Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • 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
  • G01N33/56911—Bacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
  • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
  • G01N2333/8121—Serpins
  • G01N2333/8125—Alpha-1-antitrypsin

Definitions

• the present invention is in the field of medicine. It relates to a method and kit for determining whether an infection is of bacterial or non-bacterial origin.
• Antibiotic resistance due to the inappropriate use of antimicrobials is one of the most critical public health problems worldwide.
• a major factor underlying the unnecessary use of antibiotics is the lack of rapid and accurate diagnostic tests.
• the early identification of bacterial infections and its differentiation from other types of infection is still a challenge for clinicians.
• PCT procalcitonin
• CRP C-reactive protein
• PCT is produced by the liver and peripheral blood mononuclear cells, modulated by lipopolysaccharides and sepsis- related cytokines.
• CRP is an acute-phase reactant, and CRP level measurements are frequently used to aid in the diagnosis of bacterial infections.
• CRP is synthesized by the liver, mainly in response to IL-6, which is produced not only during infection but also in many types of inflammation. It binds to polysaccharides in pathogens, activating the classical complement pathway.
• PCT and CRP-Tests are prevalent in clinical settings, their accuracy in diagnosing a bacterial infection is heavily questioned and varies between different studies. Also, the clinical benefit for diagnostics and monitoring of therapy is debated as costs for tests are very high.
• the object of the present invention is to provide new suitable markers, methods and means for differentiating a bacterial from a non-bacterial infection with high sensitivity and specificity.
• One aspect of the present invention therefore relates to a method for determining in a patient whether an infection is of bacterial or non-bacterial origin, comprising the steps of determining the concentration of antitrypsin (ATT) and/or of at least one fragment thereof, in a sample that has been taken from said patient, and determining whether said patient suffers from an infection of bacterial or non-bacterial origin based on the concentration of ATT and/or of at least one fragment thereof and designating the infection as of bacterial origin or non-bacterial origin.
• ATT antitrypsin
• the invention further relates to a kit comprising the means for determining the concentration of ATT and/or of at least one fragment thereof in a sample, and the means for comparing the determined concentration of said ATT and/or of at least one fragment thereof with reference concentrations, as well as instructions for carrying out the method and correlating the determined concentrations with the type of infection.
• fragment refers to a proteolytic cleavage product of a respective full-length protein.
• patient refers to a living human or non-human organism that is receiving medical care or that should receive medical care due to a disease, or is suspected of having a disease. This includes persons with no defined illness who are being investigated for signs of pathology. Thus the methods and assays described herein are applicable to both, human and veterinary disease.
• alpha-l-antitrypsin is also referred to as antitrypsin and "ATT”.
• Alpha 1 Antitrypsin or a- 1-antitrypsin (A1AT) is a protease inhibitor belonging to the serpin superfamily. It is generally known as serum trypsin inhibitor.
• Alpha-l-antitrypsin is also referred to as alpha-l-proteinase inhibitor (A1PI) because it inhibits a wide variety of proteases. It protects tissues from enzymes of inflammatory cells, especially neutrophil elastase, and has a reference range in blood of 1.5 - 3.5 g/l, but the concentration can rise manifold upon acute inflammation.
• A1PI alpha-l-proteinase inhibitor
• ATT is cleaved at the reactive center loop (RCL) by target proteases (e.g. neutrophil elastase), as well as non-target proteases (e.g. matrix metalloproteases) (Fig.2). Both reactions lead to a conformational change and a loss of inhibitory activity.
• C-terminal alpha-1 antitrypsin peptide is herein referred to as CAAP48.
• CAAP48 is generated through the cleavage of AAT between Phe352-Leu353 and results in a carboxyl-terminal 42-residue peptide with a mass of 4789 Da, which is bound to the cleaved complex in a non-covalent manner (Fig. 2).
• Variants of this fragment such as VIRIP or C-36 peptide have been identified in several human tissues and body fluids.
• physiological functions such as NK- cell suppression and serine protease protection (C ISPP peptide), extracellular matrix protection by reversible serine protease inhibition (SPAAT), pro- or anti-inflammatory immune modulating functions (C-36 peptide) as well as inhibition of HIV entry (VI RIP) have been reported.
• the terms "threshold”, “threshold value”, “cut-off” and “cut-off value” are used synonymously.
