Review
OF HEPATOLOGY
New virological tools for screening, diagnosis and monitoring
of hepatitis B and C in resource-limited settings
Keywords: Viral hepatitis;
Hepatitis B; Hepatitis C; POCT;
RDT; Screening; Diagnosis;
Monitoring.
Received 28 November 2017;
received in revised form 11 May
2018; accepted 14 May 2018
1
National Reference Center for
Viral Hepatitis B, C and D,
Department of Virology, Hôpital
Henri Mondor, Université
Paris-Est, Créteil, France;
2
INSERM U955, Créteil, France
⇑ Corresponding author.
Address: Department of
Virology, Hôpital Henri Mondor,
51 avenue du Maréchal de Lattre
de Tassigny, 94010 Créteil,
France. Tel.: +33 1 4981 2828;
fax: +33 1 4981 2839.
E-mail address: stephane.
chevaliez@aphp.fr
(S. Chevaliez).
JOURNAL
Stéphane Chevaliez1,2,⇑, Jean-Michel Pawlotsky1,2
Summary
Worldwide, the increasingly dominant model of laboratory testing is the centralised laboratory, in
which automation of analytical processes increases, enabling the analysis of large numbers of samples
at a relatively low cost. However, this trend does not fulfil the requirements for care of patients with
chronic hepatitis B and C in resource-limited settings. Alternative models using point-of-care (POC) tests
and dried blood spots (DBSs) are increasingly being considered for viral hepatitis screening, diagnosis
and monitoring. POC tests are small devices providing qualitative and/or quantitative determination
of viral antibodies and/or antigens. They can use original specimen matrices, such as oral fluid or blood
collected from a fingerstick. POC tests are particularly useful for large-scale screening, and to improve
access to care in regions where laboratory access is limited. New POC devices that detect and quantify
viral nucleic acids are at the developmental stage. DBSs offer the main advantage of enabling storage of
desiccated blood that can be easily transported to reference centres, where state-of-the-art molecular
and serological diagnostic tests are available. However, standardisation and better automation of DBS
handling are needed. Herein, we review alternatives to classical hepatitis B and C virological tests, exam-
ining POC tests and DBSs, as well as alternatives to nucleic acid testing. Innovations in testing
approaches resulting from the availability of these new assays are also discussed.
Ó 2018 Published by Elsevier B.V. on behalf of European Association for the Study of the Liver.
Introduction
Infections by hepatitis B virus (HBV) or hepatitis C the absolute number of infected individuals keeps
virus (HCV) affect several hundred million people growing as a result of global population growth.2
worldwide,1,2 including approximately 28 million Because chronic viral hepatitis is generally
in Europe.3 Chronic HBV and HCV infections lead asymptomatic until advanced liver disease devel-
to chronic hepatitis, cirrhosis, decompensation of ops, up to approximately 80% of infected patients
cirrhosis and/or hepatocellular carcinoma (HCC). are unaware of their infection and related liver
HBV is the number one cause of HCC, one of the disease.3,12 In low- to middle-income areas, the
most frequent cancers in areas of high endemicity vast majority of HBV or HCV infections have not
for the virus, such as sub-Saharan Africa and been diagnosed. As a result, access to care and
Asia.4–6 In industrialised countries, HCV is still antiviral therapy is denied to a large proportion
the number one cause of HCC and the main indica- of patients with chronic hepatitis B or C, who are
tion for liver transplantation.7 The annual death likely to develop severe complications and trans-
rate due to chronic viral hepatitis B or C has been mit infection. Thus, broad-scale HBV and HCV
estimated to be more than 1.3 million, a toll com- screening is required, based on local epidemiology
parable to that of human immunodeficiency virus and resources.13
(HIV) and tuberculosis.8,9 Among many obstacles, the post-screening cas-
The actual worldwide prevalence of chronic cade of care suffers from the unavailability or inac-
hepatitis B and C is unknown, mainly because cessibility of virological tests required for
the disease remains asymptomatic for many years diagnosing infection, establishing the prognosis
before the complications of chronic infection of related disease, making a treatment decision,
occur. The most recent modelling suggests that monitoring treatment outcomes and assessing
292 million individuals are chronically infected the efficacy of prevention. Classical virological
with HBV and 71 million with HCV worldwide.1,2 tests require blood sampling by venepuncture,
The implementation of birth vaccination pro- capacity for cold storage, specific infrastructure,
grammes has considerably reduced the incidence equipment and personnel training, and are often
and prevalence of HBV infection in several high unaffordable in low- to middle-income countries.
