METHOD AND APPARATUS FOR DIAGNOSING INTRACELLULAR PARASITE INFECTIONS USING AN ENZYME DETECTION SYSTEM

Abstract: The present invention involves a highly specific chemical system for detecting parasite-infected cells. The method is especially effective in detecting intraerythrocytic diseases, especially malaria. With respect to malaria, chemical agents are used to detect enzymes produced by the infecting parasites. Blood samples are combined with a plurality of substrates which are subjected to enzymatic hydrolysis. Hydrolysis is rapid and may be detected in two ways. The first method involves substrates having a fluorescent detecting group. Upon hydrolysis, the fluorescent group is cleaved and detected using a conventional fluorescent light source. Alternatively, substrates may be used having a detecting group which forms a visibly colored material when combined with a secondary reagent. These techniques produce a unique colored or fluorescent pattern which enables the rapid and accurate determination of parasite infection. Furthermore, parasite diagnosis is accomplished without the use of expensive and complex diagnostic equipment.

Description: METHOD AND APPARATUS FOR DIAGNOSING INTRACELLULAR PARASITE INFECTIONS USING AN ENZYME DETECTION SYSTEM The present invention generally relates to a method and apparatus for diagnosing intracellular parasite infections including malaria, and more particularly to a highly specific diagnosis method and apparatus designed to detect enzymes produced by parasites during disease manifestation. Many infectious diseases exist in which micro¬ organisms (e.g. bacteria, virus, ric ettsia or protozoa) invade host cells. This is especially true with respect to diseases involving the invasion of erythrocytes (red blood cells). These diseases are collectively called "intraerythrocytic parasite diseases." Malaria is a dangerous intraerythrocytic disease caused by a parasite which invades a host's liver cells and erythrocytes. Malaria is one of the most widespread infectious diseases. The World Health Organization estimates that 200,000,000 people are infected with the malaria parasite annually. These people mainly reside in the tropics. One million cases of malaria were reported in the United States in 1940. At that time, effective measures were introduced which virtually eliminated the disease, which is transmitted by the female Anopheles mosquito. However, the Anopheles mosquito is still present in many of the Southern and Western parts of the United States. During the early 1970's, there were several cases of malaria reported in Louisiana and California. These were attributed to returning veterans from the Viet Nam war who harbored the parasite. As tropical regions of the world become more accessible through improved modes of transportation, 10 travel into these areas is increasing. This has resulted in significantly more cases of malaria being reported in travelers returning from these areas. One percent (1%) of all people infected with the malaria parasite die from the disease (2,000,000 people per year). There are four -5 species of Plasmodium which infect humans and cause malaria. These include P. falciparum, P. vivax, P. ovale, and P. malariae. P. falcipariαm is the most serious species. It is responsible for cerebral malaria which is associated with a 50% mortality rate. ° There are now over 65 countries which have reported chloroquine-resistant P. falciparum, and 12 countries which have reported quinine-resistant P. falciparum. These two anti-malarial drugs were the major therapeutic agents used to treat the disease. Since 5 they are rapidly becoming entirely or partially ineffective for specific parasite species, it is essential that an accurate diagnosis of the disease be obtained in order to provide proper treatment. The diagnostic method of choice must be rapid, specific, readily available, easy to 0 perform, and easy to interpret. The life cycle of a Plasmodium parasite involves the interrelationship between an Anopheles mosquito vector and a mammalian host. When an uninfected female Anopheles mosquito bites and ingests blood from a host harboring the 5 sexual forms of the Plasmodium parasite, the parasitic life cycle begins. In the Anopheles, the male and female 1 gametocytes fuse and travel after several stages of development to the salivary glands of the mosquito. The parasite at this stage is called a "sporozoite. " If the infected mosquito bites a new host, the sporozoites are 5 injected into the host's blood. Thereafter, they travel to the liver within 30 minutes, where they enter a liver cell. In the liver cell, one sporozoite multiplies and forms about 10,000-20,000 merozoites. These merozoites are released from the liver cells in 10-12 days. Each of " ■ - 0 the released merozoites immediately invades an erythrocyte In 48 hours, each merozoite forms another 10-12 merozoites which are in turn released from the erythrocyte only to invade another 10-12 erythrocytes. The clinical manifestations of the disease ~--- include fever, headaches, sweating, vomiting, and prostration. These -manifestations occur simultaneously with merozoite release from the erythrocytes . The erythrocyte reinvasion occurs until the host dies, or until the host's immune system is able to control and 20 suppress