EP3545307A1

EP3545307A1 - Method for diagnosing different forms of malaria

Info

Publication number: EP3545307A1
Application number: EP17811878.2A
Authority: EP
Inventors: Thomas Jacobs; Maria MACKROTH; Ellis OWUSU-DABO; Christiane STEEG; Annemieke ABEL; Daniel EIBACH; Jürgen May
Original assignee: Bernhard-Nocht-Institut fur Tropenmedizin; Universitatsklinikum Hamburg Eppendorf; Universitaet Hamburg
Current assignee: Bernhard-Nocht-Institut fur Tropenmedizin; Universitatsklinikum Hamburg Eppendorf; Universitaet Hamburg
Priority date: 2016-11-25
Filing date: 2017-11-24
Publication date: 2019-10-02
Also published as: WO2018096129A1

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.

Description

METHOD FOR DIAGNOSING DIFFERENT FORMS OF MALARIA

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 con^¬ centration of the serine protease granzyme B in a body fluid sample of a subject. The concentration of granzyme B is indic^¬ ative 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 uncomplicat^¬ ed 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 bil^¬ lion people - nearly half of the world's population - are at risk of malaria. It is estimated that about 214 million malar^¬ ia 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 fe^¬ male Anopheles mosquitoes. The parasites travel from the sali^¬ va 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 af^¬ ter occurrence of the first symptoms, uncomplicated malaria can progress to a severe form, often leading to seizures, co^¬ ma, or death.

The early diagnosis of uncomplicated malaria is of utmost im^¬ portance as it increases the chances for a successful treat^¬ ment 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 pres^¬ ence of the parasite in the patient, it does not allow distin^¬ guishing 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 se^¬ vere 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 pa^¬ tients having the uncomplicated form of the disease. Based on this insight, the present invention allows providing tests that reliably diagnose severe malaria or uncomplicated malar^¬ ia .

Accordingly, the present invention provides for a diagnostic method in which the granzyme B levels in the patient are de^¬ tected 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 se^¬ vere 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 princi^¬ ple, 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 di^¬ agnosed with malaria. The malaria diagnosis can be obtained from any method suitable for confirming the presence of Plas^¬ modium protozoa in the subject, for example by PCR-based de^¬ tection 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 sub^¬ ject 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:l or an enzymatically active fragment or variant thereof.

Granzyme B having the sequence of SEQ ID NO:l 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:l are to be understood as polypeptides that differ by one or more exchanges of amino acids from the amino acid se^¬ quence of the polypeptide shown in SEQ ID NO:l. Basically, any amino acid residue of the amino acid sequence shown in SEQ ID NO:l 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 se^¬ quence 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:l are also in^¬ cluded as variants. Such insertions can be made at any posi^¬ tion of the polypeptide shown in SEQ ID NO:l. Moreover, polypeptides in which one or more amino acids are missing, in com^¬ parison with the polypeptide shown in SEQ ID NO:l are also considered to be variants of the polypeptides shown in SEQ ID NO:l. Such deletions can apply to any amino acid position of the sequence of SEQ ID NO:l. Enzymatically active fragments of the sequence shown in SEQ ID NO:l or variants thereof are to be understood as polypeptides that differ from the amino acid sequence shown in SEQ ID NO:l, 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 scien^¬ tific literature, Spaeny-Dekking et al . (1998), Extracellular granzymes A and B in humans: detection of native species dur^¬ ing 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 ELI SA 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 val^¬ ue 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 num- ber 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 malar^¬ ia. Where the method of the invention is carried out with only one type of reference value and said value reflects the uncom^¬ plicated 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 ex^¬ ceed 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 di^¬ agnosed with malaria essentially equals or only slightly devi^¬ ates from the reference value, then the tested subject is af^¬ flicted with the uncomplicated form of malaria. In that case it is preferred that the test value deviates from the refer^¬ ence 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 signifi^¬ cantly lower than the reference value. Normally, the granzyme B value measured in a subject suffering from uncomplicated ma^¬ laria will be lower than the reference value for severe malar- ia 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 di^¬ agnosed with malaria essentially equals or only slightly devi^¬ ates from the reference value, then the tested subject is af^¬ flicted with the severe form of malaria. In that case it is preferred that the test value deviates from the reference val^¬ ue 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 ref^¬ erence value which is indicative for the uncomplicated form of malaria and at least a second reference value which is indica^¬ tive for the severe form of malaria. The first and second ref^¬ erence values can be defined as outlined below. Using two dif^¬ ferent types of reference values, one for the uncomplicated form of malaria and one for the severe form, has the particu^¬ lar advantage that the test value can be compared to different reference values which further reduces the risk of misinter^¬ preting 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 popu^¬ lation 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, prefera^¬ bly 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 evi^¬ dent 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 meas^¬ ured in a subject exceeds the first reference value for uncom^¬ plicated 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 ma^¬ laria 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 regular^¬ ly show clinical symptoms that resemble those of severe malar^¬ ia. These symptoms include fever, respiratory distress, somno^¬ lence, 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 differ^¬ ent 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 ma^¬ laria, e.g. by PCR-based detection of Plasmodium-specific nu^¬ cleic 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 ob^¬ tained 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 symp^¬ toms are well known to the skilled person and include, amongst others, high fever, respiratory distress, somnolence, vomit^¬ ing, 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 hu^¬ mans, 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 tar^¬ get the detection of human granzyme B comprising the sequence of SEQ ID NO:l 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 com^¬ parison 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 meas^¬ ured 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 ma^¬ laria 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 refer^¬ ence 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 sig^¬ nificantly lower than the reference value. Normally, the granzyme B value measured in a subject suffering from uncom^¬ plicated 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%. Converse- ly, where the method of the invention is carried out only with one type of reference value reflecting the severe form of ma^¬ laria, and the test value obtained from the sample of the sub^¬ ject diagnosed with malaria essentially equals or only slight^¬ ly 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 > o ·

