Application of MXA in preparation of detection reagent for screening virus infection

Abstract

The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to application of MXA in preparation of a detection reagent for screening virus infection. The level of mucus virus resistance protein A (MXA) is detected by using a nasopharynx secretion sample, and a non-invasive diagnosis method for early stage (morbidity for 1-2 days) of common cold is carried out. The method can also be used for screening the risk of potential major viral infectious diseases (such as influenza or novel coronavirus infection and the like) with initial symptoms of common cold.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to the use of detecting myxovirus resistance protein A (MXA) in the preparation of diagnostic reagents for identifying viral infections. Background Technology

[0002] Acute upper respiratory tract infection (URTI) is a general term for acute inflammation of the nose, pharynx, or larynx caused by various viruses and / or bacteria. Viruses are the most common, accounting for about 70% to 80%. It includes the common cold, viral pharyngitis, laryngitis, herpetic pharyngitis, pharyngoconjunctival fever, and bacterial pharyngotonsitis, among others.

[0003] The common cold, also known as a "cold," is the most prevalent type of upper respiratory tract infection, characterized primarily by catarrhal symptoms in the nasopharynx. It is mostly caused by rhinoviruses, followed by coronaviruses, parainfluenza viruses, respiratory syncytial virus, echoviruses, and coxsackieviruses. The 2018 Guidelines for the Diagnosis and Treatment of Acute Upper Respiratory Tract Infections in Primary Care in my country describe the common cold as having a rapid onset, primarily manifesting as nasal symptoms such as sneezing, nasal congestion, and clear, watery nasal discharge. Other symptoms may include cough, dry throat, itchy or burning throat, and even postnasal drip. Sneezing, nasal congestion, and clear, watery nasal discharge may appear simultaneously with or several hours after the onset of symptoms. After 2-3 days, the nasal discharge thickens, often accompanied by sore throat, tearing, decreased taste, difficulty breathing, and hoarseness. Generally, there is no fever or systemic symptoms, or only low-grade fever, malaise, mild chills, and headache. Physical examination reveals nasal mucosal congestion, edema, and secretions, and mild pharyngeal congestion. The illness usually resolves within 5-7 days.

[0004] This invention defines 'early common cold' as the onset of illness 1-2 days ago, in which the patient only presents with nasopharyngeal symptoms (such as sneezing, nasal congestion, and clear watery nasal discharge) and no systemic symptoms (such as fever, muscle pain, and chills), which is consistent with the characteristics of the early stage of the common cold described in the "Guidelines for the Diagnosis and Treatment of Acute Upper Respiratory Tract Infections in Primary Care (2018)", but the guidelines do not specify the diagnostic methods for this stage.

[0005] Furthermore, influenza viruses, novel coronaviruses, and other major viral infectious diseases can all initially present with common cold-like symptoms. Therefore, diagnosing the common cold in its early stages is of great significance, with the "early stage" (1-2 days) being the optimal diagnostic window. However, existing methods are limited by low sensitivity or high invasiveness. In the early stages of the disease, viral load may be low, limiting the sensitivity of viral nucleic acid testing, and the sampling and testing process is relatively complex. Viral antigen tests (such as rapid influenza antigen tests) also generally have low sensitivity at this stage (often below 20%).

[0006] During viral infection, cellular responses involving the secretion of type I interferons (IFNs) occur, and the antiviral activity of type I IFNs can be mediated by the production of myxoviral resistance protein A (MXA). Our research team has discovered for the first time that MXA protein is stably detected and significantly elevated in nasopharyngeal samples from patients with early-stage common cold (1-2 days after onset, nasopharyngeal symptoms only), while MXA concentrations in whole blood and oropharyngeal samples from the same patients remain at background levels (usually below the diagnostic threshold). This specific distribution pattern reveals a strong local (nasopharyngeal) type I interferon response characteristic in the early stages of viral infection, surpassing the understanding of current techniques. This finding not only clarifies that the nasopharynx is the best and only effective biological sample source for detecting early viral infection-related MXA proteins, but also lays the foundation for developing a highly sensitive, highly specific, and non-invasive diagnostic method for early common cold based on nasopharyngeal MXA detection. This method can accurately identify viral common cold in the very early stage of symptom onset (1-2 days), which has important clinical significance and application value for timely symptomatic supportive treatment, reducing unnecessary antibiotic use, assessing potential severe disease risk (such as influenza, novel coronavirus, etc.) and implementing early public health intervention measures, filling the gap in the existing technology for accurate diagnosis of diseases in the very early stage. Summary of the Invention

[0007] This invention provides a non-invasive method for early diagnosis of the common cold (1-2 days after onset) by detecting the level of mucoviral resistance protein A (MXA) in nasopharyngeal secretion samples. This method can also be used to screen for the risk of potential major viral infectious diseases (such as influenza or novel coronavirus infection) presenting with initial symptoms similar to the common cold.

[0008] The first aspect of the present invention is to provide the use of MXA or a formulation specifically binding to MXA in the preparation of a diagnostic reagent for identifying the common cold, wherein if the MXA content is higher than a predetermined threshold, the common cold is diagnosed, and the diagnostic reagent has a detection cut-off value of 20 to 40 ng / mL.