merozoite activity. At some point, the merozoites (previously asexual) differentiate into male and female gametocytes. The technical and scientific basis for this transformation is an active area of current medical research. If a female Anopheles then bites a new • " host at the time of gametocyte formation, the life cycle of the parasite is completed. The most susceptible human hosts for the disease are infants and pregnant women having suppressed immunity. Recently, deaths have been reported in adult 30 male AIDS patients caused by cerebral malaria. In addition, non-immune travelers into high-risk malaria areas are also susceptible to the disease, especially with respect to chloroquine and quinine resistant malaria. There is a natural immunity to malaria which ■ - ^ develops in persons living in high-risk malaria areas. This immunity appears to depend upon the continual 1 presence of low parasite levels in the host's body. This conclusion is drawn from many studies which demonstrate that when persons living- in high-risk malaria areas leave and travel to low risk areas, they substantially lose 5 their immunity. As previously mentioned, the disease must first be properly diagnosed before treatment may be given. Ideal diagnostic methods must be specific, sensitive, accurate, and easy to implement, and require a minimum of i0 complex diagnostic equipment. Numerous approaches have been taken regarding the laboratory diagnosis of malaria. These approaches include the use of thick and thin blood smears treated with a conventional stain known as "Wright's Giemsa" and examined with a light microscope. ■ " Other methods range from fluorescent dyes to recently developed methods involving DNA probes, indirect fluorescent antibody tests, indirect haemagglutinatioπ tests, enzyme-linked irnmunosorbent assays, and gel precipitation tests as extensively discussed in Bulletin 0 of the World Health Organization: Malaria Diagnosis, 1045, 1-37 (1988). With respect to other 'malaria diagnostic procedures, U.S. Patent 3,834,874 to Geating et al involves a Plasmodia detecting apparatus consisting of a 5 pre-stained microscope slide covered with a dried mixture of methylene blue NN and cresyl violet acetate. Australian patent application 66/04,418 discloses a blood smear composition comprising a solution of methylene blue chloride, an alkali metal bicarbonate 0 (preferably NaHCO ) , eosin Y, and azure (IΙ)-eosin in an alcohol mixture. The resulting composition is designed to detect malaria parasites and may be used for all types of blood smears (thick and thin) . A similar process is disclosed in British Patent 1,183,499 which involves a biological stain for detecting malaria in which a blood film is first stained with polychrome methylene blue solution, followed by treatment with an eosin solution. U.S. Patent 4,741,898 discloses a stabilized Romanowsky-type stain composition for malaria parasites which includes a cationic eye component (methylene blue, azure A, azure B, azure C, or thionin) , an anionic dye component (eosin Y, eosin B, fluorescein, substituted fluorescein, or orange G), a 1 - 6 carbon alcohol solvent, and a stabilizer (e.g. lysine or glycine) . Further information regarding malaria staining techniques is disclosed in Bianco A.E., et al, "Plasmodium Falciparum: Rapid Quantification of Paracitemia in Fixed Malarial Cultures by Flow Cytometry, " Exp. Parasitol ■ , 62:275-282 (1986); Tanabe, K. , "Staining of Plasmodium Yoeli-Infected Mouse Erythrocytes with the Fluorescent Dye Rhodamine 123,-" J. Protozool., 30:707-710 (1983); and Makler, M.T. et al, "Thiazole Orange: A New Dye for Plasmodium Species Analysis," Cytometry, 8:568-570 (1987). The article by Makler, M.T. et al is of particular interest in that it discloses a fluorescent staining technique for Plasmodium falciparum using a membrane-permeable fluorochrome thiazole orange dye in conjunction with a fluorescent flow cytometer . A variety of other diagnosis methods exist for detecting the presence of malaria infections. For example, French patent application 2,572,528 involves a process for identifying, sorting, and counting microscopic particles (including Plasmodia) in which test samples are first prepared and deposited in succession on an advancing conveyor surface. The conveyor surface moves the samples into the viewing field of a microscope providing images which are recorded by a video camera. The recorded images are then set against a squared pattern grid so that the image corresponding to each sample may be analyzed according to standard pattern recognition techniques . European patent application 119,209 involves a method for identifying microorganisms including malaria paraεites through the use of DNA probes. The parasites are first immobilized on a solid support. DNA from the parasite sample is then subjected to hybridization with a labeled specimen of species-specific, non-cross hybridizing DNA from a known species. The parasite sample is then examined for hybridization between the first and second DNA materials. Canadian Patent 951,242 involves an immunoglobin M diagnostic reagent for detecting malaria and other diseases consisting of polystyrene particles coated with IgM. To determine IgM levels in a sample of test serum and detect disease infestation, the serum is first