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 meas^¬ ured in a subject exceeds the first reference value for uncom^¬ plicated 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 ad^¬ ministration 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 corre^¬ sponds 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 use^¬ ful 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 travellers 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 Ma^¬ laria n=5) was analyzed using a commercially available human granzyme B ELISA (Biolegend) . Significance was calculated us^¬ ing the one way ANOVA test using Graph Pad Prism.

Figure 3 shows the amino acid sequence of human granzyme B.

Figure 4 shows a box plot of the granzyme B levels in serum samples of children with malaria (n=35) , bacteraemia (n=30), or healthy controls (n=10) . The samples were analyzed by a bead-based multiplex assay using Luminex technology and the Human CD8+ T Cell panel offered by Merck.

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 de^¬ scribed 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 lympho^¬ cytes were stained with anti-Perforin and anti-granzyme B af^¬ ter 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 neg- ative 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 con^¬ trols. 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.

Example 3 : Granzyme B in patients with severe malaria

Children under the age of 5 years with either malaria or bac^¬ terial sepsis were recruited at the Agogo Presbyterian Hospi^¬ tal (Ashanti Region, Ghana) between February 2010 and May 2012. Healthy children were recruited in vaccination clinics from the same district. Case definitions of severe malaria, bacterial sepsis or healthy controls were similar to Example 2: Severe malaria was defined as a child with a negative blood culture and symptoms according to the AQUAMAT-study in Dondorp et al . (2010) . Bacterial sepsis was defined when a pathogen had been grown from a blood culture and no malaria parasites were detected. Children without a fever or history of fever were included as healthy controls. Serum concentrations of soluble granzyme B of children from all three groups were de- termined by the MILLIPLEX^® MAP Human CD8+ T Cell Magnetic Bead Panel (Luminex) according to the recommendations of the manu^¬ facturer .

The results are depicted in Figure 4. It can once again be seen that the granzyme B concentration in the serum of patients having severe malaria is much higher than in the control or sepsis group.

Claims

Method for 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 uncom^¬ plicated form of malaria.

Method according to claim 1, wherein said method comprises the comparison of the concentration obtained in step (b) with a first reference value which is indicative for the uncomplicated form of malaria and a second reference value which is indicative for the severe form of malaria.

Method according to claim 2, wherein

(i) the subject suffers from the severe form of malaria if the concentration obtained in step (b) exceeds the first reference value and equals or only slightly de^¬ viates from the second reference value; and

(ii) the subject suffers from the uncomplicated form of malaria if the concentration obtained in step (b) equals or only slightly deviates from the first ref- erence value and is significantly lower than the se^¬ cond reference value.

Method for diagnosing whether a subject is afflicted ei^¬ ther with (i) a severe form of malaria or (ii) with an un^¬ complicated form of malaria and a bacterial sepsis, com^¬ prising 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 a severe malaria or with an uncomplicated form of malaria which is combined with a bacterial sepsis.

Method according to claim 4, wherein said method comprises the comparison of the concentration obtained in step (b) with a first reference value which is indicative for the uncomplicated form of malaria and a second reference value which is indicative for the severe form of malaria.

Method according to claim 5, wherein

(ii) the subject suffers from the uncomplicated form of malaria and a bacterial sepsis if the concentration obtained in step (b) equals or only slightly deviates from the first reference value and is significantly lower than the second reference value.

7. Method according to any of claims 1-6, wherein said sample is a blood, plasma or serum sample.

8. Method according to any of claims 1-7, wherein said sub^¬ ject has been diagnosed with malaria by detection of one or more Plasmodium antigens.

9. Method according to any of claims 1-8, wherein the concentration of granzyme B is determined by an immunodiagnostic assay .

10. Method according to claim 9, wherein the immunodiagnostic assay is an enzyme-linked immunosorbent assay (ELISA) .

11. Method according to any of claims 1-10, wherein said granzyme B is human granzyme B comprising the sequence of SEQ ID NO:l or an enzymatically active fragment thereof.

12. Method according to any of claims 1-11, wherein said malaria has been caused by Plasmodium falciparum, Plasmodium ovale, und Plasmodium vivax, Plasmodium malariae or Plas^¬ modium knowlesi.

13. Method according to any of claims 1-6, wherein said first reference value which is indicative for the uncomplicated form of malaria corresponds to a blood plasma or serum concentration of granzyme B of < 250 pg/ml.

14. Method according to any of claims 1-6, wherein said second reference value which is indicative for the severe form of malaria corresponds to a blood plasma or serum concentra^¬ tion of granzyme B of > 250 pg/ml.

15. Kit for carrying out the method of any of claims 1-14, 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.