www.wpro.who.int
Training Workshop Report
3rd Intercountry Hands-on Training Workshop
on the Laboratory Diagnosis of Japanese Encephalitis
Osong, Republic of Korea
24–27 October 2011
WP/ICP/IVD/1.1/001-A
Report Series No.: RS/2011/GE/69/(KOR) English only
REPORT
THIRD INTERCOUNTRY HANDS-ON TRAINING ON THE LABORATORY DIAGNOSIS OF JAPANESE ENCEPHALITIS
Convened by:
WORLD HEALTH ORGANIZATION
REGIONAL OFFICE FOR THE WESTERN PACIFIC
Not for sale
Printed and distributed by:
World Health Organization Regional Office for the Western Pacific
Manila, Philippines
September 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 Osong, Korea from
24-27 October 2011.
SUMMARY
The third Hands-on Training on the Laboratory Diagnosis of Japanese Encephalitis (JE) for WHO-designated JE laboratories in the Western Pacific Region was held at the Korea Human Resources Development Institute for Health and Welfare, Osong, Korea from
24 to 27 October 2011 in collaboration with the Korea Centers for Disease Control and Prevention (KCDC). The training was attended by 14 participants from WHO-designated national JE laboratories from 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). In addition to support from the WHO Secretariat, temporary advisers from the United States Centers for Disease Control and Prevention (US CDC) and the National Institute of Infectious Diseases (NIID) of Japan attended the training as facilitators.
The objectives of the workshop were:
(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 discuss requirements for WHO accreditation of JE laboratories;
(3) To further familiarize participants with laboratory data management, using the WHO JE laboratory data reporting format for reporting to the Western Pacific Regional Office; and
(4) To distribute 2011 proficiency panel samples to network laboratories.
The training programme consisted of lectures on the first day, followed by three and a half days of practical sessions, country presentations and discussions.
Following a general introduction to IgM capture assay for JE, presentations on the first day covered (1) Japanese encephalitis/acute encephalitis (JE/AES) surveillance and laboratory network (Labnet); (2) Quality assurance of the JE laboratory network and (3) Introduction of JEV IgM ELISA.
On the first day and second day, practical sessions on Panbio JE Dengue Combo ELISA were performed using serum and CSF samples, respectively.
On day three, the third practical session on ELISA of serum sample was held using Panbio JE-Dengue IgM Combo ELISA kits, in-house assays, and Beixi kit. After each practical session, results were analysed and calculated and their validity determined. The ELISA results using the Panbio kit, NIHE in-house assay and Beixi kit were compared. .
On day four, the participants had the fourth practical on JEV PCR followed by a demonstration of PRNT and virus isolation.
During incubation periods, participants learnt how to calibrate and maintain ELISA equipment and micropipettes for laboratory quality assurance and control.
The post-course assessment, followed by analysis of pre- and post-course assessments, were done at the end of the workshop and the third JE proficiency test panel samples were distributed to all participating laboratories. In addition, two sets of kits were distributed to the Lao People’s Democratic Republic and Malaysia. Participants were requested to submit the results within 14 days using appropriate assays that are used in their own laboratories.
CONTENTS
Page SUMMARY
1. INTRODUCTION ... 1
1.1 Objectives ... 2
1.2 Participants ... 2
2. PROCEEDINGS ... 3
2.1 Training programme ... 3
2.2 Lecture sessions ... 3
2.3 Practical sessions ... 6
2.4 Country reports ... 6
3. CONCLUSIONS ... 14
3.1 General ... 14
3.2 Evaluation of the workshop ... 14
3.3 Main outcomes of the training ... 15
3.4 Follow-up of the workshop ... 15
ANNEXES:
ANNEX 1 - LIST OF PARTICIPANTS, TEMPORARY ADVISERS AND SECRETARIAT
ANNEX 2 - TIMETABLE
ANNEX 3 - INSTRUCTIONS AND PROTOCOLS ANNEX 4 - LECTURES AND PRESENTATIONS ANNEX 5 - COUNTRY REPORTS
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 WHO Western Pacific 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. So far, 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 Lao People's Democratic Republic and the Philippines have demonstrated some evidence of endemic JE transmission but have no vaccination
programme. Transmission may be geographically limited in some countries, as in Australia and Malaysia. Australia has a JE vaccination programme only in the Torres Strait, where JE has been endemic since 1995. Malaysia has had a JE vaccination programme in Sarawak state since 2002.
Papua New Guinea is a country in the Region with presumed endemic JE transmission but without clear disease-burden documentation.
Major progress was made in JE control in the Region in the 1990s and 2000s, and human disease has largely been eliminated in Japan and the Republic of Korea, where sustained vaccination programmes protect 11% of the total regional population at risk. China has also recorded a more than 90% decline in JE cases since the 1980s with the introduction of Government-supported, low-fee JE immunization in high-risk provinces. User fees for
Expanded Programme on Immunization (EPI) vaccinations, including JE vaccine, were abolished in China in 2005, and a decision was taken in December 2007 to introduce JE vaccine
nationwide for eligible children by 2010. A similar decision was taken by Viet Nam, which reports the second highest number of JE cases in the Region. Immunization programme expansions in these two countries will ultimately protect 82% of the Region's population from JE.
Disease burdens have yet to be established in other low-income countries because of poor disease surveillance infrastructures, but low-income countries demonstrate the greatest need for JE control support. In many countries, there is no routine surveillance and, in those that do have surveillance activities, there are few resources to introduce vaccine when JE disease is
demonstrated. Cambodia introduced routine immunization with the live attenuated SA 14-14-2 JE vaccine in three pilot provinces in 2009, with nationwide expansion planned for the coming years. The Philippines has established sentinel hospital-based surveillance. Efforts are being made to restart surveillance in the Lao People's Democratic Republic and Papua New Guinea.
The newly established JE laboratory network was formed based on the WHO poliomyelitis and measles/rubella laboratory network models, and some WHO measles/rubella laboratories have also been designated as national JE laboratories. The purpose of the JE laboratory network is to improve and standardize the capability for JE diagnosis in countries where JE is endemic.
