RU2024021C1 - Method of tuberculosis diagnosis - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves sputum sample taking off, its autoclaving, extraction with chloroform-methanol mixture, evaporation, methylation, dissolving in hexene following by chromatomass-spectrometric assay of tuberculostearic acid. Method involves also separation of organic layer after extraction with chloroform-methanol mixture, its drying over anhydrous sodium sulfate and methylation at the methylating mixture boiling point for 15-20 min. EFFECT: simplified method, shortened time of assay.

Description

 The invention relates to medicine, namely to bacteriology, and can be used to identify tuberculosis microbacteria (Mycobacterium tuberculosis).

 There are various methods for the diagnosis of tuberculosis, including the identification of microbacterium tuberculosis (MBT) in pathological material.

 A known method for detecting MBT by direct bacterioscopy (see Perelman M.I., Koryakin V.A. and Protopopova I.M. Tuberculosis. M .: Medicine, p. 32-33). To do this, a drop of saline solution and sputum of the patient are applied to fat-free glass, which are thoroughly ground to homogeneity. It is dried, fixed with alcohol and stained with fuchsin Ziehl-Nielsen. The drug is microscopic. MBTs are colored red and the surrounding background is blue.

 Also known is the bacteriological method for the diagnosis of tuberculosis, including the sowing of the test material on a Levenshtein-Jensen nutrient medium. Cultivation is carried out for 14-90 days (see Perelman M.I., Karyakin V.A. and Protopopova I.M. Tuberculosis. M .: Medicine, p. 32-33). Then the culture is subcultured on glycerol medium, incubated, followed by treatment with acids and alkalis. In conclusion, a stability test is carried out - smears are immersed in solutions: 1 - sulfuric acid, 2 - potassium hydroxide (sodium), 3 - javelin water. Then the smears are stained and microscopic.

 By the presence in the microscopic picture of colorless oblong and wrinkled rods of mycobacterium tuberculosis, tuberculosis is diagnosed.

 The disadvantages of these methods include the low sensitivity of the methods and the long periods of diagnosis due to the cultivation of bacteria.

 Closest to the technical nature of the proposed is a method for the diagnosis of tuberculosis by detecting tuberculostearic acid, which is a biomarker of mycobacterium tuberculosis (J, Clin. Investig., V. 63, N 5, 1979, p. 813-819).

 The known method is as follows.

2-4 ml Sputum patient disinfected by autoclaving (at 121 ^{° C} for 60 minutes at P = 1.1 cr / cm ²⁾ was treated with 5 ml of 3% aqueous solution of lauryl sulfate and 1% sodium hydroxide solution while stirring within 20 minutes The resulting mixture was centrifuged at 3 thousand rpm for 30 minutes, the pH of the solution was adjusted with sulfuric acid to 7.0 and lyophilized. Then the samples were transferred to a 2-ml glass ampoule and extracted overnight with a mixture of chloroform-methanol (2: 1, V / V) at room temperature. After centrifugation for 20 min at 4000 rpm, the supernatant is transferred to a new vial and concentrated to dryness in a stream of nitrogen. Then 1 ml of a 3% methanol solution of hydrogen chloride is poured and the ampoule is sealed. Methanolysis held at ⁸⁰ ° C for 20 hours, at the end of the ampoule is centrifuged at 4000 rev / min for 20 minutes. The supernatant was evaporated to dryness under a stream of nitrogen and 100 ml of n-hexane was added. The resulting mixture was treated in an ultrasonic bath for 1 min at 125 V, then the solution was analyzed by thin layer chromatography (TLC). TCX conditions: plates are prepared by applying a layer of 0.25 mm silica gel (Silicagel Go F 254, Merck AG, Darmstadt, Germany). A mixture of n-hexane and diethyl ether (9: 1) was used as a solvent. After 30-40 minutes, the spot corresponding to methyl ether was scraped off the plate and extracted with ethyl acetate. Before injection into the gas chromatographic column, the sample was evaporated to dryness and dissolved in hexane.

