Method of directly inoculating broth from a suspension source

Abstract

To provide an automated method for preparing a sample suspension.SOLUTION: The sample suspension can be used for both MALDI and antimicrobial susceptibility testing (AST). A suspension is prepared, and a portion of the suspension is removed for a first analysis (e.g., MALDI), leaving a remaining volume. The turbidity of the remaining volume is measured. If the turbidity is below a first threshold, the suspension is not used for a second analysis (e.g., AST) and is subjected to a concentration protocol to raise the turbidity of the suspension. If the turbidity is within a predetermined range, a volume of the suspension is calculated that will deliver a predetermined amount of a sample to a vessel for the second analysis. If the turbidity of the suspension is above the predetermined range, and the suspension has not been diluted a predetermined number of times, the suspension is diluted according to a dilution protocol.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application claims the interests of U.S. Patent Application No. 62 / 689,419, filed on 25 June 2018, and the disclosure of this U.S. Provisional Patent Application constitutes a part of this Specified Use by reference.

[0002] This specification discloses a method for using a single suspension for both the identification of microorganisms in a biological sample (e.g., blood) and for antimicrobial susceptibility testing. [Background technology]

[0003] As a standard part of medical diagnosis, biological samples such as blood are collected from patients and analyzed. Depending on the indication, the sample can be analyzed to determine whether or not microorganisms are present, for example, by blood culture (BACTEC® FX and BACTEC® 9000 series from Becton, Dickinson and Company, etc.) or by streaking culture on agar plates (manually or using automated instruments such as Innova® or Inoqula, sold by Becton, Dickinson and Company). If microorganisms are found to be present, there is medical and economic justification for both identifying the specific microorganism present and determining the antibiotic resistance / susceptibility of the microorganism to facilitate treatment.

[0004] Many types of microorganisms (hereinafter also referred to as microorganisms), particularly bacteria and single-celled fungi, can be identified by mass spectrometry ("mass spec") processes such as matrix-assisted laser desorption / ionization ("MALDI"). In the MALDI process, a small amount of microorganisms from colonies cultured in a conventional manner in a nutrient medium is transferred to a mass spectrometry sample support plate known as a MALDI plate, and then directly subjected to mass spectrometry, generally by MALDI time-of-flight (TOF). Mass spectrometry will show different proteins if they are present in sufficient concentration within the microorganism. The identity of the microorganism is then determined from its protein profile through a computerized search of a spectral library containing thousands of reference spectra. If a reference mass spectrum does not exist in the library of the exact species of the microorganism being examined, a computerized library search with relaxed similarity requirements can provide at least some indication of the order, family, or genus of the microorganism, because related microorganisms often contain multiple identical protein types. The MALDI process is described in more detail in International Publication No. 2009 / 065580 to Ulrich Weller, titled “Identification of Pathogens in Bodily Fluids,” the contents of which, by reference, constitute an entirety of this specification. A variety of mass spectrometers can be used for identification.

[0005] It is desirable to analyze the effectiveness of antibiotics in inhibiting the growth of microbial isolates from clinical specimens. Such analysis is known as antimicrobial susceptibility testing ("AST"). The AST technique, a background technique, is a dilution technique that involves exposing bacteria to reduced concentrations of antibiotics in a liquid medium through a series of 2-fold dilutions. The lowest concentration of antibiotic at which no visible bacterial growth occurs is defined as the minimum inhibitory concentration (MIC). The MIC is a standard measure of antibiotic susceptibility. AST instruments such as the BD Phoenix™ system, marketed by Becton, Dickinson and Company, are known in the art and perform both identification and AST.

[0006] A known instrument in the background art capable of preparing samples for such an AST process is the BD Phoenix® AP, available from Becton, Dickinson and Company. The workflow typically includes preparing the inoculum by labeling appropriate tubes, selecting microbial colonies and preparing heavy suspensions in ID broth tubes, and placing these tubes in one or more racks holding AST broth tubes. The workflow then includes performing automated turbidimetric analysis to adjust the ID tubes to 0.5 McFarland ("McF") or 0.25 McF, adding AST indicator to the AST broth, transferring a portion of the sample into the AST broth, and mixing both tubes. The workflow then includes having a healthcare professional remove the processed ID and AST tubes and place them on an inoculation station having an ID / AST panel, such as a Phoenix panel, and inoculating the panel with the sample. See also U.S. Patent Application Publication 2008 / 0072664. This U.S. patent application publication, by reference, constitutes an entire part of this specification.

[0007] The panel is then maintained for a predetermined amount of time in an ID / AST system (e.g., Phoenix instrument) with a controlled environment (e.g., controlled temperature, humidity, exposure, etc.) to promote microbial growth in the presence of an antimicrobial agent. The system typically includes an analytical function to measure microbial growth in one or more microwells without interrupting the maintenance of the controlled environment. The system may also include a function to report the analytical results to an additional device for further processing. Such a system may include both ID and AST functions, or only ID or only AST functions. Furthermore, the ID / AST system may be run for ID results only or only AST results. For panels, see, for example, U.S. Patents 5,922,593, 6,096,272, 6,372,485, 7,115,384 and 6,849,422. The contents of these U.S. patents are incorporated herein by reference in their entirety.

