ANNEX 1 W O R L D H E A L T H ORGANISATION MONDIALE ... · bureau regional du pacifique occidental 2ND INTERCOUNTRY HANDS-ON TRAINING WORKSHOP ON THE LABORATORY DIAGNOSIS OF JAPANESE

ANNEX 1

W O R L D H E A L T H

ORGANIZATION

ORGANISATION MONDIALE

DE LA SANTE

REGIONAL OFFICE FOR THE WESTERN PACIFIC BUREAU REGIONAL DU PACIFIQUE OCCIDENTAL

2ND INTERCOUNTRY HANDS-ON TRAINING WORKSHOP ON THE LABORATORY DIAGNOSIS OF JAPANESE ENCEPHALITIS IN THE WESTERN P ACIFIC REGION

Hong Kong (China) ENGLISH ONLY 15-19 November 2010

LIST OF PARTICIPANTS, TEMPORARY ADVISERS AND SECRET ARIAT

PARTICIPANTS

CAMBODIA Mr Am Chanthan Head of Immunology Unit National Institute of Public Health Lot #2 Kim Yl sung Blvd. Sangkat Boeng Kok II, Khan Tuol Kok Phnom Penh Telephone: +855 12 821196 E-mail: [email protected]

Mr Thay Kosal Laboratory Staff – Microbiology Supervisor Khmer-Soviet Friendship Hospital #32E, ST 287 Sangkat Boeng Kak II Khan Tuol Kok Phnom Penh Telephone: + 855 12 505434 E-mail: [email protected]

CHINA Dr Fu Shihong Senior Research Technician Department of Viral Encephalitis Institute for Viral Disease Control and Prevention 155 Changbai Road, Changping District Beijing 102206 Telephone: +86 10 5890 0843 E-mail: [email protected]

Dr Ma Hongxia Master Technician Henan Centers for Disease Control and Prevention Room 4302 No. 105 Nongyedonglu Zhengzhou 450016 Telephone: +86 371 6808 9290 Facsimile: +86 371 6808 9002 E-mail: [email protected]

LAO PEOPLE'S DEMOCRATIC REPUBLIC

Mr Sinakhone Xayadeth Laboratory Technician National Center for Laboratory and Epidemiology Country Surveillance Km 3, Thadeua Road Vientiane Telephone: +856 21 2336196 Facsimile: +856 21 315347 E-mail: [email protected]

Mrs Khuanta Duangmala Laboratory Technician National Center for Laboratory and Epidemiology Country Surveillance Km 3, Thadeua Road Vientiane Telephone: +856 20 99610688 Facsimile: +856 21 315347 E-mail: [email protected]

MALAYSIA Ms Nurul Asshikin bt. Ruslan Medical Laboratory Technologist Virology Unit Institute of Medical Research Ministry of Health Malaysia Jalan Pahang 50588 Kuala Lumpur Telephone: +60 3 26162675 Facsimile: +60 3 26938094 E-mail: [email protected]

PAPUA NEW GUINEA Ms Oscillah Kaminiel Laboratory Manager Central Public Health Laboratory National Department of Health P.O. Box 807, Waigani National Capital District Telephone: +67 5 3248198; +67 5 72515010 Facsimile: +67 5 3256342 E-mail: [email protected]/ [email protected]

Mr Ernest Velemu Quality Assurance Manager

Central Public Health Laboratory Private Mail Bag No. 1, Boroko National Capital District Telephone: +67 5 3248199 Facsimile: +67 5 3256342 E-mail: [email protected]

PHILIPPINES Ms Analisa Bautista Senior Science Research Specialist Research Institute for Tropical Medicine Virology Department 9002 Research Drive, FCC Compound, Alabang Muntinlupa Telephone: +63 2 8097120 Facsimile: +63 2 8097120 E-mail: [email protected]

Ms Ava Kristy Sy Bacteriologist I Research Institute for Tropical Medicine 9002 Research Drive FCC Compound, Alabang Muntinlupa City 1781

Philippines Telephone: (632) 8097120 Facsimile: (632) 8097120 E-mail: [email protected]

REPUBLIC OF KOREA Ms Jung Eun Cho Researcher Korea Centers for Disease Control and Prevention Division of Arboviruses National Institute of Health 194, Tongil-lo, Eunpyung-gu Seoul 122-701 Telephone: +82 2 3802175 Facsimile: +80 2 3801499 E-mail: [email protected]

VIET NAM

Dr DO Phuong Loan Researcher National Institute of Hygiene and Epidemiology (NIHE) No. 1 Yersin Street Ha Noi, Viet Nam

Telephone: (04) 3 9719721 Facsimile : E-mail: [email protected]

Dr Huynh Phuong Thao

Researcher Pasteur Institute

167 Pasteur Street District 3, Ho Chi Minh City, Viet Nam Telephone: (848) 38 296 351 Facsimile: (848) 38 231 419 E-mail: [email protected]

TEMPORARY ADVISERS

Dr Barbara Johnson Diagnostic and Reference Laboratory Division of Vector-borne Infectious Diseases Centers for Disease Control and Prevention 3150 Rampart Road Fort Collins, CO 80521 United States of America Telephone: +970 2663543 Facsimile: +970 22106441 E-mail: [email protected]

Dr Tomohiko Takasaki Chief Laboratory of Vector-borne Viruses National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku Tokyo 162 8640 Japan Telephone: +81 35 2581111 (ext 2526) Facsimile: +81 35 2581188 E-mail: [email protected]

Dr Wei-ling Wilina Lim Consultant Medical Microbiologist Public Health Laboratory Center 9/F Public Health Laboratory Centre 382 Nan Cheong Street, SHek Kip Mei Kowloon Hong Kong Telephone: +85 2 2319 8252 Facsimile: +85 2 2319 5989 E-mail: [email protected]

SECRETARIAT

Dr Youngmee Jee Scientist (Laboratory Virologist) Expanded Programme on Immunization World Health Organization Regional Office for the Western Pacific United Nations Avenue 1000 Manila Philippines Telephone: +63 2 528 9744 Facsimile: +63 2 526 0279 E-mail: [email protected]

Mr David Featherstone Scientist, Project Leader Global Measles Laboratory Network World Health Organization Headquarters in Geneva (HQ) Expanded Programme on Immunization Plus Avenue Appia 20 CH – 1211 Geneva 27 Telephone: +41 22 79 14405 Facsimile: +41 22 791 3111 E-mail: [email protected]

Day 1, Monday, 15 November 2010 08:30 Registration 08:45 Completion of pre-assessment questionnaire 09:00 Opening remarks Dr Wilina Lim 09:10 Workshop objectives Dr Youngmee Jee 09:20 Self-introduction of participants and administrative

announcements

Session 1 Japanese encephalitis/acute encephalitis

syndrome (JE/AES) surveillance and Laboratory Network (Lab Net)

09:30 Laboratory-based JE/AES surveillance:

progress and challenges in maintaining momentum and plans for developing sustainability

Mr David Featherstone

10:00 JE control and lab net progress in the Western Pacific Region

Dr Youngmee Jee

10:30 Coffee break Session 2 Quality assurance of the JE lab net and global

specialized laboratory (GSL) presentations

11:00 Quality assurance and proficiency in the laboratory Mr David Featherstone 11:20 Role of the United States Centers for Disease

Control and Prevention (US CDC) and WHO JE GSL: evaluation of kits

Dr Barbara Johnson

11:50 Activities of National Institute of Infectious Diseases (NIID) GSL and JE surveillance/laboratories

12:10 Discussion 12:30 Lunch break Session 3 Regional reference laboratory (RRL) and

national laboratory reports

13:30 Chinese Center for Disease Control and Prevention (China CDC)

13:50 Korea Centers for Disease Control and Prevention (KCDC)

14:10 Cambodia, Lao People's Democratic Republic, Malaysia (15-minute presentation , 5-minute Q and A)

The 2nd Intercountry Hands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Western Pacific Region

Public Health Laboratory Centre Hong Kong (China)

15 to 19 November 2010

15:10 Coffee break 15:30 Papua New Guinea, Philippines, Viet Nam (2) Session 4 Introduction of JE virus-specific

immunoglobuline M (JEV IgM) enzyme-linked immunosorbent assay (ELISA)

16:30 General Introduction to IgM assays Mr David Featherstone 17:00 Lecture/demonstration of JEV IgM ELISA (serum) Dr Barbara Johnson 17:30 Tour of laboratory and demonstration of ELISA

equipment

18:00 Adjourn for the day Day 2, Tuesday, 16 November 2010 08:30 Introduction to the ELISA practical 08:45 Laboratory practice: JEV IgM ELISA (serum)

(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)

Mr David Featherstone, facilitators and participants

12:00 Lunch break 13:30 Continuation: laboratory practice - JEV IgM ELISA Participants 15:30 Coffee break 16:00 JEV IgM ELISA reading and calculation of results Participants 17:00 Evaluation of ELISA test results and discussion Mr David Featherstone

and facilitators 17:30 Adjourn for the day Day 3, Wednesday, 17 November 2010 08:30 Discussion of Day 2 activity 09:00 Lecture and demonstration of JEV IgM ELISA –

cerebrospinal fluid (CSF)

09:30 Laboratory practice: JEV IgM ELISA (CSF) (during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)


10:00 Coffee break 10:30 Continuation: laboratory practice - JEV IgM ELISA Participants 13:00 Lunch break 14:00 Continuation: laboratory practice - JEV IgM ELISA Participants 15:00 JEV IgM ELISA reading and calculation of results Participants 15:30 Coffee break 15:45 Evaluation of ELISA test results and discussion Mr David Featherstone

and facilitators 16:15 Specimen collection, preparation and shipment for

virus isolation and serology Dr Tomohiko Takasaki

17:00 Adjourn for the day Question for the night

Day 4, Thursday, 18 November 2010 08:30 Discussion on Day 3 activity 08:45 Laboratory practice: ELISA (serum samples)



10:30 Coffee break 11:00 Continuation: laboratory practice – ELISA



12:30 Lunch break 13:30 JE PCR

Evaluation of ELISA test results and discussion PHLC

AES data management and reporting requirements Dr Youngmee Jee Practical session on data entry Participants 17:30 Adjourn for the day Day 5, Friday, 19 November 2010 08:30 Discussion on Day 4 activity (return quiz) 09:00 JE PCR continued

Global specialized laboratory testing methods - ELISA, plaque reduction neutralization testing (PRNT) and decision algorithm

PHLC Dr Barbara Johnson and Dr Tomohiko Takasaki

10:00 Consolidation of laboratory test results and classification

Mr David Featherstone and Dr Youngmee Jee

10:30 Coffee break 11:00 Course assessment and quiz 12:30 Lunch 13:30 Next steps and summary of assessment Round table 15:30 Closing ceremony: presentation of training

certificate

16:30 Distribution of proficiency panels and kits to network laboratories

PROCEDURE FOR PANBIO JE –DENGUE IgM COMBO ELISA

1) Bring all the samples and the reagents required for ELISA to room temperature.

2) Remove required number of wells and insert them into the frame.

3) Dilute positive and negative controls, JE and DENGUE calibrators, and samples using 1.5ml screw capped vials: • 10 µl of serum sample, controls, and calibrators in 1000 µl of diluent (1:100 dilution) • 25 µl of CSF sample in 225 µl of diluent (1:100 dilution)

4) Dilute 10 µl of antigen into 2.5 ml of antigen diluent in 15ml tubes (1:250 dilution)

Prepare dilutions separately for JE and DENGUE antigen.

5) Mix required amount of JE and DENGUE antigen separately with an equal amount of Mab tracer (1:1 vol/vol) in 15 ml tubes. Leave it at room temperature until use. Discard any unused antigen.

6) Within 10 minutes of mixing the antigen and Mab tracer, pipette 100 µl of diluted Pos and Neg controls and patient samples in duplicate in the JE and DEN columns in the assay plate. JE and DEN calibrators are added in triplicate to wells in either the JE or DEN column.

7) Cover the plate with aluminum foil and incubate at 37ºC for 1 hour.

8) Prepare wash buffer :

Dilute 1 part of wash buffer concentrate in 19 parts of distilled water (1:20 dilution).

9) Wash the plate 6 times with the diluted wash buffer

10) Add 100 µl of JE antigen–Mab complex and DENGUE antigen–Mab complex prepared earlier into the respective wells.

11) Cover the plate and incubate for 1 hour at 37ºC.

12) Wash the plate 6 times with the diluted wash buffer.

13) Add 100 µl of TMB into each well. Cover the plate and incubate for 10 min at room

temperature (20-25°C).

14) Add 100 µl of stop solution into each well.

15) Read the absorbance at a wavelength of 450 nm with a reference filter of 600 – 650 nm within 30 minutes.

CALCULATIONS :

1) Calculate the average absorbance of the triplicates of the calibrators.

2) Calculate the cut-off values: Cut-off value = Mean absorbance of the calibrators X calibration factor (batch specific)

3) Determine validity of test: Positive control OD/Cut-off ratio = batch-specific acceptable range If test not valid, do not continue to calculate – repeat test.

4) Calculate index value of the sample : Index value = Sample absorbance

Cut–off value

5) Calculate the Panbio units of the sample: Panbio units = Index value of sample X 10

INTERPRETATION OF PANBIO UNITS : JE Panbio units

JE IgM result

DEN Panbio units

DEN IgM result

Interpretation

< 9 Neg < 9 Neg No detectable IgM antibody.The result does not rule out the infection. An additional sample should be collected in 7-14 days and the test carried out again if early infection is suspected.

< 9 Neg 9 -11 Eqv Samples should be re-tested

9-11 Eqv < 9 Neg Samples should be re-tested

9-11 Eqv 9-11 Eqv Samples should be re-tested

< 9 Neg >11 Pos

9-11 Eqv >11 Pos

>11 Pos <9 Neg

>11 Pos 9-11 Eqv

> 11 Pos > 11 Pos

Calculate JE/ Dengue ratio : Ratio = JE Panbio units

Dengue Panbio units Interpretation of JE / Dengue ratio :

1) ≥ 1 Presence of detectable IgM antibody presumptive infection with JEV

2) < 1 Presence of detectable IgM antibody

presumptive infection with DENV

PanbioJE-DEN IgM Combo ELISA procedure

WHO JE Workshop 18-19 November 2010

World Health Organization

HANDS-ON TRAINING ON MOLECULAR

DETECTION OF

JAPANESE ENCEPHALITIS VIRUS

Protocols for Practical

18-19 November 2010

Public Health Laboratory Centre

Centre for Health Protection

Hong Kong SAR, China


Table of Contents

Thursday, 18 November

Practical 1: Extraction of Japanese encephalitis (JE) RNA

Practical 2: One-Step RT-PCR for JE virus detection

Friday, 19 November

Practical 3: Gel electrophoresis of JE PCR product (to be performed by PHLC staff)


Page 1

Objectives:

1. To have an idea on molecular testing (RNA extraction and RT-PCR) of JE virus

2. To have a brief idea of the general precautions in performing molecular testing

Location:

Practical 1: Extraction of JE virus RNA

(Location: Room 907)

Practical 2: One-step RT-PCR for the detection of JE virus

(Location: Room 817)


Page 2

Practical 1: RNA Extraction for JE virus detection

References:

� QIAamp® Viral RNA Mini Handbook, Third Edition, December 2007. Qiagen.

Materials/Reagents/Equipment provided:

2 vials (sample A & sample B) containing simulated samples to be extracted

Buffer AVL containing carrier RNA (pre-prepared by PHLC staff)*

QIAamp spin columns (2 vials)*

Buffer AW1 with ethanol*

Buffer AW2 with ethanol*

Buffer AVE*

Collection tubes*

1.5ml screw cap vials (for RNA storage)

1.5ml snap cap vials (caps already cut away by scissors)

Absolute ethanol (96-100%)

Microcentrifuge

Micropipettes

Aerosol-free pipette tips

Vortex mixer

Timer

Ice bucket

Biological safety cabinet

Worksheet for RNA extraction (Appendix 1)

*Reagents from QIAamp® Viral RNA Mini kit or reagents prepared from content of the kit.

Buffer AVL (with carrier RNA), Buffers AW1 (with ethanol added) and AW2 (with ethanol

added) will be provided. Please refer to the kit handbook for preparation of these buffers

when using the new kit.

Procedures (Room 907)

(1) Prepare worksheet for the RNA extraction.

(2) Label the vials containing 560ul of Buffer AVL with carrier RNA.

(3) In the safety cabinet add 140ul of samples to the corresponding vials.

(4) Mix by pulse-vortexing for 15 seconds. Incubate at room temperature for 10 minutes.

(5) Label the spin columns and the 1.5ml screw cap vials (for storing extracted RNA)

accordingly.

(6) Briefly centrifuge the other vials after the 10-minute incubation. Add 560µl absolute

ethanol (96-100%) to the each vial and mix by vortexing for 15 seconds.

(7) Pulse-spin all the vials.


Page 3

(8) Open the cap of the spin columns one by one and transfer 630µl of the solution from the

vials to the corresponding spin column. Close the cap and centrifuge at 6,000 × g (8,000

× rpm) for 1 minute.

(9) Place the spin columns in clean 2ml collection tubes and discard the collection tubes

containing the filtrate.

(10) Repeat steps (8) − (9).

(11) Open the cap of the spin columns one by one and add 500µl Buffer AW1. Close the cap

and centrifuge at 6,000 × g (8,000 × rpm) for 1 minute. [Note: Use a new pipette tip

when adding buffer to each column]

(12) Place the spin columns in clean 2ml collection tubes and discard the collection tubes

containing the filtrate.

(13) Open the spin columns one by one and add 500µl Buffer AW2. Close the cap and

centrifuge at 16,100 × g (13,200 × rpm) for 3 minutes. [Note: Use a new pipette tip

when adding buffer to each column]

(14) Place the spin columns in clean 2ml collection tubes and centrifuge at 16,100 × g

(13,200 × rpm) for another 1 minute.

(15) Place the spin columns in clean 1.5ml snap cap vials (caps already cut away by scissors)

and discard the collection tubes containing the filtrate.

(16) Open the caps of the spin columns and add 60µl Buffer AVE equilibrated to room

temperature.

(17) Close the cap of the spin columns and incubate at room temperature for 1 minute.

(18) Centrifuge at 6,000 × g (8,000 × rpm) for 1 minute.

(19) Remove the spin columns one by one from the 1.5ml snap cap vials and transfer the

eluted RNA into the 1.5ml screw cap vials. Keep the vials on ice.


Page 4

Practical 2: One-step RT-PCR for JE detection

References:

� QIAGEN® OneStep RT-PCR Kit Handbook. February 2008. Qiagen.

� Murakami S, Takahashi Y, Yoshida S, Fuke I, Ohmae K, Mori C, Takagi M, Takamizawa

A, Okayama H. (1994) Highly sensitive detection of viral RNA genomes in blood

specimens by an optimized reverse transcription-polymerase chain reaction. J Med Virol

43(2):175-181.


Qiagen One-Step RT-PCR kit (reagents prepared by PHLC staff)

1 working master mix for JE RT-PCR (prepared by PHLC staff)

1 vial of JE RNA control

1 vial of molecular grade water (as negative control)

Micropipettes


0.2ml PCR tubes

Vortex mixer

Tomy centrifuges (for 1.5ml and 0.2ml tubes)

Ice bucket

ABI 9700 thermal cycler

Worksheet for the RT-PCR detection of JE virus (Appendix 2)

Procedures

Preparation of master mix (prepared by PHLC staff)

Master mix will be prepared by PHLC staff in master mix preparation room according

to the following table:

Reagents Volume per test (ul)

Qiagen 5X PCR buffer * 10

dNTPs* 2

Primer JE-1 (5uM) 6

Primer JE-2 (5uM) 6

Enzyme mix (kit)* 2

RNase inhibitor 0.5

H2O* 18.5

*Reagent from Qiagen One-step RT-PCR kit


Page 5

JE-1: 5’-CACAACGAGAAGCGAGCTGATAGTA-3’

JE-2: 5’-CCCCAACTTGCGCTGAATAATTCCC-3’

RNA template addition (Room 817)

(1) Prepare worksheet for the one-step RT-PCR for JE virus.

(2) Label the 0.2ml PCR tubes.

(3) Aliquot 45ul of master mix to the labeled PCR tubes.

(4) Aliquot 5µl of extracted RNA and control to the corresponding PCR tubes accordingly.

Water (5µl) is used as a negative control.

(5) Vortex briefly to mix the content. Pulse-spin the PCR tubes. Place the PCR tubes into

thermal cycler. Start the PCR run with the PCR conditions as shown below.

Cycling parameters of RT-PCR for JE detection

50oC 30 min; 95oC 15 min; (94oC 30 sec, 55oC 30 sec, 72oC 30sec) x 40 cycles; 72oC

10min; 10oC ∞∞∞∞


Page 6

Friday 19 November

Objective:

To perform gel electrophoresis of RT-PCR product for JE virus detection obtained from

practical 2 (to be performed by PHLC staff)

Location (gel loading room):

Practical 3: Gel electrophoresis of RT-PCR product for JE virus detection obtained

from practical 2 (to be performed by PHLC staff)


2% agarose solution

Gel casting tray and combs

Microwave oven

1 vial of 1X TBE buffer

SYBR SafeTM DNA gel stain

Agarose gel tanks

Power supply

Micropipettes


Tomy centrifuge (for 0.2ml tubes)

96-well plates (U-bottomed)

1 vial of 6X loading dye

1 vial of 100bp DNA ladder

Gel documentation system

Preparation of agarose gel

Note: Gel will be set and run by PHLC staff

(1) Loosen the cap of the bottle containing 2% agarose gel. Melt the agarose gel in

microwave. Avoid overheating. Swirl from time to time.