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

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

[0011] Furthermore, the formulation 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 subject being tested.

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

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

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

[0017] Furthermore, the common cold mentioned above refers to the early stage of the common cold, which means that the course of the illness is within 1 to 2 days.

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

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

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

[0021] A second aspect of the present invention is to provide an MXA detection kit, the detection kit comprising a specific MXA-binding conjugate, a standard, and a buffer solution, and a warning line marker, the warning line marker including a detection result pattern corresponding to a sample MXA content at a cut-off value; the warning line marker further including a prominent mark corresponding to a detection result when the sample MXA content is greater than 20~40 ng / mL.

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

[0023] The beneficial effects of this invention include: In the early stages of the common cold (1-2 days, with only nasopharyngeal symptoms), nasopharyngeal swab MXA testing (threshold 30 ng / mL) demonstrates extremely high sensitivity (100%) and specificity (98.2%) for diagnosing viral common cold, significantly superior to concurrent oropharyngeal or whole blood MXA testing and rapid influenza antigen testing. Furthermore, this method also exhibits excellent early warning capabilities in the early stages of influenza virus infection (sensitivity 100%, specificity 100%). Attached Figure Description

[0024] Figure 1 Nasopharyngeal MXA testing has high sensitivity and specificity in identifying early-stage common colds. Detailed Implementation

[0025] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0026] Example 1: The experiment used an MXA detection kit as the research and verification test object. The MXA detection kit can be prepared by referring to the MXA immunoassay method (e.g., the specific antibodies used, the detection principle such as immunochromatography, etc.) described in the applicant's prior Chinese invention patent CN113866428B (Invention title: A diagnostic reagent for distinguishing viral influenza in children using MXA and SAA). By testing 264 nasopharyngeal secretion samples from healthy individuals, the normal reference range for MXA in nasopharyngeal secretions of healthy individuals was obtained, and based on this, the diagnostic threshold for MXA was determined to be 30 ng / ml.

[0027] Example 2: Using MXA levels to determine if a patient has an early common cold test The study included 32 patients with early-stage common cold (onset 1-2 days, no systemic symptoms, only nasopharyngeal symptoms such as sneezing and nasal congestion) from the Department of Infectious Diseases, Second Affiliated Hospital of Chongqing Medical University, who were confirmed to be infected with the virus by viral nucleic acid testing. Also included were 30 patients with allergic rhinitis and 26 patients with bacterial sinusitis who also presented with early-stage common cold symptoms, but were ruled out for viral infection by imaging examinations and viral nucleic acid testing.

[0028] 1. Nasopharyngeal and oropharyngeal samples were collected from 88 patients using 10µL quantitative swabs with quantitative absorption capability, and venous whole blood was also collected from 88 patients. The collected whole blood samples (10µL) and 10µL quantitative swabs were further eluted thoroughly with 100 volumes of lysis buffer containing 0.1% CHAPS, and then analyzed using an MXA detection kit. The results of MXA content detection in whole blood, nasopharyngeal secretions, and oropharyngeal secretions are recorded in Tables 1 and 2 below. 2. Based on the clinical diagnosis results of the patients in Tables 1 and 2, a statistical analysis was performed on the detection concentration results of MXA in whole blood, nasopharyngeal secretions and oropharyngeal secretions shown in Tables 1 and 2.

[0029] 3. The experimental verification results are shown in Table 1 and Table 2, following the aforementioned experimental methods.

[0030] Table 1. Results of the trial in 88 patients with early common cold symptoms

[0031] Table 2. Results of a trial involving 88 patients with early common cold symptoms and a disease course of 3-7 days.

[0032] 4. Analysis of the results in Tables 1 and 2 Statistical data in Table 1: Of the 32 patients clinically diagnosed with early-stage common cold, 30 with allergic rhinitis, and 26 with bacterial sinusitis, 32 were correctly diagnosed with early-stage common cold after nasopharyngeal MXA testing. Of the 56 patients with allergic rhinitis and bacterial sinusitis, 55 were correctly diagnosed after nasopharyngeal MXA testing. Statistical analysis of the data using SPSS software showed that 30 ng / ml could be used as the diagnostic threshold. The sensitivity and specificity of nasopharyngeal MXA testing in identifying early-stage common cold were 100% and 98.2%, respectively. Figure 1 As shown.

[0033] Meanwhile, MXA tests in the oropharynx and whole blood could not distinguish between early-stage and non-early-stage common colds, and there was no statistically significant difference between the two groups.

[0034] It is noteworthy that among the 56 patients who did not have early-stage common cold (30 with allergic rhinitis and 26 with bacterial sinusitis), one false positive occurred (patient with bacterial sinusitis, case 88, nasopharyngeal MXA 178.5 ng / mL). Furthermore, this case showed persistently elevated nasopharyngeal MXA levels, consistent with the general pattern of viral infection. Upon review of clinical data, it was found that due to the limitations of current nucleic acid testing for specific viral types or categories, there was a high probability of undetected co-infection. This positive case was included in the specificity calculation results mentioned above (98.2%).

