RU2470071C2 - CULTURE MEDIUM FOR DETERMINING DRUG SUSCEPTIBILITY OF M tuberculosis TO MAIN ANTI-TUBERCULOSIS DRUGS - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: culture medium for determining drug susceptibility of Mycobacterium tuberculosis to four main drugs - streptomycin, isoniazid, rifampin and ethambutol contains: 4.7 g dry Middlebrook 7H9 broth, 1.25 g microbioligical agar-agar, 1.25 g pancreatic digest of casein, 900 ml distilled water and 100 ml growth additive OADC. The additive contains, g/l: sodium chloride 8.5, bovine albumin (fraction 5) 50.0, glucose 20.0, catalase 0.03 and oleic acid 0.6 ml/l.

EFFECT: invention enables to obtain analysis results faster and ensures easy visual reading of results.

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Description

The invention relates to medicine, namely to the field of phthisiology and bacteriology, and can be used to determine the drug sensitivity of mycobacterium tuberculosis.

The continued increase in drug resistance of Mycobacterium tuberculosis (MBT), and in particular multidrug resistance, increases the importance of laboratory testing of clinical isolates of the pathogen. Especially important are the results of determining drug sensitivity for the choice of treatment regimen for newly diagnosed patients. Thus, an analysis of data from English-language literature for the period 1965–2007 by W.Lew et al [1] showed that treatment failure and relapse are associated with primary drug resistance. The results of the determination of drug sensitivity by the standard indirect method of absolute concentrations on Levenshtein-Jensen medium are taken into account on the 21st day after seeding of the MBT isolate. Methods for accelerated determination of drug sensitivity of MBT using liquid media shorten the time needed to obtain the data necessary for the appointment of adequate chemotherapy. Such methods include testing the sensitivity of MBT on the automated system of broth culture WASTES MGIT 960, which is by far the most effective [2, 3, 4]. But both the system itself and the reagent kits are very expensive (a set of them for determining the sensitivity of only 1 MBT strain to 4 first-line drugs costs 1,200 rubles). Therefore, the urgent task remains to quickly identify resistant strains of the Office using fast, inexpensive phenotypic methods available for practical phthisiobacteriological laboratories at any level.

Known semi-fluid medium Sotona (patent No. 2226398), which is not applicable for determining the sensitivity of mycobacterium tuberculosis to ethambutol [5]. The prototype of the proposed environment is Middlebrook's liquid nutrient medium, which is prepared from Middlebrook 7H9 commercial dry nutrient broth. Middlebrook's liquid nutrient medium requires the use of additional indirect physical or biochemical methods for detecting the growth of mycobacterial cultures [6, 7, 8, 9, 10], which increases the cost of the study, if the WASTES MGIT 960 automated system is used, to a significant extent.

Thus, the advantage of the proposed environment is 1) the speed of obtaining results is 3-8 days; 2) lower cost in comparison with other methods using Middlebrook's liquid medium; 3) the availability of any phthisiobacteriological laboratory, since additional equipment with expensive equipment is not required; 4) ease of reading the result, which is carried out visually.

The objective of the invention is the development of a nutrient medium for accelerated determination of the drug sensitivity of MBT strains to four main drugs - streptomycin, isoniazid, rifampicin, ethambutol.

The problem is solved due to the fact that microbiological agar-agar - 1.25 g and casein pancreatic hydrolyzate - 1.25 g are added to Middlebrook 7H9 liquid nutrient broth.

Preparation of culture medium for accelerated determination of drug sensitivity of the office

The basis of the medium is Middlebrook 7H9 commercial dry nutrient broth (Becton Dickinson), which is prepared in accordance with the prescription and then casein pancreatic hydrolyzate - 1.25 g and microbiological agar-agar - 1.25 g are added. The medium is close to liquid in consistency; introduction casein pancreatic hydrolyzate improves the growth properties of the nutrient medium, which leads to the rapid growth of mycobacteria. In addition, agar-agar does not allow the inoculum to sink to the bottom, increasing the local concentration of MBT and providing good aeration of these aerophilic microorganisms. All these factors determine the rapid visualization of growth: for 3-4 days when sowing suspensions with a density of 10 ^-1 and for 8-9 days at a density of 10 ^-3 . In the latter case, the growth periods coincide with those when testing drug sensitivity in the automated WASTES MGIT 960 system.

The composition of the nutrient medium

Dry broth Middlebrook 7N9 - 4.7 g, which includes:

ammonium sulfate - 0.5 g

L-glutamic acid - 0.5 g

sodium citrate - 0.1 g

pyridoxine - 0.001 g

Biotin - 0.0005 g

disubstituted sodium phosphate - 2.5 g

monosubstituted potassium phosphate - 1.0 g

lemon-ammonium iron - 0.04 g

magnesium sulfate - 0.05 g

calcium chloride - 0.0005 g

zinc sulfate - 0.001 g

copper sulfate - 0.001 g

Glycerin - 2 ml

Casein Pancreatic Hydrolyzate - 1.25 g

Microbiological agar-agar - 1.25 g

Distilled water - 900 ml

OADC Growth Supplement - 100 ml, which includes:

sodium chloride - 8.5 g

bovine albumin (fraction 5) - 50.0 g

glucose - 20.0 g

catalase - 0.03 g

oleic acid - 0.6 ml

In accordance with the commercial dry environment, distilled water is added to a portion of the powder of Middlebrook 7H9 dry broth, glycerin is added, and pancreatic casein hydrolyzate is added to enrich the broth. All components are mixed until complete dissolution and contribute 1.25 g of microbiological agar-agar. The mixture is then brought to a boil to dissolve the agar-agar. The prepared medium is sterilized at 120 ° C for 20 minutes. Then, commercial growth additive OADC (Beckton Dickinson) is aseptically introduced into the semi-liquid medium cooled to 50 ° C. The finished medium is poured into 99 ml into 4 flasks containing 1 ml of dilution of anti-TB drugs in final concentrations: streptomycin 1 μg / ml, isoniazid 0.1 μg / ml, rifampicin 1 μg / ml, ethambutol 5 μg / ml. Then the medium with the preparations is aseptically poured into 5 ml tubes. For each row consisting of 4 tubes with streptomycin, isoniazid, rifampicin, ethambutol, 2 control tubes with 5 ml of drug-free medium are attached.

The determination of the drug sensitivity of the MBT method of proportions is as follows.

A suspension of the test strain of MBT in physiological saline is prepared according to the turbidity standard of 5 units. (FGUN State Research Institute for Standardization and Control of Medical Biological Preparations named after L.A. Tarasevich of Rospotrebnadzor). Suspensions are diluted 10 (10 ^-1 ) and 1000 (10 ^-3 ) times. A suspension of MBT with a density of 10 ^{-1 is} introduced into one of the control tubes with the developed semi-liquid medium and into all test tubes with preparations, with a density of 10 ^-3 - into the second control tube. The volume of the inoculum is 0.5 ml. The suspension of MBT on a semi-liquid medium is applied superficially, by layering, with drops on the wall of an inclined tube, without stirring and shaking. Crops are incubated at 37 ° C, viewing semi-liquid media every day, starting from the third, and fixing the period of appearance of colonies of the office in the control and in test tubes with the preparations. When a culture of MBT appears on the medium with the drug, it is comparable to the growth in the control 10 ^-1 , resistance is recorded (3-5 days). Sensitivity to the drug is determined by the lack of growth - the transparency of the medium, or growth less than the growth in the control of 10 ^-3 on the 8-9th day.

The test of the proposed environment was carried out on 38 panel (obtained by the program of the Federal system of external quality assessment) and 47 clinical strains isolated from pathological material of patients with tuberculosis of various localizations. Drug sensitivity was investigated in parallel on the proposed medium, on the Levenshtein-Jensen medium by the standard method, and on prototype media: 1) semi-liquid Soton medium; 2) in the automated WASTES MGIT 960 system using the SIRE Kit.

The results of determining the growth rate of the office on the proposed environment in comparison with the prototype environments are presented in table 1.

Table 1 The growth rate of panel and clinical strains of M. tuberculosis in the proposed environment and environments prototypes. Culture medium Number of studies The growth rate in days at a suspension density (x ± I) 10 ^-1 10 ^-3 Panel strains Semi-fluid Middlebrook 38 4.13 ± 0.25 6.74 ± 0.23 * BBL MGIT 38 - 7.36 ± 0.22 Semi-fluid Sotona eighteen 3.95 ± 0.23 8.33 ± 0.56 # Clinical strains Semi-fluid Middlebrook 47 3.46 ± 0.16 8.13 ± 0.55 BBL MGIT 46 - 8.30 ± 0.31 Semi-fluid Sotona 47 3.77 ± 0.22 8.96 ± 0.72 Notes. 1) x ± I - average ± confidence interval; 2) *, # - differences between the media are significant at p <0.05.

As can be seen from the above data, the growth period of the office on the proposed environment is less than or equal to that on the prototype environments, which makes the environment promising for the accelerated determination of drug sensitivity of the office.

Thus, the studies showed that the use of the proposed semi-liquid medium of Middlebrook to accelerate the determination of the sensitivity of the office to 4 main anti-TB drugs is an alternative test method using the automated system WASTES MGIT 960. Add small agar agar to Middlebrook broth (1.25 g ) allows for a short time (3-4 days) to visually register the growth of the office (suspension density of 5 units of the turbidity standard, diluted 10 times), and the introduction of pancreatic hydrol casein isate improves the growth properties of the nutrient medium. On a semi-liquid medium, the average growth period of a suspension of mycobacteria diluted 100 times does not differ from that in the broth culture system. This makes it possible after 7-9 days to give an answer about the sensitivity of the studied strain. The coincidence of growth periods in the control tube and in the test tube with the drug with the resistance of the studied isolate allows detecting drug-resistant strains, especially the most epidemiologically dangerous cultures with multidrug resistance, for 3-7 days. Regarding isoniazid, rifampicin, streptomycin, the proposed method does not differ in its parameters from the WASTEC MGIT 960 SIRE Kit system, and even slightly exceeds it with respect to ethambutol. The method of accelerated determination of drug sensitivity of MBT using semi-liquid medium Middlebrook is not inferior to that in the automated system WASTES MGIT 960, but much cheaper, since it does not require expensive equipment and reagents. Therefore, for the accelerated determination of the sensitivity of the office to 4 main anti-TB drugs, it is advisable to use the proposed semi-liquid medium.

Claims (1)

Nutrient medium for determining the drug susceptibility of M. tuberculosis to major anti-TB drugs, including:
Dry broth Middlebrook 7N9 4.7 g Glycerol 2 ml Casein Pancreatic Hydrolyzate 1.25 g Microbiological agar agar 1.25 g Distilled water 900 ml OADC Growth Supplement 100 ml