![Secondary Structure Predictions Secondary structure of the HCV 5 9 -NCR (A) and the 3 9 -X-tail (B) of HCV 1a reference genome H77 as predicted by MFOLD [43]. Prediction was done at 50 mM salt concentration and PCR primer annealing temperature (58 8 C). Free energies D G were À 1.6 3 10 À 3 J/mol for (A) and À 5,06 3 10 À 4 J/mol for (B). 5 9 -NCR structure predictions were identical for all genotypes in the LANL genotype reference panel except genotype 2, which had one additional stem-loop element (not covered by primers, not shown in [A]). Structure predictions for all X-tail sequences (database and new sequences) were identical (not shown in [B]). (A) Binding sites of oligonucleotides used by the Roche Amplicor assay are shown in red (sense primer KY80, hybridization probe KY150, antisense primer KY78). Nucleotide variability at these binding sites is shown in Text S2). (B) Outer lines in red identify the hybridization sites of oligonucleotides used in initial X-tail RT-PCR set, yielding limited sensitivity (forward primer F5, TaqMan probe P1, reverse primer R7). Interior lines in green identify the final X-tail prototype assay (forward primer F5, reverse primer R2, probe MGB2). Nucleotide variability at these sites is shown in Text S2. doi:10.1371/journal.pmed.1000031.g002](https://www.researchgate.net/profile/Marcus_Panning/publication/24001108/figure/fig2/AS:310265380917250@1450984439564/Secondary-Structure-Predictions-Secondary-structure-of-the-HCV-5-9-NCR-A-and-the-3-9_Q320.jpg)
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A Novel Diagnostic Target in the Hepatitis C
Virus Genome
Jan Felix Drexler
1,2,3
,BerndKupfer
2
, Nadine Petersen
1
, Rejane Maria Tommasini Grotto
4
, Silvia Maria Corvino Rodrigues
4
,
Klaus Grywna
1
,MarcusPanning
1
, Augustina Annan
1
, Giovanni Faria Silva
4
, Jill Douglas
5
,EvelynS.C.Koay
6,7
,Heidi
Smuts
8
,EduardoM.Netto
3
,PeterSimmonds
5
, Maria Ine
ˆs de Moura Campos Pardini
4
,W.KurtRoth
9
, Christian Drosten
2*
1Clinical Virology Group, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 2Institute of Virology, University of Bonn, Bonn, Germany, 3Infectious
Diseases Research Laboratory, University Hospital Prof. Edgard Santos, Federal University of Bahia, Salvador, Brazil, 4University of Sa
˜o Paulo State (UNESP), Botucatu Medical
School, Blood Transfusion Centre - Molecular Biology Laboratory and Internal Medicine Department, Botucatu, Sa
˜o Paulo, Brazil, 5Virus Evolution Group, Centre for
Infectious Diseases, University of Edinburgh, Edinburgh, United Kingdom, 6Department of Pathology, Yong Loo Lin School of Medicine, National University of Singapore, 7
Molecular Diagnosis Centre, National University Hospital, Singapore, 8Division Medical Virology/National Health Laboratory Service, Department of Clinical Laboratory
Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa, 9GFE Blut mbH, Frankfurt (Main), Germany
Funding: The Brazilian study was
partially supported by a donation of
reagents from Qiagen, Germany. The
German study was supported by the
German Ministry of Health (BMGS) as
a part of funding of the National
Reference Centre for Tropical
Infections at the Bernhard Nocht
Institute and by European Union
contract number SSPE-CT-2005–
022639. The funders had no role in
study design, data collection and
analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors
have declared that no competing
interests exist.
Academic Editor: Paul Klenerman,
Oxford University, United Kingdom
Citation: Drexler JF, Kupfer B,
Petersen N, Grotto RMT, Rodrigues
SMC, et al. (2009) A novel diagnostic
target in the hepatitis C virus
genome. PLoS Med 6(2): e1000031.
doi:10.1371/journal.pmed.1000031
Received: September 8, 2008
Accepted: December 24, 2008
Published: February 10, 2009
Copyright:Ó2009 Drexler et al. This
is an open-access article distributed
under the terms of the Creative
Commons Attribution License, which
permits unrestricted use,
distribution, and reproduction in any
medium, provided the original
author and source are credited.
Abbreviations: bDNA, branched
DNA; HCV, hepatitis C virus; IU,
international units; LANL, Los
Alamos National Laboratory; LOD,
limit of detection; NAT, nucleic acid
testing; NCR, noncoding region; RT-
PCR, reverse transcriptase PCR; SD,
standard deviation; X-tail RT-PCR, X-
tail real-time RT-PCR assay
* To whom correspondence should
be addressed. E-mail: drosten@
virology-bonn.de
ABSTRACT
Background
Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and
therapeutic regimens. All molecular assays target the viral 59-noncoding region (59-NCR), and all
show genotype-dependent variation of sensitivities and viral load results. Non-western HCV
genotypes have been under-represented in evaluation studies. An alternative diagnostic target
region within the HCV genome could facilitate a new generation of assays.
Methods and Findings
In this study we determined by de novo sequencing that the 39-X-tail element, characterized
significantly later than the rest of the genome, is highly conserved across genotypes. To prove
its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test
was developed and evaluated multicentrically on a large and complete panel of 725 clinical
plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South
Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of
clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The
lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml),
suggesting applicability in donor blood screening. The upper LOD exceeded 10
9
IU/ml,
facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples,
quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly
concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail
NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based
viral loads deviated by more than 0.5 log10 from 59-NCR-based viral loads in only 12% of
samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a
Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based
protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short
turnover times (2.5 h for up to 96 samples), and without technical difficulties.
