Mycobacterium tuberculosis drug sensitive kit as well as preparation method and application thereof

Abstract

The invention provides a mycobacterium tuberculosis drug sensitivity kit and a preparation method and application thereof.The drug sensitivity condition of mycobacterium tuberculosis to 12 drugs such as bedaquiline is detected on the basis of an MGIT liquid method.The kit comprises a drug freeze-drying agent and an additive, the drug freeze-drying agent contains drugs, raffinose and glycerol, and the additive is a mixture of the drugs, the raffinose and the glycerol. 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 sensitivity detection, and in particular to a Mycobacterium tuberculosis drug sensitivity kit and a preparation 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 anti-tuberculosis 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 preparation method and application thereof, which detects the drug sensitivity of Mycobacterium tuberculosis to 12 drugs such as bedaquiline based on the MGIT liquid method. The kit includes a drug lyophilized agent and an additive, wherein the drug lyophilized agent contains drugs, raffinose and glycerol, the additive is used to culture Mycobacterium tuberculosis, acetic acid is added as a solvent for water-insoluble drugs, and a co-solvent 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 only with water. 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, raffinose and glycerol; 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 raffinose 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. Raffinose 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-drying excipient, and the mass ratio of raffinose to glycerol is 1:3.

[0014] In some embodiments, the lyophilization excipient further contains valine. The use of a combination of raffinose, 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 raffinose, 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, raffinose, 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] In another aspect, the present invention provides a method for preparing a Mycobacterium tuberculosis drug sensitivity kit, comprising the following steps:

[0026] (1) preparing a solution containing the drug, adding raffinose and glycerol, and freeze-drying;

[0027] (2) preparing an additive solution; the additive is used to culture Mycobacterium tuberculosis.

[0028] In some embodiments, the method for preparing the Mycobacterium tuberculosis drug susceptibility test kit comprises the following steps:

[0029] 1. Weigh raffinose and mannose and dissolve them in purified water;

[0030] 2. Accurately weigh the antibiotic powder according to the preparation amount and dissolve the antibiotic with purified water or acetic acid;

[0031] 3. Add raffinose and glycerol 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;

[0032] 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.

[0033] 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;

[0034] 6. Filter the drug-sensitive additive using a 0.22μm filter membrane, dispense into 30ml vials, and seal with stoppers and caps.

[0035] In some embodiments, the composition further comprises valine.

[0036] In another aspect, the present invention provides a method for drug sensitivity 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:

[0037] (1) dissolving the lyophilized drug in water to obtain a drug solution;

[0038] (2) mixing the drug solution, sample, and additives and incubating;

[0039] (3) Read the results.

[0040] 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 raffinose and glycerol; the drug lyophilized agent comprising a drug, raffinose and glycerol; 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.

[0041] In some embodiments, the composition further comprises valine.

[0042] In another aspect, the present invention provides a use of a composition for preparing a kit for improving the universality of drug sensitivity detection of Mycobacterium tuberculosis, wherein the composition comprises raffinose and glycerol; the kit comprises a drug lyophilized agent and an additive; the drug lyophilized agent contains a drug, raffinose and glycerol; the additive is used to culture Mycobacterium tuberculosis;

[0043] In some embodiments, the composition further comprises valine.

[0044] In another aspect, the present invention provides a composition for preparing an agent for improving the shape of a drug excipient, the composition comprising raffinose and glycerol; 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.

[0045] In some embodiments, the composition further comprises valine.

[0046] 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.

[0047] The beneficial effects of the present invention are:

[0048] 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.

[0049] 2. By adding raffinose and glycerol as freeze-drying excipients to the drug freeze-dried reagent, not only can the stability of the drug during long-term storage (stable storage for more than 36 months) be effectively improved, but it also helps to improve the drug's re-dissolution stability. The prepared drug freeze-dried 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.

[0050] 3. For water-insoluble antibiotics, adding acetic acid, raffinose and glycerol 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, which avoids toxic residues.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Detailed description

[0056] 1. Tuberculosis patients

[0057] 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.

