differential diagnosis of acute viral hepatitis using rapid, fully automated immunoassays

Journal of Medical Virology 33:139-150 (1991)

Differential Diagnosis of Acute Viral Hepatitis Using Rapid, Fully Automated Immunoassays Kim Eble, John Clemens, Christine Krenc, Michael Rynning, Joseph Stojak, Janice Stuckmann, Patricia Hutten, Linda Nelson, Louise DuCharme, Sally Hojvat, and Larry Mimms Hepatitis Research and Development, Abbott Laboratories, Abbott Park, Illinois

We report the development of three rapid, fully automated immunoassays allowing the differential diagnosis of acute viral hepatitis. These assays detect HBsAg, IgM antibody to hepatitis B core antigen (IgM anti-HBc) and IgM antibody to hepatitis A virus (IgM anti-HAV) using the IMx@ instrument system. All IMx assays were run in less than 45 minutes and all steps were fully automated including specimen dilution steps. Specimens from blood donors, diagnostic and hospital patients, and individuals with a variety of infectious and immune diseases were tested for IgM anti-HAV (n=1473) or for IgM anti-HBc (n=1606) or for HBsAg (n=9700) by the IMx and commercially available EIA and RIA. Each IMx assay showed 99.8% agreement with current EIA. Reproducibility in all hepatitis IMx assays was significantly better than that observed with manual or semiautomated assays; within-run and between-run 9'0 CV ranged from 2.2 to 4.8 and 3.5 to 10.3 respectively. In 29 acute hepatitis B patients studied, HBsAg and IgM anti-HBc were detected in the first available patient bleed collected from 0 to 4 weeks from the onset of symptoms. IgM anti-HBc persisted at reactive levels in the IMx assay for 1 to 24 weeks (mean 12.1 ~fr 5.3 weeks) after the patient presented with symptoms. In individuals exposed to hepatitis A, IgM anti-HAV was detectable by IMx by 40 days post exposure (average 33.5 days) and IgM had declined to unreactive levels in IMx for all patients by from 3 to 6 months post exposure. These data demonstrate the use of these rapid IMx assays for differentiation of acute hepatitis A and 6.

KEY WORDS: hepatitis A, hepatitis 6, HBsAg IgM antibodies, anti-HBc IgM

is dependent on serological identification of viral markers. To differentiate acute hepatitis A and hepatitis B, three markers have been shown to be useful: hepatitis B surface antigen (HBsAg), IgM antibodies against hepatitis B core antigen (IgM anti-HBcf, and IgM antibodies against hepatitis A virus (IgM anti-HAV) [Overby et al., 19811.

At the onset of symptoms resulting from HAV infection, antibody to HAV is almost always detectable and this early response is composed mostly of the IgM antibody subclass. Previous research indicates that in 80-90% of cases, this antibody is detectable for 3 to 6 months after the onset of illness, whereas IgG anti- HAV can persist indefinitely. Because of the transient production of IgM anti-HA?', its presence in sera indicates ongoing or recent infection and is the most valuable test for acute hepatitis A [Decker et al., 1979; Decker et al., 1981; Locarnini et al., 1977; Bradley et al., 1979; Deinhardt et al., 19781.

HBsAg is the first serological marker detected in acute hepatitis B infection. In 80-90% of cases, HBsAg is cleared 3 to 4 months after the onset of illness; however, in the remaining cases HBsAg persists indicating the existence of a carrier state. During and after antigenemia, IgG and IgM anti-HBc are detected and have been shown to be the only markers for hepatitis B infection in the serological window that often exists between the disappearance of HBsAg and the appearance of anti-HBs [Kryger et al., 1982; Overby et al., 19811. The IgM anti-HBc response has been reported to be transient (detectable for 3 to 6 months after the onset of symptoms) in contrast to IgG anti-HBc which may be detected indefinitely. [Mortimer et al., 1981; Lemon et al., 1981; Roggendorf et al., 1981; Hawkes et al., 1980; Kryger, 19851. Therefore, the IgM anti-HBc marker is the only specific marker for the diagnosis of acute hepatitis B infection.

Commercial immunoassays for the detection of IgM anti-HAV [Devine et al., 1979; Decker et al., 19811 and

INTRODUCTION Patients with acute viral hepatitis have similar

clinical symptoms and, therefore, differential diagnosis 0 1991 WILEY-LISS, INC.

Accepted for publication October 26, 1990. Address reprint requests to Larry Mimms, Hepatitis Research

and Development, Abbott Laboratories, Abbott Park, IL 60064.

140 Eble et al.

IgM anti-HBc [Chau et al., 1983; Govindarajan et al., 19841 have been available both in an RIA format since 1980 and 1984, respectively, and in an EIA format since 1982 and 1983, respectively. These assays require significant specimen dilution, multiple assay steps, and almost 2 days for a result to be obtained. In this paper we describe the performance of rapid, fully automated enzyme immunoassays for the detection of HBsAg, IgM anti-HAV and IgM anti-HBc for use with the Abbott IMx@ instrument [Fiore et al., 19881. The IMx system is a clinical laboratory instrument which uses microparticles as solid phase for capture of analyte. This system offers the advantage that all assays are run in less than 45 minutes and all steps are fully automated, including specimen dilution steps. Results demonstrate that the IMx HBsAg, IMx CORE-M, and IMx HAVAB-M assays give sensitivity, specificity, and detectability equivalent to commercially available manual assays, but give better within-run and between-run reproducibility.

