RU2268298C2 - Kit for examination of drug sensitivity and primary identification of tuberculosis mycobacteria - Google Patents

Abstract

FIELD: biotechnology, in particular microbiology, individual selection of drugs for tuberculosis treatment.

SUBSTANCE: claimed kit based on Levenshtine-Jensen medium comprises 8 flacks. Four flacks additionally contain antituberculosis drugs, namely isoniaside, rifampicin, strepromicin, and ethambutol, respectively. Two flacks additionally contain sodium salicylate and thiophenecarboxy hydrazide, respectively, and two flacks are used as control ones. Additionally kit contains one flack with Griss reagent.

EFFECT: kit for accelerated examination of drug sensitivity and simultaneous identification of tuberculosis mycobacteria.

4 tbl, 4 ex

Description

The invention relates to medical microbiology, for the production of diagnostic drugs and can be used for individual selection of drugs for the treatment of tuberculosis.

In practical laboratories in Russia, the Levenshtein-Jensen laboratory preparation with the addition of anti-tuberculosis drugs recommended for use by Order of the Ministry of Health of the Russian Federation No. 109 (Appendix 11. Instructions for unified methods of microbiological research in the detection, diagnosis and treatment of tuberculosis) is mainly used to determine the drug sensitivity of mycobacterium tuberculosis) [one]. However, the use of this medium allows to obtain data on the drug sensitivity of the pathogen only after 3-5 weeks. In addition to this, laboratory preparation media are non-standard, which leads to unreliable results.

Closest to the proposed invention is a kit for determining the drug sensitivity of Mycobiogramme tuberculosis Mycobiogramme produced by the French company BioMerieux [2], consisting of tubes with Levenshtein-Jensen medium of the following composition, g / l:

potassium dihydrogen phosphate 1.50 g magnesium sulfate 0.15 g magnesium citrate 0.38 g L-asparagine 2.25 g glycerol 7.50 ml potato starch 18.75 g malachite green 0.25 g egg mass 625 ml distilled water 375 ml

with the addition of anti-TB drugs in concentrations, mcg / ml:

isoniazid 0.1 isoniazid 1,0 rifampicin 20,0 rifampicin 40,0 streptomycin 4.0 streptomycin 10.0 ethambutol 2.0 ethambutol 3.0 PASK (paraaminosalicylic acid) 0.5 PASK 1,0 pyrazinamide 200,0

The determination of the drug sensitivity of mycobacteria using the prototype kit is carried out visually by the presence of visible growth. This process is long and takes from 14 to 28 days, which greatly complicates the timely correlation of the etiotropic treatment carried out to the patient. In addition, the kit is expensive and inaccessible to most practical laboratories in Russia.

The present invention is the creation of a test kit for the accelerated determination of drug sensitivity of Mycobacterium tuberculosis with their simultaneous primary identification by the appearance of a red color.

The problem is solved in that a test kit for determining drug sensitivity and primary identification of mycobacterium tuberculosis based on Levenshtein-Jensen medium with the addition of anti-tuberculosis drugs, characterized in that the set consists of 8 bottles with Levenshtein-Jensen medium, additionally containing sodium nitrate or potassium nitrate in a concentration of 0.6-1.2 g / l, and starch in a soluble form at a concentration of 15.0-20.0 g / l, with four vials designed to determine drug sensitivity additionally contain anti-TB drugs, the first bottle is isoniazid at a concentration of 1 μg / ml, the second bottle is rifampicin at a concentration of 40 μg / ml, the third is streptomycin at a concentration of 10 μg / ml, the fourth is ethambutol at a concentration of 2 μg / ml, two vials intended for primary identification of mycobacterium tuberculosis, one of which additionally contains sodium salicylate at a concentration of 1000 μg / ml, and the other contains 2-thiophenecarboxyhydrazide at a concentration of 2 μg / ml, two bottles are control, and additionally a set of erzhit one vial with Griess reagent to record the results from the appearance of the red color.