Conclusion
This study indicates a way to fundamentally improve HCV viral load monitoring and infection
screening. Our prototype assay can serve as a template for a new generation of viral load
assays. Additionally, to our knowledge this study provides the first open protocol to permit
industry-grade HCV detection and quantification in resource-limited settings.
The Editors’ Summary of this article follows the references.
PLoS Medicine | www.plosmedicine.org February 2009 | Volume 6 | Issue 2 | e10000310210
P
L
o
S
MEDICINE
Introduction
Hepatitis C virus (HCV) is one of the leading causes of
chronic hepatitis, liver cirrhosis, and hepatocellular carcino-
ma [1,2]. Seroprevalence studies suggest that at least 170
million individuals have been infected worldwide [3]. The
incidence of new HCV infections has decreased in affluent
countries owing to screening of blood products, but an
increase of global patient numbers is still expected [1–3]. At
present, six genotypes and more than 30 subtypes are well
characterized, with an overall nucleotide diversity of 31%–
33% between genotypes and 20%–25% between subtypes [4].
Diagnostic detection of HCV RNA identifies an infection
weeks to months before a detectable antibody response. To
prevent transmission by transfusion of viremic blood, nucleic
acid testing (NAT) of blood donors has become a routine
procedure in industrialized countries. As a marker of treat-
ment success, the decrease of virus RNA concentration (viral
load) as measured by quantitative NAT has become a clinical
gold standard [5,6]. For these molecular tests, several
generations of qualitative and/or quantitative HCV NAT
assays have been in use. The most recent improvement was
the introduction of real-time PCR-based assays [7–10].
However, even the latest versions of assays diverge in
performance between different genotypes [11–21]. This
divergence is of strategic clinical relevance as success of
therapy varies between HCV genotypes [22]. Consistently,
genotypes other than those occurring in industrialized
countries have been under-represented in clinical evaluation
studies [23]. Along with high costs for commercial assays,
genotype variation complicates the initiation of successful
treatment programmes in several less-affluent countries. In
analogy to HIV treatment, open-protocol and low-cost HCV
viral load technology would be desirable [24–28].
Unfortunately, the design of both commercial and in-house
tests for HCV suffers from properties of the viral 59-
noncoding region (59-NCR). This region is thought to be
the most conserved portion of the HCV genome [29] and is
targeted by all assays. Test manufacturers have made
substantial efforts to optimize 59-NCR target sites, keeping
them unpublished for all current commercial assays. Still,
there seem to be remaining issues with these tests, as
exemplified by the discontinuation of a new real-time PCR-
based assay in 2004 after initial introduction in blood
screening [13,30]. One of the most important issues may be
the existence of complex secondary structures due to the 59-
NCR’s function as an internal ribosomal entry site for
genome translation [31]. These secondary structures may
interfere in complex ways with primer or probe binding.
Due to notorious problems with the 59-NCR, a different
target region would be beneficial. However, viral genes
downstream of the 59-NCR are not useful as diagnostic
targets because of their nucleotide variability [4,32]. In the
mid 1990s, molecular biology studies on the HCV replication
cycle revealed that the genome carries an additional element
in its 39-UTR downstream of the poly-U tract, the so-called X-
tail [33,34]. The biological function of the X-tail is not
completely understood, but it has been shown that it is
involved in several crucial steps throughout the HCV life
cycle, involving both RNA-RNA and RNA-protein interac-
tions [35–38]. The X-tail has been suggested to be highly
conserved [35,36], but it is unclear whether other issues (e.g.,
secondary structures) might interfere with its molecular
detection. It may be due to lack of available sequence
information across all genotypes that the X-tail has been
neglected as a diagnostic target so far. In this study we
explored its utility for HCV detection and quantification by
sequencing the X-tail from a broad and complete panel of
HCV genotypes. To assess its diagnostic utility, we then
developed an X-tail-based viral load assay that fulfills the
same technical standards as commercial assays currently in
clinical use. Clinical validation involved 725 stored samples
across HCV genotypes 1–6 from patients from four con-
tinents (Germany, UK, South Africa, Singapore, and Brazil).
Technical robustness was demonstrated by implementation
of the assay in a routine viral load laboratory in Brazil.
Materials and Methods
Reference Plasma
The World Health Organization (WHO) international
standard reagent 96/798 was obtained from National Institute
for Biological Standards and Control, (Hertfordshire, UK)
[39]. It contained 50,000 international units (IU) of HCV,
genotype 1a, per milliliter. A genotype reference plasma panel
was obtained from the German Hepatitis C Reference Center
at the University of Essen, including lyophilized human
plasma containing HCV genotypes 1a, 1b, 2a, 2b, 2c, 2i, 3a,
4d, 5a, and 6e. Genotypes as determined by commercial assays
(Inno-Lipa HCV2, GEN-ETI-K DEIA) and in-house sequenc-
ing, as well as viral loads as determined by both the Roche
Amplicor 2.0 (Amplicor) and the Bayer VERSANT bDNA 3.0
(branched DNA [bDNA]) systems were provided with this
panel (http://www.uni-essen.de/virologie/hc_hcv-panel.html).
Only the samples mentioned in this paragraph were used
for the technical development of the assay.
Patient Plasma and Commercially Available Viral Load and
Genotyping Assays
For validation of the assay, a total of 725 human plasma
samples (one sample per patient) were studied. Of these, 598
were obtained from routine clinical testing in Germany, UK,
South Africa, Singapore, and Brazil (n¼319, 62, 102, 52, 46,
and 17 of HCV genotypes 1, 2, 3, 4, 5, and 6, respectively).
