JP7460131B2 - Diagnostic equipment and programs - Google Patents

Description

The present invention relates to a diagnostic device and a program.

Bacteremia is the presence of bacteria in the bloodstream and is a bacterial infection that can occur after placement of urogenital or intravenous catheters or wound treatment. Sepsis can lead to septic shock, a condition in which acute circulatory failure causes severe cellular damage and metabolic abnormalities, potentially increasing mortality.

Traditionally, patients with community-onset sepsis have often been treated with antibacterial chemotherapy using β-lactam antibiotics, assuming that Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae are the causative bacteria. On the other hand, patients with hospital-onset sepsis have sometimes been treated with a combination of β-lactam antibiotics and anti-MRSA drugs, assuming that gram-negative bacilli, including Pseudomonas aeruginosa, and multidrug-resistant gram-positive cocci, such as MRSA (methicillin-resistant Staphylococcus aureus), are the causative bacteria.

In either case, early administration of antibiotics is thought to reduce the risk of death, and antibacterial chemotherapy may be started without waiting for the primary tumor and causative bacteria to be identified by culturing blood samples, etc. There were many.

On the other hand, it is assumed that the causative bacteria differ depending on the primary tumor, and if they are identified early, they can be easily transferred to the infected organs and are proven to be more effective against the causative bacteria. antibiotics can be selected.
Therefore, in the treatment of sepsis, antibacterial chemotherapy should be started early, blood culture should be carried out in parallel, other culture specimens should be collected and cultured from the presumed infected site, and image diagnosis should be carried out as necessary. The primary tumor and the causative bacteria are identified, and de-escalation (broad-spectrum antibiotics, which were started as empiric treatment, is replaced with narrow-spectrum and single-spectrum antibiotics as soon as possible after the antimicrobial susceptibility of the causative bacteria is determined). Efforts have been made to optimize treatments, including switching from antibacterial agents to antibacterial agents (which is thought to be effective against increasing medical costs and the emergence of resistant bacteria).

However, blood culture requires a long period of time (specifically, several days) to obtain results, and in order to perform de-escalation in a shorter time, the causative bacteria can be identified more quickly. Technology is required.

Furthermore, in recent years, it has become clear that conventional blood culture may not be able to accurately diagnose bloodstream infections.
A known example of such a case is central venous catheter-associated bloodstream infection (CVCA-BSI). CVCA-BSI is thought to be caused by bacteria adhering to the inside and/or outside of a CV catheter forming a biofilm.

For those who had an indwelling CV catheter and had a fever of 38°C or higher, scanning electron microscopy of the outside of the catheter revealed a biofilm, even though both blood and catheter culture were negative. Non-patent document 1 describes a case in which adhesion of .

As described above, when a bloodstream infection occurs due to bacteria that have formed a biofilm, it may be difficult to detect and identify the causative bacteria using conventional blood cultures, etc.
There is a demand for new technology that can detect bacteremia caused by bacteria in such a biofilm state with higher sensitivity and identify the causative bacteria.

Yuka Ishihara, Michio Ohta, "Study on Biofilm Formed on Catheters in Central Venous Catheter-Associated Bloodstream Infections," Journal of the Japanese Society for Environmental Infection, February 16, 2009, Vol. 23, No. 4, pp. 258-266

In view of the above circumstances, an object of the present invention is to provide a device that can provide information for identifying the causative bacteria even if bacteremia is caused by bacteria that have formed a biofilm. . Another object of the present invention is to provide a program.

As a result of extensive research into achieving the above object, the inventors have discovered that the above object can be achieved by the following configuration.

