Patent Publication Number: US-11650213-B2

Title: Devices and assays for diagnosis of viral and bacterial infections

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This patent application is a continuation-in-part of U.S. patent application Ser. No. 15/470,849, titled “DEVICES AND ASSAYS FOR DIAGNOSIS OF SINUSITIS,” and filed on Mar. 27, 2017, which is a continuation of U.S. patent application Ser. No. 15/084,934, titled “DEVICES AND ASSAYS FOR DIAGNOSIS OF SINUSITIS,” filed on Mar. 30, 2016, now U.S. Pat. No. 9,606,118, which claims priority to U.S. Provisional Patent Application No. 62/140,405, titled “DEVICES AND METHODS FOR OBTAINING MUCOUS SAMPLES,” and filed on Mar. 30, 2015 and U.S. Provisional Patent Application No. 62/209,712, titled “DEVICES AND ASSAYS FOR DIAGNOSIS OF SINUSITIS,” filed on Aug. 25, 2015. Each of these patent applications is herein incorporated by reference in its entirety. 
    
    
     INCORPORATION BY REFERENCE 
     All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. 
     FIELD 
     The present application relates to methods and devices for the determination of the presence of one or more pathogens associated with a viral and/or bacterial infection from a collected mucus sample. 
     BACKGROUND 
     Respiratory illnesses can range from mild and self-limiting conditions, such as the common cold, to life-threatening diseases such as bacterial pneumonia. Sinusitis, defined as inflammation of the sinus tissues, usually as a complication to viral infections from the common cold. Although there are over 1 billion common colds in the U.S., a small percentage of them lead to sinusitis. In fact, 29 million people were diagnosed with sinusitis in 2011 in the US. Often antibiotics are ordered as a treatment for sinusitis and it is the 5th leading indication for the antibiotic prescriptions annually. Western EU markets are estimated to be over 43 million patients annually. The majority of these patients are initially seen by primary care physicians and then referred out to otolaryngologists, also known as ENT&#39;s if their symptoms do not resolve. Complicated cases of sinusitis eventually lead to surgery and there are 1.5 million patients in the U.S. each year that are candidates for surgical procedures, in which currently 500k patients elect to undergo some type of surgical procedure. The direct costs association with managing sinusitis amount to over $6 billion annually, with another $3 billion associated with indirect costs associated with sinusitis management. 
     The initial diagnosis of sinusitis remains a challenge for physicians. A patient presenting at a physician&#39;s office with a symptom complex of fever, headache and fatigue, also present in many different types of systemic diseases, could warrant a diagnosis of sinusitis. As a result, many patients with non-sinus related diseases such as migraine disorders, chronic fatigue, and chronic systemic disorders are misdiagnosed as sinusitis. An additional objective laboratory diagnostic testing would guide physicians as to the etiology of these common symptoms of viral upper respiratory tract infections, acute bacterial sinusitis and chronic sinusitis and lead to reduction of unnecessary antibiotic and steroid prescriptions provided to patients. 
     Currently doctors typically decide on a treatment regime without a definitive test to determine if the patient has viral sinusitis, bacterial sinusitis, upper respiratory infection, chronic fatigue, or migraines, because it is difficult to diagnose the cause of sinusitis as either viral or bacterial etiology. Treatment often involves antibiotics, which are only effective for a small amount of these conditions. The majority of sinusitis cases are viral, with some estimates that about 90% of sinusitis cases are viral. Majority of all patients receive an antibiotic that they do not need, can make their condition worse, and can lead to antibiotic resistance. If a flu virus is suspected, a rapid influenza diagnostic test (RIDT) may be performed to determine the presence of influenza virus antigens. These tests, however, are not able to determine whether a bacterial infection is present. Improved methods of diagnosing upper respiratory symptoms are needed. In particular, what is needed is a definitive, rapid test for the cause of sinusitis and other upper respiratory infections, which could save the physician time and provide timely information that will lead to fewer antibiotics being prescribed. 
     There are many advantages to determining the etiology of upper respiratory infections (e.g., as viral, bacterial, etc.), including the reduction in health care costs, decreases in antibiotic use and concomitant bacterial drug resistance, and improvements in the level of care for patients. Described herein are upper respiratory (e.g., bacterial or viral sinusitis) diagnostic apparatuses (e.g., devices, systems, kits, etc.) and methods that may address many of the needs described herein. For example, the sampling, testing, and treatment apparatuses and methods described herein may allow for rapid and definitive diagnosis of upper respiratory illnesses (e.g., bacterial sinusitis or influenza), permitting targeted treatment with optimal medications based on the specific diagnosis. Such targeted treatment may avoid unnecessary antibiotic treatments for patients not suffering from bacterial infection. A rapid diagnosis may also result in improved treatment for patients that test negative for bacterial sinusitis by instead treating the patient based on a negative test for bacterial sinusitis. 
     SUMMARY OF THE DISCLOSURE 
     Described herein are apparatuses (e.g., systems, kits, assays, including lateral flow assay kits) and methods which may allow determination of the presence of bacteria and/or viruses in a patient&#39;s mucosal sample. The methods and apparatuses can allow for detection of one or more of the three pathogens associated with over 90% of bacterial sinusitis from a collected mucus sample. Specifically, these methods and apparatuses may determine, as part of a single rapid assay, the presence of one or more of:  Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Neisseria meningitidis, Klebsiella pneumoniae  and/or methicillin resistant  Staphylococcus aureus . Additionally or alternatively, the methods and apparatuses can allow for detection of one or more viruses, such as an influenza virus (e.g., an Influenza A, B, or C) and/or a coronavirus (e.g., an alpha or beta coronavirus, MERS-CoV, SARS-CoV, SARS-CoV-2), Respiratory Syncytial Virus (RSV), Parainfluenza Virus, Human Metapneumovirus, Rhinovirus and Bocavirus. In particular, described herein are sinus collection devices for collection of mucus samples from patient sinuses; these collection devices may be included as part of the assays described herein. 
     The sinus collection devices (sampling devices) described herein are intended for use during a routine office visit to a physician. These devices may accurately and quickly (with a minimum of discomfort) allow the acquisition of a mucus sample from the middle meatus region of the sinus (while avoiding miss-targeting of the region and cross-contamination). A collected mucus sample may then be analyzed using any of the lateral flow assays described herein. If the test is positive for any of the three bacterial pathogens, the patient has bacterial sinusitis and may be prescribed an appropriate antibiotic and/or steroid regimen to address the pathogenic bacteria. If the test is negative for bacterial infection but positive for viral infection (e.g., influenza), the patient may be treated for the viral infection (e.g., flu and/or viral sinusitis) and antibiotics may not be administered. If the test is positive for any of the three bacterial pathogens and for one or more viral pathogens (e.g., influenza A and/or B), the patient may be treated for the viral infection and with antibiotics for the bacterial infection. Examples of the sampling devices and assays (e.g., lateral flow assays) are described herein. Although the majority of these examples describe apparatuses, including collection devices, that are adapted for use in the nasal cavity, any of these apparatuses and methods may be adapted for use in other regions. For example, a variation of the sampling device and/or the assay may be adapted for use in collecting mucus samples from within the sinus during sinus surgery procedures, from an ear (e.g., in the case of otitis media, which is usually caused by the same three pathogens as is bacterial sinusitis) or elsewhere. 
     As will be described in greater detail below, these assays may be configured as lateral flow assays that include a single lysis solution (e.g., lysis buffer solution) that is appropriate for use with all three types of bacteria (e.g.,  H. influenzae, M. catarrhalis  and  S. pneumoniae ) in order to expose (e.g., solubilize and/or denature) the antigens specific to each one for detection. Any of the assays described herein may be adapted for use with the lysis buffer, and may include multiple (e.g., three) pairs, or defined pools, of antigen binding agents that bind antigens (e.g., surface proteins) specific to each type of bacteria (e.g.,  H. flu, M. cat, S. pneumo ). The lysis buffer can act by lysing the bacterial cells, thereby exposing one or more antigens specific to the type of bacterial cells. Thus, the lysis buffer can be said to extract the one or more antigens. The antigen binding agents (“agents”) may be monoclonal or polyclonal antibodies, or antibody fragments (e.g., FAB fragments, etc.) or molecules including all or a portion of these. Pairs of such agents may bind to different portions of the same antigen. An agent specific to each type of bacteria (e.g.,  H. flu, M. cat, S. pneumo ) may be bound to a solid phase substrate (e.g., membrane, particle, etc.) and spatially arranged in the assay and provide specific identification of  H. influenzae, M. catarrhalis  and  S. pneumoniae  by visual detection of binding, including by binding the antigen to the tethered substrate and to a labeled agent. The pairs or pools of antibodies may be chosen to have low cross-reactivity, while allowing comparable detection of  H. influenzae, M. catarrhalis  and  S. pneumoniae.    
     The antigen binding agent (or “agent” and may also be referred to herein as an indicator) may be chosen so that they are selective for the organism of interest, binds cognate antigen specifically, have minimal cross-reactivity to common contaminating organisms and minimal cross-reactivity with commensal organisms. These antigen binding agents may also have a high affinity to the target pathogen antigen, rapid association kinetics, slow dissociation kinetics, and be sensitive to low numbers of the pathogen. Finally these antigen binding agents may be compatible with lateral flow, and compatible with a conjugate. As mentioned above, in particular the antigen binding agents may also be compatible for use with a common lysing solution for all three pathogens. 
     As will be described in greater detail herein, finding a common lysing solution that may work with multiple types of pathogens, and particularly  H. flu, M. cat  and  S. pneumo , was surprisingly difficult, as many commonly used lytic agents (detergents, enzymes, etc.) did not work with all three, resulting in incomplete lysis (clogging of the lateral flow system), lysis that was too slow (e.g., took longer than 15 minutes), or disrupted the surface proteins, including the antigens specific to each cell type. 
       Haemophilus influenzae  ( H. influenzae ) may be detected using a pair or pool of antibodies that are specific to one or more antigen binding agents that are relatively specific or characteristic of  H. influenzae . For example, the indicator for  H. influenzae  may bind with specificity to the OMP-P2 and/or OMP-P5 antigen binding site for the pathogen. As described herein, numerous primary candidate antibodies have been evaluated, and screened for cross reactivity between numerous (e.g., 30) commensal bacterial strains to assure minimal cross reactivity with the normal flora occurring in the healthy sinus. Other examples of antigen binding agents include antibodies that may be used are discussed in US20140314876, herein incorporated by reference in its entirety. 
     Similarly,  Moraxella catarrhalis  ( M. catarrhalis ) may be detected using a pair or pool of antigen binding agents that are specific to a marker for  M. catarrhalis  (see, e.g., U.S. Pat. No. 7,811,589) such as Protein C and Protein D outer member proteins. 
     One or more antigen binding agents specific for  Streptococcus pneumoniae  ( S. pneumonia ) may also be directed to  S. pneumoniae  markers such as the PsaA antigen. 
     Specifically described herein are assay kits for concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal samples. An assay kit may include: a lysis buffer to lyse cells within the sample and form a single sample solution, wherein the lysis buffer comprises between 0.01% and 5% (w/w) of the anionic surfactant and between 0.1% and 15% (w/w) of the osmotic agent; a cartridge containing one or more solid phase substrates holding a first bacterial-binding agent that that binds specifically to a first bacterial antigen specific to  H. influenzae  but not  M. catarrhalis  or  S. pneumoniae , a second bacterial-binding agent that binds specifically to a second bacterial antigen specific to  M. catarrhalis  but not  H. influenzae  or  S. pneumoniae , and a third bacterial-binding agent that binds specifically to a third bacterial antigen specific to  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae , wherein the first, second and third bacterial-binding agents are bound to specific regions of the one or more solid phase substrates in the cartridge; one or more conjugation regions within the cartridge, the one or more conjugation regions in fluid communication with the one or more solid phase substrates and comprising a fourth agent that is labeled and that binds specifically to the first bacterial antigen, a fifth agent that is labeled and that binds specifically to the second bacterial antigen, and a sixth agent that is labeled and that bind specifically to the third bacterial antigen; one or more sample inlets on the cartridge in fluid communication with the one or more conjugation regions; and one or more windows through which the specific regions of the solid phase substrate to which the first, second and third bacterial-binding agents are bound may be visualized. 
     The anionic surfactant of the lysis buffer may comprise sarkosyl and wherein the osmotic agent of the lysis buffer comprises sucrose. Any of these assay kits may include a diluting buffer, as described herein. 
     The cartridge may include a housing that encloses one or more (e.g., three, arranged in parallel) solid phase substrates. For example, a cartridge may comprise a plurality (e.g., 3) of solid phase substrates, wherein each solid phase substrate holds one of the first bacterial-binding agent, the second bacterial-binding agent or the third bacterial-binding agent. Alternatively, cartridge may comprise a single solid phase substrate holding each of the first bacterial-binding agent, second bacterial-binding agent and third bacterial-binding agent. The first bacterial antigen may be a cell surface antigen specific to  H. influenzae , the second bacterial antigen may be a cell surface antigen specific to  M. catarrhalis  and the third bacterial antigen may be a cell-surface antigen specific to  S. pneumoniae.    
     Any of these cartridge regions may include a conjugation region. The conjugation region may hold the unbound antigen binding agent, which may be marked with a marker (e.g., a visualizable marker such as a colloidal metal, colored bead, etc.). The antigen binding agent(s) in the conjugation region may be in solution (e.g., in a pre-wetted conjugation sponge or conjugation pad, a fluid conjugation chamber, etc.). Alternatively, the antigen binding agent (e.g., antibody, FAB, etc.) may be lyophilized and stored in this region, and the sample solution may re-suspend the antigen binding agent, allowing it to bind before entering the portion(s) of the solid phase substrate to which antigen binding agent(s) are bound. In variations having a single solid phase substrate with discrete regions for each of the different types of antigen binding agents binding to specific bacterial types, a single conjugation region (e.g., holding the fourth agent, fifth agent and sixth agent) may be used. Any of these cartridges may include multiple conjugation regions. In particular, cartridges having parallel fluid paths may include multiple conjugation regions, where each conjugation region holds the labeled antigen binding agent specific to one of the types of bacteria corresponding to the bound antigen binding agent on the downstream solid phase substrate. 
     Any of these kits may include a cartridge a single sample inlet. The single inlet may feed into a single fluidics line or into a plurality (e.g., 3) of parallel fluidic lines that may connect to, e.g., a sample region or chamber (e.g., sample pad), a conjugation region or chamber (e.g., conjugation pad), an incubation region or chamber (e.g., incubation pad), a solid phase substrate region (e.g., detection region, which may be combined with the incubation region or chamber or separate from it), and/or a waste chamber or region (e.g., absorbent pad). The fluid path(s) through the cartridge may include an air inlet. For example, an air inlet may be present at an opposite end of the fluid path from the sample input. 
     The one or more windows in the cartridge may allow viewing of the solid phase substrate, allowing detection (e.g., visual, optical, etc.) of binding of antigen to the solid phase substrate(s) in this region (e.g., the detection region) where the tethered/bound antigen binding agent specifically bound to the solid phase substrate. In some variations the method includes reading/detection of the binding using a reader including an optical reader (e.g., florescent reader, etc.), visual (e.g., manual or automatic) reading, etc. The cartridges described herein may be configured to be compatible with one or more readers, including optical readers such as the Quidel “Sophia” device that is an optical reader that uses fluorescent markers (see, e.g., www.quidel.com/immunoassays/sofia-tests-kits) or the Becton Dickinson “Veritor” System (see, e.g., www.bd.com/ds/veritorsystem/poctesting.asp). 
     As mentioned, any of the antigen binding agents (e.g., any or all of the first bacterial-binding agent, second bacterial-binding agent, third bacterial-binding agent, fourth agent, fifth agent, and sixth agent) may comprise an antibody or an antibody fragment. 
