Original Paper
Intervirology 2015;58:310–317
DOI: 10.1159/000441474
Is Anti-Hepatitis C Virus Antibody Level
an Appropriate Marker to Preclude the
Need for Supplemental Testing?
Kuo Zhang Lunan Wang Guigao Lin Jinming Li
National Center for Clinical Laboratories, Beijing Hospital, Beijing, PR China
Key Words
Hepatitis C · Hepatitis C antibody · Chemiluminescence
immunoassay · Signal-to-cutoff ratio · Strategy
Abstract
Objectives: In the present study, we aimed to determine
whether signal-to-cutoff (S/Co) ratios of reactive anti-HCV
samples could be used as a basis for avoiding the need for
supplemental testing in our study population. Methods: We
analyzed 901 anti-HCV-positive sera from 8 institutions in
China. The Ortho VITROS anti-HCV assay and Monolisa Plus
anti-HCV version 2 were used as screening assays to detect
anti-HCV antibodies. Recombinant immunoblot assay (RIBA)
and quantitative tests for HCV RNA were performed to vali-
date confirmed HCV infection status. Results: Receiver oper-
ating characteristic curve analyses demonstrated that 41.5%
(114/275) of true-positive samples with S/Co ratios ≤3.0
would be missed and the negative predictive value was 63.9
and 87.06%, using real-time polymerase chain reaction (RT-
PCR) and RIBA as supplemental testing, respectively. 29.8%
(90/302) of those who tested positive by RIBA samples were
missed when only RT-PCR was used as supplemental testing.
Conclusions: We determined that very low anti-HCV levels
(S/Co ≤3.0), as determined by chemiluminescence immuno-
assay, was not an appropriate marker to preclude the need
© 2016 S. Karger AG, Basel
0300–5526/16/0585–0310$39.50/0
E-Mail karger@karger.com
www.karger.com/int
Received: September 1, 2015
Accepted: October 4, 2015
Published online: January 20, 2016
for supplemental testing in our study population. A screen-
ing strategy employing a secondary HCV antibody assay us-
ing different HCV antigens from the first assay as the supple-
mental testing method should be studied further. The im-
munoblot assay, as a supplemental testing method, is still
necessary. © 2016 S. Karger AG, Basel
Introduction
Hepatitis C virus (HCV) is the causative agent of the
contagious liver disease hepatitis C. The virus can cause
both acute and chronic hepatitis infection, thus present-
ing an enormous health burden. A significant number of
people with chronic hepatitis C infection are at high risk
for consequently developing liver cirrhosis and hepato-
cellular carcinoma, which causes serious mortality and
morbidity [1–3]. It is therefore essential to identify indi-
viduals infected with HCV.
Enzyme immunoassays and chemiluminescence im-
munoassays (CIAs) are the two main screening immuno-
assays for detection of anti-HCV antibodies. Although
the CIA screening method demonstrates improved spec-
ificity compared to enzyme immunoassays [4], these anti-
HCV screening assays may generate false-positive results.
Jinming Li
National Center for Clinical Laboratories
Beijing Hospital
No. 1 Dahua Road, Dongdan, Beijing 100730 (PR China)
E-Mail jmli @ nccl.org.cn
Therefore, it is essential to validate the specificity of these
screening assays using a supplemental assay. Recombi-
nant immunoblot assay (RIBA) or HCV RNA polymerase
chain reaction (PCR) has been recommended for confir-
mation of positive anti-HCV screening tests [5]. In 2013,
due to discontinuation of RIBA, the Centers for Disease
Control and Prevention (CDC) [6] recommended that a
positive result from an initial anti-HCV screening test be
followed only by nucleic acid testing (NAT) for detection
of HCV RNA. A second round of anti-HCV screening of-
fers an alternative supplemental testing method for con-
firmation, according to the algorithm published by Ver-
meersch et al. [7].
Guidelines published by the CDC incorporated the
level of the signal-to-cutoff (S/Co) ratio relative to the
anti-HCV concentration into laboratory algorithms for
anti-HCV testing in 2003. Samples with S/Co ratios ≥8.0,
as determined by the VITROS anti-HCV assay (Ortho
Clinical Diagnostics, Raritan, N.J., USA), are considered
positive for anti-HCV antibodies [8]. Using receiver op-
erating characteristic (ROC) curve analysis to predict ap-
propriate S/Co ratios for anti-HCV testing, previous
studies have shown that very low anti-HCV S/Co ratios
of <3.0 or 4.5 are associated with a high diagnostic sensi-
tivity and negative predictive value (NPV), indicating no
risk of HCV infection and therefore requiring no addi-
