A Mycobacterium tuberculosis drug sensitivity test kit and its method and application

Abstract

The present invention provides a Mycobacterium tuberculosis drug susceptibility kit, a method, and an application thereof. The kit detects the drug susceptibility of Mycobacterium tuberculosis to 12 drugs, such as bedaquiline, based on a liquid method. The kit comprises 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. Acetic acid is added as a solvent for water-insoluble drugs, and a cosolvent or disintegrant is added for poorly soluble drugs. The prepared drug lyophilized agent can be stored for a long time and can be re-dissolved with water only. The re-dissolution stability is good, there is no residual solvent toxicity, the drug stability and drug susceptibility detection accuracy are higher, and the kit also has better universality.

Description

Technical Field

[0001] The present invention relates to the technical field of drug sensitivity detection, and in particular to a Mycobacterium tuberculosis drug sensitivity kit and a method and application thereof. Background Art

[0002] Antimicrobial drugs play a vital role in controlling bacterial diseases. However, the constant evolution of pathogens and the widespread use of antimicrobial drugs, especially their misuse for non-targeted indications, have led to the rapid development of bacterial resistance, making disease control difficult. Susceptibility testing can screen for resistant bacteria, reduce treatment errors, and facilitate personalized treatment plans for doctors, alleviating patient suffering and saving costs. It can also provide a basis for prevention and treatment for disease control departments. Resistance monitoring results can be used to control the misuse of antimicrobial drugs, reduce the emergence of resistant strains, and extend the lifespan of new drugs. It can also provide valuable information for the research and evaluation of new drugs.

[0003] Mycobacterium tuberculosis is the pathogenic bacteria that causes human tuberculosis. Tuberculosis is still a major infectious disease that seriously threatens human health. According to the WHO, there are 10 million new cases worldwide each year, causing more than one million deaths, ranking first among all infectious diseases.

[0004] Traditional solid-state drug susceptibility culture media for Mycobacterium tuberculosis (M. tuberculosis) are prepared by mixing anti-TB drugs using Roche medium or modified Roche medium. Some drugs lose potency due to the heating required during the culture medium preparation process. Some drugs, such as PZA, cannot be tested for solid-state drug susceptibility testing because solid-state culture media lack an effective pH environment. Furthermore, solid-state drug susceptibility testing is cumbersome and requires higher biosafety standards. Testing using this method typically takes four to eight weeks. In recent years, specialized systems for mycobacterial culture and drug susceptibility testing, such as the BACTEC-960 system or the BacT / ALERT 3D system, have emerged in clinical practice. These culture media contain multiple anti-TB drugs, are highly nutritious, and offer rapid culture speeds. However, their disadvantages are the high cost of the equipment, the limited selection of first-line drugs, and the relatively cumbersome operation required for single-stage culture. Both methods also share a common drawback: improper storage can lead to a decrease in the potency of some anti-TB drugs over time, potentially affecting drug susceptibility testing results and causing erroneous results. Most importantly, the drug concentrations in the finished test kits currently on the market are not uniform, and it is impossible to provide accurate clinical judgment basis by observing the results of in vitro experiments.

[0005] Several MGIT liquid drug susceptibility test kits are currently available in my country, such as those produced by Zhuhai Beisuo and Guangdong Xige. However, these kits typically only cover first-line drugs, leaving many second-line drugs untouched. Therefore, testing reagents for second-line drugs currently rely on manual preparation of pure powders of existing marketed drugs. However, the formulation of these marketed drugs can lead to unstable drug concentrations, resulting in low accuracy and poor reproducibility in drug susceptibility test results. Alternatively, improper preparation can result in concentration variations, making comparison with the WHO-recommended standard method impossible.

[0006] Existing Mycobacterium tuberculosis drug sensitivity reagents have the following problems: 1. There are few types of drugs, and new anti-tuberculosis drugs that have appeared in recent years are not covered; 2. The concentration of the antibiotic solution may be uneven due to problems such as drug solubility, residual toxicity of solvents, or precipitation or adhesion of the antibiotic solution to the wall during the detection process; 3. For poorly soluble drugs, solvents such as dimethyl sulfoxide need to be added, which has residual toxicity of the solvent and affects the test results; 4. There are problems with drug stability, which makes it difficult to store for a long time, affecting the accuracy of the test.

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

[0008] In response to the problems existing in the prior art, the present invention provides a Mycobacterium tuberculosis drug sensitivity kit, a method and an application thereof, which detects the drug sensitivity of Mycobacterium tuberculosis to 12 drugs such as bedaquiline based on a liquid method. The kit includes a drug lyophilized agent and an additive, wherein the drug lyophilized agent contains drugs, dextran, glycerol and valine, the additive is used to culture Mycobacterium tuberculosis, acetic acid is added as a solvent for water-insoluble drugs, and a cosolvent or disintegrant is added for poorly soluble drugs. The prepared drug lyophilized agent can be stored for a long time and can be re-dissolved with water only. It has good re-dissolution stability and no solvent residual toxicity. The drug stability and drug sensitivity detection accuracy are higher, and it also has better universality, providing a guarantee for accurate detection.

