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Training Workshop Report
2nd Intercountry Hands-On Training Workshop
on the Laboratory Diagnosis of Japanese Encephalitis
in the Western Pacific Region
Hong Kong (China)
15-19 November 2010
(WP)/ICP/IVD/1.1/001-A
Report series number: RS/2010/GE/71(HOK)
English only
REPORT
2nd INTERCOUNTRY HANDS-ON TRAINING WORKSHOP
ON THE LABORATORY DIAGNOSIS OF JAPANESE ENCEPHALITIS
IN THE WESTERN PACIFIC REGION
Convened by:
WORLD HEALTH ORGANIZATION
REGIONAL OFFICE FOR THE WESTERN PACIFIC
Hong Kong (China)
15-19 November 2010
Not for sale
Printed and distributed by:
World Health Organization
Regional Office for the Western Pacific
Manila, Philippines
May 2012
NOTE
The views expressed in this report are those of the participants in the Hands-on Training on the
Laboratory Diagnosis of Japanese Encephalitis and do not necessarily reflect the policies of the
World Health Organization.
This report has been prepared by the World Health Organization Regional Office for the Western
Pacific for the participants of the Hands-on Training on the Laboratory Diagnosis of Japanese
Encephalitis, which was held in Hong Kong (China) from 15-19 November 2010.
SUMMARY
The 2nd Hands-on Training on the Laboratory Diagnosis of Japanese Encephalitis (JE) for
WHO JE laboratories in the Western Pacific Region was held at the Public Health Laboratory
Center, Hong Kong, China from 15 to 19 November 2010. The training was attended by 14
participants from the WHO JE network laboratories of Cambodia, China,
the Lao People's Democratic Republic, Malaysia, Papua New Guinea, the Philippines, the
Republic of Korea and Viet Nam (Hanoi and Ho Chi Minh City). Technical support was
provided by temporary advisers from the Public Health Laboratory Center Hong Kong , United
States Centers for Disease Control and Prevention (US CDC), National Institute of Infectious
Diseases, Japan as well as from the WHO Secretariat.
The objectives of the workshop were:
(1)
to enhance knowledge and skills of national JE laboratory staff in:
(a)
performing ELISA for laboratory diagnosis of JE; and
(b)
carrying out laboratory quality assurance for JE diagnosis.
(2)
to familiarize participants with the requirements for WHO accreditation for the JE
laboratories; and
(3)
to familiarize participants with laboratory data management using the WHO JE
laboratory data reporting format for reporting to the Western Pacific Regional Office.
The training programme consisted of one day of lectures followed by four days of
practical sessions, country presentations and discussions.
On day one, four sessions consisting of general lectures followed country presentations.
Four sessions consist of 1) Japanese encephalitis/acute encephalitits syndrome surveillance
and laboratory network, 2) Quality assurance of the JE labnet and global specialized laboratory,
3) Reports from regional reference laboratories and national laboratories and 4) Introduction of
JE virus specific IgM enzyme-linked immunosorbent assay (ELISA).
The hands-on training session was conducted in the training laboratory of PHLC and
consisted of three days of practical sessions. A practical session on JE PCR procedures was
performed on 18-19 November to familiarize participants with the molecular detection method
for JE diagnosis. Participants were grouped into seven pairs.
During incubation periods between all the sessions, participants learnt how to calibrate and
maintain micropipettes and ELISA equipment for laboratory quality assurance and control.
At the end of training, the second WHO JE proficiency panel samples were distributed to
participants. Participants were requested to submit the results within 14 days using appropriate
assays which are used in their own laboratory.
This workshop contributed to further enhance the laboratory capacity of WHO network JE
laboratories in the Western Pacific Region. It is expected for participants to have enhanced
knowledge and skills in performing ELISA for laboratory diagnosis of JE, to implement
standardized approaches on quality assurance for JE diagnosis and to be familiarized with
requirements for WHO accreditation for the JE laboratories and with laboratory data
management using the WHO JE laboratory data reporting format for reporting leading to
improved JE surveillance in the Region.
CONTENTS
Page
SUMMARY
1. INTRODUCTION ....................................................................................................... 1
1.1 Objectives .......................................................................................................... 2
1.2 Opening Remarks .............................................................................................. 2
1.3 Participants ........................................................................................................ 2
2. PROCEEDINGS.......................................................................................................... 3
2.1 Training programmes ........................................................................................ 3
2.2 Lecture sessions................................................................................................. 3
2.3 Practical sessions ............................................................................................... 7
3. CONCLUSIONS ......................................................................................................... 8
3.1
3.2
3.3
3.4
General............................................................................................................... 8
Evaluation of the training workshop ................................................................. 8
Outcomes of the training ................................................................................... 9
Follow-up of the workshop................................................................................ 9
ANNEXES:
ANNEX 1 - LIST OF PARTICIPANTS, TEMPORARY ADVISERS AND
SECRETARIAT
ANNEX 2 - TIMETABLE
ANNEX 3 - PANBIO ELISA PROCEDURE AND WORKSHEET
ANNEX 4 - INSTRUCTIONS AND PROTOCOLS FOR JE PCR
ANNEX 5 - PRESENTATION FILES FROM LECTURES
ANNEX 6 - PRESENTATION FILES FROM COUNTRY REPORTS
Keywords:
Encephalitis, Japanese-diagnosis/Enzyme-linked immunosorbent assay/ Laboratory techniques
and procedures
1. INTRODUCTION
Japanese Encephalitis (JE) is an important cause of death and disability and is a pressing
public health problem for many countries in Asia. Substantial advances have been made in
recent years in the development of improved JE vaccines and in the availability of high quality
commercial diagnostics that can be used for surveillance. Subsequently, several countries in the
Region have established laboratory-supported JE surveillance. Some countries have introduced
JE vaccine into their routine vaccination programmes and enhanced surveillance activities are
needed to determine the disease burden and to monitor vaccination programmes.
In the Western Pacific Region, 1.74 billion people are at risk for JE infection in 11
countries. JE is known to be endemic in seven countries in the Region. China, Japan, Malaysia,
the Republic of Korea and Viet Nam have partially or fully controlled human disease through
vaccination while Cambodia, the Philippines and the Lao People's Democratic Republic have
demonstrated some evidence of endemic JE transmission but have no vaccination programme.
Australia has a JE vaccination programme only in Torres Strait, where JE has been endemic
beginning in 1995. Papua New Guinea is a country in the Region with presumed endemic JE
transmission but without clear disease burden documentation.
Since surveillance began in the early 1990s, there has been a decline in the number of
reported JE cases in Western Pacific Region despite the lack of data from several countries
within the Region. Vaccination for JE was introduced in various countries within the Region
from 1960 onwards and led to a drastic decline in annual cases. With the vaccine introduced into
national vaccination programmes in several countries, 11% of the total regional population at risk
was protected.
Viet Nam, which reports the second highest number of JE cases in the Region, has
introduced the vaccine nationwide to all children following a measure China took in 2010. The
introduction of a nationwide JE immunization programme in China and Viet Nam will lead to
protection over 82% of the regional population.
The JE laboratory network was established during the period 2008-2009 to improve the
capability for JE case confirmation among countries either known or suspected to be endemic for
JE in the Western Pacific Region, as recommended by the 17th Technical Advisory Group
(TAG) meeting in 2008. These countries include China, Cambodia, the Lao People's Democratic
Republic, Malaysia, the Philippines, Viet Nam and Papua New Guinea.
The newly established JE laboratory network was formed based on the WHO polio and
measles/rubella laboratory network models. Some WHO measles/rubella laboratories also were
designated as the WHO National JE Laboratory. The purpose of this network is to improve and
standardize the capability of JE diagnosis in countries where JE is endemic. The network
consists of one Global Specialized Laboratory (GSL) in Japan, two Regional Reference
Laboratories in the Republic of Korea and China and seven national laboratories in Cambodia,
the Lao People’s Democratic Republic, Viet Nam, the Philippines, Malaysia and
Papua New Guinea.
The second JE laboratory network meeting was held on 24 February 2010 as part of the
2nd Vaccine-Preventable Diseases Laboratory Network (Labnet) Meeting. Two regional
-2-
hands-on training workshops were organized in 2009 to build regional laboratory capacity for JE
testing.
There are three JE network laboratories that use their own in-house assays in the Region
and the Chinese Center for Disease Control and Prevention (China CDC) uses the
locally-produced JE kits. Six other network laboratories use the Panbio JE/Dengue IgM Combo
Enzyme-Linked Immunosorbent Assays (ELISA) kits. For quality assurance of the JE labnet,
the first proficiency tests for JE were conducted successfully in 2009 and a confirmatory testing
mechanism also was established in the Region. The second WHO JE proficiency test samples
were distributed during this second hands-on training course and results were finalized. WHO
accreditation using the WHO JE laboratory checklist was initiated in 2010.
The second hands-on training session for JE was organized for WHO JE network
laboratories at the Public Health Laboratory Centre, Hong Kong (China) from
15 to 19 November 2010 to increase the proficiency of laboratory staff in ELISA testing and
further improve the quality of laboratory performances. Fourteen participants from Cambodia,
China, the Republic of Korea, the Lao People's Democratic Republic, Malaysia, Papua New
Guinea, the Philippines and Viet Nam were invited to attend the training session. The Papua
New Guinea laboratory was the last laboratory on board among 10 laboratories in the Region and
joined the second hands-on training workshop in 2010.
1.1
Objectives
(1)
To enhance the knowledge and skills of national JE laboratory staff in:
(a)
performing ELISA for laboratory diagnosis of JE; and
(b)
carrying out laboratory quality assurance for JE diagnosis.
(2)
To familiarize participants with the requirements for WHO accreditation for the JE
laboratories.
(3)
To familiarize participants with laboratory data management using the WHO JE
laboratory data reporting format for reporting to the Western Pacific Regional Office.
1.2
Opening Remarks
Dr Thomas Tsang, Controller of the Centre for Health Protection in Hong Kong (China),
welcomed the participants and opened the hands-on training workshop with an introductory
speech. Dr Youngmee Jee, Scientist, Western Pacific Regional Office, presented the training
objectives.
1.3
Participants
The training session was attended by 14 participants from WHO-designated national JE
laboratories from Cambodia (2), China (2), the Lao People's Democratic Republic (2), Malaysia
(10), Papua New Guinea (2), the Philippines (2), the Republic of Korea (1) and Viet Nam
(Hanoi [1] and Ho Chi Minh [1]). In addition to the WHO Secretariat, temporary advisers from
United States Centers for Disease Control and Prevention (US CDC), National Institute of
Infectious Diseases (NIID) of Japan and Public Health Laboratory Centre (PHLC) Hong Kong
attended the training as facilitators (Annex 1 Participant list).
-3-
2. PROCEEDINGS
2.1
Training programmes
The training programme consisted of one day of lectures followed by four days of
practical sessions, country presentations and discussions. Proficiency panels and kits were
distributed to all participating laboratories at the conclusion of the training sessions. A USB,
including copies of all presentations made during the training, worksheets and all related
materials, was distributed upon completion of the workshop. A timetable of the training meeting
is attached as Annex 2. Instructions and protocols for the practical sessions are provided in
Annexes 3 and 4.
2.2
Lecture sessions
The presentations during these lecture sessions are attached in Annex 5 and 6.
2.2.1
Session 1: JE/AES surveillance and laboratory network
David Featherstone, WHO Headquarters, presented the progress and challenges facing
laboratory-based JE/AES surveillance. Strategies for surveillance and JE characterization were
outlined together with the development of the JE laboratory network and integration into the
wider vaccine preventable diseases (VPD) network. Challenges for maintaining a high standard
of surveillance and laboratory performance were discussed, identifying potential funding needs
and sources.
Dr Youngmee Jee gave an overview of JE and acute encephalitis syndrome (AES)
surveillance in the Western Pacific Region. The geographic distribution, seasonality and clinical
aspects of JE/AES were reviewed alongside a brief overview of the burden of disease, including
morbidity and mortality. Updates on the newly formed JE laboratory network were presented,
particularly a review of the last hands-on training session, data reporting issues and reports from
the VPD laboratory networks meeting. An outline of the quality assurance programme,
including confirmatory testing and onsite accreditation, was presented as well as challenges and
future plans for the JE network.
2.2.2
Session 2: Quality assurance of the laboratory network and Global Specialized
Laboratory (GSL) activities
David Featherstone presented an overview of the quality assurance and proficiency
programmes of the laboratory network. Methods for improving such programmes and
troubleshooting solutions were discussed. The accreditation process was described and the
importance of the quality assurance programme highlighted.
Dr Tomohiko Takahashi from the National Institute of Infectious Diseases (NIID)
presented the activities of the GSL and the national JE surveillance activities in Japan. The JE
surveillance system in Japan includes animal host surveillance as well as human case detection.
Data is analysed by a Geographic Information System. The number of annual cases in Japan has
been reduced to fewer than 10 with the introduction of the JE vaccine into the National
Immunization Programme (NIP) in 1995. Other activities of the laboratory include hands-on
training courses for prefectural laboratories and technical support through distribution of ELISA
kits and the JE vaccine quality assurance testing.
-4-
2.2.3
2.2.3.1
Session 3: GSL, Regional Reference Laboratories (RRL) and country reports
China
Dr Fu Shihong from the China CDC and Dr Hongxia Ma from the Henan CDC presented
the national JE experience and activities of the RRL. JE is one of 39 designated notifiable
diseases in China, and a nationwide immunization programme in the 1970s, with an attenuated
vaccine introduced in the 1990s, dramatically reduced the number of cases so that the disease
now has a low prevalence.
JE is endemic in 28 of 31 provinces in China and most cases occur in southwest provinces
of the country (Yunnan, Sichuan, Guizhou, Chongqing and Shanxi). Those provinces have
1>100 000 JE incidents a year. In Yunnan Province, JE accounts for over 50% of acute fever
cases in summer. Mosquitoes are collected for virus isolation. In 2008, 16 JE virus strains were
detected along with other viruses among 40 viral strains. Proficiency testing samples for
provincial laboratories are prepared and coordinated by the China CDC. The number of
laboratories participating in annual JE proficiency testing arranged by the China CDC increased
from four in 2006 to 15 provincial and six prefectural laboratories in 2010 with 100% accordance
using the Beixi kit.
-5-
2.2.3.2
Cambodia
Am Chanthan from the National Institute of Public Health Laboratory (NIPH) presented
on the current status of JE sentinel surveillance in Cambodia. Hospital-based sentinel
surveillance for suspected meningoencephalitis (ME) cases among children under 15 years old
started in 2006 by the Cambodia Centers for Disease Control and Prevention (Cambodia CDC),
NIPH, the National Immunization Programme/Ministry of Health, the Program for Appropriate
Technology in Health (PATH) and WHO.
Six selected hospitals used the Panbio JE-Dengue IgM Combo ELISA kits for testing.
Over four years, 3002 samples (sera and cerebral spinal fluid (CSF)) were collected from 1155
suspected cases of JE/AES. In order to clarify the extent of the JE disease burden, it is necessary
to strengthen the NIPH laboratory and sentinel sites through internal quality control measures as
well as external quality assurance measures such as proficiency and confirmatory testing.
2.2.3.3
The Republic of Korea
Jung-Eun Cho from the Korea Center for Disease Control and Prevention (Korea CDC)
presented the national JE experience and the activities of the JE RRL. Vaccination beginning in
the 1960s led to the rapid reduction of JE cases and only less than 10 cases were reported each
year recently. The surveillance season runs from June to October for mosquitoes and from July
to November for pigs. The same genotype 1 strain has been circulating in the Republic of Korea
since 1995. However, in 2010, one genotype 1 strain was detected from a mosquito pool
collected on 4 October in Gyenong-Nam Province. Over 20 cases were confirmed in 2010 and
there were cases until November. Age distribution of cases showed not only elderly people but
also the younger age group was affected in 2010. The Korea CDC has developed a real-time
polymerase chain reaction (RT-PCR) kit for vector surveillance that is also aimed at the regional
trial. Evaluation and implementation of the RT-PCR kit for 2011 is continuing as well as
research on the shifting age distribution of JE cases in 2010.
2.2.3.4
The Lao People's Democratic Republic
Sinakhone Xayadeth and Khuanta Duangmala from the National Center for Laboratory
and Epidemiology (NCLE) presented the JE country report from the Lao People's Democratic
Republic. There have been continued efforts to strengthen and increase JE surveillance as well
as the data management system and the quality of laboratory testing. No one from the NCLE
attended the first JE hands-on training in 2009 and two participants took part in this regional
training for the first time. Still, this laboratory participated in quality assurance measures such as
the 2010 WHO JE proficiency sent after the training and confirmatory testing. Confirmatory
testing samples were sent to NIID Japan in July 2010 and a 70.16% concordance rate (12/15)
was observed.
2.2.3.5
Malaysia
Nurul Asshikin bt. Ruslan from the Institute of Medical Research (IMR) presented the JE
country report for Malaysia. The first JE case was detected in 1952 and viral encephalitis is one
of notifiable diseases in Malaysia. However, JE cases are not quantified accurately within the
country as only passive surveillance is being conducted.
There is no national vaccination programme and only Sarawak state started statewide JE
vaccination, in 2002. In peninsular Malaysia and Sabah, JE vaccination is conducted only within
a 2 km radius when there is a JE case. More than 130 laboratories confirmed JE cases were
detected in 2010, from Selangor. Laboratory-based surveillance reports that 85% of JE cases are
-6-
in children 5-15 years old. Diagnostic methods used included the haemagglutination inhibition
test (HIT), IgM capture ELISA, viral isolation and RT-PCR and quality assurance measures are
well in place, including regular confirmatory testing. The laboratory is also undergoing
International Organization for Standardization (ISO) 15189 and participates in (external quality
assessment (EQA) from the Royal College of Pathologists of Australasia (RCPA) (Arboviruses)
as well as WHO PT.
