WO2005035783A1

WO2005035783A1 - Fluorescense based bioassay for anti-parasitic drugs

Info

Publication number: WO2005035783A1
Application number: PCT/US2003/034513
Authority: WO
Inventors: Eduardo Ortega-Barria; Yolanda Corbett Rodriguez; Liuris Herrera Valderrama; Jose Del C. Gonzalez; Todd L. Capson
Original assignee: Smithsonian Tropical Research Institute
Priority date: 2003-09-11
Filing date: 2003-10-30
Publication date: 2005-04-21
Also published as: AU2003287290A1

Abstract

A microfluorimetric assay measures the inhibition of Plasmodium falciparum and other parasitic diseases based on the detection of parasitic DNA e.g. by intercalation with PicoGreenè. The method is used to determine parasite inhibition profiles and IC50 values of known or potential anti-malarial drugs. Values for parasite inhibition with known anti-malarial drugs using the PicoGreenè assay were comparable with those determined by the standard method based upon the uptake of [3H]hypoxanthine and the Giemsa stain microscopic technique. The PicoGreenè assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for anti-malarial drug development and parasites that may be cultured axenically.

Description

Fluorescence Based Bioassay For Anti-Parasitic Drugs Background of the Invention

Field of the Invention The present invention relates to novel screening methods for the detection of anti-parasitic activity of drugs. More particularly, the invention is a fluorimetric method for drug screening utilizing a fluorescent probe that intercalates into parasite DNA in red blood cells and for parasites that can be cultured axenically (i.e., cultured in the absence of host cells). Back round and Related Art Malaria is among the most life-threatening and widespread diseases in the world, causing 250-300 million cases and about 2 million deaths annually. (Greenwood B and Mutabingwa T, 2002. Malaria in 2002. Nature 415: 670-672.) The disease is caused by four Plasmodium species (i.e. P. falciparum, P. vivax, P. ovale and P. malariae) which are transmitted to humans during the bite of the female anopheles mosquito. Malaria is endemic in many parts of the African, Asian and American continents. The growing resistance of the parasites to treatment with known anti- malarial agents such as chloroquine is of grave concern and is responsible for some of the worst cases of malaria in the tropical world. (Riddley RG, 1999. Science 285: 1502-1503). The spread of resistance of the mosquito vector to currently available insecticides and the limited success of potential anti-malarial vaccines contributes to the urgent necessity of finding new chemotherapeαtic agents for the treatment of malaria, in particular, agents effective against P. falciparum, the strain responsible of the most severe forms of malaria. The standard test for screening potential drugs for anti-plasmodial activity is a radioactivity-based method that relies upon the incorporation of [³H]hypoxanthine into the parasite's DNA in order to measure parasitic replication in red blood cells. (Desjardins RE, Canfield CJ, Haynes JD and Chulay JO, 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 16: 710-718). This method is very sensitive and it can be used to screen a large number of compounds, but employs hazardous radioactive materials that require special facilities and procedures. In developing countries most affected by malaria, regulations for the importation, use and disposal of radioactive isotopes are frequently not in place, nor is the equipment or infrastructure required for their use. Alternatives to the [³H]hypoxanthine-based methodology include a labor- intensive and time-consuming microscopic method and several colorimetric assays. (See, e.g., Makler MT and Gibbins BL, 1991. Laboratory diagnosis of malaria. Clin Lab Med 11: 941-956; Delhaes L, Lazaro JE, Gay F, Thellier M, and Danis M 1999; The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity. Annals Trop Med Parasitol 93: 31 -40; Makler MT and Hinrichs D J, 1993. Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. Am JTrop MedHyg 48: 205-210.) Existing colorimetric methods, however, are based on enzymatic activity rather than parasite replication, and in addition, may be subject to artifacts caused by pigments present in crude plant extracts that are frequently used in drug screening programs. A micromethod based on the fluorescence emitted by Plasmodium falciparum DNA in the presence of Hoechst 33258 has been previously reported (Smeijsters LJJW, ZijlstraNM, Franssen FFJ and Overdulve JP, 1996. Simple, fast, and accurate flourimetric method to determine drug susceptibility of Plasmodium falciparum in 24- well suspension cultures. Antimicrobial Agents and

