WO2008089155A2

WO2008089155A2 - Biomarker assays for the diagnosis of malaria in developing countries based on epo levels

Info

Publication number: WO2008089155A2
Application number: PCT/US2008/051021
Authority: WO
Inventors: Douglas A. Holtzman; Anna Roca; Pedro L. Alonso
Original assignee: Holtzman Douglas A; Anna Roca; Alonso Pedro L
Priority date: 2007-01-12
Filing date: 2008-01-14
Publication date: 2008-07-24
Also published as: WO2008089155A3

Abstract

Diagnostic assay systems use erythropoietin (EPO) to differentiate between a patient having severe malaria requiring hospitalization from a patient with less severe malaria or a patient with another non-malaria febrile disease.

Description

BLOMARKER ASSAYS FOR THE DIAGNOSIS OF INFECTIONS IN DEVELOPING COUNTRIES

BACKGROUND OF THE INVENTION

[0001] Infectious diseases continue to have a major impact on the health of populations in the developing world. Malaria remains a devastating global health problem, affecting close to 40% of the world population. After attempts to eradicate the disease the 1960's, malaria has resurged once again, affecting a large portion of the developing world. According to the World Health Organization, worldwide, 300- 500 million people contract malaria each year, resulting in 1 to 3 million deaths annually, most of which are children in sub-Saharan Africa.

[0002] Health workers in rural facilities in Africa are confronted every day with the diagnosis and treatment of sick children with malaria and other infectious diseases. Malaria, pneumonia and acute diarrhea are among the most important infectious diseases in developing countries in terms of morbidity and mortality burden on children. The scarcity of diagnostic tools needed to determine the etiologic origin of these infections in rural health facilities, such as those in Africa, further complicates the problem by limiting the means for determining the appropriate treatment.

[0003] One crucial factor is the lack of diagnostic tests that can be performed at low-infrastructure sites, which serve most of the rural population. Without these diagnostics, health-care workers do not know who should be treated and, just as importantly, who should not be treated. The resource limitations in rural health facilities in developing countries have a great impact on determining the design and required performance specifications of diagnostic tests.

[0004] Clinical symptoms of malaria, bacterial infections and viral infections can often overlap. For instance, the main signs and symptoms of malaria include measured or reported fever, anemia and respiratory distress, whereas frequent signs and symptoms of pneumonia in children include cough, increased respiratory rate and fever. In many developing countries, children with fever and no laboratory confirmation of parasitemia are often diagnosed with malaria, whereas pneumonia is often diagnosed in the presence of increased respiratory rate and cough. Due to the fact that fever and tachypnea can be present in both malaria and pneumonia, misdiagnosis of these diseases is thought to be prevalent in developing countries. Misdiagnosed children may receive the wrong treatment or may be treated for both diseases, wasting scarce resources and unnecessarily boosting resistance levels to antimalarials and antibiotics.

[0005] Malaria is caused by intraerythrocytic protozoa of the genus Plasmodium. Humans can be infected with P. falciparum, P. vivax, P. ovale, and P. malariae. Severe malaria is almost exclusively caused by P. falciparum. Plasmodia are primarily transmitted by the bite of an infected female Anopheles mosquito, but can also occur by exposure to infected blood, either congenitally or by transfusion.

[0006] When sporozoites are inoculated into the bloodstream, they enter hepatocytes within hours of their host invasion and begin to divide into exoerythrocytic merozoites. The release of merozoites from the liver usually takes approximately 10-14 days, whereupon the parasite begins to replicate in the red blood cells. Once inside a red blood cell, the parasite begins to multiply and eventually causes the host cell to lyse, releasing the swarm of merozoites into the blood stream, ready to infect the next blood cell. The duration of each cycle is about 48 hours. In non-severe malaria, the infected individual is ill; in cases of severe malaria the infected individual's life is at risk. In addition, multiple exposures to the malaria parasite can lead to sufficient immunity where the human can harbor parasites without noticeable clinical symptoms. Such an individual is said to be "parasitemic" without having malaria.