(2) After the gel is melted completely, leave it at room temperature for 5 minutes.

(3) Assemble the gel casting tray and together with the combs.

(4) Add 25µl of SYBR SafeTM DNA gel stain to each bottle of agarose gel. Swirl to mix.

(5) Pour the mixture into the cast and allow the gel to solidify for at least 30 minutes. Cover

the gel casting tray to protect it from light.


Page 7

(6) When the gel is solidified, remove the combs from the casting tray and put the gel

together with the casting tray into the agarose gel tank filled with TBE buffer.

Gel loading and electrophoresis

(1) Vortex and pulse-spin the 6X loading dye.

(2) Calculate the number of wells needed and add 2µl of the 6X loading dye into each well.

(3) Vortex and pulse-spin the PCR tubes.

(4) Transfer 5µl of PCR product from each PCR tube and mix with the loading dye in the

well.

(5) Load the content of each well into the wells of the agarose gel.

(6) Load 10µl of the 100bp DNA ladder one of the wells as molecular size marker.

(7) Run the gel at 140V, for 25-30 minutes (until the blue marker dye has migrated to about

5mm from the bottom of the gel).

(8) Turn off the power supply, disconnect the cable and retrieve the gel from the gel tank.

(9) Rinse the gel with water briefly.

(10) Visualize the gel and print the gel photo with gel documentation system.


Page 8

Date: _______________

Appendix 1 Worksheet for RNA extraction-QIAamp Viral RNA Mini Kit

Reagent Lot No. Expiry Date

QIAamp Viral RNA Mini kit

No. Sample ID

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Samples added by: _______________ & _______________

RNA eluted by: _______________


Page 9

Appendix 2 Detection of JE virus by One-step RT-PCR

Reagent Volume per test (µµµµl) Total volume (µµµµl)

QIAGEN 5X PCR buffer (kit) 10

dNTPs (kit) 2

Primer JE-1 (5 µµµµM) 6

Primer JE-2 (5 µµµµM) 6

Enzyme mix (kit) 2

RNase inhibitor 0.5

H2O (kit) 18.5

Total 45

RNA template 5

No Sample ID Result

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Done by: _______________ Checked by: _______________

1

Introduction of Workshop Objectives

2nd Intercountry Hands on training on the Laboratory Diagnosis of Japanese encephalitis

November 15-19 2010

Objectives of this workshop

• To further enhance knowledge and skills of national JE laboratory staff in:a) performing ELISA for laboratory diagnosis of JE,andb) carrying out laboratory quality assurance for JE diagnosis;

• To familiarize participants with the requirements for WHO accreditation for the JE laboratories; and

• To discuss laboratory data management issues and laboratory data reporting to the Western Pacific Regional Office.

Day 1

• Session 1 JE/AES surveillance and Laboratory network

Coffee break

• Session 2Quality assurance of the JE labnet and GSL presentations

Lunch

• Session 3: RRL and National Lab reports

Coffee break

• Session 4. Introduction of JEV IgM assays

– Demonstration of ELISA assay

– Tour of the laboratory

Day 2

• ELISA Practical using serum samples

Use Panbio kit

ELISA reading and calculation, interpretation

• During incubation times, calibration of micropipettes and maintenance of ELISA equipments.

Day 3

• ELISA practical using CSF samples

Use Panbio kit

(During incubation times, calibration of micropipettes and maintenance of ELISA equipments)

• Lectures on specimen collection, shipping of samples for confirmatory testing and virus isolation.

Day 4

• ELISA practical for serum samples

ELISA reading and calculation, interpretation

• (During incubation times, calibration of micropipettes and maintenance of ELISA equipments)

• Data management for JE/AES

2

Day 5

• Lecture on methods conducted in Global Specialized Labs

• Consolidation of laboratory test results

• Course assessment

• Discussions on next steps and future plans in details.

• Closing and certificate

• Distribution of proficiency panel samples (5 CSF and 6 serum samples)

Demonstration of the PanBio Japanese

encephalitis-Dengue IgM Combo ELISA procedure

Barbara W. JohnsonBarbara W. JohnsonUS Centers for Disease Control and Prevention US Centers for Disease Control and Prevention

Division of VectorDivision of Vector--Borne DiseasesBorne DiseasesArboviral Diseases Diagnostic and Reference LaboratoryArboviral Diseases Diagnostic and Reference Laboratory

The 2nd Intercountry HandsThe 2nd Intercountry Hands --on Training Workshop on the Laboratory on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Diagnosis of Japanese Encephalitis in the Western Pacific RegionPacific Region

Public Health Laboratory Centre, Hong KongPublic Health Laboratory Centre, Hong Kong15 to 19 November 201015 to 19 November 2010

PanBio JE-Dengue IgM Combo ELISA kit

� Test up to 43 samples/plate

� Included in the kit• Precoated microwell

strips• Reagents, diluents, and

buffers• Calibrators and positive

and negative controls

Principle of JE-DEN Combo IgM ELISA

� Microwells precoated with anti-human IgM

� Sample added in duplicate. Capture of all IgM in sample.

� JE or DEN antigen-Mab/HRP conjugate complex added, binds to JEV or DENV reactive IgM in sample

� Substrate binds to bound Mab/HRP

� Colorimetric measurement of JE or DEN IgM reactivity in sample

HRPHRP

HRPHRP

JE JE

JE JE

DEN

HRP

DEN

HRP

JE+ DEN-

Plate setup and worksheet for testing 11 samples

CONTROLS:

-: Negative Serum

+: Positive Serum

JC: JE Calibrator

DC: DEN Calibrator

JE DEN JE DEN

- -

+ +

JC DC

JC DC

JC DC

1 1

2 2

3 3

4 4

5 5

6 6

7 7

8 8

9 9

10 10

11 11

1. Equilibrate kit reagents and microwell strips to r oom temperature

2. Remove required number of microwell strips and inse rt them into the frame (4 strips used for 11 samples)

3. Dilute controls and samples

� Pos and neg controls, JE and DEN calibrators, and ser um dilutions: 1:100 (10 µl serum in 1000 µl diluent )

� CSF dilution: 1:10 (25 µl in 225 µl diluent)

3. Prepare JE and DEN antigen/Mab tracer mixes

� JE and DEN antigen dilutions: 1:250 (10 µl in 2500 µl diluent)

� Antigen/Mab tracer mixture: 1:1� Calculate vol needed of each antigen/Mab mixture:

� # wells X 100 ul/well

Assay Preparation Plate Washing

� Wash solution1:20 Dilution (50 ml wash buffer concentrate

into 950 ml distilled H 2O)

� Washing methods� Plate Washer� Hand Washing with squirt bottle� Hand washing with multichannel pipette

� Wash plates X6 at each wash step

+PC, NC, JC, and DC

Prepare 1/250 JE and DEN antigen dilutions

Assay

Procedure

+PC, NC, JC, and DC

Prepare 1/250 JE and DEN antigen dilutions

Dilutions:

Serum , PC, NC, JC, DC - 1:100 •10 ul sample/1ml diluent

CSF - 1:10•25 ul CSF/225 ul diluent

JE or DEN Antigen - 1:250•10 ul antigen/2.5 ml diluent

Mixtures

Antigen/Mab tracer: 1:1 vol:vol

5 control +11 sample wells = 16 wells

16 wells X 100 ul/well = 1600 ul ≈ 1800 ul

900 ul antigen + 900 ul Mab tracer = 1800 ul

To JE and DEN columns (X2)

To JE or DEN wells (X16)

All wells (X32)

All wells (

Quality control and calculations of validity

Acceptable ranges of valid

JE+ or DEN+/cut-off ratios (batch specific)

Kit expiry date

JE and DEN calibrator factors and neg Abs

(batch specific)

Information on kit box

•Kit lot # •Expiration date •JE Calibration Factor•DEN Calibration Factor•Range of acceptable Neg Control OD•Range of acceptable JE Pos C OD/cut-off ratio •Range of acceptable DEN Pos OD/cut-off ratio

Calculations:JE Cut-Off value = Mean OD of JE Calibrators X JE Calibration factorDEN Cut-Off value = Mean OD of DEN Calibrators X DEN Calibration factor

Interpretation of Validity : 1. JE Pos Control OD/JE cutoff ratio 2. DEN Pos Control OD/DEN cutoff ratio3. Neg Control OD

Determining test validity

Are these 3 values within acceptable range?

Batch-specific information on kit box:

TEST IS VALID IF: JE Pos, DEN Pos, and Neg controls are all within accept able ranges

and expiration date has not passed

If test is NOT valid, do not proceed to calculations – repeat test!

If test meets validity requirements, calculate JE an d DEN Panbio units of each sample

Calculations of Panbio Units

Cutoff value:JE: Average of 3 JE calibrator ODs X kit JE calibra tion factorDEN: Average of 3 DEN calibrator ODs X kit DEN cali bration factor

Index value:JE: Sample JE OD/JE Cutoff ValueDEN: Sample DEN OD/DEN Cutoff Value

PanBio units:JE: JE Index value X 10DEN: DEN index value X10

Interpretation

<9 Negative

9-11 Equivocal

≥11 Positive

If either JE or DEN is IgM positive calculate JE/DEN ratio

JE Panbio units ≥1 JE positiveDEN Panbio units <1 Den positive

Panbio Units IgM result

Ratio

Example: Calculations and Interpretations

Kit specifications:

Kit lot: 06193 Expiration date: 12/12/2011JE Calibration Factor: 0.63DEN Calibration Factor: 0.38Neg C OD <0.25JE PosC OD/cut-off ratio: 1.1-6.0DEN PosC OD/cut-off ratio: 3-17.0

Kit lot: 06193 Expiration date: 12/12/2011JE Calibration Factor: 0.63DEN Calibration Factor: 0.38JE PosC OD/cut-off ratio: 1.1-6.0DEN PosC OD/cut-off ratio: 3-17.0Neg C OD <0.25

Step 1: Determine validity of test

Unit calculationsJE Calibrator Avg OD = 0.574JE Cut-off = Avg JC OD X JC factor = 0.574 X 0.63 = 0.362

DEN Calibrator Avg OD = 1.331 Den Cut-off = Avg DC OD X DC factor = 1.331 X 0.38 = 0.506


JE PosC OD = 2.12 JE NegC OD = 0.063DEN PosC OD = 3.41 DEN NegC OD = 0.066

JE PosC OD/JE cut-off = 2.12/0.362 = 5.86DEN PosCOD/DEN cut-off = 3.41/0.506 = 6.74

Kit lot 06193 Expiration date 12/12/2011JE Calibration Factor: 0.63DEN Calibration Factor: 0.38JE PosC OD/cut-off ratio: 1.1-6.0DEN PosC OD/cut-off ratio: 3-17.0Neg C OD <0.25







Test valid?

Kit lot 06193 Expiration date 12/12/2011JE Calibration Factor: 0.63DEN Calibration Factor: 0.38JE PosC OD/cut-off ratio: 1.1-6.0DEN PosC OD/cut-off ratio: 3-17.0Neg C OD <0.25




Determining test validity: Yes valid test



Sample 1

JE OD = 1.493, Den OD = 1.09

JE Panbio Units = JE OD/JE Cutoff X 10 = 1.493/0. 362 X 10 =41.24 = IgM+

DEN Panbio Units = 1.09/0.506 X 10 = 21.54 = IgM+

JE/DEN Ratio= 41.24/21.54 = 1.91

Interpretation: JE Positive

Kit lot 06193 Expiration date 12/12/2011JE Calibration Factor: 0.63DEN Calibration Factor: 0.38JE PosC OD/cut-off ratio: 1.1-6.0DEN PosC OD/cut-off ratio: 3-17.0

Cut-off valuesJE Cut-off = 0.362Den Cut-off = 0.506

Sample 2

JE OD = 0.085, DEN OD = 0.641

JE Panbio Units= 0.085/0.362 X 10 = 2.35 = IgM-

DEN Panbio Units=0.641/0.506 X 10 = 12.67 = IgM+

JE/DEN ratio = 2.35/12.67 = 0.185

Interpretation: DEN Positive



Sample 3

JE OD = 0.144, Den OD = 0.102

JE Panbio Units = JE OD/JE Cutoff X 10 = 0.144/0 .362 X 10 = 3.98 = IgM-

DEN Panbio Units = 0.102/0.506 X 10 = 2.01 = IgM-

Interpretation: No detectable IgM*

*Request 2 nd sample if 1 st collected <7-10 days post-illness



Troubleshooting� Background OD� Reagents not equilibrated to room

temperature� Wells not washed properly� Incorrect incubation temperature/time� Inconsistent results� Inaccurate pipetting� Incorrect dilutions of samples/reagents� Invalid test� Kit reagents past expiry date

Before you begin …..

Checklist� Kit expiration date� Plate washer working properly� Pipettes calibrated correctly� Plate reader working � Kit equilibrated to room temperature� In-house control included

Activities of NIID GSL & JE

suveillance/laboratory activities in

JAPAN

The laboratory of VectorThe laboratory of Vector--Borne Borne

Visruses, Virology 1Visruses, Virology 1stst, National , National

Institute of Infectious Diseases, JapanInstitute of Infectious Diseases, Japan

The second Intercountry hands-on training workshop on the laboratory diagnosis

of Japanese encephalitis in the Western Pacific region.

15th to 19th November 2010

National Institute of Infectiouse DiseasesNational Institute of Infectiouse Diseases

国立感染症研究所国立感染症研究所（旧国立予防衛生研究所）（旧国立予防衛生研究所）

INTRODUCTIONINTRODUCTION

Laboratory of VVector-BBorne VViruses

• Japanese encephalitis virus

• Dengue virus

• West Nile virus

• Yellow fever virus

• Chikungunya virus

We mainly take care of following viruses.We mainly take care of following viruses.

Geographical distribution of JE cases, 2000～Mar．2010 (n=56, as of Apr 2010)

Kyushu Chugoku

Kinki

Chubu

Shikoku Kanto

Tohoku

Hokkaido

n= 7 (fatal case 1)n= 5

n= 8 (fatal case 1)n= 1n= 5 (fatal case 1)

n= 7n= 7

n= 10 (fatal case 2)n= 3

n= 3

Eight arboarboarboarbovirus centers in Iapan

Kumamoto

Miyagi

Tokyo

Aichi

MieOsaka

Kobe

Hiroshima

Activities for Arbovirus centers

• Each center held a hands-on training

course for JE and dengue etc. every two

or three years. NIID support the course

mainly technically and partly financially.

• Technical supports:

To distribute IgM captured ELISA kits and

positive control RNAs of Arboviruses* for

PCR to centers.

*Dengue, Chikungunya, West Nile, Zika etc.

Supports for prefectural institute

• Standard control serum for NT

• Challenge virus for NT；；；；PRNT & FRNT

• JE antigen for HI test can be

purchased from a vaccine company

(Denka Seiken).

NT: neutralization test, PRNT: plaque reduction neutralization test

FRNT: focus reduction neutralization test

Introduction②-Data flowSentinel surveillance system

Sentinel clinics / hosp. Clinical isolates and specimens

Local health centers

Case Report Summary Report (wk/mo)

Quarantine stations

National IDSC

（NIID）Laboratories （NIID）

MHLW

Reports

Specimens

Information dissemination

Case-based surveillance

All clinics / hosp.

Pref. Health

Depts.Pref. IDSCs

Pref. PHIs

（Laboratories）

Sentinel sites for agent surveillance

Prefectural

Level data

mngm’t

Local Level data

management

National

Level data

mngm’t

input the data to central server

input the data to central server

JE vaccination history in Japan ,2009

Japanese encephalitis HI antibody prevalence of swine in Japan 1972～2005

－National Epidemiological Surveillance of Vaccine Preventable Diseases－

yearyear(cases(cases））

Not done Not done

0%0%＜＜50%50%5050～～80%80%≧≧80%80%

19721972（（2222））

19731973（（7070））

19741974（（66））

19751975（（2727））

19761976（（1313））

19771977（（55））

19791979（（8686））

19781978（（8888））

19801980（（4040））

19821982（（2121））

19831983（（3232））

19841984（（2727））

19851985（（3939））

19861986（（2626））

19871987（（3737））

19891989（（2727））

19881988（（3232））

19901990（（5454））

19811981（（2323））

19921992（（22））

19931993（（44））

19941994（（44））

19951995（（22））

19961996（（44））

19971997（（44））

19991999（（55））

19981998（（22））

20002000（（77））

19911991（（1313））

20022002（（88））

20032003（（11））

20042004（（55））

20052005（（77））

20012001（（55））

Contribution to WHO as JEContribution to WHO as JE--GSLGSL

1. Antigen for JE IgM captured ELISA.

2. IgM Ab coated plate.

3. Secondary Abs conjugated with HRP.

4. Communication among RRC; one meeting per year.

5. FRNT technique was transferred to China CDC

The meeting among RRC of WPRO, 2010

2010/5/27Titer Method is better for

differential diagnosis

sera

x 100

x 200

x 400

x 800

x1600

x3200

X6400

X12800

JE Den

Antigen

x 10

x 20

x 40

x 80

x160

x320

X640

X1280

CSF

Anti-human IgM Ab coated plate

• Plate in house: AA

Thermo Scientific Nunc MaxiSorpTM

Surface

• Plate of FOCUSFOCUS DiagnosticsDiagnostics kit: BB

Comparison between A(NIID) and B(Focus)

A B

O.D.

Blank 0.12* 0.074

Empty 0.045 0.040

*Blocking agents is 1% BSA

IgM capture ELISAIgM capture ELISA

Virus AntigenVirus Antigen；；；；；；；；JEV antigenJEV antigenincubate overnight at 4incubate overnight at 4℃℃℃℃℃℃℃℃

Conjugate(HRP,AP,etc)Conjugate(HRP,AP,etc)AntiAnti--Flavi Mono Antibody (6B6C)Flavi Mono Antibody (6B6C)

30 min. at RT30 min. at RT

Substrate (TMB)Substrate (TMB)

Plastic Solid Phase

Test SerumTest Serumcontaining Anticontaining Anti--

Virus IgM AntibodyVirus IgM Antibody

AntiAnti--human IgM mAb human IgM mAb Capture AntibodyCapture Antibody

Blocking Blocking

1hr. at RT

Evaluation the plate by IgM positive serumEvaluation the plate by IgM positive serum

Dilution; x103Focus Diagnostics

_Plate

NIID

_Plate

1 2.138 2.043

2 1.805 1.704

4 1.168 1.144

8 0.627 0.676

16 0.320 0.342

32 0.161 0.171

Blocking reagentsinhouse

Focus 1%BSA BA

Pos

1000 1.405 1.18 1.3052000 0.872 0.792 0.8454000 0.438 0.443 0.485

8000 0.217 0.24 0.26316000 0.098 0.114 0.15732000 0.086 0.093 0.105

Neg x100 0.091 0.085 0.091Blank 0.032 0.045 0.062

BSA: bovine serum Albumin, BA:Block ACE

VBV Labo. of NIID is responsible for

the quality of JE vaccine

1. Inactivation test

2. Potency test

I am supposed to attend “Informal Meeting

of technical experts to develop Regional

Working Reference Standards (RWRS) for JE

vaccines and Polio Vaccines” next week.

Contribution to WHO; othersContribution to WHO; others

• Support the international reference

vaccine for the potency test of JE

vaccine colaborated with NIBSC and

Biken foundation.

• Supporting the dengue-NET of

WPRO.

Thank you !Thank you !

