Mycobacterium tuberculosis drug sensitivity kit and method and application thereof

Abstract

The invention provides a mycobacterium tuberculosis drug sensitivity kit and a method and application thereof, the drug sensitivity condition of mycobacterium tuberculosis to 12 drugs such as bedaquiline is detected based on a liquid method, the kit comprises a drug freeze-drying agent and an additive, the drug freeze-drying agent contains drugs, glucan, glycerol and valine, and the additive is a mixture of the drugs, the glucan, the glycerol and the valine. The additive is used for culturing mycobacterium tuberculosis, acetic acid is added to a non-water-soluble drug as a solvent, a cosolvent or a disintegrating agent is added to an indissolvable drug, and the prepared drug freeze-drying agent can be stored for a long time, can be redissolved only with water, and is good in redissolution stability, free of solvent residual toxicity and higher in drug stability and drug sensitivity detection accuracy. And meanwhile, the method also has better universality.

Description

Technical Field

[0001] The present invention relates to the technical field of drug susceptibility testing, and in particular, to a drug susceptibility test kit for Mycobacterium tuberculosis, its method and application. Background Art

[0002] Antibacterial drugs play a very important role in controlling diseases caused by bacteria. However, due to the continuous evolution of pathogenic bacteria and the widespread use of antibacterial drugs, especially the abuse of non-directed indications, the rapid emergence of bacterial drug resistance has caused difficulties in disease control. Through drug susceptibility testing, the types of bacterial drug resistance can be screened out, treatment errors can be reduced, it is convenient for doctors to choose individualized treatment plans, reduce the pain of patients, and save costs; it can provide a basis for disease control departments; it can use the results of drug resistance monitoring to control the abuse of antibacterial drugs, reduce the emergence of drug-resistant strains, and extend the use cycle of new drugs; it can also provide valuable information for the research and evaluation of new drugs.

[0003] Mycobacterium tuberculosis is a pathogenic bacterium that causes human tuberculosis. Tuberculosis remains a major infectious disease that seriously threatens human health. According to WHO reports, there are 10 million new cases globally every year, causing more than 1 million deaths, ranking first among the causes of death from various infectious diseases.

[0004] Traditional solid drug susceptibility media for Mycobacterium tuberculosis are prepared by mixing anti-tuberculosis drugs using Lowenstein-Jensen medium or modified Lowenstein-Jensen medium, etc. Some drugs have a loss of efficacy due to heating during the preparation of the medium, and some drugs such as PZA cannot be used for solid drug susceptibility tests because the solid medium cannot provide an effective working environment for pH value. Moreover, the solid drug susceptibility test is cumbersome and has higher requirements for biosafety. The detection time required for this method is approximately 4 to 8 weeks. In recent years, clinically, professional mycobacterium culture identification drug susceptibility systems have emerged, such as the BACTEC-960 system or the BacT / ALERT 3D system. The media in these systems contain a variety of anti-tuberculosis drugs, have high nutritional content, and fast culture speed. The disadvantages are that the equipment is expensive, and the types of drugs only exist in first-line drugs, with very few options for anti-tuberculosis drugs, and the operation is also relatively cumbersome, belonging to single culture. These two methods also have a common disadvantage, that is, if some anti-tuberculosis drugs are not stored properly, the potency of the anti-tuberculosis drugs will decrease over time, affecting the results of drug sensitivity tests and leading to incorrect test results. Most importantly, the drug concentrations of the commercially available finished test kits are not uniform, and it is impossible to provide a clinical accurate interpretation basis by observing the results of in vitro experiments.

[0005] In China, there are already some MGIT liquid drug susceptibility test kits, such as those produced by Zhuhai Baisuo and Guangdong Xige, etc. However, they usually only cover first-line drugs, and many second-line drugs have not been covered yet. Therefore, for the detection reagents of second-line drugs, only the pure drug powders of existing marketed drugs can be selected for manual preparation. However, for the existing marketed drugs, due to possible formulation reasons, the drug concentration may be unstable, resulting in low accuracy and poor repeatability of drug susceptibility test results, or there may be concentration differences due to non-standard preparation, making it impossible to compare with the results judged by the standard method recommended by the WHO.

[0006] The existing Mycobacterium tuberculosis drug susceptibility test reagents have the following problems: 1. The types of drugs are few, and the new anti-tuberculosis drugs emerged in recent years are not covered; 2. Due to possible problems such as drug solubility and solvent residue toxicity, or during the detection process, situations such as precipitation and wall adhesion of the antibiotic solution are likely to cause uneven concentration of the antibiotic solution; 3. For poorly soluble drugs, solvents such as dimethyl sulfoxide need to be added, and there is solvent residue toxicity, which affects the test results; 4. There are problems with drug stability, and it is difficult to store for a long time, which affects the detection accuracy.

[0007] Therefore, there is an urgent need to find a Mycobacterium tuberculosis drug susceptibility test kit that is more suitable for second-line drugs, which can effectively solve the problems existing in the existing kits in terms of reconstitution time, solubility, dimethyl sulfoxide residue amount, reagent storage, drug stability, detection accuracy, etc., and provide strong technical support for tuberculosis prevention and control. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides a Mycobacterium tuberculosis drug susceptibility test kit, its method and application. Based on the liquid method, it detects the drug susceptibility of Mycobacterium tuberculosis to 12 drugs such as bedaquiline. The kit includes a drug lyophilized agent and an additive. The drug lyophilized agent contains a drug, dextran, glycerol and valine. The additive is used to culture Mycobacterium tuberculosis. For water-insoluble drugs, acetic acid is added as a solvent, and for poorly soluble drugs, a solubilizer or disintegrant is added. The prepared drug lyophilized agent can be stored for a long time, and only needs to be reconstituted with water. The reconstitution stability is good, there is no solvent residue toxicity, the drug stability and the accuracy of drug susceptibility test are both higher, and at the same time, it also has better universality, providing guarantee for accurate detection.

