EA046276B1 - ELECTROCHEMICAL MEASUREMENT OF D-LACTATE FOR DIAGNOSIS AND PREDICTION OF INFECTIOUS DISEASE - Google Patents

Description

The present invention relates to an in vitro method for the diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease, comprising (a) providing a sample of a subject exhibiting clinical symptoms of infection and/or suspected of having an infection, (b) determining the level of D-lactate in the specified sample, (c) the level of D-lactate indicates the presence of an infectious disease, characterized in that (d) the level of D-lactate in the specified sample is determined using an electrochemical sensor system (biosensor). In embodiments, the electrochemical sensor system comprises a potentiometric or amperometric sensor. Preferably, the electrochemical system contains a D-lactate binding molecule, which is preferably immobilized on the detection (working) electrode. In embodiments, the detection electrode with the immobilized D-lactate binding molecule consists of a (disposable) test strip for insertion into a portable reader.

BACKGROUND OF THE INVENTION

Prosthetic joint infections are a serious complication associated with significant mortality and morbidity (1, 2). Timely and accurate diagnosis of infection is critical to planning adequate treatment, including arthroscopic or open surgery. In prosthetic joints, low levels of inflammation and mild clinical symptoms may preclude the diagnosis of prosthetic joint infection (PJI), which usually occurs several months to years after arthroplasty.

In addition, infectious diseases in general represent an important health problem, and early and reliable diagnosis using rapid and consistent testing modalities is urgently needed to improve therapeutic intervention in patients suspected of having or diagnosed with an infectious disease.

Currently used synovial fluid diagnostic tests do not have high sensitivity and high specificity for infection. Synovial fluid culture requires time until microbial growth begins and has limited sensitivity and specificity, especially in mild chronic PJI (3–5). Synovial fluid leukocyte counts and individual leukocyte counts (i.e., percentage of granulocytes) are highly sensitive (6) but may be elevated without infection in the setting of dislocation, periprosthetic fracture, or during the first 6 weeks after surgery due to physiological inflammatory conditions. healing process. New biomarkers in synovial fluid, such as alphadefensin, leukocyte esterase, and calprotectin (7–9), are abundant in neutrophils and therefore may not be useful for diagnosing PJI in patients with aseptic conditions associated with high leukocyte counts in synovial fluid.

Periprosthetic joint infection (PJI) is a serious complication after arthroplasty that is associated with significant morbidity and mortality. An accurate diagnosis of infection is critical to planning adequate treatment. Infection is the reason for revision surgery in more than 25% of cases in the era of orthopedic implants such as joint replacements (26). Currently used synovial fluid diagnostic tests lack both high sensitivity and high specificity for infection (27). Synovial fluid culture takes time and has limited sensitivity and specificity in mild chronic PJI (28–30). Low levels of inflammation and mild clinical symptoms may make it difficult to diagnose PJI, which can occur months or years after arthroplasty. Diagnosis is also difficult in the early postoperative period when WBC counts, CRP, and clinical signs make reliable diagnosis difficult due to local tissue inflammation (31–33).

Several attempts have been made to investigate various biomarkers, such as alpha-2macroglobulin, adenosine deaminase, procalcitonin, IL-1, IL-6, IL-1e, and alpha-defensin, which may help distinguish PJI from aseptic pathology (34–36).

D-lactate is a pathogen-specific metabolite used to diagnose bacterial infection primarily in sterile body fluids (10). L- and D-rotating isomers of lactate are products of intracellular metabolism. However, mammalian cells contain only the enzyme L-lactate dehydrogenase (LDH) and can produce almost exclusively L-lactate. The concentration of D-lactate in human serum is extremely low, ranging from nanomolar to micromolar, because it is a minor side pathway of glycolysis.

In contrast, bacterial species possess both D-LDH and L-LDH and therefore produce both D-lactate and L-lactate. As a result, during bacterial infections, D-lactate concentrations increase into the millimolar range (13, 14). Therefore, previous reports have suggested that determination of D-lactate in synovial fluid may be useful for the early diagnosis of septic arthritis, especially when compared with Gram staining and culture (11, 12).

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As early as the 1990s, several studies were conducted to measure D-lactate concentrations in certain primary sterile body fluids to distinguish infection from aseptic inflammation (40–42). D-lactate has been shown to be a promising marker for the diagnosis of infections in various body fluids, such as bacterial meningitis and septic arthritis (41, 43), including in patients receiving antimicrobial therapy (40).

However, established tests for diagnosing infectious diseases and PJI in particular have relatively low specificity. This also proved to be true when measuring the D-lactate concentration in a sample using conventional D-lactate assays, particularly spectrophotometric assays.

In the field of chemical and biological determination, the trend is toward multi-purpose devices that require little or no user training. There are many transducer options for converting a chemical recognition signal into an electrical signal: optical, mass, thermal and electrochemical sensors. Among chemical sensors, electrochemical sensors do not require external components such as bulky optical lenses or light sources and provide a high level of integration and, in many cases, a low detection limit. Moreover, a wide range of off-the-shelf components are available, making electrochemical sensors particularly attractive in environments that value portability and low cost. These sensors are typically amperometric, impedimetric, or potentiometric and have been successfully used as chemical and biological sensors (58, 59). Based on this, potentiometry provides an effective yet simple method for detecting several types of analytes such as nucleic acids, antigens, and trace metals. Potential applications include point-of-care diagnostics and personalized medicine. Among potentiometric methods, transistor-based sensors provide a good alternative to disposable sensors at low cost and reliability. If such sensors can be controlled using inexpensive and easy-to-use measurement equipment, then the sensors can be used in a wide range of environments, especially in remote and resource-poor locations, with minimal user training.

Moreover, it is not at all clear whether an electrochemical sensor for D-lactate can provide reliable and reproducible results for determining the level of D-lactate in a sample that can be used in the context of an in vitro method for diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of infectious disease.

Accordingly, there is a need in the art for a reliable in vitro method for the diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease that overcomes the limitations of known methods. In particular, a diagnostic method with high sensitivity as well as specificity is urgently needed. Preferably, such methods should provide an electrochemical sensor system that allows a simple test to be performed while providing reproducible results.

Brief description of the invention

In light of the prior art, the technical problem underlying the present invention is to provide an improved in vitro method for the diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease.

This problem is solved by using the features of independent claims. Preferred embodiments of the present invention are presented in the dependent claims.

Therefore, according to a first aspect, the present invention relates to an in vitro method for diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease, comprising:

a) providing a sample from a subject showing clinical symptoms and/or suspected of having an infection,

b) determining the level of D-lactate in the specified sample,

c) wherein the level of D-lactate indicates the presence of an infectious disease, characterized in that

d) the level of D-lactate in the specified sample is determined using an electrochemical sensor system (biosensor).

The present invention is based on the unexpected discovery that measuring D-lactate using an electrochemical sensor system (biosensor) to determine the level of D-lactate in a sample according to the method in accordance with the present invention provides a test that is characterized by surprisingly high specificity compared to similar methods, which use other means to determine D-lactate levels, such as spectrophotometric measurements.

A great advantage of the method according to the present invention is that

- 2 046276 The rate of false positive events can be dramatically reduced compared to known tests using spectrophotometric measurements. Surprisingly, it was found that the number of red blood cells as well as the hemoglobin concentration in a sample isolated from a subject exhibiting clinical symptoms and/or suspected of having an infection correlated with the level of D-lactate determined by spectrophotometric measurement. This is probably due to the fact that hemoglobin and D-lactate have interfering absorption wavelengths, i.e. 540 nm for hemoglobin and 570 nm for D-lactate. Therefore, contamination of the blood sample may lead to a false positive result in the method using spectrophotometric measurement. In contrast, it was found that the electrochemical measurement used in the context of the present invention is not affected by the presence of red blood cells, and the false positive rate and specificity of the test were significantly improved compared to known methods. It is important to note that the sensitivity of the method in accordance with the present invention is approximately equal to the very high sensitivities of known methods that use spectrophotometric measurements of D-lactate, for example, for the diagnosis of PJI.

In preferred embodiments, the electrochemical sensing system of the present invention is highly specific for the detection of D-lactate, whereas the presence of L-lactate is not detected and does not affect the determination of D-lactate levels. The detection of D-lactate is completely independent of the presence of L-lactate, since L-lactate is not recognized by the D-lactate-specific electrochemical sensor system.

According to embodiments of the present invention, the electrochemical sensor system comprises a potentiometric sensor, preferably a transistor-based potentiometric sensor.

In embodiments, the electrochemical sensor system comprises an ion sensitive field-effect transistor (ISFET). ISFET is a potentiometric sensor based on a potentiometric transistor.

In further embodiments, the electrochemical sensor system comprises an amperometric sensor.

In embodiments, the sensor of the electrochemical sensor system is a potentiometric sensor. In an alternative embodiment, the sensor of the electrochemical sensor system is an amperometric sensor.

In embodiments, the electrochemical sensing system comprises a D-lactate binding molecule, such as, for example, D-LDH. The D-lactate binding molecule is present in the electrochemical cell of the electrochemical sensor system. The D-lactate binding molecule can be immobilized on an electrochemical sensor substrate, preferably on a detection electrode. In embodiments of the present invention, a D-lactate binding molecule, such as D-LDH, can be provided in a solution, such as a buffer, that is added to the electrochemical measurement system, such as by diluting a patient sample therein.

When using a D-lactate binding enzyme, the electrochemical cell and/or sample buffer in accordance with the present invention may contain additional components necessary to perform the chemical reaction catalyzed by the D-lactate binding enzyme. In the case of D-LDH, the electrochemical system may contain nicotinamide adenine dinucleotide NAD.

According to embodiments of the present invention, the electrochemical sensing system comprises a test strip or chip with corresponding electrodes on its surface to perform electrochemical detection of D-lactate, preferably using a potentiometric or amperometric sensor. In embodiments, the test strip or chip is disposable. In further embodiments, the test strip or chip is reusable.

In preferred embodiments, the test strip or electrochemical sensor system chip may be used by insertion into a suitable reader device, such as a portable compact reader that may be battery powered. Compact, portable readers that allow mobile use and use electrochemical test strips are known in the art for other analytes such as glucose. Such devices have the advantage that measurement and determination of the level of the relevant analyte can be achieved instantly at the point of sample extraction. An example of such a device is the Freestyle Precision Pro blood glucose and β-ketone monitoring system.

In embodiments, the electrochemical sensor system preferably comprises a disposable test strip (chip) for electrochemical determination of D-lactate levels, the test strip comprising a detection electrode with an immobilized D-lactate binding molecule and preferably also a counter electrode and/or a reference electrode.

In embodiments, the measurement or determination of the D-lactate level occurs in a reader, preferably a battery-powered portable compact reader. In embodiments, the disposable test strip may be placed in

- 3 046276 preferably a battery-powered, portable, compact reader for performing D-lactate measurements. In embodiments, the reader is not a portable device, but a desktop reader.

In preferred embodiments, the electrochemical sensing system is based on at least one of amperometry, potentiometry, and field effect transistor.

In embodiments, the electrochemical sensing system comprises a D-lactate binding molecule, preferably D-lactate dehydrogenase (D-LDH).

In embodiments, the electrochemical sensing system comprises a detection (working) electrode, preferably including a carbon or gold surface.

In preferred embodiments, the D-lactate binding molecule, preferably D-LDH, is immobilized on the detection electrode.

According to further embodiments of the present invention, the electrochemical sensing system comprises a detection electrode (working electrode), preferably including a carbon or gold surface, wherein a D-lactate binding molecule, preferably a D-lactate binding enzyme, more preferably D-lactate dehydrogenase (D-LDH), is immobilized on surface of the determination electrode (working).

In preferred embodiments, the electrochemical sensing system comprises a detection electrode including an immobilized lactate binding molecule. In preferred embodiments, the lactate binding molecule is D-LDH.

In embodiments, immobilization of a D-lactate binding molecule, such as DLDH, on the surface of a detection electrode is achieved by any of adsorption, covalent binding, entrapment, encapsulation, cross-linking or thiol-gold interaction, preferably cross-linking or thiol-gold interaction.

Preferred is the use of a D-lactate binding enzyme that catalyzes the chemical reaction leading to the formation of reduced nicotinamide adenine dinucleotide NADH. Therefore, the use of D-LDH is beneficial because it catalyzes the reversible reaction of D-lactate in the presence of NAD' to pyruvate and NADH. The D-LDH enzyme to be used for immobilization on the detection electrode (working) of the electrochemical sensor system may be a commercially available D-LDH.

In embodiments, it is preferable to perform some electrode modifications prior to binding D-LDH to the electrode surface, such as metal nanoparticle coating and/or other electrode designs such as graphene electrodes using in-house produced chips, for sensitivity over the correct range of D-lactate concentrations .

NADH, released by a chemical reaction catalyzed by D-LDH (D-lactate+NAD+^pyruvate+NADH), is decomposed into NAD++H+ and ^2e- under the action of an applied voltage. 2e ^- (electrons) can be detected by a detection electrode (working), which can be located on the surface of the test strip (chip), and the electrochemical signal can be measured by a readout device in contact with the detection electrode, for example, a reader into which it is inserted test strip. Such a reader may be a battery-powered, portable compact reader. In preferred embodiments, such a reader uses amperometry. In addition, in the context of the present invention, a reading device that uses potentiometry can be used.

In the context of the present invention, it is possible to convert the electrical signal into molar concentration. For example, an electrochemical sensor system can be calibrated using test samples having known concentrations of D-LDH in a suitable environment, such as a suitable buffer. The electrochemical sensor system can be pre-calibrated. In embodiments, the system in accordance with the present invention may provide different detection modes corresponding to different sample conditions, for example, corresponding to measurements of different physiological fluids.

In the context of the present invention, immobilization of D-LDH on the surface of the detection electrode can be achieved by any of adsorption, covalent binding, entrapment, encapsulation or preferably cross-linking or through thiol-gold interaction. In embodiments, other immobilization techniques known to one skilled in the art may be used.

For adsorption, enzymes can be ensured to adhere to electrode surfaces with weak interactions to avoid denaturation of the enzyme. This has proven particularly successful when the electrode surface has been ionized first by plasma or acid treatment, covalent surface modification such as amination, or ionic polymer deposition via electropolymerization or drop-casting/spinning coating.

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Cross-linking is a commonly used technique in which dialdehydes (glutaraldehyde) often react with the amino groups of enzymes and the amino groups on aminated surfaces. Another method is the thiol-gold reaction, in which a thiol group is added to the enzyme (or cysteines in the enzyme are used), which allows the enzyme to bind to the surface by thiolation of the gold.

Encapsulation is another very common immobilization method known to one skilled in the art. Encapsulation methods include electrochemical polymerization or cross-linking of a fine network of polymer over an enzyme on a surface. Thus, the enzyme cannot leave the surface, but still provides access to substrate molecules for the enzymatic reaction.

In embodiments, the detection electrode is modified, preferably prior to immobilization of the D-lactate binding molecule, to fine-tune the detection characteristics and increase and/or adjust the sensitivity of the biosensor over a suitable range of D-lactate concentrations.

In embodiments, the electrochemical sensing system comprises a detection electrode coated with metal nanoparticles.

In embodiments, the electrochemical sensing system comprises a detection electrode containing or consisting of graphene.

Additionally, in embodiments, the system allows D-lactate levels to be determined in more than one sample in parallel. A great advantage of the present invention is that it allows multiplexing of multiple samples that can be measured in parallel.

