Application of Serologic Assays for Diagnosing Acute Hepatitis E in

National Surveillance of a Nonendemic Area

Wen-Chieh Wu,1 Chien-Wei Su,1,2,3 Jyh-Yuan Yang,4 Szu-Fong Lin,4 Jen-Yu Chen,3
and Jaw-Ching Wu3,5*
Journal of Medical Virology 86:720–728 (2014)

Presenter:
NTB
Introduction
 Hepatitis E virus or HEV is a recently discovered agent of
enterically transmitted viral hepatitis.
 HEV is Single-stranded RNA virus with four
genotypes( GT1,GT2,GT3 & GT4).
 Transmitted through the fecal–oral rout.
 Humans are the major host of HEV genotypes (GT) 1 and 2. In
contrast, HEV GT 3 and 4 can spread among humans, pigs, and
other animal species
 First outbreak of HEV infection was reported in 1955 in New
Delhi, India.
 HEV infection has High case-fatality among pregnant women.
 Acute hepatitis E infection is usually self-limiting
 cause fulminant hepatitis and cirrhosis in immunocompromised
patients.
 Symptoms are jaundice, anorexia, abdominal pain, nausea,
vomiting, Fever & hepatomegaly (an enlarged, tender liver).
 HEV Genome Structure
 HEV is None
enveloped,Spherical, 32–34
nm in diameter
 HEV has Single-stranded,
positive sense ribonucleic
acid (RNA) genome
structure.
 Surrounded by an
icosahedral capsid.
 The genome is
approximately 7200 bases in
length
 Conti…….

• HEV have three discontinuous and partially overlapping open

reading frames (ORFs) along with 5' and 3' cis-acting elements,
which have important roles in HEV replication and transcription.
 ORF1 encode a methyltransferase, protease, helicase and
replicase which participates in the replication of viral particles
and the modification of structural protein(s)
 ORF2 encode the capsid protein which is a glycoprotein of 71–
88 kDa. HEV capsid protein was used to form virus-like
particles (VLPs)
 ORF3 encodes a protein of undefined function is found between
ORF1 and ORF2
Objective
1. The present study aimed to evaluate four
different IgM/IgG assays for HEV infection for
application in national surveillance in non
endemic areas.

2. The present study attempted to determine the

best strategy to diagnose acute HEV infection
from four different IgM/IgG assays in a panel of
serum samples collected from the regular national
surveillance of non-A, non-B, non-C hepatitis,
and acute HEV infection in a period of 5 years.
MATERIALS AND METHODS
Sample Collection
 300 serum samples were collected from

patients that were stored in the Centers for

Disease Control (CDC) of Taiwan for
suspected acute HEV infection from 2004 to
2008
 18 serum samples were collected from acute

cases of HEV infection in Taipei Veteran

General Hospital from 1990 to 2005 were
evaluated
Methods of ELISAs
In this study four different ELISA methods were
chosen for the detection of HEV IgM/IgG levels
in serum samples.
1. EIAgen HEV IgG/M (Adaltis, Bologna, Italy)
2. RecomWell HEV IgG/M( Mikrogen Neuried,
Germany)
3. MP HEV IgM ELISA 3.0 and IgG ELISA (MP
Biomedicals, Singapore)
4. HEVLPs (hepatitis empty virus-like particle-
based ELISA) IgG/M In-house kit (provided
by Dr. Tian-Cheng Li
BASIC ELISA Principle
 Enzyme-linked Immunosorbent Assays (ELISAs) combine the
specificity of antibodies with the sensitivity of simple enzyme assays,
by using antibodies or antigens coupled to an easily-assayed
enzyme.
 ELISAs can provide a useful measurement of antigen or antibody

concentration.
 There are two main variations on this method:

a)The ELISA can be used to detect the presence of

antigens that are recognized by an antibody.
b) It can be used to test for antibodies that recognize by
an antigen.
INDIERCT ELISA
The type of ELISA used in this study was
indirect ELISA.
EIAgen HEV IgG/M
 The EIAgen HEV IgG, IgM is for qualitative detection of IgG or IgM antibodies to Hepatitis E
Virus in human serum or plasma
 Test based on the principle of an indirect sandwich ELISA.
 HEV specific synthetic Antigen (derived from ORF2 and ORF3 of all 4 GTs)coated wells
 Diluted serum sample with Anti HEV IgG/M are captured if present in sample
 Wash unbound antibodies
 Bound Anti HEV detected by Conjugate HRP labeled Polyclonal specific anti IgG/M
antibodies
 Wash unbound conjugate antibodies
 Specifically bound antibodies are detected by a peroxidase-catalyzed colour reaction by
addition of Substrate which is proportionate to the quantity of bound HEV IgG, IgM
antibodies
RecomWell HEV IgG/M
 The recom Well HEV IgG, IgM is a qualitative or quantitative in-vitro test for the
detection and reliable identification of IgG or IgM antibodies to Hepatitis E Virus in
human serum or plasma
 Test based on the principle of an indirect sandwich ELISA.
 Highly purified recombinant HEV-ORF2 (genotype 1 and genotype 3) virus antigens
coated wells
 Diluted serum sample with Anti HEV IgG/M are captured if present in sample
 Wash unbound antibodies
 Bound Anti HEV detected by Conjugate HRP labeled Polyclonal specific anti IgG/M
antibodies
 Wash unbound conjugate antibodies
 Specifically bound antibodies are detected by a peroxidase-catalyzed colour reaction by
addition of Substrate which is proportionate to the quantity of bound HEV IgG, IgM
antibodies
MP HEV IgM ELISA 3.0 and IgG ELISA
 The MP HEV IgM ELISA 3.0 and IgG ELISA is for
qualitative detection of IgG or IgM antibodies
to Hepatitis E Virus in human serum or plasma
 Test based on the principle of an indirect
sandwich ELISA.
 The wells are coated with highly conserved
conformational ORF2 epitope
 Diluted serum sample with Anti HEV IgG/M
are captured if present in sample
 Wash unbound antibodies
 Bound Anti HEV detected by mouse
monoclonal anti-human IgM/G antibodies
labeled with horseradish peroxidase
 Wash unbound conjugate antibodies
 Specifically bound antibodies are detected by
a peroxidase-catalyzed colour reaction by
addition of Substrate which is proportionate
to the quantity of bound HEV IgG, IgM
antibodies
 HEVLPs (hepatitis empty virus-like particle-
based ELISA) IgG/M In-house kit
 In-house hepatitis HEVLP IgG/M assays, which used highly purified empty VLPs of
HEV as an antigen expressed by a recombinant baculovirus, and self-assembled with
an ORF2-coded recombinant protein. The HEVLPs possess antigenicity similar to that
of authentic HEV particles and are highly sensitive for the detection of HEV-specific
IgM and IgG antibodies.
 Test based on the principle of an indirect sandwich ELISA.
 96-well polystyrene microplates were coated with a recombinant antigen obtained
through expression and purification of empty virus-like particles (VLPs) of HEV.
 The plates were incubated at 4°C overnight, unbound VLPs were removed, and the
wells were washed twice with Phosphate Buffered Saline They were then blocked for 1
hour with 5% skim milk at 37°C.
 Diluted serum sample is added with Anti HEV IgG/M are captured if present in sample
 Wash unbound antibodies
 Bound Anti HEV detected by horseradish peroxidase-conjugated goat antihuman IgG
or IgM
 Wash unbound conjugate antibodies
 Specifically bound antibodies are detected by a peroxidase-catalyzed color reaction
by addition of Substrate which is proportionate to the quantity of bound HEV IgG, IgM
antibodies
Diagnostic Formates of ELISA
To improve the performance of the assays, two
diagnostic formats of combination assays were
tried.
 In format A, acute HEV infection was defined if

