WO2021046343A1

WO2021046343A1 - Lysing solution for real-time determining the identification and antibiotic resistance of pathogenic microorganisms

Info

Publication number: WO2021046343A1
Application number: PCT/US2020/049394
Authority: WO
Inventors: Robert Ulrich; Victoria E. WAGNER; Lauren MCDANIEL
Original assignee: Teleflex Medical Incorporated
Priority date: 2019-09-06
Filing date: 2020-09-04
Publication date: 2021-03-11

Abstract

A lysis solution for lysing microorganisms in a biological sample includes 2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate, 20 µM to 160 µM of a Tris HCL, pH 8.5, 6 µM to 48 µM of a Magnesium chloride, 35 µM to 280 µM of a Potassium chloride, and 0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.

Description

LYSING SOLUTION FOR REAL-TIME DETERMINING THE IDENTIFICATION AND ANTIBIOTIC RESISTANCE OF PATHOGENIC MICROORGANISMS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 62/897,057, filed September 6, 2019, the disclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0001] This invention relates generally to identification of pathogenic microorganisms. More particularly, the present invention relates, for example, to a real-time identification of a pathogenic microorganism and/or its antibiotic resistance in a biological sample via nucleic acid sequence-based amplification (NASBA) of a specific RNA sequence.

BACKGROUND OF THE INVENTION

[0002] Bloodstream infections (BSIs) are major causes of morbidity and mortality. On the basis of data from death certificates, these infections are the 10th leading cause of death in the United States, and the age-adjusted death rate due to BSIs has risen by 78% over the last 2 decades. The true incidence of nosocomial BSIs is unknown, but it is estimated that approximately 250,000 cases occur annually in the U.S. Bacteremia is a BSI that occurs when various species of bacteria enter the bloodstream. In people at risk, bacteremia may result when a person's own colonizing flora, present within their digestive tract flora, enter the bloodstream. It can also occur when medical equipment (e.g., indwelling central venous catheters) or devices become contaminated with bacteria from the environment or the hands of healthcare workers. Bacteremia can be associated with an inflammatory response in the body (e.g., sepsis and septic shock). In particular, sepsis and septic shock have a relatively high mortality rate. Bacteria in the bloodstream can sometimes spread to other parts of the body.

[0003] The symptoms of bacteremia are typically not specific, and patients will most frequently present with a fever of unknown origin. Differential diagnosis of bacteremia and sepsis can be complicated by the fact that other conditions (e.g., systemic inflammatory response syndrome (SIRS)) can present with similar symptoms. Bacteremia is usually diagnosed by a combination of blood culture and post-culture testing, which also identifies the specific species. These procedures require multiple days and, in some cases, species identification can require longer than six days. However, early initiation of appropriate therapy is important for effective treatment. For example, inadequate initial antimicrobial therapy (e.g., therapy that begins too late and/or that involves administration of an inappropriate drug) is an independent predictor of mortality, and delayed therapy is also associated with an extended length of hospital stay.

[0004] Thus, there remains a need for rapid and sensitive methods, preferably requiring minimal or no sample preparation, for detecting the presence of pathogen-associated analytes for diagnosis and monitoring of diseases, including bacteremia, sepsis, and SIRS. In particular, there is a need for methods and panels that are able to simultaneously detect the presence of multiple pathogens in a sample and identify those that are present.

SUMMARY OF THE INVENTION

[0005] The foregoing needs are met, to a great extent, by the present invention, wherein aspects of a lysis solution is provided.

[0006] An aspect of the disclosure pertains to a lysis solution. The lysis solution for lysing microorganisms in a biological sample includes 2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate, 20 mM to 160 mM of a Tris HCL, pH 8.5, 6 pM to 48 pM of a Magnesium chloride, 35 pM to 280 pM of a Potassium chloride, and 0.1% v/v to 1.0% v/v of an octylphenoxypoly ethoxy ethanol.

[0007] There has thus been outlined, rather broadly, certain embodiments of the invention in order that the detailed description thereof herein may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional embodiments of the invention that will be described below and which will form the subject matter of the claims appended hereto.

[0008] In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

[0009] As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order that the invention may be readily understood, aspects of the invention are illustrated by way of examples in the accompanying drawings; however, the subject matter is not limited to the disclosed aspects.

[0011] FIG. 1 illustrates a schematic infection detection system in accordance with aspects of the invention.

[0012] Features of the infection detection system according to aspects of the invention are described with reference to the drawings, in which like reference numerals refer to like parts throughout.