In embodiments, the infectious disease of the present invention is a microbial bacterial and/or fungal infection, preferably with at least one infectious agent selected from the group consisting of Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus spp., Enterococcus spp., anaerobes , gram-negative bacteria and Candida spp.

In the context of the present invention, the infectious disease may be a joint infection, a prosthetic joint infection (PJI), meningitis, peritonitis, a pleural cavity infection, a pericardial space infection, and/or a bloodstream infection.

Particularly preferred is the use of the present invention for the diagnosis of meningitis. Surprisingly, electrochemical detection of D-lactate in cerebrospinal fluid has been found to be particularly advantageous over other methods for detecting D-lactate, since cerebrospinal fluid samples are often contaminated with blood during the sample extraction procedure.

In addition, the present invention is particularly applicable to the determination of D-lactate in blood samples that cannot be used in spectrophotometric measurements due to the large amount of red blood cells and hemoglobin. Therefore, serum or plasma samples must be taken from blood or blood samples to detect circulating D-lactate. The present invention allows the determination of D-lactate levels in a blood sample immediately after isolation, making the step of obtaining serum or plasma unnecessary.

If the infectious disease is a joint infection, synovial fluid can be used as a sample. In this context, measuring D-lactate using an electrochemical sensor system is particularly advantageous compared to spectrophotometric measurements, since joint aspirates are often contaminated with blood. In addition, the synovial fluid of prosthetic joints is often contaminated with blood, especially after surgery. Accordingly, the presence of red blood cells (RBC) or hemoglobin from lysed RBCs contaminates samples obtained from blood samples and therefore tends to produce false results when measured by spectrophotometric methods.

If the infectious disease is peritonitis, ascites isolated with or without a peritoneal catheter can be used as a sample. If the infectious disease is an infection of the pleural cavity, then pleural fluid can be used as a sample.

If the infectious disease is a pericardial space infection or pericarditis, pericardial fluid can be used as a sample.

In the case of bloodstream infection or sepsis, blood or blood-derived material can be used as a sample, and the blood can be isolated with or without an intravascular catheter.

In embodiments, an elevated D-lactate level detected by the electrochemical sensing system in the sample, compared to a corresponding control, such as a sample from a healthy subject, indicates the presence of an infectious disease.

According to another embodiment of the present invention, measuring current or voltage using an electrochemical sensor system corresponding to the level of D-lactate in

- 5 046276 in the specified sample, equal to or greater than 1.2 mmol/l, indicates the presence of an infectious disease.

In further embodiments, measuring a current or voltage using an electrochemical sensor system corresponding to a D-lactate level in said sample equal to or greater than 0.4 mmol/L, preferably 0.5 mmol/L, more preferably equal to or greater than 1.0 mmol /l, most preferably equal to or greater than 1.2 mmol/l, indicates the presence of an infectious disease.

Moreover, the method according to the present invention can be characterized by the fact that a current or voltage measurement using an electrochemical sensor system corresponding to a D-lactate level in said sample equal to or greater than 0.5 mmol/L, preferably equal to or greater than 1 .0 mmol/L, more preferably equal to or greater than 1.2 mmol/L, indicates that initiation or change in antibiotic treatment is required.

In addition, possible threshold levels in accordance with the present invention are 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0. 85, 0.9, 0.95, 1, 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1, 9, 2.0 mmol/l. Ranges covering any combination of values disclosed herein as limits are considered to represent the disclosed embodiments of the present invention.

The threshold levels disclosed herein preferably refer to measurements of D-lactate in a synovial fluid sample obtained from a patient using the D-Lactam® diagnostic kit provided by Sivital (Vitebsk, Belarus), obtained from VL-Diagnostics (Leipzig) and used in the context examples of the present invention. Accordingly, the values disclosed herein may vary to some extent depending on the detection/measurement system or set used, and the specific values disclosed herein are intended to also read corresponding values determined by other measurement systems or sets. For example, comparison of spectrophotometric measurements of D-lactate using two different test kits (provided by Sigma-Aldrich (St. Louis, MO, USA) and VL-Diagnostics (Leipzig, Germany), respectively) showed that specifically defined cutoff values also depend on test kit and method for determining D-lactate levels (Karbysheva et al. Performance of synovial fluid D-lactate for the diagnosis of acute and chronic/low-grade PJI; Abstract at EFORT Conference 2018, Barcelona, Spain).

Accordingly, according to embodiments of the present invention, the cutoff values to be used in the context of an electrochemical measurement, such as amperometry or potentiometry, may correspond to different D-lactate concentrations compared to the D-lactate threshold concentrations that were determined by spectrophotometric measurements using a specific test kit .

Electrochemical measurement data readouts include current and voltage detection. The detected values depend on the configuration of the electrochemical sensor system. Modifications to various components of the system or method in accordance with the present invention (eg, sample) affect the detected currents or voltages, respectively. Therefore, it is not possible to provide a general cutoff value or threshold value for the electrochemical measurement. However, since in embodiments the electrochemical sensing system of the present invention requires calibration with reference samples of predetermined known concentrations of D-lactate, any given electrochemical system can be provided suitable for determining D-lactate cutoff values/thresholds that correspond to a particular D-lactate concentration, which has been shown to indicate an infectious disease and not necessarily the initiation of antibiotic treatment for a particular sample type.

In preferred embodiments of the present invention, the sample is diluted with phosphate buffer. In embodiments, the pH of said phosphate buffer is 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10. Ranges covering any combination of disclosed values as limits are disclosed embodiments of the present invention. A sample buffer or solution with a pH of about 7.5-9.5 is preferred, with a Ph of about 8-9 being more preferred and a pH of 8.5 being particularly preferred. D-lactate binding molecules and D-LDH in particular have been shown to work very effectively in sensing D-lactate in the context of an electrochemical sensing system at pH 8.5. A possible buffer solution for diluting a sample such as a physiological fluid such as synovial fluid is phosphate buffer.

In embodiments, the electrochemical sensing system is calibrated using one or more calibration samples with a specific concentration of D-lactate before determining the level of D-lactate in said sample. For example, commercially available D-lactate, diluted to a known concentration with a suitable buffer solution, which may be the same as the buffer solution used to determine the level of D-lactate in the specified sample, can be used to determine the voltage corresponding to the specific concentration of D-lactate

- 6 046276 in the sample.

In some embodiments incorporating a potentiometric sensor, a voltage of approximately 85 mV corresponds to a D-lactate level of 1.2 mmol/L and may indicate the presence of an infectious disease. The voltage measurement of an electrochemical sensing system can vary depending on the fine tuning of the system and the type of sensor.

In some embodiments incorporating an amperometric sensor, a current of approximately 422 nA corresponds to a D-lactate level of approximately 1.2 mmol/L and may indicate the presence of an infectious disease. Current measurement by an electrochemical sensing system can vary depending on the fine tuning of the system and the type of sensor.

The in vitro method of any one of the preceding claims, wherein a current or voltage measurement by an electrochemical sensor system corresponding to a D-lactate level in the sample equal to or greater than 1.2 mmol/L indicates that initiation or change of treatment is required antibiotic.

However, a current or voltage corresponding to a D-lactate level of 1.2 mmol/L may represent a preferred cutoff value in the context of the present invention, particularly in the case of synovial fluid samples that can be used to detect joint infections.

Preferably, the level of D-lactate determined by the electrochemical sensor system in the context of the method according to the present invention is independent of the amount of red blood cells and/or hemoglobin present in said sample.

According to another embodiment of the present invention, the sample is selected from the group consisting of a body fluid sample, a homogenized tissue sample, a blood sample, a serum sample, a plasma sample, a urine sample, a joint aspirate, a synovial fluid sample, an ascites sample, a peritoneal fluid sample, a pleural fluid sample , pericardial fluid sample and/or cerebrospinal fluid sample.

According to another aspect, the present invention relates to an electrochemical sensor system (biosensor) for determining the level of D-lactate in a sample. The electrochemical sensor system in accordance with the present invention is a system disclosed in the context of the method and kit in accordance with the present invention.

According to another aspect, the present invention also relates to a kit for carrying out the method in accordance with the present invention, including an electrochemical sensor system for determining the level of D-lactate in a sample, reference data, such as a reference level, preferably corresponding to the level of D-lactate in the specified sample, equal to or greater than 1.2 mmol/L, wherein said reference data is optionally stored on a computer-readable medium and/or used in the form of computer executable code configured to compare certain D-lactate levels to said reference data, and optionally reagents for calibrating the electrochemical sensor system.

The present invention also relates to a kit for carrying out the method according to the present invention, comprising an electrochemical sensor system for determining the level of D-lactate in a sample, preferably including a test strip (chip) with corresponding electrodes on the surface or a transistor-based sensor for electrochemical detection, wherein the electrochemical signal is measured using a reader, preferably powered by a battery of a portable compact reader, reference data, such as a reference level, preferably corresponding to a D lactate level in said sample equal to or greater than 1.2 mmol/L, wherein said reference data optionally stored on a computer-readable medium and/or used in the form of computer-executable code configured to compare certain levels of D-lactate with specified reference data, and optionally reagents for calibrating the electrochemical sensor system.

In addition, the present invention relates to a kit for carrying out the method in accordance with the present invention, comprising:

an electrochemical sensor system for determining the level of D-lactate in a sample, wherein the electrochemical sensor system preferably comprises:

i. a test strip (chip) for electrochemical determination of the level of D-lactate, wherein the test strip contains a determination electrode with an immobilized D-lactate binding molecule and preferably also a counter electrode and/or a reference electrode, ii. and optionally a portable compact reader for inserting a test strip and performing a D-lactate measurement, reference data, such as a reference level preferably corresponding to a D-lactate level in said sample equal to or greater than 1.2 mmol/L, wherein said reference data is optional stored on a machine-readable medium and/or used in executable form

- 7 046276 computer code configured to compare specified D-lactate levels to specified reference data, and optionally reagents for calibrating the electrochemical sensor system.

Kits in accordance with the present invention may also contain instructions for use. The cutoff values provided by the kit may vary depending on the infectious disease and samples to be used to implement the method in accordance with the present invention. The kit may provide a list of suitable cutoff values for a list of infectious diseases and/or samples to be used.

Electrochemical system calibration reagents may contain D-lactate and suitable buffer solutions to create suitable calibration samples. Such samples may be provided off-the-shelf, or the base material and reagents for creating such samples may be provided as a kit to allow the user to create custom calibration samples for the specific application of the kit performed by the respective user.

According to another aspect, the present invention relates to an electrochemical sensor system (biosensor) for determining the level of D-lactate in a sample. Embodiments of an electrochemical sensor system in accordance with the present invention are disclosed in the context of a method and kit in accordance with the present invention. In a preferred embodiment, the electrochemical sensing system for determining the level of D-lactate in a sample comprises D-LDH as a D-lactate sensing component immobilized on a test strip for insertion into a portable reader.

In embodiments, the electrochemical sensor system of the present invention comprises a potentiometric sensor and/or an amperometric sensor. The system preferably contains a detection electrode (working) with a D-lactate binding molecule, preferably D-LDH, immobilized on the surface of the electrode. In preferred embodiments, the electrochemical sensing system comprises a (preferably disposable) test strip that contains a detection electrode with an immobilized D-lactate binding molecule and preferably also a counter electrode and/or reference electrodes. In embodiments, the electrochemical sensor system also includes a reader, preferably a portable handheld reader, suitable for inserting a test strip and for measuring D-lactate in the sample.

All features that have been disclosed in the context of the method in accordance with the present invention are also disclosed in the context of the kit and electrochemical sensor system in accordance with the present invention and vice versa. Accordingly, features of embodiments of a method in accordance with the present invention may also be features of embodiments of a kit and electrochemical sensor system in accordance with the present invention and vice versa.

Detailed Description of the Invention

The present invention relates to an in vitro method for the diagnosis, prognosis, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease, comprising (a) providing a sample of a subject exhibiting clinical symptoms of infection and/or suspected of having an infection, (b) determining the level of D-lactate in the specified sample, (c) the level of D-lactate indicates the presence of an infectious disease, characterized in that (d) the level of D-lactate in the specified sample is determined using an electrochemical sensor system (biosensor).

As used herein, the term diagnosis in the context of the present invention refers to the recognition and (early) detection of a clinical condition of a subject associated with an infectious disease. Also, the term diagnosis can cover the assessment of the severity of an infectious disease. The term prediction refers to the prediction of an outcome or specific risk for a subject based on an infectious disease. This may also include an assessment of the chance of recovery or the chance of an unfavorable outcome for the specified subject.

The term risk assessment and possibly subsequent patient stratification refers to the grouping of subjects into different risk groups according to their prognosis. Risk assessment also relates to stratification for the application of preventive and/or therapeutic measures. Additionally, the methods of the present invention can be used to stratify therapy, wherein the term therapy stratification specifically refers to the grouping or classification of patients into different groups, such as risk groups or treatment groups, who receive certain differentiated therapeutic measures in depending on their classification. The term therapy stratification also refers to the grouping or classification of patients with infections or symptoms of an infectious disease into a group that does not need to receive certain therapeutic measures, such as antibiotic treatment.

The methods of the present invention can also be used for monitoring. The term monitoring in the context of the method according to the present invention relating to

- 8 046276 infectious diseases, refers to the monitoring of an already diagnosed infectious disease, disorder, complication or risk, for example, to analyze the progression of a disease or the effect of a specific treatment or therapy on the progression of a disease in a seriously ill patient or an infectious disease in a patient. The terms therapy monitoring and therapeutic control as used herein refer to monitoring and/or adjusting the therapeutic treatment of a specified subject, for example, by obtaining feedback regarding the effectiveness of therapy. As used herein, the term therapeutic management refers to the administration of certain therapeutic agents, therapeutic actions or medical interventions based on the level of D-lactate determined in the context of the present invention. This includes adjusting therapy or stopping therapy.

The term infectious disease is intended to encompass and include all diseases or disorders that are associated with and/or caused by infection, such as, but not limited to, bacterial, viral and/or fungal infection. As used herein, the term infection refers to a pathological process caused by the invasion of normally sterile tissue or fluid by pathogenic or potentially pathogenic pathogens/pathogens, organisms and/or microorganisms, and preferably refers to infection(s) caused by bacteria, viruses, fungi and/or parasites. Accordingly, the infection may be a bacterial infection, a viral infection and/or a fungal infection. The infection may be local or systemic infection. For the purposes of the present invention, a viral infection can be considered as an infection caused by a microorganism.

The present invention covers nosocomial infections. Nosocomial infections are also called healthcare-associated infections because the infections may be acquired in a hospital, nursing home, rehabilitation facility, outpatient clinic, or other clinical or health care setting. Nosocomial infection can be transmitted to a susceptible patient in a clinical setting in a variety of ways. Infection can be spread by medical personnel in addition to contaminated equipment, bedding, or airborne droplets. Infection may come from the external environment, another infected patient, personnel who may be infected, or, in some cases, the source of infection cannot be determined. In some cases, the microorganism originates from the patient's own skin microbiota, becoming opportunistic after surgery or other procedures that disrupt the protective skin barrier. Although the patient may have contracted the infection through his or her own skin, the infection is still considered nosocomial because it develops in a health care setting.

In embodiments, a subject suffering from an infection may simultaneously suffer from more than one source of infection. For example, the subject may be suffering from a bacterial infection and a viral infection; viral infection and fungal infection; bacterial and fungal infection, as well as bacterial infection, fungal infection and viral infection, or may have a mixed infection involving one or more of the infections listed herein, including a potential superinfection, such as one or more bacterial infections in addition to one or more viral infections and/or one or more fungal infections.

