JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2008, p. 192–197
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 1
Evaluation of a New Assay in Comparison with Reverse Hybridization
and Sequencing Methods for Hepatitis C Virus Genotyping Targeting
Both 5? Noncoding and Nonstructural 5b Genomic Regions?
Elisa Martro ´,1,2† Victoria Gonza ´lez,1,2† Andrew J. Buckton,3Vero ´nica Saludes,1,2Gema Ferna ´ndez,1
Lurdes Matas,1,2Ramo ´n Planas,4,5and Vicenc ¸ Ausina1,6*
Microbiology Department, Hospital Universitari Germans Trias i Pujol, Universitat Auto `noma de Barcelona, Badalona, Spain1; Centro de
Investigacio ´n Biome ´dica en Red de Epidemiologı ´a y Salud Pu ´blica (CIBERESP), Barcelona, Spain2; Virus Reference Department,
Health Protection Agency, London, United Kingdom3; Liver Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain4
Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Hepa ´ticas y Digestivas (CIBEREHD), Barcelona, Spain5; and
Centro de Investigacio ´n Biome ´dica en Red de Enfermedades Respiratorias (CIBERES), Bunyola, Mallorca, Spain6
Received 14 August 2007/Returned for modification 2 October 2007/Accepted 25 October 2007
We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design
Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5? noncoding region (5?NC) (method
1; HCV genotyping analyte-specific reagent assay). This method was compared with 5?NC reverse hybridization
(method 2; InnoLiPA HCV II) and 5?NC sequencing (method 3; Trugene HCV 5?NC). Two hundred ninety-five sera
were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic
analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were
confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method
1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results,
five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2,
and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct)
and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by
methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within
genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the
possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret,
thus providing a good single-step alternative to more-time-consuming assays.
Hepatitis C virus (HCV) is the most important cause of
chronic liver disease and is the leading indication for liver
transplantation (2). It is estimated that HCV infects 3% (170
million people) of the world’s population (26). HCV possesses
a positive-sense single-stranded RNA genome of approxi-
mately 9.5 kb, which is flanked by noncoding (NC) regions.
The HCV genome encodes at least 11 proteins, which include
both structural and nonstructural (NS) proteins (5, 7).
HCV is known to have a high rate of genetic heterogeneity.
This has allowed HCV strains to be classified into a number of
genetically distinct groups, known as genotypes, subtypes, iso-
lates, and quasispecies (5). Genome sequence heterogeneity
arises due to poor fidelity of the viral polymerase during rep-
lication. Sequence variability is not evenly distributed through-
out the genome. The lowest sequence variability is found in the
5?NC region, which is the target of choice for many molecular
diagnostics assays, including genotyping tests. Nevertheless,
nucleotide sequencing coupled with phylogenetic analysis of
more-variable genomic regions has been recommended for
HCV genotyping in consensus proposals (27). As patients in-
fected with different genotypes respond differently to antiviral
drug therapy, identification of the infecting genotype has be-
come important to guide the correct dose and duration of
current combination therapy (pegylated alpha interferon plus
ribavirin) (13, 19, 23).
A new HCV genotyping method (HCV genotyping analyte-
specific reagent [ASR] assay; Abbott Molecular Inc., Des Plaines,
IL) based on real-time PCR technology has recently been com-
mercialized. This assay amplifies a portion of the HCV genome
and assigns genotype in a single step using primers and probes
targeting the NS5b region for genotypes 1a and 1b and the 5?NC
region for genotypes 2a, 2b, 3, 4, 5, and 6. The aim of our study
was to compare this new assay to two previously commercialized
genotyping methods based on the 5?NC region: the TruGene
HCV 5?NC genotyping kit (Bayer HealthCare, Berkeley, CA),
based on semiautomated sequencing; and the Inno-LiPA HCV II
assay (Innogenetics, Gent, Belgium), based on automated reverse
hybridization. Discrepant results were resolved though NS5b se-
quencing and phylogenetic analysis, and suspected mixed-geno-
type infections were confirmed through PCR cloning and pyro-
MATERIALS AND METHODS
Patients and study design. Serum specimens from 295 patients with chronic
hepatitis C submitted to the Microbiology Department for clinical HCV geno-
typing were included in the study. One hundred fifty-eight of them were retro-
* Corresponding author. Mailing address: Microbiology Depart-
ment, Hospital Universitari Germans Trias i Pujol, Ctra de Canyet, s/n.
