Streptococcal biomarker detection methods

Abstract

Methods, compositions, kits, and systems useful in the detection of Streptococcal biomarkers in non-invasively collected biological samples are provided. The disclosure describes using non-toxic, hydrolytic enzyme reagent compositions to facilitate the detection of streptococcal.

Description

Patent Application Attorney Docket No. 018.0102-WO00STREPTOCOCCAL BIOMARKER DETECTION METHODSTECHNICAL FIELD

[0001] This application claims priority to U.S. Provisional Application No. 63 / 697,399, filed on September 20, 2024, the disclosure of which is incorpoated by reference.

[0002] This invention relates to using non-toxic reagents to prepare biological specimen samples for the rapid detection of Streptococcal biomarkers.BACKGROUND

[0003] Streptococcus is a genus of gram-positive cocci or spherical bacteria. Many Streptococcus species form part of the commensal human microbiota of the mouth, skin, intestine, and upper respiratory tract. In the medical setting, the most important species of streptococci are Streptococcus pneumoniae, viridans group streptococci (VGS) (including S. mutans, S. salivarius, S. anginosus, S. mitis, S. sobrinus, and S. sanguinis) (Doern & Burnham, 2010), and the streptococci of Lancefield Groups A and B, also known as "Group A Strep" and "Group B Strep", respectively.

[0004] The most significant or clinically important streptococcal pathogen is Streptococcus pyogenes.S. pyogenes is a species of non-motile, aerotolerant, beta-hemolytic, gram-positive bacteria. These bacteria can cause Group A streptococcal infection. In particular, 5. pyogenes (Group A Strep, Strep A or GAS) is found in the throat, genital mucosa, and on the skin. Strep A is responsible for various infections, from mild conditions like pharyngitis (strep throat), impetigo and scarlet fever to severe invasive diseases such as necrotizing fasciitis, pneumonia and streptococcal toxic shock-like syndrome (STSS). Repeated GAS infections may trigger autoimmune sequelae, including rheumatic fever, which can lead to rheumatic heart disease (RHD). Rapid and accurate diagnosis of Strep A is crucial for effective treatment and prevention of complications. Importantly, traditional procedures for identification of Group A Streptococci infection involve the isolation and identification of viable organisms using culture techniques that require 24 to 48 hours or longer. Presently, rapid qualitative detection of Strep A antigen in throat swab specimens can be accomplished with a commercially available chromatographic immunoassay(s) or lateral flow assay(s) utilizing antibodies specific for a Strep A antigen, which is commonly whole cell Lancefield Group A Streptococcus carbohydrate antigen (GAC), for example.

[0005] Many streptococci, including GAS, produce a large number of cell wall-associated (e.g. M protein) and secreted virulence factors (e.g., exotoxins and degradative enzymes) to allow colonization, disseminationPatent ApplicationAttorney Docket No. 018.0102-WO00 within the host and transmission, disrupting both innate and adaptive immune responses to infection. For instance, GAS produce a wide array of secreted extracellular products, including streptokinase (SK), proteinases (e.g. C5a peptidase (ScpA), Streptococcal chemokine protease C (ScpC), Streptococcal pyrogenic exotoxin B (SpeB), S. pyogenes cell envelope proteinase (SpyCEP)), superantigens (e.g. Streptococcal pyrogenic exotoxin A (SpeA), Streptococcal pyrogenic exotoxin C (SpeC)), esterase, hemolysins (e.g. Streptolysin 0 (SLO), Streptolysin S (SLS)), CAMP factor, DNases (e.g. Streptodornase), hyaluronidases, complement inhibitor, superoxide dismutase, and immunoglobulin-degrading enzymes (e.g. IdeS / Mac-l, Mac-2 and EndoS). Some strains release exotoxins that activate certain T cells, triggering the release of cytokines which, in turn, activate the complement, coagulation, and fibrinolytic systems, leading to shock, organ failure, and death. The recent identification of clinical GAS isolates with reduced penicillin sensitivity and increasing macrolide resistance threatens both frontline and penicillin-adjunctive antibiotic treatment (Brouwer et al., 2023). Also, a significant threat to public health is S. agalactiae, or Group B Streptococcus (GBS), which is a common human pathogen that can cause pneumonia and meningitis in newborns and the elderly, with occasional systemic bacteremia.

[0006] Current diagnostic methods generally involve throat swabs, followed by the application of complex chemical treatments and detection assays. These methods have significant drawbacks, notably that the existing methods for detecting Streptococcal biomarkers require:

[0007] Toxic Reagents: Preparation of a lysis or extraction reagent by mixing two liquid components— commonly acetic acid and sodium nitrite, or citric acid and sodium nitrite— at a specified ratio to produce nitrous acid. This process is cumbersome, produces gases, and the lysis reagent is only stable and active for 15 minutes. The production of nitrous acid and associated gases poses safety risks, making it unsuitable for at- home use and not ideal for non-professional use.

[0008] Throat Swab Collection: Collecting a throat swab introduces the risk of serious injury, particularly when performed by a non-professional. This method is invasive and uncomfortable, especially for children, and can introduce variability in the results due to variability or improper collection techniques. Instructions recommend avoiding touching the tongue, cheeks, and teeth when collecting a specimen from the throat.

[0009] Application and Incubation: The swab must be immediately placed in the freshly prepared lysis reagent and incubated, which involves handling hazardous chemicals and following precise timing to avoid degradation of the sample and a failed test result.

[0010] Application to LF Assay: The lysed sample is then applied to a lateral flow (LF) assay, which, while a relatively straightforward detection step, is preceded by multiple complex and potentially unsafe preparation stages.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0011] Given the foregoing limitations, there is a critical need for a safer, simpler, and more user-friendly diagnostic method suitable for at-home use. The present inventions described below provide a novel method for detecting Streptococcal infections that overcome the limitations of current diagnostic methods by utilizing a non-invasive saliva and / or oral cavity sample and a single, non-toxic reagent.SUMMARY OF INVENTION

[0012] Methods, compositions, kits, and systems made according to the principles and illustrative embodiments of the invention offer advantages and improvements for detecting one or more biomarkers of Streptococcus species and / or strains (collectively, Streptococcal biomarkers) in a biological sample.Accordingly, the invention is useful for diagnosing and treating streptococcal infections in subjects. Moreover, the invention simplifies rapid testing to detect biomarkers of one or more Streptococcus species and offers other improvements over currently used detection methods, including dispensing without the need to use toxic, or unstable reagents. Accordingly, the methods, compositions, kits, and systems made according to the principles and illustrative embodiments of the invention provide detection and treatment benefits for patients having streptococcal infections.One aspect of the invention is directed to a method for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, the method includes:

[0013] (a) collecting the biological sample from the subject using a non-invasive technique;

[0014] (b) mixing the collected biological sample, or a portion thereof, in a vial with a composition having one or more hydrolytic enzymes resulting in a hydrolytic enzyme mixture that will carry out one or more of the following events:

[0015] (i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells;

[0016] (ii) lyse the cells to release the one or more biomarkers, wherein the one or more biomarkers are intracellular biomarkers;

[0017] (iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the the biomarkers are secreted biomarkers; and

[0018] (iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i ) and / or (b)(ii);Patent ApplicationAttorney Docket No. 018.0102-WO00

[0019] (c) incubating the biological sample and hydrolytic enzyme mixture of step (b) for a sufficient length of time to effect one or more of the events of ( b) (i-i v) to occur; and

[0020] (d) performing an assay to detect the one or more biomarkers present in the incubated sample mixture of step (c).

[0021] In some embodiments, the biological sample is an oral sample including samples collected from a surface within the oral cavity, an oral rinse, a saliva sample, or any combination thereof.

[0022] In some embodiments, the surface within the oral cavity is selected from tongue, palate, or any combination thereof.

[0023] In an embodiment, collecting the oral sample includes using a non-invasive technique.

[0024] In some embodiments, the non-invasive technique includes: absorbing the oral sample using a sponge, swab, or pad; and / or using an implement, scraper, brush, or applicator to collect the sample from inside the mouth; or spitting saliva into a collection vial or tube.

[0025] In an embodiment, the surface within the oral cavity is the tongue.

[0026] In some embodiments, the surface withinthe oral cavity is the palate.

[0027] In some embodiments, the method does not include any reagent that is nitrous acid, toxic, unstable, and / or will cause one or more toxic gases to be produced.

[0028] In some embodiments, the composition includes one or more hydrolytic enzymes having one or more amidohydrolase or protease selected from: Achromopeptidase (ACP); Pepsin; ProAlanase; Trypsin;Chymotrypsin; Proteinase K, and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Mutanolysin and Lysozyme.

[0029] In an embodiment, the composition including one or more hydrolytic enzymes includes ACP.

[0030] In some embodiments, the composition including one or more hydrolytic enzymes includes Mutanolysin and / or Labiase.

[0031] In some embodiments, the composition having one or more hydrolytic enzymes contains a buffered solution with a pH from about 2 to about 10.

[0032] In some embodiments, the pH of the buffered solution is less than about 4 or greater than about 9.

[0033] In some embodiments, the buffered solution is selected from a Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof.

[0034] In some embodiments, the method includes contacting a detergent, surfactant, and / or other nontoxic chemicals with one or more of:Patent ApplicationAttorney Docket No. 018.0102-WO00

[0035] the collected biological sample of step (a); the biological sample and hydrolytic enzyme mixture of step (b); and during incubation step (c).

[0036] In some embodiments, the method includes one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist and / or breakdown of the biological material in the sample.

[0037] In some embodiments, the method includes adding one or more enzymes to the biological sample of step (a) and / or the biological sample and hydrolytic enzyme mixture of step (b) to further break down biological material and biomarkers in the sample.

[0038] In some embodiments, the one or more enzymes includes one or more phosphodiesterases.

[0039] In some embodiments, the one or more phosphodiesterases are RNase H and / or RNase Tl.

[0040] In an embodiment, the method containing the detection of the one or more biomarkers is specific to one or more of: group A Streptococcus (GAS); group B Streptococcus (GBS); group C Streptococcus (GCS); group D Streptococcus (GDS); group E Streptococcus (GES); group F Streptococcus (GFS); group G Streptococcus (GGS); or one or more non-Lancefield Streptococci including S. pneumoniae or species from the viridans streptococci group; is a carbohydrate, a protein, or a peptide, including a streptolysin; or a streptococcal pyrogenic exotoxin.

[0041] In some embodiments, the one or more streptococcal biomarkers is a biomarker specific to GAS.

[0042] In some embodiments, the assay to detect the one or more streptococcal biomarkers is one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a fluorescent immunoassay, a western blot, a dot blot, or another reporterbased immunoassay.

[0043] In some embodiments, the immunoassay includes monoclonal or polyclonal antibodies specific for one or more streptococcal biomarkers.

[0044] In some embodiments, the method further includes diagnosing the subject as having a streptococcal infection if one or more streptococcal biomarkers are detected by the assay.

[0045] One aspect is directed to a kit for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the kit contains:

[0046] (a) a biological sample collection device, container, or a device including a container, for non- invasive sample collection;Patent ApplicationAttorney Docket No. 018.0102-WO00

[0047] (b) a container including one or more non-toxic reagents for preparing the one or more biomarkers in the biological sample to be tested;

[0048] (c) an assay device configured to detect the one or more biomarkers; and

[0049] (d) instructions for using the kit.

[0050] In some embodiments, the biological sample collection device or device including a container of (a) includes an implement, tube, scraper, brush, or applicator for collecting an oral sample.

[0051] In some embodiments, the implement, tube, scraper, brush, or applicator is designed to optimize the collection of a sample from the tongue surface or is designed to optimize the collection of a sample from the surface of the palate within the mouth.

[0052] In some embodiments, the kit further contains a sponge, swab, or pad for facilitating the non-invasive sample collection of (a), optionally wherein the sponge, swab, or pad is integrated into the biological sample collection device or device including a container.

[0053] In some embodiments, the kit includes a container, optionally a tube or vial, for receiving the collected biological sample.

[0054] In some embodiments, the biological sample collection container is a container for collecting saliva from the subject or a device including a container for collecting saliva.

[0055] In some embodiments, the kit does not include any reagent that is nitrous acid, toxic, is unstable, and / or will cause one or more toxic gases to be produced.

[0056] In some embodiments, the kit includes the one or more non-toxic reagents of component (b) contains a composition having one or more hydrolytic enzymes for carrying out one or more of the following events:

[0057] (i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells;

[0058] (ii) lyse the cells to release the one or more biomarkers, wherein the one or more cell biomarkers are intracellular biomarkers;

[0059] (iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the the biomarkers are secreted biomarkers; and

[0060] (iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii).Patent ApplicationAttorney Docket No. 018.0102-WO00

[0061] In some embodiments, the kit includes the one or more hydrolytic enzymes include one or more amidohydrolases and proteases selected from: Achromopeptidase (ACP); Pepsin; Proalanase; Trypsin; Chymotrypsin; Proteinase K and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Mutanolysin; Lysozyme; Galactosidase; and P-N-acetylglucosaminidase.

[0062] In another embodiment, the kit contains the one or more hydrolytic enzymes includes an aqueous solution or a dried down composition.

[0063] In some embodiments, the kit includes having one or more hydrolytic enzymes includes ACP.

[0064] In some embodiments, the kit includes having one or more hydrolytic enzymes includes Mutanolysin and / or Labiase.

[0065] In some embodiments, the kit includes having one or more hydrolytic enzymes includes a buffered solution with a pH from about 2 to about 10.

[0066] In some embodiments, the kit includes the pH of the buffered solution is less than about 4 or greater than about 9.

[0067] In an embodiment, the kit includes the buffered solution is selected from a Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate buffered saline (PBS) solution, or any combination thereof.

[0068] In some embodiments, the kit includes a detergent, surfactant, and / or other non-toxic chemicals, in: the biological sample collection device, container, or a device having a container of component (a); the container including one or more non-toxic reagents of component (b); and / or an additional container component.

[0069] In some embodiments, the kit contains one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with sample release from the collection tool and / or break down of the biological material in the sample.

[0070] In some embodiments, the kit includes the biological sample collection device, container, or a device having a container of component (a) and / or the container including one or more non-toxic reagents of component (b) further includes one or more enzymes to further break down biological material or biomarkers in the biological sample.

[0071] In some embodiments, the kit contains one or more enzymes to further break down biological material is one or more phosphodiesterases.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0072] In some embodiments, the kit contains one or more phosphodiesterases are RNase H and / or RNase Tl.

[0073] In some embodiments, wherein the assay device of the kit is configured to detect one or more of: group A Streptococcus (GAS); group B streptococcus (GBS); group C Streptococcus (GCS); group D Streptococcus (GDS); group E Streptococcus (GES); group F Streptococcus (GFS); group G Streptococcus (GGS); one or more non-Lancefield Streptococci including S. pneumoniae or species of the viridans streptococci group; is a carbohydrate, a protein, a streptolysin; and a streptococcal pyrogenic exotoxin.

[0074] In some embodiments, the assay device of the kit is, or configured to be used with, one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a fluorescent immunoassay, a western blot, a dot blot, or another reporterbased immunoassay.

[0075] In some embodiments, the kit includes monoclonal or polyclonal antibodies specific for the one or more streptococcal biomarkers.

[0076] In some embodiments, the kit includes one or more monoclonal or polyclonal antibodies is specific for GAS.