Evaluations of In-house and Commercial JEV IgM ELISA Kits

The 2nd Intercountry Hands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the

Western Pacific RegionPublic Health Laboratory Centre, Hong Kong


Barbara W. JohnsonU.S. Centers for Disease Control and Prevention

Division of Vector-Borne DiseasesArboviral Diseases Diagnostic and Reference Laboratory

Background:

�Standardization of JE testing by JEV IgM ELISA is needed throughout the WHO JE laboratory network (LabNet)

�Validation of commercial JEV IgM ELISA kits or reagents needed to make recommendations for use by JE LabNet

�Concerns about assay sensitivity and specificity for both CSF & serum after confirmatory testing at CDC

JEV IgM detection assays� In-house JEV ELISA IgM assays used in a number of

specialised/reference labs and also some National labs • AFRIMS, Thailand• CDC/DVBID, USA• NIID, Japan • NIMHANS, India• National Institute of Virology, India US CDC evaluation • UNIMAS, Malaysia• Viet Nam NIID, Japan limited evaluation

� Commercial JE assays available • JE-DEN IgM Combo ELISA, PanBio, Australia• JEV CheX, XCyton, India• JE Detect MAC-ELISA, Inbios, USA• Beixi JEV IgM ELISA, Beixa, China

AFRIMS and US CDC evaluations

CDC limited evaluation

Kit

Jacobson 2007

(USAMRID)

sera (n=360)

Ravi 2009

(CDC)

CSF (n=60)

Lewthwaite 2010

(VT)

sera & CSF (n=371)

Robinson 2010

(CDC)

sera & CSF (n=520)

Khalakdina 2010

(USAMRID)

sera (n=350)

% Sens % Spec % Sens % Spec % Sens % Spec % Sens % Spec % Sens % Spec

JE-DEN

Combo89 98-99 65-80 95-98

69(S)68(C)

9739(S) 20-30

(C)99 71-80 95-98

JE

Detect99 56-96

57(S) 53(C)

97

JEVChex 97 65-96 90 9857(S)75(C)

97(S)98(C)

20(S) 17(C)

97 93 89

Recent publications of JE IgM ELISA kit evaluations

CDC analysis of kit performance and

sensitivity and specificity limits

&Recommendations

Evaluation 1:

Assays evaluated:

1.JE-DEN IgM Combo ELISA, PanBio, Australia

2.JEV CheX, XCyton, India

3.JE Detect MAC-ELISA, Inbios, USA

Sample set: •438-520 acute-phase serum and CSF specimens •patients met acute encephalitis syndrome (AES/AMES) clinical case definition•Collected during AES/AMES surveillance project in India and Bangladesh

Reference assay: CDC JEV and DENV IgM ELISA and PRNT

Robinson et al. Am J Trop Med Hygiene 2010.

Kit CDC JEV IgM results

All samples

Serum

CSF + - + - + -

Panbio

+ 30 5 24 4 6 1 - 61 424 37 229 24 195

No. tested (n=520) 91 429 61 233 30 196 XCyton JEV CheX + 17 12 12 7 5 5

- 74 417 49 226 25 191 No. tested (n=520) 91 429 61 233 30 196 InBios JE Detect + 38 12 29 10 9 2 - 30 374 22 209 8 165 No. tested (n=454) 68 386 51 219 17 167

294 Sera•61 JEV IgM positive•36 DENV IgM positive (JE neg)•16 WNV IgM positive (JE neg)•181 JEV/DENV/WNV IgM negative (JE neg)

226 CSF•30 JEV IgM positive•16 DENV IgM positive (JE neg)•180 JEV/DENV/WNV IgM negative (JE neg)

Summary test results

Sample set Summary analysis of kit performance

Kit % Sensitivity(95% CI)

% Specificity(95% CI)

%Agreement

All samples combined

JE-DEN Combo 33(24-44)

99(97-99.5)

87

JE Detect 56(44-67)

97(95-98)

91

JEV CheX 19(12-28)

97(95-98)

83

CSFJE-DEN Combo 20

(10-37)99.5

(97-99.7)89

JE-DEN ComboCSF Adjusted

30(17-48)

99(96-99)

90

JE Detect 53(31-74)

99(96-99)

95

JEV CheX 17(7-34)

97(94-99)

87

SeraPanBio 39

(28-52)98

(96-99.3)86

InBios JE Detect 57(43-70)

95(92-98)

88

XCyton JEV CheX 20(12-31)

97(94-99)

81

All Sample types combined (N=68)

0

5

10

15

20

25

30

<3 3-7 7-14 >14 Unknown

Days from onset of illness to specimen collection

No

. JE

V Ig

M P

osi

tive

JE-DEN ComboJE Detect JEV CheX

CDC JEV-DEN ELISA

Sensitivity evaluation: Comparison of CDC JEV IgM+ to kit results by days post-onset of illness to specimen collection

0

5

10

15

20

25

30

35

40

45

50

Low Medium High

N0.

JE

V Ig

M+

JE-DEN ComboJE DetectJEVCheXCDC

(P/N<10) (P/N=10-20) (P/N>20)

Sensitivity evaluation: Comparison of CDC JEV IgM+ to kit results by CDC P/N ratio

All sample types combined

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

1 2 3

Sample

Rec

ipro

cal

Tite

r

CDC

Inverness PanBio

Xcyton

InBios

CDC P/N=301:400

CDC P/N=221:400

CDC P/N=12 1:12800

CDC P/N=4.4 1:12800

CDC P/N=14.611:12800

CDC P/N=301:400

Sensitivity evaluation sera:IgM detection endpoint titrations of 3 high CDC JEV IgM

positive serum samples

Sensitivity evaluation CSF:IgM detection endpoint titrations of 1 high and 1 low

CDC JEV IgM positive (1:10 dilution) CSF

0

200

400

600

800

1000

1200

4 5

Sample

Rec

ipro

cal T

iter

CDCInverness PanbioInverness Panbio AdjustedXcytonInbios

CDC P/N=92

1:10

CDC P/N=5.1

1:10

CDC P/N=4.21:1280

CDC P/N=4.2

1:80

Evaluation 2: Panbio kit with specimens collected from encephalitis surveillance in Cambodia

Background

� Cambodian JE laboratories had recently begun using the Panbio JE/DEN IgM combo ELISA kit

� Cambodian MOH needed accurate JE burden to make decisions on JE vaccination program

� Cambodian MOH requested (through WHO WIPR and PATH) confirmatory testing at CDC/DVBID

� Almost all cases had paired sera and CSF specimens for final diagnosis; very good sample set for accurate assessment of Panbio performance

Evaluation 2 cont:

Assay evaluated:

•JE-DEN IgM Combo ELISA, PanBio, Australia

Sample set: •1195 serum and CSF specimens •patients met acute encephalitis syndrome (AES/AMES) clinical case definition•Collected during AMES surveillance in Cambodia


% Sensitivity 69.8*

% Specificity 96.8

% Agreement 84.3

CDC JE + JE -/DEN + - Panbio

JE + 104 18 13

JE -/DEN + 6 82 8

Neg 39 91 755

Total 149 191 776 * JE and DEN IgM ELISA + PRNT of S2 of discordant specimens.


*CSF 78%; S1 58%; S2 72%.

Summary analysis of Panbio JE-DEN IgM Combo ELISA kit performance

Evaluation 3:

Assays evaluated:

•National Institute of Virology (NIV) India JEV IgM ELISA

Sample set: •438- acute-phase serum and CSF specimens •patients met acute encephalitis syndrome (AES/AMES) clinical case definition•Collected during AES/AMES surveillance project in India and Bangladesh


0

5

10

15

20

25

30

35

40

<5 5 to 10 >10

CDC-/NIV+

CDC+/NIV+

NIV results (sample OD/negative control OD*)

*NIV JE positive: Sample OD/NC OD ≥ 2.1

N=29 N=15 N=36

86% 40%

9%

(N= 34)Mean NIV results S OD/NC OD=4.35

(N=46)Mean NIV resultsSOD/NC OD=15.3

Specificity evaluation:Comparison of NIV JEV IgM+ results to CDC results

≥2.1

43 CDC JEV IgM-/NIV JEV IgM+; 4 CDC DENV IgM+, 5 CDC WNV IgM+ 21% (9/43).

All Samples CSF N=190 Serum N=248

% Sensitivity81 96 74

% Specificity86 96 77

% Agreement 85 96 76

CDC ResultsNIV

resultsAll Samples CSF Serum

Positive Negative Positive Negative Positive Negative

JEV IgM+ 72 50 26 7 46 43JEV IgM- 17 299 1 156 16 143

Total 89 349* 27 163 62 186


*42 JEV IgM negative/DENV IgM positive

Summary analysis of NIV assay performance

All Samples CSF N=190 Serum N=248

% Sensitivity80.9 (77.5)* 96.3 74.2 (69)

% Specificity85.7 (96) 95.7 76.9 (97)

% Agreement 84.7 (92.5) 95.8 76.2 (89.9)

CDC Results

NIV results All Samples CSF Serum

Positive Negative Positive Negative Positive Negative

JEV IgM+ 72 (69)* 50 (13) 26 7 46 (43) 43 (6)JEV IgM-

17 (20) 299 (336) 1 156 16 (19) 143 (180)Total 89 349** 27 163 62 186

*Values in parentheses are results with cutoff raised from 2.1 to 5.**42 JEV IgM negative/DENV IgM positive

CDC recommendation: Raise cuf-off to improve specificity Summary of kit evaluations

�JEV IgM ELISA kits performance with paired specimens good, but sensitivity decreased in settings with collection of single acute specimen

�Sensitivity low with very acute specimens collected <7 days post-onset of illness

�Low kit sensitivity with specimens with low CDC P/N (<10)

�Kit IgM detection in CSF near threshold of detection at 1:10 dilution

� Cut-offs may need to be modified to increase sensitivity or specificity

� Willingness or ability of manufacturers to modify and improve kits varies

� Unexpected modifications to kits by manufacturers may negativelyimpact kit performance, requiring re-evaluation

� Possible lot-to-lot variation, degradation in transport, storage requirements impact performance

� Insufficient kit production

ChallengesWay forward

� Essential that assays have undergone evaluation

� Acceptable levels of sensitivity and specificity need to be determined

� Laboratories need to be aware of kit sensitivity and specificitylimitations when making diagnosis

� Laboratories should use in-house control to monitor kit performance

� WHO JE serological reference panel (200 sera, 75 CSF) developed by CDC for evaluating and validating assays.� Preliminary panel tested by reference laboratories and results harmonized:

CDC, NIID, NIMHANS, USAMRID, UNIMAS� NIHE and IP, Vietnam and China CDC (Beixa) will bring reference panels to

labs to evaluate their in-house kits

Thank you!!

1

General Introduction to IgM General Introduction to IgM Assays Assays

WHO Vaccine Preventable Disease Lab Network

David Featherstone, EPI/IVB David Featherstone, EPI/IVB WHO GenevaWHO Geneva

The 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry Hands----on Training Workshop on on Training Workshop on on Training Workshop on on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the

Western Pacific RegionWestern Pacific RegionWestern Pacific RegionWestern Pacific Region

Public Health Laboratory Centre, Hong Kong (China)Public Health Laboratory Centre, Hong Kong (China)Public Health Laboratory Centre, Hong Kong (China)Public Health Laboratory Centre, Hong Kong (China)

15 to 19 November 2010 15 to 19 November 2010 15 to 19 November 2010 15 to 19 November 2010


Plate coated with capture antibody(anti human-IgM)

Plate coated with capture antibody(anti human-IgM)

Anti-human IgM

Plate surface


Unused binding sites blocked with proteinUnused binding sites blocked with protein

Blocking proteins

Plate surface


Mixing of antigen and conjugateMixing of antigen and conjugate

Antigen "Monoclonal Tracer" conjugate


Add serum sample Add serum sample

IgM

Wash away unbound IgG and add JE Antigen with HRP enzyme conjugate Wash away unbound IgG and add JE Antigen with HRP enzyme conjugate

JE IgM

JE antigenJE antigenAnti JE IgG Anti JE IgG conjugated to HRPconjugated to HRP

JE specific IgMJE specific IgM Non JE IgMNon JE IgM

2

Wash away unbound IgG and add JE Antigen with HRP enzyme conjugate Wash away unbound IgG and add JE Antigen with HRP enzyme conjugate

JE IgM


JE specific IgMJE specific IgM Non JE IgMNon JE IgM WHO Vaccine Preventable Disease Lab Network

Wash away unbound HRP enzyme and add substrate

Wash away unbound HRP enzyme and add substrate


Add acid to stop reactionAdd acid to stop reaction

Positive

Problems with flavivirus cross reactivityProblems with flavivirus cross reactivity


JE specific IgMJE specific IgM Dengue IgMDengue IgM

JE and Dengue IgM

Problems with flavivirus cross reactivityProblems with flavivirus cross reactivity


JE specific IgMJE specific IgM Dengue IgMDengue IgM

Dengue IgM only

Possible false positive

1


Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Progress, Challenges and Plans Progress, Challenges and Plans Progress, Challenges and Plans Progress, Challenges and Plans

Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Laboratory Based JE/AES Surveillance Progress, Challenges and Plans Progress, Challenges and Plans Progress, Challenges and Plans Progress, Challenges and Plans


David Featherstone David Featherstone

EPI EPI -- IVB WHO GenevaIVB WHO Geneva

The 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry Hands----on Training Workshop on on Training Workshop on on Training Workshop on on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Laboratory Diagnosis of Japanese Encephalitis in the Laboratory Diagnosis of Japanese Encephalitis in the Laboratory Diagnosis of Japanese Encephalitis in

the Western Pacific Regionthe Western Pacific Regionthe Western Pacific Regionthe Western Pacific Region



�


Outline of Presentation Outline of Presentation Outline of Presentation

� JE Disease

� Strategy for surveillance

� Role of the laboratory

� Progress with development of JE LabNet

� Challenges in maintaining the momentum

� Prospects for maintaining Sustainability

Clinical spectrum of JE virus infectionsClinical spectrum of JE virus infectionsClinical spectrum of JE virus infectionsClinical spectrum of JE virus infections

NeuroinvasiveNeuroinvasiveNeuroinvasiveNeuroinvasive****

Asymptomatic Asymptomatic Asymptomatic Asymptomatic infectioninfectioninfectioninfection

or or or or nonspecific nonspecific nonspecific nonspecific febrile illnessfebrile illnessfebrile illnessfebrile illness

<1%<1%<1%<1%

99%99%99%99%

****Acute encephalitisAcute encephalitisAcute encephalitisAcute encephalitis, aseptic meningitis, acute flaccid paralysis, aseptic meningitis, acute flaccid paralysis, aseptic meningitis, acute flaccid paralysis, aseptic meningitis, acute flaccid paralysis WHO Vaccine Preventable Disease Lab Network

Strategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JEStrategy for Surveillance for JE

Epidemiology and public health burden of JE are not well understood in many JE affected countries

� Primary goal to characterize the epidemiology and burden of JE

– to advocate for and guide programmatic interventions

� Where JE immunization has been introduced:

– Identify high-risk populations

– Vaccination coverage estimation

– New disease transmission,

– Document impact of control measures.


First steps in establishing JE First steps in establishing JE First steps in establishing JE First steps in establishing JE First steps in establishing JE First steps in establishing JE First steps in establishing JE First steps in establishing JE surveillancesurveillancesurveillancesurveillancesurveillancesurveillancesurveillancesurveillance

• "JE Core Working Group" established for developing surveillance standards for JE in 2002

– WHO, (HQ, SEAR & WPR), PATH, CDC, University of Liverpool

• Field Test version published 2006 and finalized 2008

• Clinical case definition (AES)Clinical case definition (AES)Clinical case definition (AES)Clinical case definition (AES)

• Laboratory criteria for confirmationLaboratory criteria for confirmationLaboratory criteria for confirmationLaboratory criteria for confirmation

• Recommended types of surveillanceRecommended types of surveillanceRecommended types of surveillanceRecommended types of surveillance

• Recommended minimum data elements Recommended minimum data elements Recommended minimum data elements Recommended minimum data elements

• Performance indicators of surveillance quality Performance indicators of surveillance quality Performance indicators of surveillance quality Performance indicators of surveillance quality

• Principal uses of data for decision makingPrincipal uses of data for decision makingPrincipal uses of data for decision makingPrincipal uses of data for decision making

CSF

Serum


Japanese Japanese

Encephalitis VirusEncephalitis VirusPolio, Polio, EnteroEntero 71 71

Coxsackie ACoxsackie A

Measles & Measles & Mumps Mumps

WNV*WNV*Dengue*Dengue*

NipahNipah

HSV, VZVHSV, VZV

OthersOthersAcute Acute

Encephalitis Encephalitis SyndromeSyndrome

Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory Requirement for Laboratory confirmationconfirmationconfirmationconfirmationconfirmationconfirmationconfirmationconfirmation

* Flaviviruses with antigenic cross reactivity with JE* Flaviviruses with antigenic cross reactivity with JE

2


Key Players in Establishing Key Players in Establishing Key Players in Establishing Key Players in Establishing Key Players in Establishing Key Players in Establishing Key Players in Establishing Key Players in Establishing JE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory SurveillanceJE Laboratory Surveillance

WHO/HQ

AFRIMS BangkokUniversity of

Liverpool

WPRO

SEARO

CDCFort Collins

PATHBMG

NIMHANSBangalore

NICD

CDCAtlantaGDD

Ad hoc Laboratory

Working Group

$$$

$

$ $$$

$$$

$

$ $$$

$$

$

$


Development of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNetDevelopment of the JE LabNet

• LabNet Structure– Tiered labs

• Standardization – Type of Assay (IgM ELISA)

– Procedures and protocols

– Reporting

– Laboratory Manual

– Training workshops

• Quality assurance– Assay assessment

– In-house controls

– External quality assurance

– Proficiency testing

– Confirmatory testing

– Accreditation

• Integration with JE programme

Gradual and stepwise process

No Big Bang !


Integration with Current WHO VPD Lab Networks

Integration with Current WHO VPD Lab Integration with Current WHO VPD Lab Networks Networks

� Polio-145 labs globally- cell culture based

� Measles/rubella – 678 labs globally

� IgM ELISA based with isolation and PCR capacity

� Yellow fever – 22 (AFR)

� IgM ELISA based� most integrated with measles & rubella

� Most labs public health based

� strong links to MoH and surveillance systems

� Tiered structure;

� Global Specialised� Regional Reference

� National

� Sub-National labs

Global Specialised

Labs

Regional Reference Labs

Serology, isolation, PCR

National LabsSerology capacity, some with virus isolation

&/or PCR+

Sub-National LabsSerology

samples

data

samples

Training

QC

Training

QC

data


Building capacityBuilding capacityBuilding capacityBuilding capacityTraining workshops

• 2 x SEAR 6 countries (JE IgM)2 x SEAR 6 countries (JE IgM)2 x SEAR 6 countries (JE IgM)2 x SEAR 6 countries (JE IgM)

• 1 x SEAR 11 participants (Bacto)1 x SEAR 11 participants (Bacto)1 x SEAR 11 participants (Bacto)1 x SEAR 11 participants (Bacto)

• 1x WPR (JE IgM) 11 participants 1x WPR (JE IgM) 11 participants 1x WPR (JE IgM) 11 participants 1x WPR (JE IgM) 11 participants from 6 labs (Seoul June 2009)from 6 labs (Seoul June 2009)from 6 labs (Seoul June 2009)from 6 labs (Seoul June 2009)

• 1x WPR (JE IgM) 14 participants 1x WPR (JE IgM) 14 participants 1x WPR (JE IgM) 14 participants 1x WPR (JE IgM) 14 participants from 9 labs (HK Nov 2010)from 9 labs (HK Nov 2010)from 9 labs (HK Nov 2010)from 9 labs (HK Nov 2010)

Regional Lab staff to CDC x 6


Challenges in maintaining the Challenges in maintaining the momentummomentum

• LabNet capacity developed

• Labs identified

• Training workshops and meetings held

• Assays assessed

• Surveillance established

• Reporting structure

• Largest cost is establishment phase

• QA programme developed


Where the resources for LabNet goWhere the resources for LabNet go……Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing Approximate Proportion of Costs for Establishing

and Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNetand Running a VPD LabNet

StandardsStandards

Assessing assaysAssessing assays

Assessment Site VisitsAssessment Site Visits

Equipping Labs Equipping Labs

TrainingTraining

MeetingsMeetings

Proficiency Test Proficiency Test ProgProg..

Confirmatory TestingConfirmatory Testing

Accreditation Visits Accreditation Visits

Establishment

Training Meetings

Kits

Consumables Shipping QAP

Need for continual SupportNeed for continual Support

3


Major PartnersMajor PartnersLaboratory Support

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

JE Funds 2000-08

YesYes Yes LabNet SupportRO Korea

?Viet Nam ADB

?NepalEnv HealthUSAID

?ChinaJE ControlJICA

VN, DPRK, INOKOICA

Liverpool School of MedWellcome Trust

?YesNepal AFRIMS

YesYesFt Collins

NoResidualChina, India, Bang.GDDCDC

YesYesYesHQ

YesYesYesWPR

YesYesYesSEAR

WHO

NoResidualYesPATH JE Project

PATH CVPBM Gates

>20102009-102006-08ProjectPartner


Projected funding needs 2009Projected funding needs 2009--20152015

• Assessment of the funding needs for all 25 countries in the JE-endemic zone to achieve "JE control" by 2015– Control target: "fewer than 0.5 confirmed JE cases per 100,000 children under the age of 15 years"

• Assumptions – These funding estimates are based on introduction of the SA 14-14-2 JE vaccine only

Japanese Encephalitis Morbidity, Mortality, and Disability Reduction and Control by 2015 PATH Document: 2009


GAVIGAVI--eligible countries eligible countries Projected Resource requirementsProjected Resource requirements



NonNon--GAVIGAVI--eligible countries eligible countries Projected Resource requirementsProjected Resource requirements


GAVI Alliance Board meeting 29 & 30 October 2008

Vaccine Costs Portfolio BGAVI Alliance Board meeting

29 & 30 October 2008

17GAVI slide

Decade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDecade of VaccinesDAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010DAVOS 29 January 2010

Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Bill and Melinda Gates Pledge $10 Billion in Call for Decade of VaccinesVaccinesVaccinesVaccinesVaccinesVaccinesVaccinesVaccines

–– Research,Research, developdevelop and and deliver vaccinesdeliver vaccines for the worldfor the world’’s poorest countriess poorest countries

–– Increase vaccination and save more than 8 million children by 20Increase vaccination and save more than 8 million children by 202020

–– Call for other to help fill critical financing gaps in both reseCall for other to help fill critical financing gaps in both research funding arch funding and childhood immunization programsand childhood immunization programs

–– Commit Commit $10 Billion over 10 years$10 Billion over 10 years,, while realizing the investment is not while realizing the investment is not sufficientsufficient

–– Confirm that the new funding is in addition to the $4.5 billion Confirm that the new funding is in addition to the $4.5 billion already already committed by BMGF to vaccine research, development and delivery committed by BMGF to vaccine research, development and delivery to to date across its entire disease portfolio since its inceptiondate across its entire disease portfolio since its inception

–– Request increased investments in vaccines by governments and theRequest increased investments in vaccines by governments and theprivate sectorprivate sector

4

By 2015 or earlier

Sustain coverage. The vaccination coverage goal reached in 2010 will have been sustained.