[0009] In one aspect, the present invention provides a Mycobacterium tuberculosis drug sensitivity test kit, comprising a drug lyophilized agent and an additive; the drug lyophilized agent contains a drug, dextran, glycerol and valine; and the additive is used for culturing 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 Mycobacterium tuberculosis growth indicator tube (MGIT). Based on the principle of proportionality, the fluorescence signals of the culture tube containing the drug and the control culture tube are simultaneously detected. 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 and thus determine whether the Mycobacterium tuberculosis in the test sample is sensitive or resistant to the drug.

[0011] Since the efficacy of drugs changes during long-term storage, thus affecting the accuracy of test results, the drugs in the test kit need to be lyophilized. The present invention has been proven through extensive research that the addition of dextran and glycerol to the drug lyophilized agent can not only serve as lyophilization excipients, achieving the best excipient effect, but also effectively improve the stability of the drug during long-term storage and contribute to improving the drug's re-dissolution stability. The prepared drug lyophilized agent only requires water for re-dissolution, and the drug performance after re-dissolution is stable. Even water-insoluble drugs can be stably dissolved without precipitation or wall adhesion, effectively improving the detection accuracy of the test kit.

[0012] The lyophilization excipients described herein are auxiliary agents used in the preparation of lyophilized formulations to provide a stable structure and morphology, maintaining the activity and stability of the drug. Dextran and glycerol are selected as excipients during the preparation of lyophilized formulations to not only protect the drug but also improve the solubility and bioavailability of the formulation.

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

[0014] In some embodiments, the lyophilization excipient further contains valine. The use of a combination of dextran, glycerol, and valine to prepare a drug lyophilized preparation not only improves the reconstitution stability of the drug during long-term storage, but also maintains the drug activity and prevents degradation.

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

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

[0017] Furthermore, when the drug is a water-insoluble antibiotic, the drug lyophilized agent contains acetic acid.

[0018] In existing drug sensitivity test kits, dimethyl sulfoxide is usually required as a solvent for poorly soluble bedaquiline and clofazimine, but dimethyl sulfoxide residues will interfere with bacterial growth.

[0019] The lyophilized pharmaceutical preparation provided by the present invention does not require the addition of dimethyl sulfoxide (DMSO). Instead, acetic acid is simply added to the lyophilized pharmaceutical preparation of water-insoluble antibiotics. Extensive screening experiments have demonstrated that the addition of acetic acid, dextran, and glycerol to the lyophilized pharmaceutical preparation ensures stable dissolution and lyophilization of water-insoluble antibiotics and effectively improves their reconstitution stability. Reconstitution is achieved with water alone, preventing solute precipitation and maintaining excellent stability, without the need for the addition of DMSO.

[0020] Furthermore, when the drug is delamanid or premanid, the drug lyophilized agent also contains a solubilizing agent; when the drug is bedaquiline or clofazimine, the drug lyophilized agent also contains a disintegrant; the solubilizing agent includes Tween 80; and the disintegrant includes cross-linked sodium carboxymethyl cellulose.

[0021] In order to improve the solubility of non-water-soluble antibiotics, cosolvents or disintegrants can be added to the drug lyophilizer according to the characteristics of different drugs. This can not only ensure solubility, but also help improve re-dissolution performance and help the drug quickly decompose and release active ingredients.

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

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

[0024] The present invention selects an additive that is more suitable for use in combination with a lyophilized drug reagent for the cultivation of Mycobacterium tuberculosis. Studies have shown that using this additive in combination with a lyophilized drug reagent for drug susceptibility testing of Mycobacterium tuberculosis is more conducive to the growth of Mycobacterium tuberculosis and can effectively inhibit the growth of other bacteria, ensuring the accuracy of test results.

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

[0026] Existing drug susceptibility test kits are prone to missing detections when used to detect the A63V mutant of Rv0678 or the rpoB B170F type, making them inapplicable. The Mycobacterium tuberculosis drug susceptibility test kit provided by the present invention has good universal applicability and is applicable not only to the wild type but also to various other existing Mycobacterium tuberculosis genotypes, and can be used to detect various Mycobacterium tuberculosis genotypes.

[0027] In another aspect, the present invention provides a method for preparing a Mycobacterium tuberculosis drug sensitivity kit, comprising the following steps:

[0028] (1) Prepare a solution containing the drug, add dextran, glycerol, and valine, and lyophilize;

[0029] (2) preparing an additive solution; the additive is used to culture Mycobacterium tuberculosis.

[0030] In some embodiments, the method for preparing the Mycobacterium tuberculosis drug susceptibility test kit comprises 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. Add dextran, glycerol, and valine solution as lyophilization excipients, add a cosolvent to delamanid, and add a disintegrant to bedaquiline and clofazimine, and then dilute to the prepared volume with purified water;

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

[0035] 5. At the same time, prepare the drug-sensitive additive: weigh bovine serum albumin, glucose, catalase, and oleic acid in purified water, stir to dissolve, and then dilute to the prepared volume with purified water;

[0036] 6. Filter the drug-sensitive additive using a 0.22μm filter membrane, dispense into 30ml vials, and seal with stoppers and caps.

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

[0038] (1) Dissolving the lyophilized drug in water to obtain a drug solution;

[0039] (2) Mix the drug solution, sample, and additives and incubate;

[0040] (3) Read the results.

