BACKGROUND

[0001] The present invention relates to field of lateral flow immunoassays. More particularly, the invention pertains to an immunoassay that rapidly detects viral infection associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viral infections using MxA proteins.

DESCRIPTION OF RELATED ART

[0002] Coronavirus disease 2019 (COVID-19), caused by novel enveloped single stranded RNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that began in the winter of 2019. Delays in the development and widespread deployment of organism identification assays has led to uncertainty surrounding overall disease burden and community spread, severely hampering containment efforts. COVID-19 illuminates the need for a tiered and coordinated diagnostic approach to rapidly identify clinically significant infections and reduce the spread of disease. Without the ability to efficiently screen patients, healthcare facilities are overwhelmed, potentially delaying treatment for other emergent conditions.

[0003] Acute respiratory infection (ARI) is responsible for more than 100 million adult ambulatory care visits and 29 million pediatric emergency department (ED) visits annually in the United States. Community- acquired bacterial pneumonia (CABP), which is a commonly associated with ARI, is the second most common cause of hospitalization and the most common infectious cause of death in the United States. Viruses cause over 85% of ARIs with seasonal influenza typically affecting 3-8% of the U.S. population each year. Common bacterial infections such as group A streptococcus pharyngitis and CABP comprise the remaining minority of ARIs. Coronavirus disease 2019 (COVID-19), caused by novel enveloped single, stranded RNA coronavirus (SARS-CoV-2), is similar to other novel coronavirus outbreaks causing ARI in humans, such as 2003 SARS-CoV and 2012 MERS-CoV. Although overall COVID-19 mortality rates are low relative to other epidemics (e.g. severe acute respiratory syndrome (SARS), Ebola), older adults and those with chronic disease are at substantially increased risk of morbidity and mortality and increasingly, younger adults (< 50) are requiring critical care hospital resources. The sudden spike in patients requiring ventilator support has overwhelmed local healthcare systems in several areas of the world.

[0004] Molecular pathogen detection using real-time polymerase chain reaction (PCR) has been established as the gold standard for confirmatory diagnosis of SARS-CoV-2 infections. This technology is designed to be a confirmatory test and is not well suited for large-scale screening efforts. Molecular tests have reported limited sensitivity during the first 7 days of symptom onset (ranging from 67-72%), which may be due to low viral loads early in the disease course. The risk for false negative results requires repeat molecular testing, potentially adding days to a confirmatory diagnosis. Furthermore, concerns about testing capacity, including reagent shortages, have resulted in the Centers for Disease Control and Prevention issuing guidelines recommending testing be restricted to only select population (e.g. high risk). This limits front line providers ability to make rapid triage decisions and further strains the healthcare system. Testing limitations can also negatively impact individual patients via delayed treatment and lack of tailored quarantine recommendations.

[0005] Molecular PCR tests require ancillary equipment, complex technical operation by trained professionals in certified labs and are therefore subject to limitations. These include the procurement of expensive materials and equipment. In addition, these assays take 2 to 3 hours to perform once the sample arrives in the lab and total turnaround time can take upwards of 24 hours if the local lab does not have molecular testing capabilities in-house. Considering that specimens often need to be transported to a testing facility, there can be a delay is establishing a diagnosis. A molecular testing strategy is further limited because many patients with, COVID-19 are asymptomatic or have mild symptoms and may never be referred for testing.

[0006] It is often challenging to differentiate viral from bacterial infections. This is especially true in the outpatient setting where access to laboratory diagnostics is expensive, time consuming, and requires several days to produce a result. More recently, many new diagnostic markers have been identified. Several of these markers show great promise to differentiate viral from bacterial infections. Two such proteins include MxA and C-Reactive Protein (CRP).

[0007] Mx proteins are members of the superfamily of high molecular weight GTPases. Accordingly, these GTPases are upregulated by type I alpha/beta or type II interferons (IFN). The Mx GTPases are expressed exclusively in IFN alpha/beta but not IFN gamma treated cells. Type I interferons play important roles in innate immune responses and have immunomodulatory, antiproliferative, and antiviral functions. Human MxA, a 78 kDa protein, accumulates in the cytoplasm of IFN treated cells and inhibits the replication of a wide range of viruses. MxA protein may offer certain advantages as a marker for viral infection over the other induced proteins such as 2’, 5’- oligoadenylate synthetase, because of its lower basal concentration, longer half-life (2.3 days) and fast induction. MxA mRNA is detectable in isolated peripheral blood white blood cells stimulated with IFN within 1 to 2 hours of IFN induction, and MxA protein begins to accumulate shortly thereafter.

[0008] Studies have shown that MxA protein expression in peripheral blood is a sensitive and specific marker for viral infection. The higher MxA levels in the viral infection group compared with the bacterial infection group can be explained by the fact that the MxA protein is induced exclusively by type I IFN and not by IFN-gamma, IL-1, TNF-alpha, or any of the other cytokines by bacterial infection. Seram type I IFN levels remain within normal limits, even in patients with severe bacterial infections. Active viral replication later results in hyperproduction type I IFN and influx of neutrophils and macrophages, which are the major sources of pro-inflammatory cytokines. With similar changes in total neutrophils and lymphocytes during COVID19, SARS-CoV-2 likely induces delayed type I IFN and loss of viral control in an early phase of infection.

[0009] Similarly, most viral infections have been reported to cause little acute phase response, and low C-Reactive Protein (CRP) concentrations have been used to distinguish illnesses of viral origin from those of bacterial etiology. Because the plasma concentration of CRP increases rapidly after stimulation and decreases rapidly with a short half-life,

CRP can be a very useful tool in diagnosing and monitoring infections and inflammatory diseases. In Scandinavia, point of care CRP testing is part of the routine evaluation of patients with respiratory infections in general practice, and its use has proved cost- effective. In general practice, CRP is found valuable in the diagnosis of bacterial diseases and in the differentiation between bacterial and viral infections. Often the diagnostic value of CRP is found superior to that of the erythrocyte sedimentation rate (ESR) and superior or equal to that of the white blood cell count (WBC).

[0010] Clinically, it can be challenging to differentiate certain systemic viral and bacterial infections. Bacterial cultures are usually performed in cases of severe infection such as pneumonia, or when the consequence of missing a diagnosis can lead to severe complications, such as with Strep throat. Often times, cultures are difficult to obtain. Unfortunately, viral cultures are not routinely performed due to the significant time delay in receiving results. New viral screening PCR panels are useful but they are expensive and do not provide information at the point of care.

[0011] Unfortunately, the rt PCR tests have experienced a high level of false negatives and in a study in China 15 out of 51 patients that were diagnosed as positive for COVID- 19 by chest x-ray were negative on a rt PCR test, but then were positive 1-7 days later (Fang Y, Zhang H, Xie J, et al. Sensitivity of chest CT for COVID-19: comparison to RT- PCR. Radiology. Published online Feb 192020; https://doi.org/10.1148/radiol.2020200432. Accessed April 28, 2020). Patients are infectious during the early stages of disease, but being missed by available testing methods.

[0012] Symptomatic patients are routinely being treated as presumed positive and put alongside other patients that do have SARS-CoV-2 infections. However, if the presumed positive patient had a bacterial infection rather a SARS-CoV-2 infection, it would be 24 hours or more before a negative result would be confirmed. In addition, a negative result might not be believed due to high number of false negatives in rt PCR testing. Therefore, many patients that do not have SARS-CoV-2 infections will be dangerously exposed to those that do. A new test that can reliably detect infection at the early symptomatic infection is needed.

SUMMARY

[0013] The nature of the problem requires a detection that is specific and sensitive to viral infection at much earlier than the stage that is currently detected by reverse transcription polymerase chain reaction (rt PCR). According to one embodiment of the present invention, a multi-tiered, diagnostic strategy incorporating: a rapid host immune response assay as an initial screening test; a rapid immunoglobulin M (IgM), immunoglobulin G (IgG) and/or immunoglobulin A (IgA) test; and molecular confirmatory testing, for example via real-time reverse transcription polymerase chain reaction is used for confirmation of SARS-CoV-2. The rapid host immune response assay provides information for early screening and triage of patients with symptoms. If a patient is positive for a rapid host immune response for viral infections is positive the treating physician can make treatment decisions quickly and responsively far faster than if pathogen specific assays alone are used. Symptomatic patients that are negative for an assay for a rapid host immune response for viral infections may be separated from SARS- CoV-2 patients. An active viral or bacterial infection may be differentiated from a subclinical/colonized state for a respiratory infection by detecting host immune responses in association with bacterial or viral specific pathogens.

[0014] The rapid immunoglobulin testing is used to assess which patients would benefit from quarantine or further testing and provide information on population exposure/herd immunity and determines if the patient’ s body is responding to a viral or bacterial infection. The strategy provides both a comprehensive screening and diagnostic testing strategy. The ability to triage patients within minutes rather than hours or days is a critical component to pandemic response as reliance on the existing single test strategy is limited by the accuracy, time to results, and testing capacity. There is an urgent need to implement a multi-tiered strategy to combat the current outbreaks. The above strategy improves response time to effective action, especially during a pandemic situation.

[0015] Optionally, additional testing for certain respiratory viruses can also be conducted. The respiratory viruses can include, but is not limited to influenza A, influenza B, respiratory syncytial virus (RSV) and severe acute respiratory syndrome (SARS). Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0016] In one embodiment, the rapid host immune response assay is a lateral flow assay, although other assay formats may be used. In a preferred embodiment, the lateral flow assay includes one strip testing for the rapid serological host immune response and immunoglobulin testing of one or more of IgG, IgM and IgA for SARS-CoV-2 combined with MxA. Alternatively, the lateral flow assay can include two strips, a first strip for the rapid MxA host immune response and a second strip for immunoglobulin testing of one or more of IgG, IgM and IgA. The strips can be read using an electronic reader or by the human eye. The strips are preferably disposable and for one-time use. The strips are also preferably readable either by electronic reader or the human eye in 10 minutes or less. In another embodiment, the strips are preferably readable either by electronic reader or the human eye within 10 minutes of collection of the sample from the patient. Either the first strip, the second strip, or portions of the first strip and the second strip can also include testing for influenza A, influenza B, SARS and/or RSV. In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or RSV in the same cassette as the first strip and the second strip. In yet another embodiment, testing for influenza A, influenza B, SARS and/or RSV is on the same strip as MxA. Other viruses can also be tested for, such as, but not limited to: parainfluenza vims, metapneumo virus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0017] In another embodiment, an oral test combines SARS-CoV-2 IgA and /or IgG and oral MxA. Additionally, the SARS-CoV-2 IgA and/or IgG could be combined with SARS-CoV-2 antigen detection. Alternatively, the lateral flow assay can include two strips, a first strip for the rapid MxA host immune response and/or direct SARS-CoV-2 antigen and a second strip for immunoglobulin testing of one or more of IgG and IgA. The strips can be read using an electronic reader or by the human eye. The strips are preferably disposable and for one-time use. The strips are also preferably readable either by electronic reader or the human eye in 10 minutes or less. In another embodiment, the strips are preferably readable either by electronic reader or the human eye within 10 minutes of collection of the sample from the patient. Optionally, either the first strip, the second strip, or portions of the first strip and the separate strip can include testing for influenza A, influenza B, SARS and/or RSV. In another embodiment, the strip containing oral MxA also includes testing for the presence of influenza A, influenza B, SARS and/or RSV. [0018] In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) in the same cassette as the first strip and the second strip.

[0019] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0020] In another embodiment, the test for MxA and immunoglobulin testing of one or more of IgG, IgM and IgA for SARS-CoV-2 is a chemiluminescent assay. In the chemiluminescent assay, beads or nanoparticles are coupled with a deactivated antigen or protein from the antigen for MxA and one or more of IgG, IgM and IgA for SARS-CoV-2, any antigen in the sample for MxA and one or more of IgG, IgM and IgA for SARS-CoV- 2 binds to the deactivated antigen of the assay and is immobilized to the plate. Enzyme labeled antibodies are added and bind to the antibodies of the sample. An enzyme substrate is added and causes a light producing chemical reaction which can then be measured. In addition, the chemiluminescent assay can include testing for influenza A, influenza B, SARS and/or RSV. In another embodiment, the assay is a fluorescent immunoassay. Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0021] In yet another embodiment, the rapid host immune response assay is a lateral flow assay, although other assay formats may be used. In a preferred embodiment, the lateral flow assay includes one strip testing for the rapid serological host immune response and detection of a specific virus. For example, a lateral flow test strip can include MxA and detection of the presence of influenza A, influenza B, SARS and/or RSV. Alternatively, the lateral flow assay can include a second test strip for at least one level of C-reactive protein (CRP). In another embodiment, the assay is a chemiluminescent immunoassay or a fluorescent immunoassay.

[0022] The rapid host immune response test uses a first biomarker to determine whether the host immune response is to bacteria and a second biomarker to determine whether the host immune response is to a virus. The first biomarker to determine whether the host immune response is to bacteria can include biomarkers such as procalcitonin (PCT), C- reactive protein (CRP), serum amyloid A, IL-6 (Interleukin- 6), Interferon Induced proteins with Tetratricopeptide repeats (IFIT), or human neutrophile lipocalin (HNL). The second biomarker to determine whether the host immune response is to a virus is MxA.

[0023] In one embodiment, the rapid host immune response is preferably determined using a combination of MxA and C-reactive protein (CRP), with MxA indicating a response to a potential bacterial infection and CRP determining a response to a potential viral infection. A first level and a second level of CRP can be detected.

[0024] In an alternate embodiment, the rapid host immune response is determined using a combination of MxA and procalcitonin.

[0025] In the multi-tiered, diagnostic strategy for determining whether a patient has SARS-CoV-2, a rapid immune host response is determined.

[0026] In another embodiment the bodily fluid tested is human tears, oral fluid such as saliva, blood, or nasal secretions (nasopharogeal).

[0027] If the rapid immune host response test indicates a bacterial infection, for example by indicating a negative result for MxA and a positive result for CRP or other bacterial biomarkers, antibiotics are considered for administration to the patient.

[0028] If the rapid immune host response test indicates a general viral infection, for example indicating a positive result for MxA, no antibiotics are administered to the patient. Then, a patient’s risk is determined. For high risk patients and low risk patients, confirmative COVID-19 molecular testing, for example via rRT-PCR is conducted. Additionally, testing for influenza A, influenza B, SARS and/or RSV could be conducted with the molecular testing. Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus. If the patient is high risk, the patient is admitted to the hospital for observation. If the patient is low risk, the patient is sent home for quarantine of at least two weeks. Additionally, if the patient has had symptoms for greater than or equal to 7 days, immunoglobulin testing can be completed. [0029] If the rapid host immune response indicates negative for bacterial and viral infection, for example by indicating negative for biomarker MxA and negative for biomarker CRP, an immunoglobulin test to COVID-19 is administered to the patient and the risk of the patient is determined.

[0030] The presence of CRP >10-20 mg/L in association with MxA > 15 ng/ml in COVID-19 patients can stratify risk and suggest worse outcome. COVID-19 patients with MxA >15 ng/ml and normal CRP are more likely to not progress to severe acute respiratory distress. However, COVID-19 patients with a presence of CRP >10-20 mg/L in association with MxA > 15 ng/ml showed more severe illness including increased and severe respiratory symptoms such as increased respiratory rate, reduced oxygen saturations, increased heart rates, as well as increased inflammatory cytokines such as procalcitonin (PCT) and white blood cells (WBC). In addition, elevated CRP predicts an increased likelihood of pulmonary involvement or pneumonia, with diffuse pulmonary infiltrates or consolidations seen on a chest x-ray.

[0031] In another embodiment a semi-quantitative amount of CRP of > 100 mg/L is used as a determination for a bacterial biomarker.

[0032] Using a quantitative assay or semi-quantitative measurements, levels or MxA and CRP and be measured overtime to assess or monitor disease respiratory infection progression.

[0033] If the patient is low risk and the immunoglobulin test is positive, confirmatory COVID-19 molecular testing is carried out with hospital observation. Additionally, testing for influenza A, influenza B, SARS and/or RSV could be conducted with the molecular testing. If the patient is low risk and the immunoglobulin test is negative, the patient is advised to home quarantine until their symptoms subside and repeat the immunoglobulin test.

[0034] If the patient is high risk and the immunoglobulin test is positive or negative, confirmatory COVID-19 molecular testing is carried out with hospital observation. Additionally, testing for influenza A, influenza B, SARS and/or RSV could be conducted with the molecular testing. [0035] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0036] In one preferred embodiment, a method determines if an infection is bacterial and/or viral by first collecting a sample. The sample is then transferred to a dual use two strip sample analysis device. The sample analysis device includes a first lateral flow chromatographic test strip with a first reagent zone and a second reagent zone. The first reagent zone includes at least one first reagent specific to a low level of C-reactive protein such that, when the sample contacts the first reagent, a first labeled complex forms if the low level of C-reactive protein is present in the sample. The second reagent zone includes at least one second reagent specific to MxA such that, when the sample contacts the second reagent, a second labeled complex forms if MxA is present in the sample. The first lateral flow chromatographic test strip also includes a first detection zone comprising a first binding partner which binds to the first labeled complex; and a second binding partner which binds to the second labeled complex. The two strip lateral flow assay device also includes a second lateral flow chromatographic test strip parallel in a lateral flow direction to the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes at least one third reagent zone including at least one third reagent specific to a high level of C-reactive protein, such that, when the sample contacts the third reagent, a third labeled complex forms if the high level of C-reactive protein is present in the sample. The third reagent on the second lateral flow chromatographic test strip only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent on the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip also includes a second detection zone with a third binding partner which binds to the third labeled complex. The sample is also analyzed for a presence of the low level of C-reactive protein, MxA, and the high level of C-reactive protein.

[0037] In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) in the same cassette as the first strip and the second strip. The additional strip can be parallel to the first or second strip. [0038] In yet another embodiment, the first strip or the second strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV).

[0039] In yet another embodiment, the testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) is on the first strip with MxA.

[0040] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus. The other viruses can be tested for on the first strip or the second strip.

[0041] In another preferred embodiment, a dual use two strip lateral flow assay device detects a bacterial and/or viral marker in a sample. The device includes a first lateral flow chromatographic test strip with a first reagent zone and a second reagent zone. The first reagent zone includes at least one first reagent specific to a low level of C-reactive protein such that, when the sample contacts the first reagent, a first labeled complex forms if the low level of C-reactive protein is present in the sample. The second reagent zone includes at least one second reagent specific to MxA such that, when the sample contacts the second reagent, a second labeled complex forms if MxA is present in the sample. The first lateral flow chromatographic test strip also includes a first detection zone comprising a first binding partner which binds to the first labeled complex; and a second binding partner which binds to the second labeled complex. The two strip lateral flow assay device also includes a second lateral flow chromatographic test strip parallel in a lateral flow direction to the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes at least one third reagent zone comprising at least one third reagent specific to a high level of C-reactive protein, such that, when the sample contacts the third reagent, a third labeled complex forms if the high level of C-reactive protein is present in the sample. The third reagent on the second lateral flow chromatographic test strip only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent on the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip also includes a second detection zone with a third binding partner which binds to the third labeled complex. The results of the two strip lateral flow assay device are preferably readable within 10 minutes of collection of the sample from the patient. [0042] In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) in the same cassette as the first strip and the second strip.

[0043] In yet another embodiment, the first strip or the second strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV). More preferably, the testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) is on the first strip with MxA.

[0044] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0045] Another preferred embodiment is a method for determining if an infection is bacterial and/or viral, and includes the step of collecting a sample. The sample is then transferred to a sample analysis device. The sample analysis device includes a sample compressor with a first reagent zone including at least one first reagent specific to a low level of C-reactive protein such that, when the sample contacts the first reagent, a first labeled complex forms if the low level of C-reactive protein is present in the sample, and at least one second reagent specific to MxA such that, when the sample contacts the second reagent, a second labeled complex forms if MxA is present in the sample, and a second reagent zone including at least one third reagent specific to a high level of C- reactive protein, where the third reagent only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent, such that, when the sample contacts the third reagent, a third labeled complex forms if the high level of C- reactive protein is present in the sample. The device also includes a first lateral flow chromatographic test strip that includes a first detection zone including a first binding partner which binds to the first labeled complex, a second binding partner which binds to the second labeled complex and a first diverting zone located upstream of the first detection zone on the lateral flow chromatographic test strip. The first diverting zone interrupts lateral flow on the first lateral flow chromatographic test strip. The device also includes a second lateral flow chromatographic test strip parallel in a lateral flow direction to the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes a second detection zone including a third binding partner which binds to the third labeled complex and a second diverting zone located upstream of the first detection zone on the lateral flow chromatographic test strip. The second diverting zone interrupts lateral flow on the second lateral flow chromatographic test strip. The device also includes a first sample application zone where sample is placed on the sample analysis device. The first sample application zone is located in a location selected from the group consisting of: i) on the first lateral flow chromatographic test strip upstream of the detection zone and ii) on the first reagent zone of the sample compressor. The device also includes a second sample application zone where sample is placed on the sample analysis device. The second sample application zone is located in a location selected from the group consisting of: i) on the second lateral flow chromatographic test strip upstream of the detection zone and ii) on the second reagent zone of the sample compressor. The sample compressor is in a different plane than the first lateral flow chromatographic test strip and the second lateral flow chromatographic test strip. The first reagent zone of the sample compressor creates a bridge over the first diverting zone and the second reagent zone of the sample compressor creates a bridge over the second diverting zone, diverting flow onto the sample compressor and returning flow to the first chromatographic test strip and the second chromatographic test strips at the end of the first diverting zone and the second diverting zone. The sample is analyzed for a presence of the low level of C-reactive protein, MxA, and the high level of C-reactive protein. The results of the two strip lateral flow assay device are preferably readable within 10 minutes of collection of the sample from the patient.

