.
ELISAs and improved sensitivity of detection of HCV antibodies using the second-generation ELISA. This newly developed ELISA detected another 11.9% of patients at high risk for HCV infection who remained undetected by the first-generation ELISA. Seventy-five percent of these cases displayed reactivity to at least one HCV recombinant antigen when also tested with the newly developed suppIementaI RIBA. Perhaps one of the most important observations of this study is that all specimens that demonstrated reactivity by RIBA were detected by the second-generation ELISA, whereas 13% of these specimens were unreactive with the first-generation ELISA. These results indicate that the addition of structural (c22-3) and nonstructural (~200) HCV antigens does appear to increase the sensitivity of detection of antibodies compared with currently available c100-3-based systems. Although the second-generation ELISA appears to be more sensitive in the high-risk setting, there does appear to be a reduction in specificity (particularly in patients at lower risk for hepatitis C exposure). In this setting we presume that most ELISA-reactivespecimens that were not confirmed with the supplemental RIBA represent false-positive results. In the lower-risk groups we studied, it is possible that interfering substances (e.g., globulin, immune complexes or prolonged storage of
24
McHUTCHISON ET AL.
HEPATOLOGY
TABLE 2. Specimens reactive with second-generationELISA only ELISA optical density Diagnosis
No. -
Acute HCV Chronic HCV IV drug use HCC Dialysis Hepatitis A or B PBC A1 CAH
20 4 8 8 4 2 3 6
First-generation
Second-generation
RIBA reactivity
~~
0.252 t 0.112 0.196 t 0.131 0.266 ? 0.138 0.298 ? 0.076 0.139 t 0.168 0.356 2 0.002 0.360 t 0.053 0.266 2 0.106
2.085 2 0.664 > 2.500 2.272 -+ 0.644 1.453 ? 0.889 > 2.500 1.186 2 0.638 1.412 i 0.988 1.326 ? 0.718
IV = intravenous; AI CAH = autoimmune CAH. "Optical density value = 490 nm; values expressed as mean ? S.D. bNumber in parentheses is the number of patients with this pattern of reactivity; underlined numbers represent the number of patients in whom second-generation ELISA optical density was a t least 2.0.
TABLE3. Pattern of antibody reactivity in RIBA-indeterminate sera Diagnosis
Acute NANB hepatitis Chronic NANB hepatitis IV drug users HCC Dialysis PBC A1 CAH Viral hepatitis A or B TOTAL
No.
8 0 6 1 2 1 0 6 24
Reactive single antigen band
~ 3 (4)", 3 ~ 2 2 - 3(4)
-
~ 3 (3); 3 ~~2 2 - 3(2); 5-1-1 (1) c33c (1) ~ 2 2 - 3(2) ~22-3 ~ 3 (5); 3 ~~1 0 0 - (1) 3 ~ 3 (13); 3 ~~ 2 2 - 3(9); ~ 1 0 0 - 3 (1);5-1-1 (1)
IV = intravenous; A1 CAH = autoimmune CAH. "Number in parentheses indicates the number of specimens displaying this pattern of reactivity.
sera) produced the reactivity in the ELISA, as has been previously described (18-21). We presume that these cases do not have actual HCV infection; as in other low-risk groups (25-271, such cases do not have detectable HCV RNA sequences. Whether a reduction in specificity of the second-generation ELISA that we have observed in patients at lower risk for HCV exposure will also apply to the blood donor population is unknown. In the United States, a large multicenter trial in volunteer blood donors has recently been completed to address this important question. We found that an optical density value of at least 2.0 accurately predicted all specimens that were reactive with the second-generation ELISA and demonstrated reactivity to at least one antigen band in the RIBA. Serum reactive only with the secondgeneration ELISA (but unreactive with RIBA) always had an optical density ratio of less than 2.0. Until supplemental or confirmatory tests become widely available, an arbitrary cutoff value of at least 2.0 with this second-generation ELISA may aid in interpreting the significance of results in individuals at a lower risk for HCV exposure and infection. Whether patients at high risk for HCV infection with a reactive second-