MATERIALS AND METHODS Serum and plasma specimens were tested with the

following commerical RIA and EIA reagents supplied by Abbott Laboratories: AUSZYME Monoclonal, AUSZYME I1 and IV, AUSRIA 11, and IMx HBsAg for HBsAg; IMx HBsAg Confirmatory and AUSZYME Monoclonal Confirmatory assays for confirmation of HBsAg reactive specimens; CORZYME and CORAB for total anti-HBc; HAVAB-M, HAVAB-M EIA, and IMx HAVAB-M for IgM anti-HAV; CORAB-M, CORZYME-M, and IMx CORE-M for IgM anti-HBc; and HAVAB for total anti-HAV.

Specimens for in-house and clinical testing were obtained from hospital and community blood banks, reference laboratories, public health laboratories, and hospital diagnostic laboratories and are described in detail below. Some serial bleeds were collected from patients presenting with acute hepatitis or from donors to a plasmapheresis center. Some anti-HAV specimens were obtained from a study conducted in which volun- teers were experimentally infected with HAV [Boggs et al., 19701.

IMx Assays All hepatitis IMx assays are based on a microparticle

enzyme immunoassay (MEIA) technology. A detailed description of the IMx instrument is given in Fiore et al. [1988]. Briefly, the MEIA technology used microparticles approximately 0.2 Fm in diameter as the solid phase and separation of bound from unbound material is accomplished by capture of the microparticles in a glass fiber matrix. The benefits of using microparticles are the increased kinetics of reaction (total assay time of less than 45 min for 24 specimens) and the ease with which they can be manipulated by instrument robotics for automation of assay protocols. The IMx instrument atuomatically performs all steps of the immunoassay for up to 24 specimens per batch with no operator intervention except the initial pipetting of the specimen into the sample well. One control, termed

the Mode 1 calibrator, is included in each run, leaving room for 23 patient samples. Different hepatitis assays must be performed in separate runs.

IMx HAVAB-M Specimen is diluted by the IMx instrument (1:200)

with TRIS buffered saline. Microparticles coated with anti-human IgM antibody (pchain) are used to capture IgM in the diluted specimen. A hepatitis A antigen (HAV Ag) solution is added and if IgM anti-HAV is present in the sample, the HAV Ag binds to the microparticle-antibody complex. Monoclonal anti- HAV: alkaline phosphatase conjugate is dispensed and binds to the microparticle antibody antigen complex. The microparticle complex captured on the matrix is washed to remove unbound material, the substrate 4-methyl umbelliferyl phosphate (MUP) is added to the matrix and a front surface fluorometer is used to quantify the enzymatic generation of fluorescent product at a rate proportional to the concentration of IgM anti-HAV captured.

Carboxylated latex particles (0.1 to 0.3 p,m diameter) (Seradyne, Indianapolis, IN) were coupled to anti- human IgM (p, chain) (Jackson Laboratories, Maine) using 1-ethyl-3-3(dimethylaminopropyl) carbodiimide chemistry [Quash et al., 19781. Monoclonal anti-HAV [described in Dawson et al. 19841 was coupled to alkaline phosphatase using a heterobifunctional crosslinking reagent (N hydroxy succinimide ester and maleimide) [Yoshitake et al., 19791.

IMx CORE M The assay for the detection of IgM anti-HBc is similar

to the IMx HAVAB-M assay in format using the same anti-human IgM microparticles to capture IgM from the specimen, but differs in that recombinant DNA derived HBcAg (from Escherichia colz) and human anti-HBc: Alkaline phosphatase are used in place of HAV Ag and anti-HAV conjugate. DEAE purified human anti-HBc (Abbott Laboratories) was coupled to alkaline phosphatase via heterobifunctional crosslinking reagents [Yoshitake et al., 19791.

IMx HBsAg Sample, anti-HBs coated microparticles and biotin-

ylated anti-HBs solution are transferred by the IMx instrument to the incubation well of the reaction cell. If HBsAg is present in the specimen, the HBsAg will form an anti-HBs coated microparticle-antigen-biotinylated anti-HBs complex. An aliquot of the reaction mixture is transferred to a glass fiber matrix, and the microparticles bind irreversibly to the matrix while unbound material flows through the matrix. The anti-biotin: alkaline phosphatase conjugate is dispensed onto the matrix and binds to the biotinylated anti-HBs-antigen- microparticle complex. After washing to remove unbound material, substrate is added and fluorescent product is measured as described above. IMx HBsAg reactives were confirmed by neutralization with human anti-HBs (IMx HBsAg Confirmatory, Abbott Lab-

Differential Diagnosis of Acute Viral Hepatitis

oratories, N. Chicago, IL). Monoclonal anti-HBs was coupled to carboxylated microparticles as described above. Affinity purified rabbit anti-biotin was conju- gated to alkaline phosphatase using the method of Nakane and Kawaoi [19741. AEnity purified goat anti- HBs was biotinylated using NHS-LC-biotin (Pierce, Boehringer-Mannheim) according to the manufactur- er’s instructions.