08916 Badalona, Spain. Phone: 34 934978 894. Fax: 34 934978 895.
† Equal contributors.
?Published ahead of print on 7 November 2007.
spectively selected from 2001 to 2004 and comprised similar numbers of com-
monly detected genotypes (1a, 1b, 2, 3, and 4), as well as all available genotype
5 and possible mixed-genotype infections. These specimens had already been
genotyped by the TruGene HCV 5?NC genotyping kit (n ? 86), the Inno-LiPA
HCV II assay (n ? 89), or both (n ? 17). For this study, sera were typed using
the real-time PCR HCV genotyping ASR method. Another 137 consecutive sera
were prospectively typed both by the TruGene HCV 5?NC genotyping kit and by
the HCV genotyping ASR assay. Seven quality control for molecular diagnostics
(QCMD) specimens (QCMD; Glasgow, Scotland) were also included.
HCV RNA extraction and RT-PCR. For 5?NC sequencing and reverse hybrid-
ization, viral RNA extraction, reverse transcription (RT), and PCR amplification
procedures were performed using 200 ?l of serum with the Cobas Amplicor
HCV test (Roche Molecular Systems, Pleasanton, CA) according to the manu-
facturer’s instructions. For the real-time PCR method, RNA was extracted from
220 ?l of serum using a QIAamp viral RNA Mini kit (Qiagen GmbH, Hilden,
Germany) following the manufacturer’s protocol. Amplification and detection
were performed on an ABI PRISM 7000 sequence detection system (Applied
Biosystems, Foster City, CA) according to the manufacturer’s instructions.
HCV genotyping. (i) Inno-LiPA HCV II reverse hybridization assay. The
Inno-LiPA HCV II reverse hybridization assay is based on the reverse hybrid-
ization principle. Briefly, biotinylated PCR product from the 5?NC region was
hybridized to specific immobilized probes. Detection was performed though an
enzymatic reaction resulting in a purple precipitate, which was visually inter-
preted. Hybridization and detection steps were performed on the Auto-LiPA
instrument (Tecan Group Ltd., Salzburg, Austria), following the manufacturer’s
instructions. The probes in this assay allow the identification of the following
types: 1, 1a, 1b, 1ab, 2, 2b, 2a/2c, 3, 4, 5a, and 6a.
(ii) TruGene HCV 5?NC genotyping assay. The TruGene HCV 5?NC geno-
typing assay is based on semiautomated sequencing. Amplified products were
purified with a QIAquick PCR purification kit (Qiagen) according to the man-
ufacturer’s instructions. Briefly, bidirectional DNA sequencing was performed
using two sequencing primers labeled with different fluorescent dyes, followed by
electrophoresis and data analysis on the OpenGene DNA sequencing system
(Bayer HealthCare). Each bidirectional sequence was automatically aligned to a
panel of reference sequences with the GeneLibrarian module of the Gene-
Objects software (Bayer HealthCare), allowing genotype assignment based on
percent sequence identity.
(iii) HCV genotyping ASR assay. The HCV genotyping ASR assay is based on
real-time PCR technology and includes probes labeled with different fluorescent
dyes specific for genotypes 1a and 1b targeting the NS5b region, as well as
primers and probes for genotypes 2a, 2b, 3, 4, 5, and 6 targeting the 5?NC region.
Genotype is determined with three reactions per specimen; the mixture for
reaction A contains primers and probes that recognize all genotypes to confirm
the presence of HCV RNA and primers and probes for genotypes 1a and 1b; the
mixture for reaction B contains primers and probes for genotypes 2a, 2b, and 3;
and the mixture for reaction C contains primers and probes to identify genotypes
4, 5, and 6. The assay was performed according to the manufacturer’s instruc-
tions. Briefly, RNA extract was added to each of three master mixes containing
primers and probes, manganese reagent, and Z05 DNA polymerase. Following
the RT step, 50 cycles of real-time PCR were performed; both steps were carried
out by use of the ABI Prism 7000 sequence detection system. Results were
analyzed using sequence genotyping software (SGS) v2.0 (Celera Diagnostics,
Alameda, CA). Two or more positive amplification signals are identified as a
mixed-genotype infection, when genotype cycle threshold values are within three
PCR cycles of each other.
