Upper respiratory infection biomarker and application thereof

Abstract

The invention provides an upper respiratory infection biomarker GP73 as well as a corresponding application and a kit product thereof. Experiments show that the GP73 marker in serum can diagnose the upper respiratory tract infection caused by influenza A virus infection, influenza B virus infection or novel coronavirus infection with high accuracy, and a new choice is provided for rapid, simple and convenient diagnosis of the upper respiratory tract infection.

Description

[0001] Cross - reference to related applications:

[0002] This application claims the priority of a Chinese patent application with the application number 202410686560.0, titled "Biomarkers for Upper Respiratory Tract Infection and Their Applications", filed with the Chinese Patent Office on May 30, 2024, and incorporates its entire content herein by reference. Field of the Invention

[0003] This application belongs to the fields of biomarkers and the diagnosis of upper respiratory diseases. Specifically, this application provides biomarkers for upper respiratory tract infection and their applications. Background Art

[0004] Upper respiratory tract infection is a common disease among people of all ages, characterized by a large number of patients, diverse symptoms, various pathogens, and an urgent need for diagnosis and treatment. Currently, the diagnostic methods based on culture and PCR detection have deficiencies in real - time performance, and the methods based on symptoms and blood routine detection may be confused with other diseases to a certain extent.

[0005] Therefore, there is a need in the art to develop biomarkers for upper respiratory tract infection that are convenient to use and accurate in judgment. Summary of the Invention

[0006] On the one hand, this application provides a biomarker for upper respiratory tract infection, and the biomarker for upper respiratory tract infection is GP73.

[0007] On the other hand, this application provides the use of a reagent for detecting the expression level of GP73 in a biological sample in the preparation of a kit for detecting upper respiratory tract infection.

[0008] On the other hand, this application provides a kit for detecting upper respiratory tract infection, and the kit contains a reagent for detecting the expression level of GP73 in a biological sample.

[0009] Further, the biological sample is blood, serum, cerebrospinal fluid, or sputum.

[0010] Further, the biological sample is serum.

[0011] Further, the upper respiratory tract infection is influenza A virus infection, influenza B virus infection, or novel coronavirus infection.

[0012] Further, the upper respiratory tract infection is an upper respiratory tract infection with an unknown pathogen.

[0013] Further, the upper respiratory tract infection is bloodstream infection.

[0014] Further, the reagent for detecting the expression level of GP73 in a biological sample is an ELISA reagent, a Western Blot reagent, or an immunohistochemical reagent.

[0015] Further, the reagent for detecting the expression level of GP73 in a biological sample is an ELISA reagent.

[0016] The influenza A virus and influenza B virus mentioned above include various known and undiscovered serotypes, and the novel coronavirus refers to COVID-19, including its various specific serotypes and strains.

[0017] In this application, GP73 is the abbreviation of Golgi transmembrane glycoprotein 73, and its specific sequence can be found in databases such as Genbank, and all its various variants are within the scope of this application.

[0018] Based on the understanding of the GP73 sequence and properties, those skilled in the art can routinely obtain reagents for detecting GP73 levels, such as ELISA reagents, Western Blot reagents, or immunohistochemical reagents using monoclonal or polyclonal antibodies. These reagents can be self-made or purchased as commercially available products. Brief Description of the Drawings

[0019] Figure 1 Serum GP73 levels in the VSV and PBS groups in the mouse model.

[0020] Figure 2 Expression of GP73 in influenza A, influenza B, COVID-19, and respiratory tract infections.

[0021] Figure 3 ROC curves of GP73 for upper respiratory tract infection patients and healthy physical examination population.

[0022] Figure 4 ROC curves of GP73 for positive blood stream infection patients and healthy physical examination population.

[0023] Figure 5 ROC curves of GP73, CRP, WBC, and NLR for the upper respiratory tract infection group and the healthy group; A: ROC curves of GP73, CRP, WBC, and NLR for the influenza A infection group and the healthy group; B: ROC curves of GP73, CRP, WBC, and NLR for the influenza B infection group and the healthy group; C: ROC curves of GP73, CRP, WBC, and NLR for the COVID-19 infection group and the healthy group; D: ROC curves of GP73, CRP, WBC, and NLR for the group with unidentified pathogen infection and the healthy group.

