anti-HD positive with both the Organon and Abbott anti-HD assays. These data reflect the lack of sensitivity of Deltassay. In no instance were HDAg and anti-HD ...
JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1988, p. 1339-1342 0095-1137/88/071339-04$02.00/0 Copyright © 1988, American Society for Microbiology
Vol. 26, No. 7
Kinetics of Delta Antigen and Delta Antibody in Acute Delta Hepatitis: Evaluation with Different Enzyme Immunoassays FREDERIC DUBOIS* AND ALAIN GOUDEAU Laboratoire de Virologie, Centre Hospitalier Régional et Universitaire, 37044 Tours Cedex, France Received 2 December 1987/Accepted 31 March 1988
The kinetics of delta antigen (HDAg) and anti-delta antibody (anti-HD) was analyzed in 22 acute delta hepatitis infections (11 coinfections and 11 superinfections), with an enzyme immunoassay developed by Organon Teknika and with two commercially available assays: Deltassay, a test for HDAg from Noctech and Abbott anti-delta enzyme immunoassay, a test for anti-HD from Abbott Laboratories. In seven cases, HDAg was detected with the Organon assay but not with Deltassay. These discrepancies were not related to the type of hepatitis delta virus infection. All samples from these patients taken beyond week 4 of illness were found anti-HD positive with both the Organon and Abbott anti-HD assays. These data reflect the lack of sensitivity of Deltassay. In no instance were HDAg and anti-HD present simultaneously when tested with the Organon assays. On the contrary, 10 sera among the 28 that were HDAg positive with the Organon assay also were found anti-HD positive with the Abbott test. We suspected that the test procedure recommended by Abbott (a one-step competitive assay) may have yielded false-positive anti-HD results when HDAg present in the sera reacted with peroxidase-labeled anti-HD of the kit. To determine the specificity of the simultaneous presence of HDAg and anti-HD, these 10 sera were retested with the Abbott anti-HD assay, but by a modified two-step procedure that avoided contact between sera and labeled antibody. For six sera a negative result was obtained with the second procedure, suggesting that a false anti-HD reaction occurred with the standard test procedure. For four sera a positive result with both procedures was indicative of the simultaneous presence of HDAg and anti-HD. In conclusion, assay for HDAg was found very convenient for the early diagnosis of acute hepatitis delta virus infections. Seroconversion to anti-HD could be used for a late diagnosis 2 to 5 weeks after the beginning of illness. However, anti-HD results obtained with one-step competitive assays have to be interpreted carefully in HDAg-positive sera.
Hepatitis delta virus (HDV) was discovered 10 years ago by indirect immunofluorescence in hepatocyte nuclei of patients with chronic hepatitis B (11). Since then, various serological tests for delta antigen (HDAg) and corresponding antibodies (anti-HD and anti-HD immunoglobulin M [IgM]), to be used alone or in combination, have been proposed for the diagnosis of acute delta hepatitis (4, 8, 12, 13, 15). In both coinfections and superinfections, tests for HDV markers have yielded conflicting results. In some studies, HDAg was not found or was found infrequently at the onset of disease even in patients who had seroconverted during convalescence (1, 3, 7, 14). A "blind period" between the negativeness of HDAg and seroconversion to anti-HD IgG was also described. This blind period was reported to last from 2 to 11 weeks, leaving anti-HD IgM as the most reliable diagnostic marker during this period (1, 5). In other studies, HDAg was found constantly in sera drawn in the early course of the disease and thus appeared to be the best diagnostic marker of acute HDV infection (2, 6). In these studies, the blind period was shorter and even co-occurrence of HDAg and anti-HD was described (2). These conflicting results may have been related to poor sensitivity of the first tests that were used (12) or to differences in the overall characteristics of the assays (4, 8, 13) or both. We have analyzed the kinetics of HDAg and anti-HD in 22 acute HDV infections diagnosed in a continuous series of 167 acute hepatitis B surface antigen-positive hepatitis patients. An enzyme immunoassay (EIA) for HDAg and antiHD developed by Organon Teknika was compared with two commercially available tests: Deltassay, a test for HDAg *