     The one or more solid phase substrates may be, for example, a membrane or other surface onto which an antigen binding agent is immobilized. The substrate may be smooth, porous, rough, etc. In some variations a single solid phase substrate is used to which each of the multiple antigen binding agents (e.g., the first, second, third, fourth, and fifth agents, each specific to an antigen of one of  M. cat, S. pneumo, H. flu , influenza A, or influenza B). Thus, in any of these variations, the one or more conjugation regions may be a single conjugation region, and the one or more sample inlets may be a single sample inlet, and the single solid phase substrate may be upstream of the single conjugation region that is upstream of the single sample inlet. 
     Any of these assay kits may also include a control region on the solid phase substrate. The control region may include an immobilized binding agent that binds to one or more of the soluble antigen binding agents in the assay (e.g., the first bacterial-binding agent, second bacterial-binding agent, third bacterial-binding agent, first viral-binding agent and second viral-binding agent) configured to bind to one or more of the fourth agent, fifth agent, sixth agent, seventh agent, or eighth agent and an absorbent pad, downstream of the specific regions of the solid phase substrate to which the first bacterial-binding agent, second bacterial-binding agent and third bacterial-binding agent, first viral-binding agent and second viral-binding agent are bound. 
     For example, described herein are assay kits for concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal sample, the assay kit comprising: a lysis buffer to lyse cells within the sample and form a single sample solution, wherein the lysis buffer comprises between 0.01% and 5% (w/w) of the anionic surfactant and between 0.1% and 15% (w/w) of the osmotic agent; a cartridge containing a solid phase substrates holding a first bacterial-binding agent that that binds specifically to a first bacterial antigen specific to  H. influenzae  but not  M. catarrhalis  or  S. pneumoniae , a second bacterial-binding agent that binds specifically to a second bacterial antigen specific to  M. catarrhalis  but not  H. influenzae  or  S. pneumoniae , and a third bacterial-binding agent that binds specifically to a third bacterial antigen specific to  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae , wherein the first, second and third bacterial-binding agents are bound to specific regions of the solid phase substrate; and a conjugation region within the cartridge, conjugation region in fluid communication with the solid phase substrate and comprising a fourth agent that is labeled and that binds specifically to the first bacterial antigen, a fifth agent that is labeled and that binds specifically to the second bacterial antigen, and a sixth agent that is labeled and that bind specifically to the third bacterial antigen; a sample inlet on the cartridge in fluid communication with the conjugation region; and one or more windows exposing the specific regions of the solid phase substrate to which the first second and third bacterial-binding agents are bound. 
     Also described herein are methods of concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal sample. For example a method of concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal sample may include: adding the sample to a lysis buffer to lyse cells within the sample and form a single sample solution, wherein the lysis buffer comprises both an anionic surfactant and an osmotic agent; adding the sample solution to a cartridge containing one or more solid phase substrates holding a first bacterial-binding agent that that binds specifically to a first bacterial antigen specific to  H. influenzae  but not  M. catarrhalis  or  S. pneumoniae , a second bacterial-binding agent that binds specifically to a second bacterial antigen specific to  M. catarrhalis  but not  H. influenzae  or  S. pneumoniae , and a third bacterial-binding agent that binds specifically to a third bacterial antigen specific to  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae , wherein the first, second and third bacterial-binding agents are bound to specific regions of the one or more solid phase substrates in the cartridge; and contacting the sample solution, either before or after it is added to the cartridge, with a fourth agent that is labeled and that binds specifically to the first bacterial antigen, a fifth agent that is labeled and that binds specifically to the second bacterial antigen, and a sixth agent that is labeled and that bind specifically to the third bacterial antigen. 
     In general, the agents that bind specifically to the antigens (e.g., first bacterial antigen, second bacterial antigen, third bacterial antigen) described herein do not bind to antigens (proteins) from the majority of other commernsural bacteria in the sinus specimen, in addition to having little or any binding to other antigens other than the intended/target antigen. For example, the antigen binding agent (e.g., antibody or antibody fragment) may bind specifically to the target first bacterial antigen (e.g., from  H. flu ), but not to non-target antigens (e.g., from  M. Cat  or  S. pneumo ). 
     In any of these methods, kits and compositions described herein, the lysis buffer may comprise between 0.01% and 5% (w/w) of the anionic surfactant and between 0.1% and 15% (w/w) of the osmotic agent. The anionic surfactant of the lysis buffer may comprise between 0.01% and 5% (w/v) of sarkosyl and the osmotic agent of the lysis buffer may comprises between 0.1% and 15% (w/w) of sucrose. 
     Any of these methods may include adding a diluting buffer to the sample solution prior to adding it to the cartridge. 
     Adding the sample solution to the cartridge may include applying a single bolus of sample or applying multiple boluses of sample. For example, adding sample solution to the cartridge may comprise dividing the sample between a plurality of regions in the cartridge, wherein each region is in fluid communication with separate solid phase substrates and wherein each solid phase substrate holds one of the first bacterial-binding agent, the second bacterial-binding agent or the third bacterial-binding agent. 
     Adding the sample solution to the cartridge may comprise adding the sample solution to a single region in the cartridge that is in fluid communication with a solid phase substrate holding each of the first bacterial-binding agent, second bacterial-binding agent and third bacterial-binding agent. In any of these methods, kits, and compositions described herein, the antigens to each bacterial type may be cell surface antigens. For example the first bacterial antigen may be a cell surface antigen specific to  H. influenzae , the second bacterial antigen may be a cell surface antigen specific to  M. catarrhalis  and the third bacterial antigen may be a cell-surface antigen specific to  S. pneumoniae.    
     Any of these methods may include passing the sample solution over the one or more solid phase substrates in the cartridge after contacting the sample solution with the fourth, fifth and sixth agents. 
     The step of contacting the sample solution with the fourth, fifth and sixth agent may comprise passing the sample through one or more portions of the cartridge upstream from the specific regions of the solid phase substrate in the cartridge to which the first, second and third bacterial-binding agents are bound. 
     Any of the methods described herein may include visually identifying which strain (e.g.,  M. cat, S. pneumo , or  H. flu ) is present in the sample solution by identifying that the fourth agent has bound to the first bacterial antigen in the solid phase substrate region where the first bacterial-binding agent was bound, and/or the fifth agent has bound to the second bacterial antigen in the solid phase substrate region where the second bacterial-binding agent was bound, and/or that the sixth agent has bound to the third bacterial antigen in the solid phase substrate region where the third bacterial-binding agent was bound. 
     The sample solution may be exposed (e.g., contacted with) the labeled antigen binding agents either before or after it is added to the cartridge. For example, the sample solution may be contacted with the fourth agent, fifth agent, and sixth agent before it is added to the cartridge, or the sample solution may be contacted with the fourth agent, fifth agent, and sixth agent after it is added to the cartridge. 
     For example, a method for concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal sample may include: adding the sample to a lysis buffer to lyse cells within the sample and form a single sample solution, wherein the lysis buffer comprises between 0.01% and 5% (w/w) of the anionic surfactant and between 0.1% and 15% (w/w) of the osmotic agent; adding the sample solution to a cartridge containing a solid phase substrate holding a first bacterial-binding agent that that binds specifically to a first bacterial antigen specific to  H. influenzae  but not  M. catarrhalis  or  S. pneumoniae , a second bacterial-binding agent that binds specifically to a second bacterial antigen specific to  M. catarrhalis  but not  H. influenzae  or  S. pneumoniae , and a third bacterial-binding agent that binds specifically to a third bacterial antigen specific to  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae , wherein the first, second and third bacterial-binding agents are bound to specific separate regions of the solid phase substrate; contacting the sample solution with a fourth agent that is labeled and that binds specifically to the first bacterial antigen, a fifth agent that is labeled and that binds specifically to the second bacterial antigen, and a sixth agent that is labeled and that bind specifically to the third bacterial antigen; and visually identifying through a window in the cartridge that the fourth agent has bound to the first bacterial antigen, the fifth agent has bound to the second bacterial antigen, or the sixth agent has bound to the third bacterial antigen. 
     According to some embodiments, the same lysis buffer used to lyse the cells and expose antigens of bacteria can be used to expose antigens from one or more viruses present in the patient&#39;s sample. The assay kit can also be adapted for concurrently detecting the presence of antigens specific to particular bacteria and viruses so that a single system can be used in the identification of particular bacteria and viruses present in the patient&#39;s sample. The assay system can use the same buffer solution to extract both bacterial and viral antigens from a specimen, which can simplify the collection/assay process, reduce errors, and provide more controlled results. Further, the assay systems can provide accurate and rapid results for distinguishing between bacterial and viral infections, as well as identifying certain types of bacterial and viral infections, making the systems/kits well suited for implementation in a point-of-care setting. 
     In some embodiments, the bacterial and viral detection assay system include a lysis buffer solution including an anionic surfactant comprising sarkosyl at a concentration of between 0.01% and 5% (w/w) and an osmotic agent comprising sucrose at a concentration of between 0.1% and 15% (w/w), as described herein. In one implementation, the lysis buffer is adapted to lyse and extract antigens specific to  H. influenzae, M. catarrhalis  and  S. pneumoniae  and extract one or both of influenza A and influenza B virus antigens (and in some cases one or more coronaviruses) within a period of time after being combined with a patient&#39;s sample (e.g., mucosal sample). In some cases, the period ranges from about 5 seconds to about 15 minutes (e.g., 5 sec to 15 mins, 5 sec to 10 mins, 10 sec to 5 mins, 30 sec to 15 min, etc.). 
     In some embodiments, an assay kit for concurrently detecting bacteria and viruses in a mucosal sample includes: a lysis buffer to lyse cells within the mucosal sample and form a single sample solution, wherein the lysis buffer comprises between 0.01% and 5% (w/w) sarkosyl and between 0.1% and 15% (w/w) sucrose, wherein the lysis buffer solution is adapted to expose an antigen specific to a particular type of bacteria (e.g., one or more of  H. influenzae, M. catarrhalis  and  S. pneumoniae ) and an antigen specific to a particular type of virus (e.g., one or more of influenza A, influenza B and a coronavirus) in the mucosal sample. The assay kit also includes a cartridge containing one or more solid phase substrates holding a bacterial-binding agent that binds specifically to the antigen specific to the particular type of bacterium (e.g., one or more of  H. influenzae, M. catarrhalis  and  S. pneumoniae ) but not to the antigen specific to the particular type of virus (e.g., one or more of influenza A, influenza B and a coronavirus), and a viral-binding agent that binds specifically to the antigen specific to the particular type of virus (e.g., one or more of influenza A, influenza B and a coronavirus) but not to the antigen specific to the particular type of bacterium (e.g., one or more of  H. influenzae, M. catarrhalis  and  S. pneumoniae ), wherein the bacterial-binding agent and the viral-binding agent are bound to specific regions of the one or more solid phase substrates of the cartridge. The assay kit additionally includes one or more conjugation regions within the cartridge, the one or more conjugation regions in fluid communication with the one or more solid phase substrates and comprising an agent that is labeled and that binds specifically to the antigen specific to the particular type of bacterium, and an agent that is labeled and that binds specifically to the antigen specific to the particular type of virus. In cases where assay kit is configured to identify multiple bacteria and/or viruses, the one or more solid phase substrates can include multiple labeled agents each configured to bind specifically to a corresponding antigen of the specific bacterium and/or virus. The assay kit further includes one or more windows through which the specific regions of the one or more solid phase substrates to which the bacterial-binding agent(s) and viral binding agent(s) are bound may be viewed. 
     In some implementations, the viral and bacterial assays can be run on a single lateral flow assay cartridge having a combined assay substrate/flow path for viruses and bacteria. In some implementations, the viral and bacterial assays are run on dual lateral flow substrates/flow paths, e.g., one substrate for detecting one or more viruses and another separate substrate for detecting one or more bacteria. In some implementations, the viral and bacterial assays can be run on one or more lateral flow assay cartridges, with individual substrates/flow paths for each type of bacterium or virus. 
     Although the kits (e.g., assay kits, systems) described herein in these examples are configured to test for the presences of three bacteria (e.g.,  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae ), any of these kits and methods may be instead configured to identify the presence of two or more than three bacteria. In particular, any of the methods and kits described herein may be configured to determine the presence of  S. pneumoniae  and/or  H. influenzae , which together account for approximately 70-75% of bacterial sinusitis. Additionally, the kits (e.g., assay kits, systems) described herein can be configured to identify the presence of one or more viruses other than influenza viruses. 
     Also described herein are nasal sampling devices that may be used by themselves or as part of a kit or system for testing a nasal (e.g., mucous) material, particularly from the middle meatus region of the sinus. 
     For example, a nasal sampling device for obtaining a sinus secretion sample from a subject&#39;s sinus may include: an elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees; a sample collector on a distal end of an extendable shaft, wherein the sample collector is configured to collect a sample of sinus fluid, further wherein the sample collector is housed entirely within the distal end of the elongate body in a retracted position; and a control coupled to the extendable shaft and configured to extend and retract the sample collector in and out of the distal end of the elongate body; wherein the nasal sampling device has a retracted configuration with the sample collector retracted and housed entirely within the distal end of the elongate body, a sampling configuration with the sample collector extended distally out of a distal opening of the distal end region of the elongate body a first distance between 0.5 cm to 3 cm, and an elution configuration with the sample collector extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance. 
     A nasal sampling device for obtaining a sinus secretion sample from a subject&#39;s sinus, wherein the nasal sampling device includes: an elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees; a sample collector on a distal end of an extendable shaft, wherein the sample collector is configured to collect a sample of sinus fluid, further wherein the sample collector is housed entirely within the distal end of the elongate body in a retracted position; and a control coupled to the extendable shaft, the control having a first set point wherein the sample collector is extended distally out of a distal opening of the distal end region of the elongate body a first distance between 0.5 cm to 3 cm, the control having a second set point, wherein the sample collector is retracted and housed entirely within the distal end of the elongate body, the control having a third set point, wherein the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance. 
     Any of these nasal sampling devices may include a spacer (which may also be a protrusion, bump, deflector, etc.) on the extendable shaft proximal to the sample collector, wherein the spacer is configured to prevent the sample collector from contacting an inner surface of the elongate body when the sample collector is retracted into the distal end of the elongate body. Centering the sample collector in this manner may prevent the sample collector from getting contaminated by other bacteria (e.g., from regions other than the sampling region) by contacting the outer housing of the elongate body, which may contact other regions; this may also prevent prematurely releasing material or limiting the amount of material held by the sample collector (e.g., swab). 
     Any of the nasal sampling devices described herein may include a releasable stop configured to prevent the control from selecting the third set point until the stop is released. Any appropriate stop may be used, including an interference region between the extendable shaft and the elongate body and/or handle, a latch, etc. For example, the stop may comprise a detachable handle configured to releasably couple to a distal end of the extendable shaft. The stop may include a releasable connector connecting the extendable shaft to the stop. 
     In general, the dimensions of the nasal sampling may be configured for use within the nasal passages (e.g., sinus) so that the sample collector may be extended at the correct region of the apparatus to reach the desired portion of the sinus (e.g. the middle meatus region, the upper meatus region, the lower meatus region, etc.). Both the angle of the distal end of the device relative to more proximal regions as well as the size and shape of the device may be configured to allow external (through the nares/nostril) application of the device to sample the mucosa. For example the distal end region of the elongate body may be between 1.5 and 3.5 cm long (e.g., between 1 and 5 cm long, between 1 and 4 cm long, between 1.5 and 4 cm long, between 2 and 3 cm long, etc.). Similarly, the proximal region of the elongate body may be greater than 1 cm long (e.g., greater than 1.5 cm, greater than 2 cm, greater than 3 cm, greater than 4 cm, greater than 5 cm, between 1 cm and 30 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, etc.). 
