UNIVERSITY OF GHANA LIBRARY QL368. H33 B63 blthr C.l G3623304 T h # B « im # L ib r a r y 3 0692 1078 6004 9 University of Ghana http://ugspace.ug.edu.gh M O L E C U L A R C H A R A C T E R IS A T IO N A N D IM M U N O - E P ID E M IO L O G Y O F PLASMODIUM FALCIPARUM IN F E C T IO N IN S C H O O L C H IL D R E N A T D O D O W A B Y JO H N S O N N Y A R K O B O A M P O N G A T H E S IS S U B M IT T E D T O T H E U N IV E R S IT Y O F G H A N A IN P A R T IA L F U L F IL M E N T O F T H E R E Q U IR E M E N T F O R T H E D E G R E E O F M A S T E R O F P H IL O S O P H Y IN Z O O L O G Y D E P A R T M E N T O F Z O O L O G Y U N IV E R S IT Y O F G H A N A L E G O N , G H A N A M A R C H , 1999 University of Ghana http://ugspace.ug.edu.gh S U P E R V IS O R S Snr Res Fellow Head. Immunology Unit N M IM R . UG. Leyon. I)i M U Wilson Snr Res Fellow Head, Parasitologs Unit N M IM R UG Leuon Dr J .A .L Kurtzhals Visiting Res Fellow Immunology Unit N M IM R . ~ U G Leizon Lecturer Zoology Department UG Leuon ii University of Ghana http://ugspace.ug.edu.gh DECLARATION This is to certify that this thesis has not been submitted for a degree to any other University. It is entirely my own work and all help has been duly acknowledged. A j t l _____ ' - ~ r r r ............. JO H N SO N N Y A R K O BO AM PO N G I I I University of Ghana http://ugspace.ug.edu.gh DEDICATION To my Uncle Kwaku Boampong and his family. iv University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I wish to express my sincere gratitude and deep appreciation to all individuals and institutions that, in diverse ways assisted me to successfully complete this work. I am especially thankful to Professor I .K Nkrumah, Director o f N M IM R for granting me permission to conduct research work at the institute and indeed the entire staff o f the institute for their warm reception, kindness and help. The work could certainly not have been completed without the patience, expert guidance, advice, constructive suggestions and criticisms from Drs. B.D . Akanmori. Head o f Immunology Unit, .I.A.L. kurtzhals, visiting Research Fellow and M .D . W ilson. Head o f Parasitology unit, all at the Noguchi Memorial Institute for Medical Research (N M IM R ). Equallv invaluable to the successful completion of the work was the experienced supervision o f Dr Edoh at Zoology Department. University o f Ghana-Legon. I am indeed very much indebted to them. I am also indebted to Messrs Ben Gyan. M Ofori. M . Osei- Atweneboana. A Hammond. Mrs Owusu. Ms V Okyere. Mrs B. Ogoe. A. Richardson and A Grace lor helping me anytime I encountered problems. The Danish International Development Agency (D A N ID A ) deserves to be thanked for providing lunds through its F.NRI'.C'A programme for this expensive work as part ol the Accra-Copenhagen Research Link I wish to thank my Uncle M r kwaku Boampong. my dearest wife Ms Georgina I hompson as well as Iriends lor their encouragement, concern and companionship University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENT T IT L E P A G E D EC LA R A T IO N D ED IC A T IO N A C K N O W L ED G EM EN T T A B L E O F C O N T EN T L IS T O F T A B L E S L IS T O F F IG U R E S A P P E N D IC E S A B S T R A C T C H A PT E R O N E IN T RO D UCT IO N 1 1 Rationale and objectives 1 2 Specific objectives C H A PT E R TW O L IT E R A T U R E R E V IE W 2 I Classification o f Plasmodium species 2.2 The life cycle of Plasmodium species 2.2 .1 Schizogony 2.2 I I Exo-Erythrocytic stages 2.2 .1 2 Erythrocytic stage University of Ghana http://ugspace.ug.edu.gh 2.2.3 Sporogony lu 2.3 Malaria in Ghana 10 2.5 Genetics of Plasmodium falciparum 12 2.6 Merozoite surface proiein (M S P 1) 14 2.6.1 The Structure o f/ ’, falciparum M S P I gene 15 2.7 Host-Parasite interaction 18 2.7.1 Natural or innate immunity 18 2.7.2 Acquired Immunity 18 2.8 Immuno-epidemiology of P falciparum malaria 21 2.9 Polymerase chain reaction (PC R ) 23 2.10 Analysis of PCR products by electrophoresis 27 2.10.1 Agarose gel electrophoresis 27 2.10.2 Polyacylamide gel electrophoresis (P A G E ) 28 2.10.3 Discontinuous non-denaturing polyacylamide 28 2.11 Enzyme-linked immunosorbent assa\ ( I -1,1S \ ) 20 C H A PT ER THR1 P. M A T E R IA L S AND M l 11IODS 3 1 3.1 Study area 31 3.2 Study population 32 3.3 Ethical Consideration 32 3.4 Study Design 33 3.4.1 Sample Collection 34 VII University of Ghana http://ugspace.ug.edu.gh 3.5 Reagents 34 3.5.1 Enzymes 34 3.5.2 Deoxyribonucleic triphosphate (dN 1 Ps) 34 3.5.3 Oligonucleotide primers 35 3.5.4 DN A molecular weight marker 35 3.6 Standard solutions 36 3.7 Isolation o f parasite D NA from blood spotted filter paper 36 3.8 PC R amplification 37 3.9 Agarose gel electrophoresis 38 3.9.1 Estimation o f DNA fragment sizes 39 3.10 Discontinuous non-denaturing pol> acylamide gel electrophoresis 39 3.10.1 Detection of DNA products using silver staining electrophoresis 40 3.11 Sensitivity o f PC R assa\ 40 3.12 Enzyme-linked immunosorbent assa\ (I-.LISA) 42 C H A PT E R FO U R R E S U L T S 44 4 1 Sensitivity o f PCR for parasite detection 44 4.2 Comparison of P falciparum detection by PCR with microscopic examination 44 4.3 A lle lic type(s) in three consecutive asymptomatic, symtomatic and asymptomatic cases ol malaria. 45 4.4 A lle lic type(s) in three consecutive asymtomatic cases 47 University of Ghana http://ugspace.ug.edu.gh 4.5 Parasitacmia levels during symplomalic and symptomatic cases 48 4.6 An ti-M SPI immunoglobulin G (IgG ) responses before and after 48 malaria attack CH A P I'ER F IV E D ISC U S S IO N 62 5.1 Methodology 62 5.2 A lle lic forms 63 5.3 Seasonal variation 65 5.4 Association of clinical malaria with new alleles 65 5.5 Synchronization of high parasitaemia with malaria 66 5.6 An ti-M SPI antibod) responses 67 CHA IM ER S IX C O N C H 'S IO N AND R EC O M M EN D A T IO N S ^0 6.1 Conclusion 70 6.2 Recommendations 70 R E F E R E N C E S 7[ University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES T A B L E T IT L E 1 DNA sequences o f oligonucleotide primers used for amplification o f allele o f MSP1 gene 2 Determination o f percentage parasitaemia by microscopy at 100X magnification in I I fields o f a single blood film 3 Relative sensitivity o f PC R to Microscopy for detection o f P. falciparum in blood samples. 4 Comparison o f M SP 1 fragment sizes from three consecutive asymptomatic, symptomatic and asymptomatic samples and their corresponding parasite densities during dry and wet seasons 5 Comparison o f molecular sizes M SP I allelic types for blood samples taken in three consecutive months from asymptomatic indi\ iduals and their corresponding parasite densities P A G E 35 50 52 53 6 Anti-M SPI antibodies in school children before and after malaria attack 56 University of Ghana http://ugspace.ug.edu.gh LIST o r F IGURES F IG U R E I i 4 5 6 1 T IT L E Schematic representation o f the life cycle of malaria parasites in human and mosquito hosts A simplified scheme illustrating theM SP I gene o f Plasmodium lalapanm i. A schematic representation o f PC R amplification process. Sensitivity o f PC R for detection of blood stage malaria parasites An illustration of the diversity o f M SP 1 allelic types found at Dodowa The distribution of M SP I variants in patients before and after malaria attack Ethidium bromide stained 1 5°o agarose gel o f P. falciparum showing M SP 1 allelic types o f symptomatic cases An illustration of MSP1 allelic type in asymptomatic subject during three consecutive samplings \i P A G E 7 17 26 ^7 ^8 59 60 University of Ghana http://ugspace.ug.edu.gh A P P E N D IC E S A P P E N D IX T IT L E P A G E 1 Standard solution 88 2 A graph of Log molecular weight 92 markers against distance travelled by the DNA fragment XII University of Ghana http://ugspace.ug.edu.gh \ H S T R A C T A longitudinal immuno-epidcmiological study was conducted between April 1994 and August 1995 among Ghanaian children within the ages o f 3-15 years, living in Dodowa, a stable malaria endemic area. A polymerase chain reaction (P C R ) typing technique based on the amplification o f polymorphic region o f merozoite surface protein 1 (M S P1 ) o f Plasmodium falciparum gene, was used to characterise parasites contained in blood spotted filter paper. The PCR assay detected as low as 2.2 parasites/^! o f blood and revealed 35 MSP1 seasonally variable allelic forms in the children. Clinical malaria could not be attributed to any specific allelic tvpe, and the acquisition o f new allelic type was not necessarily associated with clinical malaria. Malaria episode was synchronised with significant increase in parasitaemia. Also anti-M SPl|y antibodies were measured using Enzyme-linked immunosorbent assay (EL.1SA) from blood samples collected from 27 o f these children before and after malaria attack, \niibody responses to the C'-terminal o f l9kDa fragment o f Plasmodium falciparum before malaria attack showed 14 children as positive and 13 negati\e. Nine of the children were within the ages o f 3-4 years whereas eighteen were 5 years and above. Four children out of the nine aged 3-4 years were negative before malaria attack but three showed negative to positi\e sero-conversion. In contrast 2 out o f 9 children who were positive before malaria attack showed positive to negative sero-conversion. M i l University of Ghana http://ugspace.ug.edu.gh C H A P T E R O N E IN T R O D U C T IO N Malaria is a mosquito-borne protozoal disease caused by species o f the genus Plasmodium. It is characterised by acute febrile illness which may be expressed as periodic paroxysms occurring every' 48 or 72 hours with afebrile and relatively asymptomatic intervals and the tendency to recrudesce or relapse over a period o f months to many years (G illes and Warrel. 1993). Other major symptoms o f malaria are rigors/chills, vomiting, convulsion, headache, drowsiness and muscle- skeletal pain. It is a significant cause o f abortion, stillbirth, child mortality, low birth weight, death in pregnant women, impaired growth in children and loss ol productive activity in adults (TD R , 1987). Malaria is widespread and endemic throughout many parts ol the world, especially the tropics and subtropical regions, ll is estimated to kill between 1.5 and 2 7 million people every year. Another 300 to 500 million people have the disease and one third of all mankind live in zone: where they are exposed to the risk o f infection (Butler. 1997). The vast majority of malaria deaths occur among young children in Africa, especially in remote rural areas with poor access to health services. Outside tropical Africa, deaths from malaria occur principally among non-immune people who become infected with P falciparum in areas where appropriate diagnosis and treatment are not av ailable ( I heander, l c>9]) 1 he enormous number of liv es and labour lost together with the cost of treatment of patients exerts a negative impact on development and makes malaria a major economic burden ( 11)R, 1993). Chemotherapy, hitherto the most University of Ghana http://ugspace.ug.edu.gh effective control method has become less effective as a result o f the development of drug resistant malaria parasites, in addition to insecticide resistance in the anophelinc vectors (G illes and Warrel, 1993). This has led to a focus on anti­ malaria vaccine development as an additional tool for the control o f the disease. The four parasite species that infect man are P. vivax, P malarias, P ovals and P falciparum , the latter being by far the most lethal. Plasmodium vivax has the widest geographical range. It is prevalent in the tropic and subtropical regions and is also found in some temperate zones. Plasmodium malarias is patchily distributed over tropical Africa, Eastern Asia, Oceania and Amazon area. Plasmodium falciparum is the commonest malaria parasite in tropics and subtropics and is predominant over the same range as P. malarias It is mainly found in East and West Africa. Guyana and parts o f India. Plasmodium ovals is main!) found in tropical regions such as West Pacific regions, southern China. Burma and South East Asia (W l l(). 1993). Traditional malaria diagnosis is based on the standard microscopical examination of Giemsa stained blood films to identify the parasite Differential diagnosis is made possible by the morphology o f the parasites but accurate species differentiation may be difficult especially in patients with very low circulating parasitaemia and mixed infection o f/ ’, ovale and P vivax (M ilne si a!.. 1994). In malaria endemic areas, varying proportions of the population continuously earn1 low grade and asymptomatic parasitaemia but periodically show clinical episode associated with sharp rise in parasite densities (Contamin si at 1996). 