[0008] Various research devices can communicate with data management systems such as BD EpiCenter™ to provide researchers with a single location to monitor the status and results from these devices. Timely monitoring, analysis, and communication of microbiological data can directly impact patient care. However, obtaining, compiling, and communicating information from various research devices is labor-intensive. Current information systems can even make routine identification and AST testing difficult. Microbiologists, infection control officials, physicians, and pharmacists need immediate access to patient-focused information to rapidly identify and respond to ongoing resistance or HAI events.

[0009] A method and apparatus for preparing a common sample suspension for both MALDI and AST is described in U.S. Patent No. 9,180,448, issued on November 10, 2015, from an application filed on July 6, 2011, with the title "Method and Apparatus for Identification of Bacteria," assigned to Becton, Dickinson and Company. Another system for taking samples and preparing suspensions from samples for both MALDI and AST is described in U.S. Patent No. 9,556,495, issued on January 31, 2017, from U.S. Patent Application No. 14 / 388,430, filed on April 2, 2013, with the title "Automated Selection of Microorganisms and Identification Using MALDI," assigned to BD Kiestra BV. Another system is described in U.S. Patent Publication No. 2016 / 034554, filed on 27 May 2016 and assigned to BD Kiestra BV. Patents No. 448, No. 495 and No. 554 are all incorporated herein by reference. [Overview of the project]

[0010] In one embodiment, the present invention as described herein is an automated method in which a common sample suspension is used as the sample source for both a system for identifying microorganisms determined to be present in a sample and a system for testing those microorganisms for their antibiotic resistance / susceptibility. The system has a first station for preparing a common suspension for both the mass spec (e.g., MALDI) process for microbial identification and the antibiotic susceptibility testing (AST).

[0011] According to this method, the sample is inoculated into a diluent. In one embodiment, the sample is picked from a culture plate. Picking a sample from a culture plate is known to those skilled in the art and is not described in detail herein. Picking a sample from a culture plate and depositing the sample into a diluent is described in U.S. Patent Publication No. 2016 / 034554.

[0012] The picked samples are transferred to a sample diluent. Such diluents are known and will not be described in detail herein. Such diluents are described in U.S. Patent Publication No. 2016 / 034554.

[0013] Subsequently, the turbidity of the sample is measured. A turbidimeter is used to obtain the turbidity measurement. The measurement of turbidity using a turbidimeter is described in U.S. Patent Publication No. 2016 / 034554. If the measured turbidity is within a predetermined range, a first aliquot of the suspension is used for MALDI, and a second aliquot of the suspension is delivered to the broth tube for antibiotic susceptibility testing (AST). The volume of the suspension used for AST is calculated from the suspension turbidity, because AST requires a certain amount of CFU (colony-forming units) of the sample to be delivered to the AST broth tube, and the volume is calculated based on the suspension turbidity and the target amount of sample to be inoculated. A predetermined turbidity range is required due to the limits imposed by the automated pipette dispensing device. Broths used as nutrient media or culture media that enable microbial growth in AST are known to those skilled in the art and are not described in detail herein. In AST, the absence of microbial growth indicates that the microorganism being tested is susceptible to the antibiotic delivered in combination with the sample suspension. AST broth is also referred to herein as culture medium.

[0014] The amount of suspension used to inoculate a plate (e.g., a MALDI plate) for identification or a broth tube for antibiotic susceptibility testing (AST) is based on the amount of sample carried per unit volume of suspension. Once a suspension is prepared, if the concentration of the sample in the suspension (i.e., sample turbidity) is excessively high, the volume of suspension required to inoculate that amount of sample onto the MALDI plate or AST broth tube may be extremely small. Small volumes are difficult to pipette accurately. Conversely, the more "light" the suspension, the larger the volume required to inoculate the target amount of sample onto the MALDI plate or broth tube. However, the volume that can be transferred using conventional pipettes is limited.

[0015] Therefore, if the amount of sample delivered into the suspension results in a sample concentration in the suspension (as measured by suspension turbidity) that is higher than a predetermined range (e.g., about 0.2 MacFarland to about 2 MacFarland), the suspension undergoes a dilution protocol to reduce the turbidity so that the suspension turbidity falls within that predetermined range. If the amount of sample delivered into the suspension results in a suspension turbidity that is below the predetermined range, the suspension undergoes a concentration protocol. In one embodiment, the concentration protocol instructs the acquisition of additional samples to increase the concentration of the sample in the suspension. On the other hand, if additional samples are not available, the concentration protocol instructs the discarding of the suspension.

[0016] The preparation of the suspension and the inoculation of such prepared suspension into a MALDI plate are described in U.S. Patent Publication No. 2016 / 034554, which is incorporated herein by reference. The MALDI process is carried out at a second station, and the AST is carried out at a third station.

[0017] After the aliquots of the suspension are removed for MALDI, the system determines the amount of suspension to be used for inoculation into the AST panel. The volume of the suspension is determined by the total amount of sample required by a given specification to be introduced into the AST broth tube. Based on the known turbidity of the suspension and the target amount of sample for panel inoculation, the system calculates the volume of suspension required for inoculation into the AST broth tube. The system then obtains the specified volume of suspension and inoculates the panel with that volume. The systems and methods described herein do not require the turbidity of the suspension to be adjusted to a standardized MacFarland value for AST after the aliquots of the sample for MALDI are removed from the suspension tube, thereby making the methods and systems more efficient and reducing instrumentation compared to systems and methods that require the turbidity of the suspension to be standardized to a target MacFarland value before inoculation of the AST broth tube using the suspension.