tional tests. However, high anti-HCV S/Co ratios of
≥20.0, as determined by the Ortho VITROS anti-HCV
assay, were an accurate serological marker of viremia [9–
11]. In these studies, samples with high anti-HCV levels
were further evaluated by HCV RNA testing to assess vi-
remic status. Such strategies, using anti-HCV S/Co ratios
as a measure of anti-HCV concentration minimizes the
number of individuals that require supplemental testing
to some extent.
Therefore, the objective of this study was to determine
whether S/Co ratios of reactive samples could be used as
a basis of avoiding the need for supplemental testing, and
whether secondary anti-HCV testing could offer an alter-
native to the supplement or confirmation of HCV detec-
tion. We also evaluated whether RIBA could address
false-positive anti-HCV results obtained from initial
screening assays if the HCV RNA results are negative.
Materials and Methods
Ethics Statement
The study involved the use of leftover patient samples. The Eth-
ics Committee of the National Center for Clinical Laboratories ap-
proved our use of these patient samples, and we adhered to the
Anti-HCV Level as a Supplemental
Testing Marker
tenets of the Declaration of Helsinki. Since this study did not re-
quire the collection of detailed patient information, and the data
were analyzed anonymously, participants did not provide their
written informed consent.
Samples
Patient serum samples used in this study were collected from
the General Hospital of Ningxia Medical University, Peking Uni-
versity People’s Hospital, Fuzhou General Hospital of the Nan-
jing Military Area Command, Shanghai Ruijin Hospital, Shang-
dong Province Hospital, General Hospital of the Nanjing Mili-
tary Region, East Hospital of the Affiliated Hospital of Qingdao
University Medical College, and Fujian Blood Center in China.
All serum samples were assessed for the presence of antibodies to
HCV using VITROS ECi CIA (Ortho Clinical Diagnostics). S/Co
ratios were recorded directly from the automated equipment.
Samples with S/Co ratios of ≥1.0 were defined as reactive based
on the manufacturer’s recommendation. Serum samples that
showed reactive anti-HCV results were shipped on dry ice to the
National Center for Clinical Laboratories in Beijing for further
testing.
Screening Assays
Serum samples shipped to the National Center for Clinical Lab-
oratories were first retested using the VITROS ECi CIA. Sera that
were negative with VITROS ECi CIA were considered anti-HCV
negative, according to CDC guidelines [6]. Sera were subsequent-
ly tested with Monolisa Plus anti-HCV version 2 (Monolisa Plus;
Bio-Rad, Marnes-la-Coquette, France) and samples with S/Co ra-
tios ≥1.0 were considered reactive according to the manufacturer’s
instructions. These two screening assays use antigens from a dif-
ferent manufacturer.
Confirmation Assays
Quantitative HCV NAT was performed on all serum samples
with positive ECi CIA results (S/Co ratios ≥1) using the Roche
COBAS AmpliPrep/COBASTaqMan HCV Test (Roche Diagnos-
tics, Branchburg, N.J., USA) as a confirmation assay. The sensitiv-
ity (lowest limit of detection) of the quantitative HCV NAT was 15
IU HCV RNA/ml. Testing and result interpretation was performed
according to the manufacturer’s instructions. However, due to in-
sufficient sample volume, HCV NAT was not performed on 13
samples.
A third-generation RIBA (RIBA HCV 3.0; Ortho Clinical Di-
agnostics) was used to detect the HCV recombinant proteins C100
(NS4), C33c (NS3), C22p (core), and NS5 in order to define the
sample status. The results were interpreted according to the man-
ufacturer’s recommendations. Samples were deemed positive
when ≥2 bands showed reactivity, indeterminate when only 1
band was reactive, and negative when no reactivity was observed.
Definition and Statistical Analysis
Individuals with HCV-positive RNA were considered viremic.
Samples with a positive RIBA result and negative HCV RNA were
recorded as true antibody positive, nonviremic. Samples with reac-
tive anti-HCV screening test results but negative HCV RNA re-
sults and negative or indeterminate RIBA results were categorized
as falsely positive [8].
ROC curves were constructed by plotting sensitivity versus
1 − specificity, using HCV RNA and the third-generation RIBA
Intervirology 2015;58:310–317 311
DOI: 10.1159/000441474
Table 1. Categories of hepatitis C antibody levels based on S/Co ratios and the results of supplemental testing

