METHOD FOR DETECTING MYCOBACTERIUM TUBERCULOSIS

Abstract

The present invention relates to a method for detecting Mycobacterium tuberculosis, which can replace the conventional culture method that takes a long time of four to eight weeks to detect active tuberculosis, and which is a method for detecting active Mycobacterium tuberculosis by using isotopes on a sample of a patient's sputum or bronchoalveolar lavage fluid.

Claims

1. A composition for detecting Mycobacterium tuberculosis in sputum or bronchoalveolar lavage fluid, comprising urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope.

2. The composition of claim 1, wherein the Mycobacterium tuberculosis is active Mycobacterium tuberculosis.

3. The composition of claim 1, wherein the composition detects a culture product for 1 to 7 days as a target.

4. A method for detecting Mycobacterium tuberculosis, comprising the steps of: (a) treating a biological sample isolated from a patient suspected of tuberculosis with NaOH or N-acetyl-L-cysteine (NALC)-NaOH; (b) culturing the sample treated with NaOH or N-acetyl-L-cysteine (NALC)-NaOH in a liquid medium for culturing Mycobacterium tuberculosis; (c) treating a culture product cultured in the liquid medium with urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope; and (d) measuring .sup.13CO.sub.2 or .sup.14CO.sub.2 of the culture product.

5. The method of claim 4, wherein the biological sample is sputum or bronchoalveolar lavage fluid.

6. The method of claim 4, wherein the pH of the culture product cultured in the liquid medium of step (c) is pH 5 to pH 8.

7. A method for providing information for diagnosing tuberculosis, comprising the steps of: (a) treating a biological sample isolated from a patient suspected of tuberculosis with NaOH or N-acetyl-L-cysteine (NALC)-NaOH; (b) culturing the sample treated with NaOH or N-acetyl-L-cysteine (NALC)-NaOH in a liquid medium for culturing Mycobacterium tuberculosis; (c) treating a culture product cultured in the liquid medium with urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope; (d) measuring .sup.13CO.sub.2 or .sup.14CO.sub.2 of the culture product; and (e) determining the patient as a tuberculosis patient if the level of .sup.13CO.sub.2 or .sup.14CO.sub.2 is higher than a normal sample.

8. The method of claim 7, wherein the biological sample is sputum or bronchoalveolar lavage fluid.

Description

DESCRIPTION OF DRAWINGS

[0012] FIG. 1 is a mimetic diagram of the Mycobacterium tuberculosis detection process of the present invention.

[0013] FIG. 2 is the results of treating sputum without Mycobacterium tuberculosis (TB-, Control) and sputum of tuberculosis patients (TB+, Test) in the same way, and measuring changes in the concentration of .sup.13CO.sub.2 with an isotope ratio mass spectrometer (Gasbench II & Thermo Fisher Delta V advantage IRMS) within 1 day.

BEST MODE

[0014] As a result of researching a method for diagnosing Mycobacterium tuberculosis on site, the inventors of the present invention completed the present invention by confirming that active Mycobacterium tuberculosis can be detected by using isotopes in a patient's sputum or bronchoalveolar lavage sample.

[0015] The present invention provides a composition for detecting Mycobacterium tuberculosis in sputum or bronchoalveolar lavage fluid, including urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope.

[0016] As used herein, the term “tuberculosis” is a disease caused by Mycobacterium tuberculosis or the like, and it is classified into latent tuberculosis and active tuberculosis according to the status of Mycobacterium tuberculosis. The term “active tuberculosis” refers to a state in which tuberculosis symptoms and contagiousness are caused due to various causes such as reduced immunity or the like. Prompt diagnosis of active tuberculosis is of utmost importance in order to suppress the transmission of tuberculosis and to treat the same at an early stage. Currently, the culture method for culturing and confirming live Mycobacterium tuberculosis from a patient's sputum sample is used as a gold standard. However, in order to detect Mycobacterium tuberculosis by the culture method, a long time of 4 to 8 weeks is required, and in the case of a breath test using the patient's exhaled breath by using .sup.13C-urea or .sup.14C-urea, since most bacteria have the urease enzyme, it is possible to diagnose other bacteria besides Mycobacterium tuberculosis, which has a disadvantage in that the accuracy of diagnosis is sharply reduced. For example, the breath test method using .sup.13C-urea or .sup.14C-urea is also used to detect infected Helicobacter pylori.

[0017] Accordingly, the inventors of the present invention researched a method for diagnosing Mycobacterium tuberculosis with high accuracy on site, and as a result of pre-treating the sputum or bronchoalveolar lavage fluid of a patient suspected of tuberculosis, and then treating the urea composed of a carbon isotope, it was confirmed that it is possible to detect Mycobacterium tuberculosis quickly within several hours or 1 week and the accuracy is increased (Table 2 and Table 3).

[0018] In an exemplary embodiment of the present invention, the Mycobacterium tuberculosis may be active Mycobacterium tuberculosis.

[0019] In an exemplary embodiment of the present invention, the composition may detect a culture product for 1 to 7 days as a target.

[0020] In addition, the present invention provides a method for detecting Mycobacterium tuberculosis, including the steps of (a) treating a biological sample isolated from a patient suspected of tuberculosis with NaOH or N-acetyl-L-cysteine (NALC)-NaOH; (b) culturing the sample treated with NaOH or N-acetyl-L-cysteine (NALC)-NaOH in a liquid medium for culturing Mycobacterium tuberculosis; (c) treating a culture product cultured in the liquid medium with urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope; and (d) measuring .sup.13CO.sub.2 or .sup.14CO.sub.2 of the culture product.

[0021] In an exemplary embodiment of the present invention, the biological sample may be sputum or bronchoalveolar lavage fluid.