Chemotherapy 40: 835-838). However, this method requires relatively large volumes of media, parasites and reagents, and is not suitably sensitive for the detection of parasite DNA in concentrations amenable to large scale drug screening. A DNA-fluorescent method employing ethidium bromide to monitor parasite growth was documented in 1986. However, this method was also less sensitive than the traditional radioactive method (Waki S, Tamura J, Jingu M, Adachi M and Suzuki M, 1986. A new technique for drug susceptibility tests for Plasmodium falciparum by ethidium bromide fluoroassay. Trans R Soc Trop Med Hyg. 80: 47-49). Traditionally, natural products have been a rich source of anti-plasmodial drugs, including quinine and artemisinin. Many natural product based drugs are derived from flora and fauna of biodiversity-rich developing countries in many of which malaria is endemic (See for example, Klayman DL, 1993. Artemisia annua, from weed to respectable antimalarial plant, h : Human Medicinal Agents from Plants. Kinghorn AD, Balandrin MA (Eds.) American Chemical Society 242-255; Munoz V, Sauvain M, Bourdy G, Callapa J, Bergeron S, Rojas I, Bravo JA, Balderrama L, Ortiz B, Gimenez A and DeHaro E, 2000. A search for natural bioactive compounds in Bolivia through a multidisciplinary approach Part I. Evaluation of the antimalarial activity of plants used by the Chacobo Indians. J Ethnopharmacol 69: 127-137; Riddley RG, 1999. Planting the seeds of new antimalarial drugs. Science 285: 1502-1503). Since the standard anti-plasmodial assay is based on the use of radioactive isotopes, the same biodiversity-rich developing countries are often not in a position to develop anti-malarial drug discovery programs, limiting access to a large pool of scientific talent and emphasizing the need to develop cost-effective techniques that do not require the use of radioactive isotopes. (Kursar TA, Capson TL, Coley PD, Corley DG, Gupta MB, Harrison LA, Ortega-Barria E and Windsor DM, 1999. Ecologically guided bioprospecting in Panama. Pharmaceut Biol 37(Suppl): 114-126.) Summary of the Invention The present invention is a new, straightforward, efficient and accurate method for the detection of anti-malarial agents based on the intercalation of a fluorochrome or fluorophore, e.g., PicoGreen®, into Plasmodium DNA and the dsDNA of parasites that can be cultured axenically. PicoGreen® is an ultrasensitive fluorescent nucleic acid stain for measuring double-stranded DNA (dsDNA) in solution and enables the detection of quantities as low as 25 pg/mL of dsDNA with a moderately priced spectrofluorometer using fluorescein excitation and emission wavelengths [product information available from Molecular Probes (http://www.probes.com/media/pis/mp07581.pdf)]. Accordingly, the microfluorimetric method of the invention is ideally suited for anti-malarial drug discovery programs based in developing nations. The microfluorimetric assay measures the inhibition of Plasmodium falciparum based on the detection of parasitic DNA by intercalation with PicoGreen®. The method was used to determine parasite inhibition profiles and IC₅o values of known or potential anti-malarial drugs. Values for parasite inhibition with known anti-malarial drugs using the PicoGreen® assay were comparable with those determined by the standard method based upon the uptake of [³H]hypoxanthine and the Giemsa stain microscopic technique. The PicoGreen® assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for anti-malarial drug development. The microfluorimetric method for detecting anti-plasmodial compounds of the present invention has several advantages over the traditional assay that monitors the incorporation of [³H]hypoxanthine by the parasite. (Desjardin et al. 1979) The radioactivity-based method requires the use of an expensive, hazardous radioactive compound, costly liquid β-scintillation counter equipment and special local regulations for the introduction, management, and disposal of radioactive waste. The present invention overcomes disadvantages in the prior art previously thought to be insoluble. The lack of accessible and appropriate technology that would permit the efficient testing of biological materials for anti-plasmodial activity has been an impediment for the development of drug discovery programs in developing countries. Although several nonradioactivity-based methods have been developed over the years, they are cumbersome, multistep procedures. (Makler MT and Gibbins BL, 1991. Laboratory diagnosis of malaria. Clin Lab