endemicity areas, with a major impact on the inci- Additionally, the distance to a sufficient healthcare
dence of liver diseases, particularly HCC.10,11 How- service may be long and the local infrastructure
ever, vaccination programmes remain to be poor. Thus, new assays that facilitate easy and
implemented in many places, especially in low- inexpensive access and linkage to care are needed.
to middle-income countries. Thus, although the In this review article, we examine alternatives
prevalence of HBV infection diminishes globally, to classical HBV and HCV virological tests that
Journal of Hepatology 2018 vol. 69 j 916–926

use serum or plasma taken from venepuncture,
including point-of-care (POC) tests that bring
diagnostics to the site of patient care, tests based
on blood collection on filter papers known as dried
blood spots (DBSs), and alternatives to nucleic acid
testing (NAT). The article also discusses innova-
tions in testing approaches resulting from the
availability of these new assays.
Classical HBV and HCV virological tests
Screening for HBV infection is based on the detec-
tion of hepatitis B surface antigen (HBsAg) by
enzyme immunoassay, while screening for HCV
infection is based on the detection of total anti-
HCV antibodies by enzyme immunoassay.
Confirmation of infection is based on NAT, i.e.
the detection of circulating viral genomes in
serum or plasma, including HBV DNA or HCV
RNA, respectively. The European Association for
the Study of the Liver (EASL) recommends using
a real-time PCR-based or a transcription-
mediated amplification-based assay with a limit
of detection/lower limit of quantification of 10
international units (IU)/ml of HBV DNA or less,14
or 15 IU/ml of HCV RNA or less.15 However, there
is an important need for diagnostic nucleic acid
assays that are cheap (less than US$5–10) and thus
applicable for large-scale diagnosis in low-to
middle-income areas, as well as in specific settings
in high-income countries. Thus, in 2018, EASL rec-
ommended the use of qualitative, not necessarily
quantitative HCV RNA assays with a limit of detec-
tion ≤1,000 IU/ml (3.0 Log IU/ml). In the settings in
which these assays will be used, the exceptionally
low risk of a false-negative result in a small per-
centage of infected individuals is outweighed by
the benefit of scaling up access to diagnosis and
care.15 A similar recommendation for HBV DNA
detection would help to make such assays avail-
able. Although slightly less sensitive than HCV
RNA testing, the HCV core antigen has been pro-
posed as an alternative marker of viral replication
(limit of detection equivalent to 500–3,000 HCV
RNA IU/ml according to the HCV genotype).13,15,16
This assay is European Conformity (CE)-marked,
but has not yet been approved by the US Food
and Drug Administration (FDA). Notably, the HBV
DNA level is a prognostic marker of liver dam-
age,17 whereas the HCV RNA (or the HCV core anti-
gen) level is not.
HBV treatment indications depend on the HBV
DNA level, the serum alanine aminotransferase
activity and the severity of liver disease, as
assessed by liver biopsy or, more often, non-
invasive tests.14 All patients with detectable HCV
RNA have an indication for anti-HCV therapy with
current IFN-free, direct-acting antiviral drug-
based regimens.15 HCV genotype determination
is helpful to guide the choice of antiviral therapy.
However, genotype determination can be skipped
with pangenotypic anti-HCV regimens, as recently
Journal of Hepatology 2018 vol. 69 j 916–926
JOURNAL
OF HEPATOLOGY
recommended by EASL.15 Both HBV DNA and HCV
RNA are used to monitor the efficacy of antiviral
therapy: on treatment for HBV infection and 12
or 24 weeks after the end of treatment for HCV.