In the context of the present invention, the infectious disease is preferably associated with a bacterial and/or fungal infection, preferably with at least one infectious agent selected from the group consisting of Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus spp., Enterococcus spp., anaerobes, gram-negative bacteria and Candida spp.

In one embodiment, the infection to be detected or tested may be selected from Bordetella species such as Bordetella pertussis, Borrelia such as Borrelia burgdorferi, Brucella such as Brucella abortus, Brucella canis, Brucella melitensis or Brucella suis, Campylobacter such such as Campylobacter jejuni, Chlamydia and Chlamydophila such as Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium such as Corynebacterium diphtheria, Enterococcus such as Enterococcus faec alis, Enterococcus faecium, Escherichia such as Escherichia coli, Francisella such as Francisella tularensis, Haemophilus such as Haemophilus influenza, Helicobacter such as Helicobacter pylori, Legionella such as Legionella pneumophila, Leptospira such as Leptospira interrogans, Listeria such as Listeria monocytogenes , Mycobacterium such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma such as Mycoplasma pneumonia, Neisseria such as Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas such as Pseudomonas aeruginosa, Rickettsia such as Rickettsia rickettsia, Salmonella such as Salmonella typhi, Salmonella typhimurium, Shigella such as Shigella sonnei, Staphylococcus such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus such as Streptococcus agalactiae, Streptococcus pneumonia, Streptococcus pyogenes, Treponema such as Treponema pallidum, Vibrio such as Vibri o cholera, Yersinia, such as Yersinia pestis, Yersinia enterocolitica or Yersinia pseudotuberculosis.

- 9 046276

Pathogenic fungi are fungi that cause disease in humans or other organisms. Candida species are important human pathogens that are well known to cause opportunistic infections in immunocompromised hosts (eg, transplant patients, acquired immunodeficiency syndrome AIDS, cancer patients). Infections are difficult to treat and can be very severe, with 30-40% of systemic infections resulting in death. Aspergillosis is caused by another potential fungal pathogen. Aspergillus can cause disease in three main ways: through the production of mycotoxins; by inducing allergic reactions and through localized or systemic infections. For the latter two categories, the immune status of the host is critical. The most common pathogenic species are Aspergillus fumigatus and Aspergillus flavus. Aspergillus flavus produces aflatoxin, which is both a toxin and carcinogen and has the potential to contaminate food. Aspergillus fumigatus and Aspergillus clavatus can cause the disease. Cryptococcus neoformans can cause disease in humans. Cryptococcus neoformans is a major pathogen of humans and animals. Cryptococcus laurentii and Cryptococcus albidus are known to occasionally cause moderate to severe illness in immunocompromised people. Cryptococcus gattii is endemic to tropical parts of the continent of Africa and Australia and can cause disease. Histoplasma capsulatum can cause histoplasmosis in humans, dogs and cats. Pneumocystis jirovecii (or Pneumocystis carinii) can cause a form of pneumonia in people with weakened immune systems, such as premature babies, the elderly, and patients with AIDS. Stachybotrys chartarum or black mold can cause respiratory infections and severe headaches.

In one embodiment, the infection to be detected or tested may be selected from Acinetobacter baumannii, Klebsiella pneumoniae, Acinetobacter Iwoffii, Listeria monocytogenes, Aeromonas caviae, Morganella morganii, Aeromonas hydrophila, Neisseria gonorrhoeae, Aspergillus flavus, Neisseria meningitidis, Aspergillus nidulans, Pasteurella multocida, Aspergillus niger, Pasteurella pneumotropica, Aspergillus terreus, Propionibacterium acnes, Bacillus anthracis, Proteus mirabillis, Bacillus cereus, Providencia rettgeri, Bacillus subtilis, Pseudomonas aeruginosa, Bacteroides fragilis, Salmonella choleraesuis, Brucella melitens is, Serratia liquefaciens, Burkholderia cepacia, Serratia marcescens, Candida albicans, Staphylococcus aureus, Candida dubliniensis, Staphylococcus epidermidis, Candida glabrata, Staphylococcus haemolyticus, Candida krusei, Staphylococcus hominis, Candida parapsilosis, Staphylococcus saccharolyticus, Candida tropicalis, Staphylococcus warneri, Capnocytophaga can imorsus, Stenotrophomonas maltophilia, Citrobacter braakii, Streptococcus agalactiae , Citrobacter freundii, Streptococcus anginosus, Clostridium perfringens, Streptococcus bovis, Corynebacterium jeikeium, Streptococcus constellatus, Enterobacter aerogenes, Streptococcus dysgalactiae, Enterobacter cloacae, Streptococcus mutans, Enterobacter sakazakii, Streptococcus pneumoniae, Enterococcus faecas, liStreptococcus pyogenes, Enterococcus faecium, Streptococcus salivarius, Escherichia coli, Streptococcus sanguinis, Shigella spp., Streptococcus suis, Gemella haemolysans, Vibrio vulnificus, Gemella morbillorum, Yersinia enterocolitica, Haemophilus influenzae, Yersinia pestis, Kingella kingae, Yersinia pseudotuberculosis and Klebsiella oxytoca.

In embodiments of the present invention, the infectious disease is a joint infection, a prosthetic joint infection (PJI), a central nervous system infection, meningitis, peritonitis, a pleural cavity infection, a pericardial space infection, and/or a bloodstream infection.

In the context of embodiments of the present invention, the sample preferably corresponds to a physiological fluid that is in contact with a tissue or organ that is suspected of being infected. For example, in the case of a central nervous system infection or meningitis, a cerebrospinal fluid (CSF) sample may preferably be used.

Meningitis is an acute inflammation of the protective coverings around the brain and spinal cord, known collectively as the meninges. The most common symptoms are fever, headache and neck stiffness. Other symptoms include confusion or altered consciousness, vomiting, and an inability to tolerate light or loud noises. Young children often show only nonspecific symptoms, such as irritability, drowsiness, or poor appetite. If a rash is present, this may indicate a specific cause of meningitis; for example, meningitis caused by meningococcal bacteria may be accompanied by a characteristic rash. Inflammation can be caused by infection with viruses, bacteria or other microorganisms, and less commonly by certain medications. Meningitis can be life-threatening due to the inflammation's proximity to the brain and spinal cord; therefore, the condition is classified as a medical emergency. Meningitis can be diagnosed or ruled out by a lumbar puncture, in which a needle is inserted into the spinal canal to remove a sample of cerebrospinal fluid (CSF).

As used herein, the term bloodstream infection may include systemic bloodstream infection, sepsis, severe sepsis, and/or septic shock.

Joint infection, also known as septic arthritis or infectious arthritis, is

- 10 046276 is an invasion of the joint by an infectious pathogen, which leads to inflammation of the joint. Symptoms usually include redness, heat, and pain in one joint associated with decreased ability of the joint to move. The onset is usually quick. Other symptoms may include fever, weakness and headache. Sometimes more than one joint may be affected. Causes include bacteria, viruses, fungi and parasites. Risk factors include artificial/prosthetic joint, previous arthritis, diabetes and poor immune function. Joints most often become infected through the blood, but can also become infected through injury or infection around the joint. Diagnosis is usually based on joint fluid aspiration and culture. Initial treatment usually includes antibiotics such as vancomycin, ceftriaxone, or ceftazidime. Surgery may also be performed to clean out the joint. Without early treatment, long-term joint problems can occur. Septic arthritis affects approximately 5 in 100,000 people each year. It is more common in older people. With treatment, approximately 15% of people die, and without treatment, 66% die.

According to embodiments of the present invention, the method in accordance with the present invention may include the step of treating if the electrochemical determination of D-lactate indicates the presence of an infectious disease. At such a stage of treatment, one or more of the therapeutic measures disclosed herein may be administered to the appropriate patient.

In the context of the present invention, the term medical treatment or treatment includes various types of treatment and therapeutic strategies, in particular, types of treatment that are known to a qualified specialist for the corresponding diagnosed infectious disease. In the context of the present invention, treatment may include treatment with antibiotics, such as intravenous antibiotics, oral antibiotics or topical antibiotics. Medical treatment in accordance with the present invention may be antibiotic treatment, in which one or more antibiotics or antibiotic agents may be administered if an infection is diagnosed or symptoms of an infectious disease are determined.

Antibiotics or antibiotic agents in accordance with the present invention also potentially include antifungal or antiviral compounds used to treat a diagnosed infection or sepsis. Antibiotic agents commonly used in the treatment of any given infection that can be used in the context of the present invention, divided into pathogen classes, include the spectrum of action - gram-positive: penicillins (ampicillin, amoxicillin), penicillinase-resistant (dicloxacillin, oxacillin), cephalosporins ( 1st and 2nd generation), macrolides (erythromycin, clarithromycin, azithromycin), quinolones (gatifloxacin, moxifloxacin, levofloxacin), vancomycin, sulfonamide/trimethoprim, clindamycin, tetracyclines, chloramphenicol, linezolid, synercid; spectrum of action - gram-negative: broad-spectrum penicillins (ticarcillin, clavulanate, piperacillin, tazobactam), cephalosporins (2nd, 3rd and 4th generation), aminoglycosides, macrolides, azithromycin, quinolones (ciprofloxacin), monobactams (azetreonam) , sulfonamide/trimethoprim, carbapenems (imipenem), chloramphenicol; spectrum of action - Pseudomonas: ciprofloxacin, aminoglycosides, some 3rd generation cephalosporins, 4th generation cephalosporins, broad-spectrum penicillins, carbapenems;

antifungal treatments: alliamines, amphotericin B, fluconazole and other azoles, itraconazole, voriconazole, posaconazole, ravuconazole, echinocandins, flucytosine, sordarins, chitin synthetase inhibitors, topoisomerase inhibitors, lipopeptides, pradimycins, liposomal nystatin, voriconazole, echinocanidins, imidazole, triazole , thiazole , polyene;

antiviral treatments: abacavir, aciclovir, activated caspase oligomerizer, adefovir, amantadine, amprenavir (Agenerase), ampligen, arbidol, atazanavir, atripla, balavir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, double-stranded ribonucleic acid RNA , docosanol, edoxudin, efavirenz, emtricitabine, enfuvirtide, entecavir, ecoliever, famciclovir, fixed dose combination (antiretroviral), fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitor, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitor, type III interferon, type II interferon, type I interferon, interferon, lamivudine, lopinavir, loviride, maraviroc, moroxidine, metizazone, morpholinos, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogs, novir, oseltamivir (Tamiflu), peginterferon alpha/;·!, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitor (pharmacology), raltegravir, reverse transcriptase inhibitor, ribavirin, ribozymes, rifampicin, rimantadine, ritonavir, RNase H, protease inhibitors, pyramidin, saquinavir, sofosbuvir, stavudine , synergistic booster (antiretroviral), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (Valtrex), valganciclovir vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza), zidovudine.

In addition, antibiotic agents include bacteriophages to treat bacterial infections, synthetic antimicrobial peptides or iron antagonists/iron chelating agents can be used. Also therapeutic antibodies or antagonist against pathogenic

- 11 046276 structures such as antibodies against VAP (vascular adhesion protein), vaccination against resistant clones, administration of immune cells such as in vitro primed or modulated effector T cells are antibiotic agents, which are treatment options in the context of the present invention. Additional antibiotic agents/treatments or therapeutic strategies against infection or to prevent new infections include the use of antiseptics, disinfectant products, antivirulence agents such as liposomes, sanitation measures, wound care, surgery.

It is also possible to combine several of the above-mentioned antibiotic agents or treatment strategies.

In embodiments, the present invention includes administering an antibiotic suitable for treatment based on information obtained by a method disclosed herein.

The methods of the present invention are particularly advantageous because electrochemical measurement of D-lactate in patient samples has been shown to allow diagnostic tests to be performed with very high specificity and sensitivity compared to prior art methods that are suboptimal with respect to at least one of these tests. properties.

Sensitivity and specificity are statistical measures of the performance of a binary classification test, also known in statistics as a classification function, which is widely used in medicine. Sensitivity (also called true positive rate, recall, or probability of detection in some areas) measures the proportion of actual positives that are correctly identified as such (for example, the percentage of sick people who are correctly identified as having a condition). Specificity (also called the true negative rate) measures the proportion of actual negatives that are correctly identified as such (for example, the percentage of healthy individuals who are correctly identified as not having the condition). In many tests, including diagnostic medical tests, sensitivity is the degree to which actual positive results are not ignored (so there are not many false negatives), and specificity is the degree to which actual negative results are classified as such (so there are not many false positives). a lot of). Thus, a highly sensitive test will rarely ignore a true positive result (eg, show nothing untoward despite the presence of something untoward); a highly specific test will rarely record a positive classification of anything that is not the purpose of testing (for example, detecting one species of bacteria and mistaking it for another, closely related one, which is the true purpose).

As used herein, the sensitivity and specificity of a diagnostic and/or prognostic test depend not only on the analytical quality of the test, they also depend on the definition of what constitutes an abnormal result. In practice, receiver operating characteristic curves (ROC curves) are usually calculated by plotting the value of a variable against its relative frequency in normal (i.e., apparently healthy individuals without infection, and in diseased populations, such as subjects with infection). In the case of the present invention, the distribution of D-lactate levels for subjects with and without the disease/condition is likely to overlap. Under these conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy, and the area of overlap may indicate where the test cannot distinguish normal from disease. A threshold is selected below which the test is not considered normal, above which the test is considered normal, or below or above which the test is indicative of a particular condition, such as infection. The area under the ROC curve is a measure of the likelihood that a perceived measurement will correctly identify a condition. ROC curves can be used even if the test results do not necessarily produce an exact number. Provided the results can be ranked, an ROC curve can be constructed. For example, test results on disease samples may be ranked by grade (eg, 1 is low, 2 is normal, and 3 is high). This ranking can be compared with results in a normal population to create an ROC curve. These methods are well known in the art; see, for example, Hanley et al. 1982. Radiology 143: 29-36. Preferably, the threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. The term approximately in this context refers to plus/minus 5% of a given measurement.

The horizontal axis of the ROC curve represents (1 - specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Thus, for

- 12 046276 of the particular selected cutoff value can be determined (1 - specificity) and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that a measured marker level will correctly identify a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

As used herein, the term patient or subject may be a vertebrate. In the context of the present invention, the term subject includes both humans and animals, in particular mammals, and other organisms.

In accordance with the present invention, a patient exhibiting symptoms of an infectious disease is a subject who experiences, without limitation, one or more of fever, diarrhea, fatigue, muscle pain, cough, animal bite, difficulty breathing, severe headache with fever, rash or swelling, unexplained or prolonged fever, or vision problems. Other symptoms may include fever and chills, very low body temperature, oliguria, rapid pulse, rapid breathing, nausea and vomiting. In embodiments, symptoms of an infectious disease include fever, diarrhea, fatigue, muscle pain, rapid pulse, rapid breathing, nausea, vomiting, and/or cough.