Indeterminate specimens by this assay were tested with the HCV genotyping
ASR 5? NC region assay. Similarly, primers and probes targeting the 5?NC region
are designed to distinguish among 1b, 1 but non-1b, and non-1 infections in a
single PCR. Results were interpreted with the sequence genotyping 5? NC region
software v2.0 (Celera Diagnostics, Alameda, CA).
(iv) N55b sequencing and phylogenetic analysis. Samples with discrepant
results at the genotype or subtype levels were tested by NS5b sequencing. HCV
RNA was isolated from 220 ?l of serum by use of a QIAamp viral RNA kit
(Qiagen). RT was performed with 22 ?l of extracted RNA in a total reaction
volume of 40 ?l containing PCR buffer, 5 mM MgCl2,1 mM of each deoxynucle-
otide triphosphate, 0.008 U of random hexamers, 13.6 U of RNasin (Promega,
Mannheim, Germany), and 200 U of Moloney murine leukemia virus reverse
transcriptase (Invitrogen, Paisley, United Kingdom). Reaction mixtures were
incubated at 37°C for 45 min using a Gene Amp PCR system 9700 instrument
NS5b amplification was performed by nested PCR. The first round of ampli-
fication was carried out with 15 ?l of the HCV cDNA in a final reaction volume
of 50 ?l containing PCR buffer, 2.5 mM MgCl2, 10 pmol of primers p1203 and
p1204 (20), and 1 U of Taq polymerase (Invitrogen). Amplification was per-
formed on a Gene Amp PCR system 9700 under the following conditions: 94°C
for 30 s; 35 cycles at 94°C for 30 s, 54°C for 40 s, and 72°C for 50 s; and a final
elongation step at 72°C for 30 s. The second round of amplification was per-
formed adding 2 ?l of the first-round product into 48 ?l of PCR master mix
containing 46 ?l of MegaMix Blue (Microzone Limited, West Sussex, United
Kingdom) and 20 pmol of each primer (NS5-b n2? [5?TGATACCCGCTGCTT
TGACTCNACNGTCAC] and P1204). The following cycling parameters were
used: 94°C for 5 min; 30 cycles at 94°C for 30 s, 60°C for 40 s, and 72°C for 50 s;
and a final elongation step at 72°C for 30 s. PCR products were purified using a
QIAquick PCR purification kit (Qiagen) following the manufacturer’s protocol.
Amplified products were checked by electrophoresis on 2% agarose gels stained
with ethidium bromide. The concentration and purity of DNA were measured
with a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wil-
PCR products were sequenced with a BigDye Terminator cycle sequencing ready
reaction kit (Applied Biosystems) according to the manufacturer’s protocol. Three
reactions were performed for each specimen with the forward primer NS5-b n2? and
reverse primers 1204 and 123 (5?GCTCTCAGGTTCCGCTCGTCCTCC). Se-
quencing reaction products were purified with Clean Seq Dye-Terminator removal
kit (Agencourt Bioscience Corporation, Beverly, MA) according to the manufactur-
er’s instructions and then sequenced with an ABI PRISM 3100-Avant instrument
Consensus sequences were obtained using SeqScape v2.1.1 (Applied Biosys-
tems) from forward and reverse primers and aligned with genotyped reference
sequences from GenBank by the Clustal W algorithm implemented in MEGA
software package v3.1 (16). A region of 222 nucleotides within the PCR product
(positions 8316 to 8537 according to reference sequence M62321) was used for
phylogenetic analysis. Nucleotide distances were computed on MEGA by the
p-distance algorithm, and phylogenetic trees were inferred using the neighbor
joining method. The robustness of tree branches was tested by bootstrap analysis
(v) PCR cloning and pyrosequencing. Suspected multiple-genotype infections
were confirmed for previously genotyped sera by use of the methodology de-
scribed in detail in a previous study (4). Briefly, after amplification of a fragment
from the 5?NC region, products were checked by gel electrophoresis and treated
with genotype-specific restriction endonucleases to digest the dominant geno-
type. Residual amplicons were purified and subjected to PCR cloning. Ten to 15
transformant colonies were used to generate a biotinylated PCR amplicon, which
acted as the template for real-time DNA sequencing, using pyrosequencing
(Biotage, Uppsala, Sweden). Generated sequences from minority genotypes
were identified by alignment to reference sequences and BLAST analysis (3).
