LU93323B1 - Method for diagnosing different forms of malaria - Google Patents
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- LU93323B1 LU93323B1 LU93323A LU93323A LU93323B1 LU 93323 B1 LU93323 B1 LU 93323B1 LU 93323 A LU93323 A LU 93323A LU 93323 A LU93323 A LU 93323A LU 93323 B1 LU93323 B1 LU 93323B1
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a new method for diagnosing whether a subject is afflicted with a severe or uncomplicated form of malaria. The method is based on the determination of the concentration of the serine protease granzyme B in a body fluid sample of a subject. The concentration of granzyme B is indicative for either the severe form or the uncomplicated form of malaria. In a further aspect, the invention relates to a new method for diagnosing whether a subject is afflicted either with a severe form of malaria or with an uncomplicated form of malaria which is combined with a bacterial sepsis. The invention also relates to a diagnostic kit for carrying out the diagnostic method of the invention. 93323
Description
METHOD FOR DIAGNOSING DIFFERENT FORMS OF MALARIA
DESCRIPTION
The invention relates to a new method for diagnosing whether a subject is afflicted with a severe or uncomplicated form of malaria. The method is based on the determination of the concentration of the serine protease granzyme B in a body fluid sample of a subject. The concentration of granzyme B is indicative for either the severe form or the uncomplicated form of malaria. In a further aspect, the invention relates to a new method for diagnosing whether a subject is afflicted either with (i) a severe form of malaria or (ii) with an uncomplicated form of malaria and a bacterial sepsis. The invention also relates to a diagnostic kit for carrying out the diagnostic method of the invention.
FIELD OF THE INVENTION
Malaria is a serious disease that threatens a large part of the world's population. According to the WHO, about 3.2 billion people - nearly half of the world's population - are at risk of malaria. It is estimated that about 214 million malaria cases occurred in 2015 with about 438,000 people dying from the disease. Great efforts have been made in the last decades to control the disease. By improved prevention and control measures, a 60% reduction in malaria mortality rates has been reached since the year 2000.
Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans through the bites of infected female Anopheles mosquitoes. The parasites travel from the saliva of the mosquitoes via the blood of the infected human to the liver where they mature and reproduce. First symptoms of
Malaria normally occur 10-15 days after the bite and include fever, fatigue, chills, vomiting and headaches.
Malaria is classified by the WHO into either "severe malaria" or "uncomplicated malaria". If not treated within 24 hours after occurrence of the first symptoms, uncomplicated malaria can progress to a severe form, often leading to seizures, coma, or death.
The early diagnosis of uncomplicated malaria is of utmost importance as it increases the chances for a successful treatment in the patient and furthermore contributes to a reduction of malaria transmission. Malaria is typically diagnosed by the microscopic examination of blood using blood films, but these methods are burdensome and time-intensive. Moreover, although reliable PCR-based methods have been developed for diagnosing malaria, these methods are only rarely applied in regions where malaria occurs, e.g. in sub-Saharan Africa, due to cost and complexity of the test. More recently, rapid diagnostic tests have been developed which are based on the detection of Plasmodium antigens. While these tests can confirm the presence of the parasite in the patient, it does not allow distinguishing between severe malaria and uncomplicated forms of the disease. Accordingly, there is a need for methods that allow determining whether a patient develops an uncomplicated form of malaria or severe malaria.
Further problems arise from the fact that a number of symptoms that characterize severe malaria are also found in patients that suffer from bacterial sepsis, such fever, respiratory distress, somnolence, vomiting, shivers, and tachycardia. As a result, a bacterial sepsis that occurs in a patient that has been diagnosed with malaria is often not correctly diagnosed because the symptoms indicating sepsis are ascribed to a severe form of malaria. Accordingly, there is a particular need for means and methods to distinguish a severe form of malaria from the uncomplicated form which comes along with a bacterial sepsis; such means or methods would allow an early treatment of sepsis, e.g. by the administration of antibiotics.
DESCRIPTION OF THE INVENTION
The studies underlying the present invention have revealed that the determination of granzyme B in a body fluid sample of a subject, preferably in a serum sample, allows distinguishing between the uncomplicated form of malaria and the severe form of malaria. Granzyme B is a serine protease having a size of 32 kD which is expressed by cytotoxic T lymphocyte, NK cells, and dendritic cells. The amino acid sequence of human granzyme B is shown in SEQ ID NO :1 and also in Figure 3. It was found that the granzyme B levels that can be detected in samples from patients suffering from severe malaria are significantly higher than the levels of the granzyme B in samples of patients having the uncomplicated form of the disease. Based on this insight, the present invention allows providing tests that reliably diagnose severe malaria or uncomplicated malaria .