generation ELISA (yet no reactivity by RIBA) represent actual infection with this virus cannot be determined from the results of this study. Further studies are in progress to determine whether such cases have detectable HCV RNA. The patterns of antibody reactivity using the four recombinant HCV antigens incorporated in RIBA has been reported in three patients with posttransfusion hepatitis (22). Marcellin et al. (23) noted a 20% increase in detection of HCV antibodies compared with the c100-3-based ELISA. In our high-risk groups with presumed chronic HCV infection, antibodies to the structural (c22-3) and nonstructural (c33c) regions of the HCV genome were more frequently detected than were those to 5-1-1 and c100-3. Most individuals at high risk for HCV exposure did have detectable antibodies t o c33c and c22-3, whereas only two thirds of this group had detectable antibodies to 5-1-1and c100-3. We could not identify any particular patterns of antibody that corresponded with the subgroup of patients tested nor to the presumed source of exposure. Although most patients exposed to HCV had all four antibodies detected, the pattern of antibody reactivity detected in each case was variable. In acute NANB hepatitis, antibodies to c33c and c22-3 appeared before those directed toward 5-1-1and c100-3 in more than half the patients studied. As in patients with chronic HCV infection, the pattern of antibody in acute infection was also variable. No attempt can be made to determine the time of antibody appearance after the clinical episode of actue viral hepatitis from this study because our patients were not followed at regular predefined intervals for a known period of time. A significant number of individuals at high risk for HCV infection demonstrated reactivity to a single antigen band with RIBA. In these cases, the antibody detected was most often directed toward the c33c or c22-3 recombinant HCV antigens. Although the manufacturer's interpretation of such a result is "indeterminate," these patients had high optical density values when tested with ELISA. Furthermore, a number of these indeterminate specimens were from patients with
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
25
philiacs with normal transaminase levels. Ann Intern Med 1990;112:379-380. 11. Colombo M, Rumi MG, Mannucci PM. Specificity of hepatitis C antibody ELISA in patients with haemophilia. Lancet 1990;335: 1345-1346. 12. Sanchez-Tapias JM, Barrera JM, Costa J , Ercilla MG, Pares A, Comalrrena L, Soley F, et al. Hepatitis C virus infection in patients with nonalcoholic chronic liver disease. Ann Intern Med 1990; 112:921-924. 13. Kew MC, Houghton M, Choo QL, Kuo G. Hepatitis C virus antibodies in southern African blacks with hepatocellular carcinoma. Lancet 1990;335:873-874. 14. Bruix J, Barrera JM, Calvet X, Ercilla G, Costa J, Sanchez-Tapia JM, Ventura M, et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma and hepatic cirrhosis. Lancet 1989;2:1004-1006. 15. Kiyosawa K, Sodeyama T, Tanaka E, Gibo Y, Yoshizawa K, Nakano Y, Furata S, et al. 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Esteban JI, Gonzalez A, Hernandez JM, Viladamou L, Sanchez C, Lopez-Talavera JC, Lucea D, et al. Evaluation of antibodies to 22. Van der Poel CL, Cuypers HTM, Reesink HW, Weiner AJ, Quan S, DiNello R, van Boven JJP, et al. Confirmation of hepatitis C hepatitis C virus in a study of transfusion-associated hepatitis. virus infection by new four-antigen recombinant blot assay. N Engl J Med 1990;323:1107-1112. Lancet 1991;337:317-319. 5. Van der Poel CL, Reesink HW, Lelie PN, Leentvaar-Kuypers A, Choo QL, Kuo G, Houghton M. Anti-hepatitis C antibodies and 23. Marcellin P, Martinot-Peignoux M, Boyer N, Pouteau M, Aumont P, Erlinger S, Benhamou JP. Second generation (RIBA) test in non-A, non-B post-transfusion hepatitis in the Netherlands. diagnosis of chronic hepatitis C. Lancet 1991;337:551-552. Lancet 1989;2:297-298. 6. Alter MJ, Hadler S, Judson FN, Mares A, Alexander WJ, Hu PY, 24. Hopf U, Moller B, Kuther D, Stemerowicz R, Lobeck H, LudtkeHandjery A, Walter E, et al. Long-term follow-up of posttransMiller JK, et al. Risk factors for acute non-A, non-B hepatitis in the fusion and sporadic chronic hepatitis non-A, non-B and frequency United States and association with hepatitis C virus infection. of circulating antibodies to hepatitis C virus (HCV). J Hepatol JAMA 1990;264:2231-2235. 1990;10:69-76. 7. Esteban JI, Esteban R, Viladamou L, Lopez-Talavera JC, Gonzalez A, Hernandez JM, Roget M, et al. Hepatitis C virus 25. Weiner AJ, Truett MA, Rosenblatt J , Han J , Quan S, Polito AJ, Kuo G, et al. HCV testing in low-risk population. Lancet antibodies among risk groups in Spain. Lancet 1989;2:2941990;336:695. 296. 