WORLD HEALTH ORGANIZATION REGIONAL OFFICE FOR THE WESTERN PACIFIC
REPORT REGIONAL WORKSHOP ON COURSE ON LABORATORY DIAGNOSIS OF POLIOMYELITIS AND POTENCY TESTING FOR VIRUS VACCINES
Beijing, People's Republic of China 11-27 June 1990
Manila, Philippines August 1990
iNHOIWPRO LlBRARl. llaniliL Ph~
02 NOV 1990 (WP)lAB/ICP/EPI/OOI-E ENGLISH ONLY
REPORT iGIONAL TRAINING COURSE ON lABORATORY DIAGNOSIS OF POLIOMYELmS AND POTENCY TESTING FOR VIRUS VACCINES
Convened by the REGIONAL OFFICE FOR THE WESTERN PACIFIC OF THE WORLD HEALTH ORGANIZATION Beijing, People's Republic of China. 11-27 June 1990
Not for sale Printed and distributed by the Regional Office for the Western Pacific of the World Health Organization Manila, Philippines August 1990
NOTE The views expressed in this report are those of the participants in the Regional Training Course on Laboratory Diagnosis of Poliomyelitis and Potency Testing for Virus Vaccines and do not necessarily reflect the policies of the World Health Organization.
This report has been prepared by the Regional Office for the Western Pacific of the World Health Organization for governments of Member States in the Region for the participants in the Regional Training Course on Laboratory Diagnosis of Poliomyelitis and Potency Testing for Virus Vaccines held in Beijing, People's Republic of China from 11 to 27 June 1990.
CONTENrS
1. INTRODUCTION ................................................................................................................... 1
2. PlANNING OF lHE COURSE .......................................................................................... 3 3. CONCLUSION AND EVALUATION OF lHE COURSE........................................... 4 1.
2. 3. 4. 5. 6. 7.
Selection and qualification of participants ............................................................. Selection and qualification of facilitators ............................................................... Arrangements on site ................................................................................................. Evaluation of course content .................................................................................... Evaluation of training materials ......................................................................... ,..... Commentary on non-standard methods included in the course......................... Planned follow-up activities......................................................................................
4 6 6 7 8 9 11
ANNEXES: ANNEX 1 - LIST OF PARTICIPANrS ............................................................................... 7 ANNEX 2 - AGENDA ............................................................................................................ 11 ANNEX 3 - LIST OF HANDOUTS ..................................................................................... 17 ANNEX 4 - DETECTION OF POLIOVIRUS-SPECIFIC IgM ANTIBODIES BY AN IgM ANTIBODY-CAPTURE ELISA. .................................................................. 19 ANNEX 5 - EVALUATION QUESTIONNAIRE ............................................................ 23
1. INTRODUCTION
The Regional Training Course on Laboratory Diagnosis of Poliomyelitis and Potency Testing for Virus Vaccines, organized by the World Health Organization Regional Office for the Western Pacific was conducted from 11 to 27 June 1990 in Beijing, China. The specific objectives of the training course were: to review the current status of the laboratory diagnosis of poliomyelitis and (1) potency testing of virus vaccines (poliomyelitis and measles) in the Region; to review the WHO manuals on the isolation and identification of poliovirus and (2) on vaccine potency testing; (3) to review specimen collection during epidemiological surveillance (from suspected poliomyelitis cases and apparently healthy contacts); (4) to introduce standard methods and practice in laboratory techniques for isolation and identification of poliovirus (types 1-3) wild and vaccine strains; and (5) to standardize and practise the technique for potency testing of virus vaccines (poliomyelitis and measles). There were 14 participants from China, two each from the Philippines, Korea and Viet Nam, and one from New Zealand, making a total of 21 participants from five countries. The course was directed by a team from the National Institute of Public Health and Environmental Protection, Bilthoven, the Netherlands, including Dr Henk Bruning, Dr Anton van Loon, Dr Fred van Nimwegen, and Dr Paul Oostvogel. Dr Akio Hagiwara of National Institute of Health, Tokyo, Japan and Mrs Margery Kennett, Fairfield Hospital, Victoria, Australia were temporary advisers. Dr Sima Huilan, Regional Adviser, Health Laboratory Services, WHO/WPRO Manila was the operational officer. The WHO secretariat in Geneva was represented by Dr Takatoshi Kobayakawa, Medical Officer, Expanded Programme on Immunization, and Dr Julie Milstien, Scientist, Biologicals Unit. The course was opened by Dr Bernard P. Kean, WHO Representative in China, on behalf of Dr Sang Tae Han, Regional Director for the Western Pacific. Dr Kean expressed his gratitude to the Government of the People's Republic of China for agreeing to hold the training course at the Sino-Danish Biomedical Post Graduate Training Centre in Beijing. The World Health Organization's Regional Committee for the Western Pacific has resolved to make every effort to eradicate poliomyelitis in the Region by 1995. Dr Kean described the present incidence of poliomyelitis in the Region and outlined the steps required to further reduce its incidence and prevalence. This involves increasing immunization coverage using a potent vaccine and strengthening the surveillance of potential cases. Special efforts must be made to upgrade the laboratory techniques for isolating and identifying the poliovirus and for testing vaccine potency.