Med 11: 941-956; Delhaes L, Lazaro JE, Gay F, Thellier M, Danis M, 1999. The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity. Annals Trop Med Parasitol 93: 31-40.) The invention is a method for evaluating antiparasitic activity of a candidate material, such as a crude extract or a pure chemical compound, by providing a sample that contains a viable parasite that is (a) axenic and the sample is cell free, or (b) infects red blood cells and the sample incluyes red blood cells. The sample is combined with the candidate material and incubated with a fluorochrome that binds to parasite DNA within the sample and provides a fluorescent signal proportional to the extent of parasitemia. The fluorochrome has a sensitivity equivalent to conventional techniques based on radioactivity and microscopic counting between a range of 0.1 and 15% of ring stage infected erythrocytes, provides comparable sensitivity to conventional techniques for purified compounds and is qualitatively comparable to radioactive techniques when detecting anti-plasmodial activity in crude plant extracts. An example of a fluorochrome is PicoGreen®. The fluorescence of the fluorochrome in the simple is measured using a fluorescence reader, such as a fluorescent plate reader or a minifluorimeter, and a determination is made as to whether the candidate material significantly reduces the fluorescent signal. The reduction in fluorescente is indicative of antiparasitic activity. Where the parasite infects red blood cells and the sample comprises red blood cells, the fluorochrome does not bind to red blood cells. Examples of parasites include Plasmodium sp., Babesia, Leishmania sp. and Trypanosoma sp., Entamoeba sp., and Giardia lamblia. The candidate material can be incubated for about 24-48 hours before incubating with the fluorochrome, and for about 5-30 minutes with the fluorochrome. In an exemplary embodiment, a plurality of samples can be incubated simultaneously in a multivessel container, such as a 96 well plate. Where the fluorochrome is PicoGreen®, fluorescence is measured at about 485/20 nm excitation and about 528/20nm emission. Suitable fluorochrom.es can be selectively binding to dsDNA, can quantitate dsDNA at levels of about 25 pg/mL dsDNA, have a low level of interference due to signal from substances other than dsDNA, provide a high signal to noise ratio, be stable to photobleaching, have low toxicity, be safe and be available at a low cost. A control with a known antiparasitic agent. For example, where the parasite is Plasmodium, the antiparasitic agent can be an anti-malarial drug. The method is also useful as a method of assay-guided fractionation/purification of plant material or extracts and as a method for diagnosing the susceptibility of a patient to a particular antiparasitic therapy by isolating the infecting parasite and analyzing it. The invention can be provided as a kit for evaluating antimalarial drug activity, wehre the kit includes standardized preparations of Plasmodium, erythrocytes, PicoGreen, and an anti-malarial drug. Brief Description of the Drawings Figure 1 is a comparison of the results obtained from the present invention using fluorescence intensity with results obtained by microscopic counting. Figure 2 shows the results of time course experiments with P. falciparum- infected erythrocytes by microscopic counting and the microfluorimetric techniques. Figure 3 shows a graphical determination of IC_SQ values for chloroquine by the incorporation of [³H]-hypoxanthine (Figure 3a) and the microfluorimetric technique of the present invention (Figure 3b). Detailed Description of the Invention Embodiments of the invention are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected.

