Method of determining drug susceptibility of tuberculosis mycobacteria

FIELD: medical microbiology.

SUBSTANCE: invention relates to microbiological diagnostics of the drug susceptibility of tuberculosis mycobacteria to antituberculous preparations. Method provided by invention relates to mycobacteria M. tuberculosis, which is realized on nutrient medium consisting of two layers. Preparation of biphasic medium comprises placing 2 ml sterile perfluorodecaline into test tube followed by placing 2 ml Shkolnikova medium over it to form system, wherein lower phase occupies heavier perfluorodecaline and higher phase nutritive broth. To be diluted in the medium, different weighed specimens of principal antituberculous preparations are prepared from pure substances. Powders are first diluted with sterile distilled water and thus prepared solution is introduced into nutrient medium by 0.1 ml portions to provide final concentration of each preparation in medium corresponding to critical concentration. Strain being tested is placed in test tubes in the form of microbial slurry in amounts 2 ml for each test tube. Microbial growth in test tubes with antituberculous preparations is estimated in comparison with control growth with the aid of 2% solution of triphenyltetrazolium chloride, which is added into each of 5 test tubes in amounts 0.1 ml on the 8th culturing day. Test tubes are the incubated for further 24 h. During this period, colonies if mycobacteria grown in interphase border acquire vinous coloration. Culture is regarded as stable if coloration intensity of colonies grown in interphase border roughly corresponds to coloration intensity of control. When accuracy data regarding determination of drug susceptibility is compared to reference methods, the data were found to be completely the same.

EFFECT: reduced determination time (to 9 days).

2 cl, 2 tbl

 

The invention relates to medicine, namely to microbiological diagnosis of drug sensitivity of M. tuberculosis to anti-TB drugs, and can be used in bacteriological laboratories for TB control in conditions of limited funding.

A known method of determining the success of mycobacteria using a handheld fluorescent systems BBL MGIT (BD). Indication of the growth of mycobacteria in this system is a fluorescence indicator, located in the bottom part of the tube MGIT (Mycobacteria Growth Indicator Tube)containing broth Middlebrook 7H9 broth suspensions. Results in this system needs a source of ultraviolet radiation - transilluminator (wavelength 365 nm) or the device MicroMGIT. In addition, an alternative to the classical methods represent a biological system based on a broth culture using analyzers: semi-automatic radiometric, WASTES 460 and fully automated WASTES MGIT 960, which are well compatible with each other. Definition LCH using manual bacteriological system BBL MGIT or analyzers WASTES 460, WASTES 960 lasts 3-4 weeks, with testing associated with expensive equipment and reagents, which makes it unacceptable when insufficient resources common is the preservation (TB Problem. - 2002 - No. 1 - str-62).

The closest rapid and cheap way to determine LCH M.tuberculosis is a method of inoculation of microbial cells in a liquid enriched environment Shkolnikova with anti-TB drugs with subsequent determination of growth of M.tuberculosis.

(Drabkina P.O. Microbiology of tuberculosis. - M.: Medgiz, 1963, - 225 pages).

However, the known method does not provide the necessary growth of M.tuberculosis, efficiency, speed and accuracy LCH.

The purpose of the proposed method is to increase the growth rate of M. tuberculosis, improving the accuracy and reducing time determine LCH M.tuberculosis, which increases the efficiency definition LCH to drugs and as a consequence the timely use of essential anti-TB therapy and correction of the treatment.

This goal is achieved by creating a two-phase liquid medium, as the lower phase in which use perpendicular-gas-substrate - concentration of free ions of less than 10-6M, with a ratio of layers enriched environment Shkolnikova to the substrate, is equal to 1:1, followed by evaluation of metabolic activity of microbial cells with 2%triphenyl-tetrazole chloride.

The use of gas substrate provides an opportunity to improve the aeration of the growing culture and to slow the development of acidosis due to g is tabmenu between the environment and the substrate and on the topic of phases to conduct calorimetric evaluation of the presence or absence of growth of M. tuberculosis by 2%triphenyl-tetrazole chloride (hereinafter referred to TTX), which is within 24 hours stains actively growing microbial population.

In the presence of a growing mycobacteria colorless TTH changes its color to Burgundy/crimson staining micro-colonies. The value of mycobacterial growth, which is located mainly on the phase boundary, it is much easier to assess visually using TTX.

The proposed method for the determination of drug sensitivity of M. tuberculosis using a two-phase medium with PFD is as follows.

For the preparation of a two-phase medium sterile PFD in the amount of 2 ml was placed in a test tube, it was layered with 2 ml of enriched environment Shkolnikova. Thus was formed a two-phase system, the lower phase which was more severe PFD, and the top - nutrient broth - enriched environment Shkolnikova.

Prepared sample of the main anti-TB drugs of pure substances, then the powders were diluted with sterile distilled water, the solution was introduced into the nutrient medium 0.1 ml In the final concentration of each of the drugs in the environment corresponded shown in table 1.

