Kits and methods using combined samples to improve sensitivity

Abstract

A novel method for detecting an analyte in a combined sample obtained from a subject is provided. A method for detecting an analyte using combined sample collection types is disclosed. Specifically, a first sample is combined with a bioaerosol sample. A kit including components for collecting the first sample and combining it with the bioaerosol sample is also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 051116, filed July 13, 2020, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The present disclosure relates generally to medical care. More particularly, the present disclosure relates to a method for detecting an analyte using a combined swab sample collection and bioaerosol sample collection method. The present disclosure also relates to a kit for detecting an analyte using a combined swab sample collection and bioaerosol sample collection method.

[0003] Clinical and laboratory tests are used to detect analytes in samples. Clinical tests are often used to determine infection and disease. Accurate testing is important to prevent infection and transmission. Pathogen infections are often detected after infection but before the infected individual shows symptoms, a "gap period." Many analytical tests lack sensitivity and / or sensitivity during the gap period, which can result in false-negative results. If an individual tests negative, they may be cleared to return to work. A negative test can also cause an individual to reduce or remove protective measures, such as mask-wearing and social distancing. Thus, false-negative results can lead to transmission when prevention could otherwise be in place. The ability to avoid false-negative results by testing early in the course of infection can reduce transmission. Suboptimal sample collection can reduce the overall sensitivity of a test and result in false-negative test results.

[0004] Clinical and laboratory testing requires the collection of samples from patients. Various samples can be collected from patients, and specific sample collection methods exist for collecting various sample types. These include, for example, tissue (biopsy), blood, saliva, sputum, nasopharyngeal swabs, oropharyngeal (throat) swabs, throat washings, spinal fluid, and urine. For respiratory pathogen testing, nasopharyngeal swabbing is the sample collection method of choice. Oropharyngeal swabs are another acceptable method of collection for respiratory pathogen testing. Notably, the Centers for Disease Control (CDC) recommends combining nasopharyngeal and oropharyngeal swabs in one tube if both are collected. However, collecting both nasopharyngeal and oropharyngeal swabs can be painful for patients and pose risks to healthcare personnel. Others have paired saliva and nasopharyngeal samples to detect COVID-19 (see Jamal et al. Clin Infect Dis. 2021 Mar 15;72(6):1064-1066; published online June 25, 2020). Torretta et al. provide a list of all currently available diagnostic techniques reported for COVID-19 testing (see Torretta et al. Ear Nose Throat J. 2021 Apr;100(2_suppl):131S-138S; published online August 31, 2020). While the prior art discloses combining, pairing, and comparing samples, each sample type is tested separately.

[0005] Collected samples and collection methods can perform poorly due to insufficient amounts of analyte to be detected in the sample. This can result in undetectable amounts of the analyte sought to be detected. Collecting an insufficient amount of sample can also result in false negative results being reported, where the patient has the analyte, but the sample does not contain enough analyte to be detected. To address these issues, samples are often further processed after collection to concentrate the amount of analyte in the portion of the sample being tested.

[0006] One way to obtain a detectable level of an analyte to be detected is to take more sample. This may be inconvenient or impossible in certain cases. Sample treatment may also be performed to concentrate the analyte to be detected. Another method is to attempt to increase the sensitivity of the test, such as running longer test reagent incubation times, increasing the concentration of detection reagents, and running more and longer cycles. Sample treatment significantly increases the time required to perform the test and obtain test results. The need for more reagents increases the cost of the test.

[0007] Therefore, there is a need for methods to improve the detection of analytes in samples. The present disclosure accomplishes this by combining samples collected using various sample collection methods. Summary of the Invention

[0008] The present disclosure relates generally to medicine. More particularly, the present disclosure relates to methods for detecting pathogens using combined sample collection types.

[0009] In one aspect, the method includes collecting a first sample; collecting a bioaerosol sample; incorporating the first sample into a buffer, where the buffer elutes at least a portion of the first sample into the buffer to form a first elution buffer; contacting at least a portion of the first elution buffer with the bioaerosol sample, where the first elution buffer elutes at least a portion of the bioaerosol sample to form a combined sample; and analyzing the combined sample for the analyte.

