WO 2008/140608
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`PCT/US2007/086985
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`METHODS AND DEVICES FOR DETECTING METHICILLINRESISTANT
`STAPHYLOCOCCUSAUREUS
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`BACKGROUND
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`[0001] Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to the
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`antibiotic methicillin. Staphylococcus aureus, sometimes referred to simply as “staph,” or
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`“staph A” is a common bacterium found on the skin of healthy people. If staph gets into
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`the body it can cause a minor infection such as boils or pimples or serious infections such
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`as pneumonia or blood infections.
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`[0002] One antibiotic commonly used to treat staph infections is methicillin. While
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`methicillin is very effective in treating most staph infections, some staph bacteria have
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`developed a resistance to methicillin and can no longer be killed by this antibiotic. The
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`resistant bacteria are called methicillin-resistant staphylococcus aureus 0r MRSA.
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`[0003] Current methods for the detection of staph and MRSA may not be performed
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`directly from blood culture bottles. In the methods, a sample is taken from a positive blood
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`culture bottle and plated onto culture medium and grown overnight. A colony is removed
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`from the culture medium and its species determined by a coagulase test. If it is coagulase-
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`positive, the organism is staph A, if it is coagulase-negative, it is another species of
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`Staphylococcus. Once the organism is determined to be staph A, a colony is plated onto
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`culture medium containing methicillin and grown for a minimum of 24 hours. If the
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`organism grows, it is MRSA and if it does not grow, it is methicillin-sensitive
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`Staphylococcus aureus (MSSA). Thus, such methods are time-consuming and involve
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`many steps, including preparing a secondary culture plate.
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`SUMMARY
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`[0004] Provided are methods and devices to determine the presence of MRSA directly in
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`positive blood cultures. The methods comprise analyzing blood specimens taken directly
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`from positive blood culture bottles using two immunoassays to determine if the sample is
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`MRSA. The first immunoassay detects the presence of the partiucular gram positive
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`bacteria (e.g. Staphylococcus A). The second assay detects a drug resistance (e.g.
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`methicillin-resistant (positive result) or methicillin-sensitive (negative result».
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`[0005] Use of the two immunoassays provides a significant time advantage and simplicity
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`over conventional methods. Conventional methods take up to 48 hours after the blood
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`culture bottle has signaled positive on the instrument to determine if the organism is
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`MRSA. Using the two immunoassays requires a maximum of four (4) hours once the blood
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`culture bottle signals positive.
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`[0006] Further objectives and advantages of the present invention will become apparent as
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`the description proceeds. To gain a full appreciation of the scope of the present invention,
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`it will be fiirther recognized that various aspects of the present invention can be combined
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`to make desirable embodiments of the invention.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`[0007] FIGURE 1 depicts a schematic of the provided methods for determining if the
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`bacteria in a sample are MRSA, MSSA or another species of staph.
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`[0008] FIGURE 2 depicts a prototype device which may in some embodiments be used to
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`implement the provided methods. Antibodies or other molecules able to bind to molecules
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`specific to an antigen, for example, staph A antigens or penicillin—binding protein 2a are
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`conjugated to a test zone on the conjugate pad. A control zone is also present on the
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`conjugate pad.
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`[0009] FIGURE 3 depicts how to read positive or negative test results in the device of
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`FIGURE 2.
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`[0010] FIGURE 4 depicts an invalid test result as read from the device of FIGURE 2.
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`DETAILED DESCRIPTION
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`[0011] Unless defined otherwise above, all technical and scientific terms used herein have
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`the same meaning as commonly understood by one of ordinary skill in the art to which this
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`invention belongs. Where a term is provided in the singular, the inventor also contemplates
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`the plural of that term. The nomenclature used herein and the procedures described below
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`are those well known and commonly employed in the art.
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`[0012] The term “antibody” refers to an immunoglobulin, derivatives thereof which
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`maintain specific binding ability, and proteins having a binding domain which is
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`homologous or largely homologous to an immuno globulin binding domain. These proteins
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`may be derived from natural sources, or partly or wholly synthetically produced. An
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`antibody may be monoclonal or polyclonal. The antibody may be a member of any
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`immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
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`In exemplary embodiments, antibodies used with the methods and compositions described
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`herein are derivatives of the IgG class.
