Use of MXA in the preparation of detection reagents for identifying viral infections

Abstract

This invention belongs to the field of in vitro diagnostic technology, specifically the use of MXA in the preparation of a test reagent for identifying viral infections. This method utilizes nasopharyngeal secretion samples to detect myxovirus resistance protein A (MXA) levels, enabling a non-invasive diagnosis of the common cold in the early stages (1-2 days after onset). This method can also be used to screen for the risk of potentially serious viral infectious diseases (such as influenza or novel coronavirus infection) with early common cold-like symptoms.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and specifically relates to the use of detecting myxovirus resistance protein A (MXA) in preparing a detection reagent for screening viral infection. Background Art

[0002] Acute upper respiratory tract infection (URI), also known as upper respiratory infection (URI), is a general term for acute inflammation of the nose, pharynx, or throat caused by various viruses and / or bacteria. Viruses are the most common, accounting for approximately 70% to 80%. These include the common cold, viral pharyngitis, laryngitis, herpetic pharyngitis, pharyngoconjunctival fever, and bacterial pharyngitis and tonsillitis.

[0003] The common cold, commonly known as the "cold," is the most common viral infection of the upper respiratory tract, primarily manifested by catarrhal symptoms in the nasopharynx. It is most commonly caused by rhinoviruses, followed by coronaviruses, parainfluenza viruses, respiratory syncytial viruses, echoviruses, and coxsackieviruses. The 2018 Guidelines for the Diagnosis and Treatment of Acute Upper Respiratory Tract Infections in my country describe the common cold as having an acute onset, primarily manifested by nasal symptoms such as sneezing, nasal congestion, and clear, watery nasal discharge. Symptoms such as sneezing, nasal congestion, and clear, watery nasal discharge may also occur, including cough, dry throat, itchy or burning throat, and even postnasal drip. Symptoms such as sneezing, nasal congestion, and clear, watery nasal discharge may occur simultaneously or several hours after onset. Two to three days later, the nasal discharge thickens, often accompanied by sore throat, tearing, loss of taste, dyspnea, and hoarseness. Fever and systemic symptoms are generally absent, or may be limited to low-grade fever, malaise, mild chills, and headache. Physical examination reveals congestion, edema, and discharge from the nasal mucosa, as well as mild pharyngeal congestion. The disease usually takes 5-7 days to recover.

[0004] This invention defines "early common cold" as one to two days after onset, when patients only present with nasopharyngeal symptoms (such as sneezing, nasal congestion, and clear watery nasal discharge) and no systemic symptoms (such as fever, myalgia, and chills). This meets the early characteristics of the common cold described in the "Guidelines for Primary Diagnosis and Treatment of Acute Upper Respiratory Tract Infections (2018)", but the guidelines do not specify the diagnostic method for this stage.

[0005] Influenza, the novel coronavirus, and other major viral infectious diseases can all exhibit common cold-like symptoms in their early stages. Therefore, diagnosing the common cold early in the disease is crucial. The optimal diagnostic window is within the early stages (1-2 days), but existing methods are limited by low sensitivity or high invasiveness. Viral loads may be low in the early stages of illness, limiting the sensitivity of viral nucleic acid testing and the complex sampling and testing process. Viral antigen testing (such as rapid influenza antigen tests) also generally has low sensitivity (often below 20%) during this period.

[0006] Viral infection triggers a cellular response involving the secretion of type I interferons (IFNs), whose antiviral activity is mediated by the production of myxovirus resistance protein A (MXA). Our research team has demonstrated for the first time that MXA protein is consistently detected and significantly elevated in nasopharyngeal samples from patients with the early common cold (1-2 days after onset, with only nasopharyngeal symptoms), whereas MXA concentrations in whole blood and oropharyngeal samples from the same patients remain at background levels (generally below the diagnostic threshold). This specific distribution pattern reveals a robust local (nasopharyngeal) type I interferon response characteristic of the early stages of viral infection, surpassing existing understanding. This finding not only establishes the nasopharynx as the optimal and only valid biological sample source for detecting MXA proteins associated with early viral infection but also lays the foundation for the development of a highly sensitive, specific, and non-invasive diagnostic method for the early common cold based on nasopharyngeal MXA detection. Using this method to accurately identify viral common colds in the very early stages of symptom onset (1-2 days) has important clinical significance and application value for timely symptomatic supportive treatment, reducing unnecessary use of antibiotics, assessing potential risks of severe illness (such as influenza, new coronavirus, etc.), and implementing early public health intervention measures. It fills the gap in existing technologies in the accurate diagnosis of diseases in the very early stages. Summary of the Invention