[0009] On the one hand, the present invention provides a Mycobacterium tuberculosis drug susceptibility test kit, which includes a drug lyophilized agent and an additive; the drug lyophilized agent contains a drug, dextran, glycerol and valine; the additive is used to culture Mycobacterium tuberculosis.

[0010] The Mycobacterium tuberculosis drug susceptibility test kit provided by the present invention is a Mycobacterium tuberculosis drug susceptibility test kit based on the MGIT liquid method. The MGIT liquid method is a rapid liquid DST method based on the mycobacteria growth indicator tube (MGIT). According to the proportional method principle, the fluorescence signals of the culture tubes containing drugs and the control culture tubes are detected simultaneously, and the growth ratio of the drug-treated group to the drug-free control group is calculated to determine whether the drug effectively inhibits bacterial growth, so as to judge whether the Mycobacterium tuberculosis in the test sample is sensitive or resistant to the drug.

[0011] Since the drug efficacy will change during long-term storage, which affects the accuracy of the test results. Therefore, the drugs in the kit need to be freeze-dried. A large number of studies in the present invention have proved that adding dextran and glycerol to the drug lyophilizer can not only be used as a freeze-drying excipient with the best shaping effect, but also effectively improve the stability of the drug during long-term storage, and help improve the reconstitution stability of the drug, so that the prepared drug lyophilizer can be reconstituted only with water. After reconstitution, the drug performance is stable. Even for water-insoluble drugs, they can be stably dissolved without precipitation or wall sticking, effectively improving the test accuracy of the kit.

[0012] The freeze-drying excipient described in the present invention refers to an auxiliary agent used in the preparation of freeze-dried preparations, which can provide a stable structure and form, and maintain the activity and stability of the drug. Selecting dextran and glycerol as excipients can not only protect the drug during the preparation of freeze-dried preparations, but also improve the solubility and bioavailability of the preparations.

[0013] In some embodiments, the present invention optimizes the proportional relationship between the components of the freeze-drying excipient, and the mass ratio of dextran to glycerol is 1:3.

[0014] In some embodiments, the freeze-drying excipient further contains valine. Preparing a drug lyophilizer by combining dextran, glycerol and valine can not only make the drug have better reconstitution stability during long-term storage, but also maintain the drug activity and prevent degradation.

[0015] In some embodiments, the present invention optimizes the proportional relationship between the components of the freeze-drying excipient, and the mass ratio of dextran, glycerol and valine is 1:3:0.1.

[0016] Further, the drug includes water-soluble antibiotics and water-insoluble antibiotics; the water-soluble antibiotics include any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, linezolid; the water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid.

[0017] Further, when the drug is a water-insoluble antibiotic, acetic acid is contained in the drug lyophilized product.

[0018] In existing drug susceptibility test kits, for poorly soluble bedaquiline and clofazimine, dimethyl sulfoxide is usually used as a solvent, but the residue of dimethyl sulfoxide will interfere with the growth of bacteria.

[0019] The drug lyophilized product provided by the present invention does not need to add dimethyl sulfoxide, and only acetic acid needs to be added to the drug lyophilized product of water-insoluble antibiotics. A large number of screening experiments have proved that for water-insoluble antibiotics, adding acetic acid, dextran and glycerol to the drug lyophilized product can ensure the stable dissolution and lyophilization of water-insoluble antibiotics, and can effectively improve the reconstitution stability of water-insoluble antibiotics. It can be reconstituted only with water, the solute will not precipitate and has good stability, and there is no need to add dimethyl sulfoxide at all.

[0020] Further, when the drug is delamanid or pretomanid, a cosolvent is further contained in the drug lyophilized product; when the drug is bedaquiline or clofazimine, a disintegrant is further contained in the drug lyophilized product; the cosolvent includes Tween80; the disintegrant includes croscarmellose sodium.

[0021] In order to improve the dissolution performance of water-insoluble antibiotics, according to the characteristics of different drugs, a cosolvent or a disintegrant can be added to the drug lyophilized product, which can not only ensure the solubility, but also help to improve the reconstitution performance and help the drug to decompose and release active ingredients quickly.

[0022] Further, the additive includes any one or more of bovine serum albumin, glucose, catalase, oleic acid.

[0023] In some embodiments, the additive includes bovine serum albumin, glucose, catalase and oleic acid.

[0024] The present invention selects more suitable additives for use in combination with the drug lyophilized reagent for the culture of Mycobacterium tuberculosis. Research has proved that using this additive in combination with the drug lyophilized product for the drug susceptibility test of Mycobacterium tuberculosis is more conducive to the growth of Mycobacterium tuberculosis, can effectively inhibit the growth of miscellaneous bacteria, and ensure the accuracy of the test results.

[0025] Further, the genotype of the Mycobacterium tuberculosis includes wild-type Rv0678, any one or more of the RD239-deleted type (EAI family), the RD105-deleted type (modern Beijing strain), the rpoB B170F type, the RD9-deleted type, the RD702-deleted type, and the A63V mutant type of Rv0678.

[0026] In the existing drug susceptibility test kits, when used to detect the A63V mutant type of Rv0678 or the rpoB B170F type, false negative results are likely to occur, and there is a problem of inapplicability. The drug susceptibility test kit for Mycobacterium tuberculosis provided by the present invention has good universality and is applicable not only to wild-type but also to various existing genotypes of Mycobacterium tuberculosis, and can be used to detect Mycobacterium tuberculosis of various genotypes.

[0027] On the other hand, the present invention provides a method for preparing a drug susceptibility test kit for Mycobacterium tuberculosis, and the method includes the following steps:

[0028] (1) Prepare a solution containing a drug, add dextran, glycerol, and valine, and freeze-dry;

[0029] (2) Prepare an additive solution; the additive is used for culturing Mycobacterium tuberculosis.