The HCV core antigen can be used instead of
HCV RNA to assess the response to anti-HCV
therapy.13,15,16
Additional serological markers, including anti-
HBs antibodies, hepatitis B ‘‘e” antigen (HBeAg)
and anti-HBe antibodies, anti-hepatitis B core
(HBc) antibodies and anti-HBc IgM assessed by
enzyme immunoassay, are useful to classify
HBV-infected patients into the different phases of
the disease, guide the treatment decision and
characterise the response to antiviral therapy.14
Key point
Screening for HBV is based
‘‘Point-of-care’’ tests on detection of HBsAg,
POC testing is defined as testing performed close while screening for HCV is
to or near the patient, i.e. where healthcare is pro- based on detection of anti-
vided outside of traditional centralised HCV antibodies.
laboratories.
General features of point-of-care tests
In the past 20 years, POC tests for diabetes,
anaemia, pregnancy, HIV and malaria have become
common diagnostic tools in both high- and low- to
middle-income areas. They have significantly
improved the quality of care within the framework
of patient-centred approaches.18 In the setting of
infectious diseases, most existing POC tests consist
of immunoassays providing qualitative and, some-
times, quantitative determination of various mark-
ers of infection, including antigens and antibodies,
within a limited amount of time. For this reason,
they are also called ‘‘rapid diagnostic tests”
(RDTs).19 Non-immunological POC tests that detect
and sometimes quantify pathogen nucleic acids are
at earlier stages of development.
POC tests can use whole blood, serum or
plasma collected by venepuncture. However, the
main focus is on the potential use of alternative
matrices, such as tiny amounts of fingerstick
capillary whole blood or oral fluid. Fingerstick
capillary whole blood collection uses a manual or
automated lancet device that punctures the finger
(or the heel in infants in order to prevent hitting
the bone). Capillary whole blood can then be col-
lected with various materials, including a tube
with anticoagulant, a loop, a pipette, or filter paper
(DBS). Similarly, oral fluid can be simply, safely
and cheaply collected and tested.20
POC tests have been designed for use at the
Key point
sites of patient care, including physicians’ offices,
outpatient clinics, intensive care units, emergency Point-of-care tests are
rooms, medical laboratories, or even patients’ designed for use at the site
of patient care, avoiding
homes for self-testing. In low- to middle-income
many of the limitations of
countries, they are widely used in these settings, classical HBV and HCV
as well as in blood banks.21,22 POC tests enable diagnostic tests in
the delivery of results at the time of testing with- resource-limited settings.
out the need for a follow-up visit, thereby increas-
ing the proportion of individuals informed of their
917
Review
status.23 POC tests have the potential to reduce
emergency admissions, hospitalisations or the
length of hospital stay, while enabling care to be
delivered closer to patients’ homes.24 Concerns
have been raised over the analytical performance
of some POC tests, which must be carefully evalu-
ated before their use is recommended.25
Immunological point-of-care tests (rapid
diagnostic tests)
Principles, advantages and limitations of rapid
diagnostic tests
The principle of RDTs is to capture the antigens or
antibodies being investigated on a solid surface,
before attaching molecules to them that enable
their detection with the naked eye or a dedicated
reader. An ideal RDT must meet the World Health
Organization (WHO) ‘‘ASSURED” criteria
(Table 1).26 RDTs must be easy to use with mini-
mal training. A shelf life of at least one to two
years at ambient temperature, with no need for
freezing or refrigeration, is recommended. It is
important that, at most, only limited instrumenta-
tion is required, so that these tests can be per-
formed at the periphery of health systems,
particularly where there is no laboratory or elec-
tricity. These characteristics make RDTs particu-
larly adapted to low- and middle-income
countries.
A few basic technologies are offered by manu-
facturers, including: lateral-flow tests, also called
immunochromatographic strips or strip-tests;
flow-through tests, usually supplied as individual
cassettes; tests based on the agglutination of
particles; and tests based on solid-phase assays
Table 1. The WHO ASSURED criteria that must be met by an
‘‘ideal” rapid diagnostic tests.26
A = Affordable
S = Sensitive
S = Specific
U = User-friendly
R = Robust and Rapid
E = Equipment-free
D = Deliverable
WHO, World Health Organization.
