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Field Application of in Vitro Assays Sensitivity of Human Malaria Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs Leonardo K. Basco Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs Leonardo K. Basco Unité de Recherche 77, Paludologie Afro-tropicale, Institut de Recherche pour le Développement and Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, Yaoundé, Cameroon ii Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs WHO Library Cataloguing-in-Publication Data Basco, Leonardo K. Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs / Leonardo K. Basco. “Th e fi nal draft was edited by Elisabeth Heseltine”–Acknowledgements. 1.Plasmodium - drug eff ects. 2.Drug resistance. 3.Microbial sensitivity tests. 4.Antimalarials. 5.Malaria – drug therapy. I.Heseltine, Elisabeth. II.World Health Organization. III.Title. ISBN 92 4 159515 9 (NLM classifi cation: WC 750) ISBN 978 92 4 159515 5 For more information, please contact: Dr Pascal Ringwald Global Malaria Programme World Health Organization 20, avenue Appia – CH-1211 Geneva 27 Tel. +41 (0) 22 791 3469 Fax: +41 (0) 22 791 4878 E-mail: [email protected] © World Health Organization 2007 All rights reserved. Th e 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. Th e mention of specifi c 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. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. Th e responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. Th e named author alone is responsible for the views expressed in this publication. Printed in France. iii Contents Abbreviations used ..........................................................................................................................................vi Executive summary ..........................................................................................................................................1 1. In vitro assays for the sensitivity of human malaria parasites to drugs ................................................................................................................................5 1.1 Assays for the sensitivity of P. falciparum .....................................................................5 1.1.1 History of assay development ..................................................................................5 1.1.2 Microscopic or visual examination ......................................................................7 1.1.3 Radioisotope methods ..................................................................................................13 1.1.4 Non-radioactive methods ..........................................................................................20 1.1.5 Bioassays ....................................................................................................................................29 1.1.6 Ex vivo assays ........................................................................................................................32 1.1.7 Target-specifi c assays .....................................................................................................34 1.2 Assays for the sensitivity of other human Plasmodium spp. ...................35 1.2.1 P. vivax ........................................................................................................................................35 1.2.2 P. malariae .................................................................................................................................40 1.2.3 P. ovale ..........................................................................................................................................41 1.3 Factors that infl uence sensitivity to drugs ...............................................................41 1.3.1 Eff ect of culture medium and supplements .............................................41 1.3.2 Eff ect of uninfected erythrocytes: erythrocyte volume fraction ..................................................................................................................................44 1.3.3 Eff ect of infected erythrocytes .............................................................................45 1.3.4 Eff ect of leukocytes .........................................................................................................47 1.3.5 Eff ect of serum factors .................................................................................................48 1.3.6 Eff ect of serum substitutes .......................................................................................49 1.3.7 Eff ect of incubation period ......................................................................................51 1.3.8 Eff ect of gas mixtures ....................................................................................................53 1.3.9 Eff ect of previous drug intake ...............................................................................54 1.3.10 Delay after blood collection, anticoagulants and parasite viability .....................................................................................................................60 1.3.11 Sources of antimalarial drugs .............................................................................61 1.3.12 Artefactual sources .......................................................................................................61 1.4 Applications of in vitro assays .............................................................................................64 1.5 In vitro-in vivo correlation ......................................................................................................67 1.5.1 Notion of drug resistance ..........................................................................................67 1.5.2 In vitro and in vivo responses ................................................................................69 2. Practical aspects of in vitro assays ...................................................................................72 2.1 General organization of laboratory work .................................................................72 2.1.1 Th e central laboratory ...................................................................................................72 2.1.2 Organization of the central laboratory .........................................................74 2.1.3 Specialized equipment .................................................................................................74 2.1.4 Common equipment ......................................................................................................77 iv Field application of in vitro assays for the sensitivity of human malaria parasites to antimalarial drugs 2.2 Preparation of reagents for in vitro assays ...............................................................77 2.2.1 RPMI 1640 medium .....................................................................................................77 2.2.2 Antibiotics ................................................................................................................................80 2.2.3 Serum ............................................................................................................................................82 2.2.4 Serum substitutes ..............................................................................................................83 2.2.5 Uninfected human erythrocytes ..........................................................................85 2.2.6 Malaria-infected erythrocytes ...............................................................................86 2.2.7 [3H]Hypoxanthine ...........................................................................................................87 2.2.8 Monoclonal antibodies ................................................................................................88 2.2.9 Saker-Solomons urine test .......................................................................................88 2.2.10 Antimalarial drugs ........................................................................................................89 2.3 Routine procedures .................................................................................................................... 111 2.3.1 Washing blood samples ........................................................................................... 111 2.3.2 Transport of blood samples ................................................................................
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