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The training course was planned to enable participants to exchange information and ideas about establishing a regional laboratory network for poliovirus confirmation and vaccine potency testing and to practise in their own countries the techniques learned on the course. Dr Wang Chao, Deputy Director, Department of Epidemiologic Prevention, Beijing, welcomed the participants. Also present at the opening ceremony were Professor Zhang Yi Hao, former Director of the National Vaccine and Serum Institute, Beijing; Dr Yang Baoping, Chief, EPI Division, Department of Epidemiologic Prevention, Beijing, Mrs Xue Puying, Department of Foreign Affairs, Beijing; and Professor Guo Cunsan, Director, Sino-Danish Biomedical Postgraduate Training Centre, Beijing. The Sino-Danish Biomedical Postgraduate Training Centre (PTC) was an excellent location for the course as the dormitory was in the grounds of the Centre, which meant that participants had easy access to the laboratory and classrooms. The list of participants is given in Annex 1.
2. PLANNING OF THE COURSE
In preparing the timetable for the training course, due attention was given to: (a) the stated objectives of the course;
(b) the contents of similar courses on potency testing for viral vaccines in Cairo, Egypt in November/December 1989 and Coonoor, India in March/April 1990; (c) the content of the global workshop on the laboratory diagnosis of poliomyelitis at Rijksinstituut voor Volksgezondheid en Milieuhygiene (RIVM) (National Institute of Public Health and Environmental Protection), Bilthoven, the Netherlands in November 1989; (d) (e) the availability of resources in laboratories in countries of the Region; the application of several nonstandard techniques used in the Region.
Each day's programme was designed to cover presentation of principles and practice. A generous portion of time was spent on bench work and group discussions, and several relevant field visits were made. The agenda is given in Annex 2.
3. CONCLUSION AND EVALUATION OF THE COURSE
3.1
Selection and Qyalification of particwants
The course included 21 participants: 14 participants from China, two participants each from Korea, Viet Nam, the Philippines, and one from New Zealand. The course was intended for those responsible for performing vaccine potency testing, serology, and poliomyelitis
-3diagnostic techniques in production. national quality control, and national diagnostic laboratories. The mix of participants was as follows: six from production laboratories; two from quality control laboratories; and 12 from EPI/diagnostic laboratories. One participant. from Viet Nam. works in a hospital microbiology laboratory. but was sent because her director felt it would be useful to convert this laboratory to a poliomyelitis diagnostic laboratory. Of the 14 Chinese participants, three were from production laboratories, one was from the National Quality Control Laboratory and one was from the designated National Reference Laboratory for Poliomyelitis Diagnosis. The remaining nine were from nine different provinces; three of these were epidemiologists with good laboratory background. and the other six were full-time laboratory professionals. In general, the selection of participants was excellent; most of them used good laboratory techniques and had a good understanding of the techniques being used. The participant from New Zealand were exceptionally good. functioned as a co-facilitator. The two participants from Viet Nam were the weakest in understanding the theory and practice used. One had no virology experience at all. . The participants from the Philippines were both chosen from the in-process production control laboratory. It would have been useful if the proposed diagnostic laboratory Research Institute for Tropical Medicine (RITM) had been represented. In addition. of those countries who may be considered for certification of national diagnostic laboratories in the near future. Malaysia was conspicuous by its absence from the course. The course was conducted in English. with all training materials in English. This posed quite a problem for class participation and understanding of the materials covered. Some of the communication difficulties were obviated by the use of small working groups (three or four participants) with at least one person in each group fluent in both Chinese and English. The non-Chinese participants all had an acceptable working knowledge of English. except for the Vietnamese. who had some difficulty. At least half the Chinese participants also had problems working in English. 3.2 Selection and Qualification of facilitators