The network consists of one Global Specialized Laboratory in Japan, two Regional Reference Laboratories (RRLs) in China and the Republic of Korea and China, and seven national
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laboratories in Cambodia, the Lao People’s Democratic Republic (2), Malaysia, Papua New Guinea, the Philippines and Viet Nam.
Three JE network laboratories use their own in-house assays, while the Chinese Center for Disease Control and Prevention (China CDC) uses locally-produced JE kits. Six other network laboratories use Panbio JE/Dengue IgM Combo enzyme-linked immunosorbent assay (ELISA) kits. For quality assurance of the JE laboratory network, the first and second WHO proficiency tests for JE were conducted successfully in 2009 and 2010. For the quality assurance of samples tested in national laboratories, a confirmatory testing mechanism was also established in the Region and samples from national laboratories are sent to RRLs for retesting. WHO accreditation, using the WHO JE laboratory checklist, was initiated in 2010.
To build regional laboratory capacities for JE testing, two regional hands-on training workshops were organized, at the Korea Centers for Disease Control and Prevention (Korea CDC) in 2009 and at the Public Health Laboratory Centre in Hong Kong (China) in 2010. The third hands-on training session for JE was organized for WHO JE network laboratories at the Korea Human Resource Development Institute for Health and Welfare, Osong, the Republic of Korea, from 24 to 27 October 2011, to enhance the proficiency of laboratory staff in ELISA testing and further improve the quality of laboratory performance. A total of 14 participants from Cambodia, China, the Lao People's Democratic Republic, Malaysia, Papua New Guinea, the Philippines, the Republic of Korea and Viet Nam were invited to attend the training session.
The third WHO JE proficiency test samples were distributed during the third hands-on training course and the results were finalized.
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 discuss requirements for WHO accreditation of JE laboratories.
(3) To further familiarize participants with laboratory data management, using the WHO JE laboratory data reporting format for reporting to the Western Pacific Regional Office.
(4) To distribute 2011 proficiency test panel samples to network laboratories.
1.2 Participants
The training session was attended by 14 participants from WHO-designated national JE laboratories in Cambodia (1), China (3), the Lao People’s Democratic Republic (2),
Malaysia (1), Papua New Guinea (1), the Philippines (2), the Republic of Korea (2), and Viet Nam (Hanoi [1] and Ho Chi Minh City [1]). In addition to support from the WHO Secretariat, temporary advisers from the United States Centers for Disease Control and
Prevention (US CDC) and the National Institute of Infectious Diseases (NIID) of Japan attended the training as facilitators (see Annex 1 for the list of participants).
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2. PROCEEDINGS
2.1 Training programme
Dr Myung-Chan Cho, Director of the National Institute of Health, welcomed the participants and opened the hands-on training workshop with an introductory speech. The objectives of the training were presented by Dr Youngmee Jee, Scientist, WHO Western Pacific Regional Office. The training consisted of half a day of lectures followed by three and a half days of practical sessions, discussions and country presentations. The timetable of the training is attached as Annex 2.
The third proficiency test sample was distributed to all participating laboratories at the end of the training. A USB drive containing copies of all presentations made during the training, worksheets and all related materials was also distributed upon completion of the workshop.
The protocol used for the training is attached as Annex 3. The presentations made during the lecture session and country reports are attached as Annexes 4 and 5.
2.2 Lecture sessions
2.2.1 Session 1: JE/AES surveillance and laboratory network
Mr David Featherstone, Global EPI Laboratory Coordinator in EPI Immunizations, Vaccines and Biologicals (IVB), WHO Headquarters, gave a presentation on strategies for JE surveillance and steps in establishing JE surveillance; roles of the laboratory; progress and challenges in the JE Laboratory Network; and prospects for maintaining sustainability. He stressed that laboratory confirmation is needed for the differential diagnosis of flaviviruses with antigenic cross reactivity.
Strong quality assurance programmes, including evaluation of different assays, use of in-house control samples, proficiency testing, confirmatory testing and accreditation, have been implemented among WHO JE network laboratories. The assay assessment and the Quality Assurance Project (QAP) are important because a perfect assay for JE is not yet available. It is also necessary to find continuous support for maintaining the laboratory network and to ensure a manageable workload for the laboratories.
Dr Youngmee Jee, Regional EPI Laboratory Coordinator, WHO Western Pacific Regional Office, gave an overview of JE control and laboratory network progress in the Region. She presented the estimated burden of JE, the objectives and roles of JE surveillance, recommended performance indicators and data elements for JE surveillance. She also summarized the
outcomes of the fifth biregional meeting on JE prevention and control and the third JE laboratory network meeting for the WHO Western Pacific Region, which was held in Vientiane, the Lao People’s Democratic Republic, from 30 May to 1 June 2011.
The JE LabNet has grown since 2008 and 10 network laboratories consisting of one global specialized laboratory, two RRLs and seven national laboratories have now been identified and assessed in the Region. Good progress has been made in ensuring the quality of laboratory
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testing among network laboratories since 2008, and regular confirmatory testing, as well as annual proficiency testing, has been performed. As of October 2011, seven of the10 laboratories had been accredited based on on-site reviews, which started in 2010. Data-sharing is still a problem for China, although it is expected to have 10 subnational JE laboratories by the end of 2011.
Recommendations from the third JE laboratory network meeting and challenges and plans for 2012 were also presented. Challenges include sustaining funding for the network, improving communications with the surveillance group, data-sharing and reporting to the WHO Western Pacific Regional Office. The plans for 2012 include enhancing communications between laboratory and EPI/surveillance, performing quality assurance for all network laboratories, including subnational laboratories in China, and strengthening data reporting and analysis.
2.2.2 Session 2. Quality assurance of the JE laboratory network
Dr Barbara W. Johnson from the US CDC presented the results of the WHO Western Pacific Region JE LabNet proficiency testing conducted in 2009 and 2010. For the laboratories that used in-house and the non-Panbio commercial kit (NIID, Japan; China CDC; the Pasteur Institute [PI], Ho Chi Minh City, Viet Nam; and the National Institute of Hygiene and
Epidemiology (NIHE) Hanoi, Viet Nam), agreement with the US CDC in-house assay ranged from 82%-91%.