[0058] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, primarily transmitted through the air. After infection, most people remain in an asymptomatic latent state, where the bacteria are controlled by the body's immune system. However, when immunity declines, approximately 5-10% of latently infected individuals will develop active tuberculosis, characterized by persistent cough, sputum, hemoptysis, chest pain, fever, night sweats, weight loss, and fatigue. Tuberculosis can affect the lungs (pulmonary tuberculosis) or other organs (extrapulmonary tuberculosis) such as the kidneys, brain, spine, and skin. Diagnosis relies on imaging, microbiological, and immunological tests, and treatment requires long-term multidrug therapy. Drug resistance is an increasingly serious problem. Preventive measures include BCG vaccination and infection control.

[0059] 2. Mycobacterium tuberculosis

[0060] Mycobacterium tuberculosis (M. tuberculosis), also known as tubercle bacilli, is the causative agent of human tuberculosis. It is an obligate 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.

[0061] 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.

[0062] 3. Drug susceptibility test for Mycobacterium tuberculosis

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Due to the slow growth of Mycobacterium tuberculosis, 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.

[0067] The liquid culture method provided by the present invention uses the MGIT instrument of the American company BD for rapid analysis and detection. Its principle is: the bottom of the MGIT culture tube contains an oxygen-quenching fluorescent substance (such as a silica gel-embedded ruthenium compound). When Mycobacterium tuberculosis grows, it consumes oxygen in the culture medium, causing the oxygen concentration in the tube to decrease, and the fluorescent substance is activated and releases a fluorescent signal. The BDMGIT instrument (such as MGIT 960) uses an optical sensor to continuously detect the fluorescence intensity in real time without opening the lid or taking samples. Once the fluorescence signal exceeds the threshold (indicating bacterial growth), it is immediately judged as positive. Compared with traditional solid culture media (such as Roche culture medium, which takes 4-8 weeks), MGIT can shorten the culture time to 7-14 days (average 10 days). BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a photo of the prepared kit;

[0069] 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.

[0070] Figure 3 These are photos showing the precipitation in Group 2 and Group 4 in Example 4. DETAILED DESCRIPTION

[0071] 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.

[0072] Example 1: Preparation and Detection Method of the Mycobacterium tuberculosis Drug Sensitivity Kit Provided by the Present Invention

[0073] 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:

[0074] 1. Weigh 6 g of raffinose, 18 g of glycerol, and 0.62 g of valine and dissolve them in purified water to prepare 1000 mL of solution A.

[0075] 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);

[0076] 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 solution was prepared into a fixed concentration according to the drug concentrations recommended by WHO, CLSI, and the Chinese Anti-Tuberculosis Association. The antibiotic drugs used and their specific concentrations are shown in Table 1;

[0077] 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) was dispensed into 7 ml vials for lyophilization.

[0078] 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;

[0079] 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;

[0080] 7. Filter the additives using a 0.22μm filter membrane, dispense into 30ml vials, and cap and stopper them in a Class 100 environment.

[0081] Table 1. Antibiotic drug concentrations used

[0082] Serial number Drug name Drug abbreviation Concentration before freeze-drying (ug / mL) 1 Rifampin RFP 134.4 2 Isoniazid INH 13.44 3 Ethambutol EMB 672 4 Levofloxacin LFX 134.4 5 Premani PA-824 33.6 6 Moxifloxacin-High MFX 134.4 7 Amikacin AMK 134.4 8 Prothionamide PTO 336 9 Bedaquiline BDQ 134.4 10 Dramani DLM 8 11 Linezolid LZD 134.4 12 Clofazimine CFZ 134.4

[0083] The kit prepared by this embodiment (photo as Figure 1 As shown), Mycobacterium tuberculosis drug sensitivity test was performed, and the test process was as follows:

[0084] 1. Reconstitute the lyophilized drug with pure water. Add 1-4 ml (preferably 4 ml in this embodiment) 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.

[0085] 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, where the sample in the control tube was 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.

[0086] 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 cultured smoothly, 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.

[0087] 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), ΔF = 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.

[0088] Example 2: Effect of freeze-dried excipient selection on drug sensitivity testing results

[0089] 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 the combination of raffinose and glycerol had the best reconstitution effect and also enabled the reconstitution of water-insoluble drugs using only pure water, resulting in a better reconstitution effect. However, after long-term storage, its reconstitution stability and drug activity still need to be improved.