The course facilitators included four scientists from the RIVM. Netherlands. who were charged with developing and carrying out the course curriculum. two scientists from proposed regional laboratories for poliomyelitis diagnosis in the Region, who had already participated in one gIoballevel training course; and two representatives from the WHO secretariat in Geneva. The RIVM staff had developed an excellent course curriculum and the course was outstanding. A few problems arising in the diagnostic segment, which will be dealt with later, probably stem from two major factors: (1) this was the first course of this type. including curriculum topics in both control and diagnostic areas; and (2) in addition to the planned curriculum, the course covered evaluation of two alternative diagnostic methods suggested by scientists within the Region. The temporary adviser from Australia was essential to coordinate the diagnostic portion of the course, as well as the part on laboratory safety. 3.3 Arran&ements on site
The PTC is without doubt the best training site ever used for one of these courses by WHO. The fact that the participants and facilitators were in constant close contact and with ready access to the laboratory even on evenings and weekends contributed to an esprit de corps
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and to an efficient, positive working environment. The physical layout of the PTC was perfect for this course, and the facilities for photocopying. videotaping. and secretarial work were excellent. Arrangements for field trips as part of the course curriculum were excellent, and all support received by the PTC, from accommodations and transport to social activities was efficient and pleasant. The staff were always willing to do whatever was necessary to meet needs and requests. The participants would have preferred to work six-day weeks. This is important for a course involving cell culture, and it is hoped that future course agendas will take this into account. 3.4 Evaluation of course content
As this was the first course of this type to combine vaccine potency testing and serology with diagnostic methods, it was an experiment. The experiment was successful, and the course will serve as a model for future courses. The diagnostic part of the course, which was intended to be interwoven throughout the two-and-a-half-week period, but to comprise only two days of actual experimental work, was extended to four days. This was because of the evaluation of two experimental techniques was added to the curriculum: the IgM method and the microplate assay. These will be described below. The result of this extension was that the facilitators felt the need to rush through parts of the curriculum, without the leisure to put the various components into context, and to discuss them fully with the participants. It is hoped that future courses will not meet these problems. In principle, however, the facilitators felt that the combined curriculum was useful and necessary. In fact, many of the participants are doing both types of work, and need to be exposed to standard methodologies from both curricula. The one major problem in the practical work was that the preferred method for specimen processing for poliomyelitis diagnostic work, according to the WHO manual for poliomyelitis diagnostic laboratories (in press), did not work for the faecal specimens used in the course. Although this method is recommended in the manual, it has not yet been tested in a course of this type. The previous course, in the Netherlands, used chloroform extraction of faeces, a suggested alternative method. It had been intended to use the chloroform extraction procedure here. but it was discovered the night before the actual laboratory work was to be carried out that the only centrifuge tubes available were not resistant to chloroform. Thus the WHO method was substituted, without trial, in this laboratory. It did not work. 3.5 Evaluation of traininl: materials
Three types of training materials were used in the course: the BLG/EPI restricted document, LaboratOlY Methods for the Titration of Live Virus Vaccines Usina Cell Culture Techniques (BLG/EPI/89.1), which is being tested in the regions for comments prior to finalization; the EPI draft Manual for the Viroloaical Investigation of Poliomyelitis; and handouts for the practical work (Annex 3) and lectures prepared by the course facilitators. The document BLG/EPI/89.1 has been tested in two previous training courses in two other WHO regions, and, with the relevant handouts, which will be included in a subsequent revision, has been found useful and comprehensive. The Manual for the Virological Investillation of Poliomyelitis was tested in a global training course held in November in 1989 at the RIVM, and comments received from participants were included in its revision. However, the specimen treatment method noted in the manual as the preferred method has not actually been subjected to field laboratory testing; it proved unsatisfactory under the conditions of the course. The facilitators thus recommend that the description of this method be modified, either by changing the centrifugal force needed for treatment of faecal specimens prior to inoculation, or by recommending inclusion of
