Patent Application
20230167480
The present invention relates to an automatic analyzer that analyzes a sample containing bacteria and impurities.
Sepsis is a highly lethal infection, and it is important to promptly perform diagnosis and appropriate treatment based thereon. When sepsis is determined, a blood culture test is usually performed. This is to determine whether bacteria are present in blood that is a sterile sample. Generally, a smear test is then performed, and then an identification test and a sensitivity test are performed. In the identification test, a blood culture positive sample is isolated and cultured, and the type of bacteria is specified for the obtained colony. The sensitivity test measures the sensitivity of the bacteria to antimicrobials. The series of tests described above requires one day for the blood culture test, one day for the isolation and cultivation, and one day for the sensitivity test, and thus requires a total test time of two to three days. That is, it currently takes two to three days to determine whether treatment with an appropriate antimicrobial has been performed. Therefore, if an ineffective antimicrobial has been administered so far, the lethality of sepsis is extremely high.
In the blood culture test, very few bacteria, on the order of about 10 CFU/mL (CFU: colony forming unit) included in the case of sepsis, are grown in a culture bottle. In general, culture is performed for about 8 hours to overnight, and bacteria are grown until a gas component or the like generated by respiration, fermentation or the like of bacteria reaches a detectable level. It is known that a culture bottle positive for blood culture contains 106 to 1010 CFU/mL of bacteria. Since the concentration of bacteria at the time of blood culture positive varies depending on the condition of the blood of a patient, bacterial species, a blood culture test device and the like, the concentration of bacteria is in such a wide range. Main components other than bacteria in the blood culture bottle include a resin, beads, activated carbon and the like that adsorb antibiotics, in addition to blood cell components and a medium. Among them, the concentration of red blood cells and white blood cells present in blood is high, about 109 cells/mL and 107 cells/mL, respectively, and is equal to or higher than the concentration of bacteria.
In the isolation and cultivation, a sample positive for blood culture is applied to an agar medium to grow colonies. By predicting the type of bacteria from the properties of colonies and the like and more reliably identifying the type of bacteria, and by preparing a bacterial suspension from colonies, it is possible to obtain a sample having no impurities other than bacteria and having a bacterial concentration (generally 105 to 106 CFU/mL) necessary for sensitivity test.
In the sensitivity test, generally, a certain concentration of an antimicrobial is introduced into a bacterial suspension containing bacteria, and the degree of growth of the bacteria is determined according to the concentration of the antimicrobial. Since the result of the sensitivity test varies, it is important to adjust in advance the bacterial concentration in the bacterial suspension to be constant. Regarding sensitivity test, studies to speed up the time until the end of test are currently in progress. The current golden standard method measures the degree of growth of bacteria by a change in turbidity, and it takes a whole day for the test. Currently, a method of more rapidly determining turbidity change using laser light, a method of rapidly determining the degree of growth of individual bacteria by a microscope, a method of rapidly quantifying the degree of growth of bacteria by ATP (adenosine triphosphate) light emission and the like are being developed, and the time required for sensitivity test may be shortened to about several hours. On the other hand, as a pretreatment step for preparing a bacterial suspension, a method of performing isolation and cultivation for one day and diluting colonies in a liquid is still used.
In contrast, if a bacterial suspension with a certain concentration of 105 to 106 CFU/mL can be prepared in a short time by removing components other than bacteria (for example, blood cell components, impurities contained in the culture medium, and the like) from the blood culture-positive sample without performing the isolation and cultivation, the time required for the sensitivity test is further shortened by one day.
In response to such a problem, PTL 1 discloses a method of selectively destroying only blood cell components without affecting growth of bacteria by using two different surfactants. PTL 2 discloses a method of decomposing a blood cell by a protease, dilating the blood cell with a hypotonic solution, and selectively destroying only a blood cell component using a surfactant.
PTL 3 discloses a method of fluorescently labeling bacteria captured on a membrane filter and detecting the number and concentration of bacteria. According to this method, even when impurities other than bacteria are contained, the concentration of bacteria can be measured.
PTL 1: JP 2014-235076 A
PTL 2: WO 2019/097752
PTL 3: JP 2007-006709 A
However, PTLs 1 to 2 do not disclose a means for adjusting the concentration of bacteria to be constant. For example, in general, when a bacterial suspension is prepared from colonies, it is possible to adjust the concentration of bacterial suspension based on the value of turbidity. However, since an absorption wavelength of a blood cell component such as a red blood cell, a white blood cell or a platelet, hemoglobin contained in a large amount in the blood cell or the like is the same as a wavelength band used for measuring scattered light of bacteria, adjustment by turbidity is difficult.