     Similarly, the sample collector may be any appropriate size (e.g., between 0.2 and 2 cm long, between 0.4 and 1.5 cm long, between 0.5 and 1.2 cm long, etc.). The extendable shaft may be any appropriate length (e.g., greater than 2 cm, greater than 5 cm, greater than 10 cm, between 1 cm and 30 cm, between 1 cm and 20 cm, between 1 cm and 15 cm, between 1 cm and 12 cm, etc.). The extendable shaft may be configured (by operation of the control) to extend from the distal end region of the elongate body by a predetermined amount. For example, as mentioned above, in a sampling position the sample collector may be extended from the distal end by between 0.5 cm to 3 cm. In the elution configuration the extendable shaft is extended away from the elongate body further than in the sampling configuration. This may be achieved by advancing the extendable shaft relative to the elongate body, or by retracting the distal end region of the elongate body proximally, relative to the extendable shaft, or in some variation by removing all or a portion of the distal end region of the elongate shaft. For example, in some variations, the distance that the sample collector extends from the elongate body in the elution configuration (e.g., the second distance) may be 1.0 cm or greater than the first distance. 
     As described herein, in general the sample collector may be a swab, including in particular a flocked swab. It may also be beneficial to use a swab having ends which are branched (e.g., bifurcated, or multiply-divided). 
     In any of these variations, the control on the nasal sampling device may be coupled to a handle at the proximal end of the device. For example, any of these apparatuses may include a handle body extending proximally from the elongate body, wherein the extendable shaft extends through the elongate body and into an internal channel within the handle body. The extendable shaft may generally be a flexible elongate shaft. The extendable shaft may be configured to slide within the elongate body. 
     Thus, any of the devices described herein may include a control configured as a slider. Other examples of controls may include dials, knobs, switches, or the like. In some variations a control that may be included (e.g., in addition to a slider or other control) may be a finger ring. In some variations a control comprises may be a compression actuator configured to be compressed to select the third set point in which the sample collector is extended distally out of the distal opening of the distal end region of the elongate body the second distance. In general, a control may be configured to be distally advanced to select the first set point in which the sample collector is extended distally out of a distal opening of the distal end region of the elongate body the first distance. In some variations a control comprises a push button configured to be depressed to select the third set point in which the sample collector is extended distally out of the distal opening of the distal end region of the elongate body the second distance. 
     Any of these devices described herein may include a lock configured to lock the control at one or more of: the first set point, the second set point or the (optional) third set point. 
     Any of the devices described herein may include a depth gauge configured to display a position of the sample collector to a user of the device. The distal end region may be configured to have an open configuration when the sample collector is advanced out of the distal end of the elongate body, and a closed configuration when the sample collector is in the retracted position. 
     Any of these devices may also include a depth stop to prevent the sampling device from being inserted too deep into a nasal and/or sinus cavity of a subject. 
     For example, a nasal sampling device for obtaining a sinus secretion sample from a subject&#39;s sinus may include: a hollow elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees; a sample collector on a distal end of an extendable shaft, wherein the sample collector is configured to collect a sample of sinus fluid, further wherein the sample collector is housed entirely within the distal end of the elongate body in a retracted position; a control coupled to the extendable shaft, the control having a first set point wherein the sample collector is extended distally out of a distal opening of the distal end region of the elongate body a first distance between 0.5 cm to 3 cm, the control having a second set point, wherein the sample collector is retracted and housed entirely within the distal end of the elongate body, the control having a third set point, wherein the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is 1.0 cm or greater than the first distance; and a projection on the extendable shaft proximal to the sample collector, wherein the projection is configured to prevent the sample collector from contacting an inner surface of the hollow elongate body when the sample collector is retracted into the distal end of the elongate body. 
     Also described herein are methods including methods of using a nasal sampling device. For example, a method for detecting one or more nasal bacteria in a patient, using a nasal sampling device including an elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees, a sample collector on a distal end of an extendable shaft, and a control coupled to the extendable shaft, the control having a first set point wherein the sample collector is extended distally out of a distal opening of the distal end region of the elongate body a first distance, the control having a second set point, wherein the sample collector is retracted and housed entirely within the distal end of the elongate body, the control having a third set point, wherein the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance, may include: advancing the distal end region of the nasal sampling device through a nares of the patient until the distal end region is adjacent to a middle meatus of a sinus; setting the control to the first set point to extend the sample collector into the middle meatus so that it contacts a secretion fluid in the middle meatus; setting the control to the second set point to retract the sample collector entirely within the distal end; withdrawing the nasal sampling device out of the patient&#39;s nares; and testing the secretion fluid with an immunoassay test after withdrawing the nasal sampling device. 
     The secretion fluid may be tested using any of the method described above (e.g., concurrently detecting  H. influenzae, M. catarrhalis  and  S. pneumoniae  from a mucosal sample). For example, testing the secretion fluid may include setting the control to the third set point, so that the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance and contacting the sample collector with a buffer solution. Testing the secretion fluid may comprise contacting the secretion fluid with a lysing solution. For example, testing the secretion fluid may comprise contacting the secretion fluid with a lysing solution comprising both an osmotic agent and an anionic surfactant. In some variations, testing the secretion fluid comprises contacting the secretion fluid with a lysing solution comprising Sodium Lauroyl Sarcosinate and sucrose to form a sample fluid and contacting the immunoassay test with the sample fluid. Testing the secretion fluid may comprise testing the secretion fluid with one or more agents that bind to: an antigen specific to  H. influenzae , an antigen specific to  M. catarrhalis , or an antigen specific to  S. pneumoniae . Testing the secretion fluid may comprises testing the secretion fluid with one or more agents that bind to each of: an antigen specific to  H. influenzae , an antigen specific to  M. catarrhalis , or an antigen specific to  S. pneumoniae.    
     Also described herein are systems for detecting bacterial sinusitis that generally include a mucosal sampling device as described herein any any of the assays/kits described herein. For example, a system for detecting bacterial sinusitis may include a nasal sampling device for obtaining a sinus secretion sample from a subject&#39;s sinus, wherein the nasal sampling device includes: an elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees; a sample collector on a distal end of an extendable shaft, wherein the sample collector is configured to collect a sample of sinus fluid, further wherein the sample collector is housed entirely within the distal end of the elongate body in a retracted position; a control coupled to the extendable shaft, the control having a first set point wherein the sample collector is extended distally out of a distal opening of the distal end region of the elongate body a first distance, the control having a second set point, wherein the sample collector is retracted and housed entirely within the distal end of the elongate body, the control having a third set point, wherein the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance; and an immunoassay kit for detecting at least one bacterial strain associated with bacterial sinusitis infections. 
     The immunoassay kit may include a lysis buffer comprising both an anionic surfactant and an osmotic agent, such as an anionic surfactant between between 0.01% and 5% (w/w) and an osmotic agent between 0.1% and 15% (w/w). In some variations the immunoassay kit may comprises a lysis buffer comprising sarkosyl and sucrose. 
     In any of these variations, the immunoassay kit may include a cartridge, and the cartridge may include a sample inlet for depositing a sample, a sample pad onto which the sample is absorbed prior to elution, a conjugate pad containing at least one antibody complexed with a detectable marker, a detector pad comprising at least one zone, wherein the zone comprises antibodies directed to at least one bacterial antigen bound to the detector pad, and a visualization window for viewing the results of the assay. 
     The immunoassay kit may comprise a cartridge comprising a sample inlet for depositing a sample, a sample pad onto which the sample is absorbed prior to elution, a conjugate pad containing a plurality of antibodies complexed with a detectable marker, a detector pad comprising a plurality of different zones, wherein each zone comprises antibodies directed to at least one bacterial antigen bound to the detector pad, and a visualization window for viewing one or more of the zones of the detector pad. The kit may include a sampling device with a spacer on the extendable shaft proximal to the sample collector, wherein the spacer is configured to prevent the sample collector from contacting an inner surface of the elongate body when the sample collector is retracted into the distal end of the elongate body. 
     A system for detecting bacterial sinusitis may include: a nasal sampling device for obtaining a sinus secretion sample from a subject&#39;s sinus, wherein the nasal sampling device includes: an elongate body having a distal end region that is bent relative to a proximal region by between 15 degrees and 30 degrees; a sample collector on a distal end of an extendable shaft, wherein the sample collector is configured to collect a sample of sinus fluid, further wherein the sample collector is housed entirely within the distal end of the elongate body in a retracted position; a control coupled to the extendable shaft, the control having a first set point wherein the sample collector is extended distally out of a distal opening of the distal end region of the elongate body a first distance, the control having a second set point, wherein the sample collector is retracted and housed entirely within the distal end of the elongate body, the control having a third set point, wherein the sample collector is extended distally out of the distal opening of the distal end region of the elongate body a second distance that is greater than the first distance; and an immunoassay kit for detecting multiple bacterial strains associated with bacterial sinusitis infections, the kit comprising a lysis buffer comprising both an anionic surfactant between between 0.01% and 5% (w/w) and an osmotic agent between 0.1% and 15% (w/w). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is an illustration of a healthy sinus and a sinus showing symptoms of sinusitis. 
         FIG.  2    is a CT scan image of a patient exhibiting symptoms of sinusitis. 
         FIGS.  3 A- 3 F  show an example of a method for sampling a sinus in accordance with some embodiments. 
         FIGS.  4 A- 4 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  5 A- 5 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  6 A- 6 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  7 A- 7 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  8 A- 8 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  9 A- 9 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  10 A- 10 D  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  11 A- 11 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  12 A- 12 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  13 A- 13 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIGS.  14 A- 14 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. 
         FIG.  15 A  is a rendering of the lateral side of another variation of a device configured to sample from a sinus. 
         FIG.  15 B  is a rendering of the posterior side of the assembled device of  FIG.  15 A . 
         FIGS.  15 C and  15 D  show partially exploded views of the lateral side and front of the device of  FIG.  15 A . 
         FIG.  16    is an example of a proximal end of a sample collector being inserted into a distal end of the main body portion. 
         FIG.  17 A  illustrates one variation of a distal end of a sample collector and a corresponding sleeve into which the sample collector may be housed. Also shown is at least one coupler that joins the sleeve to the main body of the device. 
         FIG.  17 B  illustrates an alternative configuration for the at least one coupler that joins the sleeve to the main body of the device. 
         FIG.  18 A  shows an exploded view of one variation of a distal end of a handle and a proximal end of a main body region. 
         FIG.  18 B  illustrates a distal end of a thumb ring that is separated from a proximal end of a main body region of the device, similar to the view of  FIG.  18 A . The thumb ring controller region at the distal end (left side of  FIGS.  18 A and  18 B ) may be coupled into the distal end of the main body region. 
         FIG.  19    illustrates a sleeve for housing the distal end of the swab. Both the sleeve (protective cover) and the sample collector (including swab) are bent in a predefined manner as described herein. 
         FIG.  20    shows a sample collector proximal end coupled with the distal end of the main body. 
         FIG.  21    illustrates a proximal end region of a sample collector that couples with the handle. 
         FIG.  22    illustrates another variation of a sampling device having a coupler and releasable hold (e.g. releasable lock, or release lock) on the main body for engaging the distal handle. 
         FIG.  23 A  shows a coupler (shown as a snap-fit coupler) on the handle and a corresponding coupling channel on the main body. 
         FIG.  23 B  is an alternative view of the coupler of the handle and a corresponding coupling channel on the main body. 
         FIG.  23 C  is another view of a coupler of the handle and a corresponding coupling channel on the main body. 
         FIGS.  24 A- 24 D  show schematics of one example of a lateral flow assay (having two detection readouts, e.g., for two sets of antibodies) and components.  FIG.  24 A  shows an assay housing, a sample pad for accepting the sample, a conjugate pad containing the first antibody with complexed detector molecule, a detection pad along which the sample will run and come into contact with zones of corresponding second antibodies bound to the detection pad for each antigen of interest.  FIG.  24 B  shows a sample on the sample pad, the set of first antibodies on the conjugate pad, and zones on the detection pad holding different antibodies.  FIG.  24 C  shows an eluting solution (dark) that runs across the detection pad and brings first antibodies-detector molecule coupled to corresponding antigens in contact with the second set of antibodies.  FIG.  24 D  shows the completed assay where the first antibodies-detector molecule coupled to corresponding antigens is now also bound to the corresponding second antibodies for each different antigen of interest. 
         FIGS.  25 A- 25 K  illustrate the operation of a sample collector as described herein. 
         FIG.  26    schematically illustrates one variation of an assay similar to the assay shown in  FIGS.  24 A- 24 D  for diagnosing sinusitis. 
         FIG.  27    is a table illustrating the effectiveness of various lysis buffers on three of the types of bacteria to be concurrently examined by the apparatuses and methods described herein. 
         FIG.  28    is a table illustrating two examples of lysis buffers compatible for the concurrent detection of multiple different cell types (e.g.,  M. cat, S. pneumo  and  H. flu ) as described herein. 
         FIG.  29    is a table illustrating two exemplary dilution buffers compatible for the concurrent detection of multiple different cell types as described herein. In these examples, the lysis buffer #1 (on left of  FIG.  28   ) was used with dilution buffer #1 (on left of  FIG.  29   ), and lysis buffer #2 (on right in  FIG.  28   ) was used with dilution buffer #2 (on right in  FIG.  29   ). 
         FIGS.  30 A- 30 C  illustrate detection of each of  S. pneumo, M. cat , and  H. flu , respectively, using the kits and methods described herein. The concentration of cells detected (expressed as colony forming units (CFU)/sample) in this prototype show thresholds for visual detection from an exemplary lateral flow assay such as the one illustrated in  FIGS.  24 A- 24 D and  26   .  FIG.  30 A  illustrates that the prototype assay detected the PsaA antigen (the cell-surface marker for  S. pneumo ) at bacterial concentrations ranging from 10 3 -10 7  per 100 μl sample with resolution at 1×10 4 .  FIG.  30 B  illustrates that the prototype assay detected the CD antigen (a cell-surface marker for  M. cat ) at bacterial concentrations ranging from 10 4 -10 7  per 100 μl sample with good resolution at 1×10 5 .  FIG.  30 C  illustrates that the prototype assay detected the OMP-P5 antigen (a cell-surface marker for  H. flu ) at bacterial concentrations ranging from 10 5 -10 7  per 100 μl sample with good resolution at 2×10 5 . 
         FIG.  31    is one example of a cartridge having a single solid phase substrate (combining three separate assays, one each for a different bacterial type) that can simultaneously test for the presence of each of three different types of bacteria. 
         FIG.  32    is an example of a cartridge configured to simultaneously test for the presence of each of three different types of bacteria in parallel; the cartridge include three separate solid phase substrates and three fluidic pathways. Although the example shown in  FIG.  32    includes three separate inlet ports, a single port having three fluidic paths may be used. 
         FIG.  33    is a graph showing assay peak height readings comparing performance of a bacterial lysis buffer (ENTV Buffer) and a viral extraction buffer (AB Buffer) using a sample solution formed by bacterial specimens of  H. influenzae, S. pneumoniae , and  M. catarrhalis  combined with a bacterial lysis buffer (ENTV Buffer) or a viral extraction buffer (AB Buffer). 
         FIGS.  34 A and  34 B  are graphs showing assay peak height readings comparing performance of a bacterial lysis buffer and a viral extraction buffer against Flu A or Flu B positive archived specimens.  FIG.  34 A  shows results after ten minutes; and  FIG.  34 B  shows results after fifteen minutes. 
         FIG.  35    is an example of a cartridge configured to simultaneously test for the presence of each of three different types of bacteria and two different types of influenza virus in parallel; the cartridge includes five separate solid phase substrates and five fluidic pathways. Although the example shown in  FIG.  35    includes five separate inlet ports, a single port having five fluidic paths may be used. 
         FIG.  36    is an example of a cartridge having two solid phase substrates, each configured to simultaneously test for the presence of three different types of bacteria or two different types of influenza virus; the cartridge includes two separate solid phase substrates and two fluidic pathways. Although the example shown in  FIG.  36    includes two separate inlet ports, a single port having two fluidic paths may be used. 
