University of Ghana http://ugspace.ug.edu.gh ASSESSMENT OF SENSITIVITY OF P. FALCIPARrM TO ANTI FOLATE ANTIMALARIALS IN SOUTHERN GHANA BY POLYMERASE CHAIN REACTION (PCR) ASSAY SYSTEMS. A THESIS SUBMITTED BY EPHRAIM ELIKEM MAK-MENSAH IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE MASTER OF PHILOSOPHY DEGREE. Department of Biochemistry ,. Faculty of Science University of Ghana Legon, Accra, Ghana. July 1996 University of Ghana http://ugspace.ug.edu.gh DECLARATION, The experimental work described in this thesis was carried out by me, at the DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF GHANA, LEGON under the supervision of Prof. F.N. Gyang. I declare that this work has not been and is not being submitted for any other degree. Candidate ---------------------------------------- EPHRAIM E. MAK-MENSAH Supervisor PROF. F.N. GYANG. I University of Ghana http://ugspace.ug.edu.gh DEDICATION To my parents MR & MRS FRANCIS K. MAK-MENSAH for their love, care and financial support and especially to my sister AKPENE for her visits. i i ( ( ''v _ . ,J>. )-)(l ~,,1.18R..._ __....-;?--."\./ r University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGMENT I wish to express my profound gratitude and special thanks to Prof. F.N. Gyang, my supervisor, for his priceless guidance and comments throughout this work. My sincere thanks go to the staff of Department of Biochemistry for their interest and encouragement. I am heavily indebted to Senior Medical Officers in charge of Hospi tals wi thout whose approval and cooperation blood samples would not have been obtained for this study. The hospitals are University Hospital, Legon, Korle-Bu Teaching Hospital, Korle-Bu, Keta Government Hospital and St. Anthony's Hospital, Dzodze. The rest are E.P. Church Hospital, Adidome, Catholic Hospital, Bator, Sogakofe Comboni Clinic and Sogakofe Health Centre, Sogakofe and New Hope Clinic, Viepe-Aflao. My special thanks also go to Mr. Kwame Buah for his great assistance towards the completion of this work. I further wish to thank Miss Priscilla Boateng for the significant role she played in typing the manuscript. EPHRAIM ELIKEM MAK-MENSAH. iii r University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS ~ DECLARATION I DEDICATION i i ACKNOWLEDGMENT iii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATION x ABSTRACT xii 1.0 INTRODUCTION AND LITERATURE REVIEW 1 .1 INTRODUCTION '... 1 1 .2 LITERATURE REVIEW.. ................ 5 1.2.1 Epidemiology 5 1 . 2 . 2 Ae t i0 logy. .............. 6 1.2.3 Entomology 8 1.2.4 Life cycle of Plasmodium.................. .. 9 1.2.5 Invasion of erythrocytes by merozoite 12 1.2.6 Modification of merozoites in erythrocytes 13 1.2.7 Clinical presentation of malaria 13 1.2.8 Socio-economic aspects of malaria 16 1.2.9 The control of malaria in Ghana 17 1.2.10 Folate metabolism . 18 1.2.11 Antifolate antimalarials 22 1.2.11.1 Pyrimethamine 23 iv University of Ghana http://ugspace.ug.edu.gh 1 . 2 . 11 . 2 B 1guan ide s. ........................ ...25 1.2.12 Resistance of Plasmodium to antifolates 25 1.2.13 Mechanism of resistance to antifolates 26 1.2.14 Other causes of parasites' resistance to an t if0 1ate s ............ 28 1.2.15 Identification of Plasmodium in blood 29 1 . 2 .16 PCR.......................... 31 1.2.17 P.falciparum DNA isolation. 35 1.2.18 Gel Electrophoresis of PCR products 37 1.2.19 Staining DNA in agarose gel.. 39 1.2.20 Measurement of DNA size by Electrophoresis 39 2.0 MATERIALS AND METHODS 2.1 MATERIALS . ... 41 2.1.1 Study areas. .......................... 43 2.1.2 Sample collection.......................... .. 47 2.2 METHODS.............. 47 2.2.1 Isolation of P.falciparum DNA 47 2.2.2 Spectrophotometric determination of DNA concentrations .. 48 2.2.3 PCR assay........................ ......... ...48 2.2.4 Optimization of PCR assay 51 2.2.4.1 Units of Tag DNA polymerase................. 51 2.2.4.2 Cycling parameters............. ..52 2.2.5 Gel Electrophoresis of PCR products 52 2.2.6 Photographing the gel . • •. 54 2.2.7 Measurement of DNA size . • •.. 54 3.0 RESULTS v University of Ghana http://ugspace.ug.edu.gh 3.1 Isolation of Pi Ee Lo ipexum DNA 55 3.2 PCR assay 55 3.3 Gel Electrophoresis of PCR products 61 3.4 Measurement of DNA length 64 4.0 DISCUSSION............................ 65 5.0 CONCLUSION 73 6 . 0 REFERENCES. .. .. .. 75 7.0 APPENDIX A. .. 88 7.0.1 Solutions and buffers . ...... 88 7.1 APPENDIX B...................................... 89 7 . 1 . 1 Ca Icu Iat ions. .. 89 vi University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES TABLE 1. - Sampling sites and their corresponding number of samples . • .•.....••••....•... 43 TABLE 2. - Point mutations and antifolate susceptibility in P. falciparum .. 50 TABLE 3. - Purity & Quantity of P.falciparum DNA from the communi ties . .. 58 TABLE 4. - Assessment of sens it 1vi ty of P. falciparum to pyrimethamine 59 TABLE 5. - Assessment of sens it ivi ty of P. falciparum to cycloguanil . 60 TABLE 6. - Nested peR . .. .. 