[0010] In another aspect, the present disclosure relates to a kit comprising at least one of a swab, a saliva collection vial, a sputum collection vial, and a lavage fluid collection component; a bioaerosol collection device; a buffer; and instructions for using the kit. [Brief explanation of the drawings]

[0011] [Figure 1A] Three combinations of samples are shown: individually eluted and combined, but in separate vials. [Figure 1B] Three combinations of samples are shown. Samples eluted in the same vial are shown. [Figure 1C] Three combinations of samples are shown. Samples are shown eluted in order. [Figure 2] This graph shows false-negative swab PCR rates versus COVID transmissibility. Both viral transmission data and test false-negative rate data suggest that SARS-CoV-2 is undetectable until approximately two days before symptom onset. Daily viral load data from symptom onset, from He et al. (Nat. Med., 2020, 26:672-675), is shown in teal. Daily false-negative test rates, from Kucirka et al. (Ann. Intern. Med., 2020), are shown in magenta. The probability of transmission begins to increase approximately two days before symptom onset, at the same time that the false-negative test rate begins to decrease. (Jarvis and Kelley, Scientific Reports, 2021, 11:9221) [Figure 3] Graph showing combined swab and bioaerosol relative intensities from the day of exposure. Separately corrected swab and bioaerosol samples and the combined uncorrected signal. [Figure 4] Venn diagram showing a dynamic model of sample sensitivity, diagnostic usefulness of test method (LFA or PCR) based on pathogen temporal dynamics. [Figure 5] To reduce the impact of temporal pathogen dynamics and collection issues, a combination of sample types to increase sample sensitivity for PCR and LFA is presented. [Figure 6] 1 is a table summarizing expected results for clinical trials comparing nasopharyngeal swab (NP) testing alone, bioaerosol testing alone, and combined NP and bioaerosol testing. [Figure 7] 1 is a flow diagram for bioaerosol collection and detection. [Figure 8] 1 illustrates the components of an exemplary embodiment of a test kit. [Figure 9] This demonstrates swab collection combined with a mask insert (bioaerosol) collection method. In steps 1 and 2, a subject dons a mask with an attached mask insert. The collection base of the mask insert is removed from the mask and placed in a vial containing an elution / extraction buffer to elute / extract analytes in the bioaerosol sample collected by the collection base of the mask insert (step 3). A swab is used to collect a swab sample from the subject (step 4). The swab is then placed in the same vial with the collection base of the mask insert to elute / extract analytes from the swab (step 5). A portion of the combined sample is then analyzed (showing an LFA) (step 6), and the results are read (step 7). [Figure 10] An exemplary mask insert for bioaerosol sample collection is shown. The inner collection base is 4.25 cm long and 1.25 cm wide. The outer base (front and back) is 6.3 cm long and 2.3 cm wide. Bioaerosols pass through the outer base and reach the inner collection base, which collects bioaerosols and pathogens larger than 50 nanometers. COVID-19 is approximately 100 nm; influenza is approximately 80-120 nm; mycobacteria are <7 microns; and bioaerosols containing respiratory pathogens are primarily <10 microns. [Figure 11] The outer layer is shown removed to reveal the collection base of an embodiment of the mask insert. [Figure 12]Features of the mask insert are shown (modified from Leung et al. Nat. Med. 26, 676-680 (2020)). DETAILED DESCRIPTION OF THE INVENTION

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test this disclosure, the preferred methods and materials are described below.

[0013] Kits and methods for detecting analytes using combined samples are disclosed. In particular, the kits and methods combine a first sample obtained from a subject with a bioaerosol sample obtained from the subject. The sample can be collected from the subject by a medical professional or by the subject with or without the assistance of a medical professional. The first sample is eluted, and the eluate is then used to elute the contents of a bioaerosol sample collection device to form a combined sample. The combined sample is then analyzed.

[0014] The first sample is selected from a swab sample, a saliva sample, a sputum sample, an aspirate sample, a lavage sample, and combinations thereof. Swab samples include nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, throat swabs, middle turbinate swabs, and combinations thereof. Lavage samples include nasal washes, mouth washes, nasopharyngeal washes, oropharyngeal washes, sinus washes, tracheal washes, bronchoalveolar lavages, lung washes, and combinations thereof. Aspirate samples include nasal aspirates, nasopharyngeal aspirates, oropharyngeal aspirates, throat aspirates, tracheal aspirates, and combinations thereof.

[0015] In one embodiment, a bioaerosol sample is collected using a bioaerosol collection device, and the subject blows air into the bioaerosol collection device (by blowing, breathing, humming, singing, speaking, or a combination thereof). The air enters the bioaerosol sample collection device through an inlet. The air is conveyed to a collection base disposed within the bioaerosol collection device, where analytes contained in the air sample are captured. The bioaerosol collection device also includes an outlet that allows the air to pass out of the bioaerosol collection device after passing through the collection base. This is after collection and elution / extraction of the first sample. The eluate from the first sample is then drawn into the bioaerosol sample collection device, and the analytes captured by the collection base of the bioaerosol sample collection device are eluted to form a combined sample. The combined sample containing the analytes from the first sample and the analytes from the bioaerosol collection device sample is then analyzed. Suitable analytical methods, such as amplification (polymerase chain reaction), immunoassays, mass spectrometry, and combinations thereof, are described herein. In another embodiment, the collection base of the bioaerosol collection device is placed in a vial containing an elution / extraction buffer, and then the first sample is placed in the same vial as the bioaerosol collection base, and both the bioaerosol collection base and the first sample are eluted / extracted together in the same vial. After extraction / elution, a portion of the combined sample is analyzed.