Anti-HCV S/CO Number Viremic subjects Nonviremic subjects Subjects in whom viremia
ratio by CIA of patients (n = 586) (n = 302) was not (n = 13)
(n = 888) positive negative indeterminate positive negative indeterminate positive negative indeterminate
RIBA RIBA RIBA RIBA RIBA RIBA RIBA RIBA RIBA

1.01 – 3.00 275 30 29 40 13 72 91 2 0 0

3.01 – 20.00 204 83 7 23 44 14 33 1 3 5
≥20 409 370 0 4 33 1 1 1 0 1
Total number 888 483 36 67 90 87 125 4 3 6

test as gold standards, respectively. We determined the diagnostic

sensitivity, diagnostic specificity, positive predictive value (PPV),
NPV, and their respective exact 95% CI to predict HCV viremia 100
and RIBA status at S/Co ratios of 3.0, 8.0, and 20.0, according to S/Co = 3.00
previously published methods [8–11]. Optimal S/Co ratios were
S/Co = 8.00
identified from the analysis of ROC curves and associated data
80
[12]. We performed ROC analysis using Graphpad Prism 6 statis-
tical software. S/Co = 20.00

60
Sensitivity (%)

Results

A total of 1,017 samples were shipped to the National 40

Center for Clinical Laboratories and retested for anti-

HCV antibodies using CIA as the screening assay, and Sensitivity (%)
901 samples demonstrated reactive results (S/Co ≥1). All 20
Identity (%)
reactive samples (S/Co ≥1) were also tested using quan-
titative HCV NAT testing and third-generation RIBA.
HCV RNA test results that were <15 IU/ml (below the 0
0 20 40 60 80 100
quantifiable linear range) were deemed as ‘HCV RNA re- 100% – specificity (%)
active’ since the RIBA results illustrated that 35.8%
(63/176) of the samples demonstrated detectable HCV
RNA values of <15 IU/ml and were RIBA positive. Of
Fig. 1. RT-PCR test ROC curve based on different CIA cutoff levels
these 901 samples, HCV RNA testing was not performed for anti-HCV antibody detection. The area under the curve is 0.812
on 13 samples due to insufficient sample quantity; how- (95% CI: 0.783–0.841).
ever, 586 samples (65.0%) and 302 samples (33.5%) dem-
onstrated confirmatory HCV RNA-positive and -nega-
tive results, respectively. Furthermore, of the 901 sam-
ples, 577 (64.0%), 126 (14.0%), and 198 (22.0%) PCR test ROC curves were analyzed to determine cut-
demonstrated positive, negative, and indeterminate re- off levels based on CIA results for anti-HCV antibody
sults, respectively, after additional RIBA testing. As detection. Based on the PCR test ROC curve (fig. 1) and
shown in table 1, of the 586 samples with positive HCV associated diagnostic sensitivity, diagnostic specificity,
RNA results, 483 (82.4%) tested positive by RIBA, and of and PPV, we determined that an S/Co ratio of 20.0 was
the 302 samples with negative HCV RNA results, 90 not optimal, as demonstrated in previous studies [9, 10].
(29.8%) tested positive by RIBA. These 90 samples repre- The corresponding diagnostic sensitivity, diagnostic
sented true-positive anti-HCV results without viral rep- specificity, PPV, and NPV for HCV RNA were 63.82,
lication. 88.89, 91.67, and 56.20%, respectively (table 2). We also

312 Intervirology 2015;58:310–317 Zhang/Wang/Lin/Li

DOI: 10.1159/000441474
 Table 2. Diagnostic performance of CIA in the prediction of viremia by RT-PCR

S/Co ratio 3.0 8.0 20.0

Diagnostic sensitivity, % 82.94 (79.64 – 85.89) 75.77 (72.09 – 79.19) 63.65 (59.61 – 67.55)
Diagnostic specificity, % 57.84 (52.09 – 63.44) 78.43 (73.39 – 82.91) 88.89 (84.82 – 92.18)
PPV, % 79.02 (75.81 – 82.24) 87.06 (84.15 – 89.97) 91.67 (88.99 – 94.35)
NPV, % 63.90 (58.25 – 69.56) 62.83 (57.98 – 67.68) 56.20 (51.78 – 60.62)

Values in parentheses are the limits of the 95% CI.

