Malaria parasites are responsible for an increase in the morbidity and mortality in several tropical regions in Southern Africa. In this thesis, research was undertaken on Plasmodium, which is responsible for more than 95% of these cases. Although pharmacological prophylaxis is available, a worldwide resistance against existing drugs have been encountered. A study on chloroquineresistance in North-Eastern Transvaal in 1988, indicated that 11% of the strains in this area were resistant against chloroquine. Only 78% of these parasites were sensitive to the new drug, Mefloquine, which could serve as a substitute in chloroquine-resistant infections. Furthermore, no strain was found to be resistant or insensitive to both anti-malarial drugs. Malaria parasites can be obtained for research purposes from long term in vitro cultures. The initiation of cultures of some wild isolates of P.falciparum appears to be problematic at certain levels above sea-level. The gas composition of the medium was identified as a probable cause due to its dependence on the partial pressures of gasses at different heights above sealevel. The toxic effect of a too high oxygen concentration on the parasite, caused by the lower atmospheric pressure of the Highveld area, was prevented after the concentration of dissolved carbon dioxide in the medium was increased through equilibration with the special gas mixture. Possible shortcomings in the long-term culture method for malaria parasites that could retard optimum growth, were also investigated. Local parasite isolates could be supported on medium enriched with bovine serum although the growth rate was lower than when human serum was used. By increasing the frequency of medium replacement with progressing parasitemia, less stress were placed on the system since parasites was exposed for shorter periods and to lower concentrations of byproducts such as lactic acid. In addition, the ATP-concentration in infected cultures decreased by nearly 50 % over a growth period of 4 days. The stress in the host cell was reflected by the decrease in the total adenyl-nucleotide pool and the increase in AMP-concentration. An increase in the intracellular IMP-concentration indicated that the purine salvage pathway was inhibited which may explain the decrease in the ATP-concentration. It therefore appears that the regular replacement of medium and replenishment of erythrocytes only partially contribute to the successful establishment of malaria cultures. More research is necessary to identify the factors that are responsible for the inhibition of the purine salvage pathway. The stress placed on the human body by the parasite is complicated by the thrombocytopenia observed in some infections. Increases in the concentrations of thrombocyte-associated immunoglobulin G and M which are a characteristic for this condition, can be determined by a modified microo-method developed in our laboratory. By monitoring the parasite-infected patient over a period of time with the micro-method, the increase in thrombocytes and decrease in thrombocyte-associated antibodies were correlated with the recuperation of the patient. This method does not destroy the thrombocytes, thereby allowing displacement studies to be undertaken with purified parasite antigens of synthetic peptides. An investigation of parasite-infected erythrocytes by means of light microscopy, transmission and scanning electronmicroscopy, indicated that the local isolate could be composed of a mixture of strains, of which some have the ability to induce knobs on the erythrocyte. Furthermore, the investigation illustrated that fast fixation with gluteraldehyde is superior to slow fixation when transmission electronmicroscopy is performed. However, no difference could be observed when scanning electron microscopy was performed on infected erythrocytes that had been fixed by either of these methods. AFRIKAANS : Malaria parasiete is verantwoordelik vir 'n toenemende morbiditeit sowel as mortaliteit in verskeie tropiese streke in Suider-Afrika waarvan meer as 95 % van die gevalle deur Plasmodium falciparum veroorsaak word. Navorsing in die tesis is gevolglik op hierdie parasiet toegespits. Ten spyte daarvan dat far'makologiese profilakse toegepas word, kan malaria nog steeds opgedoen word as gevolg van n wereldwye weerstandigheid teen bestaande geneesmiddels. 