8. Tremolada F, Casarin C, Tagger A, Ribero ML, Realdi G, Alberti 26. Ulrich PP, Romeo JM, Lane PK, Kelly I, Daniel U, Vyas GN. Detection, semiquantitation, and genetic variation in hepatitis C A, Ruol A. Antibody to hepatitis C virus in post-transfusion virus sequences amplified from the plasma of blood donors with hepatitis. Ann Intern Med 1991;114:277-281. elevated alanine aminotransferase. J Clin Invest 1990;86:16099. Makris M, Preston FE, Triger DR, Underwood JCE, Choo QL, Kuo 1614. G, Houghton M. Hepatitis C antibody and chronic liver disease in 27. Kato N, Yokosuka 0, Omato M, Hosoda K, Ohto M. Detection of haemODhiha. Lancet 1990:335:1117-1119. hepatitis C virus ribonucleic acid in the serum by amplification 10. Rumi MG, Colombo M, Gringeri A, Mannucci PM. High prevawith polymerase chain reaction. J Clin Invest 1990;86:1764-1767. lence of antibody to hepatitis C virus in multitransfused hemo-
acute NANB hepatitis; on subsequent testing they had developed reactivity to two or more HCV antigens. We believe these patients do have actual HCV infection, because most have been subsequently documented to have HCV RNA sequences detected by the polymerase chain reaction (McHutchisonJ, et al., Unpublished data, 1991). In conclusion, the addition of other structural and nonstructural antigens in these newly developed assays to detect anti-HCV will allow increased sensitivity of detection of infected individuals. The final place of this second-generation ELISA and the supplemental or confirmatory four-antigen recombinant immunoblot assay in screening blood donors and caring for patients with liver disease is yet to be firmly established. Further studies comparing these tests with the detection of HCV sequences by the polymerase chain reaction should allow us to more meaningfully interpret these newer serological assays.
Sera from 483 patients at high (group 1, n = 313) and lower (group 2, n = 170) risk for exposure to hepatitis C were tested for antibodies to hepatitis C using first-generation (~100-3) and second-generation enzyme-linked immunosorbent assays and fourantigen recombinant immunoblot assay. The secondgeneration enzyme-linked immunosorbent assay and nitrocellulose-based immunoblot assay differ from c100-3-based systems in the addition of expression products from the NS3/NS4 (c33c, c200) and putative nucleocapsid (c22-3)region of the hepatitis C genome. In group 1, the sensitivity of detection of hepatitis C antibodies was 45%, 55% and 46% by the first- and second-generation enzyme-linked immunosorbent assays and recombinant immunoblot assay, respectively. In group 2, antibodies were detected by each test system in 26%, 32% and 7% of patients, respectively. Most sera (99%) reactive with the first-generation enzyme-linked immunosorbent assay were reactive with the second-generation enzyme-linked immunosorbent assay (in group 1, 89% of these specimens demonstrated reactivity to at least one antigen with the immunoblot assay, compared with only 31% in group 2). An additional 12% (group 1)and 6% (group2) of specimens demonstrated reactivity with the secondgeneration enzyme-linked immunosorbent assay only (ofthese, 75%[group 11 and 9% [group 21 demonstrated reactivity to at least one antigen with the immunoblot assay). Ninety-eight percent of specimens not reactive with both enzyme-linked immunosorbent assay test systems were also nonreactive by recombinant immunoblot assay. Antibodies to c22-3 and c33c were more frequently detected in chronic hepatitis C virus in-
Received May 9, 1991; accepted August 19, 1991. This work was supported in part by a grant provided by Ortho Diagnostic Systems, Inc., Raritan, New Jersey. Dr. Person is a naval physician currently working with the Division of Gastrointestinal and Liver Diseases at the University of Southern California. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, the Department of Defense or the US. Government. Address reprint requests to: John G. McHutchison, M.D., Scripps Clinic and Research Foundation, Division of Gastroenterology, 10666 North Torrey Pines Road, La Jolla, CA 92037. 31/1/33412
fection and appeared earlier in acute hepatitis C virus infection, compared with 5-1-1 and c100-3. These results indicate that the addition of structural and nonstructural hepatitis C virus antigens in the secondgeneration enzyme-linked immunosorbent assay improves the sensitivity of detection of hepatitis C antibodies. (HEPATOLOGY 1992;15:19-25.)