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`[0013] The term “antibody fragment” refers to any derivative of an antibody which is less
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`than fiill-length.
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`In exemplary embodiments, the antibody fi‘agment retains at least a
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`significant portion of the fiJll—length antibody’s specific binding ability. Examples of
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`antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, scFV, FV, dsFV
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`diabody, and Fd fragments. The antibody fragment may be produced by any means. For
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`instance, the antibody fragment may be enzymatically or chemically produced by
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`fragmentation of an intact antibody, it may be recombinantly produced from a gene
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`encoding the partial antibody sequence, or it may be wholly or partially synthetically
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`produced. The antibody fragment may optionally be a single chain antibody fragment.
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`Alternatively, the fragment may comprise multiple chains which are linked together, for
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`instance, by disulfide linkages. The fragment may also optionally be a multimolecular
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`complex. A filnctional antibody fragment will typically comprise at least about 50 amino
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`acids and more typically will comprise at least about 200 amino acids.
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`[0014] The terms “comprise” and “comprising” is used in the inclusive, open sense,
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`meaning that additional elements may be included.
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`[0015] The term “including” is used herein to mean “including but not limited to”.
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`“Including” and “including but not limited to” are used interchangeably.
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`[0016] The term “sample” refers to any sample potentially containing Staphylococcus
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`bacteria. For example, a sample may be a bodily fluid such as blood, urine or saliva.
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`[0017] Provided are methods and devices for determining whether a bacterium in a blood
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`sample is an antibiotic-resistant bacterium, wherein the determining is performed by
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`analyzing blood specimens taken directly from positive blood culture bottles. In certain
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`embodiments, the methods and devices are for determining whether a bacterium in a blood
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`sample is methicillin—resistant staph A.
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`[0018] In certain embodiments, the methods comprise analyzing blood specimens taken
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`directly from positive blood culture bottles using two lateral flow devices to determine if
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`the sample is MRSA.
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`[0019] In certain embodiments, the methods may comprise: (a) detecting, using a first
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`immunoassay, which detects specific gram positive bacteria in a sample from a gram
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`positive blood culture; and (b) determining, in the sample from the gram positive blood
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`culture via a second immunoassay, whether the detected species of gram positive bacteria is
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`resistant to a particular antibiotic.
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`[0020] In certain embodiments, the detected species of gram positive bacteria is a
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`Staphylococcus species. The species may be detected by using a first immunoassay that
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`detects an enzyme or other protein present on a particular species of gram positive bacteria,
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`i.e., an enzyme or other protein or molecule unique to that particular species. In certain
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`embodiments, the enzyme is coagulase.
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`[0021] Whether the species is resistant to a particular antibiotic or not may be determined
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`by a immunoassay. In certain embodiments, the assay is for a protein produced by a
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`species of gram positive bacteria resistant to a particular antibiotic. In certain
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`embodiments, the marker for antibiotic resistance is a penicillin binding protein and the
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`antibiotic is methicillin.
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`[0022] The immunoassays may comprise antibodies to detect the enzyme or other protein
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`present on a particular species of gram positive bacteria, or the protein produced by a
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`species of gram positive bacteria resistant to a particular antibiotic
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`[0023] In certain embodiments, the method may comprise the steps of: (a) detecting, using
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`a first immunoassay, whether Staphylococcus aureus or another species of Staphylococcus
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`is present in a sample from a gram positive blood culture; and (b) determining, in the
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`sample from the gram positive blood culture via a second immunoassay, whether the
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`detected species of Staphylococcus is methicillin resistant. In certain embodiments, the
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`methods may filrther comprise a preliminary step of obtaining a sample from a gram
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`positive blood culture.
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`[0024] FIGURE 1 depicts a schematic of the order in which such methods may be used to
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`determine the bacteria in a sample are MRSA, MSSA or another species of staph.
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`[0025] The steps in the methods described above may be accomplished by any
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`immunoassay format, including lateral flow, ELISA and direct fluorescence assays. In
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`some embodiments, the species detection step may be performed with one type of assay or
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`device and the antibiotic resistance detection step may be performed with another type of
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`assay or device.
