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: 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 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 E-mail: 920980@naver.com 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 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 Mr David Featherstone Dr Youngmee Jee Mr David Featherstone 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 Mr David Featherstone 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 Mr David Featherstone 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) (during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.) Coffee break Continuation: laboratory practice - JEV IgM ELISA Lunch break Continuation: laboratory practice - JEV IgM ELISA JEV IgM ELISA reading and calculation of results Coffee break Evaluation of ELISA test results and discussion Specimen collection, preparation and shipment for virus isolation and serology Adjourn for the day Question for the night Mr David Featherstone, facilitators and participants Participants Participants Participants Mr David Featherstone and facilitators Dr Tomohiko Takasaki 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) (during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.) Coffee break Continuation: laboratory practice – ELISA (during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.) Lunch break JE PCR Evaluation of ELISA test results and discussion AES data management and reporting requirements Practical session on data entry Adjourn for the day Mr David Featherstone, facilitators and participants Mr David Featherstone, 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 Dr Tomohiko Takasaki Mr David Featherstone 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 Samples should be re-tested 9-11 Eqv 9-11 Eqv Samples should be re-tested <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 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) 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. 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 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) 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) (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 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 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 Diagnosis of Japanese Encephalitis in the Western Pacific Region Public Health Laboratory Centre, Hong Kong 15 to 19 November 2010 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 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 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 Determining test validity 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 Step 1: Determine validity of test Kit lot 06193 Expiration date 12/12/2011 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 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 Determining test validity JE PosC OD = 2.12 DEN PosC OD = 3.41 JE PosC OD/JE cut-off = 2.12/0.362 = 5.86 DEN PosCOD/DEN cut-off = 3.41/0.506 = 6.74 Sample 1 Step 1: Determine validity of test Kit lot 06193 Expiration date 12/12/2011 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 Test valid? JE NegC OD = 0.063 DEN NegC OD = 0.066 Kit lot 06193 Expiration date 12/12/2011 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 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 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 JE PosC OD/JE cut-off = 2.12/0.362 = 5.86 DEN PosCOD/DEN cut-off = 3.41/0.506 = 6.74 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 Expiration date 12/12/2011 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 Kit lot 06193 Expiration date 12/12/2011 JE Calibration Factor: 0.63 DEN Calibration Factor: 0.38 JE PosC OD/cut-off ratio: 1.1-6.0 DEN PosC OD/cut-off ratio: 3-17.0 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 Public Health Laboratory Centre, Hong Kong 15 to 19 November 2010 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 Arboviral Diseases Diagnostic and Reference Laboratory 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 •patients met acute encephalitis syndrome (AES/AMES) clinical case definition •Collected during AMES surveillance in Cambodia Cambodian MOH requested (through WHO WIPR and PATH) confirmatory testing at CDC/DVBID Reference assay: CDC JEV and DENV IgM ELISA and PRNT Almost all cases had paired sera and CSF specimens for final diagnosis; very good sample set for accurate assessment of Panbio performance Evaluation 3: Summary test results 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 •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 *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 Summary test results 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 Western Pacific Region Anti-human IgM Public Health Laboratory Centre, Hong Kong (China) Plate surface 15 to 19 November 2010 David Featherstone, EPI/IVB WHO Geneva WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network Wash away unbound IgG and add JE Antigen with HRP enzyme conjugate Add serum sample IgM WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network Problems with flavivirus cross reactivity JE and Dengue IgM Positive WHO Vaccine Preventable Disease Lab Network 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 2nd Intercountry HandsHands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Western Pacific Region Challenges in maintaining the momentum Public Health Laboratory Centre, Hong Kong (China) Prospects for maintaining Sustainability 15 to 19 November 2010 David Featherstone EPI - IVB WHO Geneva WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 • WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 ! WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network • • • • • • • • • • • Train ing WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network Japanese Encephalitis Morbidity, Mortality, and Disability Reduction and Control by 2015 PATH Document: 2009 WHO Vaccine Preventable Disease Lab Network GAVIGAVI-eligible countries Projected Resource requirements Japanese Encephalitis Morbidity, Mortality, and Disability Reduction and Control by 2015 PATH Document: 2009 NonNon-GAVIGAVI-eligible countries Projected Resource requirements Japanese Encephalitis Morbidity, Mortality, and Disability Reduction and Control by 2015 PATH Document: 2009 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network Thank You! 4 Is quality assurance necessary ? Quality Assurance and Proficiency in the JE Laboratory Network The 2nd Intercountry HandsHands-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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network "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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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! WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network Precise but not accurate Accurate and precise WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network Refrigerators and freezers – Consider minimum/maximum recording thermometers Thermometers – Should be calibrated against a reference thermometer Room temperature ?? – Panbio 20-25°C WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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. WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 WHO Vaccine Preventable Disease Lab Network 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 Nitrogen content; <0.02mg/ml 1971 Nitrogen content; <0.01mg/ml 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 Western Pacific Region Public Health Laboratory Centre, Hong Kong 15 to 19 November 2010 Barbara W. Johnson Centers for Disease Control and Prevention Division of Vector-Borne Infectious Diseases Arboviral Diseases Diagnostic and Reference Laboratory 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 Real-time RT-PCR, Virus isolation Sera Real-time RT-PCR, Virus isolation, paired NT Ticks Real-time RT-PCR,Virus isolation Sera IgM ELISA, NT, Real-time RTPCR,† † virus isolation†† Liver, lung, lymph nodes Real-time RT-PCR, Virus isolation Sera IgM ELISA, NT, Real-time RTPCR,† virus isolation† Brain Real-time RT-PCR, Virus 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 Real-time RT-PCR, Virus 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 Plaque reduction neutralization test 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 Research Institute for Tropical Medicine 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 jeey@wpro.who.int WPR_EPIdata@wpro.who.int • 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 Plaque reduction neutralization 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 Results of the 2009 WPR JE Labnet proficiency testing: 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 Results of the 2009 WPR JE Labnet proficiency testing: 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 ERROR: syntaxerror OFFENDING COMMAND: --nostringval-STACK: /Title () /Subject (D:20120720102841+08’00’) /ModDate () /Keywords (PDFCreator Version 0.9.5) /Creator (D:20120720102841+08’00’) /CreationDate (Faigaor) /Author -mark- Where is PNG Papua New Guinea Oscillah Kaminiel/Ernest Velemu Central Public Health Laboratory 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) Central Public Health Laboratory Major responsibilities (CPHL) • Reference Public Health Laboratory • Diagnosis of public health diseases and surveillance • Training on new techniques/ diagnosis • Quality assurance mostly for public health diseases • Research Surveillance activities • • • • • • Measles and Rubella HIV/TB/Malaria Cholera Dengue Influenza Polio Current Situation Japanese Encephalitis • No diagnosis and surveillance done as yet • Hope fully JE testing will be established after this workshop Challengers • Minimal or no request by clinicians • Main use of clinical observation over laboratory diagnosis • Establishment of JE testing at CPHL Way Forward • Expect to gain knowledge on Laboratory diagnosis of JE at this workshop • Understand pathology of JE • Quality Assurance of JE diagnosis • Be able to establish JE diagnosis and surveillance in 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 Diagnosis of Japanese Encephalitis in the Western Pacific Region November 1515-19, 2010 Analisa N. Bautista, RMT and Ava Kristy D. Sy, RMT Research Institute for Tropical Medicine, Manila Source: National Epidemiology Center Source: National Epidemiology Center 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 Source: National Epidemiology Center Source: National Epidemiology Center 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 (mghan@nih.go.kr/sokdu@nih.go.kr) 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
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