JE and Dengue
Panbio’s new JE-Dengue IgM Combo ELISA
Overview
• Both Dengue and Japanese encephalitis (JE) belong
to the Flavivirdae family and co-circulate in many parts
of the world (Figure 1 – following slide).
• Serological distinction between these two flavivirus
infections is often necessary though difficult due to the
level of cross-reactive epitopes within the flavivirus
family.
• Accurate detection of these viruses is paramount to
treatment and surveillance programs.
• Serological tests to assist in the diagnosis of dengue
virus infection are widely available, however limited
commercial viral diagnostics for JE have been
available.
#Adapted from maps produced by the Centers for Disease Control
World distribution of Dengue – 2005 CDC http://www.cdc.gov/ncidod/dvbid/dengue/map-distribution-2005.htm
Distribution of Japanese Encephalitis in Asia, 1970-1998 http://www.cdc.gov/ncidod/dvbid/jencephalitis/map.htm
Japanese encephalitis1
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Japanese encephalitis (JE) virus: flavivirus antigenically
related to St. Louis encephalitis virus
Transmitted via mosquitoes of the Culex tritaeniorhynchus
group
Can cause acute encephalitis which may progress to
paralysis, seizures, coma and death, however the majority
of infections are subclinical
Case-fatality ratio: 30%
Serious neurologic sequela: 30%
Leading cause of viral encephalitis in Asia with 30-50,000
cases reported annually
Those at risk include:
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Residents of rural areas in endemic locations
Active duty military deployed to endemic areas
Expatriates in rural areas
Disease risk extremely low in travellers
Dengue2
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Dengue virus serotypes 1 – 4: flavivirus
Transmission via mosquito (Aedes aegypti)
Symptoms include sudden onset of fever, severe headache,
myalgias and arthralgias, leukopenia, thrombocytopenia and
hemorrhagic manifestations
Occasionally produces shock and haemorrhage, leading to
death
Incidence is variable depending on epidemic activity
Globally, there are an estimated 50 to 100 million cases of
dengue fever (DF) and several hundred thousand cases of
dengue hemorrhagic fever (DHF) per year
Average case fatality rate of DHF is about 5%
The disease is experiencing a resurgence in the tropics and
epidemics are larger and more frequent
Dengue and JE: the complicated
diagnosis
• Early in infection dengue and JE can present similarly.
– fever, headache, nausea, vomiting, and myalgia
4,5
• Both are associated with patients having a history of
mosquito exposure in endemic areas.
• Level of cross-reactivity among the Flavivirus group
has traditionally complicated serological diagnosis.
Why distinguish between JE &
dengue?
• Correct diagnosis is important for public health
surveillance
• Enable the accurate assessment of the JE burden
• Ensure correct treatment
– Although the treatment for both infections is supportive and
in no anti-viral therapy available, patients with JE may
require feeding, airway management and seizure control 4.
JE-Dengue IgM Combo ELISA
• Intended use
– The Panbio Japanese Encephalitis - Dengue IgM Combo
ELISA is for the qualitative presumptive detection of IgM
antibodies to Japanese encephalitis and dengue virus in
serum as an aid in the clinical laboratory diagnosis of
Japanese encephalitis and dengue virus infection in
patients with clinical symptoms consistent with encephalitis
or dengue fever.
– This assay is a serological aid to diagnosis of Japanese
encephalitis or dengue infection and positive results should
be confirmed by PRNT or current CDC guidelines.
Product positioning
• Laboratory market
• Offer to laboratories already using Panbio Dengue
products
• Laboratories may choose to continue using the Panbio
Dengue ELISAs or rapids to differentiate between
primary and secondary dengue infection
– JE-Dengue IgM Combo ELISA does not differentiate
between primary and secondary dengue infection
• Offer to laboratories currently referring JE testing
• Use the JE-Dengue IgM Combo ELISA for JE
diagnosis primarily
• Areas where dengue co-circulates
Specifications
Name:
Cat No:
Expiry:
Storage:
Antigen:
Antigen prep:
Sample:
Assay time:
Kit size:
Procedure:
Japanese Encephalitis - Dengue IgM
Combo ELISA
E-JED01C
12 months from date of manufacture
2 – 8°C
Recombinant Dengue 1 – 4
Recombinant JE
Dilute 1/250 prior to use
(discard any left-over diluted antigen)
Serum
2 hr 10 min total incubation time
48 tests (96 well plate provide, 2-wells
required per patient)
Standard Panbio capture ELISA format
Procedure
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Each sample is tested in
duplicate, with one well using
JE-MAb complex and the
other well using Dengue-MAb
complex in the second step.
Dilute both antigens 1/250
using the Antigen Diluent prior
to use. Remaining unused
concentrated antigen should
be stored at 2-8 ºC.
Continue as per the following
flow diagram
JE-Dengue IgM Combo ELISA
Serum antibodies combine with the antihuman IgM coated on the plate.
Simultaneously the peroxidase
conjugated MAb and antigen supplied
form complexes when incubated
together.
Washing removes residual serum
The antigen-Mab complexes bind if
antigen-specific antibodies have been
captured.
The colourless substrate,
tetramethylbenzidine/hydrogen peroxide
(TMB/H2O2) is hydrolysed to a blue
chromogen
Stopping the hydrolytic reaction
with acid turns the TMB yellow
Colour development indicates the
presence of specific antibodies in the
test sample
Interpretation #
* Results should be confirmed by PRNT or current CDC guidelines.
