RU2162709C1 - Method of indication of tuberculosis mycobacterium in atmosphere air - Google Patents

Abstract

FIELD: medicine, phthisiology. SUBSTANCE: method of indication of tuberculosis mycobacteria in air medium involves passing air sample through electric field. Bacteria are charged in the zone of negative charge and precipitated on positively charged electrode made as a changeable metallic tray covered with liquid nutrient medium for culturing tuberculosis mycobacteria. The latter are cultured for 2 days, concentrated and supernatant is removed. Obtained precipitate is resuspended and tuberculosis mycobacteria are identified by molecular-genetic method using polymerase chain reaction. Method ensures to detect tuberculosis mycobacteria in air for short time (3-5 days). Method provides to reveal minimal concentrations of tuberculosis mycobacteria that are impossible to detect by methods of bacterioscopy and cultural diagnosis. EFFECT: improved method of indication. 3 cl, 2 tbl

Description

 The invention relates to medicine, in particular sanitation and hygiene.

 The issue of determining mycobacterium tuberculosis in the air is extremely relevant, since tuberculosis is an airborne infection and infection occurs through the air. In 1962, R.L. Riley et.al. Studying the infection of the air in the office, it was shown that the air in the wards where tuberculosis patients with bacilli are located causes a generalized specific process in guinea pigs. Later studies (N.V. Chugunikhina, 1971) also showed that after isolation from a macroorganism, mycobacteria can be in the air in the droplet phase of the aerosol for several hours, and then slowly settle to the surface of various objects, where they can accumulate in significant quantities. exceeding the minimum dose of infection. Of particular interest is the contamination of the premises of the TB institution with mycobacteria, where a significant amount of tuberculous mycobacteria can be released into the environment. At the end of the last century, Cornet found that medical workers suffer from tuberculosis more often than the rest of the population, modern epidemiological data confirm this (A.E. Rabukhin, 1957, M.N. Zueva, 1996).

However, this issue of determining the presence of mycobacteria in the air has not yet been resolved due to the following reasons:
1. The bacteriological method is not sensitive enough. The concentration of mycobacteria in the air, even in the immediate surroundings of a patient with a bacterial excretion, is insufficient to detect it with bacteriological methods (bacterioscopy and culture diagnostics).

 2. Mycobacterium tuberculosis in nutrient media grows very slowly and the result becomes known after 2.5-3 months, which is unacceptable for sanitary purposes, because the result loses its meaning.

 According to M.N. Lebedeva (1963), G.M. Turkina (1982), L.A. Vinogradova (1988), EANardell et.al. (1991), the sanitary-bacteriological study of air can be carried out by two methods: sedimentation and suction. Mycobacteria were isolated from the air by the sedimentation method (by means of their natural deposition on plates with nutrient media), and recently, the aspiration method is widely used, which represents the process of pumping large volumes of air through various filters.

 The sedimentation method (according to Koch) - the sedimentation of microbes under the action of gravity - is a simple method for studying non-specific air microflora. It consists in the fact that Petri dishes with the medium are left open for a certain time (5-10 minutes for general seeding and at least 40 minutes for coccal microflora).

 Aspiration method using the Krotov apparatus. It makes it possible to explore a certain amount of air. The device is mounted in a portable box and consists of a unit for sampling air, a micromanometer and an electric motor.

 N.V. Chugunikhina (1973) conducted a comparative assessment of 3 air sampling methods for the quantitative determination of the causative agent of tuberculosis: the method of membrane filters, AFA-B-18 filters, POV-1 device (Air sampling device - 1).

 The mechanism for trapping microorganisms with the POV-1 device is based on dispersing a liquid by a current of passing air and retaining bacteria that are in the dust and droplet phases of the aerosol on drops of the trapping liquid.

 Drops of trapping liquid hit the vertical surface of the bacterial trap, flow down and disperse again. Shkolnikova's synthetic medium in an amount of 5 ml was used as a trapping liquid.

 Since the existing microbiological methods for detecting MBT in indoor air are not sensitive enough, and the study takes a long time, indirect indicators are used (determining the total microbial content and the content of Staphylococcus aureus in 1 cubic meter of air), which do not allow an objective assessment of the presence or absence of air anti-TB rooms of the Office. Therefore, it does not seem possible to purposefully carry out preventive and anti-epidemic measures in them.

 V.V.Drobenya (1980) showed that the PAB-1 device extracts 40% more microorganisms from the air than the Krotov apparatus.