[0018] The system includes a user interface and software, in which samples are tracked so that test results from a second and third station are linked to the sample and the patient from whom the sample was obtained. The system also includes a station that determines whether or not microorganisms are present in the sample, and only samples that are determined to contain microorganisms undergo further processing and testing. [Brief explanation of the drawing]

[0019] [Figure 1] This flowchart illustrates a method for using a suspension that does not require turbidity standardization before inoculation into a broth tube for AST. [Figure 2] This flowchart illustrates how to use a suspension, not requiring turbidity standardization before inoculation of broth tubes for AST, but providing a dilution protocol for preparing a sample suspension if the initial suspension is overly concentrated. [Figure 3A]A flowchart explaining the first part of a method of using a suspension that, according to another embodiment of the present invention, does not require turbidity normalization before inoculation of broth tubes for AST but provides a dilution protocol for preparing a sample suspension. [Figure 3B] A flowchart explaining the second part of a method of using a suspension that, according to another embodiment of the present invention, does not require turbidity normalization before inoculation of broth tubes for AST but provides a dilution protocol for preparing a sample suspension. [Figure 4] A diagram showing the E. coli concentration in an AST broth tube and the final turbidity of the suspension corresponding thereto, using the method shown in FIG. 2. [Figure 5] A diagram showing the E. coli concentration in an AST broth tube and the final turbidity of the suspension corresponding thereto, using the method shown in FIG. 2, for a suspension prepared using only one turbidity measurement. [Figure 6] A schematic view of AST results using suspensions prepared by the methods of FIGS. 4 and 5. [Figure 7] A system schematic diagram.

Mode for Carrying Out the Invention

[0020] In this disclosure, sample preparation devices (hereinafter, sample preparation or preparation station) are referred to as "Phoenix AP," or AST systems as BD Phoenix®, or data management systems with user interfaces as the "BD EpiCenter" system, or blood culture analyzers as "BD BACTEC®," or mass spectrometry systems as MALDI. However, it should be understood that the meaning of these terms is not limited to devices bearing these trademark names and may include devices having substantially similar functions. Such substantially similar devices may include BacT / Alert (bioMerieux) and VersaTREK (Trek) blood culture systems, and Vitek (bioMerieux) and MicroScan (Siemens Healthcare) ID / AST systems.

[0021] In one embodiment, the system described herein integrates the microbial identification function of a MALDI instrument with the AST and data processing functions of a research analysis or processing system such as the Phoenix, Phoenix AP, BACTEC, or EpiCenter system.

[0022] In another embodiment, Phoenix AP is modified to prepare the same sample for MALDI plates as well as for the Phoenix panel, rather than just for the Phoenix panel. This feature provides the benefit of automating the positive identification of samples prepared on MALDI plates by ensuring that the isolates applied to the MALDI plates are from the exact same samples used for antimicrobial susceptibility testing.

[0023] MALDI, sample preparation, AST systems, and / or blood culture equipment communicate with data management systems such as the EpiCenter system. EpiCenter provides real-time data access and analysis tools to improve patient care. EpiCenter enables timely monitoring, analysis, and communication of microbiological data, thereby directly controlling, monitoring, and improving patient care. Phoenix generates AST results, and MALDI equipment generates identification results. EpiCenter combines these results and applies expert rules to generate the final ID / AST result for the sample. An example of such a system that applies expert rules is BDXpert®.

[0024] Figure 7 shows a block diagram of a system 100 for detecting and analyzing microbial samples according to one embodiment of the present invention. The various components of the system 100 include a sample prep station 102 (e.g., Phoenix AP), a mass spec instrument 104 (e.g., MALDI-TOF), an AST system 106 (e.g., Phoenix), a blood culture system 108 (e.g., BACTEC instrument), prepared plate media 110 (e.g., manually prepared media or media prepared by a system such as Innova), a data management system 112 (e.g., EpiCenter), and a research information system 116 ("LIS") that receives data from the data management system 112 via an ID / AST link 114 and provides patient information to the data management system 112 via a PT information link 118.

[0025] In system 100, the sample preparation system is supplied with bacteria, for example, picked from a prepared plate or obtained from a blood culture vial. In one embodiment, the bacterial sample is inoculated into a cuvette from above. It is advantageous for the cuvette to be used as a sample source for both ID and AST. This ensures that not only the same patient sample but also the same isolate undergoes both ID and AST testing.

[0026] The sample preparation station 102 prepares samples for both ID and AST, while the AST system 106 generates AST results and the mass spec instrument 104 generates ID results. The data management system 112 stores the ID and AST results, optionally applies expert rules to generate final synthesized ID / AST results for the samples, and also interacts with the LIS system.