[0041] In another aspect, the present invention provides a composition for preparing a drug lyophilized agent that maintains the reconstitution stability of the drug, the composition comprising dextran, glycerol and valine; the drug lyophilized agent comprising a drug, dextran, glycerol and valine; the drug comprising a water-soluble antibiotic and a water-insoluble antibiotic; the water-soluble antibiotic comprising any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, and linezolid; the water-insoluble antibiotic comprising any one or more of prothionamide, bedaquiline, delamanid, clofazimine, and premanid; and the drug requiring only water for reconstitution.

[0042] In another aspect, the present invention provides a composition for preparing a kit for improving the universality of drug sensitivity detection of Mycobacterium tuberculosis, wherein the composition includes dextran, glycerol and valine; 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; the genotype of the Mycobacterium tuberculosis includes any one or more of wild type Rv0678, RD239 deletion type (EAI family), RD105 deletion type (modern Beijing strain), rpoB B170F type, RD9 deletion type, RD702 deletion type, and G219E type.

[0043] In another aspect, the present invention provides a composition for preparing an agent for improving the shape of a drug excipient, the composition comprising dextran, glycerol and valine; the drug comprising a water-soluble antibiotic and a water-insoluble antibiotic; the water-soluble antibiotic comprising any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin, and linezolid; the water-insoluble antibiotic comprising any one or more of prothionamide, bedaquiline, delamanid, clofazimine, and premanid.

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

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

[0046] 1. The invention provides a drug lyophilized reagent with a fixed concentration, stable product, and rapid reconstitution when used in the MGIT liquid method for second-line antibiotic drug testing. Only water is required for reconstitution of the antibiotic, and no other reagents are required. This solves the technical problem of residual toxic substances in the solvent during the general manual preparation method interfering with bacterial growth and affecting the accuracy of experimental results. It also avoids errors caused by antibiotic instability during the tedious preparation process of the antibiotic solution, thereby obtaining a drug sensitivity reagent that is storage-stable, easy to use, and of excellent quality.

[0047] 2. By adding dextran, glycerol and valine as lyophilization excipients to the drug lyophilization reagent, it can not only effectively improve the stability of the drug during long-term storage (can be stably stored for more than 36 months), but also help improve the re-dissolution stability of the drug. The prepared drug lyophilized agent only needs water to be re-dissolved, and the re-dissolution speed is faster. After re-dissolution, the drug performance is stable and no precipitation or wall adhesion will occur, which effectively improves the detection accuracy of the test kit.

[0048] 3. For water-insoluble antibiotics, adding acetic acid, dextran, glycerol and valine to the drug lyophilizer can ensure the stable dissolution and lyophilization of the water-insoluble antibiotics, and can effectively improve the re-dissolution stability of the water-insoluble antibiotics. Only water is needed for re-dissolution, the solute will not precipitate and the stability is good. There is no need to add dimethyl sulfoxide to avoid toxic residues.

[0049] 4. For Delamanid and Primanid, Tween 80 is added as a co-solvent. The required dosage is smaller, does not affect freeze-drying, and the reconstitution stability is also better.

[0050] 5. For bedaquiline and clofazimine, cross-linked sodium carboxymethyl cellulose was added as a disintegrant, and the addition ratio was optimized, which not only ensured solubility but also helped improve the re-dissolution performance and the re-dissolution speed.

[0051] 6. We screened additives that are more suitable for use in combination with drug freeze-dried reagents, which have better effects on the cultivation of Mycobacterium tuberculosis.

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

[0053] Detailed description

[0054] 1. Tuberculosis patients

[0055] The tuberculosis patients refer to patients who have symptoms such as cough, sputum, low fever, night sweats, fatigue, and weight loss due to infection with Mycobacterium tuberculosis and who have been diagnosed with tuberculosis through relevant examinations.

[0056] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, primarily transmitted through the air. After infection, most people remain in a latent state, where the bacteria are controlled by the immune system. However, when immunity declines, approximately 5-10% of latently infected individuals will develop active TB, characterized by persistent cough, sputum production, hemoptysis, chest pain, fever, night sweats, weight loss, and fatigue. TB can affect the lungs (pulmonary TB) or other organs (extrapulmonary TB) such as the kidneys, brain, spine, and skin. Diagnosis relies on imaging, microbiological, and immunological tests. Treatment requires long-term multidrug therapy, and drug resistance is an increasing problem. Preventive measures include BCG vaccination and infection control.

[0057] 2. Mycobacterium tuberculosis

[0058] Mycobacterium tuberculosis (M. tuberculosis), also known as tubercle bacilli, is the causative agent of human tuberculosis. It is an obligately aerobic bacterium that stains positively for acid fast. It lacks flagella but possesses pili and a microcapsule, but does not form spores. Its cell wall lacks either the teichoic acid of Gram-positive bacteria or the lipopolysaccharide of Gram-negative bacteria. German bacteriologist Robert Koch (1843-1910) discovered and confirmed it as the causative agent of human tuberculosis in 1882. Tuberculosis, caused by infection with this bacterium, is a serious infectious disease that threatens human life and health. After centuries of struggle, it has been gradually brought under control. However, in recent years, the disease has become increasingly severe due to a variety of factors.