Statistical analysis. Agreement between pairs of techniques was assessed
through the ? coefficient (assuming a good concordance for ? values between
0.61 and 0.80) when both methods classified specimens into the same categories.
Otherwise, Cramer’s V was computed. Spearman’s rho (?) coefficient was also
calculated to determine the correlation among all methods. Fisher’s exact test
was used to assess agreement among methods used in the retrospective study to
those of the prospective study. All statistical analyses were performed with SPSS
v14.0 software, and a significance level of 0.05 was used.
No statistically significant differences in agreement among
the three techniques (real-time PCR, reverse hybridization,
and 5?NC sequencing) were found when the results from ret-
rospective and prospective studies were analyzed separately
(P ? 0.518) (data not shown). Thus, subsequent analyses were
based on the analysis of the whole sample (n ? 295). Overall
results are shown in Table 1.
Among the 295 specimens tested by real-time PCR, a total
of 13 indeterminate results were obtained, including eight of
genotype 1, three of genotype 2, and two of genotype 3, despite
the presence of high viral loads (69,200 IU/ml to ?700,000
IU/ml). Genotype 1 specimens were retested by the real-time
PCR 5?NC region assay, and a genotype was assigned to seven
of them, while one sample remained indeterminate (subtyped
as 1b by NS5b sequencing). Thus, a genotype call was obtained
in 289 (97.9%) cases. Due to our study design, we could not
VOL. 46, 2008 NEW REAL-TIME PCR METHOD FOR HCV GENOTYPING193
compare this rate to those of the other methods in the retro-
spective study, since only specimens with a previous genotype
call by the other methods were selected. In the prospective
study, none of the 137 specimens tested by 5?NC semiauto-
mated sequencing were indeterminate.
Concordance at the genotype level. For the analysis of ge-
notype concordance between techniques, only sera with a ge-
notype call by ?2 methods (n ? 289) were included in our
analyses (indeterminate results were excluded). Results ob-
tained by real-time PCR agreed well with those obtained by
5?NC sequencing (214/220 concordant results; Cramer’s V ?
0.849; P ? 0.05) and reverse hybridization (85/86 concordant
results; ? ? 0.982; P ? 0.05). Correlation coefficients were also
high (Spearman’s ? values of 0.916 and 0.956, respectively).
Although the number of specimens processed both by 5?NC
sequencing and reverse hybridization was small, correlation
was total (17/17 concordant results; ? ? 1; ? ? 1).
Among eight (2.8%) discordant results at the genotype level
(Table 2), five mixed-genotype infections were confirmed by
cloning and pyrosequencing and/or QCMD. Two of them were
detected by reverse hybridization, two by 5?NC sequencing,
and one by real-time PCR. When specimen 2 (genotype 3/5
confirmed mixed infection shown in Fig. 1) was tested by real-
time PCR, genotype 5 amplified strongly, while genotype 3
amplified 5.8 cycles later than the dominant genotype; there-
fore, mixed infection was not identified by the software.
Among the three remaining discordant specimens, mixed-
genotype infection could not be confirmed for two 1/4 mixed
infections detected by real-time PCR, and specimen 8 had
insufficient volume for further analysis. However, when the
latter specimen was tested by real-time PCR, genotype 1b
amplified nine cycles later than genotype 4, and NS5b sequenc-
ing confirmed the presence of genotype 1b in this specimen.
The other five QCMD specimens were single-genotype in-
fections and were correctly genotyped by both 5?NC sequenc-
ing and real-time PCR (these specimens were not tested by
reverse hybridization since they belonged to the prospective
Concordance at the subtype level. Regarding genotype 1
infections, the subtyping efficiency for the real-time PCR
TABLE 1. Overall comparison of results at the genotype level according to the method used
No. of specimens of indicated genotype bya:
5?NC and NS5b real-time PCR5?NC sequencing
123451/4 Indeterminate123 1/4
aResults in boldface represent consensus between the two methods.
