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
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 General... 8
3.2 Evaluation of the training workshop ... 8
3.3 Outcomes of the training ... 9
3.4 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
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
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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).
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.
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2.2.3 Session 3: GSL, Regional Reference Laboratories (RRL) and country reports 2.2.3.1 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.
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
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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)
0 20 40 60 80 100 120 140 160
Perlis Kedah Penang Perak Selangor N Sembilan Malacca Johor Pahang Terengganu Kelantan Sabah Sarawak Kuala Lumpur Putrajaya
State
No of JE Cases
2007 2008 2009 2010
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.
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
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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
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 W O R L D H E A L T H
ORGANIZATION
ORGANISATION MONDIALE DE LA SANTE
REGIONAL OFFICE FOR THE WESTERN PACIFIC BUREAU REGIONAL DU PACIFIQUE OCCIDENTAL
2ND INTERCOUNTRY HANDS-ON TRAINING WORKSHOP ON THE LABORATORY DIAGNOSIS OF JAPANESE ENCEPHALITIS IN THE WESTERN PACIFIC REGION
Hong Kong (China) ENGLISH ONLY
15-19 November 2010
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: am.chanthan07@gmail.com Mr Thay Kosal
Laboratory Staff – Microbiology Supervisor Khmer-Soviet Friendship Hospital
#32E, ST 287
Sangkat Boeng Kak II Khan Tuol Kok Phnom Penh
Telephone: + 855 12 505434 E-mail: thay.kosal@gmail.com
CHINA Dr Fu Shihong
Senior Research Technician Department of Viral Encephalitis Institute for Viral Disease Control and Prevention
155 Changbai Road, Changping District Beijing 102206
Telephone: +86 10 5890 0843 E-mail: shihongfu@hotmail.com
Dr Ma Hongxia Master Technician
Henan Centers for Disease Control and Prevention
Room 4302
No. 105 Nongyedonglu Zhengzhou 450016
Telephone: +86 371 6808 9290 Facsimile: +86 371 6808 9002
E-mail: hongxiama2010@gmail.com LAO PEOPLE'S
DEMOCRATIC REPUBLIC
Mr Sinakhone Xayadeth Laboratory Technician
National Center for Laboratory and Epidemiology
Country Surveillance Km 3, Thadeua Road Vientiane
Telephone: +856 21 2336196 Facsimile: +856 21 315347 E-mail: tsinakhone@yahoo.com Mrs Khuanta Duangmala Laboratory Technician
National Center for Laboratory and Epidemiology
Country Surveillance Km 3, Thadeua Road Vientiane
Telephone: +856 20 99610688 Facsimile: +856 21 315347
E-mail: khuantaduangmala@yahoo.com MALAYSIA Ms Nurul Asshikin bt. Ruslan
Medical Laboratory Technologist Virology Unit
Institute of Medical Research Ministry of Health Malaysia Jalan Pahang
50588 Kuala Lumpur Telephone: +60 3 26162675 Facsimile: +60 3 26938094
E-mail: nurul_asshikin@yahoo.com 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: oscillah_kaminiel@health.gov.pg/ oscillahk@hotmail.com Mr Ernest Velemu
Quality Assurance Manager
Central Public Health Laboratory Private Mail Bag No. 1,
Boroko
National Capital District Telephone: +67 5 3248199 Facsimile: +67 5 3256342 E-mail: evelemu@gmail.com PHILIPPINES Ms Analisa Bautista
Senior Science Research Specialist Research Institute for Tropical Medicine Virology Department
9002 Research Drive, FCC Compound, Alabang Muntinlupa
Telephone: +63 2 8097120 Facsimile: +63 2 8097120 E-mail: ann31616@gmail.com
Ms Ava Kristy Sy Bacteriologist I
Research Institute for Tropical Medicine 9002 Research Drive
FCC Compound, Alabang Muntinlupa City 1781 Philippines
