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Journal of Clinical Microbiology, May 1998, p. 1461-1463, Vol. 36, No. 5
Département de Microbiologie
Médicale et Moléculaire,
Received 28 October 1997/Returned for modification 22 December
1997/Accepted 9 February 1998
Two methods for genotyping hepatitis C virus (DNA enzyme
immunoassay [DEIA] and line probe assay [Inno-LiPA HCV I and II]) were compared on 120 samples and of these 87% were assigned to the
same subtype by both assays. There were 15 subtyping discrepancies which involved 5% of type 1 isolates and 90% of type 2 isolates. Amplified products from the core and 5' untranslated regions (UTR) were
sequenced to resolve conflicts. Type 1 discordant samples had a
guanosine at position Hepatitis C virus (HCV) isolates
have diverse nucleotide sequences. They have been tentatively
classified into six major genotypes and about a dozen subtypes
(14). It is generally agreed that the HCV genotype affects
infection (10). Genotype 1b is most common in patients who
develop active chronic hepatitis C and cirrhosis (13).
Patients infected with HCV having this genotype also have higher plasma
concentrations of HCV RNA and are less responsive to interferon
(7). It is therefore very important to have reliable assays
for HCV genotyping. Two methods involving hybridization of PCR products
with genotype-specific probes have been recently described: the line
probe assay (LiPA) (15) and the DNA enzyme immunoassay
(DEIA) (9). We compared these assays, which involve
different regions of the HCV genome, by using a panel of sera from
French anti-HCV antibody-positive individuals. Amplified products were
sequenced, and the sequences were compared with the corresponding
sequences from databases to resolve any conflicts.
Sera were selected in 1994 from a consecutive series of 68,492 French
volunteers undergoing a routine medical checkup offered by the French
national health insurance system. Blood samples were sent to a single
laboratory (Institut Régional pour la Santé, Tours,
France), where alanine aminotransferase (ALT) activity was determined
and compared to a norm (N, equal to the mean value plus 2 standard
deviations) calculated for particular sex and age groups after
exclusion of values below the 2.5th percentile and above the 97.5th
percentile. Samples with ALT activities 20% above N were tested for
anti-HCV antibody by a third-generation enzyme immunoassay (Ortho HCV
3.0; Ortho Diagnostic Systems, Raritan, N.J.). It was found that 127 of
the 2,327 selected individuals were anti-HCV antibody positive.
HCV RNA was extracted from all sera testing positive in an
enzyme-linked immunosorbent assay, as described by François et al. (4). Reverse transcription-nested PCR (RT-nPCR) was
carried out for two regions of the HCV genome, the 5' untranslated
region (UTR) and core region with the primers provided by the two
genotyping kits (4). After the first PCR, amplified products
were analyzed by electrophoresis in a 1.5% agarose gel. nPCR was done
when no amplicons were detected. It was found that 121 of the 127 anti-HCV antibody-positive individuals (95.3%) were viremic as
demonstrated by RT-nPCR with primers from both the 5' UTR and the core
region. Two genotyping methods were used, LiPA (Inno-LiPA HCV
[LiPA-I] kit; Innogenetics, Zwijnaarde, Belgium) and DEIA (GEN-ETI-K
DEIA kit; Sorin, Saluggia, Italy) with amplicons from the 5' UTR and core region, respectively. The LiPA-I kit included 17 probes: generic 1 (2 probes), 1a, 1b, generic 2 (2 probes), 2a (2 probes), 2b (2 probes),
3a (4 probes), and 4/5 (3 probes) (15). The DEIA kit
included six probes: 1a, 1b, generic 2, 2a, 2b, and 3a probes. Experimental probes for genotypes 4, 5, and 6 were also evaluated. Sorin recommended that any sample reacting only with probe 2 should be
regarded as being of subtype 2c. This information is not included in
the kit leaflet.
It was found that 120 of the 121 isolates (99.2%) could be genotyped
by both methods. The overall agreement between LiPA-I and DEIA results
was satisfactory, with 105 of 120 samples (87.5%) yielding concordant
results (Table 1). There were 15 subtyping discrepancies, which involved 5% of type 1 isolates and 90%
of type 2 isolates. Four isolates were classified 1b by LiPA-I but 1a
by DEIA. One type 1 sample could not be subtyped by LiPA-I, whereas
DEIA assigned it to subtype 1b. Nine samples were classified 2a by
LiPA-I but 2c by DEIA. LiPA-I gave an ambiguous result (2a/2b) for one
isolate, whereas DEIA assigned it to subtype 2b. One sample could not
be genotyped, although the patient was viremic and PCR products were
obtained for both regions.
Samples yielding dubious LiPA-I results or results discordant with
those of DEIA were further tested with LiPA-II (Inno-LiPA HCV II;
Innogenetics). The LiPA-II kit contained 16 probes similar to those of
the LiPA-I kit and 5 additional probes, 3 of which were specific for
genotypes 4, 5, and 6 (18). LiPA-II reassigned the
unspecified type 1 sample to subtype 1b and the 2a/2b sample to subtype
2b. Both results were consistent with those obtained with DEIA. Four of
the five isolates assigned to genotype 4/5 by LiPA-I were classified
genotype 4 by DEIA; the other was classified genotype 5. The extra
probes of the LiPA-II kit gave similar results: four isolates were
classified genotype 4c/4d, and one isolate was classified genotype 5a.