2 University of Ghana http://ugspace.ug.edu.gh The development of the PCR , which is the enzymatic amplification of parasite specific D N A sequences (Saiki el a!., 1988) has made possible the detection of low parasitaemia in infected humans (Snounou el a i, 1993). Increased molecular characterisation o f a number of functional P. falciparum genes and gene families, has placed at the disposable o f researchers a suitable means o f typing the parasite. One such source o f variation suitable for typing P falciparum strains is the gene that codes for the merozoite surface protein M S P 1 . Experimental infections in humans have shown that the immunity raised to one strain of P falciparum is largely inefficient against challenge with heterologous strains (Jeffery. 1996). This raises the question whether only increased populations of parasitaemia or just a new virulent strain o f the parasite irrespective o f the density that causes clinical malaria. The erythrocytic stage o f/ ’ falciparum is responsible for the clinical malaria and as such antigen associated with this stage may be o f importance in the development of protective immunity o f the disease (G illes and Warrel. 1993). One such well characterised antigen is the /' falciparum merozoite surface protein 1 (P fM S P l) that remains attached to the merozoite during erythrocytic invasion and is also expressed by the parasite during carl)’ ring stages (Holder cl al.. 1987). Antibodies against this fragment may block merozoite invasion o f erythrocvtcs and also inhibit multiplication inside the erythrocytes (Blackman and Holder. y 1992). I he P IM S IM gene has been sequenced and it is now known to consist o f 1 ^ 3 University of Ghana http://ugspace.ug.edu.gh blocks that is either highly conserved, semiconserved or variable. The polymorphic region at the 5’ end o f the gene in block 2 varies extensively in number and in sequence detail o f repeats. This provides ideal basis to discriminate strains o f the parasite between isolates (Tanabc el a l , 1987). 1'he polymerase chain reaction is now used to distinguish P. falciparum strains using primer sequences that Hank the polymorphic region o f MSP1 gene (Conway and M cBride, 1991; Ranford-Cartwright el at, 1993). 1.1 Rationale and objectives The interaction between the human host and infecting malaria parasites is prerequisite to an understanding o f the mechanisms underlying the pathogenesis and acquisition o f immunity against malaria infection. Studies have shown that immunitv to malaria parasites has marked strain specific component. Different parasite strains have also been shown to vary in their clinical and pathogenic properties as well as their susceptibility to various drugs (Snew in el a l , 1991; Babiker el a l . 1995; Conway el a l , 1 991). The acquisition o f immunity to malaria is not rapid but rather a slow process (Theander, 1991) and mas be due to lack of exposure to a wide range of parasite strains. Another possible phenomenon that might influence immune status is the carriage ol low-grade parasitaemia in the blood of indiv iduals, which may induce clinical protection. 1 Ins has thereiore nccesMlated an investigation into clinical protection Irom malaria and to provide useful information for understanding o f epidemiolony of the disease condition. 4 University of Ghana http://ugspace.ug.edu.gh 1.2 Specific objectives 1. To determine the sensitivity of the polymerase chain reaction (P C R ) assay for parasite detection in blood collected on filter paper. 2. To use PCR to discriminate between P lalcipwum strains and to test the hypothesis that a shift from asymptomatic to symptomatic clinical malaria is caused by a new strain. 3. To investigate a possible correlation between antibody responses to M S P l iy and protection from clinical malaria. 5 University of Ghana http://ugspace.ug.edu.gh C H A P T E R T W O L IT E R A T U R E R E V I E W 2.1 C lassification of Plasmodium species Plasmodium species are unicellular, eukaryolic and parasitic organisms belonging to the phylum Protozoa, subphylum Apicomplexa, class Sporozoa, subclass Coccidia, Order Coccidiida, suborder Haemosporina. family Plasmodiidae and genus Plasmodium and Laverania (Manson-Bahr and Apted, 1987). There are four species that infect man namely, P vivax, P malarias, P. ovale and P falciparum. However this zoological classification of Plasmodium species is not universally accepted especially with regards to the differences of opinion in the taxonomic position of the parasite causing falciparum malaria. Some authors maintain that it belongs to a separate genus Laverania (G illes and Warrel. 1993) as a result o f its crecentic shape and lengths development. Others arc o f the opinion that the rejection o f the familiar name Plasmodium falciparum might be confusing and since the use o f this well known name is slill laxonomicalK permissible, it should be retained (G illes and VVarrel. 1993). 2.2 The life cycle of Plasmodium species The life cycle of all Plasmodium species infecting humans is complex and largely similar. It consists of an endogenous asexual phase (schizogony) in the human host and exogenous sexual phase (spomgony) in the female anopheline mosquito as illustrated in Figure I . 6 University of Ghana http://ugspace.ug.edu.gh Figure 1. Schematic representation o f the life cycle o f malaria parasites in human and mosquito host (reproduced from Good el a!. , 1988) &poro/o f. i * * ••««•»» : • • Jjuniii;. — C IS . . • • //"oc ,//„a a ® O f e r Jl / ' Garnctcx ylct 7 University of Ghana http://ugspace.ug.edu.gh 2.2.1 Schizogony Viable sporozoiles are inoculated into the bloodstream o f humans by an infected female Anopheles mosquito when it is taking a blood meal. W ithin thirty minutes the sporozoiles disappear from circulation. Although phagocytcs destroy most sporozoites a few enter the parenchymal cells (hepatocytes) o f the liver directly or via the Kuffer cells (G illes and Warrel, 1993). The asexual phase (schizogony) in humans involves two stages; the exo- and erythrocytic stages. 2.2.1.1 Exo-Erythrocytic stages The invasion o f hepatocytes by sporozoites leads to development and multiplication (schizogony). A fully matured exo-cnthrocytic schizont. which contains 10,000-30,000 merozoiles, ruptures and releases its contents into the bloodstream within the period of one lo three weeks after sporozoite inoculation. Some merozoiles of P vivas and /' ovale remain in hepatocytes. forming hypnozoites. which may repeat the process o f schizogony thus causing relapses weeks to years later. Plasmodium falciparum and P malariae howe\er do not have a hypnozoite stage (G illes and Warrel. 1 993). 2.2.1.2 E ry th ro cytic stage Most o f the liberated merozoiles invade ihe erythrocytes present in the sinusoids of the liver, but some are phagoeytosed The rest enter into circulation and invade other erythrocytes as described by Holder el a l (1987); Blackman and Holder (1992). Once inside the erythrocytes they form trophozoites which initially appea l / * 4% as characteristic ‘rings'. Ihe trophozoites then mature and undergo anotljtf8 K University of Ghana http://ugspace.ug.edu.gh asexual multiplication to produce mature schizonts, which contains 2000 new merozoites. The erythrocytes rupture releasing the merozoites which then invade other erythrocvtes. This erythrocytic cycle o f schizogom is repeated over and over again, leading to a progressive increase o f parasitaemia until the process is slowed down by the immune response (G illes and Warrel, 1993). Plasmodium falciparum like P. vivax and P. ovale complete the erythrocytic cycle within 48 hours, whereas the cycle o f P malariae takes 72 hours. This tends to be synchronous, producing periodic fevers with successive releases o f the merozoites. Parasite levels in the peripheral circulation are known to vary during the erythrocytic cycle. In P. falciparum only trophozoites, which are younger than 18-21 hours old, are seen in peripheral circulation. The reason for this is sequestration o f the maturing stages in the inflamed endothelia of capillaries and vessels. After several c\cles the end o f the schizogonic periodicity, sexually differentiated forms, gametocytes emerge as illustrated in Figure I In synchronous infections o f some species o f Plasmodium, gametocytes mature at night indicating an adaptation o f the parasite to nocturnal feeding habits of female anopheline mosquitoes (G illes and Warrel. 1993). Clinical malaria follows the pattern o f distribution o f miraer\throcytic stages in humans and presents a broad range of symptoms. These include repeated generalised convulsions, normocytic anaemia with haemocnt < 15%, impairment ol consciousness or unrousable coma, jaundice, hypogl\caemia. weakness, fever, rigor' chills, headache and fever. 9 University of Ghana http://ugspace.ug.edu.gh 2.2.3 Sporogony When a female Anopheles mosquito ingests the blood o f a human host, which has circulating malaria parasites, the asexual parasites are digested together with the blood cells, but gamctocytcs undergo further development. The male gamelocytes (microgametocytes) exllagellate mates with female gametocytes (macrogametocytes) to form zygotes in the mosquito midgut. The zygote then transforms into a motile ookinete, which penetrates the intestinal walls o f the midgut where it lodges to form the oocyst. It then undergoes sporogony and bursts after maturation releasing thousands o f spindle-shaped sporozoites into the haemocoele. The sporozoites migrate to the salivary glands from which they are inoculated into humans to complete the cycle 2.3 M a la r ia in Ghana Malaria still poses a major public health problem in Ghana and tops the ten most common causes of morbidity reported by Centre for Health Information Management (C H IM ) from 1990-1994 (M O II 1994). It is also the commonest cause of out-patient disease (O PD ) conditions. A ll Ghanaians are at risk o f beum inlecled but it mostly allects children under 5 years, pregnant women and non- immune visitors. It is the disease with high mortality in late infancy and early childhood (M O II 1987).The disease occurs throughout the year but more prevalent during and alter the rains. 1 he disease prevalence varies between /ones being highest in transitional savannah /ones (A lan cl a l , 1992). 10 University of Ghana http://ugspace.ug.edu.gh Plasmodium falciparum is the most common parasite in Ghana (Coulbourne and Wright, 1955). It is responsible for more than 90% o f malaria cases, followed by P. malariae which can sometimes cause more than 1 0% o f total infections in the dry seasons, especially in the coastal savannah area. Plasmodium ovate, which is rare in Ghana, has a patchy distribution and accounts for 0-15% o f infections (M OH , 1994). Mixed infections o f P. falciparum and P malariae are also generally high in dry seasons (Afari d a!., 1992). Plasmodium vivax, which hitherto has not been recorded in Ghana, was recently identified in blood sample from an American who visited Ghana (Kain el a/., 1991). Binka el al. (1994) have reported that Plasmodium falciparum constitutes 1.4% o f Plasmodium infections at Kassena-Nankani District of Upper Cast Region. Malaria transmission also varies from region to region with intense transmission rales being recorded during the season when mosquito vectors are abundant. However, even within this pattern, different transmission rates exist due to a combination o f factors, mainly environmental conditions that inlluence the distribution o f vectors and thus die disease. I he distribution of malaria within human populations in Ghana is closely linked to site-specific characteristics o f vector populations. These include abundance, susceptibility to infection, longevity and their contact with human. This is complemented by human habits that actively promote man-mosquito contact (Al'ari el al. 1994). Studies conducted in Ghana have revealed that the principal vectors o f malancr’aj^'.