RU2364629C1 - Method for determination of efficiency of disinfectants used in tb facilities - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns microbiology, sanitary hygiene. Mycobacteria culture suspensions are prepared and followed with mycobacteria contamination of test objects thereafter processed in a tested disinfectant to be then neutralised on test objects after specified disinfectant exposition on mycobacteria contaminated test objects. Then the neutralised test objects are incubated in a solid nutrient medium in a thermostat to consider and analyse experimental data. The test culture is Mycobacterium avium and Mycobacterium terrae, or strains of specified TB facilities (prevention and treatment facility). Disinfection quality level is determined by the absence of colony growth within 3-5-7-10-14 days (depending on a type) of cultivation.

EFFECT: invention allows improving and accelerating quality control of disinfectant performance in TB clinics and TB medical facilities.

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Description

The invention relates to medicine, in particular to the field of microbiology and sanitary hygiene, and is aimed at improving and accelerating the quality control of the work of disinfectants in TB dispensaries and treatment facilities of the TB profile.

It is known that at present, the tuberculocidal effectiveness of disinfectants is evaluated: in Russia, according to the guidelines “Testing methods for disinfectants to assess their safety and effectiveness”, M .: Ministry of Health of the Russian Federation 1998, 72 pp., In Europe - according to the European standard. Draft pr EN 14348, October 2002.

According to the manual “Test methods for disinfectants to assess their safety and effectiveness”, the saprophytic strain Mycobacterium B-5 is used as a test microorganism to assess the tuberculocidal effectiveness of disinfectants during their disinfection of various objects.

To obtain a culture of mycobacteria of strain B-5 and bacteriological control of samples after exposure to a disinfectant, such nutrient media as potato-glycerin broth, Petraniani medium, 2% glycerin agar are recommended.

European standard The draft pr EN 14348 for the determination of the tuberculocidal and mycobactericidal activity of disinfectants provides and regulates the use of the suspension method. The cultures of Mycobacterium avium ATCC 15769 and Mycobacterium terrae ATCC 15755 are used as test microorganisms to assess the tuberculocidal and mycobactericidal activity of disinfectants. The essence of the method is that in a test tube with 1 ml of a suspension of culture of test microorganisms (10 ⁶ mt / ml ) add 8 ml of the solution of the test disinfectant, after the exposure time expires, 1 ml of the mixture is transferred to a test tube with 8 ml of neutralizer solution and 1 ml of the resulting mixture is sown in 2 Petri dishes with Middlebrook 7H10 nutrient medium. Crops are incubated in an incubator at 37 ° C for 21 days.

The results of studies on a comparative assessment of the sensitivity of virulent (museum and clinical) and non-pathogenic strains of mycobacteria to disinfectants with different compositions of active substances convincingly showed that test mycobacteria of the saprophytic strain B-5 are less resistant (and in some cases significantly) to the effects of disinfectants than Museum and clinical strains of mycobacteria. The most adequate test microbes in this regard are, as shown by the results of these studies, mycobacteria strains of M. terrae and M. avium.

However, the approved tuberculocidal regimes of all disinfectants registered for use in Russia were developed using Mycobacteria strain B-5 as a test microbe. In this regard, there is a real threat of ineffective disinfection of objects and the spread of the causative agent of tuberculosis and mycobacteriosis both in anti-tuberculosis hospitals, where the most active bacteriostatic agents are concentrated, and in medical institutions of the general treatment network (GPI), where patients with an unknown diagnosis are referred. In hospitals, as a rule, they follow the guidelines of manufacturers in choosing tuberculocidal regimes for routine disinfection, which, for the above reason, may not provide reliable disinfection of environmental objects contaminated with the causative agent of tuberculosis. In addition, cases of the emergence of clinical strains of mycobacteria with higher resistance to disagents than museum strains of mycobacterium tuberculosis can occur in hospitals. But, since in the hospitals normative documents do not provide for targeted monitoring of the contamination of objects with mycobacteria and the study of the resistance of the latter to disinfectants, hospital epidemiologists, as a rule, do not have information and cannot adequately respond to this.

The objective of the invention is to develop a method for determining in laboratory conditions the effectiveness of disinfectants in TB facilities, which allows the most reliable and accurate with minimal time and money to assess the quality of the action of disinfectants for conditions of increased microbial load of TB facilities, using known and affordable drugs, nutrient media, technical equipment and instruments.

The problem is solved in that the quality assessment of the tuberculocidal and mycobactericidal efficacy of disinfectants used in TB facilities and the testing of disinfectants in the bacteriological laboratories of TB facilities are carried out for the presence of tuberculocidal activity in relation to cultures of mycobacteria isolated from patients undergoing treatment in these facilities, or in relation to test strains of M. terrae and / or M. avium, which are an adequate model of clinical strains of miko in terms of disinfectant resistance Actor. The determination is carried out using the method of immersion in disinfectant calico test objects of 1 × 1 cm in size, contaminated with test microorganisms using Di-Ingley broth as a universal neutralizer, when cultivated on a dense nutrient medium “New” in the amount of 4.5-5 ml in test tubes with a beveled section 8-10 cm long with placement of up to 3 test objects in one tube. The method for determining the tuberculocidal and mycobactericidal efficacy of disinfectants, which is proposed for use in TB facilities of a phthisiatric profile, provides for the sequential implementation of the preparatory stage operations, the execution of the experiment itself, and accounting and analysis of its results.

At the preparatory stage, a high-quality suspension of test mycobacteria culture is prepared, which will be used to assess the tuberculocidal (mycobactericidal) effectiveness of the disinfectant used or planned for use in the TB institution.

The following strains can be used as test mycobacteria when testing the method:

1) M. tuberculosis (especially MDR multidrug-resistant strains) isolated from patients directly at the institution;

2) M.terrae ATCC ^*) 15755;

3) M.avium ATCC 15769.

Considering that recently special neutralizing broths have appeared on the market with the optimal composition of neutralizing ingredients that are easy to prepare and are intended directly for use in disinfectant assessment experiments, the proposed method uses neutralizing broth according to Di-Engli (manufacturer of HIMEDIA "). It contains ingredients such as casein hydrolyzate, yeast extract, glucose, sodium thiosulfate, sodium thioglycolate, sodium bisulfite, lecithin, Tween-80, etc. The ready-made Di-Engli liquid nutrient medium follows, as shown by special experiments, performed at the Ural Research Institute of Phthisiopulmunology, used as a universal neutralizer for disinfectants based on Quaternary ammonium compounds (HOUR), hydrogen peroxide and aldehydes.

The “Novaya” medium used in the method is a dense egg medium — original, little known before, not used to assess the quality of disinfectants and used in the method in an amount of 5 ml in a test tube with a beveled medium 8-10 cm long for placement of up to 3 tests in vitro objects.

The composition of the nutrient medium "New" includes (in wt.%):

Monobasic potassium phosphate 0.05 Sodium Citrate 0.05 Magnesium sulfate 0.05 Sodium pyruvic acid (glycol) 0.2 Glycerol 3.6 Malachite green 0,036 Yolks 50,0 Distilled water up to 100.0

(see. Preparation and use of a new nutrient medium for the cultivation of Mycobacterium tuberculosis. Guidelines. M .: Ministry of Health of the RSFSR 1972).

The Levenshtein-Jensen medium is used as a control nutrient medium for test microorganisms - this is an international environment that is used as a standard medium for the primary isolation of the causative agent of tuberculosis and the determination of its drug sensitivity. This medium is recommended for use in all microbiological laboratories of the Russian anti-tuberculosis service to obtain comparable results (see the Instructions for the unified methods of microbiological research in the detection, diagnosis and treatment of tuberculosis. Appendix to the order of the Ministry of Health of Russia dated March 21, 2003 No. 109).

For the purity of the experiment, a ready-made nutrient medium is used in standard test tubes with screw caps.

If it is impossible to purchase a finished medium, you can cook it yourself.

The method of disinfecting test objects of the tested disinfectant and monitoring the effectiveness of disinfection involves the sequential contamination of mycobacteria with coarse calico test objects, processing them in the test disinfectant, neutralizing the disinfectant on them after a predetermined exposure of the disinfectant to mycobacteria contaminated test objects, incubating neutralized neutralized tests .

Preparation of coarse calico test objects is carried out, guided by the "Instruction for the determination of the bactericidal properties of new disinfectants" dated May 6, 1968 No. 739-68, replacing the expensive cambric, which is not in the medical facilities, with a more technological calico. A calico flap is immersed for 24 hours in cold tap water to remove starch, washed thoroughly with soap, boiled, dried and ironed. From the prepared fabric using a needle pull the threads in the longitudinal direction at a distance of 10 mm from each other and in the transverse at a distance of 10 mm. According to the prepared lines, the fabric is cut with scissors into test objects 1 × 1 cm in size and 50 pieces are laid out in Petri dishes.

Petri dishes with test objects are autoclaved for 30 minutes at 126 ° C.

Upon contamination of calico test objects, sterile calico test objects 1 × 1 cm in size (in the amount of 50-100 pieces) are poured into a Petri dish 10-20 ml of a suspension of test mycobacteria containing 10 ⁹ CFU / ml, and left evenly, wetting test objects in suspension in a closed Petri dish. After 30 minutes, coarse calico test objects contaminated with a bacterial suspension are transferred with a sterile forceps into a Petri dish on the surface of a two-layer sterile filter paper, cover them on top with sterile paper and close the cup. Leave for 10 minutes to remove excess fluid. To fix microorganisms on coarse calico test objects, the latter are transferred to the surface of a dry sterile filter paper in a Petri dish and covered with a sterile sheet of filter paper, dried in an thermostat at 37 ° C for 20 minutes.

Storage of test objects contaminated with test strains is possible at a temperature of + 4 ° C during the day.

To control the number of viable bacterial cells on a contaminated test object, the test object is immersed in a flask with 100 ml of sterile distilled water. The flask is mounted on a shaker and shaken for 30 minutes to wash the bacterial cells from the calico test object. From the resulting suspension, 0.1 ml inoculation is made into 5 tubes (5 replicates) with Levenshtein-Jensen or Novaya medium, incubated in an incubator at 37 ° C for 5-7 days. The grown colonies on the surface of the nutrient medium are counted, the average value is determined, recalculation is made taking into account the dilution. The number of viable bacterial cells in the prepared suspension on the test object should correspond to 10 ⁵ -10 ⁶ microbial bodies in 1 ml (if there is no growth of mycobacteria, when sowing a suspension of 0.1 ml, you can increase the volume to 0.2-0.3 ml and take it into account when calculating the bacterial concentration on the test object).

Then, calico test objects contaminated with the test strain are immersed with sterile forceps into test tubes with a disinfectant solution (the volume of disinfectant in the test tube should be calculated taking into account the ratio of 1.0 ml of solution to 1 test object, i.e., for example, for 9 test objects 9.0 ml of a disinfectant solution is necessary), for the time specified in the instructions for use of the disinfectant, corresponding to the concentration tested, and the time of the start of the experiment is recorded.

After the exposure time, a test object is removed from the disinfectant solution with sterile tweezers and immersed for 1-2 seconds alternately in 2 tubes with sterile neutralizer broth (Di-Engli broth).

After neutralization, a test object is placed with tweezers on an 8–10 cm long sloping surface of the Novaya nutrient medium and Levenshtein-Jensen control medium (up to 3 test objects can be placed in a test tube on a sloping surface of the nutrient medium, which can be increased without additional costs the number of samples, and hence the reliability of the results of evaluating the effectiveness of the product).

Inoculated tubes are closed with cotton-gauze plugs, placed in an incubator in an inclined position at an angle of 30 ° so that the seed is evenly distributed over the entire surface of the nutrient medium, and incubated at 37 ° C for 5-7 days with daily viewing.

The results of crops are taken into account visually by counting the grown colonies on the test object itself and on the surface of the nutrient medium. Colonies of M. tuberculosis in the control medium of Levenshtein-Jensen and Novaya have the appearance of moist or sometimes dry and rough convex formations with a yellowish tinge, which become rosettes upon growth. Colonies of M. avium on the medium of Levenshtein-Jensen and Novaya are smooth, shiny, moist, convex roundish formations with a milky, cream or yellowish tint. Colonies of M. terrae in the Levenshtein-Jensen and Novaya environments are smooth and shiny or rough and dull convex roundish formations with a milky or yellowish tint.

The presence of the growth of colonies of test mycobacteria on the test object and on the surface of the nutrient medium shows that the tested disinfectant in this mode of application (concentration of the solution and exposure time exposure) does not provide a reliable tuberculocidal (mycobactericidal) effect. Therefore, it is either advisable to refuse to use such a tool in this healthcare facility or conduct a similar test in a more stringent mode (high concentration of the working solution and exposure time), if any, in the instructions for use of the tool. Based on its results and taking into account economic and other operational indicators, decide on the use of this tool.

The lack of growth of the colonies of test mycobacteria on the test object and on the surface of the nutrient medium indicates the presence of tuberculocidal and mycobactericidal efficacy that meets the requirements for disinfectants for their practical use (to ensure that the level of contamination of the object by 10 ⁵ CFU · cm ^-2 ). Taking into account economic and other operational indicators (toxicity, corrosive properties, storage, etc.), such a tool can either be used widely or with restrictions for disinfection measures in this medical facility.

New for domestic practice in the method is the experimentally substantiated sequential use of the Di-Engli neutralizing broth as a universal neutralizer for many disinfectants based on QAS, aldehydes, as well as Novaya dense nutrient medium in test tubes with a volume of nutrient medium 5 ml and oblique with a cut of 8-10 cm when using M.avium and M.terrae as a test microbe on calico test objects 1 × 1 cm in size with up to 3 test objects placed in one tube. The use of this nutrient medium (in contrast to the Middlebrook 7H10 nutrient agar used in foreign practice when testing disinfectants in Petri dishes) allows, in combination with the proposed operations and parameters, to shorten the time for obtaining results by 2.5-3 times (i.e., reduce them to 21 days to 7-10 days), significantly reduce the consumption of the nutrient medium and minimize contamination of crops with extraneous microflora.

The use of this method in the practice of anti-TB institutions will allow chief doctors and hospital epidemiologists, as those responsible for organizing and ensuring an effective anti-epidemic mode of operation of hospitals, objectively, purposefully, with minimal time and money, to choose disinfectants in accordance with the epidemiological situation in hospitals that would ensure effective disinfection of various objects against pathogens of tuberculosis and mycobacteriosis.

The research results on the proposed method showed that museum test strains of M.avium and M.terrae mycobacteria, used as test strains in foreign practice, are more adequate to pathogens of tuberculosis and mycobacterioses in terms of resistance to disinfectants. These data confirm the advisability of using in the development of tuberculocidal regimens for the use of disinfectants of these test strains (as stipulated by the European standard), rather than mycobacteria strain B-5.

The data obtained indicate the need for testing of disinfectants in TB facilities using real tuberculosis mycobacteria isolated from patients in these hospitals, or test strains M.terrae and M.avium.

When conducting studies to evaluate the effectiveness of disinfectants using the proposed method, a comparative determination was made of the growth rate of pathogenic and non-pathogenic mycobacteria (the microbial load corresponded to 10 ⁵ -10 ⁶ microbial bodies in 1 ml) on nutrient media “Novaya”, Levenshtein-Jensen and Middlebrook 7Н10 s growth additive 10% OADC (control) after treatment with a disinfectant with neutralization with broth according to Di-Engli and without exposure to a disinfectant with neutralization. Data on the growth periods of various strains are presented in table 1 and 2.

Table 1. The growth rate of test strains of mycobacteria on various nutrient media Culture media Dates of growth of colonies of mycobacteria (day) AT 5 M. terrae M.avium H ₃₇ Rv "New" 2-3 3-4 4-5 7-8 Levenshtein-Jensen 3-4 3-5 6-7 10-12 Middlebrook 7H10 with 10% OADC No growth 8-10 14-16 21-23

From table 1 it can be seen that the growth of mycobacteria on the environment "New" without exposure to disinfectants of test strain B-5 appeared on 2-3 days; M.terrae growth - for 3-4 days; M.avium growth - at 4-5 days; growth of M. tuberculosis H ₃₇ Rv - on the 7-8th day. The growth of mycobacteria on Levenshtein-Jensen medium without exposure to disinfectants of test strain B-5 appeared on 3-4 days; M.terrae growth - for 3-5 days; M.avium growth - at 6-7 days; the growth of M. tuberculosis H ₃₇ Rv - on 10-12 days. On Middlebrook 7H10 medium with 10% OADC without exposure to disinfectants, the growth of mycobacteria of test strain B-5 was absent; the growth of M. terrae mycobacteria appeared on days 8–10; M.avium growth - on days 14-16; growth of M. tuberculosis H ₃₇ Rv - at 21-23 days.

Table 2. The growth rate of test strains of mycobacteria on various nutrient media when treated with disinfectants Culture media Dates of growth of colonies of mycobacteria (day) AT 5 M.terrae M.avium H ₃₇ Rv "New" 3-5 7-10 8-10 12-14 Levenshtein-Jensen 5-7 10-12 12-14 14-16 Middlebrook 7H10 with 10% OADC No growth 21-23 21-23 23-25

From table 2 it can be seen that the growth of mycobacteria of test strain B-5 on the Novaya medium after exposure to disinfectants and neutralization with Di-Engli broth appeared on 3-5 days; M.terrae growth - at 7-10 days; M.avium growth - on 8-10 days; growth of M. tuberculosis H ₃₇ Rv - on 12-14 days. The growth of mycobacteria on Levenshtein-Jensen medium after exposure to disinfectants and neutralization of test strain B-5 appeared on the 5-7th day; M.terrae growth - on 10-12 days; M.avium growth - on 12-14 days; the growth of M. tuberculosis H ₃₇ Rv - on the 14-16th day. On Middlebrook 7H10 medium with 10% OADC after exposure to disinfectants and neutralization, the growth of mycobacteria of test strain B-5 was absent; the growth of M.terrae and M.avium mycobacteria appeared on days 21-23; the growth of M. tuberculosis H ₃₇ Rv - at 23-25 days.

Thus, the tables show that according to the technology of the proposed method, the growth rate of mycobacteria on the Novaya environment, without exposure and after exposure to disinfectants with neutralization, is 2 times higher than on Middlebrook 7H10 medium with a growth additive of 10% OADC, and for recommended as test microorganisms M.terrae and M.avium - almost 3 times.

The growth rate of mycobacteria on Levenshtein-Jensen medium without exposure and after exposure to disinfectants with neutralization is 2-4 days lower than the growth rate of mycobacteria on Novaya medium and exceeds the growth rate of mycobacteria on Middlebrook 7H10 medium with a 10% OADC growth additive of almost 2 times.

Using the proposed method allowed us to establish that the test strain B-5, being less resistant compared to the test strains of M. terrae and M.avium against chemical disinfectants, cannot be used as a test microorganism for the development of tuberculocidal regimens for the use of disinfectants in TB facilities.

The use of the proposed method of test mycobacteria strains of M. tuberculosis (isolated from patients) or M.terrae and M.avium to assess the tuberculocidal and mycobactericidal efficacy of disinfectants used in anti-tuberculosis hospitals, using a neutralizing Di-Ingli broth and richer Novaya egg culture medium in test tubes during test control on a mowed medium with a length of 8-10 cm with up to 3 test objects placed in a test tube to control the viability of mycobacteria allows:

- to ensure compliance with the tuberculocidal regimen of the use of disinfectants of resistance to them of the real causative agents of tuberculosis;

- to increase the objectivity, reliability and reproducibility of research results by conducting extended control of the completeness of neutralization of the residual effect of the test disinfectant, monitoring the real number of viable bacterial cells in the suspension of test strains of mycobacteria and on the test object using a nutrient medium, which is available in composition for any laboratory and more effective in identifying viable mycobacteria;

- keep track of the results in a shorter time (5-7 days);

- to ensure the convenience of work and the safety of employees when conducting experiments on a nutrient medium in test tubes;

- minimize contamination of crops with extraneous microflora.

An unobvious effect of the proposed method for determining the effectiveness of disinfectants used in TB facilities is that, comprehensively using known preparations and carriers for the new purpose (test mycobacteria on calico test objects 1 × 1 cm in size, Di-Ingley broth) to neutralize contaminated test objects, the dense nutrient medium "New" in test tubes with a bevel length of 8-10 cm, with up to 3 test objects placed in a test tube), the final result is drastically (1.5-2 times) reduced time Jelenia effectiveness of disinfectants that can give a great economic and social benefits while reducing the space for placing the thermostat (tubes instead of Petri dishes) and increased personnel protection against nosocomial infections with the improvement of their working conditions, without requiring large investments of money and effort.

The application of the method allows to reliably and quickly give an objective assessment of the activity of disinfectants used in tuberculosis institutions against virulent clinical strains of mycobacteria, to ensure the optimal choice of disinfectants, increase the effectiveness of disinfection measures and reduce the risk of nosocomial spread of tuberculosis.

The method is developed and tested in a tuberculosis institution and can be recommended for the practical application of evaluation of disinfectants.

Testing of disinfectants used in hospitals, or planned for use, using the presented method is carried out when it is detected in samples-swabs that are purposefully taken in hospitals with a disinfectant solution of a disinfectant for various objects, viable pathogens of tuberculosis or mycobacteriosis. In addition, the indication for such testing is the transition to another disinfectant, which is carried out in terms of ensuring the rotation of disinfectants in order to prevent the "addiction" of mycobacteria to the disinfectant.

There are no contraindications to the use of the method.

Claims (1)

A method for determining the effectiveness of disinfectants used in TB facilities, including the sequential preparation of a suspension of mycobacterial culture, contamination of test objects with mycobacteria, processing them in a test disinfectant, neutralizing the disinfectant on test objects after a given exposure of the disinfectant to contaminated mycobacteria, test objects neutralized tests in a dense nutrient medium, in a thermostat, recording and analysis of results experiment, characterized in that Mycobacterium avium and Mycobacterium terrae are used as a culture of test mycobacteria, or strains of this anti-tuberculosis institution (MPI), while calico test objects are used as test objects for mycobacterial contamination, neutralization of contaminated test objects is carried out Di-Engli broth, and as a dense nutrient medium, Novaya medium is used in bevelled tubes, with up to 3 calico test objects placed in each test tube, and calico test objects used in size with 1 × 1 cm, with the length of the bevel of the nutrient medium in the test tube 8-10 cm, with the volume of the nutrient medium 5 ml, and the growth of the colonies is monitored for 3-5-7-10-14 days of cultivation for different types of mycobacteria after sowing with daily viewing, considering the lack of colony growth during the indicated cultivation periods as an indicator of the quality of disinfection treatment.