Accordingly, the present invention provides for a diagnostic method in which the granzyme B levels in the patient are detected and compared to reference values. The reference values, which either reflect the granzyme B levels in patients with severe malaria or those in patients with uncomplicated malaria allow the conclusion of whether the test subject from whom the test sample was derived suffers from one or the other disease. In this way, the two forms of the disease can be distinguished from each other.
Thus, in a first aspect the present invention provides for a method of diagnosing whether a subject is afflicted with a severe or uncomplicated form of malaria, comprising a) providing a body fluid sample from the subject that has been diagnosed with malaria; b) determining in said sample the concentration of granzyme B ; and c) comparing the concentration obtained in step (b) with at least one reference value; wherein the comparison of the concentration obtained in step (b) with said at least one reference value indicates whether said subject is afflicted with a severe or uncomplicated form of malaria.
The sample to be used in the above method can be, in principle, any type of body fluid obtained from the subject to be diagnosed. In a preferred aspect, the sample will be a blood sample, such as a whole-blood sample, or a plasma or serum sample. In an even more preferred aspect, the sample will be a serum sample, such as a human serum sample.
The sample originates from a subject that has already been diagnosed with malaria. The malaria diagnosis can be obtained from any method suitable for confirming the presence of Plasmodium protozoa in the subject, for example by PCR-based detection of Plasmodium-specific nucleic acid, by microscopy, or by immunodetection of one or more specific Plasmodium antigens. In a preferred aspect, the malaria diagnosis in the subject has been obtained by an immunodiagnostic assay, such as an enzyme-linked immunosorbent assay (ELISA).
The malaria diagnosed in the subject may have been caused by any Plasmodium species which is known as a causative agent for malaria, but it is preferred that the disease diagnosed in the patient is caused by one of the species which are pathogenic for humans, namely, P. falciparum, P. ovale, und P. vivax, P. malariae or P. knowlesi.
In step (b) of the above-described method, the concentration of granzyme B is determined in the sample from the patient. Preferably, the granzyme B to be determined is human granzyme B comprising the sequence of SEQ ID NO :1 or an enzymatically active fragment or variant thereof.
Granzyme B having the sequence of SEQ ID NO :1 occurs in most humans. However, a slightly modified version of granzyme B may be expressed in some individuals. Accordingly, the method of the invention also comprises the detection of such variants of granzyme B. As used herein, variants of the polypeptide of SEQ ID NO :1 are to be understood as polypeptides that differ by one or more exchanges of amino acids from the amino acid sequence of the polypeptide shown in SEQ ID NO:1. Basically, any amino acid residue of the amino acid sequence shown in SEQ ID NO :1 can be exchanged for a different amino acid, provided the resultant sequence of the variant is still an enzymatically active polypeptide with serine protease function.
In particular, variants for which up to 5%, 10%, 15%, 20%, 30%, or 40% of the amino acids differs from the amino acid sequence shown in SEQ ID NO:1 are included. Polypeptides in which one or more amino acids were inserted in the amino acid sequence of the polypeptide shown in SEQ ID NO :1 are also included as variants. Such insertions can be made at any position of the polypeptide shown in SEQ ID NO:1. Moreover, polypeptides in which one or more amino acids are missing, in comparison with the polypeptide shown in SEQ ID NO :1 are also considered to be variants of the polypeptides shown in SEQ ID NO:1. Such deletions can apply to any amino acid position of the sequence of SEQ ID NO:1.
Enzymatically active fragments of the sequence shown in SEQ ID NO :1 or variants thereof are to be understood as polypeptides that differ from the amino acid sequence shown in SEQ ID NO:1, or from the above-defined variants thereof, by the absence of one or more amino acids at the N-terminus and/or at the C-terminus of the polypeptide.
Methods for determining the concentration of granzyme B are well known in the art and have been described in the scientific literature, Spaeny-Dekking et al. (1998), Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo, J. Immunol., 160: 3610-3616; Figueiredo et al. (2009), Heat shock protein 70 (HSP70) induces cytotoxicity of T-helper cells. Blood, 113: 3008-3016). In addition, kits are commercially available for the determination of the granzyme B concentration in a sample, such as the LEGEND MAX™ Human Granzyme B ELISA Kit with Pre-coated Plates (BioLegend, San Diego, USA).