mixed with human IgM antiserum followed by combination with the polystyrene-IgM reagent. If agglutination occurs, IgM levels are normal and there is no disease. Australian patent application 87/72,041 discloses a method in which nucleotide sequences from the RESA antigen of Plasmodium falciparum are used as detection probes. Notwithstanding the methods described above, a need exists for an intracellular/intraerythrocytic parasite disease diagnosis procedure which is easily used, requires minimal amounts of instrumentation, is parasite specific, and is especially well-suited for the detection of malaria. The method must be economical, and capable of providing accurate test readings in remote geographical regions. The present invention satisfies these goals, and represents an advance in the field of disease diagnosis, as described below. Summary of the Invention It is an object of the present invenrion to provide a diagnosis method and apparatus for intraceliular parasite diseases which is characterized by a high degree of accuracy. It is another object of the invention to provide a diagnosis method and apparatus for intraceliular parasite disease which is especially effective and specific in detecting intraerythrocytic infections such as malaria. It is another object of the invention to provide a diagnosis method and apparatus for intraceliular parasite diseases which is easy and economical to use. It is a further object of the invention to provide a diagnosis method and apparatus for intraceliular parasite diseases which requires minimal amounts of labor and equipment . It is a further object of the invention to provide a diagnosis method and apparatus for intraceliular parasite diseases which is readily adaptable for use in isolated geographical areas. It is an even further object of the invention to provide a diagnosis method and apparatus for intraceliular parasite diseases which is capable of delivering rapid results . It is a still further object of the invention to provide a diagnosis method and apparatus for intraceliular parasite diseases which involves the use of a chemical detection system for enzymes produced by the infecting parasites during disease manifestation. In accordance with the foregoing objects, the present invention involves a highly specific method and apparatus for detecting infected cells in humans with particular emphasis on intraerythrocytic diseases . The method is especially effective in diagnosis malaria, and involves the use of a chemical system for detecting enzymes produced by the infecting parasites during disease manifestation. The chemical tests described herein accurately detect parasite-produced enzymes, notwithstanding the presence of endogenous enzymes from leukocytes or other elements normally present in blood. T " r *- ~ -• In a preferred embodiment, an adsorbent support medium is impregnated with a plurality of synthetic chemical substrates in numerous zones on the medium. Thereafter, a blood sample is applied directly to each of 5 the zones. Some of the substrates are designed to hydrolyze in the presence of enzymes produced by the infecting parasites. These enzymes include glycosidases, dehydrogenases, esterases, lipases, peptidases, and proteinases (e.g. hemoglobinases) . Other substrates ^ ■ 0 hydrolyze only in the presence of endogenous enzymes present in the blood from leukocytes, platelets, and other blood components . After the application of blood to the substrate zones, a specific pattern of substrate hydrolysis will 5 occur which may be used to detect parasite enzymes. Enzymatic substrate hydrolysis is rapid, and can be detected in two ways. The first method involves the use of substrates having a fluorescent detecting group. Upon hydrolysis, the fluorescent detecting group is cleaved 0 from the substrate, and is detected using a conventional fluorescent long wave (366nm) lamp. In the alternative, substrates may be used having a non-fluorescent detecting/leaving group which forms a visibly colored product when combined with a secondary 5 reagent. Enzymatic hydrolysis of the substrates produces a unique pattern on the support medium (either fluorescent or distinctively colored) which enables the rapid and accurate detection of parasite infections. The invention 0 as described herein is capable of detecting parasite infected cells (e.g. erythrocytes) with a high degree of accuracy while avoiding the use of expensive and complex diagnostic equipment. It is especially useful in isolated geographic regions of the world where such equipment is normally unavailable. 1 These and other objects, features and advantages of the invention will be described below in the following detailed description of a preferred embodiment. Detailed Description of a Preferred Embodiment The present invention involves an improved method and apparatus for detecting parasite-infected cells, with emphasis on intraerythrocytic parasite diseases (e.g. ® malaria). While the invention described herein will be primarily discussed in terms of malaria diagnosis, its applicability shall not be limited to malaria which is only one of many intracellular/intraerythrocytic parasite diseases. 