[0077] One aspect is directed to a system for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the system includes:

[0078] (a) a biological sample collection device, container, or a device including a container, for non- invasive sample collection;

[0079] (b) a container including one or more non-toxic reagents for preparing the one or more biomarkers in the biological sample to be detected; and

[0080] (c) an assay device configured to detect one or more biomarkers.

[0081] In some embodiments, the system further includes an assay apparatus configured for providing a signal in response to at least one detected Streptococcus species and / or strain biomarker in the sample; a detector device; a light source configured to transmit at least one wavelength of light capable of interacting with the signal of the assay apparatus; and a holder configured to couple the assay apparatus to the detector device in proximity to the light source, wherein the light source is positioned to illuminate at least a portion of the assay apparatus and the detector is positioned to capture at least one image of the illuminated signal.

[0082] In some embodiments, the system further including an interpretive algorithm stored in a computer- readable format that is electronically coupled to the detector device, wherein the interpretive algorithm isPatent ApplicationAttorney Docket No. 018.0102-WO00 configured to convert the at least one image of the illuminated signal to a numerical value related to the presence or amount of the at least one biomarker present in a biological sample.

[0083] In some embodiments, the assay apparatus of the system is a lateral-flow chromatographic assay cassette having at least one ligand immobilized thereon configured for capturing an analyte or biomarker of interest.

[0084] In some embodiments, the holder of the system includes an electrical connector configured to draw power from the detector device to power the light source.

[0085] In some embodiments, the system includes the biological sample collection device or device including a container of (a) includes an implement, tube, scraper, brush, or applicator for collecting an oral sample.

[0086] In some embodiments, the system includes the implement, tube, scraper, brush, or applicator is designed to optimize the collection of a sample from the tongue surface or is designed to optimize the collection of a sample from the surface of the palate.

[0087] In some embodiments, the system further includes a sponge, swab, or pad for facilitating the non- invasive sample collection of (a), optionally wherein the sponge, swab, or pad is integrated into the biological sample collection device or device including a container.

[0088] In an embodiment, the system includes a container, optionally a tube or vial, for receiving the collected biological sample.

[0089] In some embodiments, the system includes the biological sample collection container is a container for collecting saliva from the subject or a device including a container for collecting saliva.

[0090] In some embodiments, the system does not include any reagent that is nitrous acid, toxic, is unstable, and / or will cause one or more toxic gases to be produced.

[0091] In some embodiments, the system contains one or more non-toxic reagents of component (b) includes a composition including one or more hydrolytic enzymes for carrying out one or more of the following events: (i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells; (ii) lyse the cells to release the one or more biomarkers, wherein the one or more biomarkers are intracellular biomarkers; (iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the the biomarkers are secreted biomarkers; and (iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii).Patent ApplicationAttorney Docket No. 018.0102-WO00

[0092] In some embodiments, the one or more hydrolytic enzymes of the system includes one or more amidohydrolases or proteases selected from: Achromopeptidase (ACP); Pepsin; ProaAnalase; Trypsin; Chymotrypsin; Proteinase K; and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; and one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Mutanolysin, and Lysozyme.

[0093] In some embodiments, the system includes the composition including one or more hydrolytic enzymes includes ACP.

[0094] In some embodiments, the system includes the composition including one or more hydrolytic enzymes includes Mutanolysin and / or Labiase.

[0095] In some embodiments, the system includes the composition including one or more hydrolytic enzymes includes a buffered solution with a pH from about 2 to about 10.

[0096] In some embodiments, the system contains the pH of the buffered solution is less than about 4 or greater than about 9.

[0097] In some embodiments, the system includes the buffered solution is selected from a Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof.

[0098] In some embodiments, the system further including a detergent, surfactant, and / or other non-toxic chemicals, in the biological sample collection device, container, or a device including a container of component (a); the container including one or more non-toxic reagents of component (b); and / or an additional container component.

[0099] In some embodiments, the system includes one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with the release and / or breakdown of the biological material in the sample.

[0100] In some embodiments, the system contains the biological sample collection device, container, or a device including a container of component (a) and / or the container including one or more non-toxic reagents of component (b) further includes one or more enzymes to further break down biological material in the biological sample.

[0101] In some embodiments, the system includes the one or more enzymes to further break down biological material is one or more phosphodiesterases.

[0102] In some embodiments, the system includes the one or more phosphodiesterases are RNase H and / or RNase Tl.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0103] In some embodiments, the system contains the assay device is configured to detect one or more of: group A Streptococcus (GAS); group B Streptococcus (GBS); group C Streptococcus (GCS); group D Streptococcus (GDS); group E Streptococcus (GES); group F Streptococcus (GFS); group G Streptococcus (GGS); one or more of non-Lancefield Streptococci including S. pneunomiae or species of the viridans group; is a carbohydrate, a protein, a streptolysin, and a streptococcal pyrogenic exotoxin.

[0104] In some embodiments, the system includes the assay device is, or configured to be used with, one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a western blot; a dot blot, a fluorescent immunoassay, or another reporterbased immunoassay.

[0105] In some embodiments, the system includes monoclonal or polyclonal antibodies specific for one or more streptococcal biomarkers.

[0106] In some embodiments, the system includes the one or more monoclonal or polyclonal antibodies is specific for GAS.

[0107] One aspect of the invention is directed to compositions for processing a biological sample containing Gram-positive bacteria, for application to a biomarker detection assay, wherein the method includes: (A) one or more of the following hydrolytic enzymes and / or hydrolytic enzyme preparations: Achromopeptidase (ACP); a Mutanolysin preparation; Metapolyzyme; Pepsin; a Chitanase preparation; Chitosanase; Labiase; Rhamnosidase; and Proalanase; (B) one or more buffering agents with a pH from about 2 to about 10 selected from Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof); and optionally (C) a detergent selected from cationic 0.001% - 0.05% Cetyltrimethylammonium bromide (CTAB), 0.01% - 0.05% 4-dodecyl benzenesulfonic acid (4-DBS), 0.01% - 0.05% Sodium dodecyl sulfate (SDS), and 0.001% - 0.05% n-Dodecyl P-D-maltoside (DDM).

[0108] In some embodiments, a composition further contains one or more phosphodiesterase enzymes.

[0109] In some embodiments, the composition contains the phosphodiesterase enzymes, RNaseH and / or RNaseTl.BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Fig. 1A is a photograph of triplicate lateral flow assay device results of assays conducted with Strep A negative saliva samples from three healthy donors that were subjected to a general extraction method (negative saliva control). Devices photographed 5 minutes after applying extracted sample to assay devices.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0111] Fig. IB is a photograph of triplicate lateral flow assay device results of assays conducted with saliva samples from three healthy donors that were spiked with S. pyogenes strain Bruno (Strep A Bruno) at 5xl07, lxlO7, or 2xlO6Strep A Bruno cells / mL and subjected to a general extraction method (positive saliva control). Devices photographed 5 minutes after applying extracted sample to assay devices.

[0112] Fig. 2A is a photograph of triplicate lateral flow assay device results of assays conducted with Strep A negative saliva samples from three healthy donors that were subjected to an Achromopeptidase (ACP) extraction method (50 mM Tris; 0.5 mg / mL peptidase). Devices photographed 5 minutes after applying extracted sample to assay devices.

[0113] Fig. 2B is a photograph of triplicate lateral flow assay device results of assays conducted with saliva samples from three healthy donors that were spiked with S. pyogenes strain Bruno (Strep A Bruno) at 5xl07, lxlO7, or 2X10GStrep A Bruno cells / mL and subjected to an Achromopeptidase extraction method (50 mM Tris; 0.5 mg / mL peptidase). Devices photographed 5 minutes after applying extracted sample to assay devices.

[0114] Fig. 3 is a photograph of triplicate lateral flow assay device results of assays conducted with Strep A negative saliva samples from two healthy donors that were subjected to a buffer-only extraction method in 50 mM Tris. Strep A Bruno-spiked samples subjected to the buffer-only extraction method could not be assayed due to poor flow issues on the device.

[0115] Fig. 4A is a photograph showing Strep A Rapid Test Device results following enzymatic treatment of S. pyogenes cells ( Rosenbach strain Tl) with 100 units of ACP for 5 min at room temperature at different pH values, ranging from pH 5 to pH 9, using 24 mM of the indicated buffers, MES pH 5.0, MES pH 6.0, Tris pH 7.0, Tris pH 8.0, and Tris pH 9.0 from left to right, respectively.

[0116] Fig. 4B is a photograph showing Strep A Rapid Test Device results following enzymatic treatment of S. pyogenes cells (Rosenbach strain Tl) with 100 units of ACP for 5 min at room temperature at different pH values, ranging from pH 7 to pH 10, using 20 mM of the indicated buffers, MES pH 7.0, Tris pH 8.0, Tris pH 9.0, and Borate pH 10.0 from left to right, respectively.

[0117] Fig. 4C is a photograph showing Strep A Rapid Test Device results following enzymatic treatment of S. pyogenes cells (Rosenbach strain Tl) with 100 units of Mutanolysin for 5 min at room temperature at pH values ranging from pH 2 to pH 9 using the indicated buffers.

[0118] Fig. 5 contains photographs showing modified latex agglutination method results demonstrating that HONO, ACP, Metapolyzyme, and Mutanolysin released group A carbohydrate antigen (GAC) from S. pyogenes (Rosenbach strain Tl) in minutes at room temperature, leading to visible aggregation of latex particles conjugated with an antibody specific for GAC. A negative control (water treated cells) is also shown.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0119] Fig. 6A is a photograph demonstrating that ACP and Mutanolysin work synergistically to improve release of GAC from S. pyogenes (Rosenbach strain Tl) as detected with a Strep A Rapid Test Device.

[0120] Fig. 6B is a photograph showing Pepsin and Proalanase work synergistically with Mutanolysin to improve the release of GAC from 5. pyogenes (Rosenbach strain Tl) as detected with Strep A Rapid Test Device. HONO treatment results are shown as a positive control.

[0121] Fig. 6C is a photograph showing small concentrations of anionic, cationic or nonionic detergents improved ACP-mediated release of GAC from S. pyogenes (Rosenbach strain Tl) as detected with a Strep A Rapid Test Device.

[0122] Fig. 6D is a photograph showing ACP, Mutanolysin and SDS work synergistically to improve release of GAC from S. pyogenes (Rosenbach strain Tl) in saliva samples as detected with a Strep A Rapid Test Device.

[0123] Fig. 7A is a photograph demonstrating ACP and Mutanolysin retain enzymatic activity after 1 day post dry down and storage at room temperature, as measured by GAC release from S. pyogenes (Rosenbach strain Tl) and detection by a Strep A Rapid Test Device. HONO results are shown as a positive control.

[0124] Fig. 7B is a photograph demonstrating ACP and Mutanolysin retain enzymatic activity after 3 weeks post dry down and storage at room temperature, as measured by GAC release from 5. pyogenes (Rosenbach strain Tl) and detection by a Strep A Rapid Test Device. HONO results are shown as a positive control.

[0125] Fig. 7C is a photograph demonstrating ACP and Mutanolysin retain enzymatic activity after 9 months post dry down and storage at room temperature, as measured by GAC release from 5. pyogenes (Rosenbach strain Tl) and detection by a Strep A Rapid Test Device. HONO results are shown as a positive control.

[0126] Fig. 8A is a photograph showing Strep A Rapid Test Device results following enzymatic treatment of S. pyogenes cells (Rosenback strain Tl) with Mutanolysin (Mut, 50 units) in the presence of RNase H, RNase A, RNase Tl or Alkaline Phosphatase for 5 min at room temperature in 20 mM Tris pH 8. Positive control (HONO treatment) and negative control (3.5 xlO6S. pyogenes cells in water) are also shown.

[0127] Fig. 8B is a photograph showing Strep A Rapid Test Device results following enzymatic treatment of 5. pyogenes cells (Rosenback strain Tl) with ACP (25 units) in the presence of RNase H, RNase A, RNase Tl or Alkaline Phosphatase for 5 min at room temperature in 20 mM Tris pH 9. Positive control (HONO treatment) and negative control (3.5 xl0sS. pyogenes cells in water) are also shown.

[0128] Fig. 9 is a chart showing the relative abundance of bacterial genera across samples collected from various sites within the human oral cavity.

[0129] Fig. 10 is a box plot depicting a qPCR inferred abundance of bacterial DNA across samples collected from various sites within the human oral cavity.Patent ApplicationAttorney Docket No. 018.0102-WO00DETAILED DESCRIPTION

[0130] The methods, compositions, kits, and systems of the invention described herein are based on the discovery that streptococcal biomarkers are detectable in oral fluid samples and samples obtained from various oral cavity surfaces obtained from subjects that harbor Streptococci following treatment of the samples with a non-toxic, hydrolytic enzyme composition. While conventional methods for treating biological samples for assays to detect Streptococci require multiple, unstable, toxic sample preparation reagents, the methods, compositions, kits, and systems described herein simplify the preparation of biological samples for streptococcal biomarker detection analysis and diagnosing streptococcal infections, so that Streptococcal detection assays can be performed in non-clinical settings, for example, at home by lay individuals. Moreover, methods, compositions, kits, and systems of the invention provide improved streptococcal biomarker detection to permit the detection of the biomarkers in biological samples obtained non-invasively from saliva and from oral cavity surfaces like, for example, the surfaces of the tongue or the palate.

[0131] In general, a method of the invention for the detection of one or more biomarkers of one or more Streptococcus species and / or strains ("streptococcal biomarkers") in a biological sample from a subject that has, or may have, a streptococcal infection, includes steps of: (a), collecting the biological sample from the subject using a non-invasive technique; (b), mixing the collected biological sample, or a portion thereof, in a container with a composition that includes one or more hydrolytic enzymes that will carry out one or more of the following events: (i) release the one or more Streptococcal biomarkers from the cell wall surface of the one or more streptococcal cells; (ii) lyse the cells to release the one or more biomarkers, wherein the one or more Streptococcal biomarkers are intracellular biomarkers;

[0132] (iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the the biomarkers are secreted biomarkers; and

[0133] (iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i ) and / or (b)(ii); (c), incubating the biological sample and hydrolytic enzyme mixture of step (b) for a sufficient length of time to effect one or more of the events of (b)(i-iv) to occur; and (d), performing an assay to detect the one or more Streptococcal biomarkers present in the incubated sample mixture of step (c).Patent ApplicationAttorney Docket No. 018.0102-WO00

[0134] In the context of methods, compositions, kits, and systems of the invention, a subject is typically a person, but could also refer to any non-human animal, that has or is suspected of having a streptococcal infection.

[0135] The term "biological sample" can be interchangeable with terms like, but not limited to, "specimen", "patient sample", "biologic sample", "biofluid sample", "oral fluid sample", "spit sample", "saliva sample", and "oral surface sample". In some methods, compositions, kits, and systems, a biological sample is obtained from one or more surfaces of the oral cavity, such as, for example, the surfaces of the tongue, palate, gums, or any other mucosal surface of the inside of the mouth. Generally, biological samples are obtained using a non- invasive technique, but methods, compositions, kits, and systems of the invention can also accommodate biological samples obtained by invasive means, such as by a throat swab.

[0001] There are some descriptions in this disclosure of reaction conditions, amounts, periods of time, and other quantitative values, include the term, "about". As used herein, "about" means, in quantitative terms, plus or minus 5%, or plus or minus 10%, or plus or minus 15%, or plus or minus 20%.