Once the concentration of granzyme B has been measured in step (b) of the above method, the concentration is compared with at least one reference value. It is however preferred that the concentration is compared with more than one reference value, e.g. with 2, 3, 4, 5, 6 or more reference values. A skilled person will be readily able to identify and define appropriate reference values by statistical method without undue burden.
For example, the first reference value can be a reference value which is indicative for the uncomplicated form of malaria which has been obtained, e.g. as a mean value, by statistical methods from a patient population that comprised 10 or more, preferably 100 or more, more preferably 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, or 100,000 or more patients afflicted with the uncomplicated form of malaria. It will be understood that patient populations with a higher number of patients provide a more significant reference value and hence allow for a more reliable diagnosis.
The comparison of the concentration of granzyme B determined in the test sample and the reference value reveals whether the test subject is afflicted with severe or uncomplicated malaria. Where the method of the invention is carried out with only one type of reference value and said value reflects the uncomplicated form of malaria, then the tested subject is afflicted with a severe form of malaria if the test value significantly exceeds the reference value. Normally, the granzyme B value measured in a subject suffering from severe malaria will exceed the reference value for uncomplicated malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%. Conversely, where the method of the invention is carried out only with only one type of reference value reflecting the uncomplicated form of malaria, and the test value obtained from the sample of the subject diagnosed with malaria essentially equals or only slightly deviates from the reference value, then the tested subject is afflicted with the uncomplicated form of malaria. In that case it is preferred that the test value deviates from the reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5 %.
Where the method of the invention is carried out with only one type of reference value and said value reflects the severe form of malaria, then the tested subject is afflicted with an uncomplicated form of malaria if the test value is significantly lower than the reference value. Normally, the granzyme B value measured in a subject suffering from uncomplicated malaria will be lower than the reference value for severe malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%. Conversely, where the method of the invention is carried out only with one type of reference value reflecting the severe form of malaria, and the test value obtained from the sample of the subject diagnosed with malaria essentially equals or only slightly deviates from the reference value, then the tested subject is afflicted with the severe form of malaria. In that case it is preferred that the test value deviates from the reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.
According to the invention, it is particularly preferred that the diagnostic method is performed with at least a first reference value which is indicative for the uncomplicated form of malaria and at least a second reference value which is indicative for the severe form of malaria. The first and second reference values can be defined as outlined below. Using two different types of reference values, one for the uncomplicated form of malaria and one for the severe form, has the particular advantage that the test value can be compared to different reference values which further reduces the risk of misinterpreting the test value.
As stated above, the first reference value which is indicative for the uncomplicated form of malaria may be a mean value that has been obtained by statistical methods from a patient population that comprised 10 or more, preferably 100 or more, more preferably 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, or 100,000 or more patients afflicted with the uncomplicated form of malaria. Similarly, the second reference value which is indicative for the severe form of malaria may be a mean value that has been obtained by statistical methods from a patient population that comprised 10 or more, preferably 100 or more, more preferably 1,000 or more, 5,000 or more, 10,000 or more, 50,000 or more, or 100,000 or more patients afflicted with the severe form of malaria. Again, it is evident that patient populations with a higher number of patients allow for a more reliable assignment of a value obtained from a tested individual to either uncomplicated or severe malaria.
The subject suffers from the severe form of malaria if the concentration obtained in step (b) of the above method exceeds the first reference value being indicative for uncomplicated malaria and at the same time equals or only slightly deviates from the second reference value being indicative for severe malaria. Preferably, one can conclude that the subject suffers from the severe form of malaria if the granzyme B value measured in a subject exceeds the first reference value for uncomplicated malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%, and at the same time, deviates from the second reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.
Likewise, it can be concluded that the subject tested suffers from the uncomplicated form of malaria if the concentration obtained in step (b) of the above method deviates from the first reference value, which is indicative for uncomplicated malaria, by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%, and at the same time is significantly lower than the second reference value which is indicative for severe malaria, preferably by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%.