from Noctech; and Abbott anti-delta EIA, a test for anti-HD from Abbott Laboratories. MATERIALS AND METHODS Tests for hepatitis B virus (HBV). Blood samples were tested by EIA for hepatitis B surface antigen (Auzyme, Abbott Laboratories, North Chicago, Ill.; and/or Hepanostika hepatitis B surface antigen, Organon Teknika, Boxtel, The Netherlands) and for anti-hepatitis core (HBc) IgM (Corzyme M; Abbott). Tests for HDV. An EIA developed by Organon Teknika was used for HDAg and for anti-HD antibody (9). (i) The test for HDAg was based on polystyrene micro-enzyme-linked immunosorbent assay strips coated with human anti-HD and on a peroxidase-labeled human anti-HD as second antibody. The test procedure recommended by the manufacturer included treatment of sera with 3 g of Nonidet P-40 per liter. All sera were also tested without detergent treatment for comparison. (ii) The test for anti-HD was identical in principle except that sera were first adsorbed with an HDAg preparation purified from chimpanzee liver. An anti-HDpositive sample competing with the solid-phase antibody for binding of HDAg resulted in a reduction in color in comparison with a negative control. Results were controlled with two commercially available EIAs. (i) Deltassay for HDAg from Noctech is a direct sandwich EIA based on polystyrene micro-enzyme-linked immunosorbent assay strips coated with human anti-HD and on a peroxidase-labeled human anti-HD as second antibody. A delta antigen extraction buffer containing a detergent is used for treatment of the serum. (ii) Abbott anti-delta EIA
Corresponding author. 1339
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J. CLIN. MICROBIOL.
DUBOIS AND GOUDEAU
PROCEDURE I
SAMPLE WASH-OUT SOLID PHASE RESULTS INTERPRETATION
w+
FALSE POSITIVE
+ >-«
SAMPLE
2
>-
TRUE POSITIVE
>-«
P wash out
NEGATIVE
3 nil color development
PROCEDURÈII
SAMPLE WASH-OUT SOLID PHASE RESULTS INTERPRETATION
1
lt
2
o
) > -I-|NEGATIVEI
+ SIMPLE < 2 >N washl
s wash2
POSITIVE
NEGATIVE
3 nil
color development
HD Ag coated bead
>-4
labelled
anti-HD
HD Ag
o
>-
anti-HD
FIG. 1. Competitive assay for anti-HD detection. Procedure 1: One-step competitive assay (as recommended by Abbott Laboratories). Procedure 2: Two-step competitive assay. was used with two different procedures. Procedure 1 (as recommended by the manufacturer) is a competitive assay in which serum samples and peroxidase-labeled human antiHD are incubated with polystyrene beads coated with delta antigen purified from woodchuck liver. Procedure 2 involves two incubations for 2 h at 40°C; in the first step, serum samples are incubated with the HDAg-coated beads; after a washing step, peroxidase-labeled anti-HD is added. We found that simultaneous use of both procedures alleviated a number of puzzling results. For instance, if a serum sample containing HDAg is tested with procedure 1, immune complexes between serum HDAg and peroxidase-labeled anti-HD can form and be removed in the washing solution. This will result in a false-positive anti-HD result. In contrast, with the sane serum, a true negative result will be obtained with procedure 2, since HDAg will be reinoved during the first washing (Fig. 1). HDV infection was investigated in a consecutive series of 167 acute HBsAg-positive hepatitis patients addressed to our
laboratory from January 1981 to December 1986. Only patients who had a first sample drawn within 4 weeks after day 1 of icterus were included. Diagnosis of acute hepatitis was based on clinical data and a rise of alanine aminotransferase of at least 10-fold the normal value. A recent infection by HBV was assessed in 152 patients by the presence of anti-HBc IgM; the diagnosis of underlying chronic HBV was suspected in 15 patients in the absence of anti-HBc IgM (10). The first sample was available form 139 patients within the first 2 weeks of illness and within 3 or 4 wèeks for 28 others. One hundred eleven patients had a late sample drawn beyond week 4. All early samples (186 sera for 167 patients) were tested for HDAg with the Organon assay with and without detergent treatment and with Deltassay. They were also tested for anti-HD with the Organon and Abbott anti-HD assays. All late samples (130 sera for 111 patients) were tested for HDAg with the Organon assay and for anti-HD with the Organon and Abbott anti-HD assays.