 The device PAB-1 (aerosol sampler bacteriological) was designed by the Leningrad branch of the All-Union Scientific Research Institute of Medical Instrument Making. The device is based on the principle of electrical precipitation. PAB-1 consists of 2 portable units: the actual device and styling. The device itself is mounted in a portable box and consists of an air sampling unit (where a metal thread is mounted to create a zone of negative corona discharge), a micromanometer, a microammeter and an electric motor. The installation has 40 metal pallets for culture media and 20 test tubes for transporting liquid culture media. In the PAB-1 device, particles (bacteria), when air samples are sucked in, are charged in the zone of negative corona discharge, and then fall into the electric field and settle on a positively charged electrode made in the form of a replaceable metal tray, which is covered with a liquid or a dense nutrient medium. Using the PAB-1 device, you can take air samples at a speed of 150-250 liters per minute.

 Uncultured bacteria are not detected by traditional microbiological research methods. Using molecular genetic technology (PCR) revealed these mycobacteria. The phenomenon of the existence of viable bacteria in an uncultured state is of great importance in the infectious pathology of humans and animals, since it has been established that uncultured forms of bacteria retain their pathogenic potential. The discovery of uncultivated forms gave a new impetus to the study of the ecological and molecular genetic mechanisms of the existence of pathogens in nature (Romanova Yu.M., 2000).

 Of great interest both to researchers and to practical laboratories is the method for determining infectious agents using the polymerase chain reaction (PCR) method. This method of multiplication (amplification) of specific nucleotide sequences (Mullis K.V., Faloona F.A, 1987), with which you can multiply a DNA fragment with the desired nucleotide sequence millions of times in a few hours. The principle of the method was proposed and implemented in 1985 by the American scientist Cary Mulis (Mullis K.V., Faloona FA, 1987) and is now successfully used in determining the causative agents of numerous infectious diseases: tuberculosis, leprosy (Hartskeeri RA, De Wit MYL, Klatser PR, 1989), chlamydia (Griffais R., Thibon M., 1989), mycoplasmosis (Bemet C., Garrett M., de Barbeuras B. et al., 1989), viral diseases (Saito l., Servenius B., Compton T., Fox RI, 1989; Shibata D., Martin WJ, Appieman MD et al., 1988); in determining the resistance of the pathogen to drugs (Muller L. P., Grawford J.T., Shinnick T.M., 1994; Telenti A., Imboden P., Marchesi F. et al., 1993); for species identification of the pathogen (Musial C.E., Tice L.S. Stockman L., Roberts G. D., 1988; Yamamoto T., Shibagaki T., Yamori S. et al., 1993); to evaluate the effectiveness of treatment (Kennedi N., Gellespie S.H., Saruni A.O. et al. 1994); for the diagnosis of genetic disorders (Boehm C. D., 1989). The polymerase chain reaction method is designed to detect the pathogen DNA. In this way, it compares favorably with other laboratory methods that provide for the detection of indirect signs of infection (detection of antibodies), have a low level of sensitivity (serological methods) or give delayed results (for a number of infections - the cultural method). The PCR method has a particular advantage in detecting microorganisms that are latent or present in such small quantities that they cannot be detected by traditional methods, or those microorganisms that are difficult to cultivate and require a complex nutrient medium (Hartskeeri RA, De Wit MYL, Klatser PR, 1989; Pao CC, Lin SS, Wu SY, Juang WM, 1988), as well as in cases where bacteriological analysis is unsuccessful.

 PCR technology in determining the causative agent of tuberculosis is promising for the diagnosis of tuberculosis. Over the past 6-7 years, a large number of works have appeared in the foreign press in which it was shown that the PCR method provides a significant increase in the sensitivity of detection of mycobacteria by an order of magnitude higher than the culture method for culture media (Manjunath N., Shankar P., Rajan L., 1991 ; Sjobring U., Mecklenburg M., Andersen AB, 1990) with high specificity of the analysis, i.e. allows to identify the causative agent in small quantities (Andersen A.V., Thybo S., Godfi-ey-Faussett P., Stoker N.G. 1993).

 The starting material for PCR can be obtained from pleural fluid (Yilmaz G., Utku B., Kucukusta AR et al, 1995), sputum (Nolte FS, Metchock B., McGowan JE et. Al., 1993), cerebrospinal fluid (Folgueira L., Delgado R., Palengue E., Noriega AR, 1994; Kaneco K., Onodera O., Miyatake. T. et al., 1990), peripheral blood (Schluger NW, Condos R., Lewis S. , Rom WN, 1994), as well as from biopsy material, including samples of histological tissue sections (Popper N.N., Winter E., Hofler G., 1994).