• the optimal cut-off value is defined to be the concentration at which patients are classified into the bacterial or non-bacterial group with the highest sensitivity and specificity.
• a "kit” is a packaged combination optionally including instructions for use of the combination and/or other reactions and components for such use.
• Concentration measurements of AAT and its fragments in combination with the mindful analysis carried out by the inventors, surprisingly revealed an increased proteolytic activity in patients with bacterial infection resulting in high peak intensities of AAT fragments compared to infections of non-bacterial origin.
• the present invention therefore relates to a method for determining in a patient whether an infection is of bacterial or non-bacterial origin, the method comprising determining the concentration of antitrypsin (ATT) and/or of at least one fragment thereof, in a sample that has been taken from said patient, and determining whether said patient suffers from an infection of bacterial or non-bacterial origin based on the concentration of ATT and/or of at least one fragment thereof and designating the infection as of bacterial or non-bacterial origin.
• ATT fragments are substantially elevated in patients suffering from a bacterial infection, compared to patients with a non-bacterial infection (Fig. 1).
• an infection is designated as of bacterial origin if the concentration of at least one ATT fragment is above a certain cut-off value.
• the optimal cut-off value is defined to be the concentration at which patients are classified into the bacterial or nonbacterial group with the highest sensitivity and specificity.
• the infection is designated as of bacterial origin, if the concentration of said at least one ATT fragment is at least 2.5 -fold higher in the patient than a reference concentration.
• concentration is at least 3 -fold higher, more preferred, at least 4 -fold higher and most preferred at least 5 -fold higher than the reference concentration of said at least one ATT fragment thereof.
• a reference concentration in the meaning of the present invention is the median concentration of respective ATT and/or fragments thereof in healthy subjects.
• an infection is designated to be of bacterial origin if the concentration of said at least one ATT fragment is between 2.5 to 7 -fold higher than the reference concentration, more preferably between 3 to 6.5 -fold higher, more preferably between 3.5 to 6 - fold higher, preferably between 4 to 5.5 -fold higher, and most preferably between 4.5 to 5 -fold higher than the reference concentration.
• the protein and/or fragment concentration is measured via mass spectrometry.
• concentrations of said ATT and/or fragments thereof are determined via LC-MS/MS.
• One embodiment of the invention used to quantify said ATT and/or fragments thereof is detailed in the examples.
• the method of the invention can also be used to differentiate between several types of infection and determine their origin.
• said non-bacterial infection is selected from the group comprising viral and fungal infections.
• the present invention relates to a method wherein, if the concentration of said at least one ATT fragment is below a certain cut-off value, the infection is designated as of nonbacterial origin.
• Said non-bacterial origin can be, but is not limited to viral origin or fungal origin.
• the infection is designated as of non-bacterial origin if the concentration of said at least one ATT fragment is not more than 2.5. -fold higher than a reference concentration.
• concentration of the at least one ATT fragment is not more than 2 -fold higher than a reference concentration, most preferably not more than 1.5 -fold higher than a reference concentration.
• an infection is also designated to be of non-bacterial origin if the concentration of said at least one ATT fragment is between 1.2 to 2.4 -fold higher than the reference concentration, preferably between 1.5 to 2.3-fold higher, most preferably between 1.75 to 2 -fold higher than the reference concentration.
• said sample taken from a patient is selected from a group comprising a plasma sample, a serum sample, a whole blood sample, a blood sample or fractions thereof, a lymphatic fluid sample, a urine sample and an extract of any of the aforementioned samples.
• a plasma sample e.g., a plasma sample, a serum sample, a whole blood sample, a blood sample or fractions thereof, a lymphatic fluid sample, a urine sample and an extract of any of the aforementioned samples.
• the method according to the invention also relates to a method in which the concentration of CAAP48 and the SNP-variant CAAP47 is quantified via LC-MS/MS as detailed in the examples.
• the method of quantification of said ATT and/or fragments thereof, such as CAAP48 and/or CAAP47 is not limited to mass spectrometry.
• Figure 1 and 3 show the unexpected results obtained by the inventors, that the CAAP47/48 concentrations are significantly elevated in the blood of patients suffering from a bacterial infection compared to patients suffering from a non-bacterial infection.
• said ATT and/or at least one fragment thereof comprises CAAP48 and/or CAAP47.