In PTL 3, it is necessary to treat bacteria with a staining reagent for fluorescent labeling, and there is a possibility that the properties of bacteria are changed by being exposed to the reagent during the treatment, and the result of the sensitivity test is affected. Therefore, the method requiring such a staining step is difficult to apply for sensitivity test. In addition, there is also a problem that reagent cost is high and a dedicated expensive optical system by fluorescence excitation is required.
The present invention has been made in view of such a situation, and provides a technique of estimating a bacterial concentration in a sample in which impurities such as bacteria and blood cells are mixed, and adjusting the sample to a desired bacterial concentration.
An automatic analyzer according to the present invention introduces a substance that destroys impurities into a sample in which bacteria and the impurities are mixed, separates the destroyed impurities and the bacteria, and then takes out the bacteria by a filter, and estimates the concentration of bacteria in the sample according to correspondence data between the amount of impurities remaining on the filter and the concentration of bacteria in the sample.
According to the automatic analyzer of the present invention, the bacterial concentration in the sample can be estimated from the sample in which bacteria and impurities are mixed, and the sample can be adjusted to a desired bacterial concentration. As a result, the sensitivity test can be accurately performed. Problem, configuration, and effect other than those described above will be revealed from the description of the following embodiments.
In step S10, a surfactant is added to the blood sample to destroy blood cells. As the amount of blood to be treated increases, the number of bacteria finally obtained increases, and thus it is preferable to pretreat a larger sample. However, since the amount of waste liquid also increases, it is preferable to pretreat about several mL to 10 mL per sample. The surfactant is preferably (a) an anionic surfactant having hydrophilic and hydrophobic moieties and the hydrophobic moiety being a chain hydrocarbon, or (b) a surfactant having hydrophilic and hydrophobic moieties and the hydrophobic moiety having a cyclic hydrocarbon, or a combination of (a) and (b). Specifically, the former includes sodium dodecyl sulfate, lithium dodecyl sulfate, and sodium N-lauroyl sarcosine, and the latter includes saponin, sodium cholate, sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate, and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 propanesulfonate. The next step S11 may be performed immediately after the addition of the surfactant, but the reaction may be allowed to stand for about 5 to 15 minutes to wait for completion of the reaction.
In step S11, centrifugation is performed in order to remove components in the blood cells destroyed by the surfactant and flowed out, for example, hemoglobin and the like, and then, the supernatant is removed and cleaned. In this step, for example, it is preferable to perform centrifugation at 2000 G for about 5 to 10 minutes. However, it is sufficient as long as bacteria and blood cell components that have not been destroyed by the surfactant and hemoglobin and the like that have flowed out can be separated, and the centrifugation speed and the centrifugation time are not limited thereto. The cleaning is performed using pure water, physiological saline or the like, and may be performed only once or a plurality of times.
In step S12, the sample is filtered by a filter in order to further remove the blood cell component that could not be destroyed by the surfactant and impurities in the medium. By using a filter with a pore size (mesh interval) larger than that of bacteria, bacteria are allowed to pass through, and impurities other than bacteria are captured by the filter. For example, it is preferable to use a filter with a pore size of 1 to 40 μm. When the amount of impurities is large, filtration may be performed a plurality of times, such as filtration with a filter with a large filtration pore size and then filtration with a filter with a small filtration pore size. In order to prevent bacteria from being captured in the filter, it is preferable to use a filter made of a hydrophobic material. By the above steps S10 to S12, it is possible to remove impurities other than bacteria from the blood sample and to extract bacteria.
In Embodiment 1 of the present invention, a method is shown that obtains a sample having a bacterial concentration adjusted to a desired value from a blood sample containing bacteria. Note that the present embodiment is merely an example, and is not limited to this configuration. Pretreatments of S10 to S12 were performed using blood samples containing E. coli and S. aureus. The blood samples were prepared according to the following procedure. Into a blood culture bottle containing drug-adsorbing beads, 10 mL of blood derived from a healthy volunteer and 0.1 mL of a bacterial suspension whose concentration had been previously adjusted to about 150 CFU/mL from colonies were introduced to prepare blood equivalent to that of an actual sepsis patient. Thereafter, the sample was introduced into a blood culture device and cultured, and when the blood culture became positive, the sample was taken out and used for an experiment. In addition, a sample corresponding to a negative control having a blood bacterial concentration of 0 CFU/mL cultured without introducing the bacterial suspension was also prepared, and the bacterial concentration was changed by appropriately diluting the sample.