-5chloroform extraction. The recommended method should be satisfactory for all samples given the equipment found in most laboratories. A second comment on the manual was that for courses of this type, a modular format, similar to that used in BLG/EPI/89.1, would be more useful. For courses of this type, which cover a curriculum suitable for participants with mixed backgrounds (which seems to be more the rule than the exception), parts of it could then be extracted as required for the course curriculum. In its present format, it is not readily adaptable for teaching purposes. 3.6 Commentary on non-standard methods included in the course
Two non-standard methods were evaluated in the course at the suggestion of regional scientists. The first was the IgM technique for poliomyelitis diagnosis, developed by Professor Zhang Li-Bi, of the Institute of Virology, Chinese Academy of Preventive Medicine in Beijing (Annex 4). This is the laboratory designated by the Chinese authorities as the National Reference Laboratory for Poliomyelitis Diagnosis. Training in the use of this technique has been carried out by Professor Zhang throughout China, and many provincial laboratories are using it. Professor Zhang was present at the course for two days, and provided protocols, samples, and demonstrations on using the test. The entire procedure was recorded on video for future reference. Three sera were tested by this technique, and each of the six laboratory groups performed the test. Correct results were obtained by four of the six groups, and misleading results from the other two. The source of the incorrect results was thought to be the addition of sera to the wrong wells in one case ;md improper washing in a second case. However, the controls necessary for pinpointing the source of problems had not been set up. The course facilitators felt that the method shows promise, but that it should be routinely performed with more controls. In addition, crucial data on the sensitivity and specificity of the test have not yet been made available. Further questions to be answered include those relating to the interference from immunization and/or concurrent enterovirus infection, and the ability to use the test to distinguish between infection by wild or vaccine-like poliomyelitis viruses. Although the test does show promise, it is felt that it could not be used without some concurrent isolation, necessary for the characterization of circulating polioviruses. A detailed summary of comments by course facilitators is annexed (Annex 4). The second technique, the microplate method, was suggested by Japanese researchers as a means of saving on reagent costs as well as time. The test was evaluated during the course. The general concensus was that the method needs further work before it can be recommended for routine use. 3.7 Planned follow-up activities
All participants will receive a panel of samples to test and the necessary reagents to test them, to ascertain whether they can satisfactorily identify poliovirus isolates and test for poliovaccine potency after participation in this course. In addition, a kit of standard reagents will be supplied to each of the participants on request to enable them to standardize their own laboratory techniques. It is planned to contact each participant by letter at least once in the next 12-month period to determine what problems or successes they may be having in applying the course procedures, and to solicit their comments on the course and training materials. If any of the laboratories are selected to take part in the global network for laboratory support for the eradication of poliomyelitis, they will be contacted regularly after that period. WHO and the facilitators can provide technical support to the participants on request.
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ANNEX 1 LIST OF PARTICIPANTS
1)
CHINA