• said ATT and/or at least one fragment thereof is CAAP48 and/or CAAP47.
• the present invention therefore relates to a method for determining in a patient whether an infection is of bacterial or non-bacterial origin, the method comprising determining the concentration of CAAP48 and/or CAAP47, in a sample that has been taken from said patient, and determining whether said patient suffers from an infection of bacterial or non-bacterial origin based on the concentration of CAAP48 and/or CAAP47 and designating the infection as of bacterial or non-bacterial origin.
• an infection is designated as of bacterial origin if the concentration of CAAP48 and/or CAAP47 is above a certain cut-off value.
• the optimal cut-off value is defined to be the concentration at which patients are classified into the bacterial or non- bacterial group with the highest sensitivity and specificity.
• the cut-off value for determining that an infection is of bacterial origin is a concentration of CAAP48 and/or CAAP47 of at least 5 ⁇ in the blood of said patient and may deviate depending on the patient analyzed by about 20 %.
• the cut-off value is at least 5 ⁇ , but it can also be at least 6 ⁇ , at least 7 ⁇ , or at least 8 ⁇ .
• the cut-off value for determining that an infection is of bacterial origin is between 4.5 and 8 ⁇ , preferably between 5 and 7.5 ⁇ , more preferably between 5 and 7 ⁇ , also preferably between 5.5 and 6.5 ⁇ between 5.5 and 6 ⁇ , and most preferably between 5 and 5.5 ⁇ .
• the invention also relates to a method in which, if the concentration of said CAAP48 and/or CAAP47 is at least 2.5 -fold higher than a reference concentration, the infection is designated of bacterial origin.
• the concentration is at least 3 -fold higher, even more preferred, at least 4 -fold higher, and most prefera bly at least 5 -fold higher than the reference concentration of said CAAP48 and/or CAAP47.
• an infection is designated to be of bacterial origin if the concentration of said CAAP48 and/or CAAP47 is between 2.5 to 7 -fold higher than the reference concentration, more preferably between 3 to 6.5 -fold higher, more preferably between 3.5 to 6 -fold higher, more preferably between 4 to 5.5 -fold higher most preferred between 4.5 to 5 -fold higher than the reference concentration.
• the CAAP47/48 concentrations are significantly higher in patients suffering from a bacterial infection compared patients suffering from a viral infection.
• the median concentration of CAAP47/48 in the blood of patients with a bacterial infection is much higher compared to patients with a viral infection (Fig. 1).
• the inventors found that the median concentration of CAAP47/48 in the blood of patients with a bacterial infection is 7.97 ⁇ , which is a 4-fold increase compared to patients with a viral infection (Fig. 1).
• the concentration of CAAP47/48 in the blood of patients suffering from a bacterial infection is significantly higher compared patients suffering from a fungal infection.
• the inventors found that the median concentration of CAAP47/48 in patients suffering from a fungal infection is 4.5 ⁇ , compared to 7.97 ⁇ in patients suffering from a bacterial infection (Fig. 3). Therefore, in one embodiment of the method according to the invention, the cut-off value for determining that an infection is of non-bacterial origin is a concentration of CAAP48 and/or CAAP47 of less than 5 ⁇ in the blood of said patient and may deviate depending on the patient analyzed by about 20 %.
• said cut-off value for determining that an infection is of non-bacterial origin is less than 5 ⁇ , preferably less than 4.5 ⁇ , more preferably less than 4 ⁇ .
• the cut-off value for determining that an infection is of non-bacterial origin is between 3.5 and 5 ⁇ , between 3 and 4.5 ⁇ , or between 3.5 and 4 ⁇ .
• the inventors surprisingly found that the concentration of at least one ATT fragment, is clearly lower in patients suffering from a viral or a fungal infection than in patients suffering from a bacterial infection (Fig. 1 and 3).
• the concentration of at least one ATT fragment in patients suffering from a fungal infection is in between the concentration of the at least one ATT fragment in patients suffering from a bacterial and the concentration in patients suffering from a viral infection. Therefore, in one embodiment, the method according to the invention allows not only for the discrimination between bacterial and non-bacterial infections, but also whether a non-bacterial infection is of viral and fungal origin.
• an infection is designated as of fungal origin if the concentration of said at least one ATT fragment is above the cut-off value for designating an infection a viral infection and below the cut-off value for designating an infection as of bacterial origin.