Telephone: (632) 8097120 Facsimile: (632) 8097120 E-mail: avakristysy@gmail.com REPUBLIC OF KOREA Ms Jung Eun Cho
Researcher
Korea Centers for Disease Control and Prevention
Division of Arboviruses National Institute of Health 194, Tongil-lo, Eunpyung-gu Seoul 122-701
Telephone: +82 2 3802175 Facsimile: +80 2 3801499 E-mail: 920980@naver.com
VIET NAM Dr DO Phuong Loan
Researcher
National Institute of Hygiene and Epidemiology (NIHE) No. 1 Yersin Street
Ha Noi, Viet Nam
Telephone: (04) 3 9719721 Facsimile :
E-mail: loankobe@yahoo.com
Dr Huynh Phuong Thao Researcher
Pasteur Institute
167 Pasteur Street District 3,
Ho Chi Minh City, Viet Nam Telephone: (848) 38 296 351 Facsimile: (848) 38 231 419 E-mail: huynhthao196@yahoo.com
TEMPORARY ADVISERS
Dr Barbara Johnson
Diagnostic and Reference Laboratory
Division of Vector-borne Infectious Diseases Centers for Disease Control and Prevention 3150 Rampart Road
Fort Collins, CO 80521 United States of America Telephone: +970 2663543 Facsimile: +970 22106441 E-mail: bfj9@cdc.gov Dr Tomohiko Takasaki Chief
Laboratory of Vector-borne Viruses National Institute of Infectious Diseases 1-23-1 Toyama, Shinjuku-ku
Tokyo 162 8640 Japan
Telephone: +81 35 2581111 (ext 2526) Facsimile: +81 35 2581188
E-mail: takasaki@nih.go.jp Dr Wei-ling Wilina Lim
Consultant Medical Microbiologist Public Health Laboratory Center 9/F Public Health Laboratory Centre 382 Nan Cheong Street, SHek Kip Mei Kowloon
Hong Kong
Telephone: +85 2 2319 8252 Facsimile: +85 2 2319 5989 E-mail: wllim@pacific.net.hk
SECRETARIAT
Dr Youngmee Jee
Scientist (Laboratory Virologist) Expanded Programme on Immunization World Health Organization
Regional Office for the Western Pacific United Nations Avenue
1000 Manila Philippines
Telephone: +63 2 528 9744 Facsimile: +63 2 526 0279 E-mail: jeey@wpro.who.int Mr David Featherstone Scientist, Project Leader
Global Measles Laboratory Network World Health Organization Headquarters in Geneva (HQ)
Expanded Programme on Immunization Plus Avenue Appia 20
CH – 1211 Geneva 27 Telephone: +41 22 79 14405 Facsimile: +41 22 791 3111 E-mail: featherstoned@who.int
Day 1, Monday, 15 November 2010 08:30 Registration
08:45 Completion of pre-assessment questionnaire
09:00 Opening remarks Dr Wilina Lim
09:10 Workshop objectives Dr Youngmee Jee
09:20 Self-introduction of participants and administrative announcements
Session 1 Japanese encephalitis/acute encephalitis
syndrome (JE/AES) surveillance and Laboratory Network (Lab Net)
09:30 Laboratory-based JE/AES surveillance:
progress and challenges in maintaining momentum and plans for developing sustainability
Mr David Featherstone
10:00 JE control and lab net progress in the Western Pacific Region
Dr Youngmee Jee 10:30 Coffee break
Session 2 Quality assurance of the JE lab net and global specialized laboratory (GSL) presentations
11:00 Quality assurance and proficiency in the laboratory Mr David Featherstone 11:20 Role of the United States Centers for Disease
Control and Prevention (US CDC) and WHO JE GSL: evaluation of kits
Dr Barbara Johnson
11:50 Activities of National Institute of Infectious Diseases (NIID) GSL and JE surveillance/laboratories
12:10 Discussion 12:30 Lunch break
Session 3 Regional reference laboratory (RRL) and national laboratory reports
13:30 Chinese Center for Disease Control and Prevention (China CDC)
13:50 Korea Centers for Disease Control and Prevention (KCDC)
14:10 Cambodia, Lao People's Democratic Republic, Malaysia (15-minute presentation , 5-minute Q and A)
The 2nd Intercountry Hands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Western Pacific Region
Public Health Laboratory Centre Hong Kong (China)
15 to 19 November 2010
15:10 Coffee break
15:30 Papua New Guinea, Philippines, Viet Nam (2) Session 4 Introduction of JE virus-specific
immunoglobuline M (JEV IgM) enzyme-linked immunosorbent assay (ELISA)
