LU101133B1 - Method for diagnosing symptomatic malaria - Google Patents
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- LU101133B1 LU101133B1 LU101133A LU101133A LU101133B1 LU 101133 B1 LU101133 B1 LU 101133B1 LU 101133 A LU101133 A LU 101133A LU 101133 A LU101133 A LU 101133A LU 101133 B1 LU101133 B1 LU 101133B1
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Abstract
The invention relates to a new method for diagnosing sympto- matic malaria in a subject. The method is based on the deter- mination of the C2 protein concentration in a body fluid sam- ple from the subject. A C2 concentration below a defined threshold in combination with malaria parasitaemia is indica- tive for symptomatic malaria. In a further aspect, the inven- tion relates to a method for diagnosing whether a subject is afflicted either with symptomatic malaria or a bacterial blood stream infection. The invention also relates to a diagnostic kit for carrying out the diagnostic methods of the invention.
Description
P 106496 - 1 - LU101133
DESCRIPTION The invention relates to a new method for diagnosing sympto- matic malaria in a subject. The method is based on the deter- mination of the C2 protein concentration in a body fluid sam- ple from the subject. A C2 concentration below a defined threshold in combination with malaria parasitaemia is indica- tive for symptomatic malaria. In a further aspect, the inven- tion relates to a method for diagnosing whether a subject is afflicted either with symptomatic malaria or bacterial blood stream infections. The invention also relates to a diagnostic kit for carrying out the diagnostic methods 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.
- 2 — LU101133 The early diagnosis of malaria is of utmost importance as it increases the chances for a successful treatment in the pa- tient and furthermore contributes to a reduction of malaria transmission. Malaria is typically diagnosed by the microscop- ic 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 (RDT) have been developed which are based on the detection of Plasmodium antigens using labeled antibodies. While these tests can con- firm the presence of the parasite in the blood of the patient, they do not allow the conclusion that the patient is afflicted with the symptomatic form of malaria. It has been found that especially in regions where malaria is widespread, many indi- viduals are infected with malaria parasites, but do not show symptoms of the disease. In these patients, the disease is controlled by the immune system and hence asymptomatic.
This makes a reliable diagnosis problematic, in particular in children at the age of 5 and younger, as a number of symptoms that characterize malaria are also found in patients that suf- fer from e.g. a bacterial blood stream infection. For example, both malaria and a bacterial blood stream infection regularly involve fever, respiratory distress, fatigue, vomiting, shiv- ers, and tachycardia. Therefore, a bacterial blood stream in- fection that occurs in a patient with asymptomatic malaria is often not correctly diagnosed, because the presence of para- sites in the blood of said patient in combination with the oc- currence of typical malaria symptoms are ascribed to sympto- matic malaria.
- 3 = LU101133 Accordingly, there is a need for means and methods to correct- ly diagnose symptomatic malaria and in particular to distin- guish symptomatic malaria from a bacterial blood stream infec- tion. Such means and methods would allow medical practitioners to initiate the correct treatment of the patient at an early stage of the respective disease.
DESCRIPTION OF THE INVENTION The studies underlying the present invention have revealed that the determination of the concentration of the complement component 2 (C2) in a body fluid sample of a subject, prefera- bly in a serum sample, allows distinguishing between malaria and a bacterial blood stream infection. C2 is a single chain glycoprotein with a molecular mass of about 100 kDa. It func- tions as a serine protease which provides for cleavage of com- plement protein C3 to its biologically active fragments. C2 is encoded by a single gene that is on human chromosome 6p within the class III region of the major histocompatibility complex. The C2 protein 1s mainly expressed in liver hepatocytes. The amino acid sequence of human C2 is shown in SEQ ID NO:1 and also in Figure 3. It was found that the C2 protein levels that can be detected in samples from patients suffering from a bac- terial blood stream infection are higher than in patients with symptomatic malaria. The fact that symptomatic malaria and a bacterial blood stream infection are characterized by differ- ent C2 levels can be used to distinguish these diseases from each other.
Thus, the present invention allows the provision of tests and assays that reliably distinguish between symptomatic malaria and a bacterial blood stream infection. This is particularly advantageous in patients with malaria parasitaemia, i.e. pa- tients that have been diagnosed to have Plasmodium parasites in their blood. Specifically, patients with malaria . —,
— 4 - LU101133 parasitaemia and bacterial blood stream infections regularly show clinical symptoms that resemble those of symptomatic ma- laria. These symptoms include fever, respiratory distress, somnolence, vomiting, shivers, and tachycardia. As a result, a bacterial blood stream infection is not recognized and either treated too late, or treated simultaneously with broad spec- trum antibiotics. Both scenarios leave the patient at a disad- vantage; 14% of undiagnosed bacterial blood stream infection cases result in death, while unnecessary and/or empirical drug treatment leads to high drug pressure and inevitably to the development of drug resistances.