-' V ' " 11 University of Ghana http://ugspace.ug.edu.gh member species of the An. gambiae complex and An. funestus (A fari el a l, 1992). There are six recognised species o f the An. gambiae complex, namely, An. gambiae sensu strict 11, An. arabiensis, An. melas, An. quadrannulas An merus and An. bwanbav Appawu el al. (1994) have reported only three ol the six members of the An. gambiae complex; An. gambiae sensu strictu. An. arabiensis and An. melas occur in varied eco-epidemiological zones in Ghana. Anopheles arabiensis is found in the arid north o f the country and in lower densities in the coastal mangrove and swampy areas where An gambiae ss predominates. Anopheles melas plays the predominant role in malaria transmission in the coastal areas during dry season but An. gambiae ss assumes this position during the rains. The principal vector populations have the ability to maintain the transmission of different Plasmodium parasites and are able to survive in the rapidly increasing environmental changes such as creation o f stagnant water bodies in the country, thus making strategic control measures ineffective 2.5 Gcnctics of Plasmodium falciparum Chromosomes of intraerythroeytic stages of/* falciparum ha\e been determined in relation to the number of kinetochores by electron microscopy and by pulse field gel electrophoresis (PFGF ) of intact chromosomes (Prensier and Slomianny, 1986). It has been revealed that there are 14 chromosomes that range in size from 600kb to 3.5Mb. The chromosomes undergo re-assortment and crossing-over during cross fertilisation between genetically diflerent strains which co-infect the mosquito, resulting in high frequencies o f recombinant progem (Conway and 12 University of Ghana http://ugspace.ug.edu.gh McBride, 1991; Lanzer el al., 1994). A ll the chromosomes except two have been assigned 25 genetic markers (Kemp el a l. 1987). The genes of most /' falciparum antigens that have been identified so far, are located at subtelomeric regions o f chromosomes where they are prone to recombination and deletion (Lanzer el a l, 1994). Most o f these genes contain blocks o f tandemly repeated coding sequences (Kemp el al., 1987). It has been speculated that the major contributor to chromosome size polymorphism in malaria parasites is the difference in the amount o f repetitive D N A present in the genome o f an individual parasite. Lack o f strong selective pressure may permit dramatic fluctuation in the abundance o f repetitive D NA with consequential chromosome size polymorphism between parasites (Conway and McBride. 1991 ). Size polymorphisms in P falciparum do not involve intcrchromosomal exchange of large segments o f DNA In contrast. Trypanosoma hrucci genes for variant surface glycoprotein (V SG ) of the surface coat o f the parasite undergo rearrangement that regulate (heir expression and transpose large segments o f DNA from one chromosome to the other. I'his in '/’. hrucci generates marked chromosome size differences thereby displaying remarkable heterogeneity (Van der I’ loeg and Cornelissen. 1984). I he diversity o ( P. falciparum has been well demonstrated in variant forms o f antigens. proteins. enzymes, resistance/susceptibility to antimalanal drugs and sequences of many genes (Wataya el al., I 993; McBride el al 1982 l-'enlon cl a l., 1 985; Sanderson cl al.. 1981; I irasophon el al., 1994; Peters, 1987 and Lockver and Schwarz, 1987). I here is also evidence that some variants o f these characters occur at different 13 University of Ghana http://ugspace.ug.edu.gh IVcquencics in different geographical areas (W a llik c r el al., 1987). which can be discriminated using one or more genetic marker(s) that represent(s) allelic forms of each respective gene. One such antigen, which exhibits allelic polymorphism and also varies in frequencies in different geographical areas, is the P. falciparum merozoite surface protein 1 (P fM S P I) . The antigen M SP1 . also identical with the precursor to the major merozoite surface antigen (PMM SA/gp 195) and merozoite surface protein 1 (M SA-1) (Peterson cl al. 1988; M cBride cl a!.. 1985; Holder and Freeman, 1982), is encoded by a single locus gene located on chromosome 9 (Kemp el al., 1987; Walker-Jonah el a l. 1992; Anders el a l. 1993). 2.6. Merozoite surface protein 1 (M S P1 ) The merozoite surfacc is made up of multiple proteins seemingly 'fuzzy' or amorphous but organised ‘spikes' of 20-25 nm projections (Galinski and Barnwell. 1996). The MSP1 which is maj or surlace antigen o f ! ’ falciparum merozoites is a glycoprotein of 185-205 k l)a molecular weight (Holder and Freeman. 1982; Holder a al.. 1987). ll is derived from a high molecular weight precursor molecule identical with gp 190, gp 195 and P.MM SA . synthesised during schizogony and expressed on the suiTaee ol intraenihrocytie parasites (Holder and 1 reeman. 1984) Plasmodium falciparum MSP1 (P fM S P l) is proleolvticallv processed into Iragments ol 83, 42, 38, 28-30 and 1° k l ) at the end ol schizogonv. lust prior to the i el ease ol merozoites and can be lound on the surlaces ol mature merozoites However only t -terminal fragment of 19 K l)a (M S P lm ) remains on tlic 14 University of Ghana http://ugspace.ug.edu.gh merozoile surface during invasion of a new erythrocyte (Holder el al.. 1987; Holder and Freeman 1984; Blackman cl al., 1990; Blackman and Holder. 1992). When the parasite invaginates into a new erythrocyte, it forms parasitophorous \acuoles that separate it from the surrounding cytoplasm. The role o f M S I’ l is to recognise and aid adherence to the membranes o f erythrocytes (Howard and Pasloske. 1993). The proteins produced by MSP1 gene are also polymorphic in natural populations o f P falciparum The reactivities o f MSP1 strain specific monoclonal antibodies have shown a large number o f this molecule, each being encoded by different allele o f the MSP1 gene (Holder and Freeman. 1982). 2.6.1 The structure of P. falciparum M S IM gene The P fM S P l gene has been sequenced and shown to consist o f 17 blocks that are cither highly conserved, semi-conserved or variable (Figure 2). In five ol' these blocks ( I . 3. 5, 12. and 17) the I.)NA sequences are conserved in all isolates Seven regions o f the gene show extensive polymorphism (blocks 2. 4. 6. S. 10. 14. and 16), whilst the remaining parts (block 7. 9. 11, 13 and I 5) appear to consist of conserved sequences with patches of non-homologous sequence (Tanabc el al.. 1987). Surprisingly, the variable and semi-conserved regions (ie 2. 4, 6 7, 8. 9. 10, 11, 13. 14, IS , 16) exist only in two dimorphic forms. I'hc only exception to this is the polymorphic tripeptide region at the 5' end o f the gene in block 2. Hie allele K1 and MAD20 are considered as Ihe allelic prototypes from which other allelic lorms probably have been generated by intragenic recombination at the 5' 15 University of Ghana http://ugspace.ug.edu.gh end o f the gene (Tanabc et al., 1987). Using PC R analysis Certa el al. (1987) showed that a third polymorphic form o f the MSP1 gene, called R033 lacks the N- terminal tripeptide repeats. This study examined the polymorphic region (block 2) using two sets o f primers (outer and inner primers) 16 University of Ghana http://ugspace.ug.edu.gh Figure 2: A simplified scheme illustrating the MSP1 gene o f P. falciparum. 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 O , — ► O j ► -4---- N, N 2 Key A B C D E A= Conserved block B = Repetitive block 2 C = Semi-conserved block D and E = ‘Dimorphic’ non-repetitive blocks The M SP I gene is divided inlo blocks on llic basis ol' polymorphism. Block 2 contains highly polymorphic iripeplidc repeats The posilions of llic primers used for PCR in this study arc shown by arrows (sec above). The outer primers arc denoted O, and 0 : and the nested primers arc denoted N, and N : (modiHcd from Ranford-Carlwright el al.. 1993) I 7 University of Ghana http://ugspace.ug.edu.gh 2.7 Host-l’arasitc interaction In malaria endemic areas not all individuals are equally susceptible to infection by malaria parasites, and even if an infection is established, the associated morbidity is dependent on a number of intrinsic factors. These factors collectively define the resistance or immunity to malaria. The human immune response to infection with the malaria parasites is complex involving natural or innate immunitv and acquired immunity (re\ iewed by Gupta el a l 1994a). 2.7.1 Natura l or innate immunity Innate immunity is independent of previous exposure since it confers no adaptive immunological memory in response to infection. A good example is the protection against vivax malaria afforded to Duffy-negative individuals, who lack the erythrocyte surface antigen used by P.vivax mero/.oite during their invasion ol red cells (G illes and W'arrel. 1993). The geographical distribution o f malaria and certain red cell disorders such as lhalassaemia and haemoglobinopathies suggest that the deleterious anomalies may be counter-balanced b\ a relative protection against malaria (reviewed bv Ilv iid , 1995). 2.7.2 Acquired immunity The acquisition of immunity is slow, requiring mam years ol exposure to the parasite, and numerous disease episodes. The acquired immunity is practically not complete, and residents of malaria endemic areas continue to experience sporadic 18 University of Ghana http://ugspace.ug.edu.gh episodes o f clinical disease throughout life. However, the incidence and density of malaria parasitaemia decline with age. Where age-dependent acquisition o f anti- malarial immunity does occur, the slow development and fragility seemingly reflects the largely strain- and stage -specific nature o f induced immune response (reviewed by Mercereau-Puijalon cl al., 1991a; Good et al., 1988). It is believed that immunosupression during acute episodes o f malaria may interfere with development o f adequate protective immunity (reviewed by Hviid , 1995). The effector cells (natural killing cells (N K ). macrophages and granulocytes) participate in immunity through lysis and phagocytosis o f parasitized erythrocytes as the first line o f action against infection, prior to antigen specific sensitisation (Taverne el a l. 1986). The specific immune defense mechanism against malaria parasites involves the participation o f T and B cells. The T-cells occur as (1) helper cells for antibody production. (2 ) the activation o f non-specific effector cells, such as macrophages and granulocytes. (3) the producer o f substances toxic to the parasites md (4) cytotoxicity cells B-ccll responses in human malaria involve the synthesis of anliplasmodial antibodies with the immunoglobulin types IgA and IgM responses lending to be transient while IgG responses are more persistent. Immunoglobulin G is progressive!) raised in individuals infected with malaria parasites (Collins et a l., 1971). The rate of IgG synthesis is three times as high in unprotected individuals than protected ones, all living in an endemic area. It is also seven limes higher in non-immune people (reviewed b\ Theander. 1991). Both antibody-independent and antibody-dependent mechanisms appear to be involved in acquired immunit) to malaria. The antibody independent host 19 University of Ghana http://ugspace.ug.edu.gh hyperplasia with various leucocytic responses (Sheagren el a l, 1970). The immune responses providing the selection pressure lor variation in MSP1 would be even more compelling if the variable rather than conserved regions of the molecule were shown to be preferred location o f 'I- or B- cells epitopes (Anders and Smythe, 1989). The interactions between different parasites simultaneously infecting the same individual may result in antagonism between the species significantly changing the course of the infection and its potential to produce disease (Riche. 1988: Snounou cl al.. 1992). Antibody production during malaria parasite infection is age dependent in endemic population and children develop antibodies against variant epitopes whereas adults develop antibodies against less immunogenic but conserved epitopes. In addition antibodies against immunodominant epitopes often cross-read with other repeated epitopes, either within the same molecule or on other parasite proteins ( fheander, 1991. Anders. 1986). Cross-reaction interferes with the maturation of high affinity antibody directed against the parasite by causing an abnormally high proportion of somatically mutated B-eells to be preserved during clonal expansion. The cross-reacting epitopes could interfere with the absorption of antibodies on antigens th.it are not essential for the survival of the parasite, thus simply diverting immunological response from more important epitopes (Blackman el a l. 1990; Shai cl a l 1095). defense mechanism against malaria infection involves reticuloendothelial cell 20 University of Ghana http://ugspace.ug.edu.gh Antibodies against ilie ( -terminal fragment, M S P ln ; may block meiozoile invasion o f erythrocytes, and also inhibit parasite multiplication mside the erythrocytes (1 lolder and I reeman, 1984; Blackman el al., 1990). In some cases, vaccinated animals produce invasion-blocking antibody and are protected from challenge, whereas in other cases, protection develops without production of' blocking antibodies (W y le r and Pasvol, 1986). The experimental evidence for protective effect o f an ti-M SP l|9 antibody is thus inconsistent as Dodoo el al. (1999) found no significant association between the prevalence or levels o f anti-MSPl 19 antibody with protection from malaria. 