[0007] Clinical symptoms of malaria can include headache, fever, chills, diaphoresis, nausea, vomiting, extreme weakness and impaired consciousness. In laboratory results, the erythrocyte sedimentation rate, C-reactive protein, and procalcitonin are often elevated. Manifestations of severe malaria may additionally include cerebral malaria, severe anemia, hyperpyrexia, acute renal failure, pulmonary adema, hyperparasitemia, and onset of additional associated infections. Patients with severe malaria should be treated on an in-patient basis, in an intensive care unit. Clinical deterioration to severe malaria usually occurs 3-7 days after fever onset. In tropical countries with a high transmission of malaria, severe malaria is predominantly a disease of children under the age of 5. [0008] Settings with no laboratory infrastructure offer the greatest technical challenges for diagnostic test developers. The challenge is to create a test that is easy to transport, store, and administer, and which rapidly provides accurate, easy to interpret results. This challenge is further complicated by the lack of clean water, dependable electricity and trained personnel. To be successfully used in these settings, tests must also be inexpensive and capable of withstanding the grueling environmental conditions.

[0009] Rapid diagnostic tests (RDTs) or malaria rapid diagnostic devices (MRDDs), such as lateral flow immunochromatographic devices, sometimes referred to as "dipsticks," are often used in rural test centers to rapidly detect species-specific parasite antigens targeting, for example, the histidine-hch protein-2 of P. falciparum or a parasite specific lactate dehydrogenase. These antigens are present in the blood of infected patients. A color change, through the use of colohmethc detection techniques such as colloidal gold or colored latex, on the absorbing nitrocellulose strip signifies the presence of parasite antigen. The RDT may detect from one to all four species of malaria parasite which infect humans, depending on the number of antigens it is designed to detect.

[0010] RDT sensitivity can be influenced by the species of the parasite, the number of parasites present, the care employed in the storage and handling of the device, the method of administration and interpretation of the test, parasite viability, and the antigen used. Although RDT tests may enhance diagnostic speed and ease of use, they have low sensitivity below 100 parasites/μl and may show a false negative in patients with high parasitemia.

[0011] Lack of well-made or properly-stored RDTs, and accurate reference materials against which to test RDTs have impacted the reliability of tests available. Even manufacturers themselves are sometimes unsure of the performance of the tests they release for sale. With a lack of adequate manufacturing standards and over 50 manufacturers of such tests, it is often difficult for a clinician to determine if the test being administered is producing accurate results. In addition, as described above, it is possible to have parasites in the blood without suffering from clinical malaria. Furthermore, the hot humid temperatures commonly found in malaria- endemic countries contribute to the lack of reliability and susceptibility of the assays to degradation. [0012] Misdiagnosis of malaria increases the morbidity and mortality among children, and may boost resistance levels to antimalarial drugs and antibiotics. This last consequence is of particular importance given the limited availability of the new and more expensive antimalarial drugs and antibiotics in developing countries. Furthermore, the ability to determine the severity of malaria and to distinguish between malaria, bacterial infections and viral infections could have a significant impact on vaccine research in these fields. As a result, there is a need to develop new diagnostic tools for infectious diseases that can be used in conditions present in developing countries.

[0013] One approach would be to focus on the immunological response to infection. This includes production and activation of several blood cells (e.g. T-cells, B-cells, macrophages, neutrophils, denditric cells, etc.). Host immune responses are mediated by both cell-cell interactions and soluble factors that control cell proliferation and differentiation. These soluble factors include a number of specific glycoproteins called Colony-Stimulating Factors (CSF) that control the proliferation and differentiation of hemopoietic stem cells into blood cells such as granulocytes and macrophages. The molecular isolation of these protein factors - such as Granulocyte CSF (G-CSF), Macrophage CSF (M-CSF), Granulocyte/Macrophage CSF (GM-CSF) and lnterleukin 3 (IL-3), formerly known as multi-CSF - has had important implications for our understanding of hematopoiesis control.