Among the laboratories that used the Panbio JE-DEN combo ELISA kit, five laboratories (National Institute of Public Health [NIPH], Cambodia; the Naval Medical Research Unit-2 [NAMRU-2], Cambodia; the Institute for Medical Research [IMR], Malaysia; the Research Institute for Tropical Medicine [RITM], the Philippines; and the Korea CDC) obtained a 100%
score in 2009. However, the National Center for Laboratory and Epidemiology (NCLE), the Lao People’s Democratic Republic, could not participate in the training and obtained an 82%
score on the 2009 proficiency test. The results of proficiency testing in 2010 were 100% in all 10 network laboratories. Dr Johnson also provided the details of 2011 JE proficiency testing samples. All laboratories will test four cerebrospinal fluid (CSF) (45ul) and five serum specimens (25ul) as well as one in-house control sample.
Dr Tomohiko Takahashi, from the NIID Japan, presented the confirmatory test results of samples from public health institutes in Japan and national JE laboratories in Cambodia, the Lao People’s Democratic Republic and Viet Nam. Two of the 67 samples from public health institutes in Japan showed a discrepancy (97% concordance). Among the 76 samples received from the NIHE, Viet Nam, which uses its own in-house assay, 12 samples showed discrepant results (84.2%).
For confirmatory testing of samples from Cambodia and the Lao People’s Democratic Republic, the NIID used both the NIID in-house assay and the Panbio kit. Some discrepancy was observed with samples from Cambodia using the NIID in-house assay. However, when the Panbio kit was used, 100% concordance was obtained. For samples from the NCLE, the Lao People’s Democratic Republic, which uses the Panbio kit, concordance rates of 70.6% and 82.1% were obtained in 2010 and 2011 using the NIID in-house assay. Dr Takahashi also explained the titration method, which could be more accurate for differential diagnosis of JE and dengue than the ELISA method.
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Dr Johnson presented results of the evaluation of commercial kits and in-house assays using the reference serological panel for standardization of JE diagnosis. Panbio, Xcyton, InBios and Beixi kits, among the commercial JE kits, and in-house assays from the NIID, the University of Malaysia, Sarawak (UNIMAS), the PI and the NIHE were included in the evaluation. She explained the CDC testing algorithm and interpretation of the single sample, as well as the Plaque Reduction Neutralization test (PRNT) confirmatory testing algorithm for acute encephalitis syndrome (AES) cases with cerebrospinal fluid (CSF) and paired S1 and S2 sera.
The JE preliminary reference panel consisted of 253 sera and 153 CSF samples of high and low JE IgM positive and low and high dengue IgM positive and negative samples that were tested in five reference laboratories, including the US CDC. Results of preliminary panel testing by five reference laboratories showed high agreement of 88% to 95%, and all assays had high sensitivity of 95% to 99%. Samples classified as dengue infections had a high rate of
cross-reactivity with Japanese encephalitis vaccine (JEV) antigen in ELISA. Based on the results of preliminary panel samples, final reference panel samples consisting of 200 sera and CSF samples, were prepared and sent to three laboratories in the Region (the NIHE, the PI and the China CDC). All three assays had sensitivities of > 92% and specificities of 94%.
2.2.3. Session 3. Additional presentations
Dr Takahashi from the NIID gave a presentation on specimen collection, preparation and shipment for virus isolation and serology. CSF is the preferred specimen and CSF samples collected on the day of onset of encephalitis are the best samples. If CSF samples are sent within one day, it is not necessary to keep the samples at -20°C; they can be shipped at 4°C. In the NIID, all CSF samples are frozen, serum samples are divided for serological and virological testing and serum samples for serology are inactivated. Dr Takahashi also presented pathogen risk groups, categories of infectious substances, triple packaging for clinical specimens and procedures for international shipping and package labelling. He also cautioned that dry ice should not be put inside the secondary container to prevent an explosion.
Dr Takahashi also presented the various diagnostic methods used in the WHO Global Specialized JE Laboratory in the NIID. This laboratory uses IgM capture ELISA, IgG ELISA, neutralizing antibody test (plaque reduction neutralization test and focus reduction neutralization test), real-time polymerase chain reaction (RT-PCR), TaqMan RT-PCR and virus isolation using C6/36 cells, Vero cells and suckling mice. In prefectural laboratories, haemagglutination inhibition (HI) and the PRNT/Focus Reduction Neutralization test (FRNT), RT-PCR and virus isolation can be used. PRNT was compared with FRNT in the NIID. PRNT needs more space (six-well plate) and time (seven days), but it is easy to count and cheaper. FRNT can save space and time (three days for assay) and many samples can be tested in one assay, but it is expensive and counting plaques could be more difficult.
Dr Myung-guk Han from the Korea CDC gave a presentation on JE surveillance in the Republic of Korea consisting of patient, vector and animal host surveillance. The Korea CDC operates an early warning system for the public based on the monitoring results of the JE
antibody-positive ratio among pigs. For pig serosurveillance, hemagglutination inhibition assay, indirect immunofluorescent assay and dipstick (rapid immunochromatographic test) methods are used. A dipstick method developed by the Korea CDC was applied from July to October during 2009-2011 for sera from unvaccinated pigs in eight sites in eight provinces. The seroprevalence rate was 15.2% during that period.
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2.2.4 Session 4. Introduction of JEV IgM ELISA
Mr Featherstone gave a general introduction to IgM capture assay for JE. Dr Johnson from the US CDC explained the step-by-step procedures, including the preparation of kits, adding samples or reagents to wells, incubation and plate washing. After the assay, reading using ELISA readers and determining test validity, calculation of Panbio units, and interpretation were practised.
2.3 Practical sessions
Following the general introductory presentation on IgM capture assay for JE, the hands-on training session was conducted in the Korea Human Resource Development Institute for Health and Welfare, Osong, the Republic of Korea, and consisted of four days of practical sessions, during which 14 participants were grouped into seven pairs. Instructions and protocols for the practical sessions used are provided in Annex 3.
