JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1999, p. 233–234 Vol. 37, No.

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0095-1137/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Definition of False-Positive Reactions in Screening for

Hepatitis C Virus Antibodies
MATTHIAS SCHRÖTER,* HEINZ-HUBERT FEUCHT, PETER SCHÄFER,
BERNHARD ZÖLLNER, SUSANNE POLYWKA, AND RAINER LAUFS
Institut für Medizinische Mikrobiologie und Immunologie,
Universitäts-Krankenhaus Eppendorf,
20246 Hamburg, Germany
Received 21 January 1998/Returned for modification 6 May 1998/Accepted 13 October 1998

Downloaded from http://jcm.asm.org/ on October 1, 2020 by guest

The rate of false-positive hepatitis C virus enzyme immunoassay results was determined to be at least 10%
among 1,814 reactive serum samples based on (i) negative results in an independent confirmation assay, (ii)
negative PCR results, and (iii) no patients developing clinical or biochemical signs of hepatitis during a 1-year
follow-up.

In daily laboratory routine, reliable diagnosis of hepatitis C
virus (HCV) infection is not always possible by sole use of an
HCV enzyme immunoassay (EIA), since it is well known that
for a number of patients this assay produces false-positive
results (4, 8, 17). Therefore, results obtained by EIA need to be
confirmed by additional testing. However, the commercially
available assay RIBA 2.0 (Chiron Corporation) does not fulfill
the criteria defining a confirmation assay since it consists of
recombinant proteins identical to those in the EIA (1, 5, 9).
HCV PCR cannot be used for confirmation of positive EIA
results, since a negative PCR result does not exclude the pos-
sibility of HCV infection with low-level viremia (below the
limit of detection). Furthermore, PCR is too laborious and
expensive to be used regularly as a confirmatory assay. There-
fore, we have established an HCV strip immunoblot assay
(SIA) (Universitäts-Krankenhaus Eppendorf [UKE] SIA) con-
sisting of four recombinant proteins, derived from the core and
three nonstructural regions (NS3, NS4, and NS5) of HCV,
which are different from those used in the HCV EIA (5).
In the present study we compared the results of a second-
generation HCV EIA with those of the UKE SIA for 2,283
serum samples. The aim was to assess the significance of pos-
itive results in the HCV EIA to define criteria for the perfor-
mance of further tests to reliably diagnose HCV infection in
the daily laboratory routine. Sera were drawn from 2,283 per-
sons living in northern Germany around the city of Hamburg.
They were sent to our laboratory under suspicion of HCV
infection due to either elevated liver enzyme values (alanine
aminotransferase, .45 U/liter) or clinical signs of hepatitis
(jaundice and upper abdominal pain) or risk factors for par-
enterally transmitted diseases, such as chronic hemodialysis,
blood transfusion, or intravenous drug use. At the time of
investigation they tested negative for acute infection with HAV
(anti-HAV immunoglobulin M antibodies) and HBV (hepati-
tis B surface antigen). Repeated examinations were performed
as follow-up every 3 months for 1 year. For serological screen-
ing a second-generation HCV EIA (Abbott Laboratories,
North Chicago, Ill.) was performed. For confirmation of HCV
EIA results, sera were tested in parallel by the UKE SIA as
* Corresponding author. Mailing address: Institut für Medizinische
Mikrobiologie und Immunologie, Universitäts-Krankenhaus Eppen-
dorf, Martinistr. 52, 20246 Hamburg, Germany. Phone: 49-40.47173159.
Fax: 49-40.47174062. E-mail: mschroeter@uke.uni-hamburg.de.
previously described (5). The immunoblot assay was consid-
ered positive when antibodies to at least two different recom-
binant proteins were detectable. Reactivity against only a sin-
gle protein was rated as an indeterminate result. For detection
of HCV RNA reverse transcription-PCR was performed as
previously described (6, 7).
The HCV EIA was negative for 469 samples, of which 456
(97%) were also negative by UKE SIA. For 13 samples the
UKE SIA was considered indeterminate. All 469 of these sera
were negative by HCV PCR, and none of the patients devel-
oped clinical or biochemical signs of hepatitis during the fol-
low-up.
The HCV EIA was reactive for 1,814 samples, of which
1,394 (77%) were also positive by the UKE SIA (Table 1).
However, in 240 cases (13%) the reactivity in the HCV EIA
could not be confirmed by UKE SIA. Suitable specimens for
HCV PCR were available for 193 of these 240 samples, and a
positive PCR result was obtained with 13 samples. Of these,
nine became positive by UKE SIA when retested after 3
months, which suggests that these patients had acquired HCV
infection shortly prior to the first examination. In the remain-
ing four patients, who repeatedly tested PCR positive despite
a negative result by UKE SIA, immunosuppressing conditions
could be found. One had a B-cell lymphoma, one was chron-
ically hemodialyzed, and two practiced intravenous drug use. It
has been shown earlier that in patients with immunosuppres-
sive conditions, serological response is low or even absent (10,
14, 15). This could lead to negative or indeterminate results in
serological assays although the individual suffers from infec-
tion with HCV (13). Therefore, for patients with known im-
munosuppressive disorders PCR should always be performed.
The 180 initially PCR-negative subjects remained negative by
UKE SIA and HCV PCR in repeated examinations during the
follow-up. Moreover, these patients did not develop clinical or
biochemical signs of hepatitis. This indicates that in at least
these 180 samples (10%), false-positive results occurred. We
must assume that the EIA was also false positive in the spec-
imens for which no suitable material for PCR was available,
since the UKE SIA remained negative and none of the patients
developed clinical or biochemical signs of hepatitis during the
follow-up. This indicates that as long as no better screening
assays are commercially available every positive HCV EIA
result must be confirmed.
An indeterminate result in the UKE SIA was observed with