Distribution of Laboratory Confirmed JE Cases by States
(2007-Oct 2010)
Putrajaya
Kuala
Lumpur
Sarawak
Sabah
Kelantan
2010
Terengganu
Pahang
2009
Johor
2008
Malacca
N Sembilan
Selangor
Perak
Penang
Kedah
160
140
120
100
80
60
40
20
0
Perlis
No of JE Cases
2007
State
2.2.3.6
Papua New Guinea
Oscillah Kaminiel and Ernest Velemu from the Central Public Health Laboratory (CPHL)
presented their laboratory network. There is no diagnosis or surveillance for JE conducted in
Papua New Guinea, but testing is soon to be established at CPHL following this training.
Measures to set up the quality assurance programme are being implemented in order to establish
a fully functional WHO JE national laboratory in the country.
2.2.3.7
The Philippines
Analisa Bautista and Ava Kristy Sy from the Research Institute of Tropical Medicine
(RITM) presented the state of JE surveillance in the Philippines. There is no clear burden of JE
demonstrated in the country. AES, meningitis and meningococcal disease surveillance was
integrated into the Philippines Integrated Disease Surveillance and Response System in 2008 and
AES and bacterial meningitis are weekly notifiable diseases. Sentinel surveillance for
Etiological Diagnosis of Meningitis/Encephalitis/Meningoencephalitis (CNS Infections) was
implemented at five sites with 50 cases per site in 2009.
During the period 2009-2010, CSF and serum samples were collected from 212 cases and
17% of the cases were JE IgM-positive. The laboratory obtained 100% for the first proficiency
test and confirmatory testing of 64 samples conducted at the Korea CDC showed a 95%
concordance rate. In collaboration with the Virology Department, School of Medicine, Tohoku
University, Sendai, Japan, this laboratory collected adult mosquitoes in Tarlac Province, Region
III, between May 2009 and April 2010 by animal-baited trap method. Genotype III Japanese
Encephalitis Virus (JEV) was detected from Cx. tritaeniorhynchus collected in November 2009.
-7-
2.2.3.8
Viet Nam
Dr Do Phuong Loan from the National Institute of Hygiene and Epidemiology (NIHE)
Hanoi presented the status of JE and the laboratory network in northern Viet Nam. Vial
encephalitis cases are reported monthly from three sentinel sites: north (1), central (1) and south
(1), including the National Children's Hospital in Hanoi and Ho Chi Minh City. In 2009, 1042
viral encephalitis cases, 24 of them fatal, were reported in Viet Nam.
2009
Northern
Provinces
Central
Provinces
Southern
Provinces
Highland
Provinces
Total
Morbidity
699
73
234
81
1042
Mortality
5
2
16
1
24
A review of JE surveillance over the last 10 years shows a decrease in viral encephalitis
but an increase in JE, with most cases being children 10-15 years old. In 2009, JE vaccination
coverage rates for the third dose in all provinces was higher than 90%. But in 2010, JE
vaccination coverage rates for the third dose in Hanoi and Dien Bien were only 70.2% and
68.9%. In northern Viet Nam, 26 out of 29 provinces and 280 out of 325 districts introduced the
JE vaccination by 2010. This laboratory also conducts molecular detection of JE viruses and the
whole genome of genotype 1 JEV was sequenced. JE IgM antibody capture (MAC-ELISA) kits
have been produced in NIHE and provided to seven provincial preventive medicine centres and
five hospital laboratories in Viet Nam. This kit was registered under ISO 9001 in 2008.
Dr Huynh Phuong Thao from the Pasteur Institute (PI) presented the JE situation in
southern Viet Nam. Results of the last 10 years of vector and animal host surveillance were
reviewed as well as the increase in vaccination of under-5 children. A total of 794 AES cases,
30 of them fatal, were reported from southern Viet Nam in 2009 and 2010 (as of October). By
the end of 2010, all 20 provinces and 204 districts will introduce JE vaccination for under-5
children. Three sentinel sites in Binh Duong, Ben Tre and Ho Chi Minh sent AES samples to PI.
This laboratory also produces in-house JE MAC-ELISA kits and provides those kits to provincial
laboratories. While provincial laboratories in southern Viet Nam only use the PI in-house
MAC- ELISA kits for serological testing, the PI can perform hemagglutination immunization,
RT-PCR and virus isolation in addition to MAC-ELISA.
Both laboratories participated in confirmatory testing and WHO JE PT programme in 2009
and is undergoing ISO 15189 certification and completed standard operating procedures (SOPs)
and other requirements.
2.2.4
Session 4: JEV IgM ELISA
David Featherstone presented a general introduction to IgM assays for JE while
Dr Barbara Johnson, of the United States of America Centers for Disease Control and Prevention
(US CDC), outlined the step-by-step procedures for the PanBio JE-Dengue IgM combo ELISA
assay as well as an evaluation of both in-house and commercial kits.
2.3
Practical sessions
The hands-on training session was conducted in the Public Health Laboratory Centre
(PHLC) in Hong Kong (China) and consisted of three days of practical sessions. Participants
-8-
were separated into seven pairs. Instructions and protocols for the practical sessions are provided
in Annex 4.
JE PCR was added to the agenda between 18 and 19 November to familiarize the
participants with the molecular detection of JE viruses. PHLC staff explained the RT-PCR
method used in PHLC and RNA extraction and RT-PCR procedures were performed by
participants and gel electrophoresis of PCR products were performed by PHLC staff.
A practical session on the first day focused on the JEV IgM ELISA assay to test serum
samples. All participants were given serum samples consisting of JE-positive, dengue-positive
and negative samples and completed their laboratory assays where the results were then
analysed, compared and validated.
A second day practical session was conducted to process CSF samples using the ELISA
kits. Test results were analysed and evaluated. There was also a short session on specimen
collection, preparation and shipment for virus isolation and serology.
The third session processed the serum samples again to be compared with the results from
the first session. There were also short sessions on data entry, AES data management and
reporting requirements.
During incubation periods between all of the sessions, participants learnt how to calibrate
and maintain micropipettes and ELISA equipment for laboratory quality assurance and control.
At the end of training, the second WHO JE proficiency panel samples were given to
participants. The samples consisted of six serum samples (25uL) and five CSF samples (40uL).
Participants were requested to submit the results within 14 days using appropriate assays, which
are used in their own laboratory.
3. CONCLUSIONS
The main conclusions of the workshop were as follows:
3.1
General
3.1.1
The three main objectives of the training were fully achieved during five days of
intensive hands-on practical sessions, lectures and group work. By the end of the workshop,
technical capacity and knowledge of all participants was enhanced and they were able to perform
ELISA for laboratory diagnosis of JE. The participants understood laboratory quality assurance
for JE diagnosis and were fully familiarized with the requirements for WHO accreditation for the
JE laboratories and laboratory data management using the WHO JE laboratory data reporting
format for reporting to the Western Pacific Regional Office. In addition, a practical session on
JE PCR procedures was performed between 18 and 19 November to familiarize participants with
the molecular detection method.
3.2
Evaluation of the training workshop
3.2.1
The participants were positive in their feedback and the workshop met its objectives.
Administrative arrangement for the workshop by the PHLC was excellent. A locker was
-9-
allocated to everyone during the workshop and instructions to go to the laboratory or lecture
room for each session clearly was given to the participants by PHLC staff.
3.2.2
Participants were encouraged to contact each other, the facilitators from US CDC and
WHO to follow up practical issues such as quality assurance, data reporting and data
management to further strengthen JE laboratory activities in the Region.
3.2.3
With full support from PHLC staff, the training ran very smoothly throughout the
practical and lecture sessions. The participants efficiently completed all of the practical sessions
and understood issues addressed during the training. Among 14 participants, four participants
(from Cambodia, the Philippines and Viet Nam) also participated in 2009 training and 10
participants were new to the WHO JE hands-on training.
3.2.4
Between practical sessions, how to maintain ELISA readers and washers and calibrate
micropipettes were demonstrated and participants also had a chance to participate in calibration
of micropipettes.
3.2.5
The addition of JE PCR was welcomed by the participants from China, the Philippines
and Viet Nam since they are considering introducing or conducting JE PCR.
3.2.6
The training schedule provided adequate time for performing the practical procedures at
a reasonable pace and for information-sharing among the participants. The duration of each
presentation also allowed adequate time for further discussion on theoretical and technical issues.
3.3
Outcomes of the training
3.3.1
All participants were further familiarized with the Panbio JE-Dengue Combo IgM
ELISA assay, analysis and validation of results, calibration and maintenance of micropipettes
and ELISA equipment, laboratory quality assurance and quality control and laboratory data
reporting and management. They were also familiarized with JE PCR procedures.
3.3.2
Participants were familiarized with the requirements for WHO accreditation for the JE
laboratories and laboratory data management using the WHO JE laboratory data reporting format
for reporting to the Western Pacific Regional Office.
3.3.3
At the end of the workshop, the second WHO JE proficiency testing panel samples
were distributed to participants. Participants from the Lao People’s Democratic Republic also
carried PT samples for the Mahosot Hospital.
3.4
Follow-up of the workshop
3.4.1
Participants were requested to report the results of proficiency panel samples within 14
days after the samples arrive in the laboratory. Most laboratories that participated in the training
reported results within 14 working days.
3.4.2
The results of the first proficiency panel samples were received from 10 network
laboratories, including CPHL Papua New Guinea. Three laboratories in Japan and Viet Nam
(Hanoi and Ho Chi Minh City) used in-house assays and China used the Beixi kit. All other
network laboratories in the Region used Panbio kits. The results of four laboratories using
non-Panbio assays were compared with the results of laboratories using the CDC assay. All 10
laboratories scored 100%.
ANNEX 1
WORLD HEALTH
ORGANISATION MONDIALE
ORGANIZATION
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 PACIFIC REGION
Hong Kong (China)
15-19 November 2010
ENGLISH ONLY
LIST OF PARTICIPANTS, TEMPORARY ADVISERS AND SECRETARIAT
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
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
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
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
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
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
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
Private Mail Bag No. 1,
Boroko
National Capital District
Telephone: +67 5 3248199
Facsimile: +67 5 3256342
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
Ms Ava Kristy Sy
Bacteriologist I
9002 Research Drive
FCC Compound, Alabang
Muntinlupa City 1781
Philippines
Telephone: (632) 8097120
Facsimile: (632) 8097120
REPUBLIC OF KOREA
VIET NAM
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
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 :
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
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
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
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
SECRETARIAT
Dr Youngmee Jee
Scientist (Laboratory Virologist)
Expanded Programme on Immunization
Regional Office for the Western Pacific
United Nations Avenue
1000 Manila
Philippines
Telephone: +63 2 528 9744
Facsimile: +63 2 526 0279
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
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
Day 1, Monday, 15 November 2010
08:30
08:45
09:00
09:10
09:20
Registration
Completion of pre-assessment questionnaire
Opening remarks
Workshop objectives
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
JE control and lab net progress in the Western
Pacific Region
Coffee break
10:00
10:30
Session 2
Quality assurance of the JE lab net and global
specialized laboratory (GSL) presentations
11:00
11:20
Quality assurance and proficiency in the laboratory
Role of the United States Centers for Disease
Control and Prevention (US CDC) and WHO JE
GSL: evaluation of kits
Activities of National Institute of Infectious Diseases
(NIID) GSL and JE surveillance/laboratories
Discussion
Lunch break
11:50
12:10
12:30
Session 3
13:30
13:50
14:10
Regional reference laboratory (RRL) and
national laboratory reports
Chinese Center for Disease Control and Prevention
(China CDC)
Korea Centers for Disease Control and Prevention
(KCDC)
Cambodia, Lao People's Democratic Republic,
Malaysia (15-minute presentation , 5-minute
Q and A)
Dr Wilina Lim
Dr Youngmee Jee
Dr Youngmee Jee
Dr Barbara Johnson
15:10
15:30
Coffee break
Papua New Guinea, Philippines, Viet Nam (2)
Session 4
Introduction of JE virus-specific
immunoglobuline M (JEV IgM) enzyme-linked
immunosorbent assay (ELISA)
General Introduction to IgM assays
Lecture/demonstration of JEV IgM ELISA (serum)
Tour of laboratory and demonstration of ELISA
equipment
Adjourn for the day
16:30
17:00
17:30
18:00
Dr Barbara Johnson
Day 2, Tuesday, 16 November 2010
08:30
08:45
12:00
13:30
15:30
16:00
17:00
Introduction to the ELISA practical
Laboratory practice: JEV IgM ELISA (serum)
(during incubation time: calibration, maintenance of
ELISA equipment, micropipettes, etc.)
Lunch break
Continuation: laboratory practice - JEV IgM ELISA
Coffee break
JEV IgM ELISA reading and calculation of results
Evaluation of ELISA test results and discussion
17:30
Adjourn for the day
Mr David Featherstone,
facilitators and
participants
Participants
Participants
and facilitators
Day 3, Wednesday, 17 November 2010
08:30
09:00
09:30
10:00
10:30
13:00
14:00
15:00
15:30
15:45
16:15
17:00
Discussion of Day 2 activity
Lecture and demonstration of JEV IgM ELISA –
cerebrospinal fluid (CSF)
Laboratory practice: JEV IgM ELISA (CSF)
Coffee break
Lunch break
JEV IgM ELISA reading and calculation of results
Coffee break
Specimen collection, preparation and shipment for
virus isolation and serology
Adjourn for the day
Question for the night
facilitators and
participants
Participants
Participants
Participants
and facilitators
Day 4, Thursday, 18 November 2010
08:30
08:45
10:30
11:00
12:30
13:30
17:30
Discussion on Day 3 activity
Laboratory practice: ELISA (serum samples)
Coffee break
Continuation: laboratory practice – ELISA
Lunch break
JE PCR
AES data management and reporting requirements
Practical session on data entry
Adjourn for the day
facilitators and
participants
facilitators and
participants
PHLC
Dr Youngmee Jee
Participants
Day 5, Friday, 19 November 2010
08:30
09:00
10:00
10:30
11:00
12:30
13:30
15:30
16:30
Discussion on Day 4 activity (return quiz)
JE PCR continued
Global specialized laboratory testing methods ELISA, plaque reduction neutralization testing
(PRNT) and decision algorithm
Consolidation of laboratory test results and
classification
Coffee break
Course assessment and quiz
Lunch
Next steps and summary of assessment
Closing ceremony: presentation of training
certificate
Distribution of proficiency panels and kits to network
laboratories
PHLC
Dr Barbara Johnson and
and Dr Youngmee Jee
Round table
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
JE
DEN
DEN
Interpretation
Panbio IgM
Panbio
IgM
units
result units
result
<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
9-11
Eqv
9-11
Eqv
<9
Neg
>11
Pos
Calculate JE/ Dengue ratio :
9-11
Eqv
>11
Pos
>11
Pos
<9
Neg
>11
Pos
9-11
Eqv
> 11
Pos
> 11
Pos
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
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
WHO JE Workshop
18-19 November 2010
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)
WHO JE Workshop
18-19 November 2010
Objectives:
1.
2.
To have an idea on molecular testing (RNA extraction and RT-PCR) of JE virus
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 1
WHO JE Workshop
18-19 November 2010
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)
(5)
Mix by pulse-vortexing for 15 seconds. Incubate at room temperature for 10 minutes.
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.
Pulse-spin all the vials.
(7)
Page 2
WHO JE Workshop
18-19 November 2010
(8)
(9)
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.
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 3
WHO JE Workshop
18-19 November 2010
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 4
WHO JE Workshop
18-19 November 2010
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)
(3)
Label the 0.2ml PCR tubes.
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.
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.
(5)
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 5
WHO JE Workshop
18-19 November 2010
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)
(5)
Add 25µl of SYBR SafeTM DNA gel stain to each bottle of agarose gel. Swirl to mix.
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 6
WHO JE Workshop
18-19 November 2010
(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)
(3)
Calculate the number of wells needed and add 2µl of the 6X loading dye into each well.
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)
(7)
Load 10µl of the 100bp DNA ladder one of the wells as molecular size marker.
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 7
WHO JE Workshop
18-19 November 2010
Date: _______________
Appendix 1 Worksheet for RNA extraction-QIAamp Viral RNA Mini Kit
Reagent
Lot No.
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 8
Expiry Date
WHO JE Workshop
18-19 November 2010
Appendix 2 Detection of JE virus by One-step RT-PCR
Volume per test (µ
µl)
Reagent
QIAGEN 5X PCR buffer (kit)
10
dNTPs (kit)
2
Primer JE-1 (5 µM)
6
Primer JE-2 (5 µM)
6
Enzyme mix (kit)
2
RNase inhibitor
0.5
H2O (kit)
18.5
Total
Total volume (µ
µl)
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: _______________
Page 9
Objectives of this workshop
Introduction of
Workshop Objectives
2nd Intercountry Hands on training on the
Laboratory Diagnosis of Japanese encephalitis
November 15-19 2010
Day 1
• Session 1 JE/AES surveillance and Laboratory
network
Coffee break
• Session 2 Quality assurance of the JE labnet and GSL
presentations
Lunch
• Session 3: RRL and National Lab reports
Coffee break
• To further enhance knowledge and skills of national
JE laboratory staff in:
a) performing ELISA for laboratory diagnosis of JE,
and
b) 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 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.