Assay Cutoffs In all assays, results are given as fluorescent rate

values (counts/sec/sec). Specimen rates are normalized to the Mode 1 calibrator rate to give an Index value. For IMx HBsAg, the Mode 1 calibrator is recalcified human plasma testing nonreactive for HBsAg and therefore the Index value is referred to as an S/N value. Any specimen having an Index value or S/N value greater than or equal to 2.000 (i.e., a rate value greater than or equal to two times the Mode 1 calibrator rate) is considered reactive for HBsAg.

For IMx HAVAB-M, the Mode 1 calibrator is recalcified human plasma containing IgM anti-HAV diluted to a known low concentration of this antibody. Likewise for IMx CORE-M, the Mode 1 calibrator is recalcified human plasma containing IgM anti-HBc diluted to a known low concentration of the antibody. In both IgM assays, the Mode 1 is similar to a cutoff control. Specimens giving Index values (calculated as specimen rate/Mode 1 rate) between 0.800 and 1.200 are considered gray zone reactive and specimens giving Index values greater than 1.200 are considered reactive in the assay.

Statistical Methods Within-run, between-run, and total standard devia-

tions and %CV were calculated as described in Krouwer and Rabinowitz [19841.

RESULTS IMx HAVAB-M

Specificity. Specimens from 200 paid blood donors (100 sera and 100 plasma) were tested in parallel by IMx HAVAB-M and HAVAB-M EIA to demonstrate specificity of the IMx HAVAB-M assay. All samples in this donor population were unreactive by either IMx or EIA. The IMx HAVAB-M assay shows proper subclass specificity since 64 of these specimens (32%) were reactive for IgG anti-HAV as measured by HAVAB EIA (Abbott Laboratories). In addition, DEAE purified IgG anti-HAV added at high concentration (3.7 mgiml, titer was approximately 1:6400) to recalcified human plasma showed no interference in the IMx assay.

A group of 301 serum specimens sent to a reference lab for hepatitis testing were evaluated by IMx HAVAB-M, HAVAB-M EIA and RIA. In this population 5 specimens were reactive by all three IgM anti- HAV assays and the remaining 296 samples were nonreactive by all three assays. A frequency histogram of IMx results showed good separation between the negative population and gray zone reactive range (Fig.

141

1A). Similar population distributions were observed for EIA and RIA (data not shown).

An additional 288 specimens from patients with infectious diseases other than acute hepatitis A or containing other serological components which are potential interferences were tested by IMx and EIA assays. These specimens were obtained from hemodi- alysis patients (n = 30) and patients with or containing rheumatoid factor (n = 501, hyper IgG (n = lo), hyper IgM (n = 101, systemic lupus erythematosis (SLE) (n = lo), anti-nuclear antibodies (n = lo), antibodies against cytomegalovirus (CMV) (n = 121, respiratory syncytial virus (RSV) (n = 121, influenza virus (n = 21, Epstein-Barr Virus (EBV) (n = 37), Syphilis (n = lo), elevated liver enzymes (n = 221, elevated alkaline phosphatase (n = 271, and IgM anti-HBc (n = 44). Only two specimens were reactive with IMx assay and these specimens were also reactive for IgM anti-HAV by EIA and RIA as well as for total anti-HAV as measured by HAVAB. Taken together these data demonstrate the specificity of the IMx HAVAB-M assay.

Sensitivity and detectability. IMx HAVAB-M sensitivity was evaluated by testing two fold serial dilutions of 6 high titer IgM anti-HAV specimens. This IMx assay shows equivalent or 2-fold greater sensitivity than HAVAB-M EIA for all specimens tested. Sensitivity was also measured as the ability to detect IgM anti-HAV in sequential bleeds from 6 clinically diagnosed acute hepatitis A patients (n = 80). Data is plotted as a scattergram comparing IMx Index values with sample to cutoff values (S/CO) in the EIA (Fig. 2). Fifty specimens were reactive by both tests, nineteen nonreactive by both tests and eight were gray zone reactive by both tests. Two specimens were gray zone reactive in the EIA (S/CO between 0.9 and 1.1) but weakly reactive in the IMx assay and one specimen was gray zone reactive (Index 0.8 to 1.2) in the IMx but weakly reactive by the EIA procedure. Good quantita- tive correlation was observed between specimen reactivity in HAVAB-M EIA and IMx (Fig. 2). At high levels of IgM anti-HAV in sera, the IMx Index value plateaus at 4.0 to 4.5 corresponding to maximum S/CO values in the EIA of approximately 7.0.

Four serial bleeds from individuals who had been experimentally infected with HAV in a study by Boggs et al. 119701 were evaluated by IMx HAVAB-M (Fig. 3). IgM anti-HAV was detected in all 4 individuals by 40 days post inoculation shortly after the onset of clinical symptoms (average = 33.5 days post inoculation). By 120 days post inoculation, IgM levels decreased to the gray zone or negative level for the two patients from whom bleeds were available. Data from patient 2 and others (not shown) indicate that index values well above the baseline values (Index = 0.25) are observed even after 240 days indicating that low levels of IgM may persist beyond 3 to 6 months. The IMx HAVAB-M gray zone has been established so that Index values for HAV infected patients will fall below these gray zone IgM concentrations within 3 to 6 months after the onset of clinical symptoms.

