ASSESSMENT OF DIFFERENT STAINS AND STAINING PROCEDURES FOR MICROSCOPIC DETECTION OF MALARIA PARASITES By: Awad Alla Hamza Osman Kashif BSc of Medical Laboratory Sciences (1995) A thesis submitted for fulfillment requirement of master degree in Medical Laboratory Sciences (Medical Parasitology) At the Department of Parasitology & Medical Entomology Faculty of Medical Laboratory Sciences University of Khartoum Supervisor: Dr. Eldirdieri Salim Ahmed College of Medicine –University of Juba September 2004 LIST OF CONTENTS DEDICATION………………………………………………………………….. I ACKNOWLEDGEMENT.…………………………………………………….. II LIST OF TABLES……………………………………………………………... III LIST OF FIGURES……………………………………………………………. IV LIST OF APPENDICES……………………………………………………….. V ABSTRACT…………………………………………………………………….. VI ABSTRACT (ARABIC)………………………………………………………... VII 1- INTRODUCTION & LITREATURE REVIEW 1 1.1 General introduction……………………………………………………….…... 1 1.2 Historical background…………………………………………………………. 1 1.3 Malaria parasites………………………………………………………………. 3 1.4 Transmission of malaria……………………………………………………….. 4 1.5 The life cycle of malaria……………………………………………………….. 8 1.5.1 Pre-erythrocytic phase……………………………………………………….. 8 1.5.2 Erythrocytic phase…………………………………………………………… 9 1.5.3 Vector phase (Sporogony)……………………………………………….…... 9 1.6 Pathogenesis of malaria………………………………………………………... 12 1.7 Immunity to malaria…………………………………………………………… 14 1.8 Diagnosis of malaria……………………………………………………….…... 15 1.8.1 Clinical diagnosis……………………………………………………………. 15 1.8.2 Microscopic diagnosis……………………………………………………….. 16 1.8.2.1 Conventional thick and thin blood smears………………………………… 18 1.8.2.1.1 Blood drawing technique…………………………………………….….. 19 1.8.2.1.2 Preparation of the smears…………………………………………….….. 19 1.8.2.1.3 Staining procedures…………………………………………………. 21 1.8.2.1.4 Microscopic examination. ………………………………………….. 23 1.8.2.2 Quantitative Buffy Coat (QBC) Test…………………………………. 24 1.8.2. 3 Saponin lyzing technique…………………………………………….. 24 1.8.3 Rapid Diagnostic Tests (RDTs)………………………………………… 25 1.8.4 Deoxyribonucleic acid (DNA) probes and Polymerase Chain Reaction 26 (PCR). .. 1.8.5 Detection of Plasmodia specific antibodies……………………………. 26 1.9 Chemotherapy of malaria…………………………………………………. 27 1.10 Malaria in the Sudan…………………………………………………….. 29 1.10.1 Transmission …………………………………………………………... 29 1.10.2 Morbidity and mortality ………………………………………………. 30 1.10.3 Diagnosis of malaria................................................................................ 30 1.10.4 Chemotherapy of malaria........................................................................ 31 1.10.5 Control………………………………………………………………… 32 1.11 Justification……………………………………………………………… 34 1.12 Objectives………………………………………………………………... 34 1.12.1 General objective……………………………………………………… 34 1.12.2 Specific objectives…………………………………………………….. 34 2. MATERIALS & METHODS 35 2.1 Study population………………………………………………………….. 35 2.2 Study design ……………………………………………………….……… 35 2.3 Samples collection ………………………………………………………... 35 2.4 Techniques………………………………………………………………... 35 2.4.1 Giemsa stained smears………………………………………………….. 35 2.4.2 Saponin lyzed venous blood technique …………………………………. 36 2.4.3 Field’s stain smears ……………………………………………………... 37 2.4.4 Preparation of different Giemsa stains stock solution…………………... 37 2.4.5 Concentration of Giemsa working solution…...………………………… 38 2.4.6 Dilution of stock Giemsa stain with distilled or tap water……………… 39 2.4.7 Stability of Giemsa stain working solution……………………………... 40 2.5 Ethical consideration……………………………………………………… 41 2.6 Data analysis……………………………………………………………… 41 3. RESULTS 42 3.1 Giemsa stained blood smears…………………………………………….. 42 3.2 Saponin lyzed venous blood technique …………………………………... 46 3.3 Comparison between Giemsa and Field’s stain in detecting malaria 48 parasites.............................................................................................................. 3.4 Preparation of different Giemsa stains stock solutions…………………… 50 3.5 The use of different concentrations of Giemsa stain working solutions. ... 52 3.6 Preparation of Giemsa stain working solution using different diluents.... 54 3.7 Stability of Giemsa stain working solution……………………………….. 56 4. DISCUSSION 58 CONCLUSION & RECOMMENDATIONS……………………………… 63 REFERENCES………………………………………………………………. 64 APPENDICES……………………………………………………………….. 