On the first and second days, a practical session on Panbio JE Dengue Combo ELISA was performed using serum and CSF samples, respectively. On the third day, the third practical session on ELISA of serum samples was held using Panbio JE-Dengue IgM Combo ELISA kits, in-house assays and the Beixi kit. After each practical session, results were analysed and
calculated and their validity was determined. The ELISA results using the Panbio kit, the NIHE in-house assay and the Beixi kit were compared on the third day. On the fourth day, the
participants attended the fourth practical session on JEV PCR. It was followed by a
demonstration of PRNT and virus isolation. During incubation periods, participants learnt how to calibrate and maintain ELISA equipment and micropipettes for laboratory quality assurance and control.
A post-course assessment, followed by analysis of pre- and post-course assessments, were conducted at the end of the workshop, and the third JE proficiency test panel samples and kits were distributed to participants (two sets for Malaysia and the Lao People’s Democratic
Republic). Participants were asked to submit the results within 14 days using appropriate assays that are used in their own laboratories.
2.4 Country reports 2.4.1 Cambodia
Am Chanthan from the NIPH presented the current status and objectives of JE sentinel surveillance in Cambodia. Hospital-based sentinel surveillance for suspected
meningoencephalitis (ME) cases among children under 15 years of age was started in 2006 by the Cambodia CDC, the NIPH, the National Immunization Programme/Ministry of Health, PATH and WHO. Six selected hospitals use the Panbio JE-Dengue IgM Combo ELISA kits for testing. Beginning in June 2011, the sentinel sites were extended to cover the whole province in Battabang (four public and three private hospitals) and Banteaymeanchey Province (five public and five private hospitals).
From 2009 to October 2011, six sentinel sites collected paired sera and CSF samples from 432 ME cases, and 98 cases (22.7%) were positive for JE. The JE-positive rate varied from 5%
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(National Paediatric Hospital in Phnom Penh) to 26% (Kampong Cham). From six sentinel sites, 432 first sera, 298 second sera and 434 CSF samples from 439 suspected cases of ME were sent to the NIPH laboratory for JE IgM testing during the period. There were 24 laboratory-
confirmed JE cases (12%) in 2009, 39 (31.5%) in 2010 and 34 (25%) in 2011. The age
distribution of the confirmed JE cases showed that those between 1 and 5 years of age and those between 6 and 10 years were most affected.
ME and JE cases by sentinel sites 2009, 2010 to Oct- 2011
152
47 49 10
77
10 34
5 40
8 80
18
Kg Cham
Hospital Battambang Hospital Angkor
Children Hospital
National Pediatric Hospital
Svay Rieng Hospital Takeo
Hospital
ME JE
26.27%
17% 7.3% 14.7% 20% 19.7%
Japanese Encephalitis Sentinel Site Surveillancein Cambodia
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73
7
38 39
11
60 80
Under 1y 1 - 5 ys 6-10 ys 11 - 15 ys
0 10 20 30 40 50
ME cases JE possitive cases in each group
ME cases JE Positive cases
Distribution by age of JE positive cases from 2009 to Oct-2011
5.26% 25.7% 31.5% 13.8%
Japanese Encephalitis Sentinel Site Surveillancein Cambodia
105 92
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For quality assurance, in-house control is used and the laboratory participates in annual WHO proficiency testing. Confirmatory testing samples are referred to the NIID twice a year. In order to enhance the diagnosis of JE/AES, it is necessary to try to obtain CSF and second serum samples from all suspected cases and further strengthen the NIPH laboratory and sentinel sites for bacteriological culture.
2.4.2 China
Dr Cao Yuxi from the China CDC presented the national JE experience and activities of the RRL. The JE surveillance system has been established since 1950 and a nationwide immunization programme was started in the 1970s. Both inactivated and live attenuated vaccines are used in China. With the introduction of live attenuated vaccine in the 1990s, the number of JE cases decreased dramatically. In 2007, JE vaccination was integrated into the national EPI nationwide.
Various laboratory testing methods, including MAC-ELISA, virus isolation, IFA/PRNT, PCR/RT-PCR, and bio-information analysis, are used in the laboratory. For quality assurance in provincial laboratories, annual proficiency testing samples are prepared and coordinated by the China CDC. In 2010, JE proficiency test samples were sent to 15 provinces (five sera and two CSF samples) and all scored 100%. The China CDC also participates in WHO JE proficiency testing, scoring 100% using the Beixi kit in 2010. The China CDC has a strong quality assurance programme for internal quality control, including instrument calibration, maintenance of
equipment, temperature monitoring, provision of training, standard operating procedures (SOPs) revision and accident preparedness. In addition, the China CDC has also received ISO 15189 accreditation.
2.4.3 The Lao People's Democratic Republic
Virasack Som Oulay from the NCLE gave a presentation on JE surveillance and key laboratory findings in the Lao People's Democratic Republic. Acute encephalitis syndrome (AES) has been included in the national surveillance system since February 2011. From 2010 to August 2011, 174 suspected cases were reported and investigated. Among them, 101 cases were laboratory-confirmed, with a July peak, and most cases were from the northern parts of the country. Age distribution shows that children aged 1-15 years are most affected, but the age group aged 16-20 years and older are also affected. The Mahosot Hospital also receives samples for JE testing and the results are sent to the NCLE using the same WHO Western Pacific
Regional Office JE laboratory reporting format. JE vaccine has not been introduced in the Lao People’s Democratic Republic, but there are plans to introduce the vaccine in the northern provinces, which have a higher JE incidence.
The Panbio JEV-IgM Dengue Combo ELISA is used at the NCLE. The NCLE participated in WHO JE proficiency tests in 2009 and 2010. All patient information and laboratory results are recorded in the WHO JE database and reported to the WHO Country Office and Regional Office. If there is a suspected JE case, the provincial epidemiologist reports to the EPI unit of the NCLE and the investigation team collects a specimen. Samples are analysed in the NCLE laboratory and laboratory results are sent to the EPI unit. Remaining challenges are samples that are not collected from every AES case and the fact that JE/AES cases are not reported from the southern part of the country.