[0059] Mycobacterium tuberculosis can undergo variations in morphology, colonization, virulence, immunogenicity, and drug resistance. Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine strain derived by Calmette and Guerin (1908) by passaged bovine tuberculosis 230 times over 13 years in a culture medium containing glycerol, bile, and potatoes. It is now widely used for preventive vaccination.

[0060] Therefore, Mycobacterium tuberculosis has many different genotypes, and the drug sensitivity 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. Drug susceptibility test for Mycobacterium tuberculosis

[0062] The Mycobacterium tuberculosis drug susceptibility test is a test for selecting highly sensitive anti-Mycobacterium tuberculosis drugs. It is usually carried out by culturing Mycobacterium tuberculosis through bacterial culture methods, conducting drug sensitivity tests, and then selecting anti-tuberculosis drugs that are sensitive to Mycobacterium tuberculosis.

[0063] When conducting a drug sensitivity test for Mycobacterium tuberculosis, sputum or alveolar lavage fluid can be taken for culture of Mycobacterium tuberculosis. The cultured Mycobacterium tuberculosis can then be tested for drug sensitivity in order to accurately select sensitive anti-tuberculosis drugs.

[0064] The drug sensitivity test for Mycobacterium tuberculosis includes two parts: bacterial culture of Mycobacterium tuberculosis and drug sensitivity 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 identification of the Mycobacterium tuberculosis species can be performed to determine whether the bacteria in the sputum are Mycobacterium tuberculosis or non-tuberculosis mycobacteria, while the drug sensitivity test can determine whether the Mycobacterium tuberculosis is drug-resistant.

[0065] Because Mycobacterium tuberculosis grows slowly, the drug sensitivity test of solid culture medium bacterial culture takes about 8 to 16 weeks, of which the bacterial culture of pulmonary Mycobacterium tuberculosis takes 4 to 8 weeks, and the drug sensitivity test of Mycobacterium tuberculosis also takes 4 to 8 weeks.

[0066] The liquid culture method provided by this invention utilizes the BD MGIT instrument for rapid analysis and detection. The principle is that the bottom of the MGIT culture tube contains an oxygen-quenching fluorescent substance (such as a silica-gel-encapsulated ruthenium compound). As Mycobacterium tuberculosis grows, it consumes oxygen from the culture medium, causing the oxygen concentration within the tube to drop. This activates the fluorescent substance and releases a fluorescent signal. The BD MGIT instrument (such as the MGIT 960) uses an optical sensor to continuously monitor fluorescence intensity in real time, without the need to open the lid or take samples. Once the fluorescence signal exceeds a threshold (indicating bacterial growth), a positive result is immediately determined. Compared to traditional solid culture media (such as Roche medium, which requires 4-8 weeks), MGIT can shorten the culture time to 7-14 days (average 10 days). BRIEF DESCRIPTION OF THE DRAWINGS

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

[0068] Figure 2 The diagram shows the reconstitution effects of Group 1 and Group 4 after 12 months of storage using different solvents in Example 3, with the left diagram showing Group 1 and the right diagram showing Group 4.

[0069] Figure 3 These are photos showing the precipitation in Group 2 and Group 4 in Example 4. DETAILED DESCRIPTION

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

[0071] Example 1: Preparation and Detection Method of the Mycobacterium tuberculosis Drug Sensitivity Kit Provided by the Present Invention

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

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

[0074] 2. Accurately weigh antibiotic powder, dissolve water-soluble antibiotics with purified water, and dissolve water-insoluble antibiotics with acetic acid (0.3%-0.5% glacial acetic acid in pure water, preferably 0.5% glacial acetic acid in pure water in this embodiment);

[0075] 3. Solution A was then used to prepare an antibiotic solution, wherein 0.1% (mass ratio) of Tween 80 was added to delamanid and premanid, and 0.1% (mass ratio) of croscarmellose sodium was added to bedaquiline and clofazimine. The antibiotic solutions were prepared to fixed concentrations according to the drug concentrations recommended by the WHO, CLSI, and the Chinese Anti-Tuberculosis Association. The antibiotics used and their specific concentrations are shown in Table 1.

[0076] 4. After sterilizing the antibiotic solution by filtration using a 0.22 μm filter membrane, 0.625 to 2.5 ml (preferably 2.5 ml in this embodiment) of the solution was dispensed into 7 ml vials for lyophilization.

[0077] 5. After the freeze-drying is completed, the subpackaged reagents are plugged and capped in a Class 100 environment to produce 12 groups of drug freeze-dried preparations;

[0078] 6. Meanwhile, prepare the additives: weigh 500 g bovine serum albumin, 200 g glucose, 0.3 g catalase, and 6 mL oleic acid in purified water, stir to dissolve, and dilute to 10 L;

[0079] 7. Filter the additives using a 0.22μm filter membrane, dispense into 30ml vials, and cap and stopper them in a Class 100 environment.

[0080] Table 1. Antibiotic drug concentrations used

[0081]

[0082] The kit prepared by this embodiment (photo as shown) Figure 1As shown in the figure), drug susceptibility testing of Mycobacterium tuberculosis is performed. The testing process is as follows:

[0083] 1. Reconstitute the lyophilized drug with pure water. Add 1-4 ml (preferably 4 ml in this example) of pure water to each of the 12 groups of lyophilized drug to obtain 12 tubes of drug solution. At the same time, use an equal volume of pure water as a control tube.

