WO2019188924A1 - Method for detecting bacteria in blood sample - Google Patents

Abstract

The problem of the present invention is to provide a highly convenient and prompt method for detecting existence of bacteria in blood samples with high sensitivity. By using a method comprising sequential processes (a) to (d), wherein the process (a) mixes a culture of a subject-originated blood sample with ammonium chloride and obtains a hemolysed sample; the process (b) filters the hemolysed sample with a filter membrane having pore diameter in which bacteria cannot be filtered; the process (c) collects residue caught on the filter membrane after filtering and obtains a residue sample; and the process (d) analyzes existence of bacteria in the residue sample by using a mass spectrometer; the matter of bacterial infection of the subject can be detected with high sensitivity.

Description

Method for detecting bacteria in blood samples

The present invention relates to a method for detecting the presence or absence of bacteria in a blood sample derived from a subject.

In the case of bacterial infections, particularly severe infections such as sepsis, it is important to administer an appropriate antibacterial agent at an early stage from the viewpoint of healing of the bacterial infection and good prognosis. For bacterial infections, effective antibacterial drugs are selected based on the assumption of pathogenic bacteria, and empirical treatment is given. At the same time, a blood culture test is performed to clarify the pathogenic bacteria, but it takes several days until the pathogenic bacteria are revealed. For this reason, it has been required to quickly identify the pathogenic bacteria and to make appropriate use of antibacterial drugs based on empirical treatment.

Recently, mass spectrometry using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) has been performed on bacterial proteins or whole bacteria. It has been reported that it can be generated (Patent Document 1). In addition, in order to prepare a test sample for bacterial identification using mass spectrometry, a method of selectively lysing non-bacterial cells in the test sample has also been reported (Patent Document 2). However, no method has been reported so far on the method of performing hemolysis of the blood culture with ammonium chloride and then performing mass spectrometry on the residue obtained by filtration for the identification of bacteria and its effects. .

US Pat. No. 6,177,266 Special table 2012-507711 gazette

An object of the present invention is to provide a method that is excellent in simplicity and speed and can detect the presence or absence of bacteria in a blood sample with high sensitivity.

In order to solve the above problems, the present inventors filtered a subject-derived blood culture hemolyzed with ammonium chloride with a filtration membrane having a pore size that does not allow bacteria to pass through, and collected residues captured by the filtration membrane. When the bacteria in the residue sample obtained were analyzed using a mass spectrometer, it was found that whether or not bacteria were contained in the residue sample could be detected with high sensitivity. In addition, when the above hemolysis treatment is performed using ammonium chloride, bacteria in the blood sample can be compared with the case where the hemolysis treatment is performed using another hemolysis agent (for example, NP-40, Tween-20, Triton®X-100). It was also confirmed that it could be detected with high sensitivity. The present invention has been completed based on these findings.

That is, the present invention is as follows.
[1] A method for detecting bacteria in a blood sample, which comprises the following steps (a) to (d) (hereinafter sometimes referred to as “the present detection method”).
(A) a step of mixing a blood sample culture collected from a subject (hereinafter sometimes referred to as “subject-derived blood culture”) with ammonium chloride to obtain a hemolyzed sample;
(B) filtering the hemolyzed sample with a filter membrane having a pore size that does not allow bacteria to pass through;
(C) collecting the residue trapped on the filtration membrane after the filtration treatment to obtain a residue sample;
(D) analyzing the presence or absence of bacteria in the residue sample using a mass spectrometer;
[2] The detection method according to [1], wherein the mass spectrometer is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS).
[3] The detection method according to [1] or [2], wherein, in the step (c), the filtration membrane after the filtration treatment is immersed in pure water, and the residue is collected by centrifugation.
[4] The detection according to any one of [1] to [3] above, wherein in step (a), the culture of the blood sample collected from the subject is not treated with a hemolytic agent other than ammonium chloride. Method.
[5] The above [1] to [4], wherein the step (m) includes a step (m) of washing the filtered membrane after the filtration treatment with pure water between the step (b) and the step (c). The detection method in any one of.