61 TABLE 7. - DNA fragment lengths with their corrected reciprocal mobi 1i ties. ... 91 vii University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES ~ Fig.1 Current worldwide distribution of malaria 3 Fig.2 Worst case scenario for malaria mortality 4 Fig.3 Life cycle of Human Plasmodia ..•. ..11 Fig.4 Chemical structure of the folate molecule 19 Fig.5 The de novo synthesis of tetrahydrofolate in Plasmodium falciparum .•• ....... 20 Fig.6 Folate-dependent reactions in Plasmodium falciparum. •• • •..••• ....... 21 Fig.? Structures and common formulations of pyrimethamine and proguani 1 "'-. .. 24 Fig.S DNA sequence and deduced proteln sequence of the DHFR gene of paras ites and E. coli... • . • . • • • • • • . • • .27 Fig.9 Incidence of malaria by districts in Volta Region 1990-199"3' 44 Fig.10 - Map of Ghana showing three zones 45 Fig.11 - Map of Volta Region showing sampling sites 46 Fig.12 - Strategy for detect ing P. faIciparum DHFR mutat ions by mutation specific PCR amplification 50 Fig.13 - Sensitivity pattern of P. Le Lc ip ar am from the co- mmuni ties . ..56 Fig.14 - Diagram of PCR products of P. falciparum DNA and DIA3/SPl primer pair specific for Ser-10S on gel .. 62 viii University of Ghana http://ugspace.ug.edu.gh Fig.1S - Diagram of peR products of P.falciparum DNA and DIA 12/SPI primer pair specific for Asn-l08 on ge 1. .. 62. Fig.16 - A plot of molecular weight vrs reciprocal of mobility for restriction fragment of phage ¢ x 174 RFI DNA. ................ 63 ix University of Ghana http://ugspace.ug.edu.gh ABBREVIATION A (A260) absorbance (absorbance at 260nm) bp base pair Cyc-R cycloguanil-resistant parasites Cyc-S cycloguanll-sensltive parasites dATP deoxyadenosine tri-phosphate dCTP deoxycytldine tri-phosphate dGTP deoxyguanosine triphosphate dTTP deoxythymidine tri-phosphate dTMP deoxythymidine mono-phosphate dNTPs mixed deoxy nucleotide triphosphate (dATP, dCTP, dGTP, and dTTP) dUMP deoxyuridine mono-phosphate DNA Deoxyribonucleic acid DHFR Dihydrofolate-reductase Fig. Figure M molar =mol/litre = mol/dm3 Min minute mM mi llimolar mg milligramme ml millilitre PCR Polymerase Chain Reaction PNS PCR negative samples x University of Ghana http://ugspace.ug.edu.gh PPS peR positive samples Pyr-R Pyrimethamine-resistant parasites Pyr-S Pyrimethamine-sensitive parasites s Seconds TOR Tropical Diseases Research ug microgramme ul microliter uv ultraviolet xi University of Ghana http://ugspace.ug.edu.gh ABSTRACT Drug treatment of malaria is being hampered by the emergence of drug resistant strains of malaria parasites. Rapid methods for monitoring patients' response to treatment, applicable to field conditions, would aid management and control of drug resistant P. falciparum infect ions. In this study, mutation specific polymerase chain reaction assays using 3'-mismatched oligonucleotide primers which annealed to the wild or mutated parasites gene encoding dihydrofolate reductase-thymidylate synthase (DHFR-TS) were used to survey P.falciparum strains in two Regions of Southern Ghana (Volta and Greater Accra Regions). Mutations were identified directly from blood samples obtained from patients attending Out Patients Departments in Hospitals. Amplified P.falciparum DHFR-TS nucleotide sequences of 337bps were obtained when reaction proQucts were analyzed by electrophoresis. Of 151 smear positive samples analysed, 13 (S.6%) contained the Asn-10S codon AAC that confers pyrimethamine resistance, 133 (SS%) samples contained only the wild type Ser-10S codon AGC and none contained the Val-16 codon GTA found in cycloguanil-resistant pyrimethamine sensitive parasites. Out of the 13 pyrimethamine resistant cases, 10 were found in samples obtained from the Volta Region while the rest (3) were from Korle-Bu Teaching Hospital in the Greater Accra Region. No PCR product was found in 5 (3.3%) of the 151 samples. After second (nested) PCR, only one sample showed amplified product: contamination was negligible. xii University of Ghana http://ugspace.ug.edu.gh peR amplification of the DHFR-TS presents a rapid alternative to in vitro drug testing for monitoring the resistance of P.falciparum to antifolate antimalarials. xi ii University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 1.0 INTRODUCTION AND LITERATURE REVIEW 1.1 INTROPUCTION Malaria continues to -be the infectious disease with the greatest impact on mankind. It is responsible for many health problems such as fever, headache and muscular pains among others. Malaria is often described as a great imitator of other diseases, sharing various characteristics with other childhood illnesses such as influenza, tuberculosis, typhoid, brucellosis and urinary tract infection. Even more confusing, it may coexist with other diseases (Commey, 1989). Besides, it is the leading cause of morbidity and mortality in Ghana (Ahmed, 1989 & 1992). Chemotherapy is essential in malaria management for as much as it reduces mortality and morbidity by eliminating malaria parasites from human hosts.