[1] Isolated from at least one object selected from the group consisting of body fluids, secretions, and urine of the subject and placed in contact with the specimen containing membrane vesicles produced by bacteria. a measurement unit that measures at least one characteristic potential selected from the group consisting of an onset potential and a peak potential when sweeping the potential of the electrode of a biosensor having at least two electrodes; a storage unit that stores reference data that associates the specific potential with the above-mentioned specific potential; and an analysis unit that compares the reference data with the measurement results and provides information for identifying the bacteria that produced the membrane vesicles. A diagnostic device comprising:
[2] The diagnostic device according to [1], wherein the method of sweeping the potential of the electrode is such that the obtained current response has a peak shape.
[3] The storage unit further stores second reference data that associates the specific potential with a correlation between the content of the membrane vesicles in the specimen and the magnitude of the peak current. The diagnostic device according to [2].
[4] The diagnostic device according to [1], wherein the specimen further contains hydrogen peroxide, and the measurement unit applies a linear sweep voltammetry method or a cyclic voltammetry method to the biosensor.
[5] The method for sweeping the potential of the electrode is at least one method selected from the group consisting of linear sweep voltammetry, cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. 1].
[6] The diagnostic device according to any one of [1] to [5], wherein the object is at least one selected from the group consisting of blood, body cavity fluid, and urine.
[7] The diagnostic device according to any one of [1] to [6], wherein the target object is blood.
[8] The diagnostic device according to any one of [1] to [7], wherein the biosensor includes a base material and the electrode arranged on the base material.
[9] The diagnostic device according to any one of [1] to [8], wherein the bacterium is a causative bacterium of at least one disease selected from the group consisting of sepsis and catheter-related bloodstream infection.
[10] The diagnostic device according to any one of [1] to [9], wherein the bacterium is a causative bacterium of catheter-related bloodstream infection.
[11] A computer is instructed to contact a specimen containing membrane vesicles produced by bacteria and separated from at least one object selected from the group consisting of body fluids, secretions, and urine of the subject. measuring at least one characteristic potential selected from the group consisting of an onset potential and a peak potential when sweeping the potential of the electrode of a biosensor having at least two arranged electrodes; providing information for identifying the bacteria that produced the membrane vesicles by comparing the results of the measurement with reference data that associates the type of membrane vesicles with the specific potential;
[12] The program according to [10], wherein the method of sweeping the potential of the electrode is such that the obtained current response has a peak shape.
[13] From the measurement results of the peak current of the specimen with reference to second reference data that associates the characteristic potential with the correlation of the content of membrane vesicles in the specimen with respect to the magnitude of the peak current, The program according to [12], which executes the step of providing information for determining the content of membrane vesicles in the specimen.

According to the present invention, it is possible to provide an apparatus that can provide information for identifying the causative bacteria even if bacteremia is caused by bacteria that have formed a biofilm. Further, according to the present invention, a program can also be provided.

After culturing Capnocytophaga bacteria, the resulting culture solution was centrifuged to remove the bacteria, and the supernatant was then further ultracentrifuged to obtain a pellet of membrane vesicles, which was then resuspended in an electrolyte and measured by differential pulse voltammetry. FIG. 1 is a hardware configuration diagram of a diagnostic device that is an embodiment of the present invention. FIG. 2 is a perspective view of the diagnostic device. FIG. 2 is an exploded perspective view of the biosensor. FIG. 2 is an exploded perspective view of the biosensor. FIG. 2 is a functional block diagram of the diagnostic device. 4 is an operational flow of a control unit that operates according to a program. This is another operational flow of the control unit that operates according to the program.

The present invention will be described in detail below.
The following description of the components may be based on a representative embodiment of the present invention, but the present invention is not limited to such an embodiment.
In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

(Definition of terms)
As used herein, "subject" refers to pediatric and adult bacteremia patients (children), suspected patients (children), or healthy subjects (children), including, for example, wards and /Or people who seek outpatient diagnosis by a doctor.

In addition, in this specification, "body fluid" means a liquid that fills between tissues, within body cavities, and the circulatory system in a living body, and typically includes blood, lymphatic fluid, tissue fluid, and body cavity fluid (pleural fluid, ascites, cerebrospinal fluid, synovial fluid, etc.), etc., of which those that are usually sterile are preferred, and blood or body cavity fluid is more preferred.
In addition, in this specification, "secretion" means pharyngeal secretions, nasal secretions, sputum, and the like.

In addition, in this specification, "membrane vesicles (MV)" refer to those derived from a natural cell function in which the outer membrane of a bacterium protrudes outward in a budding-like manner, forming a sac-like mass and being released outside the cell, and are different from "liposomes." In this specification, MVs released from the outer membrane of gram-negative bacteria may be specifically referred to as outer membrane vesicles (OMVs). The release of MVs has been confirmed in various bacteria, and is generally considered to be a phenomenon common to all prokaryotes.

The size of MVs is not particularly limited, but is generally in the range of 20 to 400 nm. Most MVs are spherical in shape, but flat or rod-shaped MVs are also known.
It is known that many of the proteins contained in the extracellular polymeric substances (EPS) that make up biofilms are derived from MVs, and it is believed that the bacteria that make up biofilms actively produce MVs.