[0024] Figure 6The expression ratios of GP73, CRP, WBC, and NLR in the upper respiratory tract infection group, negative and positive; A: The expression ratios of GP73 and CRP, negative and positive, in each group; B: The expression ratios of GP73 and NLR, negative and positive, in each group; C: The expression ratios of GP73 and WBC, negative and positive, in each group. Detailed implementation manners

[0025] Example 1 Upper respiratory tract infection markers in a mouse model

[0026] The VSV virus (vesicular stomatitis virus) was used to simulate RNA virus infection. VSV is a common model virus that mainly infects animals through damaged skin and mucous membranes. It can also reach the liver, kidneys, and central nervous system through the blood circulation, causing non-specific inflammation. VSV can activate the RLRs signaling pathway through the mitochondrial antiviral protein MAVS, thereby triggering an antiviral immune response and inducing the production of type I interferons.

[0027] We established a VSV virus infection model using wild-type mice and collected the peripheral blood of the mice at 24 h to detect the markers therein, and detected the GP73 level in the serum by ELISA: 20-week-old C57BL / 6 male mice, weighing 21 - 23 g, provided by Spf (Beijing) Biotechnology Co., Ltd. The mice were randomly divided into two groups, and both groups were intraperitoneally injected with 1 ml of PBS (PBS group, 3 mice) and 1 ml of VSV virus (VSV group, 3 mice).

[0028] We found that the expression level of serum GP73 in the VSV group was significantly higher than that in the PBS group, and it was statistically significant ( Figure 1 ).

[0029] Example 2 Verification of the diagnostic effect in the actual population

[0030] 1. Sample collection

[0031] Specimens of patients with upper respiratory tract infections were detected in the Clinical Laboratory of the Third Medical Center of Chinese PLA General Hospital from October 2023 to January 2024. Inclusion criteria: ① According to the "Diagnosis and Treatment Plan for Influenza A (H1N1) (Trial Edition 1, 2009)" issued by the Ministry of Health in May 2009 as the inclusion criteria, a total of 128 patients with positive influenza A were collected; ② For influenza B, 75 patients with positive influenza B were collected according to the "Diagnosis and Treatment Plan for Influenza (2018 Edition)" of the National Health and Family Planning Commission and the State Administration of Traditional Chinese Medicine; ③ For COVID-19, 71 patients with positive COVID-19 were collected according to the criteria of the "Diagnosis and Treatment Plan for Novel Coronavirus Infection (Trial Version 6)"; ④ 80 patients with febrile respiratory tract infections with unclear pathogen infections; the above patients all had fever and respiratory tract infections as the main symptoms. Exclusion criteria: ① Patients with combined infections of other systems including various liver diseases, combined with severe immune system diseases, and malignant tumors; ② Patients with severe impairment of original organ functions, such as those with chronic renal insufficiency; ③ Patients with combined hematological diseases; ④ Incomplete clinical data. 83 healthy people undergoing physical examinations were collected as the control group.

[0032] Detect the changes in the expression levels of GP73 in the sera of patients with influenza A, influenza B, COVID-19, and respiratory tract infections with unclear pathogen infections. The results are as Figure 2 shown: In influenza A, influenza B, COVID-19, and respiratory tract infections with unclear pathogen infections, the expression of GP73 showed a significant upward trend compared with that of healthy people, and the expression levels were all higher than the upper limit of the normal value of 80 ng / ml, and all were statistically significant.

[0033] 2. ROC curve analysis

[0034] 2.1 Compare the ROC curves of the upper respiratory tract infection group (including influenza A, influenza B, COVID-19, and respiratory tract infection groups with unclear pathogen infections) and the healthy group. AUC = 0.8974, P < 0.0001 ( Figure 3 ). Generally, AUC = 0.5 - 0.7 indicates relatively low accuracy in the diagnostic value; AUC = 0.7 - 0.9 indicates certain accuracy in the diagnostic value; AUC > 0.9 indicates high accuracy in the diagnostic value.