[0090] In this example, a Mycobacterium tuberculosis drug sensitivity test kit was prepared according to the method provided in Example 1, wherein the lyophilized excipients were selected from the 10 components shown in Table 2 (whether alone or in combination, the content or proportion of each component was consistent with that in Example 1), thereby preparing 10 sets of Mycobacterium tuberculosis drug sensitivity test kits, which were also compared with the case 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, and a high temperature accelerated test was performed, with 3 months at 45°C equivalent to one year, and so on. The kits were reconstituted with pure water, and the reconstitution time, reconstitution stability, drug activity, and drug sensitivity test results were investigated. Since clofazimine is the most insoluble of the 12 antibiotics, the test results of the other insoluble antibiotics have similar trends to clofazimine, so this example uses the test results of clofazimine as an example. Wherein the reconstitution time refers to the time required for reconstitution of 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 placed for half an hour after reconstitution and then detected by a turbidimeter; reconstitution stability refers to the situation of the reconstituted solution after being placed for half an hour after reconstitution and observed; the drug activity detection of clofazimine adopts high performance liquid chromatography, C18 reverse phase column, mobile phase is methanol: phosphate buffer = 80: 20, detection wavelength 280nm. The sample to be tested is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and drug sensitivity detection is carried out according to the ratio method provided in Example 1. Test results are shown in Tables 2 to 5.

[0091] Table 2. Effect of lyophilized excipients (0 months)

[0092]

[0093] Table 3. Effect of lyophilized excipients (6 months)

[0094]

[0095] Table 4. Effect of lyophilized excipients (12 months)

[0096]

[0097] Table 5. Effect of lyophilized excipients (36 months)

[0098]

[0099] 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 raffinose 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 raffinose and glycerol, and the lyophilized excipient prepared containing this combination is redissoluted with water when, the drug solution prepared is the most uniform and stable, and the redissolution time is the shortest, and turbidity is the lowest, and solubility is the best, and redissolution stability is the best.

[0100] Comparing Tables 2 to 5, the reconstitution stability changes significantly with increasing storage time. As the reconstitution time increases, turbidity also begins to rise, reconstitution stability decreases, and even suspended matter appears. Within 12 months of storage, using raffinose and glycerol as excipients is also a relatively good choice. However, as storage time continues to increase, the lyophilized excipient prepared with raffinose, glycerol, and valine in Group 10 shows 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.

[0101] At the same time, as storage time increased, the drug showed significant degradation, and the GU value of the drug sensitivity test results increased significantly (a higher GU value indicates a lower sensitivity of the drug sensitivity test). Even after 36 months of storage, the GU values ​​of the drug sensitivity test results of Groups 1 to 4 and 11 exceeded 100, causing the test results to change from sensitive to resistant, a significant deviation. Groups 6 to 8 were also close to 100, and detection errors were imminent. Group 10 used a combination of raffinose, glycerol, and valine as a lyophilization excipient. The antibiotic lyophilized preparation prepared after 36 months of storage still maintained clofazimine without degradation and no significant decrease in drug activity. At the same time, the reconstitution stability was the best. Therefore, Group 10 is the most preferred lyophilization excipient.

[0102] Example 3: Effect of the selection of non-water-soluble antibiotic solvents on drug sensitivity testing results

[0103] 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.

[0104] 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 for solvents of 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 effect of Group 1 and Group 4 after 12 months is shown in Tables 6 to 7. Figure 2 (The left picture is Group 1, and the right picture is Group 4).

[0105] Table 6. Effect of non-water-soluble antibiotic solvents (0 months)

[0106]

[0107] Table 7. Effects of non-water-soluble antibiotic solvents (12 months)

[0108]

[0109] 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.

[0110] 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.

[0111] Example 4, screening of cosolvents

[0112] 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.

[0113] 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 months 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.

[0114] Table 8. Effect of Delamanid Solubility Co-solvent (0 Months)

[0115]

[0116] Table 9. Effect of Delamanid Solubility Co-solvent (12 months)

[0117]

[0118] 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.

[0119] Combining Tables 8 and 9, it can be seen that, during long-term storage, using Tween 80 as a cosolvent for delamanid also helps improve reconstitution stability, drug activity, and detection sensitivity (lower GU values ​​indicate greater sensitivity) after long-term storage. Therefore, Tween 80 is the most preferred cosolvent for delamanid.