[0084] 2. 12 drug solutions and control tubes were cultured in liquid form. 0.1 ml of drug solution was mixed with 0.5 ml of sample (sputum sample, with the sample in the control tube diluted 100-fold) and 0.8 ml of additive in a culture tube and cultured at 37°C for 10 days using a BD MGIT 960 instrument.

[0085] 3. Use the instrument to regularly monitor the fluorescent signal emitted from the bottom of the culture tube to predict bacterial growth and read the GU value. -2 mg) was inoculated into the drug-containing culture tube, and the working bacterial solution was diluted 100 times and inoculated into the blank control culture tube (the final inoculation amount was about 5×10 -4 mg), the proportional method is used for drug sensitivity testing. In fact, when using the proportional method, there is no need to predetermine the bacterial content in the sample. As long as the control tube after 100-fold dilution can be successfully cultured, it means that the sample contains the bacteria. The instrument regularly monitors the fluorescent signal emitted from the bottom of the culture tube to predict bacterial growth. When the growth unit (GU) value in the blank control culture tube reaches 400 (10 days), the instrument automatically detects the GU value in the drug-containing culture tube. If the GU value of the drug-containing tube is <100, it will be automatically judged as sensitive. If the GU value is ≥100, it will be judged as resistant.

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

[0087] Example 2: Effect of freeze-dried excipient selection on drug sensitivity testing results

[0088] Previous studies in this example revealed that using different lyophilization excipients not only affects the lyophilized shape of the drug lyophilized product but also the reconstitution stability of the drug (particularly water-insoluble drugs). A comparison of the reconstitution effects of different lyophilization excipients revealed that a combination of dextran and glycerol provided the best reconstitution effect and also enabled the reconstitution of water-insoluble drugs using only pure water, resulting in a relatively good reconstitution effect. However, after long-term storage, its reconstitution stability and drug activity still need to be improved.

[0089] In this example, a Mycobacterium tuberculosis drug susceptibility test kit was prepared according to the method provided in Example 1. The lyophilized excipients were selected from the 10 components shown in Table 2 (the contents and proportions of the components, whether used individually or in combination, were consistent with those in Example 1). Ten Mycobacterium tuberculosis drug susceptibility test kits were prepared, and comparisons were also made with kits without lyophilized excipients. The kits were stored at room temperature for 0 months (initial lyophilization and immediate use), 6 months, 12 months, and 36 months. High-temperature accelerated testing was performed, with 3 months at 45°C equivalent to 1 year, and so on. The kits were reconstituted in pure water, and the reconstitution time, reconstitution stability, drug activity, and drug susceptibility test results were evaluated. Since clofazimine was the most insoluble of the 12 antibiotics, and the test results for the other insoluble antibiotics showed similar trends to clofazimine, this example uses the test results for clofazimine as an example. The reconstitution time refers to the time required to reconstitute the drug lyophilized agent with pure water until the lyophilized agent is completely dissolved; turbidity refers to the turbidity of the reconstituted solution after being left for half an hour after reconstitution and then detected by a turbidimeter; reconstitution stability refers to the condition of the reconstituted solution after being left for half an hour after reconstitution; the drug activity of clofazimine was tested by high-performance liquid chromatography using a C18 reverse-phase column, a mobile phase of methanol:phosphate buffer = 80:20, and a detection wavelength of 280 nm. The sample to be tested was wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and drug sensitivity testing was performed according to the ratio method provided in Example 1. The test results are shown in Tables 2 to 5.

[0090] Table 2. Effect of lyophilized excipients (0 months)

[0091]

[0092] Table 3. Effect of lyophilization excipients (6 months)

[0093]

[0094] Table 4. Effect of lyophilized excipients (12 months)

[0095]

[0096] Table 5. Effect of lyophilized excipients (36 months)

[0097]

[0098] As can be seen from Table 2, the originally insoluble clofazimine, after being prepared into lyophilized agent, can effectively realize redissolution with water.But if lyophilized excipient is not added, drug stability is poor, and lyophilized shape is poor, uneven, and lyophilized excipient must be added.When adopting different lyophilized excipients, its redissolution effect to clofazimine is also completely different, compared to when adopting a kind of material such as dextran alone, the redissolution effect after combination is improved, but the lyophilized excipient prepared by different combination components also has a large difference to the redissolution effect of clofazimine.Only considering the redissolution effect, the most preferred are the 5th, 9th and 10th three groups of lyophilized excipients, whose key components are dextran and glycerol, and the lyophilized excipient prepared containing this combination is redissolved with water when, the drug solution prepared is the most uniform and stable, and the redissolution time is the shortest, the turbidity is the lowest, the solubility is the best, and the redissolution stability is the best.

[0099] Comparing Tables 2 to 5, significant changes in reconstitution stability occur with increasing storage time. As reconstitution time increases, turbidity also begins to rise, reconstitution stability decreases, and even suspended matter may appear. Within 12 months of storage, dextran and glycerol are still the preferred excipients. However, as storage time continues to increase, the lyophilized excipient prepared with dextran, glycerol, and valine in Group 10 exhibits the best reconstitution stability. Even after long-term storage (36 months), it can still be reconstituted in water to form a uniform and stable drug solution.