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Olay Rattana from Mahosot Hospital in Vientiane presented the status of JE laboratory diagnosis in the hospital. Serology (Panbio ELISA), virus culture and PCR methods are used for the laboratory diagnosis of JE. From January 2003 to March 2011, lumbar puncture was
performed for 1020 patients, and 521 patients had AES. Serological confirmation was performed for 458 (88%) of the 521 patients with AES and JE IgM was positive among 68 (15%). The JE virus was isolated from six patients (1%) and five were also positive for JEV PCR. Among the six patients with JE virus isolation, only one was JE IgM-positive. JEV was detected from AES cases in the central, northern and southern parts of the country. Most JE patients are admitted to the Mohosot Hospital during the rainy season, with a July peak.
2.4.4 Malaysia
Ms Asmah Hani Abdul Wahab from the National Public Health Laboratory (NPHL) presented the JE country report for Malaysia. The Institute of Medical Research (IMR) was designated as a WHO national JE laboratory in 2008, but the Ministry of Health is planning to transfer the WHO JE national laboratory from the IMR to the NPHL. Therefore, one staff member from the serology unit in the NPHL was invited to the training session in preparation for the transfer. The first JE case was detected in 1952 and viral encephalitis has been a notifiable disease since 1988.
There is no national JE vaccination programme; only Sarawak state has a statewide JE vaccination programme, which was started in 2002. In peninsular Malaysia and Sabah, JE vaccination is conducted only within a 2 km radius when there is a JE case. As JE vaccination coverage improved in Sarawak, the JE incidence rate decreased sharply. Immunization coverage for JE in Sarawak increased from 47.8% in 2006 to 95.8% in 2010; the number of JE patients decreased from 18 cases in 2006 to four cases in 2010. However, JE cases might be
underreported.
Before 2008, the laboratory diagnosis of JE was conducted at the University of Malaya Medical Centre (UMMC Klang Valley), at UNIMAS (Sarawak) and at the IMR. Between 1993 and 2006, 5% (571/11055) were laboratory-confirmed as JE, 0.9% (9/999) in 2007, 2.3%
(34/167) in 2008, 5.6% (18/322) in 2009 and 4% (18/447) in 2010.
Diagnostic methods used in the IMR include the HI test, IgM capture ELISA and IgM combo ELISA for antibody detection, and viral isolation using suckling mice. Severe and fatal cases are also diagnosed by HI, through virus isolation using C6/36 cells for viral isolation and by RT-PCR for molecular detection. The NPHL also can perform IgM ELISA, virus isolation using C6/36 cells and RT-PCR. For quality assurance, both the IMR and the NPHL are
undergoing ISO 15189 certification and participate in the external quality assurance programme (RCPA flavivirus serology, RT-PCR). The IMR scored 100% in WHO JE proficiency testing, and confirmatory test results showed 100% and 93% concordance in 2010 and 2011.
Remaining challenges could be underreporting of JE cases, a problem with obtaining CSF and second serum samples and smooth transition of the WHO national JE laboratory from the IMR to the NPHL by 2013. A smooth transition would be needed to ensure the quality of JE laboratory testing.
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2.4.5 Papua New Guinea
Dr Viola Asigau from the Central Public Health Laboratory (CPHL) presented the Papua New Guinea JE country report. JE was first reported in the country among the inhabitants of the Western Province between 1989 and 1998. The JE virus was isolated from mosquitoes in 1997 in the Western Province. There was an outbreak of JE in Milne Bay Province in 2003 and a confirmed JE case was reported from the Port Moresby area in 2004.
A study conducted by Port Moresby General Hospital in 2010 confirmed JE as an important cause of febrile encephalopathy in children in the country. Five JE sentinel
surveillance sites (Daru, Vanimo, Alotau, Goroka and Port Moresby General Hospital) are being established to investigate the JE disease burden and epidemiology. Since there is no JE
surveillance system, JE laboratory testing has not been performed in the CPHL.
2.4.6 The Philippines
Dr Amado O. Tandoc III from the Research Institute for Tropical Medicine (RITM) presented the JE county report for the Philippines. JE vaccination has not been included in the EPI in the Philippines. There is no clear JE disease burden in the country and the surveillance system is still being refined. Continuing surveillance systems that capture JE consist of AES surveillance by Philippine Integrated Disease Surveillance and Response (PIDSR) and
necrotizing enterocolitis (NEC) and MEMe (meningitis, encephalitis and meningoencephalitis) surveillance by RITM-WHO.
The PIDSR is symptom-based and selected diseases are notified weekly from 1664 hospitals. In contrast, the RITM operates a laboratory-based sentinel system in collaboration with WHO and reports data from five sentinel hospitals (50 cases per site) monthly. Therefore, JE surveillance consists of two parallel activities (MEMe and AES). Sera and CSF from suspected cases are sent to the RITM and analysed using a Panbio JE DEN IgM combo ELISA kit.
AES is one of 26 diseases included in the PIDSR and AES/meningitis is one of the weekly notifiable diseases. Only a few cases detected by this system have been laboratory-confirmed.
The RITM's MEMe project, run in collaboration with WHO, was initiated in 2006, but was not implemented until April 2009. There were 41(17.7%) laboratory-confirmed JE cases among 231 samples investigated from 2009 to 2011. Most JE cases were children aged 2 months to 15 years. Those aged 5-10 years were most affected, followed by 10-15 year-olds. Among the five sentinel hospitals, 28% of cases were confirmed as JE-positive from sites in Bicol and Tarlac while sites in Bulacan, Western Visayas and Manila showed 12%, 10% and 6.5% JE-positive rates, respectively.
