WO2019097752A1 - 血液検体の前処理方法 - Google Patents
血液検体の前処理方法 Download PDFInfo
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- WO2019097752A1 WO2019097752A1 PCT/JP2018/023388 JP2018023388W WO2019097752A1 WO 2019097752 A1 WO2019097752 A1 WO 2019097752A1 JP 2018023388 W JP2018023388 W JP 2018023388W WO 2019097752 A1 WO2019097752 A1 WO 2019097752A1
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- blood
- atp
- blood sample
- platelets
- pretreatment
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/008—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions for determining co-enzymes or co-factors, e.g. NAD, ATP
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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- G—PHYSICS
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- G01N2035/00465—Separating and mixing arrangements
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Definitions
- the present invention is derived from human origin, for example, when an E. coli causing infection such as sepsis is directly recovered from a sample in the state of a living cell or an E. coli is directly recovered from a blood culture bottle (blood culture sample).
- the present invention relates to a method for pretreatment of a blood sample to measure ATP of E. coli by minimizing ATP (adenosine triphosphate).
- ATP adenosine triphosphate
- E. coli viable bacteria
- drug sensitivity test In order to determine the turbidity of bacteria in the current drug sensitivity test, it is necessary to first isolate and culture E. coli bacteria, and then wait for sufficient growth of the bacteria in the drug sensitivity test. It takes a day. On the other hand, speeding up of drug sensitivity test starting from bacterial colonies has been reported by measuring ATP produced by bacteria.
- Non-patent document 1 (Non-patent document 1) According to the report, the conventional method requires 18 to 24 hours from the start of bacterial colonies, whereas ATP measurement is possible in about 6 hours. However, since the above method is a start from a colony and it takes 1 to 2 days to separate and grow the colony, it can not be said that the time from blood collection is quick.
- Patent Document 1 describes, for example, that blood cells were broken with a surfactant-treated solution consisting of 0.1, 0.2% by weight of SDS (sodium dodecyl sulfate) and 2% by weight of saponin. .
- SDS sodium dodecyl sulfate
- saponin saponin
- the present invention makes it possible to minimize human-derived ATP in a blood sample (including a blood culture sample), and to provide a pretreatment method capable of directly recovering E. coli from the blood sample in a state of viable bacteria. To aim.
- the present invention is a method for pretreatment of a blood sample for measuring ATP in E. coli in blood, which comprises the steps of preparing platelets and E. coli pellet from the blood sample, and the platelets and E. coli pellet And processing the following steps (A) to (C) in any order or simultaneously processing a plurality of steps.
- A Protease degradation treatment of platelet membrane proteins.
- B The platelets are expanded with a hypotonic solution.
- C The cell membrane of platelets is destroyed with a surfactant solution under the condition that the influence on E. coli is suppressed.
- to suppress the influence on pathogenic bacteria means that the recovery rate as living bacteria is 50% or more on average of the major 19 genera of septic bacteria.
- the pellet of the platelets and the causative bacterium is obtained by centrifuging the supernatant from which red blood cells and white blood cells have been removed from the blood sample collected by the blood collection tube containing the anticoagulant by centrifugation or separation agent.
- the anticoagulant include EDTA and the like.
- the protease used in the protease degradation process in the step (A) is a protease of microbial origin such as bacteria and fungi used for degradation of animal protein (acid protease, neutral protease, alkaline protease and Classified).
- microbial origin such as bacteria and fungi used for degradation of animal protein
- proteases of bacterial genus Bacillus origin for example, those marketed under the trade name of alloase
- proteases of fungal genus Aspergillus origin for example, those marketed under the trade name of panidase
- the hypotonic solution used in the bulging treatment in the step (B) is a solution having an osmotic pressure lower than the blood osmotic pressure, non-toxic and harmless to bacteria, and bloating or destroying only human blood cells.
- a solution having an osmotic pressure lower than the blood osmotic pressure non-toxic and harmless to bacteria, and bloating or destroying only human blood cells.
- examples thereof include 4- (2-HydroxyEthyl) -1-piperazine Ethane Sulfonic acid (HEPES), a hypotonic buffer solution containing potassium chloride and magnesium chloride as components.
- the surfactant solution used in the step (C) has an anionic surfactant (D 1 ) having a chain hydrocarbon such as a long chain alkyl group in the hydrophobic part, a structure in the hydrophobic part And a solution containing one or more of surfactants (D 2 ) having a cyclic hydrocarbon such as a steroid nucleus.
- the anionic surfactant (D 1 ) having a chain hydrocarbon such as a long chain alkyl group may be any surfactant of carboxylic acid type, sulfonic acid type and sulfuric acid ester type, and specifically, Sodium dodecyl sulfate (SDS), sodium lauryl sulfate, lithium dodecyl sulfate (LDS), N-lauroyl sarcosine sodium and the like can be mentioned. One or more of them are preferably used.
- the concentration of the total solution of an anionic surface active agent (D 1) is preferably 0.05 wt% or less.
- the surfactant (D 2 ) is a compound having a cyclic hydrocarbon such as a steroid nucleus in the structure, and specifically, saponin, sodium cholate, sodium deoxycholate, 3-[(3-cholamidopropyl) And dimethylammonio] -1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO), and the like.