All references cited herein are incorporated by reference as if each had been individually incorporated. An exemplary embodiment of the invention is discussed in detail below. While specific exemplary embodiments are discussed, it should be understood that this is done for illustration purposes only. A person skilled in the relevant art will recognize that other components and configurations can be used without parting from the spirit and scope of the invention. The method of the present invention is based upon the detection of Plasmodium or other parasitic DNA in short-term cultures using a 96-well format, allowing the efficient and quantitative measurement of anti-plasmodial activity in a large number of samples. The method employs a fluorophore that intercalates into the dsDNA of Plasmodium DNA in solution. For example, PicoGreen®, an ultrasensitive fluorophore, enables detection of as little as 25 pg/ml of dsDNA, a 400-fold increase in sensitivity compared to the DNA intercalator, Hoechst 33258 (Molecular Probes Product information). The method of the present invention is straightforward and rapid. The parasites are first incubated with the test dru^for 48 hours, followed by addition of the fluorophore, followed by a 5-30 minutes incubation period prior to the measurement of fluorescence. The assay protocol presented herein is simpler than that for Hoechst 33258 since there is no requirement to remove potentially interfering compounds such as hemoglobin and hemozoin, nor is there a chloroform extraction step to prevent quenching of fluorescence. (Smeijsters LJJW, ZijlstraNM, Franssen FFJ and Overdule JP, 1996. Simple, fast, and accurate fluorimetric method to determine drug susceptibility of Plasmodium falciparum in 24-well suspension cultures. Antimicrobial Agents Chemother 40: 835-838.) The replication of the parasite is directly proportional to the amount of fluorescence, with a linear relationship between 0.1 and 15% of parasitemia. No significant differences are observed between synchronized and non-synchronized parasites. In addition, the samples can be stored at -20°C and read at a more convenient time without a significant change in the fluorescence signal. Significantly, if a fluorescence microplate reader is not available, determination of parasite growth may be achieved with a less-expensive minifluorimeter, for example Mini-fluorimeter TKO 100 (Hoefer Scientific h st., San Francisco, CA). In comparing the microfluorimetric methodology of the invention with the conventional radioactivity-based assay in testing crude plant extracts for anti- plasmodial activity, both methods yielded identical results with respect to their ability to detect extracts with anti-plasmodial substances. However, small differences have been observed between the calculated IC50 of crude plant extracts as determined by the two methods. One possible explanation for these differences is the presence of low levels of interfering substances in the extracts. Provided the experimentalist can detect anti-plasmodial activities in plant extracts that contain compounds of interest, and obtain reliable comparisons between extracts from different species, precise IC5₀ values for plant extracts is not a necessary requirement for drug discovery. Importantly, no significant difference in IC₅₀ values were observed between the two methods when pure compounds (chloroquine and mefloquine) were tested, supporting the utility the present invention as an assay for quantifying anti-plasmodial activity of drugs. The microfluorimetric method of the invention has been used successfully to detect plant extracts that contain compounds with anti-plasmodial activity and to subsequently guide the purification of the biologically active compounds. It is hoped that the development of an effective and straightforward method for measuring anti-plasmodial activity that does not utilize radioactive isotopes will stimulate anti-malarial drug discovery programs in a number of countries, in particular, those most affected by parasitic disease. Other fluorophores can be suitable for drug screening according to the present invention. Suitable fluorophores should bind to parasite DNA within infected mammalian red blood cells, or bind to DNA from parasites in axenic culture, to provide a suitable signal. The binding will depend upon the particular parasite being studied. In exemplary embodiments, the reaction between the fluorophore and the DNA should be rapid and irreversible. Other desirable characteristics for fluorophores include: selective binding to dsDNA, high sensitivity, low level of interference due to signal from substances other than dsDNA, high signal-to-noise ratio, stability to photobleaching, low toxicity, good safety characteristics and low cost. The present invention can be used to screen drugs for the treatment of other parasitic infections as well. The parasites should be capable of being cultured in red blood cells which do not have any DNA which would otherwise interfere with the assay. In addition, the invention can be used to screen drugs using other microorganisms capable of growing in axenic cultures (i.e., cultured in the absence of host cells), including Leishmania sp., Trypanosoma sp., and Entamoeba sp. and Giardia lamblia. Babesia sp. is a tick-transmitted protozoal parasite that is the causative agent of babesiosis, a disease that may produce malaria-like symptoms and hemolytic anemia. Asplenic, elderly, and immunocompromised patients are at greatest risk for severe disease although babesiosis can also be serious in immunologically normal persons. Babesiosis is emerging as a disease of public health significance in the U.S. A, with increased reports of clinical, even fatal, cases in areas where the risk of infection with Babesia sp. was not recognized previously. Until recently, human babesial infections in the United States have been attributed to Babesia microti, derived from rodents. There is increasing evidence to suggest that human babesiosis in the United States may be caused by babesial parasites that are antigenically and genotypically distinct from Babesia microti. The epimastigote and promastigote forms of Leishmania sp. are the forms of the parasite that multiply in the digestive tract of the triatome vector and tissue of the infected host, respectively. Both the epimastigote and promastigote forms of the parasite can be can be cultured axenically. The trypomastigote of