[0046] In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) in the same cassette as the first strip and the second strip.

[0047] In yet another embodiment, the first strip or the second strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV). More preferably, the testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) is on the first strip with MxA.

[0048] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus. [0049] Another preferred embodiment is a lateral flow device for detecting an analyte in a sample. The device includes a sample compressor with a first reagent zone including at least one first reagent specific to a low level of C-reactive protein such that, when the sample contacts the first reagent, a first labeled complex forms if the low level of C- reactive protein is present in the sample, and at least one second reagent specific to MxA such that, when the sample contacts the second reagent, a second labeled complex forms if MxA is present in the sample, and a second reagent zone including at least one third reagent specific to a high level of C-reactive protein, where the third reagent only detects a level of C-reactive protein that is higher than the level of C-reactive protein detected by the second reagent, such that, when the sample contacts the third reagent, a third labeled complex forms if the high level of C-reactive protein is present in the sample. The device also includes a first lateral flow chromatographic test strip that includes a first detection zone including a first binding partner which binds to the first labeled complex, a second binding partner which binds to the second labeled complex and a first diverting zone located upstream of the first detection zone on the first lateral flow chromatographic test strip. The first diverting zone interrupts lateral flow on the first lateral flow chromatographic test strip. The device also includes a second lateral flow chromatographic test strip parallel in a lateral flow direction to the first lateral flow chromatographic test strip. The second lateral flow chromatographic test strip includes a second detection zone comprising a third binding partner which binds to the third labeled complex and a second diverting zone located upstream of the first detection zone on the lateral flow chromatographic test strip. The second diverting zone interrupts lateral flow on the second lateral flow chromatographic test strip. The device also includes a first sample application zone where sample is placed on the sample analysis device. The first sample application zone is located in a location selected from the group consisting of: i) on the first lateral flow chromatographic test strip upstream of the detection zone and ii) on the first reagent zone of the sample compressor. The device also includes a second sample application zone where sample is placed on the sample analysis device. The second sample application zone is located in a location selected from the group consisting of: i) on the second lateral flow chromatographic test strip upstream of the detection zone and ii) on the second reagent zone of the sample compressor. The sample compressor is in a different plane than the first lateral flow chromatographic test strip and the second lateral flow chromatographic test strip. The first reagent zone of the sample compressor creates a bridge over the first diverting zone and the second reagent zone of the sample compressor creates a bridge over the second diverting zone, diverting flow onto the sample compressor and returning flow to the first chromatographic test strip and the second chromatographic test strips at the end of the first diverting zone and the second diverting zone. The results of the two strip lateral flow assay device are preferably readable within 10 minutes of collection of the sample from the patient.

[0050] In another embodiment an additional strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) in the same cassette as the first strip and the second strip. Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumo virus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus.

[0051] In yet another embodiment, the first strip or the second strip can include testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV). More preferably, the testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) is on the first strip with MxA.

[0052] In another preferred embodiment, a method simultaneously detects at least one extracellular analyte and at least one intracellular analyte, by collecting a sample and transferring the sample to a sample analysis device. The sample is also lysed and the extracellular analyte and the intracellular analyte are simultaneously detected on the same sample analysis device. In one preferred embodiment, the extracellular analyte is C- reactive protein and the intracellular analyte is MxA protein.

[0053] In another preferred embodiment, a method of detecting MxA protein and C- reactive protein in a sample includes the steps of adding the sample to a mixture of an antibody to MxA protein conjugated to a first label and an antibody to C-reactive protein conjugated to a second label different from the first label, detecting a presence of MxA protein by determining whether the antibody to MxA protein has agglutinated, and detecting a presence of C-reactive protein by determining whether the antibody to C- reactive protein has agglutinated.

[0054] In another preferred embodiments, aptamers to MxA, CRP, and SARS-CoV-2 immunoglobulins can be used rather than antibodies. Additionally, mRNA for the interferon induced genes which code for MxA or genes that code for CRP, procalcitonin, serum amyloid A, IL-6, IFIT, or HNL may be used. Additionally, type I alpha/beta or type II interferons (IFN) associated with MxA can be detected and used as a viral marker to determine whether a sample has a viral infection.

[0055] In another embodiment, an immunoassay can detect mRNA for specific interferons associated with SARS-CoV-2 which were upregulated and interferon induced, such as IFI6, IFI44L, IFI27, WDR74, and OAS2.

[0056] In a preferred embodiment, a single strip has the whole blood or fingerstick blood sample applied to the test strip. An activation buffer carries the sample across binding antibodies where the first analyte captured is CRP, the second is MxA, the third is IgA, the fourth is IgM, and the fifth is IgG. Alternatively, one strip may be used for the host immune biomarkers for CRP and MxA and a second strip for the SARS-CoV-2 immunoglobulins. In other embodiments the order can be in any combination thereof, and may include either IgA, IgM, IgG or any combination thereof. CRP may be replaced with procalcitonin, HNL, or IL-6. In another embodiment, at least one of the strips includes testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV). More preferably, the testing for influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV) is on the same strip used to detect MxA.

[0057] In one embodiment, determining a presence of MxA in a sample is determined by a first test and determining a presence of one or more of influenza A, influenza B, SARS or respiratory syncytial vims in the sample is determined by a second test, which can be the same or different than the first test. The first test can be, but is not limited to an immunoassay, fluorescence immunoassay or chemiluminescence immunoassay. The second test can be, but is not limited to an immunoassay, fluorescence immunoassay or chemiluminescence immunoassay. The immunoassay can utilizing colloidal gold, colloidal selenium, colloidal carbon, colloidal gold, nanoparticles, latex beads or paramagnetic beads.

[0058] Other viruses can also be tested for, such as, but not limited to: parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Fig. 1 shows a schematic of diagnostic test categories for detecting SARS-CoV-2 and the associated host response.

[0060] Fig. 2 shows a schematic of a multi-tiered, rapid diagnostic strategy incorporating rapid host immune response, molecular pathogen detection and serology.

[0061] Fig. 3 shows rapid screening test window visual test results to distinguish viral and bacterial infections and an interpretation of those results.

[0062] Fig. 4 shows three cassettes with different colored test lines.

[0063] Fig. 5 A shows a device with a test line corresponding to the presence of a viral marker and a second, separate test line that detects the presence of a bacterial marker in an embodiment of the present invention.

[0064] Fig. 5B shows a device with a test line corresponding to the presence of a viral marker and a second, separate test line that detects the presence of a bacterial marker in another embodiment of the present invention. [0065] Fig. 6 A shows a sample analysis device including a lysis zone located between a sample application zone and a reagent zone in an embodiment of the present invention.

[0066] Fig. 6B shows a sample analysis device including a lysis zone overlapping a sample application zone in an embodiment of the present invention.

[0067] Fig. 6C shows a sample analysis device including a lysis zone overlapping a reagent zone in an embodiment of the present invention.

[0068] Fig. 6D shows a sample analysis device including a lysis zone overlapping a sample application zone and a reagent zone in an embodiment of the present invention.

[0069] Fig. 7A shows a device with a test line corresponding to the presence of a bacterial marker such as high CRP levels in an embodiment of the present invention. [0070] Fig. 7B shows a device with a test line corresponding to the presence of a bacterial marker such as high CRP levels in another embodiment of the present invention. [0071] Fig. 8A shows a sample analysis device including a lysis zone located between a sample application zone and a reagent zone in an embodiment of the present invention.

[0072] Fig. 8B shows a sample analysis device including a lysis zone overlapping a sample application zone in an embodiment of the present invention. [0073] Fig. 8C shows a sample analysis device including a lysis zone overlapping a reagent zone in an embodiment of the present invention.

[0074] Fig. 8D shows a sample analysis device including a lysis zone overlapping a sample application zone and a reagent zone in an embodiment of the present invention.

[0075] Fig. 9A shows a fully open sample analysis device with dual test strips, as well as a conjugate zone and a sample application zone on a sample compressor in a plane separate from the test strips in an embodiment of the present invention.

[0076] Fig. 9B shows the sample analysis device of Fig. 9A with part of the housing closed, but the conjugate zone still visible on the left side of the device.

[0077] Fig. 9C shows the sample analysis device of Fig. 9A after the test has been initiated.

[0078] Fig. 10A shows a test result negative for both MxA and CRP in an embodiment of the present invention.

[0079] Fig. 10B shows a test result positive for MxA in an embodiment of the present invention. [0080] Fig. IOC shows a test result positive for MxA in an embodiment of the present invention.

[0081] Fig. 10D shows a test result positive for CRP in an embodiment of the present invention.

[0082] Fig. 10E shows a test result positive for CRP in an embodiment of the present invention. [0083] Fig. 10F shows a test result positive for both CRP and MxA, indicating co- infection, in an embodiment of the present invention.

[0084] Fig. 11 A shows a fully open sample analysis device with dual test strips and a conjugate zone on a sample compressor in a plane separate from the test strips in an embodiment of the present invention.

[0085] Fig. 1 IB shows the sample analysis device of Fig. 11 A with part of the housing closed, but the conjugate zone still visible on the left side of the device.

[0086] Fig. 11C shows the sample analysis device of Fig. 11 A after the test has been initiated.

[0087] Fig. 12 shows a kit for sample analysis using a sample analysis device in an embodiment of the present invention.

[0088] Fig. 13 shows a sample analysis device with dual test strips in another embodiment of the present invention.

[0089] Fig. 14A-14B show a table of patients tested using a sample analysis device of an embodiment of the present invention.

DETAILED DESCRIPTION

[0090] In one embodiment, a multi-tiered, diagnostic strategy is disclosed. The multi tiered diagnostic strategy incorporates a rapid host immune response assay as an initial screening test; a rapid immunoglobulin test including one or more of immunoglobulin M (IgM), immunoglobulin G (IgG), and immunoglobulin A (IgA) for assessing which patients would benefit from quarantine or further testing and provide information on population exposure/herd immunity provides both a comprehensive screening and diagnostic testing strategy; and molecular confirmatory testing. The ability to triage patients within minutes rather than hours or days is a critical component to pandemic response as reliance on the existing single test strategy is limited by the accuracy, time to results, and testing capacity. There is an urgent need to implement a multi-tiered strategy to combat the current outbreaks. Early screening and triage can be achieved from the outset of a suspected pandemic with point-of-care host immune response testing which will improve response time to effective action.

[0091] Use of rapid oral screening of a combination of SARS-CoV-2 IgA, IgG, and SARS-CoV antigen would serve to quickly test and identify both infection and immunity. If IgA and/or antigen is present, an active infection is present. If only IgG is present, there is immunity.

[0092] Use of rapid serological screening of a combination of SARS-CoV-2 IgA, IgM, IgG, and MxA would serve to quickly test and identify both COVID infection, history of COVID-19 infection, other viral infection, and COVID immunity. If COVID IgA and/or IgM is present along with elevated MxA there is an active infection. If IgA or IgM is present with normal MxA there is recent infection. If MxA is normal and IgM + IgG or just IgG is present there is immunity. The addition of CRP, procalcitonin, HNL, 11-6 would confirm bacterial infection if MxA and serological IgA/IgM is negative or suggest co-infection if high CRP > 100 mg/L or PCT > 0.5 ng/ml is present.

[0093] Incorporating a rapid, host immune response assay as the first line of screening/triage, to differentiate viral from bacterial infections, would preserve healthcare resources (e.g. confirmatory testing capacity), improve operational efficiency (e.g. triage, crowding), avoid exposing the worried well and those not suffering a viral infection to actually infected patients, and aid in public health pandemic control efforts (e.g. case identification, quarantines).

[0094] Without an effective way to rapidly triage patients in the community (e.g. border control stations, mobile testing units) or healthcare settings (urgent care, EDs, occupational health services), healthcare resources are being overwhelmed. The ability to quickly and repeatedly test, both symptomatic patients and the worried, will have a significant impact on public health and resource management. Plus, during an ongoing outbreak, many patients will continue to develop more common bacterial infections like pharyngitis or pneumonia and the ability to quickly identify these patients and institute appropriate antibiotic therapy will reduce the risk of infection progression (e.g. sepsis). The ability to triage patients at the point of care and support the guidance of medical and therapeutic decisions, for viral isolation or confirmatory testing or for appropriate treatment of bacterial infections, is a critical component to our national pandemic response and there is an urgent need to implement the proposed strategy to combat the current outbreak. Furthermore, a universal strategy incorporating rapid, host immune point-of-care tests can be used now and for future pandemic planning by effectively identifying patients at risk of disease and initiating quarantine earlier in the progression of disease during the weeks and months it can take for pathogen specific confirmatory tests to be developed, validated and manufactured in sufficient quantities. Early screening and triage can be achieved with point-of-care host immune response testing which will improve response time to effective action.

[0095] COVID-19 illuminates the need for a tiered and coordinated diagnostic approach to rapidly identify clinically significant infections and reduce the spread of disease. Without the ability to efficiently screen patients, healthcare facilities will become overwhelmed, potentially delaying treatment for other emergency conditions (e.g. bacterial sepsis).

[0096] The most efficient and cost-effective approach to COVID-19 evaluation is a multi-tiered screening and diagnostic strategy. Ideally, any patients requiring evaluation can be quickly triaged as having either viral, bacterial or absent immune response to an infection and then receive rapid confirmatory testing. Rapid host immune tests can quickly identify the presence of viral or bacterial infection. Accurate ARI characterization is a critical component of optimal antibiotic stewardship, disposition planning, and quarantine procedures. When used along with serologic tests, patients who require confirmatory molecular testing can be expediently identified.

[0097] Figure 1 shows a schematic of diagnostic test categories for detecting SARS- CoV-2 and the associated host response.

[0098] The phases associated with the SARS-CoV-2 are a viral replicating phase (105) indicated by the “L” shading, which takes place from approximately days 1 through 7. The onset of symptoms starts at a high amount and decreases through day 7. The inflammatory response phase (107) indicated by diagonal cross-hatching, is at a low amount starting at day 1 on onset of symptoms and increases to a high amount as the days increase between day 1 and day 21. It is noted that the transmission risk (106) between a person infected with SARS-CoV-2 and another individual is highest at day 1 from onset of symptoms and decreases through day 10. In a first, early symptom stage (108), defined between day 1 and day 7 of onset of symptoms includes early symptoms (109) which can include, but is not limited to fever, cough, fatigue, headache, lymphopenia, thrombocytopenia, leuokopenia, and increased D-dimer. A second, pulmonary stage (110), defined between day 7 and day 14 includes symptoms (111) which can include, but is not limited to shortness of breath, hypoxia, abnormal chest imaging, high CRP and low procalcitonin (PCT). A third, acute respiratory distress syndrome (ARDS) stage (112), takes place between day 14 and day 21 and can includes symptoms (113) which can include, but are not limited to ventilation required for breathing, hyperinflammatory state and organ damage. It is noted that not all patients will experience stage three of acute respiratory distress syndrome 112.

[0099] In an embodiment of the present invention, during the early stage defined by day 1 through day 7, a rapid MxA and CRP test (102) is used to determine an early host immune response. The test can be disposable and is read in 10 minutes or less. In another embodiment, the test is read within 10 minutes of retrieving a sample from a patient. The patient sample tested can include blood, saliva, nasopharyngeal secretions or other patient fluids. The test is preferably a lateral flow assay that differentiates between viral and bacterial infections and in some embodiments can differentiate between colonization/carrier state and active infection.

[00100] It was unexpected that someone can have an active infection and be asymptomatic. Conversely, someone who is symptomatic may not have an active infection.

[00101] As described herein, “colonization” or a “carrier state” refer to a clinically insignificant local infection without an associated systemic immune or serological response. These two terms will be used interchangeably herein.

[00102] A multiplexed diagnostic device tests markers for both viral and bacterial infection and can effectively identify clinically significant infections by choosing a threshold significantly above baseline values seen in the normal population and based on the relative values of the biomarkers, and can assist in the rapid differentiation between viral and bacterial infections and/or between active infection and colonization, for example at the outpatient office or during an urgent care visit. This ability can dramatically reduce health care costs by limiting misdiagnosis and the subsequent overuse of antibiotics. Such a practice may limit antibiotic allergies, adverse events, and antibiotic resistance.

[00103] MxA is induced by interferon alpha/beta in human cells and becomes elevated in the presence of acute viral infection. The expression of MxA in peripheral blood is sensitive and is specific to biomarkers for viral infection. In the presence of severe bacterial infections, MxA remains within normal limits. In order to establish a normal range for MxA, eighty normal blood samples were obtained from the Tennessee Valley Blood Bank. The donors underwent a screening for any symptoms or recent exposure to infection using a typical blood donor questionnaire. If any of the questions on the questionnaire were answered as positive, the subjects were excluded from donating blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect MxA in peripheral blood in the collected specimens. Table 1 shows the MxA present in each specimen.

[00104] Table 1

[00105] The range of MxA found among the 80 specimens was 0.0-13.6 ng/mL, with the mean being 1.0 and the standard deviation being 5.85. Therefore, the normal range (95%) is 0.0-12.7 ng/mL and the normal range (99%) is 0.0-18.6 ng/mL. Therefore, any values of MxA higher than the mean +/- 2-3 standard deviations of the normal population are considered true positives for MxA. When identifying true MxA positives, a cutoff of at least >13-19 ng/mL should be used to avoid false positive results.

[00106] Some of the methods and devices described herein test for the presence of MxA, C-reactive protein (preferably a first level and a second level, where the second level of C- reactive protein is higher than the first level of C-reactive protein but alternatively one level of C-reactive protein may be assayed), and/or procalcitonin, or another bacterial biomarker. Testing for this unique combination of viral (MxA) and bacterial (C-reactive protein and procalcitonin or other bacterial biomarkers) immune response markers allows for a much more accurate diagnosis of a patient.

[00107] The combination of MxA, interferon, or IFIT in the presence of C-reactive protein, procalcitonin, serum amyloid A, HNL, IL-6, or another bacterial biomarker shifts the receiver operator curve to allow for higher sensitivity thresholds to be used for bacterial infection confirmation because the specificity of the bacterial biomarker is enhanced by the presence of the viral marker. Thus, if a patient has an elevated viral marker in the presence of elevated C-reactive protein and/or procalcitonin or other bacterial biomarkers, it confirms a viral infection, yet an elevation of the bacterial markers independent of the viral markers would confirm a bacterial infection. Without the presence of the viral biomarkers, the cutoff for the bacterial infection determination would need to be set much higher to generate improved specificity at the cost of sensitivity. This combination of the biomarkers dramatically improves the bacterial sensitivity by shifting the receiver operator curves in favor of higher sensitivity cutoffs.

[00108] In some preferred embodiments, a combined single diagnostic sample analysis device tests for a presence of MxA, a low level of C-reactive protein, a high level of C- reactive protein, and procalcitonin. In other preferred embodiments, a first combined diagnostic device tests for a presence of two or more of MxA, a low level of C-reactive protein, a high level of C-reactive protein, or procalcitonin and one or more additional diagnostic devices tests for a presence of at least one of MxA, a low level of C-reactive protein, a high level of C-reactive protein, or procalcitonin. [00109] In another preferred embodiment, a first combined diagnostic sample analysis device tests for a presence of MxA, a low level of C-reactive protein, and a high level of C-reactive protein, and a second sample analysis device tests for the presence of procalcitonin. In yet another embodiment, different devices test for each of MxA, a low level of C-reactive protein, a high level of C-reactive protein, and procalcitonin.

[00110] In some embodiments, obtaining results for two levels of C-reactive protein differentiates between a non-aggressive bacterial infection needing appropriate oral antibiotics (positive result for low level of C-reactive protein only in the range of 10-20 mg/L) versus an aggressive, severe bacterial infection needing aggressive therapeutic intervention such as intravenous antibiotics or other more drastic interventions (positive result for both low level and high level of C-reactive protein in the range of greater than 80 mg/L). The presence of a second higher cutoff line may also assist in identifying patients more likely requiring hospital admission. High C-reactive protein levels help determine the aggressiveness or clinical significance of a bacterial infection because of the semi- quantitative aspect of the test.