         FIG.  37    is an example of a cartridge having a single solid phase substrate configured to simultaneously test for the presence of three different types of bacteria and two different types of influenza virus; the cartridge includes one solid phase substrates and one fluidic pathway. Although the example shown in  FIG.  37    includes one inlet port, multiple ports having one fluidic path may be used. 
     
    
    
     DETAILED DESCRIPTION 
     Apparatuses (including devices, systems, kits, and assays) and methods are disclosed herein for diagnosing sinusitis, including obtaining a sample of sinus fluid from a patient and/or determining if the patient is infected with one or more of  H. influenzae  ( H. flu ),  M. catarrhalis  ( M. cat ) and  S. pneumoniae  ( S. pneumo ) and/or one or more viruses. For example, described herein are sample devices for accurately and quickly sampling sinus fluid within the sinus, such as the middle meatus or maxillary sinus, and assays for rapidly testing this sample to determine the presence of bacteria, viruses, and other diseases of interest. The fast diagnosis of the presence or absence of the diseases of interest can improve the treatment of the patient. 
       FIG.  1    illustrates a comparison between a healthy sinus and a sinus with sinusitis. The sinusitis can cause excess mucous in the frontal sinus and maxillary sinus. Other symptoms can include inflamed sinus lining and a sinus infection.  FIG.  2    illustrates a CT image of a patient with chronic sinusitis. The arrows indicate the congested sinuses typical of chronic sinusitis. 
     Testing the mucous/sinus fluid within the sinus, such as the middle meatus or maxillary sinus, can help diagnose the condition causing the discomforting symptoms in the patient. The sinus fluid can indicate a bacterial infection, viral infection, or provide other information to help diagnose and formulate an efficient and effective therapeutic treatment. Other examples of areas of the sinuses that can be tested using the devices and methods disclosed herein are the frontal sinuses, maxillary sinuses, ethmoid sinuses, and sphenoid sinuses. The devices disclosed herein can also have a tip geometry configured to be advanced in other passages within the body. For example the devices can be configured to collect a sample from the nasopharynx region, esophageal passage, from the middle ear, and other portions of the anatomy that a skilled artisan would want to sample. 
       FIGS.  3 A- 3 D  show an example of a method for sampling a sinus in accordance with some embodiments.  FIGS.  3 A- 3 D  include a schematic illustrate of a portion of a sinus  100  including the nares  102 , middle meatus  103 , ostium of the maxillary sinus  104 , maxillary sinus  106 , and sinus fluid  108  within the maxillary sinus  106 .  FIG.  3 B  is a schematic illustration of a portion of a sampling device  110 . Any of the sampling devices disclosed herein can be used as the sampling device  110  as illustrated in  FIGS.  3 A- 3 D . The sampling device  110  includes a distal portion configured to be advanced through the nares  102  to an area adjacent to the middle meatus  103  and maxillary sinus  104  as shown in  FIG.  3 B . After the sampling device  110  has been advanced to a desired area adjacent to the middle meatus  103 , the sample collector  112  can be advanced distally to contact and sample sinus fluid in the middle meatus  103  as shown in  FIG.  3 C . After the sample of the sinus fluid has been obtained by the sample collector  112 , the sample collector  112  can be retracted back into the sampling device  110 . After the sample collector  112  has been retracted back into the sampling device  110 , the sampling device  110  can be withdrawn from the nares  102  as shown in  FIG.  3 D . The sampling device  110  can be used to sample either of the nares. 
     After the sinus fluid has been sampled using the sampling device  110 , the sinus fluid sample can be tested.  FIG.  3 E  illustrates an example of a kit that can include a sampling device as described herein. The kit can include a lysis (e.g., buffer or lysis buffer) solution  150 , diagnostic test  152 , and packaging  154  in addition to the sample collector. As will be described in greater detail below, a sample collector  112  containing a sinus fluid sample can be advanced distally as described herein, and placed in contact with the lysis buffer solution  150  to form the sample solution in which bacterial cells (and particular the  H. influenzae, M. catarrhalis  and  S. pneumoniae ) will be lysed to expose markers that can be detected by the assay. Thus, an aliquot of the sample solution can be applied to the diagnostic test  152 . The diagnostic test  152  can produce a color change or other indication visible to the medical technician to indicate a positive or negative result for one or more of the bacteria tested (e.g., for sinusitis,  H. influenzae, M. catarrhalis  and  S. pneumoniae ).  FIG.  3 F  illustrates an example of a diagnostic test  156  with three different tests (one each for  H. influenzae, M. catarrhalis  and  S. pneumoniae ) and a control.  FIG.  3 F  illustrates an example of positive responses to all three different tests and the control. In general, a diagnostic test  152  can contain a plurality of immunoassay tests. The tests can provide rapid results on the order of 1-30 minutes (e.g., 5-20 min, 5-17 min, 5-15 min, etc.). Other configurations can be used for the immunoassay tests, for example multiple testing strips can be included in the diagnostic test  152  with each test strip testing for a different pathogen on each strip, or some variation of a single sequential and one or more parallel assays may be used. In some embodiments the diagnostic test includes tests for two or more pathogens. In some embodiments the diagnostic test includes tests for three or more pathogens. In some embodiments the diagnostic test includes tests for four or more pathogens. 
     In some embodiments the immunoassay tests can include common conditions implicated in sinusitis, such as strep A, influenza A, and influenza B. In some embodiments the immunoassay tests can include strep A. In some embodiments the immunoassay tests can include influenza A. In some embodiments the immunoassay tests can include influenza B. 
     In some embodiments the diagnostic tests can include bacterial sinusitis tests. Examples of bacterial sinusitis pathogens include:  Haemophilus influenzae, Moraxella catarrhalis , and  Streptococcus pneumoniae . Other examples of diagnostic tests that can be used with the devices, kits, and methods disclosed herein include U.S. Patent Publication No. 2014/0314876 to Das et al, titled “Proteomics Based Diagnostic Detection Method for Chronic Sinusitis”, the disclosure of which is incorporated by reference herein in its entirety. 
     The sample collection devices disclosed herein can include a distal tip that is configured to be advanced within the nare of the patient. The distal tip can include a bend that is configured to line up with the anatomy of most patients, such as the middle meatus. In some cases the bend has an angle of about 10 degrees to about 30 degrees relative to a major axis of the device. In some embodiments the distal tip can be flexible. The distal tip can be made out of a soft, biocompatible, and pliable material, such as a polymer. In some embodiments the distal tip can be made out of silicone. Other examples of biocompatible polymers include thermoplastic elastomer (TPE), thermoplastic vulcanizates (TPV), thermoplastic polyolefins (TPO), thermoplastic urethane (TPU) polymers, etc. Specific examples of polymers that can be used for the distal tip also include Kraton, Versaflex, Santoprene, etc. Other biocompatible polymers know by the skilled artisan can also be used. In some embodiments the distal tip can be made out of metal. It may be desirable (though not necessary) to have a material hardness of between about Durometer Shore A90 to D 85. 
     The distal tip can have an open end. In some embodiments the distal tip includes a covered or closed distal end. The covered or closed distal end can be opened with distal advancement of the sample collector. In some embodiments the covering can be designed to be punctured by the sample collector. In some embodiments the covering can be designed to open and close to reduce the chance of contamination of the sample collector. In some embodiments the covering or distal end can be designed to be resealably opened. For example, the cover or distal end can have a patterned opening. The sample collector can be pushed through the patterned opening and the patterned opening can close after the sample collector is retracted. The closed distal end or covering can prevent contamination of the sample collector when the device is advanced through the nare or retracted outside of the patient after the sample has been taken. In some embodiments the distal tip can have an open distal end. 
     The sample collector can be advanced distally past a distal end of the distal tip to take a sample of sinus fluid or other target fluid. The sample collector can be a swab or contain another absorbent material that can collect and hold fluid. The advancement of the sample collector can be done using an actuator. In some embodiments the actuator can be slider or a plurality of sliders. In some embodiments a handle portion engaged with the sample collector can be used to advance and retract the sample collector. In some embodiments the mucous sample can be collected using negative pressure. For example, the actuator can create a negative pressure in the environment surrounding the distal tip such that the mucous sample flows into the sample collector. 
     The device can include a safety or lock to reduce the inadvertent advancement of the sample collector while the device is in the nare of the patient. For example, a button or slider can be required to be pressed to allow further advancement of the actuator. In some embodiments the slider itself can be required to be depressed before it can slide. In some cases the safety can be a lock that can be deactivated prior to further advancing the sample collector. In some embodiments the slider can include two sliders that are simultaneously depressed to allow movement of the actuator. In some cases the actuator can move along a track with notches to catch or stop the actuator at the sample position and sample solution position. In some cases the actuator can move along a track with a stair type configuration that requires shifting the actuator at a stop position prior to further advancing or retracting the actuator. 
     In some embodiments the devices can be operated using a single hand. For example, one portion of the device can be held with one or more fingers while the actuator or proximal end of the device can be held and operated using the thumb. The devices can be configured for ambidextrous use. For example, the device can be ergonomically designed to accommodate use by the left hand and the right hand. The medical professional can use whichever hand they prefer to operate the device. In some embodiments the device can be operated with both hands. For example, the lab technician may prefer to use both hands to extend the sample collector for processing. 
     The device can include a marker to indicate the orientation of the device, such as the direction of the bend in the distal end. The marker can indicate the lateral direction and/or the left or right nares. The marker can include a colored portion of the device, a label on the device, or a projection on the exterior of the device indicating the orientation of the bend in the distal end. 
     The device can be used to take a sample from either nostril. The orientation of the entire device can be rotated approximately 180 degrees for use on the other nostril. In some embodiments the device can have a rotatable portion that can be rotated, e.g. by 180 degrees, such that the device can be used for the other nostril. For example, the distal portion of the tip can be rotated relative to the handle of the device. 
     The device can have a multi-piece construction. The sample collector can be part of a removable handle. In some cases a portion of the handle can be removed prior to being able to expose the sample collector to the sample solution. In some embodiments a portion of the distal cover can be removed to access the sample collector. 
     The devices described herein can be used with an endoscope to provide additional guidance and visualization to assist the healthcare professional with obtaining a sample from the desired location. 
     After obtaining the sample the device can be removed from the patient followed by contacting the sample collector with a sample solution. The sample collector can be advanced distally past the distal end to contact the sample collector with the sample solution. In some embodiments the sample collector can be withdrawn proximally through an interior of the device followed by contacting the sample collector with the sample solution. In some embodiments the distal cover can be pulled back to expose the sample collector. In some embodiments the distal cover can have a multi-piece construction such that the cover can be removed to expose the sample collector. In some embodiments a separate slider can be used to advance the sample collector to a sample solution position for contact with the sample solution. 
     The device can include a depth gauge to provide information to the user regarding the location of the sample collector, such as the distance the sample collector has been advanced. 
     In some embodiments the device can include a stop or guard configured to engage with the outside of the nose/nostril to prevent further advancement of the device. In some embodiments the stop or guard can be removed by the healthcare worker to provide additional visual guidance and clearance for endoscope 
     The devices can have a naturally retracted position. For example, a compression element could provide a resting force to keep the sample collector in the retracted position. The compression element could pull the sample collector proximally after obtaining the sample in the absence of an actuating force applied by the user. 
     In some embodiments the hand held device can be configured to be disposable after obtaining a sample fluid from the patient. In some embodiments the hand held devices can be configured to be reusable. For example, the device could be sterilized after obtaining a sample fluid and used for subsequent sample collection from a second patient. In some embodiments the handle can be designed to be reused and a new sample collector or other part can be combined with the handle to form a device for obtaining a sample from a second patient. The sample collector could be provided separately as a single use cartridge to be used with the sterilized handle. 
     The sample collector can include a structure to facilitate opening and/or closing of a distal cover. For example, fins or a shoulder can be located adjacent to the sample collector to push open the distal cover and to hold the distal cover open during retraction to prevent sample loss caused by the distal cover squeezing the sample collector. 
       FIGS.  4 - 14    illustrate aspects of various embodiments of the hand held sample collecting devices disclosed herein. 
       FIGS.  4 A- 4 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments.  FIGS.  4 A- 4 C  illustrate a hand held sample collector  200  with a distal section  202  and a proximal section  204 . The distal section  202  includes a distal end  206  configured to be introduced through the nares of the patient. The proximal section  204  is configured to slide relative to the distal section  202  to move the sample collector  210 , illustrated as a swab, relative to the distal end  206 . The device  200  is configured to be gripped with a human hand with a finger grip  212  on the distal portion  202  and a thumb grip  214  on the proximal portion  204 . The device  200  can be operated with one hand such that movement of a thumb on the thumb grip  214  can advance the proximal portion  204  relative to the distal portion  202 . The device  200  includes an opening or window  216  such that a shaft  218  of the proximal portion  204  can be observed. The window  216  can also be used to provide orientation information to the user, such as the lateral direction of the device. The illustrated device  200  includes a depth gauge  220  aid the operator in determining the position of the sample collector  210 . The distal portion  206  can be advanced through the nares to the target location followed by advancing the proximal portion  204  and sample collector  210  relative to the distal portion  206  to contact the sinus fluid. After the sample has been collected, the sample collector  210  is retracted back into the distal portion  206  of the device  200  to shield the sample collector  210  from the sinuses while withdrawing the device  200 . After the sample collector  210  has been retracted within the device  200 , the device  200  can be removed from the patient. The proximal portion  204  can be retracted proximally relative to the distal portion  202  to completely separate the proximal portion  204  and the distal portion  202  for sample testing as shown in  FIG.  4 C . The proximal portion  204  and sample collector  210  can be handled for the sample testing and processing. The sample collector  210  with the sinus sample can be tested using the rapid diagnostic testing methods disclosed herein. 
       FIGS.  5 A- 5 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments.  FIGS.  5 A- 5 C  illustrate a hand held sample collector  300  with a distal section  302  and a proximal section  304 . The distal section  302  includes a distal end  306  configured to be introduced through the nares of the patient. A compression/spring element  308  engages with a shaft  310  connected to the sample collector  312 . The distal section  302  includes positioning markers  314 . The positioning marker  314  can include a marker to provide an orientation of the device to the user, such as the “L” marking on the device  300  indicating the lateral direction. A depth gauge  315  can be included on the distal section  302  to provide depth positioning information to the user. The device  300  can be gripped using the finger grip  316  and compression element  308 . The compression element  308  can be pushed forward to advance the shaft  310  and sample collector  312  distally relative to the distal portion  302  as shown in  FIG.  5 B  to retrieve a sample of sinus fluid. The compression element  308  provides a force to retract the sample collector  312  proximally in the absence of a force applied by the user. The compression element  308  can function as an automatic retraction of the sample collector  312  after a sample of sinus fluid has been retrieved. The compression element  308  can be fully pushed forward to contact the sample collector  312  with the sample solution as shown in  FIG.  5 C . Pushing the sample collector  312  distally out of the device can minimize losses of the collected sinus fluid. 
       FIGS.  6 A- 6 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments.  FIGS.  6 A- 6 C  illustrate a hand held sample collector  400  with a distal section  402  and a proximal section  404 . The distal section  402  includes a distal end  406  configured to be introduced through the nares of the patient. The distal end  406  can be advanced and retracted by an actuator, such as the slider  408 . The slider  408  can slide along the body of the proximal section  404 . The proximal section  404  includes a finger grip  410  and a thumb grip  411 . The thumb grip  411  can pushed to advance the sample collector  412  as illustrated in  FIG.  6 B . The thumb grip  411  can retract in the absence of an applied force to retract the sample collector back within the distal portion  406 . The slider  408  can further retract the distal end  406  to expose the sample collector  412  as shown in  FIG.  6 C  for contact with a sample solution while minimizing sample loss. The slider  408  can provide orientation of the distal end  406  to the user. For example, the distal end can be curved in the same direction/side as the location of the slider as shown in device  400 . The bumps  414  on the distal portion  406  can function as a depth gauge to provide additional positioning and orientation information to the user. 