Trypanosoma sp. is found in the tissue and bloodstream of infected animals and is responsible for the spread of the infection from cell to cell. It is also the form that is transmitted by the insect vector. The methodology is readily applicable to parasite forms that can be cultured axenically for the discovery of treatments for the diseases caused by these parasites, i.e., leishmaniasis and trypanosomiasis. Collectively, malaria, Chagas' disease and leishmaniasis affect 3 billion people, most of whom survive on less than $2 a day (Gelb MH and WGJ Hoi. 2002. Drugs to combat tropical protozoan parasites. Science 297: 343-344). For most parasitic diseases, drugs remain the mainstay of control (Modabber F: Eleventh Programme Report of the UNDP World Bank/WHO Special Programme for Research and Training in Tropical Diseases, TDR, 2002). The present invention is not limited to these organisms. Persons of ordinary skill in the art will recognize other organisms for which the presently described methodology can be applied. EXAMPLES Cultivation of malarial parasites. Two chloroquine-sensitive (Sierra Leone clone D6 and Tanzania F32) strains and a chloroquine-resistant (Indochina clone W2) strain of Plasmodium falciparum were used. The D6 clone was a gift from Philip J. Rosenthal from the Division of Infectious Diseases at the University of California at San Francisco. The W2 clone was a gift from Dennis Kyle of the Division of Experimental Therapeutics at the Walter Reed Army Institute of Research, and the F32 strain was a donation from Eric DeHaro, from the IRD group in the Institution de Investigaciones Farmaco Bioqufmicas, Universidad Mayor de San Andres, La Paz, Bolivia. The three strains were maintained in vitro by a modification of the method of Trager and Jensen. (Trager W and Jensen JB, 1976. Human malaria parasites in continuous culture. Science 193: 673-675.) The culture media consisted of standard RPMI 1640 (GIBCO Laboratories) supplemented with 10% heat- inactivated human type O+ serum (Valley Biomedical, Inc.), 25 mM NaHCO₃, 2 mM Glutamine and 25 mM N-2-hydroxythylpiperazine-N-2-ethanesulfonic acid (HEPES) (Sigma Chemical Co.). Cultures were maintained in type O+ human red blood cell suspensions, obtained from healthy local donors, prepared in citrate- phosphate-dextrose anticoagulant (CPD) (Sigma Chemical Co.) at a hematocrit of

2%. The parasite density was maintained below 2% parasitemia under an atmosphere of a certified gas mixture containing 5% CO₂, 5% O₂, and 90% Ν₂ at 37°C. For each experiment, samples of stock cultures were further diluted in culture medium containing sufficient noninfected type O+ human erythrocytes to yield a final hematocrit of 2% and parasitemia of 1%. All assays were carried out in microtiter plates. For those cases in which assays were synchronized, sorbitol was employed. (Lambros C and Vanderberg JP, 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418-420.) Preparation of crude plant extracts and microtitration plates. Plant samples were prepared according to standard protocols. (Montenegro H, Gutierrez M, Romero LI, Ortega-Barria E, Capson TL and Cubilla-Rios L. 2003. Aporphine alkaloids from Guatteria sp. with antileishmanial activity. Planta Medica 69: 677- 679). Lyophilized crude extracts were provided in individual vials of 3 mg (dry weight) and stored at -20 °C until ready for testing. Crude extracts and partially- purified fractions were dissolved in DMSO (Research Organics) at a stock concentration of 50 mg/mL. Known antimalarial compounds were dissolved in distilled water or ethanol according to published methods (DeHaro E, Gautret P, Munoz V and Sauvain M, 2000. Evaluation de la actividad antimalarica in vitro de productos naturales o de sintesis. In: Tecnicas de laboratorio para la selecciόn de sustancias antimalaricas. CYTED. 51-88; Basco LK, Marquet F, Makler MT and Le Bras J, 1995. Plasmodium falciparum and Plasmodium vivax: Lactate dehydrogenase activity and its application for in vitro drug susceptibility assay. Exp Parasitol 80: 260-271). Samples were tested in 96-well plates in duplicate at final concentrations of 50, 10 and 2 μg/mL and reevaluated at higher or lower concentrations when necessary. The final dilution contained less than 0.1% DMSO, which had no measurable effect on parasite survival in this system (data not shown). DMSO at a final concentration of 0.1% in RPMI 1640 culture media was used as negative control, and represented 100% parasite viability. The positive control consisted of chloroquine at concentrations of 1.0, 0.1 and 0.01 μg/mL, and provided a measure of the parasite's susceptibility to known antimalarial drugs, hi order to measure the effect of each plant extract alone on the fluorescence signal, each extract concentration was incubated in the absence of parasites and the signal was subtracted from the value obtained in the presence of drug and parasite. Data analysis. Data analyses were performed with a pre-programmed calculus sheet on Microsoft Excel 2000 that processes the relative fluorescence units exported through the KC junior® software from the microplate fluorimeter. The calculus sheet consists of: (a) a formula that calculated the mean of the two replicates per sample condition, (b) subtraction of the respective color background of each dilution of the plant extract, (c) conversion of the mean RFU value to percentage of the response, taking as 100% the mean of the negative control, and