[0136] Sample Collection

[0137] In some methods of the invention, the biological sample is a directly collected oral fluid sample like, for example, a saliva spit sample. For example, in such methods of the invention, the device or container can receive a volume of biological sample that is sufficient for performing an assay of the invention, is preferably sterile, and, optionally, is made from a material that one of skill in the art would know to be appropriate for storing a biological sample until the sample can be assayed. In other such methods of the invention, the device is designed to collect oral fluids, such as a drool cup or suction device. In some methods, the device or container already containing the hydrolytic enzyme composition (liquid or dried) or saliva can be transferred (e.g., with a pipette) from a device or container into another container or tube containing the hydrolytic enzyme composition (liquid or dried). In some methods, for saliva, a buffer is not required. In some methods, the buffering agents are dried down along with the hydrolytic agents.

[0138] In some methods of the invention, the biological sample is an oral fluid sample that is obtained by using an absorbent device like, for example, a sponge, applicator (e.g., a swab), or pad to absorb the sample either directly from a surface inside the mouth and / or from saliva, or indirectly from a collected sample in a container, like, for example, a vial, tube, or microtube. In methods of the invention that collect biological samples from oral surfaces, a sponge, swab, pad, or other materials that are similarly absorbent, absorb fluids, biological material and biofilms from the surface of the tongue, palate, gums, inner cheeks, or teeth toPatent ApplicationAttorney Docket No. 018.0102-WO00 collect the biological sample. In one method of the invention, a sponge, swab, pad, or other materials that are similarly absorbent, is used to absorb saliva from within the mouth or from a spit sample. In such methods, the collected biological sample or portion of the sample, is transferred from the sponge, swab, pad, or other materials that are similarly absorbent to a container like a vial, tube, or microtube. In some methods, an oral sample is obtained by using an adsorbent device.

[0139] In some methods of the invention, the biological sample is collected by using an implement, scraper, brush, or applicator, designed to obtain a sample from a surface inside the mouth (i.e., oral cavity) of a subject. For example, in certain methods of the invention a scraper or brush device scrapes or brushes the surface of the tongue, palate, gums, inner cheeks to collect the biological sample. In one method of the invention, an implement or device is used to obtain a sample from the surface of teeth or plaque.

[0140] In some methods of the invention, the collected biological material, or a portion thereof, is either collected directly into, or collected and then tranferred into, a container in which the biological sample is mixed with a buffered solution, water, or other diluent before contacting the biological sample with the hydrolytic enzyme composition. Preferably, the buffered solution, water, or other diluent is sterile. In one such method of the invention, the buffered solution, water, or other diluent is mixed with a non-toxic preservative that is either added at, or near the time of sample collection, or is pre-mixed with the sterile buffered solution, sterile water, or other sterile diluent.Processing of Collected Samples

[0141] Methods of the invention do not use toxic reagents, including reagents that produce toxic or unstable products when combined, like, for example, acetic acid or citric acid and sodium nitrite, which, when combined form nitrous acid (HONO) and cause the release of toxic gasses. Rather than utilizing toxic chemicals to cause or otherwise facilitate the release of biomarkers, a method of the invention incubates biological samples with one or more hydrolytic enzymes to make streptococcal biomarkers available for detection by detection agents. Accordingly, biological samples are mixed with compositions that contain hydrolytic enzymes (or more generally, proteases).

[0142] In some methods of the invention, the biological sample is mixed with a single-hydrolytic enzyme composition, or with a hydrolytic enzyme composition that contains combinations of 2, 3, 4, 5, 6, 7, 8, or more than 8 hydrolytic enzymes. In other words, in some methods of the invention, the biological sample is mixed with either a single hydrolytic enzyme composition or a cocktail of hydrolytic enzymes. In methods of the invention that mix a biological sample with a hydrolytic enzyme cocktail, the hydrolytic enzyme composition can contain enzymes with different activities, such as a cocktail of amidohydrolases or proteases, a cocktail ofPatent ApplicationAttorney Docket No. 018.0102-WO00 glycosidases, or a cocktail of glycosidases and amidohydrolases or proteases. In some methods the method includes one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with the release of the sample from the collection tool and / or breakdown of the biological material in the sample and / or breakdown of the biological material in the sample.

[0143] In one method of the invention, the biological material is directly collected into a container, like a vial, tube, or microtube that contains either a dried or liquid hydrolytic enzyme composition. Whereas, in another method of the invention, the biological sample is collected in a first container, (e.g., a sample collection container), and then the collected biological sample or a portion thereof is transferred to a second container (e.g., a reaction container) that is pre-filled with either a dried or liquid hydrolytic enzyme composition.Incubation of Biological Samples with Hydrolytic Enzyme Compositions

[0144] In some methods of the invention, the concentration of a hydrolytic enzyme in the mixture of biological sample and the hydrolytic enzyme composition is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / L, 0.6 mg / mL, 0.7 mg / L. 0.8 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, or 3.5 mg / L.

[0145] In some methods of the invention, the amount of hydrolytic enzyme in the mixture of biological sample and the hydrolytic enzyme reagent is 0.001 unit, 0.01 unit, 0.1 unit, 1 unit, 10 units, 25 units, 50 units, 100 units, 200units, 300 units, 500 units, 1,000 units, 10,000 units, 20,000 units, and 50,000 units.

[0146] In some methods of the invention, the amount of hydrolytic enzyme in the mixture of biological sample and the hydrolytic enzyme reagent is 0.01 pg, 0.1 pg, 1 pg, 10 pg, 100 pg, and 200 pg.

[0147] In some methods of the invention, the amount of hydrolytic enzyme in the mixture of biological sample and the hydrolytic enzyme reagent is 0.001 unit / test, 0.01 unit / test, 0.1 unit / test, 1 unit / test, 10 units / test, 25 units / test, 50 units / test, 100 units / test, 200 units / test, 300 units / test, 500 units / test, 1,000 units / test, 10,000 units / test, 20,000 units / test, and 50,000 units / test

[0148] Hydrolytic enzymes of the invention are generally present in buffered solution with a pH from 2 to 10, although a practitioner of the method would know how to adjust the pH as needed to optimize the extraction of streptococcal biomarkers in a biological sample based on the particular enzyme or cocktail of enzymes used. In some preferred methods of the invention, the pH of the biological sample and hydrolytic enzyme mixture is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0149] In some methods of the invention, the hydrolytic enzymes are in a phosphate-buffered saline (PBS) solution or a 10-100 mM Tris, MES, Borate, acetate, HCI, or citrate buffer. For example, in a method of thePatent ApplicationAttorney Docket No. 018.0102-WO00 invention, the hydrolytic enzymes are present in a 10 mM, 20 mM, 30 mM, 40 mM or 50 mM Tris, MES, Borate, acetate, HCI, or citrate buffered solution.

[0150] In some methods of the invention, the hydrolytic enzyme reagent further contains a small amount of one or more detergents to facilitate detection of streptococcal biomarkers. For example, the hydrolytic enzyme compositions of some methods of the invention contain: 0.01%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05%, sodium dodecyl sulfate (SDS); 0.01%, 0.05%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05% 4-dodecyl benzenesulfonic acid (4-DBS); 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05% Cetyltrimethylammonium Bromide (CTAB); 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% n-Dodecyl |3-D- maltoside (DDM); 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% sodium lauryl sulfate (SLS);0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5% sodium deoxycholate.

[0151] In some methods of the invention, the hydrolytic enzyme composition further contains or more phosphodiesterases. For example, in one method of the invention, the hydrolytic enzyme composition further contains RNase H. In another method of the invention, the hydrolytic enzyme composition further contains RNase Tl. In another method of the invention, the hydrolytic enzyme composition further contains RNase H and RNaseTl.

[0152] As discussed above, different hydrolytic enzymes and hydrolytic enzyme combinations can be used in the methods according to the invention. Examples of hydrolytic enzymes that can be used in methods of the invention include, but are not limited to one or more amidohydrolase or protease selected from: Achromopeptidase (ACP); Pepsin; Proalanase; Trypsin; Chymotrypsin; Proteinase K; or N-Acetylmuramyl-L- alanine amidase such as found in Mutanolysin preparations; and one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Lyticase, Lysostaphin, Mutanolysin preparations, and Lysozyme. Mutanolysin preparations include Mutanolysin and one or more amidohydrolases and / or glycosidases.

[0153] As indicated above, in some methods of the invention, the hydrolytic enzymes in the hydrolytic enzyme composition are stored in a "dried down" state and then rehydrated at the time a method of the invention is performed. Briefly, a dried down process of the invention includes dissolving one or more hydrolytic enzymes in a buffered solution containing 10% sucrose and then mixing the enzyme-sucrose solution with an equal volume of 50% glycerol / 50% ethanol solution. The combined solution is divided into single-assay portioned volumes into tubes, such as 1.5 mL or microtubes, and the solutions are dried down at either RT or up to around 45°C for 1 to 12 hours, preferably about 90 minutes.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0154] In some methods of the invention, the hydrolytic enzyme in the hydrolytic enzyme composition is Achromopeptidase (ACP). For example, in such methods, the biological sample mixed with a composition that contains ACP (100-300 U) in either, PBS (pH 7.4) or 10 mM-100 mM Tris-HCI (pH 8-9), and incubated for 5 minutes at room temperature (RT). The vol / vol ratio of the hydrolytic enzyme composition and biological sample mixture is typically, but not limited to 1 : 1 or about 1 : 1. In some methods of the invention, the incubated mixture is added to the sample well of a lateral flow assay (LFA) to detect a streptococcal biomarker, optionally a Group A Streptococcus carbohydrate (GAC) biomarker. In one method of the invention, the biological sample is mixed with a composition that contains 100 U of ACP in 100 mM Tris-HCI (pH 9), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. In another method of the invention, the biological sample is mixed with a composition that contains 100 U of ACP in either, 23 mM of MES (pH 6) or Tris buffer (pH 7 or 8) or 20mM borate (pH 10) incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. GAC biomarker. GAC biomarker.

[0155] In some methods of the invention, the hydrolytic enzyme composition further contains a low concentration of one or more of Cetyltrimethylammonium bromide (CTAB), 4-dodecyl benzenesulfonic acid (4- DBS), Sodium dodecyl sulfate (SDS), and n-Dodecyl P-D-maltoside (DDM) to increase ACP-mediated extraction of streptococcal biomarkers. In one such method of the invention, the biological sample and hydrolytic enzyme composition mixture contains about 0.0125% CTAB. In another such method of the invention, the biological sample and hydrolytic enzyme composition mixture contains about 0.0125% 4-DBS. In one such method of the invention, the biological sample and hydrolytic enzyme composition mixture contains about 0.025 to 0.05% DDM. In one such method of the invention, the biological sample and hydrolytic enzyme composition mixture contains about 0.025% SDS.

[0156] In some methods of the invention, an ACP-hydrolytic enzyme composition further contains a phosphodiesterase enzyme to further facilitate extraction of streptococcal biomarkers. In one such method of the invention, the biological sample is mixed with a composition that contains 25U of ACP and 100 U of RNase H in 20 mM Tris-HCI (pH 9), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. In another such method of the invention, the biological sample is mixed with a composition that contains 25 U of ACP and 10,000 U of RNase T1 in 20 mM Tris-HCI (pH 9), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0157] In some methods of the invention, the hydrolytic enzyme in the hydrolytic enzyme composition is Mutanolysin. In some methods, Mutanolysin is mixed for 5 min at room temperature at pH values ranging from pH 2 to pH 10 using the indicated buffers. In such methods of the invention, the biological sample mixed with a composition that contains 100 U of Mutanolysin in either, 23 mM of MES (pH 6) or Tris buffer (pH 7 or 8), and incubated for 5 minutes at room temperature (RT). The vol / vol ratio of the hydrolytic enzyme composition and biological sample mixture is typically, but not limited to 1 : 1 or about 1 : 1. In some such methods of the invention, the biological sample is mixed with a composition that contains 100 U of Mutanolysin in 23 mM of MES (pH 6 or pH 7) or 100 U of Mutanolysin in Tris (pH 8), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. The biological sample in the foregoing methods of the invention can be any sample obtained from inside a subject's mouth, like a saliva or spit sample, a sample obtained from the surface of the tongue, palate, gums, or teeth.

[0158] In some methods of the invention, a Mutanolysin-hydrolytic enzyme composition further contains a phosphodiesterase enzyme to further facilitate extraction of streptococcal biomarkers. In one such method of the invention, the biological sample is mixed with a composition that contains 50 U of mutanolysin and 100 U of RNase H in 20 mM Tris-HCI (pH 8), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. In another such method of the invention, the biological sample is mixed with a composition that contains 50 U of mutanolysin and 10,000 U of RNase T1 in 20 mM Tris-HCI (pH 8), incubated for 5 minutes at RT, and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker.

[0159] In some methods of the invention, the hydrolytic composition contains ACP and Mutanolysin. In such methods of the invention, the biological sample mixed with a composition that contains 100 U of ACP and 100 U of Mutanolysin in either, 23 mM of MES (pH 6 or 7) or Tris buffer (pH 8), and incubated for 5 minutes at room temperature (RT), and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. Incubating a biological sample with a hydrolytic enzyme composition that contains a combination of ACP and Mutanolysin causes a synergistic increase in a streptococcal biomarker detection signal relative to using either ACP or Mutanolysin alone. The vol / vol ratio of the hydrolytic enzyme composition and biological sample mixture is typically, but not limited to 1 : 1 or about 1 : 1. The biological sample in the foregoing methods of the invention can be any sample obtained from inside a subject's mouth, like a saliva or spit sample, a sample obtained from the surface of the tongue, palate, gums, or teeth.

[0160] In some methods of the invention, the hydrolytic composition contains Pepsin and Mutanolysin. In such methods of the invention, the biological sample mixed with a composition that contains 100 pg of PepsinPatent ApplicationAttorney Docket No. 018.0102-WO00 and 100 U of Mutanolysin in 20 mM citric acid (pH 3), and incubated for 5 minutes at room temperature (RT), and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. Incubating a biological sample with a hydrolytic enzyme composition that contains a combination of Pepsin and Mutanolysin causes a synergistic increase in a streptococcal biomarker detection signal at pH 3 relative to using Mutanolysin alone. The vol / vol ratio of the hydrolytic enzyme composition and biological sample mixture is typically, but not limited to 1 : 1 or about 1 : 1. The biological sample in the foregoing methods of the invention can be any sample obtained from inside a subject's mouth, like a saliva or spit sample, a sample obtained from the surface of the tongue, palate, gums, or teeth.

[0161] In some methods of the invention, the hydrolytic composition contains Pepsin, Prolanase, and Mutanolysin. In such methods of the invention, the biological sample mixed with a composition that contains 100 pg of Pepsin, lpg of Proalanase, and 100 U of Mutanolysin in 20 mM citric acid (pH 3), and incubated for 5 minutes at room temperature (RT), and run on a LFA to detect a streptococcal biomarker, optionally a GAC biomarker. Incubating a biological sample with a hydrolytic enzyme composition that contains a combination of Pepsin, Prolanase, and Mutanolysin causes a synergistic increase in a streptococcal biomarker detection signal at pH 3 relative to using either of these enzymes alone. The vol / vol ratio of the hydrolytic enzyme composition and biological sample mixture is typically, but not limited to 1 : 1 or about 1 : 1. The biological sample in the foregoing methods of the invention can be any sample obtained from inside a subject's mouth, like a saliva or spit sample, a sample obtained from the surface of the tongue, palate, gums, or teeth.