[0027] Referring to Figure 1, the process begins with step 150, in which a sample is taken (usually from a culture plate or culture tube), and step 155, in which the sample is delivered to a suspension. In step 160, the turbidity of the suspension is measured, and then in step 165, the turbidity is evaluated to determine whether the measured turbidity is within a predetermined range. A predetermined range is necessary because the volume of the sample used to inoculate into the MALDI plate or broth tube is limited by the volume that can be accurately transferred by the automated pipette and within the volume constraints of the pipette. In this regard, current pipettes are configured to transfer suspensions of approximately 10 mL (i.e., 1000 μL) to approximately 12 mL (i.e., 1200 mL) or less.

[0028] If the suspension has a turbidity within the target range, in step 170, the required volume of suspension needed to inoculate the desired amount of sample into the broth tube is calculated based on the turbidity reading. In step 175, the pipette is used to aspirate the calculated volume of suspension. If the suspension is excessively light, the volume required to inoculate the MALDI plate or broth tube to deliver the target amount of sample will be excessively large for the pipette to accommodate (at least in one transfer). If the suspension is excessively heavy, only a small volume of suspension is required for inoculation into the MALDI plate or broth tube. Small volume aspiration and dispensing are difficult to control precisely, making it difficult to deliver a precise amount of sample for inoculation into the MALDI plate or broth tube. According to the method illustrated in Figure 1, in one embodiment, a given turbidity is in the range of about 0.2 MacFarland to about 2 MacFarland. If the suspension is outside this range, a correction protocol is assigned in step 180. If the suspension is excessively concentrated, it undergoes a dilution protocol 185 to reduce its concentration to a target range. Dilution protocol 185 is primarily a matter of design selection and typically consists of removing a certain volume of the suspension and replacing this suspension with a diluent. The volume of suspension to be removed is selected based on turbidity measurement. If dilution protocol 185 is successful, in step 190 the suspension is used to inoculate an AST broth tube by using the volume of suspension calculated in step 170. One exemplary example of a dilution protocol is described later in this specification. If the dilution protocol is unsuccessful, the sample is discarded in step 191.

[0029] If the suspension is determined to be excessively diluted in step 180, in step 195 the suspension undergoes a concentration protocol to increase the concentration of the sample in the suspension. Such a concentration protocol requires the addition of sample to the suspension. Concentration protocol 195 is primarily a matter of design choice and typically consists of adding some additional sample to the diluent. Since the amount of additional sample delivered into the suspension is difficult to control precisely, the concentration protocol may require additional turbidity measurements to determine the turbidity of the suspension after the concentration protocol is completed. If the protocol is successful in step 190, in step 170 the suspension is used to inoculate a broth tube by calculating the volume of suspension required to inoculate the broth tube with the target amount of sample, and in step 175 that volume of suspension is aspirated for inoculation into a target plate. If the concentration protocol is unsuccessful, the sample is discarded in step 191.

[0030] One example of a dilution protocol is described later in this specification. Both the dilution protocol and the concentration protocol are expected to require additional turbidity measurements after the suspension has been concentrated or diluted.

[0031] The amount of suspension used to inoculate a plate (e.g., a MALDI plate) for identification or a broth tube for antibiotic susceptibility testing (AST) is based on the amount of sample supported per unit volume of suspension. Once a suspension is prepared, if the concentration of the sample in the suspension (i.e., sample turbidity) is excessively high (e.g., about 2 McFarland or higher), the volume of suspension required to inoculate that amount of sample onto a MALDI plate or into an AST broth tube may be extremely small. Small volumes are difficult to pipette accurately.

[0032] Therefore, if the amount of sample delivered into the suspension results in a suspension concentration (as measured by suspension turbidity) that is higher than the upper limit of a given range (e.g., greater than 2 McFarland), the suspension undergoes a dilution protocol to reduce turbidity so that the suspension turbidity falls within the given range. If the amount of sample delivered into the suspension results in a suspension turbidity that is lower than the lower limit of a given range (e.g., less than approximately 0.2 McFarland), additional samples are taken to increase the concentration of the sample in the suspension (if additional samples are not available, the suspension is removed).

[0033] In one example, the measured turbidity of the prepared suspension is within the range of approximately 3 MacFarlands. In this example, the given turbidity range is approximately 0.2 MacFarlands to approximately 2 MacFarlands. Based on this turbidity measurement, the system determines that this suspension should undergo a dilution protocol.

[0034] The specified turbidity range is primarily a matter of design selection. Factors determining the range include i) the accuracy of the instrument used to measure turbidity (e.g., turbidimeter), ii) the reading window of the turbidimeter, and iii) the accuracy and capacity of the pipette. According to the method, a suspension with a higher MacFarland value is prepared and then diluted to reduce the turbidity to a value that delivers the target amount of sample to the ID or AST test.

[0035] In another example, a sample is obtained and inoculated into a suspension dilution. The turbidity of the suspension is measured. The measured turbidity is determined to be within a predetermined range (e.g., about 0.2 MacFarland to about 2 MacFarland). Based on the amount of sample required for inoculation, a volume of suspension containing the target amount of sample is obtained on a MALDI plate or in an AST broth tube. According to one embodiment, the suspension is prepared by picking a certain amount of colonies from a culture dish and delivering the picked sample into the suspension.