These stored samples had been selected because of their
known genotypes, and were collected at the University of
Bonn, Germany (n¼530, HCV genotypes 1 to 4, sampled from
2003 to 2007), the University of Cape Town, South Africa (n¼
46, genotype 5, sampled from 1996 to 2007), the National
University of Singapore (n¼9, genotypes 3k and 6, sampled
from 2006 to 2007), and the University of Edinburgh, UK (n¼
13, genotype 6, sampled from 1999 to 2004). Samples were
generally taken at the time of genotyping, i.e., immediately
before the beginning of therapy. We did not accept recorded
genotype information from earlier or later samples from a
given patient. An additional set of plasma samples, collected
from 2005 to 2007 at the University of Sa
˜o Paulo State, Brazil,
contained 127 nongenotyped samples from patients during
HCV therapy. All samples were anonymized and approval was
obtained by the ethical committees of the University of Bonn
Medical Centre, Bonn, Germany; the University of Sa
˜o Paulo
State (UNESP); Botucatu Medical School, Botucatu, Sa
˜o
Paulo, Brazil; and the Federal University of Bahia Medical
School, Salvador, Bahia, Brazil. Viral loads in all samples were
PLoS Medicine | www.plosmedicine.org February 2009 | Volume 6 | Issue 2 | e10000310211
Novel Diagnostic HCV Target
determined at the University of Bonn with bDNA on a
VERSANT 440 Molecular System. Samples were genotyped by
the VERSANT HCV Genotype assay (Line Probe assay [LiPA])
(Bayer Diagnostics). Genotyping of the South African and
British samples was done by restriction fragment length
polymorphism (RFLP), as previously described [40,41] and
confirmed by LiPA. The genotypes of all samples from
Singapore were confirmed by sequencing of the 59-NCR and
nonstructural protein 5b (NS5b) domains [42]. Samples were
stored at 20 8C until quantification with the X-tail real-time
reverse transcriptase (RT)-PCR assay (X-tail RT-PCR). All
Brazilian samples were tested by bDNA on a VERSANT 340
Molecular System and X-tail RT-PCR in the local laboratory
in Brazil.
Quantification of HCV Viral Load by X-Tail RT-PCR
Reactions of 50 ll contained 20 ll of RNA extract, 53
reaction buffer (Qiagen One-step RT-PCR kit), 200 lMof
each dNTP, 200 nM of primer XTF5 (GTGGCTCCATCT-
TAGCCCTAGT), 300 nM of primer HCMgR2
(TGCGGCTCACGGACCTTT), 100 nM of HCV-specific probe
HCVMGB2 (FAM-CACGGCTAGCTGTG-Black Hole Quench-
er 2/Minor grove binder), 100 nM of Internal Control-probe
YFPY (VIC-ATCGTTCGTTGAGCGATTAGCAG-Black Hole
Quencher 1), and 2 ll of Enzyme Mix. Thermal cycling was
done on Applied Biosystems 7700 or 7500 SDS instruments
under the following conditions: 55 8C, 10 min; 95 8C, 15 min;
45 cycles at 94 8C, 10 s and 58 8C, 15 s.
Computing
Statistical analyses were performed with the SPSS 13 (SPSS).
Alignments were generated using BioEdit (http://www.mbio.
ncsu.edu/BioEdit/bioedit.html). Sequences were analyzed us-
ing the Lasergene software package (DNAStar). All database
workwasperformedonlineonLosAlamosNational
Laboratory (LANL) (http://hcv.lanl.gov/content/hcv-index)
and euHCV (http://euhcvdb.ibcp.fr/euHCVdb) domains.
Supplemental Materials and Methods
A STARD diagram and checklist are included in Figure S1
and Text S1. A more detailed description of the X-tail NAT
assay including protocols for RNA extraction and data on
technical performance are provided in Text S2. A bench
protocol and instructions for requesting controls is provided
in Text S3.
Accession Numbers
Sequences determined in this study have been submitted to
GenBank (http://www.ncbi.nlm.nih.gov/Genbank) under acces-
sion numbers EU835523-EU835532.
Results
Analysis of the X-Tail Region
In order to examine the conservedness of the X-tail region,
a nucleic acid sequence alignment was generated that
contained all X-tail sequences stored in the LANL and
euHCV databases as of January 2007. These databases
comprised sequences classified as genotypes 1, 2, and 3. Sets
of sequencing primers for the ultimate 39-end of the genome
were identified, and the X-tail was sequenced from reference
plasma samples of HCV genotypes 1–6. Together with one
outlier sequence taken from the initial characterization study
on the X-tail [33] that was found later on to correspond to
genotype 4 (PS, unpublished data), these novel sequences
were added to the alignment. As shown in Figure 1, the
degree of conservedness in the X-tail was comparable to that
of the 59-NCR, which is the target of all existing viral load
assays.
Experimental Identification of a Diagnostic Target Region
within the X-Tail
The complete 39-UTR alignment was evaluated as a target
for real-time PCR design. Since primer design software was
not able to identify suitable candidate oligonucleotides, in a
first approach five forward primers, five reverse primers, and
two probes were selected upon inspection of the alignment.
All of the 50 resulting real-time PCR sets were tested
experimentally for reaction efficiency. However, even after
selection of the most effective oligonucleotides and optimi-
zation of reaction conditions, the lower limit of detection
(LOD) did not fall below 150 IU/ml (see Text S2 for additional
Figure 1. Nucleotide Similarities within HCV 59- and 39-Genome Ends
Top panel: schematic representation of the HCV reference genome H77 as given in the 2008 LANL database.