Accordingly, the present invention provides for a diagnostic method in which the C2 levels in the patient are detected and compared to one or more reference values. Where one reference value is used, said reference value refers to a split concen- tration which means that if the tested concentration is below that value, this indicates that the patients suffers from symptomatic malaria. On the other hand, if the tested concen- tration is above that value, this indicates that the patient suffers from a bacterial blood stream infection. In certain embodiments, more than one reference value is used, e.g. two reference values. A first reference value may reflect the com- mon C2 levels in patients with symptomatic malaria, and a se- cond reference value may reflect the common C2 levels in pa- tients with bacterial blood stream infection. In this way, the reference values allow to determine whether the subject from whom the test sample was obtained suffers from one or the oth- er disease.
Thus, in a first aspect the present invention provides for a method of diagnosing whether a subject is afflicted with symp- tomatic malaria or a bacterial blood stream infection, com- prising
- 5 — LU101133 a) obtaining a body fluid sample from said subject that is suspected to have either symptomatic malaria or a bacteri- al blood stream infection; b) determining the C2 protein concentration in said body flu- id sample from said subject; b) 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 symptomatic malaria or a bacte- rial blood stream infection. In step (a) of the above-described method, a body fluid sample is obtained from a subject that is suspected to have either symptomatic malaria or a bacterial blood stream infection. This means that the subject shows one or more symptoms that are known to occur both with symptomatic malaria or a bacteri- al blood stream infection. Such symptoms may include one or more, or a combination of, for example, fever, respiratory distress, somnolence, vomiting, shivers, and tachycardia. In a preferred embodiment, the subject to be tested has already been diagnosed to have malaria parasitaemia, i.e. the occur- rence of Plasmodium in the blood has already been confirmed. Malaria parasitaemia can be confirmed by any method suitable for confirming the presence of Plasmodium protozoa in the sub- ject, 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 as- pect, malaria parasitaemia in the subject is confirmed by an immunodiagnostic assay, such as an enzyme-linked immunosorbent assay (ELISA). Compared to PCR, an ELISA assay has the ad- vantage that it does not need complex equipment and can hence
- 6 - LU101133 be used in the form of a point-of-care test. The detection of malaria parasitaemia preferably includes testing for all Plas- modium species which are known to be a causative agent for ma- laria, namely, P. falciparum, P. ovale, und P. vivax, P. malariae or P. knowlesi.
The body fluid 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 se- rum sample. In an even more preferred aspect, the sample will be a serum sample, such as a human serum sample.
In step (b) of the above method, the C2 concentration is de- termined in the sample from the subject. Preferably, the meth- od involves the determination of the concentration of human C2. As used herein, the amino acid sequence of human C2 is de- picted in SEQ ID NO:1. Accordingly, it is preferred that step (b) of the method of the invention is directed to the detec- tion of a protein that comprises or consists of the sequence of SEQ ID NO:1. However, the method of the invention may also comprise the detection of a variant of the C2 protein depicted in SEQ ID NO:1, such as a splicing variant. As used herein, variants of the polypeptide of SEQ ID NO:1 are understood as polypeptides that differ from the amino acid sequence of SEQ ID NO:1 by the deletion or replacement of one or more amino acids. Preferably, the variant has an amino acid identity of 75%, 80%, 85%, 90%, 95%, or more to the amino acid sequence shown in SEQ ID NO:1. While the method of the invention could involve the detection of C2 splicing variants, it is preferred that the determination of the C2 protein concentration in a body fluid sample involves the detection of the protein de- picted in SEQ ID NO:1.
- 7 = LU101133 Methods for determining the concentration of C2 are known in the art and have been described in the scientific literature (Bhattad et al. (2015), J Clin Immunol, 35(8):777-785; Eriks- son et al. (2016), J Neuroimmunol, 295-296:130-8; Lee et al. (2015), Pediatr Int. 57(3):431-8; Wu et al. (2012), J Proteome Res, 11(9):4541-52; Inoshita et al. (2010), BMC Nephrol, 11:34; Jiang et al. (2015), 62(1):118-28). Furthermore, kits for determining the concentration of C2 are commercially available, e.g. the Human Complement C2 (C2) ELISA kit from MyBioScource (catalog number MBS925742).
Once the concentration of C2 has been measured in step (a) of the above method, the concentration is compared with at least one reference value. The reference value preferably corre- sponds to a C2 split concentration that distinguishes sympto- matic malaria from a bacterial blood stream infection. The split concentration may differ dependent on the nature of the body fluid sample. For example, the C2 concentration which is characteristic for symptomatic malaria may differ in blood and serum of an infected patient. Based on the present disclosure, a skilled person will have no problem to identify suitable reference values for different body fluid samples. For exam- ple, the reference values may be obtained, e.g. as a mean val- ue, by statistical methods from a patient population that com- prised 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 either symptomatic malaria or a bacterial blood stream infection. It will be un- derstood that patient populations with a higher number of pa- tients provide a more significant reference value and hence allow for a more reliable diagnosis.