- Surfactant (D 2) it is may not be used, either individually or in combination of two or more of use are preferred.
- the bacteria have peptidoglycan walls, they are not degraded by protease and do not swell in hypotonic solution.
- damage to bacteria is suppressed.
- platelets and red blood cells contained in minimal amounts are combined in a combination of three actions: decomposition of cell membrane proteins by protease, increase and swelling of internal pressure by hypotonic solution, and emulsification of phospholipids by surfactant solution.
- the cell membrane is broken, human-derived ATP is released to the outside of the cell, and human-derived ATP can be minimized through a washing step and the like, and a pellet of only E. coli can be recovered in a viable state.
- human-derived ATP can be suppressed to about 1 / 2500,000 or less.
- E. coli can be recovered as living bacteria, it can be subjected to the determination of the presence or absence of living bacteria in a sample and a drug sensitivity test. According to the present invention, the determination result of the drug sensitivity test can be obtained in a short time of only about 6 hours after blood collection, and it is expected that it will be useful for the treatment of the early stage of sepsis which greatly affects the prognosis.
- the operation to make platelets and pellets of E. coli from blood samples is shown.
- action is shown.
- the hypotonic solution treatment procedure and the cell swelling photograph are shown.
- action is shown.
- the washing operation of the pellet is shown.
- An example of determination of ATP measurement and drug sensitivity is shown.
- the evaluation results of the ability to recover bacteria are shown.
- (A), (b) shows the determination example of the presence or absence of viable bacteria in the clinical sample.
- (C), (d) and (e) show determination examples of drug sensitivity of clinical samples.
- the sample pretreatment method (BAMB Procedure) and the procedure of ATP measurement are shown.
- the measurement result of ATP background by the difference in the blood cell destruction step of the simple pretreatment protocol with a blood culture sample is shown.
- the determination example of the drug sensitivity of a blood culture sample is shown.
- the determination example of the drug sensitivity of a blood culture sample is shown.
- the determination example of the drug sensitivity of a blood culture sample is shown.
- the determination example of the drug sensitivity of a blood culture sample is shown.
- the determination example of the drug sensitivity of a blood culture sample is shown.
- the sample pretreatment method according to the present invention is expressed as BAMB (Bacterial ATP Measurement in Blood) Procedure.
- BAMB Bacterial ATP Measurement in Blood
- the overall flow of steps is shown in FIG.
- Blood samples are whole blood collected from patients suspected of sepsis at Toyama University Hospital and Nagaresugi Geriatric Hospital, and all procedures in the following examples are approved by Toyama University Ethics Committee and Nagase Geriatric Hospital Ethics Committee As well as with written consent from all patients, and the methods of the examples were performed according to approved guidelines.
- Step 1 Pelletizing Platelets and Bacteria from Whole Blood Remove the red blood cells and white blood cells from the blood sample and make a pellet of platelets (including very small amounts of red blood cells and white blood cells) and fulminant bacteria.
- the blood is collected, for example, with an EDTA blood collection tube containing a separating agent, and the supernatant is obtained by mild centrifugation. The supernatant is transferred to a tube and subjected to strong centrifugation to obtain platelets and E. coli pellet.
- the venous blood total 5mL was collected plasma separation EDTA tubes (Vacutainer (TM) PPT TM Plasma Preparation Tube, BD Biosciences, CA, USA).
- the blood samples were then centrifuged at 1100 ⁇ g for 10 minutes to centrifuge the blood cells, and the resulting plasma and bacterial supernatant fractions (2 mL) were used.
- the supernatant was aliquoted into two portions (1 mL each), centrifuged again at 2000 ⁇ g for 10 minutes, and then 900 ⁇ L of the supernatant fraction was carefully removed to avoid disturbing the pellet.
- Step 2 Protease treatment (proteolysis of platelet cell membrane)> Performs membrane protein degradation of platelets (and very small amounts of red blood cells and white blood cells). Bacteria have proteoglycan walls and are not degraded by proteases. For example, as shown in FIG. 2, protease is added and incubated.
- distilled water Molecular Biology Grade UltraPure TM DNase / RNase-Free distilled water, Thermo Fisher Scientific, Massachusetts, USA, hereinafter, distilled water
- ⁇ Step 3 Treatment with hypotonic solution (platelet swelling)> Increases the internal pressure of platelets (and very small amounts of red blood cells and white blood cells) to make them puffy and fragile. Bacteria do not swell because they have peptidoglycan walls. It is treated with a hypotonic hypotonic solution as shown in FIG. Specifically, distilled water such as 10 mM HEPES (pH 7.9. DOJINDO Molecular Technologies, Tokyo, Japan), 1.5 mM KCl (Wako Pure Chemical Industries, Ltd., Tokyo) and 10 mM MgCl 2 ⁇ 6H 2 O After dissolving in autoclave, autoclave it to make a hypotonic solution of pH 7.9. Then, 800 ⁇ L of a hypotonic solution was added to the pellet and pipetted 10 times, followed by incubation at room temperature for 5 minutes.