[0162] Steptococcal biomarkers refer to protein or carbohydrate antigens, or other types of molecules produced by streptococcal cells, by which an infection can be identified or characterized. Measurements of the detected levels of streptococcal biomarkers in a biological sample can reflect the severity or presence of some disease state associated with the streptococcal infection. Exemplary streptococcal biomarkers detected by methods of the invention include, but are not limited to: Lancefield grouping carbohydrate antigens such as the Group A carbohydrate ("GAC", a peptidoglycan-anchored surface rhamnose polysaccharide ( RhaPS) ); Group B carbohydrate ("GBC"); Group C carbohydrate ("GCC"); Group D carbohydrate ("GDC"); non-Lancefield carbohydrate antigens; streptolysins; streptococcal pyrogenic exotoxins; cell wall-associated virulence factors, like the M protein; secreted virulence factors; and secreted extracellular products, including streptokinase (SK), proteinases, like C5a peptidase (ScpA), Streptococcal chemokine protease C (ScpC), Streptococcal pyrogenic exotoxin B (SpeB), S. pyogenes cell envelope proteinase (SpyCEP), superantigens, like Streptococcal pyrogenic exotoxin A (SpeA), Streptococcal pyrogenic exotoxin C (SpeC), esterase, hemolysins, like Streptolysin O (SLO), Streptolysin S (SLS), CAMP factor, DNases, like Streptodornase, hyaluronidases, complement inhibitor,Patent ApplicationAttorney Docket No. 018.0102-WO00 superoxide dismutase, and immunoglobulin-degrading enzymes, like IdeS / Mac-l, Mac-2 and EndoS, or any other streptococcal protein. In some embodiments of the invention, the streptococcal biomarker is a specific marker of GAS cells. In one such embodiment, the GAS-specific biomarker is GAC.

[0163] Assays used in methods of the invention to detect the one or more streptococcal biomarkers use streptococcal biomarker-specific agents. Streptocoocal biomarker-specific agents are typically monoclonal or polyclonal antibodies. By "antibody" is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen or antigen binding ability. As used herein, the terms "antibody fragments", "fragment", or "fragment thereof" refer to a portion of an intact antibody, in particular, an immunogen- or antigen-binding portion of the antibody. Examples of antibody fragments include, but are not limited to, linear antibodies; single-chain antibody molecules; Fc or Fc' peptides, Fab and Fab fragments, and multi-specific antibodies formed from antibody fragments. In most embodiments, the terms also refer to fragments that bind an antigen of a target molecule (e.g., a protein biomarker described herein) and can be referred to as "antigenbinding fragments". It is intended that the "antibody" encompasses any immunoglobulin (e.g., IgG, IgM, IgA, IgE, IgD, IgY, etc.) obtained from any source (e.g., humans, rodents, chicken, non-human primates, caprines, bovines, equines, ovines, avian, etc.). Specific types / examples of antibodies include polyclonal, monoclonal, humanized, chimeric, human, or antibodies otherwise suitable for the detection of the targeted biomarkers.. .

[0164] Antibodies used in assays of methods of the invention generated against streptococcal antigens or markers, like any of the exemplary GAS biomarkers listed above. Assays of the invention are typically immunoassays. The assays in some methods of the invention are enzyme-linked immunosorbent assays (ELISA), while in other methods of the invention, the assay is a lateral flow (LF) immunoassay, a chemiluminescent immunoassay, a fluorescent immunoassay, or any other reporter-based immunoassay. In a preferred method of the invention, the assay includes a test strip, like a lateral flow test strip in a lateral flow device.

[0165] Lateral flow assays of the invention include a test strip composed of a membrane, preferably a cellulose membrane such as nitrocellulose, as the solid support for the immunoassay. Additional reagent pads can be used below the test line(s) for other critical reagents and sample conditioning materials. In some methods of the invention, when a biological sample is added to the test device, the solution will flow across the pads below the test lines and rehydrate the sample conditioning compound and the critical reagents for the assay, like antibodies specific for one or more streptococcal biomarker, and then pass across the specific test line and deposit a detection label which can be a visual indication (colloidal gold, carbon, colored latex or other labels known to those skilled in the art). An additional material can be added above the test line to absorb fluidPatent ApplicationAttorney Docket No. 018.0102-WO00 that passes by the test lines. The end result of a lateral flow assay of a method of the invention is the appearance or absence of a colored or non-colored line or spot, which can be compared to a control line or spot.Kits

[0166] The invention also provides assay kits. A kit of the invention for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the kit comprises: (a) a biological sample collection device, container, or a device comprising a container, for non-invasive sample collection; (b) a container comprising one or more nontoxic reagents for preparing the one or more biomarkers in the biological sample to be detected; (c) an assay device configured to detect the one or more biomarkers; and (d) instructions for using the kit.

[0167] A biological sample collection device of a kit of the invention can be any device or container that can receive a volume of biological sample that is sufficient for performing an assay of the invention, is preferably sterile, and is made from a material that one of skill in the art would know to be appropriate for storing a biological sample until the sample can be assayed.

[0168] In some kits of the invention, the biological sample is an oral fluid sample that is obtained by using an absorbent device like, for example, a sponge, applicator (e.g., a swab), or pad to absorb the sample either directly from a surface inside the mouth and / or from saliva, or indirectly from a collected sample in a container, like, for example, a vial, tube, or microtube. In kits of the invention that collect biological samples from oral surfaces, a sponge, swab, pad, or other materials that are similarly absorbent, absorb fluids, biological material and biofilms from the surface of the tongue, palate, gums, inner cheeks, or teeth to collect the biological sample. In one kit of the invention, a sponge, swab, pad, or other materials that are similarly absorbent, is used to absorb saliva from within the mouth or from a spit sample. In such kits, the collected biological sample or portion of the sample, is transferred from the sponge, swab, pad, or other materials that are similarly absorbent to a container like a vial, tube, or microtube. In some kits, the oral sample is obtained by an adsorbent device that retains an analyte on the surface of the material.

[0169] In some kits of the invention, the biological sample is collected by using an implement, scraper, brush, or applicator, designed to obtain a sample from a surface inside the mouth (i.e., oral cavity) of a subject. For example, in certain kits of the invention a scraper or brush device scrapes or brushes the surface of the tongue, palate, gums, inner cheeks to collect the biological sample. In one kit method of the invention, an implement or device is used to obtain a sample from the surface of teeth or plaque.Patent ApplicationAttorney Docket No. 018.0102-WQ00

[0170] In preferred kits of the invention, the kit does not include any reagent that is toxic, or any reagents that, either alone or in combination with another reagent, will produce an unstable, toxic product like, for example, nitrous acid, or cause one or more toxic gases to be produced. Accordingly, kits of the invention contain non-toxic reagents for preparing streptococcal cells. More specifically, a kit of the invention includes a hydrolytic enzyme composition that contains one or more hydrolytic enzymes that will carry out one or more of the following activities: (i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells; (ii) lyse the cells to release the one or more biomarkers, wherein the one or more cell biomarkers are intracellular biomarkers; (iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the the biomarkers are secreted biomarkers; and (iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii); (c), incubating the biological sample and hydrolytic enzyme mixture of step (b) for a sufficient length of time to effect one or more of the events of (b)(i- iv) to occur; and (d), performing an assay to detect the one or more Streptococcal biomarkers present in the incubated sample mixture of step (c).

[0171] Some kits of the invention, include one or more hydrolytic enzymes that target one or more of the following targets or substrates: Amide and peptidic bonds in peptidoglycans; Glycosidic bonds in peptidoglycans; glycosidic bonds in carbohydrates such as one or more of: group A Streptococcus (GAS); group B Streptococcus (GBS); group C Streptococcus (GCS); group D Streptococcus (GDS); group E Streptococcus (GES); group F Streptococcus (GFS); group G Streptococcus (GGS). In some kits of the invention the hydrolytic enzyme composition includes one or more of Achromopeptidase (ACP), Lysozyme, Mutanolysin, Chitinases, Lysostaphin, Lyticase, Pepsin, Proalanase, Labiase, Chitosanases, and Rhamnosidase. Preferred hydrolytic enzymes for inclusion in the hydrolytic enzyme compositon of a kit of the invention are ACP, Mutanolysin, Pepsin, Labiase, and Chitinases

[0172] The hydrolytic enzyme composition component of a kit of the invention is generally a buffered solution with a pH from 2 to 10. In some preferred kits of the invention, the pH of the biological sample and hydrolytic enzyme mixture is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0173] In some kits of the invention, the hydrolytic enzymes are in a phosphate-buffered saline (PBS) solution or a 10-100 mM Tris, MES, Borate, acetate, HCI, or citrate buffer. For example, in a kit of thePatent ApplicationAttorney Docket No. 018.0102-WQ00 invention, the hydrolytic enzymes are present in a 10 mM, 20 mM, 30 mM, 40 mM or 50 mM Tris, MES, Borate, acetate, HCI, or citrate buffered solution.

[0174] In some kits of the invention, the hydrolytic enzyme reagent further contains a small amount of one or more detergents to facilitate detection of streptococcal biomarkers. For example, the hydrolytic enzyme compositions of some kits of the invention contain: 0.01%, 0.05%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05%, sodium dodecyl sulfate (SDS); 0.01%, 0.05%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05% 4-dodecyl benzenesulfonic acid (4-DBS); 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045% or 0.05% Cetyltrimethylammonium Bromide (CTAB); 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% n-Dodecyl P-D-maltoside (DDM); 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% sodium lauryl sulfate (SLS); 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5% sodium deoxycholate, sodium deoxycholate.

[0175] In some kits of the invention, the hydrolytic enzyme composition further contains or more phosphodiesterases. For example, in one kit of the invention, the hydrolytic enzyme composition further contains RNase H. In another kit of the invention, the hydrolytic enzyme composition further contains RNase Tl. In another kit of the invention, the hydrolytic enzyme composition further contains RNase H and RNaseTl.

[0176] In some kits of the invention, the hydrolytic enzyme in the hydrolytic enzyme composition is Achromopeptidase (ACP). For example, in such kits, the hydrolytic enzyme composition contains 100-300 U of ACP in either, PBS (pH 7.4) or 10 mM-100 mM Tris-HCI (pH 8-9). In one kit of the invention, the hydrolytic enzyme composition contains 100 U of ACP in 100 mM Tris-HCI (pH 9). In another kit of the invention, the hydrolytic enzyme composition contains 100 U of ACP in either, 23 mM of MES (pH 6) or Tris buffer (pH 7 or 8). In some kits, the buffer is Borate (pH 10).

[0177] In some kits of the invention, the hydrolytic enzyme composition further contains a low concentration of one or more of Cetyltrimethylammonium bromide (CTAB), 4-dodecyl benzenesulfonic acid (4- DBS), Sodium dodecyl sulfate (SDS), and n-Dodecyl p-D-maltoside (DDM) to increase ACP-mediated extraction of streptococcal biomarkers. In one such kit of the invention, the enzyme composition mixture contains about 0.0125% CTAB. In another such kit of the invention, the hydrolytic enzyme composition mixture contains about 0.0125% 4-DBS. In one such kit of the invention, the hydrolytic enzyme composition mixture contains about 0.025 to 0.05% DDM. In one such kit of the invention, the hydrolytic enzyme composition mixture contains about 0.025% SDS.

[0178] In some kits of the invention, an ACP-hydrolytic enzyme composition further contains a phosphodiesterase enzyme to further facilitate extraction of streptococcal biomarkers. In one kit of thePatent ApplicationAttorney Docket No. 018.0102-WO00 invention, the hydrolytic enzyme composition contains 25U of ACP and 100 U of RNase H in 20 mM Tris-HCI (pH 9). In another such kit of the invention, the hydrolytic enzyme composition that contains 25 U of ACP and 10,000 U of RNase T1 in 20 mM Tris-HCI (pH 9).

[0179] In some kits of the invention, the hydrolytic enzyme in the hydrolytic enzyme composition is Mutanolysin. In some kits, Mutanolysin is mixed for 5 min at room temperature at pH values ranging from pH 2 to pH 10 using the indicated buffers. In such kits of the invention, the hydrolytic enzyme composition contains 100 U of Mutanolysin in either, 23 mM of MES (pH 6) or Tris buffer (pH 7 or 8). In some such kits of the invention, hydrolytic enzyme composition contains 100 U of Mutanolysin in 23 mM of MES (pH 6 or pH 7) or 100 U of Mutanolysin in Tris (pH 8).

[0180] In some kits of the invention, a Mutanolysin-hydrolytic enzyme composition further contains a phosphodiesterase enzyme to further facilitate extraction of streptococcal biomarkers. In one such kit of the invention, the hydrolytic enzyme composition contains 50 U of mutanolysinand 100 U of RNase H in 20 mM Tris-HCI (pH 8). In another such kit of the invention, the hydrolytic enzyme composition contains 50 U of mutanolysin and 10,000 U of RNase T1 in 20 mM Tris-HCI (pH 8).

[0181] In some kits of the invention, the hydrolytic composition contains ACP and Mutanolysin. In such kits of the invention, the hydrolytic enzyme composition contains 100 U of ACP and 100 U of Mutanolysin in either, 23 mM of MES (pH 6 or 7) or Tris buffer (pH 8).

[0182] In some kits of the invention, the hydrolytic composition contains Pepsin and Mutanolysin. In such kits of the invention, the hydrolytic enzyme composition contains 100 pg of Pepsin and 100 U of Mutanolysin in 20 mM citric acid (pH 3).

[0183] In some kits of the invention, the hydrolytic composition contains Pepsin, Proalanase, and Mutanolysin. In such kits of the invention, the hydrolytic enzyme composition contains 100 pg of Pepsin, 1 p g of Proalanase, and 100 U of Mutanolysin in 20 mM citric acid (pH 3).

[0184] Some kits of the invention include a hydrolytic enzyme reagent that further includes one or more detergents, such as, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% sodium dodecyl sulfate (SDS); 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% sodium lauryl sulfate (SLS); 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% sodium deoxycholate; or 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% nonyl phenoxypolyethoxylethanol (NP-40), sodium dodecyl benzene sulphonate, Triton-X, Tween, and CHAPS.

[0185] In some kits of the invention, one or more of the streptococcal biomarker-specific detection agents are monoclonal antibodies, while in other kits of the invention, the biomarker-specific detection agents arePatent ApplicationAttorney Docket No. 018.0102-WO00 polyclonal antibodies. In some kits of the invention, the streptococcal biomarker-specific detection agents are attached to a solid substrate of a device component of the kit, such as, for example, the bottom of a multi-well plate like, for example, a 96-well plate used for ELISA assays, or the test strip of lateral flow assay device. The detectable moiety component of a kit of the invention is typically a gold nanoparticle (e.g., colloidal gold), a carbon nanoparticle, a fluorescent or chemiluminescent molecule, but may also be any detectable moiety known in the art, including radioactive, up-converting particles, and enzymatic labels. A kit of the invention may also contain one or more of the buffers, reagents, detection reagents, and so forth that are useful for the practice of the methods of this invention. And certain kits of the invention may contain an assay device.

[0186] As indicated above, the assays of methods of the invention can be immunoassays. Accordingly, some kits of the invention include components required to perform either an enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a fluorescent immunoassay; or another reporter-based immunoassay.