142 Eble et al.

0 0 2 0.4 0.6 0.8 1 1.2 1 4

INDEX

180

B

110

100

90

8 0

70

60

50

40

30

20

10

0

0 0 25 0 5 0 75 1 125 1 5

INDEX

100

0.90

0.80

0 70

a 60

0.50

0.40

0.30

0.20

0.10

0 00

0 0 5 1 1 5 2 25 3

SAMPLE/NEGATlVE

Fig. 1. Frequency histogram of sera tested by IMx HAVAB-M (n = 301) A, IMx CORE-M (n = 249) B, and IMx HBsAg (n = 2440) C. Data are presented as Index values (calculated as specimen rateIMode 1 rate) for IMx HAVAB-M and IMx CORE-M or as sample rateINHP rate (SIN) for IMx HBsA . Sera for IMx HAVAB-M and CORE-M testing were obtained from a reference lab and sera for IMx H&Ag were from blood donors.

Differential Diagnosis of Acute Viral Hepatitis 143

IMx CORE M Specificity. The specificity of the IMx CORE-M

test was demonstrated similarly to testing described for IMx HAVAB-M. Two hundred forty nine specimens sent to a reference lab for hepatitis testing were evaluated by IMx CORE-M, CORZYME-M (EIA), and CORAB-M (RIA). One specimen was reactive for all 3 assays. Frequency histograms of IMx CORE-M results (Fig. 1B) show good separation between Index values for nonreactive specimens and the gray zone or retest range. Two hundred paid blood donors were also tested by IMx and EIA. No samples were reactive by IMx CORE-M even though 10% (20/200) were reactive for total anti-HBc as measured by CORZYME. These data demonstrate the proper immunoglobulin class specificity of the IMx CORE-M assay. Furthermore, IgG anti- HBc purified on a DEAE ion exchange column showed no interference in the IMx assay when added to nonreactive specimens at concentrations up to 8.4 mgiml (105,000 Paul Ehrlich Institut Units/ml) (data not shown). A variety of specimens (n = 378) from patients with acute infectious diseases other than acute hepatitis B and from subjects with other serological components which are potential interferences were also tested by IMx, CORZYME-M and CORAB-M. All 378 specimens were nonreactive by all three assays, dem- onstrating the excellent specificity of IMx CORE-M. These data also show equivalence among the three assay methods.

Sensitivity and detectability. Sensitivity of IMx CORE-M was evaluated by testing two fold serial dilutions of 2 high titer IgM anti-HBc specimens. The IMx assay gave equivalent sensitivity to EIA and RIA with endpoint titers of 1:8 and 1:32 for the two specimens.

Sensitivity and detectability were also studied using specimens from 46 acute hepatitis B patients and 76 chronic hepatitis B patients. All 46 acute patients were reactive by IMx CORE-M, with Index values ranging from 1.46 to 4.53 (Fig. 4). Seventy-four of 76 (97.4%) specimens from chronic patients were not reactive in the IMx CORE-M assay (Fig. 4). The 2 specimens reactive in IMx CORE-M had Index values of 2.63 and 2.02 and were also reactive in the EIA (S/CO 2.40 and 1.95) and RIA (S/CO 3.90 and 1.78). Sixty-seven samples from patients with serological evidence of previous hepatitis B infection (positive for both anti-HBs and anti-HBc) were also tested by IMx and EIA (Fig. 4). Overall agreement between IMx and EIA was 98.5% (66/67). All specimens were negative by both assays, except one specimen which was gray zone reactive for IMx (Index = 1.00) and elevated negative in the EIA (S/CO = 0.68). These data demonstrate that the cutoff of Index value > 1.2 for reactivity in the IMx CORE-M assay allows detectability for all acute patients. Use of this cutoff resulted in the detection of less than 3% of chronic samples as reactive and demonstrated the utility of IMx CORE-M as an aid in differentiating acute and chronic hepatitis B.

8

6

s 2 4

5 W

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0 2 4 6

IMx INDEX

Fig. 2. Correlation between HAVAB-M EIA and IMx HAVAB-M for 80 specimens from 6 clinically diagnosed acute he atitis A patients. Data is plotted as a scattergram comparing IMx In&x values to EIA SICO. A line of slope = 1 is shown.

Reproducibility. IMx HAVAB-M assay reproducibility was determined using a panel of 5 specimens containing varying concentrations of IgM anti-HAV antibody. The panel was run in replicates of 3 in fifteen separate runs on 3 instruments (Table I). The within- run, between-run, and total %CV ranged from 2.5 to 3.4, 4.0 to 5.4, and 4.9 to 6.2, respectively. These data contrast significantly with current IgM anti-HAV EIA assays which have within-run and between-run %CV’s up to 20%.

Clinical evaluation. In clinical studies, 983 specimens were tested by IMx HAVAB-M and EIA and an additional 490 specimens were tested by IMx HAVAB- M and RIA. Specimens were obtained from the following categories: blood donors, hospital patients, known reactive for IgM anti-HAV or total anti-HAV or IgM anti-HBc, patients diagnosed as potential Non-A Non- B hepatitis and patients with a wide variety of infectious diseases or containing potentially interfering substances. Agreement between IMx and EIA and between IMx and RIA was 99.8% (981/983) and 99.8% (489/490), respectively. One hundred fifty two specimens were reactive in t.hese studies by IMx and either EIA or RIA. Of the 3 discrepant samples, one was reactive by EIA but nonreactive by RIA or IMx, one was low level reactive by IMx but elevated nonreactive by EIA, and one specimen was gray zone reactive in 1Mx but borderline reactive by RIA. These data demonstrated the equivalence between IMx HAVAB-M and commercial RIA and EIA assays in sensitivity, specificity, and detectability.