Table 3. Diagnostic performance of CIA in the prediction of the presence of anti-HCV antibodies by RIBA

S/Co ratio 3.0 8.0 20.0

Diagnostic sensitivity, % 92.03 (89.51 – 94.10) 84.23 (80.99 – 87.11) 69.84 (65.92 – 73.57)
Diagnostic specificity, % 71.91 (66.68 – 76.74) 90.74 (87.05 – 93.67) 97.84 (95.60 – 99.13)
PPV, % 81.46 (84.42 – 88.54) 92.42 (90.18 – 94.67) 97.75 (96.32 – 99.17)
NPV, % 87.06 (83.60 – 90.52) 81.10 (77.63 – 84.57) 70.75 (67.13 – 74.38)

Values in parentheses are the limits of the 95% CI.

determined the diagnostic sensitivity, diagnostic specific-

ity, PPV, and NPV and their respective 95% CI for predic- S/Co = 3.00
100
tion of HCV viremia at S/Co ratios of 3.0 and 8.0 (table 2).
S/Co = 8.00
We analyzed the RIBA test ROC curve for different cutoff
levels based on CIA results for the diagnosis of HCV ex-
80 S/Co = 20.00
posure. The RIBA ROC curve (fig. 2) and associated data
demonstrated that an S/Co ratio of 20.0 corresponded to
a diagnostic sensitivity, diagnostic specificity, PPV, and
60
Sensitivity (%)

NPV for HCV antibody confirmation by RIBA of 69.84,

97.84, 97.75, and 70.75%, respectively (table 3). Tables 2
and 3 illustrate that an S/Co ratio of ≥20 better discrimi-
40
nates viremia and HCV exposure in screened anti-HCV-
positive samples compared to other S/Co ratios such as
8.0. Sensitivity (%)
The diagnostic sensitivity, diagnostic specificity, PPV, 20
Identity (%)
and NPV, as well as their respective 95% CI in predicting
HCV exposure for different cutoff levels at S/Co ratios of
3.0 and 8.0 are shown in table 3. From both the real-time 0
0 20 40 60 80 100
PCR (RT-PCR) (fig. 1) and RIBA (fig. 2) ROC curves, we 100% – specificity (%)
identified that an S/Co ratio of 3.0 was not the highest
value providing a diagnostic sensitivity of 100%. Table 1
shows that 99 out of 275 subjects (36%) with S/Co ratios
Fig. 2. RIBA test ROC curve based on different CIA cutoff levels
of <3.0 were viremic subjects, and 15 displayed HCV ex- for anti-HCV antibody detection. The area under the curve is 0.932
posure without viremia. These results illustrate that pa- (95% CI: 0.916–0.948).
tients with S/Co ratios <3.0 were not all negative for both
HCV viremia and HCV exposure.

Anti-HCV Level as a Supplemental Intervirology 2015;58:310–317 313

Testing Marker DOI: 10.1159/000441474
 Color version available online

Color version available online

% RIBA IND RIBA N RIBA P % Nonviremic Viremic
100 1 6 100
35
90 90
61
80 80
131 91
70 70 176
24
60 60
50 404 50
374
40 40
101
30 128 30
113
20 20
99
10 45 10
0 0
n = 277 n = 213 n = 411 n = 275 n = 204 n = 409
(1.01–3.00) (3.01–20.00) (–20) (1.01–3.00) (3.01–20.00) (–20)

Fig. 3. RIBA results relative to S/Co ratios according to antibody
level. Samples with an S/Co ratio ≥20, between 3.0 and 19.99, or
between 1.0 and 2.99 were classified as high antibody levels, low
antibody levels, or very low antibody levels, respectively. These
antibody levels are outlined in parentheses below each bar. The
percentages of RIBA-positive samples according to these antibody
levels were as follows: high antibody levels, 98.3% (404/411); low
antibody levels, 6.0% (128/213), and very low antibody levels,
16.2% (45/277). IND = Indeterminate; N = negative; P = positive.
Fig. 4. Distribution of HCV viremic samples according to antibody
level. Samples with an S/Co ratio ≥20, between 3.0 and 19.99, or
between 1.0 and 2.99 were classified as high antibody levels, low
antibody levels, or very low antibody levels, respectively. The per-
centages of HCV viremic samples according to these antibody lev-
els were as follows: high antibody levels, 91.4% (374/409); low an-
tibody levels, 55.4% (113/204), and very low antibody levels, 36.0%
(99/275). These antibody levels are outlined in parentheses below
each bar. Because of insufficient sample volume, 2 samples with
high antibody levels, 9 samples with low antibody levels, and 2 sam-
ples with very low antibody levels were not tested for HCV RNA.