'n Ondersoek van die klorokien-weerstandigheidstatus in die Noord-Oostelike Transvaal het in 1988 getoon dat 11 % van die stamme in die area weerstandig is teenoor klorokien. Slegs 78% van die parasiete was sensitief vir die nuwe middel, Meflokien, wat as moontlike plaasvervanger in klorokien-weerstandige parasietinfeksies gebruik kan word. Geen parasietstam was egter weerstandig of onsensitief teen beide middels nie. Langtermyn kultuurkweking van malaria parasiete is essensieël vir die verkryging van uitgangsmateriaal vir navorsing op die parasiet. Dit blyk egter dat inisiasie van kulture van wilde stamme van P. falciparum problematies by sekere hoogtes bo seespieël is. Die gassamestelling van die medium is as 'n moontlike oorsaak gëidentifiseer aangesien dit afhanklik is van die parsiële drukke van gasse by verskillende hoogtes bo seespieël. Die toksiese effek van te hoë suurstofkonsentrasies op die parasiet a.g.v. die laer atmosferiese druk op die Hoëveldstreek, is oorkom deur die konsentrasie van opgeloste koolsuurgas in die medium te verhoog deur vooraf 'n spesiale gasmengsel daardeur te borrel. Moontlike tekortkominge in die langtermyn kultuur metode vir malaria parasiete wat optimale groei kan belemmer, is ook ondersoek. Plaaslike parasietstamme kon op medium wat verryk is met beesserum onderhou word, alhoewel die groeitempo laer is as wanneer mensserum gebruik word. 'n Toenemende tempo van mediumvervanging soos die parasitemia in kulture toeneem, het getoon dat minder stres op die sisteem geplaas word deurdat die parasiete vir 'n korter periode en aan laer konsentrasies van byprodukte soos melksuur blootgestel word. Die ATP-konsentrasies in gëinfekteerde kulture daal egter met ongeveer die helfte oor 'n groeiperiode van 4 dae. Tesame hiermee, is daar ook 'n daling in die totale adeniel-nukleotied poel van die gëinfekteerde rooibloedsel en 'n styging in die AMP-konsentrasie wat die stres in die gasheersel weerspieël. 'n Verhoging in die intrasellulêre IMP-konsentrasie dui op 'n moontlike inhibisie van die purienherwinningspadweg wat die verlaagde ATP-konsentrasie mag verklaar. Uit die studie blyk dit dus of meer gereelde vervanging van medium en toevoeging van rooibloedselle slegs 'n gedeeltelike bydrae maak tot die suksesvolle vestiging van malariakulture. Die identifikasie van die faktore wat lei tot die inhibisie van die purienherwinningspadweg, verg nog verdere ondersoeke. Die stres wat die parasiet in die menslike liggaam veroorsaak, word ook weerspieël deur onderandere die trombositopenie wat in sommige infeksies waargeneem word. Verhogings in die konsentrasie van trombosiet-geassosieërde immunoglobuliene G en M wat kenmerkend is van die toestand, kan gemeet word deur 'n gemodifiseerde mikrometode wat in ons laboratoriums ontwikkel is. Deur die parasiet-gëinfekteerde pasiët oor 'n paar dae met die mikrometode te monitor, kon die verhoging in trombosiete en afname in trombosiet-geassosieërde antiliggame met herstel van die pasiënt gekorreleer word. Die metode veroorsaak geen skade aan die trombosiete nie sodat antigeenverplasingstudies ook na die tyd gedoen kan word. In 'n ondersoek van die parasiet met behulp van ligmikroskopie, deurstraal- sowel as skandeer-elektronmikroskopie, is gevind dat die plaaslike isolaat moontlik uit 'n mengsel van stamme bestaan, waarvan sommige die vermoë het om knoppe op die rooibloedsel te induseer. Verder het die ondersoek getoon dat indien deurstraal-elektronmikroskopie gebruik word, 'n vinnige fikseringsmetode met gluteraldehied beter is as 'n stadige fikseringsmetode. In vergelyking hiermee kon geen verskil waargeneem word met skandeer-elektronmikroskopie van geparasiteerde rooibloedselle wat met enige van hierdie metodes gefikseer is nie. Copyright / Dissertation (MSc)--University of Pretoria, 1992. / Biochemistry / unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/28155 |
| Date | 11 February 2013 |
| Creators | Stoltz, Anton Carel |
| Contributors | Louw, Abraham Izak, Verschoor, J.A. (Jan Adrianus), 1953-, upetd@up.ac.za |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Detected Language | Unknown |
| Type | Dissertation |
| Rights | © 1992, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria |
Page generated in 0.004 seconds