[0030] In some embodiments, the method for preparing the drug susceptibility test kit for Mycobacterium tuberculosis includes the following steps:

[0031] 1. Weigh dextran and glycerol and dissolve them in purified water;

[0032] 2. Accurately weigh the antibiotic powder according to the preparation amount, and dissolve the antibiotic with purified water or acetic acid;

[0033] 3. Then add dextran, glycerol, and valine solution as a freeze-drying excipient, wherein a solubilizer is added to delamanid, and a disintegrant is added to bedaquiline and clofazimine, and then make up the volume to the preparation volume with purified water;

[0034] 4. After filtering with a 0.22 μm filter membrane, dispense into 7 ml vials for freeze-drying, and after freeze-drying, press the stoppers and caps on the dispensed reagents;

[0035] 5. At the same time, prepare a drug susceptibility additive: weigh bovine serum albumin, glucose, catalase, and oleic acid in purified water, stir to dissolve, and then make up the volume to the preparation volume with purified water;

[0036] 6. After filtering the drug susceptibility additive with a 0.22 μm filter membrane, dispense into 30 ml vials, and press the stoppers and caps.

[0037] In another aspect, the present invention provides a method for drug susceptibility testing of Mycobacterium tuberculosis. The method uses the kit as described above or the kit prepared by the method as described above for testing, and includes the following steps:

[0038] (1) Dissolve the freeze-dried drug in water to obtain a drug solution;

[0039] (2) Mix the drug solution, the sample and the additive, and culture;

[0040] (3) Read the result.

[0041] In another aspect, the present invention provides the use of a composition for preparing a drug freeze-dried preparation for maintaining the reconstitution stability of a drug. The composition includes dextran, glycerol and valine; the drug freeze-dried preparation includes a drug, dextran, glycerol and valine; the drug includes water-soluble antibiotics and water-insoluble antibiotics; the water-soluble antibiotics include any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, linezolid; the water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid; the drug only needs water for reconstitution.

[0042] In another aspect, the present invention provides the use of a composition for preparing a kit for improving the universality of drug susceptibility testing of Mycobacterium tuberculosis. The composition includes dextran, glycerol and valine; the kit includes a drug freeze-dried preparation and an additive; the drug freeze-dried preparation contains a drug, dextran, glycerol and valine; the additive is used for culturing Mycobacterium tuberculosis; the genotypes of Mycobacterium tuberculosis include wild-type Rv0678, deletion of RD239 type (EAI family), deletion of RD105 type (modern Beijing strain), rpoB B170F type, deletion of RD9 type, deletion of RD702 type, G219E type, any one or more of them.

[0043] In another aspect, the present invention provides the use of a composition for preparing a reagent for improving the shaping of a drug. The composition includes dextran, glycerol and valine; the drug includes water-soluble antibiotics and water-insoluble antibiotics; the water-soluble antibiotics include any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, linezolid; the water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid.

[0044] In another aspect, the present invention provides the use of acetic acid for preparing a reagent for improving the reconstitution stability of water-insoluble antibiotics. The water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid.

[0045] The beneficial effects of the present invention are as follows:

[0046] 1. A freeze-dried drug reagent is provided for second-line antibiotic drug detection by the MGIT liquid method, with fixed concentration, stable product, and rapid reconstitution. Only water is required for reconstituting the antibiotic, without the need for other reagents. It solves the technical problem that the toxic substances of solvent residues interfere with the growth of bacteria and affect the accuracy of experimental results during general manual preparation. At the same time, it can avoid the errors caused by the instability of antibiotics during the cumbersome preparation process of antibiotic solutions, thereby obtaining a drug sensitivity reagent with storage stability, convenient use, and excellent quality.

[0047] 2. By adding dextran, glycerol, and valine as freeze-drying excipients to the freeze-dried drug reagent, it can not only effectively improve the stability of the drug during long-term storage (stable storage for more than 36 months), but also help improve the reconstitution stability of the drug. The prepared freeze-dried drug only needs water for reconstitution, with a faster reconstitution speed, stable drug performance after reconstitution, and no precipitation or wall sticking, effectively improving the detection accuracy of the kit.

[0048] 3. For water-insoluble antibiotics, adding acetic acid, dextran, glycerol, and valine to the freeze-dried drug can ensure the stable dissolution and freeze-drying of water-insoluble antibiotics, effectively improve the reconstitution stability of water-insoluble antibiotics, and only water is required for reconstitution, with no solute precipitation and good stability, and completely no need to add dimethyl sulfoxide, avoiding toxic residues.

[0049] 4. For delamanid and pretomanid, Tween 80 is selected as a cosolvent, with a smaller required dosage, no influence on freeze-drying, and better reconstitution stability.

[0050] 5. For bedaquiline and clofazimine, cross-linked carboxymethylcellulose sodium is selected as a disintegrant, and the addition ratio is optimized, which can not only ensure solubility but also help improve the reconstitution performance and faster reconstitution speed.

[0051] 6. More suitable additives for combination with the freeze-dried drug reagent are screened, with a better culture effect on Mycobacterium tuberculosis.

[0052] 7. The prepared kit has better universality, is applicable not only to wild types but also to various existing genotypes of Mycobacterium tuberculosis, and can be used to detect Mycobacterium tuberculosis of various genotypes.

[0053] Detailed description

[0054] 1. Tuberculosis patients

[0055] The tuberculosis patient refers to a patient who has symptoms such as coughing, expectoration, low fever, night sweats, fatigue, weight loss, etc. due to infection with Mycobacterium tuberculosis and is diagnosed with tuberculosis through relevant examinations.

