KR20150123069A - Method for enhanced diagnosis of tuberculosis - Google Patents

Abstract

The present invention relates to an improved tuberculosis diagnosis method, which ensures a diagnosis of active pulmonary tuberculosis or both active pulmonary tuberculosis and latent tuberculosis by using blood. The present invention can provide a tuberculosis diagnosis method ensuring an early diagnosis of tuberculosis, or a diagnosis of latent tuberculosis, by stimulating tuberculosis-specific and non-specific antigens by using a small amount of blood, and then calculating a ratio of a response of an induced protein 10 (IP-10) secreted due to tuberculosis-specific stimulation to a response induced by non-specific stimulation.

Description

[0001] METHOD FOR ENHANCED DIAGNOSIS OF TUBERCULOSIS [0002]

The present invention relates to a method for providing information for diagnosis of tuberculosis, and is characterized in that active tuberculosis and latent tuberculosis can be distinguished from each other.

About one-third of the world's population, 2 billion, is infected with tuberculosis, and about 9.2 million new patients develop each year, of which about 1.7 million people die from tuberculosis. Currently, the BCG vaccine, which is a preventive vaccine against tuberculosis, has been found to be ineffective in adults, and therapeutic vaccines have not been commercialized. The World Health Organization recommended tuberculosis drugs (primary and secondary drugs) have a long-term and continuous need for 6 months of treatment, significant treatment costs, frequent side effects, poor efficacy against multidrug-resistant Mycobacterium tuberculosis, Disadvantages such as efficacy have been raised. In addition, the problem of tuberculosis being more problematic in the world is that it causes serious complications and leads to death, so early diagnosis and treatment of tuberculosis are important.

1) Mantoux tuberculin skin test (TST) has been used worldwide for diagnosis of tuberculosis. This diagnostic method is a specific reaction that injects a protein derivative (PPD) obtained by purifying tuberculin into the arm and measures the degree of the reaction after 45-72 hours. However, because of the possibility of false positives such as non-tuberculosis infection, BCG vaccine, wrong TST injection, and incorrect measurement of the response, the reliability of the diagnosis of tuberculosis infection was reduced. 2) Quantiferon, a method for quantitatively analyzing IFN-g protein secreted by T-cells stimulated by Mycobacterium tuberculosis-specific antigen, has been used as a blood test method. This method is highly used in developed countries because it shows high specificity, safe, and quick results as compared to other tests because it shows immune response specific to M. tuberculosis. However, since this method is difficult to distinguish between patients with latent tuberculosis infection and those with active pulmonary tuberculosis, early diagnosis of tuberculosis is not possible and it is urgent to develop additional biomarkers in addition to the existing test target, IFN-g.

Korean Patent Application No. 10-2008-0092934 as a prior art patent discloses a screening method of a marker for diagnosing the stage of tuberculosis and a protein antigen found thereon. Korean Patent No. 10-2012-0075886 discloses a method for providing information for diagnosing tuberculosis using IP-10 mRNA target real-time reverse transcriptase polymerization and a tuberculosis diagnostic kit therefor. However, the above-mentioned patents are different from the method of diagnosing by measuring the ratio of the amount of the protein secreted by the antigen proposed in the present invention.

It is an object of the present invention to provide a method for diagnosing tuberculosis which is capable of diagnosing active pulmonary tuberculosis and latent tuberculosis as well as active pulmonary tuberculosis using a small amount of blood.

In order to solve the above-mentioned problems, the inventors of the present invention have found that, after a small amount of blood is used to stimulate tuberculosis-specific antigen and tuberculosis nonspecific antigen, the response of IP-10 secreted by tuberculosis- And to diagnose tuberculosis that can diagnose latent tuberculosis in early diagnosis of tuberculosis by calculating the ratio of response by stimulation of tuberculosis.