[00111] Some examples of assay formats for determining the presence of C-reactive protein, MxA, procalcitonin, IL-6, HNL and/or serum amyloid A include, but are not limited to, immunoassays, immunoblotting methods, agglutination reactions, a complement-fixation reaction, a hemolytic reaction, a precipitation reaction, a gold colloid method, a chromatography method, phosphorescence, chemiluminescence, radioactivity, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, fluorescent resonant emissions, or an immunostaining method. Some examples for immunoassays include, but are not limited to, immunoprecipitation, radioimmunoassays (RIA), enzyme immunoassays (EIA or ELISA), a Vidas ^® immunoassay device (Biomerieux, Hazelwood, Missouri), an i-Stat ^® portable handheld system (Abbott Laboratories, Abbott Park,

Illinois), a Philips Handheld diagnostic system (Philips Handheld Diagnostics, The Netherlands), fluorescent immunoassays (FIA), chemiluminescent immunoassays, physiochemical assays (TIA, LAPIA, or PCIA), lateral flow immunoassays, or flow cytometry. MxA monoclonal antibodies have been used in modified flow cytometry (Itazawa et ah, Increased lymphoid MxA expression in acute asthma exacerbation in children., Allergy Sep 2001 56(9): 895-8). Some preferred immunoassays for these biomarkers include, but are not limited to, ELISAs, fluorescence immunoassays, magnetic assays, paramagnetic assays, and chemiluminescent assays. In other embodiments, the mRNA or gene transcripts may be used. In some preferred embodiments, the assays are automated.

[00112] One particular example of a device to determine the presence of C-reactive protein, MxA and/or procalcitonin is a multiparametric immunoassay system that is able to detect two or more of these targets in the same device. One such device is a Vidas ^® immunoassay device (Biomerieux, Hazelwood, Missouri), which could test for the presence of one, two, three, or all four of these targets simultaneously. The Vidas ^® immunoassay device is an Enzyme Linked Fluorescent assay (ELFA) (also available in a compact version called Mini Vidas ^® ) and is widely used in clinical laboratories. Other devices that could be used include a Vitek ^® immunodiagnostic system (Biomerieux, Hazelwood, Missouri), or a Luminex ^® immunoassay system (Luminex Corporation, Austin, Texas). Another example is a device similar to an i-Stat ^® portable handheld system (Abbott Laboratories, Abbott Park, Illinois, see the devices disclosed in US Patent Nos. 5,638,828, 5,666,967, 5,653,243, 5,779,650, 6,010,463, 6,845,327, 6,896,778, 7,419,821, and 8,017,382, all herein incorporated by reference). Yet another example is a device that combines magnetic particle separation with chemiluminescent detection, such as the BioFlash multiparametric immunoassay system (Biokit, Barcelona, Spain). Another example is a Philips handheld diagnostics device (Philips Handheld Diagnostics, The Netherlands).

[00113] In a preferred embodiment, the marker for viral infection is MxA and the markers for bacterial infection are procalcitonin (PCT), and two levels of C-reactive protein. High MxA protein levels are strongly correlated with systemic viral infection and increased C-reactive protein and procalcitonin are more associated with bacterial infections. The present invention includes infectious screening tests for identifying MxA, C-reactive protein and procalcitonin in samples. MxA is present in leukocytes (white blood cells). Therefore, the sample can be taken anywhere leukocytes are available, for example in a peripheral blood sample, nasopharyngeal aspirates, saliva, tears, spinal fluid, and middle ear aspirates. In one preferred embodiment, the sample is taken from whole blood. [00114] In some embodiments, lysing buffer is used to treat the whole blood in a vacuum tube. In some embodiments, whole blood is preferably lysed before the sample is assayed for the host biomarkers. In some embodiments, a membrane and buffer are used to directly lyse the whole blood cells, separate blood into plasma/serum, and filter cellular debris, to detect a combination of intracellular and extracellular biomarkers. In some preferred embodiments, there are no external or pre-processing steps.

[00115] The C50 concentration for a particular test, where 50% of the time a visually read test is interpreted as positive, depends on an individual’s visual acuity. The C50 concentration is also known as the cut-off concentration or the threshold concentration above which the test is considered positive. Sometimes it is also called a Medical Decision Point above which a relevant decision is made by the clinician The Applicant has found the C50 values to be >13 ng/ml for MxA, >15 mg/L to 20 mg/L for low CRP (serum equivalent) and >80 mg/L to 100 mg/L for high CRP (serum equivalent). Below C50, for example at C5 there is a 5% chance the result is scored as positive. The C5 concentrations begin at 12 ng/ml for MxA, at about 10 mg/L for low CRP and about 30 mg/L for high CRP. These are not false positives because there is some analyte present in the sample.

[00116] In some preferred embodiments of testing for the presence of procalcitonin (including, but not limited to, those embodiments testing also for MxA, and/or one or both levels of C-reactive protein), the threshold concentration of procalcitonin in a sample needed to elicit a positive result is greater than approximately 0.1 ng/ml. In another preferred embodiment, the threshold concentration of procalcitonin in a sample to elicit a positive result is equal to or greater than approximately 0.15 ng/ml. In another preferred embodiment, the threshold concentration of procalcitonin in a sample to elicit a positive result is equal to or greater than approximately 0.25 ng/ml. In one preferred embodiment, the procalcitonin cut off value is defined as the mean in the normal population + 2-3.5 times the standard deviation.

[00117] In other preferred embodiments of testing for the presence of MxA (including, but not limited to, those embodiments testing also for procalcitonin, and/or one or both levels of C-reactive protein), the threshold concentration of MxA in a sample to elicit a positive result may be as low as approximately 13 ng/ml; however, the threshold concentration may by higher, in a range from approximately 15 ng/ml to approximately 400 ng/ml. In one preferred embodiment, the threshold concentration to obtain a positive result for MxA is equal to or greater than approximately 15 ng/ml. In another preferred embodiment, the threshold concentration to obtain a positive result for MxA is equal to or greater than approximately 25 ng/ml. In other preferred embodiments, a threshold concentration to obtain a positive result for MxA is equal to or greater than approximately 40 ng/ml.

[00118] The cutoff value (threshold concentration) for assaying MxA depends on whether a quantitative or qualitative assay is being performed. For example, the cutoff value for assaying MxA in lateral flow assays is preferably 20 ng/ml or 40 ng/ml because it is a qualitative assay. In some preferred embodiments, a 13 ng/ml cut off value, a 15 ng/ml cut off value or a 25 ng/ml cut off value is preferable when performing quantitative assays. Any MxA values between approximately 13 ng/ml and 25 ng/ml could preferably be used in a quantitative assay. The cut off values are preferably technology independent and the standards used may alter the cut off values slightly. The important thing is to determine whether the MxA biomarker is elevated. In one preferred embodiment, the MxA cut off value is defined as the mean in the normal population +/- 2-3.5 standard deviations.

[00119] In some preferred embodiments of testing for the presence of a low level of C- reactive protein (including, but not limited to, those embodiments testing also for MxA, procalcitonin, and/or a high level of C-reactive protein), a threshold concentration to obtain a positive result for the low level of C-reactive protein is equal to or greater than a serum equivalent of approximately 6-20 mg/L of C-reactive protein. In other preferred embodiments, the threshold concentration to obtain a positive result for the low level of C- reactive protein is equal to or greater than a serum equivalent of approximately 10 mg/L of C-reactive protein. In still other preferred embodiments, the threshold concentration to obtain a positive result for the low level of C-reactive protein is equal to or greater than a serum equivalent of approximately 20 mg/L. In one preferred embodiment, the C-reactive protein cut off value is defined as the mean in the normal population +/- 2-3.5 standard deviations.

[00120] In some preferred embodiments of testing for the presence of a high level of C- reactive protein (including, but not limited to, those embodiments testing also for MxA, procalcitonin, and/or a low level of C-reactive protein), the threshold concentration to obtain a positive result for the high level of C-reactive protein is equal to or greater than a serum equivalent of approximately 60-100 mg/L. In another preferred embodiment, the threshold concentration to obtain a positive result for the high level of C-reactive protein is equal to or greater than a serum equivalent of approximately 80 mg/L. In other preferred embodiments, the threshold concentration to obtain a positive result for the high level of C-reactive protein is equal to or greater than a serum equivalent of approximately 65 mg/L.

[00121] The threshold concentrations of each of the targets may depend on the size of the sample being applied to the assay device (for example a test strip), as well as its dilution, if applicable.

[00122] In some embodiments, the bacterial biomarker tested for is procalcitonin, with the threshold for concentration to achieve a positive result is >0.1 ng/ml. In another embodiment, the bacterial biomarker tested for is IL-6, with the threshold for concentration to achieve a positive result is > 15 pg/ml. In yet another embodiment, the bacterial biomarker is HNL, with the threshold for concentration to achieve a positive result is >100 pg/L.

[00123] In preferred embodiments, the devices and methods are intended for professional use in an outpatient office or urgent care clinic and should be used in conjunction with other clinical (laboratory or radiographic) and epidemiological information.

[00124] US Patent Publication 2010/0297611, published November 25, 2010, entitled "Method and Device for Combined Detection of Viral and Bacterial Infections", US Patent Publication 2013/0196310, published August 1, 2013, entitled "Method and Device for Combined Detection of Viral and Bacterial Infections", US Patent No. 8,962,260, issued February 24, 2015, entitled "Method and Device for Combined Detection of Viral and Bacterial Infections", and US Patent Publication 2013/0130367, published May 23, 2013, entitled "Method and Device for Combined Detection of Viral and Bacterial Infections", all incorporated herein by reference, disclose methods and devices for distinguishing between bacterial and viral infections by detecting bacterial and viral markers on lateral flow immunoassays. In some preferred embodiments of these applications, the viral marker is MxA and the bacterial marker is C-reactive protein. [00125] “Sensitivity” is the ability to detect a positive result. For example, a more sensitive test is less likely to miss a positive with a very low concentration. In a qualitative test where the results are scored either as positive or negative, the ability to determine correctly the positive samples which have low concentrations of the analyte by having a lower limit of detection is of paramount importance. This is especially true during the early time course of any infection or disease where the target analyte is generally at low concentrations. The higher the sensitivity, the lower the false negatives in the system.

[00126] "Specificity" is the ability to identify the specific analyte without interference from other components. Specificity is also the likelihood that a test will be negative when the analyte is absent from the sample. The higher the specificity, the lower the false positives in the system.

[00127] In isolation, neither MxA nor procalcitonin alone is sensitive or specific at identifying both viral and bacterial infection. Procalcitonin is specific to identify bacterial infection, but is not sensitive for viral infection. MxA is specific to identify viral infection, but it is not sensitive for bacterial infection. Using both procalcitonin and MxA together provides a sensitive and specific way to identify an immune response to a viral and/or bacterial infection.

[00128] In one preferred embodiment of a multiplexed assay using MxA and procalcitonin, the fingerstick blood pattern of test results shows a positive result with a serum equivalence to a procalcitonin cut-off between approximately 0.10 ng/ml and 0.15 ng/ml and a MxA cut-off in a range between approximately 15 ng/ml and 40 ng/ml. Alternatively, the MxA cut-off is in a range of 13 ng/ml and 40 ng/ml. These preferred values are shown in Table 2.

[00129] Table 2 [00130] Similarly, in isolation, neither MxA nor C-reactive protein alone is sensitive or specific at identifying both viral and bacterial infection. Low cut-off values of C-reactive protein show high sensitivity and low specificity for detecting bacterial infection. High cut-off values of C-reactive protein show low sensitivity and high specificity for detecting bacterial infection. MxA is specific to identify viral infection, but it is not sensitive for bacterial infection. A multiplexed pattern of results including medical decision points reflecting cut-off levels of low CRP, high CRP, and MxA together provide a sensitive and specific way to identify an immune response to a viral and/or bacterial infection. [00131] In one preferred embodiment of a multiplexed assay using MxA and two levels of C-reactive protein, the fingerstick blood pattern of test results shows a positive result with a serum equivalence to a low CRP level cut-off between approximately 10 mg/L and 20 mg/L, a serum equivalence to a high CRP level cut-off between approximately 65 mg/L and 100 mg/L, and a MxA cut-off between approximately 15 ng/ml and 40 ng/ml. These preferred values are shown in Table 3. In an alternate embodiment, the MxA cutoff is between approximately 13 ng/ml and 40 ng/ml.

[00132] Table 3

[00133] In a preferred embodiment of a multiplexed visually read qualitative assay using MxA and two levels of C-reactive protein, the blood pattern of test results shows a positive result with a serum equivalence to a low CRP level cut-off between approximately 10 mg/L and 20 mg/L, a serum equivalence to a high CRP level cut-off between approximately 60 mg/L and 100 mg/L, and a MxA cut-off between approximately 15 ng/ml and 25 ng/ml or 13 ng/ml and 25 ng/ml. [00134] In another preferred embodiment of a multiplexed assay using MxA, procalcitonin, and two levels of C-reactive protein, the fingerstick blood pattern of test results shows a positive result with a serum equivalence to a low CRP level cut-off between approximately 10 and 20 mg/L, a serum equivalence to a high CRP level cut-off between approximately 80 mg/L and 100 mg/L, a serum equivalence of procalcitonin between approximately 0.10 ng/ml and 0.15 ng/ml and a MxA cut-off between approximately 25 ng/ml and 40 ng/ml. These preferred values are shown in Table 3.

[00135] Table 4 [00136] Elevated C-reactive protein or procalcitonin levels alone are nonspecific indicators. For example, in influenza infection, there is an elevated level of C-reactive protein which may erroneously lead a clinician to prescribe antibiotics. When C-reactive protein or procalcitonin are multiplexed with MxA, the true etiology (viral or non-viral) is identified which can lead to appropriate and timely therapeutic intervention. [00137] The specificity of these tests are further enhanced by restricting the intended use.

For example, in preferred embodiments, only certain ages of the patient population are tested (preferably one year of age or older) and/or patients with specific underlying conditions that may lead to confounding factors are preferably not screened with these tests. [00138] In another preferred embodiment of a multiplexed assay using MxA and CRP, as well as influenza A, B and RSV, a patient sample showed positive results with a serum equivalence to an MxA cut-off in a range between approximately 15 ng/ml and 40 ng/ml and a CRP cut-off in a range between 20 mg/ml. Alternatively, the MxA cut-off is in a range of 13 ng/ml and 40 ng/ml. The assay can additionally detect SARS. In the multiplexed assay, MxA, CRP, influenza A, influenza B and RSV can be present on the same strip or present on two strips which are present in the same cassette insert. Additional viruses can also be tested for, for example: parainfluenza vims, metapneumovirus, rhinovirus, enterovirus, coronavirus, herpes simplex virus, cytomegalovirus, bocavirus, and Epstein-Barr virus. These preferred values are shown in Table 5.

[00139] Table 5 [00140] Colonization/carrier state versus active infection

[00141] Microbial clinical relevance is based on a host response. Overtreatment of colonizing bacteria and under-treatment of potential significant bacterial infection is thwarted with the methods described herein.

[00142] Delayed antibiotic prescription is recommended in international guidance (NICE guideline development group. Prescribing of antibiotics for self- limiting respiratory tract infections in adults and children in primary care. 2014, herein incorporated by reference). The National Institute for Health and Care Excellence (NICE) currently recommends using a strategy of either no antibiotic prescriptions or a delayed antibiotic prescription for dealing with uncomplicated acute sore throats and other respiratory infections. [00143] The NICE draft guidelines recommend considering a point-of-care C-reactive protein (CRP) test for patients presenting with lower respiratory tract infection in primary care if it is not clear after clinical assessment whether antibiotics should be prescribed. The results of the C-reactive protein test should be used to guide antibiotic prescribing as follows: • Do not routinely offer antibiotic therapy if the C-reactive protein concentration is less than 20 mg/L

• Consider a delayed antibiotic prescription (a prescription for use at a later date if symptoms worsen) if the C-reactive protein concentration is between 20 mg/L and 100 mg/L

• Offer antibiotic therapy if the C-reactive protein concentration is greater than 100 mg/L

[00144] Guidelines- IDSA and NICE

[00145] According to the Infectious Diseases Society of America (IDSA) (Caliendo AM et al. Better tests, better care: improved diagnostics for infectious diseases. Clin Infect Dis. 2013 Dec;57 Suppl 3:S 139-70, incorporated herein by reference), future diagnostic tests should have the following characteristics:

• Performed directly from accessible, minimally invasive clinical specimens, such as blood, respiratory samples, urine, and stool

• Able to rule out infection with high certainty (negative predictive value) as a first step for a variety of clinical syndromes

• Able to support differentiation of viral from bacterial infection

• Incorporating biomarkers that are either pathogen- or host-derived and capable of indicating host response to a pathogen or further classifying clinically significant infectious processes into relevant categories (e.g., bacterial or viral).

[00146] A diagnostic strategy that incorporates sensitive biomarkers (e.g., infection present yes/no) followed by pathogen-specific tests that are linked to a rapid assessment of drug resistance could not only bring antibiotic stewardship to the outpatient setting but also revolutionize sepsis management. Clinical studies that evaluated the presence of respiratory viruses in asymptomatic patients indicate that the old doctrine, which considered the presence of any respiratory virus clinically significant, is no longer true. Detected nucleic acids may be from nonviable organisms or from commensal (nonpathogenic) or colonizing bacteria or viruses that are noncontributory to the disease. Pathogen-based testing also needs to take into account colonization rates in children, especially due to their high pneumococcal colonization rates. The challenge with typical bacteria and some viral pathogens is the need to determine if the identified pathogen is colonizing or invading. Procalcitonin, MxA and C-reactive protein are promising biomarkers that can be used in addition to fever, leukocytosis, and clinical syndrome as a predictor of bacterial (PCT and/or CRP) or viral (MxA) infection.

[00147] Currently, the medical definition of colonization is the presence of a bacteria or virus without an associated immune antibody response detectable in the blood. The ability to use serology to detect antibody responses requires two patient visits, an initial visit at the start of symptoms and a subsequent visit 2-4 weeks later. Because of the inherent time delay, it is not practical to perform this testing to confirm an active infection. Thus, most doctors simply rely on antigen testing, culture, or PCR to identify the presence of a bacteria or virus instead of using paired serology, which combines identification with an antibody response. This results in the significant over-estimation of true infection and subsequent over-prescription of unnecessary antibiotics.

[00148] Traditionally the confirmation of an infection is measured against the presence or absence of microbial antigen, culture growth, or nucleic acid. However, none of these tests distinguish between colonization and active infection. In reality, more than the presence or absence of a microbial antigen is required in order to indicate infection. An active, true infection also requires an associated immune response. Without the immune response, colonization of the bacteria or vims is occurring. Only a true infection requires antibiotic therapy. Colonized bacteria are not typically contagious and do not require therapeutic intervention.

[00149] There is a challenge to define true infection from bacterial colonization or a local viral infection without a systemic host response. There needs to be a change in definition of infection, which will change the diagnostic parameters and reported performance of a test. The new definition that should be adopted and standardized for a clinically significant respiratory infection requires confirmation of the presence of a pathogen via antigen, culture, or molecular detection in association with a systemic host response.

[00150] As newly defined herein, a clinically significant infection is the local microbiological confirmation of a pathogen by cell culture, molecular techniques, and antigen in association with a systemic immune response (C-reactive protein, procalcitonin, MxA, or serological response).

[00151] It is also noted that a CRP level of greater 20 mg/L can be an indicator of severe respiratory issues that are either currently present, developing or will develop. Severe respiratory issues can include, but is not limited to increased respiratory rates, a decrease in oxygen saturation, and increase in heart rate, and an increase in inflammatory cytokines such as white blood cells and procalcitonin levels.

[00152] The details of the lateral flow assays are discussed below and can be seen in Figures 4A-13.

[00153] Referring to Figure 1, at the early stage in which early symptoms are exhibited (108), instead of testing for analytes specific to a particular bacterial or viral infection, the lateral flow assay described herein test for diagnostic markers that are specifically produced in a host in response to general, unspecified bacterial infection and general, unspecified viral infection. The diagnostic markers are preferably markers of an unspecified and/or unknown illness of bacterial or viral origin.

[00154] A combined point of care diagnostic device tests markers for both viral and bacterial infection and can effectively assist in the rapid differentiation of viral and bacterial infections, for example at the outpatient office or during an urgent care visit. This ability can dramatically reduce health care costs by limiting misdiagnosis and possibly exposing healthy individuals to SARS-CoV-2 who are not yet infected. Such a practice may limit antibiotic allergies, adverse events, and antibiotic resistance. The rapid result obtained from the test also permits a diagnosis while the patient is still being examined by the practitioner. In a preferred embodiment, the test result is obtained in under 10 minutes after applying the sample to the device, and it is preferably read at approximately 10 minutes. In samples that are highly positive, the test line is visible within approximately 1- 5 minutes. In an alternate embodiment, the test result is obtained within 10-15 minutes of extracting the sample from the patient.

[00155] For the pulmonary stage (110) and the ARDS stage (112), a serology test (103) is preferably used to test for one or more of IgG, IgM and IgA to determine the adapted host immune response. The serology test (103) is preferably a rapid test providing results in 10 minutes or less. The serology test assesses which patients would benefit from quarantine or further testing.

[00156] Additional molecular testing using polymerase chain reaction (PCR) or real-time reverse transcription polymerase chain reaction (rRT-PCR) can be used to confirm test results of the rapid MxA and CRP test. However, PCR testing requires ancillary equipment and at least 45 minutes to 3 hours for results.

