Detection of Hepatitis B Virus DNA among Chronic and potential Occult HBV patients in resource‐limited settings by Loop‐Mediated Isothermal Amplification assay

1. J Viral Hepat. 2018;1–6. wileyonlinelibrary.com/journal/jvh | 1© 2018 John Wiley Sons Ltd
1 | INTRODUCTION
Hepatitis B virus (HBV) infection is a worldwide public health prob-
lem. About one-­third of the world’s population have serological evi-
dence of past or present HBV infection, and over 350 million people
may be affected by Chronic Hepatitis B (CHB) virus infection.1
CHB infection is defined as persistence of hepatitis B surface anti-
gen (HBsAg) for 6 months or more after acute infection with HBV.
Transfusion of blood or blood products containing HBsAg is one of
the reasons for post–transfusion HBV infection. Besides this, Occult
HBV Infection (OBI) is another important cause of post–transfusion
HBV infection worldwide. OBI is defined by the absence of HBsAg
despite the presence of HBV-­DNA in the liver, blood, or peripheral
blood mononuclear cells (PBMCs), regardless of the presence of an-
tigens of HBV or other antibodies against HBV. In OBI, HBV-­DNA
is found in the liver with detectable or undetectable HBV-­DNA in
Received: 22 December 2017 | Accepted: 26 March 2018
DOI: 10.1111/jvh.12931
O R I G I N A L A R T I C L E
Detection of Hepatitis B Virus DNA among Chronic and
potential Occult HBV patients in resource-­limited settings by
Loop-­Mediated Isothermal Amplification assay
A. Akram | S. M. R. Islam | S. U. Munshi | S. Tabassum
Department of Virology, Bangabandhu
Sheikh Mujib Medical University (BSMMU),
Dhaka, Bangladesh
Correspondence
Saif Ullah Munshi, Department of Virology,
Bangabandhu Sheikh Mujib Medical
University (BSMMU), Dhaka, Bangladesh.
Email: saifmunshi@yahoo.com
Summary
Transmission of Hepatitis B Virus (HBV) usually occurs due to the transfusion of
blood or blood products from chronic HBV (CHB) or occult HBV-­infected (OBI) pa-
tients. Besides serological tests, e.g. HBsAg and anti- HBc (total), detection of
HBVDNA is necessary for the diagnosis of OBI patients. Different nucleic acid tests
(NATs) including real- time–Polymerase Chain Reaction (qPCR) are used to detect
HBV- DNA. The NATs are expensive and require technical expertise which are barri-
ers to introduce them in resource-­limited settings. This study was undertaken to
evaluate the use of Loop-­Mediated Isothermal Amplification (LAMP) assay as an al-
ternative to qPCR for the detection of HBV-­DNA in CHB and potential OBI patients
in resource-­limited settings. Following the published protocols with some modifica-
tions, a LAMP assay was developed for detection of HBV-­DNA by either using a heat
block followed by detection in an agarose gel or using a qPCR thermocycler. The
LAMP assay was applied to supernatant prepared from heat-­treated serum collected
from CHB and potential OBI patients. HBV viral load in serum was measured by
qPCR using a single-­step HBV-­DNA quantification kit. Among 200 samples tested,
qPCR was capable to detect HBV-­DNA in 25.5% of cases, whereas LAMP assay de-
tected HBV-­DNA in 43.5% cases. The qPCR was able to detect 11 (9.16%) potential
OBI cases, whereas LAMP assay identified HBV-­DNA in 43 (35.83%) cases. In addi-
tion to tests for HBsAg and/or anti-­HBc (total), detection of HBV-­DNA by LAMP
assay may aid in preventing post–transfusion HBV infection in resource-­limited
settings.
K E Y W O R D S
HBV-DNA, LAMP assay, OBI, qPCR

2. 2 | AKRAM et al.
blood.2
In cases of true OBI, the amount of HBV-­DNA in blood is
typically low.3-5
As testing liver tissue is not always feasible, OBI is
often diagnosed by testing of HBV-­DNA and other HBV markers in
the blood6
.
Worldwide, OBI is becoming increasingly recognized in several
clinical settings.7,8
The prevalence of OBI is reported to range from
41% to 90% of the population of some regions in HBV endemic East
Asia. On the contrary, the prevalence is only 5%-­20% of the pop-
ulation observed in low endemic areas of North America.9
Several
studies on blood donors have reported 0.1%-­1.05% prevalence of
OBI in HBsAg-­negative, anti-­HBc (total)-­positive blood donors from
North America, 0%-­1.59% in donors from Europe,9
0.5%-­30% from
India 10
and 8.32% from Bangladesh.11
Based on the profile of anti-
bodies against HBV, OBI can be classified into 2 groups; seropositive
OBI [anti-­HBc (total) and/or anti-­hepatitis B surface (anti-­HBs) posi-
tive] and seronegative OBI [(anti-­HBc (total) and anti-­HBs negative)].