# Refer to Instructions for use for full interpretation guidelines
Interpretation cont.
• If positive on the dengue kit, this indicates a
dengue virus infection, even if also positive on the
JE kit.
– On the Panbio JE-Dengue IgM Combo ELISA crossreactivity was predominantly only found one way i.e. at the
JE level.
– This cross-reactivity is a common feature of flavivirus
antibody assays and is typically more dramatic when native
virus preparations are used.
– The specific recombinant virus antigens used in this assay
provide distinct advantages over typical native virus
preparations especially the dengue virus antigens.
• Therefore the two-well approach is essential to
distinguish between dengue and JE.
Performance: Study sites
• Reference laboratory, Bangkok, Thailand
– Data used for package insert
• Inhouse study
– Presented at JE Bi-Regional Meeting, Bangkok,Thailand
April 2005
• AFRIMS, Thailand
– WHO Collaborating Centre
– Competitive evaluation
• Panbio vs. 2 commercially available ELISAs and an inhouse ELISA
Performance
Evaluation by a reference laboratory in Bangkok, Thailand
• Performance of the product was assessed using 360
sera collected for testing at a reference laboratory in
Bangkok, Thailand, and characterised using the
reference laboratory’s in-house IgM and IgG ELISAs.
– 121 samples JE positive
– 111 samples primary or secondary dengue samples
– 128 endemic samples seronegative for both JE and dengue
Performance cont.
Evaluation by a reference laboratory in Bangkok, Thailand
Good distinction between dengue and JE
Only 2 dengue samples incorrectly diagnosed as JE
Performance cont.
Evaluation by a reference laboratory in Bangkok, Thailand
High sensitivity and specificity
Performance
Inhouse study presented at JE Bi-Regional Meeting,
Bangkok,Thailand April 2005
• A population of 349 characterised specimens was
used to evaluate the performance of the JE-Dengue
IgM Combo ELISA.
– USA and Australian normal donor sera (n=265)
– Dengue IgM positive sera (n=19)
– JE IgM positive sera (n=65).
Clear distinction
between dengue
and JE
Performance cont.
Inhouse study presented at JE Bi-Regional Meeting,
Bangkok,Thailand April 2005
Conclusions
Inhouse study presented at JE Bi-Regional Meeting,
Bangkok,Thailand April 2005
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The development of a 2-well ELISA utilising specific
recombinant antigens provides a significant improvement in
the diagnosis of Japanese encephalitis.
Assay has a high level of dengue and JE sensitivity as well
as specificity.
Because of the high level of specificity of the dengue
recombinant antigen, a simple positive / negative
comparison of the 2-well result can be used to determine a
presumptive diagnosis.
The format of the tests allows use of common reagents with
both JE and dengue antigens, including positive and
negative control sera, enhancing ease of use.
Performance cont.
USAMC-AFRIMS competitive evaluation*
April – July 2005
Manufacturer
Sensitivity
Panbio
89.3%
99.2%
90.4%
Competitor 1
99.2%
56.2%
45.6%
Competitor 2
96.7%
65.3%
53.4%
*Study has not yet been published
Specificity
Agreement with
AFRIMS
Conclusions
USAMC-AFRIMS competitive evaluation
April – July 2005
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Smaller number of steps involved
Performed better than competitors
High sensitivity & specificity
High agreement with well recognised and established
in-house diagnostic kit
• Only commercially available kit that differentiates
between JE and dengue
• Panbio kit is advantageous in areas where dengue cocirculates
Advantages
• Simple, standardised procedure
– same format other Panbio capture ELISAs
• Short assay time (2 hr 10 min)
• Good sensitivity and specificity
– inhouse and independent assessment
• Excellent agreement with current diagnostic methods
• Two-well approach allows serological differentiation
between dengue and JE infection
• Two diseases with one kit
• Recombinant antigens
– All 4 dengue serotypes are included
• From Panbio: No. 1 in dengue diagnosis
Promotional material
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Brochure – The key to
differentiating between
dengue and JE
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Flyer – (PB0110)
Promotional material
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Poster presented at JE
Bi-Regional Meeting,
Bangkok,Thailand April
2005
Competitors
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XCyton Diagnostics - JEV CheX kit
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96 well ELISA
Currently operating only in certain parts of India
Marketed by Qualigens Diagnostics (chemical division of
GlaxoSmithKline Pharmaceuticals Ltd.)
Plans to expand the market and get exposure across India
InBios International – JE Detect
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IgM ELISA
IgG ELISA
96 well ELISA
TM
Future
• Develop protocols for use
• Lobby Departments of Health
• Evaluative comparative studies
References
1. Centers for Disease Control Japanese encephalitis fact sheet.
http://www.cdc.gov/ncidod/dvbid/jencephalitis/facts.htm
2. Centers for Disease Control Dengue fact sheet
http://www.cdc.gov/ncidod/dvbid/dengue/facts.htm
3. Valks, A. et al. (2005) Development of a Japanese encephalitis
- Dengue IgM Combo ELISA. Poster presented at JE BiRegional Meeting, Bangkok, Thailand April 2005.
4. eMedicine. Dengue Fever (Last Updated: May 24, 2005)
[http://www.emedicine.com/MED/topic528.htm]
5. eMedicine. Japanese encephalitis. (Last Updated: June 16,
2005) [http://www.emedicine.com/med/topic3158.htm]