RU2244927C2 - Method for assay of medicinal sensitivity of tuberculosis mycobacterium - Google Patents

Abstract

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 37^oC 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

Description

This invention relates to medicine, the section "bacteriology", and in particular to laboratory methods for the diagnosis of tuberculosis.

Known in practical laboratories, methods for determining the sensitivity of tuberculosis mycobacteria to anti-TB drugs are long and suggest (taking into account the yield of cultures) a period of 49-60 days. Methods for determining the sensitivity to anti-TB drugs using modern technologies are not used in all laboratories due to their high cost. In addition, in the arsenal of phthisiobacteriologists there is a method for the direct determination of drug sensitivity. This method assumes the presence of mycobacteria in the test material, confirmed by direct bacterioscopy. But he does not always allow you to get a positive result.

Thus, the search is fast enough, with a guaranteed result of the data on the drug sensitivity of mycobacterium tuberculosis to anti-TB drugs in practical laboratories, remains relevant and has the prerequisites for its improvement.

The direct determination of drug sensitivity is carried out both on liquid and solid nutrient media (T.N. Yashchenko, I.S. Mecheva, Laboratory Manual for Tuberculosis M., Medicine. - 1973. - P.60-71.)

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

Sputum is prepared and processed from sputum. The number of smears is determined by the number of anti-TB drugs and those concentrations to which it is supposed to study the resistance of mycobacteria. The treated smears are dipped with sterile forceps into bacteriological tubes into which media with different contents of anti-TB drugs are pre-poured and a control one with medium without the drug. After 10-14 days, the glass tubes are sterilized. After sterilization, smears are removed from the tubes, dried, fixed over a flame, stained according to Ziehl-Nielsen and microscopic (T.N. Yashchenko, I.S. Mecheva. Guidelines for laboratory research for tuberculosis. M., Medicine. - 1973. - С .60-71.)

2. Determination of drug sensitivity of the office to anti-TB drugs in solid nutrient media.

Diagnostic material (sputum), in the presence of a bacterioscopic method according to Ziehl-Nielsen 1-5 sticks of mycobacteria, is treated as for normal seeding.

Processing of material using sediment for sowing (T.N. Yashchenko, I.S.Mecheva Guidelines for laboratory tests for tuberculosis M., Medicine. - 1973. - P.37). For processing, take an equal amount of a 10% solution of trisubstituted phosphoric acid sodium with respect to the material, mix well and place in a thermostat for 20-24 hours, after which the mixture is centrifuged and the precipitate is inoculated into 3-4 test tubes of egg medium.

With the direct method for determining the drug resistance of tuberculosis mycobacteria on solid media, the pathological material is treated as described above and plated on solid nutrient media containing various concentrations of anti-TB drugs. Reading the results of determining sensitivity after 21 days of incubation (T.N. Yashchenko, I.S. Mecheva. Manual for laboratory tests for tuberculosis. M., Medicine. - 1973. - P.70-71.)

The primary cultivation of mycobacterium tuberculosis from diagnostic material is complicated by the fact that when sowing bacterioscopically positive diagnostic material, it is not always possible to obtain a result, i.e. This prototype is not informative enough. And as a result, clinicians get a negative result of a direct determination and are forced to wait for the culture to determine the drug sensitivity of mycobacterium tuberculosis by an indirect method, i.e. timelines for obtaining results are lengthening.

In order to increase the information content of the study, a method is proposed for determining the drug sensitivity of tuberculosis mycobacteria by plating a luminescent-positive diagnostic material on a solid nutrient medium, characterized in that before plating on a dense egg medium, with antibacterial sediment from the diagnostic material, the latter is poured with Shkolnikova's liquid medium. After growing mycobacterium tuberculosis for 3 days, the supernatant is replaced with fresh Shkolnikova's medium in an amount of 3 ml.

Material (sputum) is subjected to primary processing in order to suppress concomitant microflora, which prevents contamination of crops. Processing is carried out according to Balan V.F. chlorhexidinebigluconicum.

Diagnostic material is poured with an equal volume (1: 1) of a 0.05% solution of chlorhexidine bigluconicum. Homogenize thoroughly. Leave for 10-12 hours at room temperature. The next day, the material is centrifuged at 1500-2000 rpm for 10 minutes. Discard the supernatant, leaving a precipitate. The latter is poured into 2.0 ml of Shkolnikova's liquid medium. Incubated in a thermostat at T 37 ° C for 3 days. After this period, the supernatant of Shkolnikova’s medium is drained without centrifugation. Pour 3.0 ml of fresh medium Shkolnikova. The precipitate is mixed and inoculated with 0.2 ml in dense egg media containing anti-TB drugs. The term for the delivery of results after 3 weeks (21 days).