144 Eble et al.

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t

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x w 0 2 - 2 a > U X

Y z

0 30 6 0 9 0 120 1 5 0 180 2 1 0 240

DAYS FROM EXPOSURE

Fig. 3. Sequential bleeds from four individuals who were experimentally infected with HAV tested by IMx HAVAB-M.

TABLE I. Reproducibility of IMx HAVAB-M, IMx CORE-M and IMx HBsAg

M~~~ Index Within-run Between-run Total IMx HAVAB-M

Sample n value SD %CV SD %CV SD %CV

1 45 2.676 0.0676 2.5 0.1118 4.2 0.1306 4.9 2 45 1.897 0.0582 3.1 0.0789 4.2 0.0980 5.2 3 45 1.209 0.0404 3.3 0.0636 5.3 0.0753 6.2 4 45 0.760 0.0257 3.4 0.0397 5.2 0.0473 6.2 5 45 0.492 0.0143 2.9 0.0198 4.0 0.0244 5.0 Negative control 45 0.224 0.0057 2.5 0.0120 5.4 0.0133 5.9

IMx CORE-M M~~~ Index Within-run Between-run Total

Sample n value SD %CV SD %CV SD %CV 1 45 2.411 0.0521 2.2 0.1171 4.9 1.1262 5.3 2 45 1.790 0.0571 3.2 0.0966 5.4 0.1122 6.3 3 45 1.221 0.0352 2.9 0.0501 4.1 0.0612 5.0 4 45 0.740 0.0168 2.3 0.0373 5.0 0.0409 5.5 5 45 0.427 0.0148 3.5 0.1220 5.1 0.0265 6.2 Negative control 45 0.076 0.0036 4.8 0.0057 7.5 0.0068 8.9

IMx HBsAg

Sample (ng/mg) n value SD %CV SD %CV SD %CV Mean S,N Within-run Between-run Total

Ad (0.90) 140 Ad (0.74) 140 Ad (0.51) 140 Ad (0.41) 140 Ay (2.05) 140 Ay (0.83) 140 Ay (0.53) 140 Ay (0.30) 136 Negative control 140

4.667 2.625 2.087 1.886 5.372 2.718 2.260 1.608 1.015 -__

0.1480 0.0834 0.0549 0.0609 0.1964 0.0721 0.0740 0.0508 0.0447

3.2 3.2 2.6 3.3 3.7 2.7 3.3 3.2 4.4

0.4340 0.1964 0.1695 0.1481 0.5544 0.2735 0.1312 0.1102 0.0355

9.3 7.5 8.1 7.9

10.3 10.1 5.8 6.9 3.5

0.4585 0.2134 0.1782 0.1601 0.5881 0.2828 0.1506 0.1214 0.0571

9.8 8.1 8.5 8.6

10.9 10.4 6.7 7.5 5.6


ees, IgM anti-HAV reactive, IgM anti-HBc reactive, diagnosed Non-A Non-B patients, chronic hepatitis B patients, patients with other viral or liver diseases, and patients containing possible interfering substances. Overall 160 specimens were positive by IMx and either EIA or RIA. Of the 4 discrepant samples, 2 specimens were repeatably IMx CORE-M gray zone reactive and EIA nonreactive but elevated, one specimen was IMx CORE-M gray zone reactive and RIA nonreactive but elevated, and one specimen was IMx CORE-M reactive and RIA retest range. These results are consistent with the fact that IMx CORE-M was slightly more sensitive than the RIA test.

IMx HBsAg Specificity. The specificity of the IMx HBsAg test

was demonstrated by assaying 5484 plasma and sera specimens from blood donors and comparing the results to AUSZYME Monoclonal. For plasma specimens, (n = lOOO), 0.00% were repeat reactive by IMx HBsAg or AUSZYME Monoclonal. For serum specimens, (n = 44841, IMx HBsAg was compared to AUSZYME Monoclonal procedure C (n = 2440) and procedure A (n = 2044). Ten of 4,484 serum specimens were repeatedly reactive by IMx HBsAg and AUSZYME Mono- clonal. All were confirmed positive by neutralization with high titer human anti-HBs. Three discrepants were observed between these tests: One specimen was repeatedly reactive by IMx and confirmed by neutralization but negative by AUSZYME Monoclonal and 2 specimens were negative by IMx and reactive by AUSZYME Monoclonal. These 2 specimens did not confirm by neutralization. A serum population histogram of results obtained by IMx HBsAg showed excellent separation between the population and assay cutoff (Fig. 1C).

Random specimens from diagnostic laboratories (n = 3094) were assayed by IMx HBsAg and either AUSZYME Monoclonal, AUSZYME 11, or AUSZYME IV. Overall agreement of 99.5% was observed (39781 3094). Ninety-six (3.1%) of these specimens were reactive by IMx HBsAg. More than 1000 specimens were tested in parallel with AUSZYME Monoclonal resulting in 4 discordant results, and more than 2,000 specimens were tested in parallel with AUSZYME I1 or AUSZYME IV with 12 discordant specimens. Of these 16 discrepant results, one was borderline reactive by AUSZY ME Monoclonal and confirmed positive, three were low level IMx reactive and confirmed positive, one was AUSZY ME Monoclonal reactive, non-confirming and 11 were IMx reactive, non-confirming.