16:30 General Introduction to IgM assays Mr David Featherstone 17:00 Lecture/demonstration of JEV IgM ELISA (serum) Dr Barbara Johnson 17:30 Tour of laboratory and demonstration of ELISA
equipment
18:00 Adjourn for the day Day 2, Tuesday, 16 November 2010
08:30 Introduction to the ELISA practical
08:45 Laboratory practice: JEV IgM ELISA (serum)
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Mr David Featherstone, facilitators and
participants 12:00 Lunch break
13:30 Continuation: laboratory practice - JEV IgM ELISA Participants 15:30 Coffee break
16:00 JEV IgM ELISA reading and calculation of results Participants
17:00 Evaluation of ELISA test results and discussion Mr David Featherstone and facilitators
17:30 Adjourn for the day
Day 3, Wednesday, 17 November 2010 08:30 Discussion of Day 2 activity
09:00 Lecture and demonstration of JEV IgM ELISA – cerebrospinal fluid (CSF)
09:30 Laboratory practice: JEV IgM ELISA (CSF)
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Mr David Featherstone, facilitators and
participants 10:00 Coffee break
10:30 Continuation: laboratory practice - JEV IgM ELISA Participants 13:00 Lunch break
14:00 Continuation: laboratory practice - JEV IgM ELISA Participants 15:00 JEV IgM ELISA reading and calculation of results Participants 15:30 Coffee break
15:45 Evaluation of ELISA test results and discussion Mr David Featherstone and facilitators
16:15 Specimen collection, preparation and shipment for virus isolation and serology
Dr Tomohiko Takasaki 17:00 Adjourn for the day
Question for the night
Day 4, Thursday, 18 November 2010 08:30 Discussion on Day 3 activity
08:45 Laboratory practice: ELISA (serum samples)
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Mr David Featherstone, facilitators and
participants 10:30 Coffee break
11:00 Continuation: laboratory practice – ELISA
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Mr David Featherstone, facilitators and
participants 12:30 Lunch break
13:30 JE PCR
Evaluation of ELISA test results and discussion
PHLC
AES data management and reporting requirements Dr Youngmee Jee Practical session on data entry Participants
17:30 Adjourn for the day
Day 5, Friday, 19 November 2010
08:30 Discussion on Day 4 activity (return quiz) 09:00 JE PCR continued
Global specialized laboratory testing methods - ELISA, plaque reduction neutralization testing (PRNT) and decision algorithm
PHLC
Dr Barbara Johnson and Dr Tomohiko Takasaki 10:00 Consolidation of laboratory test results and
classification
Mr David Featherstone and Dr Youngmee Jee 10:30 Coffee break
11:00 Course assessment and quiz 12:30 Lunch
13:30 Next steps and summary of assessment Round table 15:30 Closing ceremony: presentation of training
certificate
16:30 Distribution of proficiency panels and kits to network laboratories
PROCEDURE FOR PANBIO JE –DENGUE IgM COMBO ELISA 1) Bring all the samples and the reagents required for ELISA to room temperature.
2) Remove required number of wells and insert them into the frame.
3) Dilute positive and negative controls, JE and DENGUE calibrators, and samples using 1.5ml screw capped vials:
• 10 µl of serum sample, controls, and calibrators in 1000 µl of diluent (1:100 dilution)
• 25 µl of CSF sample in 225 µl of diluent (1:100 dilution)
4) Dilute 10 µl of antigen into 2.5 ml of antigen diluent in 15ml tubes (1:250 dilution) Prepare dilutions separately for JE and DENGUE antigen.
5) Mix required amount of JE and DENGUE antigen separately with an equal amount of Mab tracer (1:1 vol/vol) in 15 ml tubes. Leave it at room temperature until use. Discard any unused antigen.
6) Within 10 minutes of mixing the antigen and Mab tracer, pipette 100 µl of diluted Pos and Neg controls and patient samples in duplicate in the JE and DEN columns in the assay plate. JE and DEN calibrators are added in triplicate to wells in either the JE or DEN column.
7) Cover the plate with aluminum foil and incubate at 37ºC for 1 hour.