As another embodiment of the present invention,
If the bacterium is detected in the residue sample after the step (a) and further including the steps (a) to (d), the subject is diagnosed as having a high possibility of being infected with the bacterium. And diagnosing whether or not the subject is infected with bacteria, comprising the step (p) of diagnosing that the subject is unlikely to be infected with bacteria if no bacteria are detected in the residue sample How to do;
Treatment of a subject diagnosed as having a high possibility of being infected with a bacterium, comprising the steps (a) to (d) and (p), using a drug such as an antibiotic against the bacterium A method of diagnosing whether or not a subject is infected with bacteria and preventing or treating the bacterial infection, comprising the step (q) of applying treatment;
Can be mentioned.

In this detection method, when the subject-derived blood culture hemolyzed with ammonium chloride is filtered with a filtration membrane having a pore size that does not allow bacteria to pass through, the bacteria contained in the subject-derived blood culture are left as residues in the filtration membrane. While captured, blood cell components such as hemoglobin pass through the filtration membrane. For this reason, bacteria contained in the subject-derived blood culture and blood cell components can be separated relatively easily without performing centrifugation. Moreover, since the said filtration process can be performed by manual operation using a syringe, even if there is no large apparatus, such as a vacuum pump, this detection method can be implemented. Further, by analyzing the bacteria in the residue sample obtained by collecting the residue captured by the filtration membrane using a mass spectrometer, whether or not the bacteria are contained in the residue sample, It can be detected with high sensitivity. For this reason, using this detection method, data useful for diagnosing whether or not a subject is infected with bacteria (especially pathogenic bacteria) can be obtained. In addition, early prevention and early treatment of bacterial infections are expected.

Blood culture tests for diagnosing sepsis are performed by culturing a blood sample collected from a subject in a culture solution, and using changes in turbidity of the blood culture, gas generation in the culture solution by bacteria, changes in pH, etc. as indicators. And confirming the presence or absence of bacterial growth for 1 to 7 days. Then, if bacterial growth is confirmed, blood culture is collected, Gram staining and subculture (secondary culture) are performed, and the antibacterial used for empirical treatment is based on the results of rough classification of bacterial species by Gram staining. Drug evaluation is performed. On the other hand, if this detection method is used, bacteria can be efficiently collected and bacterial species can be identified quickly and accurately. Leads to treatment. For this reason, this invention can contribute to improvement of the prognosis of a patient with a bacterial infection, suppression of increase of resistant bacteria, cost reduction, and the like.

In this detection method, a subject-derived blood culture is mixed with ammonium chloride to obtain a hemolyzed sample (a); a filtration membrane having a pore size that does not allow bacteria to pass through the hemolyzed sample (hereinafter simply referred to as “filtration membrane”) (B) a filtration process (b);) a step (c) of collecting the residue trapped on the filtration membrane after the filtration process to obtain a residue sample; and bacteria in the residue sample There is no particular limitation as long as the method includes steps (a) to (d) of step (d); which is analyzed (detected) using a mass spectrometer, and the detection method is derived from the subject. This method detects whether bacteria are present in the blood sample, and does not include a diagnostic action by a doctor.

The subject is not particularly limited, and is, for example, a person with unknown bacterial infection (for example, a healthy person; a non-bacterial infection patient such as a cancer patient or a diabetic patient), or a specific bacterium. Those who are unclear (for example, healthy individuals; patients with bacterial infections such as septic patients and patients with upper respiratory tract infections) can be mentioned.

[About step (a) in this detection method]
In the step (a), the subject blood culture is mixed with ammonium chloride to obtain a hemolyzed sample. Conditions such as temperature and time are such that red blood cells are destroyed and hemoglobin is released into the blood culture. The temperature may be in the range of 10 to 37 ° C., and the treatment time may be in the range of 1 second to 1 minute, for example. The method of mixing the subject-derived blood culture and ammonium chloride is not particularly limited as long as the ammonium chloride is a liquid type and the ammonium chloride is a method of stirring the entire subject-derived blood culture. In the case of a non-liquid type, the method is not particularly limited as long as ammonium chloride is stirred and dissolved in the entire subject-derived blood culture. For example, the container containing the subject-derived blood culture and ammonium chloride is moved up and down one or several times. The method of stirring by making it, the method of stirring 1 or several times with a stirring rod, the method of combining them, etc. can be mentioned.

The test subject-derived blood culture can be obtained, for example, by culturing a blood sample collected from the test subject at a culture temperature of 35 ° C. for 12 to 48 hours.