As used herein, the term sample means a biological sample obtained or isolated from a patient or subject. The term sample as used herein may, for example, refer to a body fluid sample, a homogenized tissue sample, a blood sample, a serum sample, a plasma sample, a urine sample, a joint aspirate, a synovial fluid sample, an ascites sample, a peritoneal fluid sample, a pleural fluid, pericardial fluid sample and/or cerebrospinal fluid sample. The sample is preferably isolated or obtained for the purpose of diagnosis, prognosis or evaluation of a subject of interest, such as a patient. The sample in accordance with the present invention may be a sample of a physiological fluid such as blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion, cells, cell extract, tissue sample, tissue biopsy, stool sample, and the like. . Specifically, the sample is blood, blood plasma, blood serum, or urine.

The blood sample used herein is a whole blood sample that is not processed to alter its composition. Specifically, a blood sample contains blood cells. Serum and plasma samples are obtained from blood. The term plasma in the context of the present invention is a substantially cell-free supernatant of blood containing an anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binders such as ethylenediaminetetraacetic acid EDTA or citrate, and thrombin inhibitors such as heparinates or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (eg, citrate-treated, EDTA-treated or heparinized blood), for example, for at least 15 minutes at 2000-3000 g. The term blood serum in the context of the present invention is the liquid fraction of whole blood that is collected after providing the blood with the opportunity to clot. When coagulated blood (clotted blood) is centrifuged, serum can be obtained as a supernatant.

Cerebrospinal fluid (CSF) samples are collected by puncturing body cavities filled with CSF, also called cerebrospinal fluid. CSF is primarily collected during lumbar puncture of the central canal of the spinal cord. Cerebrospinal fluid (CSF) is a clear, colorless body fluid found in the brain and spinal cord. It is produced by specialized ependymal cells of the choroid plexuses of the ventricles of the brain and is absorbed by arachnoid granulations. At one time, approximately 125 ml of CSF is contained, and in adults approximately 500 ml are produced daily. CSF acts as a cushion or buffer, providing basic mechanical and immunological protection to the brain within the skull. CSF also has a vital function in cerebral autoregulation of cerebral blood flow. CSF occupies the subarachnoid space (between the arachnoid membrane and the pia mater) and the ventricular system around and within the brain and spinal cord. It fills the ventricles of the brain, cisterns and grooves, as well as the central canal of the spinal cord. There is also a connection between the subarachnoid space and the bony labyrinth of the inner ear through the perilymphatic duct, where the perilymph communicates with the cerebrospinal fluid. The ependymal cells of the choroid plexus have several motile cilia on their apical surfaces that vibrate to move CSF through the ventricles. A CSF sample can be collected using a lumbar puncture. This can reveal intracranial pressure and also indicate diseases, including infections of the brain or surrounding meninges.

Synovial fluid samples are particularly preferred in the context of the present invention for the diagnosis of joint infections, in particular in the case of infections of prosthetic joints. Synovial fluid, also called synovium, is a viscous non-Newtonian fluid found

- 13 046276 sculpted in the cavities of synovial joints. The main role of synovial fluid is to reduce friction between the articular cartilages of synovial joints during movement. Synovial fluid is a small component of the transcellular fluid that is part of the extracellular fluid. The inner lining of synovial joints is called synovium and secretes synovial fluid into the joint cavity. Synovial fluid is an ultrafiltrate of plasma and contains plasma-derived proteins and proteins that are produced by cells in joint tissue. The fluid contains hyaluronan, secreted by fibroblast-like cells of the synovium, lubricin (proteoglycan 4; PRG4 (from English - proteoglycan 4)), secreted by superficial chondrocytes of articular cartilage, and interstitial fluid filtered from blood plasma. The fluid forms a thin layer (approximately 50 microns) on the surface of the cartilage and also seeps into microcavities and irregularities on the surface of the articular cartilage, filling all empty spaces. Articular cartilage fluid effectively serves as a reserve of synovial fluid. During movement, the synovial fluid held in the cartilage is mechanically squeezed out to maintain a layer of fluid on the surface of the cartilage (called leakage lubrication). The functions of synovial fluid include reducing joint friction, shock absorption, and transporting nutrients and waste, among others. Synovial tissue is sterile and consists of vascularized connective tissue that lacks a basement membrane. Synovial fluid can be collected with a syringe in a procedure called arthrocentesis, also known as joint aspiration.

Synovial fluid can be classified as normal, noninflammatory, inflammatory, septic, and hemorrhagic, and parameters assessed may include viscosity, clarity, color, and white blood cell count. Such parameters may be assessed in addition to D-lactate levels in the context of certain embodiments of the present invention.

The expression D-lactate level determination refers to the quantitative measurement or detection of D-lactate. The D-lactate level can be determined using various measured parameters, such as voltage or current, which correspond to a specific D-lactate concentration in the context of an electrochemical measurement. In spectrophotometric measurements, the light absorption of a chemical can be determined to determine the amount of the corresponding substance.

Lactates are salts and esters of lactic acid. Lactic acid occurs in two enantiomeric forms, so there are also two corresponding forms of its lactate anion, which are usually called the D and L forms according to their orientation in the Fischer projection. Lactic acid is a strong carboxylic acid that is highly dissociated under physiological conditions. The anion has the structural formula CH3-CHOH-COO- and is called lactate. Lactate produced in the human body is present exclusively in the L(+) form with clockwise rotation.

Lactic acid esters (CH3-CHOH-COOR) are also called lactates. Ethyl lactate (lactic acid ethyl ester) is the most important representative of these esters and is used, among other things, as a solvent. Another representative is butyl lactate (butyl ester of lactic acid).

The most common lactate found in the human body is sodium lactate. It is mainly produced in skeletal muscles. When glucose or glycogen is broken down into pyruvate by glycolysis, the coenzyme NAD+ is reduced to NADH/I+. For glycolysis to occur, it must be present in an oxidized form, such as NAD+. Only then can it act as an electron acceptor in the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase. Since in muscle fibers poor in mitochondria, not all NADH/I+ can be oxidized so quickly with increasing load, the body helps itself by reducing pyruvate to lactate. In the process, NADH/I+ is re-oxidized to NAD+. The reduction of glucose to lactate is also known as homofermentative lactic acid fermentation. In medicine, L-lactate is used as a marker of ischemia, since it is formed in tissue when there is a lack of oxygen.

Microorganisms also produce lactate during lactic acid fermentation. Unlike humans, they can also form the D-isomer using D-lactate dehydrogenase.

Lactate dehydrogenase (LDH or LD (from English - lactate dehydrogenase)) is an enzyme found in almost all living cells. LDH catalyzes the conversion of lactate to pyruvate and vice versa as it converts NAD+ to NADH and vice versa. Dehydrogenase is an enzyme that transfers hydride from one molecule to another.

D-lactate dehydrogenase (D-lactic acid dehydrogenase, D-LDH, D-specific lactate dehydrogenase, D-(-)-lactate dehydrogenase (NAD+), D-lactic acid dehydrogenase, D-lactate dehydrogenase) is an enzyme with the systematic name ^)-lactate: NAD+oxidoreductase. This enzyme catalyzes the following chemical reaction: (R)lactate + NAD + #pyruvate + NADH. D-LDH is a preferred D-lactate binding molecule in accordance with the present invention. The preferred D-LDH is D-LDH Staphy

- 14 046276 lococcus epidermidis.

In the context of the present invention, a D-lactate binding molecule is any type of molecule that specifically binds to D-lactate, but not to L-lactate or other molecules structurally related to D-lactate. In preferred embodiments, the D-lactate binding molecule is an enzyme that binds to D-lactate to catalyze a reaction that involves D-lactate as a substrate. Most preferably, the D-lactate binding molecule is D-LDH.

Additional D-lactate binding molecules useful in the context of the present invention include, but are not limited to, D-LDH, such as D-LDH of microbial origin, for example, D-LDH from Staphylococcus epidermidis; D-lactate oxidase, such as D-lactate oxidase of microbial origin, for example D-lactate oxidase from Gluconobacter or Zymomonas mobilis.

In the context of the method according to the present invention, a certain level of D-lactate may indicate the presence of an infectious disease. It can be used to compare a given level with an appropriate control, such as a control sample or a multiple sample from a control group such as healthy individuals, or with a reference value, such as a threshold value or cut-off value, with a concentration greater than that of the control sample. or equal to or greater than a reference value may indicate an infectious disease or provide prognostic value regarding the progression of an infectious disease.

If PJI is determined, the control group can be patients with prosthetic joints who do not suffer from PJI. Based on a comparison of the determined levels of D-lactate in a sample of an individual suffering from the corresponding infectious disease of interest and the levels determined for the corresponding control group, suitable reference values, such as a cut-off value or threshold value, can be obtained, with levels equal to those of D-lactate. lactate or levels exceeding D-lactate indicate the presence of a corresponding infectious disease.

In the context of the present invention, it is possible to use control values/sample or standards that provide samples with D-lactate or represent its control amounts already obtained from previous analytical tests. Reference values obtained from testing cohorts or other large numbers of subjects suffering from a given infectious disease or control group may be used. Appropriate statistical tools for analyzing and comparing such data sets are known to those skilled in the art. Control samples for positive controls (eg, from patients) or negative controls (from healthy subjects) can be used for reference values in simultaneous or non-simultaneous comparisons.

As used herein, the term electrochemical sensing system, which may also be referred to as a biosensor, refers to an analytical device used to detect a substance/analyte, in this case D-lactate, that combines a biological component with an electrochemical detector. The sensor contains a sensitive biological element, for example, tissue, microorganisms, organelles, molecule, cellular receptors, enzymes, antibodies, nucleic acids, etc., which is a biologically derived material or biomimetic component that interacts with the chemical analyte of interest, binds with it or recognizes it. Biologically responsive elements can also be created through biological engineering.

The biosensor further contains a transducer or detector element that converts the signal into an electrochemical signal resulting from the interaction of the analyte with the biological element. Based on this signal conversion, analyte levels in the sample can be easily measured and quantified.

The electrochemical system may contain or be connected to a biosensor reader that has associated electronics or signal processors for connection to a transformer. Such readers are preferably responsible for displaying the results in a user-friendly manner.

A biosensor in accordance with the present invention may comprise a biosensing section, a biotransducer component, and preferably an electronic system that includes one or more of a signal amplifier, a processor, and a display. The recognition component, often called a bioreceptor, uses biomolecules from organisms or receptors modeled after biological systems to interact with the analyte of interest. This interaction is measured by a biotransducer, which produces a measurable signal proportional to the presence of the target analyte in the sample. The overall goal of the biosensor design is to provide rapid and convenient testing at the point of concern or care (POC) where the sample was collected.

The bioreceptor is designed to interact with a specific analyte of interest

- 15 046276 to obtain the effect measured by the transducer. High selectivity for the analyte among a matrix of other chemical or biological components is a key requirement for a bioreceptor. Although the type of biomolecule used can vary widely, biosensors can be classified according to general types of bioreceptor interactions, including antibody/antigen, enzymes/ligands, nucleic acids/deoxyribonucleic acid DNA, cellular structures/cells, or biomimetic materials. In the context of the present invention, the bioreceptor is D-LDH binding molecules, such as preferably D-LDH. Accordingly, the biosensor/electrochemical sensor system in accordance with the present invention preferably involves an enzyme/ligand interaction.

The specific binding abilities and catalytic activity of enzymes make them popular bioreceptors that are advantageous for several reasons, including suitability for several different transduction modes in analyte detection. It should be noted that since enzymes are not consumed in the reactions, the biosensor can be easily used continuously. The catalytic activity of the enzymes also provides lower detection limits compared to conventional coupling techniques.

Preferably, in the context of a biosensor/electrochemical sensor system, biological elements, in this case a D-lactate binding molecule such as D-LDH, are attached to the surface of the sensor, which may be metal, polymer or glass, for example. The simplest method is to functionalize the surface to coat it with biological elements. This can be done using polylysine, aminosilane, epoxysilane or nitrocellulose in the case of silicon chips/quartz glass. Subsequently, the bound biological agent can, for example, be fixed by layer-by-layer deposition of oppositely charged polymer coatings. Alternatively, three-dimensional lattices (hydrogel/xerogel) can be used to chemically or physically capture them (chemical capture means that the biological element is held in place by a strong bond, while physically holding it in place without being able to pass through the pores gel matrix). The most widely used hydrogel is sol-gel, a glassy silica formed by the polymerization of silicate monomers (added as tetraalkyl orthosilicates such as TMOS (tetramethoxysilane) or TEOS (tetraethoxysilane)) in the presence of biological elements (along with other stabilizing polymers such as polyethylene glycol PEG) in the event of physical entrapment. Another group of hydrogels that harden under conditions suitable for cells or protein are acrylate hydrogels, which polymerize when initiated by radicals. One type of radical polymerization initiator is the peroxide radical, usually formed by combining persulfate with TEMED (tetramethylethylenediamine) (polyacrylamide gel is also commonly used for protein electrophoresis); alternatively, light can be used in combination with a photoinitiator such as DMF (2,2-dimethoxy-2-phenylacetophenone).

Electrochemical biosensors are usually based on enzymatic catalysis of a reaction that produces or consumes electrons (such enzymes are appropriately called redox enzymes). The sensor substrate typically contains three electrodes: a reference electrode, a working/sensing electrode, and a counter electrode. The target analyte participates in a reaction that occurs at the active surface of the electrode, and the reaction can either cause electron transfer through the bilayer (producing a current) or contribute to the potential of the bilayer (producing a voltage).

Accordingly, one can either measure the current (the rate of electron flow is now proportional to the analyte concentration) at a fixed potential, or one can measure the potential at zero current (this gives a logarithmic response). It should be noted that the working/detection/active electrode potential is space charge sensitive and this is often used.

The potentiometric biosensor (potential generated at zero current) gives a logarithmic response with a high dynamic range. Such biosensors are often made by screen printing electrode patterns onto a plastic substrate coated with a conductive polymer and then attaching the enzyme/biosensor. They have only two electrodes and are extremely sensitive and reliable. They enable detection of analytes at levels previously only achievable by HPLC and LC/MS and without extensive sample preparation.

All biosensors typically require minimal sample preparation because the biological sensing component is highly selective for the analyte in question. The signal is created by electrochemical and physical changes in the conductive polymer layer due to changes occurring on the surface of the sensor. Such changes may be related to ionic strength, pH, hydration and redox reactions, the latter due to the enzyme tag switching the substrate. Field effect transistors in which the gate region has been modified with an enzyme/biosensor can also detect very low concentrations of various analytes, since the binding of the analyte to the FET (field effect transistor) gate region

- 16 046276 refers to the change in current from drain to source.

Electrochemical biosensors in accordance with the present invention are used for in vitro measurements of D-lactate in a sample. The biosensor measurement can take place in a test tube, culture dish, microtiter plate, or other location outside of a living organism. The sensor uses a bioreceptor and transducer as disclosed above. Preferably, the present invention can be used as a point-of-care test (POCT), i.e. at the location where the test is required. Accordingly, it is preferable that the biosensor in accordance with the present invention is portable or portable, preferably a wearable biosensor. Eliminating the need for laboratory testing can save time and money. The POCT biosensor can be sent directly to the site and a quick and simple test can be used.

Electrochemical detection methods in accordance with the present invention include analytical chemistry techniques that examine an analyte by measuring potential (volts) and/or current (amps), preferably in an electrochemical cell containing the analyte. These methods can be broken down into several categories depending on which aspects of the cell are monitored and which are measured. The three main categories are potentiometry (measuring the difference in electrode potential), coulometry (measuring cell current over time), and amperometry, including voltammetry (measuring cell current while the cell potential is actively changing).