Although an S/Co ratio of 20.0 was not determined to
be an optimal cutoff in previous studies [9, 10], results with
an S/Co ratio ≥20.0 were still classified as possessing high
antibody levels in the present study. Those samples with an
S/Co ratio between 3.0 and 19.99 were designated as low
level. Samples with very low anti-HCV levels and S/Co ra-
tios between 1.0 and 2.99 were selected based on a previous
study showing that these samples demonstrated no risk of
having HCV infection [9]. These three antibody levels were
observed in 411 (45.62%), 213 (23.64%), and 277 (30.74%)
of the 901 samples, respectively (fig. 3). Except for 13 sam-
ples that were not tested for HCV RNA due to insufficient
sample volume, HCV viremia was confirmed by positive
HCV RNA testing in 91.4% (374/409) of samples with high
antibody levels, 55.4% (113/204) of samples with low anti-
body levels, and 36.0% (99/275) of samples with very low
antibody levels (fig.  4). A significant difference was ob-
served in the viral replication frequency between samples
with high anti-HCV antibody levels (S/Co ratios ≥20.0;
91.4%) and those in the low and very low antibody level
groups (S/Co ratios between 1.0 and 19.99; 44.3%; p <
0.001, χ2 test). In our study, 586 viremic individuals dem-
onstrated higher antibody levels (mean S/Co ratio: 19.23,
95% CI: 18.4–20.1) than individuals (90 samples) with con-
firmed serological HCV without viremia (mean S/Co ratio:
14.33, 95% CI: 12.2–16.4, p < 0.05). A mean S/Co ratio of
2.94 (95% CI: 2.51–3.37) was observed in 212 samples de-
fined as false positive for hepatitis C without viremia, and
with negative or indeterminate RIBA results.
The results of Monolisa Plus testing in 888 CIA-posi-
tive sera (S/Co ≥1) that underwent previous HCV RNA
testing are shown in table 4. The sensitivity and specific-
ity of the Monolisa Plus test were 81.8 and 85.8%, respec-
tively, compared to confirmation with RT-PCR and
RIBA. The PPV and NPV were 94.85 and 59.67%, respec-
tively. In 90 samples with confirmed serological HCV
without viremia, 16 samples (17.8%) were missed using
Monolisa Plus as a supplemental test. In the 212 individ-
uals defined as false positive for hepatitis C without vire-
mia with negative or indeterminate RIBA, 31 samples
(14.6%) were detected as false positive for anti-HCV an-
tibody using the Monolisa Plus test.

314 Intervirology 2015;58:310–317 Zhang/Wang/Lin/Li

DOI: 10.1159/000441474
 Table 4. Results of the Monolisa Plus test in 888 CIA-positive sera (1 ≤ S/Co <20)

Monolisa Plus True-positive anti-HCV False-positive anti-HCV

HCV RNA positive HCV RNA negative/ HCV RNA negative/
RIBA positive RIBA negative or indeterminate
Positive (S/Co ≥1) 94 53 29
Negative (S/Co <1) 106 16 181

The 888 CIA-positive sera were confirmed by HCV RNA test.

Table 5. Interpretation for anti-HCV results utilizing S/Co ratios and type of recommended supplemental testing

Antibody level Recommended Result Interpretation Recommendation

(S/Co ratio) supplemental testing

S/Co <1.0 None – No HCV antibody detected Notify

1.0 ≤ S/Co HCV RNA Positive Current HCV infection Notify and link to care
<20.0 Negative Nonviremic hepatitis C or Additional testing as
nonhepatitis C appropriate1
S/Co ≥20.0 HCV RNA Positive Current HCV infection Notify and link to care
Negative Nonviremic hepatitis C Notify
1 Repeat HCV RNA testing or follow-up testing for HCV antibody is recommended if the individual tested

might have been exposed to HCV within the past 6 months or has clinical evidence of HCV disease. At this time,
an immunoblot assay as supplemental testing is still necessary.