The ammonium chloride concentration in the subject-derived blood culture may be a concentration sufficient to destroy red blood cells and release hemoglobin into the blood culture, and is usually in the range of 50 to 500 mM, preferably Is 60-400 mM, more preferably 70-300 mM, more preferably 80-250 mM, even more preferably 100-220 mM, particularly preferably 110-200 mM, particularly more preferably 120-190 mM, and most preferably 130-180 mM. .

The ammonium chloride may be liquid ammonium chloride or non-liquid ammonium chloride, but liquid ammonium chloride is preferred. The liquid type ammonium chloride can be prepared by dissolving a non-liquid type ammonium chloride such as a powder in a solvent (preferably a physiological aqueous solution). Such a physiological aqueous solution may be an isotonic solution in which the concentration of salt or sugar is adjusted with sodium or potassium so as to be almost the same as the osmotic pressure of a body fluid (for example, plasma) or cell fluid. Examples include isotonic solutions such as saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), and HEPES buffered saline. In this specification, “isotonic” means that the osmotic pressure is in the range of 250 to 380 mOsm / L.

In the above step (a), it is preferable not to hemolyze the subject-derived blood culture with a substance (hemolytic agent) other than ammonium chloride, because ammonium chloride alone can be effectively hemolyzed. Here, examples of hemolytic agents other than ammonium chloride include surfactants and hypotonic solutions. Examples of such surfactants include Triton-100X-100 (Triton is a registered trademark) (polyoxyethylene (10) octylphenyl ether), Triton X-114 (polyoxyethylene (8) octylphenyl ether), Triton X-405 ( Polyoxyethylene pt-octylphenyl ether (Triton surfactant) such as polyoxyethylene (40) isooctylphenyl ether), NP-40 (Nonidet P-40) (polyoxyethylene (9) octylphenyl ether); Polyoxyethylene sorbitan fatty acid esters (Tween surfactant) such as Tween 20 (Tween is a registered trademark), Tween 40, Tween 60, Tween 80, Tween 65, Tween 85; Briji 35 (Briji is a registered trademark) (polyoxy) Polyoxyethylene alkyl ethers (Briji surfactants) such as ethylene (23) lauryl ether); Nonionic interfaces such as Dodecyl-β-D-maltose; Octyl-β-D-glucoside And anionic surfactants such as sodium dodecyl sulfate (SDS), cationic surfactants such as benzalkonium chloride, benzethonium chloride, didecyldimethylammonium salt, dodecyltrimethylammonium chloride and CHAPS (3- ( And amphoteric surfactants such as 3-cholamidepropyl) dimethylammonio-1-propanesulphonate) and alkyl polyaminoethylglycine chloride.

The hypotonic solution is not particularly limited as long as it is a hypotonic solution in which the salt or sugar concentration is adjusted with sodium or potassium so as to be lower than the osmotic pressure of a body fluid (for example, plasma) or cell fluid. As used herein, “hypotonic” means that the osmotic pressure is lower than 250 mOsm / L.

[About step (b) in this detection method]
By the filtration treatment in the step (b), blood cell components such as hemoglobin in the hemolyzed sample pass through the filtration membrane. For this reason, when bacteria are contained in the hemolyzed sample, the bacteria can be captured as a residue on the filtration membrane and separated from the blood cell component.

As the method for the filtration treatment, for example, the hemolyzed sample prepared in the step (a) is transferred to a container provided with a filtration membrane and made of a material such as plastic, metal, glass, etc., and a syringe, a vacuum pump, an aspirator, Filtration by suction or pressurization using a device such as a compressor, or filtration into a centrifuge tube or test tube made of a material such as plastic, metal, glass, etc., equipped with a filtration membrane and centrifuged From the viewpoint of more easily performing filtration without using a large-scale device, a method of performing pressure treatment or pressure reduction by manually operating a syringe and performing filtration treatment, or filtration treatment by centrifuging. Is preferred. From the viewpoint of preventing bacterial contamination, the filtration treatment is preferably performed in a closed system, and the filtration membrane is preferably disposable.