[0014] Some immunological responses are pathogen-specific. For instance, certain intracellular viral infections cause the activation of subsets of T-cells, while other infections lead to B-cell maturation, antibody production and/or antibody- mediated protective mechanisms like phagocytosis and complement-mediated lysis. As a result, cytokine blood levels may have different profiles depending on the etiology of the infection. In particular, G-CSF levels increase in response to certain bacterial infections that cause a high neutrophil turnover. Several studies have investigated the diagnostic use of G-CSF to distinguish between bacterial and viral pneumonia. It has been found that G-CSF blood levels higher than 400pg/ml were suggestive of bacterial pneumonia. Moreover, P. falciparum infections do not, in general, cause an increase in white blood cell levels, although some results suggest that G-CSF blood levels could be increased in severe malaria. [0015] In addition, one could focus on erythropoietin (EPO), the glycoprotein that regulates red blood cell production, as a substance playing an important role in the response to anemia caused by malaria. Several studies show that EPO blood levels are increased in malarial anemia. Therefore, EPO blood levels could be used as a biomarker to diagnose the severity of malaria, and as a distinguishing marker between malaria and other infectious diseases.

[0016] Another approach would be to focus on procalcitonin (PCT), a precursor protein of the hormone calcitonin. A study conducted in South Africa has suggested that an increased blood level of PCT is suggestive of bacterial infections. Therefore, PCT could be potentially used to differentiate between bacterial and viral infections. Malaria may also cause an increase of that protein, but, the use of the PCT blood level as a means to identify bacterial infections has not been evaluated in malaria endemic areas.

[0017] Studies have shown that among children less than 2 years of age admitted to hospital suggest that G-CSF may be a used as a biomarker for the differential diagnosis of children presenting with fever and clinical pneumonia. Geometric mean (GM) of G-CSF blood levels were substantially increased in children having pneumococcal bacteremia when compared to that of children with malaria parasitemia and no bacteremia. Similar comparisons were found between gram negative bacteremias and malaria.

[0018] In order to take advantage of the treatment options that are available today, it is essential to identify those individuals who require treatment. It is imperative not only to administer drugs for the appropriate diseases to those who need them, but also to prevent over treatment that will eventually result in the predominance of resistant microorganisms, which is becoming a major threat. For instance, the rapid acquisition of resistance in the malaria-causative organism Plasmodium falciparum has been partly due to overuse of the anti-malarial drug chloroquine, and has driven a shift towards a new and more expensive class of anti- malaria compounds derived from artemisinin.

SUMMARY OF THE INVENTION

[0019] One embodiment of the present claims provide a novel and improved use of a diagnostic assay system for differentiating a patient having severe malaria requiring hospitalization from a patient having a less severe form of malaria where hospitalization is not required. The present diagnostic assay system includes measuring the patient's blood erythropoietin (EPO) levels and comparing the patient's blood EPO levels to a standard value, wherein blood EPO levels above the standard value correlate to that of a patient with severe malaria, and wherein blood EPO levels below the standard value correlate to patients having less severe forms of malaria, and wherein only patients having EPO blood levels equal to or greater than the standard value require hospitalization.

[0020] In another embodiment, the present claims provide a novel use of a diagnostic assay system for differentiating a patient having severe malaria from a patient having bacterial pneumonia, comprising measuring a patient's blood EPO level and comparing the patient's blood EPO level to a standard value. If the patient's blood EPO level is above the standard value, then it correlates to a person having severe malaria. If the blood EPO level is below the standard value, then the value correlates with patients having bacterial pneumonia.

[0021] In another aspect, the present claims provide a novel use of a diagnostic assay system for differentiating a patient having severe malaria from a patient having a non-malarial febrile disease comprising measuring a patient's blood EPO levels and comparing the patient's blood EPO levels to a standard value, wherein blood EPO levels above the standard value correlate to a patient having severe malaria and blood EPO levels below the standard value correlate with patients having non- malarial febrile diseases.

[0022] In one aspect, the diagnostic assay system will use Enzyme-Linked Immunosorbent Assay (ELISA) to measure blood EPO levels. In another aspect, the diagnostic assay system will use Polymerase Chain Reaction assay (PCR) to measure EPO blood levels. In another aspect, the diagnostic assay system will use lateral flow technology to measure blood EPO levels. In another embodiment, the diagnostic assay system will use flow-through technology to measure blood EPO levels. In another embodiment, the diagnostic assay system will use latex bead technology to measure blood EPO levels. In another aspect, the diagnostic assay system will use piezoelectric technology to measure blood EPO levels. In another aspect, the diagnostic assay system will use mass spectrometry to measure blood EPO levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 depicts the distribution of patient erythropoietin (EPO) blood levels in Groupi , Group2, Group 3, and Group 5, using the diagnostic assay systems herein disclosed. Group 1 is the control group, consisting of children recruited from the community with no fever, no signs / symptoms of illness and a negative malaria parasitemia. Group 2 is the viral pneumonia group, consisting of children with infiltrates other than consolidation in the chest x-ray, negative blood cultures, negative for malaria parasitemia and positive Polymerase Chain Reaction assay (PCR) for a respiratory virus. Group 3 is the bacterial pneumonia group, consisting of children with clinically severe pneumonia, consolidation in their chest x- ray, positive blood culture, and negative malaria parasitemia. Group 5 is the inpatient malaria group, consisting of children with respiratory distress who have tested positive for malaria parasitemia, have a normal chest x-ray, negative blood culture, and have been diagnosed with malaria by an attending pediatrician. The EPO blood levels are measured in unit per liter. Median, 25^th and 75^th percentiles are indicated.

