WO2018021281A1

WO2018021281A1 - Automatic microorganism inspection device

Info

Publication number: WO2018021281A1
Application number: PCT/JP2017/026816
Authority: WO
Inventors: 桂富永; 研次岩脇
Original assignee: 日水製薬株式会社
Priority date: 2016-07-25
Filing date: 2017-07-25
Publication date: 2018-02-01
Also published as: JP2019162034A

Abstract

Provided is an automatic microorganism inspection device equipped with a mechanism which can rapidly discover developmental defects in bacteria in drug susceptibility tests. The device identifies microorganisms and tests drug susceptibility, and comprises: a specimen container storage unit for storing a specimen container; a plate storage unit for storing a culture plate; a tip storage unit for storing a dispensing tip; a tip disposal unit for storing a used tip; a plate recovery unit for storing used culture plates; a culture unit for culturing a specimen; an optical detection system for performing optical analysis; a plate transfer system; a dispensing unit; a storage device on which is stored a program for automatically performing the series of steps from the dilution of the specimen to the dispensing, culture, optical analysis and determination of the specimen, and plate recovery; and an arithmetic unit for executing this program. The device is also provided with a mechanism for performing real time optical analysis for drug susceptibility, and displaying the analysis results in a graph.

Description

Automatic microbiological testing device

The present invention relates to an automatic microbiological test apparatus used for bacterial identification test and drug sensitivity test.

∙ Identification of bacteria separated from test materials and determination of drug sensitivity are important in bacterial testing. In the flow of clinical examinations so far, one day was required for separation culture from a specimen, and bacterial identification / susceptibility tests were performed from fresh colonies on the separation medium or through enrichment culture. For this reason, it takes 3 to 4 days until an identification or sensitivity test result is obtained even in a normal test.

Therefore, development of an automatic rapid identification / sensitivity measuring device capable of reducing the work of identification / susceptibility testing and reporting the results quickly is required, and the present inventors only need to prepare a bacterial suspension. In addition to the rapid identification test and rapid susceptibility test, the system is capable of 18-hour sensitivity test in addition to the rapid identification test and rapid sensitivity test. ).

However, in the conventional system, in the drug susceptibility test, if a growth failure occurs in the control well where the bacteria should grow, a susceptibility result cannot be obtained. Until it was determined as “bad”, it could not be grasped, and there was a problem that the determination of resistant bacteria was greatly delayed.

The present invention relates to providing an automatic microbiological test apparatus equipped with a mechanism capable of early detection of bacterial growth failure in a drug sensitivity test.

The present invention is an apparatus for testing microorganism identification and drug sensitivity, a sample container storage unit for storing a sample container, a plate storage unit for storing a culture plate, a chip storage unit for storing dispensing tips, From the chip disposal unit that stores used chips, the plate recovery unit that stores used culture plates, the culture unit that cultures specimens, the optical detection system that performs optical analysis, the plate transport system, the dispensing unit, and the specimen dilution A storage device storing a program for automatically performing a series of steps from sample dispensing, culture, optical analysis and determination, and plate collection, and a computing device for executing the program, and a drug sensitivity test The present invention relates to an automatic microbiological test apparatus having a mechanism for performing optical analysis for real-time and displaying the analysis result in a graph.
The present invention also includes a diluting step for diluting the bacterial solution, a dispensing step for dispensing the sample, a culturing step for culturing the sample, and an analysis / determination step for performing a drug sensitivity test and an identification test. The present invention relates to a microorganism identification and drug sensitivity test method, which includes a step of performing optical analysis for a drug sensitivity test in real time and displaying the analysis result in a graph.

According to the automatic microbe inspection apparatus of the present invention, it is possible to test microbe identification and drug sensitivity with high accuracy and in a short time. In particular, in a drug susceptibility test, by looking at a growth curve graph that is detected and displayed in real time, a strain that cannot grow in the control well can be recognized before final determination, and a change or optimization of the drug sensitivity test method can be started in advance. Thereby, an accurate sensitivity result can be derived quickly. Further, by confirming the growth curve data of the control well and the drug-added well, it can be recognized from the growth curve graph that there is a possibility of resistant bacteria before the reference time when the computer starts the analysis process. This makes it possible to estimate whether the strain is resistant before the rapid susceptibility test results are obtained, and to perform optimal treatment such as changing the antibiotic species used for treatment earlier.