Eight hundred thirty four additional specimens were tested by IMx HBsAg and AUSZYME Monoclonal. These included specimens from obstetrics and gyneco- logical patients (n = 500), from dialysis patients (n = 169) and from patients with infectious diseases (other than Hepatitis B) or whose serological profile contains potentially interfering substances (n = 165). Overall agreement of 99.6% (831/834) was observed between these two HBsAg assays. Three discordant

5 I I 1

; i : y 1 1 - c 2

1

...... ..... .......... ............................... ........................ ............... .............................. 0 Acute HB Chronic HE Recovered HB

Fig. 4. IMx CORE-M Index values of sera from hepatitis B patients classified as acute (n = 461, chronic (n = 76), and recovered (n = 67). Acute patients were HBsAg reactive for less than 6 months from onset of symptoms compared to chronic patients who had been HBsAg reactive for greater than 6 months. Recovered atients had no detectable HBsAg but were positive for both anti-HEc and anti-HBs.

To determine the onset and duration for detection of IgM anti-HBc, serial bleeds from 29 patients with known onset of hepatitis symptoms were tested by IMx CORE-M. The results from this study, in terms of time from onset of symptoms, are presented in Figure 5. The first sample available for each patient, representing 0 to 4 weeks from the onset of symptoms, was reactive by IMx CORE-M. Duration of IMx CORE-M reactivity varied from 1 week to 24 weeks from the onset of symptoms (mean = 12.1 weeks, SD = 5.3 weeks). The range for gray zone reactive 1Mx CORE-M results was 6 to 26 weeks from the onset of symptoms (mean = 17.1 weeks, SD = 5.0 weeks). These results indicate that the IMx CORE-M assay can detect IgM anti-HBc in patient samples from approximately 4 to 7 weeks from the time when HBsAg is first detected and a t the same time that total anti-HBc is detected by CORZYME. After strong reactivity for the first 6 to 8 weeks, IMx CORE-M Index values gradually decrease, usually becoming nonreactive within 3 to 5 months from the onset of symptoms. When appropriate samples were available for evaluation, there was no evidence of IgM anti-HBc reactivity beyond 6 months from the onset of symptoms.

Reproducibility. Assay reproducibility was determined with 5 samples containing IgM anti-HBc at varying concentrations. Samples were run in replicates of 3, in multiple runs on 3 instruments. Within-run, between-run, and total %CV ranged from 2.2 to 4.8,4.1 to 7.5, and 5.0 to 8.9, respectively (Table I). Current IgM anti-HBc EIA and RIA are similar to IgM anti- HAV tests in giving significantly higher assay vari- ability (CV up to 20%).

Clinical data. IMx CORE-M clinical studies were conducted in 2 hospitals and a reference lab. A total of 1,606 specimens were tested on IMx CORE-M and EIA or RIA. Overall agreement between IMx and RIA was (538/540) 99.6% and 1Mx and EIA was (1064/1066) 99.8%. Specimens were collected from the following categories: blood donors, hospital patients and employ-

146 Eble et al.

80

f 60 -

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40 Q Q Q

r ae

0 4 1 2 16 20 24 28 32 3 6 4 0 44

TIME (weeks from onset of symptoma)

Fig. 5. Se uential bleeds from 29 patients with known onset of hepatitis symptoms tested for IgM anti-HBc bylMx CORE-M and for HBsAg by AUSRIA. IMx CORE-M Index values for each bleed and % of bleeds positive for HBsAg during each 4 week interval are plotted versus time after onset of symptoms.

specimens were all low level IMx HBsAg reactive and confirmed by neutralization.

Sensitivity and detectibility. IMx HBsAg sensitivity was determined by testing the Abbott HBsAg sensitivity panel which has members containing low concentrations of HBsAg, subtypes ad and uy. Both the IMx HBsAg and AUSZYME Monoclonal assays were able to detect HBsAg concentrations less than 0.5 ng/ml or 0.2 PEI unitsiml (Table I). The Paris subtype panel (1975) containing the 9 known HBV subtypes were each detectable at less than 0.5 ng/ml using the IMx HBsAg assay (data not shown). To examine sensitivity further, end point titers were determined by making twofold serial dilutions of 10 HBsAg carriers. IMx HBsAg gave the same end point titers as AUSZYME Monoclonal for 7 specimens, gave 1 additional dilution for 1 specimen and gave one less dilution for 2 specimens. These data indicate that the IMx and EIA assays have very similar sensitivity.

Seventy-eight specimens obtained from chronic hepatitis B patients attending a sexually transmitted disease clinic were tested by IMx and AUSZYME Monoclonal. All 78 specimens were reactive by both procedures. An additional 161 HBsAg specimens (82 diagnosed as chronic carriers, 43 diagnosed as acute hepatitis B, and 36 unclassified as to disease state) were tested by IMx HBsAg and AUSZYME Monoclonal and also were found to be reactive by both methods.