[0024] Figure 2 depicts the distribution of patient EPO blood levels in Groups 1 through 6, using the diagnostic assay systems herein disclosed. Group 1 is the control group, consisting of children recruited from the community with no fever, no signs / symptoms of illness and a negative malaria parasitemia. Group 2 is the group consisting of children with infiltrates other than consolidation in the chest x-ray, negative blood cultures, negative for malaria parasitemia and positive PCR for a respiratory virus. Group 3 is the group consisting of in-patient children with consolidation in their chest x-ray, positive blood culture, and negative malaria parasitemia. Group 4 is the group consisting of out-patient children with consolidation in their chest x-ray and negative malaria parasitemia. Group 5 is the in-patient malaria group, consisting of children with respiratory distress who have tested positive for malaria parasitemia, have a normal chest x-ray and negative blood culture. Group 6 is the group consisting of out-patient children who have tested negative for bacterial cultures an positive for malaria, but have less severe symptoms than those children requiring hospitalization. The EPO blood levels are measured in unit per liter. Median, 25^th and 75^th percentiles are indicated.

DETAILED DESCRIPTION OF THE INVENTION [0025] "Blood level" is a measure of the total amount of erythropoietin in patient blood, whether the sample is whole blood, serum, or plasma.

[0026] "Analyte" is defined as the glycoprotein or cytokine that is being measured in the diagnostic assay system.

[0027] "Diagnostic assay system" is defined as the system used to differentiate severe malaria patients from other patients with febrile diseases.

[0028] "Severe malaria" is defined as a malarial infection that requires hospitalization, wherein parasitemia is more than 5%, and which is often accompanied by hyperpyrexia, cerebral malaria, pulmonary edema, and renal and/or liver malfunction.

[0029] Currently, severe malaria is difficult to distinguish from less severe malaria and other non-malarial febrile diseases, (see, for example, Kallander, et al. ACTA Tropica, 90 (2004) 211 -214.) Parasitized erythrocytes are sometimes sequestered in tissue capillaries resulting in a falsely low parasite count in the peripheral blood. As a result, methods of diagnosis measuring the levels of parasite present in the blood are often inaccurate. Furthermore, current methods of diagnosis provide little or no information as to the development stage of the malaria.

[0030] The presently disclosed diagnostic assay systems use erythropoietin (EPO) to differentiate between a patient having severe malaria requiring hospitalization from a patient with less severe malaria or a patient with another non- malaria febrile disease.

[0031] Although examination of a blood smear is considered the traditional, reliable method of use for malarial diagnosis, advancements have been made in diagnostic methods, including Polymerase Chain Reaction assay (PCR), Enzyme- Linked Immunosorbent Assay (ELISA) and rapid "dipstick" immunoassay. Sensitivity and specificity of these methods can variety greatly. For example, tests based on PCR for species specific Plasmodium genome tend to be more sensitive than other tests but are currently not practical for use in rural areas.

[0032] The present disclosure describes the use of a diagnostic assay system for differentiating those malarial patients requiring hospitalization and immediate attention from those with less severe infections, not requiring hospitalization. The diagnostic assay system includes a flow device or other analyte detecting and measuring assay capable of detecting levels of EPO in a blood sample. The diagnostic assay system may be used by healthcare providers to distinguish severe malaria from non-severe malaria and other non-malaria febrile diseases, such as bacterial or viral pneumonia. The particular embodiments described herein are intended in all respects to be illustrative rather than restrictive. Alternative embodiments may become apparent to those skilled in the art to which the present embodiment of the invention pertains without departing from its scope.

