Application of neopterin in the detection of asymptomatic neurosyphilis

Abstract

The present invention relates to the use of neopterin in the detection of asymptomatic neurosyphilis. Specifically, the present invention provides the use of neopterin in the preparation of a product for detecting asymptomatic neurosyphilis. The present invention also provides an evaluation system for evaluating the risk of a patient suffering from asymptomatic neurosyphilis, the evaluation system comprising a detection module and an evaluation module and performing an evaluation based on the cerebrospinal fluid concentration of neopterin. The present invention can be used to detect asymptomatic neurosyphilis, and is particularly suitable for distinguishing asymptomatic neurosyphilis from latent syphilis.

Description

[0001] This application requests the priority of the invention patent whose applicant is "Beijing Ditan Hospital Affiliated to Capital Medical University", whose invention name is "Application of neopterin in detection of asymptomatic neurosyphilis" and whose application number is "202311721758.X". Technical Field

[0002] The invention relates to the field of biomedical technology, and in particular to the application of neopterin in the detection of asymptomatic neurosyphilis. Background Art

[0003] Syphilis is a systemic, chronic sexually transmitted disease caused by Treponema pallidum subspecies pallidum (also known as Treponema pallidum), which can cause damage to multiple systems and organs in the human body, produce a variety of clinical manifestations, lead to tissue destruction, dysfunction, and even endanger life. Syphilis is one of the most common sexually transmitted infections in the world. According to the World Health Organization, the number of new cases of syphilis in people aged 15 to 49 in 2016 was 6.3 million. At present, the incidence of syphilis is increasing year by year, and neurosyphilis is also increasing year by year. It is reported that the incidence of latent syphilis has increased by an average of 10.75% per year. The high incidence and high disability rate of syphilis seriously threaten people's health.

[0004] Neurosyphilis is a chronic infectious disease that causes damage to the brain, spinal cord and peripheral nerves caused by Treponema pallidum invading the nervous system. It can occur at any stage of syphilis. It is generally believed that about 20% of untreated syphilis patients may develop asymptomatic neurosyphilis, and 10% of the latter may progress to symptomatic neurosyphilis.

[0005] Currently, there is no gold standard for the diagnosis of neurosyphilis, and a comprehensive analysis is required based on the patient's medical history, symptoms, signs, laboratory, electrophysiological, neuropsychological and neuroimaging.

[0006] Patients with latent syphilis (latent syphilis) have no clinical manifestations of syphilis, nor any symptoms and signs of neurological damage, but only positive syphilis serological tests and non-treponemal serological tests. Syphilis serofast means that after syphilis patients undergo standardized anti-syphilis treatment and a certain period of follow-up (1 year for primary syphilis, 2 years for secondary syphilis, and 3 years for late syphilis), the non-treponemal serological test titers are maintained at a certain titer (generally 1:8 or below, but exceeding 1:8 is not uncommon), and reinfection, neurosyphilis, cardiovascular syphilis, and biological false positives are excluded, which is syphilis serofast. Although patients with asymptomatic neurosyphilis have no obvious symptoms and signs, they have abnormal changes in cerebrospinal fluid. Because there are currently no specific detection indicators for neurosyphilis and the existing detection methods are imperfect, asymptomatic neurosyphilis is often misdiagnosed as latent syphilis or latent syphilis serofast.

[0007] Asymptomatic neurosyphilis and latent syphilis have a common feature, that is, there are no obvious symptoms and signs. Therefore, it is easy to confuse the two during diagnosis, resulting in asymptomatic neurosyphilis not being diagnosed and treated in time, missing the best treatment opportunity, causing the disease to progress and worsen, and developing into symptomatic neurosyphilis, resulting in serious consequences, disability or life-threatening, causing serious economic and mental burdens on the country, families and patients, and increasing social instability. Therefore, the field of syphilis diagnosis technology particularly needs technology that can detect asymptomatic neurosyphilis in a timely and convenient manner.