2.8 Immuno-Epiiiem iologv of P. fa lc iparum m alaria The severity of a P falciparum infection depends on the complex interplay o f the immune status, genetic background, and possibly on parasite virulence factors such as invasive efficiency, intraerythrocytic maturation and replication rale (Contamin el al.. 1996). There is still a debate on the hypothesis that some P falciparum strains might be more virulent than others (Gupta el a l I 994b). Experimental infections in luimans have indeed indicated that some strains consistently induced more severe infections than others. It has also been shown that the onset o f symptoms in chronically infected previously asymptomatic individual may be correlated with introduction o f a new parasite strain, differing from that in the original infection (James el al., 1932; Contamin el at.. 1996). It has also been proposed that during infection with a single parasite strain all the 21 University of Ghana http://ugspace.ug.edu.gh variants are expressed within few cycles and presumably antibodies against all or most o f these variants are induccd during primary attack. Ii the immune response against any given variant declines rapidly Following its elimination, that variant may reappear later due to switching in other not yet eliminated variants (Staalsoe and Hviid, 1998). Jeffery (1996) observed that in humans immunity raised to a particular strain of P. falciparum is largely inefficient against challenge of heterologous strains. Day and Marsh (1991) showed that malaria is characterised by clinical disease and acquisition of protection is slow since a long period is required to achieve exposure to a large repertoire o f serotypes. It has been observed that in the absence o f transmission by mosquitoes in low endemic regions, malaria attack results from a single infective bite and no major allelic changes occurs, and that the parasites are propagated for a long time without major modification at loci used as markers. Intense transmission period however is marked by considerably changed genotypes and that rapid turnover ma) re lied frequent renewal of parasite populations resuliing from sporozoile inoculation (Daubersies cl al. 1996; Paul and Day. 1998). There is ample evidence that malaria parasites are polymorphic for a large number o f genetic diversity of local parasite populations. The number o f allelic types to which people are actually exposed is essentially unknown and little is known about the circulating strains in a restricted geographic area (C’onwa\ and M cBride. 1992; f'orsyth cl a l , 1989) It is believed that immunity in African children is strain specific and that clinical attacks are caused by new strains. I lie elucidation o f the contributory role that specific strains play in acquisition of clinical protection 22 University of Ghana http://ugspace.ug.edu.gh against malaria should be sought Although microscopic examination ol blood 11Inis is a cheap and reliable method to establish malaria infection, detailed studies o f the parasite necessitate the use of other methods to augumcnt its sensitivity. There are indirect fluorescent antibody test ( IFA T ), the indirect haemagglutination (1HA) lest, immuno-precipitation techniques (double gel diffusion test), the enzyme-linked immumsorbent assay (E L IS A ) and polymerase chain reaction (G illes and Warrel, 1993). To investigate /’. falciparum strains vvilh respect to their antigenic variation and the immune response o f humans PC R and E L IS A were respectively chosen in the present study. 2.9 Polymerase chain reaction (P C R ) At present infected-cell agglutination test (Brown and Brown. 1965) and variant forms of enzymes, antigens, proteins, gene sequences and drug susceptibiIii\ have been used to characterise P falciparum (Creasy a a l . K o c h /a me typing lor example (Babiker cl a l 1991; Carter and McGregor. 1973) requires substantial amount o f parasites, restricting the analysis of samples with low levels o f parasitaemia. Monoclonal antibody typing requires parasites at particular stage of development, necessitating short-term maturation o f blood stage parasite (M cBride cl a l, 1984; Comvay el a l 1991; Conway and McBride. 1992). It is neither practical nor representative to limit epidemiological studies to cases where sufficient parasite material can be obtained directlv from patients. 23 University of Ghana http://ugspace.ug.edu.gh The PC R has bccome a major diagnostic and research technique The superior sensitivity and accuracy of the PC R assay over microscopical diagnosis has been established (Snounou cl a l. 1993). It has the major advantage o f eliminating the need lor in vitro manipulation of parasites because DNA from circulating ring stage can be used to analyze a large number o f genetic loci, including those expressed al different stages or in mosquito vector. Moreover PC R generates the material to be typed, instead of consuming it, and once a gene fragment has been amplified, analysis o f the fragment can be performed by various methods (Contamin cl a l 1995). However PC R is expensive and hardly used for routine diagnosis. There is also the possibility o f contamination resulting from handling of templates and PCR product The polymerase chain reaction is a rapid procedure for in vitro exponential amplification o f a specific target DNA sequence mediated by enzymes (Saiki cl a l . 1988). It involves two oligonucleotide primers that flank the D N A fragment to be amplified. There are repealed cycles o f heat denaiuralion o f the D \ V. annealing o f the primers to iheir complementary sequences, and extension of the annealed primers with DNA polymerase as illustrated in Figure 3. The primers hybridize to opposite strands of the target sequence and are orientated so that DNA synthesis by the polymerase enzyme proceeds across the region between the primers. The extension products themselves are also complementary templates and are capable o f binding primers Successive cycles o f amplification essentially doubles the amount of the DNA synthesized in the prev ious cycle The result is the exponential accumulation ol the specific target fragment, which then can be 24 University of Ghana http://ugspace.ug.edu.gh visualised by gel electrophoresis (Saiki, 1990). The high sensitivity, specificity and yield o f PC R with the thermostable Taq DNA polymerase make it an ideal method for the isolation o f a particular genomic fragment (Saiki, 1990) The analysis o f PC R amplified DNA products encoding polymorphic protein o f P falciparum allowed the determination not only o f species, but also subspecies or strains (Paul el al., 1995). 25 University of Ghana http://ugspace.ug.edu.gh Figure 3. Schematic representation o f PC R amplification Process. A I) O r i g i n a l l ) N A 1’C R primer Neu |)N tf \ 1)N \ piimers ■ dN I I’ ■ I )N A polymerase li: I )enalure and s\ nlhesi/e ( Denature and synthesi/e D: Denature and synthesi/e DMA m he .impIi 11 c l ! is denalureJ In healing the ample. In Ihe presence o f 1 )\'A poKmer.i e\ees> ;iecv \nudcuiide inphosnhai lii-.miu ic.uiil.- ih bridize speei lle.il I \ sequenei. i m prime new l )N \ sMiihe- Depending oil the number o f e\eles manv m illioi DN.A Ir i 'inenis t a i l be g e n e ra te d (i.e. 1 bp in SQObp). It can accommodate much larger quantities 01 DNA lhan agarose gels. D N A recovered from polyacrylamide gels is extremely pure (Sambrook ci n i 1989). I here are two common types of polv acrv lamide gels which are often used i.e. non­ denaturing and denaturing 2.10.3 Discontinuous iion-dcnaluring polyacrylam ide gel Most double stranded DNA migrates through non-denaturing polyacrylamide tie Is 28 University of Ghana http://ugspace.ug.edu.gh at a rate that is approximately inversely proportional (o the log m o f their size. However, their base composition and sequence also affect their electrophoretic mobility, so that DNAs o f exactly the same size can differ in mobility up to 10%. This effect is believed to be caused by kinks that form al specific points o f double stranded DNA . Since it is impossible to know whether or not the migration o f an unknown DNA is anomalous, electrophoresis through non-denaturing polyacrylamide gels cannot be used to determine the size o f double stranded DNA (Sambrook cl al., 1989). It is therefore used to widely separate the bands, which hitherto have been close on agarose gel. 2.11 Enzyme-linked immunosorbent assay ( E L IS A ) Enzyme immunoassay was first described by Van VVecmen and Scluiurs (1972) and Engvall and Perlmann (1972). The underlying principle is the conjugation o f antibodies or antigens to enzymes such that the immunological and enzymatic activities of each moiety are maintained The degradation o f a substrate by the enzyme, measured speclrophotomeirically. proportional to the concentration of the unknown "antibody” or '‘antigen" in the test solution. Apart from it* convenience, E l ISA has the following advantages: the labelled immunoreagenis are stable for long periods, the precaution and disposal procedures required for the radioisotopes are unnecessary, the use o f chromogenic substrates for the enzyme labels permits visual interpretation of test result. Competitive and non-competitive ! I. ISA techniques are used for detection o f cither antigen or anliboJx The competitive E l. IS A detects either the antigen (Belanger cl a l 1973: Rato cl al.. 1975) or the antibody (I Iammarslrom, 1975). The non-competiti\e I M SA 29 University of Ghana http://ugspace.ug.edu.gh technique is potentially more sensitive and widely used. However there are several variations such as direct method for detection o f antibody (Engvall and Perlmann, 1972), double antibody sandwich method for detection of antigen and the antibody capture assay for detection o f class specific immunoglobulin. The 1 I ISA can be used as qualitative as well as quantitative assays for antibodies. In addition it provides a rapid method o f diagnosing a wide variety o f viral, fungal and protozoal infection with strain specificity where necessary. W H O (1974) reported that E L IS A has been used widely for the detection and measurement o f antibodies in response to erythrocytic stages o f malaria infection and Egan el al. (1995) measured antibody responses to MSP1 using the microplate E L IS A . 30 University of Ghana http://ugspace.ug.edu.gh C H A P T E R T H R E E M A T E R IA L S A N D M E T H O D S 3.1 Study area This study was conducted at Dodowa, the capital o f the Dangme West District o f the Greater Accra region o f Ghana. The district lies within longitudes 0 ° 5' and 0° 20' E and latitude 5° 40' and 6° 19' N. It is approximately 25 kilometres trom Accra, the capital town o f Ghana and is located between the coastal sav annah and the secondary forest with few streams that How most of the year. The forest is being depicted rapidly as a result of intense farming and other economic exploitation, hence the vegetation is now a mixture o f few large trees with grass covering most o f the area. Dodowa is gradually developing the characteristics ol an urban community with greater accessibility to anlimalarial drugs ( \lari cl a! 1 992 ) Dodow'a experiences two weather seasons, dry and wet. 1 he rainy season runs from April/May to October/November and the dry season from November/Deccmber to March. The area has two peaks ol rainfall during the year, the first occurs in June/July and the second in September. Rainfall is high in the forest areas and ranges between 77mm and H7mm per month. I lumidity is highest, about 90%, in the rainy season and al its lowest, about 70%, during the dry season. 31 University of Ghana http://ugspace.ug.edu.gh Malaria transmission occurs throughout the year, but is highest (.hiring or immediately after the major and minor rainy seasons, and lowest in the dry seasons. The transmission is considered as stable because it does not vary significantly from year to year. The incidence rate of clinical malaria is 106/1000 population per month and individuals are exposed to 22 infective bites per year with 98% o f the infections caused by P. falciparum (Afari el al.. 1995). 3.2 Study population Dodowa has a total population ot 6,558 based on a 1992 census (A fari el a l 1992). Children o f ages o f 5 years or below ('' 5) and, those between 6 and 9 years (6-9) constitute 17% and 16.4% o f the population respectively. The study population consisted o f a cohort o f 300 children within the ages o f 3-15 years. They were followed as part o f a longitudinal scro-epidemiological stud\ during the period April I 994 to \nyList 1995 3.3 E th ica l consider;!