Reproducibility. Assay reproducibility was determined by testing 9 specimens with varying HBsAg concentrations in duplicate at 4 sites on 6 instruments

over 3 separate days. Within-run, between-run, and total %CV ranged from 2.6 to 4.4,3.5 to 10.3, and 5.6 to 10.9, respectively (Table I).

Clinical studies review. A total of 9,700 specimens were assayed in the clinical study. Specimens tested included specimens from blood donors, diagnostic patients from a hospital and a public health laboratory, OBiGyn patients, dialysis patients, acute and chronic HBV patients, and patients with diseases other than active hepatitis B. The overall agreement between IMx HBsAg and the licensed reference method was 99.77%. Follow up testing on discordant specimens included confirmation test by neutralization and assay by AUSZYME Monoclonal or AUSRIA, if sample volume permitted. IMx HBsAg Confirmatory testing re- solved all discrepancies except one. This specimen was confirmed positive by IMx but had insufficient volume for further testing by AUSRIA.

IMx CORE-M and IMx HBsAg Testing of Serial Bleeds From Acute Hepatitis B Patients

To determine the onset and duration of IgG anti-HBc and HBsAg in acute HBV infected patients, sequential bleeds were obtained from 4 individuals who became infected with HBV while participating in a bleed study. These were tested by IMx HBsAg, IMx CORE-M, and CORZYME. Patients A, B, and D were tested for up to 12 months following the antigenemic phase of HBsAg infection. Patient C dropped out of the program during the antigenemic phase of the disease. The results from

Differential Diagnosis of Acute Viral Hepatitis

this study are presented in Figure 6 (A-D, respectively).

Patient A became HBsAg reactive on day 7 of the study and the last bleed in which HBsAg was detectable occurred 60 days later. All bleeds during this antigenemic period were reactive by both IMx HBsAg and AUSZYME Monoclonal. This patient was first detected as gray zone reactive by IMx CORE-M (Index = 0.813) 39 days after the first HBsAg positive bleed. A sample taken 9 days later was reactive by IMx CORE-M (Index = 2.197).

Patient B was low level HBsAg reactive at the beginning of the study and HBsAg levels rose to a peak 130 days into the study (IMx HBsAg S/N = 213). After this time, serum HBsAg concentrations slowly decreased until day 228 at which time HBsAg IMx was considered non reactive. Interestingly, S/N values for IMx HBsAg remained elevated up to day 248 indicating the possible presence of HBsAg in picogram/mL concentrations. Patient B was gray zone reactive by IMx CORE-M (Index = 1.069) 40 days after the first HBsAg positive bleed, and the next bleed was IMx CORE-M reactive.

Patient C dropped out of the program during the antigenemic phase while the HBsAg concentration was still rising (Fig. 6C). This patient was first detected as HBsAg positive by IMx and EIA assays on day 21 after entering the study and was initially gray zone reactive by IMx CORE-M (Index = 0.865) 27 days later (day 48 of study).

For all 3 patients the initial gray zone reactive IMx CORE-M results coincided with the first bleed detected as positive for total anti-HBc by CORZYME. The duration of IMx CORE-M reactivity (Index > 1.200) was approximately 2.5 months for Patient A and Pa- tient B, following by another month of gray zone reactive results (Index 0.800 to 1.200) in both cases. Since the last sample available for Patient C was an acute sample, the duration of IMx CORE-M reactivity could not be determined for this patient.

Patient D became IMx HBsAg reactive on day 4 of the study. A large gap existed between the time of initial HBsAg positivity and the next bleed 128 days later at which time this patient was still HBsAg positive and had become anti-HBc and IgM anti-HBc positive. This patient was followed serially for almost 1 year and finally became IMx HBsAg nonreactive 158 days after antigenemia was first detected. IgM Anti- HBc was detected as reactive by IMx CORE-M for almost 3 months; the patient specimen then became gray zone reactive and remained sporadically gray zone reactive for an additional 4 months.

DISCUSSION We demonstrate in this study that the IMx tests for

detection of HBsAg (IMx HBsAg), IgM anti-HBc (IMx CORE-M) and IgM anti-HAV (IMx HAVAB-M) show equivalent sensitivity, specificity and detectibility to current methods. A variety of specimens for patients with acute hepatitis A, acute and chronic hepatitis B,

147

random blood donors, patients with infectious diseases other than hepatitis A or B, and containing other serological components which are potential interfering materials were tested by these 3 IMx assays along with commercial EIA and RIA to determine the specificity of the IMx assays. All 3 IMx assays gave 99.8% agreement with current methods: for IMx HAVAB-M (1470/1473), IMx CORE-M (1604/1606), and IMx HBsAg (96781 9700). Results from these parallel studies clearly demonstrate the excellent correlation between the IMx assay and EIA and RIA.