[0100] At the same time, with extended storage, the drug showed significant degradation, and the GU values ​​of the drug susceptibility test results increased significantly (a higher GU value indicates a lower sensitivity of the drug susceptibility test). Even after 36 months of storage, the GU values ​​of Groups 1-4 and 11 exceeded 100, causing the test results to shift from sensitive to resistant, a significant deviation. Groups 6-8 were also approaching 100, impending test errors. Group 10, however, used a combination of dextran, glycerol, and valine as a lyophilization excipient. The antibiotic lyophilized preparation prepared maintained clofazimine without degradation and no significant decrease in drug activity after 36 months of storage. It also had the best reconstitution stability, making Group 10 the most preferred lyophilization excipient.

[0101] Example 3: Effect of the selection of non-water-soluble antibiotic solvents on drug sensitivity testing results

[0102] In the process of preparing non-water-soluble antibiotics into drug lyophilized preparations, solvents must be added to enable their smooth dissolution and preparation. However, different solvents not only affect the solubility of non-water-soluble antibiotics, but also affect their reconstitution effect after lyophilization, and even have a certain impact on the drug activity after long-term storage.

[0103] In this example, a Mycobacterium tuberculosis drug sensitivity test kit was prepared according to the method provided in Example 1, wherein four solvents as shown in Table 6 were selected as solvents for non-water-soluble antibiotics, thereby preparing four groups of Mycobacterium tuberculosis drug sensitivity test kits. The test kits were stored at room temperature for 0 months and 12 months, respectively, and subjected to high temperature accelerated experiments. 3 months at 45°C was equivalent to one year, and so on. The kits were re-dissolved with pure water, and the re-dissolution time, re-dissolution stability, drug activity and drug sensitivity test results were investigated. The test was performed according to the method provided in Example 2. Since clofazimine is the most insoluble of the 12 antibiotics, the test results of several other insoluble antibiotics have similar trends to clofazimine. Therefore, this example uses the test results of clofazimine as an example. The sample to be tested is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and drug sensitivity testing is performed according to the proportional method provided in Example 1. The test results are shown in Tables 6 to 7. The re-dissolution effects of Group 1 and Group 4 after 12 months are shown in Tables 6 to 7. Figure 2 (The left picture is Group 1, and the right picture is Group 4).

[0104] Table 6. Effect of non-water-soluble antibiotic solvents (0 months)

[0105]

[0106] Table 7. Effects of non-water-soluble antibiotic solvents (12 months)

[0107]

[0108] As can be seen from Table 6, the use of different solvents to dissolve clofazimine will directly affect its reconstitution effect after lyophilization. The use of acetic acid as the solvent has the best effect, which can significantly improve the reconstitution stability of the clofazimine lyophilized agent, and the drug activity and drug sensitivity test sensitivity after reconstitution are also the highest.

[0109] Combining Tables 6 and 7, it can be seen that using acetic acid as a solvent during long-term storage also helps improve reconstitution stability, drug activity, and detection sensitivity after long-term storage. Although dimethyl sulfoxide also has a certain degree of reconstitution stability, it is prone to residual toxicity, resulting in a decrease in GU values. Abnormal results may be due to the inhibition of some bacterial growth; drug activity is also reduced. This demonstrates that acetic acid is the most preferred solvent.

[0110] Example 4, screening of cosolvents

[0111] Because Delamanid and Primanid are very insoluble, in the process of preparing them into drug lyophilized agents, in addition to adding acetic acid, a co-solvent must also be added at the same time to ensure their smooth dissolution and preparation. The use of different co-solvents will also directly affect the re-dissolution effect of Delamanid or Primanid after lyophilization, and after long-term storage, it will also have a certain impact on drug activity and detection sensitivity.

[0112] In this example, a Mycobacterium tuberculosis drug susceptibility test kit was prepared according to the method provided in Example 1. Since the cosolvents for delamanid and premanid are similar, delamanid was used as an example. Four cosolvents, as shown in Table 8, were used in the same amounts as in Example 1 to prepare four sets of Mycobacterium tuberculosis drug susceptibility test kits, which were compared with a control group without cosolvents. The kits were stored at room temperature for 0 and 12 months, and then subjected to a high-temperature accelerated test, where 3 months at 45°C was equivalent to 1 year, and so on. The kits were reconstituted with pure water, and the reconstitution time, reconstitution stability, drug activity, and drug susceptibility test results were evaluated according to the method provided in Example 2. The drug activity of delamanid was tested using high-performance liquid chromatography (HPLC) using a C18 reverse-phase column, mobile phase A consisting of 0.1% formic acid in water, mobile phase B consisting of acetonitrile, and a detection wavelength of 290 nm. The test sample was wild-type Mycobacterium tuberculosis (susceptible to delamanid), and drug susceptibility testing was performed according to the ratiometric method provided in Example 1. The test results are shown in Tables 8 and 9. Figure 3 These are photos showing the precipitation in Group 2 and Group 4.