In this specification, the term "biofilm" refers to a complex that typically has a three-dimensional structure and is composed of bacteria attached to a solid surface and EPS produced by the bacteria.

In addition, in this specification, "bacteria" includes, but is not limited to, Streptococcus pneumoniae, Haemophilus influenzae, Legionella pneumoniae, Chlamydia pneumoniae, Mycoplasma, etc., which are associated with respiratory tract infections such as pneumonia; Staphylococcus aureus, Viridans Streptococcus, Enterococcus, etc., which are associated with intravascular infections such as endocarditis; Escherichia coli, Bacteroides fragilis, etc., which are associated with intraperitoneal infections such as peritonitis; Group A Streptococcus, and MRSA (methicillin-resistant Staphylococcus aureus) which are associated with skin and soft tissue infections such as necrotizing fasciitis. including Staphylococcus aureus; Escherichia coli, Klebsiella, Proteus, Enterobacter, and Enterococcus, which are associated with urinary tract infections such as pyelonephritis; Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, and Listeria, which are associated with central nervous system infections such as meningitis; Staphylococcus including MRSA (methicillin-resistant Staphylococcus aureus) and MRSE (methicillin-resistant Staphylococcus epidermidis), Pseudomonas aeruginosa, Enterobacter, Serratia, and Candida, which are associated with catheter-related bloodstream infections.

Furthermore, in this specification, "separation" refers to an operation for removing membrane vesicles from a target object, and includes, for example, centrifugation, filtering, and the like.
In addition, as used herein, the term "sample" refers to a liquid or solid sample containing membrane vesicles separated from a target object, for example, containing plasma, membrane vesicles, and other components. Examples include samples that Note that the specimen preferably does not contain bacteria.

Further, in this specification, "onset potential" means a potential at which a current starts to flow when the potential (or base potential) is swept. The above-mentioned "potential" means a base potential in the case of a linear voltammetry method (LSV method).
Here, "current begins to flow" means a potential (or base potential) at a position where a curve rises and a current begins to be detected in a potential (or base potential)-current function.

In addition, in this specification, the definition of "sepsis" is as follows: "JAID/JCC Infectious Disease Treatment Guidelines 2017 - Sepsis and Catheter-Related Bloodstream Infections" (Journal of the Japanese Society of Chemotherapy Vol. 66, Issue 1, 2018 (January ) p.82-117). Specifically, it refers to "a state in which life-threatening organ damage occurs due to dysregulation of the biological response to infection," or a state in which it is suspected.

In addition, in this specification, the definition of "catheter-related bloodstream infection" is as follows: "JAID/JCC Infectious Disease Treatment Guidelines 2017 - Sepsis and Catheter-related Bloodstream Infection" (Journal of the Japanese Society of Chemotherapy Vol. 66, 2018) 1 (January) p. 82-117). Specifically, it refers to a condition in which at least one percutaneously drawn blood culture and a catheter tip culture are positive, or a percutaneously drawn blood culture and a catheter tip culture are positive. .
Furthermore, even if the results of blood culture (percutaneous sampling and catheter sampling) and catheter tip culture are negative, bacteremia may have developed due to catheter placement. If suspected, it is included in the term "catheter-related bloodstream infection" herein.

[Diagnostic Device]
The diagnostic device of the present invention comprises a measurement unit that measures at least one intrinsic potential selected from the group consisting of an onset potential and a peak potential when the potential of the electrodes of a biosensor having at least two electrodes arranged to contact a specimen containing membrane vesicles produced by bacteria and separated from at least one type of object (hereinafter simply referred to as "object") selected from the group consisting of a subject's body fluids, secretions, and urine, is swept; a memory unit that stores reference data correlating the type of the bacteria with the intrinsic potential; and an analysis unit that compares the reference data with the results of the measurement to provide information for identifying the bacteria that produced the membrane vesicles.

The presumed mechanism by which the effects of the present invention can be obtained by the above-mentioned diagnostic device will be explained.
Figure 1 shows that after culturing Capnocytophaga bacteria, the resulting culture solution was centrifuged to remove bacteria, and then the supernatant was further ultracentrifuged to obtain a pellet of MV, which was resuspended in an electrolyte. The results measured by the DPV method are shown below.

