WO2013155525A1 - Système ultra rapide de culture de sang et d'essai de prédisposition - Google Patents

Système ultra rapide de culture de sang et d'essai de prédisposition Download PDF

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Publication number
WO2013155525A1
WO2013155525A1 PCT/US2013/036619 US2013036619W WO2013155525A1 WO 2013155525 A1 WO2013155525 A1 WO 2013155525A1 US 2013036619 W US2013036619 W US 2013036619W WO 2013155525 A1 WO2013155525 A1 WO 2013155525A1
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Prior art keywords
microdroplet
light
growth
sample
cartridge
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PCT/US2013/036619
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English (en)
Inventor
Gideon Eden
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Biolumix, Inc
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Publication of WO2013155525A1 publication Critical patent/WO2013155525A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present disclosure pertains to methods and systems for blood culturing and susceptibility testing.
  • the method includes the steps of enclosing at least one of the microorganism cells in a light transparent microdroplet.
  • the microdroplet contains a mixture of liquid growth media and/or at least one fluorescent indicator dye capable of indicating the presence of at least one byproduct generated by growing microorganisms, wherein the indicator dye generates visible light when excited by ultraviolet light after exposure to the metabolic by-products.
  • the method further includes the step of incubating the micro-droplet under conditions that promote rapid growth of the at least one microorganism and generation of metabolic by-products of said growth of the at least on microorganism and exposing the incubated microdroplet to a concentrated UV light emanating from a miniaturized UV light emitting diode. Visible light is generated from the at least one microdroplet and is indicative of the presence of at least one microorganism cell in the micro-droplet generating said metabolic by-products interacting with said fluorescent indicator.
  • the device includes means for enclosing at least one microorganism cell in a light transparent micro-droplet.
  • the micro-droplet also contains liquid growth media and at least one fluorescent dye capable of indicating the presence of by-products generated by growing microorganisms in which the indicator dye generates visible light when excited by ultraviolet light.
  • the device also includes an incubator device configured to maintain the temperature of the micro-droplets at a value that promotes rapid microorganism growth and production of byproducts of metabolic growth together with at least one ultraviolet light emitting diode (UV- LED) paired with a detector configured to detect light in the visible spectrum.
  • the UV-LED is configured to emit a concentrated beam of light in the UV spectrum.
  • the device also includes means for introducing the at least one micro-droplet into the beam of concentrated UV light such that the UV light interacts with the fluorescent indicator to emit visible light.
  • Figure 1 is a multi-stage diagram of an embodiment of the process and device as disclosed herein.
  • a threshold concentration of 10 6 to 10 7 CFU/ml is required in order to detect the presence of growing microorganisms. This threshold dictates culturing times of 1-2 days for fast growing organisms and up to 5 days for the slowest microorganisms.
  • the new system is predicated on the unexpected discovery that two distinct technologies that have been developed for other distinct purposes and are demonstrated to be individually operational can be integrated into the method and device disclosed herein.
  • the incorporation of the technologies in the device and method as disclosed herein will result in a new generation of rapid clinical tests that can save patients' life and reduce the growing risk of cultivating drug resisting bacteria.
  • the one such technology is the technology related to gel micro-droplets. Variations on this technology have been developed and practiced since 1986.
  • the additional technology pertains to miniaturization of Ultraviolet Light Emitting Diodes (UV LED).
  • UV LED Ultraviolet Light Emitting Diodes
  • Gel micro-droplets are spherical particles of gel that can be made by dispersing a liquid sample for example a polysaccharide such as agarose into an inert low dielectric fluid, such as mineral oil, and then cooling transiently.
  • a liquid sample for example a polysaccharide such as agarose
  • an inert low dielectric fluid such as mineral oil
  • One such non-limiting example of a method for producing gel microdroplets is appended at the end of this disclosure and is incorporated by reference herein.
  • Technology related to gel microdroplets is outlined in Weaver, et al., "Gel Microdroplets: Rapid Detection and Enumeration of Individual Microorganisms by Their Metabolic Activity" Pathology, Vol. 6, September 1986. The article was previously included as Attachment A and is incorporated by reference herein.