(inns Informed consent for participation o f the children was obtained from their parents after information had been given in the local language. The Ethical Committee o f Ministry ol Health, (ihana approved the stud_\ 32 University of Ghana http://ugspace.ug.edu.gh 3.4 Study design A target population in this longitudinal cohort study was selected after screening, using the metabisulphile test to exclude all those who had sickle cell trait or sickle cell disease. The children were followed with weekly clinical examination, monthly capillary bleeding (finger prick). Blood films (thick and thin) were prepared in the field from children with temperature > 37.5°C and at monthly bleeding by an expert microscopist. The slides were transported to Noguchi Memorial Institute for Medical Research (N M IM R ) and examined. The Plasmodium parasite density’ was determined after counting the number of parasites per 300 white blood cells (W B C ) in the Giemsa stained thick films and multiply ing by 8000/300 as an approximation o f parasite count per microlilre. A ll negative slides were re-examined by counting up to 1000 W B C The children were classified into symptomatic and asy mptomatic. An asymptomatic child had parasites in the blood in the absence o f lever or other clinical signs o f malaria such as rigors/chills vomiting, convulsion and musculo-skeletal pain. A symptomatic malaria patient had a positive blood slide together with temperature greater than 37.5°C with or without additional symptoms. Patients were excluded if they had signs o f other diseases like throat and ear infections. Uninfected children had no detectable parasitaemia. For the present study only samples having P. falciparum for three consecutive months were included. The samples were grouped as follows: children who were asymptomatic for three consecutive months (A A A ), those who were asymptomatic in the first and third month but symptomatic in the second month (A S A). 33 University of Ghana http://ugspace.ug.edu.gh 3.4.1 Sample collection hach time a blood film was prepared for diagnosis of malaria, 50|.i 1 o f blood from the individual was spotted onto filter paper (Whatman #5) after a finger prick and air dried. After drying, the samples were placed individually in small plastic bags, containing anhydrous silica gel and stored at -20°C until ready for use. Sim ilarly blood samples had been collected from malaria patients (children) al University of Ghana, Legon Hospital for preliminary studies in standardising the PC R methods and also to determine its sensitivity. Venous blood was collected in heparin, centrifuged and blood plasma taken. 3.5 Reagents Hie details ol reagents used and their sources are provided as follows 3.5.1 Enzymes Taq polymerase was supplied as a set with lOx buffer solution MgCN (50 mM ) and a 1% \ V I detergent (G IBCO BR1 1 ife Technologies. U SA ). 3.5.2 Deoxyribonucleic triphosphate (dN T Ps ) The lour dcoxyribonucleotides, deoxyadenosinc triphosphate (dATP ). deoxycvtosine triphosphate (dC I P) deoxyguanidine triphosphate (dG TP ) and deoxythymidine triphosphate (d I IP ) were obtained from Boehringer Mannheim Gmbl I, Germany. 34 University of Ghana http://ugspace.ug.edu.gh 3.5.3 O ligonucleotide primers Two sets of oligonuclcolidc primers designed to flank block 2 terminals o f merozoite surface protein (M S I5-1) gene (Fable 1) were used. The sequences o f the oligoprimcrs are the same as that published by Ranford-Cartwright el a ! (1993) and was purchased from O SW E L DNA Services, U .K . Primer ( bp) Length Sequence (5 ’ to 3’) Position 0 , 26 'C'AC A T G A A A G T T A T C A A G A A C T T G T C ' 5‘ MSP1 outer o 2 22 'G T A C G TC TA A T T C A T T T G C A C G ' 3‘ MSP1 outer N i 20 'GC’AG TAT 1 ‘G A C A G G f 1ATGG ' 5' MSP1 inner N 2 18 'G A T T G A \ A ( iG 1 A T T TG A C ' 3‘ M S P 1 inner Table 1 DNA sequences of oligonuclcolidc primers used for PC R amplification of alleles o f M SP I gene. 3.5.4 DNA molecular weight marker I he following DNA molecular weight markers were used as appropriate. 35 University of Ghana http://ugspace.ug.edu.gh 1200bp, 1300bp, 1400bp, 1 500bp, and 2072bp DNA molecular weight marker V I " This marker was supplied by Boehringer Mannheim GmbH, Germany. It consisted of a mixture o f pBR328, cleaved with B g l /, pBR328 DNA and Hm f I It generated 12 fragments of the following lengths. 154bp, 220bp, 234bp, 298bp 394bp, 453bp, 5l7bp, 653bp, 1033bp, 1230bp, 1766bp, and 2176bp 3.6 Standard solutions Details o f all solutions prepared can be found in Appendix 1. 3.7 Isolation of parasite DNA from blood spotted filter paper The method used in the extraction o f malarial parasite DNA from blood spot on filter papers was a modification of a protocol used by Walsh cl al. (1991). Eppendorf tubes ( 1 5 ml) were labelled with the corresponding recognition codes Two spots o f blood stained filter paper covering a total area o f 11 3mm were excised by a precision hole puncher and put into Eppendorf tubes containing a total volume o f 200ul o f 10% Chelex (w/v) solution which was continuously stirred while being pipetted The contents were thoroughly mixed and incubated at 56°C for 30 minutes in a water bath whilst shaking. They were then vortexed for 5 seconds and incubated in boiling water for 10 minutes They were vortexed for a further 10 seconds and spun in a microcentnfugc (Kubota, Japan) at 15,000 r p m lor 10 minutes. The supernatant was collected and the filter paper discarded. The University of Ghana http://ugspace.ug.edu.gh stirred while being pipetted. The contents were thoroughly mixed and incubated at 56°C lor 30 minutes in a water bath whilst shaking, they were then vortcxed for 5 seconds and incubated in boiling water lor 10 minutes. I hey were vortcxed for a further 10 seconds and spun in a microcentrifuge (Kubota. Japan) at 15,000 r p m. for 10 minutes. The supernatant was collected and the filter paper discarded. The supernatant obtained was centrifuged 2x and then transferred into 0.5 ml Eppendorf tubes for storage at -45°C until ready to use. Cross-contamination o f samples was avoided by treating the hole puncher and forceps used with 5M hy drochloric acid followed by 5M sodium hydroxide after each sample to pre\ent carry-over o f DN \ from one sample to another. 3.8 P C R amplification A typical reaction mix for a 100f.il reaction contained the following: lx PC R buffer (20mM fris-HCl pH 8.4. 50mM KC1). 200fiM each o f Deoxyribonucleotide triphosphate (dATP. dCTP. dGTP cl 1 IP ). 1 5mM of Magnesium chloride. 0.8jiM o f each primer. 0. 0 (v 'v ) o f detergent (W -,). 2.5 unit I'aq D N A polymerase and 5f.il o f extracted DNA solution I or the nested PC R only 1 -2f.il o f the first round PCR product was used as DNA template lo r the present study a reaction volume o f 50j.il was used and amplifications carried out in 0.5jil 1 ppendorf tubes. The contents were thoroughly mixed. overlaid with mineral oil (Sigma Chemical Corporation) and spun for 5 seconds. Mineral oil was added to prevent evaporation and relluxing of the reaction mix during ihermocycling. 37 University of Ghana http://ugspace.ug.edu.gh The PC R assays were carried out using a thermal cycler (Techne Progene, U S A ) under the following conditions; an initial cycle ol denaturation at 94 °C for 2 minutes, primer annealing al 5()°C for 25 sec and extension at 68°C for 2 minutes 30 sec. followed by 29 cycles o f denaturation at 94 °C for 25 sec, annealing at 50°C for 30 sec and extension at 68°C for 2 minutes 30 sec. The reaction was concluded with a cycle o f an extension time of 10 minutes and the reaction product was stored at 4 °C until use. The same cycling conditions were used for the nested PCR . 3.9 Agarose gel electrophoresis The agarose gel was prepared as described by (Sambrook cl a l, 1989). Briefly. 1.5 g o f agarose granules was weighed into an Erlenmever flask to which 100ml o f 1 x Tris acetate ED T A buffer (T \ Ii) was added. It was covered with a piece of perforated cling film and heated in a microwave oven for 2 minutes. Ethidium- bromide (5j.il) was added to the mixture and then cooled gently. A gel tray was prepared by placing the comb to form the wells and slicking strips o f tape to the open sides o f the tray. The agarose solution was poured into the tray and left for about an hour to solidify. The comb and tape were removed and the tray put in a horizontal M inigel electrophoretic system, (M inigel submarine, B ioRad ) containing the same buffer as the gel. The loading buffer (2 OliI 5x Orange G ) was mixed with 10|.il o f the PC R product on a piece o f paralilm before loading. About 3-5f.il o f the molecular weight markers was used. Electrophoresis was carried out at 80V for two hours and the gel viewed on an U V transilluminator (T.M-20. U SA ) and photographed using a Polaroid camera fitted with an orange filter. 38 University of Ghana http://ugspace.ug.edu.gh agarose gel using graphical method (Appendix 2) The fragment sizes o f the markers used during the study are listed in section 3.5.4. Where D N A fragments were very' close on agarose gel, polyacrylamide gel was used to further resolved them but it was not used to estimate DNA fragment sizes 3.10 Discontinuous non-denaturing po lyacrylam ide gel electrophoresis This method was used to obtain maximum separation o f bands. Electrophoresis was performed using a vertical Mini-Protean Dual Slab Cell System (ATTO , Japan). The glass plate was sandwiched and the equipment was assembled and operated according to the protocols supplied by the manufacturer. The separating gel was prepared by mixing 20 ml o f 4 ,5% gel solution and 1500fil of 1 5 % Ammonium persulphate (A P S ) (Appendix I), degassed for 15 minutes in a vacuum chamber (Nalgene, Sybron corporation) before 15f.il o f N N N N - Tetramethylethylenediamine (T EM ED ) was added Similarly the stacking gel was prepared by adding 500(.il o f 1 5% A PS to 5 % stacking gel solution (Appendix I), degassed for 15 minutes before 4j.il T EM E D was finally added The separating gel solution was poured between the two plates to the desired level and immediately overlaid with water The gel was set usually within 40 minutes and the water was drained off The stacking gel solution was poured onto the separating gel and the comb inserted immediately It was pre-inn at 1 ^OV and 30 mA lor 30 minutes in 1 x Tris borate ED TA (T H E ) buffer, after the wells had been thoroughly cleaned by washing with 39 University of Ghana http://ugspace.ug.edu.gh solution was poured between the two plates to the desired level and immediately overlaid with water. The gel was set usually within 40 minutes and the water was drained off. I he stacking gel solution was poured onto the separating gel and the comb inserted immediately. It was pre-run at 150V and 30 mA for 30 minutes in 1 x 1 ris borate ED T A (T B E ) buffer, after the wells had been thoroughly cleaned by washing with distilled water to remove unpolymerized acrylamide. The wells were loaded with a mixture of 5jil o f each of the PC R products and orange G x (1). The two outer terminal wells were loaded with 4j.il o f the desired molecular weight marker. The electrophoresis was run for 1.5 hours at 120V and 15mA. Thereafter, the apparatus was dismantled and the gel removed for silver staining. 3.10.1 Detection of DNA products using silver staining method I he polyacrylamide gel was llxed in a solution of 10% ethanol and 0.5°o glacial acetic acid for 25 minutes It was transferred to a solution o f 1 Im M AgNO ; for minutes and then washed twice with tap water. It was then developed in 15 ml of 5M NaOI-l and 0 .8% Formalin solution. When the DNA bands were visible, placing the gel in 10% glacial acetic acid stopped the reaction. Finally the stop solution was poured o ff and the gel washed with tap water and placed on a light box and photographed using a Polaroid Camera. 