The recent isolation and cloning of the genome of hepatitis C virus (HCV) has resulted in numerous reports of the prevalence, sensitivity and significance of detectable antibodies to this virus in non-A, non-B (NANB) hepatitis and other liver diseases. Worldwide, 60% to 80% of patients with chronic NANB hepatitis appear to have detectable antibodies to the recombinant HCV protein c100-3 (1-8).A high prevalence of antibody to HCV (anti-HCV)has generally been reported in other groups of individuals at risk for exposure to this virus (7, 9-11). The role of HCV in other liver diseases has not been so clearly established (12-17). Varying prevalence rates and false-positive results have been reported in some of these groups (18-21). Some of the remaining 20% to 30% of patients with NANB hepatitis without detectable c100-3antibodies do appear to have antibodies to other structural and nonstructural HCV proteins (22, 23). Whether another agent is responsible for some of these presumed infections or how many have an undetectable antibody response to these recombinant viral proteins is yet unknown. These problems in HCV infection highlight the need for improved screening test systems and a specific confirmatory test. In this report, we evaluated a second-generation ELISA and a recombinant immunoblot assay that detect antibodies to both structural and nonstructural HCV proteins in patients at high and lower risk for HCV infection. PATIENTS AND METHODS Group I : Patients at High Risk for HCV Exposure. Specimens from 56 patients with chronic NANB hepatitis were studied. All patients had at least two samples with elevated ALT values (at least two times normal, at least 6 mo apart) with no other apparent explanation for these abnormal liver 19
20
McHUTCHISON ET AL.
HEPATOLOGY
test results. Also, 106 serum samples from 49 patients with 0.600 + negative control for the first- and second-generation acute NANB hepatitis were included. Criteria for this diag- ELISAs, respectively). Sera were also tested using the newly developed fournosis included a clinical illness compatible with acute viral hepatitis, a peak ALT value at least 10 times the normal and antigen recombinant immunoblot assay (RIBA) (Chiron RIBA negative results of tests for HBsAg, HBc IgM antibody and HCV Test System; Chiron Corp., Emeryville, CAI. This antibody to hepatitis A virus IgM. Multiple samples (at least nitrocellulose stripbased assay is capable of detecting antitwo) were assayed in 37 of these 49 patients from the period of bodies to the recombinant HCV antigens 5-1-1, c100-3, c33c 6 to 25 wk after the peak ALT value. The earliest specimen and c22-3. During incubation of each strip with serum or tested was always from within 4 wk of the peak ALT value. We controls, HCV antibodies will react to the corresponding specifically included only patients with no identifiable risk nitrocellulose strip. After removal of nonspecific antibodies, factors for their presumed acute or chronic infection (i.e., all each strip is reacted with conjugated goat antihuman IgGpatients denied illicit intravenous drug use, prior blood horseradish peroxidase. Colored band patterns develop after transfusions or other percutaneous modes of transmission, as the addition of a solution containing hydrogen peroxide and well as multiple sexual contacts). Patients were excluded if any 4-chloro-1-naphthol. The intensity of the color is proportional other cause of hepatocellular injury could be identified (al- to the amount of specific antibody bound (graded as 1+ to 4 + 1. cohol, medications, hepatotoxins or other systemic diseases). Results are reported as reactive (reactivity with at least two These patients were selected from a hepatitis clinic at our antigen bands), indeterminate (reactivity with a single antigen band) or nonreactive (no reactive bands). tertiary referral center. Statistical Analysis. Student's t test and Fisher's exact test Specimens from 112 intravenous drug abusers attending a walk-in health-care clinic in the Los Angeles metropolitan area were used to compare the differences among groups and the (100 men, 12 women) were studied. Ten patients were known relative frequencies within groups. A p value 1:80) andlor ELISA only. Twenty-six were reactive when tested with antinuclear antibody with high serum globulin and high ALT RIBA, including all 22 sera reactive with both ELISA level before treatment. All patients showed a dramatic re- systems and the four patients reactive with the secondsponse to immunosuppressive treatment. Pre- and post- generation ELISA alone. A single specimen was reactive treatment sera were available for comparison in 13 cases. only with the first-generation ELISA (and was unreAnti-HCV Testing. Serum was stored at - 20" C until each active with RIBA). assay was performed. Retrospectively and prospectively HCC. Twenty-two specimens (48%) had anti-HCV gathered specimens were used in this study. Specimens were tested for anti-HCV using the first-generation (~100-3) detected by both ELISA systems, 8 demonstrated reacand second-generation (c200/c22-3) Ortho HCV ELISA test tivity with the second-generation ELISA only and 1 systems (Ortho Diagnostics, Inc., Raritan, NJ). The second- specimen was reactive with the first-generation ELISA generation assay differs from the available c100-3 based assay only. Reactivity with RIBA was demonstrated in 24 in that it detects antibodies to proteins derived from three patients, including all 21 that were reactive with both distinct regions of the HCV genome. The c200 recombinant ELISA systems. The three remaining sera