Since the redness of the filter increases according to the amount of impurities such as red blood cells contained in the sample, the reason why the result of
The specific concentration of surfactant used in the treatment can be defined in the following range. Usually, the concentration of red blood cells in blood is on the order of 109/mL, and if a 1 mL sample is pretreated, the number of red blood cells in the sample is 109. Here, the range of impurities that can be estimated from the tone of the filter shown in
Note that the range of the surfactant concentration shown here is, for example, a case where 1 mL of sample is pretreated, and when the volume to be treated increases, it is preferable to increase the concentration of the surfactant so that the destruction rate of red blood cells increases accordingly. For example, when 10 mL of sample is pretreated, a surfactant having a concentration capable of destroying 99 to 99.99% of red blood cells is required, and the concentration may vary depending on the throughput of the sample.
Specifically, the concentration of the surfactant capable of destroying 99 to 99.99% of red blood cells in blood containing bacteria is in the range of 0.05 wt % to 0.5 wt %, assuming an example of sodium dodecyl sulfate, which is an anionic surfactant having hydrophilic and hydrophobic moieties and the hydrophobic moiety being a chain hydrocarbon. Regarding other surfactants such as lithium dodecylsulfate and sodium N-lauroyl sarcosine, the concentration of the surfactant is preferably a concentration capable of destroying a desired red blood cell.
As other surfactants, for example, regarding saponin, sodium cholate, sodium deoxycholate, 3-[(3-cholamidopropyl)dimethylammonio]-1 propanesulfonate, and 3-[(3-cholamidopropyl)dimethylammonio]-2 hydroxy-1 propanesulfonate, which are surfactants having hydrophilic and hydrophobic moieties and a cyclic hydrocarbon as a hydrophobic moiety, the types of surfactants may be mixed.
In Embodiment 1, 2.7 mL of blood sample was treated with a surfactant such as sodium dodecyl sulfate having a final concentration in the range of 0.05 wt % to 0.5 wt %, capable of destroying 99 to 99.99% of red blood cells in blood containing bacteria.
In step S50, the filter is imaged, and the color of the impurities remaining on the filter is acquired. In this flowchart, since the amount of impurities is estimated based on the color of red blood cells remaining on the filter, it is not necessary to stain a sample and impurities.
In step S51, correspondence data describing correspondence between the color of the impurities remaining on the filter and the actual blood bacterial concentration is read out, and the estimated blood bacterial concentration is calculated by referring to the correspondence data using the color acquired from the filter image. Specifically, regarding the color of the impurities remaining on the filter and the actual blood bacterial concentration, for example, a calibration curve represented by a single logarithm is acquired, and the blood bacterial concentration estimated from the data of the calibration curve is calculated. The correspondence data is illustrated in
In step S52, the sample is diluted to obtain a desired bacterial concentration based on the blood bacterial concentration estimated in S51. For example, when the blood bacterial concentration estimated in S51 is 5×108 CFU/mL, if the desired bacterial concentration is 5×105 CFU/mL, 1000-fold dilution is performed. When the blood bacterial concentration estimated in S51 has not reached the desired bacterial concentration, it is preferable to proceed to step S53 and determine that the specimen is a defective specimen. When it is determined as a defective specimen, it is difficult to prepare a specimen suitable for sensitivity test, and thus the blood culture bottle is further cultured to grow the bacteria, and then the process returns to step S10. Alternatively, an identification test or a sensitivity test may be performed using colonies obtained by performing isolation and cultivation.
In step S54, an identification test and a sensitivity test are performed using the prepared bacterial suspension. Any method may be used as the inspection method. Examples thereof include such as an identification test using an automatic device, a genetic test, a sensitivity test by a microliquid dilution method, a sensitivity test by a disk method, and a rapid sensitivity test by a microscopic image, laser scattered light measurement or the like.
In Embodiment 1, the sample after destroying the blood cells is filtered by the filtration filter, and the estimated blood bacterial concentration is calculated by referring to the correspondence data between the color component of the image of the impurities remaining on the filter and the actual blood bacterial concentration. This makes it possible to create a sample having a desired bacterial concentration without performing isolation and cultivation. Therefore, the isolation and cultivation step that usually requires about one whole day can be shortened to, for example, about 30 minutes.