Bottom panel: Percent nucleotide identity along the 59-NCR and the X-tail, as calculated by a sliding window analysis with VectorNTI software. Window
size was 2 nucleotides. The alignment used for the sliding window analysis contained the complete HCV genotype reference panel (n¼60 sequences)
from the LANL HCV database (available at http://hcv.lanl.gov/content/sequence/NEWALIGN/align.html) and all X-tail sequences available in GenBank as
shown in Text S2, including the sequences determined de novo in this study (EU835523-EU835532).
doi:10.1371/journal.pmed.1000031.g001
PLoS Medicine | www.plosmedicine.org February 2009 | Volume 6 | Issue 2 | e10000310212
Novel Diagnostic HCV Target
information on technical evaluation procedures). Since
primers had a perfect match with the target sequence, the
limitation in sensitivity was ascribed to RNA secondary
structures possibly interfering with oligonucleotide hybrid-
ization.
RNA secondary structures of both the X-tail and the 59-
NCR were modeled at PCR primer annealing conditions (58
8C and 50 mM ion concentration) using MFOLD [43]. In the
59-NCR, secondary structures in all genotypes were almost
completely dissolved, but still a few stable stem-loop elements
remained. Of note, some of these fell in the conserved regions
targeted by a prototypic 59-NCR assay, the Roche Amplicor
Monitor, and probably also the later generation COBAS
TaqMan system [13]. For the X-tail, a 10-nucleotide-stem
element in the stem-loop 1 region towards the 39end of the X-
tail was predicted to be stable at the same conditions. Figure 2
shows the structure predictions with exemplified oligonucleo-
tides for X-tail and 59-NCR. It was concluded that stable stem
structures were likely to interfere with binding of all antisense
primers and most of the probes used up to this point.
Prototype Viral Load Assay Based on the HCV X-Tail
Consequently, the antisense primer and probe binding sites
were moved further upstream into the amplicon. Out of
several new candidate primers and probes, an oligonucleotide
combination was determined that provided very high
amplification efficiency (final assay oligonucleotides as shown
in the Materials and Methods section). These primers and
probe were directly adjacent, without unoccupied nucleo-
tides between them. Very few nucleotide mismatches
occurred with any of the sequences taken from GenBank or
determined from reference plasma (see Figure 2). According
to established data, these mismatches were highly unlikely to
interfere with assay performance [44–46].
To assess the diagnostic applicability of the X-tail, this PCR
set was developed into a clinical-grade prototype assay. The
input RNA volume was maximized to 20 ll in a 50-ll assay
volume, and the chemistry was adjusted for use with a semi-
automated nucleic acids extraction. In order to detect PCR
inhibition, a competitive internal control was incorporated. It
was amplified by the same primers as the diagnostic target but
was detected by a probe of different sequence composition
and different fluorescent labeling. To enhance assay stability,
control RNA was formulated to be nuclease resistant and
added at working concentration to all reactions at the lysis
buffer stage. As a reference standard for viral loads, a
noninfectious and stable copy of the HCV 1a X-tail was cloned
Figure 2. Secondary Structure Predictions
Secondary structure of the HCV 59-NCR (A) and the 39-X-tail (B) ofHCV 1a reference genome H77 as predicted by MFOLD [43]. Prediction was done at 50 mM
salt concentration and PCR primer annealing temperature (58 8C). Free energies DGwere1.6 310
3
J/mol for (A) and 5,06 310
4
J/mol for (B). 59-NCR
structure predictions were identical for all genotypes in the LANL genotype reference panelexcept genotype 2, which had one additional stem-loop element
(not covered by primers, not shown in [A]). Structure predictions for all X-tail sequences (database and new sequences) were identical (not shown in [B]).
(A) Binding sites of oligonucleotides used by the Roche Amplicor assay are shown in red (sense primer KY80, hybridization probe KY150, antisense
primer KY78). Nucleotide variability at these binding sites is shown in Text S2).
(B) Outer lines in red identify the hybridization sites of oligonucleotides used in initial X-tail RT-PCR set, yielding limited sensitivity (forward primer F5,
TaqMan probe P1, reverse primer R7). Interior lines in green identify the final X-tail prototype assay (forward primer F5, reverse primer R2, probe MGB2).
Nucleotide variability at these sites is shown in Text S2.
doi:10.1371/journal.pmed.1000031.g002
PLoS Medicine | www.plosmedicine.org February 2009 | Volume 6 | Issue 2 | e10000310213
Novel Diagnostic HCV Target
in an armored RNA phage (refer to Text S2 for synthesis and
calibration of internal control and reference standard).
Before evaluation of its technical and clinical performance,
the specificity of the assay for HCV was confirmed on cell
culture supernatants or sera containing the following
Flaviviridae species: dengue virus 1, 2, 3, and 4; yellow fever
virus; tick-borne encephalitis virus; St. Louis encephalitis
virus; West Nile virus; and hepatitis G virus (HCV)/GB virus-C.
Due to frequent co-infection of patients with HIV-1 and
HCV, an HIV-1 genotype reference panel comprising 11 HIV-
1 genotypes from groups M, N, and O [47] was acquired from
National Institute for Biological Standards and Control
(product code 01/466) and tested with X-tail RT-PCR. High
RNA content in all of these materials was confirmed by a
flavivirus genus-specific RT-PCR and an HIV-1 in-house real
time RT-PCR assay [28,48]. No nonspecific amplification
occurred with any of these materials (unpublished data).
LOD and Quantification Range
A common technical specification for the sensitivity of
NAT is the 95% LOD, i.e., the concentration down to which
.95% of iterative tests will detect virus. 95% LODs for the X-
tail-based prototype assay were determined for each individ-
ual genotype by parallel limiting dilution testing as described
in Text S2. Results indicated high sensitivity across all
genotypes (Table 1). The overall LOD was as low as 18.4 IU/
ml (Figure 3).