Discussion testing. Samples with very low hepatitis C antibody levels

Our study shows that very low anti-HCV antibody lev-
els with S/Co ratios <3.0, as determined by the VITROS
anti-HCV assay, did not identify anti-HCV false posi-
tives; therefore, this group is not an accurate marker that
can prevent the need for supplemental testing. Confirma-
tory anti-HCV testing by RIBA or secondary HCV anti-
body assay is not necessary when the S/Co ratio is >20
because of the high rate of true positives detected (PPV:
97.75%).
Lai et al. [9] reported that an S/Co ratio of 3.0 deter-
mined by the VITROS anti-HCV assay was the highest
value associated with a diagnostic sensitivity of 100% and
NPV of 100%, using either PCR or RIBA as gold stan-
dards. No positive RIBA or PCR test results were found
in samples with an S/Co ratio <3.0 in their analyses.
Therefore, it was suggested that supplemental testing was
not necessary for patient samples with S/Co ratios <3.0.
Similarly, Contreras et al. [10] and Oethinger et al. [4]
also demonstrated that very low hepatitis C antibody lev-
els were false positives and thus avoided supplemental
in the aforementioned studies were designated as having
S/Co ratios of 4.5 and 5.0, respectively [4, 10]. In the pres-
ent study, the mean S/Co ratio for false-positive hepatitis
C individuals without viremia, and with negative or inde-
terminate RIBA results (n = 212) was 2.94 (95% CI: 2.51–
3.37). However, the CIA versus RT-PCR (fig. 2) and CIA
versus RIBA (fig. 3) ROC curves illustrated that an S/Co
ratio of 3.0 was not associated with a diagnostic sensitiv-
ity or NPV of 100%, using either PCR or RIBA as gold
standards. In our population, a cutoff S/Co ratio of 3.0
would prevent detection of 41.5% (114/275) of CIA-pos-
itive samples with S/Co ≤3.0 (table 1), with either positive
RNA (n = 99) or positive RIBA (n = 15). Therefore, we
recommend that supplemental testing should still be re-
quired for patients with very low anti-HCV antibody lev-
els and S/Co ratios ≤3, as determined by CIA. Moreover,
although a previous study showed that only 1.8% of sub-
jects with an S/Co ratios <20.0 were viremic [4], we dem-
onstrated that 23.9% (212/888) of samples with the same
S/Co ratios were viremic. Therefore, performing supple-
mentary testing using HCV RNA tests on all samples, in-