DR CANG YAO-QUING Assistant Researcher Shanghai Institute of Biological Products Ministry of Public Health 1262 Yan-An Road (W), Shanghai People's Republic of China Post Code: 200052 DR HU HAN-XIAN EPI Section Hubei Provincial Anti-Epidemic Station Wu Chan&, Wuhan, Hubei Province People's Republic of China Post Code: 430070 DR JIANG YI-DE Assistant Research Fellow National Institute for the Control of Pharmaceutical and Biological Products, Ministry of Public Health Temple of Heaven, BeiiinLl People's Republic of China Zip Code: 100050 DR JIANG YONG-ZHEN Research Associate, Institute of Virology Chinese Academy of Preventive Medicine 100 Ying Xing Jie, Xuan Wu Qu BeiiinLlo People's Republic of China Zip Code: 100052 MS LI PING In-charge of Laboratory, Diagnosis of Poliomyelitis EPI Section, Health and Epidemic Prevention Station Jandong Street, Ki!n, Shanxi Province People's Republic of China Post Code: 710054 DR LlU CHUN-MEI Health and Anti-Epidemic Station of Liao-Ning Province 3 Nanjin Street, HepjnLlQu, Shenyang Liao-Ning Province People's Republic of China Post Office Box: 11 0005 DRLlUQU Inspector, Health and Anti-Epidemic Prevention Station Huaisu Street 50, ChenBdu, Sichuan Province People's Republic of China Post Code: 610031
2)
3)
4)
5)
6)
7)
- 8Annex 1 8) DR TAN SHUN-GE Assistant Researcher, Institute of Medical Biology Chinese Academy of Medical Sciences Hua Hong. Dong Xi San Qu, Kunming People's Republic of China Post Code: 650107 DR TIAN X1AOEN Chief of Section of Planning on Immunization Henan Provincial Anti-Epidemic Station Zhengzhou City, Henan Province People's Republic of China Post Code: 450003 MRWANGYAN Assistant Engineer, National Vaccine and Serum Institute Sanjianfang. Chaoyangqu, Beijing People's Republic of China Post Code: 100024 DR WU CHENG-MIN Technician in charge, EPI Section Health and Epidemic Prevention Station 176 Xingang West Road, Guana:zhou People's Republic of China Post Code: 510300 DR XU AlQIANG Doctor on Epidemiology Shandong Provincial Hygiene and Anti-Epidemic Station 20 Jingshi Road, Jinan, Shandong Province People's Republic of China Post Code: 250014 DR ZHENG KUI-CHENG Physician of Epidemiology Fujian Provincial Center of Sanitary and Epidemic Prevention Fuzhou, Fujian Province People's Republic of China Post Code: 350001 DR ZHOU YING-PING Vice Director, EPI Laboratory Prevention Station 80 Taoyuan Road, NanniDll, Guangxi Province People's Republic of China Post Code: 530013
9)
10)
11)
12)
13)
14)
NEWZEAlAND 15) MR DAVID FEATHERSTONE Scientist, Virology Laboratory New Zealand Communicable Disease Centre Kenepuru Drive Porirua, Wellington, New Zealand P.O. Box: 50-348 Porirua
-9Annex 1 PHILIPPINES 16) MRS ILUMINADA V. CRUZ Bacteriologist m Biologicals Production SelVice, Department of Health DOH Compound, Alabang 1702, Muntinlupa Metro Manila, Philippines MISS MARILOU S. PADUGA Bacteriologist I Biologicals Production SelVice, Department of Health DOH Compound, Alabang 1702, Muntinlupa Metro Manila, Philippines
17)
REPUBUC OF KOREA
18)
DR YOUN-HO CHUNG Public Health Doctor Division of Enterovirus, Department of Virology National Institute of Health 5 Nokbun-dong, Eunpyung-ku. 122-020 ~Korea
19)
MR MOON-BOO KIM Researcher, Division of Enterovirus Department of Virology, National Institute of Health 5 Nokbun-dong, Eunpyung-ku, 122-020, ~Korea
SOCIAUST REPUBUC OF VIET NAM 20) DR DANG THU DUNG Microbiologist, Institute for Clinical Research of Tropical Diseases, Bach Mai University Hospital Hanoi. Viet Nam MRS NGUYEN THI KIM TAM Chief Laboratory for Quantity Control of Poliomyelitis National Vaccine National Institute of Hygiene and Epidemiology National Centre for Control of Vaccine 1 Yersine Street, Hanoi. Viet Nam TEMPORARY ADVISERS 22) DR H. BRUNING National Institute of Public Health and Environmental Protection (RJVM) Antonie Van Leeuwenhoeklaan 9 P.O. Box 1,3270 BA Bilthoven The Netherlands
21)
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Annex 1 23)
DRAKIO HAGIWARA Department of Enteroviruses National Institute of Health Gakuen 4-7-1, Musashimurayama Tokyo 190-12, Japan DR MARGERY KENNETT Virologist, Virology Department Fairfield Hospital, Yara Bend Road Fairfield 3078, Victoria, Australia P.O. Box: 65 DR ANTON VAN LOON Virology Laboratory National Institute of Public Health and Environmental Protection (RIVM) Antonie Van Leeuwenhoeklaan 9 P.O. Box I, 3270 BA Bilthoven The Netherlands DR F. VAN NIMWEGEN National Institute of Public Health .and Environmental Protection (RIVM) Antonie Van Leeuwenhoeklaan 9 P.O. Box 1. 3270 BA Biltboven The Netherlands DR PAULUS OOSTVOGEL National Institute of Public Health and Environmental Protection (RIVM) Antonie Van Leeuwenhoeklaan 9 P.O. Box 1, 3270 BA Bilthoven The Netherlands SECRETARIAT
24)
25)
26)
27)
27)
DR SIMA HUIl.AN Regional Adviser Health Laboratory Services WHO Regional Office for the Western Pacific Manila DR T. KOBAYAKAWA Expanded Programme on Immunization Word Health Organization 1211 Geneva 27. Switzerland DR 1. MILSTIEN Scientist, Biologicals Unit World Health Organization 1211 Geneva 27, Switzerland
28)
29)
- 11ANNEX 2 AGENDA Monday. 11 June 1990 Opening Session
Introduction of participants Tea Break