8) Prepare wash buffer :
Dilute 1 part of wash buffer concentrate in 19 parts of distilled water (1:20 dilution).
9) Wash the plate 6 times with the diluted wash buffer
10) Add 100 µl of JE antigen–Mab complex and DENGUE antigen–Mab complex prepared earlier into the respective wells.
11) Cover the plate and incubate for 1 hour at 37ºC.
12) Wash the plate 6 times with the diluted wash buffer.
13) Add 100 µl of TMB into each well. Cover the plate and incubate for 10 min at room temperature (20-25°C).
14) Add 100 µl of stop solution into each well.
15) Read the absorbance at a wavelength of 450 nm with a reference filter of 600 – 650 nm within 30 minutes.
1) Calculate the average absorbance of the triplicates of the calibrators.
2) Calculate the cut-off values:
Cut-off value = Mean absorbance of the calibrators X calibration factor (batch specific)
3) Determine validity of test:
Positive control OD/Cut-off ratio = batch-specific acceptable range
If test not valid, do not continue to calculate – repeat test.
4) Calculate index value of the sample : Index value = Sample absorbance
Cut–off value 5) Calculate the Panbio units of the sample:
Panbio units = Index value of sample X 10
INTERPRETATION OF PANBIO UNITS : JE
Panbio units
JE IgM result
DEN Panbio units
DEN IgM result
Interpretation
< 9 Neg < 9 Neg No detectable IgM antibody.The result does not rule out the infection. An additional sample should be collected in 7-14 days and the test carried out again if early infection is suspected.
< 9 Neg 9 -11 Eqv Samples should be re-tested 9-11 Eqv < 9 Neg Samples should be re-tested 9-11 Eqv 9-11 Eqv Samples should be re-tested
< 9 Neg >11 Pos 9-11 Eqv >11 Pos
>11 Pos <9 Neg
>11 Pos 9-11 Eqv
> 11 Pos > 11 Pos
Calculate JE/ Dengue ratio :
Ratio = JE Panbio units Dengue Panbio units
Interpretation of JE / Dengue ratio :
1) ≥ 1 Presence of detectable IgM antibody presumptive infection with JEV
2) < 1 Presence of detectable IgM antibody presumptive infection with DENV
PanbioJE-DEN IgM Combo ELISA procedure
World Health Organization
HANDS-ON TRAINING ON MOLECULAR DETECTION OF
JAPANESE ENCEPHALITIS VIRUS
Protocols for Practical
18-19 November 2010
Public Health Laboratory Centre Centre for Health Protection
Hong Kong SAR, China
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)
Page 1
Objectives:
1. To have an idea on molecular testing (RNA extraction and RT-PCR) of JE virus 2. To have a brief idea of the general precautions in performing molecular testing
Location:
Practical 1: Extraction of JE virus RNA (Location: Room 907)
Practical 2: One-step RT-PCR for the detection of JE virus (Location: Room 817)
WHO JE Workshop 18-19 November 2010
Page 2
Practical 1: RNA Extraction for JE virus detection
References:
QIAamp® Viral RNA Mini Handbook, Third Edition, December 2007. Qiagen.
Materials/Reagents/Equipment provided:
2 vials (sample A & sample B) containing simulated samples to be extracted Buffer AVL containing carrier RNA (pre-prepared by PHLC staff)*
QIAamp spin columns (2 vials)*
Buffer AW1 with ethanol*
Buffer AW2 with ethanol*
Buffer AVE*
Collection tubes*
1.5ml screw cap vials (for RNA storage)
1.5ml snap cap vials (caps already cut away by scissors) Absolute ethanol (96-100%)
Microcentrifuge Micropipettes
Aerosol-free pipette tips Vortex mixer
Timer Ice bucket
Biological safety cabinet
Worksheet for RNA extraction (Appendix 1)
*Reagents from QIAamp® Viral RNA Mini kit or reagents prepared from content of the kit.
Buffer AVL (with carrier RNA), Buffers AW1 (with ethanol added) and AW2 (with ethanol added) will be provided. Please refer to the kit handbook for preparation of these buffers when using the new kit.
Procedures (Room 907)
(1) Prepare worksheet for the RNA extraction.