- distilled water such as 10 mM HEPES (pH 7.9. DOJINDO Molecular Technologies, Tokyo, Japan)
- Step 4 Detergent Processing> Destroy the cell membrane of platelets (and very small amounts of red blood cells and white blood cells). After hypotonic solution treatment, centrifugation is performed to pellet bacteria, but a step of adding Detergent (surfactant) solution to the hypotonic solution before centrifugation is necessary for bacterial recovery.
- Detergent surfactant
- P. aeruginosa easily adheres to the inner wall of the tube, so when it is centrifuged without Detergent, it can hardly be recovered while adhering to the inner wall. However, such problems do not occur in most other bacterial species.
- Saponin which is a kind of surfactant having cyclic hydrocarbon in the hydrophobic part, acts on cholesterol in human cell membrane so as to be trapped, and loosens (disrupts) the structure of cell membrane.
- SDS which is one type of anionic surfactant having a chain hydrocarbon in the hydrophobic part, emulsifies phospholipids of human cell membrane which is a lipid bilayer, and breaks the cell membrane.
- Step 5 Pellet washing (minimizing the background of human-derived ATP to pellet viable bacteria)> The ATP from the platelets (and very small amounts of red blood cells and white blood cells) and organelles are washed away with the detergent components, and only the fulminant bacteria are recovered in the state of viable bacteria. The flow of the operation is shown in FIG. Specifically, after the incubation of step 4, the mixture was centrifuged at 2000 ⁇ g for 5 minutes, and 1 mL of the supernatant fraction was carefully removed so as not to disturb the pellet.
- Muller-Hinton medium (Muller-Hinton II broth, Cation-adjusted, BD Biosciences) was added to the pellet, mixed gently by inverting several times, and centrifuged again at 2000 ⁇ g for 5 minutes. After centrifugation, 1 mL of the supernatant fraction was carefully removed so as not to disturb the pellet. Thereafter, this pellet washing step was repeated again. That is, 1 mL of Muller-Hinton medium was added to the pellet, mixed gently by inversion, and centrifuged again at 2,000 ⁇ g for 5 minutes. After centrifugation, 1 mL of the supernatant fraction was carefully removed so as not to disturb the pellet. These are bacterial pellets of S. pneumoniae with minimized human-derived ATP background.
- Step 6 Cultivation of E. coli (antibacterial agent ⁇ )>
- the shaking bacteria live bacteria collected from the blood sample are shake cultured in the presence and absence of an antibacterial agent.
- An example of the operation is shown in FIG. Specifically, 1 mL of Muller-Hinton medium was added to the pellet after the BAMB procedure. The mixture of these two tubes was returned to one, and divided into two again to equalize the number of bacteria. Then, shake culture was performed at 37 ° C. for 0, 2, 4, 6, 12, 24 hours in the presence and absence of 2.0 ⁇ g / ml of Levofloxacin (LVFX) (Sigma-Aldrich, USA).
- LVFX Levofloxacin
- Step 7 Measurement of viable ATP after culture of E. coli bacteria and determination of drug sensitivity> At each time point after culture, viable ATP is measured to determine drug sensitivity. Usually, drug sensitivity can be determined at a speed of about 4 hours after culture, that is, about 6 hours after blood collection (including 2 hours of the pretreatment step). An example of the operation is shown in FIG. Specifically, ATP measurement of viable bacteria by ATP bioluminescence was performed according to the following procedure. In addition, the ATP bioluminometer automatic measuring device was used for the measurement of ATP. The ATP bioluminometer automatic measurement device is a prototype model developed by Hitachi, Ltd., which enables ATP measurement at the attomole level to detect single bacteria.
- Reagent for measuring viable cell ATP was used CheckLite TM HS set (Kikkoman Biochemifa, Tokyo, Japan) and ATP standard samples (Kikkoman Biochemifa).
- the CheckLite TM HS set, ATP elimination reagent contains ATP extraction reagent, and bioluminescent reagents.
- distilled water UltraPure TM DNase / RNase-Free distilled water, Thermo Fisher Scientific
- the ATP standard curve preparation used ATP standard sample (Kikkoman Biochemifa), and prepared two dilution series for high concentration measurement and low concentration measurement.
- ATP standard curve preparation used ATP standard sample (Kikkoman Biochemifa), and prepared two dilution series for high concentration measurement and low concentration measurement.
- 100-2 ⁇ 10 9 amol / mL ATP standard samples were prepared by diluting with sterile water.
- 100 to 500 amol / mL of ATP standard sample was prepared by diluting it with sterile water or ATP extraction reagent. Sterile water or ATP extraction reagents were used as blank samples.
- ATP bioluminescence was measured in the following steps.
- ATP removal reagent was diluted to 20% (v / v) with PBS.
- 12.5 ⁇ L of 20% (v / v) ATP removing reagent was added to 12.5 ⁇ L of the bacterial sample after shaking culture. Then, it was incubated for 30 minutes to remove extracellular ATP and killed ATP. After incubation, 25 ⁇ L of ATP extraction reagent was added to bacterial samples or blank samples. The samples were incubated for 1 minute to extract intracellular ATP of viable bacteria. 10 ⁇ L of the sample after intracellular ATP extraction was added to the reaction tube and set in an automatic ATP bioluminometer.