• Session 4. Introduction of JEV IgM assays
– Demonstration of ELISA assay
– Tour of the laboratory
Day 3
• ELISA practical using CSF samples
Use Panbio kit
(During incubation times, calibration of
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
equipments)
• Data management for JE/AES
1
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)
2
PanBio JE-Dengue
IgM Combo ELISA kit
Demonstration of the
PanBio Japanese
encephalitis-Dengue IgM
Combo ELISA procedure
Test up to 43
samples/plate
Included in the kit
• Precoated microwell
strips
• Reagents, diluents, and
buffers
• Calibrators and positive
and negative controls
Barbara W. Johnson
US Centers for Disease Control and Prevention
Division of VectorVector-Borne Diseases
Arboviral Diseases Diagnostic and Reference Laboratory
The 2nd Intercountry HandsHands-on Training Workshop on the Laboratory
Public Health Laboratory Centre, Hong Kong
Plate setup and worksheet
for testing 11 samples
Principle of JE-DEN Combo IgM ELISA
HRP
HRP
HRP
DEN
JE
HRP
DEN
JE
Microwells precoated with anti-human IgM
JE DEN JE DEN
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
-
-
4
4
+
+
5
5
JC
DC
6
6
JC
DC
7
7
JC
DC
8
8
1
1
9
9
2
2
10
10
3
3
11
11
CONTROLS:
HRP
JE
Substrate binds to bound Mab/HRP
Colorimetric measurement of JE
DEN IgM reactivity in sample
-: Negative Serum
HRP
JE
JE+
+: Positive Serum
or
JC: JE Calibrator
DC: DEN Calibrator
DEN-
Assay Preparation
Plate Washing
1. Equilibrate kit reagents and microwell strips to room
temperature
2. Remove required number of microwell strips and insert
them into the frame (4 strips used for 11 samples)
3. Dilute controls and samples
Pos and neg controls, JE and DEN calibrators, and serum
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:
Wash solution
1:20 Dilution (50 ml wash buffer concentrate
into 950 ml distilled H2O)
Washing methods
Plate Washer
Hand Washing with squirt bottle
Hand washing with multichannel pipette
# wells X 100 ul/well
Wash plates X6 at each wash step
Dilutions:
Assay
+
PC, NC, JC, and DC
Prepare 1/250 JE and
DEN antigen dilutions
Procedure
Serum , PC, NC, JC, DC - 1:100
•10 ul sample/1ml diluent
CSF - 1:10
•25 ul CSF/225 ul diluent
+
PC, NC, JC, and DC
Prepare 1/250 JE and
DEN antigen dilutions
To JE and DEN columns (X2)
JE or DEN Antigen - 1:250
•10 ul antigen/2.5 ml diluent
Mixtures
To JE or DEN wells (X16)
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
Quality control and calculations of validity
Information on kit box
Kit expiry date
All wells (X32)
All wells (
Determining test validity
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
JE and DEN calibrator
factors and neg Abs
(batch specific)
Calculations:
Acceptable ranges of
valid
JE+ or DEN+/cut-off
ratios (batch specific)
1. JE Pos Control OD/JE cutoff ratio
2. DEN Pos Control OD/DEN cutoff ratio
3. Neg Control OD
JE Cut-Off value = Mean OD of JE Calibrators X JE Calibration factor
DEN Cut-Off value = Mean OD of DEN Calibrators X DEN Calibration factor
Interpretation of Validity :
Are these 3 values within
acceptable range?
Calculations of Panbio Units
TEST IS VALID IF:
JE Pos, DEN Pos, and Neg controls are all within acceptable ranges
and
expiration date has not passed
Cutoff value:
JE: Average of 3 JE calibrator ODs X kit JE calibration factor
DEN: Average of 3 DEN calibrator ODs X kit DEN calibration factor
Index value:
JE: Sample JE OD/JE Cutoff Value
DEN: Sample DEN OD/DEN Cutoff Value
If test is NOT valid, do not proceed to calculations – repeat test!
PanBio units:
If test meets validity requirements, calculate JE and DEN Panbio units
of each sample
JE: JE Index value X 10
DEN: DEN index value X10
Interpretation
Panbio Units
IgM result
<9
Negative
9-11
Equivocal
≥11
Positive
Ratio
Example: Calculations and Interpretations
Kit specifications:
Kit lot: 06193
Expiration date: 12/12/2011
JE Calibration Factor: 0.63
DEN Calibration Factor: 0.38
Neg C OD <0.25
JE PosC OD/cut-off ratio: 1.1-6.0
DEN PosC OD/cut-off ratio: 3-17.0
If either JE or DEN is IgM positive
calculate JE/DEN ratio
JE Panbio units
DEN Panbio units
≥1 JE positive
<1 Den positive
Step 1: Determine validity of test
Kit lot: 06193
Expiration date: 12/12/2011
Neg C OD <0.25
Unit calculations
JE Calibrator Avg OD = 0.574
JE 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
DEN PosC OD = 3.41
JE NegC OD = 0.063
DEN NegC OD = 0.066
JE PosC OD/JE cut-off = 2.12/0.362 = 5.86
DEN PosCOD/DEN cut-off = 3.41/0.506 = 6.74
Kit lot 06193
Expiration date 12/12/2011
Neg C OD <0.25
Unit calculations
JE PosC OD = 2.12
DEN PosC OD = 3.41
Sample 1
Kit lot 06193
Test valid?
JE NegC OD = 0.063
DEN NegC OD = 0.066
Kit lot 06193
Neg C OD <0.25
Unit calculations
Cut-off values
JE Cut-off = 0.362
Den Cut-off = 0.506
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+
Determining test validity: Yes valid test
JE PosC OD = 2.12
DEN PosC OD = 3.41
JE NegC OD = 0.063
DEN NegC OD = 0.066
DEN Panbio Units = 1.09/0.506 X 10 = 21.54 = IgM+
JE/DEN Ratio= 41.24/21.54 = 1.91
Interpretation: JE Positive
Sample 2
Sample 3
Kit lot 06193
Kit lot 06193
Cut-off values
JE Cut-off = 0.362
Den Cut-off = 0.506
Cut-off values
JE Cut-off = 0.362
Den Cut-off = 0.506
JE OD = 0.144, Den OD = 0.102
JE OD = 0.085, DEN OD = 0.641
JE Panbio Units= 0.085/0.362 X 10 = 2.35 = IgM-
JE Panbio Units = JE OD/JE Cutoff X 10 = 0.144/0.362 X 10 = 3.98 = IgM-
DEN Panbio Units=0.641/0.506 X 10 = 12.67 = IgM+
DEN Panbio Units = 0.102/0.506 X 10 = 2.01 = IgM-
JE/DEN ratio = 2.35/12.67 = 0.185
Interpretation: No detectable IgM*
Interpretation: DEN Positive
*Request 2nd sample if 1st collected <7-10 days post-illness
Before you begin …..
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
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
National Institute of Infectiouse Diseases
国立感染症研究所
（旧国立予防衛生研究所）
The laboratory of VectorVector-Borne
Visruses, Virology 1st, National
Institute 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
INTRODUCTION
Laboratory of Vector-B
Borne Viruses
Geographical distribution of JE cases, 2000～Mar．2010
(n=56, as of Apr 2010)
Kyushu
Chugoku
Hokkaido
Chubu
We mainly take care of following viruses.
•
•
•
•
•
Japanese encephalitis virus
Dengue virus
West Nile virus
Yellow fever virus
Chikungunya virus
Eight arbovirus
centers in Iapan
arbo
Miyagi
Kobe
Hiroshima
Tokyo
Aichi
Kumamoto
Osaka Mie
Kinki
Tohoku
Shikoku
Kanto
n= 3
n= 3
n= 10 (fatal case 2)
n= 7
n= 7
n= 5 (fatal case 1)
n= 1
n= 8 (fatal case 1)
n= 5
n= 7 (fatal case 1)
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.
Introduction②-Data flow
Supports for prefectural institute
Case-based surveillance
Local Level data
management
• 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
Sentinel sites for agent surveillance
Sentinel clinics / hosp.
Clinical isolates
and specimens
Summary Report (wk/mo)
Case Report
• Standard control serum for NT
Sentinel surveillance system
All clinics / hosp.
Local health centers
input the data to central server
Prefectural
Level data
mngm’t
Pref. Health
Depts.
Pref. IDSCs
input the data to central server
National
Level data
mngm’t
MHLW
Reports
Specimens
National IDSC
（NIID）
Pref. PHIs
（Laboratories）
Quarantine stations
Laboratories
（NIID）
Information
dissemination
JE vaccination history in Japan ,2009
Japanese encephalitis HI antibody
prevalence of swine in Japan 1972～2005
－National Epidemiological Surveillance of Vaccine Preventable Diseases－
year
(cases）
(cases）
1972
（22）
22）
1973
（70）
70）
1974
（6 ）
1975
（27）
27）
1976
（13）
13）
1977
（5 ）
1978
（88）
88）
1979
（86）
86）
1980
（40）
40）
1981
（23）
23）
1982
（21）
21）
1983
（32）
32）
1984
（27）
27）
1985
（39）
39）
1986
（26）
26）
1987
（37）
37）
1988
（32）
32）
1989
（27）
27）
1990
（54）
54）
1991
（13）
13）
1992
（2 ）
1993
（4 ）
1994
（4 ）
1995
（2 ）
1996
（4 ）
1998
（2 ）
1999
（5 ）
2000
（7 ）
2001
（5 ）
2002
（8 ）
2003
（1 ）
2004
（5 ）
2005
（7 ）
1997
（4 ）
Not done
0%
＜50%
50～
50～80%
≧80%
Contribution to WHO as JE-GSL
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
2010/5/27
Titer Method is better for
differential diagnosis
sera
Anti-human IgM Ab coated plate
JE Den
Antigen
CSF
x 100
x 10
x 200
x 20
x 400
x 40
x 800
x 80
x1600
x160
x3200
x320
X6400
X640
X12800
X1280
Comparison between A(NIID) and B(Focus)
• Plate in house: A
Thermo Scientific Nunc MaxiSorpTM
Surface
• Plate of FOCUS Diagnostics kit: B
A
B
O.D.
Blank
0.12*
0.074
Empty
0.045
0.040
*Blocking agents is 1% BSA
IgM capture ELISA
Substrate (TMB)
Conjugate(HRP,AP,etc)
AntiAnti-Flavi Mono Antibody (6B6C)
30 min. at RT
Virus Antigen；
Antigen；JEV antigen
incubate overnight at 4℃
4℃
Test Serum
containing AntiAntiVirus IgM Antibody
1hr. at RT
AntiAnti-human IgM mAb
Capture Antibody
Blocking
Plastic Solid Phase
Evaluation the plate by IgM positive serum
Dilution; x103
Focus Diagnostics
_Plate
NIID
_Plate
1
2
4
8
16
32
2.138
1.805
1.168
0.627
0.320
0.161
2.043
1.704
1.144
0.676
0.342
0.171
Blocking reagents
Focus
Pos
1000
2000
4000
8000
16000
32000
Neg x100
Blank
1%BSA
1.405
0.872
0.438
0.217
0.098
0.086
0.091
0.032
inhouse
BA
1.18
0.792
0.443
0.24
0.114
0.093
0.085
0.045
1.305
0.845
0.485
0.263
0.157
0.105
0.091
0.062
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.
BSA: bovine serum Albumin, BA:Block ACE
Contribution 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 !
Evaluations of In-house and
Commercial JEV IgM ELISA Kits
Background:
Standardization of JE testing by JEV IgM ELISA is needed
throughout the WHO JE laboratory network (LabNet)
The 2nd Intercountry Hands-on Training Workshop on the
Laboratory Diagnosis of Japanese Encephalitis in the
Western Pacific Region
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
Barbara W. Johnson
U.S. Centers for Disease Control and Prevention
Division of Vector-Borne Diseases
Recent publications of JE IgM ELISA kit evaluations
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
Jacobson 2007
(USAMRID)
sera (n=360)
Kit
% Sens
% Spec
Ravi 2009
(CDC)
CSF (n=60)
% Sens
% Spec
JE-DEN
Combo
89
98-99 65-80 95-98
JE
Detect
99
56-96
JEVChex
97
65-96
90
98
Lewthwaite 2010
Robinson 2010
Khalakdina 2010
(USAMRID)
(VT)
(CDC)
sera (n=350)
sera & CSF (n=371) sera & CSF (n=520)
% Sens
69(S)
68(C)
57(S)
75(C)
% Spec
% Sens
% Spec
97
39(S)
20-30
(C)
99
57(S)
53(C)
97
20(S)
17(C)
97
97(S)
98(C)
% Sens
71-80 95-98
93
CDC limited evaluation
Evaluation 1:
Assays evaluated:
CDC analysis of kit
performance and
sensitivity and specificity
limits
&
Recommendations
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.
% Spec
89
Summary analysis of kit performance
Sample set
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)
JE-DEN Combo
All samples
+
30
5
61
424
+
-
No. tested (n=520)
91
XCyton JEV CheX
+
-
No. tested (n=520)
InBios JE Detect
+
-
No. tested (n=454)
429
33
(24-44)
56
(44-67)
19
(12-28)
JE Detect
JEV CheX
CDC JEV IgM results
Panbio
Serum
+
24
4
37
229
+
6
24
61
30
233
JE-DEN Combo
1
195
JE-DEN Combo
CSF Adjusted
196
JE Detect
JEV CheX
12
417
429
12
49
61
7
226
233
5
25
30
5
191
196
38
30
68
12
374
386
29
22
51
10
209
219
9
8
17
2
165
167
Sensitivity evaluation: Comparison of CDC JEV IgM+ to kit
results by days post-onset of illness to specimen collection
%
Agreement
99
(97-99.5)
97
(95-98)
97
(95-98)
87
99.5
(97-99.7)
99
(96-99)
99
(96-99)
97
(94-99)
89
98
(96-99.3)
95
(92-98)
97
(94-99)
86
91
83
CSF
CSF
-
17
74
91
% Specificity
(95% CI)
All samples combined
Summary test results
Kit
% Sensitivity
(95% CI)
Kit
20
(10-37)
30
(17-48)
53
(31-74)
17
(7-34)
90
95
87
Sera
PanBio
39
(28-52)
57
(43-70)
20
(12-31)
InBios JE Detect
XCyton JEV CheX
88
81
Sensitivity evaluation:
Comparison of CDC JEV IgM+ to kit results by CDC P/N ratio
All Sample types combined (N=68)
30
All sample types combined
50
45
25
No. JEV IgM Positive
40
20
15
10
N0. JEV IgM+
35
JE-DEN Combo
JE Detect
JEV CheX
CDC JEV-DEN ELISA
30
JE-DEN Combo
JE Detect
JEVCheX
CDC
25
20
15
5
10
0
<3
3-7
7-14
>14
5
Unknown
0
Days from onset of illness to specimen collection
Low
Medium
(P/N<10)
Sensitivity evaluation sera:
IgM detection endpoint titrations of 3 high CDC JEV IgM
positive serum samples
13000
CDC
P/N=12
1:12800
CDC
P/N=4.4
1:12800
High
(P/N=10-20)
(P/N>20)
Sensitivity evaluation CSF:
IgM detection endpoint titrations of 1 high and 1 low
CDC JEV IgM positive (1:10 dilution) CSF
CDC
P/N=14.61
1:12800
CDC
P/N=4.2
1:80
CDC
P/N=4.2
1:1280
12000
1200
11000
1000
9000
8000
7000
CDC
6000
5000
Inverness PanBio
4000
Xcyton
3000
Reciprocal Titer
Reciprocal Titer
10000
800
CDC
Inverness Panbio
Inverness Panbio Adjusted
Xcyton
Inbios
600
400
InBios
2000
1000
200
0
1
CDC
P/N=30
1:400
2
Sample
CDC
P/N=22
1:400
3
0
4
CDC
P/N=30
1:400
5
Sample
CDC
P/N=92
1:10
CDC
P/N=5.1
1:10
Evaluation 2: Panbio kit with specimens collected
from encephalitis surveillance in Cambodia
Evaluation 2 cont:
Assay evaluated:
Background
•JE-DEN IgM Combo ELISA, PanBio, Australia
Cambodian JE laboratories had recently begun using the Panbio
JE/DEN IgM combo ELISA kit
Sample set:
Cambodian MOH needed accurate JE burden to make decisions
on JE vaccination program
•1195 serum and CSF specimens
case definition
•Collected during AMES surveillance in Cambodia
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 3:
CDC
JE -/DEN +
JE +
Assays evaluated:
-
Panbio
JE +
JE -/DEN +
Neg
Total
•National Institute of Virology (NIV) India JEV IgM ELISA
104
18
13
6
82
8
39
91
755
149
191
776
* JE and DEN IgM ELISA + PRNT of S2 of discordant specimens.
Summary analysis of Panbio JE-DEN IgM Combo
ELISA kit performance
% Sensitivity
69.8*
% Specificity
96.8
% Agreement
84.3
Sample set:
•438- acute-phase serum and CSF specimens
case definition
•Collected during AES/AMES surveillance project in India and
Bangladesh
*CSF 78%; S1 58%; S2 72%.