Potentiometry passively measures the potential of a solution between two electrodes, influencing the solution to a very small extent in the process. One electrode is called the reference electrode and has a constant potential, while the other is the determination electrode or working electrode, the potential of which varies depending on the composition of the sample. Therefore, the potential difference between the two electrodes provides an estimate of the composition of the sample. In fact, since potentiometric measurement is a non-destructive measurement, provided that the electrode is in equilibrium with the solution, the potential of the solution is measured. Potentiometry typically uses detection electrodes made selectively sensitive to the ion of interest, such as fluoride in fluoride-selective electrodes, such that the potential depends solely on the activity of that ion of interest. The time it takes for the electrode to equilibrate with the solution will affect the sensitivity or accuracy of the measurement. Platinum is often used in aqueous environments due to its high electron transfer kinetics, although an electrode made of multiple metals can be used to improve electron transfer kinetics. The most common potentiometric electrode today is the glass membrane electrode used in a pH meter. One variant of potentiometry is chronopotentiometry, which involves using direct current and measuring potential as a function of time.

A potentiometric sensor is a type of chemical sensor that can be used to determine the analytical concentration of an analyte contained in a sample. These sensors measure the electrical potential of an electrode in the absence of current. The signal is measured as the potential difference (voltage) between the detection/working electrode and the reference electrode. The potential of the working electrode should depend on the concentration of the analyte in the sample. A reference electrode is required to provide a certain reference potential.

Among various potentiometric techniques, field-effect transistor (FET) sensing has received considerable attention due to its potential for miniaturization, parallel sensing, fast response time, and seamless integration in electronic device fabrication processes such as complementary metal oxide semiconductors (CMOS). complementary metal-oxide semiconductor)). A field-effect transistor (FET) is a type of transistor that uses an electric field to control the flow of current.

The concept of the ion-sensitive field-effect transistor (ISFET) was introduced in the early 1970s and is based on the metal oxide semiconductor field effect transistor (MOSFET). effect transistor)). An ion-sensing field-effect transistor (ISFET) is a field-effect transistor used to measure the concentration of ions in a solution; when the concentration of ions changes (for example, H+, see pH scale), the current through the transistor will change accordingly. In this case, the solution is used as a gate electrode. The voltage between the substrate and the oxide surfaces arises due to the ionic shell. It is a special type of MOSFET (metal oxide semiconductor field-effect transistor) having the same basic structure, but with the metal gate replaced by an ion-sensitive membrane, an electrolyte solution, and a reference electrode. ISFET was the first biosensor FET (BioFET (from English - biosensor field-effect transistor - biosensor field-effect transistor)).

A field-effect transistor biosensor is a special potentiometric sensor, also known as a field-effect transistor biosensor (Bio-FET or BioFET), field-effect biosensor (FEB) or MOSFET biosensor, is a field-effect transistor (on

- 17 046276 new MOSFET structure), which is controlled by changes in surface potential induced by the binding of molecules. When charged molecules, such as biomolecules, bind to the gate of a FET, which is typically a dielectric material, they can change the charge distribution of the underlying semiconductor material, resulting in a change in the conductivity of the FET channel. Bio-FET consists of two main compartments: one is the biological recognition element and the other is the field effect transistor. The BioFET structure is largely based on an ion-sensitive field-effect transistor (ISFET), a type of metal oxide semiconductor field-effect transistor (MOSFET) in which the metal gate is replaced by an ion-sensitive membrane, an electrolyte solution, and a reference electrode.

Bio-FETs couple a transistor device to a biosensing layer that can specifically detect biologically relevant molecules such as enzyme substrates, nucleic acids, and proteins. The Bio-FET system consists of a semiconductor field-effect transistor that acts as a transducer, separated by an insulator layer (e.g., SiO ₂ ) from a biological recognition element (e.g., enzyme, receptors, or molecular probes) that is selective for a target molecule called an analyte . Once the analyte binds to the recognition element, the charge distribution on the surface changes with a corresponding change in the electrostatic surface potential of the semiconductor. This change in the surface potential of the semiconductor acts in the same way as the gate voltage in a traditional MOSFET, i.e. changes the amount of current that can flow between the source and drain electrodes. This change in current (or conductance) can be measured, so analyte binding can be detected. The exact relationship between current and analyte concentration depends on the area of operation of the transistor. The fabrication of a Bio-FET system involves several steps, such as the following: 1. finding a substrate suitable for use as a site for the FET and forming the FET on the substrate; 2. exposure of the FET active site from the substrate; 3. providing a layer of sensitive film on the active region of the FET; 4. providing a receptor on the sensitive film layer for use in ion detection; 5. removal of the semiconductor layer and thinning of the dielectric layer; 6. etching the remaining part of the dielectric layer to expose the active region of the FET; 7. removing the photoresist and applying a layer of sensitive film with subsequent formation of a photoresist pattern on the sensitive film; 8. etching the unprotected part of the sensitive film layer and removing the photoresist. BioFET sensors and the principles underlying them are known to the skilled person, for example from the publication Kaisti M, 2017 (Biosensors and Bioelectronics Volume 98, 15 December 2017, Pages 437-448).

Electrochemical sensor systems (biosensors), and Bio-FETs in particular, can be used for detection in areas such as medical diagnostics, biological research, environmental protection and food analysis. Conventional measurements such as optical and spectrometric measurements can also be used to analyze biological molecules. However, these traditional methods are relatively labor-intensive and expensive, involve multi-step processes, and are not compatible with real-time monitoring. In contrast, biosensors such as Bio-FETs are low mass, low mass production cost, small in size, and compatible with commercial planar processes for large-scale circuits. They can be easily integrated into digital lab-on-a-chip microfluidic devices, such as a microfluidic device that controls the transport of sample droplets, allowing biomolecule detection, signal processing, and data transfer using a versatile chip. They can also be used in portable POC devices. The methods of the present invention do not require any labeling step and simply use certain molecular properties of the sensor, preferably the sensor surface, to provide selectivity.

Coulometry is another electrochemical detection technique that can be used in the context of a system in accordance with the present invention. It uses an applied current or potential to completely convert the analyte from one oxidation state to another. It directly or indirectly measures the total current passed to determine the number of electrons passed. Information about the number of electrons passed can indicate the concentration of the analyte or, if the concentration is known, the number of electrons transferred in the redox reaction. Common forms of coulometry include bulk electrolysis, also known as potentiostatic coulometry or controlled potential coulometry, as well as various coulometric titrations.

Amperometric sensors are additional preferred electrochemical systems in accordance with the present invention. Amperometry is a term used to describe all electrochemical techniques in which current is measured as a function of an independent variable, which is usually time or electrode potential. Chronoamperometry is a technique in which current is measured at a fixed potential at different times from the onset of polarization. Chronoamperometry is usually carried out in solution without stirring and on a fixed electrode, i.e. under experimental conditions, avoiding convection as mass transfer to

- 18 046276 electrode. On the other hand, voltammetry is a subclass of amperometry in which current is measured by changing the potential applied to the electrode. Depending on the waveform, which describes the way the potential changes as a function of time, different voltammetry techniques are defined.

In single-potential amperometry, any analyte that can be oxidized or reduced is a candidate for amperometric detection. The simplest form of amperometric detection is single potential or direct current (DC) amperometry. A voltage (potential) is applied between two electrodes located in the effluent of the column. The measured current changes as the electroactive analyte is oxidized at the anode or reduced at the cathode. Single-potential amperometry has been used to detect weak acid anions such as cyanide and sulfide, which are problematic for conductometric methods. Another, perhaps more important, advantage of amperometry over other detection methods is specificity. The applied potential can be adjusted to maximize the response for the analyte of interest while minimizing the response for interfering analytes.

An extension of single-potential amperometry is pulsed amperometry, most commonly used for analytes that tend to foul electrodes. Analytes that contaminate the electrodes reduce the signal with each run and require electrode cleaning. In pulsed amperometric detection (PAD), the operating potential is applied for a short time (usually a few hundred milliseconds), followed by higher or lower potentials used to clear the electrode. The current is measured only when the operating potential is applied, then successive current measurements are processed by the detector to produce a smooth output signal. PAD is most often used for the detection of carbohydrates after anion exchange separation, but further development of appropriate techniques shows promising results for amines, reduced sulfur compounds and other electroactive compounds.

According to further embodiments of the present invention, the electrochemical sensor system is a voltammetric system. In voltammetry, a constant and/or alternating potential is applied to the surface of an electrode and the resulting current is measured using a three-electrode system. This method can reveal the reduction potential of an analyte and its electrochemical reactivity. This method is, from a practical point of view, non-destructive since only a very small amount of analyte is consumed on the two-dimensional surface of the working/determination electrode and the auxiliary electrode. In practice, analyte solutions are usually discarded because it is difficult to separate the analyte from the base electrolyte and a small amount of analyte is required for the experiment. A typical experiment may involve 1-10 ml of solution with analyte concentrations ranging from 1 to 10 mmol/L. Chemically modified electrodes are used to analyze organic and inorganic samples. Polarography is a subclass of voltammetry that uses an incident mercury electrode as the working electrode.

The electrochemical sensor system of the present invention may require calibration to provide D-lactate concentration as a measurement result. For example, the system may be calibrated either on the run, in which each test run is calibrated against the response generated by a standard sample held in a sample capillary chamber, or, for example, at the factory before shipment. The standard samples used for calibration contain a specific and known concentration of the analyte, in this case D-lactate. Additionally, in embodiments, the electrochemical sensing system may be a calibration-free system. Such systems are known in the art, for example, systems using a dual-frequency approach to achieve calibration-free operation of electrochemical biosensors that generate an output signal using square wave voltammetry to monitor binding-induced changes in electron transfer kinetics.

Electrode settings required for various electrochemical sensor systems that can be used in the context of the present invention are described in the prior art and are known to those skilled in the art. They also include preferred electrode materials and surface modification, as well as a possible method of immobilizing molecules on the surface of electrodes (see, for example, Comprehensive Nanoscience and Nanotechnology (Second Edition), Volume 3, 2019, in particular Agnieszka A. Zuber et al., 3.06 - Biosensing, Pages 105-126; Handbook of Electrochemistry, 2007, in particular, Grant A. Edwards et al., 8 - Chemically Modified Electrodes, pages 295-327; Kenneth L. Brown, Electrochemical Preparation and Characterization of Chemically Modified Electrodes , DOI: 10.5772/intechopen.81752). The selection of appropriate electrode material, electrode modifications, and/or capabilities for immobilization or attachment of molecules on the electrode depends on the appropriate application and the expected range of concentrations to be detected.

- 19 046276

In embodiments, the electrochemical sensing system comprises a test strip or chip for electrochemical detection of D-lactate. The test strip may contain or consist of a paper-based sensor for use in a point-of-care device, such as a portable reader. The test strip can be combined with electrochemical detection using small handheld electronic devices. Additionally, the test strip or chip may be or contain a flexible material such as paper, plastic, or textile as a support for a biosensing platform that may be used in the context of a microfluidic device, a lab-on-a-chip (LOC) biosensing device. - lab-on-a-chip)) or POC devices.

Electrochemical sensor systems in accordance with the present invention can be configured to detect D-lactate levels in multiple samples in parallel by providing multiple electrochemical cells, allowing multiplexing.

Graphic materials

The present invention is further disclosed with the help of the following drawings. They are not intended to limit the scope of the present invention and represent preferred embodiments of aspects of the present invention presented to more clearly illustrate the present invention disclosed herein.

Description of graphic materials

Fig. 1. Distribution of leukocytes (A), percentage of granulocytes (B) and D-lactate (C) in synovial fluid (left panels) with corresponding receiver operating characteristic (ROC) curves (right panels). AF (from English - aseptic failure) is an aseptic lesion; PJI is a periprosthetic joint infection; AUC (from English - area under the curve) represents the area under the curve.

Fig. 2. D-lactate concentration in synovial fluid, stratified by pathogen.

Fig. 3. ROC curve of synovial fluid biomarkers for PJI. D-lactate AUC, white blood cell count, and granulocyte percentage were 0.903, 0.910, and 0.861, respectively.

Fig. 4. Distribution of D-lactate (A), white blood cell count (B), and granulocyte percentage (C) in patients with aseptic lesions and PJI. Twelve cases with underlying inflammatory conditions and an elevated white blood cell count or granulocyte percentage above the threshold are represented by dark gray dots.

Fig. 5. Performance of synovial fluid D-lactate and white blood cell count tests in early postoperative PJI (A) and delayed or late PJI (B). The difference for early PJI was significant (p=0.027), whereas for delayed/late PJI it was not (p=0.572).

Fig. 6. Correlation between synovial fluid red blood cells and D-lactate concentration in patients with aseptic lesions and PJI. Note: p - Pearson correlation.

Fig. 7. Electrochemical measurement of D-lactate using potentiometry. Different concentration of D-lactate (monolithium salt) as standard calibrator and corresponding voltage. Mean values are shown, error bars represent standard deviation.

Fig. 8. Electrochemical measurement of D-lactate using amperometry. Various concentrations of D-lactate (sodium D-lactate) as a standard calibrator in phosphate buffer with pH 6.5 and appropriate current. Mean values are shown, error bars represent standard deviation.

Fig. 9. Electrochemical measurement of D-lactate using amperometry. Various concentrations of D-lactate (sodium D-lactate) as standard calibrator in phosphate buffer with pH 8.5 and appropriate current. Mean values are shown, error bars represent standard deviation.

Examples

The present invention is further described by way of examples and comparative examples. They are not intended to limit the scope of the present invention and represent preferred embodiments of aspects of the present invention presented to more clearly illustrate the present invention disclosed herein.

Example 1: Synovial Fluid D-Lactate as a Pathogen-Specific Biomarker for Accurate and Rapid Detection of Periprosthetic Joint Infection.

Materials and methods of example 1.

Study design and population. Consecutive patients aged greater than or equal to 18 years who underwent diagnostic joint aspiration of a hip, knee, and shoulder prosthesis between July 2016 and June 2018 were prospectively enrolled. Aspiration of painful joints was performed as part of a standard diagnostic procedure in the emergency department, outpatient clinic, or before incision of the joint capsule in the operating room. Patients whose aspirated synovial fluid was diluted by fluid instillation were excluded. Received institutional review board approval and entered into a public registry

- 20 046276 clinical trials www.clinicaltrials.gov (NCT02530229). Patients gave written informed consent for inclusion in the study. D-lactate results were not reported to treating physicians and did not influence treatment decisions.

Definitions. PJI was diagnosed according to the European Bone and Joint Infection Society (EBJIS) working criteria, as has been done in several studies (5, 7, 15–20). Accordingly, PJI was diagnosed when one or more of the following criteria were met: (i) presence of sinus tract or macroscopic suppuration; (ii) inflammatory-positive histopathology in the periprosthetic tissue, defined as greater than or equal to 23 granulocytes per 10 high-power fields (i.e., type II or III according to Krenn et al. (21); (iii) increased white blood cell count in synovial fluid, defined as greater than 2 x 10 ³ /μl, or a neutrophil percentage greater than 70% (6); (iv) positive culture of synovial fluid, periprosthetic tissue, or sonicated fluid. Culture of sonicated fluid was considered positive if greater than or equal to 50 colony-forming cells were detected. units (CFU)/ml, with the exception of Staphylococcus aureus, streptococci, and gram-negative rods, for which any increase (i.e., greater than or equal to 1 CFU/ml) was considered positive.(22) It should be noted that the number of leukocytes in synovial fluid is not considered a diagnostic criterion within the first 6 weeks after surgery for inflammatory joint disease and in the case of periprosthetic fracture or dislocation. In these situations, the white blood cell count could increase even in the absence of infection (19).