- Viable intracellular ATP in the sample was mixed with the bioluminescent reagent and measured for 30 seconds.
- the relative light intensity (RLU) was calculated as an average of 5 seconds from its maximum intensity within 30 seconds of mixing the sample and the bioluminescent reagent.
- Total bacterial intracellular ATP (amol) was determined by comparing and quantifying the ATP bioluminescence (RLU) of the sample with an ATP calibration curve (RLU / amol).
- FIG. 8 shows the evaluation results of human-derived ATP removing ability.
- FIG. 8 (a) shows photomicrographs of plasma pellets before and after treatment of the process (BAMB procedure) according to the present invention. After treatment it can be seen that all of the blood cells are destroyed and removed.
- FIG. 8 (b) shows the results of ATP measurement of plasma pellet before and after treatment of blood collected from 5 healthy persons. From this graph, it can be seen that human-derived ATP is reduced to about 1 / 2500,000.
- FIG. 9 (Table 1) shows the results of recovery of 19 bacteria which are frequently detected from blood culture of septic blood. The average recovery rate was 93.6% by ATP measurement and 71.9% by CFU measurement.
- Sample (e) A blood sample of a sepsis patient, in which two bacteria of Proteus mirabilis and Enterococcus faecalis sensitive to blood culture positive and antimicrobial agent LVFX are detected are shown. At 4 hours of culture after BAMB procedure, ATP elevation without LVFX and ATP suppression with LVFX were observed. That is, 4 hours after culture (6 hours after blood collection), it was determined by ATP measurement that “a blood-borne bacterium (live bacteria) is present and it is an LVFX-sensitive bacterium”.
- Steps 2 to 4 in Example 1 was changed to Steps 3, 2 and 4, and the other steps were performed three times in the same manner as in Example 1.
- the average of the background (CPS value at 0 hour in FIG. 10) was 476.
- Steps 2 to 4 in Example 1 The order of Steps 2 to 4 in Example 1 was changed to Steps 4, 2 and 3, and the other steps were performed three times in the same manner as in Example 1.
- the average of the background (CPS value at 0 hour in FIG. 10) was 500.
- step 3 of Example 1 The surfactant in step 3 of Example 1 was changed to SDS only, and the other steps were performed three times in the same manner as in Example 1. As a result, the average of the background (CPS value at 0 hour in FIG. 10) was 479.
- a method is shown in which the ATP background is minimized from the blood culture positive bottle and the E. coli is directly recovered as a viable cell. Since blood cell membranes of blood cells are already fragile after blood culture, the protease treatment in step 2 of Example 1 is not necessary, and blood cells are prepared only by a rapid and simple pretreatment method in which a hypotonic solution and Detergent A are mixed. It can destroy (including platelets). Thereafter, the recovered viable bacteria are cultured to conduct a rapid drug sensitivity test by measuring ATP.
- the protocol of this example is described below. 1.
- hypotonic solution + 0.05% SDS (without saponin) or hypotonic solution alone is sufficient to destroy blood cells. It turns out that you can not do it.
- FIG. 14 shows the results of the rapid drug sensitivity test of this example.
- the amount of bacterial ATP increases as the culture time advances regardless of the presence or absence of an antibacterial agent, and it is apparent after 4 hours of culture that the agent is resistant to the antibacterial agent (levofloxacin).
- the conventional test method detected levofloxacin resistant Escherichia coli (E. coli), and the drug sensitivity results were in agreement. From the above, accurate sensitivity results could be obtained in only 4 hours from blood culture positive.
- FIG. 15 shows the results of the rapid drug sensitivity test of this example.
- the amount of bacterial ATP increased without the antibacterial agent, and the amount of bacterial ATP decreased with the antibacterial agent (levofloxacin). That is, it is obvious after 4 hours of culture that it is sensitive to the antibiotic (levofloxacin).
- the conventional test method detected levofloxacin sensitive Streptococcus anginosus, and the drug sensitivity results were in agreement. From the above, accurate sensitivity results could be obtained in only 4 hours from blood culture positive.
- FIG. 16 shows the results of the rapid drug sensitivity test of this example.
- the amount of bacterial ATP increases as the culture time progresses regardless of the presence or absence of an antibacterial agent, and it is apparent after 4 to 6 hours of culture that the agent is resistant to the antibacterial agent (levofloxacin).
- the antibacterial agent levofloxacin
- ATP a rapid drug sensitivity test using ATP measurement in blood samples of suspected sepsis patients.
- the presence or absence of viable bacteria in a blood sample can be used to conduct a rapid determination by ATP measurement (which can compensate for weaknesses in genetic testing). It can be used to conduct a rapid drug sensitivity test using ATP measurement in blood culture samples from blood culture bottles.