(e) conversion of the percentage to IC₅₀ by Log-regression. In order to adjust for the potential contribution of the hemoglobin pigment from erythrocytes and the possible fluorescence from the intrinsic pigments present in some plant extracts, control wells were used which consisted of noninfected erythrocytes alone, and samples of diluted drugs or extracts with noninfected erythrocytes. The inhibitory concentration (IC₅₀) was defined as the drug concentration that results in 50% of the net fluorescence compared to nontreated control cultures. EXAMPLE 1 (Comparative) Radioactivity-based assay. Incorporation of [³H]hypoxanthine (specific activity 1.0 mCi/mL, American Radiolabeled Chemicals, Inc.) was used to measure growth of the parasites, as previously described by Desjardines et al. Different antimalarial compounds at final concentrations ranging from 1.95 nM to 2 μM were added in duplicate to flat- bottom 96 well microtiter plates (Corning Glass Works) in a final volume of 25 μL. A volume of 200 μL of the culture parasite was added to each well and the plate was then placed in a humidified airtight chamber (Bellco Glass Inc.) that was flushed with the gas mixture described above, sealed and stored in an incubator at 37°C for 24 hours. Each compound was tested on at least two occasions and against both chloroquine-sensitive and chloroquine-resistant strains. At the end of the incubation period, 25 μL of diluted [³H]hypoxanthine (1.5 μCi final concentration) was added to each well. The plates were then returned to the humidified airtight chamber, flushed again with the gas mixture described above, sealed, and incubated at 37°C for an additional 18 hours. The cultures were then harvested with a semi-automated PHD Cell harvester® onto fiberglass paper disks, washed with distilled water and fixed with ethanol. Each disk was placed in glass scintillation vials containing 2 mL of Micro stint scintillation cocktail

(Microscint-High Efficiency LSC-Cocktail) for 1 hour. The vials were then counted in a Packard microplate scintillation beta counter. The mean values for uptake of [³H]hypoxanthine in parasitized control and nonparasitized control erythrocytes were calculated. EXAMPLE 2 - Fluorimetric susceptibility test. Synchronized ring form cultures (hematocrit 2% and parasitemia 1%) were used to test pure compounds or serial dilutions of plant extracts in 96-well culture plates. Cultures of P. falciparum were placed in a humidified, air-sealed container, flushed with the gas mixture described under "Cultivation of parasites" and incubated at 37°C. Parasites were allowed to grow for a 48-hour incubation period, after which a 150 DL aliquot of culture was transferred to a new 96-well flat bottom plate. Fifty microliters of the fluorochrome mixture, which consists of PicoGreen®, 10 mM

Tris-HCl, 1 mM EDTA, pH 7.5 (TE buffer) and 2% Triton X-100, diluted with double distilled, DNAse-free water, was then added to liberate and label the parasitic DNA. The plates were then incubated for 5-30 min in the dark. The fluorescence signal, measured as Relative Fluorescence Units (RFU) was quantitated with a fluorescence microplate reader (FL_x 800, Bio-Tek Instruments,

Inc.) at 485/20nm excitation and 528/20nm emission. Simultaneously, the RFU from positive and negative control samples were obtained, stored, and analyzed. EXAMPLE 3 - Relationship between parasite number and fluorescence. Preliminary experiments demonstrated that serial dilutions of normal uninfected red blood cells did not emit significant amount of fluorescence when incubated in the presence of PicoGreen®, indicating that DNA from contaminating white blood cells and the hemoglobin pigment from erythrocytes does not interfere with the detection of Plasmodium DNA. In addition, serial dilutions of crude plant extracts, either alone or mixed with uninfected erythrocytes, also failed to produce significant fluorescence (data not shown) suggesting that any pigments associated with crude plant extracts do not interfere with the fluorescence signal associated with Plasmodium DNA. EXAMPLE 4 - Sensitivity. To test the sensitivity of the fluorimetric method as a means of detecting Plasmodium DNA in infected erythrocytes, the percentage of infected erythrocytes as determined by microscopic counting with results obtained from the fluorimetric technique were compared. Serial double dilutions of infected erythrocyte cultures were used to prepare Giemsa-stained thin smears and the percentage of parasitemia was then evaluated by light microscopy. Aliquots of the same or parallel cultures were mixed in a 96-well plate, with an equal volume of PicoGreen® cocktail and the amount of fluorescence was quantified. Figure 1 shows a comparison of the percentage of P. falciparum- infected erythrocytes as determined by microscopic counting with fluorescence intensity obtained from the microfluorimetric technique. A serial twofold dilution of a synchronized infected culture (15.0% ring stage) with noninfected erythrocytes was employed. Bars indicate the deviation of the mean for four independently processed samples. The inset shows the relationship below 1% of parasitemia. As can be seen in Figure 1, there is a direct relationship between the percentage of infected red blood cells and the fluorescence signal between 0.1 and