The comparison of the C2 concentration in the test sample and the reference value reveals whether the test subject is af- flicted with symptomatic malaria or a bacterial blood stream
- 8 — LU101133 infection. If the method of the invention uses a single refer- ence value that defines the split concentration, the tested subject is afflicted with symptomatic malaria if the test val- ue falls below the reference value. Conversely, the tested subject is afflicted with a bacterial blood stream infection if the test value is higher than the reference value. Accord- ing to the invention, it has been found that the C2 serum con- centration is higher in patients suffering from a bacterial blood stream infection compared to patients with malaria.
In a preferred embodiment, the sample to be tested with the method of the invention is a serum sample. It has been found that in serum, the C2 split concentration is approximately 482 ng/ml. Therefore, in a particularly preferred embodiment, the above method is performed with a serum sample from a subject, and said subject is diagnosed with symptomatic malaria, if the C2 protein concentration determined in the serum is below 480 ng/ml, more preferably below 450 ng/ml, below 400 ng/ml, below 350 ng/ml, below 300 ng/ml, below 250 ng/ml, below 200 ng/ml, below 150 ng/ml, below 100 ng/ml, below 50 ng/ml, or below 25 ng/ml, or less. On the other hand, said subject is diagnosed with a bacterial blood stream infection, if the C2 protein concentration determined in the serum is above 490 ng/ml, more preferably above 500 ng/ml, above 550 ng/ml, above 600 ng/ml, above 650 ng/ml, above 700 ng/ml, above 750 ng/ml, above 800 ng/ml, or more.
In a preferred embodiment, both parasite detection and C2 measurement are part of the same test, such as a point-of-care (POC) test. For example, a suitable test might include means for detecting P. falciparum histidine rich protein 2 (PfHRP2) and C2, optionally in combination with one or more additional markers discussed below.
- 9 - LU101133
In a particularly preferred embodiment, the invention provides a method of diagnosing whether a subject is afflicted with symptomatic malaria or a bacterial blood stream infection, said method comprising a) obtaining a serum sample from said subject that is sus-
pected to have either symptomatic malaria or a bacterialblood stream infection; b) determining the C2 protein concentration in said serumsample from said subject; wherein said subject is afflicted with symptomatic malaria if the concentration determined in step (b) is below 480 ng/ml, more preferably below 450 ng/ml or below 400 ng/ml, and where- in said subject is afflicted with a bacterial blood stream in- fection, if the concentration determined in step (b) is above 490 ng/ml, more preferably above 500 ng/ml or above 550 ng/ml.
It will be understood that the C2 concentration may also be 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 val- ues by statistical method without undue burden.
For example, the comparison may involve three different reference values, a first reference value for the C2 split concentration, a second reference value for a typical C2 concentration in a patient with symptomatic malaria, and a third reference value for a typical C2 concentration in a patient with a bacterial blood stream infection.
The method of the invention can also include the testing of additional markers.
Markers which can be used in combination with C2 for distinguishing between symptomatic malaria and a bacterial blood stream infection may include, e.g.
P. falcipa-
- 10 - LU101133rum histidine rich protein 2 (PfHRP2), human granzym B, human haptoglobin, human thrombomodulin, human interferon gamma re- ceptor 2 antibodies, human sFas Ligand, human hepcidin and hu- man procalcitonin.
The present invention also provides a test system, preferably in the form of a point-of-care (POC) assay, which allows testing of the C2 concentration in a sample from a patient simultaneously to testing of the concentration of one or more of the markers PfHRP2, granzym B, haptoglobin, thrombomodulin, interferon gamma receptor 2, sFas Ligand, hepcidin und procalcitonin.
Histidine rich protein 2 (HRP2) is a histidine and alanine rich protein that is synthesized by all natural strains and isolates of P. falciparum and accumulates in the parasite, as well as in infected red blood cell (Howard e al. (1986), J Cell Biol; 103(4):1269-77). The antigen can already be de- tected in culture supernatants of synchronized parasites 2-8 hours after ring development (i.e. young blood stage), but most of antigen is released during schizont rupture (Desakorn et al. (2005), Trans R Soc Trop Med Hyg, 99(7):517-24). HRP2 from P. falciparum (PfHRP2) has a long half-life and persists in the circulation for up to 3 weeks, even after successful treatment.