[0056] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis and is mainly transmitted through the air. After infection, most people are in an asymptomatic latent infection state, and the bacteria are controlled by the human immune system; but when the immunity declines, about 5-10% of latent infected people will develop into active tuberculosis, mainly manifested as persistent cough, expectoration, hemoptysis, chest pain, fever, night sweats, weight loss and fatigue, etc. Tuberculosis can affect the lungs (pulmonary tuberculosis) or other organs (extrapulmonary tuberculosis) such as the kidneys, brain, spine and skin, etc. Diagnosis depends on imaging, microbiology and immunology examinations, treatment requires long-term multi-drug combination therapy, and the problem of drug resistance is becoming increasingly serious. Preventive measures include BCG vaccination and infection control.

[0057] 2. Mycobacterium tuberculosis

[0058] Mycobacterium tuberculosis (M. tuberculosis) is simply referred to as tubercle bacilli and is the pathogen of human tuberculosis. It is a type of obligate aerobic bacteria and is positive for acid-fast staining. It has no flagella, has pili, has a microcapsule but does not form spores, and its cell wall has neither teichoic acid of Gram-positive bacteria nor lipopolysaccharide of Gram-negative bacteria. German bacteriologist Robert Koch (1843-1910) discovered and proved it to be the pathogen of human tuberculosis in 1882. Tuberculosis caused by the infection of this bacterium in humans is an infectious disease that seriously affects human life and health. After centuries of struggle with it, it has gradually been controlled, but in recent years, due to the influence of various factors, this disease has become increasingly serious.

[0059] Mycobacterium tuberculosis can undergo variations in morphology, colony, virulence, immunogenicity and drug resistance, etc. Bacillus Calmette-Guérin (BCG) is an attenuated live vaccine strain obtained by Calmette and Guérin (1908) through 230 subcultures of Mycobacterium bovis in a medium containing glycerol, bile and potatoes for 13 years and is now widely used for preventive vaccination.

[0060] Therefore, Mycobacterium tuberculosis has a variety of different genotypes, and the drug susceptibility reagents developed for it also need to have better universality and be able to adapt to a variety of different genotypes in order to truly achieve the effect of accurate detection.

[0061] 3. Mycobacterium tuberculosis drug susceptibility test

[0062] The drug susceptibility test of Mycobacterium tuberculosis is a test for selecting highly sensitive anti-Mycobacterium tuberculosis drugs. Usually, Mycobacterium tuberculosis is cultured by the bacterial culture method, and then the drug sensitivity test is carried out, and then the anti-tuberculosis drugs sensitive to Mycobacterium tuberculosis are selected.

[0063] When performing the drug susceptibility test of Mycobacterium tuberculosis, sputum or bronchoalveolar lavage fluid can be taken for the culture of Mycobacterium tuberculosis, and then the drug sensitivity of the cultured Mycobacterium tuberculosis is measured in order to accurately select sensitive anti-tuberculosis drugs.

[0064] The drug susceptibility test of Mycobacterium tuberculosis includes two parts: the bacterial culture of Mycobacterium tuberculosis and the drug susceptibility test of Mycobacterium tuberculosis. Through bacterial culture, it can be found whether the bacteria in the sputum are acid-fast bacilli, whether the acid-fast bacilli in the sputum are viable bacteria, and the identification of Mycobacterium tuberculosis species can be carried out to determine whether the bacteria in the sputum are Mycobacterium tuberculosis or non-Mycobacterium tuberculosis, while the drug susceptibility test can determine whether the Mycobacterium tuberculosis is drug-resistant.

[0065] Due to the slow growth of Mycobacterium tuberculosis, the drug sensitivity test of the bacterial culture medium on solid medium takes about 8-16 weeks. Among them, the bacterial culture of Mycobacterium tuberculosis in pulmonary tuberculosis takes 4-8 weeks, and the drug sensitivity test of Mycobacterium tuberculosis also takes 4-8 weeks.

[0066] The liquid culture provided by the present invention uses the MGIT instrument of BD Company in the United States for rapid analysis and detection. Its principle is: the bottom of the MGIT culture tube contains an oxygen-quenched fluorescent substance (such as ruthenium compound embedded in silica gel). When Mycobacterium tuberculosis grows, it will consume the oxygen in the culture medium, resulting in a decrease in the oxygen concentration in the tube, and the fluorescent substance is activated and releases a fluorescent signal. The BDMGIT instrument (such as MGIT 960) continuously detects the fluorescence intensity in real time through an optical sensor without opening the lid or sampling. Once the fluorescent signal exceeds the threshold (indicating bacterial growth), it is immediately judged as positive. Compared with the traditional solid medium (such as Löwenstein-Jensen medium which takes 4-8 weeks), MGIT can shorten the culture time to 7-14 days (average 10 days). Description of the Drawings

[0067] Figure 1 It is a photo of the prepared kit;

[0068] Figure 2 It is the reconstitution effect diagram of the 1st group and the 4th group placed for 12 months in Example 3 using different solvents. The left figure is the 1st group and the right figure is the 4th group;

[0069] Figure 3 It is a photo of the precipitation in the 2nd group and the 4th group in Example 4. Detailed Embodiments

[0070] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way. The reagents used in this embodiment are all known products and are obtained by purchasing commercially available products.

[0071] Example 1. Preparation and detection method of the Mycobacterium tuberculosis drug susceptibility test kit provided by the present invention

[0072] The liquid method Mycobacterium tuberculosis drug susceptibility test kit provided in this embodiment includes a drug lyophilized agent and an additive. The specific preparation process is as follows:

[0073] 1. Weigh 6 g of dextran, 18 g of glycerol and 0.62 g of valine, dissolve them in purified water, and prepare a 1000 mL solution A.

[0074] 2. Accurately weigh the antibiotic powder, dissolve the water-soluble antibiotic with purified water, and dissolve the water-insoluble antibiotic with acetic acid (0.3% - 0.5% glacial acetic acid plus purified water, 0.5% glacial acetic acid plus purified water is preferred in this embodiment).