Although most of the OBIs are seropositive, about 20% of patients
with OBI are seronegative which represent a population negative
for all serological markers of HBV infection.9
Blood free of HBsAg
but with high-­titer of anti-­HBc (total) in the absence of anti-­HBs
can transmit HBV,12
and thus only screening for HBsAg and anti-­
HBc (total) is not effective to prevent transfusion-­mediated HBV
infections. However, despite considerable improvements in eligibil-
ity criteria for blood donation and development of more advanced
screening methods, transfusion transmitted infectious agents like
HBV still present a threat to blood safety.13
Availability and safety
of blood and blood products remain a major concern in many coun-
tries around the world and countries are facing unique challenges in
ensuring self-­sufficiency in safe blood and blood products based on
voluntary non-­remunerated blood donations.14
Globally, Nucleic Acid Tests (NATs) have become a popular al-
ternative used to detect HBV in blood in the chronic phase of HBV
infection including the window period as well as OBI.13
Hence, the
introduction of highly sensitive NATs may further decrease the risk
of HBV transmission through blood transfusion.9
Among the NATs,
quantitative real-­time–Polymerase Chain Reaction (qPCR) is the
mainstay of HBV-­DNA detection even though it is time-­consuming,
expensive, requires skilled personnel and high-­tech equipment
to perform. Thus, there is need for a rapid and cost-­effective de-
tection tool for screening HBV infection in endemic as well as
resource-­limited environments.7
Nowadays, the LAMP method has
shown potential as a new molecular method in the diagnosis of var-
ious viral infections like Varicella-­zoster virus,15
Dengue,16
Herpes
Simplex Virus,17
Epstein-­Bar Virus,18
Chikungunya Virus,19
Human
Papillomavirus,20
Human Immunodeficiency Virus-­121
and HBV.2,22-
24
LAMP is a DNA amplification method that uses 4-­6 primers and
a DNA strand-­displacement process for rapid amplification under
an isothermal condition, thereby reducing the need for the use of
a thermal cycler.25
In the present study, a LAMP assay for detec-
tion of HBV-­DNA was developed targeting the Core gene (HBc)
of HBV following the methods described previously 2
by adding a
real-­time detection procedure using a fluorescence dye.26
The effec-
tiveness of LAMP assays for the detection of HBV-­DNA among the
CHB-­infected patients including potential OBI patients was studied
and its results were compared with the results of a commercially
available qPCR.
2 | MATERIALS AND METHODS
2.1 | Ethical Clearance
The present study was conducted from May 2015 to May 2016
at the Department of Virology Bangabandhu Sheikh Mujib
Medical University (BSMMU), Bangladesh. The study was ap-
proved by the Institutional Review Board (IRB), BSMMU (No.
BSMMU/2015/14411). All samples were collected from adult indi-
viduals; no minor was included in the study.
2.2 | Patient selection and sample collection
During the study period, a total of 3560 patients were tested for both
HBsAg and anti-­HBc (total), from which a total of 613 samples were
selected randomly and subdivided into 2 groups; (i) Chronic HBV group
(CHB, n = 247) which was positive for both HBsAg and anti-­HBc (total)
and (ii) potential OBI group (n = 353) which was negative for HBsAg
but positive for anti-­HBc (total). From the CHB and potential OBI pa-
tients, 80 and 120 patients were randomly selected, respectively.
After obtaining informed written consent, all data were collected in a
questionnaire. The blood specimen from each patient was collected in
a vacutainer blood collection tube and kept at room temperature for
30 minutes and centrifuged at 3,000 rpm for 15 min. The collected
serum samples were stored at −20°C until further use.
2.3 | Sample preparation for LAMP assay
All the serum samples for LAMP assay were prepared according to
the procedure previously described.2
Briefly, 25 μL of serum was di-
luted with 50 μL of nuclease-­free water, vortexed briefly and heated
at 95°C for 5 minutes and immediately again at 100°C for 5 minutes
on a heating block. The mixture was then centrifuged at 13400 × g
at room temperature for 10 minutes and the supernatant was col-
lected to perform the LAMP assay either by heat block or by qPCR
thermocycler. To observe the analytical specificity, other viral nu-
cleic acids, eg HIV, HCV and BK virus isolated from different patients
were also run together as the control.