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RESEARCH INSTITUTE FOR TROPICAL MEDICINE
MEMe Summary of Cases and Results by Sentinel sites Received from April 2009 to September, 2011
RESULTS BULACAN MEDICAL CENTER
BICOL MEDICAL CENTER NAGA
CITY
WESTERN VISAYAS MEDICAL CENTER -
ILOILO CITY
TARLAC PROVINCIAL
HOSPITAL PCMC Total
JE Positive 6 12.0% 14 28.0% 5 10.0% 14 28.0% 2 6.5% 41 17.7%
Dengue
Positive 6 12.0% 4 8.0% 10 20.0% 4 8.0% 4 12.9% 28 12.1%
H. Influenza B 3 6.0% 1 2.0% 7 14.0% 2 4.0% 0 0.0% 13 5.6%
S. pneumo 2 4.0% 5 10.0% 1 2.0% 3 6.0% 2 6.5% 13 5.6%
N. Meningitidis 0 0.0% 1 2.0% 0 0.0% 0 0.0% 1 3.2% 2 0.9%
Negative 33 66.0% 25 50.0% 27 54.0% 27 54.0% 22 71.0% 134 58.0%
TOTAL OF
CASES 50 21.6% 50 21.6% 50 21.6% 50 21.6% 31 13.4% 231 100.0%
RESEARCH INSTITUTE FOR TROPICAL MEDICINE
MEMe Summary of Cases and Results by Sentinel sites
Received from April 2009 to September, 2011
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RESEARCH INSTITUTE FOR TROPICAL MEDICINE
The RITM laboratory participated in a WHO proficiency test and confirmatory testing and obtained a 100% score on the test and a high concordance rate for confirmatory testing.
Laboratory testing data are collected monthly, but data from MEMe surveillance are not yet harmonized with JE cases in the AES surveillance system. Other challenges are low recruitment and underreporting of cases and sustainability of funding for the MEMe project, as well as harmonization of data and the format used in PIDSR and MEMe surveillance.
2.4.7 The Republic of Korea
Cho Jung-Eun from the Korea CDC presented an overview of the JE situation and the activities of the JE RRL in the Republic of Korea. The JE surveillance programme started in 1975. There was a JE epidemic in the country in 1982, with 1197 cases. After the mandatory JE vaccination of children under15 years of age started in 1983, the number of JE cases fell rapidly with fewer than eight cases every year until 2009. However, 26 JE cases were detected in 2010.
The possible reasons for the increased number are still under investigation. There were two laboratory-confirmed cases in 2011, among 410 samples. The mean age of JE cases ranged from 47-52 years between 2007 and 2011. The annual JE season is from August to October.
For serological diagnosis, the Panbio JE-DEN Duo ELISA (IgM), immunofluorescent assay, Flavivirus screening IFA kit (in-house) or the ELISA kit (commercial) and PRNT are used. Nested RT-PCR (commercial) and one-step RT-PCR methods are used for molecular analysis. In addition, virus isolation is conducted with a C6/36 cell line. The Korea CDC has developed a real-time/nested RT-PCR kit for vector surveillance, which could be used at the provincial sentinel sites for mosquitoes. Evaluation of the RT-PCR kit in 2011 was performed using 29 921 mosquitoes in 96 pools, detecting one positive sample collected on 6 September in Busan. The test kit has proven to be suitable for routine mosquito surveillance and there are plans to distribute it nationwide.
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Seroconversion surveillance of animal hosts (unvaccinated pigs) is conducted from July to October every year using a rapid immunochromatographic test. In 2011, 1297 pig serum
samples collected from eight sites in eight provinces were tested using this rapid test and 28.4%
were positive for JE. The laboratory also conducts surveillance for dengue fever, West Nile Fever and tick-borne encephalitis; 161 and 79 dengue fever cases were detected in 2010 and 2011 (as of September), respectively.
2.4.8 Viet Nam
Nguyen Thi Thu Thuy from the National Institute of Hygiene and Epidemiology (NIHE), Hanoi, presented the JE situation in northern Viet Nam and the laboratory status of the NIHE.
Antigen in first sera and CSF is detected by RT-PCR, viral isolation and cell line (C6/36). For antibody detection, MAC-ELISA is performed using an in-house kit that was licensed by the Ministry of Health. MAC-ELISA-confirmed cases in 2010 and 2011 numbered 31 and 37, respectively. Geographically, encephalitis cases were more often detected in Thai Binh Province from 2009 to 2010. It was noted that July was the peak month and that children aged 6-10 years were most affected, followed by those aged 11-15 years. The laboratory scored 82% and 100%
for 2009 and 2010, respectively. The WHO JE proficiency test and concordance rate for confirmatory testing was 94.5% in 2009 and 80% in 2010.
Bui Chi Tam from the Pasteur Institute, Ho Chi Minh City, introduced the laboratory capacity for JE diagnostics in the PI and JE sentinel surveillance, as well as the JE vaccination status in southern Viet Nam. JE sentinel sites are located in Binh Duong Provincial Hospital, Ben Tre Provincial Hospital, the Hospital of Tropical Disease and Paediatric No. 2 Hospital in Ho Chi Minh City. In 2009 and 2010, 6.9% and 12% were JE-positive among 306 cases in 2009 and 323 AES cases in 2010, respectively. MAC-ELISA, RT-PCR and the JE-DEN in-house kit are used in proficiency testing. Provincial laboratories in southern Viet Nam use MAC-ELISA distributed by the PI. The results of a proficiency test using the in-house kit were 91% and 100%
in 2009 and 2010, respectively. Confirmatory testing was 91% in 2009 and 97% in 2010. The laboratory was also ISO 15189 certified in November 2010. To expand JE surveillance in Viet Nam, some incentives for collecting samples in the provinces were proposed.
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JE-DEN detecting by ME from AES cases, 2008-2010
YEAR AES CASE CSF S1 S2
Total JE +
%
DEN +
%
JE +
%
DEN +
%
TOTAL JE + DEN + TOTAL JE + DEN +
2008 68 5 7.35
3 4.41
44 2 4.54
2 4.54
59 5 8.47
3 5.08
17 1 5.88
2 11.76
2009 306 21 6.86
16 5.22
215 12 5.58
6 2.79
296 12 4.05
9 3.04
111 7 6.30
7 6.30
2010 323 39 12.07
11 3.40
185 18 9.72
1 0.54
282 29 10.28
9 3.19
44 6 13.63
3 6.81
Sample collecting: CSF: 63.7% S1: 91.39% S2: 24,67%
3. CONCLUSIONS
3.1 General
The four main objectives of the training session were fully achieved during four days of intensive, hands-on presentations, practical sessions and discussions. By the end of the
workshop, the technical capacity and knowledge of all participants were enhanced and they were able to perform ELISA for laboratory diagnosis of JE, understood laboratory quality assurance for JE diagnosis and were fully familiarized with the requirements for WHO accreditation for JE laboratories and JE PCR procedures.