[0075] 3. Then prepare the antibiotic solution with solution A. Add 0.1% (mass ratio) of Tween 80 to delamanid and pretomanid, and add 0.1% (mass ratio) of cross-linked carboxymethylcellulose sodium to bedaquiline and clofazimine. Prepare the antibiotic solution with a fixed concentration according to the recommended drug concentrations by WHO, CLSI, the Chinese Anti-Tuberculosis Association, etc. The antibiotic drugs used and the specific concentrations are shown in Table 1.

[0076] 4. After filtering and sterilizing the antibiotic solution with a 0.22 μm filter membrane, take 0.625 - 2.5 ml (2.5 ml is preferred in this embodiment) each time and dispense it into 7 ml vials for lyophilization.

[0077] 5. After the lyophilization is completed, press the stoppers and caps on the dispensed reagents in a Class 100 environment to obtain 12 groups of drug lyophilized agents.

[0078] 6. At the same time, prepare the additive: Weigh 500 g of bovine serum albumin, 200 g of glucose, 0.3 g of catalase, and 6 mL of oleic acid in purified water, stir and dissolve, and make up to 10 L.

[0079] 7. After filtering the additive with a 0.22 μm filter membrane, dispense it into 30 ml vials and press the stoppers and caps in a Class 100 environment.

[0080] Table 1. Concentrations of antibiotic drugs used

[0081]

[0082] Using the kit prepared in this embodiment (the photo is as Figure 1As shown in the figure, mycobacterium tuberculosis drug susceptibility testing is carried out, and the testing process is as follows:

[0083] 1. Reconstitute the drug lyophilized agent with pure water. Add 1 - 4 ml (preferably 4 ml in this embodiment) of pure water to each of the 12 groups of drug lyophilized agents for reconstitution to obtain 12 tubes of drug solutions. At the same time, use the same volume of pure water as the control tube.

[0084] 2. Carry out liquid culture on the 12 kinds of drug solutions and the control tube respectively. Among them, 0.1 ml of the drug solution is mixed with 0.5 ml of the sample (sputum sample, and the sample in the control tube is the sample diluted 100 times) and 0.8 ml of the additive in the culture tube, and cultured at 37 °C for 10 days by the BD MGIT 960 instrument.

[0085] 3. Predict the bacterial growth by regularly monitoring the fluorescence signal emitted from the bottom of the culture tube by the instrument, and read the GU value. Inoculate the diluted working bacterial solution (the final inoculum size is about 5×10 -2 mg) into the drug-containing culture tube. At the same time, dilute the working bacterial solution 100 times and inoculate it into the blank control culture tube (the final inoculum size is about 5×10 -4 mg). The proportion method is used for drug susceptibility testing. In fact, when using the proportion method for testing, there is no need to pre-determine the bacterial content in the sample. As long as the control tube diluted 100 times can be successfully cultured, it means that the bacteria are contained in the sample. Predict the bacterial growth by regularly monitoring the fluorescence signal emitted from the bottom of the culture tube by the instrument. When the growth unit (GU) value in the blank control culture tube reaches 400 (10 d), the instrument automatically detects the GU value in the drug-containing culture tube. If the GU value of the drug-containing tube < 100, it will be automatically judged as sensitive. If the GU value ≥ 100, it will be judged as resistant.

[0086] Among them, the calculation formula of the GU value is: GU = k * (△F / (F max - F min )), where k is the instrument calibration constant (determined by BD company through standard strains), △F = F - F0, F is the real-time detected fluorescence intensity, F0 is the initial fluorescence intensity, F max and F min are the upper and lower limits of the fluorescence signal saturation (corresponding to 999 GU and 0 GU) respectively.

[0087] Example 2. Influence of the selection of freeze-dried excipients on the drug susceptibility testing effect

[0088] In the previous research of this example, it was found that using lyophilized excipients with different compositions not only affects the lyophilized shape of the drug lyophilized product, but also affects the reconstitution stability of the drug (especially water-insoluble drugs). After comparing the reconstitution effects of different lyophilized excipients in the early stage, it was found that the combination of dextran and glycerol has the best reconstitution effect, and it can also enable water-insoluble drugs to be reconstituted with pure water only, and the reconstitution effect is better. However, after long-term storage, there is still room for improvement in its reconstitution stability and drug activity.

[0089] In this example, the Mycobacterium tuberculosis drug susceptibility test kit was prepared according to the method provided in Example 1, and the lyophilized excipients were respectively selected from 10 components shown in Table 2 (whether alone or in combination, the content or proportional relationship of each component is the same as that in Example 1), so as to obtain 10 groups of Mycobacterium tuberculosis drug susceptibility test kits, and it was also compared with the case without lyophilized excipient. The test kits were stored at room temperature for 0 months (used immediately after initial lyophilization), 6 months, 12 months, and 36 months, and the high-temperature accelerated experiment was carried out. 3 months at 45 °C is equivalent to one year, and so on. Reconstitute with pure water, and examine its reconstitution time, reconstitution stability, drug activity and drug susceptibility test results. Since among the 12 antibiotic drugs, the most difficult to dissolve is clofazimine, and the detection result trends of other several difficult-to-dissolve antibiotics are similar to that of clofazimine, so the detection results of clofazimine are used for illustration in this example. The reconstitution time refers to the time required to reconstitute the drug lyophilized product with pure water until the lyophilized product is completely dissolved; the turbidity refers to the turbidity of the reconstituted solution detected by a turbidimeter after standing for half an hour after reconstitution; the reconstitution stability refers to observing the situation of the reconstituted solution after standing for half an hour; for the drug activity detection of clofazimine, high-performance liquid chromatography is used, with a C18 reverse-phase column, the mobile phase is methanol: phosphate buffer = 80:20, and the detection wavelength is 280 nm. The test sample is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and the drug susceptibility test is carried out according to the proportional method provided in Example 1. The test results are shown in Tables 2 to 5.