The pore size of the filtration membrane may be any pore size that does not allow bacteria to pass through, and is usually 1.5 μm or less, preferably 1.2 μm or less, more preferably 0.8 μm or less, still more preferably 0.7 μm or less, and even more preferably. Is 0.6 μm or less, most preferably 0.5 μm or less, and is usually 0.1 μm or more, preferably 0.2 μm or more, more preferably 0.25 μm or more from the viewpoint of avoiding clogging by blood cell components such as hemoglobin. More preferably, it is 0.3 μm or more, even more preferably 0.35 μm or more, and most preferably 0.4 μm or more. Therefore, the pore size of the filtration membrane used for the filtration treatment is usually within the range of 0.1 to 1.5 μm, preferably 0.2 to 1.2 μm, more preferably 0.25 to 0.8 μm, and still more preferably. It is 0.3 to 0.7 μm, even more preferably 0.35 to 0.6 μm or less, and most preferably 0.4 to 0.5 μm or less.

Examples of the material of the filtration membrane include cellulose mixed esters (MCE), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), hydrophilic PTFE, polyethersulfone (PES), and hydrophilic. Polyethersulfone (hydrophilic polyethersulfone), hydrophilic polypropylene (GHP), nylon (NYL), cellulose acetate (CA), polysulfone (PSF), acrylic copolymer (acrylic copolymer), polyamide, nylon 6, 6, polyester, polycarbonate, nitrocellulose, a mixture of nitrocellulose and cellulose ester, and the like.

[About step (c) in this detection method]
In the step (c), as a method for obtaining a residue sample, any method can be used as long as it can release and recover the residue from the filtration membrane after the filtration treatment in the step (b). However, from the viewpoint of recovery efficiency of bacteria contained in the residue, a part or all of the filtration membrane may be immersed in pure water to obtain a filtration membrane and / or pure water. Is preferably collected from the water containing the residue by centrifugation, and the centrifugation is preferably carried out in a closed system from the viewpoint of preventing bacterial contamination. Moreover, it is preferable to wash | clean the said filtration membrane using a pure water before collect | recovering a residue. The number of washings may be at least once and may be a plurality of times (2, 3, 4 etc.), but is preferably 1 in consideration of time-effectiveness and cost-effectiveness. The filtration membrane is washed by adding pure water to the filtration membrane after the filtration treatment in step (b) and passing the pure water through the filtration membrane by the same suction or pressurization method as the filtration treatment. be able to.

In the present specification, “pure water” means water from which impurities and ionic substances have been removed. Specifically, water having an electric conductivity of 10 μS / cm or less under a condition of 24 to 26 ° C. Means. Pure water can be prepared by distillation and / or ion exchange of tap water.

[About step (d) in this detection method]
In the above step (d), as a method of analysis using a mass spectrometer, the residual sample is converted into gaseous ions using an ion source (ionization), and is moved in a vacuum in the analysis unit to use electromagnetic force. Or an analysis method using a mass spectrometer capable of separating and detecting a residue sample ionized by a time-of-flight difference according to the mass-to-charge ratio is not particularly limited. Here, ionization using an ion source includes electron ionization (EI) method, chemical ionization (CI) method, field desorption ionization (FD) method, fast atom bombardment (FAB) method, matrix-assisted laser desorption. A method such as an ionization (MALDI) method or an electrospray ionization (ESI) method can be appropriately selected. In addition, as a method for separating an ionized blood sample in the analysis unit, a magnetic field deflection type, a quadrupole type, Separation methods such as an ion trap type, a time-of-flight (TOF) type, and a Fourier transform ion cyclotron resonance type can be appropriately selected. Further, tandem mass spectrometry (MS / MS) combining two or more mass spectrometry methods can be used. Further, SBP and methylcysteine can be separated and purified from impurities by gas chromatography (GC), liquid chromatography (LC), and high performance liquid chromatography (HPLC). As the mass spectrometer, a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) is preferable.

In the above step (d), whether or not bacteria are contained in the residue sample is prepared by preparing a sample for mass spectrometry from the residue sample and analyzing it using a mass spectrometer. The analysis target in the residual sample may be any substance derived from bacteria (for example, protein, peptide, compound), and its expression level in bacteria is large; it is easily ionized; peptide fragmentation is performed using a proteolytic enzyme such as trypsin. Without being easily detected by TOFMS within a molecular weight range of about 4000-15,000 Da; mass spectral patterns capable of distinguishing bacterial types are already known for many types of bacteria; Therefore, a ribosomal protein derived from bacteria is preferable.