233
234 NOTES
TABLE 1. Comparison of results of Abbott second-generation
HCV EIA and UKE SIA for 2,283 serum samples
No. (%) of samples with HCV EIA result 2.
UKE SIA result
(n 5 2,283) Positive Negative
(n 5 1,814) (n 5 469)
Positive 1,394 (77) 0 3.
Indeterminate 180 (10) 13 (3)
Negative 240 (13) 456 (97)
4.
5.
180 of the 1,814 EIA-positive samples (10%). Suitable speci-
mens for HCV PCR were obtained for 134 of these 180 sam-
ples, and HCV RNA could be detected in 58 of them. During 6.
the follow-up full seroconversion was observed in four pa-
tients. All of them initially revealed antibodies directed 7.
solely against the NS3 protein of the UKE SIA. In follow-up
samples, reactivity against additional recombinant proteins
emerged. These results support the previous assumption that
8.
antibody reactivity against NS3 plays an important role in the
early serological detection of HCV infection (5). Furthermore,
a particularly high correlation has been found between HCV
viremia and antibody reactivity against the c33c antigen of the 9.
commercially available RIBA (2). Samples with a positive re-
sult by HCV EIA and an indeterminate result by immunoblot
assay must be subjected to PCR, since we detected HCV RNA
in 43% of samples (58 of 134). The percentage of indetermi- 10.
nate results by the UKE SIA is remarkably low compared to
that by RIBA 2.0 or 3.0 (2, 3, 11, 16). One reason for this might
be that local isolates were used to establish the UKE SIA, since 11.
serological tests containing recombinant proteins of local iso-
lates have been shown to have better sensitivity and specificity
than commercially available assays (5, 12). However, this is 12.
unlikely to be the only reason, since the UKE SIA was evalu-
ated with serum samples containing a variety of HCV geno-
types as previously described (5). 13.
The diagnosis HCV positive has a deep impact on the life of
the afflicted person. Therefore, it must be reached as reliably
as possible. Our data indicate that the widely used HCV EIA
produces a high percentage (10%) of false-positive results. 14.
Compared to other screening assays, e.g., human immunode-
ficiency virus EIAs, this is unacceptably high. Therefore, con-
firmation of every positive HCV EIA result by supplemental 15.
tests is mandatory. As we have shown with our in-house UKE
SIA, one possibility for improving the reliability of HCV diag- 16.
nosis is to introduce proteins into the confirmation assay which
are different from those used in the screening assay.
17.
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