[0187] Instructional materials may also be included in a kit of the invention. The instructional materials may, in some cases, be printed on an insert included in the kits, or provided in electronic form, such as on a portable hard drive, or in a video file. Instructional materials may also refer to a website or link to an application software program, such as a mobile device or computer "App", which provides instructions. A kit may also include additional components to facilitate the particular application for which the kit is designed. For example, a kit may also contain a means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent, up-converting, or chemoluminescent labels, appropriate secondary labels such as a secondary antibody, or the like).SYSTEMS

[0188] The invention provides systems for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection. More specifically, a system of the invention can include any features described above with respect to methods and kits of the invention, and further include an assay apparatus configured for providing a signal in response to at least one detected Streptococcus species and / or strain biomarker in the sample; a detector device; a light source configured to transmit at least one wavelength of light capable of interacting with the signal of the assay apparatus; and a holder configured to couple the assay apparatus to the detector device in proximity to the light source, wherein the light source is positioned to illuminate at least a portion of the assay apparatus and the detector is positioned to capture at least one image of the illuminated signal.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0189] Some systems of the invention further include an interpretive algorithm stored in a computer- readable format that is electronically coupled to the detector device, wherein the interpretive algorithm is configured to convert the at least one image of the illuminated signal to a numerical value related to the presence or amount of the at least one streptococcal biomarker present in a biological sample.

[0190] In some systems of the invention, the assay apparatus is a lateral-flow chromatographic assay cassette having at least one ligand immobilized thereon configured for capturing an analyte or biomarker of interest.

[0191] In some systems of of the invention, the holder includes an electrical connector configured to draw power from the detector device to power the light source.EXAMPLES

[0192] The following examples exemplify certain aspects of illustrative embodiments of the inventions described herein, including compositions, methods, devices, and kits for detecting Streptococcus bacteria in biological samples. The inventions should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the reagents, compounds, and tools of the inventions described herein.Example 1: Streptococcus Group A (Strep A) Detection in Spiked Saliva

[0193] Saliva Collection: Saliva was collected from three healthy donors via spitting into respective tubes. The saliva samples were used for assays on the same day of collection and stored at 2-8 °C prior to use.Negative sample controls were prepared by not adding Strep A cells to the samples (Fig. 1A), whereas positive sample controls were prepared by spiking samples with S. Pyogenes strain Bruno 19615™ [CIP 104226] (ATCC), ("Strep A Bruno"), a whole-genome sequenced bacterial strain that was isolated from the pharynx of a child following an episode of sore throat. A stock suspension of Strep A Bruno in phosphate buffered saline (PBS) was used to spike saliva samples at 5xl07, lxlO7, or 2xl06Strep A Bruno cells / mL. See results in the left, center, and right images, respectively, in Fig. IB.

[0194] General Acid Extraction Method (HONO): Commercially available Reagent A and Reagent B, (2M sodium nitrite and 0.027M citric acid, respectively) were mixed together as per instructions for use. It is noted that the foregoing Reagents A and B could be substituted with other commercially available lysis reagents, including, for example, 2M sodium nitrite and 0.2M acetic acid. 100 pL of freshly premixed lysis reagents werePatent ApplicationAttorney Docket No. 018.0102-WO00 mixed with 100 p.L of each of the negative and positive saliva samples described above. The lysis reagent-saliva sample mixtures were incubated at room temperature for 1 minute.

[0195] Assay and Results: 100 pL of each incubated mixture was added to the sample well of a lateral flow assay device that detects a Strep Group A antigen using antigen-specific antibodies. Results were read at 5 minutes. Negative samples were negative (no test line) with strong control lines formed at 5 minutes. Robust test line signals for all Strep A concentrations tested were visible at 5 minutes. Strep A cell concentrationdependent decreases in signal from high to low concentration was observable. No flow issues were observed. Strep A was detectable in saliva samples treated with the acid extraction method and assayed using a commercially available lateral flow assay device. The detection of Strep A in the samples was a surprising result because the extraction method and device were developed for the testing of throat swab specimens.Example 2: Evaluation of Multiple Different Extraction Reagents or Buffers

[0196] Multiple different extraction reagents or buffers were tested to compare their effectiveness for preparing Strep A Bruno cells in saliva samples to make GAC antigen available for detection in an assay. These studies were conducted by spiking each of the buffers and / or lysis reagents listed in the left column of Table 1 with 5xl07cells / mL of Strep A Bruno, incubating the spiked buffers and lysis reagents for 5 minutes at room temperature, and then applying 100 pL of the incubated buffers and reagents to commercially-available lateral flow assay (LFA) Strep A Rapid Test devices. If a positive test line signal for Strep A was detected on the LFA following extraction with a Reagent / Buffer, the Reagent / Buffer in question was retested in the presence of a saliva sample that had been spiked with 5xl07cells / mL of Strep A Bruno, incubated for 5 minutes at room temperature, and then applied to new Strep A Rapid Test device. Only pre-treatment or extraction with a preparation of Achromopeptidase (ACP) in a suitable buffer resulted in the detection of a robust positive LFA signal, both in the presence and absence of spiked-saliva sample. ACP, which is also known as Lysyl endopeptidase, hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria, leading to the lysis of these bacteria.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0197] Table 1: Tested extraction reagents or buffers.EXAMPLE 3: Achromopeptidase (ACP) Extraction Method with a Lateral Flow Assay to Demonstrate Sensitivity of Group A Streptococcus Marker Detection

[0198] An extraction method that used Achromopeptidase in a Tris buffered solution as a single lysis reagent was used to prepare saliva samples, which were spiked with Strep A Bruno cells, prior to adding the sample to a lateral flow assay device or test strip for the specific detection of a Group A Streptococcus carbohydrate biomarker.

[0199] Sample Preparation: Saliva was collected from three healthy donors via spitting into respective tubes. The saliva samples were used for assays on the same day of collection and stored at 2-8 °C prior to use. Strep A cells were not spiked in negative sample controls (Fig. 2A), whereas positive samples were prepared by spikingPatent ApplicationAttorney Docket No. 018.0102-WO00 saliva samples with 5. pyogenes from a Strep A Bruno culture stock suspension of in PBS with 5xl07, lxlO7, or 2x10scells / mL. See results in the left, center, and right images, respectively, in Fig. 2B.

[0200] Achromopeptidase (ACP) Extraction Method: An ACP stock lysis buffer was prepared by dissolving ACP (Catalog No. A3547, Sigma-Aldrich) in 100 mM Tris-HCI (pH 9) by inversion mixing to a final ACP concentration of 1 mg / mL. 100 pL of the 1 mg / mL buffered ACP solution was mixed with 100 pL of each of the negative and Strep A Bruno-spiked saliva samples (final ACP concentration is 0.5 mg / mL). The ACP-treated samples were incubated at room temperature for 5 minutes.

[0201] Assay and Results: 100 pL of each incubated mixture was added to the sample well of the lateral flow assay device in a commercially-available Strep A Rapid Test. Results were read at 5 minutes. Negative samples were negative (no test line) with strong control lines formed at 5 minutes. Robust test line signals for all Strep A concentrations tested were visible at 5 minutes and no issues with sample flow were observed. Strep A cell concentration dependent decreases in signal from high to low concentration was observable, thereby demonstrating that the single enzyme composition described above could replace the conventional, toxic, two- reagent extraction (HONO) treatment step currently used routinely for rapid Strep A testing.Example 4. Buffer-only Control for the Achromopeptidase (ACP) Extraction Method

[0202] Saliva was collected from two healthy donors via spitting into a tube. The saliva samples were used for assays on the same day of collection and stored at 2-8 °C prior to use. Strep A cells were not spiked in negative control samples whereas positive samples were prepared by spiking saliva with S. pyogenes from a Strep A Bruno culture stock suspension in PBS with 5xl07cells / mL.Achromopeptidase (ACP) Buffer-only Extraction Method Control: An ACP-free stock 100 mM Tris-HCI (pH 9) buffer was mixed with 100 pL of each of the negative and Strep A Bruno-spiked saliva samples. The buffer- treated (no ACP) samples were incubated at room temperature for 5 minutes.

[0203] Assay and Results: 100 pL of each incubated mixture was added to the sample well of a lateral flow assay device in a commercially available Strep A Rapid Test. Results were read at 5 minutes (Fig. 3). Significant flow issues were observed in the absence of ACP extraction because of high sample viscosity. Negative saliva samples had very faint control lines and no test line at 5 minutes with high background. Positive saliva samples were not evaluated due to poor flow issues. Hence, this example supports the finding above in Example 3 that the treatment of saliva samples with ACP prior to lateral flow assay analysis improved and facilitated sample flow with the saliva matrix on the lateral flow assay device.Patent Application Attorney Docket No. 018.0102-WO00EXAMPLE 5: Achromopeptidase (ACP) Extraction Method with a Lateral Flow Assay to Detect Group A Streptococcus Biomarker in Multiple Donors

[0204] Sample Preparation: Saliva was collected from 14 healthy donors via spitting into respective tubes.The samples were used for assays on the same day of collection and stored at 2-8 °C prior to use. Strep A cells were not spiked in negative control samples, whereas positive samples were prepared by spiking samples with S. pyogenes. A stock suspension of Strep A Bruno cultures in PBS was used to spike saliva samples at 5xl07cells / mL.

[0205] Achromopeptidase (ACP) Extraction Method: An ACP stock lysis buffer was prepared by dissolving ACP (Catalog No. A3547, Sigma-Aldrich) in 100 mM Tris-HCI (pH 9) by inversion mixing to a final ACP concentration of 1 mg / mL. 100 pL of the 1 mg / mL buffered ACP solution was mixed with 100 pL of each of the negative and Strep A Bruno-spiked saliva samples (final ACP concentration is 0.5 mg / mL). An ACP-free stock 100 mM Tris- HCI (pH 9) buffer was also mixed with 100 pL of each of separately aliquoted negative and Strep A Bruno-spiked saliva samples. The ACP- and buffer-only-treated samples were incubated at room temperature for 5 minutes.

[0206] Assay and Results: 100 pL of each incubated mixture was added to the sample well of a lateral flow assay device in a commercially-available Strep A Rapid Test. Results were read at 5 minutes. All spiked-saliva samples evaluated with ACP extraction had clear control lines and positive test lines (Table 2). Strep A Bruno- spiked-saliva samples tested with buffer only extraction (no ACP) were viscous resulting in significant flow issues on LFAs. The majority of buffer-only treated samples LFAs had faint control lines and no test lines for Strep A.

[0207] Table 2. Detection of Strep A in saliva samples from multiple donors in presence and absence of ACP.Patent ApplicationAttorney Docket No. 018.0102-WQ00EXAMPLE 6: Impact of pH on Hydrolytic Enzymes and Strep A Detection

[0208] In the examples above, multiple different extraction reagents or buffers were tested to compare their effectiveness for preparing (treating) saliva samples to optimize the detection of GAC antigen by commercially available lateral flow assay (LFA) Strep A Rapid Test Devices. In this example, optimal pH ranges were determined for rapid and efficient hydrolytic release of this surface antigen (GAC) from S. pyogenes cultured cells ( Rosenbach strain Tl, ATCC 12344) by two different enzymes, Achromopeptidase (ACP) and Mutanolysin.

[0209] Achromopeptidase (ACP) Reaction Method: ACP enzymatic stock was prepared by dissolving ACP preparations (Catalog No. A3547, Sigma-Aldrich) in water to a final concentration of 10 U / pL. ACP enzymatic stock was added to different buffers and spiked with 4.9 - 5.0xl06S. pyogenes cells (Rosenbach strain Tl). Specifically, each 75 piL reaction contained 20-24 mM of a buffer (Tris(hydroxymethyl)aminomethane (Tris) (Catalog No. 97062-420, VWR), 2-Morpholinoethanesulfonic acid (MES) (Catalog No. 69892, Sigma-Aldrich) or boric acid (Catalog No. 470300-412, Ward's science plus), 100 units (10 pL) of ACP enzymatic stock, and 4.9-5.0 xlO6S. pyogenes cells. These reactions (ranging from pH 5 to pH 10) were incubated at room temperature (RT) for 5 minutes.Patent Application Attorney Docket No. 018.0102-WQ00

[0210] Assay and Results: After 5 minutes incubation at RT, 50 pL of each reaction (described above) was added to the sample well of a Strep A Rapid Test Device (Catalog No. STR-15CT20, BTNX Inc.), a LFA device that detects Strep A carbohydrate (GAC) antigen. Strep A Rapid Test results were read after 5 minutes (Figs. 4A, 4B, and Table 4) and interpreted on a scale of 0 to 5 using an Attribute Chart developed by OraSure Technologies, Inc. (Table 3).

[0211] Table 3. Attribute Chart for scoring the intensity of the test line on an LFA test.

[0212] Table 4. Intensities of test lines shown in Figs. 4A and 4B demonstrating GAC release in saliva samples treated with 100 U of ACP for 5 min at room temperature at various pH values.

[0213] These data demonstrated that GAC is efficiently released from Strep A cells, and detected by lateral flow immunoassay, following treatment with ACP at pH values ranging from 5 to 10 in as little as 5 minutes at RT. Interestingly, the release of GAC was consistently higher at pH 9 and pH 10, as seen by increased test line intensity, compared to pH 8. This finding was unexpected since the optimal pH of ACP has been reported to be around pH 8.5. Based on the reported optimal pH, one would expect similar activity at pH 8 and 9 and lower activity at pH 10 (lower intensity test band). The findings herein suggest that higher pH values (>8) help destabilize the cell wall and / or improve accessibility to ACP's substrate, which most likely consists of the oligopeptide crosslinks in the gram-positive cell wall's peptidoglycans.

[0214] Mutanolysin Extraction / Reaction Method: Mutanolysin enzymatic stocks were prepared by dissolving Mutanolysin preparations (Catalog No. M9901, Sigma-Aldrich) in water at a concentration of 10 U / piL.Patent ApplicationAttorney Docket No. 018.0102-WQ00Mutanolysin was added to reactions containing six different buffers spiked with 5.0 xlO6S. pyogenes cells. Specifically, each 150 pL reaction contained 10-20 mM of a buffer (HCI (Catalog No. 3710-32, Ricca), citric acid (Catalog No. 251275, Sigma-Aldrich), sodium acetate (Catalog No. 236500, Sigma-Aldrich), MES or Tris), 100 units (10 pL) of Mutanolysin enzymatic stock, and 5.0 xl0s5. pyogenes cells. These Mutanolysin reactions (ranging from pH 2 to pH 9) were incubated at RT for 5 minutes. In parallel, two negative control (Ctrl) reactions were included using 10 mM HCI and 20 mM sodium acetate buffers and Mutanolysin, but without S. pyogenes cells (Fig. 4C).