(2) Label the vials containing 560ul of Buffer AVL with carrier RNA.
(3) In the safety cabinet add 140ul of samples to the corresponding vials.
(4) Mix by pulse-vortexing for 15 seconds. Incubate at room temperature for 10 minutes.
(5) Label the spin columns and the 1.5ml screw cap vials (for storing extracted RNA) accordingly.
(6) Briefly centrifuge the other vials after the 10-minute incubation. Add 560µl absolute ethanol (96-100%) to the each vial and mix by vortexing for 15 seconds.
(7) Pulse-spin all the vials.
Page 3
(8) Open the cap of the spin columns one by one and transfer 630µl of the solution from the vials to the corresponding spin column. Close the cap and centrifuge at 6,000 × g (8,000
× rpm) for 1 minute.
(9) Place the spin columns in clean 2ml collection tubes and discard the collection tubes containing the filtrate.
(10) Repeat steps (8) − (9).
(11) Open the cap of the spin columns one by one and add 500µl Buffer AW1. Close the cap and centrifuge at 6,000 × g (8,000 × rpm) for 1 minute. [Note: Use a new pipette tip when adding buffer to each column]
(12) Place the spin columns in clean 2ml collection tubes and discard the collection tubes containing the filtrate.
(13) Open the spin columns one by one and add 500µl Buffer AW2. Close the cap and centrifuge at 16,100 × g (13,200 × rpm) for 3 minutes. [Note: Use a new pipette tip when adding buffer to each column]
(14) Place the spin columns in clean 2ml collection tubes and centrifuge at 16,100 × g (13,200 × rpm) for another 1 minute.
(15) Place the spin columns in clean 1.5ml snap cap vials (caps already cut away by scissors) and discard the collection tubes containing the filtrate.
(16) Open the caps of the spin columns and add 60µl Buffer AVE equilibrated to room temperature.
(17) Close the cap of the spin columns and incubate at room temperature for 1 minute.
(18) Centrifuge at 6,000 × g (8,000 × rpm) for 1 minute.
(19) Remove the spin columns one by one from the 1.5ml snap cap vials and transfer the eluted RNA into the 1.5ml screw cap vials. Keep the vials on ice.
WHO JE Workshop 18-19 November 2010
Page 4
Practical 2: One-step RT-PCR for JE detection
References:
QIAGEN® OneStep RT-PCR Kit Handbook. February 2008. Qiagen.
Murakami S, Takahashi Y, Yoshida S, Fuke I, Ohmae K, Mori C, Takagi M, Takamizawa A, Okayama H. (1994) Highly sensitive detection of viral RNA genomes in blood specimens by an optimized reverse transcription-polymerase chain reaction. J Med Virol 43(2):175-181.
Materials/Reagents/Equipment provided:
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
Aerosol-free pipette tips 0.2ml PCR tubes
Vortex mixer
Tomy centrifuges (for 1.5ml and 0.2ml tubes) Ice bucket
ABI 9700 thermal cycler
Worksheet for the RT-PCR detection of JE virus (Appendix 2)
Procedures
Preparation of master mix (prepared by PHLC staff)
Master mix will be prepared by PHLC staff in master mix preparation room according to the following table:
Reagents Volume per test (ul)
Qiagen 5X PCR buffer * 10
dNTPs* 2
Primer JE-1 (5uM) 6
Primer JE-2 (5uM) 6
Enzyme mix (kit)* 2
RNase inhibitor 0.5
H2O* 18.5
*Reagent from Qiagen One-step RT-PCR kit
Page 5
JE-1: 5’-CACAACGAGAAGCGAGCTGATAGTA-3’
JE-2: 5’-CCCCAACTTGCGCTGAATAATTCCC-3’
RNA template addition (Room 817)
(1) Prepare worksheet for the one-step RT-PCR for JE virus.
(2) Label the 0.2ml PCR tubes.
(3) Aliquot 45ul of master mix to the labeled PCR tubes.
(4) Aliquot 5µl of extracted RNA and control to the corresponding PCR tubes accordingly.
Water (5µl) is used as a negative control.
(5) Vortex briefly to mix the content. Pulse-spin the PCR tubes. Place the PCR tubes into thermal cycler. Start the PCR run with the PCR conditions as shown below.
