WHO/IVB/04.10 ORIGINAL: ENGLISH VII VBB Polio laboratory manual 4th edition, 2004 World Health Organization, Geneva, Switzerland Fourth edition, final revision, 31 August 2004 The Department of Immunization, Vaccines and Biologicals thanks the donors whose unspecified financial support has made the production of this document possible. Ordering code: WHO/IVB/04.10 Printed: September 2004 Copies may be requested from: World Health Organization Department of Immunization, Vaccines and Biologicals CH-1211 Geneva 27, Switzerland Fax: +41 22 791 4227; Email: [email protected] This document revises and replaces the Manual for the virological investigation of polio WHO/EPI/GEN/97.01. © World Health Organization 2004 All rights reserved. Publications of the World Health Organization can be obtained from Marketing and Dissemination, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 2476; fax: +41 22 791 4857; email: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to Publications, at the above address (fax: +41 22 791 4806; email: [email protected]). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. The World Health Organization does not warrant that the information contained in this publication is complete and correct and shall not be liable for any damages incurred as a result of its use. Printed by the WHO Document Production Services, Geneva, Switzerland ii Polio laboratory manual Contents List of figures ..............................................................................................................v List of tables ............................................................................................................vii Abbreviations and acronyms ...........................................................................................ix 1. Introduction.............................................................................................................. 1 1.1 The Polio Eradication Initiative .................................................................. 1 1.2 History of poliomyelitis and polio vaccines............................................... 4 1.3 Characterization of the pathogen................................................................ 4 1.4 Transmission of poliovirus .......................................................................... 6 2. Role and function of the laboratory in polio eradication ................................ 10 2.1 Appropriate investigations......................................................................... 10 2.2 Structure and activities of the WHO polio laboratory network ............ 11 2.3 Coordination of the network .................................................................... 13 2.4 Accreditation of network laboratories...................................................... 15 3. Laboratory quality assurance .............................................................................. 22 3.1 The basis of laboratory quality assurance................................................. 22 3.2 Standard operating procedures (SOPs)..................................................... 24 3.3 Documentation ........................................................................................... 29 3.4 Equipment and instruments....................................................................... 29 3.5 Supplies........................................................................................................ 30 3.6 Laboratory safety........................................................................................ 34 3.7 Audits .......................................................................................................... 37 4. Cell culture techniques.............................................................................................. 39 4.1 Working in the cell culture laboratory ..................................................... 39 4.2 Cell culture procedures .............................................................................. 47 4.3 Composition of media and other reagents used in cell and virus culture ......................................................................................... 59 4.4 Cell culture problems: identification and elimination............................. 62 4.5 Hoechst 33258 direct DNA stain for Mycoplasma ................................. 65 4.6 MYCO-TEST immuno assay for Mycoplasma detection....................... 66 4.7 PCR assay for Mycoplasma detection ...................................................... 68 WHO/IVB/04.10 iii 5. Evaluating cell line sensitivity.............................................................................. 73 5.1 Selection of reference standard for quality control of cell lines.............. 73 5.2 Preparation of laboratory quality-control standards............................... 74 5.3 Titration of laboratory quality control standard ..................................... 75 5.4 Calculation of the virus titre by the Kärber formula .............................. 78 5.5 Interpreting data on cell-line sensitivity and troubleshooting................ 78 5.6 Procurement of reference poliovirus strains............................................. 79 6. Specimen receipt and processing.......................................................................... 81 6.1 Receipt of specimens .................................................................................. 81 6.2 Specimen preparation ................................................................................. 82 6.3 Composition of reagents used in specimen processing ........................... 85 7. Isolation and identification of polioviruses ........................................................ 87 7.1 Recommended cell lines for the isolation of polioviruses....................... 87 7.2 Isolation of polio and other enteroviruses................................................ 88 7.3 Identification of poliovirus isolates........................................................... 91 7.4 Identification of non-poliovirus enteroviruses ........................................ 98 8. Intratypic differentiation of polio viruses ........................................................ 101 8.1 ELISA method for intratypic differentiation......................................... 101 8.2 Probe Hybridization method for intratypic differentiation ................. 108 8.3 PCR method for intratypic differentiation ............................................ 112 8.4 Protocol for use of two methods of intratypic differentiation of polioviruses ............................................................................................... 119 9. Investigating wild poliovirus contamination of cell cultures........................ 121 9.1 When to suspect wild poliovirus contamination ................................... 121 9.2 Investigating potential sources of wild poliovirus contaminants .........122 9.3 Determine if there are lapses in good laboratory practices ................... 123 9.4 Determine if samples can be tracked....................................................... 124 9.5 Communicate with the programme during the investigation............... 125 10. Specimen and isolate transport .......................................................................... 126 10.1 Transport planning ................................................................................... 126 10.2 Packaging the materials for transport ..................................................... 127 10.3 Genetically modified micro-organisms and organisms ......................... 139 10.4 Preparing the documentation and sending the package ........................ 139 11. Data management................................................................................................ 142 11.1 Recording receipt of specimens............................................................... 144 11.2 Recording laboratory results ................................................................... 147 11.3 Reporting laboratory activity and results............................................... 150 11.4 Laboratory data for certification............................................................. 151 Bibliography .......................................................................................................... 153 iv Polio laboratory manual List of figures Figure 1.1 Progress in polio eradication 1988-2002................................................ 2 Figure 1.2 Poliovirus genome................................................................................... 7 Figure 2.1 Structure of the Global Polio Laboratory Network .........................