Table 2. Respective advantages and disadvantages of rapid diagnostic tests and enzyme immunoassays.
Rapid diagnostic tests
Advantages
Easy-to-use
Whole blood, oral fluid, serum or plasma as matrices
Equipment-free
Good sensitivity and specificity
Qualitative (on/off) result
Fast delivery of the result
Long shelf life at ambient temperature
Disadvantages
No possibility to enhance the response with an enzyme reaction
No traceability
Subjective (operator-dependent) interpretation
Infectious waste disposal
Possibly high cost
918 Journal of Hepatology 2018 vol. 69 j 916–926
(so-called ‘‘dipsticks”), allowing for the detection
of one or multiple parameters with a single assay.27
The simplest and most widely used tests for
specific qualitative or semi-quantitative detection
of HBV antigens or antibodies or HCV antibodies
are lateral-flow immunoassays.28,29 After being
loaded on the absorbent pad, the analysed body
fluid moves without the assistance of external
forces through the different zones of the polymeric
strip on which molecules that can interact with
the sought antigen or antibody have been
attached. The most common readout consists of
lines appearing with different intensities, as
assessed by the naked eye or a dedicated reader.
A positive test line indicates the presence of the
antigen or antibody, while the control line con-
firms proper liquid migration through the strip
and/or the presence of a sufficient amount of
immunoglobulins in the body fluid loaded.
Lateral-flow immunoassays are supplied as indi-
vidual cassettes with all required reagents. The
main advantage of lateral-flow immunoassay-
based RDTs is the very rapid test procedure, with
results generally available in less than 20 min.
Although numerous matrices can be used, includ-
ing whole blood, serum, plasma, oral fluid, urine,
exudates or faeces, only capillary whole blood
and oral fluid are acceptable for use with POC
tests.
The advantages and disadvantages of RDTs
compared to classical enzyme immunoassays are
summarised (Table 2). The use of RDTs may be
limited by the following weaknesses: a cost that
sometimes exceeds that of traditional testing
methods; only qualitative, non-quantitative ‘‘yes
or no” result; subjective interpretation that may
be inadequate, especially in patients with a low
amount of antigen or antibodies; low throughput;
lack of sensitivity compared to existing reference-
level laboratory tests, particularly when oral fluid
or fingerstick whole blood are used (as a result
of the type of antigens used, red cell dilution in
whole blood, lower concentrations of specific anti-
bodies than in plasma, variations depending on
the method of collection, immune complex
Enzyme immunoassays
High sensitivity
High specificity (100%)
Automation (high sample throughput)
Reaction at 37 °C
Data-processing filing of the results
Cost-effectiveness for large numbers of samples
Cold chain needed
Interference from sample matrices
Equipment (centrifuges)
Longer assay development time
Poor suitability for multi-analyte applications

binding at ambient temperature, shorter reaction
times).