[0007] This invention provides a non-invasive method for diagnosing the common cold in its early stages (1-2 days after onset) by measuring myxovirus resistance protein A (MXA) levels in nasopharyngeal secretion samples. This method can also be used to screen for the risk of potentially serious viral infectious diseases (such as influenza or novel coronavirus infection) with early common cold-like symptoms.

[0008] A first aspect of the present invention is to provide the use of MXA or a preparation that specifically binds to MXA in the preparation of a detection reagent for identifying the common cold. If the MXA content is higher than a predetermined threshold, the common cold is diagnosed. The cut-off value of the detection reagent is 20-40 ng / mL.

[0009] Furthermore, the course of the common cold is 1 to 7 days.

[0010] Furthermore, the identification refers to distinguishing the common cold from allergic rhinitis and / or bacterial sinusitis.

[0011] Furthermore, the preparation that specifically binds to MXA is an antibody or antibody fragment that specifically binds to MXA.

[0012] Furthermore, the cut-off value of the detection reagent is 30 ng / mL.

[0013] Furthermore, the detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the test subject.

[0014] Furthermore, the nasopharyngeal secretion sample is a nasopharyngeal swab sample.

[0015] Furthermore, the diagnosis is based on the MXA content in the nasopharyngeal swab sample being higher than the predetermined threshold, while the MXA content in the whole blood sample and oropharyngeal swab sample of the same patient are both lower than the predetermined threshold.

[0016] Furthermore, the diagnostic reagent also includes a reagent that specifically binds to influenza virus, which is used to detect influenza virus antigens while detecting the MXA content in nasopharyngeal secretion samples, thereby screening patients for the risk of influenza virus infection while diagnosing early common colds.

[0017] Furthermore, the common cold is an early stage common cold, which means that the course of the disease is within 1 to 2 days.

[0018] Furthermore, the diagnosis of the early common cold is used to indicate whether the patient is suffering from a major viral infectious disease.

[0019] Furthermore, in the uses, diagnostic methods or diagnostic products provided by the present invention, the aforementioned diagnostic reagents can be used to detect the content of MXA by any method, including but not limited to electrophoresis, immunofluorescence, direct competition, indirect competition, ELISA, RIA, flow cytometry or immunochromatography, depending on their structure and / or composition.

[0020] Furthermore, the common cold is caused by viruses such as rhinovirus, and symptoms include runny nose and cough.

[0021] The second aspect of the present invention is to provide an MXA detection kit, which comprises a binder that specifically binds to MXA, a standard, a buffer, and a warning line mark, wherein the warning line mark includes a detection result pattern corresponding to when the MXA content of the sample is a cut-off value; the warning line mark also includes a prominent and eye-catching mark corresponding to the detection result when the MXA content of the sample is greater than 20~40ng / mL.

[0022] Furthermore, the cut-off value is 30 ng / mL.

[0023] The beneficial effects of the present invention include:

[0024] In the early stages of the common cold (1-2 days, with only nasopharyngeal symptoms), nasopharyngeal swab MXA testing (threshold 30 ng / mL) has extremely high sensitivity (100%) and specificity (98.2%) for diagnosing viral common colds, and is significantly superior to oropharyngeal or whole blood MXA testing and influenza antigen rapid tests during the same period. This method also demonstrates excellent early warning capabilities for influenza virus infection (sensitivity 100%, specificity 100%). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Sensitivity and specificity of nasopharyngeal MXA detection in identifying early common cold. DETAILED DESCRIPTION

[0026] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0027] Example 1 The MXA test kit was used as the research and verification test object

[0028] The MXA detection kit can be prepared with reference to the MXA immunoassay method (e.g., the specific antibodies used, the detection principle (e.g., immunochromatography), etc.) described in the applicant's prior Chinese invention patent CN113866428B (Invention Title: A Diagnostic Reagent for Screening Viral Influenza in Children Using MXA and SAA). By testing 264 samples of nasopharyngeal secretions from healthy individuals, a normal reference range for MXA in nasopharyngeal secretions was determined, and based on this, the diagnostic threshold for MXA was established as 30 ng / ml.