Next, the upper quantification limit was evaluated, i.e., the
maximal RNA concentration that can be measured without
technical bias contributed by the system. This is relevant
because HCV patients may show extraordinarily high viral
loads. Synthetic Armored RNA standards exceeding clinically
observed viral loads (1,890,000, 18,900,000, and 189,000,000
IU/ml) were tested. The determined quantities did not deviate
from those expected by dilution factor (unpublished data).
Accuracy in Viral Load Monitoring
Intra-assay coefficient of variation (CV) at 7,245,000 IU/ml
of HCV RNA was 5.76% with a standard deviation (SD) of
0.03 log10. Inter-assay CVs ranged from 6.11 to 18.42% (SDs,
0.03–0.09 log10) at 1,890–189,000 IU/ml (refer to Text S2).
To evaluate the utility of the X-tail in viral load monitor-
ing, the prototype assay was compared against bDNA. The
latter was chosen as a clinical gold standard because it
represented a well-established assay that is more robust
against genotype bias than other clinical assays [49–51]. All of
the 598 plasma samples, covering HCV genotypes 1–6 and all
ranges of viral loads occurring in HCV patients, were tested
with X-tail RT-PCR and results compared to bDNA. Samples
with results below or above the quantification ranges of
bDNA or X-tail RT-PCR (n¼47) were excluded from
correlation analysis, leaving 551 samples. As shown in Figure
3, X-tail RT-PCR correlated well with bDNA for all genotypes
tested, with correlation coefficients of 0.92, 0.85, 0.95, 0.91,
0.95, and 0.96 for HCV genotypes 1 to 6, respectively. All
correlations were highly significant (p,0.01 for all, Pearson’s
goodness of fit test).
Figure 4 shows differences in absolute quantification
results for genotypes 1–6. Differences of more than 0.5
log10 occurred infrequently (12.0% of samples in total;
maximally observed deviation: 0.85 log10), with a balanced
distribution to both higher and lower viral loads (5.1% and
6.9%, respectively). Mean and median log10 differences and
SDs were small for all six genotypes, and all were below
clinical significance (Figure 4). X-tail RT-PCR yielded
minimally higher viral loads than bDNA for all genotypes
except genotype 4 (mean log10 differences were 0.00, 0.01,
0.17, 0.09, 0.07, and 0.12 for genotypes 1 to 6, respectively).
In order to assess whether the slight underquantification of
genotype 4 could have been caused by a systematic error, the
two samples that showed strongest underquantification (up to
0.7 log10) were sequenced. They showed no nucleotide
mismatches at the oligonucleotide binding sites, suggesting
other reasons for the observed deviations (handling, storage,
error in the gold standard, etc.).
Clinical Sensitivity
Because correlation analysis only included samples that
were positive in both tests, it did not reflect overall clinical
sensitivity. A comparison of qualitative detection rates is
shown in Table 2. Sixteen of a total of 598 samples (2.68%)
were below the detection limit of bDNA but detectable by X-
tail RT-PCR at a median viral load of 284 IU/ml. Three of
these samples had viral loads that should have been
detectable with bDNA, according to its LOD (615 IU/ml).
Two samples were negative by X-tail RT-PCR despite RNA
detection by bDNA at viral loads of 989 and 4,681 IU/ml,
respectively.
A total of 15 samples had viral loads above the upper cut-
off of bDNA, requiring predilution and repetition of the
bDNA assay. All of them were quantifiable by X-tail RT-PCR
upon first testing.
Table 1. Lower LOD by HCV Genotype
HCV RNA Input (IU/ml)
a
Hit Rate
c
in Genotype Reference Samples
1a 1b 2a 2b 2c 2i 3a 4 5a 6 All
54 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 50/50
18 5/5 5/5 5/5 4/5 5/5 4/5 5/5 5/5 4/5 4/5 46/50
63/5 2/5 5/5 1/5 4/5 3/5 5/5 2/5 3/5 2/5 30/50
21/5 0/5 0/5 0/5 2/5 3/5 3/5 0/5 0/5 0/5 9/50
LOD
b
11.0 7.5 4.5 22.5 8.9 33.6 3.1 7.5 22.6 22.7 18.4
a
Based on Bayer VERSANT bDNA 3.0 viral load assay.
b
LOD (IU/ml) at 95% probability, Probit analysis (see Text S2).
c
nPositive test results per five parallel tests performed.
doi:10.1371/journal.pmed.1000031.t001
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Novel Diagnostic HCV Target
Potential for Implementation in Resource-Limited Settings
Affordable viral load monitoring would be desirable in
resource-limited settings with high HCV prevalence. To
evaluate whether the X-tail prototype assay would provide
adequate stability and quality in comparison to 59-NCR-based
assays, it was implemented in a diagnostic center involved in
the Brazilian HCV treatment program. Protocols, controls,
and standards for X-tail NAT were provided to the laboratory
in Brazil. All other reagents were purchased locally. Plasma
samples from 127 patients were tested by bDNA and X-tail
NAT. The correlation coefficient between viral loads obtained
with both assays was 0.97 (p,0.001) at a mean quantitative
difference of 0.05 log10 (SD 0.018). As shown in Figure 5,
almost all quantitative differences were below 0.5 log10. Only
four outlier samples occurred, with deviations to higher and
lower quantification results of up to 1 log10 in X-tail RT-PCR.
Discussion
In this study we have identified a new diagnostic target
region in the HCV genome. A prototype RT-PCR assay based
on the HCV X-tail proved to be highly sensitive (18.4 IU/ml,
95% probit probability), robust against genotype variation,
and highly precise in virus quantification. The assay was
efficiently implemented and projected to be highly cost
efficient in an emerging country setting. These data may assist
in translating state-of-the art diagnostic technology to less
affluent settings.