 The sensitivity of PCR was determined by examining the purified chromosomal DNA of mycobacteria. In various studies, the sensitivity is 10 to 100 fg DNA, which corresponds to 10 to 100 bacterial cells (Andersen A.V., Thybo S., Godfi-ey-Faussett P., Stoker N.G., 1993).

 Typically, the sensitivity of PCR was compared with cultural studies, and it ranged from 70% to 95% (Forbes BA, Hicks KE, 1993; Kaltwasser G., Garsia S., Salinas AM, 1993; Kocagoz T., Yilmaz E., Ozkara S. et., 1993). These results are directly dependent on the quality of the culture media used and the number of crops made.

The prototype is:
The PAB-1 device (aerosol sampler bacteriological) was designed by the Leningrad branch of the All-Union Scientific Research Institute of Medical Instrument Making. The device is based on the principle of electrical precipitation. PAB-1 consists of 2 portable units: the actual device and styling. The device itself is mounted in a portable box and consists of an air sampling unit (where a metal thread is mounted to create a zone of negative corona discharge), a micromanometer, a microammeter and an electric motor. The installation has 40 metal pallets for culture media and 20 test tubes for transporting liquid culture media. In the PAB-1 device, particles (bacteria), when air samples are sucked in, are charged in the zone of negative corona discharge, and then fall into the electric field and settle on a positively charged electrode made in the form of a replaceable metal tray, which is covered with a liquid or a dense nutrient medium. Using the PAB-1 device, you can take air samples at a speed of 150-250 liters per minute.

 The difference of our method is that this device was used to extract not nonspecific microflora, but mycobacterium tuberculosis in the air.

 The second prototype is a method for determining mycobacterium tuberculosis in the air, using cultural methods (Chugunikhina N.V., 1973). The disadvantage of this method is the long duration of the study (2.5-3 months) and a low threshold of sensitivity (tens of thousands of mycobacteria in the test material).

The third prototype is the PCR method for the diagnosis of mycobacterium tuberculosis in biological fluids (blood, sputum, exudate, etc.)
Our method consists in the fact that using a device based on the principle of electrical precipitation in the device, particles (bacteria) when sucking air samples are charged in the negative discharge zone, and then fall into the electric field and settle on a positively charged electrode made in the form of a replaceable metal a pan that is covered with Shkolnikova's liquid nutrient medium containing 0.1% pp V bovine albumin fraction for growing mycobacteria. The difference is that after "growing up" on Shkolnikova’s medium for 2 days, followed by the concentration of mycobacteria by centrifugation of 12000-16000g for 15 min, followed by removal of the supernatant and resuspension of the resulting precipitate in 100 μl of water, no inoculation is carried out nutrient media, as in the prototype, and standard PCR, consisting of 3 stages: extraction of mycobacteria DNA from a sample, amplification, detection of results.

Experiment
Work on sampling air was carried out in the ISTC BT in Moscow.

 In the wards, where patients with pulmonary tuberculosis with CD + are located, 12 hours before the start of the study, general and routine cleaning with the use of disinfectants was not carried out, the rooms were not ventilated and the patients were in the room during air intake. The start time of the fence is from 10 a.m.

 The air intake in the operating unit was carried out after the scheduled general cleaning using disinfectants and ultraviolet radiation.

The air intake in the smoking room was also carried out in the presence of patients, without violating the routine of the cleaning department of these premises. We established the parameters on the PAB-1 device for air intake, identical for all the test rooms: air was pumped for 20 minutes, a voltage of 40 mV was applied to the metal thread, 200 liters of air were pumped in 1 minute, i.e. within 20 minutes - 4 m ^{3 of} air.

Shkolnikova's synthetic medium was poured into removable metal pallets in an amount of 15 ml. After the device was turned off, the removable tray was removed and the liquid was specially treated to destroy the contaminating microflora by treating it with 2% sodium hydroxide with acetylcysteine (Musorger) in an amount equal to the volume of liquid extracted from the device for 15 minutes. This was followed by neutralization of the sample in phosphate buffer (pH 6.8) in a 5-fold volume of the latter. Then, centrifugation was performed at 3000 g for 15-20 minutes. After centrifugation, the precipitate was poured into specially prepared plastic tubes with a tight-fitting lid with Shkolnikova’s medium containing 0.1% pp V bovine albumin fraction. After that, the tubes were placed in a thermostat at t ^o 37 ^o C, after which the indication of mycobacteria was carried out by the method of polymerase chain reaction (PCR).