Collection of samples. Joint aspirations were performed by orthopedic surgeons using standard aseptic technique in the emergency department, outpatient department, and/or intraoperatively during revision. No patient received antimicrobial treatment before joint aspiration.

Traditional microbiological tests. Each synovial fluid sample was inoculated in 0.1 ml aliquots into tryptic soy agar with 5% sheep blood, chocolate agar, thioglycollate broth. In addition, each sample was inoculated into VersaTREK pediatric blood culture bottles, TREK Diagnostic Systems, Cleveland, Ohio, USA, at the first center and using BacTec PedsPlus/F, Beckton Dickinson and Co., Shannon, County Clare, Ireland, at the second center. All culture media were incubated at 35°C for 14 days. Identification and susceptibility testing of isolated microorganisms was performed using an automated bacteriological analyzer WalkAway 96 Plus, Beckman Coulter, Bray, California, USA) in the first center and using the automated VITEK 2 system (bioMerieux, Marcy-l'Etoile, France) in the second center.

Determination of the number and formula of leukocytes in synovial fluid. To determine the number of leukocytes and the percentage of granulocytes, 1 ml of synovial fluid was transferred into a vial containing ethylenediaminetetraacetic acid (EDTA). The clotted samples were treated with 10 μl of hyaluronidase (Sigma-Aldrich Chemie, Taufkirchen, Germany) for 10 minutes at room temperature. The test was performed by flow cytometry using an automated hematology analyzer (XE-2100, Sysmex, Norderstedt, Germany).

Measurement of synovial fluid D-lactate. A sample volume of 0.5-1 ml was placed in a sterile plastic untreated vial to determine the concentration of D-lactate spectrophotometrically using a commercial kit (D-Lactam diagnostic kit, Sivital, Vitebsk, Republic of Belarus). For each patient, a reaction mixture containing 0.025 ml of previously processed samples, 0.08 ml of the substrate mixture and 0.045 ml of the enzyme mixture, and a blank containing only the sample and substrate mixture were analyzed. A calibration curve with solutions of D-lactate (monolithium salt) in water was processed in each batch. The mixture was incubated at 37°C for 30 minutes and the absorbance was determined at 570 nm using Microplate Absorbance Reader, DYNEX Technologies MRX, Chantilly, VA, USA. The absorbance of each sample served as a measure of D-lactate concentration.

Statistical analysis. The level of significance in all hypothesis testing procedures was preliminarily determined at p less than 0.05. Quantitative data were presented as a median (range) or mean value and standard deviation (SD), depending on the situation. Mann-Whitney test and Spearman correlation were used to analyze quantitative variables. The optimal cutoff value was calculated by maximizing sensitivity and specificity. Youden's J statistic was used to determine the optimal D lactate cutoff value on the receiver operating characteristic (ROC) curve. ROC curves were calculated to determine parameters with the highest diagnostic potential; The areas under the ROC curves were assessed. All statistical analyzes were performed using MedCalc 16.4.3. (MedCalc Software bvba, Ostend, Belgium). Prism software (version 7.03; GraphPad, La Jolla, CA, USA) was used for graphics.

Example 1 results.

Patient demographics and infection characteristics. Of the 224 patients included,

- 21 046276 were diagnosed with PJI, and 137 with aseptic damage to the prosthesis were assigned to the control groups. Demographic data and affected joints from the prosthetic joints divided into aseptic group and infection group are shown in Table. 1. Hips were more often infected than knees.

Microbiology of synovial fluid. Of the 87 patients with PJI, 61 (70%) had a pathogenic microorganism growing in the synovial fluid culture (Table 2). Of the 137 patients with aseptic lesions, 9 patients with prostheses (6.6%) had positive synovial fluid cultures, which were considered contamination due to minor growth.

The number and formula of leukocytes in synovial fluid. The absolute leukocyte count in synovial fluid showed a sensitivity of 87.5% and a specificity of 95.7%. The percentage of granulocytes had a sensitivity of 80.4% and a specificity of 99.2% (Table 3).

Synovial fluid D-lactate. The optimal cutoff value for D-lactate was 1.2 mmol/L. A significantly higher mean (plus/minus SD) D-lactate concentration was found in the synovial fluid of patients with PJI compared with patients with aseptic lesions (2.33 plus/minus 0.63 mmol/L vs. 0.77 plus/minus 0.56 mmol/l), p less than 0.001, Fig. 1). The sensitivity of the D-lactate test was 97.7% and the specificity was 83.9% (Table 3).

In aseptic patients, D-lactate concentrations increased above the cutoff value in 20 patients. In 8 false-positive synovial fluid samples from patients with aseptic lesions, contamination with a skin flora pathogen was detected, since the leukocyte count was normal (varied from 127/μl to 1237/μl).

In 2 patients with PJI, D-lactate concentrations were falsely negative. In one patient with PJI, the diagnosis was based on a positive culture of synovial fluid (Staphylococcus haemolyticus) in combination with an increased number of leukocytes in the synovial fluid; in the second patient with PJI, on the presence of a confirmed sinus tract infection.

D-lactate concentration in synovial fluid depending on the pathogen. Highly virulent bacteria (S. aureus and Streptococcus spp.) had significantly higher mean D-lactate concentrations than coagulase-negative staphylococci, typical low-virulent pathogens (p = 0.019 and p = 0.004, respectively, see Fig. 2). There were no significant differences in D-lactate concentrations when comparing D-lactate concentrations between culture-negative infections and infections caused by low-virulence organisms (i.e., coagulase-negative staphylococci) (p = 0.531). One patient with PJI caused by Candida parapsilosis had D-lactate concentrations above the cutoff value (2.7 mmol/L).

Discussion of example 1.

Previous reports demonstrated that synovial fluid D-lactate was highly sensitive and specific for the diagnosis of septic arthritis (11, 12), but this biomarker has not yet been examined in PJI. In our study, synovial fluid D-lactate showed higher sensitivity than synovial fluid leukocyte count and granulocyte percentage, but lower specificity for the diagnosis of PJI. Gratacos et al. reported high diagnostic performance of synovial fluid D-lactate (AUC 0.90), high sensitivity (86%), specificity (96%), and high negative predictive value (97%) using a cutoff value of 0.05 mmol/L ( eleven). Kortekangas et al. showed that the mean D-lactate concentration was significantly higher in culture-positive synovial fluid samples compared with culture-negative synovial fluid samples from patients with extra-articular infection (p=0.006) (12).

In our study, the optimal synovial fluid D-lactate cut-off value for diagnosing PJI was 1.2 mmol/L. Highly virulent bacteria (such as S. aureus and Streptococcus spp.) had statistically higher D-lactate levels compared with low-virulent pathogens (such as coagulase-negative staphylococci), and no differences were observed in the latter group with PJI and in culture-negative infections. D-lactate concentration likely reflects the virulence of a given bacterial species and its microbial load, which explains the observed differences.

It is interesting to note that one patient with PJI caused by Candida parapsilosis had a clearly elevated D-lactate concentration (2.7 mmol/L). This unusual observation may be explained by coinfection with an unidentified additional bacterium. Alternatively, local oxygen limitation can lead to alcoholic fermentation of yeast, which produces glycerol, pyruvate, and D-lactate as the main fermentation products (23). Growth of fungal pathogens in high-glucose environments can lead to increased D-lactate production and overall lower glucose utilization efficiency, as reported for Saccharomyces cerevisiae (24).

In addition, it is important to recognize unusual disorders that cause D-lactic acidosis and increase D-lactate levels in the blood and body fluids, namely short bowel syndrome and, in particular, high-carbohydrate diets in children. Associated severe uncontrollable

- 22 046276 diabetes mellitus with insulin deficiency can also cause increased levels of D-lactate in plasma and urine (25). Future studies should examine the underlying conditions potentially affecting D-lactate concentrations, which may clarify the limited specificity of the test.

Synovial fluid D-lactate showed good diagnostic performance for the diagnosis of PJI, which was comparable to the performance of synovial fluid leukocyte count or formula. The advantages of the D-lactate test are the low volume of synovial fluid required (50 µl), short turnaround time (45 minutes) and low cost. In particular, the high sensitivity and rapid availability of results make the test particularly useful as a screening tool for PJI. To improve specificity, a confirmatory diagnostic test of synovial fluid can be included in the PJI diagnostic algorithm.

Example 2: Efficacy of synovial fluid D-lactate for the diagnosis of periprosthetic joint infection: a prospective observational study.

Material, patients and methods of example 2.

Study design and population. A prospective diagnostic cohort study included consecutive patients 18 years of age or older who were evaluated for painful prosthetic joints of the hip, knee, and shoulder and who underwent diagnostic joint aspiration before revision arthroplasty to evaluate for infection between May 2016 and March 2017. Only one (first collected) synovial fluid sample per patient was collected.

Patients with dilute synovial fluid after joint instillation, insufficient synovial fluid volume (less than 3 mL), or whose synovial fluid analysis was performed more than 48 hours after aspiration were excluded. A standardized individual case record was used to collect patient history, demographic, clinical, radiological, microbiological, histopathological and laboratory data. Each patient was examined by a multidisciplinary team consisting of orthopedic surgeons, infectious disease specialists and internists. The results of the synovial fluid D-lactate test were not shared with the treating orthopedic surgeons. The study was performed in accordance with the Declaration of Helsinki.

Diagnosis of periprosthetic joint infection. PJI was defined according to the working criteria of the European Bone and Joint Infection Society (EBJIS) (44), summarized in Table. 4. Acute infection was diagnosed if the infection occurred within 4 weeks after surgery or if the patient reported new symptoms lasting no more than 4 weeks. Infections that occurred more than 4 weeks after the last surgical procedure and were symptomatic for more than 4 weeks were defined as chronic infections. In addition, based on the interval between the last revision surgery or primary implantation and the time of aspiration, all infections were classified into early (ie, less than 3 months) and delayed or late (ie, more than 3 months) infections (45).

Obtaining and studying synovial fluid, periprosthetic tissue and implants. Synovial fluid was aspirated under sterile conditions before surgery in the outpatient department or during revision surgery before opening the joint capsule. One ml of synovial fluid was inoculated into a pediatric blood culture bottle (BacTec PedsPlus/F, Beckton Dickinson and Co), one ml was inoculated into an untreated aerobic and anaerobic culture bottle (0.1 ml each), and the remaining fluid was inoculated into thioglycollate broth for accumulation. The pediatric blood culture bottle was incubated at 36 plus/minus 1°C for 14 days or until growth was detected. Aerobic cultures were incubated at 37°C and checked daily for 7 days, and anaerobic cultures were incubated for 14 days. The morphology of microbial colonies was identified using standard microbiological methods using the automated VITEK 2 system (bioMerieux, Marcy-l'Etoile, France). To detect urate and pyrophosphate crystals, a 1-mL aliquot was sent to a pathologist to examine the synovial fluid using polarizing microscopy.

In addition, 3-5 samples of periprosthetic tissue were collected at the time of surgery at the implant-bone or cement-bone interface for microbiological and histopathological analysis if revision surgery was performed. Periprosthetic tissue cultures were considered positive if a highly virulent organism grew in more than or equal to 1 sample of synovial fluid, periprosthetic tissue, or sonicated tissue (Staphylococcus aureus, Enterobacteriaceae, Streptococcus spp., Candida spp.), or if a moderate or low virulent organism grew in more or equal 2 samples (coagulase-negative staphylococci, enterococci, Cutibacterium species [formerly known as Propionibacterium] and other skin microbiome bacteria).

The extracted prosthetic components were sent for sonication as described previously (46). Sonication was considered positive if more than or equal to 1 CFU/ml of a highly virulent organism or more than 50 CFU/ml of a low-virulence organism grew in the sonicated fluid ( 47 ).

Determination of the number of leukocytes in synovial fluid and the percentage of granulocytes. One ml of synovial fluid was transferred into a vial containing ethylenediaminetetraacetic acid

- 23 046276 (EDTC). White blood cell counts were determined by flow cytometry using an automated hematology analyzer (XE-2100, Sysmex, Norderstedt, Germany). The clotted samples were treated with 10 μl of hyaluronidase (Sigma-Aldrich Chemie, Taufkirchen, Germany) for 10 minutes at room temperature.

Determination of D-lactate in synovial fluid. D-lactate was determined spectrophotometrically from the optical density of the prepared sample. One 1-mL aliquot was transferred to an untreated vial for D-lactate determination using a commercial kit (D-lactam Kit; VLDiagnostics, Leipzig, Germany). Aliquots for D-lactate determination were stored at 4°C plus/minus 1°C and analyzed within 48 hours of aspiration. Tests were performed according to the manufacturer's instructions. The determination is based on a spectrophotometric method with a standard microplate reader absorbing at 570 nm, requiring 50 μl of synovial fluid. In the assay, D-lactate dehydrogenase (D-LDH) catalyzes the oxidation of D-lactic acid to pyruvate, along with the concomitant reduction of nicotinamide adenine dinucleotide (NAD') to NADH. NADH reacts with the fluorescent substrate to produce a colored mixture (48).

The D-lactam assay kit contains a lithium D-lactate standard to generate a calibration curve, which was run on a batch-by-batch basis. The reaction mixture contained 0.025 ml of synovial fluid sample, 0.08 ml of substrate mixture and 0.045 ml of enzyme mixture. The turbidity control mixture contained 0.025 ml of synovial fluid sample, 0.08 ml of substrate mixture and 0.045 ml of purified water. Reagents were added to a 96-well flat-bottom plate, incubated at 37°C for 30 minutes, and then read at 570 nm using a Microplate Absorbance Reader (DYNEX Technologies MRX, Chantilly, VA, USA).

Statistical analysis. Youden's J statistic was used to determine the cutoff value of D lactate in the ROC curve. The area under the ROC curve (AUC) was used to evaluate the diagnostic performance of the D-lactate test, white blood cell count, and percentage of granulocytes. A two-tailed independent sample Student's t test was used to assess the statistical significance of mean D-lactate concentrations between groups. The sample size calculation was based on the assumption that the sensitivity of D-lactate is 90%, compared with 80% for radiation diagnostic tests including white blood cell counts, periprosthetic tissue histopathology and culture, i.e. difference of 10% (80% power). The DeLong test for two correlated ROC curves was used to determine whether the difference between AUCs was statistically significant. For all statistical tests performed, a significance level of α 0.05 was chosen. The 95% confidence interval for AUC was estimated using DeLong's method, and the 95% CI for other performance measures was estimated using bootstrap resampling with 10,000 replicates (Table 6). Test for two independent medians The /2 test and Fisher's exact test were used to evaluate p-values in Table 5. Bootstrap resampling with 10,000 replicates was performed to estimate p-values between sensitivities and Figure 3. The correlation between red blood cell and D-lactate concentrations was assessed using the coefficient Pearson (ρ) IBM SPSS 22.0 (Statistical package for the Social Sciences Corporation, Chicago, IL, USA) was used for all statistical analyses.ROC and other plots were generated using the R computing environment (49).

Example 2 results.

Patient demographics. In table 5 summarizes the characteristics of 148 patients, including 103 (70%) with a prosthetic knee, 43 (29%) with a prosthetic hip, and 2 (1%) with a prosthetic shoulder. Forty-four patients (30%) were diagnosed with PJI, and 104 (70%) had aseptic lesions of the prosthetic joint. The majority of patients (n=102, 69%) underwent revision surgery, 62 with aseptic lesions and 40 with PJI.

