Evaluation and validation of methods to determine parasitemia in malaria cell cultures
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The success in the treatment of malaria is dependent on the prompt and accurate diagnosis of malaria and effectiveness of the drug regime used. The emerging of resistance to most antimalarial drug makes the treatment of malaria difficult. The need for more effective mono and combination therapy is necessary to eradicate malaria. Pheroid™ technology was used to evaluate the drug efficacy of entrapped drugs to possibly overcome this difficulty. Pheroid™ has the ability to delivery. Pheroid™ has a high affinity for cell membranes resulting in effective and fast delivery of the drug beyond the mechanism of drug efflux pump (Grobler, 2004) enhancing the therapeutic effect. To successfully evaluate the effect of Pheroid, it was necessary to validate the methods used to determine the drug efficacy. The methods used in this study was determining the percentage parasitemia giemsa stained thin blood smears through microscope evaluation, the colorimetric evaluation of pLDH and flow cytometric evaluation using the DNA specific dye acridine orange (AO). Even though microscope evaluation is relatively simple and a cheap method, it is time consuming, labour intensive and subjective. To overcome these limitations, this study focused on the development and validation of alternative methods to evaluate drug efficacy test in Plasmodium falciparum cultures. The validation is a combination of determining the selectivity, accuracy, precision, sensitivity and repeatability of these methods. The colorimetric evaluation of pLDH is a selective method to determine the presence of P. falciparum in samples. The accuracy and precision is relatively good at high parasite densities but there is a decrease in the accuracy and precision at low parasite densities. This means that the method is only sensitive at a minimum parasitemia of 1%. There are a lot of different factors, like the haemoglobin, haematocrit and Pheroid™ that have an influence on this method that can possibly lead to the low sensitivity. This method doesn't comply with the FDA standards. This method was therefore not used in determining the parasite levels in drug efficacy tests. The flow cytometric evaluation of Plasmodium infected erythrocytes used the DNA specific fluorochrome AO. The absence of DNA and RNA in erythrocytes makes it possible that AO can selectively bind to the DNA and RNA of the parasite. The accuracy and precision of the method complies with the criteria of the FDA. The method is sensitive to a quantitatively parasitemia of 0.2%. The method can be repeated on different day with high accuracy. This method does meet the specific criteria of the FDA for validation of the method. The three methods were compared with the theoretical values and with each other. The results showed that there is a very low correlation between theoretical values and the methods for the microscope evaluation (r = 0.20). The correlations for the pLDH were between 0.59 and 1.00 and for the FACS were between 0.95 and 1.0. This showed that the pLDH and FACS is more accurate than the microscope evaluation. The correlation between the slides and pLDH was 0.50 and for the FACS 0.30. This weak correlation is an indication of the low sensitivity of the microscope evaluation. The correlation between pLDH and FACS for high parasite levels was 1.0 and for the low parasite densities -0.10. This implies low sensitivity of the pLDH method. The FACS overall had the best correlation values. This method was able to successfully differentiate between the ring and schizont phase of the parasite. This method was used in drug efficacy tests of mefloquine in combination with Pheroid™ microsponges. To optimize the possible enhancement of the antimalarial drug in combination with Pheroid™ different Pheroid™ ratio was tested at different chloroquine concentrations. The results showed that Pheroid™ had an overall lower percentage parasitemia compared to the control. The best inhibition of parasite growth was at the 1:1750 ratio. There were a 55.43% and a 79.43% decreases in parasite growth at the 100 nM and 200 nM concentration respectively. A 1:1500 was used in the drug efficacy tests of mefloquine and Pheroid™. Different formulation of Pheroid™ and mefloquine was tested in vitro to determine the efficacy. As a control, mefloquine in water was tested and showed no significant decrease in the parasite growth. When no entrapment of mefloquine in the Pheroid occurred, there was only a slight decrease from 5.93% to 5.63% in the parasitemia. The mefloquine in Pheroid left for 24 hours to entrap showed a decrease of 3.96% to 3.79%. There wasn't a significant difference between the Pheroid™ mefloquine formulation and the control. Mefloquine is lipid soluble and has difficulty to dissolve in water. When an organic solvent like ethanol is added, the solubility of mefloquine may increase. The efficacy of mefloquine entrapped in Pheroid™ for 24 hours in combination with ethanol showed no difference between the formulation containing the solvent and the formulation without the solvent. Pheroid™ consists of a water and lipid phase. This makes it possible to incorporated lipid soluble drugs, like mefloquine in the Pheroid™ during manufacturing. The drug efficacy test done on this formulation showed a significant difference between the Pheroid formulation and the control. The Pheroid™ formulation showed a decrease from 2.38% at a 0 nM to a 1.03% at a 200 nM mefloquine concentration. The 50 nM showed a 30.57% inhibition in growth increasing to 46.63% at 200 nM. The IC50% value for the control was 70.53 nM and for the Pheroid formulation only 15.95 nM. This showed that mefloquine entrapped in Pheroid™ do have a better efficacy compared to the control. Pheroid™ entrapped with mefloquine enhances the effect of drug for better inhibition of parasite growth. This study concluded that the percentage parasitemia can be accurately determined by flow cytometry using acridine orange. Pheroid™ entraps the mefloquine and enhances the antimalarial effect of the drug against Plasmodium falciparum during in vitro studies. Future prospects that came to light during this study were: ♦ To incorporate other antimalarial drugs, mono and combination therapy, into the Pheroid™ to evaluate the drug efficacy. ♦ In vivo studies to determine the efficacy of the drugs in Pheroid™ and to determine pharmacokinetic properties. ♦ Toxicity studies of the drugs entrapped in Pheroid™. ♦ Optimization of the pLDH method and other dyes that can be used in flow cytometry to evaluate parasite densities. All these studies are part of the process to be able to put a new product for the treatment of malaria on the market to help fight against this global killer.
- ETD@PUK