 A polymerase chain reaction is a multiple cycle of synthesis (amplification) of a specific region of tuberculosis DNA mycobacteria in the presence of thermostable DNA polymerase, deoxynucleoside triphosphates (dNTPs), the corresponding saline buffer, oligonucleotide primers, primers that define the boundaries of the amplified mycobacterium tuberculosis DNA.

The reaction consists of three main stages:
1. Isolation of mycobacterial DNA from the sample.

 2. PCR (amplification).

 3. Detection of results.

 4. In test tubes with PCR mixture-1, add 10 μl of PCR mixture-2 to the surface of the set wax. However, it should not fall through the wax and mixed with PCR mixture-1.

 5. Top up with 1 drop of PCR oil.

Amplification
In finished tubes under oil or directly on the surface of the oil, add 10 μl of DNA samples isolated from the test material.

Control amplification reactions:
Negative control (K-) - instead of a DNA sample, a DNA buffer is used. Positive control (K +) - FFP (positive control DNA sample), diluted with DNA buffer 10 times.

 Transfer the tubes to a programmable thermostat (amplifier) and amplify according to the program specified in the table. 2 (see the end of the description).

1. Isolation of mycobacterial DNA from the test sample is carried out by the guanidine method (according to Boom)
Working solutions
1. Solution I - 6M guanidine. A weighed portion of guanidine thiocyanate equal to 21.3 g is dissolved in distilled water, 0.6 ml of a 0.5 M EDTA solution is added and the volume is adjusted to 30 ml. Dry dithiothreitol (DTT) was added to a solution of guanidine thiocyanate to a final concentration of 20 mM.

 2. Solution II - 4M guanidine. A portion of guanidine thiocyanate equal to 7.08 g is dissolved in water and the volume of the solution is adjusted to 15 ml. In a 4M guanidine solution, it is also recommended to add DTT to a final concentration of 20 mM.

 3. Solution III - 10 mm Tris HCl, pH 7.3, 3.5 mm NaCl, 50% ethanol. To 1 ml of 1 mm Tris HCl add 1 ml of 5M NaCl, bring the volume to 50 ml with distilled water and add an equal volume of distilled ethyl alcohol.

 4. Sorbent. A suspension of pre-washed sorbent (diatomaceous earth) with an equal weight by weight amount of distilled water is used.

Sample preparation
As a material, a flush from environmental objects is used. After centrifugation at 12000 - 16000g for 15 min, the supernatant is carefully removed and the resulting precipitate is resuspended in 100 μl of water.

1. To 100 μl of the wash in a 1.5 ml tube (type Eppendorf) add 300 μl of solution I and 10 μl of sorbent. Samples are incubated for 10 min at +65 ^o C twice mixing on a vortex.

 2. Samples are centrifuged for 30 seconds in a microcentrifuge at 14,000 rpm, the supernatant is aspirated.

 3. Add 100 μl of solution II to the precipitate and shake it on a vortex. Sorbent precipitated by centrifugation for 30 seconds. The supernatant is sucked off.

 4. The washing procedure is repeated 2 more times using 1000 μl of solution III.

5. Additional centrifugation followed by discarding the supernatant remove the remaining solution III. Samples are dried for 5 min at a temperature of 65 ^o C, leaving the tubes open.

6. Add 50 μl of TE buffer to the tubes with the sorbent, close them, mix with a vortex and incubate for 5 min at +65 ^o C.

 7. Samples are centrifuged for 30 seconds at 14000 rpm, the aqueous phase is taken and used as a test sample of DNA for the amplification reaction.

2. The polymerase chain reaction
Mycobacterium tuberculosis DNA is detected by polymerase chain reaction using the AmpliSens-MTB-COM (MTB-COM) Research Institute of the Epidemiology of the Ministry of Health of the Russian Federation according to the manufacturer's instructions. The kit includes reagents indicated in the table. 1 (see the end of the description).

In all kits for amplification of the AmpliSens family, a “hot start” is necessarily used, which is ensured by the separation of nucleotides and Tag polymerase by a layer of wax. Melting of wax and mixing of the reaction components occurs only at 95 ^o C, which significantly reduces the number of non-specifically seeded reactions.

 Reagent preparation.