In FIG. 1, "CO OMVs" represents the potential (VS SHE: standard hydrogen electrode)-current curve of MVs of Capnocytophaga sp., and "PG OMVs" represents the potential-current curve of Porphyromonas gingivalis sp.
It should be noted that "Idelta/μA" on the vertical axis of FIG. 1 represents the difference in current before and after the pulse.

According to Figure 1, "CO OMVs" have an onset potential at -0.1V and a peak potential at 0.1V, and "PG OMVs" have an onset potential at -0.4V and a peak potential at -0.1V. It can be seen that the peak potential is at .
Thus, the intrinsic potential of MV is specific to the type of MV, that is, the type of bacteria that produced it, and although data are not shown, the magnitude of the peak current of MV is The present inventor discovered for the first time that there is a correlation with the content, and completed the present invention.
In other words, the present inventor has obtained that the present diagnostic device can detect MV by sweeping the potential of the electrode, and that the intrinsic potential of MV is specific to the type of bacteria that produced the MV. This new knowledge is used as the basic measurement principle.

As a method for sweeping the potential of the electrode, a method in which the obtained current response has a peak shape is preferable because measurement results of peak potential and peak current can be obtained and the content of membrane vesicles can be quantified. Examples of methods in which the obtained current response has a peak shape include differential pulse voltammetry (DPV), cyclic voltammetry (CV), and square wave voltammetry (SWV), which are better. The DPV method is preferable in that a diagnostic device having the effects of the present invention can be obtained.

The specimen includes membrane vesicles separated from the object. The method for separating membrane vesicles from the target object is not particularly limited, and any known method may be used. For example, if the specimen is blood, whole blood may be centrifuged to obtain plasma, further ultracentrifuged to obtain MV as a solid content, and this solid content may be dispersed in an electrolytic solution.
Alternatively, the centrifuged plasma may be used after being filtered through a sterile membrane filter having a predetermined pore size (for example, 0.02 to 0.1 μm in many cases).

The method of centrifugation is not particularly limited, but a known centrifuge can be used, or a hand-cranked method such as that described in Powell, D. Spinning toy reinvented as low-tech centrifuge. Nature (2017) doi:10.1038/nature.2017.21273 can be used.

The sample may contain hydrogen peroxide in addition to the above. The inventors have found that the enzyme on the surface of MV catalyzes the reduction reaction of hydrogen peroxide. Under conditions in which electrons are provided to MV from the electrode, the reaction proceeds continuously, and by integrating the resulting charge, MV can be detected with higher sensitivity.
This tendency is more prominent when the CV method or the LSV method is used, and even more prominent when the LSV method is used. That is, when the CV method or the LSV method is applied, it is preferable that the sample contains hydrogen peroxide, and when the LSV method is applied, it is particularly preferable that the sample contains hydrogen peroxide.
In this case, the content of hydrogen peroxide in the specimen is not particularly limited, and may be appropriately determined with reference to the amount of hydrogen peroxide used in 3,3'-diaminobenzidine (DAB) staining or the like.

As already explained, the present diagnostic device can detect the presence of bacteria with high sensitivity by utilizing the electrochemical properties of MVs derived from bacteria contained in the target object. Therefore, even if the target object does not contain bacteria, it can detect them as long as it contains MVs.
Even if a subject has some kind of bacterial infection, particularly if the bacteria have formed a biofilm and are unlikely to be released into the blood or other fluids, this diagnostic device can provide information to determine the possibility of bacterial infection and the causative bacteria.

As already explained, it has been suggested that a large amount of MV is released (e.g., into the blood) during the biofilm formation process and/or in the state where the biofilm is formed, and according to the present diagnostic device, Information for determining the possibility that a bacterial infection may have occurred in a subject and the type of causative bacteria can be provided more easily.

Based on the information on the type of bacteria provided by this diagnostic device, the doctor can estimate the primary tumor and the causative bacteria, and perform de-escalation from an earlier stage of bacteremia treatment, for example. can.

Next, a diagnostic device according to the present invention will be described with reference to the drawings.
2 is a hardware configuration diagram of a diagnostic device 200 according to an embodiment of the present invention. The diagnostic device 200 includes a processor 201, a storage device 202, a measurement device 203, an input device 204, and an output device 205. The above devices are connected via a bus 206 and configured to be able to exchange data with each other.

The processor 201 controls the diagnostic device 200. The storage device 202 serves as a working area for the processor 201. The storage device 202 is also a non-temporary or temporary recording medium that stores various programs and data.