The X-tail region of the HCV genome has been known for
several years [33,34] but has not been used as a diagnostic
target so far. Its biological functions are now becoming
clearer, and it is likely that its role in the virus life cycle
involves both RNA and protein interactions. The three X-tail
stem-loop structures (SL1–3) seem to be involved in negative-
strand synthesis and significantly enhance HCV replication
by means of binding to ribosomal and other cellular proteins
[35–38]. A kissing-loop interaction between an X-tail stem-
loop structure (SL2) and an RNA loop within the non-
structural protein 5b (NS5b) region was shown to be essential
for HCV replication [52]. These multiple mechanisms of
interaction imply a high degree of conservedness in the X-
Figure 3. Correlation of Viral Loads as Determined by X-Tail RT-PCR (x-Axis) and bDNA (y-Axis)
Genotypes and numbers of samples (n) are given in the bottom right corner of each panel. Pearson’s bivariate correlation coefficients were 0.92, 0.85, 0.95,
0.91, 0.95, and 0.96 for HCV genotypes 1 to 6, respectively. The dashed lines represent ideal correlations. Genotype 5, 0.07/0.31; and genotype 6, 0.12/0.19.
doi:10.1371/journal.pmed.1000031.g003
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Novel Diagnostic HCV Target
tail, which could indeed be confirmed for all genotypes in this
study. As anticipated [32,53], the region proved as conserved
as the 59-NCR across all genotypes, and seemed to exhibit less
problematic structural features than the 59-NCR.
To circumvent the limitations that the 59-NCR presents in
diagnostics, we have established the HCV X-tail as a target for
molecular detection and quantification. Initially we could not
be sure about its utility for several reasons implied by its
biological functions. A possible limitation could have been its
position at the end of the genome and beyond the poly-U
tract, making the X-tail prone to nuclease degradation.
Moreover, due to its functions as a 39-element it could have
been associated strongly with viral or cellular proteins.
Although we could not investigate these issues in our study,
we are confident about its diagnostic utility from the clinical
part of our study, showing that X-tail-based viral loads were
highly concordant with results from bDNA testing. bDNA was
chosen as a gold standard because it uses multiple probes
along the 59-NCR and initial core region and has proven to be
the most robust viral load test compared to other assays [49–
51]. We used a large panel of clinical specimens that included
sufficient numbers of samples of genotypes 1–6 (n¼725 in
total, 598 genotyped samples). To our knowledge, this is the
most diversified and comprehensive panel of clinical speci-
mens used in the validation of HCV NAT so far [7,10,14,17–
21,49,54,55]. For genotypes 4–6, earlier studies relied on
sparse genotype reference samples, which have also been used
for the original design of assays and may under-represent the
genetic diversity observed in clinical samples. Our study
included field clinical samples of all genotypes in substantial
numbers, sampled in four different geographic locations. The
genotype-related robustness of the prototype X-tail assay was
at least equivalent with that of proprietary assays
[12,13,15,17–21,23,49,51,56,57]. Quantitative correlation and
lower limits of detection were highly consistent for all
genotypes by X-tail RT-PCR. The lower LOD (18.4 IU/ml)
and the upper quantification range of our assay
(.189,000,000 IU/ml) were equivalent to that of the last
generation Roche COBAS TaqMan HCV test and the Abbott
HCV RealTime assay [7–10,23,58]. Critically, X-tail-based viral
loads and bDNA results were highly congruent, making the
assay compatible with other quantification systems. This
compatibility is critical when patients switch their treating
institution, which may entail switching between viral load
tests. The observed quantitative deviations were generally
,0.5 log10, i.e., below clinical relevance.
In resource-limited settings our prototype assay could be
used instead of more costly proprietary assays in HCV
treatment and testing. It is not patented, has a simple and
accessible formulation (refer to the bench protocol and
instructions for requesting controls in Text S3), and is
appropriate for rare HCV genotypes. Several less-affluent
countries have established successful HIV treatment pro-
grams, but suffer from considerable HCV prevalence as well.
One important example is Brazil, where networks of well-
managed public laboratories conduct viral load testing as a
part of the national HIV-1 treatment program [59]. Intensive
efforts have been made to develop low-cost viral load assays
for HIV-1 in Brazil and elsewhere [24–28]. The most
important issue with such in-house assays is their robustness.
In proprietary commercial assays robustness is contributed
by advanced features like automated RNA preparation,
nuclease resistant calibrators, and synthetic internal controls.
We have incorporated all of these features in the open
protocol HCV X-tail assay. A second issue in using in-house
Table 2. Detection of HCV by X-Tail RT-PCR Versus bDNA
Sample Testing Results X-Tail RT-PCR bDNA nSamples Median Viral Load (IU/ml)
a
Range Viral Load (IU/ml)
Positive Positive 551 — —
Negative Negative 14 — —
Positive Negative 16 284 43–1,816,128
Negative Positive 2 2,835 989–4,681
Positive Overflow
b
15 16,124,130 251,662–196,797,600
Total — 598 — —
a
Median HCV RNA IU/ml in the positive assay when below LOD in compared assay (,615 IU/ml for bDNA 3.0 and ,46 IU/ml for X-tail RT-PCR).
b
Could not be quantified due to viral loads .7,692,308 IU/ml (viral load was determined a posteriori by predilution).
doi:10.1371/journal.pmed.1000031.t002
Figure 4. Quantitative Deviations between X-Tail–Based and bDNA Viral
Loads, per Genotype (y-Axis)
Genotypes are indicated below the x-axis, the number of samples tested
per genotype (n) above the x-axis. Each box shows the median,
interquartile range (box length, containing 50% of data) and whiskers
show extreme values (there were no statistical outliers). Deviations
between X-tail RT-PCR and bDNA [log10 X-tail RT-PCR log10 bDNA]
were genotype 1, 0.00/0.31 (mean/SD); genotype 2, 0.01/0.33; genotype
3, 0.17/0.32; genotype 4, 0.09/0.37; genotype 5, 0.07/0.31; and
genotype 6, 0.12/0.19.