reactive with antigen is an expression product of the putative NS3/NS4 RIBA were reactive with the second-generation ELISA region of the HCV genome and is derived from that region of only (another specimen reactive with the secondthe genome encoding both the c33c and c100-3 recombinant generation ELISA was indeterminately reactive with antigens. The c22-3 recombinant antigen is an expression product of the putative nucleocapsid region of the HCV RIBA). Intravenous Drug Users. Anti-HCV was detected by genome. All initially reactive specimens were retested in duplicate. For either assay, a reactive result was recorded if at the first- and second-generation ELISAs in 74 (66%) least two of the three optical-density values were equal to or patients. Another eight patients' sera were reactive with greater than the cutoff value (0.400 + negative control and the second-generation ELISA only. Of the 70 specimens
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
21
TABLE1. Prevalence of anti-HCV by three test systems ELISA
Diagnosis
Group 1 (high riskIb Acute HCV Chronic HCV HCC IV drug use Dialysis Group 2 (lower risky Hepatitis A or B PBC Al CAH
First-generation
Second-generation
RIBAa
49 56 46 112 50
20 23 23 74 2
29 26 30 a2 6
20 26 24 70 4
107 32 31
24 11 10
25 14 14
9 2 1
IV = intravenous; A1 CAH = autoimmune CAH. “RIBA reactivity implies at least two reactive antigen bands. Specimens classified as indeterminate (a single reactive antigen band) are not shown. b313 patients; 370 specimens. “170 patients; 184 specimens.
reactive with RIBA, 65 were reactive with both ELISA systems and 5 were reactive with the second-generation ELISA only. Two other patients with a reactive secondgeneration ELISA only were indeterminately reactive with RIBA. Hemodialysis. Two specimens (4%) were reactive with both ELISA systems and also with RIBA. An additional four specimens were reactive with only the second-generation ELISA (two were RIBA reactive and two were indeterminate). The remaining 44 sera were negative by all three tests. Viral Hepatitis A and B. Twenty-three patients were reactive with both ELISA formats, and two others were reactive with the second-generation ELISA only. Of these 25 patients, 9 were reactive, two were indeterminate and 12 were unreactive with RIBA. The specimens reactive with the second-generation ELISA only were unreactive with RIBA. Eighty-one patients were negative with both ELISA formats; 77 of these were RIBA unreactive (four were indeterminate). PBC. Eighteen (56%) of 32 patients tested were negative by all three tests. Eleven patients were reactive with both ELISA test systems, but only 2 of these 11 were RIBA reactive. Another three cases were reactive with the second-generation ELISA only (two were RIBA unreactive; one was indeterminate). Autoimmune CAH. Sera from 13patients were tested before and after successful treatment with prednisone. All were RIBA unreactive. Six patients had reactive firstand second-generation ELISA results before treatment; these later became negative. Three other patients had reactive second-generation ELISA results before treatment that remained reactive in one patient and disappeared in two patients after treatment. The remaining four patients were negative by all three assays, irrespective of treatment. In 18 additional patients with sera tested only after treatment, 12 were negative by all three assays, 4 were reactive by the second-generation ELISA (but unreactive with RIBA) and 2 were reactive with both ELISA systems (one was RIBA reactive and
one was unreactive). Thus only 1 of 31 patients evaluated was reactive with all three assays. Interrelationship Between the Three Test Systems. As shown in Figure 1, more sera were reactive with either or both ELISA systems in group 1patients at high risk for exposure to HCV. In these patients, another 12% of individuals were only reactive with the secondgeneration ELISA. In groups 1 and 2, most sera unreactive with both ELISA formats were also unreactive when tested with RIBA. Most specimens reactive with the first-generation ELISA were also reactive with RIBA in group 1 patients, but significantly fewer were confirmed as reactive with RIBA in group 2 patients (p < 0,0001, group 1 vs. 2). Note that in group 1 patients, the second-generation ELISA detected all remaining individuals who demonstrated reactivity with RIBA and most specimens that had been classified as indeterminate. Irrespective of the risk of exposure to HCV, most specimens classified as indeterminate RIBA were reactive with one or both ELISA systems. Figure 2 illustrates the results of RIBAin sera reactive by either or both the first- and second-generation ELISA. In group 1, 78% of ELISA-reactive sera were confirmed as reactive with RIBA, another 8% had a single reactive antigen band. The differences in the prevalence of RIBA-reactive results in these ELISAreactive sera in the different high-risk groups were not significant. In comparison, group 2 patients reactive with ELISA were much less likely to demonstrate reactivity with RIBA. Fifty-five specimens were found to be discordant when tested with the ELISA systems in that they were only reactive with the second-generation ELISA (Table 2). Most of these were patients at high risk for HCV exposure; of them, 50% were reactive with RIBA and another 25%were classified as indeterminate. In those cases displaying some reactivity with RIBA, the reactivity was directed toward the c33c and c22-3 antigen bands, the two HCV proteins present in the secondgeneration ELISA but not in the first-generation
22
McHUTCHISON ET AL.