  • the article outlines a new, flexible method for rapid detection and enumeration of individual microorganisms using small (e.g. 10 to 100 micron diameter) gel particles surrounded by a non-aqueous liquid with low dielectric constant.
  • small e.g. 10 to 100 micron diameter
  • Primary samples without prior cultivation can be used.
  • gel microdroplets (GMDs) surrounded by an inert oil were statistically inoculated such that GMDs had a high probability of initially containing either zero or one acid-producing microorganism.
  • GMDs retained dissociable metabolites produced by individual cells (or microcolonies) within the small GMD volume.
  • GMDs The accumulated metabolic acids led to rapid changes in pH within GMDs initially occupied by one microorganism or colony forming unit (CFU), while GMDs with zero microorganisms had unchanged pH.
  • CFU colony forming unit
  • the cumulative activity within individual GMDs was then determined using pH sensitive fluorescence indicators. This method was used to enumerate individual cell viability directly, without any prior culture, from clinically infected urine samples in about 1.5 hours for several rapidly growing pathogens, and was in agreement with much slower conventional culture methods. Because GMDs can be made readily in large numbers, and because many indicator systems can be used, GMDs used with automated measurement apparatus should have wide applicability.
  • Rapid detection and/or enumeration of viable microorganisms is of central importance to microbiological research, clinical microbiology, environmental science, food technology, toxicology and biotechnology.
  • Two major classes or assays are used.
  • the first class rapidly detects and identifies specific microorganisms directly from a primary sample, and is based on cell constituent assays, but does not determine cell viability.
  • the most widely used in this class are specific ligand binding assays, particularly immunoassays and genetic probes 5>8"10 . However, these do not distinguish between dead and viable cells, do not provide an enumeration, but instead emphasize identification. For these reasons their microbiological use is restricted to determinations in which direct assessment of the physiological state of the microorganism is irrelevant.
  • the second class of assays is used for detection and/or enumeration of viable cells, either directly from the primary sample, or from a subculture of the primary sample.
  • the most traditional and widely used method is the plate count, which allows determination of individual cell viability under many test conditions 7 ' 11.
  • An important attribute of viable plate enumeration is that the count is independent of the concentration of the microorganism in the sample, because formation of each colony proceeds from an initial individual cell.
  • GMD gel microdroplet
  • Instrumented technologies for rapidly determining cell or culture viability have been developed that partially address some of the limitations of the viable plate assay, but these instrumented methods generally require a prior culture and isolation from the primary sample, in order to first obtain a monoculture.
  • These instrumented methods include optical techniques such as those that measure the light scattering properties of a culture 12 , metabolic activity based techniques such as those that measure overall changes in pH, carbon dioxide release, electrical impedance, chemiluminescence or fluorescence.
  • a disadvantage of all of these metabolic activity methods is that they are based on the combined effects of a large, unknown number of cells, and therefore do not actually yield a count.
  • prior culture is generally required, which yields the isolates whose activity is actually measured.
  • these total population methods exhibit detection times which become significantly longer as the sample's cell concentration decreases.
  • GMDs are approximately spherical particles of gel that can be made conveniently by dispersing a liquid agarose sample into an inert low dielectric constant fluid, such as mineral oil, and then cooling transiently.
  • agarose was used, but other gel materials can also be used.
  • the resulting GMDs typically have diameters ranging from 10 to 100 microns, with corresponding volumes of which are exceedingly small.
  • Microorganisms can be readily incorporated into the matrix of GMDs as they are made. Many of the GMDs contain initial individual microorganisms where encapsulated in gel or oil, they rapidly accumulate
  • extracellular products of individual viable cells are used with optical indicators for rapid determination of individual cell viability.
  • This method combines the rapidity of total population activity approaches with the ability to detect and enumerate viable cells independently of the sample cell concentration. Both the speed and independence of cell concentration result from the basic but simple principle that GMDs confine initial individual cells within a small volume, VGMD- By surrounding the GMDs with an inert oil, hydrophilic compounds such as pyruvate and other com-GMDs, containing metabolically active cells, and therefore accumulate metabolites, can be differentially detected (using a fluorescence indicator system) compared to GMDs that do not contain cells.