83 DEDICATION To my mother, soul of my father & my brothers To all my teachers from primary school to the university I ACKNOWLEDGMENT I am greatly indebted to my supervisor Dr. Eldirdieri Salim Ahmed, Department of Microbiology & Parasitology, College of Medicine, University of Juba, for his continuous encouragement and valuable advices during the whole period of the s t u d y . My thanks are due to all those who helped me, Mr. Omer Mohamed Ali, Mr. Yousif Adam Omer, and my great thanks also to Mrs. Sara Mohamed Al Aalim, Mr. Anawar Ahmed, Mr. Al sadig Al bakheet & Mr. Yousif Manoly. My thanks to my colleagues, Mr. Said Ali Mustafa & Mr. Luai Osman Ibrahim, for their help in examination of the blood smears and my thanks also to Mrs. Enaam Husein, Mr. Mamoun Magzoub, Mr. Mohamed Abdel hadi, Mr Khaliefa Al tayeb & Mr Ahmed Baher f o r t h e i r g r e a t s h e l p s . Special thanks should go to Mr. Awad Ahmed Nasr the Head Department of Parasitology & Medical Entomology, Faculty of Medical Laboratory Sciences, University of Khartoum for his encouragement and great help to conduct this study. II LIST OF TABLES Table 1. Comparison between Giemsa , Saponin lyzed and Field’s stained thick 44 blood smear in detecting malaria parasite and in determining the parasite count/µl blood . .. Table 2. Comparison between Giemsa stained-thick blood smears and Giemsa 47 stained-Saponin lyzed venous blood smears for detecting malaria parasites examined by three technicians . Table 3. Comparison between Giemsa and Field stained thick blood smears 49 examined by three technicians in the detection of malaria parasites. Table 4. Comparison of the stain quality of thick & thin blood smears using 51 different Giemsa stain stock solutions . Table 5. Comparison between different concentrations (10%, 15% and 20%) of 53 Giemsa stain working solution in staining thick & thin blood smears . Table 6. Comparison of Giemsa stain working solution for staining blood 55 smears using different diluents . III LIST OF FIGURES Figure1. Distribution of Malaria parasites over the world . .. 7 Figure 2. Life Cycle of Malaria parasites . 11 Figure 3. Transmission of Malaria in the Sudan . .. 33 Figure 4. Age group distribution of the study population . 43 Figure 5. Relationship between detection of malaria parasites according to level 45 of parasiteamia in blood smears stained with different stain . Figure 6. The percentage of distinguishable component of blood smears (stain 57 quality) following different intervals of storage…………………………………. LIST OF APPENDICES IV Appendix 1. Relationship between detection of malaria parasites according to 83 level of parasiteamia in blood smears stained with different stains. Appendix 2. Comparison between the quality of Giemsa stain working 84 solution and time after preparation of the working solution . Appendix 3. Preparation of stock Giemsa stain (Stock I ). 85 Appendix 3a. Preparation of stock Giemsa stain (Stock I I). 86 Appendix 3b. Preparation of stock Giemsa stain (Stock III) . 87 Appendix 4. Preparation of Field’s stain solution A . .. 88 Appendix 4a. Preparation of Field’s stain solution B . .. .. .. .. 89 Appendix 5. Preparation of Saponin lyzing solution. 90 Appendix 6. Preparation of phosphate buffered solution pH 7.2 . 91 Appendix 7. Questionnaire .. .. .. .. .. .. .. .. .. .. 92 Appendix 8. Form used to assess the blood smears of study techniques . 93 Abstract V This study was carried out during the period from July 2002 to September 2003 in order to assess the use of different stains and staining procedures in microscopic diagnosis of malaria. A total of 203 individuals were included in the present study, Field-stained thick blood smear, Giemsa-stained thick and thin blood smears and Saponin lysed Giemsa- stained smears were prepared from each individual. Three different technicians examined stained blood smears independently. The result showed superiority of Giemsa-stained thick blood smears in detecting malaria parasites over others stained smears. Using Giemsa-stained thick blood smears malaria parasites were detected in 31, 32.5, 33 % whereas it was detected in 28.6, 29.5,30%, by Saponin lyzed venous blood smears and only in 26.1, 27.1%, 27.1% of Field stained smears as examined by three technicians. Further, three stocks solutions of Giemsa stain prepared by different methods were evaluated in staining blood smears, the finding showed a comparable result between the three Giemsa stain stock solutions. Unsatisfactory results were obtained when higher concentrations of Giemsa working solutions (15% & 20%) were used for shorter staining period versus convential concentration (10%). Adequate results were obtained by working solutions of Giemsa stain prepared using distilled water instead of phosphate buffered solutions, whereas poor results were obtained when a tap water was used. The study findings showed that it is possible to obtain Giemsa working solution with satisfactory staining properties, which did not decrease during the first 3 hours of preparation. اﻟﺨـــﻼﺻــﺔ VI أﺟﺮﻳﺖ هﺬﻩ اﻟﺪراﺳﺔ ﻓﻲ اﻟﻔﺘﺮة ﻣﻦ ﻳﻮﻟﻴﻮ 2002 إﻟﻰ ﺳﺒﺘﻤﺒﺮ 2003 ﻟﺘﻘﻴﻴﻢ اﺳﺘﺨﺪام أﺻﺒﺎغ و ﻃﺮق ﺻﺒﻎ ﻣﺨﺘﻠﻔﺔ ﻓﻲ ﻣﺠﺎل اﻟﻔﺤﺺ اﻟﻤﺠﻬﺮي ﻟﻄﻔﻴﻠﻴﺎت اﻟﻤﻼرﻳﺎ . أﺧﺬت ﻋﻴﻨﺎت دم ﻣﻦ 203 ﺷﺨﺺ و اﻟﺬﻳﻦ ﻳﻤﺜﻠﻮن ﺷﺮﻳﺤﺔ اﻟﺪراﺳﺔ اﻟﺤﺎﻟﻴﺔ