15 in order to survive, malaria parasites must be capable of invading host erythrocytes. Once erythrocyte invasion is accomplished, the parasites undergo a 48-hour cycle of asexual multiplication. During parasite entry into the erythrocytes and multiplication therein, the 2 ^ host's erythrocyte plasma membranes experience structural and functional changes, as discussed in Aikawa, M. , et al, "Structural Alteration of the Membrane of Erythrocytes Infected with Plasmodium falciparum," J. Protozool. , 32(3) :424-429 (1985); Kutner, S., et al . , -* ■ - *■ "Characterization of Permeation Pathways in the Plasma Membrane of Human Erythrocytes Infected With Early Stages of Plasmodium falciparum: Association with Parasite Development," Journal of Cellular Physiology, 125:521-527 (1985); and Brawn-Breton, C, et al, "Induction of the 0 Proteolytic Activity of a Membrane Protein in Plasmodium falciparum by Phosphatidyl Inositol-Specific Phospholipase C," Nature, 332:457-459 (1988) . As a result of parasitic invasion, the erythrocyte plasma membrane becomes increasingly 5 permeable, allowing enzymes produced by the parasites to leak out into the surrounding environment. A variety of f **~**i 1 ' - • •' • different enzymes are produced by the parasites, including glycosidases, dehydrogenases, eεterases, lipases, peptidases, and proteinases. Enzyme generation by malaria parasites has been well-characterized, and plays an important role in several different stages of erythrocyte infection, as described in Rosenthal, P.J., et al, "Identification of Three Stage Specific Proteinases of Plasmodium falciparum, " J. Ex . Med. , 166:816-821 (1987). For example, some of the enzymes are utilized by the parasites for attachment to host cells, while others are used for penetration through the host cell membrane and the digestion of blood proteins . In accordance with the present invention, chemical means are provided to detect the existence of these enzymes in the blood of a human subject, thereby facilitating an accurate and rapid diagnosis of intraceliular parasite infections including malaria. To accomplish this, a test kit is provided preferably consisting of an adsorbent support medium which may be shaped in a variety of ways. For example, the support medium may take the form of an elongate strip or dip stick. Exemplary materials usable for the support medium include #4 or #40 grade filter paper produced by the Schleicher & Schuell Company of Keene, New Hampshire, or conventional cellulose acetate, nylon, or cellulose nitrate sheets also available from the Schleicher & Schue11 Company. Applied to the support medium in a plurality of zones (e.g. 3-5) are synthetic chemical substrates subject to hydrolysis either by parasite enzymes or enzymes present in the blood from leukocytes, platelets, or the like. The use of chemical substrates for enzyme detection is generally discussed in Lojda, Z., "Proteinases in Pathology - Usefulness of Histochemical Methods," Journal of Histochemistry and Cytochemistry, 29:481-493 (1981); and Smith, R.E. , "Contributions of Histochemistry to the Development of the Proteolytic Enzyme Detection System in Diagnostic Medicine," Journal of Histochemistry and Cytochemistry, 31( 1A) : 199-209 (1983). Normally, each zone will be impregnated with a different substrate, possibly combined with other materials as described below. Multiple zones are used each having a different substrate in order to add internal quality controls to the procedure. In addition, the substrates selected for use in the invention are initially determined by the characterization of enzymes found in Plasmodium species and in the host's white cells and/or platelets. The enzymes of the parasite and host are different in character and therefore hydrolyze different substrates. Thus, multiple zones/substrates are used for this reason, thereby permitting a more accurate overall enzymatic analysis. Substrates usable in the invention are commercially available from the Sigma Company of St. Louis, MO, or Enzyme Systems Products Co., of Livermore, CA, and include the following as typical examples: Pyridoxal phosphate Glucose-6-phosphate 5-bromo-4-chloro-3-indoyl- beta-D-galactose Methyl umbelliferone phosphate 7-bromo-3-hydroxy-2-napht - o-anisidide The .abbreviation terminology used above with respect to the peptide substrates is standard and well-known in the art. By way of example, "Lys-Pro" stands for "lysine-proline, " etc. In addition, each of the peptide substrates usable in the invention is commercially available from several-different sources (e.g. Sigma Chemical Co. of St. Louis, MO, and Enzyme System Products of Livermore, California) with specific detecting groups thereon. These detecting groups will be discussed in further detail below, but are used to verify enzymatic hydrolysis of the substrate. For example, the peptide substrates may include fluorescent detecting groups consisting of 7-amino-methyl coumarin (AMC) or 7— mino-4-trifluoromethyl coumarin (AFC) . The fluorometric detection of AMC when cleaved from its associated substrate is readily accomplished using conventional fluorescent light sources. Excitation of AMC occurs at 380nm and emission at 460nm. With respect to AFC, excitation occurs at 400nm and emission at 505nm. In the alternative, the detecting group on the substrate may consist of a chemical which produces a colored compound (a "chromogen") when combined with a second, externally