  • a similar approach has allowed detection of individual ⁇ -galactosidase-containing bacteria 25 ' 26 , or even of individual enzyme molecules using a fluorescence assay for this enzyme's activity.
  • the gel matrix makes a microdroplet physically robust, increasing microdroplet stability and allowing physical manipulation.
  • GMDs consists of a rapid sub-division of a macroscopic sample containing cellso into many robust microscopic subvolumes (the GMDs). If the subvolumes are sufficiently small, many have a high probability of initially containing zero or one cell. For cells randomly distributed in a sample, the fraction P(n, n) of GMDs containing n cells depends on both the cell concentration and the volume of the GMDs, and is described by the Poisson distribution.
  • n is the average number of cells initially entrapped in a particular size GMD. More specifically, if the cell concentration is the average number of cells initially entrapped in a particular size GMD. More specifically, if the cell concentration is p, the average number of cells in that Thus, the probability of unoccupied GMDs (zero initial
  • CFUs colony forming units
  • Detection of extracellular changes within GMDs due to individual cells or resulting microcolonies can be accomplished by use of fluorescent or colorimetric indicators.
  • extracellular assay of common biochemical parameters such as pH, metabolites or enzyme activity all should be suitable.
  • This study demonstrated that the use of GMDs, a fluorescence pH indicator system and fluorescence microscopy allowed a viable count to be rapidly estimated by: (1) choosing GMDs in a particular size (V GMD ) range, (2) counting both the number of GMDs that exhibit a fluorescence color change and the number that does not change color in this size range, and (3) applying Poisson statistics.
  • the resulting GMDs as employed herein typically have diameters from 10 to 100 microns, with volumes of It is envisioned that the targeted or tested microorganisms in the patient's sample can be incorporated into the matrix of the GMDs as the droplets are made. Alternately, the original patient sample can initially be mixed with growth media and pre-incubated for several hours in order to increase the bacterial population after which the mixture can be incorporated into GMDs. Initially, many of the generated GMDs will each contain a few individual microorganism cells surrounded by growth media. In addition to the gel forming material and microorganism cells, fluorescence indicating substrate can be incorporated in the mixture.
  • the inoculums' concentration is low, the average initial occupation of GMDs is low, resulting in occupation by zero or perhaps one or a few cells in some of the microdroplets that are formed. It is understood that some of the micro-droplets formed will be unoccupied. This allows an enumeration based on individual cell viability that can be accomplished by counting the number of bacteria occupied and non-occupied GMDs after an additional short incubation time.
  • One significant advantage of the gel micro-droplet entrapment of a microorganism cell or cells is the very high internal microorganism concentration in spite of the low actual numerical count. Microorganism concentration is inversely proportional to the
  • GMD volume For example a GMD with 3x10 - " 8 ml can have an effective microorganism concentration of 3x10 CFU/ml although the GMD is occupied by a single microorganism cell. This concentration is high enough to generate sufficient concentration of metabolites to change the fluorescing properties of the embedded substrate within a few additional generation times of further incubation.
  • the incorporation of fluorescent dye substrates enables detection of cell- occupied GMD's by utilizing an external light source that will penetrate the transparent wall of the GMD. If UV light is employed, the UV light will interact with the fluorescent substrate present in the GMD to generate visible light in the cell-occupied GMD; while no visible light is generated in non-cell-occupied GMDs.
  • UVLED Ultraviolet Light Emitting Diodes
  • the second advantage of the LED technology is the possibility to manufacture multiple miniaturized units on a single solid state chip.
  • the most dramatic development was the replacement of the TV screens from Cathode Ray Tube (CRT) to screens constructed from LED pixels.
  • CRT Cathode Ray Tube
  • Attachment B Several commercially available arrays of UV LED are illustrated in Attachment B, the disclosure of which is incorporated by reference herein. It is contemplated that such devices can be further customized to operate in conjunction with the new invention as disclosed herein.
  • Figure 1 illustrates an embodiment of the device and methodology disclosed herein. It is illustrated in stages, each describing a serial step, associated with newly designed devices and methods.
  • Stage 1 In this step a sample is obtained by any suitable method.
  • the sample to be cultured is a blood sample
  • the blood sample is drawn by any suitable method.
  • the blood sample may be drawn, in a manner similar to those currently employed for blood culturing.
