1. Panbio’s new JE-Dengue IgM Combo ELISA
JE and Dengue
Panbio’s new JE-Dengue IgM Combo ELISA

2. 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.

3. #Adapted from maps produced by the Centers for Disease Control
World distribution of Dengue – 2005 CDC
Distribution of Japanese Encephalitis in Asia,

4. Japanese encephalitis1
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:
Residents of rural areas in endemic locations
Active duty military deployed to endemic areas
Expatriates in rural areas
Disease risk extremely low in travellers

5. Dengue2 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

6. 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.

7. 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.

8. 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.

9. 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

10. Specifications Name: Japanese Encephalitis - Dengue IgM Combo ELISA
Cat No: E-JED01C
Expiry: 12 months from date of manufacture
Storage: 2 – 8°C
Antigen: Recombinant Dengue 1 – Recombinant JE
Antigen prep: Dilute 1/250 prior to use
(discard any left-over diluted antigen)
Sample: Serum
Assay time: 2 hr 10 min total incubation time
Kit size: 48 tests (96 well plate provide, 2-wells required per patient)
Procedure: Standard Panbio capture ELISA format

11. Procedure
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

12. JE-Dengue IgM Combo ELISA
Serum antibodies combine with the anti-human 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

13. Interpretation #
* Results should be confirmed by PRNT or current CDC guidelines.
# Refer to Instructions for use for full interpretation guidelines

14. 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 cross-reactivity 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.

15. 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 in-house ELISA

16. 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

17. Performance cont.
Evaluation by a reference laboratory in Bangkok, Thailand
Good distinction between dengue and JE
Only 2 dengue samples incorrectly diagnosed as JE

18. Performance cont. High sensitivity and specificity
Evaluation by a reference laboratory in Bangkok, Thailand
High sensitivity and specificity

19. 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).

20. Clear distinction between dengue and JE

21. Performance cont. Inhouse study presented at JE Bi-Regional Meeting,
Bangkok,Thailand April 2005

22. Conclusions Inhouse study presented at JE Bi-Regional Meeting,
Bangkok,Thailand April 2005
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.

23. Performance cont. USAMC-AFRIMS competitive evaluation*
April – July 2005
Manufacturer
Sensitivity
Specificity
Agreement with AFRIMS
Panbio
89.3%
99.2%
90.4%
Competitor 1
56.2%
45.6%
Competitor 2
96.7%
65.3%
53.4%
*Study has not yet been published

24. Conclusions Smaller number of steps involved
USAMC-AFRIMS competitive evaluation
April – July 2005
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 co-circulates

25. 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

26. Promotional material
Brochure – The key to differentiating between dengue and JE
Flyer – (PB0110)

27. Promotional material
Poster presented at JE Bi-Regional Meeting, Bangkok,Thailand April 2005

28. Competitors XCyton Diagnostics - JEV CheX kit
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 DetectTM
IgM ELISA
IgG ELISA

29. Future Develop protocols for use Lobby Departments of Health
Evaluative comparative studies

30. References
Centers for Disease Control Japanese encephalitis fact sheet.
Centers for Disease Control Dengue fact sheet
Valks, A. et al. (2005) Development of a Japanese encephalitis - Dengue IgM Combo ELISA. Poster presented at JE Bi-Regional Meeting, Bangkok, Thailand April 2005.
eMedicine. Dengue Fever (Last Updated: May 24, 2005) [
eMedicine. Japanese encephalitis. (Last Updated: June 16, 2005) [

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