[0215] Assay and Results: After 5 minutes incubation at RT, 75 pL of each reaction described above was added to the sample well of a Rapid Test Device to assess Strep A carbohydrate antigen. Strep A Rapid Test results were read after 5 minutes (Fig. 4C and Table 5) and interpreted on a scale of 0 to 5 using an Attribute Chart developed by OraSure Technologies, Inc. (Table 3).Table 5. Intensities of the test lines shown in Fig. 4C demonstrating GAC release in saliva samples treated with 100 units of Mutanolysin for 5 min at room temperature at various pH values

[0216] These data demonstrated that GAC was released from Strep A cells following a short incubation at RT with Mutanolysin at pH values ranging from 2 to 9. The release of GAC was highest at pH 3 and pH 8, as seen by increased test line intensity, compared to pH 2 or pH 6 and 9. These findings suggest that Mutanolysin can effectively work across a very broad pH range and that lower pH values and / or higher pH values, similarly to ACP, may be beneficial by helping destabilize Strep A cell wall and / or improving accessibility to Mutanolysin's substrates, which consist primarily of peptidoglycans in the cell wall. Several enzymatic activities have been described in Mutanolysin preparations that could contribute to Mutanolysin being active across such a wide pH range.EXAMPLE 7: Evaluation of Numerous Hydrolytic Enzymes for GAC Release in Strep A Biomarker Detection

[0217] The cell wall of group A Streptococcus (GAS) consists of peptidoglycans conjugated with Lancefield's group A carbohydrate (GAC), the group A-defining antigenic surface polymer. This GAC-peptidoglycan linkage is highly sensitive to cleavage by nitrous acid (HONO) but resistant to mild acid conditions. Rush et al. (2022) observed that HONO deamination cleaved only 70-80% of GAC from peptidoglycan while treatment with mild acid released about 20-25% of GAC. Given the drawbacks and potential safety issues with nitrous acid, asPatent ApplicationAttorney Docket No. 018.0102-WO00 described herein (see Background), several safer and non-toxic alternatives were investigated. Specifically, numerous hydrolytic and bacteriolytic enzymes (see Table 6 below) were tested for their ability to release GAC from the cell wall of S. pyogenes during a 5-minute incubation at room temperature (RT), as measured using a modified latex agglutination method which detects the presence of GAC. For most enzymes, results were confirmed with a lateral flow assay (LFA or Rapid Test Device) that also specifically detects the group A carbohydrate antigen.

[0218] For the negative and positive control conditions of the modified latex agglutination method, 4xl085. pyogenes cells (Rosenbach strain Tl) were mixed with either 50 pL of water (negative control) or lx drop (~25 pL) of Extraction Reagent 1 plus lx drop (~25 pL) of Extraction Reagent 2 (positive control, nitrous acid extraction also known as HONO) and incubated for 5 minutes at RT. Both samples were then diluted with 5 drops (~125 pL) of Extraction Reagent 3 (neutralizing solution) [Reagents 1-3 provided in StrepPRO™ Grouping Kit (Catalog No. PLO30HD, Hardy Diagnostics)]. Finally, lx drop (~25 pL) of the resulting mixtures was mixed with lx drop (~25 pL) of StrepPRO™ Blue Latex Suspension Group A (Catalog No. PLO31HD, Hardy Diagnostics) and incubated for 1-5 minutes with shaking until agglutination or clumping of the blue latex particles was visible. In the absence of GAC release (water only control), no agglutination or clumping of the blue latex particles was visible.

[0219] For the enzymatic treatments of the modified latex agglutination method, 4xl085. pyogenes cells were added to a 50 pL reaction containing the indicated Tris buffers (see Fig. 5) and amounts of hydrolytic enzymes (ACP, Metapolyzyme and Mutanolysin) and then incubated for 5 minutes at RT. The lysates or extracts were then diluted by adding 125 pL of ddHjO and lx drop (~25 pL) of the resulting dilution was mixed with lx drop (~25 pL) of StrepPRO™ Blue Latex Suspension Group A (Hardy Diagnostics) and incubated for 5-10 minutes with shaking until agglutination or clumping of the blue latex particles was visible (or absent).

[0220] Notably, the modified latex agglutination method described above provided a rapid, sensitive, and specific method to screen enzymes capable of releasing GAC from 5. pyogenes cells. As expected, treatment of cultured S. pyogenes cells with HONO (nitrous acid) resulted in rapid agglutination of the latex particles, following release of GAC antigen (Fig. 5). In contrast, water did not release GAC from S. pyogenes cells, as evidenced by the absence of agglutination (Fig. 5). Interestingly, ACP, Mutanolysin, and Metapolyzyme were all shown to lead to significant release of GAC from cultured S. pyogenes after a short incubation at RT, as demonstrated by agglutination or clumping of the latex particles (Fig. 5). The speed of this agglutination was directly tied to the amount of GAC released by each treatment, with HONO providing the fastest results, followed by ACP, Mutanolysin, and Metapolyzyme. A summary of all the enzymes tested with this modifiedPatent ApplicationAttorney Docket No. 018.0102-WO00 agglutination method is presented in Table 6. In most instances, release of GAC from 5. pyogenes by these enzymes was confirmed with a lateral flow assay (Table 6).Table 6: Summary of the ability of various hydrolytic enzymes (or mixtures thereof) to release group A carbohydrate (GAC) from 5. pyogenes after a short incubation at room temperature (RT), as measured by a modified latex agglutination method. For some of the conditions, GAC release (or lack thereof) was confirmed by lateral flow assay (LFA) specific for GAC.Patent ApplicationAttorney Docket No. 018.0102-WO00+++++: rapid and intense agglutination observed;++++: intense agglutination observed;+++: strong agglutination observed;++: visible agglutination observed;+: weak agglutination observed;0: no agglutination observed.Patent ApplicationAttorney Docket No. 018.0102-WQ00* Indicates enzymes that were shown to release limited amounts of GAC upon longer treatments (e.g., ~60 minutes) at elevated temperatures (e.g., 37°C).

[0221] As seen in Table 6 above, GAC can be rapidly released from the cell wall of S. pyogenes cells by a number of different enzymes which lack the drawbacks and safety concerns of nitrous acid (HONO). A short 5- 10-minute incubation at RT was intentionally set in these experiments to mimic as closely as possible the current standard of care (SOC) for GAC detection using nitrous acid (HONO).

[0222] Generally, proteolytic enzymes (ACP, Proalanase, Pepsin) and Muramidase (Mutanolysin, Labiase) appeared to quickly release GAC, potentially by attacking the amide and glycosidic bonds in the gram-positive cell wall oligopeptides and glycans, respectively. Mutanolysin, through its N-Acetylmuramyl-L-alanine amidase activity, may also digest the amide bond between glycans and oligopeptides. Other enzymes such as Chitinases, Labiase, Chitosanase or a-Rhamnosidase, on the other hand, can release GAC rapidly, potentially through their ability to digest glycosidic bonds between the rhamnose, glucosamine and / or N acetyl glucosamine residues, found in the GAC molecule, to release it directly from the cell wall (Rush et al., 2022). Notably, some enzyme preparations (Chitinases, Labiase, a-Rhamnosidase) had relatively low activity per unit of volume, so only a limited amount of enzyme (<15 units) could be added to each reaction, compared to ACP and Mutanolysin where >100 units could easily be used. The higher affinity of the proteolytic enzymes ACP and Proalanase for residues such as alanine, glycine, and lysine, appeared to increase GAC release. The increased release of GAC could have been driven by the improved abilities of these enzymes to target alanine, glycine and / or lysine amino acids typically found in S. pyogenes and other gram-positive bacteria peptidoglycans (Vollmer et aL, 2008).

[0223] Pepsin, a broad-spectrum endopeptidase, also showed limited ability to release GAC during short incubations at RT (Table 1), while Trypsin, which like ACP cuts after lysine residues, failed to release detectable amounts of GAC during short incubations at RT. These findings suggest that beyond substrate specificity other factors may be at play for these enzymes (e.g. activity at lower temperature, ability of the active site to accommodate s, pyogenes oligopeptides, steric hindrance, optimal pH, enzyme purity and specific activity, etc.). Notably, for a number of the enzymes tested (Rhamnosidase, Chitinases, and Labiase), longer incubations (e.g., 60 minute) or higher temperatures (e.g., 37°C) increased GAC release, compared to short incubations at RT (Table 6). Moreover, limited GAC release was detectable following a 60-minute incubation at 37°C for proteases such as Trypsin, Chymotrypsin and Proteinase K (Table 6), indicating that other common proteases can release GAC from S. pyogenes cells, but they are far less effective than ACP, Proalanase and Pepsin. Similarly, large amounts (>4000 units) of Lysozyme and a 60-minute incubation at 37°C were required toPatent ApplicationAttorney Docket No. 018.0102-WQ00 release detectable amounts of GAC (Table 6). This observation highlights Lyzozyme's much lower ability to release GAC from S. pyogenes, compared to Mutanolysin, another muralytic enzyme, which only requires 100 units and a few minutes at RT.EXAMPLE 8: Use of Multiple Hydrolytic Enzymes and Detergents for the Detection of Strep A Biomarkers in Saliva Samples (and Spiked Samples)

[0224] Multiple different hydrolytic enzymes, tested alone or in combination with other enzymes, with or without detergent, were evaluated for their ability to release GAC from the cell wall of S. pyogenes during a 5- minute incubation at room temperature (RT), as detected by a commercially available lateral flow assay (LFA), Strep A Rapid Test Device (Catalog No. STR-15CT20, BTNX Inc.). The following studies showed that certain enzymes and small amounts of detergent facilitated Strep A detection in samples spiked with S. pyogenes cells, in the presence or absence of saliva, using this LFA. In particular, the combination of lytic enzymes and small amounts of detergent increased GAC release and improved detection of Strep A. Additionally, the mucolytic activity of these enzymes and detergents was shown to improve sample flow on the LFA strip in the presence of biological material such as saliva.

[0225] For each reaction (100 pL final), 100 U of ACP and / or Mutanolysin were added to 23 mM of MES (pH 6) or Tris buffer (pH 7 or 8) and spiked with 4.9xlO6S. pyogenes cells (Rosenbach strain Tl). These reactions were incubated at RT for 5 minutes before loading 50 pL into the sample well of this LFA device to detect group A carbohydrate (GAC) antigen. Strep A Rapid Test results were read after 5 minutes and interpreted on a scale of 0 to 5 using the Attribute Chart (See Table 3, Fig. 6A and Table 7, below).

[0226] These LFA results demonstrated that at each of the pH values tested, the intensity of the test line was more intense when ACP and Mutanolysin were combined than when each enzyme was used individually. This finding suggested that these two enzymes acted synergistically to increase the release of GAC from S. pyogenes cells, by hydrolyzing different bonds of the peptidoglycans anchoring GAC on the cell wall.Table 7. Scores of test line intensity (GAC release) following enzymatic treatment of S. pyogenes cells with ACP, Mutanolysin, or ACP and Mutanolysin for 5 minutes at room temperature at increasing pH values.

[0227] Next, for each reaction (200 pL final), 100 U of Mutanolysin and / or 1 pg Proalanase and / or 100 pgPepsin was added to 20 mM citric acid (pH 3) and spiked with 1.3xl07S. pyogenes cells. These reactions werePatent ApplicationAttorney Docket No. 018.0102-WO00 incubated at RT for 5 minutes and then 75 L of each reaction was loaded into the sample well of an LFA device to assess GAC release. LFA test results were read after 5 minutes and interpreted on a scale of 0 to 5 using an Attribute Chart Table 3, Fig. 6B and Table 8).

[0228] The data in Fig. 6B demonstrated that Proalanase and Pepsin released little GAC on their own; however, the combined use of these enzymes resulted in an increase in the release of GAC (i.e., a more intense test line was visible), especially in the presence of Mutanolysin. The detection of GAC by LFA improved when Proalanase was combined with Mutanolysin and when Pepsin was combined with Mutanolysin, compared to Mutanolysin alone. Interestingly, the combination of both Proalanase and Pepsin with Mutanolysin also increased GAC release, as demonstrated by the more intense test lines, compared to Mutanolysin alone(Fig. 6B and Table 8). Together, these results demonstrated that these three enzymes also acted synergistically to increase the release of GAC from the Strep A cell wall, possibly by hydrolyzing different bonds in S. pyogenes cell wall peptidoglycans.Table 8. Scores of test line intensity (GAC release) following enzymatic treatment of S. pyogenes cells with Proalanase, Pepsin, Mutanolysin, and various combinations of these enzymes for 5 minutes at room temperature in acidic conditions.

[0229] Next, the impact of small amounts of detergent or surfactant added to ACP-based reactions was tested. Specifically, 100 units of ACP, with and without increasing amounts (0.0125-0.1% w / v) of anionic (Sodium dodecyl sulfate (SDS), Catalog No. D0996, TCI; 4-dodecyl benzenesulfonic acid (4-DBS), Catalog No. 44198-250ML, Sigma-Aldrich), cationic (Cetyltrimethylammonium bromide (CTAB), Catalog No. 219374- 100GM, Millipore), and non-ionic detergents (n-Dodecyl [3-D-maltoside (DDM), Catalog No. D4641-1G, Sigma- Aldrich), were added to reactions (75 pL final) containing 13 mM Tris buffer (pH 8) and spiked with 5xl06S. pyogenes cells. These reactions were incubated at RT for 5 minutes and then 50 pL of each reaction was loaded into the sample well of an LFA device to assess GAC release. LFA test results were read after 5 minutes and interpreted on a scale of 0 to 5 using an Attribute Chart (Table 3, Fig. 6C and Table 9).Patent ApplicationAttorney Docket No. 018.0102-WQ00

[0230] These data showed that low concentrations of CTAB (0.0125%), 4-DBS (0.0125%), SDS (0.025%), and DDM (0.025 to 0.05%) increased ACP-mediated GAC release, as demonstrated by the presence of more intense test lines, compared to the controls without detergent (Fig. 6C and Table 9). However, higher concentrations of these detergents (>0.05%) were detrimental to enzymatic activity and led to a decrease in the signal intensity as seen by fainter or absent test lines. Detergents likely increased GAC release by destabilizing the bacterial cell wall structure and making ACP's substrate more accessible and / or by increasing enzymatic activity, as previously reported for other proteases, such as Proteinase K and Trypsin (( Hilz et al., 1975) [DOI: 10.1111 / j.1432-1033.1975.tb02211.x]; Tsai et al. (2021)). At higher concentrations of detergent, the ACP was likely denatured and lost the ability to release GAC from the S. pyogenes cell wall.Table 9. Scores of test line intensity (GAC release) following enzymatic treatment of S. pyogenes cells with ACP in the presence of increasing concentrations of anionic, cationic or non-ionic detergents.

[0231] Finally, the impact of a biological sample such as saliva, which introduces a complex and mucinous matrix, on the release and subsequent detection of Strep A biomarkers such as GAC was assessed. Specifically, 100 units of ACP and / or Mutanolysin were added to reactions containing 9.3 mM of the indicated buffer (pH 8, 9 or 10; see Fig. 6D), 5xl06S. pyogenes cells, 33.3 pL saliva (~30% vol / vol) and in the presence or absence of 0.025% SDS in a final reaction volume of 108 pL. After a 5-minute incubation at RT, 100 pL of the reaction mixture or lysate was loaded on an LFA to assess GAC release. LFA test results were read after 5 minutes and interpreted on a scale of 0 to 5 using an Attribute Chart (Table 3, Fig. 6D and Table 10).

[0232] The LFA results showed that, in the presence of saliva, ACP treatment led to more effective detection of GAC from Strep A than Mutanolysin, likely through ACP's ability to proteolytically cleave abundant saliva proteins such as mucin. Unlike ACP, Mutanolysin preparations have limited proteolytic activity and cannot degrade proteins / mucin in the samples efficiently (Yokogawa et al. (1974) [DOI: 10.1128 / AAC.6.2.156]). As such, treatment with ACP decreased sample viscosity, thereby improving the flow of the lysate on the LFA, leading to better performance and detection. Improved flow rate correlated with increased intensity of the control line in ACP-treated sample, compared to Mutanolysin-treated samples (Fig. 6D). Interestingly, addition of low concentrations of SDS (0.025% w / v) improved the detection of GAC from Strep A following treatmentPatent ApplicationAttorney Docket No. 018.0102-WO00 with Mutanolysin alone by decreasing the viscosity of the samples and / or increasing enzymatic activity. Importantly, ACP, Mutanolysin and low concentrations of SDS acted synergistically in the presence of saliva, to increase release of GAC, as well as breakdown of the saliva viscous matrix, to improve detection of Strep A in LFA (Fig. 6D). These data demonstrated that in the presence of biological and mucinous material, the presence of a proteolytic enzyme(s) and / or detergent was beneficial for GAC detection by LFA as it provides mucolytic activity.Table 10. Scores of test line intensity (GAC release) following enzymatic treatment of saliva spiked with S. pyogenes and treated with ACP and / or Mutanolysin in the presence or absence of a low concentration of SDS.