  • the sample may be introduced into a suitable container that includes suitable microbial growth media.
  • the container can also include at least one fluorescing indicator substrate that is capable of interacting with metabolic products of growing
  • microorganisms to produce a detectable light emission upon exposure to ultra violet light.
  • the indicator substrate is activated by ultraviolet light and fluoresces in the visible spectrum.
  • a sample such as a blood sample can be directly drawn into a suitably configured container.
  • the suitably configured container can be equipped with suitable material such as growth medium and/or indicator substrate(s) such as material(s) that reacts with by-products of metabolic processes of microorganisms that may be present in the introduced sample.
  • This drawing bottle may have vacuum in its head space to allow a direct blood draw of predetermined volume if desired or required.
  • Stage 2 The drawing bottle or other suitable container containing the mixture of the blood sample, the growth media and/or the indicator substrate is placed into an incubator device that maintains the resulting mixture at an optimal temperature for bacterial growth.
  • the vessel may be shaken or otherwise stirred to enable efficient oxygenation of the liquid.
  • the incubation time may differ for different kinds of organisms that may be present in the sample. For very fast growing organisms (for example, microorganisms exhibiting 20-35 minute doubling times) relatively short incubation time (for example 2-3 hours) should provide a sufficient number of cells in the liquid. For slower growing organisms, longer incubation times may be required. Since it is unknown what types of organisms are present in many particular given situations, several mixtures can be incubated for distinctive durations of incubation time.
  • Stage 3 In this stage microorganisms in the sample are encapsulated in GMDs by a suitable method and the GMD-encapsulated microorganisms are introduced into a suitably configured reading cartridge.
  • Encapsulation can progress by a suitable method.
  • a portion of the previously incubated mixture is drawn from the bottle or other suitable container via a line that flows through a microdroplets generation station.
  • the generated droplets can encapsulate microorganisms as they flow by.
  • the flow rate and the generation rate of the droplets are set so that each individual droplet may encapsulate 0-3 microorganisms. It is understood that the majority of the droplets may remain empty depending upon the microbial concentration in the sample after the incubation stage.
  • a suitable reading cartridge is a device that comprises a flat container defining a well or suitable sampler receiving area capable of containing a thin layer of liquid (for example, a device that receives sample to a width or depth of 0.2-0.5).
  • a suitable device will be one having a sample receiving area that is transparent to UV light. It is understood that at this stage, one or more reading cartridges can be prepared. For example, at this stage similar reading cartridges can be filled, with some of the cartridges containing droplets with additional anti microbial agents (antibiotics) of different chemical compounds at various concentrations. These agents can be introduced by the same
  • injector/mixer station during the sample's flow through the station in certain embodiments.
  • Stage 4 In this stage each reading cartridge is incubated for predetermined time to allow several new bacterial generations to form. Within a few generations of growth cycles, the microorganisms contained in a respective microdroplet will exceed the concentration detection threshold, and the metabolites produced as a result of microorganism activity will interact with the fluorescent indicator substrate.
  • Stage 5 After incubation has been completes ⁇ d, the specific reading cartridge is placed into an optical reader that can identify and enumerate fluorescing micro-droplets.
  • the optical reader as disclosed herein incorporates a specially designed array of UV pixels that can be activated one at a time. This UV LED screen provides illumination, one pixel at a time, to the transparent section of the reading cartridge.
  • a photo detector with a wide reception area is placed on the other side of the reading cartridge.
  • suitable photo detectors include either a wide area Photo Multiplying Tube (PMT) or a sensitive CCD camera.
  • a UV filter can be placed between the cartridge and the photo detector to prevent direct UV light from activating the photo detector.
  • Stage 6 If no fluorescing droplets are detected, the sample is negative, and no further action is required at this point in time (excluding repeat of the same test for longer incubated samples of Stage 2). If this (reference) sample is positive, the corresponding cartridges containing the anti-microbial agents as discussed previously are tested as well.
  • results may be available in several hours. For slower growing microorganisms, results may be available during the second day.
  • the original sample can be further incubated to provide identification (ID) of the organisms if further diagnostic steps are required.
  • ID identification

