Method for assay of medicinal sensitivity of tuberculosis mycobacterium

FIELD: medicine, phthisiology, microbiology.

SUBSTANCE: diagnostic material is poured preliminary with chlorohexidine bigluconium solution, homogenized, kept at room temperature for 10-12 h and centrifuged. Precipitate is poured with Shkolnikova's liquid medium, incubated at 37oC for 3 days, supernatant part of Shkolnokova's medium is removed, fresh Shkolnikova's medium is added, and precipitate is stirred and inoculated on the dense cellular egg media. Sensitivity of the strain is determined in 3 weeks by the presence of growth in the control tube only. Invention provides enhancing precision and reducing time for assay. Invention can be used in assay for medicinal sensitivity of tuberculosis mycobacterium.

EFFECT: improved assay method.

3 ex

 

This invention relates to the field of medicine, section of bacteriology”, namely the laboratory diagnosis of tuberculosis.

Known in practical laboratory methods for determining the sensitivity of Mycobacterium tuberculosis to anti-TB drugs long and assume (taking into account the output of crops) date 49-60 days. Methods for determining sensitivity to anti-TB drugs using modern technologies used due to its high cost, not all laboratories. In addition, in the Arsenal of fisiopatologia there is a method for direct determination of drug sensitivity. This method assumes the presence of mycobacteria in the studied material, confirmed by direct microscopy. But it is not always possible to get a positive result.

Thus, the search is quite fast, with a guaranteed result of drug sensitivity of Mycobacterium tuberculosis to anti-TB drugs in terms of practical laboratory retains its relevance and has the prerequisites for its improvement.

Direct method of drug susceptibility testing is performed on both liquid and on solid nutrient media (Tonawanda, I.S. Mechev Guide to laboratory issledovaniya M. tuberculosis, Medicine. - 1973. - P.60-71.)

1. Determination of drug sensitivity of Mycobacterium tuberculosis to anti-TB drugs in liquid nutrient media.

From sputum prepare and treat strokes. The number of strokes is determined by the number of anti-TB drugs and those concentrations, which is expected to study the resistance of mycobacteria. Treated swabs dipped sterile forceps in germ tubes, which are pre-poured medium with different amounts of anti-TB drugs and control with medium without drug. After 10-14 days the tubes with glass sterilized. After sterilization, the swabs are removed from the test tubes, dried, fixed on the flames painted on ZIL-Nielsen and mikroskopiruyut (Tonawanda, I.S. Mechev. Guide to laboratory tests for tuberculosis. M, Medicine. - 1973. - P.60-71.)

2. Drug susceptibility testing office to anti-TB drugs on solid nutrient media.

Diagnostic material (sputum), in the presence of bacterioscopic method of ZIL-Nielsen 1-5 sticks mycobacteria, process as well as for normal sowing.

Processing material using for seeding sludge (Tonawanda, Issaeva guidelines on laboratory studies of tuberculosis is e M, Medicine. - 1973. - P.37). To handle take equal with respect to a material amount of 10% solution translesanas phosphoric acid sodium, mixed well and placed in the incubator for 20-24 hours, after which the mixture is centrifuged and the precipitate sow 3-4 tubes of egg environment.

When the direct method of determining drug resistance of Mycobacterium tuberculosis in dense environments pathological material is processed as described above and inoculated on solid nutrient medium containing different concentrations of anti-TB drugs. Read the results of the determination of sensitivity after 21 days of incubation (Tonawanda, I.S. Mechev. Guide to laboratory tests for tuberculosis. M, Medicine. - 1973. - P.70-71.)

Primary cultivation of Mycobacterium tuberculosis from the diagnostic material is complicated by the fact that when sowing bakteriostaticheski positive diagnostic material is not always possible to get the result, i.e. this prototype is insufficiently informative. And as a consequence clinicians receive a negative result direct determination and have to wait for the culture to determine drug sensitivity of Mycobacterium tuberculosis by an indirect method, i.e. the time lengthened.

With the aim of increasing the explanatory issledovaniia method for determining drug susceptibility of Mycobacterium tuberculosis by planting fluorescent-positive diagnostic material on the solid nutrient medium characterized in that before sowing on thick egg environment, with antibacterial drugs precipitate with diagnostic material last pour the liquid medium Shkolnikova. After rearing of M. tuberculosis within 3 days replacement of the supernatant to a fresh environment Shkolnikova in the amount of 3 ml.

Material (sputum) is subjected to a primary treatment to inhibit accompanying flora that prevents contamination of crops. The processing is carried out by Balan SCI-hlorgeksidinbiglyukonat.