• the infection is designated as to be of fungal origin if the concentration of said at least one ATT fragment is at least 1.5 -fold higher but not more than 2.5. -fold higher than a reference concentration.
• an infection is designated as to be of viral origin if the concentration of said at least one ATT fragment is not more than 1.5 -fold higher than a reference concentration.
• Figure 1 shows, the CAAP47/48 concentrations are significantly higher in patients suffering from a bacterial infection compared patients suffering from a viral infection.
• the cut-off value for determining that an infection is of viral origin is a concentration of CAAP48 and/or CAAP47 of less than 3 ⁇ in the blood of said patient and may deviate depending on the patient analyzed by about 20 %.
• the cut-off value for determining that an infection is of viral origin is less than 3 ⁇ , more preferably less than 2 ⁇ and most less than 1.5 ⁇ .
• the cut-off value for determining that an infection is of viral origin is between 1 and 3 ⁇ , more preferably between 1.5 and 2.5 ⁇ and most prefera bly 2 ⁇ .
• the infection is designated as of viral origin, if the concentration of said CAAP48 and/or CAAP47 is less than 1.5 fold higher than a reference concentration.
• concentration to designate an infection as of viral origin is about the same value as the reference concentration of said CAAP48 and/or CAAP47.
• the CAAP47/48 concentrations are significantly higher in patients suffering from a bacterial infection compared patients suffering from a fungal infection and even higher than in patients suffering from a viral infection.
• the inventors found that the median concentration of CAAP47/48 in the blood of patients with a fungal infection is 4.5 ⁇ .
• an infection is designated as of fungal origin if the concentration of CAAP47/48 is below the cut-off value of designating an infection as to be of bacterial origin and above the cut-off value of designating an infection as to be of viral origin. Therefore, in one embodiment of the method according to the invention, an infection is designated as to be of fungal origin if the concentration of CAAP48 and/or CAAP47 in the blood of said patient is between 1.5 ⁇ and 5 ⁇ and may deviate depending on the patient analyzed by about 20 %.
• the concentration of CAAP48 and/or CAAP47 for designating an infection as to be of fungal origin is between 5 ⁇ and 1.5 ⁇ , preferably between 2 ⁇ and 4.5 ⁇ , more preferably between 2.5 ⁇ and 4 ⁇ and most preferably between 3 ⁇ and 3.5 ⁇ .
• the infection is designated as of fungal origin, if the concentration of said CAAP48 and/or CAAP47 is less than 2.5 -fold higher than a reference concentration but more than 1.5 -fold higher than a reference concentration.
• the infection is designated as of fungal origin if the concentration of CAAP47/48 is 2 -fold higher than a reference concentration.
• a sample is taken at one or more of the following time points: when the subject is first admitted to a medical institution or in the ambulance, when the subject is in the emergency room, when the subject is in the intensive care unit, before treatment, after initiation of treatment, 24 hours after initiation of treatment, 48 hours after initiation of treatment and/or 72 hours after initiation of treatment.
• the method according to the invention can also be used to monitor the success of treatment of a patient suffering from an infection.
• it can be used to monitor the success of antimicrobial treatment, e.g. with antibiotics.
• the method according to the invention relates to a method for monitoring the success of treating a patient suffering from an infection with a certain drug, comprising the steps of determining the concentration of antitrypsin (ATT) and/or of at least one fragment thereof, in a sample that has been taken from said patient, and associating the levels of ATT and/or fragments thereof with a positive or a negative response to said certain drug.
• ATT antitrypsin
• Said positive response is given if the level of ATT increases during drug treatment and/or if the level of ATT fragments decreases during drug treatment.
• said drug can be an antimicrobial drug, e.g. an antibiotic.
• Said ATT fragments may comprise CAAP47/48.
• the method according to the invention relates to a method for monitoring the success of treating a patient that suffering from an infection with a drug, comprising the steps of:
• Said positive response is given if the level of ATT increases during drug treatment and/or if the level of ATT fragments decreases during drug treatment.
• said drug can be an antimicrobial drug, e.g. an antibiotic.
• Said ATT fragments may comprise CAAP47/48.