HEPATOLOGY
GROUP 1 (HIGH RISK) 151 GEN
ELISA (c-100-3)
No. 01
ELSA REACTIVIN
2nd GEN ELISA
RlEA RESULT
SPECIMENS
POS
lsland 2nd GEN.
155
2nd GEN. ONLY
44
111 GEN. ONLY
NEGATIVE
Reactivity
IN0
NEG
135
5
15
22
11
11
2
0
0
2
169
0
1
168
GROUP 2 (LOWER RISK) 1st GEN ELISA (c-100-3)
No. of
ELSA REACTlVlW
+
2nd GEN. ELSA
-
Reactivity
RlEA RESULTS
SPECIMENS
POS
IN0
NEG
1sl and 2nd GEN
44
12
2
30
2nd GEN. ONLY
11
0
1
10
1st GEN. ONLY
1
0
0
1
NEGATIVE
128
0
4
124
Reactivily
FIG.1. Comparison between the three test systems in patients at high risk (group 1) and lower risk (group 2) for exposure to HCV. Boxed numbers represent the relationship between the first- and second-generation ELISAs. RIBA results are shown as positive or reactive (POS), indeterminate (IND) and negative or unreactive (NEG). More sera were reactive with the second-generation than the first-generation ELISA (254 vs. 202; p < 0.005).
1
croup 1
I
7Croup 2 1
100%
v)
[I
i
._
80%
V W
a v)
W
.-2
60%
V
0
? I Q
40%
J
w
r
0
20%
? a 0% Acute NANB Chronic NANB IV drug use
RIBA non--rmctive RIBA indeterminate
Dialysis (n=6)
PBC (n=14)
Hap A & 8 (n=26)
(n=16)
19%
0%
79x
57x
94%
3%
33%
77.
8%
78%
67%
14Z
35%
OX 6%
(n=55)
(n=Z7)
__
(n=82)
Liver Cancer (n=31)
25% 15% 60%
4%
9%
0% 96%
6% 85%
1
RIBA rmctive
RIBA indelerminale
0RIBA non-raoctive
A1 CAH
I
FIG.2. Results of the four-antigen recombinant immunoblot assay in specimens reactive by one or both ELISA test systems (n = 257). Data are shown as the percentage in each subgroup that was reactive, indeterminate or unreactive with the four-antigen RIBA. More group 1 sera were reactive with RIBA than were group 2 sera (78% vs. 12%;p < 0.0001).
ELISA. In comparison, most group 2 patients who were discordant with ELISA testing demonstrated no reactivity with RIBA. In both groups, none of the specimens with an optical density value of less than 2.0 in the second-generation ELISA were reactive or indeterminate when tested with RIBA. Three other patients were discordant in that they were reactive with only the first-generation ELISA (one had chronic NANB hepatitis; one had HCC and one had viral hepatitis B). All three were unreactive with RIBA.