[0120] Example 5: Screening of Bedaquiline and Clofazimine Disintegrants

[0121] 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.

[0122] The present embodiment prepares the Mycobacterium tuberculosis drug sensitivity test kit according to the method provided in Example 1, wherein for the disintegrant of bedaquinoline and clofazimine, respectively select 3 kinds of disintegrants as shown in Table 10, and the dosage is consistent with Example 1, thereby preparing 4 groups of Mycobacterium tuberculosis drug sensitivity test kits, and compared with the situation that does not add or adds cosolvent. The kit is stored at room temperature for 0 month and 12 months respectively, and is carried out with high temperature accelerated experiment, and 45 ℃ 3 months is equivalent to one year, and so on. It is redissolved with pure water, and its redissolution time, redissolution stability, drug activity and drug sensitivity test result are investigated, and the method provided in Example 2 is detected. Because clofazimine is more insoluble, and the test result trend of bedaquinoline is similar to clofazimine, therefore the present embodiment is illustrated with the test result of clofazimine, and the sample to be tested is wild-type Mycobacterium tuberculosis (sensitive to clofazimine), and drug sensitivity test is carried out according to the ratio method provided in Example 1, and test result is as shown in Table 10~11.

[0123] Table 10. Effect of disintegrant (0 month)

[0124]

[0125] Table 11. Effect of disintegrants (12 months)

[0126]

[0127] 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.

[0128] 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.

[0129] 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 reconstitution stability after long-term storage, drug activity, and detection sensitivity (lower GU values ​​indicate greater sensitivity). Therefore, croscarmellose sodium is the most preferred disintegrant.

[0130] 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.

[0131] 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 achieved the best effect.

[0132] Example 6: Universality comparison

[0133] 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.

[0134] 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.

[0135] Table 12. Comparison of universality test results

[0136]

[0137] 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.

[0138] 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 sensitivity test kit.

[0139] 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 freeze-dried agent and an additive; the drug freeze-dried agent contains drugs, raffinose and glycerol; and the additive is used for culturing Mycobacterium tuberculosis.

2. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 1, wherein 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.

3. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 2, wherein When the drug is a water-insoluble antibiotic, the drug lyophilized agent contains acetic acid.

4. The Mycobacterium tuberculosis drug susceptibility test kit according to claim 2, wherein When the drug is delamanid or premanid, the drug lyophilized agent further contains a solubilizing agent; when the drug is bedaquiline or clofazimine, the drug lyophilized agent further contains a disintegrant; the solubilizing agent includes Tween 80; and the disintegrant includes cross-linked sodium carboxymethyl cellulose.

5. 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.

6. A method for preparing a Mycobacterium tuberculosis drug sensitivity test kit, characterized in that: The following steps are involved: (1) preparing a solution containing the drug, adding raffinose and glycerol, and freeze-drying; (2) preparing an additive solution; the additive is used to culture Mycobacterium tuberculosis.

7. A method for drug sensitivity testing of Mycobacterium tuberculosis, characterized in that: The test is performed using the kit according to any one of claims 1 to 5 or the kit prepared by the method according to claim 6, comprising the following steps: (1) dissolving the lyophilized drug in water to obtain a drug solution; (2) mixing the drug solution, sample, and additives and incubating; (3) Read the results.

8. A composition for preparing a lyophilized drug that maintains the stability of drug reconstitution, characterized in that: The composition comprises raffinose and glycerol; the drug lyophilized agent comprises a drug, raffinose and glycerol; the drug comprises a water-soluble antibiotic and a water-insoluble antibiotic; the water-soluble antibiotic comprises any one or more of rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, amikacin and linezolid; the water-insoluble antibiotic comprises any one or more of prothionamide, bedaquiline, delamanid, clofazimine and premanid; and the drug only requires water for reconstitution.

9. A composition for preparing an agent for improving the shape of a drug excipient, characterized in that: The composition includes raffinose and glycerol; 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.

10. Use of acetic acid for preparing a reagent for improving the reconstitution stability of water-insoluble antibiotics, characterized in that: The water-insoluble antibiotics include any one or more of prothionamide, bedaquiline, delamanid, clofazimine, and premanid.

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