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Abstract
Description
現行の薬剤感受性試験は菌の濁度で判定するため、先ず起炎菌を分離培養し、次に薬剤感受性試験にて菌の十分な増殖を待たなければならず、通常、採血から2~3日を要する。
それに対し、菌が産生するATPを測定することによって、細菌コロニーからスタートする薬剤感受性試験の迅速化が報告されている。(非特許文献1)
同報告によれば従来法が細菌コロニーのスタートから18~24時間要するのに対して、ATP測定ではおよそ6時間で可能とされている。
しかし上記方法はコロニーからのスタートであり、このコロニーを得るまでの分離培養に1~2日間は必要であるため、採血からの時間としては迅速であるとは言いがたい。
しかし、SDSの濃度が0.1%,0.2%のように高濃度であると、例えば敗血症の起炎菌として度々検出される[Streptococcus agalactiae]の場合に殆どが死滅して生菌として回収できなかった。
一方、SDSの濃度を0.05%まで下げると、上記菌の生菌回収率が向上したが、血液中の血小板は殆ど破壊されないため、ヒト由来のATPを最小化することができず生菌をATP測定できなかった。
(A)血小板の細胞膜蛋白をプロテアーゼ分解処理する。
(B)血小板を低張液にて膨化処理する。
(C)起炎菌への影響を抑えた条件下で、血小板の細胞膜を界面活性剤溶液にて破壊処理する。
本発明にて起炎菌への影響を抑えるとは、生菌としての回収率が敗血症起炎菌の主要19菌属の平均で50%以上であることをいう。
抗凝固剤としては、例えばEDTAなどが挙げられる。
具体的には、細菌のバシラス属起源のプロテアーゼ(例えば、アロアーゼの商品名で市販されているもの)、真菌のアスペルギルス属起源のプロテアーゼ(例えば、パンチダーゼの商品名で市販されているもの)などが挙げられる。
それらは、1種あるいは2種以上の使用が好ましい。
また、陰イオン性界面活性剤(D1)の全溶液中の濃度は、0.05重量%以下であることが好ましい。
界面活性剤(D2)は使用しなくてもよいが、1種あるいは2種以上の使用が好ましい。
また、界面活性剤溶液による処理においては、細菌へのダメージを抑えている。
これらにより、血小板及び極小量含まれている赤血球や白血球が、プロテアーゼによる細胞膜蛋白の分解、低張液による内圧の上昇と膨化、界面活性剤溶液によるリン脂質の乳化、の3つの作用の組み合わせにて細胞膜が破壊され、ヒト由来のATPが細胞外に放出され、洗浄工程などを経て、ヒト由来のATPを最小化し起炎菌のみのペレットを生菌の状態で回収することができる。
本発明においてヒト由来のATPを最小化するとは、起炎菌のATP測定に影響を与えない程度にヒト由来のATPを除去することをいう。
本発明においては、ヒト由来のATPを約1/2,500,000以下に抑えることができる。
また、起炎菌を生菌として回収できるので、検体中の生菌の有無判定や薬剤感受性試験に供することができる。
本発明により、採血から僅か約6時間程度の短時間で薬剤感受性試験の判定結果が得られるようになり、予後を大きく左右する敗血症早期の治療に役立つことが期待される。
なお、本発明に係る検体前処理方法をBAMB(Bacterial ATP Measurement in Blood)Procedureと表現した。
全体のステップの流れを図12に示す。
ここで、ステップ2~4の順序は問わない。
血液検体は、富山大学病院と流杉老人病院において、敗血症が疑われる患者から採取した全血であり、以下の実施例のすべての手技は、富山大学倫理委員会および流杉老人病院倫理委員会の承認並びにすべての患者から文書による同意を得てられ行われ、また実施例の方法は、承認されたガイドラインに従って実施された。
<ステップ1:全血からの血小板および細菌のペレット化>
血液検体から赤血球・白血球を取り除き、「血小板(極少量の赤血球や白血球を含む)と起炎菌のペレット」を作る。
図1に示すように、例えば分離剤入りEDTA採血管で採血し、軽度の遠心操作にて上清を得る。
この上清をチューブに移し、強い遠心操作を行うことで血小板と起炎菌のペレットを得る。
具体的には、血漿分離EDTAチューブ(Vacutainer(登録商標)PPTTM Plasma Preparation Tube、BD Biosciences、CA、USA)に合計5mLの静脈血を採取した。
その後、血液サンプルを1100×gで10分間遠心分離して血球を遠心分離し、得られた血漿および細菌である上清画分(2mL)を使用した。
上清を等分して2つの部分(それぞれ1mL)に分け、2000×gで10分間再度遠心分離し、次いで上清画分900μLを慎重に取り出してペレットを乱さないようにした。
血小板(および極少量の赤血球や白血球)の細胞膜蛋白の分解を行う。
細菌はプロテオグリカン壁を持つため、プロテアーゼでは分解されない。
図2に示すように例えば、プロテアーゼを加え、インキュベートする。