applied material known as a "post-coupling reagent." An exemplary detecting group for this purpose includes 4-methoxy-2-naphthylamine (MNA) . This material releases a naphtol group which may be coupled to a variety of known post-coupling reagents including the following diazonium salts, all of which are available from the Sigma Chemical Company of St. Louis, Missouri: 1. Fast Red TR Salt: (4-chloro-2-amino-toluene (meta)—4-chloro-2-methylbenzendediazonium chloride hemi [zinc chloride] salt. 2. Fast Blue B Salt: (o-dianisidine (tetrazotized)) ; and 3. Fast Blue BB Salt: (diazotized 4'-amino-2'- 5'-diethoxybenzanilide [zinc chloride salt]) In addition, several substituted aldehyde post-coupling reagents may be used which are also available from the Sigma Chemical Company, including para dimethylaldehyde and paradimethyl trans cinnamaldehyde. A variety of enzymatic inhibitors may also be combined with one or more of the substrates, if appropriate. In the present invention, the inhibitors may include leupeptin, chymostatin, pepstatin, p-chloro mercuriphenyl sulphonate, cysteine, dithiothreitol, ethylene diamine-tetraacetic acid, N-methyl malemide, all of which are available from the Sigma Chemical Company. They are used in order to inhibit the specific parasite enzyme under consideration so that the sensitivity of the test , procedure is ensured. The selection of specific inhibitors therefore depends on the parasite enzyme which the test is designed to detect. Thus, the use of inhibitors will vary accordingly. The substrate solutions are ultimately dissolved in an isotonic buffer (e.g. saline in dithiothreitol) in order to obtain a pH range of 5.5-8.0, depending on the parasite enzyme to be detected. In addition, the final concentration of the substrate solutions is preferably about 1.0 mM. After impregnation of each zone on the support media with a 10 microliter supply of the selected substrate solutions, the media is allowed to air dry, followed by the application of a blood sample (10 microliters; 1/100 saline) to each zone. If substrates are used having a fluorescent detecting group, the support medium after the addition of blood is incubated in a humidifying chamber at 37°C for 15-30 minutes and thereafter excited with long wave (366nm) fluorescent light. The indication of a fluorescent yellow-green color from the support medium provides a positive test for the presence of enzymatic hydrolysis. If substrates are used having a non-fluorescent detecting group (e.g. MNA) , a selected post-coupling reagent (e.g. 10 microliters of a lOmg/lOOml solution) is applied to each test zone ' after blood addition. A positive result will be indicated by the formation of a chromogen from the post-coupling reagent and the cleaved detecting group. The resulting chromogen is distinctively colored, and readily visible. In an alternative embodiment of the invention, a plurality of standard microhematocrit tubes (each having a 200 microliter capacity) may first be individually filled with approximately 10 microliters of a selected substrate solution prepared as described above having fluorescent detecting groups. Thereafter, blood (10 microliters) can be added to the microhematocrit tubes, followed by agitation (cent ifugation) using a conventional microcentrifuge unit operating at about 2000 rpm over a 5-minute period. After centrifugation, the resulting column of packed erythrocytes is observed with a long wave (366nm) fluorescent light. The presence of an interspersed green fluorescence will indicate enzymatic hydrolysis and the presence of parasites. Using the procedures described above, hydrolysis of the substrates is rapid, usually taking less than fifteen minutes. The unique colors or fluorescent patterns generated during the tests may be used as standards to enable the rapid and accurate determination of parasitic infection. Furthermore, the invention is highly specific, in that selection of the proper substrates enables an accurate chracterization of parasite infection, notwithstanding the existence of endogenous enzymes from the host's leukocytes, platelets, and the like. In order to specifically illustrate use of the substrates, inhibitors, post-coupling reagents, and other materials described above, the following Examples are provided relative to detection of the cysteine protease enzyme produced by malaria parasites. EXAMPLE 1 A small amount of blood (10 microliters - 1/100 saline) was applied to four zones on a portion of No. 40 filter paper. Each zone was previously impregnated with a 10 microliter quantity of the following materials: Zone 1 — l.O M solution of Ala-Arg-Arg buffered to pH 6.0 using an isotonic saline/dithiothreitol solution (0.ImM) . Zone 2 — l.OmM solution of Ala-Arg-Arg buffered to pH 6.0 using an isotonic saline/dithiothreitol solution (O.l M). This material was combined with 10 microliters of a l.OmM solution of leupeptin (inhibitor) buffered to pH 6.0 again using an isotonic saline/ dithiothreitol solution (O.lmM). Zone 3 — l.OmM solution of methyl umbelliferone phosphate or 7-bromo-3-hydroxy-2 naphth-o- anisidide buffered to pH 6.0 using an isotonic saline/dithiothreitol solution (O.lmM). These materials are both substrates which are hydrolyzed by host enzymes. II these substrates are hydrolyzed, it indicates a