In a still further aspect, the present invention is used for providing a differential diagnosis between two states of malaria which are show highly similar symptoms and can be easily be confused with each other. Specifically, patients suffering both from an uncomplicated form of malaria and sepsis regularly show clinical symptoms that resemble those of severe malaria. These symptoms include fever, respiratory distress, somnolence, vomiting, shivers, and tachycardia. As a result, sepsis is not recognized and often treated too late. The fact that uncomplicated and severe malaria are characterized by different granzyme B levels can be used to distinguish uncomplicated malaria with sepsis from severe malaria.
Accordingly, in a second aspect, the invention provides a method for diagnosing whether a subject is afflicted either with (i) a severe form of malaria or (ii) an uncomplicated form of malaria and a bacterial sepsis. The method comprises the following steps: a) providing a body fluid sample from the subject that has been diagnosed with malaria and shows evident symptoms of severe malaria; b) determining in said sample the concentration of granzyme B ; and c) comparing the concentration obtained in step (b) with at least one reference value; wherein the comparison of the concentration obtained in step (b) with said at least one reference value indicates whether said subject is afflicted with either (i) a severe malaria or (ii) an uncomplicated form of malaria and a bacterial sepsis.
The method is based on the use of a body fluid sample. As stated above, any type of body fluid can be used in the course of the claimed method, but the sample will preferably be a blood sample from the subject to be tested, such as a whole-blood sample, or a plasma or serum sample. In an even more preferred aspect, the sample will be a serum sample, such as a human serum sample.
The subject to be tested has already been diagnosed with malaria, e.g. by PCR-based detection of Plasmodium-specific nucleic acid, by microscopy, or by immunodetection of one or more specific Plasmodium antigens. In a preferred aspect, the malaria diagnosis in the subject to be tested has been obtained by an immunodiagnostic assay, such as an enzyme-linked immunosorbent assay (ELISA). In addition, the subject to be tested shows evident symptoms of severe malaria. These symptoms are well known to the skilled person and include, amongst others, high fever, respiratory distress, somnolence, vomiting, shivers, and tachycardia.
The malaria diagnosed in the subject to be tested may have been caused by any Plasmodium species, and preferably it has been caused by one of the species which are pathogenic for humans, which means P. falciparum, P. ovale, und P. vivax, P. malariae or P. knowlesi.
As described above, the granzyme B concentration measurement can be conducted by methods known in the art, and it can target the detection of human granzyme B comprising the sequence of SEQ ID NO :1 or an enzymatically active fragment or variant thereof as described elsewhere herein.
The comparison of the value measured for the sample obtained from the subject to be tested is compared with the reference value as outlined above. The method can be based on the comparison with one or more than one reference values. Where the method of the invention is carried out with only one type of reference value and said value reflects the uncomplicated form of malaria, then the tested subject is afflicted with a severe form of malaria if the test value significantly exceeds the reference value. As outlined above, the granzyme B value measured in a subject suffering from severe malaria may exceed the reference value for uncomplicated malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%. Where the method is carried out only with only one type of reference value reflecting the uncomplicated form of malaria, and the test value obtained from the sample of the subject diagnosed with malaria essentially equals or only slightly deviates from the reference value, then the tested subject is afflicted with the uncomplicated form of malaria combined with sepsis. In that case it is preferred that the test value deviates from the reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.
Where the method is carried out with only one type of reference value and said value reflects the severe form of malaria, then the tested subject is afflicted with an uncomplicated form of malaria combined with sepsis if the test value is significantly lower than the reference value. Normally, the granzyme B value measured in a subject suffering from uncomplicated malaria will be lower than the reference value for severe malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%. Conversely, where the method of the invention is carried out only with one type of reference value reflecting the severe form of malaria, and the test value obtained from the sample of the subject diagnosed with malaria essentially equals or only slightly deviates from the reference value, then the tested subject is afflicted with the severe form of malaria. In that case it is preferred that the test value deviates from the reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.
Also for this method, it will be preferred that the value measured in the sample of the subject is compared with two different reference values, one reflecting the severe form of malaria and one reflecting the uncomplicated form of malaria. The subject suffers from the severe form of malaria if the concentration obtained in step (b) of the above method exceeds the first reference value being indicative for uncomplicated malaria and at the same time equals or only slightly deviates from the second reference value being indicative for severe malaria. Preferably, one can conclude that the subject suffers from the severe form of malaria if the granzyme B value measured in a subject exceeds the first reference value for uncomplicated malaria by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%, and at the same time, deviates from the second reference value by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%.