As one aspect of the present invention, the present invention can provide a method for providing information for diagnosis of tuberculosis including the following steps:

(One) Collecting a blood sample of the subject;

(2) Treating the blood sample with a tuberculosis common antigen (A) and a non-specific antigen (B), respectively;

(3) Reacting the blood sample with an antigen;

(4) Measuring the value of IP-10 in the blood sample; And

(5) Obtaining a resultant value by the following Reaction Scheme 1;

 [Reaction Scheme 1]

B is the concentration of IP-10 secreted by the stimulation of the tuberculosis nonspecific antigen, C is the concentration of IP-10 secreted by the stimulation of tubercle common antigen, and C is the concentration of secreted IP- 10 < / RTI >

If the D value is greater than 0.8244, it can be considered as active tuberculosis.

If the D value is less than 0.8244, it can be considered latent tuberculosis.

As another aspect of the present invention, the present invention can provide an information providing apparatus for diagnosing tuberculosis comprising:

(One) An input unit for inputting the concentration of secreted IP-10 after treating the tubercle common antigen (A) and the tubercle nonspecific antigen (B), respectively, in the blood sample;

(2) A calculation unit for calculating a predicted value of tuberculosis using data input to the input unit; And

(3) And outputting the calculation result.

[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of tubercle nonspecific antigen, C is the concentration of IP-10 secreted without antigen stimulation Concentration value.

If the D value is greater than 0.8244, it can be considered as active tuberculosis.

If the D value is less than 0.8244, it can be considered latent tuberculosis.

As another aspect of the present invention, the present invention can provide a diagnostic kit for initial diagnosis of tuberculosis. The diagnostic kit includes IP-10 as a tuberculosis common antigen and a tuberculosis nonspecific antigen as an antigen, and a tuberculosis biomarker.

The present invention is a value obtained by dividing the amount of protein secretion by tubercle common antigen divided by the amount of secretion by nonspecific mitogen, that is, the ratio thereof. The diagnosis has improved sensitivity and specificity, and it is effective to distinguish latent tuberculosis from active tuberculosis by more than 90%.

Figure 1 is a graph showing the reactivity of IFN-g with IP-10. The graph shows the reactivity of IFN-g and IP-10 and the ratio (C) thereof after the blood was reacted with the common tubercle antigen (A) and the mitogen (B).
Figure 2 is a graph of ROC curves for comparing the efficacy of IFN-g and IP-10 as biomarkers.
Figure 3 is a graph showing the sensitivity and specificity in IFN-g and IP-10.
4 is a flowchart showing an information providing method for diagnosing tuberculosis according to the present invention.
FIG. 5 is a block diagram showing the configuration of a diagnosis device for tuberculosis according to the present invention.

Hereinafter, the present invention will be described in detail.

It is an object of the present invention to provide a method for diagnosing tuberculosis by using a small amount of blood to stimulate tuberculosis common antigen and tuberculosis nonspecific antigen and then calculating the ratio of the response of IP-10 secreted by tuberculosis-specific stimulation to the response by nonspecific stimulation , And to provide an initial diagnosis of tuberculosis, that is, a diagnosis of tuberculosis that can diagnose latent tuberculosis.

The 'active phase' in tuberculosis refers to the active proliferation of Mycobacterium tuberculosis. It is a time when a large amount of Mycobacterium tuberculosis is detected from the serum of an animal suffering from tuberculosis. The term "latent phase" or inactivity refers to a stage in which a tubercle bacillus has been infected, but its proliferation is inhibited and no tubercle bacilli are detected. In general, tubercle bacilli enter the incubation period without active period when infected with normal animals. The proliferation of Mycobacterium tuberculosis is inhibited by the treatment of host immune response or antibiotics, and depending on the immune status of the host or the degree of treatment, the incubation period can be continued for a very wide range of time. At this time, the tubercle bacilli are not detected or an insufficient amount is detected.

As used herein, the term " diagnosis " means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain whether latent tuberculosis is present.