具体的には、分子生物学研究グレードの蒸留水(UltraPureTM DNase / RNase-Free蒸留水、Thermo Fisher Scientific、Massachusetts、USA、以下、蒸留水)で溶解したプロテアーゼ(Aroase NP-10:ヤクルト製薬工業、東京、日本)1mL(10%w/v)をペレットに添加し、10回ピペッティングした後、37℃で10分間インキュベートした。
インキュベーション後、混合物を2000×gで5分間遠心分離し、次いでペレットを乱さないように上清画分1mLを注意深く除去した。
血小板(および極少量の赤血球や白血球)の内圧を高め、膨化させ壊れ易くする。
細菌はペプチドグリカン壁を持つため、膨化されない。
図3に示すように低浸透圧の低張液にて処理する。
具体的には、10mM HEPES(pH7.9.DOJINDO Molecular Technologies、Tokyo、Japan)、1.5mM KCl(和光純薬工業(株)、東京)および10mM MgCl2・6H2Oとなるように蒸留水で溶解した後、オートクレーブしてpH7.9の低張液を作成する。
そして、低張液800μLをペレットに加えて10回ピペッティングした後、室温で5分間インキュベートした。
血小板(および極少量の赤血球や白血球)の細胞膜を破壊する。
低張液処理後、菌をペレット化するために遠心操作を行うが、遠心前に低張液にDetergent(界面活性剤)溶液を加える工程が細菌の回収にとって必要である。
例えば、緑膿菌はチューブ内壁に付着しやすいため、Detergent無しで遠心すると、内壁に付着したままで殆ど回収できない。
但し、他の殆どの菌種ではそのような問題は生じない。
疎水性部分に環状炭化水素を有する界面活性剤の1種であるサポニンはヒト細胞膜のコレステロールに作用して楔を打つように入り込み、細胞膜の構造を緩める(破壊する)。
一方、細菌の細胞壁はペプチドグリカン層であるため、サポニンは作用しない。
疎水性部分に鎖状炭化水素を有する陰イオン性界面活性剤の1種であるSDSは脂質二重層であるヒト細胞膜のリン脂質を乳化して細胞膜を破壊する。
SDSは細菌の細胞壁をも障害するが、例えばSDS濃度を0.05%まで薄めれば、Detergent処理で死滅し易いStreptococcus agalactiaeでさえ殆ど死滅しない。
0.05%の低濃度で処理することで、細菌への影響を最小化してヒト細胞膜の破壊を主とした作用をもたらす。
その操作の流れを図4に示す。
具体的には、ステップ3のインキュベーション後、2%サポニン(SERVA Electrophoresis GmbH、Heidelberg、Germany)および0.05%SDS(和光純薬工業(株)製)をpH7.4のリン酸緩衝食塩水(PBS:Thermo Fisher Scientific)で溶解したDetergent A 200μLを低張液・ペレット混合液に加え、混合物を10回ピペッティングした後、2,000×gで5分間遠心分離し、ペレットを乱さないように注意深く1mLの上清画分を除去した。
次に、1mLのDetergent Aをペレットに再度加え、混合物を20回ピペッティングし、続いて室温で15分間インキュベートした。
血小板(および極少量の赤血球や白血球)由来のATPや細胞小器官をDetergent成分と共に洗い流し、起炎菌のみを生菌の状態で回収する。
その操作の流れを図5に示す。
具体的には、ステップ4のインキュベーション後、混合物を2000×gで5分間遠心分離し、ペレットを乱さないように上清画分1mLを注意深く除去した。
次に、ペレットにMuller-Hinton培地(Muller-Hinton IIブロス、Cation-adjusted、BD Biosciences)1mLを加え、数回穏やかに転倒混和した後、再び2000×gで5分間遠心分離した。
遠心分離後、ペレットを乱さないように、1mLの上清画分を注意深く除去した。
その後、このペレット洗浄工程を再度繰り返した。
すなわち、ペレットに1mLのMuller-Hinton培地を加え、穏やかに転倒混和した後、再び2,000×gで5分間遠心分離した。
遠心分離後、ペレットを乱さないように、1mLの上清画分を注意深く除去した。
これらは、ヒト由来のATPバックグラウンドを最小化した敗血症起炎菌の細菌ペレットである。
血液検体より回収された起炎菌(生菌)を抗菌薬存在下、および非存在下で振盪培養する。
その操作例を図6に示す。
具体的には、BAMB procedureの後、1mLのMuller-Hinton培地をペレットに添加した。
これら2チューブの混合物を1つに戻し、再び2等分に分割して菌数を揃えた。
その後、Levofloxacin(LVFX)(Sigma-Aldrich、USA)2.0μg/ml存在下、非存在下の各々の条件で、37℃、0,2,4,6,12,24時間の振盪培養を行った。
培養後の各タイムポイントで生菌ATPを測定し、薬剤感受性を判定する。
通常、培養後4時間程度、すなわち採血後6時間程度(前処理工程の2時間含む)の迅速さで薬剤感受性が判定可能。
その操作例を図7に示す。
具体的には、ATPバイオルミネッセンスによる生菌のATP測定を以下の手順で行った。
なお、ATPの測定にはATPバイオルミノメーター自動測定装置を使用した。