2.4 | LAMP assay followed by detection in Agarose
Gel and qPCR thermocycler
The LAMP reaction was carried out according to the procedure
described previously.2
Briefly, 25 μL of LAMP reaction contained
20 pmol of each backward inner primer (BIP) and forward inner
primer (FIP), 10 pmol of forward loop primers (Loop F) and backward
loop primers (Loop B), 5 pmol of each forward outer primers (F3) and
backward outer primers (B3), 1X Thermopol Buffer (New England
Biolabs Inc., United States), 10 μmol/L dNTPs (Geneaid, Taiwan), 5 M

3. | 3AKRAM et al.
Betaine (Sigma Aldrich, St. Louis, MO, USA), 100 mmol/L MgSO4
(New England Biolabs Inc., the United States), 8U of Bst DNA poly-
merase (New England Biolabs Inc., United States) and 5 μL of super-
natant collected from heat-­treated template. This reaction mixture
was incubated at 63˚ for 1 hour in the heat block and terminated by
keeping the reaction tube at 85°C for 5 minutes. The LAMP assay was
performed in duplicate for each sample and nuclease-­free water was
used instead of the supernatant of serum in the no template control
(NTC). Ten microliters of HBV-­LAMP products were electrophoreti-
cally analysed on 2% agarose (1 × TBE) stained with ethidium bro-
mide at 100 volts for 40 min and visualized with a UV transilluminator
at 302 nm. The LAMP products which showed typical step ladder-­
like pattern in agarose gel were considered as positive for HBV-­DNA
(Figure 1A,B,C). For performing LAMP assay on qPCR thermocycler,
supernatant collected from heat-­treated serum (described in the
sample preparation section) was serially diluted 10-­fold, and 4 μL of
1/100 diluted sample was used to set the LAMP reaction. Real-­time
LAMP assay was performed in a thermocycler (StepOne™, Applied
Biosystems; USA) using the same reaction mixture described above,
except for the addition of 20X EVAGreen™ (Biotium, Inc. CA, USA:
Catalog No-­31000) as the intercalating dye.26
The reactions were
subjected to 40 cycles at 63°C for 1 minute followed by 85°C for
5 minute. During real-­time amplification, the fluorescence data were
obtained on the 6-­carboxyfluorescein (FAM) channel (Figure 1D).
2.5 | Measurement of HBV viral load by qPCR
To measure HBV viral load, single-­step HBV quantification was per-
formed using an HBV-­DNA quantification kit (Genebio, Norwell, MA,
USA) according to manufacturer’s protocol. Briefly, 5 μL of serum was
added to the reaction mixture containing 38 μL of HBV PCR mixture
and 2 μL of enzyme mixture. Quantification of HBV was performed
using a qPCR thermocycler (Applied Biosystem 7300, Foster City,
California, USA). A standard curve was prepared by serial 10-­fold dilu-
tion of standard HBV-­DNA from 101
to 108
copies/reaction.
2.6 | Routine laboratory tests
All serum samples were tested by commercially available ELISAs for
HBsAg using Murex HBsAg confirmatory version 3 (Diasorin, S.p.A,
Vercelli, Italy) and HBeAg using ETI-­EBK PLUS (HBeAg) (Diasorin,
Italy). Tests for anti-­HBc (total) and anti-­HBs were performed using
chemiluminescent immunoassays (CLIA) kits on the LIAISON®
sys-
tem (Diasorin S.p.A, Vercelli, Italy).
2.7 | Statistical analysis
All the data were analysed using Microsoft ®
Excel (version-­15.29.1,
USA), where quantitative data were expressed as number, percent-
age and mean ± SD.
3 | RESULTS
In the present study, the anti-­HBs titer was undetected (100 IU/mL) in
all CHB patients and the majority (67.5%) of the OBI patients (Table-1).