TABLE 2. Specimens with discordant results at the genotype level among the three compared methods and corresponding
Compared methodConfirmatory method
5?NC sequencing5?NC and NS5b real-time PCR
a-, not performed.
bGenotype 3 amplified 5.8 cycles later than genotype 5.
cGenotype 1b amplified nine cycles later than genotype 4. None of these results was given as a mixed-genotype infection by SGS v2.0 software.
dNA, not applicable.
194 MARTRO ´ ET AL. J. CLIN. MICROBIOL.
method was 100% (163/163 genotype 1 infections), while the
5?NC sequencing method assigned a subtype to 102 of 132
genotype 1 infections (77.3%); the remaining 30 specimens
were 1a (n ? 14), 1b (n ? 15), or indeterminate (n ? 1) by
real-time PCR (the last of these was confirmed as genotype 1b
by NS5b sequencing). Similarly, among 46 genotype 1 infec-
tions by reverse hybridization, only 34 (73.9%) were assigned a
subtype; the other 12 specimens were genotype 1ab by this
method, and all were 1a by real-time PCR.
All specimens with a subtype assigned by ?2 genotyping
methods were compared, and discordant subtypes were con-
firmed by NS5b sequencing. Among genotype 1 specimens with
a subtype assigned both by real-time PCR and 5?NC sequenc-
ing (n ? 101), 11 (10.9%) gave discordant results (Table 3).
One of them was correctly typed as 1b by 5?NC sequencing and
typed as 1a/1b by real-time PCR. Among the other specimens,
the real-time PCR method was predominantly correct (9/10
cases) according to the NS5b sequencing. Similarly, one of the
two discordant results obtained for the 34 specimens subtyped
both by real-time PCR and reverse hybridization was typed as
1a/1b by the former assay. The other one was identified as a
1a/4 mixed infection by reverse hybridization that was con-
firmed as a 1/4 mixed infection by cloning and pyrosequencing,
but genotype 1 in that specimen actually belonged to subtype
1b instead of 1a according to NS5b sequencing and real-time
Regarding the genotype 2 infections, subtyping efficiencies
were 9/20 (45%) for 5?NC sequencing, 12/13 (92.3%) for re-
verse hybridization, and 27/27 for real-time PCR. Three out of
the 30 specimens genotyped as genotype 2 by either reverse
hybridization or 5?NC sequencing were indeterminate by real-
time PCR. NS5b sequencing and phylogenetic analysis showed
that two of these specimens clustered together and did not
group with any of the confirmed genotype 2 subtypes. This
potentially reveals that they may belong to a putative new
subtype within genotype 2. Further investigations are ongoing.
For the other specimen, there was an insufficient volume of
serum remaining to permit further analyses. Comparison of
subtype discrimination across all specimens showed that 16
were discordant (Table 4). NS5b sequencing revealed that
probes 2a and 2ac from the real-time PCR and the reverse
hybridization assays, respectively, lead to misclassification of
subtypes 2c, 2i, and 2j.
Since HCV genotype is one of the most important factors
determining the outcome of antiviral therapy (13), most clin-
ical laboratories routinely perform HCV genotyping. HCV
FIG. 1. Confirmatory cloning and pyrosequencing results for a
specimen with a mixed 3/5 infection. (A) Genotype 5 (ATAAACCC
GCTCAATGCCCGGAGATTTGGGCGTGCCCCCGCG). (B) Ge-
notype 3 (AGCAAACCCGCTCAATACCCAGAAATTTGGGCGT
GCCCCCGCG). Pyrograms are shown for both HinfI restriction-
digested (A) and undigested (B) PCR products from the same
specimen. The y axis represents signal intensity in relative light units,
and the x axis shows time in minutes and the nucleotide dispensation
order. E, dispensation of enzyme mix; S, dispensation of lumogenic
substrate. Peak heights represent pyrophosphate release when nucle-
otides are incorporated to the cDNA strand and are proportional to
the numbers of each of the four nucleotides incorporated.
TABLE 3. Genotype 1 specimens with discordant results at the
subtype level among the three compared methods and
corresponding confirmatory results
5?NC and NS5b
a-, not performed.
bMixed-genotype infection confirmed by cloning and pyrosequencing.