Previous studies have demonstrated that an IgM anti-HBc is the only specific marker for the diagnosis of acute hepatitis B but this diagnosis depends on the proper assay sensitivity [Chau et al. 1983; Perrillo et al. 19831. If the assay is too sensitive IgM anti-HBc may be detectable for a year or longer after acute infection [Sjogren and Hoofnagle, 1985; Gerlich et al., 1986; Hoofnagle et al., 1985; Surrenti et al., 19861. The serial bleed data from Figures 5 and 6 indicate the utility of IgM anti-HBc for the diagnosis of acute hepatitis B infection and confirms the work of Gerlich et al. [1986] that there is a cutoff level for IgM anti-HBc which allows for differentiation of chronic and acute hepatitis B patients. As shown in Figure 4, all 46 acute hepatitis B patients had reactive IgM anti-HBc levels (Index > 1.2) whereas only 2 of 76 chronic carriers had levels above this value. The percentage of chronic hepatitis B patient samples that are reactive or gray zone reactive can vary and appears to be highest in HBeAg positive, chronic patients or chronic patients undergoing severe episodes of HBV reactivation compared to healthy HBsAg carriers [F. Bonino, personal communication].

In the IMx CORE-M assay, specimens testing in the gray zone reactive range (Index between 0.8 and 1.2) were observed most frequently very early in HBV infection or during the decline of IgM anti-HBc after infection. These data indicate that patients exhibiting gray zone levels of IgM anti-HBc should be closely monitored over time to distinguish rising IgM anti-HBc levels associated with acute infection from falling or unchanging IgM anti-HBc levels associated with recov- ery (Fig. 6). HBsAg was the first serological marker detected in four plasmapheresis patients tested pro- spectively. Total anti-HBc and IgM anti-HBc were detected early in infection during rising or peak levels of HBsAg in these patients. The rate of decline in HBsAg level measured by IMx HBsAg varied dramat- ically among patients. In Patient A HBsAg concentration dropped from peak level to negative in less than one month, contrasting with Patient D who showed a slow decline in HBsAg concentrations over a period of 100 days. Many laboratories have used HBsAg exclusively for the diagnosis of acute hepatitis B, but two types of errors can occur if IgM anti-HBc is not also used. In the first case, if the patient presents with symptoms during the late acute phase in which the serological window between disappearance of HBsAg and appearance of anti-HBs when anti-HBc IgG and IgM are the sole markers, then the false diagnosis of

148 Eble et al. 1000 7 30

1000

100

m 4

: 10

1

- ?

1

1

Days

Days

Fig. 6. A-D: Sequential bleeds from four plasmapheresis donors who became infected with hepatitis B. HBsAg (circles) and IgM anti-HBc (s uares) were measured at each time oint using IMx HBsAg and h x CORE-M. Data for IMx HBsAg and I K x CORE-M are presented as SIN and Index value, respectively. Filled symbols indi-

acute Non-A Non-B hepatitis infection may be made. The second error would be to overlook a superimposed non-B hepatitis infection in a chronic carrier. In several studies [Gerlich et al., 1986; Papaevangelou et al., 1984; Perrillo et al., 19831 a significant percentage of the HBsAg positive cases had no detectable IgM anti- HBc and therefore were not acute hepatitis B infections but may have been a superimposed delta or Non-A Non-B hepatitis infection since a high risk of acquired

cate values considered positive in the assay and open s mbols indicate values considered negative in the assay. Half-fillel squares show values considered gray zone reactive in the IMx CORE-M assay. The black bar indicates time of total anti-HBc reactivity measured by CORZYME.

Non-A Non-B hepatitis was known for some of the patients tested [Gerlich et al., 1986; Sjogren and Hoofnagle, 1985; Wang et al., 19841.

The best marker for acute hepatitis A infection is also an IgM response, IgM anti-HAV. Serial bleed studies from naturally and experimentally infected individuals indicate a sharp rise in IgM anti-HAV concentration from 30 to 35 days post infection with a more gradual decline in IgM concentration over a period of 3 to 4


Days

100 30

D 28

1

1

Days

Figure 6 (continued).

months during resolution of the disease (Fig. 3). Specimens testing in the gray zone reactive range

(Index between 0.8 and 1.2) for IMx HAVAB-M assay were observed exclusively early during acute HAV infection or during the decline of IgM anti-HAV post infection. Like IgM anti-HBc, the sensitivity of the assay has been tightly controlled to limit the detection of IgM anti-HAV to the acute phase of the illness (0 to 6 months). IgM anti-HAV concentration may decrease slowly with time after infection so that very sensitive assay with cutoffs set inappropriately low may detect

IgM anti-HAV up to 1 year or longer after infection. These data taken together confirm the utility of the

IMx HAVAB-M for the diagnosis of acute hepatitis A and combined usage of IMx CORE-M and IMx HBsAg for the diagnosis of acute hepatitis B infection. The major advantages of the IMx assays over conventional bead or microtiter tests are the speed in obtaining results (24 tests in less than 45 minutes), the full automation including specimen dilution steps required for the IgM assays, and the within-run and between- run reproducibility (Table I).

150 Eble et al.

ACKNOWLEDGMENTS We thank Joe Carlos, Shaun Helgesen, Scott Det-

wiler, Kathy O’Rourke, Marian Heavey, and William Phillips for their help in developing the IMx HAVAB-M and CORE-M assays. We also appreciate the hard work of Sue Washer, Kurt Wicklund, Christy Hillebrand, Wes Schmidt, and Bill Black in the development of IMx HBsAg. Tracy Wilson, Karen Sachs, and Willie Kiang performed essential clinical data analysis and support work. Finally, we thank Laurie Metz and Pat Haffer- kamp for typing the manuscript.

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differential diagnosis of acute viral hepatitis using rapid, fully automated immunoassays

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