Anti-HCV Level as a Supplemental Intervirology 2015;58:310–317 315

Testing Marker DOI: 10.1159/000441474
cluding those with low antibody levels, is not cost-effec-
tive (table 5).
Interestingly, the ROC curve analysis in our study
demonstrated that an S/Co ratio of 20.0 was not an opti-
mal cutoff, as has been suggested in previous studies [9,
10]. The differences in the findings could be attributed to
the following three reasons. First, the previous study [9]
assumed that all samples with positive results, deter-
mined by RT-PCR testing, would also be positive by
RIBA. The present study demonstrated that 6.14% (6/586)
and 11.4% (67/586) of samples with positive RT-PCR re-
sults displayed negative and indeterminate results by
RIBA, respectively. Second, the study populations were
different. Lai et al. [9] proposed an algorithm for HCV
testing based on the results in a population of veterans.
Oethinger et al. [4] conducted the study using blood do-
nor samples. However, the population in our study came
from three groups: patients from a liver disease clinic
(14%, 127/901), patients from other disease clinics (83%,
748/901), and blood donors (3%, 26/901). The difference
in the prevalence of anti-HCV antibodies in the various
study populations might account for the differences in
optimal S/Co ratio cutoffs. Third, the distribution of the
predominant HCV subtype varies regionally; for exam-
ple, HCV-1b and 2a are the most common subtypes in
China [13]. This might have caused the varying results
between the studies for the VITROS anti-HCV assay.
Our values for diagnostic specificity (91.67%) and PPV
(88.89%) in predicting HCV viremia using the VITROS
anti-HCV assay at an S/Co ratio of 20.0 were higher than
the values (58.8 and 81%, respectively) reported by Lai et
al. [9], but were lower than those (96.6 and 93.7%) re-
ported by Contreras et al. [10]. The results demonstrated
that the proportion of our population with an S/Co ratio
of ≥20.0 but with HCV viremia was between the propor-
tions of populations studied by Lai et al. [9] and Contreras
et al. [10]. Diagnostic sensitivity (75.77%) and NPV
(62.83%) at an S/Co ratio of 8.0 were much lower than
comparable results in the two previous studies, which
suggest that we identified a greater proportion of our
population with S/Co ratios ≥8.0 but who also have HCV
viremia.
The present study showed that 586 viremic individuals
had higher antibody levels (mean S/Co ratio: 19.23, 95%
CI: 18.4–20.1). We also showed that of the 409 samples
tested for HCV RNA with S/Co ratios ≥20.0, based on
CIA, 374 were positive for HCV RNA (91.4%). The RNA
positivity rate was different from that reported in previ-
ous studies: 81% [9], 90% [14], 93% [10], 81% [4], and
>60% [15]. Of the 34 samples with S/Co ratios ≥20.0 and
316 Intervirology 2015;58:310–317
DOI: 10.1159/000441474
with negative HCV RNA, 33 samples were RIBA positive
and 1 sample was RIBA indeterminate. Of the two sam-
ples not tested for HCV RNA due to insufficient volume,
one was RIBA positive and the other was RIBA indeter-
minate. The true-positive rate was at least 99.5% (408/410).
The results also showed that 99.8% (409/410) of samples
with S/Co ratios ≥20.0 were reactive, as determined by
the Monolisa Plus test.
In our study, using ROC curve analysis, the high diag-
nostic specificity and PPV values for the prediction of ei-
ther viremia by RT-PCR or presence of anti-HCV anti-
bodies by RIBA showed that an S/Co ratio ≥20.0 strong-
ly indicates HCV exposure. Therefore, we recommend, at
least for our study population, that samples with S/Co
ratios ≥20.0 should not undergo supplemental RIBA test-
ing or secondary immunoassay testing. This would be un-
necessary as these samples with such high S/Co ratios are
confirmed by positive anti-HCV RIBA results ≥98%.
Upon evaluation for antiviral therapy, these samples
should directly proceed to NAT to assess HCV viremic
status (table 5).
A strategy for HCV antibody testing using two enzyme
immunoassays in a routine clinical laboratory has been
validated, and the sensitivity and specificity of confirma-
tion of the second enzyme immunoassay were 98.15 and
98.33%, respectively [7]. The CDC recently recommend-
ed that testing be done with a second HCV antibody assay
that is different from the initial antibody assay used for
diagnosis of HCV infection when the result of HCV RNA
is negative, as a result of the discontinuation of HCV
RIBA [6]. In the present study, samples were also tested
by Monolisa Plus. The results showed that 17.8% of sam-
ples (16/90) with confirmed serological HCV without vi-
remia were missed when Monolisa Plus was used as the
supplemental testing method. Immunodeficiency might
be the common cause of false-negative anti-HCV results
in chronic HCV-infected patients [16]. Our previous
study of blood donors also showed that even with two
screening assays in addition to NAT, some anti-HCV-
positive samples were still missed, suggesting that there
may be no suitable combination to achieve a 100% sensi-
tivity rate and avoid viral transmission [17]. In this study,
14.6% of samples (31/212) were detected as false positive
for anti-HCV by Monolisa Plus. The results show that a
strategy employing secondary HCV antibody assay test-
ing with different HCV antigens from the first assay as a
supplemental screening method was not an excellent
strategy for accurate HCV screening in our population.
Therefore, although there are many disadvantages,
such as high cost, requirement of specialized equipment
Zhang/Wang/Lin/Li
and qualified personnel, extended execution time, and in-
determinate results, the immunoblot assay as supplemen-
tal testing is still necessary, especially for the nonviremic
individual with false-negative anti-HCV results. A previ-
ous study [18] evaluated the sensitivity of five anti-HCV
immunoblot assays licensed in France and found that the
results were less divergent across assays with more uni-
form criteria for interpretation. The RIBA HCV 3.0 assay
is no longer available; therefore, other immunoblot assays
should be selected for supplemental testing. When the S/
Co ratio based on the VITROS anti-HCV assay is between
1.0 and 20.0, additional testing should be performed as
appropriate, i.e. an immunoblot assay for samples with-
out viremic hepatitis (table 5).
Our interpretation of anti-HCV results utilizing S/Co
ratios and the type of recommended supplemental testing
is summarized in table 5. If the S/Co ratio is <20.0, based
on CIA, HCV RNA testing should also be performed as a
confirmatory test for discrimination of nonviremic hepa-
titis C or nonhepatitis C. Repeat HCV RNA testing or
follow-up testing for HCV antibodies is recommended if
the person tested might have been exposed to HCV with-
in the past 6 months or has clinical evidence of HCV dis-
ease. For positive samples without viremia, immunoblot
assays as supplemental testing is still necessary. If the S/
Co ratio is ≥20.0, based on CIA, then performing RT-
PCR could further assess the presence of HCV viremia.
Acknowledgments
We gratefully acknowledge all of the listed institutions in Mate-
rial and Methods for the provision of samples.