Diagnostic material is filled with an equal volume (1:1) 0.05% solution hlorgeksidinbiglyukonat. Thoroughly homogenized. Leave for 10-12 hours at room temperature. The next day, the material is centrifuged at 1500-2000 rpm / min for 10 minutes Decant the supernatant, leaving the sediment. Last pour of 2.0 ml of a liquid medium Shkolnikova. Incubated in a thermostat at T 37°C for 3 days. After this period, the supernatant part of the environment Shkolnikova drained, not centrifuger. Fill in 3.0 ml of fresh medium Shkolnikova. The precipitate is stirred and seeded with 0.2 ml of a dense egg-based media containing anti-TB drugs. Term results after 3 weeks (21 days).

Division semi-synthetic environment Shkolnikova (Tonawanda, I.S. Mechev Guides the laboratory studies with M. tuberculosis, Medicine. - 1973. - P.153):

Potassium phosphate monobasic-acid - 1.5 g

Sodium Doonbeg phosphoric acid and 2.5 g

Magnesium sulfuric-acid - 05 g

Sodium citric acid (average) 1.5 g

Iron lemon collegeamerica - 0.005 g

L-asparagine - 1 g

Distilled water 1 l

Table 1
The comparison in terms of output and growth rate of the cultures of Mycobacterium tuberculosis parisianne with liquid media Shkolnikova on egg medium
The timing of re-sowing with a liquid medium to denseThe release dates of crops on egg medium (days, environments. arithm.)The intensity of growth on egg medium
After 3 days15,5+++
After 5 days15,5++
After 7 daysof 17.5+
(Growth intensity MW was estimated in the crosses: ++ up to 20 colonies; +++ more than 20 colonies)

When mycobacterial growth only in the test tubes strain is sensitive. When there is a growth of more than 20 colonies of mycobacteria in vitro with varying concentrations of the drug culture is regarded as stable.

Table 2
The comparison in terms of output and growth rate of cultures of M. tuberculosis without replacement and replace it with fresh fluid when reseeding on egg medium
 The release dates of crops on egg medium (days, environments. arithm.)The intensity of growth on egg medium
Without replacement by fresh liquid medium Shkolnikova15,5+
Replace it with fresh fluid Shkolnikova12,5+++

It is proved that the optimal time of incubation with native sediment material is 3 days. In table. 1 shows comparative data on the timing of the output of the cultures of Mycobacterium tuberculosis and growth rate of mycobacteria depending on the time of reseeding sediment diagnostic material with a liquid medium egg. From the presented data it follows that the optimal period reseeding with liquid egg after 3 and 5 days of growth. But when the term “3 days” egg environment is marked by more intensive growth of the cultures of mycobacteria.

Table 3
The growth of M. tuberculosis in the determination of drug sensitivity is eljnosti proposed and compared methods
The group of PatientsThe growth of M. tuberculosis AC, %Growth of tubercle bacilli not AC, %
Control186
N=2475,0%25,0%
Experienced22-
N=25100,0% 

After 3-day incubation of sediment required is to replace the environment Shkolnikova on a fresh environment. The conducted experiments proved that, if re-seeding sludge diagnostic material without replacing the liquid with a fresh release dates cultures of mycobacteria can grow up to 15.5 days and the intensity of growth on egg medium is evaluated to 1 cross. If the environment in which produced incubation for 3 days drained and replaced with fresh, marked shortening of the maturity of the crops on average up to 12.5 days and most importantly the intensity of the Mycobacterium growth is estimated at 3 of the cross, which is essential when assessing the growth of mycobacteria on egg medium when determining drug sensitivity (table 2). This manipulation helps to remove the toxic products of metabolism of mycobacteria, formed during the lag-phase of microbe reproduction.

The proposed method of determining Lekarstvo the th sensitivity of Mycobacterium tuberculosis to anti-TB drugs tested on 49 patients with different forms of tuberculosis, installed fluorescent-positive smear (24 - control group, 25 - experienced). In the control group of patients with drug sensitivity was determined in dense environments experienced by the proposed method. The research results are summarized in table 3.

In patients with fluorescent-positive smear-the use of the proposed method for determining drug susceptibility to anti-TB drugs with advanced growing mycobacteria in liquid nutrient medium allowed us to get a result in 100% of cases. Our proposed method for determining drug susceptibility simple to execute, available for any Microbiology laboratory.

The method for determining drug susceptibility of Mycobacterium tuberculosis by culture results on thick egg nutrient medium with antibacterial drugs, wherein the pre-diagnostic material pour an equal volume of 0.05%aqueous solution of hlorgeksidinbiglyukonat, homogenize, leave for 10-12 hours at room temperature, centrifuged for 10 minutes at 1500-2000 rpm, the sediment fill in 2.0 ml of a liquid medium Shkolnikova, incubated at 37°C for 3 days, the supernatant is poured part of the environment Shkolnikova, pour 3.0 ml of the fresh creditscoremooi, the residue is stirred, inoculated with 0.2 ml of a dense egg-based medium and the strain sensitivity determined after 3 weeks in the presence of growth only in a test tube.



 

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