Patterns of anti-HCV Reactivity. One hundred sixty-nine specimens were reactive when tested with RIBA (at least two reactive antigen bands), and 24 were interpreted as indeterminate (one reactive band). In the 124 group 1patients with reactive immunoblots whom we presume had chronic HCV infection (not including the acute NANB hepatitis group), 100% had detectable antibodies to c33c, 94% had detectable antibodies to c22-3, 69%had detectable antibodies to 5-1-1and 66% had detectable antibodies to the c100-3 HCV protein
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
(Fig. 3). Twelve group 2 patients were also reactive with RIBA, and the prevalence of antibodies detected to these four recombinant HCV antigens was similar to that in group 1 cases (c33c, 100%; c22-3, 92%; 5-1-1, 67%; c100-3, 67%). Most (53%) of the 136 patients with reactive immunoblots had antibodies detected to all four recombinant HCV antigens. There was no consistent pattern of antibody observed in cases with three detectable antibodies; when antibody was detected to only two of these HCV proteins, they were usually c33c and ~22-3. In evaluating the first specimen that displayed a reactive antigen band with RIBA in acute NANB hepatitis, development of antibodies to c33c and c22-3 preceded the development of antibodies to 5-1-1 and c100-3 in 11 of the 22 cases that displayed reactive or indeterminate RIBA results. In the other 11patients, a variable pattern of antibodies to these structural and nonstructural regions of the HCV genome was observed first. In only one case were antibodies to 5-1-1 and c100-3 detected first. There was no pattern evident in assessing the first detectable antibody in acute HCV infection (i.e., c22-3, c33c or both). Although most patients in this group who were serially followed appeared to have constant or increasing amounts of detectable antibody to each HCV antigen, antibody reactivity occasionally decreased or disappeared with time. Twenty-four patients had a single reactive antigen band on RIBA; they were classified as indeterminate. As shown in Table 3, most of these patients were at high risk for HCV infection and displayed reactivity to either the c33c or c22-3 recombinant antigen. On subsequent testing of the eight patients with acute NANB hepatitis indeterminate on RIBA, reactivity developed in four (50%)to two or more HCV antigens. DISCUSSION
The prevalence of anti-HCV detected by the firstgeneration ELISA in our subgroups of patients at high and lower risk for HCV infection is comparable to that in previous reports. With the newer test systems, we were able to detect anti-HCV in approximately 50% of patients with chronic sporadic NANB hepatitis -a lower prevalence than those seen in other reports (1,2, 6,241. Whether some of these individuals lack an immune response detectable by these assays, are infected with another NANB agent or have some other viral or nonviral cause for the observed liver injury is unknown. Despite the fact that we excluded patients with known percutaneous risk factors for HCV infection, we found a high percentage of patients with other types of liver diseases that were reactive with ELISA only and that presumably represent false-positive results. In particular, in autoimmune CAH, most sera reactive with ELISA were unreactive when tested with RIBA. Reactivity in these cases disappeared after successful immunosuppressive therapy. The results of this study demonstrate a high rate of concordance between the first- and second-generation
23
100%
80% U z D ._
6
L
60%
1
u
OI
1 L
.Z
40%
L
(D C
2 a
20%
0%
(n=26)
(n=24)
_
~
_
_
Dialysis (n=4)
75:
c 100-2
7551
5-1-1 ~22-3
,
92%
93%
100%
100%
c100-3
5-1-1
1005 100% _____
0c22-3
100%
c33c
1
FIG. 3. Pattern of reactivity to each of the recombinant HCV antigens in group 1 patients. Bars = percentage of patients in each group with each antibody detected. Only patients displaying reactivity to a t least two HCV antigens are included.
ELISAs and improved sensitivity of detection of HCV antibodies using the second-generation ELISA. This newly developed ELISA detected another 11.9% of patients at high risk for HCV infection who remained undetected by the first-generation ELISA. Seventy-five percent of these cases displayed reactivity to at least one HCV recombinant antigen when also tested with the newly developed suppIementaI RIBA. Perhaps one of the most important observations of this study is that all specimens that demonstrated reactivity by RIBA were detected by the second-generation ELISA, whereas 13% of these specimens were unreactive with the first-generation ELISA. These results indicate that the addition of structural (c22-3) and nonstructural (~200) HCV antigens does appear to increase the sensitivity of detection of antibodies compared with currently available c100-3-based systems. Although the second-generation ELISA appears to be more sensitive in the high-risk setting, there does appear to be a reduction in specificity (particularly in patients at lower risk for hepatitis C exposure). In this setting we presume that most ELISA-reactivespecimens that were not confirmed with the supplemental RIBA represent false-positive results. In the lower-risk groups we studied, it is possible that interfering substances (e.g., globulin, immune complexes or prolonged storage of
24
McHUTCHISON ET AL.