[0029] Example 2 Using MXA Levels to Determine Whether a Patient Has an Early Common Cold

[0030] The trial involved 32 patients with early common cold symptoms (1-2 days after onset, with no systemic symptoms, only nasopharyngeal symptoms such as sneezing and nasal congestion) admitted to the Department of Infectious Diseases at the Second Affiliated Hospital of Chongqing Medical University. Viral infection was confirmed by viral nucleic acid testing. The trial also included 30 patients with allergic rhinitis and 26 patients with bacterial sinusitis who also presented with early common cold symptoms. Viral infection was ruled out by imaging examinations and viral nucleic acid testing.

[0031] 1. Nasopharyngeal and oropharyngeal specimens were collected from 88 patients using a 10µL quantitative swab with quantitative absorption capacity. Venous whole blood was also collected from 88 patients. The 10µL whole blood sample and the 10µL quantitative swab were further eluted with 100 volumes of lysis buffer containing 0.1% CHAPS and then tested with the MXA assay kit. The MXA levels in whole blood, nasopharyngeal secretions, and oropharyngeal secretions are reported in Tables 1 and 2 below.

[0032] 2. Combined with the clinical diagnosis results of the patients in Tables 1 and 2, statistical analysis was performed on the MXA concentration results in whole blood, nasopharyngeal secretions, and oropharyngeal secretions shown in Tables 1 and 2.

[0033] 3. According to the above test method, the test verification results are shown in Tables 1 and 2.

[0034] Table 1 Test results of 88 patients with early common cold symptoms

[0035]

[0036] Table 2 Test results of 88 patients with early common cold symptoms with a course of 3-7 days

[0037]

[0038] 4. Analysis of the results in Tables 1 and 2

[0039] Statistical Table 1 data:

[0040] 32 patients with early common cold, 30 patients with allergic rhinitis, and 26 patients with bacterial sinusitis were clinically diagnosed. After nasopharyngeal MXA testing, 32 patients were correctly diagnosed with early common cold. 56 patients with allergic rhinitis and bacterial sinusitis were correctly diagnosed after nasopharyngeal MXA testing. The data were statistically analyzed using SPSS software, and 30 ng / ml can be used as the diagnostic threshold. The sensitivity and specificity of identifying early common cold after nasopharyngeal MXA testing were 100% and 98.2%, respectively. Figure 1 shown.

[0041] At the same time, oropharyngeal and whole blood MXA tests could not distinguish between early common cold and non-early common cold, and there was no statistically significant difference between the two groups of data.

[0042] Of note, among 56 patients with non-early common cold conditions (30 with allergic rhinitis and 26 with bacterial sinusitis), one false-positive case occurred (bacterial sinusitis patient, case 88, nasopharyngeal MXA 178.5 ng / mL). This patient's nasopharyngeal MXA levels remained elevated, consistent with a general pattern of viral infection. A review of the clinical data revealed the high likelihood of an undetected concurrent viral infection, likely due to limitations in the types or categories of viruses currently tested for nucleic acid detection. This positive case is included in the aforementioned specificity calculation (98.2%).

[0043] The data in Statistical Table 2 also demonstrate that MXA protein can still be stably detected in nasopharyngeal samples from patients in the middle to late stages of the common cold (days 3-7), with concentrations significantly elevated compared to those in the early stages. MXA protein concentrations in nasopharyngeal samples do not decline as the common cold progresses. Furthermore, oropharyngeal and whole blood MXA assays still cannot distinguish between common and non-common colds, with no statistically significant difference between the two groups.