       FIGS.  7 A- 7 C  illustrate aspects of a device configured to sample sinus fluid in accordance with some embodiments. The hand held sample collector  500  includes a distal section  502  and a proximal section  504 . The distal section  502  includes a distal end  506  with an outer covering  508  having a patterned cut distal end section  510  that allows the sample collector  512  to advance distally past the covering  508 . The covering  508  can be made out of a flexible and biocompatible material such as silicone. The device  500  includes a finger grip  514  and thumb grip  516 . The thumb grip  516  can be advanced to push the sample collector  512  distally past the covering  508  as shown in  FIG.  7 B . The sample collector  512  advances past the patterned cut distal end section  510  to contact the sinus fluid. The sample collector  512  can be retracted and covered while removing the device from the user to prevent contaminating the sample collector with mucous from areas besides the targeted sinus fluid. The thumb grip  516  can include markings or a colored section  518  to provide orientation information to the user, such as the direction of the bend in the distal section  502 . The ridge  520  on the distal section  502  can function as a depth gauge to provide additional positioning and orientation information to the user. The covering  508  can be pulled back to expose the sample collector  512  to the sample solution as shown in  FIG.  7 C . The covering  508  can be patterned or scored to fold back as it is pulled back away from the sample collector  512 . Retracting the covering  508  relative to the sample collector  512  can help minimize the sample loss from the sample collector  512 . 
       FIGS.  8 A- 8 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held sample collector  600  includes a distal section  602  and a proximal section  604 . The distal section has a two piece construction with a first distal sleeve portion  606  and second distal sleeve portion  608 . The proximal section  604  includes a finger grip section  610  and a thumb grip  612 . The finger grip section  610  includes a marking  611  to label the lateral side of the device to let the user know the orientation of the bend in the distal section  602  of the device  600 . The thumb grip  612  can be pushed distally to expose the sample collector  614  to collect a sample of sinus fluid as shown in  FIG.  8 B . The thumb grip  612  can be retracted to retract the sample collector  614  having the sinus fluid sample back within the distal section  602  prior to removing the device  600  from the patient to avoid contaminating the collected sample. The sample collector  614  can be processed after the device  600  is removed from the patient. The sample collector  614  can be exposed to the sample solution by removing the first distal sleeve portion  606  and second distal sleeve portion  608  as shown in  FIG.  8 C . 
       FIGS.  9 A- 9 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held device  700  has a pen type shape with a distal section  702  and a proximal section  704 . The distal section  702  includes a distal tip  706  with a patterned opening  708 . The sample collector  710  can be advanced past the patterned opening by advancing the slider  712 . The device  700  includes two sliders  712  on a central portion  713  of the device. The body of the device  700  can include a marker  714  to provide information on the orientation of the device to the user, such as the direction of the bend of the distal section  702 . The device  700  can include a handle  716  on the proximal section  704 . The device  700  can be gripped by the user using the thumb and middle finger. The slider  712  can have multiple positions. A retracted position is shown in  FIG.  9 A . A partially advanced slider  712  position is shown in  FIG.  9 B  with the sample collector  710  advanced distally past the patterned opening  708 . The slider  712  can be advanced further to expose the sample collector  710  to the sample solution as shown in  FIG.  9 C . The slider  712  has a retracted position, sample position, and testing position. The device  700  can include a lock at each of the retracted position, sample position, and testing position. The slider  712  can be configured such that both sliders  712  are pushed to allow movement of the slider  712 . The device  700  can be used for either nostril by rotating by 180 degrees. The distal tip  706  can be made out of a soft polymer material like silicone to be comfortable for the patient. The shape of the engagement  720  between the distal tip  702  with a handle portion can be contoured as shown in  FIGS.  9 A- 9 C  such that the contour of the engagement  720  can provide depth and orientation information to the user of the device. The patterned opening  708  can open like an alligator jaw to protect the sample collector  710  from being contaminated by nasal mucous or bacterial or cross contamination while the device is advanced in the nostril or retracted from the nostril after sampling. The device can include a shoulder or fin  718  adjacent to the sample collector  710  to facilitate opening of patterned opening  708 . The shoulder or fin  718  can push open the patterned opening  708  and also assist with pushing open and holding open the patterned opening  708  when the sample collector  710  is retracted to minimize sample loss. 
       FIGS.  10 A- 10 D  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held device  800  has a distal section  802  and proximal section  804 . The proximal section  804  includes a handle  806  with a slider  812 . The distal section  802  includes a distal portion  808 . The distal portion  808  includes an orientation marker  810  to display the orientation of the bend in the distal portion  808 . The distal portion  808  can be rotated 180° relative to the handle  806  so that the device  800  can be used for either nostril. The distal portion  808  can be made out of a soft material such as silicone to improve patient comfort. The slider  812  can be advanced to the sample mark  814  to distally advance the sample collector  816  past the distal portion  808  as shown in  FIG.  10 C . The sample collector  816  can be further advanced using the slider  812  as shown in  FIG.  10 D  to contact the sample solution. 
       FIGS.  11 A- 11 C  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments.  FIGS.  11 A- 11 C  illustrate a hand held sample collector  900  with a distal section  902  and a proximal section  904 . The distal section  902  includes a distal end  906  configured to be introduced through the nares of the patient. The proximal section  904  is configured to slide relative to the distal section  902  to move the sample collector  910 , illustrated as a swab, relative to the distal end  906 . The device  900  is configured to be gripped with a human hand with a finger grip  912  on the distal portion  902  and a thumb grip  914  on the proximal portion  904 . The device  900  includes a marking  916  to indicate the orientation of the bend in the distal end  906 . The device can include a collar  908  to prevent further advancement of the distal end  906  after it has been inserted in the nares. The device  900  can be operated with one hand such that movement of a thumb on the thumb grip  914  can advance the proximal portion  904  relative to the distal portion  902 . The distal end  906  can be advanced through the nares to the target location followed by advancing the proximal portion  904  and sample collector  910  relative to the distal portion  906  to contact the sinus fluid as shown in  FIG.  11 B . After the sample has been collected the device  900  can be removed from the patient and the proximal portion  904  can be retracted proximally relative to the distal portion  902  to completely separate the proximal portion  904  and the distal portion  902  as shown in  FIG.  11 C . The sample collector  910  with the sinus sample can be tested using the rapid diagnostic testing methods disclosed herein. 
       FIGS.  12 A- 12 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held device  1000  has a pen type shape with a distal section  1002  and a proximal section  1004 . The distal section  1002  includes a distal tip  1006  with a patterned opening  1008 . The sample collector  1010  can be advanced past the patterned opening by advancing the slider  1012 . The device  1000  includes two sliders  1012  on a central portion  1013  of the device. The body of the device  1000  can include a removable depth gauge  1018 . The depth gauge  1018  can be rotated 90 degrees to a major axis of the device  1000  to maximize visualization of the nasal cavity.  FIG.  12 B  illustrates the device  1000  with the depth gauge  1018  removed. The body of the device  1000  can include a marker  1014  to provide information on the orientation of the device to the user, such as the direction of the bend of the distal section  1002 . The device  1000  can include a handle  1016  on the proximal section  1004 . The device  1000  can be gripped by the user using the thumb and middle finger. 
     The slider  1012  can have multiple positions. A retracted position is shown in  FIGS.  12 A and  12 C . A partially advanced slider  1012  position is shown in  FIG.  12 D  with the sample collector  1010  advanced distally past the patterned opening  1008 . The slider  1012  can be advanced further to expose the sample collector  1010  to the sample solution as shown in  FIG.  12 E . Thus the slider  1012  has a retracted position, sample position, and testing position. The device  1000  can include a lock at each of the retracted position, sample position, and testing position. The slider  1002  can be configured such that both sliders  1012  are pushed to allow movement of the slider  1012 . The device  1000  can be used for either nostril by rotating by 180 degrees. The distal tip  1006  can be made out of a soft polymer material like silicone to be comfortable for the patient. The patterned opening  1008  can open like an alligator jaw to protect the sample collector  1010  from being contaminated by nasal mucus or bacterial or cross contamination while the device is advanced in the nostril or retracted from the nostril after sampling. The device can include a depth gauge  1018  adjacent to the sample collector  1010 . The device  1000  can optionally include a safety that makes it difficult to accidentally fully push the sample collector past the sample configuration ( FIG.  12 D ) while the device is deployed in the patient. The device  1000  can allow for improved endoscopic camera access for the user. For example, the depth gauge  1018  can be removed to improve clearance for a camera or for visualization as shown in  FIG.  10 B . 
       FIGS.  13 A- 13 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held device  1100  has a distal section  1102  and a proximal section  1104 . The distal section  1102  includes a distal tip  1106  with a patterned opening  1108 . The sample collector  1110  can be advanced past the patterned opening by advancing the handle  1113 . The body of the device  1100  can include a removable depth gauge  1118 .  FIG.  13 B  illustrates the device  1100  with the depth gauge  1118  removed. The different appearance of the body of the device  1100  and the distal section  1102  can also provide depth and orientation information to the user. The body of the device  1100  can include a marker  1114  to provide information on the orientation of the device to the user, such as the direction of the bend of the distal section  1102 . The device can be held by the handle  1113  and finger grips  1112 . A retracted position is shown in  FIGS.  13 A and  13 C . An advanced handle  1113  position is shown in  FIG.  13 D  with the sample collector  1110  advanced distally past the patterned opening  1108 . The handle  1113  can be removed as shown in  FIG.  13 E . The bottom side of the device  1100  includes a slider  1120  configured to advance the sample collector  1110  to contact the sample solution as shown in  FIG.  13 E . The device  1100  can be used for either nostril by rotating by 180 degrees. The device  1100  can optionally include a safety that makes it difficult to accidentally fully push the sample collector past the sample configuration while the device is deployed in the patient. The device  1100  can allow for improved endoscopic camera access for the user. 
       FIGS.  14 A- 14 E  illustrate aspects of a device configured to sample a sinus in accordance with some embodiments. The hand held device  1200  has a distal section  1202  and a proximal section  1204 . The distal section  1202  includes a distal tip  1206  with a patterned opening  1208 . The sample collector  1210  can be advanced past the patterned opening  1208  by advancing the handle  1213 . The handle  1213  can be advanced and retracted by the user&#39;s thumb. The body of the device  1200  can include a removable depth gauge  1218 .  FIG.  14 B  illustrates the device  1200  with the depth gauge  1218  removed. The different appearance of the body of the device  1200  and the distal section  1202  can also provide depth and orientation information to the user. The body of the device  1200  can include a marker  1214  to provide information on the orientation of the device to the user, such as the direction of the bend of the distal section  1202 . The device can be held by the handle  1213  and finger grips  1212 . A retracted position is shown in  FIGS.  14 A and  14 C . An advanced handle  1213  position is shown in  FIG.  14 D  with the sample collector  1210  advanced distally past the patterned opening  1208 . The handle  1213  can be removed as shown in  FIG.  14 E . The bottom side of the device  1200  includes a slider  1220  configured to advance the sample collector  1210  to contact the sample solution as shown in  FIG.  14 E . In some embodiments the handle  1213  can be configured such that it has to be removed prior to being able to advance the slider. The device  1200  can be used for either nostril by rotating by 180 degrees. The device  1200  can optionally include a safety that makes it difficult to accidentally fully push the sample collector past the sample configuration while the device is deployed in the patient. The device  1200  can allow for improved endoscopic camera access for the user. 
       FIGS.  15 A- 21    illustrates devices configured to sample a sinus in accordance with some embodiments. For example, in  FIGS.  15 A- 15 D , hand held device  1300  has a distal section  1302  and a proximal section  1304 . Hand held device  1300  includes a sample collector  1310  housed within a sleeve  1305  with a sleeve opening  1307 , a main body  1330 , a (thumb ring) handle  1313  and a depth stop  1318 . Sleeve  1305  couples to main body  1330  via a couplers,  1332 . Couplers  1332  can have a double tip lock feature as shown in  FIG.  17 A  or a single locking tip feature as shown in  FIG.  17 B . 
     Sample collector  1310  (e.g., swab) can be advanced past sleeve opening  1307  by the ring handle  1313 , which may be configured to extend the sample collector a predetermined distance from the distal end. This first predetermined distance is configured to extend into the correct sample region (e.g., the sinus, such as the middle meatus or maxillary sinus), while avoiding regions distal to these regions which may otherwise contaminate the sample. Handle ring  1313  includes a connector (shown as a handle ring cavity)  1315  that is able to engage a proximal end of the sample collector  1310  while both elements are retained within main body  1330 . Sample collector includes at least one notch  1311  for coupling to handle ring  1313  as shown in  FIG.  16   .  FIG.  21    shows an alternative design for coupling sample collector  1310  with handle  1313 , which includes one or more notch  1311  (two are shown in this example) that is vertically arranged and parallel to the longitudinal axis of sample collector  1310 . Sample collector  1310  can be advanced past sleeve opening  1307  the first predetermined distance (e.g., between 5 and 20 mm (e.g., between 7 mm and 15 mm, between 8 mm and 12 mm, etc.) by advancing handle  1313 . Handle  1313  can be advanced and retracted by the user&#39;s thumb. Having the handle  1313  limited to only advancing the sample collector  1310  the first pre-determined distance from sleeve opening  1307  may prevent a longer portion of sample collector  1310  from being extended further into the sinus of the subject and causing discomfort and pain, and/or contamination. Once a sample has been collected on sample collector  1310 , sample collector  1310  may be retracted within sleeve  1305 , and device  1300  removed from subject&#39;s sinus and nasal cavity, the sample can be processed for assaying. 
     In both the designs shown in  FIGS.  18 A and  18 B , handle  1313  can be removed. Removal may also release a locking mechanism that prevents the second actuator/control (shown as slider  1312  in  FIG.  15 A ) from extending the sample collector. Removal may be achieved with minimal force by pulling it in an opposite lateral direction from the body of device  1300 . Once handle  1313  has been removed from device  1300 , slider  1312 , shown disposed longitudinally along device  1300  in  FIG.  15 A  can advance the sample collector  1310  beyond sleeve opening  1307  for processing a second predefined distance that is typically further than the first predefined distance. Thus, the distance that slider  1312  can advance sample collector  1310  may be greater than the distance sample collector  1310  can be extended with handle  1313 . This second predetermined distance may allow the sample collector  1310  to be inserted into a sample (e.g., lysis buffer) tube for processing the sample, as described in greater detail below. 
     Any of these devices may also include a centering element  1340  (which may also be referred to a spacer or centering element) between the sample collector and the main body. Spacer  1340  in  FIG.  17 A  is a protrusion on either the sample collector (e.g., near the distal end) or the main body that prevents sample collector  1310  from contacting the inner sides of sleeve  1305  when sample collector  1310  is extended and retracted from sleeve  1305 . Preventing sample collector  1310  from contacting the internal sides of sleeve  1315  is especially important when retracting sample collector  1310  because if the sample-containing sample collector  1310  scrapes past the internal sides of sleeve  1305  as it is being retracted, a portion of the sample collected on that surface of sample collector  1310  will be lost, thus diminishing the amount of sample (e.g., cells or bacteria) available for processing and detection. 
     In the variations shown in  FIG.  22   , hand held sample device  1400  has a distal section  1402  and a proximal section  1404 . Hand held device  1400  includes a sample collector  1410  housed within a sleeve  1405  with a sleeve opening  1407 , a main body  1430 , a (thumb ring) handle  1413  and a depth stop  1418 . 