3.2 Evaluation of the workshop
3.2.1 Overall, the participants were positive in their feedback; the workshop met its
objectives and the schedule and administrative arrangements were well organized by the Korea Human Resource Development Institute for Health and Welfare. The workshop participants were encouraged to contact each other, the facilitators and the WHO Regional Office to follow up on practical issues, such as quality assurance, confirmatory testing and data reporting, to further strengthen JE laboratory capacities in the Region.
3.2.2 The training ran smoothly throughout the practical and presentation sessions. The participants were keen to complete all the tasks and to understand the topics addressed during the training. The topics covered throughout the workshop were relevant to the needs of the
participants.
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3.2.3 The Korea Human Resource Development Institute for Health and Welfare has a good facility for the training, and the accommodation arranged by the institute for the trainees was conveniently located within the complex.
3.3 Main outcomes of the training
3.3.1 All participants became familiar 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.
3.3.2 Participants were further familiarized with the requirements for WHO accreditation for JE laboratories and laboratory data management using the WHO JE laboratory data-reporting format for reporting to the WHO Western Pacific Regional Office.
3.3.3 The training also provided a chance for participants to be familiarized with JE RT-PCR.
3.3.4 At the end of the workshop, the third WHO JE proficiency testing panel samples were distributed to participants. Participants from the Lao People’s Democratic Republic and Malaysia carried two sets of proficiency test samples for the IMR and the Mahosot Hospital, respectively.
3.4 Workshop follow-up
3.4.1 At the end of the workshop, participants with the necessary import permits carried home the third proficiency panel samples. Participants were requested to report the results of proficiency panel samples within 14 days after the samples arrive in the laboratory.
3.4.2 The results of the first proficiency panel samples were received from 11 network laboratories, except for the Korea CDC and two additional laboratories in the
Lao People’s Democratic Republic (the Mahosot Hosiptal) and Malaysia (the NPHL). The Korea CDC received and tested the third JE proficiency test samples in advance, and the results of the Korea CDC were used as a reference for other laboratories that used the Panbio kit.
Most laboratories, except for two, reported the results within 14 days. Six laboratories, including two provincial laboratories in China that participated in the training, used the non-Panbio kit, and eight laboratories used the Panbio kit. Three laboratories in Japan and Viet Nam (Hanoi and Ho Chi Minh City) used in-house assays and three laboratories in China used the Beixi kit. The results were compared with the results of laboratories using the US CDC in-house assay. The results of seven laboratories that used the Panbio kit were compared with the results of the Korea CDC.
3.4.3 Results were finalized by the end of December 2011. Five of the six laboratories that used the non-Panbio assay obtained 100% and the remaining one laboratory scored 89%. Four of the seven laboratories that used the Panbio kit scored 100% and the remaining three laboratories scored 89%.
ANNEX 1 W O R L D H E A L T H
ORGANI ZATI ON
ORGANI SATI ON M ONDI ALE DE LA SANTE
REGI ONAL OFFI CE FOR THE WESTERN PACI FI C BUREAU REGI ONAL DU PACI FI QUE OCCI DENTAL
3rd I NTERCOUNTRY HANDS-ON TRAI NI NG WORKSHOP ON THE LABORATORY DI AGNOSI S OF JAPANESE ENCEPHALI TI S I N THE WESTERN PACI FI C REGI ON
Osong, Korea ENGLI SH ONLY
24-27 October 2011
LI ST OF PARTI CI PANTS, TEM PORARY ADVI SERS AND SECRETARI AT
PARTI CI PANTS
CAM BODI A M r Am Chanthan
Head, Immunology Unit (Laboratory) National Institute of Public Health Lot #2 Kim Yi Sung Blvd.
Sangkat Boeng Kok II, Khan Tuol Kok Phnom Penh
Telephone: (855) 12 881 196 Fax no. : (855) 23 882 889
E-mail : am.chanthan07@gmail.com
CHI NA M r Ke Changwen
Senior Doctor
Chinese Center for Disease Control and Prevention
#176, Xingang Road West Haizhu District
Guangzhou City
Telephone: (8620) 8445 7751
E-mail : keew1965@yahoo.com.cn Dr Cao Yuxi
Assistant Professor
Department of Viral Encephalitis
Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention 155 Changbai Road, Changping District
Beijing
Telephone: (8610) 5800 0842 Fax no. : (8610) 5890 2661 E-mail : yuxicao@hotmail.com
M rs Hu M ei
Associate Senior Technologist
Chinese Center for Disease Control and Prevention No. 6 Zhongxue Road, Wuhou District
Chengdu , Sichuan
Telephone: (8628) 8558 9063 Fax no. : (8628) 8558 9093 E-mail : flemingyang@sina.com LAO PEOPLE' S
DEM OCRATI C REPUBLI C
M r Virasouk Som Oulay
Serology and Virology Laboratory
National Center for Laboratory and Epidemiology No. 157, Unit 10 Thaphalanxay Village
Sisattanak District Vientiane
Telephone: (856)21 312850 Fax no. : (856)21 315858
E-mail : virasacksom@gmail.com M rs Olay Rattana
Technical Laboratory Staff University of Health Sciences Vientiane
Telephone: (856) 21 250752 Fax no. : (856) 21 242168 E-mail : olay@tropmedres.ac M ALAYSI A M s Asmah Hani Abdul Wahab
Science Officer (Mircobiology) National Public Health Laboratory Lot 1853 Kampung Melayu Sungai Buloh Selangor
Telephone: (603) 6126 1200 ext. 1318 Fax no. : (603) 6140 2249