In Embodiment 2, an automatic analyzer for obtaining a sample having a bacterial concentration adjusted to a desired value from a blood sample containing bacteria is shown. Note that the present embodiment is merely an example, and is not limited to this configuration.
The sample into which the surfactant is introduced is introduced into a centrifugal separator 102. The blood cells in the sample are destroyed by the surfactant. The centrifugal separator 102 separates the eluted hemoglobin and the like from bacteria, blood cells that could not be destroyed, and the like. As a result, the separation step of S11 is performed.
The sample that has been centrifuged is introduced into a cleaning unit 103. The cleaning unit 103 removes the supernatant of the sample, and cleans the sample with, for example, about 1 mL of physiological saline or pure water, or a cleaning liquid such as a medium. A cleaning pipette 104 aspirates the supernatant. A portion from the bottom of the sample container to a certain reference height may be treated as the supernatant. As a result, the rest of S11 is performed. More strictly, it is preferable to provide a liquid position sensor or the like, detect an interface between a portion where bacteria and blood cells are coagulated into a pellet form and a liquid portion, and treat a portion up to the vicinity of the interface position as the supernatant.
The cleaned sample is introduced into a filtration filter unit 105. The filtration filter unit 105 consists of, for example, a disposal filtration filter, a syringe for capturing impurities in the filter, and the like. S12 is performed by the filtration filter unit 105. In some cases, the sample may be filtered using the centrifugal separator 102.
A camera 106 (corresponding to a sensor that detects the amount of impurities) images impurities remaining on the filtration filter unit 105. A storage unit 107 stores the correspondence data described in
The filtered sample is introduced into a dilution unit 108. The dilution unit 108 adjusts the dilution ratio so as to obtain a desired bacterial concentration on the basis of the blood bacterial concentration estimated by the computer 110. A dilution pipette 109 introduces the diluent according to its dilution ratio. Accordingly, S52 is performed.
The computer 110 automatically performs the above steps by controlling each unit included in the automatic analyzer 100. It is preferred that the computer 110 includes an input/output device and the operator can instruct the computer 110 about the type of bacteria, the desired bacterial concentration, and the like. The computer 110 can change the correspondence data to be referred according to the input bacteria type, and can also change the dilution ratio by the dilution unit 108 according to the desired bacterial concentration. The blood bacterial concentration value estimated by the computer 110 may be output to an output device such as a display, and when the blood bacterial concentration value is equal to or less than the desired bacterial concentration, a flag indicating a defective specimen may be displayed (S53).
The automatic analyzer 100 may include a sensitivity test device 111 in addition to the above configuration. The computer 110 automatically performs the sensitivity test by controlling the sensitivity test device 111. As a result, all processes from S10 to S54 can be automatically performed. As the contents of the sensitivity test, in addition to those described in Embodiment 1, the minimum inhibitory concentration and the like described in Examples described later can also be measured.
It is considered that the method for adjusting the blood bacterial concentration in the blood sample described in Embodiments 1 to 2 is influenced to some extent by the amount of red blood cells contained in the original blood. The concentration of human red blood cells varies depending on sex and health condition, but is about 3×109 to 6×109/mL, and the variation is extremely small as compared with a bacterial concentration range of 106 to 1010/mL. Therefore, it is considered that the influence on the result of the estimated blood bacterial concentration is small. However, in a case where another detection result of the amount of impurities such as the red blood cell concentration or the hematocrit value is obtained in advance by another blood cell analysis or the like, the result of step S51 can be corrected using the value. As a result, the blood bacterial concentration estimated more accurately can be calculated. As the correction procedure, for example, the following ones are conceivable.
(Correction procedure 1) When the blood bacterial concentration estimated by the operator or the computer 110 performing the procedure of
(Correction procedure 2) The operator or the computer 110 performs the procedure of
In the correction procedure, the correspondence data is referred using another detection result obtained by measuring the amount of impurities. Therefore, the correspondence data needs to describe correspondence between the amount of impurities and the actual blood bacterial concentration. For example, the saturation value of the filter image and the amount of impurities corresponding thereto can be described together in the correspondence data, or a conversion formula between the saturation value and the amount of impurities can be defined in advance, and another detection result can be converted into the saturation value using the conversion formula. That is, the correspondence data may describe correspondence between a value representing the amount of impurities remaining on the filter in some form and the actual blood bacterial concentration in the blood sample.