     Unlike the previously discussed embodiment where the handle is coupled to the sample collector with the main body, and where upon obtaining a sample, the user can disengage the handle by pulling the handle laterally away from the rest of the device, in the variations shown in  FIG.  17   , the device  1400  may include an additional safety feature to prevent inadvertent uncoupling of handle  1413  from sample collector  1410  and main body  1430 . In this variation, main body  1430  includes a channel  1439 . Channel  1439  is of a predetermined length with respect to main body  1430 . In operation, handle  1413  couples with sample collector  1410  and channel  1439  provides is of a predetermined length that handle  1413  can only slide a given distance within main body  1430  when coupled. The predetermined distance corresponds to the distance sample collector  1410  can extend from sleeve  1405 . In any of the device variations described herein, the handle may include a release  1442 . Release  1442 , when engaged with channel  1439 , may allow handle  1413  to extend and retract sample collector  1410  (e.g., the predetermined first length). To release handle  1413 , a user presses and slide release element  1442  to allow release element  1442  to disengage from main body  1430  of device  1400 . Then, similar to other embodiments, device  1400  includes a slider  1420  that can extend sample collector  1410  a second predetermined distance (that may be greater than the first predetermined distance possible when sample collector  1410  is extended by handle  1413 ). 
     Methods of Using Extraction Device 
     Also disclosed herein are methods of using the devices described above, as well as sinusitis assays using them. 
     For example, the following steps can be taken to obtain a sample of a patient&#39;s infected mucous. When the patient is seated or laying down, place the sampling device (or other embodiment of the device) at approximately forty five degrees with respect to the floor. Insert sleeve of the device (e.g., the distal end) into the subject&#39;s nasal passage, ensuring that the angle of sleeve is pointed downward to follow the natural curvature of the nasal passage. Discretion should be used where user experiences resistance when inserting the device into the nasal cavity of the subject. The depth stop on the device may provide a safety measure and prevent the user from inadvertently inserting more of the device into the subject&#39;s nasal cavity than is needed or safe. 
     Next, user pushes the slider forward to expose the swab&#39;s distal end to the sinus middle meatus. In other embodiments, the thumb ring is coupled to the swab element. There, the user may use the thumb ring by inserting his thumb through the thumb ring aperture and sliding the thumb ring forward to expose the distal end of the swab for sampling the sinus region. Once a sample has been collected, the user can retract the sample collector distal end using the slider. In other embodiments, the user can retract the distal end of the swab back into the sleeve by pulling the thumb ring proximal end away from the main body. The device then can be removed from the nasal cavity of the subject and the sample collected can be now tested for bacterial presence. 
       FIGS.  25 A- 25 K  illustrate another method of operating the sinus mucosal sample devices described herein. In  FIG.  25 A , the sinus collection device is inserted into the patient&#39;s nose by placing the device (probe) at an approximately 45 degree angle relative to the floor (shown by arrows), with the patient in an upright, sitting position. As shown in  FIG.  25 B , the collection device is then placed into the sinus by inserting the sinus collection device tip through the nasal cavity and into sinus middle meatus until the depth stop of the device is engaged (e.g., the stop touches nasal entry), or until a slight tissue resistance is felt.  FIG.  25 C  illustrates the collection device properly positioned into the middle meatus region. 
     After positioning the collection device, as shown in  FIG.  25 D , the thumb ring may be pushed to expose the swab tip and collect specimen, by pushing in the direction of the arrow to expose swab tip to sinus fluids. The tip of the swab is extended a first predetermined distance into the proper region of the sinus, as shown in  FIG.  25 E , showing the swab tip exposed for fluid collection. Thereafter, the swab tip may be retracted into collection device by reversing the thumb motion and retracting the swab tip into collection device, as shown in  FIG.  25 F .  FIG.  25 G  shows the swab tip fully retracted into Collection device. Thereafter, the collection device may be removed from the nose, as shown in  FIG.  25 H . The collection device, once removed from nose, is ready for assay testing, which may be performed in a physician&#39;s office or other lab area. 
     For processing, the distal controller, e.g., the distal handle or thumb loop, may be removed, as shown in  FIG.  25   i   . Removing the handle in any of these devices may release a limiter or lock that prevents the swab from extending distally. The collection device may be fully or partially extended before testing the sample. For example,  FIG.  25 K  shows a device with the swab fully extended for processing; the swab portion (circled) was fully extended by sliding the second (distal) controller distally. Once fully extended, the swab may be processed by inserting into a buffer (e.g., lysing buffer) as described in greater detail below. 
     Sample Assay 
     Also described herein are assays that can be used to detect and diagnose bacterial sinusitis. More specifically, the assays may utilize antigen binding agents (e.g., antibodies, antibody fragments, etc.) for detecting markers specific to the types of bacteria contained with the sinus secretions of the subject. The methods disclosed herein may allow detection of signature antigens that are associated with specific bacterial pathogens within the paranasal sinus cavity, and may thus allow a caregiver better insight as to whether prescribing an antibiotic is beneficial. The assays may also provide information that aids a caregiver in deciding which antibiotic regimen would provide the most favorable outcome and most importantly, reduce the use of broad-spectrum antibodies in cases where such treatment would not effective. 
     These assays may utilize biomarkers that are specific antigens indicating the presence of the organism or pathological process of interest. “Biomarkers” are naturally occurring molecule, gene, or characteristic by which a particular pathological or physiological process, disease, or the like can be identified or characterized. The term “biomarker” may refer to a protein measured in sample whose concentration reflects the severity or presence of some disease state. Biomarkers may be measured to identify risk for, diagnosis of or progression of a pathological or physiological process, disease or the like. Exemplary biomarkers include proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA and combinations thereof. Although the examples of assays described herein are specific to antigen binding agents such as antibodies in a sandwich-type lateral flow assays, other assays, including nucleotide hybridization, enzymatic and ligand-receptor type assays may also or alternatively be used. 
     In some variations, the assay is capable of detecting at least one biomarker, and more preferably two biomarkers, including biomarkers from each of a plurality of bacterial types linked to sinusitis. The assays can be further modified to detect greater than two biomarkers (e.g., preferably three). Furthermore, detecting the biomarkers can mean detecting a portion of the proteins, hormones, prohormones, lipids, carbohydrates, DNA, RNA and combinations thereof. The biomarkers may also be a biologically active variant of the naturally occurring molecule of interest. For example, a protein or DNA biomarker can have at least 65%, at least 70%, at least 80%, at least 85%, 86%, 87%, 88%, or 89%, and more typically 90%, 91%, 92%, 93%, 94%, and most common, 95%, 96%, 97%, 98% or 99% conformity or sequence identity to the native molecule. 
     Assays and Kits 
     Any of the assays described herein may be part of a kit that allow a user to easily perform the assays for detecting antigens that are the primary cause bacterial sinusitis. The kits may include the sampling device that is described above. The kits can also include a means for lysing the cells in order to expose the target antigens of interest, such as a lysis buffer, and a means for delivering the lysed supernatant to the assay portion of the kit. 
     A first critical step in obtaining accurate results is in properly processing the sample extracted from the sampling device. Proper processing includes formulating an appropriate lysis buffer. While finding a lysis buffer that can lyse one particular type of cell is fairly straightforward, it is much more challenging to prepare a buffer composition that is able to lyse multiple bacterial cells of interest while protecting the bioactivity of the antigens of interest and while lysing in a reasonable amount of time (e.g., less than 15 minutes). Most cells can be lysed by mechanical means, such as sonification or freeze/thaw cycles, but such methods may require additional equipment. Thus, in some instances, it is preferable to use milder methods, such as detergents, for disrupting the cell membrane. Detergents may disrupt the lipid layer surrounding cells by solubilizing proteins and interrupting the lipid-lipid, protein-lipid, and/or protein-protein interactions. The appropriate detergent composition also depends upon the type of cells to be lysed, be it animal, bacteria, or yeast. 
     In the developing the “triple” assay for detecting one or more of  M. catarrhalis, S. pneumoniae , and  H. influenzae  described herein, various lysis buffers were tested for their ability to lyse all of the bacterial cells of interest, namely  M. catarrhalis, S. pneumoniae , and  H. influenzae . For example, while N-Lauroylsarcosine effectively lysed NTHI and exposed antigens for recognition by their antigens, it did not effectively lyse  M. catarrhalis . Next, TritonX-100, a commonly used lysis buffer was also tested and appeared to lyse  M. catarrhalis , but did not work well with NTHI (nontypeable  Haemophilus influenzae ) The inventors determined that the addition of sucrose to a lysis buffer containing N-Lauroylsarcosine (e.g., Sarkosyl) was effective in lysing all three bacterial cell lines of interest. Without being limited to a particular theory of operation, the addition of an appropriate percentage of sucrose to the N-Lauroylsarcosine lysis buffer may provide an osmotic shock to the cell membranes of the more lysis-resistant cell membranes to achieve appropriate lysing. 
       FIG.  27    illustrates various examples of lysis buffers that have been examined, indicating their effectiveness with different types of the bacterial strains of interest. Although many of the buffers identified are effective in lysing one of the types of bacteria, in  FIG.  27   , only the 7% sucrose with 1.3% Sarkosyl was effective (and efficient) in lysing all three of  M. catarrhalis, S. pneumoniae , and  H. influenzae . For example, 0.1% Triton X-100 with lysozyme effectively/efficiently lysed  M. catarrhalis  but not  S. pneumoniae  or  H. influenzae , while 1% Sarkosyl effectively/efficiently lysed  H. influenzae  but not  S. pneumoniae , and  M. catarrhalis . None of B-PER reagent, 0.1% Triton X-100 without lysozyme, RIPA buffer, 0.1% Tween 20, 0.1% IGEPAL, 0.1% Tergitol or 0.1% Brij  35  was sufficiently effective in lysing any of  M. catarrhalis, S. pneumoniae , and  H. influenzae . Thus, it was surprising that only the combination of sucrose with Sodium lauroyl sarcosinate, (e.g., 7% sucrose with 1.3% Sarkosyl) was able to lyse all three types of bacterial cells at near-comparable levels. Although only 7% sucrose with 1.3% Sarkosyl, other combinations of sucrose (e.g., between 3% and 15% sucrose, and between 0.5% and 3% Sarkosyl, etc.) may be effective. Thus, the combination of osmotic and anionic detergent disrupting cell wall appears to be most effective. 
     Thus, in one variation, a lysis buffer appropriate for use with all of  Haemophilus influenzae, Moraxella catarrhalis  and  Streptococcus pneumoniae  includes between 5-15% sucrose (e.g., 7% sucrose), EDTA, PMSF, 1.3% sarkosyl (Sodium lauroyl sarcosinate), 50 mM Tris at a pH of 8.0. 
     The assay may include antigen binding agents (e.g., antibodies, antibody fragments, etc.) that specifically bind the protein biomarker of interest and components for immunoassay to detect the protein biomarkers using associated antibodies. The kits can also contain instructions on carrying out the sampling, performing the assay, and any of the methods associated with this invention. 
     The present invention provides for methods for detecting at least one biomarker that is specific to a biofilm protein profile for a pathogenic bacteria. In general, immunological methods are well-known in the art, and performed routinely for diagnostic and research purposes. ELISA (enzyme-linked immunosorbent assay) is a powerful tool in studying antibodies and antigens and their concentrations in a sample. ELISA can be used to detect the presence of antigens that are recognized by an antibody or conversely, ELISA can be used to test for antibodies that recognize an antigen. An immunoassay that utilizing the ELISA platform is the two antibody sandwich ELISA. 
     Sandwich ELISA is used to determine antigen concentration in unknown samples. If a pure antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. Sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or a batch of affinity-purified polyclonal antibodies. A purified first antibody (the capture antibody) is bound to a solid phase. A sample containing the corresponding first bacterial antigen is added and allowed to complex with the bound first antibody. Unbound first bacterial antigen is washed away and a second antibody with a label (the detecting antibody) is allowed to bind to the first bacterial antigen, thus forming a “sandwich”. The assay is then either quantitative or qualitative amount of the second/detecting antibody bound. It is also possible to first bind the antigen to the labeled/detecting antibody and then expose the antigen-labeled antibody complex to the bound antibody. 
     The present invention allows for greater than one sandwich ELISA assay, in order to concurrently detect one or more of  Haemophilus influenzae, Moraxella catarrhalis  and  Streptococcus pneumoniae , which together may account for &gt;90% of bacterial sinusitis. In some variations, additional pathogens may also be detected (e.g.,  Pseudomonas aeruginosa ). In some embodiments, the assay contains two, three, or more distinct antigen and antibody pairings such that more than one antigen can be detected with one single assay. The results of the presence of at least one or more antigen can be qualitatively obtained, meaning that there is a threshold concentration of the targeted antigen or antigens within the sample. Also, the assay can provide more quantitative measure of the presence of one or more antigens by comparison with a standard or reference. A standard or reference refers to a sample that has a known antigen and biomarker, and in some cases, a known concentration of the known antigen and biomarker or antigens and corresponding biomarkers of interest. 
     An optical (including, but not limited to visual) indicator may be used to indicate the presence of an antigen. The visual indicator is typically displayed on a region of the assay. The visual indicator can be colorimetric. The visual indicator can also be a symbol, such as a line, that indicates the presence of a particular antigen. The immunoassay may contain labeling next to the regions where different indicators for the presence of various antigens will be shown. Having the labeling will alert the user as to which particular antigen is or antigens are present with the sample. A caregiver user will then have a better knowledge as to which antibiotic, if any, should be provided to the subject. 
     The immunoassay can be of any suitable format. In some examples, the immunoassay can be performed using a dipstick format where the sample solution is drawn up the “dipstick” type assay with capillary action. The immunoassay can also have a largely horizontal format such as a lateral flow assay. In this latter formats, the sample extracted from the subject&#39;s nose is treated such that the cells are lysed in an appropriate buffer, freeing the proteins and providing the sample solution. An aliquot of the sample solution then can be placed in a sample reservoir on the assay or other region noted on the assay and migrated across regions of the assay that contain bound antibodies. Furthermore, the dipstick or flat format immunoassays can have a solid support made of any suitable material, such as nitrocellulose or polyvinylidene difluoride (PVDF) or other membranes, dipstick, wells, or tubes. 
     In this present example, a combined immunoassay for three different antigens are described. The three antigens of interest and described below are all associated with bacterial sinusitis, but the overall concept of having a multiple antigen test on one assay can be applied to other antigen/antibody systems as well. An example of a lateral flow assay  1500  is shown in  FIGS.  24 A- 24 D . Lateral flow assay  1500  includes a housing  1509  having a sample inlet  1501  (e.g., sample port) for depositing an aliquot of sample and detection window  1511  for visualizing the results of the assay. A sample pad  1503  a conjugate pad  1505 , and a detection pad  1507  are included within a housing  1509 . Sample pad  1503  will hold the sample to be run through the assay. Sample pad  1503  is in contact with conjugate pad  1505 . Conjugate pad  1505  contains the first bacterial antigen binding agent (e.g., shown here as an antibody) specific for at least one antigen. In some examples, the conjugate pad may contain two distinct antigen binding agent (e.g., antibodies) that recognize and bind to two distinct antigens. In other examples, the conjugate pad may contain three distinct antigen binding agents that recognize and bind three distinct antigens. When a sample is run through sample pad  1503  past conjugate pad  1505 , any antigen or antigens of interest will bind to corresponding antibodies. The antibodies contained in conjugate pad  1505  may be linked to a detector molecule. The complex or complexes of antigen with detector-labeled antigen binding agent is then delivered across detection pad  1507  where a second bacterial antigen binding agent (e.g., antibody or antibodies) corresponding to the one more antigens are immobilized on a solid support. In this example, distinct antibodies are affixed in strips across detection pad  1507  as shown in  FIGS.  24 B and  24 C . Thus, when the corresponding antigen complexed with detector-labeled antibody is eluted past the different regions of detection pad  1507 , the antigen complexed with detector-labeled antibody will bind to the corresponding second immobilized antibody and produce a signal as shown in  FIGS.  24 C and  24 D . In any variation described herein, the pattern of the immobilized antigen binding agent may be any appropriate pattern and/or density. For example, one (or preferably all) of the bound antigen binding agent may be arranged on the solid phase substrate into a character, symbol, letter, word, pictogram, etc. In some variations the antigen binding agent is arranged into a letter (e.g., spelling the initial or type of bacteria (e.g.,  H. flu, M. cat, S. pneumo , etc.). 