[0016] In one embodiment, a bioaerosol sample is collected using a mask or mask insert, and the subject wears the mask for a period of time sufficient to allow analytes contained in the air expelled by the subject (through normal breathing, forced breathing, coughing, speaking, sneezing, and combinations thereof) to contact and be captured by the mask insert. After collection of a first sample, the first sample is placed in an elution buffer to elute / extract the analytes contained in the first sample. The eluate from the first sample is then used to elute the analytes captured by the capture base of the mask insert, and the analytes captured by the capture base of the mask insert are eluted to form a combined sample with the first sample. The combined sample containing the analytes from the first sample and the analytes from the mask insert sample is then analyzed. Suitable analytical methods, such as amplification (polymerase chain reaction), immunoassays, mass spectrometry, and combinations thereof, are described herein.

[0017] In one embodiment, a bioaerosol sample is collected using a bioaerosol collection device, in which a subject delivers an air sample into the bioaerosol collection device. It should be understood that the bioaerosol collection sample can be delivered by the subject blowing into the bioaerosol collection device, coughing into the bioaerosol collection device, humming into the bioaerosol collection device, speaking into the bioaerosol collection device, singing into the bioaerosol collection device, and other methods that allow the subject to deliver a breath sample toward the inlet of the bioaerosol collection device. In particular, the subject delivers air into the inlet of the bioaerosol collection device. The bioaerosol collection device is configured to deliver the exhaled air sample to a collection medium (or base, also referred to herein as a "capture base") contained in the bioaerosol collection device, which captures, attracts, and collects analytes contained in the breath exhaled by the subject. The bioaerosol collection device is also configured to allow the air sample to pass through an outlet to avoid pressure differences that could move and / or affect the positioning of the collection base. In one embodiment, a bioaerosol sample is collected using a bioaerosol collection device comprising a hollow housing including an inlet; a capture base; and an outlet. The collection base is positioned in the bioaerosol collection device such that the bioaerosol sample of interest flows through, over, and / or toward the collection base, and analytes contained in the bioaerosol sample are captured by the collection base. The collection base can also be removed from the bioaerosol collection device. Removing the collection base allows other components of the bioaerosol collection device to be reused. When the bioaerosol collection device is reused, the device is disinfected and sterilized, and a new collection base is placed into the bioaerosol collection device. Removing the collection base also allows the collection base to be placed into a vial for storage, transportation, processing, testing, and combinations thereof.To avoid or minimize contamination of the collection base when transferring the collection base to the vial, tools such as a swab, tweezers, and / or probe can be used to move the collection base from the bioaerosol collection device and into the vial. The swab used to collect the swab sample can be used to transfer the collection base from the bioaerosol collection device into the vial, and the swab can then be placed in the same vial with the collection base. Alternatively, the swab used to collect the swab sample can be used to transfer the collection base from the bioaerosol collection device into the vial, and the swab is then placed in a different (second) vial. Additionally or alternatively, the bioaerosol collection device can be configured to detach the collection base from its position within the bioaerosol collection device without or with minimal use of tools. For example, the bioaerosol collection device can include a "locking mechanism" to ensure that the collection base is "locked" to the bioaerosol collection device when the device is delivered to the clinician. After the subject provides a bioaerosol sample to the bioaerosol collection device, the clinician can perform an action on the device (such as a twisting action, a pulling action, and the like) to detach the collection base from the bioaerosol collection device and remove the collection base from the bioaerosol collection device. As described herein, the vial can contain a solution (transport medium, storage medium, elution buffer, extraction buffer, and combinations thereof) or can be empty. The vial can also contain a desiccant.

[0018] In some embodiments, the bioaerosol sample is collected using a breath droplet collection device. Suitable methods for collecting breath droplet samples include, for example, a droplet collection device such as a mask and mask insert.