Reduce morbidity and mortality. Global childhood morbidity and mortality due to vaccine-preventable diseases will have been reduced by at least two thirds compared to 2000 levels.

Ensure access to vaccines of assured quality. Every person eligible for immunization included in national programmes will have been offered vaccination with vaccines of assured quality according to established national schedules.

Introduce new vaccines. Immunization with newly introduced vaccines will have been offered to the entire eligible population within five years of the introduction of these new vaccines in national programmes.

Global Immunization Vision and StrategyGoals 2015

Global Immunization Vision and StrategyGoals 2015

GIVSGIVSGIVSGIVS

By 2015 or earlier

Ensure capacity for surveillance and monitoring. All countries will have developed the capacity at all levels to conduct case-based surveillance of vaccine-preventable diseases, supported by laboratory confirmation where necessary, in order to measure vaccine coverage accurately and use these data appropriately.

Strengthen systems. All national immunization plans will have been formulated as an integral component of sector-wide plans for human resources, financing and logistics.

Assure sustainability. All national immunization plans will have been formulated, costed and implemented so as to ensure that human resources, funding and supplies are adequate.

Global Immunization Vision and StrategyGoals

Global Immunization Vision and StrategyGoals

GIVSGIVSGIVSGIVS


Challenges Challenges Challenges

� JE LabNet newly established – Functioning reasonably well– Progress is fragile

� Resources – LabNet establishment phase mostly over– Finding support for maintaining the LabNet is critical– Where possible countries should take responsibility for supporting JE surveillance integrated with other surveillance programmes

– Possibility of support through Decade of Vaccines and GIVS...but many others looking for support too

� Development of appropriate diagnostic tools – The perfect assay yet to be found! – Assay assessment and QAP important new facet

� Ensure labs' workload manageable– Surveillance aim; not to detect and sample every case – Outbreaks vs sporadic


AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

Lab Working Group

Nalini Ramamurty

Ravi Vasanthapuram

Barbara Johnson

Jaimie Robinson

Susan Hills

Tom Solomon

Asheena Khalakdina

Penny Lewthwaite

Youngmee Jee

Tomohiko Takasaki

David Featherstone

Thank You! Thank You!

1

Quality Assurance and Proficiency in the JE Laboratory Network

Quality Assurance and Proficiency in Quality Assurance and Proficiency in the JE Laboratory Networkthe JE Laboratory Network


The 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry HandsThe 2nd Intercountry Hands----on Training Workshop on on Training Workshop on on Training Workshop on on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the the Laboratory Diagnosis of Japanese Encephalitis in the

Western Pacific RegionWestern Pacific RegionWestern Pacific RegionWestern Pacific Region



Is quality assurance necessary ? Is quality assurance necessary ?


Do we need quality assurance in the lab?Do we need quality assurance in the lab?

� Lab expected to provide critical information to the disease programme

– Confirming suspected cases as positive or negative– Providing differential diagnosis– May help with adverse events following immunization

� Lab results can lead to decisions being made which may cost $ millions and may have huge impact on disease process

� One incorrect or untimely lab result may be remembered longer than 1,000 accurate and timely results!

� Critical that results from lab are accurate and timely


Limitations to accurate resultsLimitations to accurate results

� Sensitivity of assay– Number of false negatives detected

� Specificity of assay– Number of false positives detected

� Very few assays can be 100% sensitive and 100% specific

� Added complication of flavivirus antigen cross reactivity, slow IgM response and subclinical disease


Sensitivity and Specificity of AssaysSensitivity and Specificity of Assays

� Regular assessment of available assays using well validated panels of sera by independent assessor

� Assays need regular validation in the lab – Meet all QA indicators – In-house controls used regularly

– Proficiency test assessment

� Essential that kit is stored and used according to manufacturer's guidelines


JE – PanBio AssayJE – PanBio Assay

Some limitations

� Serum heat inactivation not recommended

� "It is advised that icteric or lipaemic sera, or sera exhibiting haemolysis or microbial growth not be used"

� Developed and assessed for serum (plasma not evaluated)

� CSF recently evaluated and OK with revised cut off

2


All AssaysAll Assays

Some limitations

� If samples frozen-thawed, ensure well mixed before testing

� IgM sensitive to freeze-thawing– Minimize number of freeze-thaw cycles

– If sterile, serum stable at +4°C for several weeks


Quality assurance requirementsQuality assurance requirements

� All procedures will have some variable components which may impact on the final result, e.g.

– Volume– Temperature– Time– Personnel– Others

� Need for standardisation and documentation of the variables in any procedure

� Need to minimize the chances of variation– Calibrating the calibration devices– Regular maintenance of equipment


Maintenance of equipmentMaintenance of equipment

� ELISA reader – Correct filters being used– Light path is clear

– Machine is kept clean– Power stabiliser used


Absorbance of TMB at different wavelengths

Using appropriate filter in ELISA reader is

critical


Maintenance of equipmentMaintenance of equipment

� ELISA washer – Wash buffer contains NaCl which can crystallize and block

wash-lines if not rinsed after use with distilled water– Check all channels working

– Appropriate number of washes and dwell time– Appropriate wash buffer used

– Electricity stabiliser for current fluctuations


Quality assurance - MicropipettesQuality assurance - Micropipettes

� Micropipettes– Precision and accuracy need to monitored

– Appropriate size used for appropriate volumes– Ensuring the tips are "pre-wetted"

– Tips appropriate for the purpose and micropipette– Calibrated using gravimetric methods at regular intervals

• Check both the pipette and the user!

3


Major causes of inaccuracy or poor precision

�O-ring damaged

�Tip holder worn

�Reagents drawn into pipette barrel

�Both easily and cheaply replaced


Quality assurance - MicropipettesQuality assurance - Micropipettes

� Reason for calibration

Accurate but not Accurate but not preciseprecise

Precise but not Precise but not accurate accurate

Accurate and Accurate and preciseprecise


Gravimetric determination of Micropipettes

Gravimetric determination of Micropipettes

Gravimetric determination– 1 ml (distilled water) = 1 gram

• At (1°C and 1013 hPa barometric pressure)

• but 1.0029 gram at 20°C and 1013 hPa

– Or 10 µl = 10 mg etc (or 10.029 mg at 20°C)– Need 3- 4 decimal place balance

– For volumes below 20 µl may have to consider humidity to account for evaporation problems

– Test at least 10 replicas of each volume tested to find accuracy and precision


Quality assurance - TemperatureQuality assurance - Temperature

� Incubators

� Refrigerators and freezers– Consider minimum/maximum recording thermometers

� Thermometers – Should be calibrated against a reference thermometer

� Room temperature ??– Panbio 20-25°C


Temperature Chart - In UseTemperature Chart - In UsePolio Laboratory Network

Daily 37 °C Incubator Temperature Log

Date

+

37°C

-

Initials

Asset Number 3456 Month June Year 2006

Acceptable Range 37 °C ± 1°C

1 2 3 4 5 6 7 8 9 1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

1 2 3 4 5 6 7 8 9 1

0

1

1

1

2

1

3

1

4

1

5

1

6

1

7

1

8

1

9

2

0

2

1

2

2

2

3

2

4

2

5

2

6

2

7

2

8

2

9

3

0

3

1

D

F

D

F

D

F

D

F

A

M

A

M

D

F

B

C

A

M

D

F

A

M

D

F

A

M

D

F

D

F

D

F

D

F

D

F

D

F

D

F

A

M

A

M

A

M


Quality assurance – Reagents and Kits Quality assurance – Reagents and Kits

� Ensure expiry date of reagents is recorded and respected

� Consider inappropriate handling during shipment

� Ensure to expedite collection of reagents/ kits after arrival atairport

� Plotting OD of Positive controls and calibrators– In-house and kit controls– Useful for kit to kit, day to day, person to person comparability

� Proficiency test (PT)– Annually

4


Quality assurance - ProceduresQuality assurance - Procedures

Procedures

� Follow manufacturers recommendations in Product Insert – And check for updates…

� Develop SOPs for all processes

� Document all processes– Date of test, kit details, expiry, operator, supervisor, OD readings

recorded – Comprehensive worksheet– OD output reading should be added to worksheet– Calculate standard deviation of calibrators

� Wash cycles are critical – PanBio requires 6 x


Lab ManagementLab ManagementLab ManagementLab ManagementLab ManagementLab ManagementLab ManagementLab Management

� Monitoring kit usage and other consumables

� Monitoring staff performance

� Ensuring timeliness indicators are met

� Maintaining staff safety and motivation


Quality AssuranceQuality Assurance

� Proficiency tests– Critical monitor of quality of laboratory and personnel– But a snapshot indicator of testing quality

� Referral of samples to Reference lab– Regular confirmatory testing of routine samples – Long term indicator of a lab's capability

� Performance indicators– Use of validated assay – Timeliness, accuracy and quality of testing and reporting

� Accreditation– Comprehensive annual assessment of all performance indicators– Onsite review by WHO Lab Coordinator or nominated specialist virologist


Assuring performanceAssuring performanceAssuring performanceAssuring performanceAssuring performanceAssuring performanceAssuring performanceAssuring performance

� LabNet will monitor quality of kits both in-house

and commercial through validation panels

� Labs will monitor quality of kits and their own

performance through:

– Proficiency testing: annual panel of samples

– Confirmatory testing programme: continual review of

performance indicators


Trouble shooting Trouble shooting

Check lab manual for comprehensive guide

� High absorbances– Incubation time too long – or temperature too high

� Low absorbances– Incubation times too short– Incubation temperature too low

� Unusual colour change– All yellow: Contaminated substrate, poor washing– No colour change: conjugate not added or added too dilute– Edge effect- evaporation during incubation

� Unexpected/unusual results. – All samples positive during period of low incidence– Check compatibility of results between paired sampl es, Serum 1 & 2, &/or CSF


Accreditation ProcessAccreditation Process

� Annual assessment covering 6 quality indicators

– Test results reported within 14 days of receipt (>80%)

– Minimum workload (>50 ELISA assays per year))

– Samples referred to the Reference Laboratory within 180

days: (≥ 80%)

– Confirmatory concordance results with RRL (≥ 80%)

– The score on the most recent WHO JEV IgM proficiency

test is ≥ 80%:

– On-site review of laboratory operating procedures and

practices (≥ 80%)

5


Validation of results by RRLValidation of results by RRL

� Part of the QA process and an Accreditation indicator

– To strengthen the quality of LabNet

– National Labs expected to have accuracy of 90% for samples confirmed by RRL

– Representative 10% of samples tested with a minimum of 10 per quarter to be sent to RRL

� Essential that OD results are provided with samples

– ODs of antigen and control antigen

– To help to identify possible causes of discrepant results ASAP


Summary Summary Summary Summary Summary Summary Summary Summary

� Lab plays a critical role in JE surveillance

� Essential that labs perform to a high level of

quality and meet timeliness needs of programme

� QA programme is not to criticise labs but to

identify where support can be provided to

strengthen lab processes

� Regular communication is critical:

– Share experiences, both good and not so good!

– Problems should be communicated to Dr Jee ASAP

Thank You !

Specimen collection, preparation

& shipment for virus isolation

and serology

Our policyOur policy

Contact with the patientContact with the patient’’s doctor !s doctor !

Tomohiko Takasaki MD, National Institute of Infectious Diseases, JAPAN

17th, Nov. 2010 in Public Health Lab. Centre, Hong Kong

Typical Specimens

• Blood / serum (serology, virus detection)

• CSF (serology, virus detection)

• Stool (virus isolation)

• Swabs: nose, throat, rectal (virus isolation)

• Urine (virus detection)

• Dried blood spot (serology)

• Virus cultures (characterisation)

• Cell lines (cell culture)

• Proficiency panels

Collection of CSF

• CSF on the onset of encephalitis is best.

• If it takes within one day for

transportation, sample is not necessary

to freeze at -80℃ for viral isolation. It

should be sent under 4℃.

For serological test & virological test

• If a sample is devided for

serological test & virological test.

One sample for serology can be

inactivated.

• However CSF samples are

important, we freeze all of them.

Active approach

• Samples are sometimes stored in a laboratory of hospital.

• Japanese neurologist often stored CSFs of AME patients in -80℃．

• Contact the private laboratory testing services Inc. which Dr. sent specimens.

A JE case in Yamaguchi prefecture,2010 A JE case in Yamaguchi prefecture,2010

JE10-10 IgM P/NIgM P/NIgM P/NIgM P/N Collection date

/1:serum 3.173.173.173.17 9/6/1:csf NTNTNTNT 9/6

/2:serum 9.129.129.129.12 9/7/3:serum 11.411.411.411.4 9/8/4:serum 14.814.814.814.8 9/9

/4:csf 18.218.218.218.2 9/9/5:serum 15.215.215.215.2 9/13/6:serum 16.716.716.716.7 9/21/7:serum 18.218.218.218.2 10/21

Pathogen Risk GroupsPathogen Risk Groups

1. No or low individual & community risk

2. Moderate individual risk, low community risk

3. High individual risk, low community risk

4. High individual & community risk

WHO Laboratory Biosafety Manual ,3rd ed 2004

Risk Groups and Categories of Infectious

Substances

• Classification of microorganisms into risk

groups is based on the risk in the laboratory

environment.

• Infectious substances are classified into two

categories based on scientific assessment of

risk during transportation.

Exempt SpecimensExempt Specimens

•• Minimal likelihood that pathogens are Minimal likelihood that pathogens are

present (ex. present (ex. Convalescent serum, CSF)Convalescent serum, CSF)

•• Inactivated or neutralised pathogensInactivated or neutralised pathogens

•• Dried blood spotsDried blood spots

•• Environmental samples considered to not Environmental samples considered to not

pose a significant risk eg, food or soilpose a significant risk eg, food or soil

Triple Packaging for Clinical Specimen

••Sufficient AbsorbentSufficient Absorbent

••Tough plastic bagTough plastic bag

••Biohazard markBiohazard mark

Shipment Responsibilities: Sender _ International

• Contact consignee to confirm that they can accept the shipment

- avoid public holidays, weekends

• Book shipment through a courier company or airline

• Appropriate packaging and paperwork• Notify consignee of shipment details

- airway bill number (AWB), flight details

International PackagingPackaging Labels

BIOLOGIC

AL SUBSTANCE

CATEGORYB

UN3373 Category BPI 650

UN2814 Category API 602

Dry Ice

Shipment for a lot of samples &

Long distant transportation

The reminder of Isolation by C6/36

• Human sera, Pig sera （mammalian sera） give

toxicity to C6/36 cells.

20 uL serum

10 uL

5 uL

2.5 uL

Next day, if C6/36 is damaged, the well can not use for viral isolation !

JE vaccine in the worldJE vaccine in the world

•Inactivated vaccines

1.Mouse brain derived JE-VAX

(Beijing-1, Nakayama st.)

2.Cell-derived Vaccine

•Live attenuated vaccine

1.SA-14-14-2(Chengdue Inst. Biological

Product, China)

2.live-attenuated yellow fever-JE chimeric

vaccine (ChimeriVax-JE; Sanofi Pasteur)

The history of JEThe history of JE--VAX development in Japan VAX development in Japan

1954 The mouse brain derived JEThe mouse brain derived JEThe mouse brain derived JEThe mouse brain derived JE----VAX (Nakayama VAX (Nakayama VAX (Nakayama VAX (Nakayama strain) was produced in the market firstly.strain) was produced in the market firstly.strain) was produced in the market firstly.strain) was produced in the market firstly.

: Five : Five : Five : Five ％％％％ JEV infected mouse brain after JEV infected mouse brain after JEV infected mouse brain after JEV infected mouse brain after centrifugationcentrifugationcentrifugationcentrifugation

1957 Two % JEV infected mouse brain after centrifugation.JEV infected mouse brain after centrifugation.JEV infected mouse brain after centrifugation.JEV infected mouse brain after centrifugation.

Nitrogen content; <0.4mg/ml

1965 The mouse brain emulsion was treated with alcohol, protamine and purified by ultracentrifugation

Nitrogen content; <0.02mg/ml

1971 Nitrogen content; <0.01mg/ml

1989 The seed virus had been changed fromNakayama-NIH strain to Beijing-1 strain.

Inactivated vaccines, Vero cell culture

derived in the world

– SA-14-14-2 based: Ixiaro (Intercell, Austria)

JEV is categolized to biosafety level 3 in Europe.

– Beijing-1 based:

• BK-VJE (Biken; Japan)

• KD-287 (Kaketsuken; Japan)← This vaccine have

applied to pmdapmda to get a licence for the

production in this month.

– P3 based: in China

PHK cells ⇒change ⇒ Vero cells

19

Vero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in JapanVero cell derived JE vaccine in Japan

• Same strain of JE virus as mouse brain JE vaccine : Beijing-1

• No mouse brain components• Lyophilized: stable• No thimerosal • No critical adverse events

Vero cell was established by prof. Yasumura of Vero cell was established by prof. Yasumura of Chiba universityChiba university in 1962 in Japanin 1962 in Japan

20

Vero cell cultureVero cell cultureVero cell cultureVero cell culture：：：：CytodexCytodexCytodexCytodex↓↓↓↓

Inoculation with JEV (BeijingInoculation with JEV (BeijingInoculation with JEV (BeijingInoculation with JEV (Beijing----1)1)1)1)↓↓↓↓

Harvest culture fluidHarvest culture fluidHarvest culture fluidHarvest culture fluid↓↓↓↓

Filtration of culture fluidFiltration of culture fluidFiltration of culture fluidFiltration of culture fluid↓↓↓↓

ConcentrationConcentrationConcentrationConcentration↓↓↓↓

Formaline treatmentFormaline treatmentFormaline treatmentFormaline treatment↓↓↓↓

Protamine sulfate treatmentProtamine sulfate treatmentProtamine sulfate treatmentProtamine sulfate treatment↓↓↓↓

Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose density gradient 2x density gradient 2x density gradient 2x density gradient 2x

↓↓↓↓Collecting virion fractionCollecting virion fractionCollecting virion fractionCollecting virion fraction

↓↓↓↓FiltrationFiltrationFiltrationFiltration

↓↓↓↓Bulk vaccineBulk vaccineBulk vaccineBulk vaccine

↓↓↓↓LyophilizedLyophilizedLyophilizedLyophilized

↓↓↓↓VaccneVaccneVaccneVaccne

Production procedures

MiceMiceMiceMice↓↓↓↓

Inoculation with JEV (BeijingInoculation with JEV (BeijingInoculation with JEV (BeijingInoculation with JEV (Beijing----1)1)1)1)↓↓↓↓

Harvest infected brainsHarvest infected brainsHarvest infected brainsHarvest infected brains↓↓↓↓

Preparation of virus fluidPreparation of virus fluidPreparation of virus fluidPreparation of virus fluid↓↓↓↓

Protamine sulfate treatmentProtamine sulfate treatmentProtamine sulfate treatmentProtamine sulfate treatment↓↓↓↓

Formaline treatmentFormaline treatmentFormaline treatmentFormaline treatment↓↓↓↓

Ammonium Sulfate treatmentAmmonium Sulfate treatmentAmmonium Sulfate treatmentAmmonium Sulfate treatment↓↓↓↓

Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose Ultra centrifugation (Sucrose density gradient 2xdensity gradient 2xdensity gradient 2xdensity gradient 2x

↓↓↓↓Collecting virion fractionCollecting virion fractionCollecting virion fractionCollecting virion fraction

↓↓↓↓FiltrationFiltrationFiltrationFiltration

↓↓↓↓Bulk vaccineBulk vaccineBulk vaccineBulk vaccine

↓↓↓↓LiquidLiquidLiquidLiquid／／／／LyophilizedLyophilizedLyophilizedLyophilized

↓↓↓↓VaccineVaccineVaccineVaccine

Mouse brainMouse brainMouse brainMouse brain Vero cellsVero cellsVero cellsVero cells

21

Micro-carrier tank and Vero cells on micro-carrier

CDC/DVBD diagnostic laboratory testing methods

The 2nd Intercountry Hands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the

Western Pacific RegionPublic Health Laboratory Centre, Hong Kong


Barbara W. JohnsonCenters for Disease Control and PreventionDivision of Vector-Borne Infectious Diseases