TABLE 4. Genotype 2 specimens with discordant results at the
subtype level among the three compared methods and
corresponding confirmatory results
5?NC and NS5b
a-, not performed.
bSpecimen classified as a putative new subtype.
VOL. 46, 2008NEW REAL-TIME PCR METHOD FOR HCV GENOTYPING195
genotyping methods, such as automated reverse hybridization
(29) and semiautomated sequencing (10, 11), need a previously
amplified genomic product as starting material. Since 5?NC
amplification is regularly performed for HCV molecular diag-
nosis and viral load quantitation, genotyping methods based on
this highly conserved region are convenient. However, discrim-
ination among subtypes (especially 1a versus 1b and 2a versus
2c) and certain genotypes (genotype 6 isolates have been iden-
tified as genotype 1) is not always reliable using this region, and
this often leads to subtyping errors (6, 27, 30, 32). Nucleotide
sequencing followed by phylogenetic analysis of more-variable
genomic regions, such as NS5b or core, has been recom-
mended for HCV genotyping in consensus proposals (27).
Nevertheless, this procedure is considered impractical for most
clinical laboratories because it is time-consuming and techni-
cally challenging and does not readily permit detection of
mixed-genotype infections. The purpose of this study was to
compare a novel commercial assay based on real-time PCR,
targeting both the 5?NC and NS5b regions, with two commonly
used genotyping assays in the clinical setting (reverse hybrid-
ization and semiautomated sequencing, both based on the
Overall, the real-time assay performed well compared to
results obtained by the two other methods. Among the 295
specimens tested by real-time PCR assay, 13 (4.4%) were pos-
itive with the universal HCV probe but not reactive to any
genotype/subtype-specific probes and were therefore classified
as indeterminates. Upon the retesting of genotype 1 specimens
with the HCV genotyping ASR 5?NC region assay, the rate of
indeterminates decreased to 2%. This is lower than previously
reported for this and other real-time-based techniques (8, 18).
The amount of HCV RNA did not apparently account for
these results, since 14 specimens with a genotype call had viral
loads lower than those of indeterminate specimens.
We report a high level of concordance between the results
of the three methods, although correlation was higher between
the two 5?NC region-based techniques, as expected. Among
the discordant results at the genotype level (eight specimens
[2.8%]), five were confirmed as mixed-genotype infections by
cloning and pyrosequencing and/or QCMD. Mixed-genotype
infections have been widely reported, particularly in those mul-
tiply exposed to HCV infection (1, 24, 31). Detection rates vary
depending on the patient population studied and the HCV
genotyping method used (9, 12, 15). In our study, not all con-
firmed mixed infections could be detected by all the methods
used. These results may reflect different sensitivities of the
three compared methods at detecting minority types or differ-
ent sensitivities of each method according to genotype. The
criterion used by the SGS v2.0 software to interpret results
obtained by the real-time PCR method as mixed infections
(the cycle thresholds of the amplified genotypes must be within
three cycles of one another) should be reoptimized at least for
some of the primer/probe sets. We found this software was too
restrictive, and this limited the detection of mixed infections; in
a specimen with a 3/5 infection according to QCMD and clon-
ing, the weaker amplification signal was not detected as a
mixed-genotype infection. Currently, the most widely accepted
methodology to confirm mixed-genotype infection is cloning
RT-PCR products and sequencing individual clones. In the
method we used, the predominant type was cleaved by geno-
type-specific restriction endonuclease previous to cloning and
pyrosequencing. Thus, this method allowed an effective iden-
tification of minority variants without the need for screening a
large number of clones. Moreover, pyrosequencing is faster
and simpler than standard sequencing. However, two 1/4 mixed
infections in our study could not be confirmed by this method.
This fact does not completely exclude the presence of a true
mixed infection because of several limitations (4): (i) this
method is not able to detect a mixed infection when the viral
load of the minority genotype is less than 1:10,000 of that of the
dominant genotype; (ii) in some cases, the number of clones
obtained to screen by pyrosequencing is limited; and (iii) 1/4
mixed infections are more difficult to confirm with this method-
ology because there is no appropriate restriction endonuclease to
specifically cleave genotype 4. Conversely, the two 1/4 mixed in-
fections detected by real-time PCR that could not be confirmed
could have been due to cross-reactivity between genotypes 1 and
4 in the real-time assay, as suggested by Cook et al. (8). Our
reviewed in search of mixed-genotype infections.