ENZYME IMMUNOASSAYS FOR HDV MARKERS
VOL. 26, 1988
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TABLE 1. Kinetics of HDAg and anti-HD with the different assays in 69 serum samples from 22 acute HDV infections Coinfections (n = 11)
Superinfections (n = 11) Wk from day 1 of icterus
No. of
No. of
Organon HDAg
samples tested
Noctech Deltassay
1 3-4 >4
7b 4
7 4 6
2 0
23
Organon anti-HD assay
assay
2
Anti-HD
HDAg
Anti-HD
HDAg
5 1 1 0
Abbott anti-HD assay'
samples
1 1 5 23
il 4 4 10
0 0 3 23
Organon HDAg
tested
Noctech
DeItassa
assay
10 0 0 0
il
2 2 0
Organon
Abbott anti-HD assaya
0 0 1 10
0
anti-HD assay
2 3 10
a Positive with procedures 1 and 2. b Results are given as number positive.
RESULTS Twenty-two patients had HDAg in their first sample when tested by the Organon HDAg assay. Of these, 15 were also positive with Deltassay. No patient had an HDAg-positive first sample with Deltassay that was HDAg negative with the Organon HDAg assay. Some 145 patients were HDAg negative with both tests. Of 111 patients for whom a late sample was available, 14 were found anti-HD positive with both Organon and Abbott anti-HD assays. All of them had been found HDAg positive in their first sample, 8 with both Organon HDAg assay and Deltassay and 6 only with the Organon HDAg assay. Of 22 HDV infections, 11 were HBVMHDV superinfections in patients with evidence of chronic HBV infection (IgM anti-HBc negative); 11 were HBV/HDV coinfections in patients with serological markers of a recent exposure to HBV (IgM anti-HBc positive). A total of 69 blood samples were available from these 22 H-DV patients: 36 had been drawn within the first 4 weeks of illness and 33 were drawn after week 4 (Table 1). Twenty-eight samples were found HDAg positive with the Organon HDAg assay. Results were identical whether the test was used with a detergent treatment of sera (recommendation of the manufacturer) or without detergent treatment. Only 17 of these 28 samples were also found HDAg positive by Deltassay. In no instance was Deltassay positive for a serum found HDAg negative with the Organon HDAg assay. Due to lack of sensitivity of Deltassay, seven HDV infections (five superinfections and two coinfections) would have been missed by this test. HDAg was detected for a longer period of time with Organon HDAg assay than with Deltassay. During week 1 of
illness, 15 of 18 samples found HDAg positive with the Organon HDAg assay were also positive with Deltassay, while during weeks 3 and 4 more samples were HDAg positive with the Organon HDAg assay (4 of 10) than with Deltassay (1 of 10). None of 33 late samples taken after week 4 of illness was HDAg positive. HDAg and anti-HD were never detected simultaneously when tested with the Organon assays. On the contrary, 10 of 28 early samples that were HDAg positive with the Organon HDAg assay were also found anti-HD positive with the Abbott anti-HD assay when test procedure 1 (one step) was used (Table 2). These 10 sera were tested again with the Abbott anti-HD assay, but using test procedure 2 (two steps). In six sera (five patients), discrepancies between results of procedures 1 and 2 (situation 1, Fig. 1) were indicative of false-reactive results due to the presence of HDAg. In four sera (four patients), both procedures yielded positive results but with a difference in intensity of the reaction (90 to 100% inhibition in procedure 1 down to 65 to 70% in procedure 2). These data may be interpreted as evidence for the co-occurrence of HDAg and anti-HD. A late sample was available for eight of these patients and showed seroconversion to anti-HD with both the Organon and Abbott anti-HD assays. A difference in the level of inhibition between procedures 1 and 2 was not observed with samples which were HDAg negative with the Organon HDAg assay and positive with both the Organon and Abbott anti-HD assays. In the early weeks of HDV infection, the Abbott anti-HD assay appeared more sensitive than the Organon anti-HD assay (Table 1). Beyond 4 weeks, all sera were found anti-HD positive with both assays.