Lecture: Regional overview on polio eradication (Dr Sima HuiIan, WHO/WPRO) Lunch Lecture: Laboratory safety (Mrs Margery Kennett) Practice: Preparation for the next day Cell counting Pretreatment fllter discs Tuesday. 12 June 1990 Film: EPI Documentary - Recognize the Disease
Lecture: The EPI vaccine (Dr Julie Milstien) Tea Break
Lecture: Introduction measles haemagglutination (HA) and HA-inhibition (m) Practice: Pretreatment of sera Performing m-assay Performing HA-assay Lunch Tuesday. 12 ,1une 1990 (afternoon) Practice: Reading and reporting results Preparation for the next day Wednesday. 13 ,1une 1990 Presentation by a participant Lecture: Introduction polio serum neutralization test Tea Break
Practice: Polio serum neutralization assay (Micro)
- 12Annex 2 Lunch Practice: Preparation for the next day Thursday_ 14 June 1990 Introduction OPV-potency testing (cpe) Tea Break Practice: Add Hep2 cells to polio SN plates Trivalent oral polio vaccine potency testing CCID50 Micro Lunch Lecture: Preparing and testing anti-sera (Dr Henk Bruning) Practice: First reading and reporting - polio SN assay Preparation for the next day Lecture: Difference between plaque assay and cpe test (Dr Henk Bruning) Friday. IS June 1990 Lecture: Introduction measles-cpe test Practice: Measles cpe test (CCID50) Tea Break Lecture: Adverse Reactions (Dr Julie Milstien) Lunch Practice: Reading and reporting results - fIrst week Monday_ 18 June 1990 Lecture: Collection. transportation and registration of faecal specimens (Dr Paul Oostvogel) Preparation of faecal specimens and inoculation of cell cultures (Mrs Margery Kennett) Tea Break Preparation of specimens Lunch Practice: Inoculation of cell cultures with faecal suspensions
- 13 Annex 2 Lecture: Introduction yellow fever plaque assay Practice: Yellow fever potency testing (plaque assay) Tuesday. 19 .June 1990 Lecture: Isolation and identification from polio and other enteroviruses from faeces Rapid simultaneous isolation and identification of polio viruses (Mrs Margery Kennett)
Film:
The Polio Virus Tea Break
Practice: Isolation and identification from polio and other enteroviruses from faeces Lunch Practice: Addition of cells Examination and registation of previous experiments Tea Break Lecture: Staining (Dr Henk Bruning) Wednesday. 20 .June 1990 Lecture: Introduction single IgM for the diagnosis of poliomyelitis Practice: Single IGM for the diagnosis of poliomyelitis (Dr Zhang Li Bi) Tea Break Practice: Single IGM for the diagnosis of poliomyelitis Examination and registration of previous experiments Lunch Wednesdu. 20 .June 1990 (afternoon) Field Visit:National Laboratory for the Control of Pharmaceuticals and Biologicals. Beijing Tea Break Lecture: Preparing and testing of anti-sera (continued) (Dr Henk Bruning)
-14 Annex 2 Thursday. 21 June 1990 Lecture: Use of filter discs for blood collection (Dr Henk Bruning) The EPI laboratory network (Dr Paul OoslVogel) The WPRO laboratory networkk (Dr Julie Milstien) Tea Break Practice Single IGM for the diagnosis of poliomyelitis Examination and registration of previous experiments Lunch Practice: Single IGM for the diagnosis of poliomyelitis Lecture: Reading and calculation of microtitre and plaque assays (Dr Henk Bruning) Tea Break Lecture: Interpretation of microtitre tests for polio and enterovirus typings. Friday. 22 .June 1990 Field Visit:A routine immunization session -Tong County Lunch Practice: Examination and registration of previous experiments Yellow fever plaque assay Tea Break Lecture: Calculation of experimental results Monday. 2S .June Field visit: National Vaccine and Serum Institute, Beijing Lunch Lecture: lntratypic differentation: State of the art at a reference laboratory (Dr A.M. Van Loon) Cell banking (Dr H. Bruning) Acceptance of results (Dr J. Milstien)
- 15 Annex 2 Tea Break Practice: Examination and registration of previous experiments
Tuesday_ 26 .June Return of course examination Evaluation of practical work Discussions on the examination results Tea Break Evaluation - WHO Manual Lunch Closing Ceremony
- 17· ANNEX 3 LIST OF HANDOUTS
1. 2.
EPI Update May 1989 Poliomyelitis Global Eradication by the year 2000 Regional Overview of Poliomyelitis eradication in the Western Pacific Region Report on WHO/RIVM Workshop on Laboratory Diagnosis of Poliomyelitis Cell Counting Requirements for EPI vaccines Adverse Reactions Preparation of specimens for isolation of polio and other entero virus Identification of Polio and other enterovirus How to test suitability of antipolio sera Cpe versus plaque tests Optional test circumstances Timetable for polio laboratory development Intratype differentiation of polio virus isolates - Dr Sima Huilan - Mrs M. Kennett • Dr H. Bruning - Dr I.Milstien • Dr I. Milstien • Mrs M. Kennett • Mrs M. Kennett - Dr H. Bruning • Dr H. Bruning - Dr H. Bruning - Dr P. Oostvogel • Dr J. Milstien • Dr A. van Loon
3. 4. 5.
6. 7. 8. 9. 10. 11. 12.
13.