When bacteria are detected in the residue sample in the step (d), the subject can acquire data for diagnosing that there is a high possibility that the subject is infected with bacteria. If no is detected, the subject can acquire data for diagnosing that the possibility of being infected with bacteria is low. Therefore, as another embodiment of the present invention, it is diagnosed whether or not the subject is infected with bacteria, including the above steps (a) to (d), and further including the step of acquiring these data The method of collecting data for can be mentioned.

Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples. The blood sample culture (blood culture) used in this example is a blood sample collected from a patient with severe infection between June 2016 and April 2017 under the condition of a culture temperature of 35 ° C. Among the cultures obtained by culturing for 12 to 48 hours under the above, it is a culture (n = 120) that became culture positive by an automated blood culture analyzer (BACTEC FX SYSTEM, BD Bioscience).

1. Method 1-1 Case Detection Method The case detection method was carried out according to the following procedures [1] to [6].
[1] 100 μL of 1.55 M ammonium chloride-containing hemolytic agent (BD Bioscience) was diluted 10-fold with 900 μL of PBS solution, added to and mixed with 1.5 mL of blood culture, and immediately the pore size was increased. It added on the 0.45 micrometer filtration membrane (37-mm quality monitor [made by Japan PALL)].
[2] A blood sample containing 155 mM ammonium chloride was filtered by manual operation with a syringe attached to a 37 mm quality monitor.
[3] 2 mL of distilled water was added on a 37 mm quality monitor, and was filtered and washed by manual operation using a syringe attached to the 37 mm quality monitor.
[4] The 37 mm quality monitor after washing was taken out, immersed in 1 mL of distilled water, stirred for 30 seconds using a vortex mixer, and then centrifuged (20600 g × 3 minutes, room temperature) to remove the supernatant.
[5] Several μg of the obtained precipitate is placed on a MALDI sample target (manufactured by Bruker Daltonics), 1 μL of 70% formic acid and 1 μL of a matrix solution (2,5-dihydroxybenzoic acid, 80 mg / mL) , 30% acetonitrile, 0.1% trifluoroacetic acid) was added, and crystallization was performed by a drying treatment. It is reported that the crystallized sample can be measured most stably when the humidity of the sample before the drying treatment is reduced to 40% (references “Umemura, H. et al., Clinica”). Chimica Acta. 2010, 411 2109-2111 "), and in this example, a crystallized sample having a humidity of 40% was also prepared.
[6] 1 μL of α-Cyano-4-hydroxycinnamic Acid (CHCA) matrix reagent (manufactured by Bruker Daltonics) is added to the sample after drying, and is introduced into MALDI-TOF MS (Microflex [manufactured by Bruker Daltonics]) after drying. Then, double measurement was performed and the average value was calculated. Mass spectra were acquired using Flex Control software (Bruker Daltonics), and bacterial species identification based on ribosomal protein fingerprints (mass spectrum patterns) was performed using MALDI Biotyper 3.1 software (Bruker Daltonics). Made by the company). When the score value is 2.0 or more, it is evaluated that the bacterial species level is high, and when the score value is 1.7 to less than 2.0, it is evaluated that the genus level is high, and the score value is 1 If less than .7, it was evaluated as unidentifiable.

1-2 Prior Art Detection Method The prior art detection method was performed according to the following procedures [1] to [5].
[1] A 1.5 mL blood culture was placed in a sterile vacuum blood collection tube (Insepack [registered trademark] II; manufactured by Sekisui Medical Co., Ltd.) containing a plasma separating agent, and centrifuged (1710 g × 5 minutes).
[2] The supernatant (plasma fraction) was removed, 1 mL of pure water was added, transferred to a 1.5 mL tube, and then centrifuged (15000 rpm × 3 minutes).
[3] The supernatant (pure water fraction) was removed, 0.9 mL of ethanol was added, and the mixture was centrifuged (15000 rpm × 3 minutes).
[4] The supernatant (ethanol fraction) was removed, 50 μL of 70% formic acid and 50 μL of acetonitrile were added, and centrifuged (15000 rpm × 3 minutes).
[5] The supernatant was collected, introduced into MALDI-TOF MS (Microflex [manufactured by Bruker Daltonics)], double measurement was performed, and the average value was calculated. Acquisition of mass spectrum and identification of bacterial species were carried out according to the method described in the procedure [6] in the item “1-1” above.