Among the CHB patients, 50% (40/80) were positive for HBeAg and
were also positive for HBV-­DNA by qPCR. On the other hand, among
potential OBI patients, only 2 (1.6%) patients were positive for HBeAg,
but 11 (9.16%) were positive for HBV-­DNA by qPCR including the 2
HBeAg positive patients. Among the CHB patients, HBV-­DNA levels
detected by qPCR ranged between 102
and 104
IU/mL in 20% (16/80)
FI G U R E 1 Detection of HBV-­DNA
LAMP assay products (performed in
the heat block) in an agarose gel under
UV light (A, B, C), and using a real-­time
thermocycler (D). A: Lane 1-­5 amplified
HBV DNA of CHB patients, Lane-­6
negative template controls (NTC); B: Lane
1-­4 amplified HBV DNA of OBI patients,
Lane 5 NTC; C: Lane-­1 HBV-­DNA, lane-­2
HIV-­RNA, lane-­3 HCV-­RNA and lane 4 BK
virus-­DNA, lane-­5 NTC. Lane-­M contains
the 100-­bp DNA ladder. D: Graph of
the LAMP assay performed in a qPCR
thermocycler where the upper curves
represent amplified HBV DNA of CHB and
OBI patients and the lowest curve that of
NTC
M 1 2 3 4 5 6 1 2 3 4 5
M 1 2 3 4 5
(A) (B)
(C) (D)

4. 4 | AKRAM et al.
and 105
IU/mL in 30% (24/80) of samples. All these HBV-­DNA posi-
tive CHB samples were also positive by HBV-­LAMP assays. In addition,
the LAMP assay detected 4 more CHB samples which were HBeAg
negative [total 44 (55%)] and undetectable by qPCR.
Among the potential OBI subjects, HBV-­DNA was detected only
in 11 (9.16%) samples by the commercial qPCR but the LAMP assay
detected HBV-­DNA in 43 (35.8%) samples including those detected
by qPCR. Among all the study participants, HBV-­DNA was detected
in 25.5% (51/200) of samples by qPCR while the LAMP assays de-
tected HBV-­DNA in 43.5% (87/200) samples irrespective of the
HBV infection status. None of the other viral nucleic acids, ie HIV,
HCV and BK virus was detected in any of the LAMP assays. The
LAMP assay performed either in a heat-­block or qPCR thermocycler
showed similar results.
4 | DISCUSSION
In many countries with limited-­resources, detection of HBV in blood
is achieved by screening of HBsAg. This practice poses a great threat
of misdiagnosis and transmission of HBV. To exclude possible OBI in
case of blood and solid organ donors, patients undergoing haemo-
dialysis or immunesuppressive therapy, molecular testing is required
for detection of HBV-­DNA. Current technologies, like nested-­PCR,
real-­time PCR and transcription-­based mediated amplification (TMA)
are used for this purpose. However, some cases positive for anti-­
HBc (total) and/or HBV-­DNA are sometimes not identified through
the current screening. In the present study, a LAMP assay was de-
veloped targeting the HBc gene of HBV to test its performance for
detection of HBV-­DNA in CHB and OBI patients in resource-­limited
settings. The results of this study showed good promise of the HBV
LAMP assay as a screening or diagnostic test for HBV-­DNA. We
demonstrated that this LAMP assay was able to detect HBV in sam-
ples which was undetectable by qPCR. In addition, this assay was
able to detect HBV-­DNA as low as 102
IU/mL and below. In earlier
studies, better performance, ie 10-­to 1000-­fold higher sensitiv-
ity of the LAMP assay than PCR was observed for the detection of
Arcobacter species 27
and Toxoplasma gondii.28
In the present study, HBV-­DNA was detectable in 100 times di-
luted heat-­treated serum by running the LAMP assay for 40 minutes
in both CHB and OBI samples in a qPCR thermocycler. Since the
LAMP method has the ability to detect even 6 copies of the HBV
target within 45 minutes,29
it was possible to detect HBV-­DNA es-
pecially among OBI patients, where detection of HBV-­DNA could be
missed using qPCR as a NAT in blood screening or in diagnostics. To
observe the analytical specificity, nucleic acids of other blood-­borne
viruses like HIV, HCV and BK virus were used in the run as disease
control but no viral nucleic acids were detected in HBV LAMP as-
says, indicating that the LAMP assay is specific for HBV-­DNA detec-
tion. This specificity of the LAMP assay is maintained due to use of 6
primers in the assay. In the initial steps of the LAMP assay, 4 primers
recognize 6 distinct sequences of the target and during the subse-
quent steps 2 primers recognize 4 distinct sequences which ensures
high specificity of target amplification. Moreover, these 4 primers
of the LAMP assay initiate DNA synthesis simultaneously from the
original unamplified DNA to generate a stem–loop DNA for subse-
quent LAMP cycling, during which the target is recognized by 4 se-
quences. Therefore, target selectivity is higher than those obtained
by PCR.25
In addition, the LAMP assay shows better performance
because it is less affected by different inhibitors in clinical sam-
ples. This avoided the HBV-­DNA isolation step2
and increased the
amount of DNA in the supernatant. Considering the high sensitivity
and specificity obtained for the detection of HBV-­DNA in CHB and
OBI serum samples, this study suggests that the LAMP assay could
be useful for screening clinical samples with heat-­treated serum, and
may be a better alternative of qPCR to screen blood before blood
transfusion or for diagnosis in the laboratory.