[0022] In an exemplary embodiment of the present invention, the pH of the culture cultured in the liquid medium in step (c) may be pH 5 to pH 8, but the present invention is not limited thereto.

[0023] In addition, the present invention provides a method for providing information for diagnosing tuberculosis, including the steps of (a) treating a biological sample isolated from a patient suspected of tuberculosis with NaOH or N-acetyl-L-cysteine (NALC)-NaOH; (b) culturing the sample treated with NaOH or N-acetyl-L-cysteine (NALC)-NaOH in a liquid medium for culturing Mycobacterium tuberculosis; (c) treating a culture product cultured in the liquid medium with urea (.sup.13C-urea or .sup.14C-urea) composed of a carbon isotope; (d) measuring .sup.13CO.sub.2 or .sup.14CO.sub.2 of the culture product; and (e) determining the patient as a tuberculosis patient if the level of .sup.13CO.sub.2 or .sup.14CO.sub.2 is higher than a normal sample.

[0024] In an exemplary embodiment of the present invention, the biological sample may be sputum or bronchoalveolar lavage fluid.

Modes of the Invention

[0025] Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Detection of Mycobacterium tuberculosis

[0026] As with the standard method (culture method) used for the existing diagnosis of active tuberculosis, sputum or bronchoalveolar lavage from tuberculosis patients was used as diagnostic samples. The used samples were confirmed to contain Mycobacterium tuberculosis by the standard method (culture method) (Tuberc. Respir. Dis. 2015; 78: 64-71; Yon Ju Ryu, M.D.). As shown in FIG. 1, the patient's sample was collected in a sealable container. 5 to 10 mL of NaOH solution or N-acetyl-L-cysteine (NALC)-NaOH (KS Seongnam, Korea) was injected into the container, mixed and reacted at room temperature for 15 minutes. 40 to 50 mL of distilled water or buffer was injected and mixed to neutralize the solution. Through this process, all other bacteria except for Mycobacterium tuberculosis were killed, leaving only live Mycobacterium tuberculosis. For the smooth metabolic activity and culture of Mycobacterium tuberculosis, the solution was replaced with a liquid medium for tuberculosis culture. In order to prevent the loss of Mycobacterium tuberculosis in the sample, it was purified by using a filter membrane having a pore size of 1 μm or less, and recovered as 5 to 10 mL of a liquid medium for culturing Mycobacterium tuberculosis. Depending on the condition of the sample, the next detection step was performed immediately or after culturing within 1 week. Various samples were evaluated as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Classification Sample name Sample content Control group 1 control (TB − sputum) 2 control (TB − sputum) 3 control (TB − sputum) 4 control (TB − sputum) Experimental 5 TB + sputum group 6 TB + sputum 7 TB + sputum 8 TB + sputum Control group 9 control (TB − bronchoalveolar lavage fluid) 10 control (TB − bronchoalveolar lavage fluid) 11 control (TB − bronchoalveolar lavage fluid) 12 control (TB − bronchoalveolar lavage fluid) Experimental 13 TB + bronchoalveolar lavage group fluid 14 TB + bronchoalveolar lavage fluid 15 TB + bronchoalveolar lavage fluid 16 TB + bronchoalveolar lavage fluid

[0027] .sup.13C-urea was added to the samples in Table 1 and reacted within 1 hour. In contrast to the control group (sputum bronchoalveolar lavage sample which was not infected with Mycobacterium tuberculosis), the concentration of .sup.13CO.sub.2 generated in the container was evaluated by using mass spectrometry or infrared spectroscopy. If an increase in the .sup.13CO.sub.2 concentration or an increase in the .sup.13CO.sub.2/.sup.12CO.sub.2 value was confirmed, the presence of live Mycobacterium tuberculosis in the patient's sample was diagnosed as active tuberculosis. When the .sup.13CO.sub.2 concentration of the experimental group compared to the control group was measured by using the carbon dioxide carbon isotope ratio analyzer (POCone, Otsuka electronics Co.) equipment used in the existing breath test method, it was confirmed that the .sup.13CO.sub.2 level increased in all the experimental groups such that live Mycobacterium tuberculosis was detected in the samples (Table 2).

[0028] In addition, sputum without Mycobacterium tuberculosis (TB-, Control) and sputum of tuberculosis patients (TB+, Test) were treated in the same way, and changes in the concentration of .sup.13CO.sub.2 were measured by using an isotope ratio mass spectrometer (Gasbench II & Thermo Fisher Delta V advantage IRMS) within 1 day. As a result, significant increases in the .sup.13CO.sub.2 concentration were confirmed in the sputum samples of tuberculosis patients, which confirmed that Mycobacterium tuberculosis could be detected (FIG. 2). The measured values were derived as relative values in accordance with the Vienna Pee Dee Belemnite (VPDB) international standards.

[0029] This indicates that when Mycobacterium tuberculosis is detected by using the conventional culture method, it takes about 4 weeks, but the method according to the present invention takes only 1 day to 1 week.

TABLE-US-00002 TABLE 2 Name of measurement sample .sup.13CO.sub.2 change rate Sample No. 1 Sample No. 2 (Sample No. 2 (control (experimental .sup.13CO.sub.2/Sample Measurement group) group) No. 1 .sup.13CO.sub.2) results 1 2 0.0 TB − (negative) 1 5 1.7 TB + (positive) 2 6 2.0 TB + (positive) 3 7 2.2 TB + (positive) 4 8 1.0 TB + (positive) 9 13 1.9 TB + (positive) 10 14 0.9 TB + (positive) 11 15 2.4 TB + (positive) 12 16 3.9 TB + (positive)

[0030] The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains will understand that it may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the exemplary embodiments described above are illustrative in all respects and not restrictive.