As one aspect of the present invention, the present invention can provide a method for providing information for diagnosis of tuberculosis including the following steps:

(One) Collecting a blood sample of the subject;

(2) Treating the blood sample with a tuberculosis common antigen (A) and a tuberculosis nonspecific antigen (B), respectively;

(3) Reacting the blood sample with an antigen;

(4) Measuring the value of IP-10 in the blood sample; And

(5) Obtaining a resultant value by the following Reaction Scheme 1;

[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of tubercle nonspecific antigen, C is the concentration of IP-10 secreted without antigen stimulation Concentration value.

A method of providing information for diagnosis of tuberculosis will be described with reference to the schematic diagram of Fig. As shown in FIG. 4, blood is collected from the subject (100). The collected blood sample is divided into three samples and prepared (200). Three blood samples are treated with tuberculosis common antigen (A) and tuberculosis nonspecific antigen (B), respectively, and the other one is not treated with antigen (c). Here, the tuberculosis nonspecific antigen (B) is a positive control and the untreated antigen is a negative control. The reactions proceed in each of the three blood samples (300). The reaction is carried out at 37 DEG C for 24 hours. After the reaction, the concentration of IP-10, a diagnostic marker for tuberculosis, is measured (400). The concentration of IP-10 can be analyzed by a cytometric bead array or any assay capable of quantifying a protein such as an assay using an ELISA or Luminex. The measured values are put into Reaction 1 to perform an operation (500).

In the present specification, 'tuberculosis common antigen' means ESAT-6, CFP-10, TB7.7, Ag85A, PstS-3, Mtb32 and PPD, but is not limited thereto.

As used herein, the term " tuberculosis nonspecific antigen " means a chemical substance that non-specifically stimulates the activity of T cells. In the present invention, a tuberculosis nonspecific antigen is used to measure the degree of activation of immune cells in response to an antigen specific to tuberculosis. In particular, according to the present invention, the tuberculosis nonspecific antigen is a mitogen. The mitogens include, but are not limited to, phytohaemagglutinin (PHA), concanavalin A (conA), and pokeweed mitogen (PWM).

As an embodiment according to the present invention, the mitogen is a positive control, for example, it may contain phytohaemagglutinin (PHA). From this positive control, we can see a nonspecific immune response contrasted with tuberculosis common antigens

On the other hand, as a negative control, the concentration of IP-10 in the non-antigen treated sample can be referred to. This can improve the accuracy of the predicted value by correcting it to the negative control, since the reaction may occur even if it is not stimulated by the antigen.

'IP-10' is a small-sized chemokine expressed by an antigen presenting cell and is a major inducer of an inflammatory immune response. Secretion of IP-10 is initiated when T-cells recognize the antigen-presenting cells present on the cell surface. Since IP-10 is known to be expressed in greater amounts than IFN-γ, targets targeted for existing IFN-γ, such as children, HIV-infected individuals, and elderly people with sensitivity problems, Is a major biomarker that has recently been actively studied.

According to the present invention, it is possible to diagnose whether the active tuberculosis or the latent tuberculosis is caused by the ratio of the protein calculated by the reaction formula 1.

The D value calculated according to the reaction formula 1 was 0.8244, confirming that 90% or more of the latent tuberculosis and active pulmonary tuberculosis can be distinguished (see FIG. 3C). 0.8244 or less is considered to be latent tuberculosis, and more than one can be classified into active pulmonary tuberculosis, which has a sensitivity and specificity of 90% or more.

[Reaction Scheme 2]

Active pulmonary tuberculosis>0.8244> latent tuberculosis

According to another aspect of the present invention, there is provided an apparatus for providing information for diagnosis of tuberculosis including an input unit, an arithmetic unit, and an output unit as described below.

(One) An input unit for receiving the concentration of IP-10 secreted by the tuberculosis common antigen and the tubercle nonspecific antigen stimulated by the blood sample;

(2) A calculation unit for calculating a predicted value of tuberculosis using data input to the input unit; And

(3) An output unit for outputting the calculation result.

5, the analyzer according to the present invention will be described in detail.