The proposed test kit compared to the prototype is characterized by short periods of 2-7 days for determining the drug sensitivity of mycobacteria by the appearance of a red color, the ability to identify mycobacteria at the same time. This is achieved by

1) introducing Levenshtein-Jensen starch in a soluble form, which improves the growth properties of the medium, increases the growth rate of mycobacteria, reduces the time taken to account for results, and does not affect the critical concentrations of anti-TB drugs (Tables 1, 2),

2) the introduction of sodium or potassium nitrate into the medium, and into the composition of the Griss reagent test kit, which allows taking into account the results of the appearance of a red color, without waiting for the visible growth of mycobacteria on the medium.

3) the use in the test kit of one bottle with sodium salicylate and one bottle with 2-thiophenecarboxyhydrazide, the combination of which allows the primary identification of the analyzed culture of mycobacteria (table 3).

Table 1
The effect of the form of starch used on the growth rate of M. tuberculosis Mycobacteria The cultivation time at 37 ° C (days) Control bottle (Levenshtein-Jensen medium with sodium or potassium nitrate) soluble starch potato starch (from prototype) M. tuberculosis 1 7 Red coloring No coloring M. tuberculosis 2 7 Red coloring Pink color M. tuberculosis 3 7 Pink color No coloring M.tuberculosis 4 6 Red coloring Pink color M.tuberculosis 5 5 Pink color No coloring M.tuberculosis 6 5 Pink color No coloring M.tuberculosis 7 6 Red coloring No coloring M.tuberculosis 8 6 Red coloring No coloring M.tuberculosis 9 four Pink color No coloring Red color - characterizes the intensive growth of culture
Pink color - characterizes weak growth of culture
No color - lack of culture growth

table 2
The influence of the form of starch on the results of determining the drug susceptibility of M. tuberculosis Test isolate Observation period Starch The control The control Isoniazid Rifampicin Streptomycin Etam-butol Sodium Salicylate TKG M. tuberculosis H37Ra 7 Mortar the form + + - - - - - + 7 Kart. + + - - - + - + M. tuberculosis Academy 7 Mortar the form + + - - - - - + 7 Kart. + + + - + - - + M. tuberculosis 138 7 Mortar the form + + - - + - - + 7 Kart. + + - - + + - + M. tuberculosis 8 7 Mortar the form + + + - - - - + 7 Kart. + + + + - - - + M.tuberculosis 118 7 Mortar the form + + - + - - - + 7 Kart. + + + + - + - + * + - the presence of red color in the bottles
- - lack of red color in bottles

Example 1

Preparation of a nutrient medium. Samples of the following nutrient medium ingredients:

potassium dihydrogen phosphate 1.50 g sodium nitrate 1.00 g magnesium sulfate 0.15 g magnesium citrate 0.38 g L-asparagine 2.25 g glycerol 7.50 ml soluble starch 18.75 g malachite green 0.25 g

dissolved in the indicated sequence in 375 ml of distilled water when heated, not bringing to a boil, and sterilized in an autoclave for 20 minutes at a temperature of 121 ° C.

After cooling, 625 ml of egg mass is added to the base of the nutrient medium, filtered.

Example 2

The nutrient medium is prepared as described in example 1, but the ingredients that distinguish the medium from Levenshtein-Jensen medium: potassium nitrate and starch in soluble form, are used in the following concentrations:

potassium nitrate 1.2 g soluble starch 20.0 g

Example 3

The nutrient medium is prepared as described in example 1, but sodium nitrate and starch in soluble form are used in the following concentrations:

potassium nitrate 0.6 g soluble starch 15.0 g

Example 4

The prepared nutrient medium in accordance with examples 1, 2, 3 is poured equally into eight sterile flasks.