     For example, an antigen profile for NTHI may include outer membrane proteins (OMP), specifically, OMP P5 and OMP P2. It has been verified that presence of OMP P5 and OMP P2 within NTHI biofilm supernatant and thus detection of OMP P5 and OMP P2 with a sample is indicative of NTHI infection. Corresponding antibodies were developed to both OMP P5 and OMP P2. For  M. catarrhalis , antibodies to Protein C and Protein D outer member proteins (OMP-CD) may be used. For  S. pneumoniae , the PsaA (pneumoccal surface adhesion A) protein may be a viable antigen to indicate the presence of  S. pneumoniae . PsaA is a surface-exposed common 37-kilodalton multi-functional lipoprotein detected on all known serotypes of  Streptococcus pneumoniae.    
       FIGS.  31  and  32    illustrate example of other types of cartridge configurations that may be useful. For example  FIG.  31    illustrates schematically how three different assays for different bacteria may be combined into a single assay, using a single lysis buffer for all three types of bacteria. In this example, the cartridge  3100  includes a single port  3103 . The cartridge also includes a window  3109  into an inner region of the cartridge, showing the solid phase substrate on which the assay is being run. 
       FIG.  32    is another variation of cartridge in which each assay is run in parallel and the report out includes three separate windows and/or three separate solid phase substrates. Both of the examples shown in  FIGS.  31  and  32    include a control band that is formed as a positive control that the labeled antigen binding agent has diffused through the device. As mentioned above, any of these device may include a vent or opening at the end opposite to the cartridge. 
     Example 1 (Lysis Buffer) 
     As shown in  FIG.  27   , different lysis buffers were tested to determine the best buffer composition for successfully lysing the cells corresponding to  Haemophilus influenzae  ( H. flu ),  Moraxella catarrhalis  ( M. cat ) and  Streptococcus pneumoniae  ( S. pneumo ). Some of the potential candidates tested included N-Lauroylsarcosine, Triton X100, Sarkosyl, Sarkosyl and sucrose, and Bugbuster (Novogen). Standardized samples with known concentrations of  Haemophilus influenzae, Moraxella catarrhalis  and  Streptococcus pneumoniae  were used to test the effectiveness of each of these buffers. Based on the lysing data, the sucrose and Sarkosyl lysis buffer composition appeared to be the most effective in being able to lyse all three cell types. Importantly, many combinations (including combinations not including sarkosyl and sucrose) did not work for all three cell types and therefore may not be compatible with a combined assay for detection of all three cell types. 
     Thus, in general, only lysis buffers having an osmotic agent (e.g., sucrose) and an anionic surfactant (sarkosyl, sodium lauroyl sarcosinate, which may be referred to as an ionic surfactant, anionic detergent or ionic detergent) was compatible with the assays for all three of  M. cat, S. pneumo , and  H. flu . In particular, lysis buffers having an osmotic agent between 0.1% and 15% (w/w or w/v, e.g., between 0.5% and 12%, 0.5% and 10%, etc.) and an anionic surfactant between 0.01% and 5% (w/w or w/v, e.g., 0.05% and 5%, 0.1% and 5%, 0.05% and 3%, etc.) were effective, whereas other lysis buffers having non-ionic detergents/surfactants, enzymatic agents, or either osmotic agents alone or anionic/ionic surfactants alone were not effective. For example the lysis buffer may include an osmotic agent within a range having a lower value of 0.1%, 0.2%, 0.3%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, and an upper value of 1.3%, 1.5%, 1.7%, 2%, 2.5%, 3%, 4%, 5%, 7.5%, 10%, 12%, 15%, 20%, etc., where the lower value is less than the upper value, and an anionic detergent within a range having a lower value of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc., and an upper value of 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, etc., where the lower value is less than the upper value. Examples of anionic surfactants (e.g., detergents) include alkylbenzenesulfonates, sulfates, sulfonates, and phosphate esters, including in particular sarkosyl (sodium lauroyl sarcosinate). Surprisingly, only lysis buffers containing the combination of an osmotic agent and an anionic surfactant within the specified ranges were compatible for use in the assay looking for epitopes specific to each of the three cell types ( M. cat, S. pneumo, H. flu ). 
       FIG.  28    illustrates two exemplary lysis buffer that may be used (and were used to generate exemplary readings in a lateral flow assays, such as the ones shown in  FIGS.  30 A- 30 C , and may be used as part of the assay (e.g., kits, systems, etc.) described herein.  FIG.  29    illustrates two exemplary dilution buffers used as part of the assay (e.g., kits, systems, etc.) described herein. 
     Example 2 (Swabbing Material Selection) 
     Experiments testing the optimal sampling swab material was also performed. Because the region where the sample is to be collected, a subject&#39;s nasal and sinus cavity, is a fairly sensitive area, it is important to be able to quickly and effectively gather enough sample material for assaying. Also, it would be desirable only sample the subject&#39;s nasal and sinus cavities once because repeated sampling can cause irritation to the subject&#39;s nose and sinus cavities. In addition, any attempts to gather sample after a first try may elicit an autonomic response of excess mucous in the nasal passage that may dilute the sample collected or blood. While materials as cotton swabs and gauze can be used, two commercially available swab materials were tested for their ability to quickly take up sample. Hydraflock, Ultraflock, and Purflock were tested for their ability to take up water, ATS-M (artificial test soil) lab soil containing mucin, and bacteria in ATS lab soil in a given time interval. In addition to the uptake analysis, materials were also analyzed for their ability to release sample. Hydraflock showed 100% recovery and release at 10 and 30 seconds. In contrast materials such as Purflock showed only 85% recovery and release. 
     Thus, in general, the sample collector (swab or swab material) may be a flocked material that is coupled to a shaft (e.g., an extendable and/or slideable shaft, rod, member, etc.). Flocked materials may minimize entrapment of the cells, while efficiently absorbing them for later release. In particular, flocked swabs having split (e.g., bifurcated, or multiply-split) distal ends of the fibers, such as the hydroflock material described above work surprisingly well, even compared to other flocked materials. 
     Example 3: Assay 
     Following sampling with a device as described above for the collection device, the swab with the sample is inserted into the lysis buffer by fully extending the swab tip and inserting into an appropriate volume of lysis buffer, as shown in  FIG.  26   . In this example, the swab positive end goes into a tube with the lysis buffer (the swab may be the swab of a sample device, such as shown in  FIG.  25 K ). The swab may be mixed or agitated in the buffer, and removed (e.g., after between 1-30 sec). The bacteria may be lysed within the sample buffer by the action of the lysing buffer. See  FIGS.  28  and  29    for examples of lysing buffer ( FIG.  28   ) and dilution buffer ( FIG.  29   ). The sample buffer may then be directly applied to the assay or it may be diluted (e.g., 1:10) in the lysis buffer or a second buffer (a dilution buffer, e.g., a Tris buffer), which may aid in wicking on the membrane. 100-200 microliters of sample may then be loaded into the port on a lateral flow cartridge, possibly followed by loading an additional volume (e.g., 100-200 microliters) of buffer to induce wicking (as a “chaser”). An assay configured similar to that shown in  FIGS.  24 A- 24 D  and described above may be used (e.g., a lateral flow assay) in which there are three separate regions arranged in sequence (adjacent each other) followed by a control region. This configuration is one variation of a multiplexed arrangement. Alternatively or additional one or more of the bound antibody-containing regions (sensing/detection regions) may be present in a parallel region that is fed by the same or a different port. In these examples the antigen binding agents are antibodies, including a pair of antibodies is directed to each antigen specific to one of the bacterial types; a tethered (e.g., in the detection region) antibody (e.g., antigen binding agent) and a soluble (detection) antibody (antigen binding agent) which may be linked to a visible marker (readout) such as colloidal gold or a visible dye (e.g., colored latex bead). In some variations the assay may be read in about 5 minutes (e.g., following 2-3 min of lysis or less and 3-5 min on the cartridge or cassette). The time to a positive or negative result (as shown by the positive control, which may be cross-reactive to the detection antibody, e.g., tethered anti-mouse antibody within a downstream control region), may depend on the wicking of the sample in the membrane. Typically after adding the sample to the assay device (“cartridge” or “cassette”), the sample wicks through a sample pad into a conjugate pad, then into the membrane, where the markers may be captured by capture antibody, concentrating antibody also bound to the marker (antigen) and having a readout (e.g., colloidal gold) so that it can be visualized. 
     As mentioned, the capture antibodies may be laid down on the membrane in different characteristic positions (e.g., marked/labeled), and may depend on the wicking capacity of the membrane. 
       FIGS.  30 A- 30 C  illustrate proof-of-concept data showing sensitivity ranges for assays as described herein for detection of each of  S. pneumo, M. cat , and  H. flu , respectively. 
     In  FIG.  30 A , an assay using a first bacterial antigen binding agent that is labeled and that binds to OMP-P2 and/or OMP-P5 and a fourth tethered antigen binding agent that binds specifically to another region of the same antigen (e.g., OMP-P2 and/or OMP-P5) were used. The first and fourth antigen binding agents specific to  H. influenzae  but not  M. catarrhalis  or  S. pneumoniae . In this example, a lateral-flow assay was prepared with these antigen binding agents, a mucosal sample (which may be taken using a sampling device such as the ones shown in  FIGS.  15 A- 23 C  and the steps of  FIGS.  25 A- 25 K ). To generate the curve shown, the sample used may be from a cultured example of the bacteria, so that the concentration may be determined accurately. The sample was re-suspended in a lysis buffer such as the buffer shown in  FIG.  28    (e.g., lysis Buffer #1), so that all three types of bacteria being sampled (e.g.,  H. flu, M. cat , and  S. pneumo ) were lysed appropriate following approximately 30 sec to 1 min of lysis. The solution was then diluted and applied to the sample port  3205  of a lateral flow cartridge such as the one shown in  FIG.  32   , so that it may be fluidically channeled to a conjugation region (e.g., chamber or bead) containing the labeled and untethered first bacterial antigen binding agent. In this example, the antigen binding agent is a monoclonal antibody conjugated to a particle (e.g., colloidal gold or dyed bead) that can be visualized through the window of the cartridge after travelling (e.g., via capillary action) from the conjugation region onto and/or across the portion of the solid phase structure to which the fourth antigen binding agent is tethered. As illustrated in  FIG.  30 A , different concentrations of sample bacteria ( H. flu ) were used to generate the sensitivity curve. In  FIGS.  30 A- 30 C , the detection was performed manually and visually, however higher sensitivity may be achieved by using a reader (e.g., optical reader) as mentioned above. 
     For viruses, such as influenza A and B, the antigen binding agent may include antibodies, F(ab) or F(ab′)2 fragments, aptamers, or fluorescent or biotin labeled nucleic acid probes. 
     In this example, as described above, a single lysis buffer was used for lysing the sample so that multiple (e.g.,  M. cat, H. flu  and  S. pneumo ) bacterial types could be simultaneously tested for from the same sample, even after a very brief lysis (e.g., between 5 seconds and 15 minutes, between 5 seconds and 10 minutes, between 5 seconds and 5 minutes, between 5 seconds and 4 minutes, between 5 seconds and 3 minutes, between 5 seconds and 2 minutes, between 5 seconds and 1 minute, between 5 seconds and 45 seconds, etc., or less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, etc.). The particular composition (and combination) of lysing agents described herein are surprisingly effective at quickly, completely and gently lysing the multiple different types/classes of bacteria without disrupting the antigens or their ability to be recognized by the antigen binding agents used. 
     In  FIG.  30 B ,  M. cat  was detected in parallel (e.g., by loading a sample of the lysed solution into the second port  3207  of the cartridge shown in  FIG.  32   , and  S. pneumo  was detected by loading the solution into the third port  3209  in  FIG.  32   . Alternatively concurrent detection may be performed using a cartridge  3100  such as the one shown in  FIG.  31   . With respect to detection of  Moraxella catarrhalis  ( M. cat ) a pair of antigen binding agents (second and fifth antigen binding agents or just agents) that are specific to the antigen Protein C (an outer member protein) at different portion of the antigen were used; the second bacterial antigen biding agent is labeled as described above (e.g., using colloidal gold), and the fifth antigen binding agent is tethered to a specific region of the solid phase substrate (e.g., a membrane within the cartridge). Similarly detection of  Streptococcus pneumoniae  ( S. pneumo ) in  FIG.  30 C  may be performed using the lateral flow cartridge such as the one shown in  FIG.  32    (applying the sample into the port  3209 ). The antigen in this example is a PsaA antigen that is recognized by both the third and sixth antigen binding agents, where the third bacterial-binding agent is labeled and the sixth is tethered. Thus, the second bacterial-binding agent binds specifically to a second bacterial antigen specific to  M. catarrhalis  but not  H. influenzae  or  S. pneumoniae . Similarly, the third and sixth agents bind specifically to the third bacterial antigen specific to  S. pneumoniae  but not  M. catarrhalis  or  H. influenzae.    
     In  FIGS.  30 A- 30 C , the sensitivities for detection of cells (expressed as colony forming units (CFU)/sample) show thresholds for visual detection between 10 3 -10 5  per 100 μl sample. As mentioned above, this sensitivity may be increased, for example by using a reader to read the cartridge. 
     In some embodiments, the apparatuses (e.g., systems, kits, assays, including lateral flow assay kits) and methods described herein can be used to determine the presence of one or more viruses, such as one or more influenza viruses and/or coronaviruses. Surprisingly, the lysis solutions having the range of anionic surfactant and osmotic agent concentrations described herein have been found to be effective in exposing (also referred to herein as extracting) antigens from one or more types of viruses. In one implementation, the lysis buffer can extract influenza A antigens and/or influenza B antigens from a patient&#39;s specimen (e.g., mucosal sample). When a patient&#39;s specimen is combined with the lysis buffer solution, the lysis buffer can disrupt the viral particles and expose internal nucleoproteins. Thus the same buffer used to lyse and expose antigens to all three of the  H. influenzae, M. catarrhalis  and  S. pneumoniae  cells can also be used to expose antigens specific to one or more types of viruses (e.g., influenza A antigens and/or influenza B antigens). This can be advantageous since the same sample solution (containing the combined patient sample with the lysis solution) can be used for assay detection, thereby simplifying the sample collection process. 
     The time required to sufficiently extract the one or more virus antigens can be within the timeframe for lysing of multiple bacterial types described herein. For example, virus extraction may occur within the brief lysis period for lysing  H. influenzae, M. catarrhalis  and  S. pneumoniae , such as ranging from about 5 seconds and 15 minutes (e.g., 5 seconds to 15 minutes, 5 seconds to 10 minutes, 5 seconds to 5 minutes, 5 seconds to 4 minutes, 5 seconds to 3 minutes, 5 seconds to 2 minutes, 5 seconds to 1 minute, or 5 seconds to 45 seconds) or less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, or less than 30 seconds. a single lysis buffer may be used can be tested for from the same sample, after a very brief lysis (e.g., between 5 seconds and 15 minutes, between 5 seconds and 10 minutes, between 5 seconds and 5 minutes, between 5 seconds and 4 minutes, between 5 seconds and 3 minutes, between 5 seconds and 2 minutes, between 5 seconds and 1 minute, between 5 seconds and 45 seconds, etc., or less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, etc.). The particular composition (and combination) of lysing agents described can be effective at quickly and gently lysing the multiple different types/classes of viruses without disrupting the antigens or their ability to be recognized by the antigen binding agents used in, for example, a lateral flow assay. 