Specificity evaluation:
Comparison of NIV JEV IgM+ results to CDC results
NIV
results
40
35
9%
30
(N= 34)
Mean NIV results
S OD/NC OD=4.35
20
CDC Results
CSF
All Samples
Positive
CDC-/NIV+
25
JEV IgM+
JEV IgMTotal
Negative
Positive
Serum
Negative
Positive
Negative
72
50
26
7
46
43
17
89
299
349*
1
27
156
163
16
62
143
186
15
10
86%
40%
*42 JEV IgM negative/DENV IgM positive
CDC+/NIV+
(N=46)
Mean NIV results
SOD/NC OD=15.3
5
0
≥2.1 <5
5 to 10
>10
N=29
N=15
N=36
NIV results (sample OD/negative control OD*)
Summary analysis of NIV assay performance
All Samples
CSF N=190
Serum N=248
81
96
74
86
85
96
96
77
76
% Sensitivity
% Specificity
43 CDC JEV IgM-/NIV JEV IgM+; 4 CDC DENV IgM+, 5 CDC
WNV IgM+ 21% (9/43).
*NIV JE positive: Sample OD/NC OD ≥ 2.1
% Agreement
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
CDC Results
NIV results
All Samples
Positive
JEV IgM+
JEV IgMTotal
CSF
Negative
Positive
Serum
Negative
Positive
Negative
Sensitivity
72 (69)*
50 (13)
17 (20)
299 (336)
89
349**
26
7
46 (43)
43 (6)
1
156
16 (19)
143 (180)
27
163
62
186
All Samples
CSF N=190
Serum N=248
80.9 (77.5)*
96.3
74.2 (69)
85.7 (96)
84.7 (92.5)
95.7
95.8
76.9 (97)
76.2 (89.9)
low with very acute specimens collected <7 days postonset of illness
Low
Kit
kit sensitivity with specimens with low CDC P/N (<10)
IgM detection in CSF near threshold of detection at 1:10 dilution
% Sensitivity
% Specificity
% Agreement
*Values in parentheses are results with cutoff raised from 2.1 to 5.
**42 JEV IgM negative/DENV IgM positive
Challenges
Way forward
Cut-offs may need to be modified to increase sensitivity or specificity
Essential that assays have undergone evaluation
Willingness or ability of manufacturers to modify and improve kits
varies
Acceptable levels of sensitivity and specificity need to be
determined
Unexpected modifications to kits by manufacturers may negatively
impact kit performance, requiring re-evaluation
Laboratories need to be aware of kit sensitivity and specificity
limitations when making diagnosis
Possible lot-to-lot variation, degradation in transport, storage
requirements impact performance
Insufficient kit production
Thank you!!
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
Plate coated with capture antibody
(anti human-IgM)
General Introduction to IgM
Assays
The 2nd Intercountry HandsHands-on Training Workshop on
the Laboratory Diagnosis of Japanese Encephalitis in the
Anti-human IgM
Public Health Laboratory Centre, Hong Kong (China)
Plate surface
David Featherstone, EPI/IVB
WHO Geneva
WHO Vaccine Preventable Disease Lab Network
Unused binding sites blocked with protein
Mixing of antigen and conjugate
"Monoclonal
Tracer" conjugate
Antigen
Blocking proteins
Plate surface
Wash away unbound IgG and add JE
Antigen with HRP enzyme conjugate
Add serum sample
IgM
JE IgM
JE antigen
Anti JE IgG
conjugated to HRP
JE specific IgM
Non JE IgM
1
Wash away unbound IgG and add JE
Antigen with HRP enzyme conjugate
Wash away unbound HRP enzyme and
add substrate
JE IgM
JE antigen
Anti JE IgG
conjugated to HRP
JE specific IgM
Non JE IgM
Add acid to stop reaction
Problems with flavivirus cross reactivity
JE and Dengue IgM
Positive
JE antigen
Anti JE IgG
conjugated to HRP
JE specific IgM
Dengue IgM
Problems with flavivirus cross reactivity
Dengue IgM only
Possible false positive
JE antigen
Anti JE IgG
conjugated to HRP
JE specific IgM
Dengue IgM
2
Laboratory Based JE/AES Surveillance
Progress, Challenges and Plans
Outline of Presentation
JE Disease
Strategy for surveillance
Role of the laboratory
Progress with development of JE LabNet
the Laboratory Diagnosis of Japanese Encephalitis in
the Western Pacific Region
Challenges in maintaining the momentum
Prospects for maintaining Sustainability
David Featherstone
EPI - IVB WHO Geneva
Clinical spectrum of JE virus infections
Strategy for Surveillance for JE
Neuroinvasive*
Neuroinvasive*
<1%
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
Asymptomatic
infection
or
nonspecific
febrile illness
Where JE immunization has been introduced:
99%
– Document impact of control measures.
*Acute encephalitis,
encephalitis, aseptic meningitis, acute flaccid paralysis
First steps in establishing JE
surveillance
•
Requirement for Laboratory
confirmation
"JE Core Working Group" established for developing
surveillance standards for JE in 2002
Japanese
Encephalitis Virus
HSV, VZV
– WHO, (HQ, SEAR & WPR), PATH, CDC,
University of Liverpool
•
– Identify high-risk populations
– Vaccination coverage estimation
– New disease transmission,
Field Test version published 2006 and finalized 2008
Acute
Encephalitis
Syndrome
Others
• Clinical case definition (AES)
• Laboratory criteria for confirmation
Polio, Entero 71
Coxsackie A
CSF
Measles &
Mumps
Serum
• Recommended types of surveillance
• Recommended minimum data elements
Nipah
• Performance indicators of surveillance quality
Dengue*
WNV*
• Principal uses of data for decision making
* Flaviviruses with antigenic cross reactivity with JE
1
Key Players in Establishing
JE Laboratory Surveillance
Development of the JE LabNet
SEARO
$
$
$
CDC
Ad hoc
Laboratory
Working Group
$
$
PATH
BMG
$
$
$
$
NICD
NIMHANS
• Quality assurance
– Tiered labs
$
$
$
Fort Collins
• LabNet Structure
WHO/HQ
WPRO
–
–
–
–
–
Type of Assay (IgM ELISA)
Procedures and protocols
Reporting
Laboratory Manual
Training workshops
Bangalore
$
$
$
$
CDC
$
Atlanta
GDD
$
–
–
–
–
–
–
• Standardization
Assay assessment
In-house controls
External quality assurance
Proficiency testing
Confirmatory testing
Accreditation
• Integration with JE
programme
AFRIMS
Bangkok
University of
Liverpool
$
$
Gradual and stepwise process
No Big Bang !
Integration with Current WHO VPD Lab
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
Building capacity
Training workshops
data
sam
Global
Specialised
Labs
ple
s Regional Reference
Labs
data
sam
Serology, isolation, PCR
National Labs
pleSerology capacity, some with virus isolation
s
&/or PCR
+
Sub-National Labs
Regional Lab staff to CDC x 6
QC
Serology
Challenges in maintaining the
momentum
LabNet capacity developed
Labs identified
Training workshops and meetings held
Assays assessed
Surveillance established
Reporting structure
Largest cost is establishment phase
QA programme developed
2 x SEAR 6 countries (JE IgM)
1 x SEAR 11 participants (Bacto)
1x WPR (JE IgM) 11 participants
from 6 labs (Seoul June 2009)
• 1x WPR (JE IgM) 14 participants
from 9 labs (HK Nov 2010)
QC
Train
ing
•
•
•
•
•
•
•
•
•
•
•
Train
ing
Where the resources for LabNet go…
go…
Approximate Proportion of Costs for Establishing
and Running a VPD LabNet
Proficiency Test Prog.
Prog.
Consumables
Shipping
QAP
Confirmatory Testing
Accreditation Visits
Kits
Standards
Assessing assays
Establishment
Assessment Site Visits
Equipping Labs
Training
Meetings
Training
Meetings
Need for continual Support
2
Major Partners
Partner
JE
Funds 2000-08
Project
BM Gates
WHO
CDC
Laboratory Support
2006-08
2009-10
>2010
PATH CVP
Yes
PATH JE Project
Yes
Yes
Residual
No
SEAR
Yes
Yes
Yes
Yes
WPR
Yes
Yes
Yes
Yes
HQ
Yes
Yes
Yes
Yes
GDD
Yes
China, India, Bang.
Residual
No
Yes
Ft Collins
Yes
Yes
AFRIMS
Nepal
Yes
Yes
Wellcome Trust
Liverpool School of Med
Yes
KOICA
VN, DPRK, INO
Yes
JICA
JE Control
Yes
China
USAID
Env Health
Yes
Nepal
ADB
Viet Nam
Yes
RO Korea
LabNet Support
?
Projected funding needs 20092009-2015
• 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
?
?
?
Yes
Yes
Yes
Japanese Encephalitis Morbidity, Mortality, and Disability
Reduction and Control by 2015 PATH Document: 2009
GAVIGAVI-eligible countries
Projected Resource requirements
NonNon-GAVIGAVI-eligible countries
Projected Resource requirements
Decade of Vaccines
Vaccine Costs Portfolio B
GAVI Alliance Board meeting
29 & 30 October 2008
DAVOS 29 January 2010
Bill and Melinda Gates Pledge $10 Billion in Call for Decade of Vaccines
– Research, develop and deliver vaccines for the world’
world’s poorest countries
– Increase vaccination and save more than 8 million children by 2020
2020
– Call for other to help fill critical financing gaps in both research
research funding
and childhood immunization programs
– Commit $10 Billion over 10 years,
years, while realizing the investment is not
sufficient
– Confirm that the new funding is in addition to the $4.5 billion already
committed by BMGF to vaccine research, development and delivery to
date across its entire disease portfolio since its inception
– Request increased investments in vaccines by governments and the
private sector
GAVI slide
GAVI Alliance Board meeting
29 & 30 October 2008
17
3
Global Immunization Vision and Strategy
Goals 2015
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 Strategy
Goals
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.
GIVS
Challenges
GIVS
Acknowledgements
JE LabNet newly established
Lab Working
Group
– Functioning reasonably well
– Progress is fragile
Nalini Ramamurty
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
Ravi Vasanthapuram
Barbara Johnson
Jaimie Robinson
Susan Hills
Development of appropriate diagnostic tools
Tom Solomon
– The perfect assay yet to be found!
– Assay assessment and QAP important new facet
Asheena Khalakdina
Penny Lewthwaite
Ensure labs' workload manageable
– Surveillance aim; not to detect and sample every case
– Outbreaks vs sporadic
Youngmee Jee
Tomohiko Takasaki
David Featherstone
Thank You!
4
Is quality assurance necessary ?
Quality Assurance and Proficiency in
the JE Laboratory Network
the Laboratory Diagnosis of Japanese Encephalitis in the
Do we need quality assurance in the lab?
Limitations to accurate results
Lab expected to provide critical information to the disease
programme
Sensitivity of assay
– Confirming suspected cases as positive or negative
– Providing differential diagnosis
– May help with adverse events following immunization
Specificity of assay
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
Sensitivity and Specificity of Assays
Regular assessment of available assays using well
validated panels of sera by independent assessor
– Number of false negatives detected
– 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
JE – PanBio Assay
Some limitations
Serum heat inactivation not recommended
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
"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
1
All Assays
Some limitations
Quality assurance requirements
All procedures will have some variable components
which may impact on the final result, e.g.
–
–
–
–
–
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
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
Absorbance of TMB at different wavelengths
Maintenance of equipment
ELISA reader
–
–
–
–
Correct filters being used
Light path is clear
Machine is kept clean
Power stabiliser used
Using appropriate filter
in ELISA reader is
critical
Maintenance 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 - 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!
2
Quality assurance - Micropipettes
Major causes of inaccuracy or poor precision
Reason for calibration
O-ring damaged
Tip holder worn
Reagents drawn into pipette barrel
Both easily and cheaply replaced
Accurate but not
precise
Precise but not
accurate
Accurate and
precise
Gravimetric determination of
Micropipettes
Quality assurance - Temperature
Incubators
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
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 Use
Quality assurance – Reagents and Kits
Polio Laboratory Network
Daily 37°C Incubator Temperature Log
1
Date
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
Ensure expiry date of reagents is recorded and respected
2
4
2
5
2
6
2
7
2
8
2
9
3
0
3
1
Consider inappropriate handling during shipment
+
Ensure to expedite collection of reagents/ kits after arrival at
airport
37°C
-
Plotting OD of Positive controls and calibrators
1
Initials
Asset Number
2
D
D F
F
3
4
5
6
7
8
D D A A D B
F F M M F C
3456
9
1
0
A D
M F
Month June
Acceptable Range 37 °C ± 1°C
1
1
A
M
1
2
D
F
1
3
A
M
1
4
D
F
1
5
D
F
1
6
D
F
1
7
D
F
1
8
D
F
Year
1
9
D
F
2
0
D
F
2006
2
1
A
M
2
2
A
M
2 2
3 4
A
M
2
5
2
6
2
7
2
8
2
9
3
0
3
1
– In-house and kit controls
– Useful for kit to kit, day to day, person to person comparability
Proficiency test (PT)
– Annually
3
Quality assurance - Procedures
Procedures
Lab Management
Monitoring kit usage and other consumables
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
Monitoring staff performance
Ensuring timeliness indicators are met
Maintaining staff safety and motivation
Wash cycles are critical
– PanBio requires 6 x
Quality 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
Assuring 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
Accreditation
– Comprehensive annual assessment of all performance indicators
– Onsite review by WHO Lab Coordinator or nominated specialist virologist
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.
Accreditation 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%)
– All samples positive during period of low incidence
– Check compatibility of results between paired samples, Serum 1 & 2, &/or CSF
4
Validation 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
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 !
5
Specimen collection, preparation
& shipment for virus isolation
and serology
Our policy
Contact with the patient’
patient’s doctor !
Typical Specimens
•
•
•
•
•
•
•
•
•
Blood / serum
CSF
Stool
Swabs: nose, throat, rectal
Urine
Dried blood spot
Virus cultures
Cell lines
Proficiency panels
(serology, virus detection)
(serology, virus detection)
(virus isolation)
(virus isolation)
(virus detection)
(serology)
(characterisation)
(cell culture)
Tomohiko Takasaki MD, National Institute of Infectious Diseases, JAPAN
17th, Nov. 2010 in Public Health Lab. Centre, Hong Kong
Collection of CSF
For serological test & virological test
• 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℃.
• 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
A JE case in Yamaguchi prefecture,2010
• 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.
JE10-10
/1:serum
/1:csf
/2:serum
/3:serum
/4:serum
/4:csf
/5:serum
/6:serum
/7:serum
IgM P/N
3.17
NT
9.12
11.4
14.8
18.2
15.2
16.7
18.2
Collection date
9/6
9/6
9/7
9/8
9/9
9/9
9/13
9/21
10/21
Pathogen Risk Groups
1. No or low individual & community risk
2. Moderate individual risk, low community risk
Risk Groups and Categories of Infectious
Substances
• Classification of microorganisms into risk
groups is based on the risk in the laboratory
environment.
3. High individual risk, low community risk
4. High individual & community risk
WHO Laboratory Biosafety Manual ,3rd ed 2004
Exempt Specimens
• Minimal likelihood that pathogens are
present (ex. Convalescent serum, CSF)
• Inactivated or neutralised pathogens
• Dried blood spots
• Environmental samples considered to not
pose a significant risk eg, food or soil
Shipment Responsibilities:
Sender _ International
• Infectious substances are classified into two
categories based on scientific assessment of
risk during transportation.
Triple Packaging for Clinical Specimen
•Sufficient Absorbent
•Tough plastic bag
•Biohazard mark
International
Packaging Packaging
Labels
• Contact consignee to confirm that they
can accept the shipment
- airway bill number (AWB), flight details
LO
G
C ICA
A
TE L
G SU
O B
R S
Y TA
B
N
BI
O
• Book shipment through a courier
company or airline
• Appropriate packaging and paperwork
• Notify consignee of shipment details
CE
- avoid public holidays, weekends
UN2814 Category A
PI 602
UN3373 Category B
PI 650
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 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 JEJE-VAX development in Japan
1954
1957
The mouse brain derived JEJE-VAX (Nakayama
strain) was produced in the market firstly.
: Five ％ JEV infected mouse brain after
centrifugation
Two % 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
1971
1989
The seed virus had been changed from
Nakayama-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 pmda to get a licence for the
production in this month.