[0036] In another example, a sample is obtained and inoculated into a suspension diluent. The measured turbidity is determined to be below a predetermined range (i.e., less than approximately 0.2 McFarland). In this example, an additional sample is taken and inoculated into the suspension to increase the sample turbidity. The turbidity of the suspension is measured again. If the turbidity is within the predetermined range, a specified volume of suspension is obtained to deliver the target amount of sample onto a MALDI plate or into an AST broth tube. If the adjusted turbidity is excessively high, a dilution protocol is used to dilute the suspension. If the adjusted turbidity is still excessively low, the process is repeated (if there is still an additional sample to be taken). If no additional sample is available, the suspension is not used and is removed from the automated process. If repeated attempts to obtain a suspension with turbidity within the predetermined range are unsuccessful, the suspension is removed from the automated process.

[0037] The volume of the suspension diluent is primarily a design choice issue. The suspension volume cannot be excessively low, because this would result in the suspension's turbidity being far above the target turbidity range, requiring multiple dilution steps to obtain a suspension with the target turbidity. The suspension volume cannot be excessively high, because if this volume is excessively high, the turbidity of the inoculated suspension will be excessively low, requiring multiple steps to obtain a suspension concentration within the target turbidity range.

[0038] In one embodiment, the volume of the suspension diluent into which the sample is initially inoculated is approximately 200 μL to approximately 400 μL. Alternatively, the volume of the suspension diluent can range from approximately 250 μL to approximately 350 μL. In one example, the volume of the suspension diluent into which the sample is inoculated is approximately 300 μL.

[0039] Examples of workflows with specific concentration / dilution protocols are shown in Figures 2 and 3. Generally, a sample is taken in step 200. If the sample is determined to be a mucoid sample in step 210, the sample is mixed with a diluent in step 220, and its turbidity (in MacFarland units) is measured in step 270.

[0040] If the sample is not a mucoid sample, the turbidity of the sample is determined in step 230 before dilution. In step 240, if the initial McFarland value of the suspension for a normal sample is higher than 2 McFarland values, or if the initial McFarland value of the suspension for a sample suspected to contain streptococcus is higher than approximately 1 McFarland value, the suspension proceeds to step 250, in which deionized water is added to the suspension and the suspension is mixed. If the initial McFarland value for a normal sample is approximately 2 or less (or, for a sample suspected to contain streptococcus, the initial McFarland value is 1 or less), the sample is ready to be mixed and the sample proceeds to step 220.

[0041] In step 250, in an automated system with a robotic pipette, the robotic pipette picks up a 1000 μL pipette tip and dispenses 950 pi of deionized water into a cuvette containing the suspension. If the sample is suspected to contain streptococci, the robotic pipette picks up a 1000 μL pipette tip and dispenses 495 μL of deionized water into a cuvette containing the suspension. In the manual procedure, a 1000 μL pipette tip is obtained and the amount of deionized water described above is dispensed into the suspension.

[0042] In step 220, a 1000 μL aliquot suspension is obtained and used to mix the sample by aspirating and dispensing a series of approximately 250 μL suspensions about five times. The pipette tip is then discarded.

[0043] In step 260, any deionized water that was not discharged into the suspension in step 250 is discharged into the waste section. Furthermore, if the volume of the diluted sample exceeds the volume limit, the excess volume of the suspension is removed.

[0044] In step 270, the turbidity of the diluted suspension is measured using a turbidimeter. The measurement of turbidity and the devices used to measure turbidity are known to those skilled in the art and are not described in detail herein. A method and apparatus for measuring turbidity is described in U.S. Patent Publication No. 2016 / 034554. In the protocols of Figures 2 and 3, any step that causes a change in the concentration of the sample in the suspension requires a new turbidity measurement, because the sample turbidity value determines how the suspension is handled in the workflow illustrated in Figures 2 and 3.

[0045] In step 280, the measured sample turbidity is evaluated. If the McFarland value of the cuvette suspension is lower than 0.2, the suspension cannot be used to inoculate the AST broth tube. The system tags the sample with an error flag to ensure that the suspension is not used to inoculate the AST broth tube. The system is updated with this information and the suspension is removed. If the McFarland value of the suspension is higher than 0.2, the suspension can potentially be used to inoculate the AST broth tube.

[0046] In step 290, if the McFarland value of the suspension is 0.2 or greater but 2 or less (if the sample is thought to contain streptococci, the range is 0.2 or greater but 1 or less), the method proceeds to step 295, in which the inoculation volume of the suspension to deliver the target amount of sample into the AST broth tube is calculated.

[0047] The volume is calculated for typical AST broth using the following relationship: The volume to be added = (0.55 ÷ (measured McFarland value of the suspension)) × 47.5 μL (1) For example, if the measured turbidity of the suspension is 2.3, the volume of suspension used to inoculate the AST broth tube is (0.55 ÷ 2.3) × 47.5 μL = 11.36 μL. If the sample is a streptococcal AST sample, the broth is inoculated in the volume according to the following relationship. The volume to be added = (0.55 ÷ (measured McFarland value of the suspension)) × 25.00 μL (2)

[0048] If the McFarland value is higher than 2 (higher than 1, if the suspension is thought to contain streptococci), step 296 determines the number of times the sample has been diluted. If the number of dilutions is less than 3, the suspension is returned to step 250 for further dilution and mixing (and reduction of suspension volume if further dilution is required). If the number of dilutions is 3, an error message is issued because the sample has exceeded the maximum number of dilution steps allowed.

[0049] Figure 3 illustrates the process by which the suspension is used to inoculate a broth tube without standardizing the turbidity of the suspension. The process begins with a prepared suspension, from which a small amount has been removed, and which is to be used to inoculate a MALDI plate. Referring to Figure 3A, the suspension is obtained in step 300. In step 310, the volume of the suspension after MALDI spotting is determined. The actual volume is determined according to the following relationship: Actual volume = V s (For example, 330 μL) - ((Number of target plate spots × Number of suspension layers) × V) spot [For example, 3 μL]) - (Number of layers × Buffer margin) - (Evaporation rate × Deck life (in hours)) (3) In the above equation, V s V is the suspension volume, spotis the volume per spot of suspension to be inoculated onto the MALDI plate. For example, a cuvette containing a sufficient volume of suspension for one spot and four layers on a target plate will have an actual cuvette suspension volume of 330 μL - ((1 × 4) × 3 μL) - (4 × 3) - (10 × 3) = 276 μL. This ensures that the suspension has a deck life of 3 hours. In step 320, the sample is evaluated to determine whether or not it is a mucoid sample. If the sample is mucoid, the sample proceeds to step 370, in which the sample is diluted, the suspension is mixed, and its turbidity is measured. If the sample is not mucoid, the sample proceeds to step 330. The initial turbidity determination of the suspension is used to initiate the processing of the heavy suspension first, thereby allowing the suspension to be inoculated into a broth tube for AST.

[0050] In step 340, if the initial turbidity of the suspension is greater than or equal to a predetermined threshold (e.g., 0.75 McFarland), in step 350, deionized water is added to the suspension and the suspension is mixed. If the initial turbidity of the suspension is lower than a predetermined threshold (e.g., 0.75 McFarland), the method proceeds to step 370 for that suspension. As mentioned elsewhere in this specification, the turbidity and volume thresholds described in the description of Figures 2 and 3 are illustrative and not limiting.

[0051] In step 350, a pipette tip (1000 μL) is obtained and a certain volume of deionized water is dispensed into the target cuvette. This can be done manually or using a robotic pipette mounted on a gantry. The volume of deionized water dispensed is calculated by the following formula: Volume of deionized water = ((Initial MacFarland value ÷ 0.75) × Actual cuvette volume) - Actual cuvette volume (4) According to Equation 4, this ratio between the actual McFarland value and the threshold McFarland value is used to determine the volume of deionized water to be added. If the actual McFarland value is below the upper limit, no deionized water is added to the suspension. Using the above example with the initial McFarland value from 1.4, the volume of deionized water is ((1.4 ÷ 0.75) × 276 μL) - 276 μL = 239 μL. In step 360, if the calculated volume is lower than the maximum volume (e.g., 950 μL for a 1000 μL pipette tip), the calculated volume of deionized water is added to the sample (indicated as "Use the calculated volume" in Figure 3A). If the amount of deionized water to be added exceeds 950 μL, only the maximum volume of 950 μL is added (indicated as "Set the maximum volume" in Figure 3A).

[0052] In step 370, a pipette tip (1000 μL) is used to mix the sample by a series of approximately five aspirations and dispensings of about 250 μL of suspension. After the fifth cycle, the pipette tip is discarded.

[0053] In step 380, it is determined whether an additional volume (i.e., deionized water) needs to be added to the suspension. If an additional volume is required, the suspension proceeds to step 390, in which additional diluent (deionized water) is added to the suspension. If the additional volume causes the suspension to exceed the volume limit, the suspension is removed to reduce the suspension volume so that the volume is below the volume limit. If no additional diluent is required, the method proceeds to step 400 (Figure 3B), in which a turbidimeter is used to measure the turbidity of the suspension. Subsequent processing of the suspension is determined by the measured turbidity (in this embodiment, the measured turbidity is measured in MacFarland units).

[0054] Specifically, if the McFarland value is lower than 0.2, the suspension cannot be used, and an error message is issued as a result. See step 410 in Figure 3B. If the McFarland value is 0.2 or greater, in step 420, if the McFarland value is between 0.2 and 0.3 (which is 0.25 McFarland value ± 20 percent), the suspension is used as a sample source for the AST panel.

[0055] If the McFarland value is higher than 0.3, in step 430 it is determined whether the McFarland value is within the range of 0.5 to 0.6 (which is 0.55 McFarland value ± 10 percent). Such a suspension is deemed suitable to be used as a sample source for inoculation into broth for use in the AST panel. If the turbidity of the suspension is outside the range of 0.5 to 0.6 McFarland in step 430, the sample proceeds to step 440, where further processing of the suspension is determined by the number of pre-dilutions of the suspension. If the suspension has been diluted five times, an error message is issued for that suspension, and the suspension is not used as a sample source for AST broth inoculation.

[0056] If the suspension has been diluted less than five times, in step 450, the suspension is returned to step 360 for further dilution in step 370. For suspensions that are determined to have a McFarland value within a predetermined range, making them suitable for use as a sample source for AST broth inoculation, the suspension is diluted without further turbidity measurements according to the following schedule in Table 1.

[0057] [Table 1]

[0058] To obtain a suspension with a target MacFarland value from a sample having turbidity within the range described in Table 1 above, the sample is diluted according to the following relationship. The volume added = ((Measured McFarland value (step 370) ÷ Target McFarland value) × Actual volume) - Actual volume (5) For example, using the cuvette volume in step 350, if the initial measured MacFarland value of the suspension is 1, the volume to be added is equal to ((1 ÷ 0.75) × 276) - 276, which is 92 μL of diluent (e.g., deionized water) that will be added to the suspension to obtain a suspension with a target MacFarland value of 0.75. However, if the amount of volume to be added exceeds 950 μL, only 950 μL will be added to the sample.

[0059] In step 370, once the volume to be added is determined, the specified volume of diluent (e.g., deionized water) is added to the sample. A 1000 μL pipette tip is obtained for this purpose (in an automated environment, a robotic pipetter obtains the pipette tip, and then the pipetter is translated via a gantry to position the pipette directly above the suspension). The pipette tip is used to mix the suspension by aspirating a certain volume of the suspension and then dispensing that volume of suspension from the pipette tip. The last 50 parts (e.g., 50 μL) are dispensed using the pipette tip over the suspension, ensuring that the pipette tip is completely empty.

[0060] If the target MacFarland value is 0.55 or 0.25, and the total volume of the suspension in the cuvette after dilution is greater than 1500 μL, a certain volume is removed from the suspension so that the suspension does not exceed its maximum volume. Referring to step 390, the excess volume is removed according to the following formula: Volume to be removed = Actual volume (μL) - 1500 μL (6)

[0061] However, if the number of dilutions exceeds five, the sample will be excessively diluted and cannot be used for subsequent processing (follow-up) in the automated AST. In such cases, an error message will be issued as a result. If the number of pre-dilutions is less than five, the suspension returns to step 360 for dilution as described above.

[0062] The above method, in which the volume of AST inoculant was calculated according to the method described above (rather than adjusting the McFarland value of the heavy suspension prepared for MALDI and inoculating a predetermined volume into the AST panel based on the turbidity (e.g., McFarland) specification), was evaluated to determine the effectiveness of AST broth inoculation. For this determination, the reproducibility of the calculation of the volume of inoculant required to achieve an acceptable concentration of the organism in the AST broth tube was tested using E. coli QC strain (BACTEC A25922). The predetermined range of concentration was 2 × 10⁻⁶ for E. coli (BACTEC A25922). 5 CFU / mL ~ 8 × 10 5 The concentration is CFU / mL. BACTEC A25922 was also used to compare the method described herein (in which the suspension is evaluated and the volume of the suspension is determined based on the suspension turbidity) with the process used to prepare a heavy suspension for MALDI, which was then diluted to a target turbidity (either 0.5 to 0.6 McFarland or 0.2 to 0.3 McFarland), and a certain volume of the suspension was inoculated into an AST broth tube. Twenty samples (40 samples in total) were tested using both methods, and plate counts were used to measure bacterial concentrations in the AST broth tubes. AST results from the processed AST panel were also analyzed.

[0063] As reported in Figure 6, for a variety of initial McFarland values, all 40 samples were 2 × 10⁻⁶. 5 CFU / mL ~ 8 × 10 5resulted in an AST broth concentration within CFU / mL. For all 40 samples, all of the minimum inhibitory concentrations exactly matched. Therefore, the process of diluting the resuspension and then determining the volume of the suspension required to deliver the target amount of sample to the AST broth is reproducible and leads to the conclusion that it functions equivalently to the dilution process to the standardized turbidity value (McFarland) for this QC strain.

[0064] Referring to FIG. 4, the E. coli concentration in the AST broth tubes and the final turbidity of the suspension used to inoculate the AST broth tubes using direct AST are shown. The error bars represent the standard deviation of nine plate counts prepared for each sample. The upper dashed line and the lower dashed line indicate the limits of the acceptable concentration range (2×10 5 CFU / mL to 8×10 5 CFU / mL), and the central dashed line represents the center of the range (5×10 5 CFU / mL).

[0065] Referring to FIG. 5, the E. coli concentration in the AST broth tubes and the final turbidity of the suspension used to inoculate the AST broth tubes using a dilution method that dilutes the suspension to the target McFarland value (0.5 McFarland to 0.6 McFarland) are shown. The error bars represent the standard deviation of nine plate counts prepared for each sample. The upper dashed line and the lower dashed line indicate the limits of the acceptable concentration range (2×10 5 CFU / mL to 8×10 5 CFU / mL), and the central dashed line represents the center of the range (at 5×10 5 CFU / mL).

[0066] Referring to FIG. 6, the minimum inhibitory concentration (MIC) for each antibiotic was the same regardless of whether the turbidity of the suspension was adjusted within the target turbidity or whether the volume of the inoculum was adjusted to inoculate the target amount of sample into the AST broth.

[0067] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments, and that other configurations can be devised without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The process involves inoculating a biological sample into a sample diluent to form a biological sample suspension, wherein the biological sample is thought to contain one or more microorganisms. To determine whether the aforementioned biological sample is thought to contain a mucoid sample or a non-mucoid sample, If the biological sample is a non-mucoid sample, first measure the turbidity of the biological sample suspension containing the non-mucoid sample, If the biological sample is a mucoid sample, before first measuring the turbidity of the mucoid sample suspension, the mucoid sample is mixed with the sample diluent to form the mucoid biological sample suspension. To determine whether the measured turbidity of the biological sample suspension or the mucoid biological sample suspension is within a predetermined range, The volume of the biological sample suspension or mucoid biological sample suspension required for inoculation into the culture medium is calculated based on a value obtained by multiplying the ratio of a predetermined target turbidity of the biological sample suspension or mucoid biological sample suspension for inoculation into the culture medium to the measured turbidity, by the volume of the biological sample suspension or mucoid biological sample suspension required to deliver a predetermined amount of biological sample to the culture medium. The calculated volume of the biological sample suspension or the mucoid biological sample suspension is transferred onto the culture medium. An automated method for preparing a sample suspension to provide a target amount of sample for use in microbial identification tests and antimicrobial susceptibility tests, including, The aforementioned microbial identification test is an automated method, specifically a mass spectrometry process.

2. If the measured turbidity of the biological sample suspension or the mucoid biological sample suspension is below the predetermined range, a concentration protocol is performed to increase the turbidity of the biological sample suspension or the mucoid biological sample suspension, and the turbidity of the biological sample suspension or the mucoid biological sample suspension is remeasured. If the measured turbidity is higher than the predetermined range, a dilution protocol is performed to reduce the turbidity of the biological sample suspension or the mucoid biological sample suspension, and the turbidity of the biological sample suspension or the mucoid biological sample suspension is remeasured. The automated method according to claim 1, further comprising:

3. The automated method according to claim 2, wherein the concentration protocol includes adding an additional sample to the biological sample suspension or the mucoid biological sample suspension before remeasuring the turbidity of the biological sample suspension or the mucoid biological sample suspension.

4. The automated method according to claim 2, wherein the dilution protocol includes adding an additional diluent to the biological sample suspension or the mucoid biological sample suspension before remeasuring the turbidity of the biological sample suspension or the mucoid biological sample suspension.

5. The aforementioned enrichment protocol is To determine whether the biological sample suspension or the mucoid biological sample suspension is in a volume greater than a predetermined specification, To provide a prepared sample suspension within the predetermined volume specifications before adding the additional sample to the biological sample suspension or the mucoid biological sample suspension, the excess volume is removed from the container of the biological sample suspension or the mucoid biological sample suspension. The automated method according to claim 3, further comprising:

6. The aforementioned dilution protocol is To determine whether the biological sample suspension or the mucoid biological sample suspension is in a volume greater than a predetermined specification, To provide a biological sample suspension or mucoid biological sample suspension within the predetermined volume specifications, remove excess volume from the container of the biological sample suspension or mucoid biological sample suspension before adding the additional diluent to the biological sample suspension or mucoid biological sample suspension. The automated method according to claim 4, further comprising:

7. If the remeasured turbidity of the biological sample suspension or the measured turbidity of the mucoid biological sample suspension is within a predetermined range of turbidity values, the volume of the biological sample suspension or the mucoid biological sample suspension required to inoculate the culture medium is calculated based on a value obtained by multiplying the ratio of the remeasured turbidity to a predetermined target turbidity for the sample suspension for inoculation into the culture medium by the volume of the biological sample suspension or the mucoid biological sample suspension required to deliver a predetermined amount of biological sample to the culture medium using the biological sample suspension or mucoid biological sample suspension having the predetermined target turbidity, The calculated volume of the biological sample suspension or the mucoid biological sample suspension is transferred onto the culture medium. The automated method according to claim 5, further comprising:

8. If the remeasured turbidity of the biological sample suspension or the remeasured turbidity of the mucoid biological sample suspension is within a predetermined range of turbidity values, the volume of the biological sample suspension or the mucoid biological sample suspension required to inoculate into the culture medium is calculated based on a value obtained by multiplying the ratio of the remeasured turbidity of the biological sample suspension or the mucoid biological sample suspension by the volume of the biological sample suspension or mucoid biological sample suspension with the predetermined target turbidity required to deliver a predetermined amount of biological sample to the culture medium, The calculated volume of the biological sample suspension or the mucoid biological sample suspension is transferred onto the culture medium. The automated method according to claim 6, further comprising:

9. The automated method according to claim 5, further comprising repeating the concentration protocol if the remeasured turbidity is not within a predetermined range of turbidity values.

10. The automated method according to claim 6, further comprising repeating the dilution protocol if the remeasured turbidity is not within a predetermined range of turbidity values.

11. The automated method according to claim 9, further comprising discarding the biological sample suspension or the mucoid biological sample suspension when the concentration protocol has been repeated a predetermined number of times.

12. The automated method according to claim 10, further comprising discarding the biological sample suspension or the mucoid biological sample suspension when the dilution protocol has been repeated a predetermined number of times.

13. The automated method according to claim 1, further comprising mixing the mucoid biological sample suspension with a diluent before first measuring the turbidity.

14. The automated method according to claim 1, further comprising first measuring the turbidity of the mucoid biological sample suspension before dilution.

15. The automated method according to claim 1, wherein the biological sample suspension contains streptococci.

16. The automated method according to claim 1, wherein the mass spectrometry process is a matrix-assisted laser desorption / ionization method.

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