[0008] Neopterin is a decomposition product of adenosine triphosphate, and it is reported that it can be used as a marker for activation of the cellular immune system, but its role in the diagnosis and treatment of syphilis patients has never been reported, especially it has not been reported that it can be used for the detection of asymptomatic neurosyphilis, and it has not been reported that it can be used to distinguish asymptomatic neurosyphilis from latent syphilis. The inventors have found through research that neopterin can be used to detect asymptomatic neurosyphilis, and is particularly suitable for distinguishing latent syphilis from asymptomatic neurosyphilis, thereby completing the present invention. Summary of the invention

[0009] A first aspect of the present invention provides a kit for detecting asymptomatic neurosyphilis, the kit comprising a first detection reagent and an optional second detection reagent, the first detection reagent being a detection reagent for detecting the concentration of neopterin in cerebrospinal fluid, and the second detection reagent being a detection reagent for detecting the white blood cell count in cerebrospinal fluid.

[0010] The second aspect of the present invention provides the use of neopterin in preparing a product for detecting asymptomatic neurosyphilis.

[0011] A third aspect of the present invention provides an evaluation system for evaluating the risk of a patient suffering from asymptomatic neurosyphilis, the evaluation system comprising a detection module and an evaluation module; wherein:

[0012] The detection module includes a first detection module for detecting the concentration of neopterin in cerebrospinal fluid and a second detection module for detecting the white blood cell count in cerebrospinal fluid; the evaluation module includes a readable carrier storing evaluation rules;

[0013] The evaluation rule is: when the neopterin concentration and / or the white blood cell count of the cerebrospinal fluid is detected to be higher than the corresponding cutoff value, the syphilis patient is evaluated as having a high risk of suffering from asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of suffering from asymptomatic neurosyphilis.

[0014] Compared with the prior art, the present invention can distinguish asymptomatic neurosyphilis from other types of syphilis, especially latent syphilis, thereby achieving early identification and early treatment of asymptomatic neurosyphilis and preventing it from developing into symptomatic neurosyphilis with serious consequences. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The difference in the expression level of neopterin in two groups, latent syphilis (LS, n = 29) and asymptomatic neurosyphilis (ANS, n = 30), is shown. Each box plot includes the lower quartile (lower horizontal line), median (middle horizontal line) and upper quartile (upper horizontal line). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney U test was used to compare the non-normally distributed data. p value ≤ 0.05 was considered statistically significant.

[0016] Figure 2 Figure 2 shows the difference in the expression level of neurofilament light chain (NF-L) in the LS (n=29) and ANS (n=30) groups. Each box plot includes the lower quartile (lower horizontal line), median (middle horizontal line), and upper quartile (upper horizontal line). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney U test was used to compare the non-normally distributed data. p values ​​≤ 0.05 were considered statistically significant.

[0017] Figure 3The difference in the expression level of the clinical parameter cerebrospinal fluid leukocyte count (CSF wbc) between the LS (n=29) and ANS (n=30) groups is shown. Each box plot includes the lower quartile (lower horizontal line), median (middle horizontal line) and upper quartile (upper horizontal line). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney U test was used to compare the non-normally distributed data. p value ≤ 0.05 was considered statistically significant.

[0018] Figure 4 The difference in the expression level of clinical parameter cerebrospinal fluid protein (CSF protein) level between LS (n = 29) and ANS (n = 30) groups is shown. Each box plot includes the lower quartile (lower horizontal line), median (middle horizontal line) and upper quartile (upper horizontal line). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney Utest test was used to compare the non-normally distributed data. p value ≤ 0.05 was considered statistically significant. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. However, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments described in this application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0020] As described above, the present invention provides, in a first aspect, a kit for detecting asymptomatic neurosyphilis, the kit comprising a first detection reagent and an optional second detection reagent, the first detection reagent being a detection reagent for detecting the concentration of neopterin in cerebrospinal fluid, and the second detection reagent being a detection reagent for detecting the white blood cell count in cerebrospinal fluid.

[0021] The inventors have found through research that the area under the curve (AUC) of the receiver operating characteristic curve (ROC) drawn according to the cerebrospinal fluid concentration of neopterin is significantly greater than 0.7 and the corresponding p value is significantly less than 0.05. Therefore, the cerebrospinal fluid concentration of neopterin is particularly suitable for detecting whether a syphilis patient with no symptoms of nervous system damage suffers from asymptomatic neurosyphilis.

[0022] The inventors also found that the area under the curve (AUC) of the receiver operating characteristic curve (ROC) drawn based on CSF wbc was also significantly greater than 0.7 and the corresponding p value was significantly less than 0.05. Therefore, CSF wbc is also suitable for detecting whether syphilis patients with no symptoms of nervous system damage suffer from asymptomatic neurosyphilis.