There is a linear relationship between the PfHRP2 concentration and the number of infected red blood cells (iRBCs). This correlation is shown in Figure 1. The average value of one parasite corresponds to about 1.012 fg of Pf£HRP2 (Kifude et al. (2008), Clin Vaccine Immunol, 15(6):1012-8). The relationship between parasite load and PfHRP2 concentra- tion implies, that the higher the measured amount of HRP2 in a patient sample is, the higher the number of parasites per ul blood.
Hence, the probability of an acute malaria infection (i.e. symptomatic malaria) increases with increasing HRP2 con- centration.
- 11 - LU101133 Granzyme B is a serine protease having a size of about 32 kDa and is expressed by cytotoxic T lymphocytes, NK cells, and dendritic cells. It was found that granzyme B levels in sam- ples of patients suffering from severe malaria are considera- bly higher than in samples of patients with uncomplicated ma- laria or bacterial blood stream infections. This correlation signifies that high granzyme B concentrations stand for an acute and a severe malaria infection.
Outside of erythrocytes, hemoglobin (Hb) is highly toxic and dangerous for cell integrity. Its prosthetic group haem is lipophilic and intercalates as well as disrupts the lipid bi- layer of membranes. Haptoglobin (Hp) is a unique acute phase protein that binds released Hb and is generally present in >400 molar excess, compared to free Hb, in serum (Bowman €& Kurosky (1982), Adv Hum Genet, 12:189-261, 453-4). One of the severe clinical consequences of malaria can be malarial asso- ciated anemia. The general diagnosis of chronic or acute ane- mia is, among other diagnostic means, through detection of de- creased haptoglobin levels in the blood. It was found that haptoglobin levels were considerably lower in children with malaria compared to bacterial bloodstream infections. This translates into low haptoglobin levels in children with malar- ia and higher levels in children with bacterial blood stream infections.
Thrombomodulin is an integral membrane protein and present on the surface of endothelial cells. It acts as an anticoagulant by preventing thrombin from exerting its procoagulant effects (e.g. converting fibrinogen to fibrin). In a bacterial blood stream infection, the expression of thrombomodulin on endothe- lial cells is strongly downregulated (Levi & van der Poll (2013), Minerva Anestesiol, 79(3):294-8). It was found that median expression levels of thrombomodulin were lower for bac- terial patients with a bacterial blood stream infection com- Fg
- 12 - LU101133 pared to those suffering from malaria. As a result, higher thrombomodulin concentrations are indicative of an acute ma- laria infection, whereas lower levels are indicative for a bacterial blood stream infection.
Interferons (IFNs) are a group of signaling proteins with imu- nostimulatory effect. Upon induction, IFN-y binds to its recep- tors, two homodimeric subunits made up of two alpha and two beta chains (IFNGRa/IFNGR1 and IFNGRR/ IFNGR2) which ulti- mately leads to the transcription of interferon-stimulated genes. High levels of autoantibodies were found in children with malaria compared to children with bacterial bloodstream infections. It was found that the median induction levels of antibodies against IFNGR2 were lowest in controls, followed by bacterial blood stream infections caused by bacterial species. Antibody levels were highest in patients with malaria. Hence, the expression levels of antibodies against IFNGR2 would be high in samples from patients with severe malaria and lower in samples from patients with a bacterial blood stream infection. Procalcitonin (PCT) is a marker for bacterial meningitis and regarded as a better diagnostic and prognostic marker for a bacterial blood stream infection than, e.g. customary C- reactive protein (CRP). In healthy individuals, the PCT levels are low (about 0.05 ng/mL). During inflammation, PCT is pro- duced by two alternative mechanisms, a direct pathway induced by microbial lipopolysaccharides (LPS) or other toxic metabo- lites and an indirect pathway induced by inflammatory media- tors (e.g. IL-6 and TNF-a). The PCT levels rapidly elevate be- tween 2-6 h and reach a peak within 6-24 h after bacterial in- fections. Further, the PCT levels return to normal when the patient responds to treatment. The PCT levels are lower in pa- tients with malaria compared to patients with a bacterial blood stream infection.
- 13 - LU101133 Fas or FasR, also known as apoptosis antigen 1, is a death re- ceptor on the surface of cells. Fas ligand (FasL or CD95L) is a type-II transmembrane protein that belongs to the tumor ne- crosis factor (TNF) family, and FasL binding with its receptor FasR induces apoptosis. Soluble Fas ligand is generated by cleaving membrane-bound FasL at a conserved cleavage site by the external matrix metalloproteinase MMP-7. FasL has a good negative predictive value for the diagnosis of severe blood stream infections (Chu et al. (2016), Cell Mol Biol, 62(11):32-37). It was found that elevated levels of soluble FasL in samples from patients suffering from malaria can be detected compared to bacterial blood stream infections. Hepcidin serves to block iron absorption from the diet and al- so to route iron in the body into macrophages and away from the serum. High levels of hepcidin suppress iron absorption whereas low hepcidin levels have the contrary effect. Hepcidin plays a complex but vital role in iron restriction that occurs during malaria infection. It has been found that hepcidin lev- els increase after infection with malaria parasites. Accord- ingly, the levels of hepcidin will be higher in samples from patients suffering from malaria compared to bacterial blood stream infections.