The input unit may include an analyzer for collecting blood and the like from an examinee and analyzing the blood and the like, and may be connected to a diagnostic kit including a tuberculosis common antigen and a tuberculosis nonspecific antigen. Alternatively, the input unit may be an input device such as a keyboard or a mouse.

And the arithmetic operation unit performs the arithmetic operation according to the following equation (1). The operation unit may be implemented by a computer or the like.

[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of tubercle nonspecific antigen, C is the concentration of IP-10 secreted without antigen stimulation Concentration value.

If the D value is greater than 0.8244, it can be considered as active tuberculosis.

If the D value is less than 0.8244, it can be considered latent tuberculosis.

The output unit determines whether the tuberculosis is active tuberculosis or latent tuberculosis based on the value of D according to the reaction formula, and outputs the result. The output unit may be implemented as an output device such as a monitor or an LCD panel.

As another aspect of the present invention, the present invention can provide a diagnostic kit for initial diagnosis of tuberculosis. The diagnostic kit includes IP-10 as a tuberculosis common antigen and a tuberculosis nonspecific antigen as an antigen, and a tuberculosis biomarker.

The diagnostic kit can diagnose tuberculosis according to the following reaction formula (1).

[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of the tuberculosis nonspecific antigen, C is the secreted IP-10 .

If the D value is greater than 0.8244, it is characterized by active tuberculosis.

If the D value is less than 0.8244, it is a latent tuberculosis.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

Collection and collection of blood samples

The aim of this study is to establish a biomarker that can distinguish patients with tuberculosis using blood samples from patients and control subjects. The clinical samples used in this study are summarized in Table 1 below.

Blood samples used in this example were collected for three months in blood samples from the patients and control group of the clinical study of Severance Hospital. Control means 26 individuals without any bacilli, no symptoms of X-ray on TB, and no contact with TST negative or tuberculosis patients. LTBI means 20 people with TST positive who have contact with active pulmonary tuberculosis for more than a month. Active TB patients represent 33 patients diagnosed by bacterial detection and X-ray results.

[Table 1]

Detection of protein amount by antigen-antibody reaction

Specifically, 1) In the same manner as in Quantiferon, 1 ml of blood was placed in a tube containing three kinds of antigens ESAT-6, CFP-10 and TB7.7 known as tuberculosis common antigens and reacted for 24 hours. A mitogen tube was used as a positive control for this, including phytohaemagglutinin (PHA). 2) Cytometric Bead Array was prepared by taking 25 μl of the supernatant of the reactant for 24 hours. 3) The amount of various proteins specifically secreted from M. tuberculosis, the amount of nonspecifically secreted proteins due to mitogen, and the ratio thereof were calculated by the following equation (1).

Bio diagnosis for tuberculosis Marker  Test for navigation

The inventors evaluated 11 biomarkers to find other biomarkers to replace IFN-g, and the list is shown in Table 2 below.

[Table 2]

As shown in Table 2, various cytokines and chemokines were tested and interesting results were observed in IFN-g and IP-10 (interferon inducible protein-10) (FIG. 1).

In this study, the cytokine bead assay kit from BD Biosciences was used. In addition, BD can perform quantitative analysis of cytokines as well as BD. Not only bead products, but also ELISA and Luminex assays are available.

As a result, as shown in FIG. 1A, both of IFN-g and IP-10 were highly expressed in tuberculosis patients, and normal patients and tuberculosis patients, The difference between latent tuberculosis and active pulmonary tuberculosis was statistically significant (see Figure 1A). However, despite these statistically significant differences, IFN-g and IP-10 concentrations in the tuberculosis common antigen were unable to distinguish latent tuberculosis from active pulmonary tuberculosis. Interestingly, in the nonspecific reaction with the mitogen, IFN-g levels were lower in active pulmonary tuberculosis than in normal and latent tuberculosis, and IP-10 concentrations were not significantly different between the three groups (FIG. 1B). Based on the results of these new non-specific IFN-g and IP-10 observations, we found that IFN-g and IP-10 levels when stimulated with the common tubercle antigen and IFN- g and IP-10 concentrations were calculated and compared (Fig. 1C). In the case of IFN-g ratio, there was a statistically significant difference between normal and tuberculosis patient, latency tuberculosis and active pulmonary tuberculosis, similar to the result of stimulating the common tubercle, but it still did not clearly distinguish latent tuberculosis from active pulmonary tuberculosis. On the other hand, IP-10 ratio could be distinguished from normal pulmonary tuberculosis patient and active pulmonary tuberculosis.