Arboviral Diseases Diagnostic and Reference LaboratoryFort Collins, Colorado

DVBID Arboviral Diseases Branch

•Japanese encephalitis virus•West Nile virus•Saint Louis encephalitis virus•Tick-borne encephalitis viruses•Yellow fever virus•Dengue viruses (Dengue Branch San Juan, Puerto Rico)•Zika virus•Equine encephalitis viruses (EEEV, WEEV, VEEV)•Chikungunya virus•LaCrosse virus and other Bunyaviruses

WHO Collaborating Center for Reference and Research

CDC Arboviral Diseases Diagnostic Laboratory provides reference and confirmatory diagnostic

testing for suspected arboviruses

�Travelers/physicians

�US State public health laboratories

�International public health laboratories

�National and international outbreak investigations

�Identification of unknown viruses

�Serosurveys, following epidemic or after vaccination campaigns

�Other CDC divisions

Serum, CSFMosquito pools, tissues,

serum, CSF

Virus DetectionAssays

IgM ELISAMicrosphere immunoassay

IgG ELISAPlaque reduction neutralization test

Viral RNA detectionNucleotide sequencing

Virus isolationImmunofluorescence assays

Antigen detection assays

Arboviral Diseases Diagnostic Assays

SerologicalAssays

Factors that determine the CDC/DVBD arbovirus diagnostic testing algorithm:

•Geographical origin of specimen

•Clinical symptoms

•Specimen type and timing of collection

•Age of patient

•Time of patient in endemic area (resident vs traveler)

•Characteristics of the virus infection and immune response

•Volume and condition of sample

Geographical Origin of SpecimenSpecimens are tested for all possible arboviruses from geographical

region, based on clinical information and volume of sample

First priority testing method based on characteristics of the virus infection and immune response

DAYS POST ONSETDAYS POST ONSET

1 2 3 4 5 6 7 8 9 10-14 to -2 0

IgMELISAELISAP/NP/N#pfu/ml#pfu/ml

100

CNS illnessCNS illness

2

20

Serology AssaysVirus Assays

IgGNeutralizing Ab


1 2 3 4 5 6 7 8 9 10-14 to -2 0

IgMIgMELISAELISAP/NP/N#pfu/ml#pfu/ml

DEN

viremia

10000


DENV Human Viremia & Immune Response

2

20

Serology AssaysSerology AssaysVirus Assays

IgGNeutralizing Ab

IgGNeutralizing Ab


1 2 3 4 5 6 7 8 9 10-14 to -2 0

IgM


1 2 3 4 5 6 7 8 9 10-14 to -2 0


WNviremia

100


2

20


IgGNeutralizing Ab


1 2 3 4 5 6 7 8 9 10-14 to -2 0


WNviremiaWNviremia

100


2

20

Serology AssaysSerology AssaysVirus AssaysVirus Assays

IgGNeutralizing Ab

IgGNeutralizing Ab

JEV Human Viremia & Immune Response


1 2 3 4 5 6 7 8 9 10-14 to -2 0


WNviremia

100


2

20


IgGNeutralizing Ab


1 2 3 4 5 6 7 8 9 10-14 to -2 0


WNviremiaWNviremia

100


2

20

Serology AssaysSerology AssaysVirus AssaysVirus Assays

IgGNeutralizing Ab

IgGNeutralizing Ab

JEV Human Viremia & Immune Response

ELISAELISAP/NP/N#pfu/ml#pfu/ml

100


2

20


IgGNeutralizing Ab


1 2 3 4 5 6 7 8 9 10-14 to -2 0


DEN

viremia

10000


DENV Human Viremia & Immune Response

2

20

Serology AssaysSerology AssaysVirus Assays

IgGNeutralizing Ab

IgGNeutralizing Ab

Current Laboratory Testing Strategy for Arboviruses

� Human Infection• Acute antibody (IgM) in serum and/or csf

� IgM ELISA or Microsphere Immunoassay� Confirmation by plaque reduction neutralization test (PRNT)

• Seroconversion in paired specimens� IgG ELISA and/or 4-fold rise in neutralizing antibody by PRNT

• Detection of virus and/or viral RNA in serum and/or csf� Real time RT-PCR, Consensus RT-PCR + sequencing, or

virus isolation

� Environmental Surveillance• Detection of virus and/or viral RNA in mosquito vectors or

amplifying hosts� Real time RT-PCR, Consensus RT-PCR + sequencing,

or virus isolation

Principles of plaque reduction neutralization assayMix patient serum dilutions + 100 PFU of virus; incubate with cells. 100 plaques = no virus antibody present90% reduction of virus plaques (≤10 PFU/well) = virus antibody present

neutralization

Calculation of neutralizing antibody titer: Reciprocal of end-point specimen dilution that reduces the

challenge virus plaque count by 90%.

End point dilutionneutralization titer = 20

Partial neutralization

Serum dilution 1:40 1:20 1:10

1:320 1:160 1:80

No neutralization

Acute Specimen

IgM ELISA

IgG ELISA

PRNTNo Interpretation

ID Virus

No Interpretation

No Interpretation

Possible 2°Infection

ConsensusRT-PCR

Real-TimeRT-PCR

VirusIsolation

Nucleic acidsequencing

ID Virus

POS POS

RT-PCR orIFA

ID Virus

POS

ID Virus

Interpretations of test results for a single acute specimen

IgM ELISA, NT, Real-time RT-PCR/virus isolation

CSF

Real-time RT-PCR, virus isolation

Brain

Isolation requires biosafety level 3 containment

MIA/IgM ELISA, NT, Real-time RT-PCR†

SeraSt. Louis encephalitis

IgM ELISA, NT, Real-time RT-PCR, Virus isolation

Horse sera

Real-time RT-PCR,Virusisolation

Mosquitoes

IgM ELISA, NT, Real-time RT-PCR, † Virus isolation†

CSF

Real-time RT-PCR, Virus isolation

Brain

Isolation of Venezuelan equine encephalitis virus requires biosafety level 3 containment

IgM ELISA, NT, Real-time RT-PCR,† virus isolation†

SeraEastern equine encephalitis/Venezuelan equine encephalitis/Western equine encephalitis


Liver, lung, lymph nodes

IgM ELISA, NT, Real-time RT-PCR,† † virus isolation††

SeraDengue 1-4


Ticks

Real-time RT-PCR, Virus isolation, paired NT

Sera

Do not freeze samples for CTF virus isolation.


Whole blood/clotColorado tick fever


Mosquitoes

IgM ELISA, NT, Real-time RT-PCR,† Virus isolation†

CSF


Brain

Except where noted freeze specimens for virus isolation at –65oC (dry ice)

IgM ELISA, NT SeraCalifornia encephalitis/La Crosse encephalitis

COMMENTSMETHOD OF CONFIRMATION OR IDENTIFICATION**

SPECIMEN(S) TO COLLECT

AGENT OR DISEASE

IgM ELISA, NT, Real-time RT-PCR/virus isolation

CSF


Brain



SeraSt. Louis encephalitis

IgM ELISA, NT, Real-time RT-PCR, Virus isolation

Horse sera


Mosquitoes

IgM ELISA, NT, Real-time RT-PCR, † Virus isolation†

CSF


Brain

Isolation of Venezuelan equine encephalitis virus requires biosafety level 3 containment

IgM ELISA, NT, Real-time RT-PCR,† virus isolation†

SeraEastern equine encephalitis/Venezuelan equine encephalitis/Western equine encephalitis


Liver, lung, lymph nodes

IgM ELISA, NT, Real-time RT-PCR,† † virus isolation††

SeraDengue 1-4


Ticks

Real-time RT-PCR, Virus isolation, paired NT

Sera

Do not freeze samples for CTF virus isolation.


Whole blood/clotColorado tick fever


Mosquitoes

IgM ELISA, NT, Real-time RT-PCR,† Virus isolation†

CSF


Brain

Except where noted freeze specimens for virus isolation at –65oC (dry ice)

IgM ELISA, NT SeraCalifornia encephalitis/La Crosse encephalitis

COMMENTSMETHOD OF CONFIRMATION OR IDENTIFICATION**


AGENT OR DISEASE

CDC/DVBID Diagnostic Testing Algorithm for Medically Important Arthropod-Borne Viral Diseases in North America *

Real-time RT-PCR, Virus isolationMosquitoes

Real-time RT-PCR , Virus isolation, histopathology

Liver

Isolation requires biosafety level 3 containment with HEPA filtered exhaust air flow;Yellow fever immunization required

IgM ELISA, NT, Real-time RT-PCR,†Virus isolation†

SeraYellow fever§

Real-time RT-PCR, Virus isolation,Dipstick, RAMP

Mosquitoes


CSF

Real-time RT-PCR, Virus isolation Brain, brain stem, spinal cord



SeraWest Nile virus

COMMENTSMETHOD OF CONFIRMATION OR IDENTIFICATION


AGENT OR DISEASE

Real-time RT-PCR, Virus isolationMosquitoes

Real-time RT-PCR , Virus isolation, histopathology

Liver

Isolation requires biosafety level 3 containment with HEPA filtered exhaust air flow;Yellow fever immunization required


SeraYellow fever§

Real-time RT-PCR, Virus isolation,Dipstick, RAMP

Mosquitoes


CSF

Real-time RT-PCR, Virus isolation Brain, brain stem, spinal cord



SeraWest Nile virus

COMMENTSMETHOD OF CONFIRMATION OR IDENTIFICATION


AGENT OR DISEASE

*See Appendix 22-3 for definitions of acronyms.**Listed in order of priority.†If specimen is acute and volume allows for both serology and molecular testing.‡ In acute specimens up to 7 days post-onset of fever.§Imported cases only; international travel history to yellow fever endemic areas.

Example:CDC Testing algorithm for WN or JE Virus

Specimen 1st Choice Other Comments

Human serum and CSF

Serology: WN (JE) and SLE (DEN) ELISA + PRNT

WN-specific qRT-PCR,flavirus RT-PCR + sequencing,virus isolation

WN qRT-PCR sensitivity: 57% acute CSF, <10% serum

Human tissue

WN-specific qRT-PCR

Virus isolation,IHC

Fatal WN cases: WN qRT-PCR sensitivity~ 100%

Non-Human

1st Choice 2nd Choice

Avian tissue

WN-specific qRT-PCR, Virus isolation

VecTest/ antigen capture ELISA, flavivirus RT-PCR

Ag.-based tests require1000 PFU

Mosquito pool

WN qRT-PCR, flavivirus RT-PCR,virus isolation

VecTest/Ag. Cap. ELISA/RT-PCR

P/N: O.D. patient serum on P/N: O.D. patient serum on viral antigen/O.D. viral antigen/O.D. negative control serum on negative control serum on viral antigenviral antigen

�� P/N > 3 = positiveP/N > 3 = positive

�� P/N < 2 = negativeP/N < 2 = negative

�� P/N 2P/N 2--3 = equivocal3 = equivocal

Ref = pos control serumRef = pos control serum

N = normal control serumN = normal control serum

• OD for the test specimen must be ≥ twice the mean OD of the test specimen reacted on normal antigen. If this requirement is not met, non-specific background is being generated, and the result MUST be reported as uninterpretable.

CDC IgM ELISA differential diagnosticsWN (JE) antigen

SLE (DEN) antigen

��ELISA Assay must be standardized in ELISA Assay must be standardized in each labeach lab

S1S1

S2

S3

S4

S5

S6

S7 Ref

N

ViralAntigen

NormalAntigen

S8

S1S1

S2

S3

S4

S5

S6

S7 Ref

NS8

JE DEN1:40 1:20 1:10 1:40 1:20 1:10

1:320 1:160 1:80 1:320 1:160 1:80

Plaque reduction neutralization test

InterpretationPRNT titer ≥ 10 = presence of neutralizing antibody

PRNT titer ≥ 4-fold over heterologous flavivrius = virus-specific neutralizing antibody

PRNT titer 4-fold difference between acute and convalescent specimen= evidence of acute infection

and Prevention

Flavivirus Cross-reactivities of IgM from WN Patient Serum*

P/N > 3 = positiveP/N < 2 = negativeP/N 2-3 = equivocal

Serum SLE JE WN DEN2 YF POW

1 4.96 7.75 16.74 2.45 1.82 1.56

2 4.8 13.77 16.68 4.13 2.14 1.75

3 5.45 9.67 16.08 4.09 1.61 1.44

4 4.76 10.07

17.19 3.32 1.62 1.3

Positive Control 6.5 8.2 6.34 7.45 3.96 4.5

* 1:400 screening dilutionCenters for Disease Control

P/N P/N P/N P/N P/N P/N

Complete Serological Analysis: Differential diagnosis

Interpretation of ELISA Results Interpretation of PRNT results

P/N: O.D. patient serum/O.D. negative control serum reacted on viral antigen


PRNT titer > 10 = positive antibody

PRNT titer =4-fold over heterologousflavivirus = positive for specific antibody

orPRNT titer = 4-fold between acute and convalescent = evidence of acute infection

PatientDays P.I.

IgM(JE)

IgM(WN)

JE WN DEN2 SLE

CSF 8 26.91 1.78 nd nd nd nd

S1 9 9.1 4.16 160 20 <10 10

S2 34 6.7 4.62 1280 20 <10 20

Positive Control

n.a. 9 6.5 >5120 2560 2560 320

90% neutralization titerPatient

Days P.I.

IgM P/N

(JE)

IgMP/N

(DEN)JE WN DEN2 SLE

CSF 8 26.91 1.78 nd nd nd nd

S1 9 9.1 4.16 160 20 <10 10

S2 34 6.7 4.62 1280 20 <10 20

Positive Control

n.a. 9 6.5 >5120 2560 2560 320

90% neutralization titer

Days IgM P/N IgG P/N PRNTSample post-onset JE DEN JE DEN JE DEN

Case interpretation: Primary JE Infectionacute serum 8 12.75 4.00 1.37 2.04 <1:10 <1:10conv. serum 31 11.35 4.21 6.38 5.76 1:1280 1:80Case interpretation: Secondary Flavivirus infection?acute serum 4 1.59 1.42 3.12 2.62 1:20 1:80conv. serum 15 9.01 3.96 10.00 9.90 1:640 1:320

JE Case Interpretation based on SerologyJE Case Interpretation based on Serology

Interpretation of ELISA Results Interpretation of PRNT results

P/N: O.D. patient serum/O.D. negative control serum reacted on viral antigen


PRNT titer > 10 = positive antibody

PRNT titer =4-fold over heterologousflavivirus = positive for specific antibody

orPRNT titer = 4-fold between acute and convalescent = evidence of acute infection

All sample types (%)

CSF (%)

Sera (%)

Total samples tested 1195 439 756

JEV IgM+ 348 108 240JE-DEN IgM cross-reactive 170 (49) 55 (51) 115 (48)Classification resolved by PRNT

155 (45) 54 (50) 101 (88)

Confirmed JE positve 48 24 24Confirmed DEN positive 107 30 77

IgM JEV and/or DENV equivocal

43 16 27

Confirmed JE positive 1 0 1IgM- (no neutralization titer) 17 7 10

Increasing specificity and resolving IgM equiv resultsExample: CDC Confirmatory testing of Cambodian specimens

Testing algorithm: CSF, S1, and S2 JEV and DENV ELISA , followed by S2 JEV and DENV PRNT90 if CSF, S1 or S2 IgM pos or equiv. If no S2 or S1 available, PRNT on CSF.

All Sample

Types (%)

No. CSF (%)

No. Sera (%)

Total samples tested 520 226 294

JEV IgM+ 103 34 69JEV IgM Cross-Reactive (DEN/WN)

36 (35) 10 (29) 26 (38)

Classification resolved by PRNT

32 (31) 2 (6) 21 (30)

Confirmed JE+ 16 0 15Confirmed DEN+ 7 2 5Confirmed WN+ 9 0 1

Increasing specificity by PRNT

Example: 3 kit evaluation with samples from India and Bangladesh


1 2 3 4 5 6 7 8 9 10-14 to -2 0

IgM

IgG

ELISAELISAP/NP/N#pfu/ml#pfu/ml

106

Simplified Depiction of CHIK Human Viremia & Immune ResponseSimplified Depiction of CHIK Human Viremia & Immune Response

2

20

Neutralizing Ab

CHIKviremia

Serological & RT-PCR Test Results of CHIK Infected Returning Travelers

Serological Testing Algorithmfor Chikungunya Virus Infection

single acute patient serum

IgM Capture ELISA IgG ELISA

RT-PCR (<10day)

IgM POS

PRNT

IgM NEG(<10day)

NoInterpretation

IgM NEG(>10day)

NEG

RT-PCR orIsolation POS

POSPOS

CDC/DVBD JE serological testing of a single acute specimen

Acute human serum (1:400) or CSF (1:10)*

IgM ELISA JEV & DENV(WNV sera)

POS

(P/N >3) or

EQUIV

(P/N=2 - 3)

NEG

(P/N<2)

PRNT with:

JEV + DENV(WNV sera)

Interpreted as either NEG or IgM not yet

present

Action : Report as negative, or test

convalescent specimen if available

Interpretation

Confirmed positive:IgM positive or equivocal

AndPRNT titer ≥1:4 (CSF) or 1:10 (sera)

And ≥4-fold over heterologous flavivirus

Presumptive positive:IgM positive or equivocal

AndPRNT titer ≥1:4 (CSF) or 1:10 (sera)

And<4-fold over heterologous flavivirus

OrIgM positive

AndPRNT negative

NegativeIgM negative or equivocal

AndPRNT negative

*All specimens must meet clinical acute encephalitis syndrome case definition.

Testing algorithm

Thank you!

1

JE Lab Data reporting

• Started from July 2009 using MS excel format

• MS Access format distributed in 2010

• Malaysia, two Vietnam labs (3) switched to MS Access

• Cambodia, Laos, Philippines, Korea (4) use MS excel format

Japanese Encephalitis Surveillance in CAMBODIA-2010

JENegNegPosPos28-Jan-201013-Jan-2010Kg Cham10KC 59RY RAKSMEY8

NegNegNegNegNegNeg28-Jan-201010-Jan-2010Kg Cham12KC 58PHEAP SOPHEAK7

NegNegNegNeg28-Jan-20105-Jan-2010Kg Cham6KC 57SUS MAKARA6

DENNegPosNegNegNegNeg28-Jan-20104-Jan-2010Kg Cham4mKC 56THANN VITHEY5

NegNeg28-Jan-201030-Dec-2009

Svay

Rieng20m211KUTH RACHANA4

JENegNegPosPos

28-Jan-201029-Dec-2009

Svay

Rieng5119PUTH SAMBO3

NegNeg28-Jan-201015-Jan-2010Takeo13m1401/87OU SOK MEAS2

JENegNegNegPosPosNeg28-Jan-201031-Dec-2009Takeo72912/72SAT LETH1

January

S2S1CSFS2S1CSFFMFinal Result

Result DENResult JE

Testing

dateColl.dateHospital

Age

Patient

IDNAMENº

3JE Equi

2DEN Equi

11DEN Positive

29JE Positive

59Serum 2

96Serum 1

96CSF

99Total Patients

2

JE Cases lab reported in 2010-Cambodia

00541024930000675811419299014490144Cambodia

00212500004201125555OctoberCambodia

00200200001400001414SeptemberCambodia

00207100000430061010151015AugustCambodia

00802130000109002013201320JulyCambodia

00102416000099124516271627JuneCambodia

0071311000089013211171117MayCambodia

00400400004400004747AprilCambodia

00942150000149071315261526MarchCambodia

00200300003300003333FebruaryCambodia

00824140000106032712201220JanuaryCambodia

Dengue

JECSF

Serum

CSF

Serum

CSF

Serum

CSF

Serum

CSF

Serum

CSF

Serum

CSF

Serum

Cases

referred to another lab

Cases

with pendin

g results

Cases negati

ve to both

JE and Dengu

e

Cases with positive results

Cases tested

Specimens with

results ≤ 7

days of receipt in

lab

Specimens pending

lab results

Specimens

negative to both JE

and Dengue

Specimens

Dengue positive

Specimens

JE positive

Specimens tested

Specimens received

CASE DETAILSSPECIMEN DETAILS

MonthCountry

25.8%

Total

Dece

mber

Nove

mber

00000000000000000000

Octob

er

00021300000003002133

Septe

mber

0011251830??13318011251818

Augu

st

00501621403014420050162121July

00101202010122June

0May

0April

0

Marc

h

0

Febru

ary

0

Janua

ry

Deng

ue

positive

JE

positive

samples

with results ≤

7 days of

reception

in lab

specime

ns

pending

lab results

specimen

s

negative

to both

JE and

Dengue

spec

ime

ns

Den

gue

positive

specim

ens

JE

positive

speci

mens

tested

specim

ens

received

sample

s with

results

≤ 7

days of

recepti

on in lab

speci

mens

pendi

ng

lab

results

specim

ens

negati

ve to

both

JE and

Dengue

specim

ens

Dengu

e

positiv

e

speci

mens

JE

positive

speci

mens

tested

speci

mens

received

Number

of

negativ

e cases

referred

to

another

lab (specify)