[0019] The collection base (e.g., mask insert) of the bioaerosol collection device and the exhaled droplet collection device is suitably made of synthetic fibers, natural fibers, and combinations thereof. The fibers used to form the collection base include hydrophobic fibers, hydrophilic fibers, and combinations thereof. Hydrophobic fibers include, for example, polylactone, poly(caprolactone), poly(L-lactic acid), poly(glycolic acid), similar copolymers, poly(alkyl acrylate), polybutadiene, polyethylene, polystyrene, polyacrylonitrile, polyethylene(terephthalate), polysulfone, polycarbonate, poly(vinyl chloride), and combinations thereof. Hydrophilic fibers include, for example, linear poly(ethyleneimine), cellulose, cellulose acetate and other grafted cellulose derivatives, poly(hydroxyethyl methacrylate), poly(ethylene oxide), polyvinylpyrrolidone, poly(acrylic acid), poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl acetate), poly(acrylamide), proteins, poly(vinylpyrrolidone), poly(styrenesulfonic acid), and combinations thereof.Other suitable fiber materials include, for example, acrylonitrile / butadiene copolymer, cellulose, cellulose acetate, chitosan, collagen, DNA, fibrinogen, fibronectin, nylon, poly(acrylic acid), poly(chlorostyrene), poly(dimethylsiloxane), poly(etherimide), poly(ethersulfone), poly(ethyl acrylate), poly(ethyl vinyl acetate), poly(ethyl-co-vinyl acetate), poly(ethylene oxide), poly(ethylene terephthalate), poly(lactic-co-glycolic acid), poly(methacrylic acid), poly(methyl methacrylate), poly(methylstyrene), poly(styrenesulfonate), poly(styrenesulfonyl fluoride), poly(styrene-co-acrylonitrile), poly(acrylic acid), poly(chlorostyrene), poly(ethylene terephthalate), poly(ethylene terephthalate), poly(lactic-co-glycolic acid), poly(methacrylic acid), poly(methyl methacrylate), poly(methylstyrene), poly(styrenesulfonate), poly(styrenesulfonyl fluoride), poly(styrene-co-acrylonitrile), poly(acrylic acid), poly(acrylic acid), poly(chlorostyrene), poly(ethylene terephthalate ... nitrile), poly(styrene-co-butadiene), poly(styrene-co-divinylbenzene), poly(vinyl acetate), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene fluoride), polyacrylamide, polyacrylonitrile, polyamic acid (PAA), polyamide, polyaniline, polybenzimidazole, polycaprolactone, polycarbonate, polydimethylsiloxane-co-polyethylene oxide, polyether ether ketone, polyethylene, polyethyleneimine, polyimide, polyisoprene, polylactic acid, polypropylene, polystyrene, polysulfone, polyurethane, polyvinylpyrrolidone, proteins, SEBS copolymer, silk, and styrene / isoprene copolymer. For example, polymer blends such as poly(vinylidene fluoride)-blend-poly(methyl methacrylate), polystyrene-blend-poly(vinyl methyl ether), poly(methyl methacrylate)-blend-poly(ethylene oxide), poly(hydroxypropyl methacrylate)-blend-poly(vinylpyrrolidone), poly(hydroxybutyric acid)-blend-poly(ethylene oxide), protein blend-polyethylene oxide, polylactic acid-blend-polyvinylpyrrolidone, polystyrene-blend-polyester, polyester-blend-poly(hydroxyethyl methacrylate), poly(ethylene oxide)-blend-poly(methyl methacrylate), and poly(hydroxystyrene)-blend-poly(ethylene oxide).The fiber material used to form the capture base can be selected to allow the analyte (including the carrier containing the analyte) to be transported from one portion of the capture base to another portion of the capture base. The fiber material used to form the capture base can be selected to allow the analyte (including the carrier containing the analyte) to be transported from one portion of the capture base out of the capture base and into the analysis base and / or collection vial.

[0020] Another suitable capture base can be an electret (including thermal electrets and fibrillated electret films). Particularly suitable electrets include polypropylene and polylactic acid. Electrets are dielectric materials that have a semi-permanent electric charge or oriented polarization. Electrets can be obtained from commercially available raw materials. Electrets can be fabricated by heating a material and simultaneously exposing it to an electric field, thereby orienting many dipoles in the material in a preferred direction. After heating, the material can be "frozen" and retain the location of its electric dipoles for an extended period of time. Suitable materials for fabricating electrets include materials that can be readily used to make thermal electrets, including organic materials such as hard rubber, naphthalene, polymethylmethacrylate, and many polymers, as well as inorganic materials such as sulfur, quartz, glass, steatite, and some ceramics. Electret fibrous membranes are particularly suitable. Polyvinylidene fluoride (PVDF) / polytetrafluoroethylene (PTFE) NP electret nanofiber membranes can be formed by electrospinning. Also suitable are the fibrillated electret films disclosed in van Turnhout (U.S. Pat. No. 3,998,916). One collection method is disclosed in PCT / US2007 / 061082 to Kanzer. Kanzer discloses a filtering face mask with a biological sample collection material attached to the mask that traps pathogens exhaled by the wearer. Another suitable mask is disclosed in U.S. Pat. No. 6,119,691 to Angadjivand. Angadjivand discloses a mask with an electret filter material.

[0021] The collection base can be an immunochromatographic test base, a colloidal gold test base, and combinations thereof. The base can include, for example, a quantum dot-labeled test base, a colloidal gold-labeled test base, a colloidal selenium-labeled test base, an upconversion phosphorescent-labeled test base, a nano-rare earth fluorescent complex-labeled test base, a time-resolved chromatography test base, a chemiluminescent test base, and other test bases.