[Comparative Example 1]

The existing diagnostic method and the diagnostic method developed in this task Biomarker  Validity comparison review

In order to compare the efficacy of two biomarkers, IFN-g and IP-10, the ROC curve was plotted to obtain FIG. When IFN-g and IP-10 concentrations were used as a biomarker, both area and p-value were statistically significant. However, IFN-g ratio and IP-10 ratio The concentration of IFN-g and IP-10 produced during the stimulation of the tuberculosis common antigen were compared with the concentrations of IFN-g produced during mitogen stimulation IP-10 concentrations were used as biomarkers to verify the biomarkers that can dramatically improve the sensitivity and specificity of the diagnosis of tuberculosis. Especially, IP-10 ratio can be used as a diagnostic biomarker with the most sensitivity and specificity.

The efficiency as a biomarker that can diagnose tuberculosis using the ratio of the cytokine responsiveness by the common biomarker of tuberculosis, which is currently being used, and the cytokine responsiveness to the common antigen stimulus and mitogen stimulation found in the present study The results of this study are summarized in Table 3. The results of this study suggest that the biomarkers developed in this study can be used not only for diagnosis of tuberculosis patients but also for patients with tuberculosis and latent tuberculosis, Marker.

[Table 3]

The cut-off value was obtained from the ROC curve, and it was confirmed how much it can distinguish between latent tuberculosis infection and active pulmonary tuberculosis (Fig. 3). In the case of IFN-g ratio, neither the sensitivity nor the specificity were found to be more than 90%. If the cut-off value was set at a point where sensitivity was 90% or more, the value was calculated as 0.04135. In this case, only 70% of latent tuberculosis could be classified and 90% of active pulmonary tuberculosis could be classified as <3A>. When the cut-off value was set at a point with a specificity of 90% or more, the value was calculated as 0.4833. At this time, latency tuberculosis was classified as 90%, whereas active tuberculosis only 27% 3B. On the other hand, for the IP-10 ratio, the cut-toff is 0.8244, which is the point where both sensitivity and specificity are more than 90% at the same time. In this case, more than 90% of latent tuberculosis could be distinguished, and more than 90% of patients with active pulmonary tuberculosis could be identified.

As a result of the calculation of the reaction formula 1, it was confirmed that 90% or more of the latent tuberculosis and the active pulmonary tuberculosis can be distinguished based on the value of 0.8244 (see FIG. 3C). 0.8244 or less is considered to be latent tuberculosis, and more than one can be classified into active pulmonary tuberculosis, which has a sensitivity and specificity of 90% or more.

[Reaction Scheme 2]

Active pulmonary tuberculosis>0.8244> latent tuberculosis

[ Comparative Example  2]

Comparing the degree of distinction between groups using the existing diagnostic method and the diagnostic method developed in this study

In order to compare the biomarkers that can diagnose tuberculosis with the ratio of existing biomarkers diagnosed with tuberculosis reactivity to mitogen reactivity developed in this study, The cut-off values were used to compare the extent to which latent tuberculosis infection can be distinguished from active pulmonary tuberculosis (see Table 4). In the case of biomarkers stimulated only with tuberculous common antigen, cut-off values that could satisfy both sensitivity and specificity could not be determined for both IFN-g and IP-10. In the case of IFN-g, the cut-off value was 90% or more, and the value was 183.1. Based on this value, 0 (zero) (40%) and active pulmonary tuberculosis (30%). When the cut-off value was defined as 90% or more, the value was 2005, 0 (0%) normal, 2 (10%) for latent tuberculosis, 10 (12.1%) could be distinguished. In the case of IP-10, when the cut-off value is set to 90% or more, the value is 7398, and 0 (0%) indicates normal value indicating positive value based on this value. (45%), and active pulmonary tuberculosis (30%). If the cut-off value is defined as 90% or more, the value is 17394, and 0 (0%) is normal and 2 (10%) of latent tuberculosis is positive. , And active pulmonary tuberculosis (69.7%). However, because there are still many overlapping parts between groups, it is impossible to distinguish them using numerical values.