Numbe

r of

cases

with

pendin

g results

Nu

mbe

r of

case

s

neg

ativ

e to

bot

h JE

and

Dengue

№ of cases with positive results

Total

number

of cases

of cases

tested

CSFSerum


Month

Laos reporting

JE Cases lab reported in 2010-Laos

0017423447430031704223744744Lao PDR

00000000000000000000OctrLao PDR

00021303000002010303SeptLao PDR

00112518318000110215318318AugustLao PDR

00501621420003500116421421JulyLao PDR

00101202000100010202JuneLao PDR

00000000000000000000MayLao PDR

00000000000000000000AprilLao PDR

00000000000000000000MarchLao PDR

00000000000000000000FebLao PDR

00000000000000000000JanLao PDR

Dengue

JECSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

Cases

referred to

another lab

Cases with

pendin

g results

Cases

neg

ative to both JE and

Dengue

Cases with positive results

Cases

tested

Specimens with results ≤ 7 days of receipt in

lab

Specimens pending lab

results

Specimens negative to

both JE and Dengue

Specimens Dengue positive

Specimens JE positive

Specimens tested

Specimens received


MonthCountry

52.3%

011082330141290126527158158261832731143144Tot

al

0070310707031010700821010Sep

009191900808161600111122424Aug

00144322*10172322220017232323July

0032381050277104241010Jun

0027623500330235350023623131May

001530181301710181813014201616Apr

001335217015152121407341515Mar

01731120091111111112034Feb

001314180015031818006141111Jan

Den

gue

positive

JE

posit

ive

sample

s with

results

≤ 7

days of

recepti

on in lab

spe

cim

ens

pen

din

g

lab

results

speci

mens

negat

ive to

both

JE

and

Dengue

spe

cim

ens

Den

gue

posi

tive

spe

cim

ens

JE

positive

speci

mens

tested

spe

cim

ens

rec

eived

sample

s with

results

≤ 7

days

of

recepti

on in lab

spe

cim

ens

pen

din

g

lab

results

speci

men

s

nega

tive

to

both

JE

and

Dengue

spe

cim

ens

Den

gue

posi

tive

spec

ime

ns

JE

positive

speci

men

s

test

ed

speci

men

s

recei

ved

Number

of cases

referred

to

another

lab

(specify)

Num

ber

of

cases

with

pendi

ng

resul

ts

Nu

mb

er

of

cas

es

neg

ativ

e to

bot

h JE

and

Den

gue

№ of cases

with

positive

results

Tota

l

num

ber

of

case

s of

case

s

tested

CSFSerum


Mon

Research Institute for Tropical Medicine

PHILIPPINESPhilippines reporting

JE Cases lab reported in 2010-Philippines

021142732175333002134867302931170150170151Philippines

01642124401832320127127OctrPhilippine

s

0070310770070083210101010SeprPhilippine

s

009191900008110181216241624AugPhilippine

s

0014432210001717223322232223JulyPhilippine

s

0032381100540224710710JunePhilippine

s

0027623500003323062235313531MayPhilippine

s

001530181313001714120018161816AprilPhilippine

s

001335217400157135421152115MarchPhilippine

s

01731120101911210113114FebPhilippine

s

001314180000156013418111811JanPhilippine

s

Dengue

JECSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

Cases

referred to

another lab

Cases

with

pending

results

Cases

negativ

e to both JE and Dengue

Cases with positive resultsCas

es tested


lab


results


both JE and Dengue



Specimens tested

Specimens received


MonthCountry

18.3%

North Vietnam reporting

3

JE Cases lab reported in 2010-North Vietnam

001192221421021470086131221614102147103148Viet Nam, North

00102313000100121313OctViet Nam,

N

00201322001200102222Sepr

Viet Nam,

N

00711828001810102828AugViet Nam,

N

009011148003700104848July

Viet Nam,

N

002501540283500182500101028352835JuneViet Nam,

N

002112231626001424122216261626May

Viet Nam,

N

001800181719001719000017191719AprilViet Nam,

N

001200121116001115000011161116March

Viet Nam,

N

001300131318001318000013181319FebViet Nam,

N

00110011812008120000812912JanViet Nam,

N

Dengue

JECSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

Cases

referred to

another lab

Cases

with

pending

results

Cases

negativ



es tested


lab


results


both JE and Dengue



Specimens tested

Specimens received


MonthCountry

15.5%

South Vietnam reporting

JE Cases lab reported in 2010-South Vietnam

001475151671211660011615116917142195143195Viet Nam, South

00512835004501125858OctrViet Nam, S

00402634003500225757SepViet Nam, S

001203151013001112001412161216AugViet Nam, S

002932342636002532031226372637JulyViet Nam, S

001802201826001723001318261826JuneViet Nam, S

002010211627001625120017281728MayViet Nam, S

001902211822001821001319242024AprilViet Nam, S

002001211926001925001120262026MarchViet Nam, S

00801976002200108787FebViet Nam, S

00120012110011000012161216JanViet Nam, S

Dengue

JECSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

CSFSerum

Cases

referred to

another lab

Cases

with

pending

results

Cases

negativ



es tested


lab


results


both JE and Dengue



Specimens tested

Specimens received


MonthCountry

9%

Korea Reporting

Total

0

Decem

ber

Novem

ber

Octobe

r

Septe

mber

August

0000361700001722000001924July

00000110000011160000016June

00000100000010220000022May

000004000004110000011April

00000100000010170000017March

0000080000086000006

Februa

ry

05005800000850500516

Januar

y

Deng

ue

positive

JE

positive

samples

with results ≤ 7

days of

reception in

lab

speci

mens

pendi

ng

lab

results

specime

ns

negativ

e to

both JE

and Dengue

speci

mens

Deng

ue

posit

ive

speci

mens

JE

positive

speci

men

s

tested

speci

men

s

received

samples

with results

≤ 7 days of

reception in lab

speci

mens

pendi

ng

lab

results

specim

ens

negati

ve to

both

JE and

Dengue

speci

mens

Deng

ue

positi

ve

speci

mens

JE

positive

speci

men

s

tested

speci

men

s

received

Number of

cases

referred to

another lab (specify)

Numbe

r of

cases

with

pendin

g results

Num

ber

of

cases

nega

tive

to

both

JE

and

Dengue

№ of cases

with positive results

Total

numbe

r of

cases

of

cases tested

CSFSerum


Month

Problems

• Current MS Access feedforward files need to be converted by data managers in WPRO

• Laboratory data only sent to data manager in WPRO

Recommendations

• Use MS excel format (old format)

• Please send the report to

[email protected]@wpro.who.int

[email protected][email protected]

• Report by 10th every month

4

AdditionalOngoing JE/AES/MEME surveillance in

the Region• Sentinel surveillance started in selected hospitals in Cambodia and Lao

PDR(2006-7) and in PHL and PNG (2008):

– Cambodia

• NIPH: ELISA testing for JE/Dengue (Panbio)Sentinel sites: Latex agglutination test for the Bacterial agents from 2008 (Wellcogen® Bacterial Antigen Test Kits)

• Namru 2 performs real time PCR for Hib, Meningo, Str pneumo– Philippines

• Laboratory diagnosis in the RITM and sentinel hospitals (5)

• JE/Dengue Panbio and Latex aggutination /real time PCR for bacterial Ag

– Laos

• samples are tested in Mahosot hospital labs bacterial culture and JE/dengue ELISA

• NCLE recently on board-need to communicate with sentinel hospitals

– PNG

• Port Moresby General Hospital sent samples to VIDRL(Melbourne): Panbio Dengue/JE combo test kit

• CPHL now on board

Sentinel sites for AES surveillance

Mahosot Hospital, Vientiane

Luang Nam tha provincial hospital

Lao (2)

National Pediatric Hospital, Pnomh Penh

Angkor Hospital (Siam Reap)

Battambang Provincial hospital

Kampong Cham Provincial hospital

Svay Rieng

Takeo

Cambodia (6)

North (1), Central (1), South (1) ; Thai Binh, Quang Ngai

National hospitals in Hanoi, HCM City

Vietnam (3)

Bulacan provincial hospital

Iloilo provincial hospital

Cebu provincial hospital

Tarlac Hospital

Philippine general hospitalPhillipines (5)

Hubei province (six sentinel hospitals in Yichang perfecture)

Shandong Province (six sentinel in Jinan Perfecture)China (12)

WHO designated labLaboratory testing used Country

Referred to VIDRL(Panbio)

RITM : training from AFRIMS, in house anti-dengue/anti-JE ELISA

JE/Dengue Combo IgM capture ELISA – Panbio : St Luke hospital

San Lazaro hospital-AFRIMS in house ELISA: dengue and JE

Xcyton/Panbio JE IgM capture ELISA (Mahosot hospital)

JE IgM capture ELISA (Panbio), Pasteur Institute in house test (Battambang site)

JE specific IgM capture ELISA,, PCR

HI(IMR, UMMC, UNIMAS)

Viral Isolation: Suckling mice, C6/36 (IMR, NPHL, UMMC, UNIMAS)

Molecular Detection: IMR, NPHL, UMMC, UNIMAS

JEV IgM capture ELISA: Shanghai Beixi and Yueda

PCR

PRNT:

JEV isolation

Panbio ELISA, IFA/HI/CF

PRNT

Viral RNA detection – RT-PCR

Virus isolation – suckling mouse inoculation, mosquito cell culture

NIHE / PI in-house MAC ELISA, Panbio kits in pilot provinces

PRNT, HI

RT-PCR assay for CSF

Viral isolation

In-house IgM capture (MAC) ELISA for JE, IgG ELISA,

PRNT, HI

TaqMan RT-PCR

Virus Isolation

PNG

Philippines

Laos

Cambodia

Malaysia

China

Korea

VTN

Japan

CPHL

RITM-Panbio

NCLE-Panbio

NIPH-Panbio

IMR -Panbio from 2009

Institute of Viral Disease Control, China CDC -Beixi

Korea CDC -Panbio

NIHE, PI - in house

NIID -in house

Regional status of JE diagnosis

Types of JE vaccines and schedules used in WPR

Country Vaccine used Schedule

Australia MBD vaccine

2 doses 7-10 days apart 1 year and 3rd dose

one year later, boosters every 3-5 years in

areas with demonstrated transmission

China MBD vaccine 8months, 7-10days, 2 years, 6 years

Live attenuated 8 mo, 2 years

Japan MBD vaccine->Vero cell derived Y1,+1-2W, +1 Y, Y6, Y12

Malaysia MBD vaccine

2 doses 7-10 days apart 1 year and 3rd dose

one year later, boosters every 3-5 years till

15 years of age, in Sarawak

South Korea MBD vaccine/Live attenuated M12-24, +1-2W, +1Y, Y6, Y12

Vietnam MBD vaccine (locally produced) Y1, +2W, +1Y

Confirmatory testing

8 Aug

27 May

31 March

2 April

8 April

15 April

Last shipping in 2010

NIIDFebruary2011

CambodiaNIID Japan(PHLC, HK)

NIID or KCDCFebruary2011

Malaysia

NIIDDecember2010

PhilippinesCDC, Korea

NIIDJanuary2011

Vietnam South

NIIDJanuary2011

Vietnam North

NIIDDecember 2010

LaosNIID Japan

Next shippingNMLReferral lab

Please send every 4 months from 2011!

Focus reduction neutralization assay for Japanese

encephalitis with peroxidase anti-peroxidase method

Department of Virology I, National Institute of Infectious Disease

Chang-Kweng Lim, D.V.M., PhD., Tomohiko Takasaki, M.D., PhD., Ichiro Kurane, M.D., PhD.

Diagnosis of Japanese encephalitis by laboratory te st

Collect spacemen

Vector spacemenHuman spacemen

� Viral isolation and nucleic

acid detection

• viral genome detection using

RT-PCR

• virus isolation using vero cells

and suckling mouse

� serology

• IgM capture-ELISA

• Nutralizing antibody titration

• HI test

� Viral isolation and nucleic

acid detection

• viral genome detection using

RT-PCR

• virus isolation using vero

cells and suckling mouse.

� PRNT assay requires large amounts of

• sample sera,

• indicator cells,

• medium,

• and incubator space.

� The entire assay requires 7 days.

Plaque reduction neutralization test (PRNT)

� FRNT assay, using peroxidase anti-peroxidase (PAP) method, requires small amounts of • sample sera, • indicator cells,• medium, • and incubator space. � The entire assay requires as few as 3 days. � Neutralizing antibody titers obtained with FRNT assays show good agreement with those obtained by plaque reduction neutralization test (PRNT).

Peroxidase anti-peroxidase (PAP) method for focus reduction neutralization test (FRNT)

x108

72404080

x204040404080

x404040404080

x804040404080

x1604040404080

x3204040404080

V.C.-

40404080

N.C.-

8080

-80

2-fold dilutionSample serum10%FCS-MEM

sub-totalJEV (100FFU/25µl)

total

40

The methods for FRINT assay

• 37℃℃℃℃ ｘｘｘｘ 1hr for nutralizing reaction

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

x 10

x 20

x 40

x 80

x160

x320

V.C.

N.C.

a b c d e f

• 37℃℃℃℃ ｘｘｘｘ 1h for incubation• Add 100µl/well 10%FCS-MEM, and incubate at 37 ℃℃℃℃ for another 45hrs

Vero Osaka2 x 104/well/100µ

Anti Japanese encephalitis rabbit antibody

Anti rabbit goat antibody Peroxidase anti-peroxidase rabbit antibody complex

1st antibody 2nd antibody PAP complex (rabbit)

The model of peroxidase anti-peroxidase (PAP) method

Anti-JEV rabbit antibody

JEV Anti-rabbit goat antibody

PAP complex peroxidase

Vero cell

The model of peroxidase anti-peroxidase (PAP) methodThe model of PAP (peroxidase anti-peroxidase)

method

anti-JEV rabbit

antibody（（（（1st antibody ））））

anti-rabbit goat antibody

（（（（2nd antibody)

PAP complex

peroxidase

Vero cell

JEV

Focus formation by JEV

PRNT (plaque) FRNT (PAP Method)

Analysis for a result of potency test for JE Vaccine by Parallel line assay method

Using Bioassay Assist

Bioassay assist is the statistic analysis software for the quality control of biological products

Vaccine sampleReference vaccine

PRNTPRNT（plaque) vsvs FRNTFRNT(PAP)PRNTPRNT FRNTFRNT

1) Need space（6 well plate）

2) 7 days assay

1) Save space in CO2incubator (96 well plate)

2) Easy to test many samples

3) It takes 3 days for one assay

3) Plaque is largerand easy to count

4) Cheaper

4) Focus is smaller

5) Expensive (need antibodies)

Japanese Encephalitis Virus

Taq Man primers & probe

TaqMan primers & probe for JEVTaqMan primers & probe for JEV

JEV prim ers & probs S eq. 5'-3'JEen585p599s622c A C T R A A C A C T G A A G C G TJEen562s-585pset C T G G A Y T G T G A R C C A A G G AJEen623c-585pset G A H C C C A C G G T C A T G A

G enotype1& 3

Universal set (Envelope region)

JEV prim ers & probs Seq. 5'-3'M odified byAB051292

JE1&3Env1052F-1082Pset ATG G G AATTAYTC AG C G C AAG TJE1Env1082P C TC AAG C AG C AAAJE1Env 1119R-1082Pset G G G AG C G TTTG G AG TTAC AG TAA

JE&3Env1052F-1082Pset ATG G G AATTAYTC AG C G C AAG TJE3Env1082P C C C AG G C G G C AAAJE3Env 1119R-1082Pset AG G AG C ATTG G G TG TTAC TG TAAA

JE1とJE3のsence　prim erは共通

Genotype1

G enotype3

Genotyping set (Envelope region)

References

Ito M, Takasaki T, Yamada K et al: Development and evaluation fluorogenicTaqMan reverse transcriptase PCR assays for detection of dengue virustypes 1 to 4. J Clin Microbiol 2004;42(12): 5935-5937.

RNA stable tube at RT

Virological diagnosis in Virological diagnosis in JapanJapan

<Methods in NIID><Methods in NIID>1.1. TaqMan RTTaqMan RT--PCRPCR2.2. Conventional RTConventional RT --PCR ( nested PCR)PCR ( nested PCR)3.3. Virus isolation (C6/36, Vero9013 cells)Virus isolation (C6/36, Vero9013 cells)

<Methods in provincial level>1. Conventional RT1. Conventional RT --PCR ( nested PCR)PCR ( nested PCR)2. Virus isolation (2. Virus isolation ( C6/36 cellsC6/36 cells , Vero9013 cells), Vero9013 cells)

CDC PanbioResults Laboratory number/results

Sample # Type 5** 6** 7** 8** 9** 10**10

retest** 11**1 CSF DEN DEN DEN DEN DEN DEN DEN NEG2 CSF NEG NEG NEG NEG NEG NEG NEG NEG3 CSF NEG NEG NEG NEG NEG NEG NEG NEG4 CSF JE JE JE JE JE JE JE NEG

5 CSF NEG NEG NEG NEG NEG NEG JE equiv JE NEG6 serum DEN DEN DEN DEN DEN DEN DEN DEN

7 serum JE JE JE JE JE JEDEN equiv JE JE

8 serum JE JE JE JE JE JE JE JE9 serum NEG NEG NEG NEG NEG NEG NEG NEG

10 serum NEG NEG NEG NEG NEG NEG NEG NEG11 serum DEN DEN DEN DEN DEN DEN DEN DEN

Kit usedPanbio Panbio Panbio Panbio Panbio Panbio Panbio Panbio

Reporting date June 25 June 30 June 30 Jul-03 July 7 August 22 NA August 26

%Agreement with CDC Panbio results

100 100 100 100 100 91 91 82

**Compared against CDC Panbio results .

Difference between CDC and national laboratory Panbio results

Results of the 2009 WPR JE Labnet proficiency testing:Panbio JE-DEN Combo ELISA kit

CDC ELISA and PRNT results

CDC updated results(2/17/10) Laboratory Number/results

Sample # Type 1* 2* 3* 4*1 CSF JE PRESUMPTIVE JE PRESUMPTIVE JE NEG NEG NEG

2 CSF NEG NEG NEG NEG NEG NEG

3 CSF NEG JE PRESUMPTIVE

difficult to

distinguish JE JE JE

4 CSF JE PRESUMPTIVE JE PRESUMPTIVE JE JE JE JE

5 CSF JE PRESUMPTIVE JE PRESUMPTIVEJE suspected

JE JE JE

6 serum DEN POSITIVE DEN POSITIVE DEN NEG DEN NEG

7 serum JE POSITIVE JE POSITIVE JE JE JE JE

8 serum JE PRESUMPTIVE JE PRESUMPTIVE JE JE JE JE

9 serum NEG NEG NEG NEG NEG NEG

10 serum DEN PRESUMPTIVE DEN PRESUMPTIVE NEG NEG NEG NEG

11 serum DEN POSITIVE DEN POSITIVE DEN JE DEN JE

Kit used In-house

Non Panbio

Commercial In-house In-house

Reporting date July 2 July 2 July 5 July 6

%Agreement with CDC results 100 82 91 82

*Compared against CDC JEV and DENV IgM ELISA + JEV and DENV PRNT results.

Difference between national kit and CDC resultsDifference between CDC DEN and national kit negative results

Updated results: Difference between CDC results 6/5/09 and 2/17/10

Results of the 2009 WPR JE Labnet proficiency testing:In-house and non-Panbio commercial kits

1

AM CHANTHANImmunology unit

Hand on training Workshop on Laboratory Diagnosis of Japanese Encephalitis on 15-19 Nov.2010, Hong Kong

Japanese encephalitis Sentinel Site Surveillance in Cambodia

NATIONAL PUBLIC HEALTH LABORATORYOrganization Chart of NPH Laboratory

Hematology and Blood Parasite Unit

Microbiology and Parasitology Unit

Biochemistry Unit

Immunology and Molecular Unit

Quality Assurance Unit

OPD and Public Relation Unit

NPH LaboratoryNPH LaboratoryNPH LaboratoryNPH Laboratory

Immunology and Molecular performances

• Serology analysis• Flow Cytometry analysis( CD4/CD8)• HIV Drug resistance study• Measles and Rubella analysis• JE and DEN analysis• Illness like influenza surveillance (ILI)• Syndrome Acute Respiratory Infection

surveillance and Influenza outbreak response .

JE Background in Cambodia

• In Cambodia, JE has been recognized as a serious disease for

many years. However information on the extent of JE

disease burden has been limited. A small number of hospital-

based studies showed about 20%-30% encephalitis cases

among children were due to JE.

• In May 2006, the Cambodian CDC, NIPH, NIP / MOH, PATH,

and WHO started JE Surveillance Project, which is hospital-

based sentinel site surveillance for JE among children under

15 years of age with suspected meningo-encephalitis.

• 6hospitals were selected and the Panbio JE-Dengue IgM

COMBO ELISA kit was used.

Meningo-encephalitis Case Definition

A person with acute onset of fever (≥≥≥≥38 C) and one of the

following: neck stiffness, altered consciousness, other meningeal sign at all ages.

• Suspected in children <1 year of age when fever is accompanied by a bulging fontanelle.

• Does not include cases suspected to be caused by chronic infections such as tuberculosis or HIV.