15%ι of ring stage infected erythrocytes (r=0.99). EXAMPLE 5 - Time course for the assessment of parasitemia. Time course experiments were then performed in which cultures of P. falciparum- infected erythrocytes were initiated at 0.5% of parasitemia and the number of parasites was determined at different time intervals by both microscopic counting and the microfluorimetric technique. Figure 2, showing analyses at 24 and 48 hours, shows that both methods of detection are equally effective in detecting the presence of infected erythrocytes. Bars indicate the standard deviation of the mean for two independently processed samples. No differences were observed when nonsynchronized or D-sorbitol-synchronized Plasmodium cultures were used

(data not shown), nor were differences observed when chloroquine-sensitive (F32 and D6) or chloroquine-resistant (W2) strains were tested (data not shown). Based upon these experiments, a time point of 48 hours was chosen for the evaluation of potential anti-plasmodial compounds. EXAMPLE 6 - Determination of ICsn values of known antimalarial drugs.

The microfluorimetric method was used to determine the effect of known antimalarial drugs on the growth of P. falciparum by testing the effect of chloroquine and mefloquine on the growth on the F32 strain, a chloroquine- susceptible parasite. Cultures of P. falciparum W2 strain-infected erythrocytes were initiated at 0.5% of parasitemia, incubated with different concentrations of chloroquine and the number of parasites determined at 48h. From dose-response experiments, an IC₅₀ of 31 ± 0.7 nM for chloroquine was determined using the microfluorimetric method, which is comparable to the previously reported value of 29 ± 9 nM determined by [³H]hypoxanthine incorporation. The IC50 for mefloquine was 15 ± 3.7 nM, comparable to the value of 9.2 ± 4.2 nM that was determined with the radioactivity-based method. Figure 3 shows the dose response curves obtained with the radioactivity-based (Fig. 3 A) and microfluorimetric (Fig. 3B) methods for measuring the effect of chloroquine on the growth of the chloroquine-resistant W2 clone. No significant difference in the IC₅₀ values determined by either method was observed: the radioactive assay yielded an ICs₀ value of 86.5± 9 and the present fluorimetric assay yielded an IC₅₀ value of 88.7± 0.72 nM for the radioactivity-based and microfluorimetric methods, respectively. Bars indicate the deviation from the mean for four independently processed samples. The IC₅₀ values determined for chloroquine in these experiments is comparable to the published value of 128=1=73 nM for the chloroquine-resistant strains. (Delhaes L, Lazaro JE, Gay F, Thellier M, Danis M, 1999. The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity. Annals Trop Med Parasitol 93: 31-40;

Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, Hinrichs DJ, 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg 48: 739-741.) EXAMPLE 7 - Drug discovery. Natural products from plants have been a rich source of anti-parasitic compounds. (Klayman DL, 1993; Mufioz V et al.

2000) Therefore, the ability of the microfluorimetric method to detect plant extracts with anti-plasmodial activity and to assess its utility as a systematic and efficient means of screening large numbers of crude extracts was evaluated. Table 1 shows IC50 values for crude plant extracts as measured by uptake of [³H]hypoxanthine, microscopic counting of Giemsa thin blood smears, and the microfluorimetric technique of the present invention. Plant extracts with IC₅₀ values <50 μg/mL were considered "active." Table 1 shows a near perfect correlation between the radioactivity-based, microscopic, and microfluorimetric techniques with respect to their ability to detect plant extracts with anti-plasmodial activity (9/14). While the IC₅₀ levels of crude extracts measured by the radioactivity-based and microscopic methods tend to be lower than those values measure by the microfluorimetric assay, no differences were observed in IC₅₀ values when pure compounds were evaluated (Figure 3). Complementary experiments in which plants shown to be inactive by the radioactivity-based method were tested in the microfluorimetric assay. In every case, plants that were inactive in the radioactivity-based assay were also inactive in the microfluorimetric method (6/14), an observation relevant to the use of the latter method for drug discovery (Table 1). Table 1.