Concordance at the subtype level was studied for genotypes
1 and 2, since the real-time method does not include subtype-
specific probes for genotypes 3 to 5. Regarding genotype 1,
subtyping efficiencies were 100%, 77.2%, and 73.9% for the
real-time PCR method and the 5?NC sequencing and reverse
hybridization assays, respectively. These data are consistent
with other studies that have compared HCV genotyping results
based on the 5?NC region (14, 33). The real-time PCR method
agreed more frequently with NS5b sequencing than 5?NC re-
gion-based methods, as we expected, since discrimination be-
tween subtypes within genotype 1 is based on the NS5b region.
There were two specimens in which both 1a and 1b subtypes
gave a positive signal by real-time PCR. These results could
point to the presence of a mixed infection comprising both
subtypes, which could not be confirmed by the methods used,
recombination, or a low percentage of unspecific binding of
primers to their respective subtypes, as previously suggested
(8). Regarding genotype 2, most specimens were subtype dis-
cordant between techniques, and none of the genotyping tech-
niques assigned a reliable subtype call. Only methods based on
the use of more-variable regions, such as NS5b sequencing and
phylogenetic analysis, should be relied upon for accurate sub-
typing of genotype 2 specimens. This method also offered the
opportunity to identify new genotype 2 variants. Studies to
confirm these results are in progress. Concerning genotypes 3
to 5, no comparison was performed at the subtype level be-
cause the real-time PCR assay has only a single probe for each
of these genotypes regardless of subtype. Regarding reverse
hybridization and 5?NC sequencing, only two specimens be-
longing to genotype 3 and none belonging to genotypes 4 and
5 were tested by both methods.
The limitations of reverse hybridization and 5?NC sequenc-
ing methods for discrimination among subtypes are related to
the high degree of conservation of the 5?NC region. Genotypes
1a and 1b differ by a single nucleotide (A/G) at position ?99
within this region, and subtypes 2a and 2b differ by only two
nucleotides at positions 124 and 164. Moreover, a reliable
discrimination of subtype 2a and 2c isolates is not possible
based upon sequence analysis of the 5?NC region alone (27,
28). While the clinical significance of the infecting HCV sub-
196MARTRO ´ ET AL.J. CLIN. MICROBIOL.
type remains unknown, accurate subtyping is necessary for
epidemiological purposes, such as outbreak studies, and vac-
cine trials. Our results confirm that NS5b-based genotyping
methods are preferable to 5?NC region-based tests, as previ-
ously reported (17, 21, 25). Next-generation HCV genotyping
diagnostics are attempting to investigate these more-informa-
tive regions. Indeed, a new version of the reverse hybridization
assay used in this study (Versant HCV genotype 2.0 assay;
Siemens Medical Solutions Diagnostics) has recently been
commercialized; this assay comprises probes targeting core
and 5?NC regions to improve genotypic precision (22).
In conclusion, although only sequencing-based methods pro-
vide the possibility of identifying new HCV variants, the real-
time PCR method is suitable for genotyping in routine clinical
laboratories. The test is rapid (results available within 2.5 h
excluding RNA extraction), straightforward, and simple to in-
terpret, thus providing a good single-step alternative to more-
complex assays. Moreover, correlation with the other methods
at the genotype level was high, and being based on the NS5b
for genotype 1 identification, the real-time PCR method per-
formed better than the other two at assigning subtypes within
This work was supported by grant PI051131 from Instituto de Salud
Carlos III—Fondo de Investigaciones Sanitarias and grant CD05/
00258 (contratos postdoctorales de perfeccionamiento) from the Min-
isterio de Sanidad y Consumo, within the Plan Nacional de Investiga-
cio ´n Cientı ´fica, Desarrollo e Innovacio ´n Tecnolo ´gica (I?D?I).
Reagents were partially funded by Abbott Molecular.
We thank the Virus Reference Department, Health Protection
Agency, for the development of the oligonucleotide primer n2?. We
also thank Eduardo Padilla for his help in study design as well as Sonia
Molinos for her help in specimen processing. Finally, we thank Anna
Espinal for useful support in statistical analysis.
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