Schematic diagram of the front of the device of the present invention Schematic view of the back of the device of the present invention Schematic view around the dispensing unit of the device of the present invention Schematic diagram of sample storage and chip storage Schematic diagram of plate storage / culture unit Flow from preparation of bacterial solution to identification of microorganisms and determination of drug sensitivity Growth curve of MRSA clinical isolates (0-1080 min) Growth curve of MRSA clinical isolates (0-420 min) Growth curve of MSSA clinical isolates (0-1080 min) Growth curve of MSSA clinical isolates (0-420 min)

In the automatic microbiological examination apparatus of the present invention, the preparation of the bacterial solution (primary sample), the input of the sample information and the plate information, and the set of the dedicated reagent are performed only by the measurer, and subsequent dilution, dispensing, culture, Calculation and reporting of test results and plate collection are fully automated. FIG. 5 shows a flow from preparation of the bacterial solution to identification of the microorganism and determination of drug sensitivity.

Here, the dedicated reagent includes a culture plate on which an identification substrate and an antibacterial agent for drug sensitivity test are dried and fixed, sterilized water for preparing a bacterial solution, bouillon (diluent) used for dilution in a drug sensitivity test, drug Pipette tips for dispensing special supplements (growth-supporting substances) and samples to culture plates that are required when testing strains with strict growth requirements in susceptibility tests, etc. Is mentioned.

Sample information includes sample number, sample type (eg, sputum, blood, etc.), bacteria number, and the like. The plate information includes the plate name, drug type, drug concentration, measurement mode, and the like. These pieces of information are input to storage means in the apparatus, and output in the form of a barcode, QR code (registered trademark), or the like.

The test bacteria to be used as specimens are cultured in a non-selective medium with test materials and purely cultured. A small amount of such pure cultures are suspended in sterilized water to prepare a bacterial solution (primary sample). Is done. The prepared bacterial solution is affixed with a label in which the above-described information is registered, and is set in the specimen storage unit.

1 to 3 show an outline of the automatic microorganism identification susceptibility measuring apparatus of the present invention (FIG. 1: front, FIG. 2: back, FIG. 3: around the dispensing unit).
1 is a sample storage unit for storing a container containing a bacterial solution or a diluent, 2 is a plate storage unit for setting a culture plate, 3 is a chip storage unit for storing a chip, and 4 is injecting a sample into the culture plate Dispensing area for storing 5 is a chip discarding unit for storing used chips, 6 is a plate collecting unit for storing used plates, 7 is a culture unit for culturing specimens (identification test / drug sensitivity test), 8 Is a plate transport system for transporting the plate, 9 is a dispensing unit for sucking and discharging the sample and dispensing it into the container, and 10 is an optical detection system.

The specimen storage unit 1 is a structure for storing a container in which a bacterial solution (primary specimen) is stored and a container in which a diluent (bouillon) is stored (FIG. 4). The structure of the specimen storage unit is not particularly limited as long as it is possible to set a plurality of specimen storage containers (for example, for 10 specimens) and a diluent storage container corresponding thereto, and may be cylindrical or the like.

The culture plate has a plurality of (for example, 96 wells) independent reaction wells, and the identification substrate and the antimicrobial agent for drug sensitivity test are dried and fixed. When rapid identification / sensitivity test is performed, the identification substrate is dried and fixed in 32 wells in 1 to 4 rows of the microplate, and the antimicrobial drug for drug sensitivity test is dry fixed in 64 wells in 5 to 12 rows. A combination plate is used. When performing CLSI-compliant 18 hour method sensitivity measurement, an MIC plate in which antimicrobial agents for drug sensitivity test are dried and fixed in all wells is also used. Each plate is affixed with a label in which information such as drug type, concentration, and arrangement is registered.

In the plate storage unit 2, a plurality of (for example, 10) plates are set according to the number of samples. The plate storage portion can be set by stacking the plates. When the hook-shaped member is operated, it can be sequentially taken out from the lowermost plate. *

The chip storage part 3 has a hole so as to support the taper part of the chip, and stores a necessary number of chips.

The dispensing unit 9 has a mechanism for attaching and detaching the chip to and from the chip nozzle and a mechanism for sucking and injecting the sample, and includes a dispensing arm unit that can move freely in the XZ axis direction.
For example, the bacterial solution is diluted by moving the dispensing unit to the chip stored in the chip storage unit 3 and mounting the chip, then moving the dispensing unit onto the bacterial solution and sucking the bacterial solution, and then diluting. It is performed by moving onto the liquid and discharging the bacterial liquid into the diluent.