Blood-stage malaria parasites synthesize lactate dehydrogenase (LDH) as the terminal enzyme in their glycolytic pathway. It is a highly conserved molecule across all human malaria spe- cies that can be recognized by monoclonal antibodies. This ap- proach is used in the LDH-based malaria rapid diagnostic test (RDT) detection method which is routinely employed for clini- cal malaria diagnostics. Almost all of the available malaria RDTs currently on the market use antibodies to detect malaria parasites. There are two types of anti-LDH antibodies that differ with respect to their reactivity. One antibody can rec- ognize four species of human malaria, but it cannot differen-
- 14 — LU101133 tiate between those species (panspecific antibody). The other antibody recognizes only LDH isoforms of P. falciparum.
The above method renders possible a differential diagnosis be- tween two diseases, malaria and a bacterial blood stream in- fection, which show highly similar symptoms and can be easily be confused with each other. The insight regarding the C2 concentration levels also allows the reliable diagnosis of symptomatic malaria. Accordingly, in a second aspect, the invention provides a method for diagnos- ing symptomatic malaria in a subject, said method comprising a) determining malaria parasitaemia in a body fluid sample from said subject; b) determining the C2 protein concentration in a body fluid sample from said subject; C) comparing the concentration obtained in step (b) with at least one reference value; wherein malaria parasitaemia in combination with a C2 concen- tration below said reference value indicates that said subject is afflicted with symptomatic malaria. The method for diagnosing symptomatic malaria is based on the use of a body fluid sample. As stated above in the context with differential diagnosis, any type of body fluid can be used for carrying out the claimed method, but the sample will preferably be blood 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.
- 15 - LU101133 In step (a) of the above method for diagnosing symptomatic ma- laria, it is determined in a body fluid sample from said sub- ject whether the subject has a malaria parasitaemia. This means that the subject is tested, e.g. by PCR-based detection of Plasmodium-specific nucleic acid, by microscopy, or by immunodetection of one or more specific Plasmodium antigens, for the presence of Plasmodium parasites in the blood. In a preferred aspect, the malaria parasitaemia diagnosis in the subject to be tested is performed by an immunodiagnostic as- say, such as an enzyme-linked immunosorbent assay (ELISA). The subject from whom the body fluid sample to be tested by the above method has been derived may show symptoms of sympto- matic malaria. These symptoms may include, amongst others, high fever, respiratory distress, somnolence, vomiting, shiv- ers, and tachycardia. The malaria parasitaemia 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. In step (b) of the above method, the C2 protein concentration is determined in a body fluid sample from said subject. The sample may be the same sample that was used in step (a) of the method, or it can be a different sample. In one embodiment, the sample used in step (a) is a blood sample, and the sample used in step (b) is a serum sample. As described above, the C2 concentration measurement can be conducted by methods known in the art, and it preferably targets the detection of human C2 comprising the sequence of SEQ ID NO:1 as described above. In step (c) of the above method, the C2 protein concentration obtained in step (b) is compared with at least one reference
A 07 Ae
- 16 - LU101133 value. The reference value has preferably been determined from a large group of patients that are known to be afflicted with symptomatic malaria. The reference value may represent a maxi- mum C2 concentration that was found in patients with sympto- matic malaria. This means that the symptomatic malaria can be assumed in a subject, if the subject is afflicted with malaria parasitaemia and the test value for the C2 concentration is lower than the reference value. For example, where the method of diagnosing symptomatic malaria is carried out in a serum sample of a subject, the reference value is approximately 482 ng/ml.
In this case, symptomatic malaria can be assumed in a subject, if said subject shows malaria parasitaemia and the C2 protein concentration in the serum sample is lower than 480 ng/ml, lower than 450 ng/ml, lower than 400 ng/ml, lower than 350 ng/ml, lower than 300 ng/ml, lower than 250 ng/ml, lower than 200 ng/ml, lower than 150 ng/ml, lower than 100 ng/ml, lower than 50 ng/ml, or lower than 25 ng/ml. In a particularly preferred embodiment of the invention, a method for diagnosing symptomatic malaria in a subject is pro- vided, said method comprising a) determining malaria parasitaemia in a body fluid sample from said subject; b) determining the C2 protein concentration in a body fluid sample from said subject; wherein malaria parasitaemia in combination with a C2 concen- tration of less than 480 ng/ml, and more preferably less than 450 ng/ml or less than 400 ng/ml indicates that said subject is afflicted with symptomatic malaria. >
- 17 - LU101133 In a preferred embodiment, the malaria parasitaemia is one that involves between 10,000 up and 100,000 parasites/ul of the patient's blood which is considered a moderate malaria parasitaemia according to WHO standards. In yet another pre- ferred embodiment, the malaria parasitaemia is one that in- volves more than 100,000 parasites/ul of the patient's blood which is considered a high malaria parasitaemia according to WHO standards. 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 C2 concentration; 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 C2 concentration.