[0033] The present disclosure may include an assay for detecting and measuring blood EPO levels using, for example, ELISA, lateral flow technology, flow-through technology, latex bead technology, piezoelectric technology, or mass spectrometry.

[0034] To detect the amount of analyte present in the subject sample, the diagnostic assay system may use various detection techniques, including, but not limited to spectral, colorimethc, fluohmetric, and electrophoretic analysis. Detection of analyte using any of the foregoing techniques provides information as to the amount of analyte present in the blood sample, which can be compared to the standard value and used to distinguish patients having severe malaria and requiring hospitalization from those having a less severe form of malaria and not requiring hospitalization.

[0035] The present disclosure may include an assay for detecting and measuring the blood levels of EPO which may include flow-through and/or lateral flow diagnostic technology, often used in determining the levels of a particular protein in a sample. The flow-through and/or lateral flow diagnostic technology may include the following steps: (1 ) obtaining a sample from the subject being diagnosed, (2) contacting the sample with a particular membrane, wherein the subject analyte of interest is bound, and (3) a method of detection is used to determine the presence and quantity of analyte present in the subject sample.

[0036] Forney, et al. (J. Clin. Microbiol. 2001 ; 39(8):2884-2890) and Makler, U.S. Pat. No. 5,124,141 , are herein incorporated by reference, particularly with respect to their description of the "dipstick" assays often employed in the detection of Plasmodium falciparum (P. falciparum), the malarial parasite most often responsible for the onset of severe malaria. Techniques like those described in Forney et al. and Makler are well-known by those skilled in the art and involve antigen capture technology, lateral flow or flow-through systems using a nitrocellulose strip, and detection technology to determine the presence of a particular analyte in a blood sample.

[0037] The present disclosure describes a diagnostic assay system wherein techniques described in Forney et al. and Makler may be used to diagnose and differentiate severe malaria requiring hospitalization from less severe malaria. It has further been discovered that the techniques described in Forney et al. and Makler may be used to differentiate patients with severe malaria from patients with other non-malarial febrile diseases, such as pneumonia and diarrhea.

[0038] The present embodiment provides a diagnostic assay system that may include techniques for measuring and detecting EPO. Lee et al., U.S. Pat. No. 7,312,089, is herein incorporated by reference, particularly with respect to its description of techniques for detecting and measuring the amounts of EPO in a blood sample. Techniques like those described in Lee et al. are well-known by those skilled in the art. It has been discovered that these techniques for detecting blood EPO levels may be used to differentiate patients with severe malaria requiring hospitalization from those with less severe malaria. It has further been discovered that the techniques described in Lee et al. may be used to diagnose and differentiate patients with severe malaria from patients with other non-malarial febrile diseases, such as pneumonia and diarrhea.

[0039] The present embodiment provides a diagnostic assay system that may include an enzyme-linked immunosorbent assay for measuring EPO blood levels, similar to that described in "Alpco Diagnostic EPO EIA Protocol" (www.alpco.com) and Fibi et al., U.S. Pat. No. 5,712,370, herein incorporated by reference.

[0040] The present embodiment provides a diagnostic assay system that may include latex bead suspension and flow cytometry techniques, similar to those described in Hechinger et al., U.S. Pat. No. 6,159,748, herein incorporated by reference, particularly with respect to its description of methods of detecting antigens in a sample solution.

[0041] The present disclosure also provides a diagnostic assay system that may include a piezoelectric chemical sensing device, similar to that described in Josse et a/., U.S. Pat. No. 5,852,229, herein incorporated by reference, particularly with respect to its description of a device used to measure concentrations of a particular analyte.

[0042] Scholl et al. (Am. J. Trap. Med. Hyg., 75(5):546-551 ; 2004) and Demirev et al., U.S. Pat. No. 7,270,948, are herein incorporated by reference, particularly with respect to their description of mass spectrometry and its use in detecting malarial parasites.