Cycling parameters of RT-PCR for JE detection
50oC 30 min; 95oC 15 min; (94oC 30 sec, 55oC 30 sec, 72oC 30sec) x 40 cycles; 72oC 10min; 10oC ∞∞∞∞
WHO JE Workshop 18-19 November 2010
Page 6
Friday 19 November
Objective:
To perform gel electrophoresis of RT-PCR product for JE virus detection obtained from practical 2 (to be performed by PHLC staff)
Location (gel loading room):
Practical 3: Gel electrophoresis of RT-PCR product for JE virus detection obtained from practical 2 (to be performed by PHLC staff)
Materials/Reagents/Equipment provided:
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
Aerosol-free pipette tips
Tomy centrifuge (for 0.2ml tubes) 96-well plates (U-bottomed) 1 vial of 6X loading dye 1 vial of 100bp DNA ladder Gel documentation system
Preparation of agarose gel
Note: Gel will be set and run by PHLC staff
(1) Loosen the cap of the bottle containing 2% agarose gel. Melt the agarose gel in microwave. Avoid overheating. Swirl from time to time.
(2) After the gel is melted completely, leave it at room temperature for 5 minutes.
(3) Assemble the gel casting tray and together with the combs.
(4) Add 25µl of SYBR SafeTM DNA gel stain to each bottle of agarose gel. Swirl to mix.
(5) Pour the mixture into the cast and allow the gel to solidify for at least 30 minutes. Cover the gel casting tray to protect it from light.
Page 7
(6) When the gel is solidified, remove the combs from the casting tray and put the gel together with the casting tray into the agarose gel tank filled with TBE buffer.
Gel loading and electrophoresis
(1) Vortex and pulse-spin the 6X loading dye.
(2) Calculate the number of wells needed and add 2µl of the 6X loading dye into each well.
(3) Vortex and pulse-spin the PCR tubes.
(4) Transfer 5µl of PCR product from each PCR tube and mix with the loading dye in the well.
(5) Load the content of each well into the wells of the agarose gel.
(6) Load 10µl of the 100bp DNA ladder one of the wells as molecular size marker.
(7) Run the gel at 140V, for 25-30 minutes (until the blue marker dye has migrated to about 5mm from the bottom of the gel).
(8) Turn off the power supply, disconnect the cable and retrieve the gel from the gel tank.
(9) Rinse the gel with water briefly.
(10) Visualize the gel and print the gel photo with gel documentation system.
WHO JE Workshop 18-19 November 2010
Page 8
Date: _______________
Appendix 1 Worksheet for RNA extraction-QIAamp Viral RNA Mini Kit
Reagent Lot No. Expiry Date
QIAamp Viral RNA Mini kit
No. Sample ID 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Samples added by: _______________ & _______________
RNA eluted by: _______________
Page 9
Appendix 2 Detection of JE virus by One-step RT-PCR
Reagent Volume per test (µµµµl) Total volume (µµµµl)
QIAGEN 5X PCR buffer (kit) 10
dNTPs (kit) 2
Primer JE-1 (5 µµµµM) 6
Primer JE-2 (5 µµµµM) 6
Enzyme mix (kit) 2
RNase inhibitor 0.5
H2O (kit) 18.5
Total 45
RNA template 5
No Sample ID Result 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Done by: _______________ Checked by: _______________
1
Introduction of Workshop Objectives
2ndIntercountry Hands on training on the Laboratory Diagnosis of Japanese encephalitis
November 15-19 2010
Objectives of this workshop
• To further enhance knowledge and skills of national JE laboratory staff in:
a) performing ELISA for laboratory diagnosis of JE, 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 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
• Session 4. Introduction of JEV IgM assays – Demonstration of ELISA assay
– Tour of the laboratory
Day 2
• ELISA Practical using serum samples Use Panbio kit
ELISA reading and calculation, interpretation
• During incubation times, calibration of micropipettes and maintenance of ELISA equipments.
Day 3
• ELISA practical using CSF samples Use Panbio kit
(During incubation times, calibration of micropipettes and maintenance of ELISA equipments)
• Lectures on specimen collection, shipping of samples for confirmatory testing and virus isolation.