high level of "non-specific" host enzymes. Zone 4 — l.OmM solution of Lys-Pro buffered to pH 6.0 using an isotonic saline/ dithiothreitol solution (O.lmM). This is also a substrate hydrolyzed by host enzymes. The substrates in zones 1, 2, and 4 all incorporated the fluorescent detecting group AFC or AMC. The substrates in zone 3 both hydrolyze to produce fluorescent byproducts without the need for a specific fluorescent detecting group. After the application of the blood to zones 1-4, the filter paper was incubated in a warm environment (approximately 37°C) for about 5-60 minutes. It was then examined with a long wave (366nm) ultraviolet lamp for the presence of a distinct fluorescent product. A positive test for malarial cysteine protease is shown in Table I. TABLE 1 Blood Having Non-infected Parasite-Infected Blood Containing Erythrocytes Leukocytes, Platelets and other Endogenous Enzyme-Producinσ Components Zone 1 (Ala-Arg-Arg) Zone 2 (Ala-Arg-Arg + leupeptin) - Zone 3 (Methyl umbelliferone - + phosphate or 7- brσmo-3-hydroxy- 2-naphth-o-anisidide) Zone 4 (Lys-Pro) - + (+ = fluorescence) The data shown in Table 1 illustrates how parasite infected blood may be enzymatically detected and distinguished from normal blood having leukocytes and platelets which generate their own enzymes. Thus, the results in Table 1 may be used as a standard for accurately detecting malaria parasite infections. EXAMPLE 2 The same materials were used as those in Example 1, except for the absence of the fluorescent detecting groups AFC or AMC attached to the substrates in zones 1, 2 and 4. Instead, the detecting group MNA (4-methoxy-2-naphthylamine) was used. Also, zone 3 and the chemicals associated therewith were eliminated entirely. After the application of blood to the appropriate zones, each zone was spotted with 10 microliters of a lOOmg/lOOml solution of a diazonium salt (e.g. Fast Blue B) . A positive result was indicated by the formation of a chromogen and a negative result produced no color. The same substantive results were achieved as indicated above in Table 1. This test has an advantage over the test of Example I in that an external fluorescent illumination system is not required. The present invention represents an advance in the field of diagnostic medicine. Tests performed using the methods and materials described above are highly accurate and sensitive. In addition, diagnosis is accomplished without the use of expensive and complex analytical equipment which may not be available in remote geographical areas including West Africa and Southeast Asia. Furthermore, pre-prepared test media may be manufactured by impregnation of an appropriate support medium (e.g. paper or cellulose acetate) with selected substrate solutions, followed by storage thereof until testing is desired. Likewise, standard microhematocrit tubes may be filled with substrate solutions and sealed until needed. These pre-prepared units will have a shelf life of approximately 3-4 months, and represent a sir-.pie - •• - and inexpensive system for conducting highly accurate diagnoses of intraerythrocytic parasite infections including malaria. While the present invention is described above with particular reference to the treatment of malaria, it is also prospectively applicable to other intraceliular/ intraerythrocytic diseases as previously noted. These disease include Babesiosis (caused by the protozoan Babesia microti), Trypanosomiasis (caused by the protozoans Trypanosoma Brucei rhodesiense and qambiense) , as * well as Listeriosis (caused by Listeria monσcytogenes, and various rickettsial diseases. Having herein described a preferred embodiment of the invention, it is anticipated that suitable modifications may be made by those skilled in the art. Thus, the invention shall be construed only in accordance with the following claims:

Claims: WHAT IS CLAIMED IS: 1. A method for the detection of intraceliular parasite diseases in a warm-blooded animal wherein said parasite produces enzymes in the cells of said animal and releases said enzymes therefrom, said method comprising the steps of: obtaining a sample of blood from said animal; combining said sample of blood with at least one chemical substrate capable of being hydrolyzed by said enzymes, said substrate releasing a chemical detecting group upon hydrolysis; and analyzing said blood after said combining with said substrate for the presence of said detecting group, whereby the presence of said detecting group indicates the existence of said enzymes from said parasite. 2. The method of claim 1 wherein said substrate is applied to a portion of adsorbent media material prior to said combining thereof with said blood. 3. The method of claim 2 wherein said adsorbent media material is constructed of a material selected from the group consisting of paper, cellulose acetate, cellulose nitrate, and nylon. 4. The method of claim 1 wherein said substrate is combined with at least one enzyme inhibitor . 5. The method of claim 1 wherein said blood and said substrate are combined in a substantially transparent vessel. 6. The method of claim 1 wherein said detecting group comprises a fluorescent compound. 