DESCRIPTION OF THE FIGURES Figure 1 shows the correlation of the PfHRP2 concentration with parasite numbers in human blood. Figure 2 shows a box plot of the C2 concentration in patient sera stratified by disease group. A split at concentration
482.63 ng/ml correctly classifies the patients for their re- spective disease, i.e. malaria or a bacterial blood stream in- fection. “i
— 18 - LU101133 Figure 3 shows the amino acid sequence of human C2.
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: The C2 concentration in serum samples from children aged £5 was analyzed. Patient samples were collected between February 2010 and August 2015 at the Agogo Presbyterian Hospital (APH) within the framework of two different studies (Marks et al.,
2017. Lancet Glob. Health. 5(3):e310-e323; Hogan et al., 2018. Clin. Infect. Dis. 66(12):1838-1845). The studies had been ap- proved by the Committee on Human Research, Publications and Ethics, School of Medical Science, Kwame Nkrumah University of Science and Technology, in Kumasi, Ghana, and the Ethics Com- mittee of the Arztekammer Hamburg in Germany. Malaria case definitions were based on children admitted to the hospital with a fever (2 37.5°C) and a P. falciparum positive slide, in the absence of a positive bacterial blood culture. Case defi- nitions for bacteraemia patients were hospital admitted chil- dren with a fever (2 37.5°C), a positive bacterial blood cul- ture and a negative malaria slide. Children who did not pre- sent any symptoms of an infection were recruited at the vacci- nation centre of the hospital as healthy controls.
org
- 19 — LU101133 Thirty-eight children suffering from malaria, 30 children with bacterial bloodstream infection and 10 healthy controls were subjected to the C2 concentration analysis. The C2 concentration levels were measured using XMAP cytokine detection kits (MerckMillipore) and measured using a Luminex 200 xPONENT® 3.1 system. Serum samples were diluted 1:2 in se- rum matrix or sample buffer, and further diluted if indicated in the kit manual. Beads were diluted 1:1.5. Figure 2 shows the C2 concentration stratified by the respective disease group. It can be seen that the median C2 concentration is clearly predictive when used for distinguishing between symptomatic malaria and bacterial a bacterial blood stream infection.
SEQUENCE LISTING <110> Bernhard-Nocht-Institut für Tropenmedizin <120> METHOD FOR DIAGNOSING SYMPTOMATIC MALARIA <130> P 106496 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 752 <212> PRT <213> Homo sapiens <400> 1 Met Gly Pro Leu Met Val Leu Phe Cys Leu Leu Phe Leu Tyr Pro Gly 1 5 10 15 Leu Ala Asp Ser Ala Pro Ser Cys Pro Gln Asn Val Asn Ile Ser Gly Gly Thr Phe Thr Leu Ser His Gly Trp Ala Pro Gly Ser Leu Leu Thr 40 45 Tyr Ser Cys Pro Gln Gly Leu Tyr Pro Ser Pro Ala Ser Arg Leu Cys Ze.