 The sample was taken using a mass spectrometer (Varian Assaciatis, Instrument Dis, Palo Alto, Calif, MaT model 112) connected to a gas chromatograph (Varian. Associates, model 112).

The chromatographic conditions are as follows: glass column, ext. diameter 2 mm, filled with 3.7% OS-17 on Chromosorb W, HP 80/100 mesh column length - 3 m injector temperature -. 270 ^C, the furnace temperature - 230 ° ^C. Carrier gas - helium, its speed - 20 ml / min. The operating conditions of the mass spectrometer are as follows. Separator Temperature - ^{250 °} C Ion source temperature - 230 ^{° C,} electron energy 70 eV. The measurement was carried out during detection by one ion, respectively, by 312 m / z (molecular ion) and by 167 m / z. By the presence in the obtained mass spectrum of a characteristic molecular ion of tuberculostearic acid, tuberculosis is diagnosed. The total analysis time is 4 days. The disadvantages of this method include the duration and multi-stage operations preceding the chromato-mass spectrometric study of the sample.

 The aim of the proposed diagnostic method is to reduce the determination time and simplify the method.

 This goal is achieved in that a method for the diagnosis of tuberculosis is carried out by sampling, autoclaving, extraction with chloroform-methanol mixture, evaporation, methylation, dissolution in hexane and chromatography-mass spectrometric determination of tuberculostearic acid.

 Comparative analysis with the prototype shows that the distinguishing features of the proposed method are: separation of the organic layer after extraction, its drying over anhydrous sodium sulfate and methylation at the boiling temperature of the methylating mixture for 15-20 minutes

 These differences allow us to conclude that the claimed technical solution meets the criteria of the invention of "novelty."

A comparison of the proposed solution with other technical solutions shows that the method of separating the organic layer from the aqueous phase of the sample is widely used in organic chemistry, as well as the removal of traces of water from the organic extract using anhydrous Na ₂ SO ₄ salt.

 However, these techniques have not been used previously to prepare an anhydrous sample for chromatography-mass spectrometric analysis. The introduction of these techniques in the inventive method eliminated the technically difficult technique - freeze drying of the biosubstrate and thereby reduce the total analysis time by 4-6 hours.

The authors of the proposed method experimentally found that in the proposed method, the methylation process occurs in 15-20 minutes The necessary condition is to conduct the reaction at the boiling point mixture methylates (t = 65-70 ° ^C) to reflux. This method allows to reduce the time of diagnosis for a day, as in the known method the methylation process is within 20 hours (t = 80 ° ^C).

 Thus, the authors of the proposed diagnostic method established a new property of the analyzed mixture - the methylation process takes 15-20 minutes, which allows us to conclude that the technical solution meets the criterion of "significant differences".

 The claimed method for the diagnosis of tuberculosis is as follows.

For analysis, 2 to 10 mg of sputum biosubstrate, the contents of the pleural cavity or drainage fluid are taken. Biosubstrates autoclaved at 121 ^{° C} for 60 minutes, thereto was added 5 ml of sodium hydroxide (4%) is shaken for 10 minutes, neutralized with dilute H ₂ SO ₄ to pH 7.0 and extraction mixture is poured - chloroform: methanol, taken in 2: 1 ratio. The ratio of biosubstrate and extractant is 1: 1. Extraction is carried out at room temperature for 20 hours. Then the mixture is centrifuged for 15 minutes at 3000 rpm, the lower organic layer is separated, which is dried over Na ₂ SO ₄ (anhydrous) . After which the extract is filtered off from Na ₂ SO ₄ and evaporated to dryness in a stream of nitrogen. The residue is methylated in a 3% HCl solution in methanol at the boiling point of 65-70 ^{° C} for 15-20 min. Then the solution is evaporated in a stream of nitrogen, the residue is dissolved in 10 ml of hexane and analyzed on a chromatography-mass spectrometer (XMS) by selective ion detection (SIM) at m / z 312 and recording the complete mass spectrum of C ₁₉ acid methylate. The main fragmented ions of the mass spectrum of tuberculostearic (TbS) acid: (m / z) 312, 269, 199, 171, 167, 149, 141, 113.