Day 4
• ELISA practical for serum samples
ELISA reading and calculation, interpretation
• (During incubation times, calibration of micropipettes and maintenance of ELISA equipments)
• Data management for JE/AES
2
• Lecture on methods conducted in Global Specialized Labs
• Consolidation of laboratory test results
• Course assessment
• Discussions on next steps and future plans in details.
• Closing and certificate
• Distribution of proficiency panel samples (5 CSF and 6 serum samples)
Demonstration of the PanBio Japanese encephalitis-Dengue IgM Combo ELISA procedure
Barbara W. Johnson Barbara W. Johnson
US Centers for Disease Control and Prevention US Centers for Disease Control and Prevention
Division of Vector
Division of Vector--Borne DiseasesBorne Diseases Arboviral Diseases Diagnostic and Reference Laboratory Arboviral Diseases Diagnostic and Reference Laboratory
The 2nd Intercountry Hands
The 2nd Intercountry Hands--on Training Workshop on the Laboratory on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the
Diagnosis of Japanese Encephalitis in the WesternPacific RegionPacific Region Public Health Laboratory Centre, Hong Kong Public Health Laboratory Centre, Hong Kong
15 to 19 November 2010 15 to 19 November 2010
PanBio JE-Dengue IgM Combo ELISA kit
Test up to 43 samples/plate
Included in the kit
• Precoated microwell strips
• Reagents, diluents, and buffers
• Calibrators and positive and negative controls
Principle of JE-DEN Combo IgM ELISA
Microwells precoated with anti-human IgM
Sample added in duplicate. Capture of all IgM in sample.
JE or DEN antigen-Mab/HRP conjugate complex added, binds to JEV or DENV reactive IgM in sample
Substrate binds to bound Mab/HRP
Colorimetric measurement of JE or DEN IgM reactivity in sample
HRP HRP
HRP HRP
JE JE
JE JE DEN
HRP
DEN HRP
JE+ DEN-
Plate setup and worksheet for testing 11 samples
CONTROLS:
-: Negative Serum +: Positive Serum JC: JE Calibrator DC: DEN Calibrator
JE DEN JE DEN
- -
+ + JC DC JC DC JC DC 1 1 2 2 3 3
4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11
1. Equilibrate kit reagents and microwell strips to 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:
# wells X 100 ul/well
Assay Preparation
Plate Washing
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
Wash plates X6 at each wash step
Assay Procedure
CSF - 1:10
•25 ul CSF/225 ul diluent JE or DEN Antigen - 1:250
•10 ul antigen/2.5 ml diluent
Mixtures
Antigen/Mab tracer: 1:1 vol:vol 5 control +11 sample wells = 16 wells 16 wells X 100 ul/well = 1600 ul
≈1800 ul
900 ul antigen + 900 ul Mab tracer
= 1800 ul
To JE and DEN columns (X2)
To JE or DEN wells (X16)
All wells (X32)
All wells (
Quality control and calculations of validity
Acceptable ranges of valid JE+ or DEN+/cut-off ratios (batch specific) Kit expiry date
JE and DEN calibrator factors and neg Abs
(batch specific) Information on kit box
•Kit lot #
•Expiration date
•JE Calibration Factor
•DEN Calibration Factor
•Range of acceptable Neg Control OD
•Range of acceptable JE Pos C OD/cut-off ratio
•Range of acceptable DEN Pos OD/cut-off ratio
Calculations:
JE Cut-Off value = Mean OD of JE Calibrators X JE Calibration factor DEN Cut-Off value = Mean OD of DEN Calibrators X DEN Calibration factor
Interpretation of Validity : 1. JE Pos Control OD/JE cutoff ratio 2. DEN Pos Control OD/DEN cutoff ratio 3. Neg Control OD
Determining test validity
Are these 3 values within acceptable range?
Batch-specific information on kit box:
TEST IS VALID IF:
JE Pos, DEN Pos, and Neg controls are all within acceptable ranges and
expiration date has not passed
If test is NOT valid, do not proceed to calculations – repeat test!
If test meets validity requirements, calculate JE and DEN Panbio units of each sample
Calculations of Panbio Units
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 PanBio units:
JE: JE Index value X 10 DEN: DEN index value X10