[0023] In some embodiments, the first detection reagent is an anti-neopterin antibody or antibody pair.

[0024] In some preferred embodiments, the antibody is a monoclonal antibody against neopterin.

[0025] In other preferred embodiments, the antibody pair is a combination of a coating antibody for detecting neopterin and a detection antibody.

[0026] The second aspect of the present invention provides the use of neopterin in the preparation of a product for detecting asymptomatic neurosyphilis.

[0027] In some preferred embodiments, the product is any one of a kit, a detection chip and a detection array.

[0028] In some more preferred embodiments, the kit is the kit described in the first aspect of the present invention.

[0029] In other preferred embodiments, the detection chip is a detection chip loaded with a first detection reagent and an optional second detection reagent.

[0030] In some other preferred embodiments, the detection array is a detection array loaded with a first detection reagent and an optional second detection reagent. The first detection reagent is a detection reagent for detecting the concentration of neopterin in cerebrospinal fluid; the second detection reagent is a detection reagent for detecting the white blood cell count in cerebrospinal fluid. That is to say, the detection chip and the detection array are at least loaded with the first detection reagent, and may further be loaded with the second detection reagent.

[0031] A third aspect of the present invention provides an evaluation system for evaluating the risk of a patient suffering from asymptomatic neurosyphilis, the evaluation system comprising a detection module and an evaluation module; wherein:

[0032] The detection module includes a first detection module for detecting the concentration of neopterin in cerebrospinal fluid and a second detection module for detecting the white blood cell count in cerebrospinal fluid; the evaluation module includes a readable carrier storing evaluation rules;

[0033] The evaluation rule is: when the neopterin concentration and / or the white blood cell count of the cerebrospinal fluid is detected to be higher than the corresponding cutoff value, the syphilis patient is evaluated as having a high risk of suffering from asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of suffering from asymptomatic neurosyphilis.

[0034] The cutoff value, also known as the cutoff value, i.e., the judgment standard, is the boundary value for determining the positive and negative of the test, that is, determining the normal value of a certain index to distinguish between normal and abnormal. The most commonly used method for determining the cutoff value is to draw the receiver operating characteristic curve (receiver operating characteristic curve, ROC curve). The point on the ROC curve with the shortest distance from the upper left corner is defined as the critical point, which is the point where the tangent line of the ROC curve intersects with the curve, and its sensitivity and specificity are higher. The determination of the cutoff value is completely within the capabilities of those skilled in the art, and can be obtained, for example, by conventional SPSS software.

[0035] In some embodiments, the detection module includes the first detection module. In this case, the evaluation rule is: when the neopterin concentration in the cerebrospinal fluid is detected to be higher than the corresponding cutoff value, the syphilis patient is evaluated as having a high risk of asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of asymptomatic neurosyphilis.

[0036] In some embodiments, the detection module further includes the second detection module. In this case, the detection module includes not only the first detection module but also the second detection module.

[0037] During detection, the first detection module may be started first to detect the cerebrospinal fluid concentration of neopterin, and then the second detection module may be started to detect CSF wbc; of course, the second detection module may be started first to detect CSF wbc, and then the first detection module may be started to detect neopterin; in addition, the first detection module and the second detection module may be started at the same time to detect cerebrospinal fluid neopterin and white blood cell count simultaneously.

[0038] During the evaluation, if the result of the evaluation based on neopterin is consistent with the result of the evaluation based on the white blood cell count, the consistent evaluation result shall be used as the final evaluation result. If the result of the evaluation based on neopterin and the result of the evaluation based on the white blood cell count are inconsistent, either the result of the evaluation based on neopterin shall prevail, or another test and evaluation shall be performed. In the case of another test and evaluation, if the result of the re-evaluation based on neopterin is consistent with the result of the re-evaluation based on the white blood cell count, the consistent re-evaluation result shall be used as the final evaluation result. If the result of the re-evaluation based on neopterin is consistent with the result of the re-evaluation based on the white blood cell count, the consistent result of the evaluation based on neopterin and the white blood cell count shall prevail. If the results of the two evaluations based on neopterin and the white blood cell count are consistent, the result of the re-evaluation based on neopterin shall prevail.

[0039] Example

[0040] The present invention will be illustrated below by examples, but the protection scope of the present invention is not limited to these examples.