On the other hand, it can be concluded that the subject tested suffers from the uncomplicated form of malaria combined with a bacterial sepsis if the concentration obtained in step (b) of the above method deviates from the first reference value, which is indicative for uncomplicated malaria, by less than 100%, preferably less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5%, and at the same time is significantly lower than the second reference value which is indicative for severe malaria, preferably by at least 100%, preferably by at least 200%, at least 300%, at least 400%, at least 500%, or more, such as by at least 800%, at least 1000%, or at least 1500%. In such a situation, the administration of antibiotics is advisable.
The methods set out above refer to reference values which are indicative for the uncomplicated form of malaria or the severe form of malaria. Preferably, the first reference value which is indicative for the uncomplicated form of malaria corresponds to a blood plasma or serum concentration of granzyme B of less than 250 pg/ml, more preferably between 50-240 pg/ml, between 100-200 pg/ml, between 75-150 pg/ml, such as about 100 pg/ml. Still more preferably, the second reference value which is indicative for the severe form of malaria corresponds to a blood plasma or serum concentration of granzyme B of more than 250 pg/ml, more preferably between 300-3000 pg/ml, between 400-2000 pg/ml, between 500-1000 pg/ml, such as about 750 pg/ml.
In a third aspect, the invention provides a kit for carrying out the methods described herein above, comprising: (a) means for determining whether a subject is afflicted with malaria; (b) means for determining the concentration of granzyme B; and (c) optionally, buffers and diluents.
In one embodiment, the kit contains antibodies which are useful for the detection of Plasmodium antigens, e.g. by an ELISA. The kit may also include suitable immunologic reagents for determining the granzyme B concentration.
DESCRIPTION OF THE FIGURES
Figure 1 shows the production of cytotoxic molecules by CD4+ T cells from travelers infected with P. falciparum.
Figure 2 shows the granzyme B levels in serum analyzed by ELISA. The granzyme B content of frozen plasma samples from different individuals (Control n=10; Sepsis n=10, severe Malaria n=5) was analyzed using a commercially available human granzyme B ELISA (Biolegend). Significance was calculated using the one way ANOVA test using Graph Pad Prism.
Figure 3 shows the amino acid sequence of human granzyme B.
EXAMPLES
The invention is described in the following on the basis of examples, for the purpose of illustration, without limiting the invention. It will be evident to a person skilled in the art that modifications and variations of the examples described are possible without deviating from the idea of the invention.
Example 1 : Determination of granzyme B in malaria patients
Whole blood from patients (travelers) suffering from malaria and healthy donors were stained with anti-CD4, anti-PD-1 and CD107a. After fixation, red blood cells were lysed and lymphocytes were stained with anti-Perforin and anti-granzyme B after permeabilization. Cells were recorded on the LSRII flow cytometer (Becton Dickinson) and analyzed using the flow jow software.
The results are depicted in Figure 1. The upper panel depicted the intracellular expression of granzyme B in CD4+ T cells from a patient with a very high parasitemia. The lower panel showed a summary of data from malaria patients and healthy controls showing that CD4+ T cells from patients have the degranulation marker CD107a on the surface and expressed perforin and granzyme B intracellular at higher levels than healthy controls.
Example 2 : Granzyme B in patients with severe malaria
Serum samples were obtained from children below 15 years and above one months of age presenting with fever >38°C at the Agogo Presbyterian Hospital (Ashanti Region, Ghana). Healthy children were recruited in vaccination clinics from the same district. Sera from three different groups have been selected, which are (i) patients with bacterial sepsis, (ii) patients with severe malaria, (iii) healthy controls with no parasitae-mia. When a bacterial pathogen has been grown from a blood culture and no parasitemia has been detected, the child was considered to have a bacterial sepsis. Environmental bacteria and bacteria belonging to the skin flora (e.g., coagulase negative staphylococci, Corynebacterium spp. and Bacillus spp. ) were classified as contaminants. Severe malaria was defined as a child with a negative blood culture according to a previous ly published definition (Dondorp et al. 2010, Lancet 376, 1647-1657). Children under 15 years of age without fever (< 37.5°C), any respiratory symptom in the past 14 days and without any history of antipyretic, antibiotic or antimalarial treatment in the past 14 days were included as healthy controls. The concentrations of soluble granzyme B in the sera of all children of the three study groups were determined by the LEGEND MAX™ Human Granzyme B ELISA Kit with Pre-coated Plates (BioLegend, San Diego, USA) according to the recommendations of the manufacturer.