[0022] The collection base can also be a multi-layer base. A multi-layer collection base has an inner collection base protected on all sides by additional layers. The inner collection layer is made of a material with a high surface area for collecting the analyte to be detected. The inner collection base can also be cut and / or separated from the protective layer. The protective layer and inner collection base can be made of various materials designed for the specific purpose of the layer (e.g., protection and sample collection). For example, the inner collection base can be a corrugated double-sided polyester swab material. The inner collection base can be coated with a reagent to retain the collected sample volume. The inner collection base and / or protective layer(s) can also be coated with a reagent to stabilize the sample. The protective layer can be an air-permeable, touch-protective coating.

[0023] The disclosed method combines a sample obtained using a swab with a bioaerosol collection device. In another embodiment, the disclosed method can combine a swab sample and a bioaerosol sample with a third sample collection method. Suitable third samples include, for example, saliva, sputum, nasal wash, mouthwash, mouth swab, oropharyngeal swab, bronchoalveolar lavage fluid, blood, urine, stool, and combinations thereof. The method includes obtaining a swab sample, obtaining a bioaerosol sample, obtaining a third sample, eluting the swab sample to form a swab eluate, using at least a portion of the swab eluate to elute the bioaerosol sample to form a combined eluate, analyzing the combined eluate, and analyzing the third sample.

[0024] In another embodiment, the combined swab sample eluate and bioaerosol sample eluate (also referred to herein as the "combined sample") are placed in a sterile transport container. The combined swab sample eluate and bioaerosol sample eluate can then be stored and / or transported for analysis at a facility for laboratory testing. After collection, the sample can be placed in a sterile transport container containing a transport medium. Suitable transport media include, for example, viral transport medium (VTM), universal transport material (UTM), Amies transport medium, and sterile saline. Delivery vehicles can include, for example, RNase inhibitors, DNase inhibitors, protease inhibitors, preservatives, stabilizers, antimicrobial additives such as silver-containing antimicrobial agents and antimicrobial polypeptides, anesthetic compounds such as lidocaine, antibiotics such as neomycin, thrombogenic compounds, nitric oxide-releasing compounds such as sydnonimines and NO complexes, bactericidal compounds, fungicidal compounds, bacteriostatic compounds, other pharmaceutical compounds, adhesives, fragrances, odor-absorbing compounds, preservatives, and nucleic acids, including deoxyribonucleic acid, ribonucleic acid, and nucleotide analogs.

[0025] In another embodiment, the swab is placed in a first vial and the bioaerosol collection base is placed in a second vial. In another embodiment, the swab and bioaerosol collection base are placed in the same vial. The vial in which the swab and / or bioaerosol collection base is placed can further contain a transport medium. Suitable transport media include saline, phosphate buffered saline, HEPES, Tris-buffered saline, water, commercially available transport media (such as UTM viral transport medium commercially available from COPAN Diagnostics, Murrietta, CA), and combinations thereof.

[0026] Particularly suitable elution buffers contain non-ionic detergents. Suitable detergents include TRITON X100, TRITON X100 reduced, TRITON X114, TRITON X45, Nereid, polyoxyethylene lauryl ether, sodium dodecyl sulfate (SDS), sodium lauryl sulfate (SLS), NP-40, polysorbate, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid), DOC, cetyltrimethylammonium bromide (CTAB), Brij 52, and combinations thereof. The elution buffer can be a solution of about 0.25% saline to about 0.9% saline containing a detergent. The elution buffer can further contain a pH buffer, salt, chelating agent, and combinations thereof. As used herein, "eluting" and "extracting" are used to describe removing at least a portion of an analyte from a sample. For example, if the sample is collected using a swab, the elution buffer removes at least a portion of the sample from the swab. The elution buffer can also disrupt the sample in a manner that allows components of the sample to be detected. For example, the elution buffer can disrupt the membrane of a microorganism so that the microorganism's DNA or RNA can be transferred into the buffer, and the microorganism's DNA and / or RNA can then be detected to determine the presence of the microorganism in the sample obtained from the subject.

[0027] Any method known in the art for analyzing the samples of the present disclosure is suitable for analyzing the combined sample. Suitable methods include, for example, DNA and RNA amplification methods (e.g., PCR, RT-PCR, qPCR, loop-mediated isothermal amplification (LAMP)), lateral flow assays (rapid lateral flow), vertical flow assays, optical immunoassays (e.g., Biostar, Inc., Boulder, CO), Western blot analysis, direct fluorescent antibody assays, immunoassays such as antigen-enzyme immunoassays and enzyme-linked immunosorbent assays (ELISAs), chromatography such as high-performance liquid chromatography (HPLC) and gas chromatography, capillary pheresis, 2D and 3D gel electrophoresis, mass spectrometry, and combinations thereof.

[0028] The combined sample can be analyzed by devices such as lateral flow assays and vertical flow assays, which use strips containing one or more capture zones: a control line that detects the presence of any antibody in the sample, and a test line that reacts specifically with the analyte to be detected.