[0041] 1. Subjects’ conditions

[0042] In the embodiments of the present invention, the inventors recruited 59 patients from Beijing Ditan Hospital affiliated to Capital Medical University according to the 2018 Health Industry Standard of the People's Republic of China (WS273-2018), including 29 patients with latent syphilis (LS) and 30 patients with asymptomatic neurosyphilis (ANS). None of these patients were diagnosed with AIDS. Among them, the specific criteria for latent syphilis patients and asymptomatic neurosyphilis patients are as follows:

[0043] 1.1 Criteria for latent syphilis (LS)

[0044] (1) History of syphilis infection;

[0045] (2) positive serum non-treponemal test;

[0046] (3) positive serum Treponema pallidum test;

[0047] (4) No symptoms or signs of neurological damage; (5) No abnormalities were found in CSF.

[0048] 1.2 Criteria for diagnosis of asymptomatic neurosyphilis (ANS)

[0049] (1) History of syphilis infection;

[0050] (2) positive serum Treponema pallidum test;

[0051] (3) No symptoms or signs of neurological damage;

[0052] (4) CSF VDRL (TRUST) test is positive; or CSF VDRL (TRUST) test is negative, and CSF Treponema pallidum test is positive or CSF white blood cells > 5 / μl or CSF protein > 45 mg / dl.

[0053] The examples of the present invention were approved by the Ethics Committee of Beijing Ditan Hospital Affiliated to Capital Medical University, and all participants provided signed informed consent.

[0054] 2. Sample Collection

[0055] Patient CSF samples were collected as part of routine diagnostic workup. After lumbar puncture, 2 mL CSF samples were obtained. The supernatant of CSF was extracted by centrifugation at 1500 rpm for 10 minutes and stored at -80°C until analysis.

[0056] 3. Determination of CSF concentrations of neopterin and neurofilament light chain (NF-L)

[0057] The concentrations of CSF neopterin and NF-L were measured using commercial ELISA kits (IBL, Hamburg, Germany) according to the manufacturer's instructions. Optical density was measured at 450 nm using a microplate reader (Spectra Max M2, England). CSF neopterin levels were expressed in nmol / L and NF-L levels were expressed in pg / mL.

[0058] 4. Statistical analysis

[0059] Statistical analysis of all data was performed using the R program (https: / / cran.r-project.org / ), SPSS (IBM Corporation, New York, NY, USA), or GraphPad Prism7 (GraphPad Software, La Jolla, CA, USA). Data were presented as mean ± SD for normally distributed data, and as median (interquartile range) for non-normally distributed data. Student's T-Test was used to compare normally distributed data between two groups, and Mann-Whitney U test was used to compare non-normally distributed data. p values ​​≤ 0.05 were considered statistically significant.

[0060] 5. Data Processing

[0061] (1) Detection of intergroup differences

[0062] GraphPad Prism 7 software was used to perform statistical analysis of the differences in different parameters between the two groups. For normally distributed data, the data were expressed as mean ± standard deviation, while for non-normally distributed data, the data were expressed as median (interquartile range). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney Utest test was used to compare the non-normally distributed data. p value ≤ 0.05 was considered statistically significant.

[0063] (2) ROC analysis

[0064] SPSS software was used to perform ROC analysis to evaluate the diagnostic accuracy of each parameter. AUC was used to evaluate the discriminatory ability of each parameter.

[0065] 6. Results Analysis

[0066] Figures 1 to 4 The differences in expression levels of CSF neopterin, NF-L and clinical parameters white blood cell count and protein levels in latent syphilis (LS, n = 29) and asymptomatic neurosyphilis (ANS, n = 30) are shown. Each box plot includes the lower quartile (lower horizontal line), median (middle horizontal line) and upper quartile (upper horizontal line). Student's T-Test was used to compare the normally distributed data between the two groups, and Mann-Whitney U test was used to compare the non-normally distributed data. p value ≤ 0.05 was considered statistically significant.

[0067] from Figures 1 to 4 It can be seen that the expression levels of CSF neopterin and clinical parameters white blood cell count and protein level in the latent syphilis and asymptomatic neurosyphilis groups were significantly different, but the expression level of NF-L in the latent syphilis and asymptomatic neurosyphilis groups was not statistically significant.

[0068] Table 1 shows the data of receiver operating characteristic curve analysis for neopterin, NF-L, and clinical parameters white blood cell count and protein levels.