• the method according to the invention can be used to determine the appropriate treatment and/or therapy. For example, it can be used to determine the right medication before and during treatment. Therefore, the method according to the invention may prevent harmful side- effects, toxicity and allergic reactions due to inappropriate drug treatment and/or therapy. Moreover, serious complications can be prevented in patients and the healing process can be accelerated. This may also significantly reduce the costs of therapy. In case of treating a patient suffering from an infection, the inappropriate prescription of antibiotics may be prevented, thus preventing the spread of antibiotic resistance.
• the invention also relates to a method of medical decision making for individual patient therapy in a subject suffering from an infection, comprising the steps of, determining the concentration of antitrypsin (ATT) and/or of at least one fragment thereof, in a sample that has been taken from said patient, and associating the levels of ATT and/or fragments thereof with a certain choice of therapy.
• ATT antitrypsin
• the method according to the invention can be used in medical decision making for individual patient therapy in a subject suffering from an infection.
• it can be used to determine whether a patient should be treated with antibiotics.
• the invention also relates to a method of medical decision making for individual patient therapy in a subject related to the severity of the disease.
• the invention relates to a method of medical decision making for individual patient therapy related to the severity of the disease by monitoring therapy response in a subject to a certain drug.
• drug can be an antimicrobial-drug, e.g. antibiotics. Therefore, the invention also relates to a method of medical decision making for individual patient therapy in a subject suffering from an infection, comprising the steps of,
• Said positive response to therapy is given if the level of ATT increases during drug treatment and/or if the level of ATT fragments decreases during drug treatment.
• said therapy may comprise the treatment with antimicrobial drugs, which can be antibiotics.
• Said ATT fragments may comprise CAAP47/48.
• the present invention also relates to a kit for determining whether an infection is of bacterial or non-bacterial origin, comprising means for determining the concentration of ATT and/or of at least one fragment thereof in a sample, means for comparing the determined concentration of said ATT and/or of at least one fragment thereof with reference concentrations, and instructions for carrying out the method and relating the determined concentrations with the type of infection.
• the means for determining the concentration of said ATT and/or of at least one fragment thereof is an antibody specific for ATT and/or for at least one fragment thereof.
• said fragment is CAAP48 and/or CAAP47 and said antibody is specific for CAAP48 and/or CAAP47.
• the means for comparing the determined concentrations comprise standard data showing the correlation between the concentration of said ATT and/or at least one fragment thereof contained in samples and the origin of infection.
• the present invention also relates to a kit as described above, wherein said infection of nonbacterial origin is an infection selected from the group comprising viral and fungal infections.
• the present invention also relates to a kit according to the invention, wherein said sample is selected from a group comprising a plasma sample, a serum sample, a whole blood sample, a blood sample or fractions thereof, a lymphatic fluid sample, a urine sample and an extract of any of the aforementioned samples.
• the method and kit according to the invention can be used not only for determining if an infection is of bacterial or non-bacterial origin, but also for the monitoring of treatment of a patient.
• Such treatment may comprise the treatment of an infection such as a bacterial, viral or fungal infection.
• the monitoring of treatment can relate to the monitoring of treatment of a patient with an antimicrobial drug, such as antibiotics.
• the invention also relates to a kit for diagnosis of a bacterial infection and/or predicting the outcome of treatment with a certain drug and/or the monitoring of treatment of a patient suffering from an infection comprising, an antibody, an aptamer, or another target specific molecule specific for ATT and/or fragments thereof, standard data showing the correlation between the amounts of ATT and/or fragments thereof contained in samples and prognosis, and a manual.
• the invention relates to an LC-MS/MS kit for diagnosing a bacterial infection and/or predicting the outcome of treatment in a patient with a certain drug and/or the monitoring of treatment of a patient suffering from an infection, wherein the concentrations of ATT and/or fragments thereof are re guaranteed by LC-MS/MS. These concentrations are compared to standard data sets. Examples
• Example 1 Patient groups and characteristics
• HIV patients Nine, six and eight HIV patients were enrolled with virus titers of ⁇ 100, 100 - 10000 and > 10000 cop/ml, respectively. Healthy volunteers were included for AAT genotype and phenotype correlation and for the cell experiments. Patients less than 18 years old, pregnant or lacking informed consent were excluded. The study and all protocols were approved by the local ethics committee.
• Peptides were synthesized by solid phase peptide synthesis and purified by HPLC with greater 95% purity.