Efficiency of traditional tests and microbiological examination. The results of diagnostic tests are shown in table. 6. White blood cell count in synovial fluid showed a sensitivity of 80%. However, in 12 patients, the absolute or relative leukocyte count was elevated due to aseptic conditions, including rheumatologic joint disease (n = 3), recurrent dislocation (n = 2), early postoperative condition (n = 2), trauma (n equal to 2)), crystal arthropathy (n equal to 1), periprosthetic fracture (n equal to 1) and metallosis with crystals (n equal to 1). There were 21 cases (48%) of culture-negative PJI. Significant microbial growth was observed in 23 PJI patients (52%), whereas formal contamination (i.e., minor growth) was found in 8 PJI cases and 19 aseptic lesions. In table 7 shows PJI pathogens. A total of 23 culture-positive PJIs were caused by low-virulence pathogens in 10 episodes (43%) and high-virulence pathogens in 13 episodes (57%).

Efficacy of synovial fluid D-lactate. The optimal cutoff value for D-lactate was calculated to be 1.263 mmol/L. The sensitivity and specificity of the D-lactate test were 86.4% and 81.7%, respectively (Table 6). In 19 cases of aseptic lesions, the D-lactate concentration was above the cut-off value, including 12 cases of aseptic lesions with white blood cell count and formula below the threshold value and 7 cases with uninterpretable cell count due to the underlying

- 24 046276 inflammatory condition. In 2 cases of false-positive D-lactate samples, contamination with a skin flora pathogen was recorded. D-lactate showed a negative result in 6 patients diagnosed with PJI according to the applied definition criteria. However, in 2 cases, the diagnosis of PJI was based on only one available criterion (increased number of leukocytes in the synovial fluid or positive histopathology); in the remaining 4 cases, the diagnosis of PJI was based on multiple fulfilled criteria, including one case with a sinus tract. The mean D-lactate concentration was significantly lower in aseptic lesions than in PJI cases (p less than 0.001). The commercial D-lactate test kit required 50 μl of synovial fluid. Processing time in both tests was 30-45 minutes.

Comparison of synovial fluid D-lactate and white blood cell count. No significant differences were observed in any pairwise comparisons of AUC between the synovial fluid biomarkers examined (D-lactate AUC vs _WBC AUC p=0.8; FIG. 3). The distribution of D-lactate and leukocyte counts in PJI and aseptic lesions is shown in Fig. 4. In 12 cases of aseptic lesions with nondiagnostic elevated white blood cell counts due to underlying inflammatory conditions, 7 cases were D-lactate positive and 5 cases were D-lactate negative. Of these 12 patients, 11 underwent revision surgery, and 6 of 12 ultimately underwent a complete diagnostic evaluation confirming aseptic pathology.

In acute PJI, the sensitivity of D-lactate and white blood cell count was 100%, while in chronic PJI the sensitivity decreased to 81% and 72%, respectively (p=0.268). The effectiveness of D-lactate and leukocyte counts in early and delayed/late infections are shown in FIG. 5. While D-lactate showed higher sensitivity compared with white blood cell count, white blood cell count was more specific for both groups. In patients presenting early after surgery, tests showed similar sensitivity (67% versus 58%; p = 0.572), whereas in delayed/late situations, D-lactate was more sensitive (94% versus 84% ; p equals 0.027).

Synovial fluid D-lactate concentration and microbiological examination. Culture-negative PJI had a significantly lower mean D-lactate concentration than culture-positive PJI (0.915 mmol/L vs. 2.421 mmol/L; p = 0.004). The mean D lactate concentration in culture-negative PJI was significantly higher than in cases with aseptic contamination (0.915 mmol/L versus 1.40 mmol/L; p less than 0.001). No significant difference in D-lactate concentration was observed when comparing PJI caused by low-virulent and high-virulent microorganisms (2.047 mmol/L vs. 2.586 mmol/L; p = 0.074) or early and delayed or late infections (1.459 mmol/L vs. from 1.217 mmol/l); p equals 0.196).

Correlation between red blood cells in synovial fluid and D-lactate concentration. A positive correlation was observed between red blood cells and D-lactate overall (p equal to 0.185, p equal to 0.02), as well as in the subgroup with aseptic lesions (p equal to 0.339, p less than 0.01). In the subgroup with PJI, a negative correlation was found, but it did not reach significance (p equals -0.199, p equals 0.195) (Fig. 6). The difference between the aseptic and PJI subgroups was significant (p less than 0.01).

Discussion of example 2.

In recent years, several biomarkers have been investigated as a diagnostic test for PJI (34, 35, 50). However, none of them have been evaluated solely for their ability to detect low-grade infections and early postoperative infections, which are difficult to distinguish from aseptic conditions. The performance of diagnostic tests is highly dependent on the criteria used to define infection. Most studies used the Musculoskeletal Infection Society (MSIS) criteria (51), which missed some cases of low-grade infection due to a high threshold for confirming infection (44). In this study, we used lower threshold criteria for diagnosing PJI, also detecting low-grade PJI (44, 52). In contrast to the MSIS criteria, C-reactive protein CRP and erythrocyte sedimentation rate ESR were not considered diagnostic criteria for PJI because they have limited utility in low-grade infections and are not specific for PJI (33). In addition, leukocyte esterase was not included because it gives reliable results only in samples not contaminated with blood (50).

Delayed infections are known to cause only mild clinical signs and symptoms, most likely due to a low microbial load. Although bacterial metabolism decreases as the biofilm matures, detectable amounts of D-lactate are still produced. A statistically significant difference in D-lactate concentration was observed between culture-negative PJI and aseptic lesions, supporting a septic etiology in culture-negative specimens. In addition, D-lactate concentration appears to depend on the number of bacteria, as D-lactate concentration was higher in culture-positive PJI than culture-negative PJI.

In a study by the present inventors, a false negative test for synovial fluid D-lactate was observed in 6 patients with chronic PJI, two of whom had a positive result.

- 25 046276 result (1 polymicrobial infection with the sinus tract and coagulase-negative staphylococci in the synovial fluid). In four of them the number of leukocytes in the synovial fluid was also normal, and in 3 of them the infection was confirmed only by a positive histopathological examination of the periprosthetic tissue. It remains unclear whether these cases are truly PJI or whether they represent overdiagnosed cases of PJI. In one case, a sinus tract was present, which, as previously described, altered diagnostic markers in the synovial fluid due to persistent drainage of inflammation. While D-lactate production has been described for several bacterial species, including Staphylococcus spp., Streptococcus spp., Escherichia coli, Klebsiella pneumoniae, and Bacteroides fragilis, as well as Lactobacillales and gut microbiota (40, 42, 53), production data D-lactate is limited to other bacteria in body fluids. The effect of bacterial virulence on D-lactate concentration cannot be assessed from the data of the present invention and the literature (41).

D-lactate concentrations were above the cutoff value in 19 patients with aseptic lesions. Based on the positive correlation between red blood cells and D-lactate in the aseptic group, we hypothesized that hemoglobin may cause a false-positive D-lactate test result due to similar absorption wavelengths, i.e. 540 nm for hemoglobin and 570 nm for D-lactate (54). In patients with PJI, the slightly negative correlation may be explained by a significant source of D-lactate from bacterial metabolism, with no other factors influencing the concentration. The present inventors have not assessed whether centrifugation of a synovial fluid sample could potentially improve the specificity of the D-lactate test.

In conclusion, synovial fluid D-lactate is an accurate diagnostic test for diagnosing PJI, comparable to the synovial fluid leukocyte count. It requires only 50 μl of synovial fluid, has a short processing time and is inexpensive. Modifications to the test could potentially improve its specificity or could be combined with a confirmatory test with higher specificity.

Example 3 Measurement of D-lactate using a potentiometric electrochemical sensor.

Material and methods of example 3.

Manufacturing of a biosensor. A potentiometric electrochemical sensor system was used. The system was designed with three electrodes: a working/sensing electrode (a gold electrode purchased from Genefluidic (CA, USA) with a 0 2.5 mm sensor), a platinum wire as a counter electrode, and an Ag/AgCl electrode (BASi) as a reference electrode.

The preparation of the sensitive layers of the working electrodes was performed as previously disclosed (A polyaniline based ultrasensitive potentiometric immunosensor for cardiac troponin complex detection. Qi Zhang a, n, Alok Prabhu a, Avdar San a, Jafar F. Al-Sharab b, Kalle Levon, Biosensors and Bioelectronics 72 (2015) 100106). Working electrodes were coated with 20 μl of 1.5 wt.% PANI (polyaniline)/DNNSA (dinonylnaphthalene sulfonic acid) dissolved in chloroform and dried in an oven at 60°C for 2 hours. PANI/DNNSA coated electrodes were immersed in CP buffer with 2.5 wt.% glutaraldehyde (GA) cross-linking reagent (Sigma-Aldrich, MO, USA) at room temperature for 1.5 hours, followed by thorough rinsing with deionized water.

Enzyme immobilization. A commercially available kit for the determination of Dlactic acid was used. Reagent 1 (16 ml) contains D-lactate dehydrogenase greater than or equal to 60 kU/L plus buffer, pH 9.0, and reagent 2 (4.5 ml) contains NAD' greater than or equal to 20 mmol/L. 50 μl of the reagent mixture was placed on the working electrode, pre-treated as previously disclosed, overnight at 4°C to immobilize the enzyme.

Preparation of D-lactate calibration samples. Lyophilized lithium D-lactate (SigmaAldrich, MO, USA) was diluted with deionized water to various concentrations (0, 25, 50, 75 and 100%).

Electrochemical measurements. Place 100 μl of samples containing different concentrations of D-lactate on the surface of the working electrode and measure the voltage corresponding to a certain concentration of D-lactate in the calibrated sample at room temperature. Open circuit potentiometry (OCP) was performed using a CHI 660d electrochemical workstation (CH Instruments).

Example 3 results.

The measured concentrations and corresponding voltages from two independent experiments are shown in FIG. 7 and in table. 8. When using D-lactate at concentrations below 1.2 mM, voltages were below 85 mV (interpreted as a negative result), whereas concentrations above this cutoff value, as determined by spectrophotometric measurements, consistently showed voltage measurements above 85 mV.

Discussion of example 3.

The effect of the dose response of the method based on potentiometric electrochemical

- 26 046276 sensor demonstrates a proof of concept for a spectrophotometry-independent measurement that is independent of other components of biological samples (e.g., synovial fluid), such as red blood cells, which can give false positive results to spectrophotometric methods due to the similarity of the absorption wavelength to the hemoglobin. Therefore, the specificity of the potentiometric electrochemical sensor method according to the present invention will be higher than that of other currently available methods. This feature of the new test is important because false-positive results can lead to excessive antimicrobial and surgical treatment with negative consequences for the patient.

Example 4 Measurement of D-lactate using an amperometric electrochemical sensor.

Material and methods of example 4.

The inventors of the present invention performed a study using an amperometric electrochemical sensor, which contains a test strip (chip) with a working electrode, a counter electrode and reference electrodes on the surface for electrochemical detection and an electrochemical potentiostat for measuring the electrical signal. The test strip can be combined with small portable electronic devices for electrochemical detection.

Preparation of D-lactate calibration samples. Commercially available lyophilized sodium D-lactate (Sigma-Aldrich, MO, USA) was reconstituted from the lyophilisate by adding the required amount of deionized water to achieve a final concentration of 0.01, 0.03, 0.1, 0.3, 1.0, 3 ,0, 10.0 and 30.0 mM.

Preparation of the enzyme mixture. Commercially available lyophilized D-lactate dehydrogenase from Staphylococcus epidermidis (Sigma-Aldrich, MO, USA) was diluted with phosphate buffer to achieve a final concentration of 100 U/ml. Commercially available lyophilized NAD-free acid (Sigma-Aldrich, MO, USA) was diluted to a final concentration of 20 mmol/L. The reagents were dissolved in phosphate buffer with two different pH concentrations (pH 6.5 and pH 8.5).

Electrochemical measurements. Two experiments were performed using phosphate buffer with different pH (pH 6.5 and pH 8.5). 100 μL of a mixture containing phosphate buffer (pH 6.5 or pH 8.5), 10 units of D-LDH, 20 mmol/L NAD, and various concentrations of D-lactate was placed on the surface of the chip. Measurement of the current corresponding to a specific concentration of D-lactate in a calibrated sample was carried out at room temperature using chronoamperometry with a standard potentiostat (CompactStat.h-Standard, Ivium Technologies, Eindhoven, the Netherlands).

Example 4 results.

The measured concentrations of D-lactate in phosphate buffer pH 6.5 and the corresponding current are shown in FIG. 8 and in table. 9. The measured concentrations of D-lactate in phosphate buffer pH 8.5 and the corresponding current are shown in Figure 9 and Table. 10. When using D-lactate at a concentration below 1.2 mM, the current was below 422 nA (interpreted as a negative result), while concentrations above this cut-off value, which was determined by spectrophotometric measurements, each time showed current measurements above 422 nA.

Discussion of example 4.

The amperometric electrochemical sensor showed a dose-response effect when measuring different concentrations of D-lactate regardless of the pH of the buffer used, demonstrating proof of concept for spectrophotometry-independent measurement of D-lactate. Moreover, by using a phosphate buffer with pH 8.5, the present inventors were able to determine the D-lactate concentration with a higher current, which provides better sensitivity of the biosensor for determining the D-lactate concentration in unknown samples.

Example 5 Measurement of D-lactate in synovial fluid samples using an electrochemical sensor.

Material and methods of example 5.

The inventors of the present invention performed a study using an amperometric electrochemical sensor (biosensor) including a test strip (chip) with a working electrode, a counter electrode and reference electrodes on the surface for electrochemical detection and an electrochemical potentiostat for measuring the electrical signal.

Synovial fluid samples. In this study, the present inventors used the synovial fluid samples from Example 2. In the cohort of the present invention, 10 patients had prosthetic joint infection (PJI). Thirty patients were diagnosed with aseptic lesion (AF) of the prosthetic joint, 20 of whom were tested false-positive in a previous study using spectrophotometric methods.

Preparation of the enzyme mixture. Commercially available lyophilized D-lactate dehydrogenase from Staphylococcus epidermidis (Sigma-Aldrich, MO, USA) was diluted with phosphate buffer (pH 8.5) to achieve a final concentration of 100 U/ml. Commercially available

- 27 046276 NAD-free acid (Sigma-Aldrich, Missouri, USA) was diluted with phosphate buffer (pH 8.5) to a final concentration of 20 mmol/L.

Electrochemical measurements. 90 μL of a mixture containing phosphate buffer (pH 8.5), 10 units of D-LDH, 20 mmol/L NAD, and 10 μL of synovial fluid samples was placed on the surface of the chip. Current measurements were performed at room temperature using chronoamperometry with a standard potentiostat (CompactStat.h-Standard, Ivium Technologies, Eindhoven, The Netherlands).

Example 5 results.

Using electrochemical measurement of D-lactate, as disclosed herein, it was possible to identify all patients with AF and distinguish them from patients with PJI. Lower current measurements were observed for all AF patients than for PJI patients. Therefore, a suitable cutoff value (current) can be applied, allowing identification of PJI with very high specificity and sensitivity.

Discussion of example 5.