In Embodiments 1 to 2, the saturation value of the filter image is used to detect red blood cells remaining in the filter. Instead of the camera 106, an optical sensor that detects absorption or reflection of a specific wavelength corresponding to red can also be used. That is, by using an optical sensor capable of detecting at least the largest RGB component of impurities remaining on the filter, information similar to the saturation value of the image captured by the camera 106 can be obtained. In this case, the correspondence data also needs to describe a numerical value measured by the optical sensor instead of the saturation value. Alternatively, a person may visually measure the amount of impurities based on a color sample, and refer to the correspondence data based on the measurement result. Furthermore, the color sample itself may describe the correspondence between the color of the impurities and the blood bacterial concentration.
In Example 1 of the present invention, the superiority of the pretreatment method according to the present invention will be described together with a comparative example. In Example 1, comparison was made between concentration adjustment using turbidity measurement and concentration adjustment according to the present invention, respectively adjusting the recommended bacterial concentration range of the sensitivity test. In the case of turbidity measurement, absorbance measurement at a wavelength of 600 nm was used. In the case of concentration adjustment according to the present invention, the pretreatment method shown in
When the bacterial concentration in the blood sample is 108 CFU/mL or more, it is possible to adjust the bacterial concentration to the vicinity of a desired concentration range, but when the blood bacterial concentration in the blood sample is 106 to 107 CFU/mL, the number of bacteria after the adjustment is reduced by 1 to 2 digits. This is because hemoglobin that could not be removed in steps S11 and S12, fine particles contained in the medium and the like contribute to an increase in scattered light, so that the value of turbidity increases even though the bacterial concentration is low. Therefore, since the bacterial concentration is estimated excessively, it is difficult to adjust the bacterial concentration by using turbidity measurement. This is also apparent from the fact that the plot of
Example 2 of the present invention shows an example in which bacterial concentration was adjusted from a blood culture positive sample by the pretreatment method according to the present invention using E. coli. As a comparative example, a growth rate in the case of preparing bacterial suspension from colonies by isolation and cultivation for a whole day is used. The bacterial concentration in a blood culture-positive sample was adjusted to a final bacterial concentration of 5×105 CFU/mL using three different bacterial concentrations up to 107 to 109 CFU/mL. Even when bacterial suspension was prepared from colonies, the bacterial suspension was adjusted using turbidity measurement so that the final bacterial concentration was 5×105 CFU/mL. 50 μL of the sample was dispensed into a 96-well plate, and 50 μL of Mueller-Hinton medium adjusted to twice the concentration was also dispensed. Thereafter, the 96-well plate was placed in an incubator at 35 to 37° C., and the state of growth was observed with a bright field microscope. Regarding the growth rate, a temporal change in the growth rate was calculated using the area of a region determined to be a bacterium in a microscopic image as an index of the growth rate.
In Example 3 of the present invention, the results when E. coli in Example 2 was changed to S. aureus will be described.
Example 4 of the present invention shows a result of performing a drug sensitivity test on both blood culture positive samples and a sample created from colonies. In the sensitivity test, the lowest concentration (Minimum Inhibitory Concentration: MIC) among the concentrations at which the drug exerts an antibacterial action on bacteria is measured. Here, a microliquid dilution method was used for the sensitivity test. The bacterial suspension and different concentrations of the drug were mixed, and the MIC was determined by visually determining turbidity of each well of the cultured 96-well plate after 18 hours.
In any case, the MIC in the case of pretreating the blood culture sample falls within the range of ±1 tube (twice or half) of the MIC in the case of the sample created from the colonies, indicating that the sensitivity test can be correctly performed even from the sample obtained by pretreating the blood culture sample.
In the above embodiment, it was described that the correspondence data is referred using the average value of the saturation values of the filter region 20, but a maximum value or a mode value may be used instead of the average value. Alternatively, instead of the saturation value, the amount of impurities remaining on the filter may be represented by a feature amount represented by at least two of hue, lightness, and saturation.
In the above embodiment, an example in which blood cells contained in the blood sample are detected as impurities has been described, but the present invention can also be used for other bacterial samples. That is, the present invention can be used for a sample having correspondence between image information obtained by imaging the impurities remaining on the filter and the bacterial concentration in the sample. The substance added to destroy the impurities may be appropriately changed depending on the type of impurities.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/018899 | 5/12/2020 | WO |