[0233] EXAMPLE 9: Dry Down Method for Hydrolytic Enzymes of the Present Invention to Improve Longterm Stability or Storage

[0234] In previous examples herein, several hydrolytic enzymes dissolved in buffered solutions were shown to release GAC from the cell wall of S. pyogenes following a 5-minute incubation at room temperature (RT), as detected by an LFA. It is uncertain how long these enzyme preparations will remain active in liquid form at RT. For prolonged storage, manufacturers of these enzymes, such as Sigma-Aldrich and Creative Enzymes, recommend they be stored as lyophilized powders at -20°C, which is not practical for at-home use in test kits or shipping to customers, clinicians, or laboratories.

[0235] This example discloses a method to dry down liquid preparations of ACP and Mutanolysin, to enable long-term storage of these enzymes at ambient temperature, without significantly impacting their enzymatic activity. This example also highlights that the dried-down preparations of ACP and Mutanolysin readily go back in solution when mixed with the appropriate buffer to support the detection of GAC following a 5-minute incubation with S. pyogenes at RT.

[0236] In the following method, ACP and Mutanolysin were first dissolved in 10% sucrose at a concentration of 40 and 10 U / pL, respectively, and then mixed with an equal volume of 50% glycerol / 50% ethanol solution. Next, 5 to 20 pL of the resulting enzyme suspensions (100 units total) were transferred to 1.5 mL tubes and dried down at room temperature (RT) or 45°C. ACP suspensions took approximately 90 minutes to dry at RTPatent ApplicationAttorney Docket No. 018.0102-WO00 and 45 minutes at 45°C, while Mutanolysin suspensions required overnight incubation at room temperature to fully dry down.

[0237] Enzymatic activity of these dried down preparations was assessed after 1 day (Fig. 7 A), 3 weeks (Fig. 7B), and 9 months (Fig. 7C) storage at room temperature. At each time point, the dried enzymes were reconstituted in 50 pL of 20 mM Tris pH 9 (for ACP) or 20 mM MES pH 6 (for Mutanolysin), followed by the addition of 25 pL of 2 x 108CFU / mL S. pyogenes culture (Rosenbach strain Tl). After a 5-minute incubation at RT, 50 pL of the mixture was loaded on an LFA to assess GAC release, compared to the "Control" stock enzyme (stored in solution at -20°C). HONO treatment was also included as a control.

[0238] These data demonstrate that dried-down preparations of ACP and Mutanolysin retain their enzymatic activity for up to 9 months following dry down and storage at RT in 1.5 mL Eppendorf tubes. Following reconstitution in the appropriate buffer, the dried-down enzymes released GAC at all timepoints following a 5- minute incubation at RT to a similar extent as the enzyme stocks (i.e., stock enzyme controls stored frozen at - 20°C), as demonstrated by the presence of a test line for each condition (Figs. 7A-7C). These data also demonstrate that the dried-down enzyme preparations readily go back in solution following addition of the appropriate buffer solution, without the need for additional mechanical steps (pipetting, vortexing, or extensive mixing, etc.). Hence, this dry-down method for enzymes, such as ACP and Mutanolysin, supports long-term stability facilitating storage of enzyme mixtures required for safe, at-home use in test kits (e.g., StrepA LFA), as well as during shipping to customers, clinicians, and laboratories.EXAMPLE 10: Evaluation of Phosphodiesterase Enzymes in Streptococcal Biomarker Hydrolysis and Detection

[0239] The effects of phosphodiesterase enzymes on the hydrolytic release of biomarker GAC from 5. pyogenes in the presence of ACP and Mutanolysin were investigated. Three different phosphodiesterase enzymes were tested, namely RNase H (Catalog No. M0297L, New England Biolabs), RNase A (Catalog No. EN0531, Thermo Fisher), and RNase Tl (Catalog No. EN0541, Thermo Fisher), as well as Alkaline phosphatase (AP) as a control (Catalog No. EF0651, Thermo Fisher). Specifically, each 100 pL reaction contained 20 mM Tris (pH 9 for reactions with ACP; pH 8 for reactions with Mutanolysin), 25 units ACP or 50 units Mutanolysin, and 3.5 xlO6S. pyogenes cells (Rosenbach strain Tl), in the presence or absence of 10-20 pL of phosphodiesterase enzyme or phosphatase (RNase H, 100 units; RNase A, 200 pg; RNase Tl, 10,000 units; AP, 20 units). Lower amounts of ACP and Mutanolysin were used to lower signal intensity and better capture impact of the phosphodiesterases. In parallel reactions, a negative control (cells only, no hydrolytic enzyme) and a positive control (HONO treatment) were included (Figs. 8A and 8B). These reactions were incubated at room temperature (RT) for 5 minutes and then the whole reaction (100 pL) was added to the sample well of a Strep APatent ApplicationAttorney Docket No. 018.0102-WO00Rapid Test Device (Catalog No. STR-15CT20, BTNX Inc.). Strep A test results were read after 5 minutes (Figs. 8A and 8B and Table 11) and interpreted on a scale of 0 to 5 using an Attribute Chart developed by OraSure Technologies, Inc. (see Table 3 ).Table 11. Scores of test line intensity (GAC release) following enzymatic treatment of S. pyogenes cells with ACP and / or Mutanolysin in the presence or absence of phosphodiesterase enzymes or phosphatase.

[0240] These data demonstrate that phosphodiesterase enzymes like RNase H and RNase T1 can increase the release of GAC (i.e., higher intensity of the test line) when combined with other enzymes, such as Mutanolysin and ACP at pH 8 and 9, respectively. The findings also suggest that RNase T1 and RNase H can hydrolyze the phosphodiester bond linking GAC glucosamine residue to the peptidoglycan N-acetyl muraminic acid. Other phosphodiesterase enzymes with higher affinity for the phosphodiester bond between an N- acetyl muraminic acid and a glucosamine residue, which attaches GAC to the S. pyogenes cell wall, would also be expected to work.

[0241] Interestingly, unlike RNases H and Tl, RNase A did not increase Mutanolysin- or ACP-mediated release of GAC, suggesting that only certain nucleases / phosphodiesterases can cleave or hydrolyze this specific phosphodiester bond. Alkaline phosphatase, which has been reported to display phosphodiesterase activity in addition to phosphatase activity, did not increase the release of GAC in the presence of Mutanolysin or ACP either. RNase H and RNase Tl did not improve Labiase-mediated GAC release (data not shown) when the lysis reaction was performed at pH 5, which is well below the optimal pH range of RNase H and RNase Tl (pH 7.5-9). Taken together, this data demonstrates that phosphodiesterase enzymes, such as RNase H and RNase Tl, can assist with the release of GAC from S. pyogenes, provided that they are combined with enzymes able to start hydrolyzing the cell wall (e.g. Mutanolysin and ACP) thereby improving accessibility to GAC molecules and / or phosphodiester bonds.EXAMPLE 11: Bacterial Content and Streptococcal Species Relative Abundance in the Oral Cavity.

[0242] Presently, the standard of care for detecting and diagnosing a Strep A infection involves the collection of one or more throat swab samples by a trained medical professional, which is an invasive process. To provide a rapid diagnostic test for Strep A that individuals can use at home, an easy, non-invasive sample collectionPatent ApplicationAttorney Docket No. 018.0102-WO00 method is essential. In this example, samples were collected from different sites within the oral cavity of healthy individuals and then the relative abundance of bacterial DNA, as well as the relative abundance of Streptococcal species was determined. In particular, "spit" saliva samples were compared with swab samples taken from the tongue or gums.

[0243] Saliva (1 mL) was collected from 10 healthy individuals in OM-505 Kits (DNA Genotek, Inc., Canada), whereas gum / plaque samples and tongue samples were collected from 15 healthy individuals using the flocked swabs provided in QMR-110 and QMR-120 Kits, respectively (DNA Genotek, Inc., Canada). After the manufacturer's recommended Proteinase K treatment step at 50°C (for gum and tongue samples) or 50°C incubation (for saliva samples), DNA was extracted from a 250 pL aliquot of each sample using the QIAamp™ PowerFecal™ Pro DNA QIAcube Kit (Catalog No. 51826, Qiagen), as per manufacturer's instructions.

[0244] Metagenomics libraries were generated from the extracted DNA and sequenced using the BoosterShot™ methodology (Diversigen, Inc.). The resulting FASTQs were aligned to a curated database containing all representative genomes in RefSeq for bacteria with additional manually curated strains. Alignments were made at 97% identity against all reference genomes. Every input sequence was compared to every reference sequence in the Diversigen proprietary Venti database using fully gapped alignment with BURST. Ties were broken by minimizing the overall number of unique Operational Taxonomic Units (OTUs). For taxonomy assignments, each input sequence was assigned the lowest common ancestor that was consistent across at least 80% of all reference sequences tied for best hit. Samples with fewer than 10,000 sequences were discarded. OTUs accounting for less than one millionth of all strain-level markers and those with less than 0.01% of their unique genome regions covered (and < 0.1% of the whole genome) at the species level were discarded. The number of counts for each OTU was normalized to the OTU's genome length.

[0245] As depicted in Fig. 9, the extracted DNA that was sequenced using shotgun sequencing, with resulting FASTQs being mapped against a bacterial database, showed clear differences in the most abundant genera across the three oral sample types. Fig. 9 data shows the average relative abundance of each genus across the entire cohort (n=10 or 15 individuals) for each type of oral sample. Interestingly, tongue and gum / plaque swab samples had a much higher relative abundance of Streptococcus genus (27% and 34%, respectively) than saliva (19%), indicating that the tongue and gum microenvironments can adequately support commensal Streptococcal species closely related to Strep A, and potentially the S. pyogenes (Strep A) bacteria itself.Additionally, higher relative abundance of Streptococcal bacteria in tongue and gum / plaque samples could also increase the probability of detecting Streptococcal organisms of interest, such as Strep A at these sites, since one would also expect increased relative abundance of biomarkers of interest.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0246] Next, the relative bacterial DNA content was quantified in the extracted DNA from each sample. Bacterial 16S copy number was determined by qPCR, using an assay targeting variable region 3 of the 16S RNA gene (Muyer et aL, 1993), and relative bacterial DNA content was estimated based on 16S copy number / ng of extracted DNA. The box plots (Fig. 10) show inferred bacterial content for oral samples collected from gum or tongue, as well as saliva, with a line at the median.

[0247] Surprisingly, as shown in Fig. 10, tongue swab samples had a dramatically higher relative bacterial content (median at 85%), compared to gum (median at 11%) and saliva samples (median at 4%). Taken together, this data indicates that the absolute number of Streptococcal cells in tongue swab samples is at least 5x greater than in saliva (spit) or gum swab samples. This suggests that Streptococcal biomarkers of interest could be orders of magnitude more abundant in samples readily collected from the tongue, compared to other sites. As such, the tongue may represent a prime candidate site in the oral cavity for the detection of Streptococcal biomarkers, including those specific for Strep A in infected individuals.EXAMPLE 12: Use of Tongue and Palate Samples in Strep A Biomarker Detection

[0248] The previous example showed that Streptococcal biomarkers could be orders of magnitude more abundant in samples readily collected from the surface of the tongue. As such, the tongue may represent a prime candidate site in the oral cavity for the detection of Streptococcal biomarkers, including those specific for Strep A in infected individuals. Spit saliva, tongue swab samples, and palate swab samples were compared with matched standard of care (SOC) throat swab samples using HONO lysis or extraction, followed by a rapid LFA test for Strep A detection.

[0249] First, 20 patients, already confirmed positive for Strep A, provided spit saliva and an additional throat swab sample. These samples were evaluated using a commercially available Strep A LFA test and interpreted on a scale of 0 to 5 using an Attribute Chart developed by OraSure Technologies, Inc. (Table 3 o).

[0250] The results, shown in Table 12 indicate 80% (16 / 20) agreement in LFA test results for the saliva samples (spit) and throat swab samples; however, the intensity of the test line was significantly lower for 70% (14 / 20) of saliva samples compared to throat swab samples. Additionally, a number of saliva samples had a very weak signal (<3) in LFA tests, detectable only by a trained individual. This is likely indicative of lower abundance of Strep A in saliva and / or interference of saliva with the lateral flow assay.Table 12. Strep A LFA test results following HONO treatment for matched spit saliva samples and throat swab samples collected from patients previously confirmed positive for Strep A.Patent ApplicationAttorney Docket No. 018.0102-WO00

[0251] Second, matched tongue and throat swab samples as well as matched palate and throat swab samples were collected from patients previously confirmed positive for Strep A using a standard of care (SOC) throat swab rapid LFA test. These samples were evaluated using a commercially available Strep A LFA test following HONO lysis and interpreted on a scale of 0 to 5 using an Attribute Chart (Table 3 ). Table 13 shows Strep A LFA test results for matched tongue swab samples and throat swab samples, as well as matched palate swab samples and throat swab samples, collected from patients previously confirmed positive for Strep A using a standard of care (SOC) LFA test.Patent ApplicationAttorney Docket No. 018.0102-WO00Table 13. Strep A LFA test results for matched tongue and throat swab samples, as well as matched palate and throat swab samples.

[0252] Surprisingly, these results indicate 100% (8 / 8 donors) agreement between tongue and the standard of care (SOC) throat swab samples. Furthermore, GAC levels were consistently high in tongue swab samples, with rapid LFA test results exhibiting visible lines that are near or above the intensities that are easily detectable by untrained users (i.e., >3). Results also show that palate samples have a 100% correlation with the standard of care (SOC) throat samples (2 / 2), but the GAC signal was significantly weaker than that seen for tongue or throat samples. Two palate samples had a 0.1 score, a result only detectable by a trained technician. Importantly, tongue swab samples appear to be a viable, non-invasive sample type for the accurate diagnosis of Strep A infection, as they consistently provide robust signals comparable to those from throat swab samples.

Claims

Patent ApplicationAttorney Docket No. 018.0102-WQ00The claimed invention is:

1. A method for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the method comprises:(a) collecting the biological sample from the subject using a non-invasive technique;(b) mixing the collected biological sample, or a portion thereof, in a vial with a composition comprising one or more hydrolytic enzymes resulting in a hydrolytic enzyme mixture that will carry out one or more of the following events:(i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells;(ii) lyse the cells to release the one or more biomarkers, wherein the one or more biomarkers are intracellular biomarkers; and(iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are secreted biomarkers; and(iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii);(c) incubating the biological sample and hydrolytic enzyme mixture of step (b) for a sufficient length of time to effect one or more of the events of (b)(i-ii i) to occur; and(d) performing an assay to detect the one or more biomarkers present in the incubated sample mixture of step (c).

2. The method of claim 1, wherein the biological sample is an oral sample comprising samples collected from a surface within the oral cavity, an oral rinse, a saliva sample, or any combination thereof.

3. The method of claim 2, wherein the surface within the oral cavity is selected from tongue, palate, or any combination thereof.

4. The method of claim 2, wherein collecting the oral sample comprises using a non-invasive technique.Patent ApplicationAttorney Docket No. 018.0102-WO005. The method of claim 4, wherein the non-invasive technique comprises: absorbing the oral sample using a sponge, swab, or pad; and / or using an implement, scraper, brush, or applicator to collect the sample from inside the mouth; or spitting saliva into a collection vial or tube.