The processor can be, for example, a CPU (central processing unit), a GPU (graphics processing unit), or the like.
As the storage device 202, for example, ROM (Read Only Memory), RAM (Random Access Memory), HDD (Hard Disk Drive), flash memory, SSD (Solid State Drive), etc. can be used.

The measuring device 203 is configured to allow the attachment and detachment of a biosensor 207 having at least two electrodes arranged to contact a specimen containing membrane vesicles separated from a target object. The measuring device 203 is a unit including a connection terminal for connecting the electrodes of the biosensor 207; a temperature control block for controlling the temperature of the specimen during measurement; a potentiostat/galvanostat capable of adjusting the potential of the electrodes, etc.

The input device 204 is a device for inputting information, such as measurement start, stop, measurement conditions, and a symbol for identifying a sample.
The input device 204 may be, for example, a keyboard, a button, a mouse, a touch panel, a numeric keypad, and a scanner.
The output device 205 is a device for outputting, for example, measurement data, progress of the measurement, operation procedures, and the status of the diagnostic device.
The output device 205 includes, for example, a display and a printer.

FIG. 3 is a perspective view of the diagnostic device 200. The diagnostic device 200 has a button 302, a display 303, and an insertion port 304 for loading a biosensor 207, which are arranged on a main body 301.
Button 302 is input device 204 and display 303 is output device 205.

Further, a circuit board (not shown) is arranged inside the main body 301. A processor 201, a storage device 202, and a measurement device 203 are arranged on the circuit board.
The measurement device 203 can be electrically connected to the biosensor 207 inserted through the insertion port 304.

The user can insert the biosensor 207 through the insertion port 304, operate the button 302 according to the instructions displayed on the display 303, and perform a desired measurement.

Figures 4 and 5 are exploded perspective views of the biosensor 207. The biosensor 207 is typically disposable, and more accurate measurements can be performed by using a new biosensor for each measurement.

The biosensor 207 has a region where a sample is introduced and measurement is performed (hereinafter, this region is also referred to as a "cell" and is indicated as "CELL" in FIG. 4), and a region where the sample is introduced into the cell. It has a pair of electrodes 401 (consisting of a first electrode 401a and a second electrode 401b) arranged at .

The cell is an area defined by the upper surface of the support 402, the side surface of the recessed part of the capillary base material 403 placed on the support 402, and the lower surface of the cover 404, and the sample is introduced from the inlet 405. and is introduced into the cell by flowing through the capillary 406. The analyte flows into the cell through capillary 406 and is brought into contact with a pair of electrodes 401 .

When the biosensor 207 is inserted through the insertion port 304 shown in FIG. 3, the first electrode 401a and the second electrode 401b are electrically connected to the connection terminals of the measuring device 203 arranged on the circuit board arranged in the main body 301, making it possible to perform measurements by the LSV method and the DPV method.

The thickness of the support 402, capillary substrate 403, and cover 404 is not particularly limited and can be selected as appropriate, but from the viewpoint of ease of handling, a thickness of 0.1 μm to 10 mm is typically preferred.

The embodiment of the diagnostic device according to the present invention is not limited to the above, and the biosensor 207 and the diagnostic device 200 may be configured as one unit. When the biosensor and the diagnostic device are integrated, the structure of the measuring device is simpler, making it easier to manufacture the measuring device.

Here, diagnostic device 200 has button 302 as input device 204, but the embodiment of the diagnostic device according to the present invention is not limited to the above, and input device 204 does not have to be a button. For example, display 303 may have a touch panel function and serve as input device 204. In this case, an instruction such as to start measurement from an operator may be received via a GUI (Graphical User Interface) displayed on display 303.
Measurement may also be configured to begin automatically when the biosensor 207 is loaded into the insertion port 304 .

Since the diagnostic device 200 has a display 303 as an output device 205, a series of steps from setting measurement conditions to displaying diagnostic results can be carried out with one device, and the type of bacteria can be determined with simple operations. Information for identifying (the type of bacteria that produced MV) can be provided.

Returning to FIG. 4, the electrodes will now be described. The first electrode 401a of the diagnostic device 200 is a working electrode, and the second electrode 401b is a counter electrode. These two electrodes (electrode pair) are formed on a substrate.