ATPバイオルミノメーター自動測定装置は日立製作所が開発したプロトタイプモデルであり、単菌を検出するためのattomoleレベルでのATP測定を可能とする。
CheckLiteTM HSセットには、ATP除去試薬、ATP抽出試薬、および生物発光試薬が含まれている。
洗浄液と希釈用緩衝液として、蒸留水(UltraPureTM DNase / RNase-Free蒸留水、Thermo Fisher Scientific)を用いて、それぞれ細菌試料とATP標準サンプルの希釈系列を作成した。
高濃度測定用には100~2×109amol/mLのATP標準サンプルを滅菌水で希釈することによって調製した。
低濃度測定用には100~500amol/mLのATP標準サンプルを滅菌水またはATP抽出試薬で希釈することにより調製した。
ブランクサンプルとして滅菌水またはATP抽出試薬を使用した。
ATP生物発光は以下のステップで測定した。
まず、反応チューブ内に滅菌水またはATP抽出試薬で希釈したATP標準サンプル10μLを、試薬チューブ内に生物発光試薬1mLを、それぞれATPバイオルミノメーター自動測定装置にセットした。
そして、ATPバイオルミノメーター自動測定装置により50μLの生物発光試薬をサンプルに添加した。
まずATP除去試薬をPBSで20%(v/v)に希釈した。
次に振盪培養後の細菌試料12.5μLに20%(v/v)ATP除去試薬12.5μLを添加した。
そして細胞外ATPおよび死菌ATPを除去するために30分間インキュベートした。
インキュベーション後、25μLのATP抽出試薬を細菌試料またはブランク試料に添加した。
生菌の細胞内ATPを抽出するために、試料を1分間インキュベートした。
細胞内ATP抽出後の試料10μLを反応チューブに加え、ATPバイオルミノメーター自動測定装置にセットした。
試料中の生菌細胞内ATPを生物発光試薬と混合し、30秒間測定した。
相対光強度(RLU)は、試料と生物発光試薬を混合した後30秒以内にその最大強度から5秒間の平均値として計算した。
サンプルのATP生物発光(RLU)をATP検量線(RLU/amol)で比較定量することにより、全細菌細胞内ATP(amol)を決定した。
図8(a)は、本発明に係るプロセス(BAMB procedure)の処理前と処理後の血漿ペレットの顕微鏡写真を示す。
処理後には、血球のすべてが破壊され、除去されているのが分かる。
図8(b)は、健常人5人から採血した血液の処理前後における血漿ペレットのATP測定結果を示す。
このグラフから、ヒト由来のATPが約1/2,500,000に低下しているのが分かる。
その平均回収率はATP測定で93.6%,CFU測定にて71.9%であった。
検体(a),(b):敗血症疑い患者の血液検体で、血液培養陰性であった例を示す。
BAMB procedure後の培養時間経過でATPの上昇は認められず、血液中に生菌がいないことが分かる。
検体(c),(d):敗血症患者の血液検体で、血液培養陽性、抗菌薬のLVFX(レボフロキサシン)に耐性のE.coli(c)とMRSA(d)とが検出された例を示す。
BAMB procedure後の培養4時間でLVFX±に関わらずATPの上昇が認められた。
つまり、培養後4時間(採血後6時間)で「血液中に起炎菌(生菌)が存在し、それがLVFX耐性菌である」ことがATP測定で判定された。
検体(e):敗血症患者の血液検体で、血液培養陽性、抗菌薬のLVFXに感受性のあるProteus mirabilisとEnterococcus faecalisの2菌が検出された例を示す。
BAMB procedure後の培養4時間でLVFX無しでATP上昇、LVFXありでATP抑制が認められた。
つまり、培養後4時間(採血後6時間)で、「血液中に起炎菌(生菌)が存在し、それがLVFX感受性菌である」ことがATP測定で判定された。
その結果、バックグラウンド(図10において0時間のCPS値)の平均は476であった。
その結果、バックグラウンド(図10において0時間のCPS値)の平均は500であった。
その結果、バックグラウンド(図10において0時間のCPS値)の平均は479であった。
本実施例では、血液培養陽性となったボトルからATPバックグラウンドを最小化して起炎菌を生菌の状態で直接回収する方法を示す。
血液培養した後では血球の細胞膜が既に壊れ易くなっているため、実施例1のステップ2におけるプロテアーゼ処理を必要とせず、低張液とDetergent Aを混合した迅速・簡便な前処理方法のみで血球(血小板を含む)を破壊することができる。
その後、回収した生菌を培養してATP測定による迅速薬剤感受性試験を行う。
以下、本実施例のプロトコールを記載する。
1.培養検体(血液培養陽性化から1時間後)100μlを1.5mlのエッペンチューブに採る。
2.低張液800μl+Detergent A 200μlを添加し、20回ピペッティング。それから遠心(2000×G,10分間,室温)して菌のペレット化を行う。
3.上清1000μlを除去
4.ミューラーヒントン培地を用いて3000倍希釈
5.1.5mlのエッペンチューブ2本それぞれに希釈した産物を270μlずつ添加