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Novel Diagnostic HCV Target
assays is quality control, which is shifted largely to the
production process in commercial kits, but can be managed
at the laboratory level if appropriate quality control
procedures are in place. In the context of HIV viral load
monitoring, we and others have demonstrated that in-house
testing in emerging countries is feasible in well-managed
laboratories [24,25,27,28]. The potential for translation of the
assay was demonstrated by implementation in a Brazilian
laboratory and on-site comparison against the Bayer bDNA
assay. X-tail RT-PCR had short turnover times (2.5 h for up to
96 samples) and was implemented from provided protocols
without technical difficulties. Overall results suggest that X-
tail RT-PCR is suitable for the Brazilian setting, even though
genotyping was not feasible at the study site. The overall HCV
genotype distribution in Sa
˜o Paulo state is known to be
approximately 65%, 5%, and 30% of genotypes 1, 2, and 3,
respectively [60]. Reaction costs of the assay ranged around
US$8.70 per sample, or 8.1% of the current viral load costs in
Brazil. It should be mentioned that under European
conditions, an additional service license fee would apply,
increasing the cost of one assay to US$18 per sample (refer to
Table 3 for an estimation of costs).
Finally, HCV is an important issue for blood safety in
emerging countries. Again, Brazil provides an example
representative of many other emerging countries. An urgent
federal decree in 2003 demanded the general testing of
donated blood for HCV by NAT [61], but this strategy has not
yet been implemented. According to estimates by the Brazil-
ian Ministry of Health, it would cost about US$40 million to
screen 4 million blood donations annually [62].
We previously demonstrated the feasibility of in-house
testing of blood donors for HCV by NAT methods [63]. Due
to the high sensitivity of X-tail RT-PCR (18.4 IU/ml), the
prototype assay could be used for testing pooled blood donor
samples. With the well-established approach of pooling 24
donors prior to NAT testing [64], the projected LOD would
be 441 IU/ml per donor (18.4 IU/ml 324). This projection
would match the sensitivity achieved with pooled blood
donor screening in Europe [65,66] and North America [64,67].
Calculating the cost for one X-tail NAT assay at US$10
(US$8.70 for RT-PCR according to Table 3, plus US$1.30 for
extra consumables due to pooling), and assuming 20%
additional costs for controls and confirmatory testing, 24-
donor pool testing by X-tail RT-PCR would amount to US$2
Table 3. Approximate Pricing of HCV Viral Load Assays, US Dollars, without Taxes
Item X-Tail RT-PCR Germany
a
Roche Cobas Amplicor, Brazil
b
Bayer bDNA, Brazil
b
RNA preparation 2.70
a
Included
c
Included
c
Reagents 4.20 Included
c
Included
c
Consumables and controls 1.80 0.80 0.80
License fees
d
10.00 Included
c
Included
c
Total 18.70 106.80 106.80
a
Based on current conditions in Germany.
b
Current conditions for the Sa
˜o Paulo State Viral Hepatitis Network (unpublished data based on personal communications [RMTG and MIMCP]; see also [68]). Purchase by laboratories
outside this privileged network may involve higher prices up to about US$190 per test.
c
These items are included in the cost of the commercial assay.
d
Service licence fees for using the generic 59-nuclease probe format.
doi:10.1371/journal.pmed.1000031.t003
Figure 5. Results Obtained with X-Tail Viral Load Monitoring, Implemented in Brazil
127 samples were measured in a Brazilian HIV-1 viral load monitoring centre by Bayer VERSANT 3.0 HCV bDNA assay and by X-tail in-house PCR. (A)
Correlation of viral loads between X-tail RT-PCR (x-axis) and Bayer bDNA 3.0 (y-axis). The dashed line represents an ideal correlation. Pearson’s bivariate
correlation coefficient was 0.97.
(B) Quantitative differences. The box shows the median and interquartile range (box length). The whiskers represent an extension of the 25th or 75th
percentiles by 1.5 times the interquartile range. Datum points beyond the whisker range are considered as outliers and marked as asterisks.
doi:10.1371/journal.pmed.1000031.g005
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Novel Diagnostic HCV Target
million per 4 million donations annually (4 million/24 3
US$10 31.2). This means a reduction down to 5% of the
current economic estimate. In view of the technical robust-
ness and transferability of the assay described in this report,
we believe that such a strategy could be a realistic approach
in emerging countries aiming to increase blood safety
through the introduction of HCV NAT.
Supporting Information
Alternative Language Abstract S1. Translation of the Abstract into
French by Felix Drexler
Found at doi:10.1371/journal.pmed.1000031.sd001 (29 KB DOC).
Alternative Language Abstract S2. Translation of the Abstract into
German by Felix Drexler and Christian Drosten
Found at doi:10.1371/journal.pmed.1000031.sd002 (29 KB DOC).
Alternative Language Abstract S3. Translation of the Abstract into
Portuguese by Felix Drexler and Maria Inez Pardini
Found at doi:10.1371/journal.pmed.1000031.sd003 (31 KB DOC).