• peptides were synthesized automatically by a standard Fmoc [N-(9- fluorenyl)methoxycarbonyl] protocol by manual SPPS or on an EPS221 peptide synthesizer (Intavis Bioanalytical Instruments AG, Cologne, Germany).
• Fmoc-amino acids 5 equiv
• HBTU equiv
• N-methylmorpholine/DMF (1:1) for 5—15 min (double couplings).
• Fmoc removal was effected by treating the resin twice with piperidine in DMF (20 %). All CAAP48 an immunomodulatory sepsis biomarker deprotection and coupling steps were followed by intensive washings with DMF and dichlorometane (CH2CI2), alternately. Peptide cleavage and deprotection was accomplished with reagent K (trifluoroacetic acid (TFA)/water/phenol/thioanisole/ethanedithiol 82.5:5:5:5:2.5) for 4 h at room temperature. The crude peptides were precipitated with diethyl ether, centrifuged, and washed several times with diethyl ether.
• TFA trifluoroacetic acid
• the yields of the crude peptides varied between 60 - 70 %.
• the crude peptides were purified by semipreparative reversed-phase HPLC with a Shimadzu LC- 8A system equipped with a C18 column (Knauer Eurospher 100, Berlin, Germany).
• the gradient elution system was 0.1 % TFA in water (eluent A) and 0.1 % TFA in acetonitrile/water (9:1, eluent B).
• the peptides were eluted with a gradient of 0 - 50 % eluent B in 120 min and a flow rate of 10 ml/min. The peaks were detected at 220 nm.
• the peptide was eluted with solvent A (H20 + 0.1% FA) and solvent B (AcN + 0.1% FA), starting at 90% A for 2 min, followed by a 6 min linear gradient to 40% A, 5 min 40% A, 1 min linear gradient to 90% A, 6 min 90% A with a flow rate of 200 ⁇ /min.
• the column oven was set 38°C.
• Two peptides were chosen for fragmentation: the 5 fold charged CAAP48 peptide (4789.7 Da) at 958.9 Da and the 6 fold charged CAAP48 peptide at 799.3 Da.
• transitions could be detected at high intensities: 799.3/873.6, 799.3/243.4, 799.3/314.3, 958.9/1091.8.
• MS parameters were CXP:27, DP:60, EP:10, CAD:12, CU :22, GS1:40:, GS2:50, IS:5500, TEM :600.
• CE was optimized for the single fragments (874.4: 27, 1091.5: 33, 243.4: 39, 314.3: 28).
• For detection of the CAAP47 peptide (4775.7 Da) equivalent transitions were chosen at 797.0/ 873.6, 797.0/ 243.4, 956.167/ 1088.4 and 797.0/ 314.3.
• Recovery was determined by spiking three standard concentrations in EDTA plasma of donor plasma. Autosampler stability was determined by measuring three samples 8 times in a time range of 24 hours. Freeze/thawCAAP48 an immunomodulatory sepsis biomarker stability was determined by thawing and freezing two samples on 8 consecutive days. Parallelism was determined by diluting a sample consecutively in buffer and referring the concentrations of the dilutions to the original concentration.
• Validation resulted in a linear calibration curve in a range of 2 - 200 ng ⁇ l with a coefficient of determination of 1.
• the LOD was 2.03 ng/ ⁇
• Intraday accuracy was determined to be 0.98, 1.13 and 0.88 for the three respective concentrations.
• Interday accuracy was determined to be 1.07, 1.01 and 0.95.
• Intraday precision was 4, 11 and 11%, interday precision 15, 13 and 10%.
• FIG. 1 CAAP47/48 concentrations are significantly elevated in the blood of patients suffering from a bacterial infection.
• CAAP47/48 concentrations are significantly higher in patients suffering from a bacterial infection compared to patients suffering from a viral infection.
• the median concentration for bacterial infection is 7.97 ⁇ , which is a 4-fold increase compared to patients with a viral infection.
• Fig. 2 Cleavage sites in the AAT reactive center loop (RCL).
• Fig. 3 Median CAAP47/48 concentrations are significantly lower in the blood of patients suffering from a fungal infection than in patients suffering from a bacterial infection.
• CAAP47/48 concentrations are significantly higher in patients suffering from a bacterial infection compared to patients suffering from a fungal infection.
• the median concentration for fungal infection is 4.5 ⁇ .

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