• Samples collection Serum 1,Serum 2 and CSF

Structure for JE Laboratory

Project coordinator

Project assistance

Staff analysisStaff analysis Staff analysis

2

Patient

Provincial Epidemiology Surveillance

Unit

NIP Center

NPH Laboratory

WHO Lab network

STORAGE -75°C Analysis

MOH

Structure Routine Sentinel site Surveillance For Menin go-Encephalitis in Cambodia

Provincial Hospital

National Hospital

JE in CAMBODIA

JE sentinel sites in Cambodia

Based on geographical locations, capacity, and human resources,

six sites were selected at national and provincial hospitals

1. National Pediatric Hospital

2. Angkor Children Hospital

3. Takeo Provincial Hospital

4. Kampong Cham Provincial

Hospital

5. Svay Rieng Provincial

Hospital

6. Battambang Provincial

Hospital just started in 2009

Samples Analysis

• The National Institute of Public Health (NIPH) laboratory

in Phnom Penh receives samples from sentinel sites

weekly; they are analyzed within a week and feedback is

provided to sites the week after.

• ELISA method is used for detecting Japanese Encephalitis

IgM antibody by Panbio JE-Dengue IgM COMBO ELISA kit;

the kit also tests for dengue IgM, as dengue virus is a cross-

reacting flavivirus that also circulates in Cambodia.

• Paired sera (Serum-1 and Serum-2) and CSF are used.

• Remaining samples are stored in freezer at -75°°°°C in NIPH

Laboratory for future need.

Testing Algorithm For JE

CSF or Serum 1and Serum 2

JE & Dengue IgM Capture ELISA

NegPos

Samples CollectionSamples CollectionSamples CollectionSamples Collection

� From 2006 to Oct-2010 : 3002 samples (from 1155 patients) were received from 6 hospital sites (NPH, Kg Cham, AHC, SVR, TK and BB)

� The hospitals send the samples to NIPH Laboratory as soon as possible after collecting.

� Cool boxes are used for transporting Samples from all sites

3

WORKLOAD JE AND DEN FROM 2006TO Oct-2010

1155

167

211

TOTAL CASE

JE positive

DEN positive

Amount sample by site2006-Oct-2010

342

264

234

162127

1155

BB, 26

ACH

NPH

KCM

Takeo

SVR

BB

Tot al

Samples received from 6 JE sentinel sites

From 2006 to Oct 2010

0

500

1000

1500

2000

2500

3000

3500

Year 2006 2007 2008 2009 2010

Serum 1 179 275 376 201 96 1127

Serum 2 110 180 291 131 59 771

CSF 176 267 364 201 96 1104

TOTAL 465 722 1031 533 251 3002

1 2 3 4 5 6

JE and DEN Result Positive form 2006 to 2009 JE and DEN Result Positive form 2006 to 2009 JE and DEN Result Positive form 2006 to 2009 JE and DEN Result Positive form 2006 to 2009 distributed by Hospitalsdistributed by Hospitalsdistributed by Hospitalsdistributed by Hospitals

342

4967

162

4253

234

4153

127

2519

264

22

4426

2 1

AHCAHCAHCAHC KampongKampongKampongKampong

ChamChamChamCham

NPHNPHNPHNPH

Total patientsTotal patientsTotal patientsTotal patients

JE positiveJE positiveJE positiveJE positive

Den PositiveDen PositiveDen PositiveDen Positive

53

69

12

33

9

29

41

21

0

20

40

60

80

Under 1y 1 - 5 ys 6-10 ys 11 - 15 ys

0

10

20

30

40

50

JE cases % of cases in each group

JE cases % of JE

Distribution by age of JE positive cases from 2006 to 2009

JE and DEN Negative control Lot#9159

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

JE NEG DEN NEG

4

JE and DEN POSITIVE IQC Lot# 9159

0.000

0.500

1.000

1.500

2.000

2.500

3.000

3.500

4.000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

JE PO DEN POS

Mean Calibration kit of lot #9159

0.000.100.200.300.400.500.600.700.800.90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mean Cal JE Mean Cal JE

JE and DEN CALLOT#9159

0.0000.1000.2000.3000.4000.5000.6000.7000.8000.900

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

JE CA DEN CAL

Challenges

– Quality of some samples are not good (Heamolysis etc..)

– Inadequate volume– Difficulties in second serum collection– Not in house control

Technical Requirement

Internal Quality Control 1. Internal quality control (IQC) – a set of

procedures for continuously assessing laboratory work and the emergent results; immediate effect, should actually control release of results.

External Quality control

1- In 2006-2007 :1195 samples from 451 ME cases received for testing JE confirmatory testing at US CDC

2- In 2010 : 63 samples (37 serum and 26 CSF) to HK

3- PT panel samples

5

Future Challenges

• JE surveillance will shift to ME surveillance which

NIP will be responsible for. More responsibility is

expected to surveillance system.

• The CSF will be tested by PCR and bacterial culture

for other pathogens such as Hib, pneumo and

• N-meningitidis

• NIPH Laboratory needs more technical and

financial support from WHO laboratory network.

• The laboratories of sentinel sites need to be

strengthened for basic bacteriology including

culture

JE surveillance in mainland ChinaJE surveillance in mainland China

Dr. Fu Shihong

Department of Viral Encephalitis and Arbovirus

Institute for Viral Disease Control and Prevention

China CDC

15 Nov 2010 Hong 15 Nov 2010 Hong kongkong ChinaChina

Introduction of JE

Name in Chinese: 乙脑乙脑乙脑乙脑�Japanese encephalitis (JE) is an acute epidemic disease of the central nervous system (CNS) caused by infection with the Japanese encephalitis virus (JEV). JE mainly affects children and adolescents. JEV is transmitted by mosquitoes and the genus Culex, which is major vector. It is a perennial disease in tropical area, but is clearly seasonal in temperate zones, with a peak incidence period between June and October each year.�Historically, JE prevalence has been high in China, where major outbreaks occurred in 1960s and 1970s. After the nationwide vaccination program initiated in the 1970s, the number of reported cases dramatically decreased and maintains a relative lower level of prevalence rate these years.

2012-7-20 3

Mosquito-born disease

Encephalitis

Area of prevalent

JE transmission cycle and possible control points

2012年7月20日 Arboviruses in China 4

JE - major encephalitis in china

JE in 2006

�Highly: >1/100,000 �Moderately: 0.5/100,000 and 1/100,000 �Low: 0.1/100,000 and 0.5/100,000 �Non: No JE cases

�Endemic in 28 provinces�The major cases: south-west China

JE distribution China 2005

1 dot = 1 case

JE cases are reducing dramatically

in China, special in recent years

�JE has been reported in China since 1951�In the history, big outbreaks 180 000 cases in 1971�In-activated vaccine was developed in 1960’s in China�Attenuated vaccine was developed in 1990’s in China

0

5

10

15

20

25

1950

1951

1952

1953

1954

1955

1956

1957

1958

1959

1960

1961

1962

1963

1964

1965

1966

1967

1968

1969

1970

1971

1972

1973

1974

1975

1976

1977

1978

1979

1980

1981

1982

1983

1984

1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

年

病病

发/10万

JE in China historically

↑ ↑

In 2007, JE vaccine has been integrated into national immunization program in whole country

Conclusion:

“Human vaccination is the only effective long-term control measure against JE. All at-risk residents should receive a safe and efficacious vaccine as part of their national immunization program.”

Consensus statements from Global JE meetings

1995, 1998, and 2002

JE surveillance in

serology in China

Serum and CSF sample collection

and tests

�In 2008 summer, 3127 specimens of acute serum and CSF were collected from 2292 cases of viral encephalitis in 6 provinces and all samples tested in national lab in 2009.

�Specimens were tested for 15 viruses, including JE and Enterovirus, etc. infections by several serological and molecular techniques, including ELISA, IFA, virus isolation and PCR.

Yunnan:747 / 853Xinjiang:641/641Guizhou:649/720Gansu:134/208Liaoning:121/121Sichuan:600/600)

1 2 3 4 5 6 7 8 9 10 11 12

ABCDEFGH

ELISA assay IFA assay

Yunnan samples tests (1)

0

5

10

15

20

25

30

35

40

45

50

55

3月 4月 5月 6月 7月 8月 9月 10月 11月

发病月份

阳性

病例

数

JEV

COX

Echo

HSV

MuV

DENV

BAV

�Positive rate of JE is the highest, over 50%

�JE infection was concentrated in Jun-Sep, account for 60% of fever cases

�Several viral infection existed in Yunnan, including JEV, Mumps, Enterovirus, and HSV, etc.

Yunnan samples tests (2)

Moreover, IgM antibodies to dengue virus, Tahyna virus, Ross river virus and Barmah forest virus were firstly detected in specimens collected in these areas from cases, indicating that several emerging pathogens of viral encephalitis existed in China.

ConclusionIt is of great public health significance to enhance surveillances of pathogenic spectrum in cases of unknown encephalitis and will have great impact on control and prevention of viral infectious diseases.

JEV surveillance

In mainland China

JEV surveillance in China –Mosquito collection

61303 mosquitoes collected from 8 provinces in 2008: Jiangxi, Sichuan, Gansu, Liaoning, Guizhou, Qinghai Xinjiang, Inner Mongolia

Mosquito collection Mosquito collection

Virus isolation and identification

Liquid nitrogen

Inoculated to CellsAnimal test

Supernatant Homogenize

Virus isolation and identification

CPE in BHKCell lines CPE in C6/36

IFA test with virus-antibody PCR/Real-time PCR Sequence/Bioinformatic

Virus isolation

�All processes were conducted in BSL - 2 lab�50-100 mosquitoes in each pool

Virus isolation

�Mosquitoes homogenized in shakers, Centrifuged with 12 000 rpm/ 20 min

The virus isolation and dentification result

40 virus strains were isolated and 16 of 40 was identified in JEVJEV(16), GATV(8),BANV(1), LNV(2), TAHV(2), and unidentified strains

�Jiangxi 4 JEV / 11916�Sichuan 4 JEV / 8000�Gansu 6 JEV / 13739�Liaoning 1JEV etc./ 9145�Guizhou 1 JEV 8 GETV / 9300�Qinghai 8 dsRNA virus / 7838 �Xinjiang 2 LNV / 4630 �Inner Mongolia 6 TAHV / 5780

2 395 specimens from cases were collected in 7 provinces 2009

In 2009, 82 428 mosquitoes, 2 592 ticks, 50 sandflies were collected from 11 provinces

In 2009, specimens from human cases and vectors were collected and all detection are under way

Jiangxi:5905Sichuan:8122Xizang:4089Shangdong:9859Hubei:9424Guizhou:14516Yunnan:17790Qinghai:7623Xinjiang:1500，，，，shadflies 50Liaoning:3600Heilongjiang:2592 ticks

Yunnan: 568 cases / 720Guizhou: 279 cases / 359Qinghai:661cases/ 801 animao:364Jiangxi: 80 cases / 80Tibet: 248 human，，，，pigs 66Liaoning:187 humanXinjiang:190 animal

JE proficiency testing in

Mainland ChinaLocal CDC 2006 2007 2008 2009 2010

Shandong Province CDC 100 100 100 100 100

Jinan Prefecture CDC 100 100 100 100 100

Hubei Province CDC 100 100 100 100 100

Yichang Prefecture CDC 100 100 100 100 100

Hebei Province CDC 100 100 100 100

Shijiazhuang Prefecture CDC 100 100 100 100

Guangxi Province CDC 100 100 100 100

Guigang Prefecture CDC 100 100 100 100

Other 11 Province CDC Other 2 Prefecture CDC

100 100100

JE-PT in China

JE-PT, WHO WPR, 2009

Name of lab: Dep. of Viral Encephalitis, IVDC, CCDCName of the assay: Beixi kit (China) Plate ID: FUBatch No. of the kit: EVI-002M Date: 2009/06/28Lot No. of the kit: 0905-1 Assay performed by: Fu ShihongExpiry date of the kit: 2010/05/08

No 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Mark BLK Neg. Pos.Sample

1Sample

2Sample

3Sample

4Sample

5Sample

6Sample

7Sample

8Sample

9Sample

10Sample

11

R D 0.043 0.054 1.242 0.049 0.052 0.734 1.108 0.683 0.090 1.112 0.667 0.092 0.058 0.180

M BLK 0.000 0.011 1.199 0.006 0.009 0.691 1.065 0.640 0.047 1.069 0.624 0.049 0.015 0.137

P / N 0.12 0.18 13.82 21.30 12.80 0.94 21.38 12.48 0.98 0.30 2.74

Result Neg Neg Pos Pos Pos Neg Pos Pos Neg Neg Pos

Note: 1.Intepretation: Mark: the name of wells; R D: is raw data; M BLK: Minus Blank value;2. Control System: Pos. OD / Neg. OD ≥ 2.13. Result Interpretation: P / N Value (Sample) ＝ Sample OD / Neg. OD (or 0.05); Positive: P/N≥2.1, Negative: P/N＜2.1;4. When the Neg. control OD value is below 0.05, use 0.05 to calculate the ratio. So in this test, 0.05 is used to calculate P/N ratio.

The results were reported to WHO WPR in 2 July (within two weeks after receive)

CDC ELISA and PRNT results

CDC updated results(2/17/10) Laboratory Number/results

Sample # Type 1* 2* 3* 4*1 CSF JE PRESUMPTIVE JE PRESUMPTIVE JE NEG NEG NEG2 CSF NEG NEG NEG NEG NEG NEG

3 CSF NEG JE PRESUMPTIVEdifficult to distinguish JE JE JE

4 CSF JE PRESUMPTIVE JE PRESUMPTIVE JE JE JE JE

5 CSF JE PRESUMPTIVE JE PRESUMPTIVEJE

suspected JE JE JE6 serum DEN POSITIVE DEN POSITIVE DEN NEG DEN NEG

7 serum JE POSITIVE JE POSITIVE JE JE JE JE8 serum JE PRESUMPTIVE JE PRESUMPTIVE JE JE JE JE9 serum NEG NEG NEG NEG NEG NEG10 serum DEN PRESUMPTIVE DEN PRESUMPTIVE NEG NEG NEG NEG11 serum DEN POSITIVE DEN POSITIVE DEN JE DEN JE

Kit used In-houseNon PanbioCommercial In-house In-house

Reporting date July 2 July 2 July 5 July 6

%Agreement with CDC results 100 82 91 82*Compared against CDC JEV and DENV IgM ELISA + JEV and DENV PRNT results.

Difference between national kit and CDC resultsDifference between CDC DEN and national kit negative results

Updated results: Difference between CDC results 6/5/09 and 2/17/10

Results of the 2009 WPR JE Labnet proficiency testing:In-house and non-Panbio commercial kits

26

Thanks And Welcome to

new campus

NATIONAL CENTER FOR

LABORATORY AND

EPIDEMIOLOGY, LAO PDR

National Center for Laboratory & Epidemiology (NCLE)

NATIONAL CENTER FOR

LABORATORY AND EPIDEMIOLOGY

� Reference Laboratory

� Conduct Research and Study of Outbreak potential

infectious diseases.

� Provide teaching and training to provincial Hospitals.

� Epidemiology

� Surveillance and Study.

� Outbreak investigation and rapid response.

CAPACITIES

� ELISA� Dengue

� Japanese encephalitis virus

� Measles

� Rubella

� HIV

� Hepatitis A virus

� Leptospira

� PCR� Influenza

� Dengue serotyping

� Luminex Technologies� Upper Respiratory virus

� Cell Culture� Influenza

JE LABORATORY

� National Center for Laboratory (NCLE)

� Mahosoth Hospital (Welcome Trust)

Diagnostic Methods Used Diagnostic Methods Used Diagnostic Methods Used Diagnostic Methods Used

----NCLE: NCLE: NCLE: NCLE: Panbio JEV-IgM Dengue Combo ELISA

----Mahosot Hospital.Mahosot Hospital.Mahosot Hospital.Mahosot Hospital.

� Panbio JEV-IgM Dengue Combo ELISA

� Xcyton JEV CheX

� Hapalyse Dengue JE MP PA kit-Pentax

EQUIPMENT AVAILABLE (NCLE)

• ELISA Reader :

� Multiskan EX

� Humareader

� BIORAD 550

• ELISA washer

• Conventional PCR machine

• Real time PCR machine

• Luminex PCR machine

• Freezers for specimens

• Refrigerators for specimens

JE TESTING ALGORITHM

Specimen SourceSpecimen SourceSpecimen SourceSpecimen SourceNumber Number Number Number ReceivedReceivedReceivedReceived

Number Number Number Number TestedTestedTestedTested

Result (JEResult (JEResult (JEResult (JE----IgM)IgM)IgM)IgM)

NegativeNegativeNegativeNegative PositivePositivePositivePositiveEquivocaEquivocaEquivocaEquivoca

llll

Bokeo 8 8 4 1 3

Oudomxay 4 4 4 0 0

LuangPrabang 1 1 1 0 0

Sayaboury 1 1 0 1 0

Vientiane Capital 3 3 2 1 0

TotalTotalTotalTotal 17171717 17171717 11111111 3333 3333

NUMBER OF SAMPLES TESTED FOR JE IN NUMBER OF SAMPLES TESTED FOR JE IN NUMBER OF SAMPLES TESTED FOR JE IN NUMBER OF SAMPLES TESTED FOR JE IN 2009 AT NCLE2009 AT NCLE2009 AT NCLE2009 AT NCLE

NUMBER OF SPECIMEN TESTED

FOR JE

AS 06 OCTOBER 2010 (NCLE)

Province Sample

Tested by

ELISA

Positive IgM

ELISA

%

Oudomxay 23 12 52.17%

Bokeo 04 02 50.00%

Huaphanh 16 06 37.50%

Xayaboury 06 02 33.33%

Vientiane Capital 01 0 0%

Total 50 22 44%

QUALITY ASSURANCE

� Sending Specimen to NIID for confirmation (2009

= 17 samples)

� Received PT from Hong Kong (2009)

FUTURE PLANS

� Strengthening and expanding JE surveillance in all provinces

� Strengthening on Laboratory data management system at NCLE.

� Improve the quality of Laboratory Testing

� Strengthening of NCLE laboratory to be JE reference Lab in the country.

� Strengthening close collaboration with Mahosoth

Hospital.

CHALLENGES AND REQUEST

� Limited space to perform the test (NCLE renovation)� Insufficient space for keeping specimens (freezer and refrigerator)

� Staff needs more technical and data management training

� Provincial and District staff require more training on field collection of blood specimens and CSF

� It would be good to have some long term technical on data management assistance

Thank you

Country Report – Malaysia

Japanese Encephalitis

Virology UnitInstitute for Medical Research

Kuala Lumpur

15 November 2010

Introduction

� First confirmed case of JE in Malaysia was in 1952

� 1954 serological surveys in man and animals showed JE is endemic in Malaysia

� Viral encephalitis is a notifiable disease (no specific aetiological agent recorded)

� Therefore, cases of JE cannot be quantified accurately

Outbreak

Surveillance

(1) IMR

(2) NPHL

MOH

Clinical

Cases

(1)UMMC –Klang Valley

(2)UNIMAS -Sarawak

MOEMOH

IMR

Laboratories Performing Tests for JE Diagnostic Methods� Antibody Detection:

Haemagglutination Inhibition Test*

JE IgM Capture ELISA (1991)

� Viral Isolation:

Suckling mice (till 1993)

C6/36*

� Molecular Detection

rRT-PCR*

* for severe and fatal cases

Testing Algorithm For JE (Virology Unit, IMR)

CSF and/or Serum

JE & Dengue IgM Capture ELISA

Neg

Request for 2nd Sample

JE IgM Capture ELISA

Pos Neg

RT-PCR

Viral IsolationPos

Request for 2nd Sample

JE IgM Capture ELISA

Pos Neg

HI

+

Part of AES investigations

QA Measures Implemented

1. ISO 15189 (NATA, Australia)

-in progress

2. External Quality Assurance program

RCPA (Arboviruses), WHO/WPR

Number of Sample Tested for JE IgM Ab(2007-Oct 2010)

0

100

200

300

400

500

600

700

800

2007 2008 2009 Till Oct 2010

Year

No o

f Sam

ple

Serum CSF

69.9%

30.1%

44.2%

55.8%

35.2%

64.8%

64.3%

35.7%

Distribution of Laboratory Confirmed JE Cases(1999-Oct 2010)

0

10

20

30

40

50

60

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

Till

Oct

201

0

Year

No o

f JE

Cas

es

% of Samples Tested Positive for JE IgM(2007-Oct 2010)

0123456789

10

2007 2008 2009 Till Oct 2010

Year

% P

ositi

ve

Serum+CSF Serum CSF

38/637

20/224

18/41334/717

21/317

13/400

18/322

17/225

1/97

16/292

14/166

2/126

Age Distribution of Laboratory Confirmed JE(2007-Oct 2010)

0

5

10

15

20

25

30

0-04 '05-15 16-24 25-59 >59

Age (Yrs)

Num

ber of

JE

Cas

es

2007 2008 2009 2010

Distribution of Laboratory Confirmed JE Cases by States (2007-Oct 2010)

020406080

100120140160

Per

lis

Ke

dah

Pe

nan

g

Per

ak

Se

lang

or

N S

em

bila

n

Mal

acc

a

Joh

or

Pa

han

g

Te

reng

gan

u

Kel

ant

an

Sa

bah

Sar

aw

ak

Ku

ala

Lum

pur

Pu

traja

ya

State

No

of J

E C

ases

2007 2008 2009 2010

Distribution of Laboratory Confirmed JE Cases by Month (2007-Oct 2010)

0

10

20

30

40

50

60

70

80

90

100

Jan'0

7

Mar

'07

May

'07

Jul'0

7

Sep'07

Nov'07

Jan'0

8

Mar'08

May

'08

Jul'0

8

Sep'08

Nov'08

Jan'0

9

Mar

'09

May

'09

Jul'0

9

Sep'09

Nov'09

Jan'1

0

May

'10

Sep'10

Pos Number of Sample Tested

JE Vaccination Programme

� Sarawak-Started in 2002 (EPI)-Age: 9mths, 10mths

Boosters: 18mths of age, 3yrly till age 15 yrs

� Peninsular Malaysia & Sabah-Vaccination given within 2 KM radius when there is a case of JE

Immunisation Coverage for JE (First Dose) Sarawak

0

20

40

60

80

100

120

2005 2006 2007 2008 2009

Year

% V

acci

nate

d

27,981 / 42,180

19,744 / 41,340

35,680 / 41,360

66.3%

47.8%

85.0%

41,784 / 42,150

99.1%

40,850 / 42,292

96.6%

HEAD UNIT

TISSUE CULTURE LAB

HEPATITIS LAB

HIV LAB

MOLECULAR DIAGNOSTIC

LAB

SEROLOGY LAB

EVALUATION/ QC LAB

EXOTIC DISEASES

LAB

ELECTRON MICROSCOPY

LAB

BSL-3 LAB

Virology Unit, IMRDiagnostic Laboratory

Head Unit and Clinical Virologist

Pathologist 1

Senior Research Officer 4

Research Officer 3

Senior Medical Laboratory Technologist 3

Medical Laboratory Technologist 22

Health Assistant 5

Human Resources:

Challenges:

� JE is not a specific notifiable disease

� Problem in getting CSF sample

� Problem in getting 2nd sample

� Shortage of manpower

� Expensive (~12USD)

Request:Currently, IMR performed passive surveillance

Require funding for active surveillance, ???? WHO

Thank You

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Papua New Guinea

Oscillah Kaminiel/Ernest Velemu

Central Public Health Laboratory

Department of Health

Where is PNG

Demography • Over 850 indigenous languages.