Prescription of the semisynthetic environment of Shkolnikova (T.N. Yashchenko, I.S. Mecheva, Laboratory Manual for Tuberculosis M., Medicine. - 1973. - P.153):

Potassium monobasic phosphoric acid - 1.5 g

Sodium dibasic phosphoric acid - 2.5 g

Magnesia Sulfuric Acid - 05 g

Sodium citric acid (medium) - 1.5 g

Ammonium citric acid iron - 0.005 g

L-asparagine - 1 g

Distilled water 1 l

Table 1 Comparison of the yield and growth rate of cultures of Mycobacterium tuberculosis seeded with Shkolnikova’s liquid medium on egg media Duration of reseeding from liquid to dense The timing of the output of crops on the egg environment (days, Wednesday. Arithm.) Egg growth rate In 3 days 15,5 +++ In 5 days 15,5 ++ In 7 days 17.5 + (MVT growth rate was estimated in crosses: ++ up to 20 colonies; +++ more than 20 colonies)

In the presence of growth of mycobacteria only in control tubes, the strain is considered sensitive. In the presence of growth of more than 20 colonies of mycobacteria in test tubes with one or another concentration of the drug, the culture is regarded as stable.

table 2 Comparison of the yield and growth rate of cultures of tuberculosis mycobacteria without replacement and with replacement with fresh liquid medium when replanting on egg media The timing of the output of crops on the egg environment (days, Wednesday. Arithm.) Egg growth rate Without replacement for Shkolnikova’s fresh fluid 15,5 + With a replacement for fresh liquid medium Shkolnikova 12.5 +++

It is proved that the most optimal period of incubation of sediment from native material is 3 days. In the table. Figure 1 shows comparative data on the yield of cultures of mycobacterium tuberculosis and the growth rate of mycobacteria depending on the timing of reseeding of the sediment of diagnostic material from the liquid medium to the egg. From the above data it follows that the most optimal time for reseeding from a liquid medium to the egg after 3 and 5 days of growth. But with the term “after 3 days” on the egg medium, a more intensive growth of cultures of mycobacteria is noted.

Table 3 Growth of Mycobacterium tuberculosis in determining drug sensitivity by proposed and compared methods Patient Groups The growth of mycobacterium tuberculosis A.h.,% There is no growth of mycobacterium tuberculosis A. hours,% Control eighteen 6 N = 24 75.0% 25.0% Experienced 22 - N = 25 100.0%

After a 3-day incubation of the sediment, it is mandatory to replace Shkolnikova’s medium with fresh medium. The experiments have shown that if reseeding of diagnostic material without replacing the liquid medium with a fresh yield of mycobacterial cultures is lengthened to 15.5 days and the growth rate on the egg medium is estimated to be 1 cross. If the medium in which the incubation was performed for 3 days is drained and replaced with fresh, then the crop yield is shortened to an average of 12.5 days and the most important growth rate of mycobacteria is estimated at 3 crosses, which is the main one when assessing the growth of mycobacteria on egg medium when determining drug sensitivity (Table 2). This manipulation helps to remove the toxic metabolic products of mycobacteria formed during the lag phase of microbial reproduction.

The proposed method for determining the drug sensitivity of tuberculosis mycobacteria to antituberculosis drugs was tested on 49 patients with various forms of tuberculosis, with a luminescent positive sputum smear established (24 - control group, 25 - experimental). In the control group of patients, drug sensitivity was determined on solid media in the experimental proposed method. The research results are shown in table 3.

In patients with a luminescent-positive sputum smear, the use of the proposed method for determining drug sensitivity to anti-TB drugs with preliminary growth of mycobacteria in a liquid nutrient medium allowed to obtain a result in 100% of cases. Our proposed method for determining drug sensitivity is simple to implement, available for any microbiological laboratory.

Claims (1)

A method for determining the drug sensitivity of tuberculosis mycobacteria by seeding diagnostic material on a solid egg nutrient medium with antibacterial drugs, characterized in that the preliminary diagnostic material is poured with an equal volume of a 0.05% chlorhexidine bigluconicum solution, homogenized, left for 10-12 hours at room temperature, centrifuged for 10 min at 1500-2000 rpm, the precipitate was poured into 2.0 ml of Shkolnikova's liquid medium, incubated at 37 ° C for 3 days, the supernatant of medium Ш was drained olnikovoy, poured 3.0 ml of fresh medium Shkolnikova, the precipitate was stirred, seeded with 0.2 ml per egg dense medium and the strain sensitivity was determined after 3 weeks of growth in the presence of only the control tube.