There were several advantages of the HBV-­LAMP assay offered
over the HBV qPCR assay besides comparatively better results in
detecting HBV-­DNA. To perform the LAMP assay, no sophisticated
equipment was required and the reagents were relatively cheaper.30
It was possible to perform the LAMP assay on a simple digital heat-­
block or even on a water bath (data not shown), without the need
TA B LE 1 Serological and molecular data of study population
(N = 200)
CHB (n = 80)
Potential OBI
(n = 120)
HBsAg +ve
80 (100%) 00 (00%)
Anti-­HBc (total) +ve
80 (100%) 120 (100%)
Anti-­HBs titer
100 (IU/mL) 0 (0%) 100 (83.33%)
100 (IU/mL) 0 (0%) 20 (16.66%)
HBeAg +ve
40 (50%) 02 (1.6%)
Total HBV-­DNA
+ve in qPCR
51 (25.5%)
102
-­105
IU/mL 16 (20%) 11 (9.16)
105
IU/mL 24 (30%) 0 (0%)
Total 40 (50%) 11 (9.16%)
Total HBV-­DNA
+ve in LAMP
87 (43.5%)*
44 (55%) 43 (35.8%)
Age: Average (Range)
31.96 (18-­65) 37.76 (20-­62)
Gender: n (%)
Male 47 (58.75%) 75 (65.4%)
Female 33 (41.25%) 45 (34.61%)
CHB, Chronic Hepatitis B; OBI, Occult HBV Infection.
*Results of the LAMP assay were similar for all samples; whether it was
detected in heat block followed by agarose gel electrophoresis or by
qPCR.

5. | 5AKRAM et al.
of any high technical expertise. In this study, the LAMP assay was
performed with a heat block followed by detection on gel electro-
phoresis and by qPCR. When performed in the heat block, the LAMP
assay for HBV-­DNA needed 1 hour for amplification of CHB samples
but for OBI samples it required 2-­hour time to get a typical step lad-
der band pattern on an agarose gel. When using the heating block
(or water bath), 30 minutes extra time was required for visualization
of the LAMP product on agarose gel. However, when the detection
steps of the LAMP assay were performed in a qPCR thermocycler,
only 40 minutes were required for all the samples including samples
of OBI patients to amplify and detect. The LAMP assay used in this
study can be performed using a turbidimeter or naked eye visualiza-
tion which may reduce the duration of assay time.
In the present study, serum samples collected from HBV-­
infected individuals were heat-­treated and subjected to the LAMP
assay avoiding the traditional DNA isolation step, indicating that un-
processed or partly processed samples can be used as HBV-­DNA
template for the assay. This not only contributed to reducing assay
time and cost, but also simplified the detection process. Hence, the
LAMP assay developed in the present study was found to be a sim-
ple, cost-­effective and robust technique which may be useful for
resource-­limited settings. Similar observations were demonstrated
for detection of the malaria parasite in field settings using the LAMP
assay.30,31
The World Health Organization (WHO) has recommended
the LAMP assay for the diagnosis of pulmonary tuberculosis (TB) in
peripheral health centres.32
In conclusion, the findings of this study demonstrate that the
HBV LAMP assay may play a crucial role in aiding the prevention
of post–transfusion HBV infection by the detection of HBV-­DNA,
in addition to tests for HBsAg and/or anti-­HBc (total) prior to blood
transfusion or organ/tissue donation. Although further research for
evaluation of the LAMP assay for application in clinical settings is
required, the method is potentially adaptable for POC settings and
disease surveillance in HBV-­endemic areas which may contribute to
the approach of nearing HBV transmission to zero.
CONFLICT OF INTEREST
We declare that we do not have any conflict of interest.
ORCID
S. U. Munshi http://orcid.org/0000-0002-9661-6592
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How to cite this article: Akram A, Islam SMR, Munshi SU,
Tabassum S. Detection of Hepatitis B Virus DNA among
Chronic and potential Occult HBV patients in resource-­
limited settings by Loop-­Mediated Isothermal Amplification
assay. J Viral Hepat. 2018;00:1–6. https://doi.org/10.1111/
jvh.12931