E-mail : hani4804@moh.gov.my PAPUA NEW GUI NEA Dr Viola Asigao
Pathology Registrar
Central Public Health Laboratory National Department of Health P.O. Box 807
Waigani
Telephone: (675) 7677 0301 Fax no. : (675) 323 6108
E-mail : vida_asigao@hotmail.com PHI LI PPI NES Dr Amado O. Tandoc I I I
Medical Specialist III
Research Institute for Tropical Medicine 9002 Research Drive, FCC Compound Alabang, Muntinlupa City
Telefax : (632) 809 7120
E-mail : amado.tandocMD@gmail.com M r Joseph M . Bonifacio
Science Research Analyst
Research Institute for Tropical Medicine 9002 Research Drive, FCC Compound Alabang, Muntinlupa City
Telefax : (632) 809 7120
E-mail : jsphbonifacio@gmail.com
REPUBLI C OF KOREA M s Jung Eun Cho Researcher
Division of Arboviruses National Institute of Health
Centers for Disease Control and Prevention Yeonje-ri, Gangoe-myeon
Chungbuk 363-951 Seoul
Telephone: (82) 43 719 8502 Fax no. : (82) 43 719 8519 E-mail : 920980@naver.com M s Go Woon Cha
Researcher
Division of Arboviruses National Institute of Health
Centers for Disease Control and Prevention Yeonje-ri, Gangoe-myeon
Chungbuk 363-951 Seoul
Telephone: (82) 43 719 8504 Fax no. : (82) 43 719 8519 E-mail : gounfairy@naver.com
VI ET NAM M rs Nguyen Thi Thu Thuy
Chief of Arbo-Laboratory-virology Department
National Institute of Hygiene and Epidemiology (NIHE) No. 1 Yersin Street
Ha Noi
Telephone: (844) 9 3622 8286 Fax no. : (844) 9 3972 4514 E-mail : ticun_2002@yahoo.com
M r Bui Chi Tam Researcher
Pasteur Institute 167 Pasteur Street District 3,
Ho Chi Minh City
Telephone: (848) 3829 6351
E-mail : chitam0301@yahoo.com
TEM PORARY ADVI SERS
Dr Barbara Johnson
Diagnostic and Reference Laboratory Arbovirus Diseases Branch
Division of Vector-borne Infectious Diseases Centers for Disease Control and Prevention 3150 Rampart Road, Building 401, Room 3-322 Fort Collins, CO 80521
United States of America Telephone: (1970) 2663543 Fax no. : (1970) 2216441 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) Fax no. : (81) 35 2581188
E-mail : takasaki@nih.go.jp
SECRETARI AT
Dr Youngmee Jee (Responsible Officer) Scientist (Laboratory Virologist)
Expanded Programme on Immunization World Health Organization
Regional Office for the Western Pacific United Nations Avenue
1000 Manila, Philippines Telephone: (632) 528 9744 Fax no. : (632) 521 1036 E-mail : jeey@wpro.who.int
M r David Alexander Featherstone Global Coordinator
Measles and Rubella Laboratory Network World Health Organization Headquarters Avenue Appia 20
CH-1211 Geneva 27, Switzerland Telephone: (4122) 791 4405 Fax no. : (4122) 791 3111 E-mail : featherstoned@who.int
ANNEX 2
Day 1, Monday, 24 October 2011
08:30 Registration
08:45 Completion of pre-assessment questionnaire
09:00 Welcoming remarks NIH Dr Myung-Chan Cho
09:10 Workshop objectives WPRO Dr Youngmee Jee
09:15 Self-introduction of participants and administrative announcements
Group Photo
Session 1 Japanese encephalitis/acute encephalitis syndrome (JE/AES) surveillance and Laboratory Network (LabNet)
09:30 Laboratory-based JE/AES surveillance:
progress, challenges and plans for sustain the JE LabNet Mr David Featherstone 10:00 JE control and lab net progress in the Western Pacific
Region Dr Youngmee Jee
10:30 Coffee break
Session 2 Quality assurance of the JE LabNet
11:00 Proficiency testing 2009-2010 Dr Barbara Johnson
11:15 Confirmatory testing 2010-2011 and RRL activities NIID Japan
Korea CDC China CDC
Dr Tomohiko Takasaki Dr Myung-guk Han China CDC
12:00
Reference serological panel for Standardization of JEDiagnosis and Evaluation of JE assays
Dr Barbara Johnson 12:20 Discussion
12:30 Lunch break
Session 3 Introduction of JE virus-specific immunoglobulin M (JEV IgM) enzyme-linked immunosorbent assay (ELISA)
13:30 General introduction to IgM assays Mr David Featherstone 13:45 Introduction to the ELISA practical Dr Barbara Johnson
The 3rd Intercountry Hands-on Training Workshop on the Laboratory Diagnosis of Japanese Encephalitis in the Western Pacific Region
Korea Center for Disease Control and Prevention
Korea Human Resource Development Institute for Health & Welfare Osong, Korea
24 to 27 October 2011
14:00 Laboratory practice: JEV IgM ELISA (serum)
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Facilitators & participants
15:30 Coffee break
15:50 Continuation: laboratory practice - JEV IgM ELISA
JEV IgM ELISA reading and calculation of results Participants 18:00 Adjourn for the day
Welcoming reception by WHO Day 2, Tuesday, 25 October 2011
08:30 Review of Day 1 results. Discussion of Day 2 activity Mr David Featherstone 09:00 Lecture and demonstration of JEV IgM ELISA –
cerebrospinal fluid (CSF) Dr Barbara Johnson
09:30 Laboratory practice: JEV IgM ELISA (CSF)
(during incubation time: calibration, maintenance of ELISA equipment, micropipettes, etc.)
Facilitators & participants
10:00 Coffee break
10:30 Continuation: laboratory practice - JEV IgM ELISA 13:00 Lunch break
14:00 Continuation: laboratory practice - JEV IgM ELISA 15:00 JEV IgM ELISA reading and calculation of results 15:30 Coffee break
15:45 Evaluation of ELISA test results and discussion Mr David Featherstone and facilitators
16:15 Specimen collection, preparation and shipment for virus
isolation and serology Dr Tomohiko Takasaki
16:30 Global specialized laboratory testing methods- ELISA, plaque reduction neutralization testing (PRNT) and decision algorithm
US CDC
NIID Dr Barbara Johnson
Dr Tomohiko Takasaki
Reception by Korea CDCDay 3, Wednesday, 26 October 2011