TABLE 2. Reassessment of anti-HD results when sera were tested with the Abbott anti-HD assay by one-and two-step procedures Anti-HD Interpretation HDAg Time (days after Patient Abbott anti-HD assay Organon Organon onset) samples no. Noctech Anti-HD anti-HD HDAg HDAg were taken Deltassay 2 i steps' step" assay assay 4 5
8 3
5
21
7
8
8 9 10
il 3 10
15
24
18 22
13 15
+ + + + + + + + + +
a Standard one-step Abbott anti-HD competitive assay. b Two-step Abbott anti-HD competitive assay.
-
-
+
+
+
+
-
+ +
+ +
+
-
+
+
-
+
+
-
-
-
+ -
+
+
+
+
+
-
+
-
-
-
+
+
+
+
-
-
+ +
-
+ +
-
+
-
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J. CLIN. MICROBIOL.
DUBOIS AND GOUDEAU
ACKNOWLEDGMENTS
DISCUSSION In view of a rather heterogeneous literature, it is not easy to make a sensible choice among various serum markers for the diagnosis of acute HDV infection (1, 2, 5, 14). The possibility of conflicting results between different assays for HDAg was illustrated in our study in which only 17 of 28 samples positive with the Organon HDAg assay were also positive for Deltassay. These discrepancies were not related to the type of HDV infection but appeared to reveal a greater sensitivity of the Organon test over Deltassay as assessed by three observations. (i) Seven of 22 patients were positive with the Organon HDAg assay but negative with Deltassay. For six of these patients, a late sample was available and showed a seroconversion to anti-HD. (ii) No patients were Deltassay positive and Organon HDAg negative. (iii) Only patients who had been HDAg reactive with the Organon assay during the first few weeks of illness developed a seroconversion to anti-HD in late serum samples. Various studies have reported an inconstant or very transient appearance of HDAg that left a blind period before the seroconversion to anti-HD IgG. During this period, some authors have recommended testing for anti-HD IgM, which is also transient but less inconstant (1, 5). We also found that a blind period could occur (between weeks 2 and 4 of illness) when Organon HDAg and anti-HD assays were used. However, with these tests, HDAg was always detected in sera taken within week 1 of illness and the blind period did not extend beyond week 4 of evolution. With the Abbott antiHD assay, we have observed a disappearance of the blind period and even, as already described (2), a co-occurrence of HDAg and anti-HD. However, when the Abbott anti-HD assay was used as recommended by the manufacturer (onestep procedure), 6 of 28 sera that were HDAg positive with the Organon assay gave false anti-HD results. These problems were probably due to the occurrence of immune complexes between labeled anti-HD from the test and HDAg of the serum and could be avoided by a two-step procedure. It may be noteworthy that five of these six sera were HDAg negative when tested with Deltassay (Table 2). These intriguing results are perhaps due to different specificities in the HDAg/anti-HD system. The Organon anti-HD assay based on sandwich inhibition yielded no false-positive reactions but appeared less sensitive in the early weeks of
evolution. A combination of the Abbott anti-HD assay, used with procedure 2, and the Organon HDAg assay suppresses the HDAg/anti-HD blind period. The diagnosis of acute HDV coinfection is thus possible at any time and can be based on the simultaneous presence of anti-HBc IgM and one of the HDV markers. For acute HDV superinfection (anti-HBc IgM negative), early diagnosis will rely on the presence of HDAg, but delayed diagnosis will be more delicate since anti-HD in a serum sample taken a few weeks after onset could reflect a previous exposure to HDV. In such cases, only persistence and rise in titers of anti-HD will differentiate recent from past HDV infections. In conclusion, an early and reliable diagnosis of acute HDV infections is possible. Tests for HDAg are preferred during the first 2 weeks of illness provided a sensitive assay is used. Late diagnosis, 2 to 5 weeks after the beginning of illness, can be based on seroconversion to anti-HD IgG.
