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 ControlWorld distribution of Dengue – 2005 CDC http://www.cdc.gov/ncidod/dvbid/dengue/map-distribution-2005.htmDistribution of Japanese Encephalitis in Asia, 1970-1998 http://www.cdc.gov/ncidod/dvbid/jencephalitis/map.htm

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

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

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

SpecificationsName: Japanese Encephalitis - Dengue IgM

Combo ELISA Cat No: E-JED01C Expiry: 12 months from date of manufactureStorage: 2 – 8°CAntigen: Recombinant Dengue 1 – 4

Recombinant JEAntigen prep: Dilute 1/250 prior to use

(discard any left-over diluted antigen)Sample: SerumAssay time:2 hr 10 min total incubation timeKit size: 48 tests (96 well plate provide, 2-wells

required per patient) Procedure: Standard Panbio capture ELISA format

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

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 reactionwith 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 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.

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

Performance

• 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

Evaluation by a reference laboratory in Bangkok, Thailand

Performance cont.

Good distinction between dengue and JE

Only 2 dengue samples incorrectly diagnosed as JE

Evaluation by a reference laboratory in Bangkok, Thailand

Performance cont.Evaluation by a reference laboratory in Bangkok, Thailand

High sensitivity and specificity

Performance

• 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).

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

Clear distinction between dengue and JE

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

Conclusions

• 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.

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

Performance cont.USAMC-AFRIMS competitive evaluation*April – July 2005

Manufacturer Sensitivity Specificity Agreement with AFRIMS

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

Conclusions

• 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

USAMC-AFRIMS competitive evaluationApril – July 2005

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• Brochure – The key to

differentiating between dengue and JE

• Flyer – (PB0110)

Promotional material• Poster presented at JE

Bi-Regional Meeting, Bangkok,Thailand April 2005

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– 96 well ELISA

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

References1. 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 Bi-Regional 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]