[0113] Table 8. Effect of Delamanid Solubility Co-solvent (Month 0)

[0114]

[0115] Table 9. Effect of Delamanid Solubility Co-solvent (12 months)

[0116]

[0117] As can be seen from Table 8, the use of different co-solvents to dissolve delamanid directly affects the reconstitution effect of delamanid after lyophilization. Compared with several other co-solvents, the use of Tween 80 as a co-solvent has the best effect, significantly improving the reconstitution stability of the lyophilized delamanid. The drug activity and sensitivity of drug susceptibility testing after reconstitution are also the highest.

[0118] Combining Tables 8 and 9, it can be seen that using Tween 80 as a cosolvent for delamanid during long-term storage also helps improve reconstitution stability, drug activity, and detection sensitivity (lower GU values ​​indicate greater sensitivity). Therefore, Tween 80 is the most preferred cosolvent for delamanid.

[0119] Example 5: Screening of Bedaquiline and Clofazimine Disintegrants

[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 ensure 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] In this example, a Mycobacterium tuberculosis drug sensitivity test kit was prepared according to the method provided in Example 1, wherein the disintegrants for bedaquiline and clofazimine were selected as shown in Table 10, respectively, and the dosage was the same as in Example 1, thereby preparing three groups of Mycobacterium tuberculosis drug sensitivity test kits, and compared with the case where no disintegrant was added or a cosolvent was added. The kit was stored at room temperature for 0 months and 12 months, respectively, and a high temperature accelerated test was performed, with 45°C for 3 months equivalent to one year, and so on. It was reconstituted with pure water, and its reconstitution time, reconstitution stability, drug activity and drug sensitivity test results were investigated, and the test was performed according to the method provided in Example 2. Since clofazimine is more difficult to dissolve, and the test result trend of bedaquiline is similar to that of clofazimine, this example is illustrated by the test results of clofazimine. The test sample is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and the drug sensitivity test was performed according to the ratio method provided in Example 1. The test results are shown in Tables 10 and 11.

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

[0123]

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

[0125]

[0126] As shown in Table 10, even with the use of a cosolvent, the poorly soluble antibiotic clofazimine can be prepared, but its reconstitution is very poor, making it difficult to achieve reconstitution in pure water. Therefore, a disintegrant must be added to dissolve it to improve its reconstitution stability. Furthermore, the simultaneous addition of a cosolvent and a disintegrant reduces its drug stability, making long-term storage unfavorable. Therefore, direct addition of a disintegrant is most preferred.

[0127] Using different disintegrants to dissolve clofazimine will directly affect the reconstitution effect of clofazimine after lyophilization. Compared with several other disintegrants, the use of cross-linked sodium carboxymethyl cellulose as a disintegrant has the best effect and can significantly improve the reconstitution stability of clofazimine lyophilized preparation. The drug activity and sensitivity of drug sensitivity detection after reconstitution are also the highest.

[0128] Combining Tables 10 and 11, it can be seen that during long-term storage, the use of croscarmellose sodium as a disintegrant for clofazimine also helps improve post-storage reconstitution stability, drug activity, and detection sensitivity (lower GU values ​​indicate greater sensitivity). Therefore, croscarmellose sodium is the most preferred disintegrant.

[0129] Similarly, this embodiment also proves through experiments that the most preferred disintegrant for bedaquiline is also cross-linked carboxymethyl cellulose sodium. The detailed experimental data are omitted.

[0130] In addition, in confirming the proportion of disintegrants added to bedaquiline and clofazimine, this example added 0.05%, 0.1%, 0.2%, 0.3%, and 0.4% of cross-linked sodium carboxymethyl cellulose to the two drug lyophilized preparations, respectively. It was found that adding 0.1% cross-linked sodium carboxymethyl cellulose could achieve the best effect.

[0131] Example 6: Universality comparison

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

[0133] The present embodiment adopts the mycobacterium tuberculosis drug sensitivity test kit prepared by embodiment 1 respectively, with existing several mycobacterium tuberculosis drug sensitivity test kits, and the test kit adopting different additives, lyophilized excipients or solvent to carry out the comparative analysis of universality, because major difference is on the A63V type mutant of Rv0678 gene, A63V type mutant is shellfish and reaches quinoline drug-resistant type bacterium, but existing drug sensitivity test kit is due to the low problem of universality, easily cause A63V type mutant to also have the situation of being detected as not resistant to drugs, and easily occur inconsistent test result through repeated detection, and detection accuracy is not high.Therefore the present embodiment mainly enumerates the test result for this genotype mutant bacterium. Because major difference is shellfish and reaches quinoline, the present embodiment carries out universality research to the drug sensitivity test result of shellfish and reaches quinoline for A63V type mutant, and the test kit of preparation uses immediately, without long-term preservation.Mainly investigate the accuracy of drug sensitivity test result, every group carries out 20 repeated experiments, calculates the accuracy ratio, and compares with wild-type Rv0678 detection accuracy, thereby analyzes universality. The test was performed according to the method provided in Example 2. The sample to be tested was a sample solution containing the A63V mutant Mycobacterium tuberculosis, and the drug sensitivity test was performed according to the ratio method provided in Example 1. The test results are shown in Table 12.