The XMS analysis is performed on an instrument with a LKB 20-91 chromatography-mass spectrometer with an RDP1134 computer. Chromatography conditions: glass capillary column (l = 25) with a stationary liquid phase SE-30; injector temperature - 350 ^o C; thermostat temperature - 140 - >> 320 ^о С, 4 '/ min; carrier gas - He, V _He - 20 ml / min.

Mass spectrum removal conditions: separator temperature - 320 ^о С; ion source temperature - 320 ^{° C;} the energy of ionizing electrons is 70 eV. The ion current was recorded for one ion, m / z 312, i.e. in selective ion detection mode. The minimum detectable concentration of TbS acid in the sample was 100 mg / ml. The total analysis time took 2 days.

PRI me R 1. Taken to the study of sputum from patient G., admitted to the purulent department of the center of a surgical infection with a preliminary diagnosis of lung gangrene (N case history 5850, date of analysis 20.12.90). 10 ml sputum was autoclaved at 121 ^{° C} for 30 min, R = 1.1 kp / cm ^2. 5 ml of a 4% NaOH solution was added to the disinfected biosubstrate, the contents were shaken for 10 min, then neutralized with diluted H ₂ SO ₄ to pH 7.0 and 15 ml of chloroform-methanol (2: 1) was added with the extractant and left on night at room temperature. After centrifugation, the lower organic layer was separated and dried over anhydrous Na ₂ SO ₄ for 20 minutes. The extract was filtered off from Na ₂ SO ₄ and evaporated to dryness in a stream of nitrogen. The dry residue was methylated with a 3% solution of hydrochloric acid in methanol prepared by adding 5 ml of acetyl chloride to 100 ml of dry MeOH. Methylation was carried out with ten ml of methylating agent while heating to boiling point (under reflux) for 15 minutes. The methylated product was evaporated to dryness under nitrogen flow, the residue was dissolved in 100 μl of hexane and analyzed on an IKB 2091 chromatography-mass spectrometer with an RDP-1134 computer using a glass capillary column l = 25 m with a stationary liquid phase SE-30. Chromatography was performed at a temperature of 140-320 ^about C, 4 ^about / min; carrier gas Not; injector temperature - 350 ^o C; ion source temperature - 320 ^{° C;} separator temperature - 320 ^{° C;} the energy of ionizing electrons is 70 eV. The TBS acid mass spectrum was recorded at a maximum intensity of 1357. As a result of the analysis, the presence of tuberculostearic acid in the sample was established.

PRI me R 2. Sponge was taken from patient C. with a preliminary diagnosis of bilateral abscessed pneumonia (N case history 1899). 10 ml of sputum were autoclaved (121 ^° C, 30 min, P = 1.1 cr / cm ² ) with 5 ml of 4% aqueous NaOH solution added to it, the contents were shaken for 10 min, the solution was neutralized with diluted H ₂ SO ₄ to pH 7.0 and filled with extractant chloroform-methanol (2: 1, V / V) in an amount of 15 ml and left overnight at room temperature. After centrifugation, the lower organic layer was separated and dried over 15 g of anhydrous NaSO ₄ for 20 minutes. The extract was filtered and evaporated to dryness in a stream of nitrogen. The dry residue was methylated with a 3% solution of HCl in methanol. Methylation was carried out with 10 ml of methylating agent while heating to boiling point (under reflux) for 20 minutes. The methylated product was evaporated to dryness under a nitrogen stream, the residue was dissolved in 100 μl of hexane and analyzed on a chromatography-mass spectrometer (XMS analysis conditions are given above). The mass spectrum was recorded in the yield range of TbS acid at an intensity of 2857. No tuberculostearic acid was detected.