- 20 — LU101133 50 55 60 Lys Ser Ser Gly Gln Trp Gln Thr Pro Gly Ala Thr Arg Ser Leu Ser 65 70 75 80 Lys Ala Val Cys Lys Pro Val Arg Cys Pro Ala Pro Val Ser Phe Glu 85 90 95 Asn Gly Ile Tyr Thr Pro Arg Leu Gly Ser Tyr Pro Val Gly Gly Asn 100 105 110 Val Ser Phe Glu Cys Glu Asp Gly Phe Ile Leu Arg Gly Ser Pro Val 115 120 125 Arg Gln Cys Arg Pro Asn Gly Met Trp Asp Gly Glu Thr Ala Val Cys 130 135 140 Asp Asn Gly Ala Gly His Cys Pro Asn Pro Gly Ile Ser Leu Gly Ala 145 150 155 160 Val Arg Thr Gly Phe Arg Phe Gly His Gly Asp Lys Val Arg Tyr Arg 165 170 175 Cys Ser Ser Asn Leu Val Leu Thr Gly Ser Ser Glu Arg Glu Cys Gln 180 185 190 Gly Asn Gly Val Trp Ser Gly Thr Glu Pro Ile Cys Arg Gln Pro Tyr 195 200 205 Ser Tyr Asp Phe Pro Glu Asp Val Ala Pro Ala Leu Gly Thr Ser Phe 210 215 220 Ser His Met Leu Gly Ala Thr Asn Pro Thr Gln Lys Thr Lys Glu Ser 225 230 235 240 Leu Gly Arg Lys Ile Gln Ile Gln Arg Ser Gly His Leu Asn Leu Tyr 245 250 255 Leu Leu Leu Asp Cys Ser Gln Ser Val Ser Glu Asn Asp Phe Leu Ile 260 265 270 Phe Lys Glu Ser Ala Ser Leu Met Val Asp Arg Ile Phe Ser Phe Glu 275 280 285 Ile Asn Val Ser Val Ala Ile Ile Thr Phe Ala Ser Glu Pro Lys Val 290 295 300 Leu Met Ser Val Leu Asn Asp Asn Ser Arg Asp Met Thr Glu Val Ile 305 310 315 320 Ser Ser Leu Glu Asn Ala Asn Tyr Lys Asp His Glu Asn Gly Thr Gly 7 Hg ~
- 21 - LU101133 325 330 335 Thr Asn Thr Tyr Ala Ala Leu Asn Ser Val Tyr Leu Met Met Asn Asn 340 345 350 Gln Met Arg Leu Leu Gly Met Glu Thr Met Ala Trp Gln Glu Ile Arg 355 360 365 His Ala Ile Ile Leu Leu Thr Asp Gly Lys Ser Asn Met Gly Gly Ser 370 375 380 Pro Lys Thr Ala Val Asp His Ile Arg Glu Ile Leu Asn Ile Asn Gln 385 390 395 400 Lys Arg Asn Asp Tyr Leu Asp Ile Tyr Ala Ile Gly Val Gly Lys Leu 405 410 415 Asp Val Asp Trp Arg Glu Leu Asn Glu Leu Gly Ser Lys Lys Asp Gly 420 425 430 Glu Arg His Ala Phe Ile Leu Gln Asp Thr Lys Ala Leu His Gln Val 435 440 445 Phe Glu His Met Leu Asp Val Ser Lys Leu Thr Asp Thr Ile Cys Gly 450 455 460 Val Gly Asn Met Ser Ala Asn Ala Ser Asp Gln Glu Arg Thr Pro Trp 465 470 475 480 His Val Thr Ile Lys Pro Lys Ser Gln Glu Thr Cys Arg Gly Ala Leu 485 490 495 Ile Ser Asp Gln Trp Val Leu Thr Ala Ala His Cys Phe Arg Asp Gly 500 505 510 Asn Asp His Ser Leu Trp Arg Val Asn Val Gly Asp Pro Lys Ser Gln 515 520 525 Trp Gly Lys Glu Phe Leu Ile Glu Lys Ala Val Ile Ser Pro Gly Phe 530 535 540 Asp Val Phe Ala Lys Lys Asn Gln Gly Ile Leu Glu Phe Tyr Gly Asp 545 550 555 560 Asp Ile Ala Leu Leu Lys Leu Ala Gln Lys Val Lys Met Ser Thr His 565 570 575 Ala Arg Pro Ile Cys Leu Pro Cys Thr Met Glu Ala Asn Leu Ala Leu 580 585 590 Arg Arg Pro Gln Gly Ser Thr Cys Arg Asp His Glu Asn Glu Leu Leu , A / CL
- 22 - LU101133 595 600 605 Asn Lys Gln Ser Val Pro Ala His Phe Val Ala Leu Asn Gly Ser Lys 610 615 620 Leu Asn Ile Asn Leu Lys Met Gly Val Glu Trp Thr Ser Cys Ala Glu 625 630 635 640 Val Val Ser Gln Glu Lys Thr Met Phe Pro Asn Leu Thr Asp Val Arg 645 650 655 Glu Val Val Thr Asp Gln Phe Leu Cys Ser Gly Thr Gln Glu Asp Glu 660 665 670 Ser Pro Cys Lys Gly Glu Ser Gly Gly Ala Val Phe Leu Glu Arg Arg 675 680 685 Phe Arg Phe Phe Gln Val Gly Leu Val Ser Trp Gly Leu Tyr Asn Pro 690 695 700 Cys Leu Gly Ser Ala Asp Lys Asn Ser Arg Lys Arg Ala Pro Arg Ser 705 710 715 720 Lys Val Pro Pro Pro Arg Asp Phe His Ile Asn Leu Phe Arg Met Gln 725 730 735 Pro Trp Leu Arg Gln His Leu Gly Asp Val Leu Asn Phe Leu Pro Leu 740 745 750 Ae
Claims (15)
1. Method for diagnosing whether a subject is afflicted with symptomatic malaria or a bacterial blood stream infection, comprising a) obtaining a body fluid sample from said subject that is suspected to have either symptomatic malaria or a bacterial blood stream infection; b) determining the C2 protein concentration in said body fluid sample from said subject; 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 symptomatic malaria or a bacterial blood stream infection.