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ANNEX 4 DETECTION OF POLIOVIRUS-SPECIFIC IgM ANTIBODIES BY All IBM A.~TIBODY-cAPTURE ELISA (Prof Dr Zhang-Li bi) Principle'
~~
~
anei-Hu-IgM --lgM --Poliovirus aneigen (serum)
--An~
i-'pol 10 --Anti-rabbie virus IgG IgG-HRP (rabbie) (goat)
Reagencs used· Anei-Hu-IgM, Affinity-purified polyclonal (Sigma) . Monoclonal (hoole-ma.de). ant!s~ra
Poliovirus. anelgen, Sonicated tissue culcure supernaean1: fro", VERa cells infected yith Sabin strains. ' • Control antigen: Idem from uninfeceed cells
Anei-poliovirus IgG: Antibodiesagainse poliovirus eype 1. 2 or (rabbit) 3 raised in rabbits by immunizacion wieh purified pOliovirus. Antibodies are sa.Id to be eype specific. Anti rabbit IgG (HRP) ,Home-made: HRP conjugation method noe (goat) given. Other reagen~s:
See annex ,
Note: Concen~raeion ahd buffer competition of the subscrate (OPO) soiucion were n01: given; instead data yere given for preparaeion of the subs1:race for alKaline phosphaease conjugates. Procedure: See annex 2.
Results: Results are measured as optical densieies and are expressed as ehe raeio beeyeen the reaction wieh the poliovirus antigen (P) and ehe control antigen (N). A PIN raeio > 2.' is considered to indicate the presence of poliovirus (type) specific IgM. Kineeics of the assay syseem· Details on ehe kinetics of ehe assay syseem Yere noe given and may well be not known very well (personal communication Prof. Zhang Li-bi). This is pareiculariy regrettable with the respece to che correlacion between serumdilution and op1:ical density in infants vs. children and adults.
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Annex 4 D~~~ction
rar.e: pa~ients
I
poliovirus-specific.lgM antibody was detecte~ in between 13~ (6/46) and 92.3~ (48/52) of patients. It is remarkable ~ha~ IgM antibody to ,nora than one type .. as found in only one out of 367 positive patients (annex 3, table 5). The relation to the onse~ of illness (paralytic?) was wtudied in more than 200 suspected cases. Results are given in table 1. ·The numbers of patients in each group are not known. suspec~ed
In
T~ble
1. Correlation between days after onset of illne~s in patients suspec~ed of pollnmyelitis and the detection or sp~ci£ic
pol. lovirus
Igl1
..dl\. loodies
Days atce:r ·ons€:'t 0-3 4-21
percentage IgM 6'~ 7o~
posi~ive
22-25 25-55
., 0'n.;) O~
rjD-r..:a
)55 Sensi~ivi~v:
comparison with viru$ i$olacion. So Car, data regarding sensitivity as rel~t~d to virus
iSOlation are available from tWO laboratories. Data from Beijing and Shan dong shOW that IgM antibodies to poliovirus were detected in 76.9~ and 80~, respectively, of suspected patients. The figures for virusisolation arGo 42.2>t an.:I 45'',;. r •• sp.~ctively (table 2). Comparison of ~he isolated virustype and t~. type specificity of the IgM response showed infection by the same virus in 39 OUt of 42 patients (annex 3, table 4) Table 2. Correlation between virus iSOlation and detection of poliovirus-specific IgM in susp~c~ed patients. IgM d",c •• ct ion Virus isola.tion
.. ~oer.:
Beijing (n=52) Chan •
..
don~
(n=201
18
5
7 9
2
22 i [' i c i 1:
7
v:
A PIN ra~io ) 2.1 is considered to indica~e a positive IgM reaction. However, no da~a regarding the frequency distribution of PIN ratio's in a sufficiently lar~e number (>100) ot nega~ive pa~ients are available. A blocking assay wi~h different enterovirus antigens was carried out ~o confirm ~he specificity of a posi~ive IgM reaction in the poliovirus type 1 IgM test. Reactivi~y was neutralized by the homologous antigen only (annex 3 tabla 2). Additional data on sp~cificity were obtained by examining a few sera from patients with other enterovirus infections as demonstrated by detection of a specific I~M
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Annex 4
l'esponse. No cross-react i v it y was obsr:rv,,,d in t h .. pol iovirus type 1 IgM ant ibody. (."nnex 3, table 1)
""s"£"y
for
Conclusion Oet .. ctlon of cype-specit"ic Igrl antioodielil Co poliovil'u,z ..... may tacilitat~ and complement ~he dia,nosis Of poliomyelitis in suspected patients. The test is relatively simple and .. asy to pertorm and to standardi:!e. obviates tile need for tis:!>ue culeure facilities and results may be obtained within 24 hour~ after receiving specimens. However. it can not complet~ly r ... plac~ virus isolat ion Which is abSOlUtely need<i!d I-or .san,lot Ic and anci.enetic analysis of virus £trains. In addition. the I,K assay cannot (yet) discriminate between antibody induced by wild or vaccine type viruses. This last problem may be of limited importance as one may expect the Lgl1 response after wild type virus infeceion to be restrict .. d to on .. type. Th .. presently available data on sensitivity and sp<i!cificity appear promiSing but are clearly insu£Cicient for r~comm~ndation ot the method to diagnose poliomyelitis. In addition. data on tecllnical aspe·cts of the assay ",r", ne.;-.rly c,;,mpletely lc.cl<.ing. Therefor £ur~her scudies ~re needed to d~t~rmine th~ v~lue of ~he teChnique (or diagnosis of poliomyelitis. These studies should at l .. ast address the followin.e; points: T.. chnical ~spects:
- Dose response relationships. nOtably serumdilution vs. optic ... l denSity in ser... £1'0'"' infants. - Effect. of using wild type viral antigens endemic to the region - Frequency distribut ion of sera t-ro'l Pd.t ients with a.nd without poliovirus infection - Developm .. nt of a confirmatory assay (blocking test, immunoblotting) Sensitivity and specificity: - Performance of the test in specimens from well characterized patients with poliovirus as well as other infections (not only by enteroviruses) , preferably in a prospective stud),. - Follow-up studies in patients and vaccineesto determine the type specific IgM response in relation to .onset ot" disease and to Study persistence o( Igl'I ani: ibody. In addition, it is recommended to develop a similar a$$ay tor detection of IgA antibodies to poli9virus. This m~y be useful for $.=rologlcal diagnosis of pOliomyelitis in partially immunized patients. 8eijing. 24/06/90 A.I'\. vam Loon.