HEPATOLOGY
TABLE 2. Specimens reactive with second-generationELISA only ELISA optical density Diagnosis
No. -
Acute HCV Chronic HCV IV drug use HCC Dialysis Hepatitis A or B PBC A1 CAH
20 4 8 8 4 2 3 6
First-generation
Second-generation
RIBA reactivity
~~
0.252 t 0.112 0.196 t 0.131 0.266 ? 0.138 0.298 ? 0.076 0.139 t 0.168 0.356 2 0.002 0.360 t 0.053 0.266 2 0.106
2.085 2 0.664 > 2.500 2.272 -+ 0.644 1.453 ? 0.889 > 2.500 1.186 2 0.638 1.412 i 0.988 1.326 ? 0.718
IV = intravenous; AI CAH = autoimmune CAH. "Optical density value = 490 nm; values expressed as mean ? S.D. bNumber in parentheses is the number of patients with this pattern of reactivity; underlined numbers represent the number of patients in whom second-generation ELISA optical density was a t least 2.0.
TABLE3. Pattern of antibody reactivity in RIBA-indeterminate sera Diagnosis
Acute NANB hepatitis Chronic NANB hepatitis IV drug users HCC Dialysis PBC A1 CAH Viral hepatitis A or B TOTAL
No.
8 0 6 1 2 1 0 6 24
Reactive single antigen band
~ 3 (4)", 3 ~ 2 2 - 3(4)
-
~ 3 (3); 3 ~~2 2 - 3(2); 5-1-1 (1) c33c (1) ~ 2 2 - 3(2) ~22-3 ~ 3 (5); 3 ~~1 0 0 - (1) 3 ~ 3 (13); 3 ~~ 2 2 - 3(9); ~ 1 0 0 - 3 (1);5-1-1 (1)
IV = intravenous; A1 CAH = autoimmune CAH. "Number in parentheses indicates the number of specimens displaying this pattern of reactivity.
sera) produced the reactivity in the ELISA, as has been previously described (18-21). We presume that these cases do not have actual HCV infection; as in other low-risk groups (25-271, such cases do not have detectable HCV RNA sequences. Whether a reduction in specificity of the second-generation ELISA that we have observed in patients at lower risk for HCV exposure will also apply to the blood donor population is unknown. In the United States, a large multicenter trial in volunteer blood donors has recently been completed to address this important question. We found that an optical density value of at least 2.0 accurately predicted all specimens that were reactive with the second-generation ELISA and demonstrated reactivity to at least one antigen band in the RIBA. Serum reactive only with the secondgeneration ELISA (but unreactive with RIBA) always had an optical density ratio of less than 2.0. Until supplemental or confirmatory tests become widely available, an arbitrary cutoff value of at least 2.0 with this second-generation ELISA may aid in interpreting the significance of results in individuals at a lower risk for HCV exposure and infection. Whether patients at high risk for HCV infection with a reactive second-
generation ELISA (yet no reactivity by RIBA) represent actual infection with this virus cannot be determined from the results of this study. Further studies are in progress to determine whether such cases have detectable HCV RNA. The patterns of antibody reactivity using the four recombinant HCV antigens incorporated in RIBA has been reported in three patients with posttransfusion hepatitis (22). Marcellin et al. (23) noted a 20% increase in detection of HCV antibodies compared with the c100-3-based ELISA. In our high-risk groups with presumed chronic HCV infection, antibodies to the structural (c22-3) and nonstructural (c33c) regions of the HCV genome were more frequently detected than were those to 5-1-1 and c100-3. Most individuals at high risk for HCV exposure did have detectable antibodies t o c33c and c22-3, whereas only two thirds of this group had detectable antibodies to 5-1-1and c100-3. We could not identify any particular patterns of antibody that corresponded with the subgroup of patients tested nor to the presumed source of exposure. Although most patients exposed to HCV had all four antibodies detected, the pattern of antibody reactivity detected in each case was variable. In acute NANB hepatitis, antibodies to c33c and c22-3 appeared before those directed toward 5-1-1and c100-3 in more than half the patients studied. As in patients with chronic HCV infection, the pattern of antibody in acute infection was also variable. No attempt can be made to determine the time of antibody appearance after the clinical episode of actue viral hepatitis from this study because our patients were not followed at regular predefined intervals for a known period of time. A significant number of individuals at high risk for HCV infection demonstrated reactivity to a single antigen band with RIBA. In these cases, the antibody detected was most often directed toward the c33c or c22-3 recombinant HCV antigens. Although the manufacturer's interpretation of such a result is "indeterminate," these patients had high optical density values when tested with ELISA. Furthermore, a number of these indeterminate specimens were from patients with
Vol. 15, No. 1, 1992
IMPROVED DETECTION OF HCV ANTIBODIES
25
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