either IgG or IgM from the same manufacturer was

Positive.
 In the format B, it was defined if one of the

Serologic Assays for Hepatitis E Virus IgM assays

from two different manufacturers was
positive.
PCR (Polymerase Chain Reaction)
Methods of RT-PCR (Reverse Transcriptase
Polymerase Chain Reaction)& genome
sequencing
 Serum HEV RNA was detected
by RT-PCR
 Two sets of paired primers
were synthesized for nested
PCR
 Viral RNA templates were
extracted from 50 ml of serum
and were reverse transcribed.
 Nested PCR was then carried
out by using the paired primers
in a thermal cycler, beginning
at 95°C for 5 min
 Followed by 35cycles (each
cycle: 95°C for 40 sec, 55°C for
40 sec, 72°C for 2 min)
 Amplification ending at 72°C
for10 min.
 The PCR products were
analyzed in 1.5% agarose gel,
followed by ethidiumbromide
staining.
 The sensitivityof RT-PCR for
detecting HEV RNA was 10–100
viral genome copies/50 ml
Genome Sequencing
 The amplified PCR products were
inserted into the plasmid pCR2
vector (Original TA Cloning1
Kit,Invitrogen, Carlsbad, CA)
according to the manufacturer’s
instruction.
 The ligation mixture was used to
transform the competent Escherichia
coli strain DH5a (Gibco-BRL, Life
Technologies, Gaithersburg,MD)
 Incubated overnight in LB agar plate
containing 100 mg/ml ampicillin
and 30 mg/ml X-gal at 37°C.
 The successful ligation clones in
blue white screening were picked-up
and cultured overnight in 3ml LB-
Amp broth at 37°C
 Plasmid DNA was purified by
QIAprep Spin MiniPrep Kit
 The plasmid DNAs were subjected to
sequence from both ends by
thermocycle sequencing
RESULTS
 A total of 318 serum samples were tested with four IgM/IgG HEV
ELISAs for anti-HEV and RT-PCR for HEV RNA
 Because of insufficient blood sample volumes, nine samples stored
in the CDC were excluded as these did not complete all ELISAs.
 The 309 enrolled patients had a median age of 51 years and male
predominance (70.6%)
 There were 15 patients with HEV viremia detected by RT-PCR,while
the remaining 294 patients had negative HEV RNA
 The genotype distribution was as follows: GT 1, 5 (33.3%); GT 3, 1
(6.7%), and GT 4, 9 (60%).
 The median duration between initial development of symptoms and
examinations for HEV infection was 13 days (range, 2–20 days)
Conti……
 Most assays exhibited moderate to high
sensitivity rates (66.7–93.3%).
 Among four different types of ELISA

RecomWell IgM had the highest

sensitivity(93.3%) and specificity rates
(86.1–95.6%).
Conti…………
 Combining Two Assays for Diagnosing Acute
HEV Infection
 Format A, which applied the same brand of IgG

andIgM, showed improved screening, with

higher sensitivity, However, specificity was
lower.
 Format B, which involved combining the IgM

assay of different brands, exhibited improved

performance.The recomWell IgM, performance
in terms of screening and diagnosis were all
satisfactory.
Discussion

 The present study showed that recomWell IgM, which had

the highest sensitivity (93.3%) and negative predictive value
(100%), exhibited the best overall performance for first-line
screening acute HEV infection.

 All anti-HEV IgM ELISAs in this study were better than anti-
HEV IgG assays for the purpose of first-line screening for
the national surveillance of acute HEV infection in
nonendemic areas because of excellent
 NPV (is the proportion of true negative results from the false
positive results ) was 98.9–100%.
 Specificity of the test was 86.1–95.6%.
 Higher PPV than anti-HEV IgG ELISA (17.4–40% vs. 5.8–
15.2%) which could improve cost-effectiveness.
Conclusion
In conclusion, anti-HEV IgM ELISA is a good
screening test for the national surveillance of acute
HEV infection in nonendemic areas and not limited
by inconsistent performances of sensitivity and
specificity
among different assays. The combination of
anti-HEV IgM ELISA with anti-HEV IgG or another
different anti-HEV IgM ELISA could provide better
screening performance.