7. The method of claim 6 wherein said analyzing of said blood after said combining with said substrate comprises the step of applying fluorescent light from a fluorescent light source thereto, said fluorescent light causing said detecting group after said release from said substrate to visibly fluoresce. 8. The method of claim 1 wherein said analyzing of said blood after said combining with said substrate comprises the step of adding at least one chemical coupling reagent to said blood and said substrate, said coupling reagent reacting with said detecting group after said release from said substrate to produce a visibly colored product. 9. The method of claim 5 wherein said detecting group comprises a fluorescent compound. 10. The method of claim 9 wherein said analyzing of said blood after said combining with said substrate in said vessel comprises the step of applying fluorescent light from a fluorescent light source to said vessel, said fluorescent light causing said detecting group after said release from said substrate to visibly fluoresce. 11. A method for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said method comprising the steps of: obtaining a sample of blood from said animal; combining said blood with at least one chemical substrate capable of being hydrolyzed by said enzymes from said malaria parasites, said substrate releasing a chemical detecting group upon hydrolysis; and analyzing said blood after said combining with said substrate for the presence of said detecting group, whereby the presence of said detecting group indicates the existence of said enzymes from said malaria parasites. 12. The method of claim 11 wherein said sbustrate is applied to a portion of adsorbent media material prior to said combining thereof with said blood. 13. The method of claim 12 wherein said adsorbent media material is constructed of a material selected from the group consisting of paper, cellulose acetate, cellulose nitrate, and nylon. 14. The method of claim 11 ' wherein * said substrate is combined with at least one enzyme inhibitor. 15. The method of claim 11 wherein said blood and said substrate are combined in a substantially transparent vessel . 16. The method of claim 11 wherein said detecting group comprises a fluorescent compound. 17. The method of claim 16 wherein said analyzing of said blood after said combining with said substrate comprises the step of applying fluorescent light from a fluorescent light source thereto, said fluorescent light causing said detecting group after said release from said substrate to visibly fluoesce. 18. The method of claim 11 wherein said analyzing of said blood after said combining with said substrate comprises the step of adding at least one chemical coupling reagent to said blood and said substrate, said coupling reagent reacting with said detecting group after said release from said substrate to produce a visibly colored product. 19. The method of claim 15 wherein said detecting group comprises a fluorescent compound. 20. The method of claim 19 wherein said analyzing of said blood after said combining with said substrate in said vessel comprises the step of applying fluorescent light from a fluorescent light source to said vessel, said fluorescent light causing said detecting group after said release from said substrate to visibly fluoresce. 21. A method for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said method comprising the steps of: obtaining a sample of blood from said animal; providing a portion of adsorbent media material; impregnating said media material with first, second, third, and fourth chemical solutions, each solution being applied to a separate zone on said media material, said first solution comprising a substrate selected from the group consisting of ala-arg-arg- (7-amino-4-methyl coumarin) and ala-arg-arg-(7-amino- trifluoromethyl coumarin) , said second solution comprising leupeptin and a substrate selected from the group consisting of ala-arg-arg-(7-amino-4-methyl coumarin) and ala-arg-arg-(7-amino-4-triflouromethyl coumarin), said third solution comprising a substrate selected from the group consisting of 7-bromo-3-hydroxy-2-naphth-o-anisidide and methyl urabelliferone phosphate, and said fourth solution comprising a substrate selected from the group consisting of lys-pro-(7-amino-4-methyl coumarin) and lys-pro-(7-amino-4-trifluoromethyl coumarin) ; applyi ' ng an equal portion of said blood onto each zone impregnated with said solutions; and applying fluorescent light from a fluorescent light source to said media material to enable the observation of a fluorescent pattern on said media material, said pattern being used to detect the presence of said enzymes from said malaria parasites. 22. A method for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said method comprising the steps of: obtaining a sample of blood from said animal; providing a portion of adsorbent media material; impregnating said media material with first, second, and third chemical solutions, each solution being applied to a separate zone on said media material, said first solution comprising ala-arg-arg-(4-methoxy-2- naphthylamine) , said second solution comprising leupeptin and ala-arg-arg-(4-methoxy-2-naphthylamine) , and said third solution comprising lys-pro-(4-methoxy-2- naphthylamine) ; applying an equal portion of said blood onto each zone impregnated with said solutions; and applying at least one chemical coupling reagent to each zone in order to form a colored pattern on said media material, said pattern being used to detect the presence of said enzymes from said malaria parasites. ' 23. The method of claim 22 wherein said coupling reagent is selected from the group consisting of para dimethylaldehyde, paradimethyl trans cannamaldehyde, 4-chloro-2-amino-toulene (rneta)-4-chloro-2-methyl- benzenediazonium chloride, o-dianisidine, and 4'-amino- 2 '-5 '-diethoxybenzanilide. 