The plate transport system 8 has a hand for holding the culture plate and an elevating unit for storing it in the culture tank. To the plate storage unit, dispensing area, culture unit, optical detection system, and plate recovery unit The culture plate is moved and the upper lid is placed on the culture plate. The lid is put on the plate in advance, and when dispensing, the lid is lifted by a suction cup.

In the culture unit 7, culture for identification test and drug sensitivity test is performed. Therefore, the culture section is provided with a temperature adjustment mechanism 11 capable of adjusting the temperature to 34 to 36 ° C. (FIG. 5).

An optical detection system 10 includes a fluorescence detector and an absorption detector.
As the fluorescence detector, any substance capable of detecting a fluorescent substance such as 4-methylumbelliferone can be used. As the light source, a halogen lamp can be used. However, in terms of the light source life and energy saving, an LED lamp is used. It is preferable to use it. As a suitable LED lamp, for example, a new model NCSU275 (U365) (maximum 150 mW) manufactured by Nichia Corporation may be mentioned.
The light absorption detector is not limited as long as it can detect light-absorbing substances such as metanil yellow, but an LED lamp is preferably used as the light source. Suitable LED lamps include, for example, LXML-PR01-0500 manufactured by Philips Lumileds.

The automatic microbiological test apparatus of the present invention has at least the above-described configuration, and dilution of the sample (dilution step), sample dispensing (dispensing step), culture, optical analysis and determination (analysis / determination step), In addition, a storage device storing a program for automatically performing a series of steps up to plate collection and an arithmetic device for executing the program are provided.

Below, the main process performed automatically in the automatic microbiological test | inspection apparatus of this invention is demonstrated.
1) Dilution step This step is a step of preparing a cultivatable sample (secondary sample) from a bacterial solution (primary sample) for a drug sensitivity test. Specifically, after the dispensing unit 9 is operated and the tip is mounted on the tip nozzle, it moves onto the bacterial solution in the sample storage unit 1 to aspirate the bacterial solution, and then moves onto the diluted solution, By discharging the liquid, a cultivatable secondary specimen is prepared.

2) Dispensing step In this step, the primary sample and the secondary sample are automatically dispensed into each reaction well of the culture plate. In the dispensing operation, the plate transport system 8 is driven, the culture plate is moved to the dispensing area 4, the dispensing unit 9 is operated, and the primary sample solution and the secondary sample solution are respectively aspirated. , Injected into each well in the plate.

3) Culture process The culture plate into which the specimen has been dispensed is transported to the culture unit 7 by the plate transport system 8, and culture for a drug sensitivity test and an identification test is performed.
The culture is performed until the culture time set for each specimen.
In the present invention, in order to grasp the growth state of the bacteria in real time, the culture plate is transported from the culture unit to the optical detection system 10 at a preset interval (for example, every 15 minutes), and optical measurement is performed.

4) Analysis / determination step This step is a step of measuring and analyzing the growth state of the bacteria independently and individually by optical changes occurring in a plurality of reaction wells by at least two different assays.
One of the assays is an absorbance assay for drug sensitivity testing and another assay is a fluorescence assay for identification testing. The absorbance assay is performed with an absorbance detector and the fluorescence assay is performed with a fluorescence detector.

i) Identification test The biochemical test items used in the identification test are composed of a total of 32 items: an item using a fluorescent substrate and an item using a fluorescent substance as an indicator.
In the item using a fluorescent substrate, the amount of fluorescence of a fluorescent substance (for example, 4-methylumbelliferone, 7-amino-4-methylcoumarin) that is released when the bacterial cell enzyme of the bacterium decomposes the substrate is measured.
In the item using a fluorescent substance as an indicator, the change in pH of the medium with the metabolism of the bacteria is measured by increasing or decreasing the amount of fluorescence. For example, when bacteria decompose sugar and produce acid, the pH decreases, and the amount of fluorescence that decreases with the pH is measured. The amount of fluorescence is measured with an excitation wavelength of 360 nm ± 25 nm (half-value width) and a fluorescence wavelength of 450 nm ± 32.5 nm (half-value width), and is quantified as relative fluorescence intensity.

For these fluorescence intensities, a fixed cutoff value is set for each biochemical test item, positive reaction (+) and negative reaction (-) are judged, and identification is made in advance based on these judgment results Based on the database held by the system, the bacterial name of the unknown strain is obtained by computer analysis processing (probabilistic identification method).