One of the following preparations is introduced into six flasks with stirring:

isoniazid - 0.125 mg,

rifampicin - 5 mg,

streptomycin - 1.25 mg,

ethambutol - 0.25 mg,

sodium salicylate - 125 mg,

2-thiophenecarboxyhydrazide (TCH) - 0.25 mg

to obtain final concentrations of isoniazid - 1 μg / ml, rifampicin - 40 μg / ml, streptomycin - 10 μg / ml, ethambutol - 2 μg / ml sodium salicylate - 1000 μg / ml, TKG - 2 μg / ml. Then the contents of all eight flasks are poured into sterile vials and thermocoagulation is performed.

The test kit is completed in accordance with the composition:

Culture medium (control) - 2 bottles Culture medium with 1 μg / ml isoniazid - 1 bottle Culture medium with 40 μg / ml rifampicin - 1 bottle Culture medium with 10 μg / ml streptomycin - 1 bottle Culture medium with 2 μg / ml ethambutol - 1 bottle Culture medium with 1000 μg / ml sodium salicylate - 1 bottle Culture medium with 2 μg / ml TKG - 1 bottle Griss reagent - 1 bottle.

Storage of the test kit is carried out in a dark place at a temperature of 2-8 ° C for no more than 5 months.

Example 5

The determination of drug sensitivity of cultures of mycobacteria is carried out in parallel on the proposed test kit and on the prototype kit. On the test kit, primary identification of mycobacterium tuberculosis is additionally carried out. The determination of drug sensitivity and the primary identification of mycobacterium tuberculosis is carried out by a method based on the ability of mycobacterium tuberculosis to restore nitrates to nitrites. Nitrates are easily detected using the Griss reagent, which causes the appearance of a red color. Determination of drug sensitivity of mycobacteria using a prototype kit is carried out by the method of proportions by the presence of visible growth.

Eight bottles with nutrient media of the proposed test kit are seeded with cultures of tuberculosis mycobacteria in accordance with the Order of the Ministry of Health of the Russian Federation No. 109. Test tubes with nutrient media of the prototype kit are seeded in accordance with the instructions for use.

Analysis of the results on the prototype is carried out after 14-28 days of incubation of crops according to the presence of visual growth of mycobacteria, on the proposed test kit - after 2-7 days for color change by applying Griss reagent in the form of an aqueous 7.5% Griss reagent solution in an amount of 0, 5 ml or in dry form in the amount of several crystals in each of the 8 bottles of the test kit. A culture is considered sensitive to an anti-TB drug if a red color does not occur in the bottle with this drug (with intense color in the control bottle). The culture is considered stable when a red color appears in a vial with an anti-TB drug.

The primary identification of mycobacterium tuberculosis is carried out by the appearance of a red color in a bottle with TKG and the absence of a red color in a bottle with sodium salicylate. Non-tuberculous mycobacteria, which have the ability to reduce nitrates to nitrites, cause the simultaneous appearance of a red color in bottles with sodium salicylate and with TKG.

The test results of the three examples of the test kit were identical and are presented in tables 3.4.

Table 3
Determination of drug susceptibility and primary identification of mycobacteria Test isolate Observation period The control Sodium Salicylate TKG Isoniazid Rifampicin Streptomycin Etam-butol M. tuberculosis H37Ra 7 * + - + - - - - M. tuberculosis 135 7 + - + + + + + M. tuberculosis 6 + - + + - - -

Test isolate Observation period The control Sodium Salicylate TKG Isoniazid Rifampicin Streptomycin Etam-butol 138 M. kansasii 413 7 + + + + - + + M. fortuitum 12 2 + + + + + + + + - presence of discoloration in bottles
- - no discoloration in the bottles

The data presented in the tables allow us to characterize the studied cultures, as a strain of M. tuberculosis H37Ra, sensitive to four drugs; clinical isolate of M. tuberculosis 135 resistant to isoniazid, rifampicin, streptomycin, ethambutol; clinical isolate of M. tuberculosis 138, resistant to isoniazid, and also M. kansasii 413, resistant to isoniazid, streptomycin, ethambutol and M. fortuitum 12, resistant to all four anti-TB drugs, which completely coincides with the results obtained in the prototype kit.