– P3 based: in China
PHK cells ⇒change ⇒ Vero cells
Vero 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
Chiba university in 1962 in Japan
19
Micro-carrier tank and Vero cells on micro-carrier
21
Production procedures
Mouse brain
Vero cells
Mice
↓
Inoculation with JEV (Beijing(Beijing-1)
↓
Harvest infected brains
↓
Preparation of virus fluid
↓
Protamine sulfate treatment
↓
Formaline treatment
↓
Ammonium Sulfate treatment
↓
Ultra centrifugation (Sucrose
density gradient 2x
↓
Collecting virion fraction
↓
Filtration
↓
Bulk vaccine
↓
Liquid／
Liquid／Lyophilized
↓
Vaccine
Vero cell culture：
culture：Cytodex
↓
Inoculation with JEV (Beijing(Beijing-1)
↓
Harvest culture fluid
↓
Filtration of culture fluid
↓
Concentration
↓
Formaline treatment
↓
Protamine sulfate treatment
↓
Ultra centrifugation (Sucrose
density gradient 2x
↓
Collecting virion fraction
↓
Filtration
↓
Bulk vaccine
↓
Lyophilized
↓
20
Vaccne
CDC/DVBD diagnostic laboratory
testing methods
The 2nd Intercountry Hands-on Training Workshop on the
Laboratory Diagnosis of Japanese Encephalitis in the
Barbara W. Johnson
Centers for Disease Control and Prevention
Division of Vector-Borne Infectious Diseases
Fort 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
Arboviral Diseases Diagnostic Assays
Serum, CSF
Serological
Assays
Mosquito pools, tissues,
serum, CSF
Virus Detection
Assays
National and international outbreak investigations
Identification of unknown viruses
Serosurveys, following epidemic or
after vaccination campaigns
IgM ELISA
Microsphere immunoassay
IgG ELISA
Plaque reduction neutralization test
Viral RNA detection
Nucleotide sequencing
Virus isolation
Immunofluorescence assays
Antigen detection assays
Other CDC divisions
Geographical Origin of Specimen
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
Specimens are tested for all possible arboviruses from geographical
region, based on clinical information and volume of sample
Current Laboratory Testing Strategy
for Arboviruses
JEV Human Viremia & Immune Response
Serology Assays
Virus Assays
#pfu/ml
ELISA
P/N
IgM
100
20
IgG
WN
viremia
Neutralizing Ab
2
-14 to -2
0 1 2 3 4 5 6 7 8 9 10
CNS illness
DAYS POST ONSET
First priority testing
method based on
characteristics of the
virus infection and
immune response
• 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
DENV Human Viremia & Immune Response
Serology Assays
Virus Assays
Virus Assays
#pfu/ml
ELISA
P/N
IgM
100
10000
20
IgG
Neutralizing Ab
DEN
viremia
0
1
CNS illness
2 3
4
5
6 7
8
9
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
2
-14 to -2
Human Infection
10
DAYS POST ONSET
Principles of plaque reduction neutralization assay
Interpretations of test results for a single acute specimen
Acute
Specimen
Mix patient serum dilutions + 100 PFU of virus; incubate with cells.
100 plaques = no virus antibody present
90% reduction of virus plaques (≤10 PFU/well) = virus antibody present
Calculation of neutralizing antibody titer:
Reciprocal of end-point specimen dilution that reduces the
challenge virus plaque count by 90%.
Serum dilution 1:40
1:20
1:10
neutralization
No
Interpretation
1:320
1:160
IgG
ELISA
IgM
ELISA
PRNT
No
Interpretation
Possible 2°
Infection
Consensus
RT-PCR
Real-Time
RT-PCR
Virus
Isolation
POS
POS
POS
Nucleic acid
sequencing
ID
Virus
RT-PCR or
IFA
1:80
Partial neutralization
No neutralization
ID
Virus
ID
Virus
No
Interpretation
ID
Virus
End point dilution
neutralization titer = 20
Example:
CDC Testing algorithm for WN or JE Virus
CDC/DVBID Diagnostic Testing Algorithm for Medically
Important Arthropod-Borne Viral Diseases in North America *
AGENT OR
DISEASE
California
encephalitis/
La Crosse
encephalitis
Colorado tick
fever
Dengue 1-4
Eastern equine
encephalitis/
Venezuelan
equine
encephalitis/
Western equine
encephalitis
St. Louis
encephalitis
SPECIMEN(S)
TO COLLECT
METHOD OF
CONFIRMATION OR
IDENTIFICATION**
Sera
IgM ELISA, NT
Brain
Real-time RT-PCR, virus
isolation
CSF
IgM ELISA, NT, Real-time RTPCR,† Virus isolation†
Mosquitoes
Real-time RT-PCR, Virus
isolation
Whole blood/clot
isolation
Sera
isolation, paired NT
Ticks
Real-time RT-PCR,Virus
isolation
Sera
IgM ELISA, NT, Real-time RTPCR,† † virus isolation††
Liver, lung, lymph
nodes
isolation
Sera
IgM ELISA, NT, Real-time RTPCR,† virus isolation†
Brain
isolation
CSF
IgM ELISA, NT, Real-time RTPCR, † Virus isolation†
Mosquitoes
Real-time RT-PCR,Virus
isolation
Horse sera
IgM ELISA, NT, Real-time RTPCR, Virus isolation
Sera
MIA/IgM ELISA, NT, Realtime RT-PCR†
Brain
Real-time RT-PCR, virus
isolation
CSF
IgM ELISA, NT, Real-time RTPCR/virus isolation
COMMENTS
AGENT OR
DISEASE
West Nile virus
Except where noted freeze
specimens for virus
isolation at –65oC (dry
ice)
Yellow fever§
Do not freeze samples for
CTF virus isolation.
SPECIMEN(S) TO
COLLECT
METHOD OF CONFIRMATION OR
IDENTIFICATION
Sera
MIA/IgM ELISA, NT, Real-time RTPCR†
Brain, brain stem, spinal
cord
Real-time RT-PCR, Virus isolation
CSF
IgM ELISA, NT, Real-time RT-PCR,†
Virus isolation†
Mosquitoes
isolation,Dipstick, RAMP
Sera
IgM ELISA, NT, Real-time RT-PCR,†
Virus isolation†
Liver
Real-time RT-PCR , Virus isolation,
histopathology
Mosquitoes
Real-time RT-PCR, Virus isolation
*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.
Isolation of Venezuelan
equine encephalitis virus
requires biosafety level 3
containment
Isolation requires
biosafety level 3
containment
COMMENTS
Specimen 1st Choice
Other
Comments
Isolation requires biosafety level
3 containment
Isolation requires biosafety level
3 containment with HEPA filtered
exhaust air flow;
Yellow fever immunization
required
Human
serum
and CSF
Serology: WN (JE) WN-specific qRT-PCR, WN qRT-PCR
and SLE (DEN)
flavirus RT-PCR +
sensitivity: 57% acute
ELISA + PRNT
sequencing,
CSF, <10% serum
virus isolation
Human
tissue
WN-specific qRTPCR
Virus isolation,
IHC
NonHuman
1st Choice
2nd Choice
Avian
tissue
WN-specific qRTPCR,
Virus isolation
VecTest/ antigen
capture ELISA,
flavivirus RT-PCR
Mosquito
pool
WN qRT-PCR,
VecTest/Ag. Cap.
flavivirus RT-PCR, ELISA/RT-PCR
virus isolation
Fatal WN cases: WN
qRT-PCR sensitivity
~ 100%
Ag.-based tests
require
1000 PFU
CDC IgM ELISA differential diagnostics
WN (JE) antigen
P/N: O.D. patient serum on
viral antigen/O.D.
negative control serum on
viral antigen
P/N > 3 = positive
P/N < 2 = negative
P/N 22-3 = equivocal
JE
1:20
1:40
S1
S3
S5
S7
Ref
S2
S4
S6
S8
N
1:320
Ref = pos control serum
N = normal control serum
1:10
1:160
1:40
1:80
1:320
DEN
1:20
1:10
1:160
1:80
SLE (DEN) antigen
• 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.
Interpretation
S1
S3
S2
S4
S5
S7
Ref
S8
N
PRNT titer ≥ 10 = presence of neutralizing antibody
PRNT titer ≥ 4-fold over heterologous flavivrius
= virus-specific neutralizing antibody
S6
PRNT titer 4-fold difference between acute and convalescent specimen
= evidence of acute infection
ELISA Assay must be standardized in
each lab
Viral
Antigen
Normal
Antigen
Flavivirus Cross-reactivities of IgM from
WN Patient Serum*
Serum
P/N
SLE
P/N
JE
1
4.96
7.75
2
P/N
WN
P/N
DEN2
P/N
YF
POW
16.74
2.45
1.82
1.56
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
17.19
3.32
1.62
1.3
Positive
Control
6.5
10.0
7
8.2
6.34
7.45
3.96
4.5
Cente rs for Dise ase Control
and Pre vention
Sample
IgM P/N
JE
DEN
IgG P/N
JE
DEN
PRNT
JE
DEN
Case interpretation: Primary JE Infection
acute serum
8
12.75 4.00
1.37
2.04 <1:10 <1:10
conv. serum
31
11.35 4.21
6.38
5.76 1:1280 1:80
Case interpretation: Secondary Flavivirus infection?
acute serum
4
1.59
1.42
3.12
2.62
1:20
1:80
conv. serum
15
9.01
3.96 10.00 9.90 1:640 1:320
Interpretation of ELISA Results
P/N: O.D. patient serum/O.D. negative
control serum reacted on viral antigen
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
Days
P.I.
IgM
IgM
P/N
(JE)
IgM
IgM
P/N
(DEN)
(WN)
CSF
8
26.91
S1
9
S2
Positive
Control
90% neutralization titer
JE
WN
DEN2
1.78
nd
nd
nd
nd
9.1
4.16
160
20
<10
10
34
6.7
4.62
1280
20
<10
20
n.a.
9
6.5
>5120
2560
2560
320
Interpretation of ELISA Results
P/N: O.D. patient serum/O.D. negative
control serum reacted on viral antigen
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
JE Case Interpretation based on Serology
Days
post-onset
Patient
SLE
P/N
* 1:400 screening dilution
P/N > 3 = positive
P/N < 2 = negative
P/N 2-3 = equivocal
Complete Serological Analysis: Differential diagnosis
Interpretation of PRNT results
PRNT titer > 10 = positive antibody
PRNT titer =4-fold over heterologous
flavivirus = positive for specific antibody
or
PRNT titer = 4-fold between acute and
convalescent = evidence of acute infection
Increasing specificity and resolving IgM equiv results
Example: CDC Confirmatory testing of Cambodian specimens
Total samples tested
JEV IgM+
JE-DEN IgM cross-reactive
Classification resolved by
PRNT
Confirmed JE positve
Confirmed DEN positive
All sample
types (%)
1195
CSF
(%)
439
Sera
(%)
756
348
170 (49)
155 (45)
108
55 (51)
54 (50)
240
115 (48)
101 (88)
48
107
24
30
24
77
43
16
27
1
17
0
7
1
10
Interpretation of PRNT results
PRNT titer > 10 = positive antibody
PRNT titer =4-fold over heterologous
flavivirus = positive for specific antibody
or
PRNT titer = 4-fold between acute and
convalescent = evidence of acute infection
IgM JEV and/or DENV
equivocal
Confirmed JE positive
IgM- (no neutralization titer)
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.
Simplified Depiction of CHIK Human Viremia & Immune Response
Increasing specificity by PRNT
Example: 3 kit evaluation with samples from India and
Bangladesh
All
Sample
Types (%)
Total samples tested
JEV IgM+
JEV IgM Cross-Reactive
(DEN/WN)
Classification resolved by
PRNT
Confirmed JE+
Confirmed DEN+
Confirmed WN+
No. CSF
(%)
106
#pfu/ml
226
294
103
36 (35)
34
10 (29)
69
26 (38)
32 (31)
2 (6)
21 (30)
16
7
9
0
2
0
15
5
1
IgM
CHIK
viremia
No. Sera
(%)
520
ELISA
P/N
20
IgG
Neutralizing Ab
2
-14 to -2
0 1
2
3 4
5 6
7 8
9 10
DAYS POST ONSET
Serological & RT-PCR Test Results of
CHIK Infected Returning Travelers
Serological Testing Algorithm
for Chikungunya Virus Infection
single acute
patient serum
IgM Capture ELISA
IgG ELISA
RT-PCR (<10day)
IgM POS
IgM NEG
(<10day)
IgM NEG
(>10day)
No
Interpretation
NEG
RT-PCR or
Isolation POS
PRNT
POS
CDC/DVBD JE serological testing of a single acute
specimen
Interpretation
Testing algorithm
Acute human serum (1:400)
or CSF (1:10)*
IgM ELISA JEV & DENV
(WNV sera)
Confirmed positive:
IgM positive or equivocal
And
PRNT titer ≥1:4 (CSF) or 1:10 (sera)
And
≥4-fold over heterologous flavivirus
Presumptive positive:
POS
(P/N >3)
or
NEG
(P/N<2)
EQUIV
(P/N=2
- 3)
Interpreted as either
NEG or IgM not yet
present
PRNT with:
JEV + DENV
(WNV sera)
Action : Report as
negative, or test
convalescent specimen
if available
IgM positive or equivocal
And
PRNT titer ≥1:4 (CSF) or 1:10 (sera)
And
<4-fold over heterologous flavivirus
Or
IgM positive
And
PRNT negative
Negative
IgM negative or equivocal
And
PRNT negative
*All specimens must meet clinical acute encephalitis syndrome case definition.
Thank you!
POS
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
Age
Nº
NAME
Patient
ID
M
Result JE
Result DEN
Coll.date
Testing
date
CSF
S1
S2
CSF
S1
S2
Takeo
31-Dec-2009
28-Jan-2010
Neg
Pos
Pos
Neg
Neg
Neg
Takeo
15-Jan-2010
28-Jan-2010
Neg
Neg
Neg
Hospital
F
Final Result
January
1
SAT LETH
2912/72
2
OU SOK MEAS
1401/87
3
PUTH SAMBO
119
4
KUTH RACHANA
211
7
13m
5
20m
5
THANN VITHEY
KC 56
4m
6
SUS MAKARA
KC 57
6
7
PHEAP SOPHEAK
KC 58
12
8
RY RAKSMEY
KC 59
10
Svay
Rieng
29-Dec-2009
28-Jan-2010
Svay
Rieng
30-Dec-2009
28-Jan-2010
Kg Cham
4-Jan-2010
28-Jan-2010
Pos
Neg
Kg Cham
5-Jan-2010
28-Jan-2010
Neg
Neg
10-Jan-2010
28-Jan-2010
Neg
Neg
Kg Cham
13-Jan-2010
28-Jan-2010
Pos
Pos
99
CSF
96
Serum 1
96
Serum 2
59
JE Positive
29
DEN Positive
11
DEN Equi
2
JE Equi
3
JE
Neg
Kg Cham
Total Patients
Pos
Neg
Neg
JE
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Pos
Neg
Neg
Neg
DEN
Neg
JE
1
Laos reporting
JE Cases lab reported in 2010Cambodia
SPECIMEN DETAILS
Country
Month
SPECIMEN DETAILS
CASE DETAILS
Specimen
s received
Specimens
tested
Specimen
s
JE
positive
Specimen
s
Dengue
positive
Specimens
negative
to both JE
and
Dengue
Specimens
pending
lab results
Specimens
with
results ≤ 7
days of
receipt in
lab
Ser
um
CS
F
Seru
m
CS
F
Ser
um
CS
F
Se
ru
m
CS
F
Ser
um
CS
F
Ser
um
CS
F
Ser
um
CS
F
Month
Cas
es
tes
ted
Cases with
positive
results
JE
De
ng
ue
Cases
negati
ve to
both
JE and
Dengu
e
Cases
with
pendin
g
results
Cases
referr
ed to
anoth
er lab
speci
mens
recei
ved
Janua
ry
0
Febru
ary
0
Cambodia
January
20
12
20
12
7
2
3
0
6
10
0
0
0
0
14
4
2
8
0
0
Cambodia
Februar
y
3
3
3
3
0
0
0
0
3
3
0
0
0
0
3
0
0
2
0
0
Cambodia
March
26
15
26
15
3
1
7
0
9
14
0
0
0
0
15
2
4
9
0
0
Marc
h
0
Cambodia
April
7
4
7
4
0
0
0
0
4
4
0
0
0
0
4
0
0
4
0
0
April
0
0
May
Cambodia
May
17
11
17
11
2
3
1
0
3
1
7
16
27
16
5
4
2
1
9
9
0
0
0
0
16
4
2
10
0
0
June
2
2
1
0
1
0
2
0
20
13
0
2
0
0
9
10
0
0
0
0
13
2
0
8
0
0
July
21
21
16
0
5
0
20
4
Cambodia
August
15
10
15
10
10
6
0
10
0
2
0
Total
number
of cases
of cases
tested
№ of cases with
positive results
Deng
ue
positi
ve
JE
positiv
e
Nu
mbe
r of
case
s
neg
ativ
e to
bot
h JE
and
Den
gue
Numbe
r of
cases
with
pendin
g
results
Number
of
negativ
e cases
referred
to
another
lab
(specify)
speci
mens
teste
d
specim
ens
JE
positiv
e
spec
ime
ns
Den
gue
posi
tive
specimen
s
negative
to both
JE and
Dengue
specime
ns
pending
lab
results
samples
with
results ≤
7 days of
reception
in lab
2
1
0
1
0
0
4
1
0
3
0
4
21
16
0
5
0
0
0
0
Septem
ber
Augu
st
18
18
5
2
11
0
18
3
3
1
0
3
18
5
2
11
0
0
Cambodia
4
1
4
1
0
0
0
0
4
1
0
0
0
0
2
0
0
2
0
0
Septe
mber
3
3
1
2
0
0
3
0
0
0
0
0
0
0
3
1
2
0
0
0
Cambodia
October
5
5
5
5
2
1
1
0
2
4
0
0
0
0
5
2
1
2
0
0
144
90
144
90
29
19
14
1
58
67
0
0
0
0
93
24
10
54
0
0
Octob
er
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Cambodia
7
0
specim
ens
receive
d
13
0
11
sample
s with
results
≤7
days of
recepti
on in
lab
27
0
0
speci
mens
pendi
ng
lab
resul
ts
20
0
0
specim
ens
negati
ve to
both
JE and
Dengu
e
June
4
0
specim
ens
Dengu
e
positiv
e
July
3
0
speci
mens
JE
positi
ve
Cambodia
0
8
speci
mens
teste
d
CSF
Cambodia
0
9
CASE DETAILS
Serum
?
?