In the identification test, measurement is started 3 hours after culturing, and when it is determined that the accuracy of the identification result obtained by the computer analysis process is high and reliable, the identification is completed. If it is determined that the identification accuracy of the obtained results is low, extended culture (4 hours, 5 hours, 6 hours, 8 hours, 12 hours, maximum 18 hours) is appropriately performed. The measurement time can be arbitrarily set at a maximum of 8 measurement points per hour.

ii) Drug sensitivity test The antimicrobial drug for drug sensitivity test dried and fixed on the plate includes a drug and a redox indicator that have been serially diluted, and the redox indicator includes 2- (4-indophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt (water soluble tetrazolium, hereinafter referred to as WST-1) has been used. WST-1 is reduced in the process of growth and growth of the fungus to form water-soluble yellow formazan, which is colored in the well and causes a highly sensitive change in absorbance (410 to 455 nm). By measuring the absorbance at 10 ± 2 nm (half width), a MIC (Minimum Inhibitory Concentration) value is calculated.

The absorbance of the control well (no drug added) is compared with the absorbance of the drug added well from the change in the absorbance value obtained, and the bacterial name information determined by the identification test of i) above, the antibacterial spectrum and the drug efficacy are considered in advance. A MIC (Minimum Inhibitory Concentration) value is calculated by computer analysis processing (calculation conditions) consisting of the created bacterial species / drug combination.
Here, if the preset condition cannot be cleared, the culture is extended. For example, if the absorbance (growth index) of the control well is not reached, it is assumed that the growth of the strain is insufficient and the culture is extended.

The drug sensitivity test in the apparatus of the present invention can be carried out by a rapid method and a method based on CLSI (equivalent to 10 ⁵ cfu / mL). The rapid method is a method of inoculating 10 times the amount of bacteria (corresponding to 10 ⁶ cfu / mL) with respect to the CLSI method.
In the present invention, the absorbance at 410 to 455 nm is measured at a predetermined interval (for example, at an interval of 15 minutes) until the determination is completed in the rapid method, and in the culture period of 18 to 24 hours (or 48 hours) in the CLSI standard method. And the said measurement result is sequentially displayed by a graph as a growth curve. Thereby, the growth situation in the control well and the drug-added well can be grasped in real time, and the result can be predicted before the result is obtained by the apparatus from the curve data.

In the conventional method, for example, when a poor growth occurs even in a control well that should not grow, the drug is not fixed, a sensitivity result cannot be obtained. This growth failure has been determined as “poor growth” by computer analysis after culturing for 18 to 48 hours, for example. Even in the rapid susceptibility test, if the culture time and the control well do not show a certain growth (absorbance), the MIC is not calculated by the computer analysis process, and as a result, the determination of the resistant bacteria expected by the customer may be delayed. .

According to the apparatus of the present invention, a strain that cannot grow in the control well can be recognized before final determination by looking at the growth curve graph, and the sensitivity test method can be changed and optimized in advance. As a result, an accurate sensitivity result can be quickly derived, which is highly clinically significant.
In addition, by confirming the growth curve data of the control well and drug-added wells (especially the concentration range for determining resistant bacteria), the growth curve indicates that there is a possibility of resistant bacteria before the reference time when the computer starts the analysis process. It can be recognized from the graph. By this, it can be estimated whether it is a resistant bacterium before a quick drug susceptibility test result is obtained, and optimal treatment such as change of antibiotic species used for treatment can be performed earlier (Example 1).

One mode of the operation in the case of examining bacteria identification and drug sensitivity for one specimen using the apparatus of the present invention will be described below.
1) Start.
2) Bacteria solution barcode reading, culture plate barcode reading (lowermost plate).
3) The dispensing unit 9 moves onto the bacterial solution corresponding to the culture plate selected in 2) and aspirates the bacterial solution.
4) The dispensing unit 9 moves onto the diluent and discharges the bacterial solution into the diluent.
5) The dispensing unit 9 moves again onto the bacterial solution, and sucks the bacterial solution in a volume that allows a predetermined amount to be dispensed continuously into 32 wells.
6) The plate transport system 8 is operated, and the culture plate selected in 2) is moved to the dispensing area 4.
7) The dispensing unit 9 moves to the identification test area (32 wells) of the culture plate set in 6) and discharges a predetermined amount into each well.
8) The dispensing unit 9 moves onto the bacterial solution and discharges the remaining bacterial solution from the chip.
9) After the dispensing unit 9 moves onto the diluted solution and is aspirated / discharged and agitated, a sufficient amount of the bacterial solution that can be continuously dispensed into 32 wells is aspirated.
10) The dispensing unit 9 moves to the sensitivity test area (64 wells) of the culture plate set in 6), and discharges and injects a predetermined amount into each well of the 32 well area.
11) Repeat steps 9) to 10) to discharge and inject a predetermined amount into the remaining 32 wells.
12) The dispensing unit moves to the tip disposal section 5 and discards the tip.
13) The plate transport system 8 is operated, a lid is placed on the culture plate, and transported to the culture unit 7.
14) Start culture. Until the end of the culture, the culture plate is transported to the optical detection system 10 at predetermined intervals and optically analyzed to display a growth curve in a graph.
15) Output of determination results after culturing.
16) The plate transport system 8 is operated, and the used culture plate is moved to the plate collection unit 6.