[0090] Table 2. Influence of lyophilized excipient (0 months)

[0091]

[0092] Table 3. Influence of lyophilized excipient (6 months)

[0093]

[0094] Table 4. Influence of lyophilized excipient (12 months)

[0095]

[0096] Table 5. Influence of lyophilized excipient (36 months)

[0097]

[0098] As can be seen from Table 2, the originally poorly soluble clofazimine can be effectively redissolved in water after being prepared into a freeze-dried preparation. However, if no freeze-drying excipient is added, the drug stability is poor, and the freeze-dried shape is poor, uneven, so freeze-drying excipients must be added. When different freeze-drying excipients are used, their redissolution effects on clofazimine are also completely different. Compared with using only one substance such as dextran alone, the redissolution effect after combination is improved, but there are also significant differences in the redissolution effects of the freeze-drying excipients prepared with different combination components on clofazimine. Only considering the redissolution effect, the most preferred are the 5th, 9th, and 10th groups of freeze-drying excipients. Their key components are dextran and glycerol. The freeze-dried preparation prepared with this combination is the most uniform and stable when redissolved in water, with the shortest redissolution time, the lowest turbidity, the best solubility, and the best redissolution stability.

[0099] Comparing Tables 2 to 5, with the extension of the storage time, the redissolution stability changes significantly. The redissolution time increases, the turbidity also begins to rise, the redissolution stability decreases, and even suspended matter may appear. Within 12 months of storage, using dextran and glycerol as excipients is also a better choice. However, as the storage time continues to extend, the freeze-dried excipient prepared with dextran, glycerol, and valine in the 10th group has the best redissolution stability. Even after long-term storage (36 months), it can still be redissolved in water to form a uniform and stable drug solution.

[0100] At the same time, with the extension of the storage time, the drug shows obvious degradation, and the GU value of the drug sensitivity test results shows a significant increase (the higher the GU value, the lower the sensitivity of the drug sensitivity test). Even after 36 months of storage, the GU values in the drug sensitivity test results of the 1st to 4th and 11th groups exceed 100, resulting in an obvious deviation in the test results from the sensitive type to the drug-resistant type; the 6th to 8th groups are also close to 100, and the situation of test errors is about to occur. For the 10th group using the combination of dextran, glycerol, and valine as the freeze-drying excipient, the prepared antibiotic freeze-dried preparation can still maintain that clofazimine does not degrade and the drug activity does not decrease significantly after 36 months of storage. At the same time, the redissolution stability is also the best. Therefore, the 10th group is most preferably used as the freeze-drying excipient.

[0101] Example 3. Influence of the selection of non-water-soluble antibiotic solvents on the drug sensitivity test effect

[0102] In the process of preparing a drug freeze-dried preparation from a non-water-soluble antibiotic, a solvent must also be added to make it dissolve and be prepared smoothly. However, different solvents not only affect the dissolution effect of the non-water-soluble antibiotic, but also affect its redissolution effect after freeze-drying. Even after long-term storage, it also has a certain impact on the drug activity.

[0103] In this example, a drug susceptibility test kit for Mycobacterium tuberculosis was prepared according to the method provided in Example 1. For the solvents of water-insoluble antibiotics, 4 solvents shown in Table 6 were selected respectively, and 4 groups of drug susceptibility test kits for Mycobacterium tuberculosis were prepared. The kits were stored at room temperature for 0 months and 12 months respectively, and the high-temperature accelerated experiment was carried out. 3 months at 45°C is equivalent to one year, and so on. It was redissolved with pure water, and its redissolution time, redissolution stability, drug activity and drug susceptibility test results were investigated, and the detection was carried out according to the method provided in Example 2. Since among the 12 antibiotic drugs, the most difficult to dissolve is clofazimine, and the detection result trends of other several poorly soluble antibiotics are similar to those of clofazimine, the detection results of clofazimine were used for illustration in this example. The test sample was wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and the drug susceptibility test was carried out according to the proportional method provided in Example 1. The detection results are shown in Tables 6-7. The redissolution effects of the 1st group and the 4th group after being placed for 12 months are shown in Figure 2 (The left figure is the 1st group, and the right figure is the 4th group).

[0104] Table 6. Influence of water-insoluble antibiotic solvents (0 months)

[0105]

[0106] Table 7. Influence of water-insoluble antibiotic solvents (12 months)

[0107]

[0108] It can be seen from Table 6 that using different solvents to dissolve clofazimine will directly affect its redissolution effect after lyophilization. When acetic acid is used as the solvent, the effect is the best, which can significantly improve the redissolution stability of the clofazimine lyophilized agent, and the drug activity and drug susceptibility test sensitivity after redissolution are also the highest.

[0109] Combining Table 6 and Table 7, it can be seen that during long-term storage, using acetic acid as the solvent is also helpful to improve the redissolution stability, drug activity and detection sensitivity after long-term storage. Although dimethyl sulfoxide also has certain redissolution stability, it is easy to remain toxic, resulting in a decrease in the GU value, and the abnormal results may come from the inhibition of the growth of some bacteria; at the same time, the drug activity also decreases. Thus, it can be proved that the most preferred solvent is acetic acid.

[0110] Example 4. Screening of solubilizers

[0111] Since delamanid and pretomanid are very difficult to dissolve, during the process of preparing the drug lyophilized agent, in addition to adding acetic acid, solubilizers must also be added at the same time to make it dissolve and prepare smoothly. Selecting different solubilizers will also directly affect the redissolution effect of delamanid or pretomanid after lyophilization. After long-term storage, it also has a certain impact on drug activity and detection sensitivity.