[0069]

[0070] As can be seen from Table 1, there are significant differences in the neopterin concentration and white blood cell count in CSF between latent syphilis and asymptomatic neurosyphilis (p values ​​are all less than 0.05), and the AUC of neopterin is significantly greater than 0.7. Therefore, the neopterin concentration and white blood cell count in CSF can be used to detect whether syphilis patients with no symptoms of neurological damage have asymptomatic neurosyphilis, especially to distinguish latent syphilis from asymptomatic neurosyphilis. However, the AUC of neopterin is higher than the white blood cell count in CSF. Therefore, compared with the white blood cell count, the CSF concentration of neopterin is more advantageous in distinguishing latent syphilis from asymptomatic neurosyphilis. However, the CSF NF-L concentration and protein level cannot distinguish latent syphilis from asymptomatic neurosyphilis, which is consistent with the results of clinical CSF parameter detection.

[0071] The inventors further randomly selected 41 patient sample measurement data from the above 59 patient sample measurement data as the test group, and the remaining 18 patient sample data as the verification group (the ratio of the number of samples in the test group and the verification group was 7:3). The Cut-off values ​​of neopterin and CSF protein were calculated using the 41 sample measurement data of the test group, and then the Cut-off values ​​were used to judge the remaining 18 patient sample measurement data (the random grouping operation was repeated 5 times), and the sensitivity, specificity and accuracy results are shown in Table 2 below.

[0072] Table 2. Sensitivity, specificity, and accuracy of partial factorial group validation.

[0073] factor Random number Sensitivity Specificity Accuracy Neopterin 1 0.8 0.875 0.833333 2 0.916667 0.833333 0.888889 3 0.818182 0.857143 0.833333 4 0.857143 0.818182 0.833333 5 0.846154 0.8 0.833333 CSF proteins 1 0.7 0.875 0.777778 2 0.6 0.75 0.666667 3 0.5 0.875 0.666667 4 0.428571 0.818182 0.666667 5 0.555556 0.777778 0.666667

[0074] From the data in Table 2, it can be seen that the neopterin concentration in cerebrospinal fluid can be used to detect whether syphilis patients with no symptoms of neurological damage have asymptomatic neurosyphilis, with high sensitivity, specificity and accuracy. Although the CSF protein level has a high specificity, its sensitivity is low.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of neopterin in the preparation of a product for detecting asymptomatic neurosyphilis.

2. The use according to claim 1, characterized in that: The application is the application of neopterin as a molecular marker for asymptomatic neurosyphilis.

3. The use according to claim 1, characterized in that: The product is any one of a test kit, a detection chip and a detection array.

4. The use according to claim 3, characterized in that: The kit includes a first detection reagent for detecting the concentration of neopterin in cerebrospinal fluid; the detection chip is a detection chip loaded with the first detection reagent; and the detection array is a detection array loaded with the first detection reagent.

5. The use according to claim 4, characterized in that: The kit also includes a second detection reagent for detecting the leukocyte count of cerebrospinal fluid; The detection chip is also loaded with the second detection reagent; or The detection array is also loaded with the second detection reagent.

6. An assessment system for assessing a patient's risk of developing asymptomatic neurosyphilis, characterized in that The evaluation system comprises a detection module and an evaluation module; wherein: The detection module includes a first detection module for detecting the concentration of neopterin in cerebrospinal fluid and a second detection module for detecting the white blood cell count in cerebrospinal fluid; the evaluation module includes a readable carrier storing evaluation rules; The evaluation rule is: when the neopterin concentration and / or the white blood cell count of the cerebrospinal fluid is detected to be higher than the corresponding cutoff value, the syphilis patient is evaluated as having a high risk of suffering from asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of suffering from asymptomatic neurosyphilis.

7. The evaluation system according to claim 6, characterized in that: The evaluation rule is: when the neopterin concentration in the cerebrospinal fluid is detected to be higher than the corresponding cutoff value, the syphilis patient is evaluated as having a high risk of suffering from asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of suffering from asymptomatic neurosyphilis.

8. The evaluation system according to claim 6 or 7, characterized in that: The evaluation rule is: when the neopterin concentration and the white blood cell count of the cerebrospinal fluid are detected to be higher than their respective corresponding cutoff values, the syphilis patient is evaluated as having a high risk of suffering from asymptomatic neurosyphilis; otherwise, the syphilis patient is evaluated as having a low risk of suffering from asymptomatic neurosyphilis.

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