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`[0026] For use in the methods described above, kits and devices for the practice of the
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`above—described methods are also provided. Devices for practice of the methods include
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`lateral flow devices (wherein the reagents employed in the reaction may be dried or
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`immobilized onto a chromatographic support contained within the device), a test strip, or
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`other support for practice of the methods. A kit for the practice of the above methods may
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`include a support, reagents and wash and incubation buffers. Such kits and devices can
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`contain any number or combination of reagents or components. The kits can comprise one
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`or more of the above components in any number of separate containers, tubes, vials and the
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`like or such components can be combined in various combinations in such containers. Kit
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`components may be packaged for either manual or partially or wholly automated practice of
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`the foregoing methods. Further, instructions for the use of a device or kit may be included
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`with the device or kit. Such kits and devices may have a variety of uses, including, for
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`example, diagnosis, therapy, and other applications.
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`EXEMPLIFICATION
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`[0027] The invention, having been generally described, may be more readily understood by
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`reference to the following examples, which are included merely for purposes of illustration
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`of certain aspects and embodiments of the present invention, and are not intended to limit
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`the invention in any way. All headings are for the convenience of the reader and should not
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`be used to limit the meaning of the text that follows the heading, unless so specified.
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`[0028] Example 1: Exemplary MRSA Test Procedures Using a Prototype Device
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`[0029] In one embodiment, the methods comprisc assaying for staph A first as a screen and
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`then assaying for penicillin-binding protein 2a using two lateral flow devices. A device as
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`shown in FIGURE 2 may be used to implement the lateral flow devices as follows:
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`[0030] A. Staph A Test Procedure
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`[0031] l. Aspirate the sample directly from a positive blood culture bottle, via syringe.
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`[0032] 2. Centrifiige the sample for 10 minutes at 10,000 x g.
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`[0033] 3. Slowly dispense lOO uL of the supernatant sample directly onto the sample pad of
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`the device.
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`[0034] 4. Allow the sample to flow to the bottom of the test strip.
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`[0035] 5. Peel off the brown adhesive strip and close the device.
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`[0036] 6. Read the result within 15-30 minutes after closing the device. Ifthe sample is
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`positive, proceed to the PBP2a assay.
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`[0037] B. PBP2a Test Procedure (#1)
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`[0038] l. Aspirate sample from a blood culture bottle, via syringe, if the sample tests
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`positive on the staph A assay.
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`[0039] 2. CentrifiJge the sample for 5 minutes at 3,000 rpm.
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`[0040] 3. Remove 1 .0 mL ofthe supernatant. Add 100 uL ofthe extraction buffer and mix.
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`[0041] 4. Slowly dispense lOO uL ofthe sample directly onto the sample pad ofthe device.
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`[0042] 5. Allow the sample to flow to the bottom ofthe test strip.
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`[0043] 6. Peel off the brown adhesive strip and close the device.
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`[0044] 7. Read the result within 15-30 minutes after closing the device.
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`[0045] C. PBP2a Test Procedure (#2)
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`[0046] In this alternative procedure, the device has a wash pad in place of the plastic shim.
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`[0047] l. Aspirate sample from a blood culture bottle, via syringe, if the sample tests
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`positive on the staph A assay.
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`[0048] 2. Add 100 uL of the extraction buffer and mix.
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`[0049] 3. CentrifiJge the sample for 10 minutes at 10,000 x g.
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`[0050] 4. Slowly dispense 100 uL of the sample directly onto the sample pad of the device.
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`[0051] 5. Allow the sample to flow to the bottom of the test strip.
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`[0052] 6. Add 4 drops of wash buffer to the wash pad.
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`[0053] 6. Peel off the brown adhesive strip and close the device.
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`[0054] 7. Read the result within 15-30 minutes after closing the device.
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`[0055] For any of the procedures, the test results may by read as indicated in FIGURE 3
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`and FIGURE 4.
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`EQUIVALENTS
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`[0056] Those skilled in the art will recognize, or be able to ascertain using no more than
`routine experimentation, many equivalents to the specific embodiments of the invention
`described herein. While specific embodiments of the subject invention have been
`discussed, the above specification is illustrative and not restrictive. Many variations of the
`invention will become apparent to those skilled in the art upon review of this specification.
`The filll scope of the invention should be determined by reference to the claims, along with
`their filll scope of equivalents, and the specification, along with such variations. Such
`equivalents are intended to be encompassed by the following claims.
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