[0029] In another embodiment, the combined sample can be analyzed using an antigen test. As known in the art, an antigen test contains an antibody on a test device that will bind to an antigen contained in the sample. In another embodiment, the combined sample can be analyzed with an antibody test. As known in the art, an antibody test contains an antigen on a test device that will bind to an antibody contained in the sample. In both antigen and antibody tests, the binding of the antibody and antigen is the clue that provides a visible result indicating that the subject is infected.

[0030] The analysis can be automated using commercially available platforms such as the Cobas Amplicor (Roche Molecular Diagnostics, Pleasanton, CA).

[0031] Once placed in the transport container, the collected and combined sample can be stored until later analysis or can be processed for analysis, which can include lysis, extraction, and other known processing steps for detecting pathogens from the collected sample.

[0032] The analysis can use point-of-care and / or off-site high-throughput platforms.

[0033] Analytes include microorganisms, biological molecules, and chemical molecules. Particularly suitable microorganisms to be detected are pathogens. As used herein, "pathogen" refers to microorganisms such as, for example, bacteria, fungi, and viruses. The term "pathogen" also refers to viroids, prions, and proteins.

[0034] Suitable analytes are contained in gases exhaled by a user and in aerosol droplets in the air. Suitable analytes include microorganisms, chemicals, proteins, nucleic acids, and combinations thereof. Suitable microorganisms include bacteria and viruses.

[0035] Particularly suitable microorganisms include pathogens. The term "pathogen" is used in its ordinary sense to refer to bacteria, viruses, and other microorganisms that directly or indirectly cause disease. Exemplary pathogens include, for example, Yersinia, Klebsiella, Providencia, Erwinia, Enterobacter, Salmonella, Serratia, Aerobacter, Escherichia, Pseudomonas, Shigella, Vibrio, Aeromonas, Streptococcus, Staphylococcus, Micrococcus, Moraxella, Bacillus, Clostridium, Corynebacterium, Eberthella, Francisella, Haemophilus, Bacillus Bacteroides, Listeria, Erysipelothrix, Acinetobacter, Brucella, Pasteurella, Flavobacterium, Fusobacterium, Streptobacillus, Calymmatobacterium, Leuconostoc, Legionella, Treponema, Borrelia, Leptospira, Actinomyces, Nocardia, Rickettsia, Micrococcus, Mycobacterium, Neisseria, Campylobacter, e.g. papillomavirus, parvovirus,Pathogenic viruses such as adenovirus, herpesvirus, vaccine virus, arenavirus, coronavirus, rhinovirus, respiratory syncytial virus, influenza virus, picornavirus, paramyxovirus, reovirus, retrovirus, rhabdovirus, human immunodeficiency virus (HIV), tapeworms (Taenia), Hymenolepsis, Diphyllobothrium, Echinococcus, Fasciolopsis, Heteropsis hyes, Metagonimus, Clonorchis, Fasciola, Paragonimus, Schistosoma, Enterobius, Trichuris, Ascaris, Ancylostoma, Necator, Wuchereria, Brugia, Loa, Onchocerca, Dracunculus, Naegleria, Acanthamoeba, Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba, Giardia, Isospora, Cryptosporidium, Enterocytozoon, Strongyloides ), Trichinella, such as tinea, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, mucormycosis, candidiasis, dermatomycosis, protothecia, pityriasis, mycetoma, paracoccidioidomycosis, phaeohyphomycosis, pseudoalleliciosis, sandworm, fungi causing pneumocystis, adenovirus, coronavirus (coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OCL43),Human metapneumovirus, human rhinovirus, enterovirus, influenza A, influenza B, Middle East respiratory syndrome coronavirus (MERS-CoV), parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumonia, Mycoplasma pneumoniae, and combinations thereof.

[0036] Particularly suitable chemicals include ketones, nicotine, cocaine, opioids, marijuana, benzodiazepines, amphetamines, barbiturates, and combinations thereof.

[0037] Samples can also be analyzed for protein, DNA and RNA.

[0038] A sample can be analyzed simultaneously for multiple different analytes (e.g., a multiplex assay). For example, the disclosed methods are particularly suitable for detecting and distinguishing RNA from SARS-CoV-2, influenza A virus, and influenza B virus in upper or lower respiratory tract samples in a multiplex assay.

[0039] [Example] [Example 1] This example outlines the testing of various sample collection methods and combinations of sample collection methods.

[0040] 1.Device: Mask insert

[0041] Use Case: Essential Workers

[0042] Detection: Sputum at multiple sites vs. PCR (mouth + NP) swabs vs. bioaerosols by PCR

[0043] Pathogens tested: COVID+TB

[0044] Inclusion criteria: suspected positive

[0045] Scientific Need: Test longer collection periods and non-localized sample collection to screen for COVID+TB to protect essential workers.

[0046] Actions for bioaerosol collection: Wear a standard surgical mask with a collection base. Speak, cough, and breathe normally.