The results are depicted in Figure 2. It can be seen that the concentration of granzyme B in the serum is about 35 times higher in the group of patients having severe malaria than in the control or sepsis group.
SEQUENCE LISTING <110> Bernhard-Nocht-Institut für Tropenmedizin Universitätskiinikum Hamburg-Eppendorf
<120> METHOD FOR DIAGNOSING DIFFERENT FORMS OF MALARIA <130> P 99907 <160> 1 <170> Patentln version 3.5 <210> 1 <211> 247
<212> PRT <213> Homo sapiens <400> 1
Met Gin Pro lie Leu Leu Leu Leu Ala Phe Leu Leu Leu Pro Arg Ala 15 10 15
Asp Ala Gly Glu lie lie Gly Gly His Glu Ala Lys Pro His Ser Arg 20 25 30
Pro Tyr Met Ala Tyr Leu Met lie Trp Asp Gin Lys Ser Leu Lys Arg 35 40 45
Cys Gly Gly Phe Leu Ile Arg Asp Asp Phe Val Leu Thr Ala Ala His 50 55 60
Cys Trp Gly Ser Ser lie Asn Val Thr Leu Gly Ala His Asn lie Lys 65 70 75 80
Glu Gin Glu Pro Thr Gin Gin Phe lie Pro Val Lys Arg Pro lie Pro 85 90 95
His Pro Ala Tyr Asn Pro Lys Asn Phe Ser Asn Asp lie Met Leu Leu 100 105 110
Gin Leu Glu Arg Lys Ala Lys Arg Thr Arg Ala Val Gin Pro Leu Arg 115 120 125
Leu Pro Ser Asn Lys Ala Gin Val Lys Pro Gly Gin Thr Cys Ser Val 130 135 140
Ala Gly Trp Gly Gin Thr Ala Pro Leu Gly Lys His Ser His Thr Leu 145 150 155 160
Gin Glu Val Lys Met Thr Val Gin Glu Asp Arg Lys Cys Glu Ser Asp 165 170 175
Leu Arg His Tyr Tyr Asp Ser Thr lie Glu Leu Cys Val Gly Asp Pro 180 185 190
Glu lie Lys Lys Thr Ser Phe Lys Gly Asp Ser Gly Gly Pro Leu Val 195 200 205
Claims (15)
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LU93323A LU93323B1 (en) | 2016-11-25 | 2016-11-25 | Method for diagnosing different forms of malaria |
PCT/EP2017/080433 WO2018096129A1 (en) | 2016-11-25 | 2017-11-24 | Method for diagnosing different forms of malaria |
EP17811878.2A EP3545307A1 (en) | 2016-11-25 | 2017-11-24 | Method for diagnosing different forms of malaria |
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WO2010099607A1 (en) * | 2009-03-02 | 2010-09-10 | Fio Corporation | Diagnostic test panel for the diagnostic of malaria and severe bacterial infections |
US20110117107A1 (en) * | 2009-04-29 | 2011-05-19 | Morehouse School Of Medicine | Compositions and methods for diagnosis, prognosis and management of malaria |
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WO2010099607A1 (en) * | 2009-03-02 | 2010-09-10 | Fio Corporation | Diagnostic test panel for the diagnostic of malaria and severe bacterial infections |
US20110117107A1 (en) * | 2009-04-29 | 2011-05-19 | Morehouse School Of Medicine | Compositions and methods for diagnosis, prognosis and management of malaria |
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HERMSEN C.C ET AL: "Circulating concentrations of soluble granzyme A and B increase during natural and experimental Plasmodium falciparum infections", CLINICAL & EXPERIMENTAL IMMUNOLOGY, 1 June 2003 (2003-06-01), Oxford, UK, pages 467 - 472, XP055393063, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1808730/pdf/cei0132-0467.pdf> [retrieved on 20030601], DOI: 10.1046/j.1365-2249.2003.02160.x * |
WAN-CHUNG HU: "Human immune responses to Plasmodium falciparum infection: molecular evidence for a suboptimal TH[alpha][beta] and TH17 bias over ideal and effective traditional TH1 immune response", MALARIA JOURNAL, BIOMED CENTRAL, LONDON, GB, vol. 12, no. 1, 5 November 2013 (2013-11-05), pages 392, XP021176901, ISSN: 1475-2875, DOI: 10.1186/1475-2875-12-392 * |
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