2. Method according to claim 1, wherein said method comprises the comparison of the concentration obtained in step (Db) with a single reference value.
3. Method according to claim 2, wherein (1) the subject suffers from a bacterial blood stream in- fection if the concentration obtained in step (b) ex- | ceeds the reference value; and (ii) the subject suffers from symptomatic malaria if the concentration obtained in step (b) is lower than the reference value. _ en
- 24 — LU101133
4. Method according to any of claims 1-3, wherein the subject has malaria parasitaemia.
5. Method according to any of claims 1-4, said method com- prising a) obtaining a serum sample from said subject that is suspected to have either symptomatic malaria or a bacterial blood stream infection; b) determining the C2 protein concentration in said se- rum sample from said subject; wherein said subject is afflicted with symptomatic malaria if the concentration determined in step (b) is below 480 ng/ml, more preferably below 450 ng/ml or below 400 ng/ml, and wherein said subject 1s afflicted with a bacterial blood stream infection, if the concentration determined in step (b) is above 490 ng/ml, more preferably above 500 ng/ml or above 550 ng/ml.
6. Method for diagnosing symptomatic malaria in a subject, said method comprising a) determining malaria parasitaemia in a body fluid sam- ple from said subject; b) determining the C2 protein concentration in a body fluid sample from said subject; c) comparing the concentration obtained in step (b) with at least one reference value;
- 25 — LU101133 wherein malaria parasitaemia in combination with a C2 con- centration below said reference value indicates that said subject is afflicted with symptomatic malaria.
7. Method according to claim 6, wherein said method comprises the comparison of the concentration obtained in step (b) with a single reference value.
8. Method according to any of claims 6-7, said method com- prising a) determining malaria parasitaemia in a body fluid sam- ple from said subject; b) determining the C2 protein concentration in a body fluid sample from said subject; wherein malaria parasitaemia in combination with a C2 con- centration of less than 480 ng/ml indicates that said sub- ject is afflicted with symptomatic malaria.
9. Method according to any of claims 1-8, wherein the C2 con- centration is determined by an immunodiagnostic assay, preferably an enzyme-linked immunosorbent assay (ELISA).
10. Method according to any of claims 1-9, wherein said C2 is human C2 comprising the sequence of SEQ ID NO:1 or a frag- ment thereof.
11. Method according to any of claims 1-10, wherein said ma- laria is caused by Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae or Plasmodium knowlesi.
N = Pa
- 26 - LU101133
12. Method according to any of claims 2-3 and 7, wherein said reference value is approximately 482.63 ng/ml.
13. Method according to any of claims 4-5, wherein said first reference value which is indicative for symptomatic malar- ia corresponds to a C2 blood plasma or serum concentration of <482.63 ng/ml.
14. Method according to any of claims 4-5, wherein said second reference value which is indicative for a bacterial blood stream infection corresponds to a C2 blood plasma or serum concentration of >482.63 ng/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 C2 concentration; and (c) optionally, buffers and diluents.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150111776A1 (en) * | 2013-09-23 | 2015-04-23 | Assaypro, Llc | Immunoassays using over-labeled fluorescent probes |
WO2018096129A1 (en) * | 2016-11-25 | 2018-05-31 | Universitätsklinikum Hamburg-Eppendorf | Method for diagnosing different forms of malaria |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150111776A1 (en) * | 2013-09-23 | 2015-04-23 | Assaypro, Llc | Immunoassays using over-labeled fluorescent probes |
WO2018096129A1 (en) * | 2016-11-25 | 2018-05-31 | Universitätsklinikum Hamburg-Eppendorf | Method for diagnosing different forms of malaria |
Non-Patent Citations (2)
Title |
---|
GELFAND E W ET AL: "Inherited deficiency of properdin and C2 in a patient with recurrent bacteremia", AMERICAN JOURNAL OF MEDICINE, EXCERPTA MEDICA, INC, UNITED STATES, vol. 82, no. 3, 23 March 1987 (1987-03-23), pages 671 - 675, XP026850104, ISSN: 0002-9343, [retrieved on 19870323] * |
KARLEE L. SILVER ET AL: "Complement driven innate immune response to malaria: fuelling severe malarial diseases : Complement in malaria", CELLULAR MICROBIOLOGY, vol. 12, no. 8, 25 June 2010 (2010-06-25), GB, pages 1036 - 1045, XP055599422, ISSN: 1462-5814, DOI: 10.1111/j.1462-5822.2010.01492.x * |
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