The amperometric electrochemical sensor demonstrates excellent sensitivity and specificity in the diagnosis of PJI, providing proof of concept for a spectrophotometry-independent measurement. This method does not rely on components of biological samples (eg, synovial fluid), such as red blood cells, which can give false-positive results in spectrophotometric methods due to absorption wavelengths similar to those of hemoglobin. Therefore, the specificity of the electrochemical method in accordance with the present invention will be higher than that of other currently available methods. This feature of the new test is important because false-positive results can lead to excessive antimicrobial and surgical treatment with negative consequences for the patient.

In planned embodiments, electrical signal detection will be accomplished using a battery-powered, portable, compact reader like a glucometer (Freestyle Precision Pro, Abbott, North Chicago, IL, USA) that is used to obtain quantitative information about the analyte.

Tables.

Example 1 tables.

Table 1 Demographics and characteristics of 224 patients with periprosthetic joints, stratified by ________aseptic . and infectious pathology________

Characteristics P.J.I. A.F. value Joint prostheses (n = 224), (%) 137 (61) 87 (39) Age, median (range), years 67 (33-94) 64 (30-89) 0.578 Gender, number of men (%) 51 (37) 49 (63) 0.356 Type of affected joint, number (%) Knee Hip 83 (61) 54 (39) 41 (47) 47 (54) 0.054 Time from initial implantation of the prosthesis to aspiration, median (range), months 67 (6-240) 34 (0.2-180) 0.001

PJI - periprosthetic joint infection, AF - aseptic lesion.

table 2

Microbiological study of prosthetic joint infection

Pathogen Prosthetic joint infection (n = 87) ² S. aureus 16 (18) Coagulase-negative staphylococci 27 (31) Streptococcus spp. 6(6) Enterococcus spp. 4(5) Anaerobes 4(5) Gram-negative bacteria 3(3) Others ¹ 2(2) Negative culture 26 (30)

¹ Candida parapsilosis (n=1), Corynebacterium spp. (n=1).

² One patient with PJI had a mixed infection with S. aureus and S. pyogenes.

- 28 046276

Table 3

Analytical performance of synovial fluid tests

Tests Cutoff P.J.I. A.F. AUC Sensitivity, % Specificity, % PPV, % NPV, % (95% CI) D-lactate, mmol/l > 1.2 76/78 20/137 (95% CI) 97.7 (91.9-99.7) 83.9 (76.7-89.7) 79.4 (70.5-86.6) 98.3 (93.9-99.3) Leukocytes, x 10 ³ / µl >2 70/78 5/137 0.96 (0.93-0.98) 87.5 (78.7-93.6) 95.7 (91.0-98.4) 92.8 (84.9-97.3) 92.5 (86.9-96.2) Percentage of granulocytes, % > 70 63/78 2/137 0.96 (0.93-0.98) 80.4 (70.6-88.2) 99.2 (96.1-99.9) 98.6 (92.4-99.8) 89.2 (83.2-93.6) Positive culture - 61/78 9/137 - 78.2 (67.4-86.8) 93.4 (87.9-97.0) 87.1 (78.1-92.8) 88.3 (83.2-92.0)

PJI - periprosthetic joint infection, AF - aseptic lesion, AUC - area under the curve, PPV (positive predictive value) - predictive value of a positive result, NPV (negative predictive value) - predictive value of a negative result, CI - confidence interval.

Example 2 tables.

Table 4 According to the working criteria of the European Bone and Joint Infection Society (EBJIS), periprosthetic joint infection is defined as a criterion greater than or equal to 1.

Test Criteria Clinical signs Sinus tract (fistula) or visible purulent discharge around the prosthesis G histology Acute inflammation in periprosthetic tissue ¹ Number of cells in 2 joint aspirate more than 2000/µg leukocytes or more than 70% granulocytes Microbiological examination Microbial growth in • synovial fluid, or • greater than or equal to 2 tissue samples ³ , or • sonicated fluid (greater than or equal to 50 CFU/ml) ⁴

¹ Acute inflammation is defined as more than or equal to 23 granulocytes per high-power field, corresponding to type II or III according to Krenn and Morawitz (56).

² Leukocyte cutoff values are not considered diagnostic within 6 weeks of surgery, in active rheumatic joint disease, periprosthetic fracture, joint trauma, or dislocation.

³ Periprosthetic tissue culture was considered positive if a highly virulent organism grew in more than or equal to 1 sample (Staphylococcus aureus, Enterobacteriaceae, Streptococcus spp., Candida spp.), or a low virulent organism grew in more than or equal to 2 samples (coagulase-negative staphylococci, enterococci, Cutibacterium [ formerly known as Propionibacterium spp. and other skin microbiome bacteria).

⁴ Sonication was considered positive if more than or equal to 1 CFU/ml of a high-virulence organism or more than 50 CFU/ml of a low-virulence organism grew in the sonicated fluid (47).

- 29 046276

Table 5

Patient characteristics

All patients (n = 148) Patients with PJI (n = 44) Patients with aseptic lesions (n = 104) value Median (range) age of patients (years) 69.5 (29-93) 69.0 (41-89) 69.5 (29-93) 0.857 Gender, number (%) 0.032 men 81 (55) 30 (68) 51 (49) Joint, number (%) 0.006 Knees 103 (70) 24 (55) 79 (76) Hips 43 (29) 18 (41) 25 (24) Shoulder 2(1) 2(4) 0(0) Patients undergoing revision surgery, number (%) 102 (69) 40 (91) 62 (60) <0.001 Date joint aspiration after primary surgical intervention, number (%) 0.765 Early (less than 3 months) 19/138 (14) 7/43 (16) 12/95 (13) Deferred (3-24 months) 55/138 (40) 16/43 (37) 39/95 (41) Late (more than 24 months) 64/138 (46) 20/43 (47) 44/95 (46)

Table 6

Performance of non-microbiological and microbiological tests in accordance with the proposed criteria__________________________________________ EPJIC______________________________

Positive results Aseptic lesion (n= 104) RL* (n = 44) AUC (%) (95% CI) Sensitivity (%) (95% CI) Specificity (%) (95% CI) PPV (%) (95% CI) NPV (%) Ί (95% CI) C accuracy %) 95% CI) Non-microbiological tests Clinical signs ¹ 0 19 - 43.2 (29.5-56.8) 100 100 80.6 (77.0-84.6) 83.1 (79.1-87.2) D-lactate synovial liquids more than 1.263 mmol/l 19 38 90.3 (85.7-95.0) 86.4 (75.0-95.5) 81.7 (74.0-88.5) 66.7 (57.8-76.6) 93.5 (88.7-97.5) 83.1 (77.0-89.1) Leukocyte count in 9 35 91.0 (85.1-96.8) 79.5 (68.2-90.9) 91.3 (85.6-96.2) 80.0 (69.4-90.2) 91.4 (86.8-96.0) 87.8 synovial fluid more than 2000/µl ² (82.4-92.6) Percentage of granulocytes in 8 25 86.1 (79.4-92.9) 56.8 (40.9-70.5) 92.3 (86.5-97.1) 75.9 (62.9-88.9) 83.5 (78.8-88.3) 81.8 synovial fluid more than 70% ² (75.7- 87.2) White blood cell count or granulocyte percentage ³ 9 35 ^Ξ 79.5 (68.2-90.9) 89.4 (83.7-95.2) 76.2 (66.0-87.2) 91.3 (86.5-95.9) 86.5 (81.1-91.9) Histopathology of periprosthetic tissue 0/43 25/34 - 73.5 (58.8-88.2) 100 100 82.7 (75.4-91.5) 88.3 (81.8-94.8) Microbiological tests Culture of synovial fluid Culture of periprosthetic tissue ⁴ Culture of sonicated fluid ⁴ 8 7/63 5/49 20 17/41 17/39 - 45.5 (31.8-61.4) 41.5 (26.8-56.1) 43.6 (28.2-59.0) 100 100 100 100 100 100 81.2 (77.6-86.0) 72.4 (68.8-77.8) 69.0 (63.6-75.4) 83.8 (79.785.5) 76.9 (71.2-82.7) 75.0 (68.2-81.8) Sample of any crop 19 23 - 52.3 (38.6-65.9) 100 100 83.2 (79.4-87.4) 85.8 (81.8-89.9)

- 30 046276

Note. If the denominator is shown, the test was not performed on all patients. *PJI was confirmed when at least one of the following criteria was present: clinical signs (ie, macroscopic pus in the synovial fluid or surrounding the prosthesis, or the presence of a sinus tract, elevated white blood cell count in the synovial fluid (more than 2000 leukocytes/μl or more 70% granulocytes), histopathological evidence of inflammation in the periprosthetic tissue with a largely positive microbiological examination. ¹ Eleven patients had visible pus in the synovial fluid, 1 patient had a sinus tract, and 7 patients had both. ² In 12 of 148 patients, the white blood cell count (n = 9) or granulocyte percentage (n = 8) was elevated but was not diagnostic for PJI due to concomitant crystalline arthropathy (n = 1), recurrent dislocation (n = 2), rheumatologic joint disease (n = 3), early postoperative condition (n = 2), trauma (n = 2), periprosthetic fracture (n = 1), or metallosis with crystals (n = 1). ³ False-positive results were interpreted as positive for efficacy assessment. In 3 cases, the white blood cell count and granulocyte percentage did not exceed the cutoff value, although they were determined to be uninterpretable. ⁴ Growth of a low-virulence organism in only one specimen was not sufficient to diagnose PJI.

Table 7

Isolated organisms from 23 culture-positive PJI patients

Pathogen Quantity (%) Coagulase-negative staphylococci ¹ 11 (48) Staphylococcus aureus 5(22) Streptococcus spp. ² 3 (13) Gram-negative rods ³ 3 (13) Enterococcus spp. 14) Bacteroides fragilis 14)

Table 8

Example table 3.

Dlactate concentration Corresponding ID lactate (mM) Control Example 1 Example 2 Average voltage (mV) Interpretation* 0% 0.0 0 0 0 0 Negative 25% 0.9 0 41 71 56 Negative thirty% 1.2 0 - - 85 Positive (cutoff value) 50% 1.8 0 273 155 214 Positive 75% 2.7 0 217 141 179 Positive 100% 3.7 0 276 159 218 Positive

Average voltage (cB) was calculated from 2 experiments. *Based on cutoff value determined by spectrophotometry.

- 31 046276

Example 4 tables.

Table 9

Amperometric measurement at pH 6.5

Corresponding D-lactate (mM) Control Example 1 Example 2 Average current (nA) Interpretation* ο,ο 0 0 0 0 Negative 0.01 0 7 5 6 Negative 0.03 0 eleven eleven eleven Negative 0.1 0 35 25 thirty Negative 0.3 0 42 65 53.5 Negative 1.2 0 108 114 111 Positive (cutoff value) 3.0 0 156 161 158.5 Positive 10.0 0 288 270 279 Positive 30.0 0 490 510 500 Positive

The average current (nA) was calculated from 2 experiments.

*Based on cutoff value determined by spectrophotometry.

Table 10

Amperometric measurement at pH 8.5

Corresponding D-lactate (mM) Control Example 1 Prime P2 Average current (nA) Interpretation* 0.0 0 0 0 0 Negative 0.01 0 16 20 18 Negative 0.03 0 44 50 47 Negative 0.1 0 90 106 98 Negative 0.3 0 184 223 204 Negative 1.2 0 439 405 422 Positive (cutoff value) 3.0 0 740 843 792 Positive 10.0 0 1386 1803 1595 Positive 30.0 0 2455 2600 2528 Positive

The average current (nA) was calculated from 2 experiments.

*Based on cutoff value determined by spectrophotometry. Links.

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Claims (18)

CLAIM 1. An in vitro method for diagnosing, prognosticating, risk assessment, monitoring, therapeutic management and/or therapeutic control of an infectious disease, including: a) providing a sample from a subject showing clinical symptoms and/or suspected of having an infection, b) determining the level of D-lactate in the specified sample, c) wherein the level of D-lactate indicates the presence of an infectious disease, characterized in that d) the level of D-lactate in the specified sample is determined using an electrochemical sensor system (biosensor). 2. The in vitro method of claim 1, wherein the electrochemical sensor system comprises a potentiometric sensor, preferably a transistor-based potentiometric sensor. 3. In vitro method according to claim 1 or 2, where the electrochemical sensor system contains an ion sensitive field effect transistor (ISFET). 4. In vitro method according to any one of claims 1 to 3, where the electrochemical sensor system contains an amperometric sensor. 5. In vitro method according to any one of claims 1 to 4, wherein the electrochemical sensor system contains a D-lactate binding molecule, preferably D-lactate dehydrogenase (D-LDH). 6. In vitro method according to any one of claims 1 to 5, where the electrochemical sensor system contains a detection electrode (working), preferably including a surface of carbon or gold. 7. In vitro method according to claim 5, wherein the D-lactate binding molecule is immobilized on the detection electrode. 8. In vitro method according to any one of claims 1 to 7, wherein the electrochemical sensor system preferably contains a disposable test strip (chip) for electrochemical determination of the level of D-lactate, wherein the test strip contains a detection electrode with an immobilized D-lactate binding molecule and preferably also a counter and/or reference electrode. 9. An in vitro method according to any one of claims 1 to 8, wherein a disposable test strip is used which is placed in a preferably battery powered portable compact reader to perform the D-lactate measurement. 10. In vitro method according to any one of claims 1 to 9, wherein immobilization of the D-lactate binding molecule, which is D-LDH, on the surface of the biosensor detection electrode is achieved by any of adsorption, covalent binding, entrapment, encapsulation, cross-linking or interaction thiol-gold, preferably cross-linking or thiol-gold interaction. 11. In vitro method according to any one of claims 1 to 10, wherein the system allows parallel determination of D-lactate levels in more than one sample. 12. In vitro method according to any one of claims 1 to 11, where the infectious disease is a microbial bacterial and/or fungal infection, preferably caused by at least one infectious agent selected from the group consisting of Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus spp. , Enterococcus spp., anaerobes, gram-negative bacteria and Candida spp. 13. In vitro method according to any one of claims 1 to 12, wherein the infectious disease is a joint infection, prosthetic joint infection (PJI), meningitis, peritonitis, pleural cavity infection, pericardial space infection and/or bloodstream infection. 14. In vitro method according to any one of claims 1 to 13, wherein an increased level of D-lactate, determined using an electrochemical sensor system in the specified sample compared to an appropriate control, such as a sample from a healthy subject, indicates the presence of an infectious disease. 15. In vitro method according to any one of claims 1 to 14, wherein the measurement of current or voltage using an electrochemical sensor system corresponding to a D-lactate level in the specified sample equal to or greater than 1.2 mmol/L indicates the presence of an infectious disease and /or indicates the need to start or change antibiotic treatment. 16. An in vitro method according to any one of claims 1 to 15, wherein the electrochemical sensor system is calibrated using one or more calibration samples with a certain concentration of D-lactate before determining the level of D-lactate in the specified sample. 17. In vitro method according to any one of claims 1 to 16, wherein the level of D-lactate determined by the electrochemical sensor system is independent of the amount of red blood cells and/or hemoglobin present in said sample. 18. In vitro method according to any one of claims 1 to 17, where the sample is selected from the group consisting of a physiological fluid sample, a homogenized tissue sample, a blood sample, a blood serum sample, a plasma sample, a urine sample, a joint aspirate, a synovial fluid sample, a sample ascites, peritoneal fluid sample, pleural fluid sample, pericardial fluid sample and/or cerebrospinal fluid sample. -