3.1 1 Sensitivity o l 'P C R assay The aim ol this experiment was to determine the lowest level o f parasitaemia that 40 University of Ghana http://ugspace.ug.edu.gh the PC R could delect under our laboratory conditions. This was done before analysis o f the samples lrom Dodowa. A small drop from blood which was collected in 10 ml containers lrom patients who reported at the University of Ghana hospital, Legon, was placed on a glass slide (25 mm x 75 mm) and another slide was used to spread the blood evenly to produce a thin smear which was air dried. The thin film preparation was fixed by immersing the slide in absolute methyl alcohol for 30 seconds and the 5 % Giemsa stain solution was then poured on the slide whilst it was placed Hat on two glass rods and left for 1 5 minutes. The thick film was also prepared by placing three small drops o f blood in the middle o f a slide and the drops were spread to form a film o f blood. It was allowed to dry at room temperature and stained with 5 % Giemsa. The stain on both slides was gently washed off with tap water and the slide was left upright to dry. The thick film was however not fixed. Both the thick and thin blood films were examined using an Olympus CH-2. microscope. Infected as well as uninfected red blood cells (R B C ) were counted in eleven oil immersion fields o f standardised thin blood film at a magnification of lOOOx and the parasitaemia was calculated as ratio o f the infected to uninfected RBC counted. The total number o f R B C was estimated using a modified protocol described b> Lynch el a l (1969). The results obtained were later checked with that using an automated haematological analyzer counter (M ic roD iff 1 8, Coulter. I ISA ). fo r manual estimation, Sahli's pipette was used to take 0.02 ml blood and washed into 4 ml of diluting solution (40% formaldehyde and 3% (w/v) trisodium citrate) to give a 1 in 201 dilution. The diluted blood was then mixed thoroughly using a 41 University of Ghana http://ugspace.ug.edu.gh whirl mixer (Dcnlcy Greiner, England) and the counting chamber was filled using a capillary lube. Eighty squares o f the counting chamber were examined by microscop' The total number of R BC was estimated using the formula R x 200 x 1/0.02. where R is the dilution factor The percentage parasitaemia was then calculated. Once the parasitaemia has been determined the infected blood was then serially diluted in ten steps with blood of non-immune uninfected Danish nationals. Fifty microlitre blood o f each o f infected and unifected blood as well as the serially diluted was spotted on filter papers (Whitman #5). air dried and stored at -45°C DNA o f the parasite was then extracted from each filter paper and PC R carried out using the methods and the reaction conditions described (sections 3.7 and 3.8). For all reactions both positive and negative controls were carried out and the results of the PCR were analysed as previously described (section 3.9). 3.12 Enzyme-linked immunosorbent assay ( E L IS A ) The 1 LISA, assay performed to measure the an li-M SPI antibodies against /’ falciparum antigens (M S P lm ) was a modification of the protocol described by Riley cl al. (1991). The modified method is routinely used in the Immunology Unit of the Noguchi Memorial Institute for Medical Research, and for the present study' was targeted at the 1’ fM S l’ 1 m. Dynalech Microlitre plates were coated with l|ig/ml ol the antigen in carbonate buffer (pi 1-9.6) (constructed with glutathione) donated by Dr I leanor Riley, ol the University o f I dinburgh. Wells A-l and A-2, 42 University of Ghana http://ugspace.ug.edu.gh which were used as blanks, were not coated. The plates were incubated at 4 °C overnight, washed three times with phosphate buffered saline (PBS)/Tween (pH- 7.4) using an autowasher (F.l 404 B IO T E K , U SA ), and then blocked with 5 % bovine serum albumin (B S A ) in tween-20. The blocking was performed at 37°C for 2 hours followed by three times washing with P B S tween. Plasma samples were diluted 1:100 in B SA . added in duplicates and then incubated at 37 °C for 1 hour. After washing three more limes, a 1:2000 dilution o f anti-human IgG alkaline phosphatase conjugate (Sigma, U SA ) was added and the plates incubated at 37°C for 1 hour. The plates were washed three limes and then developed with I|.ig/ml p-Nitrophenyl Phosphate (PN P ). The reaction was stopped with 3M NaOH after incubating for 25 minutes al room temperature in the dark. The absorbance was read al 405 nm using microplate reader MTP-32 (Corona Electric. Japan). The minimum significant value was established from the mean ol the negative controls (x) + 2 standard deviation. The cut-off point o f optical density (OD )< 0 3 indicating negative and ()L)>0.4 as positive were used in the analysis that sought to determine whether there was any association between increased antibodies and clinical protection from malaria In this study, negative control sera were obtained from non-immunc and uninfected Danish nationals who have never been exposed to malaria. The positive control sera were obtained from patients who have had several malaria attacks. 43 University of Ghana http://ugspace.ug.edu.gh C H A P T E R FO U R R E S U L T S 4.1 Sensitivity of P C R for P. falciparum detection The aim o f this experiment was to determine the least detectable number of Plasmodium falciparum from filter paper blood using PC R assay based on the amplification of the MSP1 gene As shown in Figure 4, the bands represent a D NA fragment size o f 600 bp. Initially the blood contained 218250 parasites per microlitre but was diluted. The same DNA fragment size was expressed but the band gradually became faint with increasing dilution Consequently beyond 1: 100,000 dilution o f the test blood no band was observed in the gel (Figure 4). Based on the dilution the sensitivity o f the PC R technique employed in this studv was calculated to be 22 parasites/jul o f blood using the outer primers for amplification and agarose gel for analysis but with polyacrylamide gel analvsis as low as 2 2 parasites/pl of blood could be revealed 4.2 Comparison of P C R and microscopy in detection of P falcipasrum The PCR assay was assessed using microscopically positive and negative samples as shown in Table 2 A total o f 197 samples obtained from a subpopulation o f 38 children were analysed to establish the sensitivity ol the PC R assay using outer and inner primers (Table 3 ) -u University of Ghana http://ugspace.ug.edu.gh Microscopic examination o f 197 giemsa-stained blood films showed that 194 contained Plasmodium falciparum and 3 had no parasites. The PC R assay confirmed 160 microscopically positive samples when outer primers were used and missed 34 samples. Sim ilarly outer and nested PC R confirmed 1 out o f 3 microscopically negative samples while 2 were positive (Table 3). This gave a relative specificity o f 33.3%. In all, the outer primer showed 161 samples positive and 36 samples negative. The nested PC R performed on 34 microspically positive but nagative when outer primers were used for amplification indicated that 32 o f the samples were really nagative and two were positive. In summary therefore, a total of 192 out of 194 microscopically positive samples were PC R positive representing 99% sensitivity. 4.3 A lle lic typc(s) in individuals w ith consecutivc asymptomatic (A |) symptomatic (S ) and asymptomatic (A ?) eases of m alaria status The purpose ol' this work was to find out if symptomatic cases o f malaria are limited to certain MSP1 allelic types. To determine this, blood on filter paper o f individuals who exhibited consecutive asymptomatic, symptomatic and asymptomatic malaria in the dry and wet seasons were used, lable 4A and 4B summarise the results obtained lor the two seasons and showed the sizes o f the amplified M S P ! DNA fragment sizes and their corresponding parasite densities during wet and dry seasons. Different MSP1 types (Figures 5 and 7) were associated with malaria episodes. In general there was a switch of allelic type (determined by size differences) when 45 University of Ghana http://ugspace.ug.edu.gh an asymptomatic child suddenly became symptomatic. In all, 8 children showed this A |S A 2 phenomenon of which 6 (75% ) had allelic type switch over when they experienced malaria episode. The list showing the switch over o f the corresponding children is provided in Tables 4A and 4B; namely l (A B C ) - 546bp to 596bp, 13 (ABC ) - 615bp to 602bp, 2 0 (EFG ) - 6 I5bp to 596bp, 219 (CDE) - 423bp to 376bp, 276(DEF) - 447bp to 531 bp and 124(ABC)- 596bp to 631 bp. However 2/8 (25% ) children did not show the switch over when they experienced a clinical malaria episode but maintained the same allelic types. The DNA fragment sizes o f the samples belonging to these children were 284 (EFG ) - 477bp and 139 (A BC ) - 582bp. It was observed that the allelic type(s) corresponding to 596bp was associated with malaria attacks in two children l (A B C ) and 20 (EFG ). However an allelic type of the same size was also found in an asymptomatic child 124(ABC). Children 13(ABC) and 20 (EFG ) had allelic type o f 615bp in the asymptomatic blood samples that preceded the malaria episode but their corresponding symptomatic blood showed different DNA fragment sizes of 602bp and 596bp. respectively. In the child identified as 13(ABC). the same DNA fragment size o f 602bp that caused clinical attacks remained even after recover)’. Whereas allelic type o f DNA fragment size of 477bp was found in asymptomatic blood sample o f child 276(DEF), the ensuing symptomatic blood sample showed different allele o f D NA fragment size o f 531 bp. Sim ilarly the allele o f DNA fragment sizes o f 477bp in the asymptomatic blood of 284(1 T 'G ) caused clinical malaria the following month. I lie same DNA lragment size remained a month after recovery. The MSP1 allele 46 University of Ghana http://ugspace.ug.edu.gh of 596bp was associated with clinical malaria during the wet season but was associated with asymptomatic malaria in the following dry season in sample 124 (ABC ). The DNA fragment size o f 582bp in the asymptomatic blood sample of 139 (A BC ) remained unchanged the following month. However it changed to 613bp the next month lie recovered. There were instances where reversion to allelic type o f the preceding asymptomatic phase showed up after malaria attack. Figure 6 illustrates this clearly in children identified as 1 and 13. In other instances, such as in child 13, the allelic type found in attack phase was the same as that found when the child was asymptomatic (Figure 8). There were no specific MSP1 allelic types associated with a particular season. Nonlheless there were diverse forms o f the MSP1 gene (Figure7) Generally there was an increase in parasite densities when previously asymptomatic children experienced clinical malaria except I24 iA BC ') who showed a slight decrease in parasite densih The result (see Table 4A and 4B ) indicates a significant increase (p = 0.017. Mann Whilne\ rank test and p< 0.05. Student Newman Keuls method), l'here was however no association (7/ (Yates corrected), p = 0.1 34 j of clinical malaria with acquisition of new parasite strain. 4.4 M SIM allclic typc(s) in individuals w ith tlircc consecuti\e asymptomatic malaria status The details ol the results were shown in fable 5A and 5B. In all 23 children were examined ol w’hicli 6 showed no change at all in allelic types Switch over involving two allelic types were observed in sample from child 13 and in samples 47 University of Ghana http://ugspace.ug.edu.gh obtained from other three children, all the alleles were different. Multiple infections o f two alleles in blood samples were observed in five instances (Table 5A and 5B) 4.5 Parasitaem ia levels during symptomatic and asymptomatic m alaria cases Generally there was an increase in parasite densities when previously asymptomatic children experienced clinical malaria (Table 4A and 4B). This was observed in the samples belonging to the following l (A B C ) 400-17469. 13 (ABC ) 2773-4507, 20 (EFG ) 160-1093. 284 (EFG ) 773-7573,139 (ABC ) 240-933. However there was only one sample I2 4 (A BC ) which showed slight decrease in parasite density. There was a significant increase (p=0.17. Mann Whitney rank test and p< 0.05 Student Neuman Keuls method) In parasitaemia during symptomatic malaria status o f the individuals. In three consecutive asymptomatic cases, there was generally no increase in parasite densities during the three months period (see Tables 5A and 5B). There was no significant increase (p=0 105 Friedman repeated A N O VA on ranks) in parasites densities between any of the months. 