(IC₅₀ μg/mL) ND= Not done EXAMPLE 8 - Assay Guided Separation. The microfluorimetric assay was used to guide the purification of a compound with axύi-Plasmodium activity from the plant, Coccoloba parimensis. Initial screening of a crude extract of leaves of C. parimensis demonstrated significant anti-plasmodial activity (IC₅₀ = 6-12 μg/mL). The extract was subjected to liquid-liquid partition with hexane, ethyl acetate methanol and water, a technique used to separate the chemical constituents on the basis of their relative polarity (Montenegro H et al.) and the fractions were tested for anti-plasmodial activity using the fluorimetric method of the present invention. Samples showing activity were further purified. Purification of the sample resultant from the ethyl acetate fraction (IC₅₀ = 10 μg/mL) led to the isolation of the methyl ester of gallic acid which showed IC₅₀ values of <2 μg/mL. (Westenburg HE, Lee KJ, Lee SK, Fong HHS, Van Breemen RB, Pezzuto JM, Kinghorn DA, 2000. Activity-Guided Isolation of Antioxidative Constituents of Cotinus coggygria. J Nat Prod 63: 1696-1698.) The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the present invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

WE CLAIM: 1. A method for evaluating antiparasitic activity of a candidate material comprising: providing a sample comprising a viable parasite, (i) the parasite being axenic and the sample being cell free, or (ii) the parasite infecting red blood cells and the sample comprising red blood cells; combining the candidate material with the sample, incubating the sample with a fluorochrome that binds to parasite DNA within the sample and provides a fluorescent signal proportional to th.e extent of parasitemia, the fluorochrome having a sensitivity equivalent to conventional techniques based on radioactivity and microscopic counting between a range of 0.1 and 15% of ring stage infected erythrocytes and of comparable sensitivity to conventional techniques for purified compounds and qualitatively comparable to radioactive techniques when detecting anti-plasmodial activity in crude plant extracts, measuring fluorescence of the fluorochrome using a fluorescence reader, and determining whether the candidate material significantly reduces the fluorescent signal, the reduction indicating antiparasitic activity.

2. The method of paragraph 1, wherein the candidate material is a crude extract or a pure chemical compound.

3. The method of paragraph 1, wherein the parasite infects red blood cells and the sample comprises red blood cells, the fluorochrome not binding to red blood cells.

4. The method of paragraph 1 , wherein the parasite is a Plasmodium.

5. The method of paragraph 1, wherein the parasite is axenic.

6. The method of paragraph 1, wherein the parasite is selected from Babesia, Leishmania sp. and Trypanosoma sp., Entamoeba sp., and Giardia lamblia.

7. The method of paragraph 1, wherein the fluorochrome is

PicoGreen®.

8. The method of paragraph 1, wherein the candidate material is incubated for about 24-48 hours before incubating with the fluorochrome

9. The method of paragraph 1, wherein the fluorochrome is incubated for about 5-30 minutes.

10. The method of paragraph 1 , wherein a plurality of samples are incubated simultaneously in a multivessel container.

11. The method of paragraph 10, wherein the multivessel container is a

96 well plate.

12. The method of paragraph 1 , wherein the fluorochrome is PicoGreen and fluorescence is measured at about 485/20 nm excitation and about 528/20nm emission.

13. The method of paragraph 1 , wherein the fluorochrome has a property selected from selectively binding to dsDNA, ability to quantitate dsDNA at levels of about 25 pg/mL dsDNA, low level of interference due to signal from substances other than dsDNA, a high signal to noise ratio, stability to photobleaching, low toxicity, safe characteristics and low cost.

14. The method of paragraph 1, wherein the reader is either a fluorescent plate reader or a minifluorimeter.

15. The method of paragraph 1, further comprising running a control with a known antiparasitic agent.

16. The method of paragraph 15, wherein the parasite is Plasmodium and the antiparasitic agent is an anti-malarial drug.

17. A method of assay-guided fractionation/purϊfication comprising the method of paragraph 1.

18. A method for diagnosing the susceptibility of a patient to a particular antiparasitic therapy comprising isolating the infecting parasite and analyzing it according to the method of paragraph 1.

19. The method of paragraph 17, wherein the parasite is Plasmodium.

20. A kit for evaluating antimalarial drug activity comprising standardized preparations of Plasmodium, erythrocytes, PicoGreen, and an antimalarial drug.