24. An apparatus for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said apparatus comprising: a portion of adsorbent media material; and at least one chemical substrate applied to said media material and impregnated therein, said substrate being subject to hydrolysis by said enzymes from said malaria parasites, said substrate releasing a fluorescent detecting group upon hydrolysis. 25. The apparatus of claim 24 wherein said adsorbent media material is constructed of a material selected from the group consisting of paper, cellulose acetate, cellulose nitrate, and nylon. 26. An apparatus for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said apparatus comprising: a portion of adsorbent media material; "and at least one chemical substrate applied to said media material and impregnated therein, said substrate being subject to hydrolysis by said enzymes from said malaria parasites, said substrate releasing a chemical detecting group capable of being combined with at least one coupling reagent to produce a fisibly colored product upon hydrolysis. 27. The apparatus of claim 26 wherein said adsorbent media material is constructed of a material selected from the group consisting of paper, cellulose acetate, cellulose nitrate, and nylon. 28. An apparatus for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said apparatus comprising: a portion of adsorbent media material; and first, second, third and fourth chemical solutions, each solution being applied to a separate zone on said media material, said first solution comprising a substrate selected from the group consisting of ala-arg-arg-(7-amino-4-methyl coumarin), and ala-arg-arg-(7-amino-4-trifluoromethyl coumarin), said 10 second solution comprising leupeptin and a substrate selected from the group consisting of ala-arg-arg-(7-amino- 4-methyl coumarin), and ala-arg-arg-(7-amino-4- trifluoromethyl coumarin) , said third solution comprising a substrate selected from the group consisting of methyl 15 umbelliferone phosphate and 7-bromo-3-hydroxy-2-naphth-o- anisidide, and said fourth solution comprising a substrate selected from the group consisting of lys-pro-(7-amino-4- methyl coumarin), and lys-pro-(7-amino-4-trifluoromethyl 0 coumarin) . 29. An apparatus for the detection of malaria parasites in a warm blooded animal wherein said parasites .produce enzymes in the erythrocytes of said animal and _ release said enzymes therefrom, said apparatus comprising: a portion of adsorbent media material; and first, second and third chemical solutions, each solution being applied to a separate zone on said media material, said first solution comprising ala-arg-arg- Q (4-methoxy-2-naphthylamine) , said second solution comprising leupeptin and ala-arg-arg-(4-methoxy-2- naphthylamine) , and said third solution comprising lys-pro-(4-methoxy-2-naphthylamine) . 30. An apparatus for the detection of intra¬ celiular parasite diseases in a warm blooded animal wherein said parasite produces enzymes in the cells of said animal and releases said enzymes therefrom, said apparatus comprising: a portion of adsorbent media material; and at least one chemical substrate applied to said media material and impregnated therein, said substrate being subject to hydrolysis by said enzymes. 31. An apparatus for the detection of malaria parasites in a warm blooded animal wherein said parasites produce enzymes in the erythrocytes of said animal and release said enzymes therefrom, said apparatus comprising: a plurality of transparent vessels; and at least one chemical substrate contained within each of said vessels, said ' substrate being subject to hydrolysis by said enzymes, said substrate releasing a fluorescent detecting group upon hydrolysis. 32. The apparatus of claim 31 wherein said vessels are divided into first, second, third and fourth vessels, said first vessel containing a chemical solution comprising a substrate selected from the group of ala-arg-arg-(7-amino-4-methyl coumarin) and ala-arg-arg- (7-amino-trifluoromethyl coumarin), said second vessel containing a solution comprising leupeptin and a substrate selected from the group consisting of ala-arg-arg- (7-amino-4-methyl coumarin) and ala-arg—arg-(7-amino-4- trifluoromethyl coumarin), said third vessel containing a solution comprising a substrate selected from the group consisting of methyl umbelliferone phosphate and 7-bromo-3-hydroxy-2-naphth-o-anisidide, and said fourth vessel containing a solution comprising a substrate selected from the group consisting of lys-pro-(7-amino- 4-methyl coumarin) and lys-pro-(7-amino-4-trifluoromethyl coumarin) .

Inventors: MAKLER MICHAEL T, MAKLER, MICHAEL, T.
Applicants: MAKLER MICHAEL T, TOTEM HEALTH SCIENCES INC, MAKLER, MICHAEL, T., TOTEM HEALTH SCIENCES, INC.
Application year: 1989
Main classification: C12Q1
IPC codes: A61K49, C12M1, C12Q1