     The lysis buffer is found to be more effective at extracting both bacterial and viral antigens compared to conventional extraction buffers.  FIG.  33    illustrate results from lateral flow assay cartridges used to detect  H. influenzae, S. pneumonia , and  M. catarrhalis , comparing results using two different types of buffers. The result shown in  FIG.  33    are derived from running assays each using one of three sample solutions formed by combining a lysis buffer solution described herein (having 0.01% to 5% (w/w) sarkosyl and 0.1% and 15% (w/w) sucrose (ENTV Buffer) with bacterial specimens of  H. influenzae  (HI),  S. pneumoniae  (SP), and  M. catarrhalis  (MC). Each of the three sample solutions were assayed on single port cartridges (80 μL assay for 20 minutes), which include binding agents adapted to identify the presence of antigens specific to each of  H. influenzae, S. pneumoniae , and  M. catarrhalis , as described herein. A fourth cartridge was run with the lysis buffer solution without a target bacterium (negative solution) as a control. Results indicate that the ENTV lysis buffer solution sufficiently lysed/extracted all three bacteria types ( H. influenzae, S. pneumoniae , and  M. catarrhalis ). 
       FIG.  33    also shows results from running assays on three sample solutions formed by combining bacterial specimens of  H. influenzae, S. pneumoniae , and  M. catarrhalis  with a commercially available influenza A and B extraction solution (i.e., AB Extraction Buffer, AccessBio). Each of the bacterial sample solutions were assayed on single port cartridges which include binding agents adapted to identify the presence of antigens specific to each of  H. influenzae, S. pneumoniae , and  M. catarrhalis , as described herein. A fourth cartridge was run with the viral extraction solution without a target bacterium (negative solution) as a control. Results were obtained using ENTV-DX LFR reader after 15 minutes. These results indicate that the commercially available influenza A and B extraction solution was able to extract antigens specific to  S. pneumoniae , and  M. catarrhalis  bacteria, but was not able to sufficiently extract antigens for the  H. influenzae  bacteria for lateral flow assay detection/identification. The results from the analysis of  FIG.  33    show that a conventional viral extraction solution can extract antigens specific to influenza A and B and may be able to lyse the cells of and extract antigens specific to some bacteria (e.g.,  S. pneumoniae  and  M. catarrhalis ) but not able to lyse/extract antigens of other bacteria (e.g.,  H. influenzae ). 
       FIGS.  34 A and  34 B  show charts indicating assay peak height readings with different run times for samples combined with the lysis buffer assay system (labeled “ENTV Buffer”) described herein compared to samples combined with a conventional viral extraction assay system (labeled “AB Buffer”). Triplicates of archive Flu specimens were run using the ENTV Buffer and results were compared to samples processed using the AB Buffer. Results were obtained using ENTV-DX LFR reader after 10 and 15 minutes. An archive flu specimen (labeled DLS0116241 Flu A(H3)) showed a significant signal increase when processed using the ENTV Buffer. The other samples showed comparable performance between the two buffer systems. The graph of  FIG.  34 B  indicates that there was a slight increase in overall positive signal after an additional 5 minutes of runtime. Visual observation of the AB Buffer after the 15 minutes showed no nonspecific binding, and the reader results back up the observation. 
     The results of  FIGS.  33  and  34 A- 34 B  demonstrate that, surprisingly, the lysis solution when combined with the bacterial and/or viral samples provide a sample solution with conditions that allow all three of the bacterial antigen binding agents (for  H. influenzae, S. pneumoniae , and  M. catarrhalis ) and both viral antigen binding agents (for influenza A and B) for reading in the lateral flow assays. This is surprising since the sensitivity of the binding agents in the solid phase substrates may be highly dependent on the conditions of the solution being run. For example, the pH and composition of one buffer may be optimized for running on an assay having binding sites for particular viruses and another buffer may be optimized for running on an assay having binding sites for particular bacteria. The lysis buffer solutions described herein are found to sufficiently expose antigens in bacteria and viruses, and be successfully run on assays having binding sites for bacteria and viruses. 
     In some implementations, the assay kits are used in point-of-care clinical settings to determine the type of viral and/or bacterial pathogens within a patient&#39;s sample. For example, a patient may come into the clinic with signs and symptoms that may point to either a viral infection (e.g., the flu), bacterial infection (e.g., causing bacterial sinusitis) or both. One or more mucosal samples can then be processed in the lysis buffer (and in some cases, along with dilution buffer) and run on a lateral flow assay, such as described in Example 3 above. One of the advantages of a lysis buffer than can lyse/extract antigens for one or more viruses and one or more bacteria is that a patient&#39;s sample can be combined with a single buffer solution to form a single sample solution for running the assay, thereby simplifying and reducing errors during the collection process. Further, the same patient sample can be used to run bacterial and viral assays, thereby providing results under better controlled conditions compared to assays run with different samples (even if from the same patient). Additionally, one 
     The assays can include any antigen binding agents that bind antigens (e.g., surface proteins) specific to each type of bacteria and/or viruses. In one implementation, the antigen binding agent(s) includes monoclonal or polyclonal antibodies, or antibody fragments (e.g., F(ab) or F(ab′)2 fragments, etc.) or molecules including all or a portion of these. In some cases, pairs of such agents may bind to different portions of the same antigen. An agent specific to each type of bacteria (e.g.,  H. flu, M. cat, S. pneumo ) and/or virus (e.g., influenza A or B) may be bound to a solid phase substrate (e.g., membrane, particle, etc.) and be spatially arranged in the assay and provide specific identification of the bacterium/bacteria and/or virus(es) by visual detection of binding, including by binding an antigen to the tethered substrate and to a labeled agent. In some cases, pairs or pools of antibodies may be chosen to have low cross-reactivity, while allowing comparable detection of the bacterium/bacteria and/or virus(es). A pair or pool of antibodies specific to one or more antigen binding agents can be relatively specific or characteristic of a bacteria or virus. As described above, for example, the binding agent/indicator for  H. influenzae  may bind with specificity to the OMP-P2 and/or OMP-P5 antigen binding site for the pathogen. As another example, the binding agent/indicator for influenza A may bind with specificity to the nucleoprotein antigen binding site of influenza A. Likewise, the binding agent/indicator for influenza B may bind with specificity to the nucleoprotein antigen binding site of influenza B. In another example, the binding agent/indicator for coronavirus (e.g. SARS-CoV, MERS-CoV, or SARS-CoV-2) may bind with specificity to the a nucleoprotein and/or Spike (S1, S2, or RBD subunits) antigen binding site of coronavirus (e.g. SARS-CoV, MERS-CoV, or SARS-CoV-2). 
     The assay kits described herein can have a variety of different cartridge configurations. In some cases, a kit includes one or more viral cartridges, such as one or more of the influenza A and B assay cartridges in  FIGS.  35 - 37   , and one or more separate bacterial assay cartridges. In some embodiments, the kit includes a single cartridge with includes a flow medium/media for viral and bacterial assays. 
       FIG.  35    shows an example of a cartridge configured to test for the presence of two different types of viruses and three different types of bacteria in parallel. The cartridge includes five separate windows and solid phase substrates for five fluidic pathways with corresponding five sample ports. In particular, a first port  3502  can provide access to a first window/solid phase substrate configured to provide a fluidic pathway for detecting the presence of antigens for a first bacteria (e.g.,  H. influenzae ), a second port  3504  can provide access to a second window/solid phase substrate for a second bacteria (e.g.,  M. catarrhalis ), a third port  3506  can provide access to a third window/solid phase substrate for a third bacteria (e.g.,  S. pneumoniae ), a fourth port  3508  can provide access to a fourth window/solid phase substrate for a first virus (e.g., influenza A) and a fifth port  3510  can provide access to a fifth window/solid phase substrate for a second virus (e.g., influenza B). The first solid phase substrate can hold a first bacterial-binding agent that binds specifically to a first bacterial antigen (e.g., of  H. influenzae ) but not to second bacterial antigen, third bacterial antigen, first viral antigen and second viral antigen (e.g., of  M. catarrhalis, S. pneumoniae , influenza A, or influenza B). The second solid phase substrate can hold a second bacterial-binding agent that binds specifically to a second bacterial antigen (e.g., of  M. catarrhalis ) but not to the first bacterial antigen, third bacterial antigen, first viral antigen and second viral antigen (e.g., of  H. influenzae, S. pneumoniae , influenza A, or influenza B). The third solid phase substrate can hold a third bacterial-binding agent that binds specifically to a third bacterial antigen (e.g., of  S. pneumoniae ) but not to the first bacterial antigen, second bacterial antigen, first viral antigen and second viral antigen (e.g., of  H. influenzae, M. catarrhalis , influenza A, or influenza B). The fourth solid phase substrate can hold a first viral-binding agent that binds specifically to a first viral antigen (e.g., of influenza A) but not to the first bacterial antigen, second bacterial antigen, third bacterial antigen, and second viral antigen (e.g., of  H. influenzae, M. catarrhalis, S. pneumoniae , or influenza B). The fifth solid phase substrate can hold a fifth agent that binds specifically to a second viral antigen (e.g., of influenza B) but not to the first bacterial antigen, second bacterial antigen, third bacterial antigen, and first viral antigen (e.g., of  H. influenzae, M. catarrhalis, S. pneumoniae , or influenza A). As described above, the agents (e.g., first bacterial-binding agent, second bacterial-binding agent, third bacterial-binding agent, first viral-binding agent and second viral-binding agent) can be bound to specific regions of the one or more solid phase substrates in the cartridge, and can include corresponding conjugation regions with corresponding agents that are labeled. Although the example shown in  FIG.  35    includes five separate inlet ports, the cartridge may include any number of ports. For example, in some embodiments the cartridge includes a single port providing access to the fluidic paths. 
       FIG.  36    shows another variation of a cartridge that is similar to the cartridge of  FIG.  35    except that the report out includes two separate windows and two solid phase substrates: one for a combined bacterial assay and another for a combined viral assay. In particular, a first port  3602  can provide access to a first window/solid phase substrate configured to provide a fluidic pathway for detecting the presence of antigens for three different bacteria (e.g.,  H. influenzae, M. catarrhalis  and  S. pneumoniae ) and a second port  3604  can provide access to a second window/solid phase substrate configured to provide a fluidic pathway for detecting the presence of antigens for two different viruses (e.g., influenza A and influenza B). Although the example shown in  FIG.  36    includes two separate inlet ports, the cartridge may include any number of ports. For example, in some embodiments the cartridge includes a single port providing access to the fluidic paths. 
       FIG.  37    shows another variation of a cartridge that is similar to the cartridge of  FIGS.  35  and  36    except that the report out includes a single window and solid phase substrate for combined bacterial and viral assays. In particular, a sample port  3702  can provide access to a window/solid phase substrate configured to provide a fluidic pathway for detecting the presence of antigens for three different bacteria (e.g.,  H. influenzae, M. catarrhalis  and  S. pneumoniae ) and for two different viruses (e.g., influenza A and influenza B). Although the example shown in  FIG.  37    includes one inlet port, the cartridge may include any number of ports. For example, in some embodiments the cartridge includes a two or more sample ports. 
     The examples of  FIGS.  35 - 37    can each include a control band that is formed as a positive control that the labeled antigen binding agents have diffused through the device, as described herein. As mentioned above, any of the device may include a vent or opening at the end opposite to the cartridge. 
     It should be noted that although examples of  FIGS.  33 - 37    show embodiments configured to detect influenza A and B viruses and  H. influenzae, M. catarrhalis  and  S. pneumoniae  bacteria, the apparatuses and methods described herein are not limited only to these viruses and bacteria. For example, in some embodiments, the lysis buffer solution may additionally or alternatively be configured to lyse/extract antigens from one or more bacteria different than  H. influenzae, M. catarrhalis  or  S. pneumoniae  and/or extract antigens from one or more viruses different than influenza A or B. Similarly, the solid phase substrate(s) may additionally or alternatively include antibody and/or antibody fragments for binding to antigens of one or more bacteria different than  H. influenzae, M. catarrhalis  or  S. pneumoniae  and/or antigens of one or more viruses different than influenza A or B. In one implementation, the lysis buffer, solid phase substrate(s) and cartridge(s) is configured to detect the presence of one or more coronaviruses (e.g., in addition to one or more of  H. influenzae, M. catarrhalis  or  S. pneumoniae , influenza A and influenza B). Further, the apparatuses and methods described herein can include those that are configured to detect a subset of  H. influenzae, M. catarrhalis  and  S. pneumoniae  bacterial and/or only one of influenza A and B viruses. 
     Another unexpected advantage of the methods and systems described herein is that the same lysis buffer solution can be used to store the patient&#39;s sample for later testing. For example, in some cases sarkosyl is found to stabilize DNA and/or RNA within a patient&#39;s sample for a period of time without the use of a separate DNA or RNA stabilization agent (e.g., RNAprotect manufactured by Qiagen). In some instances, the DNA and/or RNA of the bacteria and/or viruses can be stabilized and stored for weeks or months (e.g., at least 2 weeks refrigerated and at least 6 months when frozen at ≤−65° C.). This may be useful in situations where a backup or confirmation assay is warranted, or if the sample is to be transferred to a laboratory for more extensive testing (e.g., molecular testing, such as a large respiratory panel testing). 
     Example 4: (Point-of-Care Assay) 
     A patient exhibits symptoms that may be indicative of the viral and/or bacterial sinusitis. A mucosal sample is taken from the patient using one of the collection devices described herein and/or an alternative collection device (e.g., a nasopharyngeal swab for influenza testing). The mucosal sample is added to a lysis buffer solution (e.g., having 0.01-5% (w/w) sarkosyl and 0.1-15% (w/w) sucrose) to form a sample solution. After a period of 1 second to about 15 minutes the samples is either applied directly to one or more sample ports of one or more lateral flow assay cassettes (e.g., one of  FIGS.  35 - 37   ), or diluted with one or more additional buffers (e.g., a dilution buffer, such as a Tris buffer) to aid wicking prior to addition to the one or more sample ports, as described herein. Having a single sample solution can simplify the collection/assay process and reduce the chance of errors during the collection/assay process. The results of the combined assay can be used to inform a diagnosis of the flu and/or bacterial sinusitis. The result may be used to determine (or include recommendations for) appropriate medications for treatment. For instance, if the test is positive for any of the three bacterial pathogens and negative for any of the influenza viruses, this may indicate that the patient has bacterial sinusitis and not viral sinusitis. In this case, the patient may be prescribed an appropriate antibiotic and/or steroid regimen to address the pathogenic bacteria. If the test is negative for any of the three bacterial pathogens and positive for any of the influenza viruses, this may indicate that the patient has the viral sinusitis and not bacterial sinusitis. In this case, the patient may not be administered antibiotics and be treated for viral infection symptoms, which may include an antiviral regimen and/or steroid regimen. If the test is positive for any of the three bacterial pathogens and positive for any of the influenza viruses, this may indicate that the patient has viral and bacterial sinusitis. In this case, the patient may be treated with antibiotics, an antiviral regimen and/or a steroid regimen. Thus, these methods can improve antibiotic stewardship as well as provide timely and accurate diagnosis of influenza and/or bacterial sinusitis. In one example, the sample solution is stored and used to perform a confirmation assay using another lateral flow assay device or a different assay device. In another example, the results from the lateral flow assay do not indicate the presence of  H. influenzae, M. catarrhalis, S. pneumoniae , influenza A or influenza B. The sample solution (containing the lysis solution combined with the patient&#39;s sample) is packaged and sent to a laboratory for a large respiratory panel testing to validate result of the lateral flow assay test and determine whether other bacteria and/or viruses are present. 
     When a feature or element is herein referred to as being “on” another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being “directly on” another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being “connected”, “attached” or “coupled” to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being “directly connected”, “directly attached” or “directly coupled” to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature. 
     Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “/”. 
     Spatially relative terms, such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature&#39;s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise. 
     Although the terms “first” and “second” may be used herein to describe various features/elements, these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention. 
     As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/−0.1% of the stated value (or range of values), +/−1% of the stated value (or range of values), +/−2% of the stated value (or range of values), +/−5% of the stated value (or range of values), +/−10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. 
     Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims. 
     The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.