4.6 A n ti-M SP l Immunoglobulin G (IgCI) responses and protection from malaria attack The purpose o f this experiment was lo find out whether anti-MSPl antibodies status of individuals are altered during malaria attacks and correlates with clinical protection. An ti-M SP l antibody responses to the 19KDa C-terminal part o f the MSP1 in school children before and alter malaria attack showed that 14 out o f the total 27 children were positive ( fable 6 ). Nine o f the children fell w ithin the ages 48 University of Ghana http://ugspace.ug.edu.gh of 3 to 4 years whereas eighteen were > 5 years, four children out o f the 9 children aged 3-4 years had no anti -MSP1 antibodies before malaria attack but three o f them produced anti-MSIM antibodies after malaria attack indicating sero­ conversion o f 75% for this age group. The five children who were positive for anti-MSPl antibodies before attack had no change in their anti M SP1 IgG litres. Nine out o f the eighteen children > 5 years were positive and the other 9 negative for an li-M SPl antibodies before malaria attack. However after malaria attack two children become negative for IgG (OD^onm = 0.984 to 0.300. and 0.489 to 0.326) constituting 22%. In all 75% negative to positive sero-conversion and 22% positive to negative sero-conversion were shown. 49 University of Ghana http://ugspace.ug.edu.gh T A B L E S Table 2. Determination o f percentage parasitaemia by microscopy (100X magnification) in 11 fields o f a single blood film A. Determination o f parasitaemia by microscopy Field Total number of red cells Infected red cells 1 116 5 2 1 21 4 3 1 0 0 7 4 6 8 4 5 71 2 6 106 6 7 6 8 4 8 67 4 9 1 0 0 6 10 109 4 11 125 5 Total 1051 51 B. Determination o f P C R sensitivity Using the formula, Percentage parasiteamia = No. o f infected R B C X 100 Total no. o f R B C 50 University of Ghana http://ugspace.ug.edu.gh = 51 X 100% 1051 = 4.85% Parasite density = % parasitaemia x Total R B C count 4.85% x 4.50 x 106/|.il 218250 /(.il Positive Negative Dilution 1 10' 1 102 10° 10'4 10'5 10'6 10‘7 Parasite density 21850 21825 2183 218 22 2.2 0.22 0.022 The P C R method can detect parasites in blood samples with 2.2 parasites per microlitre. 51 University of Ghana http://ugspace.ug.edu.gh Tab le 3: Relative sensitivity o f P C R to M icroscopy for detection o f P. fa lciparum in blood samples. Microscopic Single P C R (O 1+O2) Nested P C R (N ,+N2) Examination Negative Positive Negative Positive Positive (n=194) 34 160 2 32 Negative (n=3) 2 1 1 1 Total (n=197) 36 161 3 33 Oi and O2 = Outer primers N |andN 2= inner primers 52 University of Ghana http://ugspace.ug.edu.gh Tab le 4A and 4B : Comparison o f M SPlfragm ent sizes from three consecutive asymptomatic, symptomatic and asymptomatic samples and their corresponding parasite densities during dry and wet seasons. 4A- wet season Study number Molecular Weight (bp) of M SP 1 allelic types A j S A 2 Parasite density. (No. /pi blood) A. S A 2 New allelic type 1 (A BC ) 546 596 546 400 17469* 133 Yes 13 (A BC ) 615 602 602 2773 4507* 320 Yes 20 (E FG ) 615 596 589 160 37707* 1040 Yes 219 (CDE)» 422 376 398 27 880* 133 Yes 276 (D E F ) . 447 531 516 27 1093* 160 Yes 284 (EFG )« 477 477 477 773 7573* 453 No 4B- Dry season Study number Molecular Weight (bp) Parasite density'. New allelic type A| S A ’ A| S A 2 124 (A BC ) 596 631 6 6 8 1707 1 147* 27 Yes 139 (A BC ) 582 582 613 240 933* 1 147 Yes • = Nested PCR using inner primers, A| = Asymptomatic cases before malaria episode, S = Symptomatic cases, A 2 = Asymptomatic cases after malaria episode. * = There was statistically significant difference (p = 0.017) using Mann Whitney Rank sum test and also A ll Pairwise Multiple Comparison (Student Newman Keuls Method, p < 0.05) for the parasite densities. There was no association of clinical malaria with acquisition (x 2 Yates corrected p = 0 .134). The alphabets such as A, B. C etc correspond to the months during which blood samples were taken The first month was taken as A. 53 University of Ghana http://ugspace.ug.edu.gh Tab le 5 Comparison o f molecular sizes o f MSP1 allelic types for blood samples taken in three consecutive months from asymptomatic individuals and their corresponding parasite densities. 5A Wet season Study number Molecular Weight (bp) A| A 2 A j Parasite density. A ,* A 2* A 3* New allelic type 20 (A BC ) 614 602 602 480 12400 269 Yes/No 106 (D EF ) 562 562 516 560 80 267 No/Yes 158 (A BC ) 562 562 546 213 2 0 0 0 33947 No /Yes 159 (A BC ) 624 638 638 8347 2267 9200 Yes /No 359 (D FE ) 579 579 579 320 640 2987 No 64 (D FE ) 562 562 562 160 53 13 No 114 (D FE ) 631 631 6 6 8 3707 80 9200 No Yes 345 (CD E ) 6 6 8 6 6 8 749/63 1 267 1040 2720 No 'Yes 351 (A BC ) 596 596/562 596 15093 6400 17333 (No)(Yes) A, =Asymptomatic cases before malaria S =Symptomatic malaria cases A 2 =Asymptomatic cases after malaria 54 University of Ghana http://ugspace.ug.edu.gh 5B- Dry season: Study number Molecular Weight (bp) A| A2 Aj Parasite density. A|* A 2* A 3* New allelic type 72 (ABC) 562 562 649 720 80 3067 No /Yes 192 (ABC) 596 596 596 373 667 453 No 240 (DEF) 596 596 596 240 53 1040 No 266 (DFE) 596 569 596 540 80 160 Yes 284(ABC) 559 649/559 631/559 4667 560 2080 (Yes)(No) 285(ABC) 560 617/519 560 640 640 53 Yes 360(ABC) 638 638 575 320 9760 560 Yes/No 421 (ABC) 646 646 646 9040 80 1547 No 422(ABC) 596 596 582 1173 1973 347 No/Yes 85 (CDE) 546 579 546 80 1040 187 Yes 92 (ABC) 579 579 546 80 267 933 No/Yes 214(ABC) 550 550 582 1867 902 373 No/Yes 291(ABC) 649 649 649 213 427 267 No/Y es 354(ABC) 562 562 516 1040 2560 160 No/Yes * - There was 110 statistically significant difference (p = 0.105) in parasite densities using Friedman repeated ANOVA on ranks. 55 University of Ghana http://ugspace.ug.edu.gh Table 6: Anti-MSP-1 antibodies in different age groups o f school children before and after malaria attack. Age Number Antibody Status Sero-Conversion(%) Years Tested Positive Negative -/+ +/- 3-4 9 5 4(3)b 3/4(75%) 0 >5 18 9(2)c 9 0 2/9(22%) Total 27 14(2)c 13(3)b 3/13 2.'14 a = Presence or absence of anti M SP I antibodies before and after malaria attack, b = Number of individual with anti M SP I antibodies after malaria attack. -/+ = Production of anti M SP I antibodies in previously negative individuals. +/- = Loss of anti M SP I antibodies in previously positive individuals, c = Number of individuals without anti-MSP I antibodies after malaria attack. Optical density (OD45o„m) = (OD < 0.3 - negative, OD > 0 *4 - positive) There was no statistically significant difference (p = 0 .182) using McNemar's test 56 University of Ghana http://ugspace.ug.edu.gh Figure 4: Sensitivity of P C R for detection o f blood stage malaria parasites. Amplifications were performed on serially diluted samples o f known parasitaemia using outer j.u irn cK S . Lanes M show makers. Lanes 2- 8 represent ten foid serial dilution. Lanes 9 and 10 are positive and negative controls respectively. 57 University of Ghana http://ugspace.ug.edu.gh M 1 :• 4 5 6 7 8 9 10 11 12 13 14 15 M Figure 5: An illustration o f the diversity o f M S P allelic types found at Dodowa. Samples were obtained from three patients in both wet and dry seasons. Lanes 3-9 show patient number 20, i 0-12 represent patients number 13 and 13-15 patient number 1. Lanes land 2 are negative and positive controls respectively. Lanes 5,6 and 15 are dry season samples whereas 3,4,7,8,9,10,11,12,13 and 14 are wet season samples. Lanes 11 and 14 from patient showed clinical malaria. 58 University of Ghana http://ugspace.ug.edu.gh ft A i. S A M 1 2 3 4 5 6 7 ■ r * V 2 1 7 5 t i ; " ■ j - 6 5 3 bp —^ I54hf Figure 6: The distribution o f M S P I variants in two patients before and after malaria attacks. Lanes 1,2 and 3 are M S P I alleles o f patient 13 and 4,5,6 are those of patient 1 A = Asymptomatic. S = Symptomatic, M = Marker 59 University of Ghana http://ugspace.ug.edu.gh I - lOObp Figure 7: Ethidium bromide stained 1.5% agarose gel o f P C R product o f P falciparum showing MSP1 allelic types o f symptomatic cases n = 7. Lanes M show marker, 1,2,3 and 7 are dry season samples. Lanes 4,8 and 10 are asymptomatic cases. The gel shows different allelic types o f M SP1 gene that were associated with malaria attacks. 60 University of Ghana http://ugspace.ug.edu.gh 1 2 3 4 5 6 M Figure 8: An illustration o f M SP1 allelic type in an asymptomatic subject during three consecutive sampling. The first three lanes represent amplification using outer primers and ensuing three lanes show amplification using inner primers. 61 University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE D ISC U S S IO N 5.1 Methodology 11 has been shown that the P C R method was used with success to dclecl P falciparum in blood spots on filter paper using a rapid and simple method o f boiling to isolate parasite D NA . The results obtained even showed ‘hat the fust round o f PC R using only the outer primers for M SP1 alleles detection was sensitive enough to analyse most o f the samples obtained by simple blood spot on filter paper. As shown in Figure 2 as low as 22 detect P. fa lciparum paras ites/jil were detectable in agarose gel and 2.2 parasites /pi in polyacrylamide gel confirming the superiority o f polyacrylamide gels over agarose gels (Dawson et al., 1996). In summary, the observed limits o f detection were between 0.1% - 0.01% parasitaemia using the outer primers. However, the minimum delectable number of I ' falciparum using outer primers was 25/pl obtained by others (N loum i et at. 1995). The nested P C R detected even lower levels o f parasitaemia than the first round PC R in conformity with Foley et a l.( 1992) who showed through a nested P I R the scaling down o f the minimum detection level from 500 parasite* (.d to 20(1 parasites/pl using the MSP1 as primer set. Using microscopy as the reference standard the PC R gave a relative sensitivity o f 99% and a relative 33.3% specificity when parasitaemia was as 2.2 - 62 University of Ghana http://ugspace.ug.edu.gh 22 parasites per microlitre o f blood. The nested P C R missed only two (2) microscopy - positive specimen (with 650 and 1400 parasites/|il). The reason for this is unclear as other samples with relatively lower levels o f parasitaemia were successfully amplified. There are several possible explanations as to why the P C R was not successful in the first instance. First, there could have been possible degradation o f genomic DNA . Secondly, the concentration o f the isolated parasite DNA might have been scanty below the detection limit o f PC R . The negative sample could have resulted from a mutation in the sequence targeted by the primers but was not investigated. It could also be attributed to very low levels o f parasitaemiae which was overestimated. There was one sample which was both negative by microscopy and PC R indicating the absence o f parasitaemiae in the blood sample. Two microscopy negative samples were found positive and this confirmed the superiority o f PC R in detecting parasitaemia (Snounou el a l., 1993). To summarise, 99% relative sensitivity and 33.3% specificity were obtained when PCR was performed using isolated parasite D N A from dried blood on filter paper. These levels were adequate for the present study. The use o f dried blood spot was an improvement over other studies that achieved similar results but used whole blood that needed thawing. 5.2 A lle lic forms PC R typing method was used to distinguish the different MSP-1 allelic types in the locality as shown in figures 5-7. It W'as observed that there were 35 MSP-1 allelic types similar to the 34 distinct MSP-1 alleles that were found in Senegalese children (Contamin el al., 1996). In addition and the most common allele o f D NA 63 University of Ghana http://ugspace.ug.edu.gh size (596bp) did not exceed 15% o f the total allelic types. This reflects the local transmission intensities o f circulating P. fa lciparum strains. It is possible that two parasites o f the same allelic size polymorphism would differ from each other when more than one locus is examined. It is also possible that certain alleles, which were identical in size, could have varied D N A sequences. Conversely, the allelic types observed could be invalidated by placing the same allelic type into different size classes, stemmed from the estimation o f the D N A fragment sizes o f P C R products in agarose gel with an accuracy range within 10- 15bp (Ranford-Cartwright el a l, 1993). The absolute sizes o f the P C R products were not critical to the interpretation o f the main study. Comparison between three consecutive blood samples (either asymptomatic, symptomatic and asymptomatic or three consecutive asymptomatic cases) were carried out by examining the P C R products run in adjacent tracks o f a single gel. Despite the limitation in estimating the DNA fragment sizes from a gel, the PC R products exhibited a wider range o f sizes from 513bp to 749bp when outer primers were used, the nested PC R ranged from 376bp to 531 bp. It was observed that 12.9% o f the samples showed two multiple fragments size (Tables 5A and 5B). These were all asymptomatic samples, a situation consistent with the switching over model where infection with a new strain leads to clinical episode (Jeffery, 1996). The multiple allelic infections in individuals (Results 4.3) are probably o f high transmission intensity in an area has been postulated by (Daubersies el al., 1996) and Paul el al. (1998). It might also be due to the chronic nature o f asymptomatic infections. 