However, LiPA-II did not resolve any other discordant genotype
assignments.
5' UTR-based LiPA-I and LiPA-II rely on a single nucleotide difference
between 1a and 1b subtypes (position 5' UTR-based LiPA-I and LiPA-II cannot distinguish the 2a and 2c
subtypes because their sequences are identical in this domain (2). In contrast, the core regions of the 2a and 2c subtypes differ by 20 nucleotides in the region covered by the DEIA amplicons. Nine of our isolates were subtyped 2c by DEIA but assigned to 2a by
LiPA-I and to 2a/2c (n = 8) or generic type 2 (n = 1) by LiPA-II. The 5' UTR and core regions of
eight samples were sequenced. All eight samples had the same 5' UTR
sequence but had divergent core sequences. A comparison with the
consensus 2a and 2c sequences showed that only two samples had
sequences more similar to that of 2c than 2a (patients 7 [P < 0.01] and 11 [P < 0.02]).
Phylogenetic analyses of these eight samples and 55 published sequences
including those of isolates representative of the 2a to 2f subtypes and type 2 sequences of unidentified subtype were carried out by using a
neighbor-joining algorithm (11), the maximum-likelihood
method (options QFYG; fdnaml program of G. J. Olsen, University of
Illinois, Urbana), and the maximum-parsimony method (PAUP version 3.0s
for the Macintosh computer; heuristic search). The robustness of each topology was checked by a neighbor-joining method and 500 bootstrap replications. Our isolates clearly belonged to genotype 2 but could not
be grouped into the previously defined subtypes (Fig. 1). These data confirm that type 2 is
frequently mistyped by rapid-genotyping methods, including LiPA and
DEIA (19). Contrary to the recommendations of DEIA's
manufacturer, samples which react only with generic probe 2 should not
be considered subtype 2c but unspecified type 2 (6, 20).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Comparison of DNA Enzyme Immunoassay and Line Probe
Assays (Inno-LiPA HCV I and II) for Hepatitis C Virus
Genotyping
ABSTRACT
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99 in the 5' UTR, a characteristic of genotype
1b, and a core region typical of subtype 1a. The eight isolates
classified as 2a/2c by LiPA and as subtype 2c by DEIA belonged to type
2.
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TABLE 1.
Comparison of the two genotyping assays
99). This tenuous distinction
may lead to the mistyping of 2 to 10% of genotype 1 samples (1,
5, 17). In contrast, the core regions of the 1a and 1b subtypes
differ by 17 nucleotides, and DEIA probes cover 4 of these
subtype-specific nucleotides. To determine the subtypes of our
discordant type 1 samples, we sequenced part of the 5' UTR (207 nucleotides [272 to 66]) and the core region (203 nucleotides
[159 to 361]) by the dideoxynucleotide chain termination method
(12). The sequencing of three of the samples classified as
1a by DEIA but as 1b by LiPA-I and LiPA-II showed that they had a
guanosine at position 99 in the 5' UTR, a characteristic of genotype
1b, but that their core sequences were more similar to the subtype 1a
consensus sequence (97%) than to the subtype 1b consensus sequence
(90%; P < 0.01).
View larger version (28K):
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FIG. 1.
Unrooted phylogenetic tree of 63 HCV type 2 isolates
derived from an analysis of partial core sequences (nucleotides 159 to
361) retrieved from the EBI database or by using the Entrez software
from the National Center for Biotechnology Information (about 600 sequences). The consensus sequences of types 2a, 2b, and 2c were
obtained from Bukh et al. (2), and sequences 1F.03, 1F.48,
and RC.12 were from Cammarota et al. (3). Sequences were
aligned manually, and preliminary phylogenetic analyses were used to
identify a subset of 120 sequences representative of each monophyletic
taxon. More detailed analyses were restricted to type 2 sequences. The
topology was obtained by a maximum-likelihood method (double asterisks
indicate branches at P < 0.01). Monophyletic taxons
also retrieved in the most-parsimonious tree are indicated by a plus
sign. Percentages show branches identified according to a bootstrap
resampling (500 replicates) by neighbor-joining analysis. Sequences
obtained in this study are shaded.
Some reports have assigned new HCV type 2 subtypes after limited phylogenetic analyses performed with only a few sequences (8, 16). Our analysis was based on about one-third of the HCV core region and gave a clear separation of genotypes 2a and 2b. These results are consistent with previous studies covering the entire core region (2). In contrast, subtype 2c seems to be a largely paraphyletic group, making any assignment quite weak. Our isolates that supposedly belonged to subtype 2c intermingled with various other type 2 subtypes such as 2d, 2e, and 2f. Investigations covering other domains of the HCV genome such as E1 and NS5 and more complete phylogenetic analyses involving a large number of type 2 sequences are required for accurate classification of this heterogeneous HCV type.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Association pour la Recherche sur le Cancer, Villejuif, France (ARC 415/94), and the Association Recherche & Partage des Caisses d'Epargne Ecureuil, Paris, France.
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FOOTNOTES |
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* Corresponding author. Mailing address: Département de Microbiologie Médicale et Moléculaire CNRS EP 117-CHU Bretonneau, 2 Boulevard Tonnellé, 37044 Tours Cedex, France. Phone: (33) 2 47 47 69 97. Fax: (33) 2 47 47 36 10. E-mail: gondeau{at}med.univ-tours.fr.
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Journal of Clinical Microbiology, May 1998, p. 1461-1463, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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