2. Results The identified Gram-negative and Gram-positive bacteria are shown in Tables 1 and 2, respectively. For Gram-negative bacteria, the number evaluated as having high agreement between the species levels was 44 when using the detection method of the prior art, whereas it was 50 when using this detection method. The detection sensitivity of the present detection method was about 1.14 times higher than the detection sensitivity of the conventional detection method (see “≧ 2.0” in Table 1). In addition, the number of gram-positive bacteria evaluated as having a high match at the bacterial species level was 23 when the conventional detection method was used, while 51 when the detection method was used. Yes, the detection sensitivity of the present detection method was about 2.2 times as high as the detection sensitivity of the conventional detection method (see “≧ 2.0” in Table 2). Percentage of gram-positive bacteria identified that are the most pathogenic and are known to cause diseases such as skin infections, pneumonia, endocarditis, osteomyelitis, especially those detected in blood culture (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus simulans, and Staphylococcus haemolyticus) (literature journal of Japanese Society of Clinical Microbiology Vol.22 No.1 2012.13-19) And the document “Shinshu Medical Journal, 54 (5): 257-263, 2006”), the number that was evaluated as having high coincidence of bacterial species levels was 14 when the detection method of the prior art was used. On the other hand, when this detection method is used, the detection sensitivity is 32, and the detection sensitivity of this detection method is about 2.3 times higher than the detection sensitivity of the conventional detection method. Was Tsu (see "≧ 2.0" in Table 2).
These results show that this detection method can detect bacteria in blood culture with higher sensitivity compared to the detection method of the prior art, and unlike the detection method of the prior art, without centrifugation, Blood cell components can be separated from bacterial cells, and samples for MALDI-TOF MS can be prepared in a relatively short time. It is shown that.

[Comparison with hemolytic agents other than ammonium chloride]
In the present detection method described in Example 1, in place of ammonium chloride, three types of hemolytic agents (NP-40, Tween 20, and Triton X-100) were used, and enteric bacteria (Klebsiella pneumoniae) in the blood sample were used. Was similarly detected. Note that the concentrations of NP-40, Tween 20, and Triton X-100 after addition to the blood culture are 0.032 mol / L, 0.016 mol / L, and 0.03 mol / L, respectively.

As a result, when Triton X-100 and Tween 20 were used, the peak of the mass spectrum derived from Klebsiella pneumoniae was not detected, whereas when NP-40 was used, it was the same as when ammonium chloride was used. Thus, the score value was 2 or more, and it was shown that it can be identified at the bacterial species level (see Table 3).

Next, the detection target bacteria (Klebsiella pneumoniae) were replaced with Staphylococcus aureus and detected in the same manner. As a result, when NP-40 was used, the number of specimens identified at the bacterial species level (score value ≧ 2) was 1 out of 3 specimens (see Table 4), and the mean value of the score values ± standard deviation Was 1.868 ± 0.14, but when ammonium chloride was used, all three samples could be identified at the bacterial species level (see Table 4), and the average score value The standard deviation was 2.138 ± 0.03.
The above results show that in the present detection method, when ammonium chloride is used, bacteria in the blood sample are less than when a hemolytic agent other than ammonium chloride (eg, NP-40, Tween 20, Triton X-100) is used. It shows that it can be detected with high sensitivity.

The present invention contributes to diagnosis, early prevention or early treatment of bacterial (especially pathogenic bacteria) infections in subjects.

Claims (5)

A method for detecting bacteria in a blood sample, comprising the following steps (a) to (d):
(A) mixing a culture of a blood sample collected from a subject with ammonium chloride to obtain a hemolyzed sample;
(B) filtering the hemolyzed sample with a filter membrane having a pore size that does not allow bacteria to pass through;
(C) collecting the residue trapped on the filtration membrane after the filtration treatment to obtain a residue sample;
(D) analyzing the presence or absence of bacteria in the residue sample using a mass spectrometer; The detection method according to claim 1, wherein the mass spectrometer is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS). The detection method according to claim 1 or 2, wherein in step (c), the filtration membrane after the filtration treatment is immersed in pure water, and the residue is collected by centrifugation. The detection method according to any one of claims 1 to 3, wherein, in the step (a), the culture of the blood sample collected from the subject is not treated with a hemolytic agent other than ammonium chloride. The process (m) according to any one of claims 1 to 4, further comprising a step (m) of washing the filtered membrane after the filtration treatment with pure water between the step (b) and the step (c). Detection method.