[00157] A multi-tiered, rapid diagnostic strategy of an embodiment of the present invention incorporates a rapid host immune response assay as an initial triage test and confirmatory molecular testing and a rapid IgM/IgG test for assessing which patients would benefit from quarantine or further testing provides both a comprehensive screening and diagnostic testing strategy shown in further detail in Figure 2.

[00158] Fig. 2 shows a schematic of a multi-tiered, rapid diagnostic strategy incorporating rapid host immune response, molecular pathogen detection and serology.

[00159] Patients that present with symptoms of acute respiratory infection (ARI) such as fever, cough, sore throat or shortness of breath (121) are administered a dual biomarker point-of-care (POC) lateral flow test (122) capable of rapidly assesses the body’s host immune response to an ARI and helping differentiate viral from bacterial infections. The dual biomarker (MxA/CRP) test (122) is a single use, 10-minute, POC test that (i) identifies host immune response to infection and (ii) aids in the differentiation of viral and bacterial ARI through the simultaneous detection of both Myxovirus resistance protein A (MxA) and C-reactive protein (CRP) directly from a blood sample obtained via fingerstick. Although other secretions from the patient can be used, such as whole blood, human tears, saliva, or nasal secretions (nasopharogeal).

[00160] When viral pathogens induce a clinically significant host immune response, MxA, a biomarker of the body’s innate response to a viral infection, will elevate. An elevation of MxA with or without an associated elevation in CRP is consistent with a viral infection. MxA with an associated rise in CRP may suggest a more severe underlying viral infection. Furthermore, early data published from the COVID-19 pandemic in China showed that CRP was significantly elevated in patients who progressed to severe illness or death compared to patients who experienced clinical improvement/stabilization (38.9 [14.3, 64.8] mg/L vs. 10.6 [1.9, 33.1] mg/L, U=1.315, P=0.024). In the context of SARS- CoV-2 screening efforts, MxA can identify if a patient has a host-response to a viral infection while MxA in addition to CRP may provide insight to risk of decompensation. While an elevation in CRP without MxA can help to identify a patient with a true bacterial infection who may benefit from antibiotic therapy.

[00161] If the lateral flow test visually indicates either through visually reading by the human eye or via a reader a negative result for MxA and a positive result for CRP (123), antibiotics should be considered for administration to the patient (133). The threshold concentration of CRP in a sample needed to elicit a positive result is approximately 6-15 mg/L. In other preferred embodiments, the threshold concentration of MxA in a sample to elicit a positive result may be as low as approximately 13 ng/ml; however, the threshold concentration may by higher, in a range from approximately 15 ng/ml to approximately 250 ng/ml.

[00162] If the lateral flow test visually indicates a positive result for MxA (124) and symptoms of ARI have been present for 7 or more days (147), no antibiotics are administered (134). Further determination of whether the patient is high risk or low risk is carried out. The determination of low risk and high risk if preferably dependent on one or more factors such as age, overall health, and preexisting medical conditions. If the patient is at high risk (135), confirmatory COVID molecular testing (137), such as PCR testing (104) is conducted and the patient is preferably admitted to the hospital for further observation (138). If the patient is at low risk (136), confirmatory COVID molecular testing (137), such as PCR testing (104) is conducted and the patient is preferably sent home and quarantined for at least 2 weeks (139). Additional testing after the quarantine can take place. Additionally, other testing can be conducted, such as a respiratory panel including at one or more of influenza A, influenza B, SARS, and RSV at the same time confirmatory COVID molecular testing takes place.

[00163] If the lateral flow test visually indicates a negative result for MxA and a negative result for CRP (125), further determination of whether the patient is high risk or low risk is carried out. The determination of low risk and high risk if preferably dependent on one or more factors such as age, overall health, and preexisting medical conditions. [00164] If the patient is at low risk (126), serological testing for one or more of IgM, IgA and IgG is conducted. The serological testing can take place via the same lateral flow device or assay as the MxA and CRP detection, on the same strip or on a separate strip.

For patients who test positive for one or more of COVID IgM, IgA and IgG (128), additional confirmatory COVID molecular testing (140) via PCR should be considered and the patient admitted to the hospital for observation (141). Antibiotics are not administered (134).

[00165] If the patient is at low risk (126) and conducted serological testing for one or more of IgM, IgA and IgG is negative (129), the patient is home quarantined until symptom free (132) and repeating of the COVID serological test should be considered if symptoms persist for 7 or more days (147). Antibiotics are not administered (134).

[00166] If the patient is at high risk (127), and conducted serological testing for one or more of IgM, IgA and IgG is positive (130) or if the serological testing for one or more of IgM, IgA and IgG is negative (131), additional confirmatory COVID molecular testing (144) via PCR should be considered and the patient admitted to the hospital for observation (145). Antibiotics are not administered (134).

[00167] The multi-tiered, rapid diagnostic strategy provides an ability to detect intracellular host proteins and serum based acute phase proteins and/or immunoglobulins on a lateral flow test strip within 5-30 minutes with visual detection or through use of an accessory reader.

[00168] Two multicenter, U.S. clinical trials, found that MxA elevated in clinically significant viral infections caused by the following pathogens: Adenovirus, Rhinovirus, Influenza A, Influenza B, Metapneumovirus, Parainfluenza Virus 1-4, Respiratory Syncytial Virus, Herpes Simplex Virus, Epstein-Barr Virus, Cytomegalovirus, and Coronavirus (types 229E, OC43, NL63, and HKU1). While there is no direct evidence SARS-CoV-2 elevates MxA, based on the response generated from other strains, it is probable that COVID- 19 will elicit an MxA response. A prospective multi-center U.S. clinical trial found the dual biomarker test to be 95% sensitive, 94% specific and have a negative predictive value of 99% to exclude a bacterial infection and a positive predictive value of 90% to confirm viral infection in febrile ARI patients. The dual biomarker test has also been shown to influence clinical management in up to 90% of cases and reduced unnecessary use of antibiotics by 80-90%. As such, the presence of elevated MxA could support a general triage strategy to rapidly identify patients with an acute viral infection and only refer positive or high-risk cases for further confirmatory testing. The proposed paradigm, which uses 10-minute screening tests, would result in increased throughput and decrease crowding in clinics, urgent cares, and emergency departments (EDs). Additionally, by triaging patients using rapid point of care tests, confirmatory testing, molecular testing capacity can be further augmented to alleviate backlogs and reagent shortages while simultaneously reducing costs (i.e. material and labor).

[00169] Another rapid POC lateral flow immunoassay detects one or more of IgM, IgA and IgG antibodies to SARS-CoV-2 virus within 15 minutes. Typically, IgM takes 7-10 days to develop a detectable response but if present would confirm new infections. The presence of IgM/IgG would suggest recent infection while IgM negative and IgG positive would suggest a previous infection. This testing strategy would be most effective 1-2 weeks after the initial onset of symptoms and would also help to help determine herd immunity and the risk of a new infection for those returning from quarantine. The testing sensitivity ranges 28.7% (symptom onset 1-7 days) and increases to 73.3 % (symptom onset 8-14 days) and 94.3% by greater than 15 days of symptom onset. Another study reported an overall sensitivity of 88.66% and specificity was 90.63% in clinically suspected SARS-CoV-2 confirmed cases (date of symptom onset not collected). Use of rapid serological screening of a combination of SARS-CoV-2 IgA, IgM, IgG, and MxA would serve to quickly test and identify both COVID infection, history of COVID-19 infection, other viral infection, and COVID immunity. If COVID IgA and/or IgM is present along with elevated MxA, the patient has an active infection; If IgA or IgM is present with normal MxA, the infection is a recent infection; If MxA is normal and IgM + IgG or just IgG is present, the patient has immunity. The addition of CRP, Procalcitonin, HNL, and 11-6 would confirm bacterial infection if MxA and serological IgA/IgM is negative or suggest co-infection if high CRP > 100 mg/L or PCT > 0.5 ng/ml is present.

[00170] Therefore, the point of care multi-tiered, rapid diagnostic strategy of an embodiment of the present invention incorporates (i) a rapid host immune response assay as an initial triage test and (ii) confirmatory molecular testing and (iii) a rapid IgM/IgG/IgA test for assessing which patients would benefit from quarantine or further testing. As a first step, the host immune response assay can differentiate the cause of the infection as viral or bacterial. Patients with a viral positive host response would have swabs sent for pathogen-specific molecular testing. Patients confirmed as bacterial infection could undergo additional evaluation (e.g. chest imaging) and be started on appropriate antibacterial therapy. Symptomatic patients that test negative for MxA or positive for MxA and present with symptoms for more than 7 days would be tested with a rapid COVID-19 IgM/IgG/IgA test to rule out prior history of COVID-19; confirmatory molecular testing could be performed on all viral positive patients that are negative on the rapid antibody testing to minimize the risk of missing an infection. Deploying this rapid, multi-tiered screening strategy, could preserve healthcare resources for those who need them most while containing spread of disease more effectively with targeted quarantine.

[00171] It should be noted that while MxA and CRP are discussed as determining the early host immune response, other biomarkers can also be used, such as, but not limited to procalcitonin, serum amyloid A, IL-6 (Interleukin- 6), IFIT, or human neutrophile lipocalin (HNL). Some device examples include, but are not limited to, lateral flow devices, ELISA, fluorescence, or chemiluminescence. The results may be qualitative or quantitative or a combination thereof. The test may represent a single use disposable format or a portable or desktop analyzer. Other examples are also described herein.

[00172] In another embodiment, the test for MxA and immunoglobulin testing of one or more of IgG, IgM and IgA for SARS-CoV-2 is a chemiluminescent assay. In the chemiluminescent assay, beads or nanoparticles are coupled with a deactivated antigen or protein from the antigen for MxA and one or more of IgG, IgM and IgA for SARS-CoV-2, any antigen in the sample for MxA and one or more of IgG, IgM and IgA for SARS-CoV- 2 binds to the deactivated antigen of the assay and is immobilized to the plate. Enzyme labeled antibodies are added and bind to the antibodies of the sample. An enzyme substrate is added and causes a light producing chemical reaction which can then be measured. In addition, the chemiluminescent assay can include testing for influenza A, influenza B, SARS and/or RSV. In another embodiment, the assay is a fluorescent immunoassay.

[00173] In yet another embodiment, the rapid host immune response assay is a lateral flow assay, although other assay formats may be used. In a preferred embodiment, the lateral flow assay includes one strip testing for the rapid serological host immune response and detection of a specific virus. For example, a lateral flow test strip can include MxA and detection of the presence of influenza A, influenza B, SARS and/or RSV. Alternatively, the lateral flow assay can include a second test strip for at least one level of C-reactive protein (CRP). In another embodiment, the assay is a chemiluminescent immunoassay or a fluorescent immunoassay.

[00174] The rapid host immune response test uses a first biomarker to determine whether the host immune response is to bacteria and a second biomarker to determine whether the host immune response is to a virus. The first biomarker to determine whether the host immune response is to bacteria can include biomarkers such as procalcitonin (PCT), C- reactive protein (CRP), serum amyloid A, IL-6 (Interleukin- 6), Interferon Induced proteins with Tetratricopeptide repeats (IFIT), or human neutrophile lipocalin (HNL). The second biomarker to determine whether the host immune response is to a virus is MxA.

[00175] In another embodiment, the immunoassay detects for a presence of MxA through detection of mRNA or determining a presence of MxA in a sample through the precursors of MxA including one or more of type 1 alpha interferons, type 1 beta interferons and type II interferons. The immunoassay can also detect for a presence of CRP through detection of mRNA of CRP. It is noted that instead of CRP, the immunoassay can determine a presence of mRNA of serum amyloid A, IL-6, IFIT, or HNL. In addition, the immunoassay can include testing for the presence of influenza A, influenza B, SARS and/or respiratory syncytial virus (RSV). Furthermore, the immunoassay can include determining a presence of specific interferons associated with SARS-CoV-2. The specific interferons can include proteins from genes IFI6, IFI44L, IFI27, WDR74, and OAS2. According to Mick et al. (“Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2” https://www.medrxiv.org/content/10.1101/2020.05.18.20105171v 4 ; Published May 2020), which is hereby incorporated by reference, found that, for example IFI27 was induced by SARS-CoV-2 significantly more than by other viruses, even at low viral load and that the most significant genes upregulated by SARS-CoV-2 were interferon inducible, including IFI6, IFI44, IFI27 and OAS2. The assay format used can include, but is not limited to, immunoprecipitation, radioimmunoassays (RIA), enzyme immunoassays (EIA or ELISA), a Vidas ^® immunoassay device (Biomerieux, Hazelwood, Missouri), an i- Stat ^® portable handheld system (Abbott Laboratories, Abbott Park, Illinois), a Philips Handheld diagnostic system (Philips Handheld Diagnostics, The Netherlands), fluorescent immunoassays (FIA), chemiluminescent immunoassays, physiochemical assays (TIA, LAPIA, or PCIA), lateral flow immunoassays, or flow cytometry. MxA monoclonal antibodies have been used in modified flow cytometry (Itazawa et al., Increased lymphoid MxA expression in acute asthma exacerbation in children., Allergy Sep 2001 56(9): 895- 8). Some preferred immunoassays for these biomarkers include, but are not limited to, ELISAs, fluorescence immunoassays, magnetic assays, paramagnetic assays, and chemiluminescent assays. In other embodiments, the mRNA or gene transcripts may be used. In some preferred embodiments, the assays are automated.

[00176] In another preferred embodiments, aptamers to MxA, CRP, and SARS-CoV-2 immunoglobulins can be used rather than antibodies. Additionally, mRNA for the interferon induced genes which code for MxA or genes that code for CRP, procalcitonin, serum amyloid A, IL-6, IFIT, or HNL may be used. Additionally, type I alpha/beta or type II interferons (IFN) associated with MxA can be detected and used as a viral marker to determine whether a sample has a viral infection.

[00177] In another embodiment, an immunoassay can detect mRNA for specific interferons associated with SARS-CoV-2 which were upregulated and interferon induced, such as IFI6, IFI44L, IFI27, WDR74, and OAS2.

[00178]

[00179] In a preferred embodiment of the present invention, the lateral flow immunoassay device of the present invention includes a sample-transporting liquid, which can be a buffer, and a chromatographic test strip containing one or several fleece materials or membranes with capillary properties through which sample flows. Some preferred materials and membranes for the test strip include, but are not limited to, Polyethylene terephthalate (PET) fibers, such as Dacron® fibers, nitrocellulose, polyester, nylon, cellulose acetate, polypropylene, glass fibers, and combinations of these materials and their backings. In some embodiments of the invention, it is unnecessary to lyse the cells in the sample or treat the sample in any way prior to applying it to the test strip. [00180] One preferred method of the present invention uses a sample analysis device, for example a chromatographic test strip, to determine if an infection is bacterial or viral. In this method, a sample is collected, and transferred to the chromatographic test strip. In a preferred embodiment, the sample is a sample including leukocytes. The test strip includes a reagent zone. The reagent zone preferably includes at least one first reagent specific to a bacterial marker such that, when the bacterial marker present in the sample contacts the first reagent, a first labeled complex forms. The reagent zone also preferably includes at least one second reagent specific to a viral marker such that, when the viral marker present in the sample contacts the second reagent, a second labeled complex forms. A detection zone includes both a bacterial marker binding partner which binds to the first labeled complex and a viral marker binding partner which binds to the second labeled complex. The sample is then analyzed for the presence of the viral marker and/or the bacterial marker.

[00181] A preferred embodiment of a device of the present invention includes a sample application zone. The device also includes a reagent zone, which includes at least one first reagent specific to a bacterial marker such that, when a bacterial marker present in the sample contacts the first reagent, a first labeled complex forms and at least one second reagent specific to a viral marker such that, when a viral marker present in the sample contacts the second reagent, a second labeled complex forms. A detection zone on the device includes a bacterial marker binding partner which binds to the first labeled complex and a viral marker binding partner which binds to the second labeled complex. One example of a device that could be used is a chromatographic test strip. In other preferred embodiments, some of the zones of the device are on one or more chromatographic test strips, while other zones (for example, the reagent zone, the sample application zone, and/or the control binding partner) are on a sample compressor, separate from and in a different plane than the chromatographic test strip.

[00182] In a preferred embodiment, the presence of the viral marker or the bacterial marker is indicated by a test line visible to the naked eye. The presence of the viral marker may be indicated by a first test line while the presence of the bacterial marker is indicated by a second test line. In some embodiments, the first test line displays a first color when positive and the second test line displays a second color different from the first color when positive. In embodiments where both the first test line and the second test line are located in the same space on the sample analysis device, a third color is preferably formed when both the first test line and the second test line are positive. In other embodiments, the two test lines are spatially separate from each other on the device.

[00183] Viral and bacterial infections are highly contagious and difficult to clinically differentiate due to a significant overlap in signs and symptoms, which often leads to the over prescription of systemic antibiotics and fosters antibiotic resistance. In developed countries, acute respiratory infections are the leading cause of morbidity, accounting for: 20% of medical consultations, 30% of absences from work, and 75% of all antibiotic prescriptions. In the U.S., there are approximately 76 million physician office visits annually for acute respiratory infection. The ability to detect an immune response to an infection aids in the clinical diagnostic ability to differentiate infections resulting from a viral and/or bacterial etiology.

[00184] In one preferred embodiment, the bacterial marker is CRP. In another preferred embodiment, the viral marker is MxA. In some preferred embodiments, the detection zone also includes a control line that is visible to the naked eye when the device is working.

[00185] In one preferred embodiment, the marker for viral infection is MxA and the marker for bacterial infection is C-reactive protein (CRP). High MxA protein levels are strongly correlated with systemic viral infection and increased CRP is more associated with bacterial infections. The present invention includes a rapid infectious screening test for identifying MxA and CRP in samples. MxA is present in leukocytes (white blood cells). Therefore, the sample can be taken anywhere leukocytes are available, for example in a peripheral blood sample, nasopharyngeal aspirates, saliva, tears, spinal fluid, and middle ear aspirates.

[00186] In some preferred embodiments with a single test strip containing CRP and MxA, the threshold concentration of CRP in a sample needed to elicit a positive result is approximately 6-15 mg/L. In other preferred embodiments, the threshold concentration of MxA in a sample to elicit a positive result may be as low as approximately 13 ng/ml; however, the threshold concentration may by higher, in a range from approximately 15 ng/ml to approximately 250 ng/ml. The threshold concentration may depend on the size of the sample being applied to the test strip, as well as its dilution, if applicable. [00187] In some embodiments, the devices and methods described herein allow for the rapid, visual, qualitative in vitro detection of both MxA and CRP directly from peripheral whole blood. In one preferred embodiment, the test measures an immune response to a suspected viral and/or bacterial infection in patients older than one year that present within seven days of onset of a fever, with respiratory symptoms consistent with respiratory disease, and with a suspected diagnosis of acute pharyngitis or community acquired pneumonia. Negative results do not necessarily preclude respiratory infection and should not be used as the sole basis for diagnosis, treatment, or other management decisions. In some embodiments, the use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, viral polymerase chain reaction, and radiography) and clinical presentation is preferably additionally used to confirm whether a specific lower respiratory or pharyngeal pathogen exists.

[00188] In addition, there are some conditions that lead to erroneous false positives or negatives. These include, but are not limited to, current use of immunosuppressive drugs by the patient providing the sample, current use of oral anti-infective drugs by the patient providing the sample, current use of interferon therapy (e.g. for multiple sclerosis, human immunodeficiency virus (HIV), Hepatitis B (HBV), Hepatitis C (HCV)) by the patient providing the sample and live viral immunization within the last 30 days by the patient providing the sample. Both false negatives and false positives are possible since the levels can fluctuate due to therapy.

[00189] In preferred embodiments, the devices and methods are intended for professional use in an outpatient office or urgent care clinic and should be used in conjunction with other clinical (laboratory or radiographic) and epidemiological information.

[00190] In preferred embodiments, a dual-use dual chromatographic test strip assay detects the body’s immune response to viral and/or bacterial infections in patients using a multiplexed pattern of results. In one specific preferred embodiment, the assay tests for Myxo virus resistance A (MxA), low levels of C-reactive Protein (“low” CRP), and high levels of C-reactive Protein (“high” CRP). Two test strips are preferably used. In some embodiments, a sample compressor in a different plane from the chromatographic test strips is also used. The first test strip assays for MxA and low levels of C-reactive Protein, and the second test strip is an assay for high levels of C-reactive Protein. The first test strip and/or the sample compressor include reagents to detect MxA protein and a low level of C-reactive protein. The second test strip and/or the sample compressor include reagents to detect a high level of C-reactive protein. The two test strips are preferably ran side-by- side, and each strip also preferably includes a control line. The control reagents are preferably either on the test strips or on the sample compressor. These tests detect and classify biological infections as viral, bacterial, or a co-infection of virus and bacteria. In some preferred embodiments, the dual-use dual chromatographic test strip assay is used to detect samples from patients with a febrile respiratory illness. In some embodiments, one of the two test strips can include testing for one or more of influenza A, influenza B,

SARS and/or RSV. More preferably, the tests for influenza A, influenza B, SARS and/or RSV are on the same strip as MxA.