DETAILED DESCRIPTION

[0013] FIG. 1 shows an schematic representation of an exemplary infection detection system 10 in accordance with aspects of the invention. The infection detection system is configured to process a sample and to determine whether the sample contains one or more predetermined pathogens. The infection detection system in accordance with embodiments of the invention includes a sampling device 20, a lysing chamber 30, a filter 40, a meter 50, a nucleic acid sequence-based (NASBA) fluidic network 60, and an instrument 70. The infection detection system also includes a sample processor 80, such as a cartridge, which at least includes the NASBA fluidic network and may include any or all of the lysing chamber, the filter, and the meter. The sample processor is configured to connect to the sampling device and to receive and process a sample contained within the sampling device. The sample processor may be disposable and replaceable, and may be adapted to process the collected sample using at least one NASBA assay. The infection detection system may process the sample and determine whether the sample contains one or more predetermined pathogens rapidly, for example within an hour, thirty minutes, or less. The infection detection system may process the sample and determine whether the sample contains one more predetermined pathogens at the point-of-care, for example within the same building, room, etc. as the patient. The infection detection system thus eliminates the need for time-wasting intermediary treatment, storage, and/or extraneous transport of the sampling device. According to aspects of the invention, the entirety of the sample processing may occur within the various components of the infection detection system thereby obviating the need of direct user intervention with the sample after the sample is collected. The infection detection system may accordingly be used by a user of low skill and may be readily transported to and applied in a variety of environments (e.g., the home, a hospital room, etc.). As a result, infection in a patient may be rapidly detected and identified, which may improve the prognosis of the patient.

[0014] The sampling device of the infection detection system may be adapted to collect a sample, such as blood (e.g., whole blood), urine, fecal matter, purulence/pus, etc. Whole blood, as used herein, means blood drawn directly from a patient from which none of the components, such as plasma, platelets, or pathogens, has been removed. The sampling device may collect the sample from a medical device (not shown). For example, the sampling device may be exposed for a predetermined and/or extended period of time to an internal space or lumen in the medical device so as to collect a sample of any pathogen which may form in said space and/or lumen. The medical device may be an external communicating device used for treating a patient, such as a Foley catheter, a vascular catheter, a suction catheter, a bronchial scope, a urinary drain line, a respiratory suction catheter, a Bronco- Alveolar-Lavage Catheter, etc. The sampling device may additionally or alternatively be adapted to collect a sample directly from a sample source such as urine, fecal matter, purulence/pus, a suspected infection site (such as a surgical dressing, wound, and/or an insertion site), etc. The sampling device may additionally or alternatively be adapted to collect a sample intravenously, subcutaneously, or intraosseously. The sampling device may be disposable and replaceable. According to aspects of the invention, the sampling device may include a sample collection tube. The sample collection tube may be a standard blood collection vacuum tube containing a whole blood sample. Additionally or alternatively, the sampling device may be a standard syringe containing a whole blood sample.

[0015] The lysing chamber may be any chamber configured to receive the whole blood sample and lyse the whole blood sample into a lysate. The lysing chamber may be in fluid communication with the sampling device. Fluid communication, as used herein, may mean that the structures in question are fluidly connected via any of a number of structures such as tubing, conduits, etc., that allow fluid to travel from one structure to another. In embodiments of the invention, flow of the whole blood sample from the sampling device to the lysing chamber may be operatively connected to the instrument and may be controlled and/or driven by the instrument. Throughout this disclosure, reference is made to the instrument controlling and/or driving fluid flow, for example flow of the whole blood sample from the sampling device to the lysing chamber. The instrument may control and/or drive fluid flow when operatively connected to a fluid pathway and via any number of known fluid control systems, which may for example include pumps, valves, conduits etc. Further, the instrument may control and/or drive fluid flow without physically contacting the fluid. Accordingly, the sample collector and/or the sample processor maybe disposed and replaced while the instrument may be used repeatedly without contaminating the samples.

[0016] The lysing chamber may include all of the materials for lysing the whole blood sample and pathogen cells contained therein and for extracting and purifying pathogen messenger RNA (i.e., dissolve targeted mRNA and remove inhibitors that could interfere with nucleic acid amplification). For example, the lysing chamber may include a lysing agent, such as a lyophilized Acris lysing chemistry, that is configured to lyse the whole blood sample into the lysate. Additionally or alternatively, the lysing chamber may physically lyse the whole blood sample ultrasonically or by freezing the whole blood sample.