[0047] Collection time: at least 4 hours

[0048] Sample handling: The POC worker removes the bioaerosol sample collector from the mask and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0049] 2.Device: Mask insert

[0050] Use Case: Essential Workers

[0051] Detection: (mouth + NP) swabs by PCR at multiple sites vs. bioaerosols by PCR

[0052] Pathogens tested: Respiratory group

[0053] Inclusion criteria: Cough or shortness of breath or sore throat of any duration

[0054] Scientific Need: Examine longer collection periods and non-localized sampling to screen for respiratory pathogens to protect essential workers.

[0055] Actions for bioaerosol collection: Wear a standard surgical mask with a collection base. Speak, cough, and breathe normally.

[0056] Collection time: at least 4 hours

[0057] Sample handling: The POC worker removes the bioaerosol sample collector from the mask and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0058] 3.Device: Blow tube

[0059] Use Case: General Population

[0060] Detection: (mouth + NP) swabs by PCR at multiple sites vs. bioaerosols by PCR vs. sputum

[0061] Pathogens tested: COVID+TB (single elution protocol needs to be determined)

[0062] Inclusion criteria: suspected positive

[0063] Scientific Need: Compare rapid bioaerosol collection vs. NP swab collection methods, ideally from day -2 to +7 after symptom onset for COVID; sputum for TB.

[0064] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0065] Collection time: 10 to 15 minutes

[0066] Sample handling: The POC worker removes the bioaerosol sample collector and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0067] 4.Device: Blow tube

[0068] Use Case: General Population

[0069] Detection: (mouth + NP) swabs by PCR at multiple sites vs. bioaerosols by PCR

[0070] Pathogens tested: Respiratory group

[0071] Inclusion criteria: Cough or shortness of breath or sore throat of any duration

[0072] Scientific Need: Compare rapid bioaerosol collection versus NP swab collection methods, ideally from day -2 to +7 after symptom onset in a group of respiratory illnesses.

[0073] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0074] Collection time: 10 to 15 minutes

[0075] Sample handling: The POC worker removes the bioaerosol sample collector and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0076] 5.Device: Blow tube

[0077] Use Case: General Population

[0078] Detection: (mouth + NS) swab + bioaerosol by PCR at multiple sites vs. NP swab by PCR

[0079] Pathogens tested: COVID+TB

[0080] Inclusion criteria: suspected positive

[0081] Scientific Need: Compare the NP swab collection method in combination with rapid bioaerosol collection on days -2 to +7 of COVID symptom onset.

[0082] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0083] Collection time: 10 to 15 minutes

[0084] Sample handling: The POC worker removes the bioaerosol sample collector and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0085] 6.Device: Blow tube

[0086] Use Case: General Population

[0087] Detection: (mouth + NS) swab + bioaerosol by PCR at multiple sites vs. NP swab by PCR

[0088] Pathogens tested: Respiratory group

[0089] Inclusion criteria: Cough or shortness of breath or sore throat of any duration

[0090] Scientific Need: Compare the NP swab collection method in combination with rapid bioaerosol samples, ideally from day -2 to +7 after symptom onset for respiratory groups.

[0091] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0092] Collection time: 10 to 15 minutes

[0093] Sample handling: The POC worker removes the bioaerosol sample collector and places it in a 5 mL Eppendorf. 1 mL UTM. Mechanical shaking for 10 seconds, 30 rotations, and obtains the sample in buffer.

[0094] 7.Device: Blow tube

[0095] Use Case: General Population

[0096] Detection: (oral + NP) swabs with PCR at multiple sites vs. bioaerosols (visual and reader) with rapid detection tests (RDTs)

[0097] Pathogen tested: COVID

[0098] Inclusion criteria: suspected positive

[0099] Scientific Need: Compare qualitative testing with rapid bioaerosol sample-only RDTs versus quantitative NP swab collection tested with reference PCR, ideally on days -2 to +7 of symptom onset in the respiratory group.

[0100] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0101] Collection time: 10 to 15 minutes

[0102] Sample handling: Patient performs the action. The POC dispenses the required elution / extraction buffer into the bioaerosol base.

[0103] 8.Device: Blow tube

[0104] Use Case: General Population

[0105] Detection: (mouth + NP) swabs by PCR at multiple sites vs. NS swabs + bioaerosols by RDT (visual and reader)

[0106] Pathogen tested: COVID

[0107] Inclusion criteria: suspected positive

[0108] Scientific Need: Compare qualitative testing by RDT of rapid bioaerosol samples combined with NS samples versus quantitative NP swab collection tested by reference PCR, ideally from day -2 to +7 of symptom onset in the respiratory group.

[0109] Actions for bioaerosol collection: 20 deep breaths, 10 coughs, count to 20.