[0112] In this example, a drug susceptibility test kit for Mycobacterium tuberculosis was prepared according to the method provided in Example 1. For the solubilizers for delamanid or pretomanid, since the situations are relatively similar, delamanid was selected for illustration. Four solubilizers shown in Table 8 were respectively selected, and the dosages were the same as those in Example 1, so as to prepare 4 groups of drug susceptibility test kits for Mycobacterium tuberculosis, and they were compared with a control group without solubilizer. The kits were stored at room temperature for 0 months and 12 months respectively, and high-temperature accelerated experiments were carried out. Three months at 45 °C is equivalent to one year, and so on. They were reconstituted with pure water, and their reconstitution time, reconstitution stability, drug activity and drug susceptibility test results were investigated, and the detection was carried out according to the method provided in Example 2. For the drug activity detection of delamanid, high-performance liquid chromatography was used, with a C18 reversed-phase column, mobile phase A being 0.1% formic acid aqueous solution, mobile phase B being acetonitrile, and the detection wavelength being 290 nm. The test sample was wild-type Mycobacterium tuberculosis (sensitive to delamanid), and the drug susceptibility test was carried out according to the proportional method provided in Example 1. The test results are shown in Tables 8 to 9. Figure 3 Photos showing the precipitation in the 2nd and 4th groups.

[0113] Table 8. Influence of delamanid solubilizers (0 months)

[0114]

[0115] Table 9. Influence of delamanid solubilizers (12 months)

[0116]

[0117] It can be seen from Table 8 that dissolving delamanid with different solubilizers will directly affect the reconstitution effect of delamanid after freeze-drying. Compared with several other solubilizers, using Tween 80 as the solubilizer has the best effect, can significantly improve the reconstitution stability of delamanid freeze-dried preparation, and the drug activity and drug susceptibility test sensitivity after reconstitution are also the highest.

[0118] Combining Tables 8 and 9, it can be seen that during long-term storage, using Tween 80 as the solubilizer for delamanid also helps to improve the reconstitution stability, drug activity and detection sensitivity after long-term storage (the lower the GU value, the more sensitive). Therefore, the most preferred solubilizer for delamanid is Tween 80.

[0119] Example 5. Screening of disintegrants for bedaquiline and clofazimine

[0120] Bedaquiline and clofazimine are extremely insoluble. In the process of preparing drug lyophilized agents, in addition to adding acetic acid, disintegrants must also be added at the same time to enable their smooth dissolution and preparation. The selection of different disintegrants will also directly affect the re-dissolution effect of bedaquiline and clofazimine after lyophilization, and after long-term storage, it will also have a certain impact on drug activity and detection sensitivity.

[0121] The present embodiment prepares a Mycobacterium tuberculosis drug sensitivity test kit according to the method provided in Example 1, wherein the disintegrants for bedaquinoline and clofazimine are respectively selected as shown in Table 10, and the dosage is consistent with Example 1, thereby preparing 3 groups of Mycobacterium tuberculosis drug sensitivity test kits, and compared with the case where no or cosolvent is added. The kit is stored at room temperature for 0 months and 12 months, respectively, and a high temperature accelerated experiment is performed, and 45°C for 3 months is equivalent to one year, and so on. Redissolve with pure water, investigate its resolubility time, resolubility stability, drug activity and drug sensitivity test results, and detect according to the method provided in Example 2. Since clofazimine is more difficult to dissolve, and the trend of the test results of bedaquinoline is similar to clofazimine, this embodiment is illustrated by the test results of clofazimine, and the sample to be tested is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and drug sensitivity detection is performed according to the ratio method provided in Example 1, and the test results are shown in Tables 10 to 11.

[0122] Table 10. Effect of disintegrant (0 month)

[0123]

[0124] Table 11. Effect of disintegrant (12 months)

[0125]

[0126] It can be seen from Table 10 that for the poorly soluble antibiotic clofazimine, even if the cosolvent can be barely prepared, the re-dissolution effect is very poor and it is difficult to achieve re-dissolution in pure water. Therefore, a disintegrant must be added to dissolve it in order to improve the re-dissolution stability. At the same time, if a cosolvent and a disintegrant are added at the same time, the drug stability decreases, which is not conducive to long-term storage. Therefore, it is most preferred to directly add a disintegrant.

[0127] Using different disintegrants to dissolve clofazimine will directly affect the reconstitution effect of clofazimine after freeze-drying. Compared with several other disintegrants, the effect is best when cross-linked sodium carboxymethyl cellulose is used as a disintegrant, which can significantly improve the reconstitution stability of clofazimine freeze-dried preparation, and the drug activity and sensitivity of drug sensitivity detection after reconstitution are also the highest.

[0128] As can be seen from Table 10 and Table 11, during long-term storage, using sodium carboxymethylcellulose cross-linked as the disintegrant for clofazimine also helps to improve the redissolution stability, drug activity, and detection sensitivity after long-term storage (the lower the GU value, the more sensitive). Therefore, the most preferred disintegrant is sodium carboxymethylcellulose cross-linked.

[0129] Similarly, this example has also been experimentally proven that the most preferred disintegrant for bedaquiline is also sodium carboxymethylcellulose cross-linked. The detailed experimental data are omitted.

[0130] In addition, during the confirmation of the proportion of the disintegrant added to bedaquiline and clofazimine, in this example, 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% of sodium carboxymethylcellulose cross-linked were added to the lyophilized preparations of the two drugs respectively, and it was found that adding 0.1% of sodium carboxymethylcellulose cross-linked could achieve the optimal effect.

[0131] Example 6. Universal comparison

[0132] Existing drug susceptibility test kits for Mycobacterium tuberculosis sometimes have the problem of low universality, while the present invention can better solve this problem and effectively improve the universality.