[0044] Example 3: Experiment on using MXA level to warn of influenza virus infection

[0045] A prospective study was conducted among patients with a history of close contact with influenza at the Department of Infectious Diseases, Second Affiliated Hospital of Chongqing Medical University. Based on final influenza nucleic acid diagnostic results, 22 patients were confirmed to have influenza. Ten patients with allergic rhinitis and bacterial sinusitis who presented with common cold symptoms and had viral infection ruled out by imaging and viral nucleic acid testing served as non-influenza controls. All 32 patients underwent MXA testing of whole blood, nasopharyngeal secretions, and oropharyngeal secretions during the early common cold symptom phase (within 1-2 days of onset, no systemic symptoms, only nasopharyngeal symptoms such as sneezing and nasal congestion). The cut-off value was 30 ng / ml; values ​​greater than or equal to 30 ng / ml were considered positive, and values ​​less than 30 ng / ml were considered negative. In addition, the 32 patients with early common cold symptoms were tested simultaneously using commercially available influenza antigen test strips.

[0046] Table 3 Experimental results of 32 influenza patients with early common cold symptoms

[0047]

[0048] Statistical Table 3 shows that among 22 clinically diagnosed influenza patients and 10 non-influenza controls, 22 patients were correctly diagnosed with influenza using nasopharyngeal MXA during the early stages of common cold symptoms. Of the 10 non-influenza controls, 10 were correctly diagnosed using nasopharyngeal MXA. Using a diagnostic threshold of 30 ng / ml, the sensitivity and specificity of nasopharyngeal MXA testing for early warning of influenza virus infection during the early stages of common cold symptoms was 100%.

[0049] Commercially available influenza antigen test strips were used to test 22 patients with influenza and early common cold symptoms, and four were correctly diagnosed as influenza. A total of 10 non-influenza patients were also tested, and all 10 were correctly diagnosed. The sensitivity and specificity for distinguishing influenza from non-influenza were 18.2% and 100%, respectively.

[0050] At the same time, in the early common cold symptom period of influenza, oropharyngeal and whole blood MXA tests could not distinguish between influenza and non-influenza, and there was no statistically significant difference between the two groups of data.

[0051] These clinical validation results fully demonstrate that, in the early stages of illness (1-2 days, with only nasopharyngeal symptoms), nasopharyngeal swab MXA testing (threshold 30 ng / mL) has extremely high sensitivity (100%) and specificity (98.2%) for diagnosing viral common colds, significantly outperforming oropharyngeal or whole blood MXA testing and influenza antigen rapid tests performed during the same period. Furthermore, this method demonstrates excellent early warning capabilities (sensitivity 100% and specificity 100%) for influenza virus infection in the early stages.

[0052] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. Use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for identifying early common colds, characterized in that: If the MXA content is higher than a predetermined threshold, it is diagnosed as an early common cold. The detection reagent has a cut-off value of 20-40 ng / mL, where the early common cold refers to a disease course of 1-2 days. The detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the test subject; the diagnosis is based on the MXA content in the nasopharyngeal swab sample being higher than the predetermined threshold, while the MXA content in the whole blood sample and oropharyngeal swab sample of the same subject are both lower than the predetermined threshold.

2. The use according to claim 1, characterized in that The preparation that specifically binds to MXA is an antibody or antibody fragment that specifically binds to MXA.

3. The use according to claim 1 or 2, characterized in that The cut-off value of the detection reagent is 30 ng / mL.

4. Use of MXA or a preparation specifically binding to MXA in the preparation of a detection reagent for influenza early warning, characterized in that: If the MXA content is higher than a predetermined threshold, potential influenza is diagnosed. The detection reagent has a cut-off value of 20-40 ng / mL. The detection reagent is used to detect the MXA content in the nasopharyngeal secretion sample of the test subject. The diagnosis is based on the MXA content in the nasopharyngeal swab sample being higher than the predetermined threshold, while the MXA content in the whole blood sample and oropharyngeal swab sample of the same subject are both lower than the predetermined threshold.

Images