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Abstract

La présente invention concerne un procédé et un dispositif qui réduisent significativement le temps de diagnostic associé à la détection de micro-organismes dans un échantillon biologique associant (a) l'encapsulation microbactérienne et (b) la technologie améliorée des diodes électroluminescentes à ultraviolet (DEL UV).
PCT/US2013/036619 2012-04-13 2013-04-15 Système ultra rapide de culture de sang et d'essai de prédisposition WO2013155525A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11204350B2 (en) 2013-03-15 2021-12-21 Ancera, Llc Systems and methods for bead-based assays in ferrofluids
US11268124B2 (en) 2016-01-25 2022-03-08 Bio-Rad Europe Gmbh Digital microbiology
US11285490B2 (en) 2015-06-26 2022-03-29 Ancera, Llc Background defocusing and clearing in ferrofluid-based capture assays
US11383247B2 (en) 2013-03-15 2022-07-12 Ancera, Llc Systems and methods for active particle separation

Citations (3)

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US6806058B2 (en) * 2001-05-26 2004-10-19 One Cell Systems, Inc. Secretions of proteins by encapsulated cells
WO2005010169A2 (fr) * 2003-07-23 2005-02-03 Diversa Corporation Criblage a haut rendement ou sur la base de capillaires pour bioactivite ou biomolecule
US8030095B2 (en) * 2008-03-04 2011-10-04 Crystal Bioscience Inc. Gel microdrop composition and method of using the same

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Publication number Priority date Publication date Assignee Title
US6806058B2 (en) * 2001-05-26 2004-10-19 One Cell Systems, Inc. Secretions of proteins by encapsulated cells
WO2005010169A2 (fr) * 2003-07-23 2005-02-03 Diversa Corporation Criblage a haut rendement ou sur la base de capillaires pour bioactivite ou biomolecule
US8030095B2 (en) * 2008-03-04 2011-10-04 Crystal Bioscience Inc. Gel microdrop composition and method of using the same

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Title
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EUN ET AL.: "Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation", ACS CHEMICAL BIOLOGY, vol. 6, no. 3, 18 March 2011 (2011-03-18), pages 260 - 266 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11204350B2 (en) 2013-03-15 2021-12-21 Ancera, Llc Systems and methods for bead-based assays in ferrofluids
US11383247B2 (en) 2013-03-15 2022-07-12 Ancera, Llc Systems and methods for active particle separation
US11285490B2 (en) 2015-06-26 2022-03-29 Ancera, Llc Background defocusing and clearing in ferrofluid-based capture assays
US11833526B2 (en) 2015-06-26 2023-12-05 Ancera Inc. Background defocusing and clearing in ferrofluid-based capture assays
US11268124B2 (en) 2016-01-25 2022-03-08 Bio-Rad Europe Gmbh Digital microbiology
US11952610B2 (en) 2016-01-25 2024-04-09 Bio-Rad Europe Gmbh Digital microbiology

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