[0035] Similarly, the data in Table 2 indicate that in patients in the middle and late stages of the common cold (3-7 days), MXA protein can still be stably detected in nasopharyngeal samples, and the concentration is significantly higher than in the early stages. The concentration of MXA protein in nasopharyngeal samples does not decrease as the common cold improves. Furthermore, MXA detection in the oropharynx and whole blood still cannot distinguish between the common cold and non-common colds; there is no statistically significant difference between the two groups.

[0036] Example 3: Experiment using MXA level for early warning of influenza virus infection A prospective study was conducted on patients with a history of close contact with influenza at the Department of Infectious Diseases, Second Affiliated Hospital of Chongqing Medical University. Based on the final influenza nucleic acid diagnosis results, 22 cases were confirmed as influenza. Ten patients with allergic rhinitis and bacterial sinusitis presenting with common cold symptoms were excluded from viral infection by imaging examinations and viral nucleic acid testing, serving as a non-influenza control group. All 32 patients underwent MXA testing of whole blood, nasopharyngeal secretions, and oropharyngeal secretions during the early common cold symptom stage (1-2 days after onset, without 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, commercially available influenza antigen test strips were also used to test these 32 patients during the early common cold symptom stage.

[0037] Table 3. Experimental results of 32 influenza patients in the early stage of common cold symptoms.

[0038] Table 3 shows the data: Of the 22 clinically diagnosed influenza patients and 10 non-influenza control patients, 22 were correctly diagnosed with influenza after nasopharyngeal MXA testing during the early stage of common cold symptoms. Of the 10 non-influenza control patients, all 10 were correctly diagnosed after nasopharyngeal MXA testing. Using 30 ng / ml as the diagnostic threshold, the sensitivity and specificity of nasopharyngeal MXA testing in predicting influenza virus infection during the early stage of common cold symptoms was 100%.

[0039] Commercially available influenza antigen test strips were used to test 22 patients with early common cold symptoms, correctly diagnosing 4 cases of influenza. A control group of 10 non-influenza patients was also tested, and all 10 were correctly diagnosed. The sensitivity and specificity for differentiating between influenza and non-influenza were 18.2% and 100%, respectively.

[0040] Meanwhile, during the early stages of influenza and the common cold symptom period, MXA testing in the oropharynx and whole blood could not distinguish between influenza and non-influenza, and there was no statistically significant difference between the two groups.

[0041] The above clinical validation results fully demonstrate that in the early stage of the disease (1-2 days, with only nasopharyngeal symptoms), nasopharyngeal swab MXA detection (threshold 30 ng / mL) has extremely high sensitivity (100%) and specificity (98.2%) for diagnosing viral common cold, and is significantly superior to concurrent oropharyngeal or whole blood MXA detection and rapid influenza antigen detection. Simultaneously, this method also demonstrates excellent early warning capability in the early stage of influenza virus infection (sensitivity 100%, specificity 100%).

[0042] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. Use of MxA or an agent specifically binding to MxA for the manufacture of a test reagent for the differential diagnosis of common cold, characterized in that, If the MXA content is higher than a predetermined threshold, the common cold is diagnosed, and the detection reagent has a cut-off value of 20-40 ng / mL in the detection, and the detection reagent is used for detecting the MXA content of a nasopharyngeal secretion sample of the subject to be tested.

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

3. 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 an agent specifically binding to MxA for the manufacture of a test reagent for the discrimination of an early common cold, characterized in that, If the MXA content is higher than a predetermined threshold, the early common cold is diagnosed, and the detection reagent has a cut-off value of 20-40 ng / mL in the detection, wherein the early common cold refers to a disease course of 1-2 days.

5. Use according to claim 4, characterized in that, The preparation specifically binding to MXA is an antibody or an antibody fragment specifically binding to MXA.

6. Use according to claim 4 or 5, characterized in that, The cut-off value of the detection reagent is 30 ng / mL.

7. Use according to claim 4 or 5, characterized in that, The detection reagent is used for detecting the MXA content of a nasopharyngeal secretion sample of the subject to be tested.

8. Use of MxA or an agent specifically binding to MxA for the manufacture of a test reagent for the early warning of influenza, characterized in that, If the MXA content is higher than a predetermined threshold, the potential influenza is diagnosed, and the detection reagent has a cut-off value of 20-40 ng / mL in the detection, and the detection reagent is used for detecting the MXA content of a nasopharyngeal secretion sample of the subject to be tested.

9. Use according to claim 8, characterized in that, The diagnosis is based on that the MXA content in the nasopharyngeal swab sample is higher than the predetermined threshold, while the MXA content in the whole blood sample and the oropharyngeal swab sample of the same subject is lower than the predetermined threshold.

10. An MXA detection kit, characterized by, The detection kit comprises a binder specifically binding to MXA, a standard and a buffer, and a warning line mark, which comprises a detection result pattern corresponding to the MXA content of the sample being the cut-off value; the warning line mark further comprises a highlighted warning mark corresponding to the detection result of the MXA content of the sample being higher than the cut-off value of 20-40 ng / mL.

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