1. Warm the test tube with wax in a thermostat at + 95 ^o C until complete melting.

 2. Dispense 5 μl of PCR mixture-1 into microtube for PCR.

 3. Pip on top of 12-15 μl of molten wax so that it completely covers the liquid.

30 seconds after starting the program (when the temperature in the cell of the amplifier reaches 80 - 95 ^o C and the program starts to pause) place the tubes in the cells of the amplifier, close the lid of the device, remove the pause and wait for the end of the reaction (about 2.5 hours on the regulator with regulation temperature in the matrix). PCR results are taken into account by the study of amplification products by electrophoresis in a 2% agarose gel.

 The length of the amplified specific fragments of Mycobacterium tuberculosis / bovis is 390 nucleotide pairs.

3. Registration of results
Pour into a 1xTAE electrophoresis apparatus buffer prepared with distilled water.

 To 2.0 g of agarose, add 2 ml of 50xTAE buffer and 100 ml of distilled water.

 Agarose is melted on an electric stove or in a microwave oven. 5 μl of ethidium bromide solution are added to 100 ml of molten agarose. Mixed.

Pour agarose gel according to the instructions for the apparatus for horizontal electrophoresis. For pouring agarose gel it is convenient to use covers for immunological plates of suitable sizes. To obtain pockets on an agarose gel, a comb is used. Cool the agarose to a temperature of 50 ^o - 60 ^o C. After solidification of the agarose, carefully remove the comb from the gel.

 Add 5 μl of sample buffer to each amplification tube.

 Put in the pockets of the gel 10-15 μl of amplification, pre-mixed with sample buffer (6xTAE with 40% sucrose and 0.25% bromophenol blue).

 An electrophoretic camera is connected to a power source and a voltage corresponding to an electric field strength of 10-15 V / cm is set. Conduct electrophoretic separation of amplification products for 30-45 minutes

 The gel is removed from the mold and transferred to a glass of a transilluminator.

 Fragments of amplified DNA appear as luminous orange-red stripes.

 Analysis of the results.

 Negative control - the strip should be absent.

 Positive control - a strip is detected.

 The analyzed sample - the absence of a band at the level of the corresponding control indicates a negative response. The presence of a band corresponding to the positive control in electrophoretic mobility confirms the presence of mycobacterial DNA in the sample.

 The appearance of a band in the negative control indicates contamination of the components of the kit. The results can be documented by photographing the gels using orange or interference (594 nm) filters.

In total, 30 air studies were done in 4 types of rooms:
1. Places of constant location of bacillary patients (hospital wards).

 2. Premises where bacillary patients are sporadic (corridors, smoking rooms, dining room).

 3. Premises where the stay of bacillary patients is excluded (pharmacy, administrative premises).

4. Premises of a strict disinfection regime (operating room) after treatment with an ultraviolet lamp and disinfectants
The following results were obtained:
1. In the places where the bacillary patients are permanently located (hospital wards) - 9 samples - 9 positive PCR results (by seeding, MBT could not be detected).

 2. In rooms where bacillary patients are occasionally (corridors, smoking rooms, dining room) - 5 samples - 5 positive PCR results (by seeding method, it was not possible to detect the office).

 3. In rooms where the stay of bacillary patients is excluded (pharmacy, administrative premises), 12 samples - 12 negative PCR results (by seeding method, MBT could not be detected).

 4. In the premises of a strict disinfection regime (operating room) after treatment with an ultraviolet lamp and disinfectants, 4 samples — 4 negative results (by seeding, MBT could not be detected).

 The method allows to determine mycobacterium tuberculosis (MBT) in the air in a short time (3-5 days), in contrast to detection using the culture method (2.5-3 months). Moreover, the method is highly sensitive and allows you to identify the minimum concentration of MBT in the air, which are not determined using bacterioscopy and culture method. Using this method will solve the issue of sanitary-hygienic assessment of air in the rooms where patients with tuberculosis are.

Claims (3)

 1. A method of indicating mycobacterium tuberculosis in the air, characterized in that the air sample is passed through an electric field, while the bacteria are charged in the negative discharge zone and deposited on a positively charged electrode made in the form of a removable metal tray coated with a liquid nutrient medium for growing mycobacteria tuberculosis, the latter are cultured for 2 days, concentrated, the supernatant is removed, and the resulting precipitate is resuspended, then the mycobacterium tuberculosis is determined by lecularvirological genetic method (PCR).  2. The method according to claim 1, characterized in that the Mycobacterium tuberculosis is concentrated by centrifugation of 120 000 - 16 000 g for 15 minutes  3. The method according to claim 1 or 2, characterized in that the precipitate is resuspended in 100 μl of water.

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