We are grateful to G. Lesage and P. Roingeard for assistance in the preparation of the manuscript. We thank S. Romand for skillful typing. LITERATURE CITED 1. Aragona, M., S. Macagno, F. Caredda, O. Crivelli, C. Lavarini, E. Maran, P. Farci, R. H. Purcell, and M. Rizzetto. 1987. Serological response to the hepatitis delta virus in hepatitis D. Lancet. i:478-480. 2. Buti, M., R. Esteban, R. Jardi, J. L. Esteban, and J. Guardia. 1986. Serological diagnosis of acute delta hepatitis. J. Med.
Virol. 18:81-85. 3. Craxi, A., G. Raimondo, G. Longo, G. Giannuoli, R. De Pasquale, M. Caltagirone, S. Patti, G. Squadrito, and L. Pagliaro. 1984. Delta agent infection in acute hepatitis and chronic HBsAg carriers with and without liver disease. Gut 25:12881290. 4. Crivelli, O., M. Rizzetto, C. Lavarini, A. Smedile, and J. L. Gerin. 1981. Enzyme-linked immunoabsorbent assay for the detection of antibody to the hepatitis B surface antigen-associated delta antigen. J. Clin. Microbiol. 14:173-177. 5. Dimitrakakis, M., M. J. Walters, A. Wootton, and I. Gust. 1986. Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus. J. Med. Virol. 20:305311. 6. Dubois, F., A. Goudeau, D. Leturcq, S. Margoux, A. Marguilies, J. L. Guilmot, and P. Choutet. 1987. Diagnostic précoce des hépatites Delta par la recherche de l'AgHD. Gastroenterol. Clin. Biol. 11:68-69. 7. Farci, P., A. Smedile, C. Lavarini, P. Piantino, O. Crivelli, N. Coporaso, M. Toti, F. Bonino, and M. Rizzetto. 1983. Delta hepatitis in inapparent carriers of hepatitis B surface antigen: a disease simulating acute hepatitis B progressive to chronicity. Gastroenterology 85:669-673. 8. Hansson, B. G., T. Moestrup, A. Widell, and E. Nordenfelt. 1982. Infection with delta agent in Sweden: introduction of a new hepatitis agent. J. Infect. Dis. 146:472-478. 9. Heijtink, R., J. Kruining, L. Kuipers, A. Jacobs, C. Geudens, and G. Wolters. 1983. An Elisa for Delta markers in hepatitis B infection, p. 263. In G. Verme, F. Bonino, and M. Rizzetto (ed.), Viral hepatitis and viral infection. Alan R. Liss, New
York. 10. Perillo, R. P., K. H. Chau, L. R. Overby, and R. H. Decker. 1983. Anti-hepatitis B core immunoglobulin M in the serologic evaluation of hepatitis B virus infection and simultaneous infection with type B, delta agent, and non-A, non-B viruses. Gastroenterology 85:163-167. 11. Rizzetto, M., M. G. Canese, S. Arico, O. Crivelli, C. Trepo, F. Bonino, and G. Verne. 1977. Immunofluorescence detection of
12.
13.
14. 15.
new antigen/antibody system (delta/anti-delta) associated to hepatitis B virus in liver and serum of HBsAg carriers. Gut 18:997-1003. Rizzetto, M., J. W.-K. Shih, and J. L. Gerin. 1980. The hepatitis B virus-associated delta antigen: isolation from liver, development of solid phase radioimmunoassay for delta antigen and anti-delta and partial characterization of delta antigen. J. Immunol. 125:318-324. Shattock, A. G., and B. M. Morgan. 1984. Sensitive enzyme immunoassay for the detection of Delta antigen and anti-Delta, using serum as the delta antigen source. J. Med. Virol. 13:73-82. Smedile, A., P. Dentico, A. Zanetti, E. Sagnelli, E. Nordenfelt, G. Actis, and M. Rizzetto. 1981. Infection with the Delta Agent in chronic HBsAg carriers. Gastroenterology 81:992-997. Smedile, A., C. Lavarini, O. Crivelli, G. Raimondo, M. Fassonne, and M. Rizzetto. 1982. Radioimmunoassay detection of IgM antibodies to the HBV associated delta antigen, clinical significance in Delta infection. J. Med. Virol. 9:131-138.