Nove
mber
25.8%
Dece
mber
Total
Philippines reporting
PHILIPPINES
JE Cases lab reported in 2010-Laos
CASE DETAILS
SPECIMEN DETAILS
Serum
SPECIMEN DETAILS
Specimens
received
Specimens
tested
Specimens
Dengue
positive
Specimens
JE positive
Specimens
negative to
both JE and
Dengue
Specimens
pending lab
results
Specimens
with results
≤ 7 days of
receipt in
lab
Cases with
positive
results
Cas
es
test
ed
Cas
es
neg
ativ
e to
bot
h JE
and
Den
gue
Cas
es
wit
h
pen
din
g
res
ults
Cas
es
refe
rred
to
ano
ther
lab
Country
Month
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Lao PDR
Jan
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lao PDR
Feb
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Ser
um
CSF
Ser
um
JE
Den
gue
Lao PDR
March
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lao PDR
April
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lao PDR
May
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lao PDR
June
2
0
2
0
1
Lao PDR
July
21
4
21
4
16
1
0
0
5
3
0
0
20
Lao PDR
August
18
3
18
3
5
1
2
0
11
0
0
0
18
Lao PDR
Lao PDR
Sept
Octr
Lao PDR
3
0
3
0
1
0
0
0
2
0
1
0
CSF
CASE DETAILS
0
0
0
0
0
0
0
2
3
0
speci
men
s
nega
tive
to
both
JE
and
Deng
ue
spe
cim
ens
pen
din
g
lab
res
ults
sample
s with
results
≤7
days
of
recepti
on in
lab
spe
cim
ens
rec
eiv
ed
speci
mens
teste
d
Jan
11
11
4
1
6
0
0
18
18
Feb
4
3
0
2
1
1
1
11
11
spe
cim
ens
JE
posi
tive
spe
cim
ens
pen
din
g
lab
res
ults
sample
s with
results
≤7
days of
recepti
on in
lab
JE
posit
ive
Den
gue
posi
tive
Number
of cases
referred
to
another
lab
(specify)
spe
cim
ens
Den
gue
posi
tive
speci
mens
negat
ive to
both
JE
and
Deng
ue
3
0
15
0
0
18
4
1
13
0
0
1
1
9
0
0
12
1
3
7
1
0
Mar
15
15
4
3
7
0
4
21
21
5
1
15
0
7
21
5
3
13
0
0
Apr
16
16
0
2
14
0
13
18
18
0
1
17
0
13
18
0
3
15
0
0
May
31
31
2
6
23
0
0
35
35
2
0
33
0
0
35
2
6
27
0
0
4
21
16
0
5
0
0
Jun
10
10
4
2
4
0
1
7
7
2
0
5
0
1
8
3
2
3
0
0
18
5
2
11
0
0
July
23
23
3
2
17
0
0
22
22
3
2
17
0
1
22*
3
4
14
0
0
Aug
24
24
12
1
11
0
0
16
16
8
0
8
0
0
19
9
1
9
0
0
Sep
10
10
2
8
0
0
7
10
10
3
0
7
0
7
10
3
0
7
0
0
Tot
al
144
143
31
27
83
1
26
158
158
27
5
126
0
29
141
30
23
108
1
0
2
0
0
0
spe
cim
ens
Den
gue
posi
tive
Num
ber
of
cases
with
pendi
ng
resul
ts
3
1
0
spec
ime
ns
JE
posi
tive
Nu
mb
er
of
cas
es
neg
ativ
e to
bot
h JE
and
Den
gue
1
3
1
speci
men
s
test
ed
№ of cases
with
positive
results
2
0
0
speci
men
s
recei
ved
Mon
Tota
l
num
ber
of
case
s of
case
s
test
ed
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
44
7
44
7
23
2
4
0
17
3
0
0
43
7
44
23
4
17
0
0
52.3%
JE Cases lab reported in 2010-Philippines
SPECIMEN DETAILS
Country
Specimens
received
Specimens
tested
Specimens
JE positive
Specimens
Dengue
positive
Specimens
negative to
both JE and
Dengue
Specimens
pending lab
results
Ser
um
CSF
Ser
um
Ser
um
Ser
um
Ser
um
CSF
Ser
um
Ser
um
Month
CSF
CSF
CSF
North Vietnam reporting
CASE DETAILS
Specimens
with results
≤ 7 days of
receipt in
lab
CSF
Cas
es
test
ed
CSF
Cases with
positive
results
JE
Den
gue
Cas
es
neg
ativ
e to
bot
h JE
and
Den
gue
Cas
es
wit
h
pen
din
g
res
ults
Cas
es
refe
rred
to
ano
ther
lab
Philippine
s
Jan
11
18
11
18
4
3
1
0
6
15
0
0
0
0
18
4
1
13
0
0
Philippine
s
Feb
4
11
3
11
0
1
2
1
1
9
1
0
1
0
12
1
3
7
1
0
Philippine
s
March
15
21
15
21
4
5
3
1
7
15
0
0
4
7
21
5
3
13
0
0
Philippine
s
April
16
18
16
18
0
0
2
1
14
17
0
0
13
13
18
0
3
15
0
0
Philippine
s
May
31
35
31
35
2
2
6
0
23
33
0
0
0
0
35
2
6
27
0
0
Philippine
s
June
10
7
10
7
4
2
2
0
4
5
0
0
1
1
8
3
2
3
0
0
0
Philippine
s
July
23
22
23
22
3
3
2
2
17
17
0
0
0
1
22
3
4
14
0
Philippine
s
Aug
24
16
24
16
12
8
1
0
11
8
0
0
0
0
19
9
1
9
0
0
Philippine
s
Sepr
10
10
10
10
2
3
8
0
0
7
0
0
7
7
10
3
0
7
0
0
Philippine
s
Octr
Philippines
7
12
7
12
0
2
3
2
3
8
1
0
4
4
12
2
4
6
1
0
151
170
150
170
31
29
30
7
86
134
2
0
30
33
175
32
27
114
2
0
18.3%
2
JE Cases lab reported in 2010-North
Vietnam
SPECIMEN DETAILS
Country
CASE DETAILS
Specimens
received
Specimens
tested
Specimens
JE positive
Specimens
Dengue
positive
Specimens
negative to
both JE and
Dengue
Specimens
pending lab
results
Specimens
with results
≤ 7 days of
receipt in
lab
Ser
um
Ser
um
Ser
um
Ser
um
Ser
um
Ser
um
Ser
um
Month
CSF
CSF
CSF
CSF
South Vietnam reporting
CSF
CSF
Cas
es
test
ed
CSF
Cases with
positive
results
JE
Den
gue
Cas
es
neg
ativ
e to
bot
h JE
and
Den
gue
Cas
es
wit
h
pen
din
g
res
ults
Cas
es
refe
rred
to
ano
ther
lab
Viet Nam,
N
Jan
12
9
12
8
0
0
0
0
12
8
0
0
12
8
11
0
0
11
0
0
Viet Nam,
N
Feb
19
13
18
13
0
0
0
0
18
13
0
0
18
13
13
0
0
13
0
0
Viet Nam,
N
March
16
11
16
11
0
0
0
0
15
11
0
0
16
11
12
0
0
12
0
0
Viet Nam,
N
April
19
17
19
17
0
0
0
0
19
17
0
0
19
17
18
0
0
18
0
0
Viet Nam,
N
Viet Nam,
N
May
26
16
26
16
2
2
2
1
24
14
0
0
26
16
23
2
1
21
0
0
June
35
28
35
28
10
10
0
0
25
18
0
0
35
28
40
15
0
25
0
0
Viet Nam,
N
July
8
4
8
4
0
1
0
0
7
3
0
0
8
4
11
1
0
9
0
0
Viet Nam,
N
Aug
8
2
8
2
0
1
0
1
8
1
0
0
8
2
8
1
1
7
0
0
Viet Nam,
N
Sepr
2
2
2
2
0
1
0
0
2
1
0
0
2
2
3
1
0
2
0
0
Viet Nam,
N
Oct
3
1
3
1
2
1
0
0
1
0
0
0
3
1
3
2
0
1
0
0
148
103
147
102
14
16
2
2
131
86
0
0
147
102
142
22
2
119
0
0
Viet Nam, North
15.5%
JE Cases lab reported in 2010-South
Vietnam
SPECIMEN DETAILS
Country
SPECIMEN DETAILS
Specimens
tested
Specimens
JE positive
Specimens
Dengue
positive
Specimens
negative to
both JE and
Dengue
Specimens
pending lab
results
Specimens
with results
≤ 7 days of
receipt in
lab
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Ser
um
CSF
Cas
es
test
ed
Cases with
positive
results
JE
Den
gue
Serum
Cas
es
neg
ativ
e to
bot
h JE
and
Den
gue
Cas
es
wit
h
pen
din
g
res
ults
Cas
es
refe
rred
to
ano
ther
lab
Viet Nam, S
Jan
16
12
16
12
0
0
0
0
1
1
0
0
1
1
12
0
0
12
0
0
Viet Nam, S
Feb
7
8
7
8
0
1
0
0
2
2
0
0
6
7
9
1
0
8
0
0
Viet Nam, S
March
26
20
26
20
1
1
0
0
25
19
0
0
26
19
21
1
0
20
0
0
Viet Nam, S
April
24
20
24
19
3
1
0
0
21
18
0
0
22
18
21
2
0
19
0
0
Viet Nam, S
May
28
17
28
17
0
0
2
1
25
16
0
0
27
16
21
0
1
20
0
0
Viet Nam, S
June
26
18
26
18
3
1
0
0
23
17
0
0
26
18
20
2
0
18
0
0
speci
men
s
teste
d
speci
mens
JE
positi
ve
speci
mens
Deng
ue
positi
ve
specim
ens
negati
ve to
both
JE and
Dengu
e
speci
mens
pendi
ng
lab
result
s
Januar
y
16
5
0
0
5
Februa
ry
6
0
0
0
0
March
17
0
0
0
April
11
0
0
0
May
22
0
0
June
16
0
0
July
24
19
0
Month
July
37
26
37
26
2
1
3
0
32
25
0
0
36
26
34
2
3
29
0
0
August
Viet Nam, S
Aug
16
12
16
12
4
1
0
0
12
11
0
0
13
10
15
3
0
12
0
0
Septe
mber
Viet Nam, S
Sep
7
5
7
5
2
2
0
0
5
3
0
0
4
3
6
2
0
4
0
0
Viet Nam, S
Octr
8
5
8
5
2
1
1
0
5
4
0
0
5
3
8
2
1
5
0
0
195
143
195
142
17
9
6
1
151
116
0
0
166
121
167
15
5
147
0
0
CSF
speci
men
s
recei
ved
Viet Nam, S
Viet Nam, South
CASE DETAILS
CASE DETAILS
Specimens
received
Month
Korea Reporting
Total
numbe
r of
cases
of
cases
tested
№ of cases
with positive
results
JE
positi
ve
Deng
ue
positi
ve
Num
ber
of
cases
nega
tive
to
both
JE
and
Deng
ue
Numbe
r of
cases
with
pendin
g
results
0
samples
with results
≤ 7 days of
reception in
lab
speci
men
s
recei
ved
speci
men
s
teste
d
speci
mens
JE
positi
ve
speci
mens
Deng
ue
posit
ive
specime
ns
negativ
e to
both JE
and
Dengue
speci
mens
pendi
ng
lab
resul
ts
samples
with
results ≤ 7
days of
reception in
lab
0
5
8
0
0
0
0
0
8
5
0
0
5
0
6
8
0
0
0
0
0
8
0
0
0
0
0
0
0
17
10
0
0
0
0
0
10
0
0
0
0
0
0
0
11
4
0
0
0
0
0
4
0
0
0
0
0
0
0
0
22
10
0
0
0
0
0
10
0
0
0
0
0
0
0
0
16
11
0
0
0
0
0
11
0
0
0
0
0
0
0
0
0
22
17
0
0
0
0
17
36
0
0
0
0
Number of
cases
referred to
another lab
(specify)
Octobe
r
Novem
ber
Decem
ber
0
9%
Problems
• Current MS Access feedforward files need
to be converted by data managers in
WPRO
• Laboratory data only sent to data manager
in WPRO
Total
Recommendations
• Use MS excel format (old format)
• Please send the report to
[email protected]
[email protected]
• Report by 10th every month
3
Ongoing JE/AES/MEME surveillance in
the Region
Additional
• 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
China (12)
Shandong Province (six sentinel in Jinan Perfecture)
Regional status of JE diagnosis
Country
Laboratory testing used
WHO designated lab
Japan
In-house IgM capture (MAC) ELISA for JE, IgG ELISA,
PRNT, HI
TaqMan RT-PCR
Virus Isolation
NIID -in house
VTN
NIHE / PI in-house MAC ELISA, Panbio kits in pilot provinces
PRNT, HI
RT-PCR assay for CSF
Viral isolation
NIHE, PI - in house
Korea
Panbio ELISA, IFA/HI/CF
PRNT
Viral RNA detection – RT-PCR
Virus isolation – suckling mouse inoculation, mosquito cell culture
Korea CDC -Panbio
China
JEV IgM capture ELISA: Shanghai Beixi and Yueda
PCR
PRNT:
JEV isolation
Institute of Viral Disease
Control, China CDC -Beixi
Malaysia
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
IMR -Panbio from 2009
Cambodia
JE IgM capture ELISA (Panbio), Pasteur Institute in house test (Battambang site)
NIPH-Panbio
Laos
Xcyton/Panbio JE IgM capture ELISA (Mahosot hospital)
NCLE-Panbio
Philippines
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
RITM-Panbio
PNG
Referred to VIDRL(Panbio)
CPHL
Hubei province (six sentinel hospitals in Yichang perfecture)
Phillipines (5)
Philippine general hospital
Tarlac Hospital
Cebu provincial hospital
Iloilo provincial hospital
Bulacan provincial hospital
Vietnam (3)
North (1), Central (1), South (1) ; Thai Binh, Quang Ngai
National hospitals in Hanoi, HCM City
Cambodia (6)
National Pediatric Hospital, Pnomh Penh
Angkor Hospital (Siam Reap)
Battambang Provincial hospital
Kampong Cham Provincial hospital
Svay Rieng
Takeo
Lao (2)
Mahosot Hospital, Vientiane
Luang Nam tha provincial hospital
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
MBD vaccine->Vero cell derived
Japan
Malaysia
MBD vaccine
Y1,+1-2W, +1 Y, Y6, Y12
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
Vietnam
MBD vaccine/Live attenuated
MBD vaccine (locally produced)
M12-24, +1-2W, +1Y, Y6, Y12
Y1, +2W, +1Y
Confirmatory testing
Referral lab
NML
Last shipping
in 2010
Next shipping
NIID Japan
Laos
15 April
December
2010
NIID
Vietnam North
8 April
January
2011
NIID
Vietnam South
2 April
January
2011
NIID
Philippines
31 March
December
2010
NIID
Malaysia
27 May
February
2011
NIID or KCDC
Cambodia
8 Aug
February
2011
NIID
CDC, Korea
NIID Japan
(PHLC, HK)
Please send every 4 months from 2011!
4
Diagnosis of Japanese encephalitis by laboratory test
Focus reduction neutralization
assay for Japanese
encephalitis with peroxidase
anti-peroxidase method
Collect spacemen
Human spacemen
Vector spacemen
Viral isolation and nucleic
Viral isolation and nucleic
acid detection
acid detection
• viral genome detection using
RT-PCR
Department of Virology I,
National Institute of Infectious Disease
• viral genome detection using
RT-PCR
• virus isolation using vero cells
and suckling mouse
• virus isolation using vero
cells and suckling mouse.
serology
Chang-Kweng Lim, D.V.M., PhD., Tomohiko
Takasaki, M.D., PhD., Ichiro Kurane, M.D., PhD.
• IgM capture-ELISA
• Nutralizing antibody titration
• HI test
(PRNT)
PRNT assay requires large amounts of
• sample sera,
• indicator cells,
• medium,
• and incubator space.
The entire assay requires 7 days.
The methods for FRINT assay
2-fold dilution
Sample serum
10%FCS-MEM
sub-total
JEV (100FFU/25µl)
total
•
x10
8
72
40
40
80
x20
40
40
40
40
80
x40
40
40
40
40
80
x80 x160
40
40
40
40
40
40
40
40
80
80
x320
40
40
40
40
80
V.C.
40
40
40
80
N.C.
80
80
80
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).