[Table 4]

The ratio of IP-10 reactivity developed by the common tuberculosis antigen to the mitogen-induced tuberculosis was suggested to be used as a biomarker capable of distinguishing latent tuberculosis from active pulmonary tuberculosis at a level satisfying both sensitivity and specificity. It is considered to be a much improved biomarker than the previous method.

Claims (17)

(1) collecting a blood sample of a subject;
(2) treating the blood sample with a tuberculosis common antigen (A) and a non-specific antigen (B), respectively;
(3) reacting the blood sample with an antigen;
(4) measuring the value of IP-10 in the blood sample; And
(5) obtaining the result according to the following Reaction Scheme 1;
The method comprising the steps of:
[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of the tuberculosis nonspecific antigen, C is the secreted IP-10 .
The method according to claim 1, wherein the D-value is greater than 0.8244.
The method of claim 1, wherein the D value is less than 0.8244.
The information providing method according to claim 1, wherein the TB common antigen is at least one selected from the group consisting of ESAT-6, CFP-10, TB7.7, Ag85A, PstS-3, Mtb32 and PPD.
The method of claim 1, wherein the nonspecific antigen stimulates T cell activity.
6. The method according to claim 5, wherein the non-specific antigen is mitogen.
The method according to claim 6, wherein the mitogen is one or more selected from the group consisting of phytohemagglutinin (PHA), concanavalin A (conA), and fowwight mitogen (PWM) Delivery method.
(1) an input unit for receiving the concentration of secreted IP-10 after treating each of tuberculosis common antigen and tuberculosis nonspecific antigen in a blood sample;
(2) an operation unit for calculating a predicted value of tuberculosis using data input to the input unit; And
(3) An output unit
And an information providing device for diagnosing tuberculosis.
The information providing apparatus according to claim 8, wherein the step (2) is calculated by the following equation (1).
[Reaction Scheme 1]

B is the concentration of IP-10 secreted by the stimulation of tuberculous nonspecific antigen, C is the concentration of secreted IP-10 without antigen stimulation, A is the concentration of IP- to be.
The information providing apparatus according to claim 9, wherein if the D value is greater than 0.8244, [10] The information providing apparatus according to claim 9, wherein the D value is less than 0.8224.
9. The method according to claim 8, wherein the TB common antigen is at least one selected from the group consisting of ESAT-6, CFP-10, TB7.7, Ag85A, PstS-3, Mtb32 and PPD.
9. The method according to claim 8, wherein the non-specific antigen is mitogen.
14. The method according to claim 13, wherein the mitogen is one or more selected from the group consisting of phytohemagglutinin (PHA), concanavalin A (conA), and fowwight mitogen (PWM) Delivery method.
As a diagnostic kit for the initial diagnosis of tuberculosis, it is characterized by including tuberculosis common antigen and tuberculosis nonspecific antigen as an antigen and IP-10 as a tuberculosis biomarker,
A diagnostic kit characterized by diagnosing tuberculosis according to the following reaction formula (1).
[Reaction Scheme 1]

A is the concentration of IP-10 secreted by the stimulation of the tubercle common antigen, B is the concentration of IP-10 secreted by the stimulation of the tuberculosis nonspecific antigen, C is the secreted IP-10 .
The diagnostic kit according to claim 1, wherein the D-value is greater than 0.8244.
2. The diagnostic kit according to claim 1, wherein the D value is less than 0.8244.

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