HBV rapid diagnostic tests
Several RDTs exist for HBsAg detection, including
tests that are CE-marked, but none has been
FDA-approved or WHO-prequalified. The existing
HBsAg RDTs and their respective specifications
are shown (Table 3). Most of them can use whole
blood in addition to conventional matrices such
as serum or plasma. A recent meta-analysis of 30
studies performed between 1996 and 2015 in
23,176 individuals showed overall pooled sensitiv-
ity and specificity of HBsAg RDTs of 90% (95% CI
89%–91%) and 99% (95% CI 99%–99%), respec-
tively.30 The sensitivity of individual tests varied
(range: 50%–100%), whereas specificity estimates
were more robust (range: 86%–100%). The Deter-
mineTM and VIKIA tests had the highest pooled sen-
sitivity and specificity; their characteristics are
shown (Table 4).30 Overall, the performance of
HBsAg RDTs had improved compared to previous
meta-analyses.31 The accuracy of three HBsAg
RDTs was assessed in the French general popula-
tion. Their sensitivity varied between 90% and
96%, while their specificity was 99%–100%. How-
ever, the tests were performed in health centres
using whole blood collected from venepuncture,
i.e. not in the conditions of their use in a screening
situation in low- or middle-income countries.32
The accuracy of three HBsAg RDTs was evaluated
in a cross-sectional study in The Gambia: speci-
ficity was excellent, while sensitivity was around
JOURNAL
OF HEPATOLOGY
90%.33 Finally, HBsAg RDTs showed altered perfor-
mance in patients coinfected with HIV compared
to HIV-seronegative patients, with pooled sensi-
tivities of 72% (95% CI 68%–76%) and 93% (95%
CI: 90%–95%), respectively.30 This could be
because of lower quantities of HBsAg in patients
receiving antiretroviral therapy. Nevertheless, the
clinical impact should be modest, because most
patients undergoing dual HIV-HBV screening will
not receive antiretroviral treatment. These results
justify careful assessment of the clinical perfor-
mance of HBsAg RDTs prior to recommending
their broad use for HBV screening. Most studies
were performed in specific populations at various
facilities (blood donors, hospitalised patients, HIV-
infected individuals), but the tests performed bet-
ter in industrialised countries than in developing
countries, possibly a result of the variable preva-
lence of HBV infection across different areas.
Rapid tests for other serological markers of
HBV infection, including anti-HBs antibodies,
anti-HBc antibodies and HBeAg, are currently used
in some parts of the world. None of them is
approved by the US FDA, while some have
received product license in Europe. Only few stud-
ies assessing their diagnostic performance are
available,32,34 warranting further assessment.
The WHO recommends the use of a serological
assay (in either RDT or laboratory-based
immunoassay format) that meets minimum qual-
ity, safety and performance standards with regard
to both analytical and clinical sensitivity and
specificity for HBsAg detection.13 However, the

Table 3. Specifications of available HBsAg rapid diagnostic tests.

Test Manufacturer Nature of device Matrices Volume Time to result CE- FDA- WHO-
needed marked approved prequalified
DetermineTM Alere, Waltham, MA Lateral flow Whole blood, 50 ll 15 min No No No
HBsAg serum, plasma
VIKIAÒ HBsAg bioMérieux, Lateral flow Whole blood, 75 ll 15–30 min Yes No No
Marcy-l’Étoile, France serum, plasma
DRW HBsAg Diagnostics for the Lateral flow Serum, plasma 80 ll 30 min Yes No No
rapid Test Real World, San Jose,
CA
Toyo HBsAg Turklab, Izmir, Turkey Flow-through Whole blood, 100 ll 5–15 min Yes No No
Rapid Test serum, plasma
Assure HBsAg MP Biomedicals, Flow-through Whole blood, 50 ll 15–20 min No No No
Rapid Test Singapore serum, plasma (whole blood) or
75 ll (serum,
plasma)
First Response Premier Medical Flow-through Serum, plasma 50 or 75 ll 5–10 min Yes No No
HBsAg Card Test Corporation, Daman,
India
SD Bioline HBsAg Standard Diagnostics, Flow-through Whole blood, 100 ll 20 min No No No
Yongin, Korea serum, plasma
CE, European Conformity; FDA, US Food and Drug Administration; HBsAg, hepatitis B surface antigen; WHO, World Health Organization.

Table 4. Performance of DetermineTM HBsAg and VIKIAÒ HBsAg for the detection of HBsAg (from30).
Test Matrices Pooled specificity (95% CI) Pooled sensitivity (95% CI) Positive LR (95% CI) Negative LR (95% CI)
DetermineTM HBsAg Plasma, serum 99.1 (98.9–99.4) 90.8 (88.9–92.4) 239.2 (17.1–33,339.4) 0.077 (0.035–0.168)
VIKIAÒ HBsAg Plasma, serum 99.9 (99.8–100) 82.5 (77.5–86.7) 1,072.3 (376.1–3,057.2) 0.108 (0.026–0.458)
HBsAg, hepatitis B surface antigen; LR, likelihood ratio.

Journal of Hepatology 2018 vol. 69 j 916–926 919