[0133] In this example, the drug susceptibility test kit for Mycobacterium tuberculosis prepared in Example 1 was used to conduct a comparative analysis of universality with several existing drug susceptibility test kits for Mycobacterium tuberculosis, as well as test kits using different additives, lyophilized excipients, or solvents. Since the main difference lies in the A63V-type mutant bacteria of the Rv0678 gene, the A63V-type mutant bacteria are bedaquiline-resistant bacteria, but due to the problem of low universality of the existing drug susceptibility test kits, it is easy to cause the A63V-type mutant bacteria to also be detected as drug-susceptible, and inconsistent test results are likely to occur after multiple repeated tests, and the test accuracy is not high. Therefore, this example mainly lists the test results for this genotype of mutant bacteria. Since the main difference lies in bedaquiline, in this example, a universality study was conducted on the drug susceptibility test results of the A63V-type mutant bacteria to bedaquiline. The prepared test kit was used immediately without long-term storage. The accuracy of the drug susceptibility test results was mainly investigated. 20 repeated experiments were carried out for each group, the accuracy ratio was calculated, and it was compared with the detection accuracy of the wild-type Rv0678 to analyze the universality. The detection was carried out according to the method provided in Example 2. The test sample was a sample solution containing A63V-type mutant Mycobacterium tuberculosis, and the drug susceptibility test was carried out according to the proportion method provided in Example 1. The test results are shown in Table 12.

[0134] Table 12. Comparison of universal detection results

[0135]

[0136] As can be seen from Table 12, for the Mycobacterium tuberculosis drug susceptibility test kits prepared with different formulations, there are differences in their universality among different genotypes. For the kit prepared in Example 1, the repeated detection accuracy can reach 100% in both the wild-type Rv0678 and the A63V mutant type, showing better universality.

[0137] In this example, the kit prepared in Example 1 was also used to verify the detection universality for genotypes such as the RD239 deletion type (EAI family), the RD105 deletion type (modern Beijing strain), the rpoB B170F type, the RD9 deletion type, and the RD702 deletion type. It was found that the universality for each genotype is very good, with an accuracy of 100%, which is significantly better than the existing Mycobacterium tuberculosis drug susceptibility test kits.

[0138] All patents and publications mentioned in the specification of the present invention indicate that these are publicly known technologies in the field and can be used in the present invention. All patents and publications cited herein are equally listed in the references, just as each publication is specifically individually referenced. The present invention described herein can be implemented in the absence of any one or more elements, one or more limitations, where such limitations are not specifically stated. For example, in each example herein, the terms "comprising", "consisting essentially of", and "consisting of" can be replaced by the other two of the three terms. The so-called "a" herein only means "one", and does not exclude including only one, nor does it exclude including more than two. The terms and expressions used herein are for descriptive purposes and are not limiting, and there is no intention to indicate that the terms and interpretations described herein exclude any equivalent features, but it can be understood that any suitable changes or modifications can be made within the scope of the present invention and the claims. It can be understood that the embodiments described in the present invention are all preferred embodiments and features, and any person of ordinary skill in the art can make some changes and variations based on the essence described in the present invention, and these changes and variations are also considered to be within the scope of the present invention and the scope limited by the independent claims and the dependent claims.

Claims

1. A Mycobacterium tuberculosis drug susceptibility test kit, characterized in that, It includes a freeze-dried drug and an additive; the freeze-dried drug contains a drug, dextran, glycerol and valine; the additive is used for culturing Mycobacterium tuberculosis.

2. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 1, wherein The drug includes water-soluble antibiotics and water-insoluble antibiotics; the water-soluble antibiotics include any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, linezolid; the water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid.

3. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 2, characterized in that, When the drug is a water-insoluble antibiotic, the freeze-dried drug contains acetic acid.

4. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 3, characterized in that When the drug is delamanid or pretomanid, the freeze-dried drug also contains a cosolvent; when the drug is bedaquiline or clofazimine, the freeze-dried drug also contains a disintegrant; the cosolvent includes Tween 80; the disintegrant includes croscarmellose sodium.

5. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 1, wherein The additive includes any one or more of bovine serum albumin, glucose, catalase, oleic acid.

6. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 1, wherein, The genotype of the Mycobacterium tuberculosis includes any one or more of wild-type Rv0678, ΔRD239 type, ΔRD105 type, rpoB B170F type, ΔRD9 type, ΔRD702 type, A63V mutant type of Rv0678.

7. A method for preparing a drug susceptibility test kit for Mycobacterium tuberculosis, characterized in that, It includes the following steps: (1) Prepare a solution containing the drug, add dextran, glycerol and valine, and freeze-dry. (2) Prepare an additive solution; the additive is used for culturing Mycobacterium tuberculosis.

8. A drug susceptibility testing method for Mycobacterium tuberculosis, characterized in that, Using the kit described in any one of claims 1 to 6 or the kit prepared by the method described in claim 7 for testing, it includes the following steps: (1) Dissolve the freeze-dried drug in water to obtain a drug solution. (2) Mix the drug solution, the sample and the additive, and culture. (3) Read the result.

9. Use of a composition for preparing a freeze-dried pharmaceutical preparation for maintaining the reconstitution stability of a drug, characterized in that, The composition includes dextran, glycerol and valine; the freeze-dried drug includes a drug, dextran, glycerol and valine; the drug includes water-soluble antibiotics and water-insoluble antibiotics; the water-soluble antibiotics include any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, linezolid; the water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, pretomanid; the drug only needs to be reconstituted with water.

10. Use of a composition for preparing a kit for improving the universality of drug susceptibility testing of Mycobacterium tuberculosis, characterized in that, The composition includes dextran, glycerol and valine; the kit includes a freeze-dried drug and an additive; the freeze-dried drug contains a drug, dextran, glycerol and valine; the additive is used for culturing Mycobacterium tuberculosis; the universality means that the kit is applicable to different genotypes of Mycobacterium tuberculosis; the different genotypes of Mycobacterium tuberculosis include any one or more of wild-type Rv0678, ΔRD239 type, ΔRD105 type, rpoB B170F type, ΔRD9 type, ΔRD702 type, A63V mutant type of Rv0678.

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