[0035] Collect the sera of patients with positive blood cultures to further verify the accuracy of the biomarker. The blood culture operation follows the standardized procedure of WS / T 503—2017 "Clinical Microbiology Laboratory Blood Culture Operation Specification". The inclusion criteria for patients with positive blood flow are: patients have systemic infection symptoms, fever (body temperature ≥ 38°C) or hypothermia (body temperature ≤ 36°C), chills, and abnormal white blood cell counts; the exclusion criteria are: ① Patients with immune function deficiencies; ② Severe diseases such as endocarditis and pyelonephritis; ③ Incomplete clinical data.

[0036] The ROC curve of patients with positive bloodstream infections and the healthy group. AUC = 0.9749 ( Figure 4)。

[0037] 2.2 Comparison of the ROC curves of patients with influenza A, influenza B, COVID-19, and infections with unknown pathogens

[0038] In view of the fact that when comparing the overall upper respiratory tract infection group with the healthy group, many interfering factors will be introduced. For example, in the comprehensive comparison, the upper respiratory tract infection group contains various different pathogen infection types, and there may be certain similarities or overlaps between these types, which increases the difficulty of the model in distinguishing these different infection types, thereby reducing the overall AUC value; at the same time, the sample size and distribution will also affect the AUC value. In the comprehensive comparison, the sample size of the influenza A infection group is relatively large, and the sample distribution of different infection types may be unbalanced, which will lead to the prediction performance of the model for some infection types being "diluted" by other types, thus affecting the overall AUC value; different infection types may have different characteristic manifestations, and these characteristics may be masked or interfered by the characteristics of other types during the comprehensive comparison - for example, although both influenza A and influenza B belong to influenza viruses, there are differences in some biomarkers or clinical manifestations between them, which may affect the accuracy of the AUC.

[0039] For the above reasons, we further analyzed each etiology separately to improve the accuracy of each etiology judgment.

[0040] In influenza A infection, the GP73 AUC = 0.9712, the CRP (C-reactive protein) AUC = 0.9996, the NLR (neutrophil lymphocyte ratio) AUC = 0.9428, the WBC (white blood cell count) AUC = 0.5076, P < 0.0001( Figure 5 in A);

[0041] In influenza B infection, the GP73 AUC = 0.9779, the CRP AUC = 0.9832, the NLR AUC = 0.9360, the WBC AUC = 0.5319, P < 0.0001( Figure 5 in B);

[0042] In COVID-19 infection, the GP73 AUC = 1.000, the CRP AUC = 0.9650, the NLR AUC = 0.9555, the WBC AUC = 0.5220, P < 0.0001( Figure 5 in C);

[0043] In infections with unknown pathogens, the GP73 AUC = 0.9189, the CRP AUC = 0.9808, the NLR AUC = 0.9379, the WBC AUC = 0.6220, P < 0.0001( Figure 5 in D).

[0044] Generally, an AUC of 0.5 - 0.7 indicates relatively low accuracy in the diagnostic value; an AUC of 0.7 - 0.9 indicates a certain degree of accuracy in the diagnostic value; an AUC > 0.9 indicates high accuracy in the diagnostic value. GP73 has a similar diagnostic efficacy compared to CRP and NLR, and the diagnostic efficacy of GP73 is higher than that of the traditional infection indicator WBC.

[0045] 3. Positive expression ratio and negative expression ratio

[0046] Collect the sera of patients with influenza A, influenza B, COVID-19, and those with infections of unknown pathogens. Detect the expression of CRP in the sera of each group of patients, and compare the positive expression ratios and negative expression ratios of GP73 and CRP in each group of patients ( Figure 6 as shown in A). In the serum, GP73 > 80 ng / ml is considered positive, and CRP > 8 mg / L in the serum is considered positive. When the expression of GP73 is positive and the expression of CRP is also positive, the proportion is 55.9%, that is, when CRP reflects the infection status of the patient, GP73 can also reflect the infection status of the patient in 55.9% of the cases; when the expression of CRP is negative, the proportion of positive expression of GP73 is 31.5%, that is, when CRP cannot reflect the infection status of the patient, GP73 can reflect the infection status of the patient in 31.5% of the cases; when CRP is positive, the negative expression ratio of GP73 is 6.7%, that is, when CRP can reflect the infection status of the patient, GP73 cannot reflect the infection status of the patient in 6.7% of the cases; when the expressions of both GP73 and CRP are negative, the proportion is 5.9%, that is, in 5.9% of the cases, neither GP73 nor CRP can reflect the infection status of the patient.