[00191] In some preferred embodiments with two test strips, on the first test strip, a threshold concentration of CRP (“low” CRP level) of approximately 6-15 mg/L (serum cut-off value) in the sample is needed to elicit a positive result and a threshold concentration of at least 13 ng/ml MxA in a sample is needed to elicit a positive result. In other preferred embodiments, the threshold concentration for MxA may be in a range from approximately 15 ng/ml to approximately 250 ng/ml to elicit a positive result. The threshold concentration may depend on the size of the sample being applied to the test strip, as well as its dilution, if applicable. In one preferred embodiment, the threshold concentration of low CRP, for example in extracellular serum from a blood sample, is 7 mg/L for a fingerstick cut-off value, which is equivalent to 10 mg/L for a serum cut-off value. In one preferred embodiment, the threshold concentration of MxA, for example in peripheral blood mononuclear cells from a blood sample, is 15 ng/ml for a fingerstick cut off value, which is equivalent to a 15 ng/ml venous blood cut-off value. On the second test strip, a threshold concentration of CRP (“high” CRP level) of approximately 60-100 mg/L in the sample is needed to elicit a positive result in some preferred embodiments. In one particularly preferred embodiment, a threshold concentration of high CRP on the second test strip is approximately 80 mg/L on a fingerstick cut-off value.

[00192] In other embodiments, other markers for viral infection and/or bacterial infection may be used. For example, approximately 12% of host genes alter their expression after Lymphocytic Choriomeningitis Vims (LCMV) infection, and a subset of these genes can discriminate between virulent and nonvirulent LCMV infection. Major transcription changes have been given preliminary confirmation by quantitative PCR and protein studies and are potentially valuable candidates as biomarkers for arenavirus disease. Other markers for bacterial infection include, but are not limited to, procalcitonin, urinary trypsin inhibitor (uTi) , lipopolysaccharide, IL-1, IL-6, IL-8, IL-10, ESR and an elevated WBC count (increased bands), Lactate, Troponin, vascular endothelial growth factor, platelet derived growth factor, cortisol, proadrenomedullin, macrophage migratory inhibitory marker, activated protein C, CD 4,8,13,14, or 64, caspase, placenta derived growth factor, calcitonin gene-related peptide, high mobility group 1, copeptin, natriuretic peptides, lipopolysaccharide binding protein, tumor necrosis factor alpha, circulating endothelial progenitor cells, complement 3a, and triggering receptor expressed on myeloid cells (trem- 1).

[00193] Lateral flow devices are known, and are described in, e.g., U.S. Published Patent Application Nos. 2005/0175992 and 2007/0059682. The contents of both of these applications are incorporated herein by reference. Other lateral flow devices known in the art could alternatively be used with the systems and methods of the present invention.

[00194] U.S. Published Patent Application No. 2007/0059682 discloses detecting an analyte and a sample which can also contain one or more interfering substances. This publication teaches separating the analyte from the interfering substances by capturing the interfering substances on the chromatographic carrier, and detecting the analyte on the carrier separated from the interfering substances.

[00195] U.S. Published Patent Application No. 2005/0175992 discloses a method for detecting targets, such as pathogens and/or allergy-associated components, in a human body fluid where the body fluid sample is collected by a collection device, such as a swab member. The samples are transferred from the swab member to a sample analysis device, on which an analysis of the targets can occur by immunochemical or enzymatic means.

The test result is capable of being displayed within a very short period of time and can be directly read out by the user. This enables point-of-care testing with results available during a patient visit. The inventions disclosed in this application are particularly advantageous for the diagnosis of conjunctivitis.

[00196] In a method of the invention, the sample to be analyzed is applied to a chromatographic carrier. The carrier can be made of one single chromatographic material, or preferably several capillary active materials made of the same or different materials and fixed on a carrier backing. These materials are in close contact with each other so as to form a transport path along which a liquid driven by capillary forces flows from an application zone, passing a reagent zone, towards one or more detection zones and optionally a waste zone at the other end of the carrier. In other embodiments, the liquid passes the reagent zone prior to flowing into the sample application zone. In an especially preferred embodiment, the carrier is a chromatographic test strip. In other preferred embodiments, the sample may be applied to a sample compressor in a different plane from the chromatographic test strip, and then transferred to the chromatographic test strip by the sample compressor.

[00197] In some embodiments, the sample is directly applied to the carrier by dipping the carrier's application zone into the sample. Alternatively, application of the sample to the carrier may be carried out by collecting the sample with a dry or wetted wiping element from which the sample can be transferred, optionally after moistening, to the carrier's application zone. Usually, the wiping element is sterile and may be dry or pretreated with a fluid before the collection step. Materials suitable for wiping elements according to the invention may comprise synthetic materials, woven fabrics or fibrous webs. Some examples of such wiping elements are described in German Patents DE 44 39429 and DE 19622 503, which are hereby incorporated by reference. In other embodiments, the sample may be collected by a collection receptacle, such as a pipette, and transferred directly to the carrier.

[00198] Depending on the type of detection method, different reagents are present in the carrier's reagent zone, which, in some embodiments, is preferably located between the application zone and the detection zone or, in other embodiments, is preferably located before the application zone. In yet other embodiments, the reagents may be on a sample compressor separate from and in a different plane than the carrier including the detection zone.

[00199] In a sandwich immunoassay, it is preferred to have a labeled, non-immobilized reagent in the reagent zone that is specific to each bacterial and viral marker that is being detected. Thus, when a viral or bacterial marker present in the sample contacts the corresponding labeled viral or bacterial reagent present in the reagent zone, a labeled complex is formed between the marker and the corresponding labeled reagent. The labeled complex in turn is capable of forming a further complex with an immobilized viral or bacterial marker binding partner at a test line in the detection zone. In a competitive immunoassay, the reagent zone preferably contains a labeled, non-immobilized marker analogue which competes with the marker for the immobilized marker binding partner in the detection zone. The marker binding partners in the reagent zone and in the detection zone are preferably monoclonal, polyclonal or recombinant antibodies or fragments of antibodies capable of specific binding to the corresponding marker.

[00200] In a preferred embodiment, the present invention provides for the reduction of interfering substances that might be present in the sample to be tested. Since an interfering substance, e.g. a human anti-mouse antibody (HAMA), may also be capable of forming a complex with the labeled, non-immobilized reagent of the reagent zone and the immobilized binding partner of the detection zone, thus indicating a positive test result in the immunoassay, the carrier may further include at least one capturing zone. Each capturing zone contains an immobilized capturing reagent specifically binding to a certain interfering substance, thereby immobilizing the interfering substance in the capturing zone. As the capturing zone is separated from the detection zone by space, and the sample starts to migrate over the reagent zone and the capturing zone before reaching the carrier's detection zone, the method allows a separation of the interfering substance or substances from the analyte or analytes of interest. Preferably, the capturing zone is located between the reagent zone and the detection zone. However, the capturing zone may also be located between the application zone and the reagent zone.

[00201] Detection of the marker may be achieved in the detection zone. The binding molecule immobilizes the labeled complex or the labeled marker- analogue by immune reaction or other reaction in the detection zone, thus building up a visible test line in the detection zone during the process. Preferably, the label is an optically detectable label. Forming a complex at the test line concentrates and immobilizes the label and the test line becomes visible for the naked eye, indicating a positive test result. Particularly preferred are direct labels, and more particularly gold labels which can be best recognized by the naked eye. Additionally, an electronic read out device (e.g. on the basis of a photometrical, acoustic, impedimetric, potentiometric and/or amperometric transducer) can be used to obtain more precise results and a semi-quantification of the analyte. Other labels may be latex, fluorophores or phosphorophores.

[00202] In one embodiment, the sensitivity of visually read lateral flow immunoassay tests is enhanced by adding a small quantity of fluorescing dye or fluorescing latex bead conjugates to the initial conjugate material. When the visible spectrum test line is visibly present, the test result is observed and recorded. However, in the case of weak positives that do not give rise to a distinct visual test line, a light of an appropriate spectrum, such as a UV spectrum, is cast on the test line to excite and fluorescent the fluorescing latex beads which are bound in the test line to enhance the visible color at the test line.

[00203] In a preferred embodiment, the reagents are configured such that the visible test line corresponding to the presence of the viral marker will be separate from the test line corresponding to the presence of the bacterial marker. Therefore, it can be readily determined whether the sample contained bacterial or viral markers (or both) simply by the location of the development of the test lines in the detection zone. In another preferred embodiment, the reagents may be chosen such that differently colored test lines are developed. That is, the presence of a viral marker will cause the development of a differently colored line than that developed by the presence of a bacterial marker. For example, the label corresponding to the reagent recognizing the viral marker may be red, whereas the label corresponding to the reagent recognizing the bacterial marker may be green. Differently colored labels that may be attached to the non-immobilized reagents are well known. Some examples include, but are not limited to, colloidal gold, colloidal selenium, colloidal carbon, colloidal gold, nanoparticles, latex beads, paramagnetic beads, fluorescent and chemiluminescent labels and mixtures thereof.

[00204] Figures 5A and 5B show a chromatographic test strip (400) with a test line (402) corresponding to the presence of a viral marker and a second, separate test line (403) that detects the presence of a bacterial marker. The sample is applied to the application zone (401) of the chromatographic test strip (400). As shown in Figure 5A, the sample then passes a reagent zone (460) containing at least one labeled viral binding partner and at least one labeled bacterial binding partner that is eluted by and then able to migrate with a sample transport liquid (e.g. a buffer solution). Alternatively, as shown in Figure 5B, the reagent zone (460) is located upstream of the sample application zone (401) such that the labeled binding partners in the reagent zone are eluted by the sample transport liquid and travel to the sample. The labeled viral binding partner is capable of specifically binding to a viral marker of interest to form a complex which in turn is capable of specifically binding to another specific reagent or binding partner in the detection zone. The labeled bacterial binding partner is capable of specifically binding to a bacterial marker of interest to form a complex which in turn is capable of specifically binding to another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier backing, a housing, and an opening in the housing for result read out, may optionally also be a component of the test strip (400) in these embodiments.

[00205] The test strip (400) also includes a detection zone (405) containing at least one first section for detection of a viral marker, e.g. a test line (402), including an immobilized specific binding partner, complementary to the viral reagent complex formed by the viral marker and its labeled binding partner. Thus, at the test line (402), detection zone binding partners trap the labeled viral binding partners from the reagent zone (460) along with their bound viral markers. This localization of the viral marker with its labeled binding partners gives rise to an indication at the test line (402). At the test line (402), the presence of the viral marker is determined by qualitative and/or quantitative readout of the test line (402) indication resulting from the accumulation of labeled binding partners.

[00206] The detection zone (405) also includes at least one second section for detection of a bacterial marker, e.g. a test line (403), including an immobilized specific binding partner, complementary to the bacterial reagent complex formed by the bacterial marker and its labeled binding partner. Thus, at the test line (403), detection zone binding partners trap the labeled bacterial binding partners from the reagent zone (460) along with their bound bacterial markers. This localization of the bacterial marker with its labeled binding partners gives rise to an indication at the test line (403). At the test line (403), the presence of the bacterial marker is determined by qualitative and/or quantitative readout of the test line (403) indication resulting from the accumulation of labeled binding partners. While test line (402) is upstream of test line (403) relative to the direction of flow (408) in the figures, in alternative embodiments, test line (403) is upstream of test line (402). In still other embodiments, test lines (402) and (403) are located in the same location on the test strip.

[00207] Optionally, the detection zone (405) may contain further test lines to detect other viral and/or bacterial markers, as well as a control line (404). The control line (404) indicates that the labeled specific binding partner traveled through the length of the assay, even though it may not have bound any viral or bacterial markers, thus confirming proper operation of the assay. As shown in Figures 5A through 5B, the control zone (404) is preferably downstream of the test lines (402) and (403). However, in other embodiments, the control zone (404) may be located upstream of either or both of the test lines (402) and (403).

[00208] In a preferred embodiment, the control line (404) includes an antibody or other recombinant protein which binds to a component of the elution medium or other composition being used in the test. In embodiments where nucleic acids are the targets, the control line (404) preferably includes a nucleic acid complementary to the labeled nucleic acid being used as a binding partner for the target nucleic acid.

[00209] Although only one test line is shown in the figures for each of the viral and bacterial markers, multiple test lines for both or either of the viral and bacterial markers may be used within the spirit of the invention. In some embodiments where there are multiple bacterial and/or viral targets, the presence of each target preferably corresponds to a separate test line (402) or (403). In other embodiments, both the bacterial marker and the viral marker are detected on a single test line. In these embodiments, the presence of both a bacterial marker and a viral marker on the same test line has different characteristics than the presence of either a bacterial or viral marker alone. For example, the presence of both a bacterial marker and a viral marker on the same test line may be visually indicated by a different color than the presence of either a bacterial marker or a viral marker alone.

[00210] Fresh whole blood samples of patients showing symptoms of viral infections (flu like symptoms and fever of >100.5°F) were tested to determine what levels of MxA in the blood could be detected with the lateral flow tests described herein. The lateral flow assays used in these experiments had a similar configuration as the device shown in Figure 5B described above, without a second test line for the presence of a bacterial marker. More specifically, the test strip included a reagent zone upstream of a sample application zone. The reagent zone included mobilizable antibodies to MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan) labeled with colloidal gold. The test strip also included a test line in a detection zone. The test line included an immobilized antibody for MxA (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The control line in the detection zone included rabbit anti chicken antibody plus rabbit Ig (for an extra stabilizing effect), which binds to mobilized chicken IgY labeled with blue or black latex beads.

[00211] The whole blood samples were collected with EDTA as the anticoagulant. In these tests, the amount of MxA protein in the blood samples was determined using an MxA Protein ELISA Test kit (Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan). The blood was lysed 1:10 with lysing solution provided in the kit, prior to being applied to the test strip. 100 pi of lysed blood was tested in the ELISA test. 10 mΐ of lysed blood was used as the sample in the MxA lateral flow test.

[00212] The lysed blood samples were applied to the application zone of the test strip. The labeled MxA antibodies in the reagent were eluted by the sample transport liquid and travelled to the blood samples. At the test line, the immobilized MxA antibody trapped any labeled MxA antibody from the reagent zone bound to MxA. This localization of the MxA with its labeled antibody gave rise to a red visual indication at the test line if there was a sufficient concentration of MxA.

[00213] Figures 14A-14B shows the test results for 29 patients of the 48 patients between the ages of 29-96 who were tested for SARS-COV2 using CRP and MxA on a test strip and for influenza A, B, and RSV. The results were confirmed using PCR. The signs and symptoms presented were recorded along with the duration in days of said symptoms. Chest x-ray results were also documented. Most patients exhibited either pulmonary infiltrates indicating a white abnormal area on the lungs exhibited on the chest x-ray, which can be located in either one lung or both lungs or consolidation in which a region of normally compressible lung tissue is filled with liquid instead of air. As shown in the results, the multiplexed assay had all tests run accurately as indicated by the control and accurately detected positive cases of SARS-COV-2 as confirmed by OCR testing. Additionally, the patients all tested negative for influenza A, influenza B and RSV.

[00214] Table 6

[00215] Table 6 shows the MxA ELISA kit standards run per the test instructions. As shown in Table 6, an MxA concentration of 24 ng/ml produced a positive result in the lateral flow test. The kit standard was used to generate the standard curve from which the MxA concentrations were determined.

[00216] Table 7 shows the results of clinical fresh whole blood samples of patients showing symptoms of viral infections (flu like symptoms and fever of > 100.5°F)).

[00217] Table 7 [00218] The OD (optical density) values were used in combination with the standard curve from the kit’s standard in order to determine the MxA concentration in the samples. The concentration (ng/ml) column was the concentration as diluted with the lysing agent. The concentration x dilution (lOx) (ng/ml) column was the actual concentration in the whole blood sample. As shown in the table, the lateral flow test produced a positive result for MxA in samples C and F, which had approximately 105 ng/ml of MxA and approximately 22 ng/ml of MxA, respectively, in the samples. [00219] Table 8 shows the results of frozen whole blood samples from normal individuals from the Tennessee blood bank. None of the blood samples had any discernible amounts of MxA, and all of them were negative in the lateral flow test.

[00220] Table 8

[00221] Table 9 shows freshly frozen whole blood samples from BioReclamation (BioReclamation, Hicksville, NY) of patients showing apparent symptoms of viral infections (flu like symptoms and fever of >100.5°F)). None of these patients had ODs that corresponded to MxA levels higher than approximately 8 ng/ml. These samples were all negative in the lateral flow test. [00222] Table 9

[00223] The results of these tests indicate that the lateral flow tests described herein can detect MxA levels at least as low as approximately 20 ng/ml in a 10 pi sample (diluted 1:10).

[00224] One example of a rapid screening test for distinguishing viral and bacterial infection is shown in Figure 3. As discussed above, MxA is a diagnostic marker for viral infection, while CRP is a diagnostic marker for bacterial infection. In this example, a control line in A-D of the Figure, which could be blue, represents the control. Another line represents a C-reactive protein (CRP) level > 15 mg/L (“CRP test” in A-D of the figure) and could, for example green. Another line represents an MxA level > 15 ng/ml (“MxA test” in A-D of the figure) and could be indicated by a red line. A positive result for the MxA protein, with a negative result for the CRP protein indicates only a viral infection (Visual Test Result A). A positive result for the (CRP) with a negative result for the MxA protein indicates only a bacterial infection (Visual Test Result B). A positive result for both MxA and CRP indicates co-infection (infection with both a bacteria and a virus) (Visual Test Result C). No bacterial or viral infection is indicated by a negative result for both MxA and CRP (Visual Test Result D). While particular color lines are discussed in this example, other colors, or the same colors at different locations on the test strip to indicate viral or bacterial markers, are within the spirit of the present invention. It is noted that the amount of MxA of >235 ng/ml does not include a correction factor which is accounting for aggregation of MxA that can occur. Other amounts of MxA take into account the correction factor and are within 2-3 standard deviations of the average normal range. [00225] When development of different colored lines is utilized, the lines may or may not be separated by space. In the latter instance, the labels are chosen such that the color seen when both markers are present is different from the colors seen when the individual markers are present. For example, the presence of the viral marker may be indicated by a red line; the presence of the bacterial marker by a blue line; and the presence of both by a purple line (combined red and blue).

[00226] The use of two colors to distinguish acute and chronic infection is shown in Figure 4. In the first cassette, only IgM antibodies are present, which indicates an acute infection. In this cassette, the test line is red. In the second cassette, the test line is blue because the immunoglobulins are IgG. The third cassette shows an intermediate case, where both IgM and IgG antibodies are present. Consequently, the test line is purple. While this example is shown to test for IgMs and IgGs, the same concept is alternatively used with a single line which detects both viral and bacterial markers for infection.

[00227] In another preferred embodiment, the test strip may also include a control section which indicates the functionality of the test strip. Figure 3 shows a control line. Figure 4 shows an example where there is a control section for all three cassettes. If present, the control section can be designed to convey a signal to the user that the device has worked. For example, the control section may contain a reagent (e.g., an antibody) that will bind to the labeled reagents from the reagent zone. In one preferred embodiment, rabbit anti chicken is used as the control line and chicken IgY conjugated to a label, for example blue or black latex beads, is the control conjugate. Alternatively, the control section may contain an anhydrous reagent that, when moistened, produces a color change or color formation, e.g. anhydrous copper sulphate which will turn blue when moistened by an aqueous sample. As a further alternative, the control section could contain immobilized viral and bacterial markers which will react with excess labeled reagent from the reagent zone. The control section may be located upstream or downstream from the detection zone. A positive control indicator tells the user that the sample has permeated the required distance through the test device.

[00228] In an example, 48 consecutive symptomatic ARI patients presented in the hospital or emergency department setting with a suspected COVID-19 infection. A POC lateral flow test testing for at least MxA and CRP was used to rapidly assess the patient’s immune response to an ARI and differentiate viral from bacterial infection. The lateral flow test identifies host immune response to infection and differentiates viral and bacterial ARI through the simultaneous detection of both Myxovirus resistance protein A (MxA) and C-reactive protein (CRP) directly from a fingerstick blood sample. MxA is specifically induced by the production of Type I interferon (IFN) a/b. The IFN system is a key component of the innate host response to viral infections and has immune modulating and antiviral functions. Type I IFNs are produced by many different cell types, specifically monocytes and macrophages, in response to a wide range viral infections and are found to be elevated in the presence of most acute viral infections 5,7,8 C-reactive protein (CRP) is a nonspecific, acute-phase protein that is upregulated due to acute inflammation, including response to infection. CRP is predominately produced by the liver in response to inflammatory cytokines such as IL-6 and assists in pathogen recognition and phagocytosis by macrophages and bacterial infection is a potent stimulus of CRP.

[00229] When viral pathogens induce a clinically significant host immune response, MxA, a biomarker of the body’s innate response to a viral infection, will elevate. While a bacterial infection is associated with an elevated CRP in the absence of MxA. Consistent with previous studies, an elevation of MxA with or without elevation in CRP was interpreted as a viral infection; and an elevation in CRP without MxA was interpreted as a bacterial infection. The lateral flow test of FebriDx ^® from Rapid Pathogen Screening, Inc. of Sarasota Florida was used according to the manufacturer package insert and results were verified by two physicians.