Alternative Language Abstract S4. Translation of the Abstract into
Spanish by Oscar Martinez Martinez
Found at doi:10.1371/journal.pmed.1000031.sd004 (27 KB DOC).
Figure S1. STARD Diagram
Found at doi:10.1371/journal.pmed.1000031.sg001 (11 KB PDF).
Text S1. STARD Checklist
Found at doi:10.1371/journal.pmed.1000031.sd005 (18 KB PDF).
Text S2. Detailed Description of the X-tail NAT Assay
Found at doi:10.1371/journal.pmed.1000031.sd006 (696 KB DOC).
Text S3. Bench Protocol and Instructions for Requesting Controls
Found at doi:10.1371/journal.pmed.1000031.sd007 (86 KB PDF).
Acknowledgments
We are grateful to technical staff at the involved institutions.
Author contributions. JFD and CD conceived and designed the
experiments. JFD, BK, NP, RMTG, SMCR, KG, MP, AA, GFS, JD,
ESCK, HS, PS, MIdMCP, and WKR performed the experiments. JFD,
EMN, and CD analyzed the data. BK, RMTG, SMCR, GFS, JD, ESCK,
HS, PS, MIdMCP, and WKR contributed materials. JFD and CD wrote
the paper.
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Novel Diagnostic HCV Target
Editors’ Summary
Background. About 3% of the world’s population (170 million people)
harbor long-term (chronic) infections with the hepatitis C virus (HCV) and
about 3–4 million people are newly infected with this virus every year.
HCV—a leading cause of chronic hepatitis (inflammation of the liver)—is
spread through contact with the blood of an infected person. Globally,
the main routes of transmission are the use of unscreened blood for
transfusions and the reuse of inadequately sterilized medical instru-
ments, including needles. In affluent countries, where donated blood is
routinely screened for the presence of HCV, most transmission is through
needle sharing among drug users. The risk of sexual and mother-to-child
transmission of HCV is low. Although HCV infection occasionally causes
an acute (short-lived) illness characterized by tiredness and jaundice
(yellow eyes and skin), most newly infected people progress to a
symptom-free, chronic infection that can eventually cause liver cirrhosis
(scarring) and liver cancer. HCV infections can be treated with a
combination of two drugs called interferon and ribavirin, but these drugs
are expensive and are ineffective in many patients.
Why Was This Study Done? An effective way to limit the global spread
of HCV might be to introduce routine screening of the blood that is used
for transfusions in developing countries. In developed countries, HCV
screening of blood donors use expensive, commercial ‘‘RT-PCR’’ assays
to detect small amounts of HCV ribonucleic acid (RNA; HCV stores the
information it needs to replicate itself—its genome—as a sequence of
‘‘ribonucleotides’’). All the current HCV assays, which can also quantify
the amount of viral RNA in the blood (the viral load) during treatment,
detect a target sequence in the viral genome called the 59-noncoding
region (59-NCR). However, there are several different HCV ‘‘genotypes’’
(strains). These genotypes vary in their geographical distribution and,
even though the 59-NCR sequence is very similar (highly conserved) in
the common genotypes (HCV genotypes 1–6), the existing assays do not
detect all the variants equally well. This shortcoming, together with their
high cost, means that 59-NCR RT-PCR assays are not ideal for use in many
developing countries. In this study, the researchers identify an alternative
diagnostic target sequence in the HCV genome—the 39-X-tail element—
and ask whether this sequence can be used to develop a new generation
of tests for HCV infection that might be more appropriate for use in
developing countries.
What Did the Researchers Do and Find? The researchers determined
the RNA sequence of the 39-X-tail element in reference samples of the
major HCV genotypes and showed that this region of the HCV genome is
as highly conserved as the 59-NCR. They then developed a prototype X-
tail RT-PCR assay and tested its ability to detect small amounts of HCV
and to measure viral load in genotype reference samples and in a large
panel of HCV-infected blood samples collected in Germany, the UK,
Brazil, South Africa, and Singapore. The new assay detected low levels of
HCV RNA in all of the genotype reference samples and was also able to
quantify high RNA concentrations. The viral load estimates it provided
for the clinical samples agreed well with those obtained using a
commercial assay irrespective of the sample’s HCV genotype. Finally, the
X-tail RT-PCR assay gave similar results to a standard assay at a fraction of
the cost when used to measure viral loads in a Brazilian laboratory in an
independent group of 127 patient samples collected in Brazil.
What Do These Findings Mean? These findings suggest that the HCV
39-X-tail element could provide an alternative target for screening blood
samples for HCV infection and for monitoring viral loads during
treatment, irrespective of HCV genotype. In addition, they suggest that
X-tail RT-PCR assays may be stable and robust enough for use in
laboratories in emerging countries. Overall, these findings should
stimulate the development of a new generation of clinical HCV assays
that, because the protocol used in the X-tail assay is freely available,
could improve blood safety in developing countries by providing a
cheap and effective alternative to existing proprietary HCV assays.
Additional Information. Please access these Web sites via the online
version of this summary at http://dx.doi.org/10.1371/journal.pmed.
1000031.
The World Health Organization has a fact sheet about hepatitis C (in
English and French)
The US Centers for Disease Control and Prevention provides
information on hepatitis C for the public and for health professionals
(information is also available in Spanish)
The US National Institute of Diabetes and Digestive and Kidney
Diseases provides basic information on hepatitis C (in English and
Spanish)
The MedlinePlus Encyclopedia has a page on hepatitis C; MedlinePlus
also provides links to further information on hepatitis C (in English and
Spanish)
PLoS Medicine | www.plosmedicine.org February 2009 | Volume 6 | Issue 2 | e10000310220
Novel Diagnostic HCV Target