[0017] According to one aspect of the invention shown in FIG. 1, the lysing chamber may include a plurality of chambers, for example, a first chamber and a second chamber, The first chamber may include a lysing chemistry, such as, the lyophilized Acris lysing chemistry. The lysing chemistry contained within the first chamber may be in the form of a reagent plug(s) having dried lysis reagents. The first chamber may be in fluid communication with the sampling device and may receive the whole blood sample from the sampling device. In embodiments of the invention, the instrument may control and/or drive flow of the whole blood sample from the sampling device to the first chamber. Additionally or alternatively, the whole blood sample may be driven from the sampling device to the first chamber via gravity, capillary flow, etc. The second chamber may include a diluent and may be in fluid communication with the first chamber. The diluent may be driven from the second chamber to the first chamber to form the lysate. The instrument may control and/or drive the flow of the diluent from the second chamber to the first chamber. The lysate formed in the first chamber may contain the lysing agent, the diluent, and the whole blood sample. The diluent may be driven to the first chamber in advance of the arrival of the whole blood sample to prepare the lysate. Alternatively, the diluent and the whole blood sample may be driven to the first chamber simultaneously.

[0018] The filter is in fluid communication with the lysing chamber and is configured to filter the lysate into a filtered lysate. The filter may filter out large, opaque structures from the lysate (e.g., hemoglobin) while allowing a target sequence (e.g., genetic material from pathogen targets) within the lysate to pass through the filter for subsequent processing and analysis. In embodiments of the invention, the instrument may control and/or drive the flow of the lysate from the lysing chamber and to through the filter to form the filtered lysate. While FIG. 1 discloses a filter downstream from the lysing chamber, a filter may additionally or alternatively be provided upstream of the lysing chamber. Accordingly, in embodiments of the invention a filter may be provided to filter the whole blood sample from the sampling device.

[0019] The meter is in fluid communication with the filter and is configured to meter a predetermined amount of filtered lysate for the NASBA analysis. The predetermined amount may, for example, be between 1 and 3 ml. In embodiments of the invention, the instrument may control and/or drive the flow of the filtered lysate from the filter and to the meter to collect the predetermined amount of filtered lysate. While FIG. 1 discloses the meter downstream from the lysing chamber and the filter, in embodiments of the invention an additional or alternative meter may be provided upstream of the lysing chamber and/or of the filter. Accordingly, in embodiments of the invention a meter may be provided to meter the whole blood sample and/or a filtered whole blood sample from the sampling device.

[0020] The NASBA fluidic network may be in fluid communication with the meter and may receive the predetermined amount of filtered lysate from the meter. The NASBA fluidic network may include all of the materials (e.g., reagents, structures, etc.) necessary to perform predetermined NASBA-based nucleic-acid assays for mRNA and/or DNA on the predetermined amount of filtered lysate. The NASBA fluidic network may include a plurality of reaction tubes that are each directly or indirectly in fluidic communication with the meter and that are configured to receive filtered lysate from the meter. In embodiments of the invention, the instrument may control and/or drive flow of the filtered lysate from the meter to each of the plurality of reaction tubes.

[0021] Each of the plurality of reaction tubes may include all of the materials for processing the filtered lysate for isothermal amplification of predetermined pathogen target sequence (e.g., targeted mRNA to identify the presence of specific genes). Specific examples of materials that may be included in each of the plurality of reaction tubes include lysing buffers, mRNA-dependent DNA polymerase, mRNA primers, DNA primers, amino acids, and the like. Each of the plurality of reaction tubes may at least include an enzyme, a primer, and a beacon for performing an NASBA assay on a pathogen target sequence within the filtered lysate. Each of the plurality of reaction tubes may include one or more of the following three enzymes: Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Ribonuclease H (RNase H), and a T7 RNA polymerase. Each of the plurality of reaction tubes may include two or more oligonucleotide primers. The enzyme(s) and the primer(s) may amplify a predetermined genetic sequence in the predetermined pathogen target sequence. The beacon provided in each of the plurality of reaction tubes may be configured to attach to the predetermined pathogen target sequence. The beacon may include a fluorophore that emits light when attached to the predetermined genetic sequence and when excited by an excitation source (e.g., a laser). Each reaction tube may include at least one window such that the instrument may detect light emitted from the beacon when attached to a predetermined pathogen target sequence. Each reaction tube may be provided with a beacon that is different from the beacons provided in each of the other reaction tubes. Accordingly, the NASBA fluidic network may detect as many different predetermined pathogen target sequences as there are reaction tubes.

[0022] The NASBA fluidic network may include a chamber containing an NASBA diluent. The chamber may be in fluid communication with each of the plurality of reaction tubes. In embodiments of the invention, the instrument may control and/or drive flow of the diluent from the chamber to each of the plurality of reaction tubes. The diluent contained within the chamber may be fluidly communicated to each of the plurality of reaction tubes a predetermined period (e.g., 5 minutes) before introduction of the filtered lysate. After expiration of the predetermined period, the filtered lysate may be distributed to each of the plurality of reaction tubes to induce the NASBA reactions and the results of the NASBA reactions may be analyzed by the instrument.