The model of peroxidase anti-peroxidase (PAP) method
1st antibody
2nd antibody
PAP complex (rabbit)
37℃
℃ ｘ 1hr for nutralizing reaction
1
2
3
4
5
6
7
8
9 10 11 12
A
x 10
B
x 20
C
D
E
x 40
a
b
c
d
e
f
x 80
x160
F
x320
G
V.C. Vero Osaka
N.C. 2 x 104/well/100µ
H
•
•
40
Peroxidase anti-peroxidase (PAP) method for focus
reduction neutralization test (FRNT)
37℃
℃ ｘ 1h for incubation
Add 100µl/well 10%FCS-MEM, and incubate at 37℃
℃ for another 45hrs
Anti Japanese
encephalitis
rabbit antibody
Anti rabbit goat antibody
Peroxidase
antiperoxidase
rabbit
antibody
complex
The model of peroxidase anti-peroxidase (PAP) method
PAP complex
The model of PAP (peroxidase anti-peroxidase)
method
peroxidase
PAP complex
Anti-rabbit goat
antibody
JEV
peroxidase
anti-rabbit goat
antibody
（2nd antibody)
Vero cell
Anti-JEV rabbit
antibody
Focus formation by JEV
anti-JEV
rabbit
antibody
（1st antibody）
）
JEV
Vero cell
Analysis for a result of potency test for JE Vaccine by Parallel line
assay method
Using Bioassay Assist
Vaccine sample
Reference vaccine
PRNT (plaque)
FRNT (PAP Method)
PRNT（plaque)
vs FRNT(PAP)
PRNT
FRNT
PRNT
FRNT
1) Need space
（6 well plate）
2) 7 days assay
1) Save space in CO2
incubator (96 well plate)
2) Easy to test many samples
3) It takes 3 days for one assay
3) Plaque is larger
and easy to count
4) Cheaper
4) Focus is smaller
5) Expensive (need antibodies)
Bioassay assist is the statistic analysis software for the quality control of
biological products
TaqMan primers & probe for JEV
Universal set
Japanese Encephalitis Virus
Taq Man primers & probe
(Envelope region)
J E V prim ers & probs
J E en585p599s622c
G enotype
J E en562s-585pset
1& 3
J E en623c-585pset
S eq. 5'-3'
A C TR A A C A C TG A A G C G T
C TG G A YTG TG A R C C A A G G A
G AH C C C AC G G TC A TG A
Genotyping set (Envelope region)
JEV prim ers & probs
JE1&3Env1052F-1082Pset
G enotype
JE1Env1082P
1
JE1Env 1119R -1082Pset
JE&3Env1052F-1082Pset
G enotype
JE3Env1082P
3
JE3Env 1119R -1082Pset
M odified by
Seq.5'-3'
A B 051292
A TG G G A A TTA YTC A G C G C A A G T
C TC A A G C A G C A A A
G G G A G C G TTTG G A G TTA C A G TA A JE1とJE3の
sence　prim er
A TG G G A A TTA YTC A G C G C A A G T は共通
C C C AG G C G G C AAA
A G G A G C A TTG G G TG TTA C TG TA A A
RNA stable tube at RT
References
Ito M, Takasaki T, Yamada K et al:
Development and evaluation fluorogenic
TaqMan reverse transcriptase PCR
assays for detection of dengue virus
types 1 to 4.
J Clin Microbiol 2004;42(12): 5935-5937.
Virological diagnosis in
Japan
<Methods in NIID>
1. TaqMan RTRT-PCR
2. Conventional RTRT-PCR ( nested PCR)
3. Virus isolation (C6/36, Vero9013 cells)
<Methods in provincial level>
1. Conventional RTRT-PCR ( nested PCR)
2. Virus isolation (C6/36
(C6/36 cells,
cells, Vero9013 cells)
Results of the 2009 WPR JE Labnet proficiency testing:
Panbio JE-DEN Combo ELISA kit
CDC Panbio
Results
Sample #
1
2
3
4
Type
CSF
CSF
CSF
CSF
5
6
7
8
9
10
11
Laboratory number/results
10
retest**
DEN
NEG
NEG
JE
5**
DEN
NEG
NEG
JE
6**
DEN
NEG
NEG
JE
7**
DEN
NEG
NEG
JE
8**
DEN
NEG
NEG
JE
9**
DEN
NEG
NEG
JE
10**
DEN
NEG
NEG
JE
CSF
serum
NEG
DEN
NEG
DEN
NEG
DEN
NEG
DEN
NEG
DEN
NEG
DEN
serum
serum
serum
serum
serum
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
DEN
JE equiv
DEN
DEN
equiv
JE
NEG
NEG
DEN
Kit usedPanbio
Panbio
Panbio
Reporting date June 25
11**
NEG
NEG
NEG
NEG
JE
NEG
DEN
JE
JE
JE
NEG
NEG
DEN
Panbio
Panbio
Panbio
June 30
June 30
Jul-03
July 7
August 22
Panbio
NA
August 26
Panbio
100
100
100
100
91
91
82
%Agreement with CDC Panbio
results
100
**Compared against CDC Panbio results .
Difference between CDC and national laboratory Panbio results
In-house and non-Panbio commercial kits
CDC
ELISA and PRNT results
Sample # Type
1
CSF
2
CSF
CDC updated
results(2/17/10)
JE PRESUMPTIVE
NEG
JE PRESUMPTIVE
NEG
3
4
CSF
CSF
NEG
JE PRESUMPTIVE
JE PRESUMPTIVE
JE PRESUMPTIVE
5
6
CSF
serum
JE PRESUMPTIVE
DEN POSITIVE
JE PRESUMPTIVE
DEN POSITIVE
7
8
9
10
11
serum
serum
serum
serum
serum
JE POSITIVE
JE PRESUMPTIVE
NEG
DEN PRESUMPTIVE
DEN POSITIVE
JE POSITIVE
JE PRESUMPTIVE
NEG
DEN PRESUMPTIVE
DEN POSITIVE
Laboratory Number/results
1*
JE
NEG
difficult to
distinguish
JE
JE suspected
Kit used
Reporting date
2*
NEG
NEG
3*
NEG
NEG
4*
NEG
NEG
JE
JE
JE
JE
JE
JE
DEN
JE
NEG
JE
DEN
JE
NEG
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
JE
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
JE
In-house
July 2
Non Panbio
Commercial
July 2
In-house In-house
July 5
July 6
%Agreement with CDC results
82
91
82
100
*Compared against CDC JEV and DENV IgM ELISA + JEV and DENV PRNT results.
Difference between national kit and CDC results
Difference between CDC DEN and national kit negative results
Updated results: Difference between CDC results 6/5/09 and 2/17/10
NATIONAL PUBLIC HEALTH LABORATORY
Organization Chart of NPH Laboratory
Quality Assurance Unit
Japanese encephalitis Sentinel
Site Surveillance in Cambodia
OPD and Public Relation Unit
Immunology and Molecular Unit
NPH Laboratory
Biochemistry Unit
Hand on training Workshop on Laboratory Diagnosis of Japanese
Encephalitis on 15-19 Nov.2010, Hong Kong
Microbiology and Parasitology Unit
Hematology and Blood Parasite Unit
AM CHANTHAN
Immunology unit
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 .
Meningo-encephalitis
Case Definition
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 hospitalbased 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 hospitalbased 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.
Structure for JE Laboratory
A person with acute onset of fever (≥
≥38 C) and one of the
following: neck stiffness, altered consciousness, other meningeal
sign at all ages.
•
Project coordinator
Suspected in children <1 year of age when fever
is accompanied by a bulging fontanelle.
Project assistance
•
•
Does not include cases suspected to be caused by
chronic infections such as tuberculosis or HIV.
Samples collection Serum 1,Serum 2 and CSF
Staff analysis
Staff analysis
Staff analysis
1
Structure Routine Sentinel site Surveillance For Meningo-Encephalitis in Cambodia
NPH
Laboratory
STORAGE -75°C
Analysis
JE in CAMBODIA
Patient
National
Hospital
Provincial
Hospital
Provincial
Epidemiology
Surveillance
Unit
WHO Lab
network
NIP Center
MOH
Samples Analysis
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
• 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 crossreacting flavivirus that also circulates in Cambodia.
• Paired sera (Serum-1 and Serum-2) and CSF are used.
6. Battambang Provincial
Hospital just started in 2009
• Remaining samples are stored in freezer at -75°
° C in NIPH
Laboratory for future need.
Testing Algorithm For JE
Samples Collection
CSF or Serum 1and Serum 2
JE & Dengue IgM Capture ELISA
Pos
Neg
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
2
Am ount s am ple by site
2006-Oct-2010
WORKLOAD JE AND DEN FROM 2006TO Oct-2010
211
167
ACH
342
TOTAL CASE
NPH
264
KCM
JE positive
Tak eo
1155
DEN positive
SVR
234
BB
162
127
Tot al
BB, 26
1155
JE and DEN Result Positive form 2006 to 2009
distributed by Hospitals
Samples received from 6 JE sentinel sites
From 2006 to Oct 2010
342
3500
264
3000
234
2500
2000
Total patients
JE positive
Den Positive
162
1500
127
1000
500
0
67
49
1
2
3
4
5
6
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
4153
4253
44
2519
22
26
21
AHC
Kampong
Cham
JE and DEN Negative
control Lot#9159
Distribution by age of JE positive cases
from 2006 to 2009
JE cases
% of JE
80
NPH
50
69
0.100
0.090
0.080
60
53
40
41
30
29
40
0.060
0.050
33
20
21
20
0.070
12
0.040
0.030
10
9
0.020
0.010
0
0
Under 1y
1 - 5 ys
JE cases
6-10 ys
% of cases in each group
11 - 15 ys
0.000
1
2
3
4
5
6
7
JE NEG
8
9
10
11
12
13
14
15
DEN NEG
3
Mean Calibration kit of lot #9159
JE and DEN POSITIVE
IQC Lot# 9159
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
4.000
3.500
3.000
2.500
2.000
1.500
1.000
0.500
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0.000
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean Cal JE
JE PO
Mean Cal JE
DEN POS
JE and DEN CAL
LOT#9159
Challenges
– Quality of some samples are not good
(Heamolysis etc..)
– Inadequate volume
– Difficulties in second serum collection
– Not in house control
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
1
2
3
4
5
6
7
JE CA
8
9
10
11
12
13
14
15
DEN CAL
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
4
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
5
Introduction of JE
Name in Chinese: 乙脑
JE surveillance in mainland China
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.
Dr. Fu Shihong
Department of Viral Encephalitis and Arbovirus
Institute for Viral Disease Control and Prevention
China CDC
15 Nov 2010 Hong kong China
JE transmission cycle
and possible control points
Mosquito-born disease
JE - major encephalitis in china
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
Encephalitis
JE distribution China 2005
Endemic in 28 provinces
The major cases: south-west
China
Area of prevalent
2012-7-20
3
2012年7月20日
Arboviruses in China
4
JE in 2006
JE cases are reducing dramatically
in China, special in recent years
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.”
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
25
Consensus statements from
Global JE meetings
1995, 1998, and 2002
发病病/10万
20
JE in China historically
15
10
5
In 2007, JE vaccine has been integrated into national immunization program in whole country
2006
2005
2004
2003
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
1992
1991
↑
1990
1989
1988
1987
1986
1985
1984
1 dot = 1 case
1983
1982
1981
1980
1979
1978
1977
1976
1975
1974
1973
1972
1971
1970
↑
1969
1968
1967
1966
1965
1964
1963
1962
1961
1960
1959
1958
1957
1956
1955
1954
1953
1952
1951
1950
年
0
Serum and CSF
sample collection
and tests
JE surveillance in
serology in China
Yunnan:747 / 853
Xinjiang:641/641
Guizhou:649/720
Gansu:134/208
Liaoning:121/121
Sichuan:600/600)
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.
IFA assay
ELISA assay
1
2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
55
JEV
COX
Echo
HSV
MuV
DENV
BAV
50
45
40
阳性病例数
Yunnan samples tests
(1)
35
30
25
20
15
10
5
0
3月
4月
5月
6月
7月
8月
发病月份
9月
10月
11月
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.
Conclusion
It 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 China
–Mosquito collection
JEV surveillance
In mainland China
Mosquito collection
61303 mosquitoes collected from 8 provinces in 2008:
Jiangxi, Sichuan, Gansu, Liaoning, Guizhou, Qinghai
Xinjiang, Inner Mongolia
Mosquito collection
Virus isolation and identification
Virus isolation and identification
Cell lines
Liquid nitrogen
CPE in C6/36
Homogenize
Supernatant
IFA test with virus-antibody
Inoculated to Cells
CPE in BHK
Animal test
PCR/Real-time PCR
Sequence/Bioinformatic
Virus isolation
Virus isolation
All processes were conducted in BSL - 2 lab
50-100 mosquitoes in each pool
The virus isolation and dentification result
40 virus strains were isolated and 16 of 40 was identified in JEV
JEV(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
Mosquitoes homogenized in shakers,
Centrifuged with 12 000 rpm/ 20 min
In 2009, specimens from human cases and vectors were
collected and all detection are under way
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
Yunnan: 568 cases / 720
Guizhou: 279 cases / 359
Qinghai:661cases/ 801 animao:364
Jiangxi: 80 cases / 80
Tibet: 248 human，
， pigs 66
Liaoning:187 human
Xinjiang:190 animal
Jiangxi:5905
Sichuan:8122
Xizang:4089
Shangdong:9859
Hubei:9424
Guizhou:14516
Yunnan:17790
Qinghai:7623
Xinjiang:1500，
， shadflies 50
Liaoning:3600
Heilongjiang:2592 ticks
JE-PT in China
JE proficiency testing in
Mainland China
Local 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
100
100
100
Other 11 Province CDC
Other 2 Prefecture CDC
JE-PT, WHO WPR, 2009
In-house and non-Panbio commercial kits
Name of lab: Dep. of Viral Encephalitis, IVDC, CCDC
Name of the assay: Beixi kit (China)
Plate ID: FU
Batch No. of the kit: EVI-002M
Date: 2009/06/28
Lot No. of the kit: 0905-1
Assay performed by: Fu Shihong
Expiry date of the kit: 2010/05/08
CDC
ELISA and PRNT results
CDC updated
results(2/17/10)
JE PRESUMPTIVE
NEG
JE PRESUMPTIVE
NEG
CSF
CSF
NEG
JE PRESUMPTIVE
JE PRESUMPTIVE
JE PRESUMPTIVE
5
6
CSF
serum
JE PRESUMPTIVE
DEN POSITIVE
7
8
9
10
11
serum
serum
serum
serum
serum
JE POSITIVE
JE PRESUMPTIVE
NEG
DEN PRESUMPTIVE
DEN POSITIVE
Sample # Type
1
CSF
2
CSF
3
4
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Mark
BLK
Neg.
Pos.
Sample
1
Sample
2
Sample
3
Sample
4
Sample
5
Sample
6
Sample
7
Sample
8
Sample
9
Sample
10
Sample
11
RD
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 ≥ 2.1
3. 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)
Laboratory Number/results
2*
NEG
NEG
3*
NEG
NEG
4*
NEG
NEG
JE
JE
JE
JE
JE
JE
JE PRESUMPTIVE
DEN POSITIVE
1*
JE
NEG
difficult to
distinguish
JE
JE
suspected
DEN
JE
NEG
JE
DEN
JE
NEG
JE POSITIVE
JE PRESUMPTIVE
NEG
DEN PRESUMPTIVE
DEN POSITIVE
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
JE
JE
JE
NEG
NEG
DEN
JE
JE
NEG
NEG
JE
Kit used
Reporting date
In-house
July 2
%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 results
Difference between CDC DEN and national kit negative results
Updated results: Difference between CDC results 6/5/09 and 2/17/10
Thanks And Welcome to
new campus
Non Panbio
Commercial In-house In-house
July 2
July 5
July 6
26
National Center for
Laboratory & Epidemiology
(NCLE)
NATIONAL CENTER FOR
LABORATORY AND
EPIDEMIOLOGY, LAO PDR
NATIONAL CENTER FOR
LABORATORY AND EPIDEMIOLOGY
Reference Laboratory
CAPACITIES
ELISA
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.
PCR
Luminex Technologies
Cell Culture
National Center for Laboratory (NCLE)
Mahosoth Hospital (Welcome Trust)
Diagnostic Methods Used
-NCLE: Panbio JEV-IgM Dengue Combo ELISA
-Mahosot Hospital.
Panbio JEV-IgM Dengue Combo ELISA
Xcyton JEV CheX
Hapalyse Dengue JE MP PA kit-Pentax
Influenza
Dengue serotyping
JE LABORATORY
Dengue
Japanese encephalitis virus
Measles
Rubella
HIV
Hepatitis A virus
Leptospira
Upper Respiratory virus
Influenza
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
NUMBER OF SAMPLES TESTED FOR JE IN
2009 AT NCLE
JE TESTING ALGORITHM
Specimen Source
NUMBER OF SPECIMEN TESTED
FOR JE
AS 06 OCTOBER 2010 (NCLE)
Province
Oudomxay
Bokeo
Huaphanh
Xayaboury
Vientiane Capital
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
Total
17
17
11
3
3
QUALITY ASSURANCE
Sample
Tested by
ELISA
23
04
16
06
01
Positive IgM
ELISA
%
12
02
06
02
0
52.17%
50.00%
37.50%
33.33%
0%
50
22
44%
Total
Result (JE(JE-IgM)
Number Number
Received Tested Negative Positive Equivoca
l
Sending Specimen to NIID for confirmation (2009
= 17 samples)
Received PT from Hong Kong (2009)
FUTURE PLANS
CHALLENGES AND REQUEST
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.