References
1 World Health Organization. Hepatitis C fact
sheet No. 164. Updated July 2013. http://
www.who.int/mediacentre/factsheets/fs164/
en/.
2 Di Bisceglie AM: Hepatitis C. Lancet 1998;69:
213–216.
3 Lauer GM, Walker BD: Hepatitis virus infec-
tion. N Engl J Med 2001;345:41–52.
4 Oethinger M, Mayo DR, Falcone J, Barua PK,
Griffith BP: Efficiency of the Ortho VITROS
assay for detection of hepatitis C virus-specif-
ic antibodies increased by elimination of sup-
plemental testing of samples with very low
sample-to-cutoff ratios. J Clin Microbiol
2005;43:2477–2480.
5 Chapko MK, Sloan KL, Davison JW, Dufour
DR, Bankson DD, Rigsby M, et al: Cost effec-
tiveness of testing strategies for chronic hepa-
titis C. Am J Gastroenterol 2005;100:607–615.
6 Centers for Disease Control and Prevention
(CDC): Testing for HCV infection: an update
of guidance for clinicians and laboratorians.
MMWR Morb Mortal Wkly Rep 2013; 62:
362–365.
7 Vermeersch P, Van Ranst M, Lagrou K: Vali-
dation of a strategy for HCV antibody testing
with two enzyme immunoassays in a routine
clinical laboratory. J Clin Virol 2008;42: 394–
398.
8 Alter MJ, Kuhnert WL, Finelli L; Centers for
Disease Control and Prevention: Guidelines
for laboratory testing and result reporting of
antibody to hepatitis C virus. Centers for Dis-
ease Control and Prevention. MMWR Re-
comm Rep 2003;52:1–13.
9 Lai KK, Jin M, Yuan S, Larson MF, Dominitz
JA, Bankson DD: Improved reflexive testing
algorithm for hepatitis C infection using sig-
nal-to-cutoff ratios of a hepatitis C virus anti-
body assay. Clin Chem 2011;57:1050–1056.
10 Contreras AM, Ochoa-Jiménez RJ, Celis A,
Méndez C, Olivares L, Rebolledo CE, Her-
nandez-Lugo I, Aguirre-Zavala AI, Jiménez-
Méndez R, Chung RT: High antibody level:
an accurate serologic marker of viremia in
asymptomatic people with hepatitis C infec-
tion. Transfusion 2010;50:1335–1343.
11 Contreras AM, Tornero-Romo CM, Toribio
JG, Celis A, Orozco-Hernández A, Rivera PK,
Méndez C, Hernández-Lugo MI, Olivares L,
Alvarado MA: Very low hepatitis C antibody
levels predict false-positive results and avoid
supplemental testing. Transfusion 2008; 48:
2540–2548.
12 Akobeng AK: Understanding diagnostic tests
3: receiver operating characteristic curves.
Acta Paediatr 2007;96:644–647.
13 Zhuang H, Tracy L, Cui Y: Study on hepatitis
C virus genotyping in some parts of China (in
Chinese). Zhonghua Liu Xing Bing Xue Za
Zhi 2001;22:99–101.
14 Dufour DR, Talastas M, Fernandez MD, Har-
ris B: Chemiluminescence assay improves
specificity of hepatitis C antibody detection.
Clin Chem 2003;49:940–944.
15 Dufour DR: Lot-to-lot variation in anti-hep-
atitis C signal-to-cutoff ratio. Clin Chem
2004;50:958–960.
16 Fabrizi F, Poordad FF, Martin P: Hepatitis C
infection and the patient with end-stage renal
disease. Hepatology 2002;36:3–10.
17 Zhang K, Wang L, Sun Y, Zhang R, Lin G, Xie
J, Li J: Improving the safety of blood transfu-
sion by using a combination of two screening
assays for hepatitis C virus. Transfus Med
2014;24:297–304.
18 Couroucé AM, Noel L, Barin F, Elghouzzi
MH, Lunel F, North ML, Smilovici W: A com-
parative evaluation of the sensitivity of five
anti-hepatitis C virus immunoblot assays.
Vox Sang 1998;74:217–224.

Anti-HCV Level as a Supplemental Intervirology 2015;58:310–317 317

Testing Marker DOI: 10.1159/000441474