- 23ANNEX 5 EVALUATION QUESTIONNAIRE
1. Educational sains No 1.1 Were the following objectives met? (a) To review the current status of laboratory diagnosis of poliomyelitis and potency testing of virus vaccines (polio and measles) in the Region To review the WHO manuals on isolation and identification of polio virus and on vaccine potency testing; To review the specimen collection during epidemiological surveillance (from suspected poliomyelitis cases and apparently healthy contacts); To introduce standard methods and practice on laboratory techniques for isolation and identification of polio virus (by types 1-3 and wild, vaccine-strain); Two people would have liked to learn more of identification techniques of polio wild strains and vaccine strains. One of them wants to learn more of polymerised chain reaction in detail. (e) To standardize and practice the technique for potency testing of virus vaccines (OPV and measles) Y§
N!l
~
100%
(b)
100%
(c)
100%
(d)
90% 10%
100%
If no, please describe: 1.2 Have new skills or concepts been learned at the course? 1.3 Can these skills and concepts be applied in your country? 2. Process and outcome 86% 14% 100% 100%
2.1 Were you able to express your ideas or poblems at the course?
- 24Annex 5 2.2 Was there enough opportunity to exchange knowledge and experience with other participants? 86%
14%
If response to any of the above is !lQ, give comments as appropriate: 2.3 Were you satisfied with all training materials provided? 76% 5% 9%
If no, please explain for specific paper(s): 2.4 Specify which of the training materials distributed for the course are suitable for wider distribution: a)
Laboratory methods for the titration of live virus vaccines Laboratory techniques for isolation and identification of polio virus Polio and measles handouts Examination items Reference antisera for Polio I, II and III Reference OPV and measles vaccines Polio virus video
52% 9.5% 9.5% 9.5% 9.5% 9.5% 9.5% 62% 33% 5%
b)
c) d) e) f) g)
2.5 Did you have enough time to study the training materials? One person replied: 'Sometimes yes but sometimes no because of our heavy schedule during the weekdays, even weekend but I think I would rather review this manual when I come back to my country.' 2.6 Were methods of introduction and presentation of different topics satisfactory? 2.7 Were you fully satisfied with the
66%
34%
a) b)
formal discussions? laboratory discussions?
100% 86%
14%
If no, please explain:
\
I
I
-25 Annex 5 2.8 Fjeld Visits 2.8.1 IT there were field visits as part of the course, were they useful to meet the objectives? IT no, explain your response: 3. Organizatjon of the course Were the duration and scheduling of different activities - lectures, benchwork practice, disicussions, etc. - satisfactory? 4. Administratjve aspect Are organization or administrative arrangements for travel, accommodation, per diem, meeting room and secretarial support satisfactory? 5. Is there any better way to achieve the course objectives?
90%
10%
90%
10%
81%
5%14%
5% 43%
One participant said: "With the help of video and film." Another one said: "Yes", but did not specify how. 6. What follow-up activities, if any would you recommend? a) National government 2 people - not specified 1 person - to set up the National Control Authority 1 person - EPI meetings about methods with laboratory and epidemiology staff 1 person - training for the staff who are involved in laboratory diagnosis of poliomyelitis and those in the production and control of vaccines 10% 5%
5%
5%
b)
WHO 8 people - not specified 1 person - continue training of methods expanding DPT 38% 5%
c)
By other agencies 1 person 5%