64 University of Ghana http://ugspace.ug.edu.gh 5.3 Seasonal variation As shown in Figure 5 and 7 there was seasonal variation o f allelic types. There were many allelic types during both dry and wet seasons. This confirms that there is substantial changes in composition o f the parasite population in peripheral circulation irrespective o f the season (Daubersies et a l., 1996, Paul et al., 1998). Interestingly, the estimated D N A fragment sizes o f the MSP-1 gene indicated no single allelic type contrasting with Toxoplasma gondii where the genetic make up o f virulent parasite is remarkably homogenous (S ib ley and Bothroyd, 1992). 5.4 Association of clin ical malaria w ith new allele The allelic types harboured by the children who initially were asymptomatic but suddenly became symptomatic in the subsequent month and then turned asymptomatic again after treatment revealed a change o f allelic types compared with the initial asymptomatic sample. However there is no significant association (X2, p= 0.134) o f acquisition o f new allelic type with clinical attack. This contradicts the assertion that the onset o f symptoms in chronically infected, previously asymptomatic individual, may be due to the introduction o f new parasite (Contamin et a!., 1996). The clinical attack might have culminated from the random feeding habits of the female Anopheles mosquito and lack specific virulence strains. It is also influenced by different parasite interaction in the individual which change the course o f infection (Riche, 1988, Snounou et al, 1992). It could be speculated that clinical malaria is mainly influenced by the cross-rcaction o f immunodominant epitopes and other repeated epitopes either within the same molecule or other parasite proteins (Theander, 1991). The cross­ 65 University of Ghana http://ugspace.ug.edu.gh reacting epitopes could interfere with the absorption o f antibodies that are not essential for the survival o f the parasite but divert immunological response from more important epitopes. This therefore promotes random advantage to any o f the infecting allele. Figure 4A for example supports the allelic changes that occur during malaria. Sample l (C B A ) showed reversion to the original asymptomatic allelic type a month after the treatment was observed. The possible but alternate interpretations other than renewed inoculation, is that the allelic type originally present was either relatively resistant to the anti malarial drug or it was in competition with allelic types w'hich were dominant during malaria attack and hence escaped PC R detection. Sim ilarly, in 13 (CBA ) the D N A fragment size o f the original asymptomatic sample was different from the symptomatic sample. However one month after the treatment the DNA fragment size found in the blood sample had the same molecular size as the symptomatic sample. In addition, recrudescence as a result o f drug resistance or inoculation o f the same allelic type granting that equal molecular weight sizes correspond to genetic homogeneity could also be the cause. 5.5 Synchronization of high parasitaem ia w ith malaria The striking revelation from this study was the association o f malaria episode w'ith high parasitaemia (see Tables 4A, 4B, 5A and 5B). The observation is compatible 66 University of Ghana http://ugspace.ug.edu.gh with the interpretation that clinical protection reduces with increased parasite density. This could stem from differences in the growth rate o f various allelic types, resulting from the poor fitness o f some types in certain children, such as poor invasion efficiency, impaired intraerythrocytic maturation, or slow replication rate and intense specific immune pressure, which would restrict parasite multiplication of certain allelic types while leaving other allelic types unaffected (Contamin et al., 1996). Increased parasitaemia is significantly associated with clinical malaria (p= 0.017). This implies that the activities o f non­ specific and specific immune responses lag behind the unrestrained multiplication of the parasite. No significant increase in parasite densities o f three consecutive asymptomatic infections (p= 0.105) supports that symptomatic cases are synchronised with high parasitaemia. However other factor such as age, cross-reacting epitopes, immunosupression, previous exposure can influence the course o f infection and the potential to cause clinical attacks (Theander, 1991; Ntoumi et a l, 1995). 5.6 A n ti-M SP l antibodies responses Elucidation o f the respective contribution o f species-specific and nonspecific responses to control parasite propagation is a key element in our understanding o f protection against malaria. To date, this has been difficult to study because the immune effectors contributing to parasite clearance in humans are unclear, and as a consequence, the MSP-1 target antigen supposedly offering clinical protection (R iley et a l, 1991; 1993) was investigated to unravel the biological role merozoite surface protein-119 plays in the development o f clinical protection from malaria 67 University of Ghana http://ugspace.ug.edu.gh attack. Antibody production during malaria parasite infection is age dependent in endemic population (Ntoumi et al., 1995). Children develop antibodies against variant epitopes whereas adults develop antibodies against less but conserved epitopes (reviewed Theander, 1991). This explains why three children in the age group o f 3-4 years showed 75% negative to positive sero-conversion in contrast to 22% positive to negative sero-conversion showed by those in the age group Z 5 years after malaria attack. It confirms Theander (1991) assertion that the rate of IgG synthesis is three times as high in unprotected individuals than protected one, in endemic areas and also seven times higher in non-immune people. It is believed that those in the age group o f > 5 years have relatively matured non­ specific immune systems. This accounts for the reduced anti-M SPl production after malaria attack because the parasitized cells can be eliminated mainly by various leucocytic responses (Sheagren, et a !., 1970). Another possible explanation to the lost anti-MSPl antibody production after malaria attack in two children in the age group > 5years could be attributed to low immunogenicity of epitopes within C-terminal region o f MSP1 or lack o f adequate T-cell help for antibody production (Andrea et al., 1997; George et a l., 1996; Venkatachalam et al., 1995). Also the tendency to respond or not to respond to the C-terminal part o f the MSP1 could depend on the host factors and the parasite ability to suppress the host immune response. Some MSP1 antibodies serve as smoke screen preventing the body from recognising the particular part o f the molecule involved in 68 University of Ghana http://ugspace.ug.edu.gh protection (Bouharoun-Tayoun and Druilhe, 1992). It may be possible that cross­ reaction between different parasites or within the same parasite molecule simultaneously infecting the same individual (R iche, 1988; Snounou et al., 1992, Anders, 1986) resulted in rapid decline o f the immune responses to produce antibodies (Staalsoe and Hviid, 1998). It could be speculated that those who lost antibody when they experienced malaria attack might have had the episode caused by mixed infection. In this case there were cross-reactions that interfered with the maturation o f the high affinity antibodies directed against M S P I 19 and with the absorption o f antibodies on the antigen, thus simply diverting immunological responses from more important epitopes (Theander, 1991). In summary, there was no significant correlation (p= 0.182) o f anti-MSPl antibodies production with adequate protective immunity. 69 University of Ghana http://ugspace.ug.edu.gh C H A P T E R S IX C O N C L U S IO N S A N D R E C O M M E N D A T IO N S 6.1 Conclusion In conclusion, the PCR-based assay showed a high relative sensitivity o f 99% with 33.3% specificity. The least detectable parasites per microliter o f blood was 2.2 using outer primer set. None o f the 35 M SP1 allelic types could be said to cause clinical malaria at all times. Though clinical malaria was associated with significantly high parasitaemia, there was no correlation o f acquisition o f new MSP1 allelic type with clinical malaria (p= 0.134). Similarly, anti-MSPl IgG antibody production was not associated with protection from malaria (p= 0 182) 6.2 Recommendations It is therefore recommended that: 1. Change in allelic type alone cannot does not appear to account for clinical malaria therefore any investigation related to strain specific malaria should take into account all other factors. 2. allelic type specific antigens should be developed to assay antibodies in order to determine the relationship between strain specific responses and clinical malaria. 70 University of Ghana http://ugspace.ug.edu.gh R E F E R E N C E S . Afari E .A ., Dunyo S.K ., Koram K .A . and Nkrumah F.K . (1992). Epidem iology o f malaria with special emphasis on transmission, morbidity, mortality and disease control in Ghana. 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Walsh S.P., Metzger and Higuchi R.( 1991). Chelex as a medium for simple extraction o f D NA for PCR-Based Typing from Forensic material Biotechniques. 10: 4: 506 - 513. World Health Organisation (W HO )( 1974).Serological testing in malaria Bu lle tin . 5: 209. World Health Organisation (WHO )(1993) Weekly I pidemiological Records J : I 7- 21. Wyler I)..I and I’asvol ( . j . (1986). Malaria: Perspective on recent developments M edical M icrobiology I7. 65-109. 87 University of Ghana http://ugspace.ug.edu.gh Appendix 1 Standard Solution. The following standard solution were prepared using sterile double distilled water (sddw). Where appropriate, the solutions were autoclavcd at 121 lbs for 15 minutes in Eyela Autoclave(Rikikkaki, Tokyo). DNA Extraction. C H E L E X (20% ) 20g o f Chelex dissolved in some amount o f sddw and made up to 100ml. 5M HC1 7.68ml o f concentrated 11 Cl was measured and diluted to 100ml with sddw. 5M NaOH 4g of NaOH weighed and dissolved in sddw to make 100ml Solutions for Electrophoresis. Agarose gels. 1 Ox T A E buffer 242g In s base. 57.1ml glacial acetic acid. 100ml 0.5M I D TA . pi I adjusted to 7.7 (with glacial acetic acid) and the volume made to 1000ml ddw 1 Ox T B F 100g/l 1 ris base 55g/l Boric acid acid 9.3g/l N a ; ED TA .2H :0 in water pH 83 is reached when diluted to 1\ working solution (stored at room temperature) 88 University of Ghana http://ugspace.ug.edu.gh Polyacrylamide gel. 15. lg/l Tris base, 72.0g/l glycine in double distilled water (stored at room temperature) pH 8.3 was reached when diluted lx working solution. 30% Acrylamide /0.8 Bisacrylamide solution 90g Acrylamide. 2.4g bisacrylamide in 300ml sddw. Filtered and stored at 4°C . 5% Stacking gel solution (50ml). 8ml 30% Acrylamide / 0 .8% Bisacrylamide 6.25ml Tris-HCl, pH 6.8. stored at 4 °C 12.5% Separating (Resolving) gel solution (30ml). 15.0ml 30% Acrylamide/0.8% Bisacrylamide solution. 3.75m! Tris HC1 (pH 8.8), 30mg Ammonium Persulphate. 20j.il T EM ED . Gel loading Buffer 89 University of Ghana http://ugspace.ug.edu.gh 5xOrange G. 20% (vv/v) Ficoll, 25mM ED TA , 2 .5% (w/v) orange G. Stored at room temperature. S ilver staining solution. Fixative 10% EtOH: 0.5% glacial acetic acid. Stored at room temperature. Staining solution. 0.984g AgNO i in 500ml d d w (l Im M ). stored in the dark at room temperature Developing solution 15ml 5M NaOH, 0.8ml Formalin made up to 100ml with sddw (Freshly prepared). Quenching solution 10% glacial acetic acid. Stored at room temperature. Solutions for E L IS A . Coaling buffer (Bicarbonate Buffer pi 1 9.8) NajCO;, l - 5 g NaHCO- 2-93g NaN^ 0.20g Distilled water to 1000ml (stored al 4 °C .) Phosphate buffer saline- 1 ween ( washing buffer pH 7.4) 90 University of Ghana http://ugspace.ug.edu.gh NaCl 8.0g K lU ’O, 0.2g Nal II’O , 1211,0 2.9g KC1 0.2g NaNj 0.2g Tween-20 0.5g Distilled water to 1000ml (stored at 4°C.) 91 University of Ghana http://ugspace.ug.edu.gh Log .M ol. V /ei ght . Append ix 2 A graph o f Log molecular weight against distance(s)/ cm travelled by the DNA fragments. 3.5 3.0 2.5 2 0 D i s t a n e e / c m 92 University of Ghana http://ugspace.ug.edu.gh