Example 1
When Staphylococcus aureus is isolated from clinical specimens at the clinical laboratory site, methicillin-resistant Staphylococcus aureus (MRSA) or not greatly affects the selection of antibacterial agents for treatment. In this example, MRSA and a clinical isolate of methicillin-sensitive Staphylococcus aureus (MSSA) were used, and a drug sensitivity test for cefoxitin (CFX) effective for differentiating MRSA was measured by the rapid method of the instrument.
Differentiation of MRSA requires that the MIC of CFX be ≧ 8 μg / mL. Therefore, if growth is observed in a well of CFX 4 μg / mL, it is determined as MRSA. When MRSA clinical isolates were tested with the rapid method of the device, the time for the absorbance of the control wells to exceed a preset value (eg 1.5) was 300 minutes after incubation. At this time, the absorbance of each drug concentration well of CFX is compared with a predetermined threshold value, and if it is equal to or higher than the threshold value, it is regarded as “growth”. This time, wells of 4 μg / mL or less became “growth” and were determined to be MRSA.
FIG. 7 is a growth curve of MRSA clinical isolates cultured for up to 1080 minutes (18 hours). When this growth curve is monitored in real time on the monitor of the apparatus, attention can be paid to the growth of 4 μg / mL wells as shown in FIG. The possibility of MRSA can be predicted and will be useful for early treatment.
On the other hand, when the MSSA clinical isolate was tested by the rapid method of the apparatus, the time when the control well absorbance exceeded, for example, 1.5 was 285 minutes after culturing, as described above. Wells that are less than the threshold are considered “non-developed”. This time, wells of 4 μg / ml or more became “non-developed” and were determined to be MSSA.
FIG. 9 is a growth curve of MSSA clinical isolates cultured for 1080 minutes (18 hours). When the growth curve is monitored on the monitor as in FIG. 8, paying attention to the growth of the 4 μg / mL well as shown in FIG. 10, it is confirmed that there is no change in the curve at an early stage (195 minutes in culture). The possibility of MSSA can be predicted before the CFX MIC is determined by this apparatus.

DESCRIPTION OF SYMBOLS 1 Sample storage part 2 Plate storage part 3 Chip storage part 4 Dispensing area 5 Chip discard part 6 Plate collection | recovery part 7 Incubation part 8 Plate conveyance system 9 Dispensing unit 10 Optical detection system 11 Temperature control mechanism

Claims

A device for inspecting microorganism identification and drug sensitivity, including a sample container storage unit for storing a sample container, a plate storage unit for storing a culture plate, a chip storage unit for storing dispensing tips, and a used chip Dispose of the sample from the chip disposal unit to be stored, the plate recovery unit to store the used culture plate, the culture unit to culture the sample, the optical detection system for optical analysis, the plate transport system, the dispensing unit, and the sample dilution. Optical analysis for drug susceptibility testing, including a storage device that stores a program that automatically performs a series of steps from injection, culture, optical analysis and determination, and plate collection, and a computing device that executes this program An automatic microbiological testing device equipped with a mechanism for performing analysis in real time and displaying the analysis results in a graph.
The automatic microbiological test apparatus according to claim 1, wherein optical analysis performed in real time is performed every 15 minutes after the start of culture.
It includes a dilution process for diluting a bacterial solution, a dispensing process for dispensing a specimen, a culturing process for culturing a specimen, and an analysis / judgment process for performing a drug sensitivity test and an identification test. A method for testing sensitivity comprising the steps of performing optical analysis for drug sensitivity testing in real time and displaying the analysis results in a graph.