Table 4
Terms for determining the drug sensitivity of mycobacterium tuberculosis Test material Duration of observation (day) Match results (%) in sensitivity test set prototype M tuberculosis 129 7 28 one hundred M. tuberculosis 131 6 28 one hundred M. tuberculosis 106 7 28 one hundred M. tuberculosis 112 5 28 one hundred M. tuberculosis 107 5 21 one hundred

Test material Duration of observation (day) Match results (%) in sensitivity test set prototype M. tuberculosis 104 7 28 one hundred M. tuberculosis 156 6 28 one hundred M. tuberculosis 161 7 28 one hundred M. tuberculosis 130 7 28 one hundred M. tuberculosis 147 7 28 one hundred M. fortuitum 12 2 fourteen one hundred M. tuberculosis H37Ra 7 21 one hundred M. tuberculosis 135 5 21 one hundred M. tuberculosis 138 5 28 one hundred

From the above data it is seen that the use of the proposed test kit reduces the time for determining the drug sensitivity of mycobacteria to 2-7 days compared to 14-28 days on the prototype kit with 100% coincidence of the results. In addition to determining drug sensitivity, the proposed test kit allows for additional primary identification of mycobacterium tuberculosis.

The proposed test kit is not difficult and convenient to use, does not require additional equipment and special training of personnel, and, importantly, is cheap, and in terms of determining drug sensitivity, it approaches automatic systems of the WASTES type.

The technology was developed and industrial production of the test kit in convenient packaging for transportation and subsequent work was organized. Industrial production with the implementation of input control of raw materials and control of the final product allows the production of standard inexpensive test kits. Clinical trials of the test kit were carried out in five regions of the country in the study of about 1000 clinical isolates of mycobacterium tuberculosis, and positive results were obtained. A package of scientific and technical documentation for the production of a test kit has been issued, which is being approved by the Pharmacopoeia Committee.

The use of the proposed test kit of industrial manufacture in phthisiobacteriological laboratories will accelerate the determination of the drug sensitivity of mycobacteria, increase the reliability of the results, and will also make it possible to obtain comparative data by region for an accurate assessment of the epidemic situation in the country. A convenient form of release in hermetically sealed vials and a sufficient shelf life allows you to deliver and use test kits in the most remote regions of the country.

Information sources

1. On the improvement of anti-TB measures in the Russian Federation. Order of the Ministry of Health of the Russian Federation No. 109 of 03/21/03

2. Culture Media Manual, bio Merieux, 1994 Edition.

Claims (1)

Test kit for determining drug sensitivity and primary identification of Mycobacterium tuberculosis based on Levenshtein-Jensen medium with the addition of anti-TB drugs, characterized in that the set consists of 8 bottles with Levenshtein-Jensen medium, additionally containing sodium nitrate or potassium nitrate in a concentration of 0.6 -1.2 g / l, and starch in a soluble form at a concentration of 15.0-20.0 g / l, while four bottles designed to determine drug sensitivity additionally contain anti-tuber Aesthetic preparations, the first bottle is isoniazid at a concentration of 1 μg / ml, the second bottle is rifampicin at a concentration of 40 μg / ml, the third is streptomycin at a concentration of 10 μg / ml, the fourth is ethambutol at a concentration of 2 μg / ml, two bottles are intended for primary identification of mycobacterium tuberculosis, one of which additionally contains sodium salicylate at a concentration of 1000 μg / ml, and the other contains 2-thiophenecarboxyhydrazide at a concentration of 2 μg / ml, two bottles are control, and the kit additionally contains one bottle with Griss reagent for register tion of results on the appearance of a red color.