6. The method of claim 2, wherein the surface within the oral cavity is the tongue.

7. The method of claim 2, wherein the surface within the oral cavity is the palate.

8. The method of any one of claims 1-7, wherein the method does not comprise any reagent that is nitrous acid, toxic, unstable, and / or will cause one or more toxic gases to be produced.

9. The method of any one of claims 1-8, wherein the composition comprising one or more hydrolytic enzymes comprises one or more amidohydrolase or protease selected from: Achromopeptidase (ACP); Pepsin; ProAlanase; Trypsin; Chymotrypsin; Proteinase K, and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Mutanolysin and Lysozyme.

10. The method of claim 9, wherein the composition comprising one or more hydrolytic enzymes comprises ACP.

11. The method of claim 9, wherein the composition comprising one or more hydrolytic enzymes comprises Mutanolysin and / or Labiase.

12. The method of any one of claims 1-11, wherein the composition comprising one or more hydrolytic enzymes comprises a buffered solution with a pH from about 2 to about 10.

13. The method of claim 12, wherein the pH of the buffered solution is less than about 4 or greater than about 9.Patent ApplicationAttorney Docket No. 018.0102-WO0014. The method of claim 12, wherein the buffered solution is selected from a Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof.

15. The method of any one of claims 1-14, further comprising contacting a detergent, surfactant, and / or other non-toxic chemicals with one or more of: the collected biological sample of step (a); the biological sample and hydrolytic enzyme mixture of step (b); and during incubation step (c).

16. The method of claim 15, wherein one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with the release from the collection tool and / or breakdown of the biological material in the sample.

17. The method of any one of claims 1-16, further comprising adding one or more enzymes to the biological sample of step (a) and / or the biological sample and hydrolytic enzyme mixture of step (b) to further break down biological material and biomarkers in the biological sample.

18. The method of claim 17, wherein the further added one or more enzymes comprise one or more phosphodiesterases.

19. The method of claim 18, wherein the one or more phosphodiesterases are RNase H and / or RNase Tl.

20. The method of any one of claims 1-19, wherein the detection of the one or more biomarkers is specific to one or more of: group A Streptococcus (GAS); group B Streptococcus (GBS); group C Streptococcus (GCS); group D Streptococcus (GDS); group E Streptococcus (GES); group F Streptococcus (GFS); group G Streptococcus (GGS); or one or more non-Lancefield Streptococci comprising S. pneumoniae or species from the viridans streptococci group; is a carbohydrate a protein or a peptide comprising a streptolysin; or a streptococcal pyrogenic exotoxin.Patent ApplicationAttorney Docket No. 018.0102-WO0021. The method of claim 20, wherein the one or more streptococcal biomarkers is a biomarker specific to GAS.

22. The method of any one of claims 1-21, wherein the assay to detect the one or more streptococcal biomarkers is one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a fluorescent immunoassay, a western blot, a dot blot, or another reporter-based immunoassay.

23. The method of claim 22, wherein the immunoassay comprises monoclonal or polyclonal antibodies specific for one or more streptococcal biomarkers.

24. The method of any one of claims 1-23, wherein the method further comprises diagnosing the subject as having a streptococcal infection if one or more streptococcal biomarkers are detected by the assay.

25. A kit for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the kit comprises:(a) a biological sample collection device, container, or a device comprising a container, for non-invasive sample collection;(b) a container comprising one or more non-toxic reagents for preparing the one or more biomarkers in the biological sample to be detected;(c) an assay device configured to detect the one or more biomarkers; and(d) instructions for using the kit.

26. The kit of claim 25, wherein the biological sample collection device or device comprising a container of (a) comprises an implement, tube, scraper, brush, or applicator for collecting an oral sample.

27. The kit of claim 26, wherein the implement, tube, scraper, brush, or applicator is designed to optimize the collection of a sample from the tongue surface or is designed to optimize the collection of a sample from the surface of the palate within the mouth.Patent ApplicationAttorney Docket No. 018.0102-WO0028. The kit of any one of claims 25-27, wherein the kit further comprises a sponge, swab, or pad for facilitating the non-invasive sample collection of (a), optionally wherein the sponge, swab, or pad is integrated into the biological sample collection device or device comprising a container.

29. The kit of any one of claims 25-28, wherein the kit comprises a container, optionally a tube or vial, for receiving the collected biological sample.

30. The kit of claim 29, wherein the biological sample collection container is a container for collecting saliva from the subject or a device comprising a container for collecting saliva.

31. The kit of any one of claims 25-30, wherein the kit does not comprise any reagent that is nitrous acid, toxic, is unstable, and / or will cause one or more toxic gases to be produced.

32. The kit of any one of claims 25-31, wherein the one or more non-toxic reagents of component (b) comprises a composition comprising one or more hydrolytic enzymes for carrying out one or more of the following events:(i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells;(ii) lyse the cells to release the one or more biomarkers, wherein the one or more cell biomarkers are intracellular biomarkers; and(iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are secreted biomarkers; and(iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii).

33. The kit of claim 32, wherein the one or more hydrolytic enzymes comprise one or more amidohydrolases and proteases selected from: Achromopeptidase (ACP); Pepsin; Proalanase; Trypsin; Chymotrypsin;Proteinase K and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, and Mutanolysin; Lysozyme;Galactosidase; and p-N--acetylglucosaminidase.Patent ApplicationAttorney Docket No. 018.0102-WO0034. The kit of claim 33, wherein the one or more hydrolytic enzymes comprises an aqueous solution or a dried down composition.

35. The kit of claim 33, wherein the composition comprising one or more hydrolytic enzymes comprises ACP.

36. The kit of claim 33 or 34, wherein the composition comprising one or more hydrolytic enzymes comprises Mutanolysin and / or Labiase.

37. The kit of any one of claims 25-36, wherein the composition comprising one or more hydrolytic enzymes comprises a buffered solution with a pH from about 2 to about 10.

38. The kit of claim 37, wherein the pH of the buffered solution is less than about 4 or greater than about 9.

39. The kit of claim 37 or 38, wherein the buffered solution is selected from a Tris-buffered, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate buffered saline (PBS) solution, or any combination thereof.

40. The kit of any one of claims 25-39, further comprising a detergent, surfactant, and / or other non-toxic chemicals, in: the biological sample collection device, container, or a device comprising a container of component (a); the container comprising one or more non-toxic reagents of component (b); and / or an additional container component.

41. The kit of claim 40, wherein one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with the release from the collection tool and / or breakdown of the biological material in the sample.

42. The kit of any one of claims 25-41, wherein the biological sample collection device, container, or a device comprising a container of component (a) and / or the container comprising one or more non-toxic reagents ofPatent ApplicationAttorney Docket No. 018.0102-WO00 component (b) further comprises one or more enzymes to further break down biological material or biomarkers in the biological sample.

43. The kit of claim 42, wherein the one or more enzymes to further break down biological material is one or more phosphodiesterases.

44. The kit of claim 43, wherein the one or more phosphodiesterases are RNase H and / or RNase Tl.

45. The kit of any one of claims 25-44, wherein the assay device is configured to detect one or more of: group A streptococcus (GAS); group B streptococcus (GBS); group C streptococcus (GCS); group D streptococcus (GDS); group E streptococcus (GES); group F streptococcus (GFS); group G streptococcus (GGS); one or more non-Lancefield Streptococci comprising S. pneumoniae or species of the viridans streptococci group; is a carbohydrate, a protein, a streptolysin; and a streptococcal pyrogenic exotoxin.

46. The kit of any one of claims 25-45, wherein the assay device is, or configured to be used with, one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a fluorescent immunoassay, a western blot, a dot blot, or another reporterbased immunoassay.

47. The kit of claim 46, wherein the kit comprises monoclonal or polyclonal antibodies specific for the one or more streptococcal biomarkers.

48. The kit of claim 47, wherein the one or more monoclonal or polyclonal antibodies is specific for GAS.

49. A system for detecting one or more biomarkers of one or more Streptococcus species and / or strains in a biological sample from a subject that has, or may have, a streptococcal infection, wherein the system comprises:(a) a biological sample collection device, container, or a device comprising a container, for non-invasive sample collection;(b) a container comprising one or more non-toxic reagents for preparing the one or more biomarkers in the biological sample to be detected; andPatent ApplicationAttorney Docket No. 018.0102-WO00(c) an assay device configured to detect one or more biomarkers.

50. The system of claim 49, wherein the system further comprises an assay apparatus configured for providing a signal in response to at least one detected Streptococcus species and / or strain biomarker in the sample; a detector device; a light source configured to transmit at least one wavelength of light capable of interacting with the signal of the assay apparatus; and a holder configured to couple the assay apparatus to the detector device in proximity to the light source, wherein the light source is positioned to illuminate at least a portion of the assay apparatus and the detector is positioned to capture at least one image of the illuminated signal.

51. The system of claim 50, further comprising an interpretive algorithm stored in a computer-readable format that is electronically coupled to the detector device, wherein the interpretive algorithm is configured to convert the at least one image of the illuminated signal to a numerical value related to the presence or amount of the at least one biomarker present in a biological sample.

52. The system of any one of claims 50-52, wherein the assay apparatus is a lateral-flow chromatographic assay cassette having at least one ligand immobilized thereon configured for capturing an analyte or biomarker of interest.

53. The system of any one of claims 49-52, wherein the holder includes an electrical connector configured to draw power from the detector device to power the light source.

54. The system of any one of claims 49-53, wherein the biological sample collection device or device comprising a container of (a) comprises an implement, tube, scraper, brush, or applicator for collecting an oral sample.

55. The system of claim 54, wherein the implement, tube, scraper, brush, or applicator is designed to optimize the collection of a sample from the tongue surface or is designed to optimize the collection of a sample from the surface of the palate.

56. The system of any one of claims 49-55, wherein the system further comprises a sponge, swab, or pad for facilitating the noninvasive sample collection of (a), optionally wherein the sponge, swab, or pad is integrated into the biological sample collection device or device comprising a container.Patent ApplicationAttorney Docket No. 018.0102-WO0057. The system of any one of claims 49-56, wherein the system comprises a container, optionally a tube or vial, for receiving the collected biological sample.

58. The system of claim 57, wherein the biological sample collection container is a container for collecting saliva from the subject or a device comprising a container for collecting saliva.

59. The system of any one of claims 49-58, wherein the system does not comprise any reagent that is nitrous acid, toxic, is unstable, and / or will cause one or more toxic gases to be produced.

60. The system of any one of claims 49-59, wherein the one or more non-toxic reagents of component (b) comprises a composition comprising one or more hydrolytic enzymes for carrying out one or more of the following events:(i) release the one or more biomarkers on the cell wall surface of the one or more streptococcal cells;(ii) lyse the cells to release the one or more biomarkers, wherein the one or more biomarkers are intracellular biomarkers; and(iii) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are secreted biomarkers; and(iv) induce one or more conformational or structural changes in the one or more biomarkers that will facilitate their binding to detection agents, wherein the biomarkers are released into the extracellular compartment as described in (b)(i) and / or (b)(ii).

61. The system of any one of claims 49-60, wherein the one or more hydrolytic enzymes comprise one or more amidohydrolases or proteases selected from: Achromopeptidase (ACP); Pepsin; ProaAnalase; Trypsin; Chymotrypsin; Proteinase K; and N-acetylmuramoyl-L-alanine amidase found in Mutanolysin preparations; one or more glycosidases selected from Labiase, Rhamnosidase, Chitinase, Chitosanase, Mutanolysin, and Lysozyme.

62. The system of claim 61, wherein the composition comprising one or more hydrolytic enzymes comprises ACP.Patent ApplicationAttorney Docket No. 018.0102-WO0063. The system of claim 61 or 62, wherein the composition comprising one or more hydrolytic enzymes comprises Mutanolysin and / or Labiase.

64. The system of any one of claims 49-63, wherein the composition comprising one or more hydrolytic enzymes comprises a buffered solution with a pH from about 2 to about 10.

65. The system of claim 64, wherein the pH of the buffered solution is less than about 4 or greater than about 9.

66. The system of claim 62 or 63, wherein the buffered solution is selected from a Tris-buffered, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof.

67. The system of any one of claims 49-66, further comprising a detergent, surfactant, and / or other non-toxic chemicals, in: the biological sample collection device, container, or a device comprising a container of component (a); the container comprising one or more non-toxic reagents of component (b); and / or an additional container component.

68. The system of claim 67, wherein one or more of the: hydrolytic enzymes; and / or detergent, surfactant, and / or other non-toxic chemicals have mucolytic activity to assist with the release and / or breakdown of the biological material in the sample.

69. The system of any one of claims 49-68, wherein the biological sample collection device, container, or a device comprising a container of component (a) and / or the container comprising one or more non-toxic reagents of component (b) further comprises one or more enzymes to further break down biological material in the biological sample.

70. The system of claim 69, wherein the one or more enzymes to further break down biological material is one or more phosphodiesterases.Patent ApplicationAttorney Docket No. 018.0102-WQ0071. The system of claim 70, wherein the one or more phosphodiesterases are RNase H and / or RNase Tl.

72. The system of any one of claims 49-71, wherein the assay device is configured to detect one or more of: group A streptococcus (GAS); group B streptococcus (GBS); group C streptococcus (GCS); group D streptococcus (GDS); group E streptococcus (GES); group F streptococcus (GFS); group G streptococcus (GGS); one or more of non-Lancefield Streptococci comprising S. pneunomiae or species of the viridans group; is a carbohydrate, a protein, a streptolysin; and a streptococcal pyrogenic exotoxin.

73. The system of any one of claims 49-72, wherein the assay device is, or configured to be used with, one of the following immunoassays: enzyme-linked immunosorbent assay (ELISA); a lateral flow (LF) immunoassay; a chemiluminescent immunoassay; a western blot; a dot blot, a fluorescent immunoassay, or another reporterbased immunoassay.

74. The system of claim 73, wherein the system comprises monoclonal or polyclonal antibodies specific for one or more streptococcal biomarkers.

75. The system of claim 74, wherein the one or more monoclonal or polyclonal antibodies is specific for GAS.

76. A composition for processing a biological sample comprising Gram-positive bacteria, for application to a biomarker detection assay, wherein the method comprises:(A) one or more of the following hydrolytic enzymes and / or hydrolytic enzyme preparations: Achromopeptidase (ACP); a Mutanolysin preparation;Metapolyzyme;Pepsin; a Chitinase preparation;Chitosanase;Labiase;Rhamnosidase; andProalanase; andPatent ApplicationAttorney Docket No. 018.0102-WQ00(B) one or more buffering agents with a pH from about 2 to about 10 selected from Tris, MES, borate, acetate, citrate, HEPES, CAPS, CAPSO, MOPS, BES, phosphate-buffered saline (PBS) solution, or any combination thereof); and optionally(C) a detergent selected from cationic 0.001% - 0.05% Cetyltrimethylammonium bromide (CTAB), 0.01% - 0.05% 4-dodecyl benzenesulfonic acid (4-DBS), 0.01% - 0.05% Sodium dodecyl sulfate (SDS), and 0.001% - 0.05% n-Dodecyl P-D-maltoside (DDM).

77. The composition of claim 76 further comprising one or more phosphodiesterase enzymes.

78. The composition of claim 76 or 77, wherein the phosphodiesterase enzymes are RNaseH and / or RNaseTl.

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