[0047] Detect the expression of NLR in the sera of each group of patients, and compare the positive expression ratios and negative expression ratios of GP73 and NLR in each group of patients ( Figure 6 as shown in B). In the serum, GP73 > 80 ng / ml is considered positive expression, and NLR > 2.5 or NLR < 1.75 in the serum is considered positive expression. When the expression of GP73 is positive and the expression of NLR is also positive, the proportion is 72.5%, that is, when NLR reflects the infection status of the patient, GP73 can also reflect the infection status of the patient in 72.5% of the cases; when the expression of NLR is negative, the proportion of positive expression of GP73 is 14.2%, that is, when NLR cannot reflect the infection status of the patient, GP73 can reflect the infection status of the patient in 14.2% of the cases; when NLR is positive, the negative expression ratio of GP73 is 11.1%, that is, when NLR can reflect the infection status of the patient, GP73 cannot reflect the infection status of the patient in 11.1% of the cases; when the expressions of both GP73 and NLR are negative, the proportion is 1.6%, that is, in 1.6% of the cases, neither GP73 nor NLR can reflect the infection status of the patient.

[0048] Detect the expression of WBC in each group of patients, and compare the positive expression ratio and negative expression ratio of GP73 and WBC in each group of patients ( Figure 6 in C), GP73 > 80 ng / ml in serum is positive expression, and WBC < 4 × 10 9 / L or WBC > 10 × 10 9 / L is positive expression. When the proportion of positive expression of GP73 and WBC is 21.3%, that is, when WBC reflects the infection status of patients, GP73 can also reflect the infection status of patients in 21.3% of the cases; when the expression of WBC is negative, the proportion of positive expression of GP73 is 65.7%, that is, when WBC cannot reflect the infection status of patients, GP73 can reflect the infection status of patients in 65.7% of the cases; when WBC is positive, the negative expression ratio of GP73 is 2.8%, that is, when WBC can reflect the infection status of patients, GP73 cannot reflect the infection status of patients in 2.8% of the cases; when the proportion of negative expression of both GP73 and WBC is 9.4%, that is, in 9.4% of the cases, neither GP73 nor WBC can reflect the infection status of patients.

[0049] Obviously, the above embodiments are only examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A biomarker for upper respiratory tract infection, characterized in that: The upper respiratory tract infection biomarker is GP73.

2. Application of a reagent for detecting GP73 expression levels in biological samples in the preparation of a kit for detecting upper respiratory tract infection.

3. A kit for detecting upper respiratory tract infection, characterized in that: The kit comprises a reagent for detecting the expression level of GP73 in a biological sample.

4. The use according to claim 2 or the kit according to claim 3, wherein the biological sample is blood, serum, cerebrospinal fluid or sputum.

5. The use or kit according to claim 4, wherein the biological sample is serum.

6. The use according to claim 2 or the kit according to claim 3, wherein the upper respiratory tract infection is influenza A virus infection, influenza B virus infection or new coronavirus infection.

7. The use or kit according to claim 6, wherein the upper respiratory tract infection is an upper respiratory tract infection caused by an unknown pathogen.

8. The use according to claim 2 or the kit according to claim 3, wherein the upper respiratory tract infection is a bloodstream infection.

9. The use according to claim 2 or the kit according to claim 3, wherein the reagent for detecting the expression level of GP73 in a biological sample is an ELISA reagent, a Western Blot reagent or an immunohistochemistry reagent.

10. The use or kit according to claim 9, wherein the reagent for detecting the expression level of GP73 in a biological sample is an ELISA reagent.

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