EXAMPLES

Example 1

[0043] Child subjects under the age of 5 in Mozambique are divided into four groups. Group 1 is the control group, consisting of children recruited from the community with no fever, no signs / symptoms of illness and a negative malaria parasitemia. Group 2 is the viral pneumonia group, consisting of children with infiltrates other than consolidation in the chest x-ray, negative blood cultures, negative for malaria parasitemia and positive PCR for a respiratory virus. Group 3 is the bacterial pneumonia group, consisting of children with clinically severe pneumonia, consolidation in their chest x-ray, positive blood culture, and negative malaria parasitemia. Group 5 is the in-patient malaria group, consisting of children with respiratory distress who have tested positive for malaria parasitemia, have a normal chest x-ray, negative blood culture, and have been diagnosed with malaria by an attending pediatrician.

Table 1

[0044] Figure 1 depicts the distribution of patient EPO blood levels using the diagnostic assay systems herein disclosed. The EPO blood levels are measured in unit per liter. Median, 25^th and 75^th percentiles are indicated. EPO blood levels are significantly elevated severe malaria patients and may be used to differentiate patients with severe malaria, requiring hospitalization, from those with no fever as well as from those with bacterial or viral pneumonia. Median EPO blood levels for the Group 5 is 510.6 U/L, almost 7 times higher than the median EPO blood levels for any other group and 28 times higher than the median for the control group. Table 1 depicts a summary of the resulting data.

Example 2

[0045] Child subjects under the age of 5 in Mozambique are divided into six groups. Group 1 is the control group, consisting of children recruited from the community with no fever, no signs / symptoms of illness and a negative malaria parasitemia. Group 2 is the group consisting of children with infiltrates other than consolidation in the chest x-ray, negative blood cultures, negative for malaria parasitemia and positive PCR for a respiratory virus. Group 3 is the group consisting of in-patient children with consolidation in their chest x-ray, positive blood culture, and negative malaria parasitemia. Group 4 is the group consisting of out-patient children with consolidation in their chest x-ray and negative malaria parasitemia. Group 5 is the in-patient malaria group, consisting of children with respiratory distress who have tested positive for malaria parasitemia, have a normal chest x-ray and negative blood culture. Group 6 is the group consisting of out-patient children who have tested negative for bacterial cultures an positive for malaria, but have less severe symptoms than those children requiring hospitalization.

[0046] Figure 2 depicts the distribution of patient EPO blood levels using the diagnostic assay systems herein disclosed. The EPO blood levels are measured in unit per liter. Median, 25^th and 75^th percentiles are indicated. EPO blood levels are significantly elevated in patients with severe malaria and may be used to differentiate patients with severe malaria, requiring hospitalization, from those with no fever, those with less severe malaria, not requiring hospitalization, those with bacterial pneumonia or consolidations, and those with viral pneumonia. Median EPO blood levels for the Group 5 group is 12 times higher than the median EPO blood levels for patients with less severe malaria, and 28 times higher than the median for the control group. Table 2 depicts a summary of the resulting data.

Table 2

[0047] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0048] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0049] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0050] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0051] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0052] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

We claim:

1. The use of a diagnostic assay system for differentiating a patient having severe malaria requiring hospitalization from a patient having a less severe form of malaria where hospitalization is not required comprising measuring the patient's erythropoietin (EPO) blood level and comparing the patient's EPO blood level to a standard value, wherein patient EPO blood levels above the standard value correlate to a patient having severe malaria and EPO blood levels below the standard value correlate with patients having less severe forms of malaria and wherein only patients having EPO blood levels equal to or greater than the standard value require hospitalization.

2. The use of a diagnostic assay system for differentiating a patient having severe malaria from a patient a patient having non-malarial febrile diseases comprising measuring a patient's erythropoietin (EPO) blood level and comparing the patient's EPO blood level to a standard value, wherein patient EPO blood levels above the standard value correlate to a patient having severe malaria and EPO blood levels below the standard value correlate with patients having non-malarial febrile diseases.

3. A diagnostic assay system according to claim 2, wherein EPO blood levels above the standard value correlate to a patient having severe malaria and EPO blood levels below the standard value correlate with patients having bacterial pneumonia.

4. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using enzyme-linked immunosorbent assay.

5. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using polymerase chain reaction assay.

6. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using lateral flow technology

7. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using flow-through technology.

8. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using latex bead technology.

9. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using piezoelectric technology.

10. A diagnostic assay system according to claims 1 or 2, wherein EPO blood level is measured using mass spectrometry.