[00230] Patients meeting the inclusion criteria were offered the POC FebriDx ^® test at the same time as the nasal and pharyngeal swab for viral PCR testing (COVID-19, Influenza A, Influenza B and Respiratory Syncytial Virus (RSV)). The patients additionally had standard routine blood tests and tests for procalcitonin (PCT). Patients who had recent onset of symptoms and clinical course consistent with COVID-19 infection along with bilateral infiltrates/pneumonia on a chest x-ray and lymphocytopenia were categorized as highly likely COVID-19 infection. This was confirmed by two independent physicians. Others were categorized as having a low likelihood of COVID-19 infection. Any patients with both FebriDx ^® positive and COVID-19 (rRT-PCR) negative tests, were considered clinically to have COVID-19 infection, if the clinical suspicion of COVID-19 was high. [00231] For this study, the COVID-19 infection per the Public Health England (PHE) was as follows:

-has either clinical or radiological evidence of pneumonia or -acute respiratory distress syndrome or

-influenza-like illness (fever >37.8°C and at least one of the following respiratory symptoms, which must be of acute onset: persistent cough (with or without sputum), hoarseness, nasal discharge or congestion, shortness of breath, sore throat, wheezing, sneezing.

[00232] Inclusion criteria and exclusion criteria are as follows:

Inclusion Criteria:

1. Ago 16 years;

2. Patients requiring admission for at least one night with suspected COVID-19 infection; and

3. Inpatients who meet the PHE criteria for swab testing for COVID-19 infection which includes inpatients with new respiratory symptoms or fever without another cause or worsening of a pre-existing respiratory condition.

Exclusion Criteria:

1. Patients who do not consent for pinprick testing;

2. Patients who do not meet the PHE criteria for swab testing for COVID-19;

3. Live vaccination or antiviral treatment in the last 14 days;

4. Symptoms of more than 7 days duration; and

5. Patient on any immunosuppressive therapy or systemic corticosteroids.

[00233] The primary aim of the study was to determine the positive predictive value (PPV) of FebriDx ^® in identifying patients who have positive viral swab test or a very high clinical likelihood of COVID-19 infection. Test results were logged and tallied against the viral swab results, as well as the clinical likelihood of COVID-19 infection. A secondary aim of the study was to determine the negative predictive value (NPV) of FebriDx ^® in excluding COVID-19 infection in hospitalized patients with respiratory tract symptoms during a pandemic situation.

[00234] The data from the study was summarized using descriptive statistics and results are reported as medians and interquartile ranges or means and standard deviations as appropriate. Categorical variables are summarized numerically and as percentages.

[00235] The study was conducted from March 26, 2020 through April 7, 2020. Seventy- five consecutive patients were assessed, 26 patients excluded. Twenty-five of the 26 patients excluded were not considered due to their history of symptoms being longer than 7 days in duration. One of the 26 patients excluded was not considered due to their immunosuppressed immune system. Forty-nine patients were tested with FebriDx ^® , 48 tests were completed and 1 could not be done due to inability to obtain enough blood. Of the 48 patients enrolled, 66.7% (32/48) males and 33.3% (16/68) female and 54.2% were older than 65 years with a median age of 67 years. Patients reported symptoms for 2-7 days with a mean, median of 3.8 days and 3-day symptom onset, respectively. Fever was present at the time of testing in 85.4% (41/48) patients.

[00236] Of the 48 subjects enrolled, 8.3% (4/48) had a final diagnosis categorized as non-infections, 16.7% (8/48) bacterial infection, 2.1% (1/48) as non-COVID viral infection and 72.9% (35/48) had confirmed COVID infection.

[00237] Of the confirmed COVID infections, 68.6% (24/35) of the patients presented with a clinical picture highly suggestive of COVID-19 infection. FebriDx ^® test results were positive for a viral infection in 72.9 % (35/48) of cases, all of which were confirmed COVID infection, bacterial infection in 22.9% (11/48) of case and non-infectious in 4.2% (2/48) of cases. Final disposition amongst the cohort was 31.3% (15/48) of patients were sent home, 66.7 % (32/48) were still admitted to the hospital and 1 patient died. Cohort characteristics are described in Table 10.

[00238] Table 10

[00239] Overall, 35 patients had FebriDx ^® test positive, and all 35 patients had either positive real-time reverse transcription polymerase chain reaction (rRT-PCR) for COVID- 19 (n=30) or a clinical picture highly suggestive of COVID-19 infection (n=5), which gives a PPV of 100% in this pandemic situation. In all 13 patients, out of the 48 tested, in which FebriDx ^® yielded negative results for a viral infection, but positive results for either a bacterial lower respiratory tract infection (LRTI) or bacterial pneumonia, rRT-PCR for COVID-19 was also negative, confirming the negative results of FebriDx ^® . In one case of lower respiratory tract infection (LRTI), it was not possible to determine the exact cause of the infection and a viral infection could not be excluded despite negative viral test results from the FebriDx ^® test, rRT-PCR for COVID-19, influenza and RSV. Including this one patient, the NPV of FebriDx ^® for COVID-19 was 92%, exceeding the NPV of rRT-PCR, which is 72.2%.

[00240] In this study, the overall sensitivity of FebriDx ^® for COVID-19 infections was 100% for 97.2 % of the patients that tested positive for a viral infection, compared to 85.7 % which tested positive for a viral infection via rRT-PCR. The specificity of both FebriDx ^® and rRt-PCR was 100%. Overall diagnostic performance is summarized in Table 11.

[00241] Table 11

[00242] It is noted that the sensitivity, specificity, PPV and NPV are calculated as follows:

. . . TruePositive

Sensitivity -

TruePositive + FalseNegative

_„ . TrueNegative

Specificity -

FalsePositive + FalseNegative

TruePositive

PPV =

TruePositive + FalsePositive

[00243] More specifically, in the viral diagnostic performance of identifying COVID-19 by FebriDx ^® versus the clinical truth of the diagnosis is shown in Table 12.

[00244] Table 12

[00245] Based on the study results, in a SARS-CoV-2 pandemic situation, any suspected COVID-19 case with FebriDx ^® test results indicating ‘viral positive’ (+MxA), should be treated as ‘positive COVID-19’ and cohorted with other COVID-19 positive patients. This would help avoid unnecessary exposure to other suspected patients who may turn out to be negative on confirmatory rRT-PCR testing. If FebriDx ^® test results indicate ‘viral negative’ , alternative diagnoses should be considered at the outset.

[00246] For bacterial diagnostic performance in identifying bacterial infections of patients using FebriDx ^® versus the clinical truth of the diagnosis is shown in Table 13.

[00247] Table 13 [00248] FebriDx ^® correctly identified 8 of 8 bacterial infections as bacterial with a sensitivity of 100%. FebriDx ^® had 3 false positives for bacterial infection in two non- infectious patients and one clinically indeterminate patient suggesting a specificity for bacterial infection of 92.7 % (37/40). Avoiding mixing these patients with other suspected COVID-19 patients whilst awaiting the results of rRT-PCR, would avoid unnecessary exposure to COVID-19 positive patients.

[00249] For viral diagnostic performance in any viral infections of COVID-19 and non- COVID viruses of patients using FebriDx ^® versus the clinical truth of the diagnosis is shown in Table 14.

[00250] Table 14

[00251] For viral diagnostic performance for identifying COVID-19 using rRT-PCR versus clinical truth is shown in Table 15.

[00252] Table 15

[00253] rRT-PCR results take at least 48 hours to process and were not a sensitive to COVID-19 as FebriDx ^® .

[00254] In some preferred embodiments, the devices and methods of the present invention include a lysis zone to help differentiate viral and bacterial infections. In these embodiments, the sample that has been collected is not lysed prior to collection and transfer to the sample analysis device. This decreases the number of steps needed to collect and prepare the sample for analysis. One situation where a lysis agent improves assay efficiency is in assaying for the presence of MxA. As discussed herein, the presence of this protein can help to distinguish between bacterial and viral infection in febrile children. In situ lysis using a combination of 1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volume NP40 as the lysis agent improves detection of MxA in fresh or frozen whole blood.

[00255] In the embodiments utilizing a lysis agent, following sample loading, the sample traveling with the transport liquid (buffer) will encounter the lysis agent. The lysis agent will have preferably been pre-loaded onto the test strip and is eluted by the transport liquid. In some preferred embodiments the lysis agent has been dried into the test strip. Alternatively, the lysis agent may be pre-dried by freeze drying or lyophilizing and then pre-loaded into the test strip. In other embodiments, the lysis agent may be absorbed, adsorbed, embedded or trapped on the test strip. The initially dried lysis agent is preferably localized between the sample application zone and a reagent zone. In embodiments where the reagent zone is upstream of the sample application zone, the lysis zone is downstream of the sample application zone. The lysing agent is preferably soluble in the sample transport liquid, and the lysing agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains both lysing agent in solution or suspension and sample components in suspension. Any lysis- susceptible components in a sample, then being exposed in suspension to the lysing agent, are themselves lysed in situ. The running buffer then carries the analyte, including any lysis-freed components, to the detection zone.

[00256] The location where the lysis agent is pre-loaded and dried can be varied as needed. In order to maximize the time that the sample has to interact with the lysis agent as well as to minimize the amount of lysis agent reaching the detection zone, the dried, absorbed, adsorbed, embedded, or trapped lysis agent may be located in or just downstream of the sample application zone. Or, in order to minimize the distance along which the lysis product must travel before reaching the reagent zone, the dried lysis agent may be located closer to the reagent zone. In other embodiments, the lysis agent may be included in the running buffer.

[00257] The concentration of lysis agent pre-loaded onto a test strip is preferably between 0.001% and 5% weight/volume. The volume to be pre-loaded depends on where the lysis agent is pre-loaded. Appropriate ranges are 1 to 10 microliters when pre-loaded into the sample collector fleece (the sample application zone) or 5 to 50 microliters when pre-loaded into the absorbent pad or into other locations within the test strip. Ideally, the amount pre-loaded should be approximately 3 microliters pre-loaded into the sample collector fleece or approximately 10 microliters pre-loaded into the absorbent pad or into other locations within the test strip.

[00258] Selection of a specific lysing environment and agent will depend on the viral and bacterial markers and the assay. The pH and ionic strength are key to the lysing environment. As to pH established by the lysis agent, a pH below 4.0 tends to precipitate materials, especially proteins. Higher pH, above approximately 10.0, tends to lyse materials such as proteins and cells walls. Therefore, a pH of approximately 10.0 or above is preferable for many applications. Alternatively, lower pH may be preferred for nucleic acid targets.

[00259] As to ionic strength established by the lysis agent, both the high and low ionic strength may be used to lyse. For example, a lower ionic strength (hypotonic) tends to break up erythrocytes. For example, water by itself can lyse erythrocytes. Higher ionic strength environments may be used to rupture certain cell walls and membranes.

[00260] As to specific lysis agents, they may be grouped and selected based on their properties: salts, amphoteric and cationic agents, ionic and non-ionic detergents. The salt, Ammonium Chloride (NH4CI), lyses erythrocytes. Other salts, including, but not limited to, high concentrations of Sodium Chloride (NaCl) and Potassium Chloride (KC1), may rupture certain cell walls and membranes. Other lysis agents are amphoteric agents including, but not limited to, Lyso PC, CHAPS, and Zwittergent. Alternatively, cationic agents including, but not limited to, C16 TAB and Benzalkonium Chloride may be used as a lysis agent. Both ionic and non-ionic detergents are often used to break or lyse the cell wall or cell membrane components such as lipoproteins and glycoproteins. Common ionic detergents include, but are not limited to, Sodium Dodecyl (lauryl) Sulfate (SDS), Cholate, and Deoxycholate. Ionic detergents are good solubilizing agents. Antibodies retain their activity in 0.1% SDS or less. Common non-ionic detergents include, but are not limited to, Octylglucoside, Digitonin, C12E8, Lubrol, Triton X-100, Noniodet P-40, Tween 20, and Tween 80. Non-ionic and mild ionic detergents are weaker denaturants and often are used to solubilize membrane proteins such as viral surface proteins. Additional lysis agents include, but are not limited to, urea and enzymes. Combinations of different lysis agents may be used to optimize the lysing environment.

[00261] Surfactants are generally wetting agents and lower the surface tension of a liquid. This then allows easier spreading by lowering the interfacial tension between liquids. So, surfactants can interfere with the natural binding of antigen and antibody or ligand and receptors. The concentrations are, therefore, experimentally chosen for each class of lysis agent. Once lysis occurs, it is important that the desired binding reactions not be hindered. Generally, 0.001% lysis agent concentration is considered the lower limit, and the upper limit is approximately 1%. There is an additive or synergistic effect when combinations of lysis agents are used. This expands the working range of concentration to ran from approximately 0.001% to 1%. Finally, some undesirable non-specific binding may be prevented at a Tween 20 concentration of 5%. In all cases, the total amount of lysis agent pre-loaded onto all locations of an individual test strip must be sufficient to lyse barriers to immunodetection, permitting practical operation of the test strip.

[00262] The lysis agent itself should not interfere with any other assay detector or indicator agents and thus does not interfere with any other assay interactions and reactions to such an extent as to prevent practical operation of the assay. A lysis agent should have sufficient shelf life to allow manufacture, distribution and storage before use of a test strip in point-of-care testing.

[00263] In preferred embodiments where MxA is the viral marker, in situ lysis using a combination of 1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volume NP40 as the lysis agent is preferably used. As a more specific example, 2 microliters of 100 mM HEPES buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA, and 0.1% Sodium Azide (all percentages weight/volume) are dried onto a lysis zone of a test strip.

[00264] In a preferred embodiment, as shown in Figures 6A through 6D, the sample is applied to the application zone (201) on a chromatographic test strip (200). The sample passes a lysis zone (250), where a lysis agent will have preferably been pre-loaded onto the test strip and is eluted by the transport liquid. The lysis agent lyses any lysis- susceptible components in the sample in situ. [00265] The chromatographic test strip contains a sample application zone (201), a lysis zone (250) containing a lysis agent, and a reagent zone (260) containing at least one labeled binding partner that binds to a viral marker and at least one labeled binding partner that binds to a bacterial marker that are eluted by and then able to migrate with a sample transport liquid (e.g. a buffer solution). While the reagent zone (260) is shown downstream of the sample application zone in these figures, in alternative embodiments, the reagent zone (260) could be upstream of the sample application zone (see Figure 5B), as long as the reagents encounter the sample at some point after the sample reaches the lysis zone and is effectively lysed. The labeled binding partners are capable of specifically binding to a viral or bacterial marker of interest to form a complex which in turn is capable of specifically binding to another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier backing, a housing, and an opening in the housing for result read out, may optionally also be a component of the test strip (200) in these embodiments.

[00266] In a preferred embodiment, the lysis agent is localized in the lysis zone (250) between the sample application zone (201) and the reagent zone (260). The lysis agent is preferably soluble or miscible in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains both lysis agent in solution or suspension and sample components in suspension. Any lysis-susceptible components in a sample, then being exposed in suspension to the lysis agent, are themselves lysed in situ. The running buffer then carries the sample, including any lysis-freed components, to the detection zone (205).

[00267] The lysis zone (250) is preferably located between the sample application zone (201) and the reagent zone (260), as shown in Figure 6A. In other embodiments, the lysis zone (250) overlaps the sample application zone (201), the reagent zone (260) or both the sample application zone (201) and the reagent zone (260) as shown in Figures 6B, 6C, and 6D, respectively. Note that the figures are schematic, and are not drawn to scale. The amount of overlap between the different zones (as shown in Figures 6B through 6D) may be highly variable. [00268] The test strip (200) also includes a detection zone (205) containing a first section for detection of at least one bacterial marker, e.g. a test line (203), including an immobilized specific binding partner, complementary to the bacterial conjugate formed by the bacterial marker and its labeled binding partner. Thus, at the test line (203), detection zone binding partners trap the bacterial labeled binding partners from the reagent zone (260) along with their bound bacterial markers. This localization of the bacterial markers with their labeled binding partners gives rise to an indication at the test line (203). At the test line (203), the presence of a bacterial marker is determined by qualitative and/or quantitative readout of the test line (203) indication resulting from the accumulation of labeled binding partners.

[00269] The detection zone (205) also includes a second section for detection of at least one viral marker, e.g. a test line (202), including an immobilized specific binding partner, complementary to the viral conjugate formed by the viral marker and its labeled binding partner. Thus, at the test line (202), detection zone binding partners trap the viral labeled binding partners from the reagent zone (260) along with their bound viral markers. This localization of the viral markers with their labeled binding partners gives rise to an indication at the test line (202). At the test line (202), the presence of a viral marker is determined by qualitative and/or quantitative readout of the test line (202) indication resulting from the accumulation of labeled binding partners. While test line (203) is upstream of test line (202) relative to the direction of flow (208) in the figures, in alternative embodiments, test line (202) is upstream of test line (203). In still other embodiments, test lines (202) and (203) are located in the same location on the test strip.

[00270] Optionally, the detection zone (205) may contain further test lines to detect other bacterial and/or viral markers, as well as a control line (204). The control line (204) indicates that the labeled specific binding partner traveled through the length of the assay, even though it may not have bound any markers, thus confirming proper operation of the assay. As shown in Figures 6A through 6D, the control zone (204) is preferably downstream of the test lines (203) and (202). However, in other embodiments, the control zone (204) may be located upstream of either or both of the test lines (203) and (202).

[00271] In a preferred embodiment, the control line (204) includes an antibody or other recombinant protein which binds to a component of the elution medium or other composition being used in the test. In embodiments where nucleic acids are the targets, the control line (204) preferably includes a nucleic acid complementary to the labeled nucleic acid being used as a binding partner for the target nucleic acid.

[00272] Although only one test line is shown in the figures, multiple test lines are within the spirit of the invention. In some embodiments where there are multiple targets, the presence of each target preferably corresponds to a separate test line (202). In other embodiments where there are multiple targets, the presence of multiple targets may be indicated on the same test line such that the presence of more than one target has different characteristics than the presence of a single target. For example, the presence of multiple targets on the same test line may be visually indicated by a different color than the presence of each of the targets alone.

[00273] In other embodiments, it is possible to have one or more mild lysis agents in the running buffer itself. In these embodiments, there is no adverse effect on the reagent zone which will be downstream and the sample can either be upstream or downstream of the reagent zone. A lysing enzyme in the running buffer can “target” its substrate and cut it to open up the cell membrane or cell wall. As an example, penicillin can excise or “punch a hole” in a susceptible bacteria. In other embodiments, when the lysis agent is applied to the sample collection material, then the reagent zone may be upstream of the sample application zone.

[00274] As an example, one or more lysis agents are dried onto the sample application zone of a lateral flow strip. On a per strip basis, the lysis agent is made of approximately 2 microliters of 100 mM HEPES buffer (pH 8.0) containing 5% CHAPS and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA, and 0.1% Sodium Azide (all percentages weight/ volume). Up to 10 microliters of whole blood are then added to the sample application zone to be lysed in situ. MxA protein is released from inside white blood cells to react with an MxA monoclonal antibody on a visual tag (colloidal gold or visible latex beads). This complex traverses with a running buffer containing Triton X-100 and is captured by MxA monoclonal antibodies immobilized at the test line of the nitrocellulose membrane. This binding at the test line gives rise to a visible indication.

[00275] Sample Analysis Device with Bimodal Dual Test strips [00276] MxA is a derivative of interferon alpha/beta cells that becomes elevated in the presence of viral infections but is not specific for a particular type of virus. MxA protein expression in peripheral blood is a sensitive and specific marker for viral infection.

[00277] MxA inhibits the replication of a wide range of viruses. MxA has a low basal concentration [less than 50 ng/ml] and a fast induction [1-2 hours]. It peaks at 16 hours and remains elevated in the presence of elevated interferon. MxA also has a long half-life [2.3 days] and constant titres in presence of interferonemia. Viral infections elevate MxA levels while only having a modest increase in CRP levels.

[00278] In one prospective clinical trial using ELISA (Towbin H et al. J Interferon Res 1992;12:67-74, herein incorporated by reference), the trial enrolled 87 normal healthy adults. The MxA levels were measured to be < 5 ng/ml in 66% of the adults, between 5- 50 ng/ml in 29% of the adults, and above 50 ng/ml in 5% of the adults.

[00279] Another prospective clinical trial using ELISA (Chieux V et ak, J Virol Methods 1998;70:183-191, herein incorporated by reference) enrolled 174 children. 45 of these children had acute fever (respiratory infection and/or gastroenteritis) and there were 30 age-matched controls. The MxA values were 7 ng/ml ± 7 ng/ml in the 30 age-matched controls. The MxA values were 10 ng/ml ± 6 ng/ml in 13 confirmed bacterial infections.

[00280] Another prospective clinical trial using ELISA enrolled 60 patients (Kawamura M et al. J Clin Lab Anal 2012;26:174-183, herein incorporated by reference). 42 of the patients had acute fever (respiratory infection and/or gastroenteritis) and there were 18 age matched controls. The median MxA value was 110.0 ng/ml in 31 confirmed viral infections. The median MxA value was 10.6 ng/ml in 11 confirmed bacterial infections. The median MxA value was 2.0 ng/ml in the 18 age matched controls. The ELISA test had a sensitivity of 87.1% and a specificity of 90.9% for differentiating viral from bacterial infection, but only tested MxA values to make this determination. Patients with viral infection were sharply distinguished from the healthy controls with 100% sensitivity and specificity. A cut-off of 36.7 ng/ml MxA was used to determine viral infection by ELISA in this study.