Table 1

Critical (low) concentration of drug (ßg/ml) in test tubes

Environment to determine LCH Anti-TB drugs contained in the media
 StreptomycinIsoniazidRifampicinEthambutol
Shkolnikova0,8*0,1*1,0*3,5*

Table 2 presents all the necessary in the formulation of the test components that were made in 4 test tubes with anti-TB drugs and 1 tube to control growth.

Table 2

Drug susceptibility testing of M.tuberculosis using a two-phase system

MedicationPFDEnriched environment SHK.Aq medicationDistil. waterMicrobial suspensionTTX
Streptomycin2,02,00,1 0,20,1
Isoniazid2,02,00,1 0,20,1
Rifampicin2,02,00,1 0,20,1
Ethambutol2,02,00,1 0,2 0,1
The growth control (without drug)2,02,0 0,10,20,1

The object of the study were three cultures of mycobacteria:

M.tuberculosis H37Rv laboratory strain-sensitive

anti-TB drugs;

M. tuberculosis clinical isolates resistant to isoniazid;

M. tuberculosis clinical isolates resistant to streptomycin,

isoniazid, rifampicin and ethambutol.

From each culture were prepared microbial suspension in saline with 0.05% tween-80 standard turbidity risk corresponding to the 5×108microbial cells/ml suspension was then diluted 1:10. The test strain in the form of microbial suspension was introduced into each test tube 0.2 ml

Microbial growth in test tubes with anti-TB drugs was assessed by comparison with the growth in the control using a 2% solution of TTX, which was added 0.1 ml into each of 5 tubes on the 8th day of cultivation. Then the tubes were incubated for another 24 hours, during this period the colonies of mycobacteria grown on the phase boundary, was painted in Burgundy. Culture is considered sustainable if the intensity of staining of colonies grown on the phase boundary, in vitro drug were similar intensity of staining in control.

The obtained result is t determine drug sensitivity were compared with reference methods, which is regulated by the Ministry of health of the Russian Federation No. 109 (2003): on the solid l÷wenstein-Jensen (L-J) and using the equipment BBL MGIT AST, which uses a liquid medium and the fluorescent method.

On the solid l÷wenstein-Jensen (L-Th) detect drug-resistant mutants in the culture of M. tuberculosis within 3-4 weeks. The most significant drawback of this method is the long term study, significantly complicating the timely use of essential anti-TB therapy and correction of the treatment. The disadvantage of using equipment BBL MGIT AST, which uses a liquid medium and the fluorescent method is its high cost, because it requires expensive equipment and expensive reagents.

Example 1 implementation definition LCH M.tuberculosis in the liquid two-phase environment.

To determine LCH M.tuberculosis selected 30 isolates of M.tuberculosis isolated from patients with various forms of pulmonary tuberculosis. 16 cultures of M.tuberculosis were sensitive, and 14 are resistant to anti-TB drugs of the first row.

The introduction of anti-TB drugs: streptomycin (S), isoniazid (I), rifampicin (R) and ethambutol (E), and inoculation of the studied culture of M.tuberculosis in a two-phase system of cultivation was made in accordance with the CX is my above.

The results obtained with the developed method was compared with the data definition LCH obtained by the classical method of absolute concentrations in dense environments Levenshtein-Jensen medium and method of using the equipment BBL MGIT AST, which was considered as reference methods. Received full match results sensitivity of Mycobacterium tuberculosis to anti-TB drugs. However, in dense environments the study took 28 days in liquid media using equipment BBL MGIT AST - 20 days, but the proposed method is only 9 days, with this method, low-cost and easy to execute.

Most important for treatment and prognosis of the disease is the identification of multiresistentsuse, i.e. determination of the sensitivity of M. tuberculosis to rifampicin and isoniazid.

Thus, the proposed method does not require expensive equipment and reagents, is comparable to the results with traditional methods and allows you to define LCH cultures of M.tuberculosis, most importantly, to rifampicin and isoniazid for 9 days, which is several times faster than other methods, and allows for the timely use of essential anti-TB therapy and correction of the treatment.

1. The method for determining drug susceptibility of Mycobacterium t is tuberculosis by inoculation of microbial cells in a liquid enriched environment Shkolnikova with anti-TB drug, characterized in that the planting is carried out in a double-Wednesday, bottom layer which is perpendicular with a concentration of free ions of less than 10-6M and the number adaptirovannyh impurities less of 0.003 wt.%, and the top layer is enriched environment Shkolnikova when the ratio of layer 1:1, followed by evaluation of metabolic activity of microbial cells, and the degree of metabolic activity determine the sensitivity or resistance of mycobacteria to anti-TB drug.

2. The method according to claim 1, characterized in that the metabolic activity was determined by staining of microbial cells solutions triphenyl-tetrazole chloride.



 

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EFFECT: enhanced growth of mycobacteria.

1 tbl

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