From the presented materials it follows that the claimed method allows to reduce the total analysis time by 2 times (2 days together 4 according to the prototype method) and significantly simplify it by eliminating the methods of lyophilization. To clearly identify the differences between the compared technical solutions, their comparative analysis is given:
The known method 1. Taking bioassays (sputum) 2. sample Autoclaving at 121 ^{° C} for 60 min 3. Treatment of 3% aqueous sodium lauryl sulfate and 1% aqueous sodium hydroxide solution under agitation for 20 min at 4. Centrifugation 3 thousand about in minutes for 30 minutes 5. Bringing the pH to 7.0 (using sulfuric acid) 6. Lyophilization 7. Extraction with a mixture of chloroform - methanol (1: 1) for 20 hours 8. Centrifugation at 4 thousand vol. min during 20 min 9. The concentration of nitrogen stream (to dryness) 10. Addition of 1 ml of 3% methanolic acid solution 11. methanolysis at ⁸⁰ ° C for 20 h 12. Centrifugation at 4 th. vol. per min for 20 min 13. Evaporation of the supernatant in a stream of nitrogen 14. Addition of 100 ml of n-hexane 15. Processing of the mixture in an ultrasonic bath for 1 min at 125 V 16. Analysis by thin-layer chromatography on silica gel, n-hexane-diethyl eluent ether (9: 1) 17. Scraping off the stain after 30-40 minutes, corresponding to methyl ether and extracting it with ethyl acetate 18. Evaporation to dryness 19. Dissolution in hexane 20. Analysis by chromatography-mass spectrometer 21. Chromatography conditions: glass column, l = 3 m, internal d = 2 mm, filled with 3.7% OU-17 on chrome osorbe WHP 80/100 mesh,
t injector = 270 ^about C.

t thermostat = 230 ^о С,
carrier gas is helium,
V _He = 20 ml / min 22. The operating conditions of the mass spectrometer:
separator t = 250 ^о С,
t ion source = 230 ^about With
E = 70 eV, the measurement was carried out with selective detection by ions m / z 312 and 167.

Total analysis time - 4 days
The inventive method 1. + 2. + 3. Processing 4% aqueous sodium hydroxide solution, shaking for 10 minutes 4. - 5. + 6. - 7. +, solvent ratio 2: 1, the ratio of extract to extractant 1: 1, + 8. +, at 3 thousand about. per minute for 15 minutes, the separation of the lower organic layer, drying it over anhydrous sodium sulfate, filtering 9. + 10. + 11. + at 65-70 ^{° C} for 15-20 min 12 - 13 + 14. +, 10 ml of n-hexane 15. - 16. - 17. - 18. - 19. - 20. + 21. Chromatography conditions: glass capillary column, l = 25 m, filled with 5% SE-30 on chromosorb,
t injector 350 ^o With
t thermostat 140-320 ^{C, of} 4 / min,
carrier gas is helium,
V _He = 20 ml / min 22. The operating conditions of the mass spectrometer:
separator t = 320 ^о С,
t ion source = 320 ^about With
E = 70 eV, the measurement was carried out with selective ion detection by ion m / z 312.

Total analysis time - 2 days
The main distinguishing methods of the claimed method / providing movement of the goal / are drying the organic layer over anhydrous sodium sulfate and conducting the methylation process for 15-20 minutes at 65-70 ^about C.

Claims (1)

DIAGNOSTIC TUBERCULOSIS METHOD, including sputum sampling, autoclaving at 121 ^o C for 60 min at a pressure of 1.1 cr / cm ² , extraction with chloroform-methanol mixture taken in a 2: 1 ratio, centrifugation, evaporation of the supernatant in a stream of nitrogen, adding to the dried supernatant of a 3% methanolic hydrogen chloride solution and incubating the resulting mixture at 80 ^o C, re-centrifuging the supernatant and evaporation, dissolving the latter in hexane followed by determination tuberkulostearinovoy acid chromate graphic and mass spectrometric techniques, characterized in that, in order to accelerate and simplify the process, the organic layer was separated after centrifugation and before evaporation it was dried over anhydrous sodium sulfate and incubation was carried out at 65 - 70 ^o C for 15 - 20 min.