• Three official languages (English, Pidgin, Motu)

• Extremely rugged, mountainous and dense rain forest (Difficult to develop transportation infrastructure)

• Access to widely scattered rural communities (82%) is often difficult, slow and expensive.

Demographic Data Total

Area (1000 sq km) 462,84

Estimated population (in 2008) 6,621,132

Province 20 ( 2 newly legislated)

Districts 89

Annual Population growth (%) 2.7

Urban population (%) 18

Laboratory NetworkNational (CPHL)

BTS

Clinical Labs

(PMGH)

(3)

Province

(19)

District

(~130)

Central Public Health Laboratory

Major responsibilities (CPHL)

• Reference Public Health Laboratory

• Diagnosis of public health diseases and

surveillance

• Training on new techniques/ diagnosis

• Quality assurance mostly for public health

diseases

• Research

Surveillance activities

• Measles and Rubella

• HIV/TB/Malaria

• Cholera

• Dengue

• Influenza

• Polio

Current Situation Japanese

Encephalitis

• No diagnosis and surveillance done as

yet

• Hope fully JE testing will be established

after this workshop

Challengers

• Minimal or no request by clinicians

• Main use of clinical observation over

laboratory diagnosis

• Establishment of JE testing at CPHL

Way Forward

• Expect to gain knowledge on Laboratory

diagnosis of JE at this workshop

• Understand pathology of JE

• Quality Assurance of JE diagnosis

• Be able to establish JE diagnosis and

surveillance in PNGTHANK YOU

Japanese Encephalitis Country Report : The Philippines

22ndnd InterInter--country Handscountry Hands--on Training Workshop on the Laboratory on Training Workshop on the Laboratory

Diagnosis of Japanese Encephalitis in the Western Pacific RegionDiagnosis of Japanese Encephalitis in the Western Pacific Region

November 15November 15--19, 201019, 2010

Analisa N. Bautista, RMT and Ava Kristy D. Sy, RMTAnalisa N. Bautista, RMT and Ava Kristy D. Sy, RMT

Research Institute for Tropical Medicine, ManilaResearch Institute for Tropical Medicine, Manila

Status of JE Surveillance in the Philippines

Source: National Epidemiology Center

Source: National Epidemiology Center Source: National Epidemiology Center

AES Cases by Agegroup & Sex (N=34)Philippines, 2008

<01

1-4

5-14

15-24

25-39

40-64

65 & above

Age

gro

up

(Yea

rs)

051015 0 5 10 15

Female Male

No. of Cases


>49 yrs

31-40 yrs

21-30 yrs

11-20 yrs

1-10 yrs

<1 yr


Status of JE Surveillance in the Philippines

� Sentinel Surveillance for Etiological Diagnosis of Meningitis / Encephalitis / Meningoencephalitis in the Philippines (CNS Infections) has been implemented in 2009Source of Funding: World Health Organization

� 5 sentinel sites –Bulacan and Tarlac, Region 3 Naga, Region 5 Iloilo, Region 6 Quezon City, National Capital Region

� 50 cases/site

Sentinel Surveillance for Etiological Diagnosis of Meningitis/Encephalitis/Meningoencephalitis

(MEMe) in the Philippines

• Sample collection started in March 2009 from Bulacan, and Tarlac (Region 3), Iloilo (Region 6), Naga (Region 5),

NCR to start this December 2010

• Testing began in June 2009 when JE/Dengue IgM Combo ELISA kits became available

• Panbio JE/Dengue IgM Combo ELISA

Type of specimen:

• CSF

• Serum

Summary of Samples Received, 2009-2010 (n=212)

Summary of Results, 2009-2010 (n=212)

* Discrepant result: Serum 1 =Dengue positive and Serum 2 = JE positive**Not tested for Bacterial pathogens

RESULTS BULACAN % NAGA % ILOILO % TARLAC %Other

Hospitals**% Total %

JE Positive6 13.0% 14 36.8% 5 10.0% 7 28.0% 4 7.5% 36 17.0%

Dengue Positive5 10.9% 2* 5.3% 10 20.0% 3 12.0% 7 13.2% 27 12.7%

H. influenza B3 6.5% 1 2.6% 7 14.0% 1 4.0% 0 0.0% 12 5.7%

S. pneumoniae2 4.3% 4 10.5% 1 2.0% 1 4.0% 0 0.0% 8 3.8%

N. meningitidis0 0.0% 1 2.6% 0 0.0% 0 0.0% 0 0.0% 1 0.5%

Negative30 65.2% 16 42.1% 27 54.0% 13 52.0% 42 79.2% 128 60.4%

TOTAL OF CASES46 100.0% 38 100.0% 50 100.0% 25 100.0% 53 100.0% 212 100.0%

• Proficiency Panel• Scored 100%

• Confirmatory / Validation of Samples in RRL

• Sent 64 samples (31 CSF, 33 Serum Samples)• 95% concordance

Quality Assurance

Challenges• Low recruitment of cases

1. Refusal of the parents to sign the Informed consent, hence, physician cannot collect CSF

2. No full time physician in charge of MEMe Surveillance in the sites

3. Lack of political support

• Non-submission of Case Report Forms (CRFs)

Where is JE in EPI in the Philippines?

• JE vaccine not introduced in EPI• Possible introduction?

– Requires sufficient disease burden data for decision makings

– Requires an expert committee to compare this to other vaccines

– Requires extensive advocacy at all levels, starting with Secretary and including Senate/Congress

Surveillance of Infection with Japanese Encephalitis virus in mosquitoes in Northern Philippines

In collaboration with Virology Department, School of Medicine, Tohoku University, Sendai,

Japan

Objectives:

1. To determine the spatial and temporal distribution of JEV in mosquito vectors in the Philippines

2. To determine molecular characterization of JEV detected from mosquito vectors in this study area

Study Site

• Barangay Moriones & Lubigan

Minicipality of San Jose,

Tarlac Province, Region III

-Population; 4,437

-Household; 876 (2007)

• EnvironmentEcology suitable for the

transmission of JEV such as breeding of Culex spp, pig farm and the rice field all within 1km radius

Tarlac province

RITM

Collection methodology

• Adult mosquitoes were collected from May2009 to April2010 by animal baited trap method.No collection in September 2009 due to the typhoon.

Processing of collected mosquitoes

Indentify the mosquito by the species (Cx. tritaeniorhynchus, Cx. visinui,

Cx. gelidus, Cx. fuscocephala) and pool with 50-100 head/tube

↓↓↓↓

Homogenate the mosquitoes with MEM and Centrifuge

↓↓↓↓

Filter the supernatant with 0.45 uM filter

↓↓↓↓

RT-PCR and Sequencing（RITM/Tohoku)

RT-PCR and Sequencing（RITM/Tohoku)

Population of the collected mosquitoes

2009-2010

May June July Aug Oct Nov Dec Jan Feb Mar Apr Total

Cx.

tritaeniorhynchus948 671 300 162 200 818 287 253 56 92 19 3806

Cx.vishinui 2213 2060 1052 670 417 740 770 1001 732 143 40 9838

Cx.gelidus 179 39 16 129 74 49 342 114 15 5 1 963

Cx. fuscocephala 290 587 123 325 110 1028 1051 527 198 83 12 4334

Cx. spp 4816 1155 1431 1191 874 4045 4857 4631 4156 248 416 27820

Total 8,446 4,512 2,922 2,477 1,675 6,680 7,307 6,526 5,157 571 488 46761

Phylogenetic analysis of the JEV from partial of Envelope gene (326bp)

Genotype 3

Genotype 2

Genotype 1

Genotype 4

CH1392/Mo/1990/China/AF254452

TPC0706a/Mo/2007/Taiwan/GQ260626

T1P1-S1/Mo/1997/Taiwan/AY303791

JaGAr 01/Mo/1959/Japan/AF069076

ML17/Hu/1981/Japan/AY508812

81P241/Sw/1981/Japan/FJ943464

SA14/Hu/1959/China/AY243842

SA14-14-2/Va/2000/China/AF315119

JaOArS982/Mo/1982/Japan/M18370

PhAn1242/Sw/1984/Philippines/U70417

CC27-L3/Mo/1983/China/AY303796

Phi09/Mo/2009/Philippines

Beijing-1/Hu/1949/China/L48961

Nakayama/Hu/1935/Japan/EF571853

FU/Hu/1995/Australia/AF217620

JKT5441/Mo/1981/Indonesia/1981/U70406

VN78/Mo/2002/Vietnam/AY376467

JaNAr13-04/Mo/Japan/2002/FJ185146

JKT6468/Mo/1981/Indonesia/AY184212

WNV NY2002 Clinton/DQ164193

95

81

66

8882

92

91 42

93

51

8067

41

79

59

0.02

Neighbor-Joining methodAt bootstrap value of 1,000 replicates

Summary

• More than 40,000 collected by this method and majority were Culex spp.

• JEV was detected from Cx. tritaeoniorhynchuscollected in Nov 2009

• Philippine strain was belonged to genotype 3

Thank you for your attention!

1

Report from JE RRL

Japanese Encephalitis in Korea

Division of Arboviruses National Institute of Health

Korea Centers for Disease Control and Prevention

Cho, Jung-Eun

15 November, 2010

2

Overview of JE in Korea

1960 - 70s 1980 - 90s 2000s1930s 1940 - 50s

First case report in 1933 in Japan (1,924 cases)

Notifiable disease in 1949 (5,548 cases/2,429 deaths)

Largest outbreak in 1958 (6,897 cases)

Vaccine import (1968)

JE surveillance program (1975)

Last epidemic in 1982 (1,197 cases)

Mandatory vaccination < 15 age

Annual JE cases < 8

(1983)

Designated as JE RRL (2009)

3

Laboratory diagnosis of JE in Korea

1. Serology•Panbio’s JE-DEN Duo ELISA (IgM)

•Immunofluorescent assay for whole Ab titer check

•Cross reactions are screened by the use of Flavivirus screening IFA kit (in-house) or each ELISA kit (commercial)

•PRNT is conducted for differential diagnosis from other flaviviruses

2. Molecular work•First choice : Nested RT-PCR (commercialized)•One step real-time RT-PCR (mainly for mosquito surveillance)

3. Virus isolation• Cell culture (C6/36 cell line)

4

JE cases in Korea

Year 2003 2004 2005 2006 2007 2008 2009 2010

No. of patient 1 0 6 0 7 6 6 24

No. of sample 171 217 202 253 258 311 324 314

• Annual JE cases

* As of November 10, 2010. One case is from foreigner aged of 28.

• Age pattern of JE patients • Annual JE seasonYear Mean age Range

2007 52 32–85

2008 52 45–69

2009 50 36-69

2010 54 39–77

*One foreigner case was excluded.

Year Period

2007 1 SEP – 1 NOV

2008 22 AUG – 19 OCT

2009 13 SEP – 25 OCT

2010 11 AUG – 21 OCT

*

*

5

Vector and animal host surveillance in 2010

• Specimen: C. tritaeniorhynchus

• Period: June – October

• Locations: 11 sites (9 provinces)

• Method: real time or nested RT-PCR

• No. of mosquitoes tested in 2010

-27,769 mosquitoes (366 pools)

Virus detection from mosquitoes

ProvinceNo. of

detection*Mosquito

collection date

Gyeong-Nam 1 4 OCT

*Detected JEV belongs to the genotype 1

6

Vector and animal host surveillance in 2010

Sero-conversion of animal host• Specimen: Unvaccinated pig’s sera

• Period: July – November

• Locations: 8 sites (8 provinces)

• Method: Immunochromatographic test

• No.of samples tested in 2010: 1,474

• Positive rates : 32.8% (need to be confirmed by PRNT)Year No.of test No.of positive Positive rate (%) No.of JE case

2005 2,515 1,043 41.5 6

2006 1,850 481 26.0 0

2007 2,809 305 10.9 7

2008 1,479 91 6.2 6

2009 1,535 115 10.9 7

2010 1,474 483 32.8 24

7

Other flavivirus surveillance in Korea

Year 2003 2004 2008 2006 2007 20082009 2010

No. of cases* 22 16 28 63 159 94 88 161

No. of sample 59 74 109 177 290 280 269 429

• Dengue fever

• West Nile feverYear 2003 2004 2005 2006 2007 20082009 2010

No. of cases 0 0 0 0 0 0 0 0

No. of sample 0 7 4 14 9 19 84 44

• Tick-borne encephalitisYear 2006 2007 2008 2009 2010

No. of cases 0 0 0 0 0

No. of sample 17 47 76 145 1398

Publications in 2009-2010

Development and field evaluation of a nested RT–PCR kit for detecting Japanese encephalitis virus in mosquitoes.

J Virol Methods. In press, accepted

9

Suggestions & Future plan

•Results of flavivirus surveillance (Dengue, West Nile) conducted in 2010 will be presented at the next meeting

•Multiplex real-time RT-PCR kit is now being evaluated and will be released in 2011

• From 2011, sero-survey in adult population will be implemented to cope with the age shift phenomenon in JE patients

• We hope that nested RT-PCR and immunochromatographic test kit could be distributed to JE endemic regions

10

Thank you for your attention!

Any Questions or Suggestions:

Dr. Han or Mr. Jeong ([email protected]/[email protected])

COUNTRY REPORT

VIET NAM

Arbovirus Laboratory

National Institute of Hygiene and Epidemiology

Hong Kong, November, 2010

Viral encephalitis surveillancein Vietnam

• Viral encephalitis cases were reported monthly.

• 3 sentinel sites: North (1), Central (1), South (1)

• National hospitals in Hanoi, HCM City.

• Some other provincial preventive medicine centre.

Viral Encephalitis in Vietnam, 2009

Northern Provinces

Central Provinces

Southern Provinces

Highland Provinces

Total

Morbidity 699 73 234 81 1,042

Mortality 5 2 16 1 24

Viral encephalitis in Northern Vietnam, 2009 – Oct, 2010

699

194

13

496

124

22

0

100

200

300

400

500

600

700

2009 2010

Viral Encephalitis caseCollected sample caseJE possitive case

JE case in Northern Vietnam, 2010

2

0

6

9

5

0

1

2

3

4

5

6

7

8

9

0-1 1 - 4 5- 10 10 - 15 >15

Age group

JE cases in 2 sentinel sites(2009 – 10/2010)

JE (+)/ Viral Encephalitis

2009 2010 (10/2010)

Total

Thai Binh 5/175(2.9%)

7/79(8,7%)

12/254(4,7%)

Quang Ngai 7/23(30%)

1/9(11,1%)

8/32(25%)

Confirmatory test

• Total samples sent: 74- Positive: 23- Negative: 51• Agreement result: 70• Disagreement result: 4 (2 cases)

JE vaccination in Northern Vietnam 2009 - 2010

Year Province District

2009 25/28 275/325

2010 26/29 280/325

JE vaccination in Vietnam 2009

Northern Provinces

Central Provinces

Southern Provinces

Highland Provinces

Total

1st and 2nd dose

94.3% 96.5% 92.9% 90.7% 93.1%

3rd dose 94.8% 98.1% 93.8% 94.0% 94.8%

JE vaccination in Northern Vietnam,January - September, 2010

Province 2 doses 3 doses

Total Vaccinated Ratio Total Vaccinated Ratio

1 Hanoi 109.198 91.548 83,8 104.296 73.179 70,2

2 Nam Dinh 33.811 32.940 97,4 32.700 31.981 97,8

3 Bac Giang 20.897 20.462 97,9

4 Ha Giang 10.678 9.668 90,5 9.791 9.229 94,3

5 Dien Bien 10.983 7.642 69,6 9.427 6.502 68,9

Some other activities

• Surveillance the molecular biology characteristic of JE in

some provinces in Vietnam and sequencing the whole

genome of the first JE isolation from human genotype 1

in Vietnam.

• Produce enough MAC- ELISA kits for the laboratory and

the provincial PMCs (preventive medicine centre),

hospital’s laboratories in Vietnam.

• Complete SOPs and other requirements to register ISO

15189.

Production of MAC – ELISA kits

• Total production October, 2010:- 2 x 8 test/kit: 164 kits.- 2 x 48 test/kit: 37 kits.• Provincial Medicine Centre: 7

centre. • Hospitals: 5 hospitals.• Register ISO 9001 - 2008

SOPs

– Sample (Collection, reception, storage)

– Equipment.

– Reagents and materials.

– Experiments.

Thank you!

JAPANESE ENCEPHALITIS IN SOUTHERN VIETNAM

Country report

Hong Kong - 2010

Laboratory capacity for JE Diagnosticsin Southern Viet Nam

2

Provincial Laboratory– Serology

• MAC-ELISAPasteur Institute

– Serology– Virology– RT-PCR– Realtime RT-PCR

3

MAC-ELISA� Using the in-house KIT

(modif. protocol CDC ).

� AES samples were tested DEN- IgM and JE-IgM.

Laboratory JE Diagnostics

Real-time RT-PCR

ISOLATION OF VIRUS ON C6/36 CELL

JE sentinel sites in

Vietnam South

-Sentinel site: Binh Duong provincal Hospital

-Sujected:

1.Ben Tre provincal Hospital

2.Ho Chi Minh city (Hospital of Tropical Disease and Pediatric 2 Hospital)

Bến Tre

Ho Chi Minh

Binh Duong

JE surveillance by MAC-ELISA 2007- 2009

5

Year No. of cases CSF 1st serum 2nd serum

No. of samples (+)

No. of samples (+)

No. of samples (+)

2007 242 96 3 - 1 151 2 - 8 11 0

2008 78 52 2 - 2 71 4 - 4 19 1 - 3

2009 288 182 11 - 6 250 12 - 9 112 7 - 6

Total 608 330 16 - 9 472 18 - 21 142 8 - 9

JE - DEN

Case identification: WHOUsing the Japanes encephalitis- Dengue IgM in-house KIT

6

JE surveillance by MAC-ELISA , 2007- 2009

7

JE surveillance by MAC-ELISA , 2007- 2009

0

20

40

60

80

100

120

140

160

180

200

2007 2008 2009

0

2

4

6

8

10

12

14

No. of samples JE(+) DEN(+)

0

20

40

60

80

100

120

2007 2008 2009

0

2

4

6

8

10

12

14

No. of samples JE(+) DEN(+)

CFS

S1

S2

Epidemiology of Acute Encephalitis Syndrome

Month

Epidemiology of Acute Encephalitis Syndrome

Month

JE Vaccination Status

YearNo. of

provinces

No. of district

No. of children under 5 year-olds

with JE vac. coverage

Ratio (%)

2001 3 3

2002 5 7

2003 11 20 88,157 85.60

2004 16 42 138,828 95.90

2005 18 60 235,136 95.70

2006 19 78 235,241 87.40

2007 20 102 289,488 94.10

2008 20 136 279,290 87.80

2009 20 152 448,114 92,93

2010 (%)

20 (100%)

204 (100%)

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ANNEX 1 W O R L D H E A L T H ORGANISATION MONDIALE ... · bureau regional du pacifique occidental 2ND INTERCOUNTRY HANDS-ON TRAINING WORKSHOP ON THE LABORATORY DIAGNOSIS OF JAPANESE