[0134] Table 12. Comparison of universality test results

[0135]

[0136] As can be seen from Table 12, the universality of the combined mycobacterium susceptibility test kits prepared using different formulas varies in different genotypes. The kit prepared using Example 1 can achieve 100% repeated detection accuracy in both the wild type Rv0678 and the A63V mutant, showing better universality.

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

[0138] All patents and publications cited in this specification are intended to indicate that they are state of the art and that the present invention may be used. All patents and publications cited herein are incorporated by reference in their entirety, as if each publication were specifically incorporated by reference. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations, unless otherwise specified. For example, in each instance, the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The term "a" or "an" herein simply means "one" and does not exclude the inclusion of only one or more. The terms and expressions used herein are intended to be descriptive, not limiting, and are not intended to exclude any equivalent features. However, it is understood that any suitable changes or modifications may be made within the scope of the present invention and the appended claims. It is understood that the embodiments described herein are preferred embodiments and features, and that modifications and variations can be made by persons of ordinary skill in the art based on the spirit of the present invention. Such modifications and variations are considered to be within the scope of the present invention and the scope of the independent and appended claims.

Claims

1. A Mycobacterium tuberculosis drug sensitivity test kit, characterized in that: The invention comprises a drug lyophilized agent and an additive; the drug lyophilized agent contains a drug, dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the mass ratio of the dextran, glycerol and valine is 1:3:0.1; the additive is used for culturing Mycobacterium tuberculosis; the drug is clofazimine; and the preparation method of the Mycobacterium tuberculosis drug sensitivity test kit is as follows: (1) preparing a clofazimine drug solution containing 0.3-0.5% acetic acid and 0.1% cross-linked carboxymethyl cellulose sodium, adding dextran, glycerol, and valine, and lyophilizing; the mass ratio of the dextran, glycerol, and valine is 1:3:0.1; (2) preparing an additive solution; the additive is used to culture Mycobacterium tuberculosis.

2. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 1, wherein The additives include any one or more of bovine serum albumin, glucose, catalase, and oleic acid.

3. A Mycobacterium tuberculosis drug sensitivity test kit, characterized in that: The invention comprises a drug lyophilized agent and an additive; the drug lyophilized agent contains a drug, dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the mass ratio of the dextran, glycerol and valine is 1:3:0.1; the additive is used for culturing Mycobacterium tuberculosis; and the drug is clofazimine.

4. A method for preparing a Mycobacterium tuberculosis drug sensitivity test kit, characterized in that: The following steps are involved: (1) preparing a clofazimine drug solution containing 0.3-0.5% acetic acid and 0.1% cross-linked carboxymethyl cellulose sodium, adding dextran, glycerol, and valine, and lyophilizing; the mass ratio of the dextran, glycerol, and valine is 1:3:0.1; (2) preparing an additive solution; the additive is used to culture Mycobacterium tuberculosis.

5. A composition for preparing a lyophilized drug for maintaining the reconstitution stability of the drug, characterized in that: The composition comprises dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the drug lyophilized agent comprises the drug, dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the mass ratio of the dextran, glycerol and valine is 1:3:0.1; the drug is clofazimine; and the drug only requires water for reconstitution.

6. A composition for preparing a kit for improving the universality of drug sensitivity detection of Mycobacterium tuberculosis, characterized in that: The composition includes dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the kit includes a drug lyophilized agent and additives; the drug lyophilized agent contains drugs, dextran, glycerol, valine, 0.3-0.5% acetic acid and 0.1% cross-linked sodium carboxymethyl cellulose; the mass ratio of the dextran, glycerol and valine is 1:3:0.1; the drug is clofazimine; the additives are used to culture Mycobacterium tuberculosis; the additives include bovine serum albumin, glucose, catalase and oleic acid; 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, RD239 deletion type, RD105 deletion type, rpoB B170F type, RD9 deletion type, RD702 deletion type, and Rv0678 A63V mutant type.

7. A method for preparing a Mycobacterium tuberculosis drug sensitivity test kit, characterized in that: The following steps are involved: (1) Weigh 6 g of dextran, 18 g of glycerol, and 0.62 g of valine and dissolve them in purified water to prepare 1000 mL of solution A. (2) Accurately weigh antibiotic powder, dissolve water-soluble antibiotics with purified water, and dissolve water-insoluble antibiotics with 0.3%-0.5% glacial acetic acid added to purified water; (3) Antibiotic solution was prepared using solution A, wherein 0.1% by weight of Tween 80 was added to delamanid and premanid, and 0.1% by weight of cross-linked sodium carboxymethyl cellulose was added to bedaquiline and clofazimine. The antibiotic drugs used and their specific concentrations are shown in the table below; (4) After sterilizing the antibiotic solution by filtration using a 0.22 μm filter membrane, 0.625–2.5 ml of the solution was dispensed into 7 ml vials for lyophilization. (5) After the freeze-drying is completed, the subpackaged reagents are plugged and capped in a Class 100 environment to produce 12 groups of drug freeze-dried preparations; (6) At the same time, prepare the additives: 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 to dissolve, and dilute to 10 L; (7) After filtering the additive using a 0.22 μm filter membrane, dispense it into 30 ml vials and cap them in a Class 100 environment; 。

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