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
Introduction
Country Report – Malaysia
Japanese Encephalitis
First confirmed case of JE in Malaysia was
in 1952
1954 serological surveys in man and
animals showed JE is endemic in Malaysia
Virology Unit
Institute for Medical Research
Kuala Lumpur
Viral encephalitis is a notifiable disease
(no specific aetiological agent recorded)
Therefore, cases of JE cannot be
quantified accurately
15 November 2010
Laboratories Performing Tests for JE
Outbreak
Clinical
Surveillance
Cases
Diagnostic Methods
Antibody Detection:
Haemagglutination Inhibition Test*
JE IgM Capture ELISA (1991)
MOH
MOH
Viral Isolation:
MOE
Suckling mice (till 1993)
C6/36*
(1) IMR
IMR
(1)UMMC –
Klang Valley
(2) NPHL
(2)UNIMAS Sarawak
Testing Algorithm For JE (Virology Unit, IMR)
Molecular Detection
rRT-PCR*
* for severe and fatal cases
QA Measures Implemented
CSF and/or Serum
Part of AES
investigations
1. ISO 15189 (NATA, Australia)
JE & Dengue IgM Capture ELISA
-in progress
Pos
Neg
Request for 2nd Sample
JE IgM Capture ELISA
Pos
Neg
+
HI
RT-PCR
Viral Isolation
Request for 2nd Sample
JE IgM Capture ELISA
Pos
Neg
2. External Quality Assurance program
RCPA (Arboviruses), WHO/WPR
Distribution of Laboratory Confirmed JE Cases
(1999-Oct 2010)
800
60
700
50
No of JE Cases
600
55.8%
64.8%
35.7%
69.9%
64.3%
2009
Till Oct 2010
44.2%
10
2008
2009
2008
2007
Year
Serum
Year
CSF
Age Distribution of Laboratory Confirmed JE
(2007-Oct 2010)
% of Samples Tested Positive for JE IgM
(2007-Oct 2010)
10
9
8
7
6
5
4
3
2
1
0
Serum
2007
CSF
2008
2009
2010
30
20/224
14/166
17/225
21/317
38/637
18/322
18/413
16/292
34/717
13/400
2/126
1/97
Number of JE Cases
% Positive
Serum+CSF
25
20
15
10
5
0
2007
2008
2009
0-04
Till Oct 2010
'05-15
16-24
25-59
>59
Age (Yrs)
Year
Distribution of Laboratory Confirmed JE Cases by States
(2007-Oct 2010)
Distribution of Laboratory Confirmed JE Cases by Month
(2007-Oct 2010)
100
90
2008
2009
2010
80
70
60
50
40
30
20
State
0
Ja
n'
0
M 7
ar
'0
M 7
ay
'0
7
Ju
l'0
S 7
ep
'0
7
N
ov
'0
7
Ja
n'
0
M 8
ar
'0
M 8
ay
'0
8
Ju
l'0
S 8
ep
'0
8
N
ov
'0
8
Ja
n'
0
M 9
ar
'0
M 9
ay
'0
9
Ju
l'0
S 9
ep
'0
9
N
ov
'0
9
Ja
n'
1
M 0
ay
'1
S 0
ep
'1
0
Putrajaya
Kuala
Lumpur
Sabah
Sarawak
Kelantan
Pahang
Terengganu
Johor
Malacca
Selangor
N Sembilan
Perak
Penang
Perlis
10
Kedah
No of JE Cases
2007
160
140
120
100
80
60
40
20
0
Till Oct 2010
2007
2006
1999
0
0
2005
35.2%
100
20
2004
200
30.1%
2003
300
30
2002
400
40
2001
500
2000
No of Sample
Number of Sample Tested for JE IgM Ab
(2007-Oct 2010)
Pos
Number of Sample Tested
Immunisation Coverage for JE (First Dose)
Sarawak
JE Vaccination Programme
Sarawak
Peninsular Malaysia & Sabah
-Vaccination given within 2 KM radius when
there is a case of JE
120
100
% Vaccinated
-Started in 2002 (EPI)
-Age: 9mths, 10mths
Boosters: 18mths of age, 3yrly till age 15
yrs
80
60
35,680 /
41,360
27,981 /
42,180
66.3%
40
41,784 /
42,150
40,850 /
42,292
99.1%
96.6%
2008
2009
85.0%
19,744 /
41,340
47.8%
20
0
2005
2006
2007
Year
Virology Unit, IMR
Diagnostic Laboratory
Human Resources:
HEAD UNIT
TISSUE
CULTURE LAB
MOLECULAR
DIAGNOSTIC
LAB
HIV LAB
SEROLOGY
LAB
HEPATITIS
LAB
BSL-3 LAB
ELECTRON
MICROSCOPY
LAB
EVALUATION/
QC LAB
Head Unit and Clinical Virologist
Pathologist
Senior Research Officer
Research Officer
Senior Medical Laboratory Technologist
1
4
3
3
Medical Laboratory Technologist
Health Assistant
22
5
EXOTIC
DISEASES
LAB
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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-mark-
Where is PNG
Papua New Guinea
Oscillah Kaminiel/Ernest Velemu
Department of Health
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 Network
National (CPHL)
BTS
Clinical Labs
(PMGH)
(3)
Province
(19)
District
(~130)
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 PNG
THANK YOU
Japanese Encephalitis Country Report :
The Philippines
Status of JE Surveillance in the
Philippines
2nd InterInter-country HandsHands-on Training Workshop on the Laboratory
November 1515-19, 2010
Analisa N. Bautista, RMT and Ava Kristy D. Sy, RMT
Research Institute for Tropical Medicine, Manila
Source: National Epidemiology Center
AES Cases by Agegroup & Sex (N=34)
Philippines, 2008
Male
Female
>49 yrs
<01
31-40 yrs
Agegroup (Years)
1-4
21-30 yrs
5-14
11-20 yrs
15-24
1-10 yrs
25-39
<1 yr
40-64
65 & above
15
10
5
0
5
10
15
No. of Cases
Status of JE Surveillance in the
Philippines
Sentinel Surveillance for Etiological Diagnosis
of Meningitis/Encephalitis/Meningoencephalitis
(MEMe) in the Philippines
Sentinel Surveillance for Etiological Diagnosis of
Meningitis / Encephalitis / Meningoencephalitis in the
Philippines (CNS Infections) has been implemented in
2009
• Sample collection started in March 2009 from Bulacan,
and Tarlac (Region 3), Iloilo (Region 6), Naga (Region 5),
NCR to start this December 2010
Source of Funding: World Health Organization
• Testing began in June 2009 when JE/Dengue IgM Combo
ELISA kits became available
5 sentinel sites –Bulacan and Tarlac, Region 3
Naga, Region 5
Iloilo, Region 6
Quezon City, National Capital Region
50 cases/site
Summary of Samples Received,
2009-2010 (n=212)
•
Panbio JE/Dengue IgM Combo ELISA
Type of specimen:
•
CSF
Serum
•
Summary of Results, 2009-2010
(n=212)
RESULTS
Quality Assurance
%
TARLAC
%
Other
Hospitals**
%
Total
%
5
10.0%
7
28.0%
4
7.5%
36
17.0%
5.3%
10
20.0%
3
12.0%
7
13.2%
27
12.7%
2.6%
7
14.0%
1
4.0%
0
0.0%
12
5.7%
10.5%
1
2.0%
1
4.0%
0
0.0%
8
3.8%
BULACAN
%
NAGA
%
6
13.0%
14
36.8%
5
10.9%
2*
3
6.5%
1
2
4.3%
4
ILOILO
• Proficiency Panel
• Scored 100%
JE Positive
Dengue Positive
H. influenza B
S. pneumoniae
N. meningitidis
0
0.0%
1
2.6%
0
0.0%
0
0.0%
0
0.0%
1
0.5%
30
65.2%
16
42.1%
27
54.0%
13
52.0%
42
79.2%
128
60.4%
46
100.0%
38
100.0%
50
100.0%
25
100.0%
53
100.0%
212
100.0%
Negative
TOTAL OF CASES
* Discrepant result: Serum 1 =Dengue positive and Serum 2 = JE positive
**Not tested for Bacterial pathogens
• Confirmatory / Validation of Samples
in RRL
• Sent 64 samples (31 CSF, 33 Serum Samples)
• 95% concordance
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
Study Site
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
Collection methodology
• Adult mosquitoes were collected from May2009
to April2010 by animal baited trap method.
No collection in September 2009 due to the typhoon.
• Barangay Moriones &
Lubigan
Minicipality of San Jose,
Tarlac Province, Region III
-Population; 4,437
-Household; 876 (2007)
Tarlac province
RITM
• Environment
Ecology suitable for the
transmission of JEV such as breeding
of Culex spp, pig farm and the rice
field all within 1km radius
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)
Phylogenetic analysis of the JEV from partial of
Envelope gene (326bp)
Population of the
collected mosquitoes
CH1392/Mo/1990/China/AF254452
2009-2010
May
Cx.
tritaeniorhynchus
Cx.vishinui
Cx.gelidus
Cx. fuscocephala
Cx. spp
Total
June
July
Aug
Oct
Nov
Dec
Jan
Feb
Mar
Apr
Total
Neighbor-Joining
method
At bootstrap value of
1,000 replicates
59 TPC0706a/Mo/2007/Taiwan/GQ260626
T1P1-S1/Mo/1997/Taiwan/AY303791
79
948
671
300
162
2213 2060 1052 670
179
39
16
129
290 587 123
325
4816 1155 1431 1191
8,446 4,512 2,922 2,477
200
818
287
253
56
92
19
3806
417
74
110
874
1,675
740
49
1028
4045
6,680
770
342
1051
4857
7,307
1001
114
527
4631
6,526
732
15
198
4156
5,157
143
5
83
248
571
40
1
12
416
488
9838
963
4334
27820
46761
JaGAr 01/Mo/1959/Japan/AF069076
ML17/Hu/1981/Japan/AY508812
41
92
81P241/Sw/1981/Japan/FJ943464
93
SA14/Hu/1959/China/AY243842
Genotype 3
SA14-14-2/Va/2000/China/AF315119
67
JaOArS982/Mo/1982/Japan/M18370
80
PhAn1242/Sw/1984/Philippines/U70417
51
91
42
CC27-L3/Mo/1983/China/AY303796
Phi09/Mo/2009/Philippines
Beijing-1/Hu/1949/China/L48961
88
82
81
Nakayama/Hu/1935/Japan/EF571853
FU/Hu/1995/Australia/AF217620
JKT5441/Mo/1981/Indonesia/1981/U70406
66
VN78/Mo/2002/Vietnam/AY376467
95
JaNAr13-04/Mo/Japan/2002/FJ185146
JKT6468/Mo/1981/Indonesia/AY184212
Genotype 2
Genotype 1
Genotype 4
WNV NY2002 Clinton/DQ164193
0.02
Summary
• More than 40,000 collected by this method and
majority were Culex spp.
• JEV was detected from Cx. tritaeoniorhynchus
collected in Nov 2009
• Philippine strain was belonged to genotype 3
Thank you for your attention!
Overview of JE in Korea
Report from JE RRL
Vaccine import (1968)
JE surveillance program (1975)
Last epidemic in 1982 (1,197 cases)
Japanese Encephalitis in Korea
Mandatory vaccination < 15 age
(1983)
1930s
15 November, 2010
1940 - 50s 1960 - 70s 1980 - 90s
2000s
Annual JE cases < 8
Designated as JE RRL (2009)
Largest outbreak in 1958 (6,897 cases)
Cho, Jung-Eun
Division of Arboviruses
National Institute of Health
Korea Centers for Disease Control and Prevention
Notifiable disease in 1949 (5,548 cases/2,429 deaths)
First case report in 1933 in Japan (1,924 cases)
1
2
Laboratory diagnosis of JE in Korea
JE cases in Korea
1. Serology
• Annual JE cases
•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)
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
* As of November 10, 2010. One case is from foreigner aged of 28.
•PRNT is conducted for differential diagnosis from other flaviviruses
• Age pattern of JE patients
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)
3
•
•
•
•
•
•
No. of
Mosquito
detection* collection date
1
Year
2007
52
32–85
2007
1 SEP – 1 NOV
Period
2008
52
45–69
2008
22 AUG – 19 OCT
2009
50
36-69
2009
13 SEP – 25 OCT
2010 *
54
39–77
2010
11 AUG – 21 OCT
4
Sero-conversion of animal host
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)
Gyeong-Nam
Range
Vector and animal host surveillance in 2010
Virus detection from mosquitoes
Province
Mean age
*One foreigner case was excluded.
Vector and animal host surveillance in 2010
•
•
•
•
•
• Annual JE season
Year
4 OCT
*Detected JEV belongs to the genotype 1
5
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 (%)
2005
2,515
1,043
41.5
No.of JE case
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
6
Other flavivirus surveillance in Korea
Publications in 2009-2010
• Dengue fever
2008
2006
2007
2008
2009
2010
No. of cases*
Year
2003 2004
22
16
28
63
159
94
88
161
No. of sample
59
74
109
177
290
280
269
429
• West Nile fever
Year
2003 2004 2005 2006 2007 2008 2009 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 encephalitis
Year
2006 2007 2008 2009 2010
No. of cases
0
0
0
0
0
No. of sample
17
47
76
145
139
J Virol Methods. In press, accepted
7
Development and field evaluation of a nested RT–PCR kit for
detecting Japanese encephalitis virus in mosquitoes.
8
Suggestions & Future plan
• We hope that nested RT-PCR and immunochromatographic test kit
could be distributed to JE endemic regions
•Multiplex real-time RT-PCR kit is now being evaluated and will
be released in 2011
•Results of flavivirus surveillance (Dengue, West Nile) conducted in
2010 will be presented at the next meeting
Thank you for your attention!
• From 2011, sero-survey in adult population will be implemented
to cope with the age shift phenomenon in JE patients
Any Questions or Suggestions:
Dr. Han or Mr. Jeong
([email protected]/[email protected])
9
10
Viral encephalitis surveillance
in Vietnam
COUNTRY REPORT
• Viral encephalitis cases were reported
monthly.
• 3 sentinel sites: North (1), Central (1),
South (1)
• National hospitals in Hanoi, HCM City.
VIET NAM
Arbovirus Laboratory
National Institute of Hygiene and Epidemiology
• Some other provincial preventive medicine
centre.
Hong Kong, November, 2010
Viral Encephalitis in Vietnam, 2009
Northern
Central
Southern Highland
Provinces Provinces Provinces Provinces
Total
Viral encephalitis in Northern Vietnam,
2009 – Oct, 2010
700
699
600
496
500
Morbidity
699
73
234
81
1,042
400
300
Viral Encephalitis case
Collected sample case
JE possitive case
194
200
Mortality
5
2
16
1
24
124
100
22
13
0
2009
JE case in Northern Vietnam, 2010
7
6
JE cases in 2 sentinel sites
(2009 – 10/2010)
JE (+)/ Viral
Encephalitis
9
9
8
6
Thai Binh
5
2009
2010
(10/2010)
Total
5/175
7/79
(8,7%)
12/254
(4,7%)
1/9
(11,1%)
8/32
(25%)
(2.9%)
5
4
3
2010
Age group
2
2
1
Quang Ngai
0
0-1
1-4
7/23
(30%)
0
5- 10
10 - 15
>15
JE vaccination in Northern Vietnam
2009 - 2010
Confirmatory test
•
•
•
Total samples sent: 74
Positive: 23
Negative: 51
Agreement result: 70
Disagreement result: 4 (2 cases)
Year
Province
District
2009
25/28
275/325
2010
26/29
280/325
JE vaccination in Northern Vietnam,
January - September, 2010
JE vaccination in Vietnam 2009
Northern
Central
Southern Highland Total
Provinces Provinces Provinces Provinces
Province
2 doses
Total
1st and
2nd dose
3rd dose
94.3%
94.8%
96.5%
98.1%
92.9%
93.8%
90.7%
94.0%
93.1%
94.8%
1
Hanoi
2
3
Vaccinated
3 doses
Ratio
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.
Ratio
91.548
83,8 104.296
73.179
70,2
Nam Dinh
33.811
32.940
97,4
32.700
31.981
97,8
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
Production of MAC – ELISA kits
• Surveillance the molecular biology characteristic of JE in
genome of the first JE isolation from human genotype 1
Vaccinated
109.198
Some other activities
some provinces in Vietnam and sequencing the whole
Total
•
•
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!
Laboratory capacity for JE Diagnostics
Country report
in Southern Viet Nam
JAPANESE ENCEPHALITIS IN
SOUTHERN VIETNAM
Provincial Laboratory
– Serology
• MAC-ELISA
Pasteur Institute
Hong Kong - 2010
–
–
–
–
Serology
Virology
RT-PCR
Realtime RT-PCR
2
JE sentinel sites in
Vietnam South
Laboratory JE Diagnostics
MAC-ELISA
Using the in-house KIT
(modif. protocol CDC ).
AES samples were
tested DEN- IgM and JEIgM.
Binh Duong
Ho Chi Minh
Real-time RT-PCR
Bến Tre
-Sentinel site: Binh Duong provincal Hospital
-Sujected:
1.Ben Tre provincal Hospital
ISOLATION OF VIRUS ON C6/36 CELL
2.Ho Chi Minh city (Hospital of
Tropical Disease and Pediatric 2 Hospital)
3
JE surveillance by MAC-ELISA ,
2007- 2009
JE surveillance by MAC-ELISA
2007- 2009
Year
2007
No. of cases
242
CSF
No. of
samples
96
1st serum
(+)
No. of
samples
3-1
151
2nd serum
(+)
No. of
samples
(+)
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: WHO
Using the Japanes encephalitis- Dengue IgM in-house KIT
5
6
JE surveillance by MAC-ELISA ,
2007- 2009
200
Epidemiology of Acute Encephalitis Syndrome
14
CFS
180
12
160
10
140
120
8
100
6
80
60
4
S1
40
2
20
0
0
2007
2008
No. of samples
2009
JE(+)
DEN(+)
120
14
S2
100
12
10
80
8
60
Month
6
40
4
20
2
0
0
2007
2008
No. of samples
2009
JE(+)
DEN(+)
7
Epidemiology of Acute Encephalitis Syndrome
Month
www.themegallery.com
JE Vaccination Status
No. of children
under 5 year-olds
with
JE vac. coverage
Ratio (%)
20
88,157
85.60
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%)
Year
No. of
province
s
No. of
district
2001
3
3
2002
5
7
2003
11
2004

Laboratory Network - WPRO IRIS

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