6.エッペンチューブ2本それぞれにATP消去試薬30μlを添加
7.エッペンチューブの1本にはレボフロキサシン(LVFX 20μg/ml)を33.3μl添加、もう1本には超純水33.3μlを添加して、それぞれ35℃で培養
8.測定ポイントは2時間,4時間,6時間,(24時間)
9.培養後、1ポイントあたり10μlずつ3回サンプルを採取
10.ATP抽出試薬10μlを添加してボルテックス後にATP量をそれぞれ測定
本プロトコールにおいてもDetergent Aは必須である(図13)。
表2に示すように、健常人血液を4時間血液培養してヒト由来ATPバックグラウンドを測定した結果、低張液+0.05%SDS(サポニン無し)、或いは低張液のみでは血球を十分破壊することは出来ないことが分かる。
菌ATP量は抗菌薬有り・無しに関わらず培養時間が進むにつれて増加しており、抗菌薬(レボフロキサシン)耐性であることが培養4時間後には明らかである。
実際、従来の検査法ではレボフロキサシン耐性のEscherichia coli(大腸菌)が検出され、薬剤感受性結果は一致した。
以上により、血液培養陽性時より僅か4時間で正確な感受性結果を得ることが出来た。
実施例5と同じプロトコール(但し血液培養陽性化から4時間後の培養検体を使用)で、実施例5とは別な敗血症患者の血培ボトルから迅速薬剤感受性試験を行った。
図15に本実施例の迅速薬剤感受性試験結果を示す。
培養時間が進むにつれ、抗菌薬無しでは菌ATP量は増加、抗菌薬(レボフロキサシン)有りでは菌ATP量は減少した。
つまり、抗菌薬(レボフロキサシン)感受性であることが培養4時間後には明らかである。
実際、従来の検査法ではレボフロキサシン感受性のStreptococcus anginosusが検出され、薬剤感受性結果は一致した。
以上により、血液培養陽性時より僅か4時間で正確な感受性結果を得ることが出来た。
実施例5と同じプロトコール(但し血液培養陽性化から5時間後の培養検体を使用)で、実施例5,6とは別な敗血症患者の血培ボトルから迅速薬剤感受性試験を行った。
図16に本実施例の迅速薬剤感受性試験結果を示す。
菌ATP量は抗菌薬有り・無しに関わらず培養時間が進むにつれて増加しており、抗菌薬(レボフロキサシン)耐性であることが培養4~6時間後には明らかである。
但し抗菌薬有り・無しで菌ATP量の増加に差があるため、抗菌薬耐性菌と感受性菌とが混合していた可能性がある。従来の検査法ではレボフロキサシン耐性のStaphylococcus capitis or Staphylococcus capraeが検出され、薬剤感受性結果は一致した。
以上、血液培養陽性時より僅か4~6時間で正確な感受性結果を得ることが出来た。
血液検体中の生菌の有無について、ATP測定により迅速判定(遺伝子検査の弱点を補える)の実施に利用できる。
血液培養ボトルからの血液培養検体における、ATP測定を用いた迅速薬剤感受性試験の実施に利用できる。
血液検体以外にも、脳脊髄液(細菌性髄膜炎)、心嚢水(心膜炎)、胸水(胸膜炎)、腹水(腹膜炎)、関節包液(整形外科の術後感染症)、眼房水(眼内炎)、肺胞洗浄液(肺炎)、尿(尿路感染症)、術後のドレーン排液(術後感染症)、CVカテーテル先端(長期臥床患者のカテ先バイオフィルムによる敗血症)などで、生菌の有無の迅速判定、および迅速薬剤感受性試験等の実施に利用できる。
Claims (6)
- 血液中の起炎菌をATP測定するための血液検体の前処理方法であって、
血液検体から血小板と起炎菌のペレットを作成するステップと、
前記血小板と起炎菌のペレットを、下記(A)~(C)のステップを任意の順であるいは、
複数のステップを同時に処理するステップとを、
有することを特徴とする血液検体の前処理方法。
(A)血小板の細胞膜蛋白をプロテアーゼ分解処理する。
(B)血小板を低張液にて膨化処理する。
(C)起炎菌への影響を抑えた条件下で、血小板の細胞膜を界面活性剤溶液にて破壊処理する。 - 前記血小板と起炎菌のペレットは、抗凝固剤入り採血管で採血した血液検体から遠心操作又は分離剤により、赤血球及び白血球を除いた上清を遠心操作にてペレット化したものである請求項1記載の血液検体の前処理方法。
- 前記ステップ(A)のプロテアーゼ分解処理に用いるプロテアーゼは、微生物起源のプロテアーゼである請求項1又は2記載の血液検体の前処理方法。
- 前記ステップ(B)に用いる低張液は、浸透圧が、血液浸透圧より低い液である請求項1~3のいずれかに記載の血液検体の前処理方法。
- 前記ステップ(C)に用いる界面活性剤溶液は、疎水性部分に鎖状炭化水素を有する陰イオン性界面活性剤(D1)、疎水性部分に環状炭化水素を有する界面活性剤(D2)のうち1種以上である請求項1~4のいずれかに記載の血液検体の前処理方法。
- 血液培養検体の前処理においては、請求項1に記載の前処理において(A)のステップを行わず、(B)と(C)とのステップのみを同時に処理する簡易ステップであることを特徴とする血液培養検体の前処理方法。
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