[0023] The instrument of the infection detection system may be adapted to receive the sample processor, to initiate and/or control aspects of processing of the sample within the sample processor, and to analyze the processed sample. As discussed in detail above, the instrument may control and/or drive fluid flow (e.g., whole blood flow, diluent flow, lysate flow, filtered lysate flow, etc.). In addition, the instrument may include a heater and/or a heat exchanger that may maintain the sample processor within a predetermined temperature range necessary for isothermal amplification of predetermined pathogen target sequences during the NASBA assays. The predetermined temperature range may be within 35-50 degrees Celsius. In embodiments, the predetermined temperature range may be within 40 - 42 degrees Celsius.

[0024] In embodiments of the invention, the instrument may be configured to perform any suitable NASBA-based nucleic-acid assay on the sample utilizing the reagents. For example, the instrument may be configured to perform any steps for lysing pathogen cells and extracting and purifying pathogen messenger RNA (i.e., dissolve targeted mRNA and remove inhibitors that could interfere with nucleic acid amplification). In another example, the instrument may be configured to perform any steps for processing the output solution from the extraction and purification steps for isothermal amplification of targeted mRNA to identify the presence of specific genes. [0025] In accordance with a variety of embodiments of the present disclosure, pathogenic microorganisms and/or sequences related to antibiotic resistance are detected in a biological sample obtained from a patient. For the purposes of this disclosure, a biological sample includes whole blood, serum, plasma, cerebrospinal fluid (CSF), urine, synovial fluid, breast milk, sweat, tears, saliva, semen, feces, vaginal fluid or tissue, sputum, nasopharyngeal aspirate or swab, lacrimal fluid, mucous, or epithelial swab (buccal swab), and tissues (e.g., tissue homogenates), organs, bones, teeth, among others). For the purposes of this disclosure, a pathogenic microorganism includes, for example, one or more of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida parapsilosis, Streptococcus pneumoniae, Enterobacter cloacae complex, Haemophilus influenzae, Neisseria meningitidis, and Enterobacter aerogenes. For the purposes of this disclosure, an antibiotic resistance includes, for example, resistance to one or more of Fluconazole, Methicillin, Carbapenem, and Vancomycin.

[0026] A lysing solution suitable for use in the infection detection system 10 quickly lyses microbial cell wall and membranes. In a particular example, the lysing solution may facilitate this lysis at room temperature and without physio-mechanical cell disruption. In addition, the lysing solution may be benign to RNA and stable at room temperature. A specific example of a suitable lysing solution is found in Table I:

Table

[0027] It is an advantage of the lysing solution according to Table I that it is suitable for use in lysing a variety of gram positive, gram negative, and fungal microorganisms. In addition, it is an advantage of the lysing solution according to Table I that it has a viable shelf life of greater than 1 year of storage at room temperature. In addition, it is an advantage of the lysing solution according to Table I that it has a viable shelf life of greater than 1 year of storage at negative twenty degrees Celsius (-20°C). Of note, IGEPAL CA-630^® is a nonionic, non denaturing detergent having the IUPAC name of octylphenoxypoly ethoxy ethanol.

[0028] The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.

Claims

What is claimed is:

1. A lysis solution for lysing microorganisms in a biological sample, the lysis solution comprising:

2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate;

20 pM to 160 pM of a Tris HCL, pH 8.5;

6 pM to 48 pM of a Magnesium chloride;

35 pM to 280 pM of a Potassium chloride; and

0.1% v/v to 1.0% v/v of an octylphenoxypolyethoxyethanol.

2. An infection detection system comprising: a sampling device configured to contain a whole blood sample containing a pathogen target sequence; a lysing chamber configured to be in fluid communication with the sampling device to receive the whole blood sample, the lysing chamber being configured to lyse the whole blood sample into a lysate using a lysing solution, the lysing solution comprising:

2 mM to 16 mM of a Quanidinium thiocyanate/guanidine thiocyanate;

20 mM to 160 pM of a Tris HCL, pH 8.5;

6 pM to 48 pM of a Magnesium chloride;

35 pM to 280 pM of a Potassium chloride; and

0.1% v/v to 1.0% v/v of an octylphenoxypoly ethoxy ethanol; a filter configured to be in fluid communication with the lysing chamber and to filter the lysate into a filter lysate; a meter configured to be in fluid communication with the filter and configured to meter a predetermined amount of filtered lysate from the filtered lysate; a NASBA fluidic network configured to be in fluid communication with the meter to receive the predetermined amount of filtered lysate, the NASBA fluidic network comprising: an enzyme and a primer for amplifying a predetermined genetic sequence in the pathogen target sequence contained within the predetermined amount of filtered lysate, the primer including the oligonucleotide sequence; and a beacon that is configured to attach to the pathogen target sequence, the beacon including the oligonucleotide sequence; and an analytical instrument configured to excite the beacon when the beacon is attached to the pathogen target sequence to signal a presence of the pathogen target sequence.