[00281] Another prospective clinical trial using ELISA enrolled 174 children (Nakabayashi M et ak, Pediatr Res 2006;60:770-774, herein incorporated by reference, data corrected for recalibrated ELISA standard). 122 of the children had acute fever (respiratory infection and/or gastroenteritis) and there were 52 age-matched controls. The mean MxA value was 123.7 ng/ml ± 83.0 ng/ml in 95 confirmed viral infections. The mean MxA value was 12.3 ng/ml ± 10.0 ng/ml in 27 confirmed bacterial infections. The mean MxA value was 14.5 ng/ml ± 11.0 ng/ml in the 52 age matched controls. The test showed a 92.6 % specificity and a positive likelihood ratio of 13.1 for accurately identifying viral infection. A cut-off of 36.7 ng/ml MxA was used to determine viral infection by ELISA in this study.

[00282] The cut-off in the ELISA test is artificial and is picked to discriminate between positive and negative. Therefore, it is preferable to routinely assign 10% CV from this cut off. In a point of care test, 100% of the people can visibly see the test line at >40 ng/ml, but some people can see a positive result at lower levels.

[00283] CRP becomes elevated in the presence of bacterial infections but is not specific for a particular type of bacteria. CRP is a nonspecific indicator for the presence of acute inflammation and is elevated in the presence of bacterial infections. CRP is an acute- phase protein synthesized by the liver. IL-6 is the primary mediator of CRP production. Bacterial infection is a potent stimulus of marked CRP elevation. Following antibiotic treatment, CRP levels fall rapidly. Bacterial infections dramatically elevate CRP levels while MxA levels remain low. Bacterial infection is a potent stimulus of CRP with marked elevation in serum CRP levels occurring within a few hours. CRP levels elevate within 4-6 hours after stimulation and peak after 36 hours. The serum concentration of CRP is normally less than 3 mg/L. With severe infection or inflammation, CRP can rise above 500 mg/L.

[00284] Pneumonia has elevated serum CRP levels (> 10 mg/L). The serum CRP levels are typically greater than 100 mg/L for severe pneumonia. 32% of patients with pneumococcal bacteremia had serum CRP less than 60 mg/L. Serum CRP is not usually elevated above 10 mg/L in viral infection. Invasive Adenovirus and Influenza can raise CRP to 10-80 mg/L. Very infrequently, the CRP levels exceed 60 mg/L in viral infections.

[00285] Metadata analysis of ten studies (Aouifi et ak, Crit care Med. 2000, 28:3171-6; Hatherill et ak, Arch Dis Child 1999: 81: 417-21; Muller et ak, Crit Care Med. 2000, 28: 977-83; Penel et al., Rev Med Interne 2001:22: 706-714; Rothenberger et al., Clin Chem Lab Med, 1999, 37: 275-9; Schwarz et al., Crit Care Med 2000, 28: 1828-32; Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et al., Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care Med 1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26: 1082-8, all herein incorporated by reference) that looked at a single value for serum CRP to be used as a cut-off for bacterial disease resulted in a bimodal outcome. Three of the studies (Aouifi et al., Crit care Med. 2000, 28:3171-6; Penel et al., Rev Med Interne 2001:22: 706-714; Schwarz et al., Crit Care Med 2000, 28: 1828-32) recommended that the CRP cut-off value be set at 6-15 mg/L, while the other seven studies (Hatherill et al., Arch Dis Child 1999: 81: 417-21; Muller et al., Crit Care Med. 2000, 28: 977-83; Rothenberger et al., Clin Chem Lab Med, 1999, 37: 275-9; Selberg et al., Crit Care Med 2000, 28: 2793-8; Suprin et al., Intensive Care Med 2000, 26: 1232-8; Ugarte et al., Crit Care Med 1999, 27: 498-504; Viallon et al., Intensive Care Med 2000, 26: 1082-8) recommended a cut-off of 60-100 mg/L.

[00286] In isolation, neither MxA nor CRP alone is sensitive or specific at identifying both viral and bacterial infection. Low cut-off values of CRP show high sensitivity and low specificity for detecting bacterial infection. High cut-off values of CRP show low sensitivity and high specificity for detecting bacterial infection. MxA is specific to identify viral infection, but it is not sensitive for bacterial infection. A multiplexed pattern of results including medical decision points reflected cut-off levels of low CRP, high CRP, and MxA together provide a sensitive and specific way to identify an immune response to a viral and/or bacterial infection.

[00287] In one preferred embodiment of a multiplexed lateral flow immunoassay, the fingerstick blood pattern of test results shows a positive result with a serum equivalence to a low CRP level cut-off of approximately 10 mg/L, a serum equivalence to a high CRP level cut-off of approximately 80 mg/L, and a MxA cut-off of approximately 40 ng/ml. These preferred values are shown in Table 16. [00288] Table 16

[00289] The specificity of the test is further enhanced by restricting the intended use.

For example, in preferred embodiments, only certain ages of the patient population are tested (preferably one year of age or older) and/or patients with specific underlying conditions that may lead to confounding factors are preferably not given this test.

[00290] A rapid, point-of-care MxA immunoassay was developed and compared to the MxA ELISA in 25 peripheral blood samples from patients with a febrile respiratory illness, as shown in Table 16. Table 17 sorts the same data from lowest to highest amounts of MxA in the ELISA test.

[00291] The MxA ELISA cut-off value was 36.7 ng/ml (+/- 10%CV = 33 ng/ml to 40/5 ng/ml). Patient 19 had a positive result in the MxA rapid point of care test, even though the ELISA results were much less than 40 ng/ml (15 ng/ml). However, the MxA immunoassay demonstrated 100% (9/9) sensitivity and 94% (15/16) specificity. [00292] Table 17

[00293] Table 18 [00294] Rapid, point-of-care low-CRP level and high-CRP level immunoassays were developed and compared to the CRP ELISA in 25 peripheral blood samples from patients with a febrile respiratory illness. These patients are the same patients that were tested for MxA in Tables 17 and 18. The results are shown in Table 19.

[00295] Table 19

[00296] Patient number 6 and 8 showed a positive CRP result even though the ELISA results were below 80 mg/L. Patient 23 showed a negative result even though the ELISA results were exactly 80 mg/L. Patient number 9 showed a negative low CRP test result even though the ELISA results for that patient were above lOmg/L. But, overall, the CRP values correlated well with the CRP ELISA at both cut-off values. [00297] Using an MxA and CRP ELISA, RPS analyzed 25 healthy, normal blood bank samples for the presence or absence of elevated MxA and CRP. The average CRP concentration in plasma was shown to be 1.6 mg/L. CRP levels were shown to range from 0.1 to 3.7 mg/L. The results are shown in Table 20.

[00298] Table 20

[00299] The bimodal dual test strips can be used to differentiate bacterial and viral infection in humans, but also may be used in veterinary applications for animals. Since CRP differs depending upon the species, there are not common antibodies to CRP between species. Therefore, the veterinary tests need to include CRP specific to the particular species being tested. MxA is well conserved among species, so it is possible to use human MxA in veterinary tests. However, MxA to a particular species could alternatively be used to try to further increase specificity. Veterinary tests using the bimodal dual test strips described herein may be developed for a specific species, including, but not limited to, cats, dogs, rabbits, pigs, sheep, horses, cows, monkeys, chimpanzees, baboons, and orangutans.

[00300] The strip with MxA and low CRP could be made with any configuration, for example the configurations shown in Figures 5 A and 5B, or Figures 6 A through 6D, where MxA is the viral marker being detected and relatively low levels of CRP is the bacterial marker being detected. In other embodiments, the MxA test line and the CRP test line could overlap, or be in the same location on the test strip. In these embodiments, the presence of low CRP and MxA on the same test line has different characteristics than the presence of either a bacterial or viral marker alone. For example, the presence of both low CRP and MxA on the same test line may be visually indicated by a different color than the presence of either MxA or low CRP alone. In these embodiments, a positive result for MxA would give a different color or indication than a positive result for low CRP, so that the person reading the assay could distinguish between a completely negative result, a positive result for MxA, a positive result for low CRP, and a positive result for both MxA and low CRP. For example, a positive result for MxA could result in a red test line, and a positive result for low CRP could result in a blue test line. So, when a sample is positive for both MxA and low CRP, the line is visibly purple.

[00301] Some embodiments for lateral flow assay devices to detect high levels of CRP are shown in Figures 7A-7B and 8A-8D. These configurations are similar to the configurations shown in Figures 5A-5B and 6A-6D, without a test line for a viral marker, and the same reference numerals are used for the same components of the strip (600), (700).

[00302] Figures 7 A and 7B show a chromatographic test strip (600) with a test line (623) that detects the presence of a bacterial marker, such as high levels of CRP. The sample is applied to the application zone (401) of the chromatographic test strip (600). As shown in Figure 7 A, the sample then passes a reagent zone (660) containing at least one labeled bacterial binding partner that is eluted by and then able to migrate with a sample transport liquid (e.g. a buffer solution). Alternatively, as shown in Figure 7B, the reagent zone (660) is located upstream of the sample application zone (401) such that the labeled binding partners in the reagent zone are eluted by the sample transport liquid and travel to the sample. The labeled bacterial binding partner is capable of specifically binding to a bacterial marker of interest, for example high levels of CRP, to form a complex which in turn is capable of specifically binding to another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier backing, a housing, and an opening in the housing for result read out, may optionally also be a component of the test strip (600) in these embodiments.

[00303] The test strip (600) also includes a detection zone (605) containing a section for detection of a bacterial marker, e.g. a test line (623), including an immobilized specific binding partner, complementary to the bacterial reagent complex formed by the bacterial marker and its labeled binding partner. Thus, at the test line (623), detection zone binding partners trap the labeled bacterial binding partners from the reagent zone (660) along with their bound bacterial markers. This localization of the bacterial marker with its labeled binding partners gives rise to an indication at the test line (623). At the test line (623), the presence of the bacterial marker is determined by qualitative and/or quantitative readout of the test line (623) indication resulting from the accumulation of labeled binding partners.

[00304] Optionally, the detection zone (605) may contain further test lines to detect other bacterial and/or viral markers, as well as a control line (404). The control line (404) indicates that the labeled specific binding partner traveled through the length of the assay, even though it may not have bound any bacterial markers, thus confirming proper operation of the assay. As shown in Figures 7A through 7B, the control zone (404) is preferably downstream of the test line (623). However, in other embodiments, the control zone (404) may be located upstream of the test line (623).

[00305] In a preferred embodiment, the control line (404) includes an antibody or other recombinant protein which binds to a component of the elution medium or other composition being used in the test. In embodiments where nucleic acids are the targets, the control line (404) preferably includes a nucleic acid complementary to the labeled nucleic acid being used as a binding partner for the target nucleic acid.

[00306] In other preferred embodiments to test for a bacterial marker, such as high CRP levels, as shown in Figures 8A through 8D, the sample passes a lysis zone (250), where a lysis agent will have preferably been pre-loaded onto the test strip and is eluted by the transport liquid. The lysis agent lyses any lysis-susceptible components in the sample in situ.

[00307] The chromatographic test strip (700) contains a sample application zone (201), a lysis zone (250) containing a lysis agent, and a reagent zone (760) containing at least one labeled binding partner that binds to a bacterial marker, for example high levels of CRP, that is eluted by and then able to migrate with a sample transport liquid (e.g. a buffer solution). While the reagent zone (760) is shown downstream of the sample application zone in these figures, in alternative embodiments, the reagent zone (760) could be upstream of the sample application zone (see Figure 7B), as long as the reagents encounter the sample at some point after the sample reaches the lysis zone and is effectively lysed. The labeled binding partner is capable of specifically binding to a bacterial marker of interest, for example high levels of CRP, to form a complex which in turn is capable of specifically binding to another specific reagent or binding partner in the detection zone. Although not shown in these Figures, an absorbent pad, as well as other known lateral flow immunoassay components including, but not limited to, a waste zone, a carrier backing, a housing, and an opening in the housing for result read out, may optionally also be a component of the test strip (700) in these embodiments.

[00308] In a preferred embodiment, the lysis agent is localized in the lysis zone (250) between the sample application zone (201) and the reagent zone (760). The lysis agent is preferably soluble or miscible in the sample transport liquid, and the lysis agent is solubilized and activated upon contact with the sample transport liquid. The sample transport liquid then contains both lysis agent in solution or suspension and sample components in suspension. Any lysis-susceptible components in a sample, then being exposed in suspension to the lysis agent, are themselves lysed in situ. The running buffer then carries the sample, including any lysis-freed components, to the detection zone (705).

[00309] The lysis zone (250) is preferably located between the sample application zone (201) and the reagent zone (760), as shown in Figure 8A. In other embodiments, the lysis zone (250) overlaps the sample application zone (201), the reagent zone (760) or both the sample application zone (201) and the reagent zone (260) as shown in Figures 8B, 8C, and 8D, respectively. Note that the figures are schematic, and are not drawn to scale. The amount of overlap between the different zones (as shown in Figures 8B through 8D) may be highly variable.

[00310] The test strip (700) also includes a detection zone (705) containing a section for detection of at least one bacterial marker, e.g. a test line (723), including an immobilized specific binding partner, for example, a specific binding partner for a high level of CRP, complementary to the bacterial conjugate formed by the bacterial marker and its labeled binding partner. Thus, at the test line (723), detection zone binding partners trap the bacterial labeled binding partners from the reagent zone (760) along with their bound bacterial markers. This localization of the bacterial markers with their labeled binding partners gives rise to an indication at the test line (723). At the test line (723), the presence of a bacterial marker is determined by qualitative and/or quantitative readout of the test line (723) indication resulting from the accumulation of labeled binding partners.

[00311] Optionally, the detection zone (705) may contain further test lines to detect other bacterial and/or viral markers, as well as a control line (204). The control line (204) indicates that the labeled specific binding partner traveled through the length of the assay, even though it may not have bound any markers, thus confirming proper operation of the assay. As shown in Figures 8A through 8D, the control zone (204) is preferably downstream of the test line (723). However, in other embodiments, the control zone (204) may be located upstream of the test line (723).

[00312] In a preferred embodiment, the control line (204) includes an antibody or other recombinant protein which binds to a component of the elution medium or other composition being used in the test. In embodiments where nucleic acids are the targets, the control line (204) preferably includes a nucleic acid complementary to the labeled nucleic acid being used as a binding partner for the target nucleic acid.

[00313] One preferred configuration for a bimodal dual test strip sample analysis device is shown in Figures 9A through 9C. The sample analysis device or test card (800) includes a closable housing (835) with two sides (836), (837) and a spine or hinged portion (831). In one preferred embodiment, the test card (800) is approximately 11.5 cm long (L) x 7 cm wide (W) when the two sides (836), (837) are closed. However, any size test card (800) that accommodates all of the components may be used. Within the first side (836) of the housing (835), there are two test strips (815), (825), each including a receiving pad (845), a diverting zone (850), a transfer pad (855) and a detection zone (805). The first side (836) also includes an absorbent pad (840) and preferably a waste pad (860). The first test strip (815) preferably includes a detection zone (805) with an MxA test line (802), a low CRP test line (803) and a control line (804). The second test strip (825) preferably includes a detection zone (805) with a high CRP test line (823) and a control line (804). All of the test lines are visible through the windows (865) on the second side (837) of the housing (835) when the housing (835) is closed. The absorbent pad (840) is preferably a single pad that the running buffer is added to start lateral flow. Similarly, the waste pad (860) is preferably a single pad that collects excess running buffer at the end of the test. However, in other embodiments, each strip could have a separate absorbent pad (840) and/or waste pad (860).

[00314] The second side (837) of the housing (835) includes three separate sections (838), (839) and (870). The middle portion, a sample compressor or flap (870), preferably includes two conjugate zones (872), (874), each including a labeled binding partner for at least one analyte, and a labeled control. A window (843) is located in the lower portion (838) of the second side (837) of the housing so that the buffer can be added to the absorbent pad (840) when the housing (835) is closed. The viewing windows (865) for the detection zones (805) are on the upper portion (839) of the second side (837) of the housing (835).

[00315] The upper portion (839) and the lower portion (838) of the second side (837) of the housing (835) also preferably each include at least one knob, peg or protrusion (875) that mates with one or more holes (895) so that the upper and lower portions (838), (839) may be easily fastened onto the first side (836) of the housing (835). In a preferred embodiment, there are two pegs (875) on the lower portion (838) that mate with two holes (895) flanking the absorbent pad (840) on the first side (836) of the housing (835) and two pegs (875) on the upper portion (839) that mate with two holes (895) flanking the waste pad (860) on the first side (836) of the housing (835). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the pegs (875) are on the first side (836) of the housing (835). In yet other embodiments, other reversible fastening mechanisms could be used to secure the upper portion (838) and/or lower portion (839) of the second side (837) of the housing (835) to the first side (836) of the housing (835). In other embodiments, the upper and lower sections (838), (839) are permanently closed, for example using an adhesive, before use.

[00316] The flap (870), also known as a sample compressor, on the second side (837) of the housing includes two conjugate zones (872), (874) and two sample application zones (873), (876), and can be easily opened and closed. The flap (870) also preferably includes at least one knob, peg or protrusion (875) that mates with one or more holes (895) so that the flap (870) is easily correctly closed onto the first side (836) of the housing (835) after sample has been added to the sample application zones (873), (876). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the pegs (875) are on the first side (836) of the housing (835). In yet other embodiments, other reversible fastening mechanisms could be used to secure the flap (870) to the first side (836) of the housing (835).

[00317] The conjugate zones (872), (874) and the sample application zones (873), (876) preferably overlap. In preferred embodiments, the conjugate zones (872), (874) are colored due to the dyes in the sample conjugates and control conjugates, and the sample is placed directly on the colored portion of the flap (870). In one preferred embodiment, the conjugate zone (872) that is used for the first test strip (815) contains an MxA binding partner that is labeled with a red dye, a low CRP binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (872) appears purplish. The other conjugate zone (874) contains a high CRP binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (874) appears bluish.

[00318] The diverting zone (850) preferably includes a gap or barrier that interrupts lateral flow, diverting the running buffer up into the flap (870) that includes the conjugate zones (872), (874) and the sample application zones (873), (876).

[00319] In operation, the upper and lower portions (838), (839) of the second side (837) of the housing (835) are preferably snapped closed before use by securing the pegs (875) to the holes (895). The sample analysis device, or test card (800) is preferably placed on a flat surface. If the flap (870) is not already open, the user opens it to access the sample application zones (873), (876). A blood sample to be tested is taken from the patient. The sample may be taken by any procedure known in the art. In a preferred embodiment, a sample of 5pl of blood is added to each of the sample application zones (873), (876) and then the flap (870) is closed. Each of the 5 mΐ samples is preferably collected independently of each other. The blood samples are preferably added directly to the device (800), without any pretreatment.

[00320] To ensure that the sample compressor or flap (870) has been closed correctly, pressure is preferably applied to the housing (835) above the pegs (875) to snap the pegs (875) closed. The top of the flap (870) needs to be flush with the top of the rest of the second side (837) of the housing (835) for the test to ran properly. Running buffer is added to the absorbent pad (840), which initiates lateral flow (885). In preferred embodiments, the running buffer includes one or more lysis agents, for example detergents, to lyse the blood sample and expose the intracellular MxA in the sample.

When the running buffer reaches the diverting zone (850), it is diverted up into the flap (870). It travels through the conjugate zones (872), (874), collecting any complexes formed between the MxA binding partner and MxA in the sample, the low CRP binding partner and low levels of CRP in the sample, the high CRP binding partner and high levels of CRP in the sample, as well as the control conjugate.

[00321] Since the conjugate zones (872), (874) bridge the diverting zone (850) on the lateral flow test strips (815), (825), the running buffer, which now contains sample, conjugate, and the complexes described above, then travels into the transfer pad (855), and to the detection zones (805) on each of the test strips (815), (825). If MxA is present in the sample, the MxA test line (802) on the first test strip (815) will be red. If a threshold low level of CRP is present in the sample, the low CRP test line (803) on the first test strip (815) will be black. If a threshold high level of CRP is present in the sample, the high CRP test line (823) on the second test strip (825) will be black. If the test is run correctly, the control lines (804) on both the first strip (815) and the second test strip (825) will be blue. In preferred embodiments, the levels of detection are 40 ng/ml for MxA, 10 mg/L for low CRP on the first test strip (815) and 80 mg/L for high CRP on the second test strip (825). The results of the test should be visible after approximately 5-20 minutes, preferably within about 10 minutes. [00322] Since the control binding partner is on the sample compressor or flap (870) and not on either of the test strips (815), (825), there is a true procedural control to this configuration. If the flap (870) is not closed properly, nothing will show up in the detection zone (805), indicating that the test was run improperly.