When the biosensor 207 having the above configuration is connected to the diagnostic device 200, the potential of the first electrode 401a (working electrode) can be changed and the resulting current value can be measured.
Note that although the biosensor 207 has a working electrode (first electrode 401a) and a counter electrode (second electrode 401b), this arrangement may be reversed. That is, it may have a first electrode 401a that is a counter electrode and a second electrode 401b that is a working electrode.
Moreover, in addition to the above, it is preferable to have another electrode (third electrode) in contact with the specimen. In this case, the third electrode is preferably a reference electrode. When the biosensor has a reference electrode, the electrode potential can be measured, so that a diagnostic device having better effects of the present invention can be obtained.

In FIG. 4, a pair of electrodes combined in a hook shape is shown, but the shape of the electrodes is not particularly limited to the above, and may be an interdigit electrode. These electrodes can be manufactured by known methods, and for example, the electrodes can be arranged in a pattern on a support by photolithography, plating, printing, etc. The distance between the electrodes is also not particularly limited, and may be a distance known for electrochemical cells. In particular, the area of the electrodes in contact with the sample is preferably 1 ^cm2 or less, since measurement can be performed with good sensitivity even with a smaller amount of sample (specifically, 0.001 to 5 ml).

The electrode material is not particularly limited, and known electrode materials can be used. Examples of electrode materials include carbon, gold, platinum, silver, molybdenum, cobalt, nickel, palladium, and ruthenium, and may be indium tin oxide, etc.

The reference electrode may be a known reference electrode, such as a silver/silver chloride electrode. The counter electrode may be a known counter electrode.

Returning to FIG. 4 and FIG. 5, the support 402, the capillary base material 403, and the cover 404 will now be described.
As already explained, a cell is formed by combining the above-mentioned components, and each of the components is preferably made of an insulating material.

Examples of the insulating material include organic materials, inorganic materials, composites thereof, and more specifically resins, paper, etc.; glass, etc.
Examples of the resin include thermoplastic resins such as polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE); thermosetting resins such as polyimide resin and epoxy resin; and the like.
Examples of the insulating material include glass and paper.

The size of the cell is not particularly limited, but can be selected appropriately depending on the amount of sample, and a capacity of approximately 0.0001 to 5 ml is preferable.

Figure 6 is a functional block diagram of the diagnostic device 200. The diagnostic device 200 has a control unit 601, an operation unit 602, an output unit 603, a measurement unit 604, a memory unit 605, and an analysis unit 606.

The control unit 601 includes the processor 201 and controls the operation unit 602, the output unit 603, the measurement unit 604, the memory unit 605, and the analysis unit 606 to realize the functions of the diagnostic device 200.

The measurement unit 604 is configured to include the measurement device 203. The measurement unit 604 is a function realized by the measurement device 203 controlled by a program stored in the storage device 202 executed by the processor 201.

Here, the electrode section 607 to which the measurement section 604 is connected is configured to include the electrode 401 of the biosensor 207. The electrode section 607 is controlled by the measurement section 604.
In addition, the sample capture section 608 is composed of the inlet 405 of the biosensor 207, the capillary 406, and a cell, and has the function of capturing the sample introduced into the biosensor 207 within the cell and holding it in contact with the electrode 401.

The measurement unit 604 sweeps the potential of the electrode of the biosensor and measures the inherent potential of the specimen.

The storage unit 605 is configured to include the storage device 202. The storage device 202 stores in advance a program for the processor 201 to control each section, a program for performing calculations, reference data, and the like.

This reference data is data that associates the type of bacteria with the specific potential. It is preferable that the reference data is prepared by the following procedure.

Procedure for generating reference data First, a test sample containing MVs produced by a known bacterium is prepared.
Next, the electrode potential of the test sample is swept using this diagnostic device (or another device capable of realizing the same measurement conditions as this diagnostic device) to measure the intrinsic potential.

The obtained characteristic potential is combined with information on the type of bacteria that produced MV contained in the test specimen to form one record.
Note that in this specification, each combination of a type of bacteria and a corresponding characteristic potential is referred to as a record.

Note that the record may include at least information on the type of bacteria that produced the MV and the corresponding characteristic potential, and may also include other information.

It is preferable that the reference data is configured as a data collection having a plurality of the above-mentioned records for different bacteria.
Note that in this specification, data configured as a group of multiple records is referred to as a table.
That is, it is preferable that the reference data is a table consisting of a plurality of records.

In addition, when the method for sweeping the electrode potential is a method in which the current response obtained has a peak shape, such as the DPV method, the memory unit 605 may store second reference data that correlates the intrinsic potential with the correlation between the magnitude of the peak current and the content of membrane vesicles in the sample.

・Procedure for creating the second standard data Prepare multiple test specimens that contain MV produced by the same bacteria and differ only in their content.
Next, for those test specimens, the potential of the electrodes is swept using this diagnostic device (or other device that can realize measurement conditions similar to this diagnostic device), and the peak potential and peak current ( (size). Note that the onset potential may also be measured at this time.

The inventors have found that there is a correlation between the magnitude of the peak current and the MV content, and this correlation can be calculated from the data on the peak current magnitude and the MV content of multiple test samples.
The second reference data is data relating the above correlation to the intrinsic potential.

Returning to FIG. 6, the storage unit 605 has a function that allows the storage device 202 controlled by the processor 201 to read and write these data.

The analysis unit 606 is a function realized by executing a program stored in the storage device 202 by the processor 201. The analysis unit 606 compares the characteristic potential of the specimen obtained by the measurement unit 604 with reference data stored in the storage device 202, and provides information for identifying the type of bacteria that produced the MV.

The operation unit 602 includes the input device 204 and has a function of accepting user operations.
The output unit 603 includes the output device 205 and has a function of displaying various types of information and data.

(Operation of diagnostic device)
Next, the operation of the diagnostic device 200 will be explained.
The diagnostic device 200 operates as follows according to the program. FIG. 7 is an operation flow of the control unit 601 that operates according to the above program.

When the biosensor 207 is inserted into the insertion port 304 of the main body 301 and the biosensor 207 and the circuit board housed in the main body 301 are connected, measurement is performed according to a user's instruction (or when the insertion of the biosensor is detected). is started.

First, the control unit 601 controls the measurement unit 604 to sweep the potential of the electrodes of the biosensor 207 and measure the intrinsic potential (step S701).
Next, the control unit 601 controls the analysis unit 606 to compare the intrinsic potential with reference data (step S702).

Here, an example of a method for comparing the reference data with the measured intrinsic potential (hereinafter sometimes referred to as the "measurement result") will be described.
First, the measurement result is compared with the characteristic potential of each record included in the table. When the measurement result is compared with the characteristic potential of each record and the measurement result belongs to any of the records (step S703: YES), the control unit 601 controls the output unit 603 to display information on the type of bacteria on the output device 205 (step S704).

It is preferable that the criteria for attribution of the measurement results are determined in advance and stored in the storage unit.
The criteria for the attribution may be, for example, that if the difference (absolute value) between the measurement result and the intrinsic potential of each data is equal to or less than a predetermined value, the measurement result is attributable to that record.

On the other hand, if the measurement result is not assigned to any record of the reference data (step S703: NO), the measurement ends.
Note that at this time, the control unit 601 may control the output unit 603 and display this on the output device 205.

The flow in Figure 7 provides information for identifying the bacteria that produced the MVs contained in the sample. In cases of infections caused by biofilms, the bacteria themselves may not be easily released into the blood. By applying the above flow to this device, even in such cases, it is possible to provide useful information for inferring bacterial infection and the causative bacteria.

FIG. 8 is another operational flow of the control unit 601 of the diagnostic device 200.
First, when the biosensor 207 is inserted into the insertion port 304 of the main body 301 and the biosensor 207 and the circuit board housed in the main body 301 are connected, the biosensor 207 is connected to the circuit board housed in the main body 301. , measurement is started.

First, the control unit 601 controls the measurement unit 604 to sweep the potential of the electrode of the biosensor 207 by a method that produces a peak-shaped current response, and measures the intrinsic potential including at least the peak potential and the magnitude of the peak current (step S801).
Next, the control unit 601 controls the analysis unit 606 to compare the intrinsic potential with the reference data (step S802).

The measurement result is compared with the characteristic potential of each record, and if the measurement result is attributed to one of the records (step S803: YES), the control unit 601 next controls the analysis unit 606 to calculate the result obtained from the measurement. The unique potential thus obtained is compared with the unique potential of each record of the second reference data, and a corresponding correlation is obtained (step S804).

Next, the control unit 601 controls the analysis unit 606 to apply the measurement result of the magnitude of the peak current to the above correlation, and calculates the content of MV in the sample (step S805).
Next, the control unit 601 controls the output unit 603 to display information on the type of bacteria and the content of MV on the output device 205 (step S806).

On the other hand, if the measurement result does not belong to any record of the reference data (step S803: NO), the measurement ends.
At this time, the control unit 601 may control the output unit 603 to display a message to that effect on the output device 205 .

According to the flow shown in FIG. 8, in addition to information for determining the type of bacteria that produced MV contained in the specimen, it is also possible to provide information for determining the content of MV in the specimen. The content of MV in the sample is considered to be related to the activity state of the bacteria that produced the MV in the subject's body, such as forming a biofilm and proliferating. By applying the above flow to this device, more useful information can be provided for formulating treatment plans, etc.

This diagnostic device can quickly provide the information necessary to identify the causative bacteria of a bacterial infection by simply collecting at least one type of fluid selected from the group consisting of body fluids, secretions, and urine from a subject, obtaining a sample containing membrane vesicles from the above, and loading a biosensor to which this is applied.

200: Diagnostic device 201: Processor 202: Storage device 203: Measurement device 204: Input device 205: Output device 206: Bus 207: Biosensor 301: Main body 302: Button 303: Display 304: Insertion port 401: Electrode 401a: First Electrode 401b: Second electrode 402: Support body 403: Capillary base material 404: Cover 405: Inlet port 406: Capillary 601: Control section 602: Operation section 603: Output section 604: Measurement section 605: Storage section 606: Analysis section 607 : Electrode part 608 : Sample capture part

Claims (13)

1. A diagnostic device for diagnosing a bacterial infection in a subject, comprising:
a measuring unit for measuring at least one intrinsic potential selected from the group consisting of an onset potential and a peak potential when the potential of the electrodes of a biosensor having at least two electrodes arranged to be in contact with a specimen that is separated from at least one object selected from the group consisting of a body fluid, a secretion, and a urine of the subject and that contains membrane vesicles produced by bacteria, is swept;
a storage unit in which reference data correlating the type of bacteria with the specific potential is stored;
and an analysis unit that compares the reference data with the results of the measurement to provide information for identifying the bacterium that produced the membrane vesicles. The diagnostic device according to claim 1, wherein the method of sweeping the potential of the electrode is such that the obtained current response has a peak shape. The storage unit further includes:
The characteristic potential;
a correlation between the content of the membrane vesicles in the specimen and the magnitude of the peak current;
The diagnostic device according to claim 2, wherein second reference data for associating the two is stored. the sample further contains hydrogen peroxide;
The diagnostic device according to claim 1 , wherein the measurement section applies a linear sweep voltammetry method or a cyclic voltammetry method to the biosensor. The diagnostic device according to claim 1, wherein the method for sweeping the potential of the electrode is at least one method selected from the group consisting of linear sweep voltammetry, cyclic voltammetry, square wave voltammetry, and differential pulse voltammetry. The diagnostic device according to any one of claims 1 to 5, wherein the object is at least one selected from the group consisting of blood, body cavity fluid, and urine. The diagnostic device according to any one of claims 1 to 6, wherein the object is blood. The diagnostic device according to any one of claims 1 to 7, wherein the biosensor has a substrate and the electrode disposed on the substrate. The diagnostic device according to any one of claims 1 to 8, wherein the bacteria is a causative bacterium of at least one disease selected from the group consisting of sepsis and catheter-associated bloodstream infections. The diagnostic device according to any one of claims 1 to 9, wherein the bacteria are causative bacteria of catheter-related bloodstream infections. 1. A program for diagnosing a bacterial infection in a subject, comprising:
On the computer,
A step of measuring at least one intrinsic potential selected from the group consisting of an onset potential and a peak potential when sweeping the potential of a biosensor having at least two electrodes arranged to contact a specimen containing membrane vesicles produced by bacteria, the specimen being separated from at least one object selected from the group consisting of a body fluid, a secretion, and a urine of the subject;
and comparing the results of the measurement with reference data relating the type of bacteria to the intrinsic potential to provide information for identifying the bacteria that produced the membrane vesicles. 12. The program according to claim 11, wherein the method of sweeping the potential of the electrode is such that the obtained current response has a peak shape. measuring the magnitude of the peak current when sweeping the potential of the electrode;
obtaining the correlation corresponding to the measured intrinsic potential from second reference data that correlates the intrinsic potential with the correlation of the content of membrane vesicles in the specimen with respect to the magnitude of the peak current;
13. The program according to claim 12, applying the magnitude of the measured peak current to the correlation to calculate and provide the content of the membrane vesicles in the sample.