[0110] Collection time: 10 to 15 minutes

[0111] Sample handling: The patient performs the bioaerosol collection action in the device. The POC worker takes a swab from the patient with an NS swab and elutes it in the RDT elution / extraction buffer as usual. The POC dispenses the required elution / extraction buffer into the bioaerosol base for testing by the RDT.

[0112] While the embodiments described herein involve combining a sample obtained using a bioaerosol collection device with a sample collected using a swab collection method, it should be understood that a sample obtained using a bioaerosol collection device can be analyzed by itself (without combining with a second sample). Advantageously, the disclosed methods make available a viral load at a density that can be used in a variety of tests, such as amplification, antibody, antigen, and other paper-based tests. The disclosed methods are particularly advantageous when a sample collected using a bioaerosol collection device is combined with a swab sample, as the combination of the samples inherently increases the availability of viral load.

[0113] The disclosed method advantageously allows for the integration of test sensitivity and reduces false-negative test results. In particular, combining exhaled droplet samples with swab samples can maximize test sensitivity and reduce the amount of laboratory resources used to analyze samples. Furthermore, combining bioaerosol samples and released viruses collected using a bioaerosol collection device with other samples collected using different collection methods advantageously increases the amount of collected virus that can be tested, thereby increasing test sensitivity. Combining bioaerosol samples and released viruses collected using a bioaerosol collection device with samples collected using a swab collection method can also advantageously overcome any signal reduction inherent in collecting samples from the upper respiratory tract, compared to, for example, saliva samples. Combining a bioaerosol collection device with samples collected using swab collection can also advantageously facilitate downstream test handling. Combining a bioaerosol collection device with samples collected using swab collection can advantageously increase the amount of pathogens collected using at least two different sample collection methods, thereby reducing false negatives.

Claims

1. 1. A method for detecting an analyte in a combined sample obtained from a subject, comprising: collecting a first sample; collecting bioaerosol samples; taking up the first sample in a buffer, the buffer eluting at least a portion of the first sample into the buffer to form a first elution buffer; contacting at least a portion of the first elution buffer with the bioaerosol sample, wherein the first elution buffer elutes at least a portion of the bioaerosol sample to form a combined sample; and analyzing the combined sample for the analyte. A method comprising:

2. The method of claim 1 , wherein the bioaerosol sample is collected using a droplet collection device.

3. 3. The method of claim 2, wherein the droplet collection device is selected from a mask, a mask insert, an exhaled droplet collection strip, an electret filter material, and combinations thereof.

4. 2. The method of claim 1, wherein the bioaerosol collection is performed using a bioaerosol collection device, and the subject blows into the bioaerosol collection device, coughs into the bioaerosol collection device, hums into the bioaerosol collection device, speaks into the bioaerosol collection device, or combinations thereof.

5. 3. The method of claim 2, wherein the bioaerosol sample is collected using a material capable of trapping pathogens exhaled by the subject.

6. 10. The method of claim 1, wherein the first sample is selected from a swab sample, a saliva sample, a sputum sample, an aspirate sample, a lavage sample, and combinations thereof.

7. The method of claim 1 , wherein the assay is selected from amplification, immunoassay, and combinations thereof.

8. The method of claim 7 , wherein the analysis comprises a polymerase chain reaction.

9. The method of claim 1 , wherein the analysis is selected from lateral flow analysis, vertical flow analysis, and combinations thereof.

10. 10. The method of claim 1, wherein the combined sample is analyzed by subjecting at least a portion of the combined sample to a lateral flow assay, a vertical flow assay, or a combination thereof.

11. The method of claim 1 , wherein the analyte is a pathogen.

12. 10. The method of claim 1, wherein the bioaerosol collection device includes a collection base, and the collection base is moved from the bioaerosol collection device into a vial.

13. The method of claim 12 , wherein the vial contains a buffer.

14. 13. The method of claim 12, wherein the vial contains the first elution buffer.

15. A kit comprising: at least one of a swab, a saliva collection vial, a sputum collection vial, and a lavage fluid collection component; Bioaerosol collection devices; buffer; and Instructions for using said kit Including, The instructions include the following instructions: collecting a first sample using the at least one of the swab, saliva collection vial, sputum collection vial, and lavage fluid collection component; collecting a bioaerosol sample using a bioaerosol collection device; taking up a first sample in said buffer and eluting at least a portion of said first sample into said buffer to form a first elution buffer; contacting at least a portion of the first elution buffer with the bioaerosol sample to elute at least a portion of the bioaerosol sample to form a combined sample; and analyzing the combined sample; Kit includes instructions.

16. 16. The kit of claim 15, further comprising an analytical device.

17. 17. The kit of claim 16, wherein the analytical device is selected from the group consisting of a lateral flow assay, a vertical flow assay, and a combination thereof.

18. 16. The kit of claim 15, wherein the bioaerosol collection device is a mask insert.

19. The kit of claim 15, wherein the bioaerosol collection device is a blow-tube device.

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