[00323] Figures 10A through 10F show test results using the device (800) shown in Figures 9A through 9C, with two test strips (815), (825) side by side, where a first test strip (815) tests for the presence of both MxA and low levels of CRP and the second test strip (825) tests for high levels of CRP.

[00324] Figure 10A shows a negative result at the MxA test line (802) and a negative result at the low CRP test line (803) on the first test strip (815), as well as a negative result at the high CRP test line (823) on the second test strip (825). More specifically, the only visible lines in the detection zone (805) of the lateral flow assay (800) are the two blue control lines (804). This result indicates that the sample is negative for both viral and bacterial infection.

[00325] Figures 10B and IOC are positive for viral infection. In Figure 10B, the presence of two blue control lines (804) and a red MxA line (802) indicate a viral infection. In Figure IOC, the presence of two blue control lines (804) and a red MxA line

(802) indicate a viral infection. Since there is also a black low CRP line (803) in Figure IOC, there is a possibility of bacterial co-infection, although there is an absence of a high CRP line (823).

[00326] Figures 10D and 10E are positive for bacterial infection. In Figure 10D, the presence of two blue control lines (804) and a black low CRP line (803) indicates a bacterial infection. In Figure 10E, the presence of two blue control lines (804), a black low CRP line (803), and a black high CRP line (823) also indicates a bacterial infection. The MxA line is absent in both Figures 10D and 10E, indicating an absence of a viral infection.

[00327] Figure 10F indicates co-infection (both bacterial and viral infection). The presence of two blue control lines (804), a red MxA line (802), a black low CRP line

(803), and a black high CRP line (823) indicates the presence of both viral and bacterial infection. [00328] Another preferred configuration for a bimodal dual test strip sample analysis device (1000) is shown in Figures 11A through 11C. This configuration is similar to the configuration (800) shown in Figures 9A through 9C, but the sample application zones (1073), (1076) are located on each of the test strips (1015), (1025), downstream of the diverting zone (850). The sample analysis device or test card (1000) includes a closable housing (835) with two sides (836), (837) and a spine or hinged portion (831). In one preferred embodiment, the test card (1000) is approximately 11.5 cm long (L) x 7 cm wide (W) when the two sides (836), (837) are closed. However, any size test card (1000) that accommodates all of the components may be used. Within the first side (836) of the housing (835), there are two test strips (1015), (1025), each including a receiving pad (845), a diverting zone (850), a transfer pad (1055) and a detection zone (805). The first side (836) also includes an absorbent pad (840) and preferably a waste pad (860). The first test strip (1015) preferably includes a detection zone (805) with an MxA test line (802), a low CRP test line (803) and a control line (804). The second test strip (1025) preferably includes a detection zone (805) with a high CRP test line (823) and a control line (804). All of the test lines are visible through the windows (865) on the second side

(837) of the housing (835) when the housing (835) is closed. The absorbent pad (840) is preferably a single pad to which the running buffer is added to start lateral flow. Similarly, the waste pad (860) is preferably a single pad that collects excess running buffer at the end of the test. However, in other embodiments, each strip could have a separate absorbent pad (840) and/or waste pad (860).

[00329] The second side (837) of the housing (835) includes three separate sections

(838), (839) and (1070). The middle portion, or flap (1070), also known as a sample compressor, preferably includes two conjugate zones (872), (874), each including a labeled binding partner for at least one analyte, and a labeled control. A window (843) is located in the lower portion (838) of the second side (837) of the housing so that the buffer can be added when the housing (835) is closed. The viewing windows (865) for the detection zones (805) are on the upper portion (839) of the second side (837) of the housing (835).

[00330] The upper portion (839) and the lower portion (838) of the second side (837) of the housing (835) also preferably each include at least one knob, peg or protrusion (875) that mates with one or more holes (895) so that the upper and lower portions (838), (839) may be easily fastened onto the first side (836) of the housing (835). In a preferred embodiment, there are two pegs (875) on the lower portion (838) that mate with two holes (895) flanking the absorbent pad (840) on the first side (836) of the housing (835) and two pegs (875) on the upper portion (839) that mate with two holes (895) flanking the waste pad (860) on the first side (836) of the housing (835). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the pegs (875) are on the first side (836) of the housing (835). In yet other embodiments, other reversible fastening mechanisms could be used to secure the upper portion (838) and/or lower portion (839) of the second side (837) of the housing (835) to the first side (836) of the housing (835). In other embodiments, the upper and lower sections (838), (839) are permanently closed, for example using an adhesive, before use.

[00331] The flap (1070) on the second side (837) of the housing includes two conjugate zones (872), (874) and can be easily opened and closed. The flap (1070) also preferably includes at least one knob, peg or protrusion (875) that mates with one or more holes (895) so that the flap (1070) is easily correctly closed onto the first side (836) of the housing (835) after sample has been added to the sample application zones (1073), (1076) on the test strips (1015), (1025). In other embodiments, the holes (895) are on the second side (837) of the housing (835) and the pegs (875) are on the first side (836) of the housing (835). In yet other embodiments, other reversible fastening mechanisms could be used to secure the flap (1070) to the first side (836) of the housing (835).

[00332] In preferred embodiments, the conjugate zones (872), (874) are colored due to the dyes in the sample conjugates and control conjugates. In one preferred embodiment, the conjugate zone (872) that is used for the first test strip (1015) contains an MxA binding partner that is labeled with a red dye, a low CRP binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (872) appears purplish. The other conjugate zone (874) contains a high CRP binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (874) appears bluish. [00333] The diverting zone (850), which preferably includes a gap or barrier, interrupts lateral flow, diverting the running buffer up into the flap (1070) that includes the conjugate zones (872), (874).

[00334] In operation, the upper and lower portions (838), (839) of the second side (837) of the housing (835) are preferably snapped closed before use by securing the pegs (875) to the holes (895). The sample analysis device, or test card (1000) is preferably placed on a flat surface. If the flap (1070) is not already open, the user opens it to access the sample application zones (1073), (1076). The sample application zones (1073), (1076) may be located in any portion of the transfer pad (1055). A blood sample to be tested is taken from the patient. The sample may be taken by any procedure known in the art. In a preferred embodiment, a sample of 5pl of blood is added to each of the sample application zones (1073), (1076) zones and then the flap (1070) is closed. Each of the 5 mΐ samples is preferably collected independently of each other. The blood is preferably added directly to the device (1000), without any pretreatment. In preferred embodiments, an arrow (1002) or other indication (shown in Figure 11 A), for example the words “add sample here” shows the user where to place the sample on the test strips (1015), (1025).

[00335] To ensure that the flap (1070) has been closed correctly, pressure is preferably applied to the housing (835) above the pegs (875) to snap the pegs (875) closed. The top of the flap (1070) needs to be flush with the top of the rest of the second side (837) of the housing (835) for the test to ran properly. Running buffer is added to the absorbent pad (840), which initiates lateral flow (885). In preferred embodiments, the running buffer includes one or more lysis agents, for example detergents, to lyse the blood sample and expose the intracellular MxA in the sample. When the running buffer reaches the diverting zone (850), it is diverted up into the flap (1070). It travels through the conjugate zones (872), (874), collecting the MxA binding partners, the low CRP binding partners, and the high CRP binding partners, as well as the control conjugate.

[00336] Since the conjugate zones (872), (874) bridge the diverting zone (850) on the lateral flow test strips (1015), (1025), the running buffer, which now contains conjugate, then travels into the transfer pad (1055), which includes the sample application zones (1073), (1076), and to the detection zones (805) on each of the test strips (1015), (1025). If MxA is present in the sample, the MxA test line (802) on the first test strip (1015) will be red. If a threshold low level of CRP is present in the sample, the low CRP test line (803) on the first test strip (1015) will be black. If a threshold high level of CRP is present in the sample, the high CRP test line (823) on the second test strip (1025) will be black. In preferred embodiments, the levels of detection are 40 ng/ml for MxA, 10 mg/L for low CRP on the first test strip (1015) and 80 mg/L for high CRP on the second test strip (1025). The results of the test should be visible after approximately 5-20 minutes, preferably within about 10 minutes. If the test was run correctly, the control lines (804) on both the first strip (815) and the second test strip (825) will be blue.

[00337] Since the control binding partner is on the flap (1070) and not on either of the test strips (1015), (1025), there is a true procedural control to this configuration. If the flap (1070) is not closed properly, nothing will show up in the detection zone (805), indicating that the test was ran improperly.

[00338] In an alternative embodiment, the sample application zones (1073), (1076) are located on the receiving pad (845), before the diverting zone (850). In this embodiment, the running buffer travels through the sample application zones (1073), (1076), and then is diverted into the flap (1070).

[00339] In preferred embodiments of the configurations shown in Figures 9A through 9C and 11A through 11C, greater than approximately 1.2 ml of running buffer is placed on the absorbent pad (840). If less than 1.0 ml is added in embodiments where the diverting zone (850) is a gap, the buffer gets stalled at the gap because the gap holds approximately 1.0 ml.

[00340] As shown in Figure 12, in one preferred embodiment, a kit (1100) includes the sample analysis device (800), (1000), a lancet (1102), one or more pipettes (1101), and a running buffer (1103). The lancet (1102) is used to make a skin puncture and one or more pipettes (1101) are used to collect the blood from the puncture site. In a preferred embodiment, 5 ul of blood is transferred from a first pipette (1101) to the first conjugate zone (872) and another 5 pi of blood is transferred from a second pipette (1101) and added to the second conjugate zone (874). The flap (870) is closed, and the running buffer (1103) is added to the absorbent pad (840), as described in the description of Figs. 9A through 9C and 11A through 11C. [00341] The diverting zone (850) preferably includes at least one feature that interrupts flow in the plane in which flow is occurring. The diverting zone may include a barrier, a gap, a ditch, or any combination of these features. The barrier is preferably an impermeable membrane (or substantially impermeable membrane) that may be made of any material that prevents the flow of liquid from continuing to flow in the same plane. Some materials for the barrier include, but are not limited to, inert materials, semi- permeable materials, plastics, hydrocarbons, metal, hydrophobic materials, Sephadex, Sepharose, cellulose acetate, a hygroscopic material (for example CaCh, CaSC or silica gel), or hydrogels. The gap or ditch is any break in the plane of the lateral flow test strip that extends to a depth sufficient to stop flow. In one preferred embodiment, the gap is preferably at least approximately 0.1 mm deep.

[00342] The diverting zone (850) in Figures 9A through 9C and 11 A through 11C delays or completely stops flow until the sample compressor/flap (870), (1070) is brought into contact with the rest of the device, and creates a bridge along which the fluid can flow.

The sample compressor (870), (1070) acts as a bridge and redirects flow into a different plane. Flow is diverted into the sample compressor (870), (1070). This increases collection of the reagents on the sample compressor (870), (1070). For example, in embodiments where the conjugate is on the sample compressor (870), (1070), collection of the conjugate increases in devices with a diverting zone (850). In embodiments where both the sample application zones (873), (876), (1073), (1076) and the conjugate are on the sample compressor (870), (1070), the sample and conjugate both encounter the running buffer when it is diverted into the sample compressor (870), (1070), and a ½ sandwich or full sandwich (depending upon where the second binding partner for the analyte is located on the sample analysis device) is formed before the running buffer is diverted back to the test strips if the analyte is present in the sample. Embodiments with a diverting zone (850) and a sample compressor (870), (1070) increase speed, allow for better interactions between the conjugate and the sample, and allow for more sensitivity because more conjugate is placed into the fluid. In these embodiments, all of the fluid preferably interacts with the conjugate. This is a significant improvement over compressor embodiments without redirection, where approximately 20-30% of the fluid interacts with the conjugate. [00343] Another preferred configuration for a bimodal dual test strip sample analysis device (1200) is shown in Figure 13. This configuration is similar to the configurations (800), (1000) shown in Figures 9A through 9C and Figures 11A through 11C, without a second section (837) of the housing (1235) or a diverting zone (850). Instead, all of the components of the test are located in the same plane and flow proceeds laterally from the absorbent pad (840) to the waste pad (860). Note that this embodiment could also include a housing with a window to facilitate application of the buffer to the absorbent pad (840), a window located above each sample application zone (1273), (1276) for applying sample to the device (1200), and viewing windows for the detection zone (805). In one preferred embodiment, the sample analysis device (1200) is approximately 11.5 cm long (L) x 7 cm wide (W). However, any size test card (1200) that accommodates all of the components may be used. There are two test strips (1215), (1225), each including a receiving pad (845), a conjugate zone (1272), (1274), a transfer pad (1240) containing a sample application zone (1273), (1276), a detection zone (805) and a waste pad (860). The device (1200) also preferably includes an absorbent pad (840) and a waste pad (860). While the conjugate zones (1272), (1274) are shown upstream of the sample application zones (1273), (1276) in this figure, in other embodiments, one or both of the conjugate zones (1272), (1274) are located downstream of the sample application zones (1273), (1276).

The detection zone (805) of the first test strip (1215) preferably includes an MxA test line (802), a low CRP test line (803) and a control line (804). The detection zone (805) on the second test strip (1225) also preferably includes a high CRP test line (823) and a control line (804). The absorbent pad (840) is preferably a single pad that the running buffer is added to start lateral flow. Similarly, the waste pad (860) is preferably a single pad that collects excess running buffer at the end of the test. However, in other embodiments, each strip could have a separate absorbent pad (840) and/or waste pad (860).

[00344] In preferred embodiments, the conjugate zones (1272), (1274) are colored due to the dyes in the sample conjugates and control conjugates. In one preferred embodiment, the conjugate zone (1272) that is used for the first test strip (1215) contains an MxA binding partner that is labeled with a red dye, a low CRP binding partner that is labeled with a black dye, and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (1272) appears purplish. The other conjugate zone (1274) contains a high CRP binding partner that is labeled with a black dye and a control binding partner that is labeled with a blue dye. In this embodiment, the conjugate zone (1274) appears bluish.

[00345] In operation, a blood sample to be tested is taken from the patient. The sample may be taken by any procedure known in the art. In a preferred embodiment, a sample of 5 pi of blood is added to each of the sample application zones (1273), (1276). Each of the 5 mΐ samples is preferably collected independently of each other. In preferred embodiments, an arrow (1002) or other indication (shown in Figure 10A), for example the words “add sample here” shows the user where to place the sample on the test strips (1215), (1225).

[00346] The blood is preferably added directly to the device (1200), without any pretreatment. Running buffer is added to the absorbent pad (840), which initiates lateral flow (1285). In preferred embodiments, the running buffer includes one or more lysis agents, for example detergents, to lyse the blood sample and expose the intracellular MxA in the sample. It travels through the conjugate zones (1272), (1274), collecting the MxA binding partners, the low CRP binding partners, the high CRP binding partners, as well as the control conjugate.

[00347] The running buffer, which now contains conjugate, then travels into the transfer pad (1255), which includes the sample application zones (1273), (1276), and to the detection zones (805) on each of the test strips (1215), (1225). If MxA is present in the sample, the MxA test line (802) on the first test strip (1215) will be red. If a threshold low level of CRP is present in the sample, the low CRP test line (803) on the first test strip (1215) will be black. If a threshold high level of CRP is present in the sample, the high CRP test line (823) on the second test strip (1225) will be black. In one embodiment, the levels of detection are 40 ng/ml for MxA, 10 mg/L for low CRP on the first test strip (1215) and 80 mg/L for high CRP on the second test strip (1225). In another embodiment, the levels of detection are 15 ng/ml for MxA, 10 mg/L for low CRP on the first test strip (1215) and 80 mg/L for high CRP on the second test strip (1225). The results of the test should be visible after approximately 5-20 minutes, preferably within about 10 minutes. If the test was run correctly, the control lines (804) on both the first strip (1215) and the second test strip (1225) will be blue.

[00348] In an alternative embodiment, the sample application zones (1273), (1276) are located upstream of the conjugate zones (1272), (1274). In this embodiment, the running buffer travels through the sample application zones (1273), (1276), and then to the conjugate zones (1272), (1274). In still other embodiments, the conjugate zones (1272), (1274) overlap the sample application zones (1273), (1276). In still other embodiments, the conjugate zones (1272), (1274), and/or the sample application zones (1273), (1276) may be located in the receiving pad (845).

[00349] In preferred embodiments of the configurations shown in Figures 5A through 9C, 11A through 11C and 13, the control is rabbit anti-chicken and the control conjugate is blue latex beads coupled to chicken IgY. In other preferred embodiments, there is at least one lysis agent, preferably a detergent, in the running buffer.

[00350] Simultaneous Detection of intracellular and extracellular proteins

[00351] There are extracellular analytes and there are intracellular analytes. The detection of each is often a separate event. The intracellular analyte has to be extracted by lysing the cells so the internalized analyte is externalized and available for testing.

[00352] Methods disclosed herein simultaneously detect at least one extracellular analyte and at least one intracellular analyte. An “intracellular” target or analyte, as described in this embodiment, is an analyte that is inside a cell and does not touch anything within the cell (such as surface proteins, the cellular wall, or internal surfaces). An “extracellular” target or analyte, as described in this embodiment, is a completely extracellular analyte, that does not contact anything outside the cell. For example, the extracellular analyte is in the plasma, which does not contain cells. The cell can be removed completely, and the extracellular analyte can still be collected.

[00353] In contrast, viral particles, while being outside the cell, are attached to the cellular wall. It is well known in the art to blend a cocktail of antibodies to detect an intracellular bound fraction and a surface bound fraction. But, the methods described herein are different, and detect a lysed, intracellular portion and a disassociated serum protein.

[00354] In one preferred embodiment, the extracellular analyte is C-reactive protein and the intracellular analyte is MxA protein. This is a serological test for the detection of CRP and MxA antigens. MxA is an intracellular protein inside the white blood cells. CRP is an extracellular protein found in whole blood, plasma and serum. [00355] In one preferred method, glass fibers, such as Whatman GD filters, physically trap the erythrocytes. Specific erythrocyte-binding lectins and/or antibodies can be added to physically bind to the erythrocytes in the glass fiber matrix. Leukocyte lysing solutions, which lyse the leukocytes to release the intracellular MxA, may be incorporated into the glass fiber filters.

[00356] In some preferred embodiments, the whole blood sample is added to the glass fiber filter. The liquid blood dissolves the embedded lysing agents, which lyses the leukocytes. Lateral flow immunochromatography is initiated by adding the running buffer. The running buffer then carries the suitable antibody conjugates which bind to the extracellular CRP and the newly released intracellular MxA. The entire complex moves into the detection zone where immobilized specific antibodies capture the respective complexes to form the sandwich. Different test lines are formed with MxA and CRP. These test lines can be visual, fluorescent, phosphorescent, chemiluminescent, paramagnetic, or any combinations of these. Different dyes may be incorporated to distinguish MxA and CRP test lines. Any of the lateral flow assays and configurations described herein could be used to simultaneously detect MxA and CRP or other intracellular and extracellular analytes.

[00357] Agglutination of MxA and CRP

[00358] Some embodiments include a simple agglutination test for MxA and CRP in blood. As an analogy, the present inventors believe that the cement in a brick wall is holding the bricks apart instead of holding them together. The rationale is that if the cement is removed, the bricks coalesce and fall together into a heap. Therefore, if the cement is “inactivated” or removed, individual bricks coalesce together.

[00359] Gold conjugates and latex beads are colloidal particles that repel one another and hence are brought together in suspension. If the repulsive force is removed, or individual colloidal particles are cross-linked together, they coalesce and one can visualize the agglutinated particles. The cross linking to overcome this natural repulsion is accomplished by the presence of the antigen analyte in question.

[00360] In one preferred embodiment, MxA monoclonal KM 1124 and/or KM 1135 is conjugated to red colloidal gold particles. Anti-CRP monoclonal antibodies are conjugated to green latex beads of suitable size. In the presence of MxA, the monoclonal antibody KM 1124 and/or KM 1135 binds and, since there is a multiplicity of the epitopes recognized by KM 1124 and/or KM 1135, a natural cross linking of the colloidal gold takes place and one sees the clumping of the red colloidal gold particles. This process is antigen-dependent and fairly rapid, occurring generally within a minute or two. The same phenomenon takes place with the green colloidal latex beads coated with suitable monoclonal antibodies in the presence of a CRP analyte.

[00361] Clumping of red gold particles means at least a threshold amount of MxA is in the sample, indicating a viral infection. Clumping of green beads means at least a threshold amount of CRP is in the sample, indicating a bacterial infection. Clumping of both red and green particles mean either a co-infection or is indicative of an indeterminate result. Absence of any clumping indicates that the sample is negative for viral and bacterial infection.

[00362] In one preferred embodiment, the threshold concentration of C- reactive protein is equal to or greater than approximately 6-15 mg/L of C-reactive protein and the threshold concentration of MxA protein is equal to or greater than a serum equivalent of approximately 13-250 ng/ml. Since the MxA is an intra-cellular biomarker, the blood sample is preferably lysed during the assay to lyse the white blood cells and externalize the MxA antigen. In other embodiments, the sample is lysed prior to performing the assay. Any immunoassay format known in the art could be used for the agglutination assay. The reagents are added, and then the user waits to see if the reagents agglutinate in the presence of the sample h

[00363] Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention.