RU2792782C1 - Method for determining the sensitivity of mycobacterium tuberculosis cultures to pyrazinamide - Google Patents

Abstract

FIELD: medical microbiology.

SUBSTANCE: invention can be used for diagnosing tuberculosis pathogen sensitivity to pyrazinamide. A method for determining the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide is proposed, which consists in carrying out a color reaction of the oxidation of ferrous iron to ferric iron in the presence of pyrazic acid synthesized from pyrazinamide under the action of the mycobacterial enzyme pyrazinamidase. The color reaction is carried out directly in the liquid nutrient medium for cultivation, while ensuring the incubation time of the culture for 1 day. The concentration of the culture is carried out by precipitation, and the registration of the result of the color reaction over the entire reaction volume is carried out for 15 minutes.

EFFECT: increasing the sensitivity and reliability of the detection of the result while expanding the arsenal of technical means that can be used to determine the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide.

2 cl, 2 dwg

Description

SUBSTANCE: invention relates to medical microbiology and can be used for diagnosing tuberculosis pathogen sensitivity to pyrazinamide.

The proposed method makes it possible to determine the activity of the mycobacterial enzyme pyrazinamidase, which causes the sensitivity of Mycobacterium tuberculosis (MBT) to pyrazinamide, by carrying out a color reaction directly in a liquid nutrient medium for cultivating mycobacteria in the presence of pyrazinamide and Mohr's salt.

Pyrazinamide is a first-line anti-tuberculosis drug (TAP). Its use makes it possible to reduce the treatment time for tuberculosis from 9-12 to 6 months, since it is the only anti-TB drug that is active against non-degradable MBT in the acidic environment of the lesion [1, 2]. WHO recommends including pyrazinamide in treatment regimens for drug-resistant tuberculosis (including MDR), provided that sensitivity to this drug is maintained [3].

Pyrazinamide, a synthetic pyrazine analog of nicotinamide, is a prodrug. In the active form - pyrazic acid (ROA), pyrazinamide is converted under the action of mycobacterial enzyme - pyrazinamidase. In the neutral environment of the cytoplasm of the cell, ROA does not have antimicrobial activity and is removed from the cell by the active transport system of the membrane. In the acidic environment of the focus of inflammation, ROA is protonated (HROA). Then the protonated form can re-penetrate into the MBT cell and, releasing protons, acidifies the intracellular environment, leading to MBT death [2].

Culture methods are the gold standard for determining the sensitivity of microorganisms to antibacterial drugs.

The classical culture method for determining drug susceptibility of Mycobacterium tuberculosis is based on taking into account the growth of a culture on a medium with an anti-tuberculosis drug compared to the growth of a control culture on a medium without a drug.

Traditionally, for the cultivation of MBT and the determination of their drug sensitivity, dense egg media of Levenshtein-Jensen and Finn-II are used, which have an acidity value close to neutral [4].

Pyrazinamide has an antimycobacterial effect only in an acidic environment, therefore, under conditions of neutral pH, the effect of the drug will not manifest itself even if the pathogen is sensitive to it.

A known method for testing the sensitivity of Mycobacterium tuberculosis to drugs [RU 2573917, C2, C12Q 1/18, 10.05.2014], including the following steps:

a) obtaining enriched samples containing Mycobacterium tuberculosis;

b) adding liquid culture medium to enriched samples to obtain liquid culture samples and, if necessary, performing enrichment culturing for up to 7 days;

c) heating and melting the solid medium to obtain a molten solid medium and lowering the temperature of the molten solid medium to the appropriate temperature for culturing samples of liquid culture or to room temperature, while the solid medium has a melting temperature of 50-95°C and a solidification temperature of 25-45°C for solid media containing agar or agarose and nutrients;

d) mixing liquid culture samples with a molten solid medium to obtain liquid samples for drug susceptibility testing;

e) solidifying liquid drug susceptibility samples to obtain solid drug susceptibility samples using drug sensitive paper strips;

f) culturing solid samples for drug susceptibility testing for 5-15 days, then adding an indicator to the solid sample;

g) observation of the zone of inhibition of the drug by the level of indication of the indicator and determination of the sensitivity of Mycobacterium tuberculosis to drugs.

This technical solution is relatively complex. In particular, according to this method, after a series of cultivation of mycobacteria on a liquid nutrient medium, to obtain a sufficient bacterial mass, additional cultivation of mycobacterium tuberculosis is carried out on agar with added preparations for up to 15 days, which significantly increases the time for obtaining the result.

In addition, this technical solution does not indicate which anti-tuberculosis drugs can be tested in this way. However, given the mechanism of action of pyrazinamide, it becomes obvious that the proposed technical solution is not suitable for testing drug sensitivity to pyrazinamide, because. the agar medium offered as a nutrient base for the growth of mycobacteria is not acidified, and therefore, inhibition of culture growth in the presence of pyrazinamide will not occur.

Also known is a nutrient medium with pyrazinamide for determining the drug resistance of tuberculosis pathogens and their identification [RU 2099417, C1 C12Q 1/18, 20.12.1997], containing monosubstituted potassium phosphate, magnesium sulfate, brilliant green, glycerin, homogenized egg mass and water, different the fact that it additionally contains sodium citrate, iron (III) ammonium sulphate, iron (II) ammonium sulphate, aminoacetic acid, a-aminosuccinic acid, succinic acid, ammonium succinic acid, monosubstituted sodium phosphate, hydrolysin, penicillin, pyrazinamide in the following quantitative ratio components:

Potassium phosphate monosubstituted, g 4.0-9.0;

Magnesium sulfate, g 0.2-0.6;

Sodium citrate, g 0.3-0.9;

Iron (III) ammonium sulphate, g 0.01-0.04;

Iron (II) ammonium sulphate, g 0.01-0.04;

Aminoacetic acid g 0.5-1.3;

α-aminosuccinic acid, g 0.3-0.9;

Succinic acid, g 0.8-1.6;

Ammonium succinic, g 1.2-2.4;

Sodium phosphate monosubstituted, g 1.3-2.4;

Brilliant green, g 0.01-0.04;

Glycerin, ml 7.0-17.0;

Hydrolysin, ml 15.0-50.0;

Homogenized egg mass, ml 200-500;

Pyrazinamide, mcg/ml 50-200;

Penicillin, units/µl 100000-300000;

Water l. up to 1.0.

However, this method has a relatively limited scope, because. according to the Order of the Ministry of Health of the Russian Federation No. 109, the determination of sensitivity to pyrazinamide on dense nutrient media is not allowed [4]

There is also a method recommended by WHO for determining the phenotypic sensitivity to pyrazinamide, known as the method of proportions on the Middlebrook 7H9 liquid medium in the automated detection system for the growth of mycobacteria VASTEC MGIT 960 (BD, USA) [5].

The disadvantage of this method is its complexity and high material costs, because. its implementation requires expensive equipment and consumables; in particular, a drug sensitivity test for pyrazinamide requires the purchase of a specially designed set of MGIT tubes and reagents [6].

A known method for detecting pyrazinamide-resistant isolates of Mycobacterium tuberculosis [RU 2552214, C2, C12N 15/00, 10.10.2014] by determining the presence of mutations in the rpsA gene associated with the formation of resistance to pyrazinamide, by performing real-time PCR using HRM analysis, according to which amplification is carried out on a mixture of an equal amount of test DNA and wild-type DNA using primers:

heteroduplexes are formed due to the simultaneous co-amplification of wild-type DNA and the tested DNA, at the same time, at the first stage of PCR, the concentration is equalized using quantitative PCR using the same pair of primers (Pncl8U/Pncl5R) with the construction of a calibration curve and using a regression equation.

The disadvantage of this method is its relatively high complexity, because implies the need for labor-intensive procedures that require highly qualified specialists, the availability of expensive equipment (for example, an amplifier with an optical module and the ability to plot melting curves), which limits its wide application.

In addition, at present there is no unequivocal confirmation that any mutation at the pncA level leads to disruption of the enzyme and, consequently, to MBT resistance to pyrazinamide.

Closest to the claimed is a method for determining sensitivity to pyrazinamide [Wayne LG. Simple pyrazinamidase and urease tests for routine identification of mycobacteria. Am Rev Respire Dis. 1974 Jan; 109 (l): 147-51], which is based on the fact that the transfer of the drug to the active form occurs under the action of pyrazinamidase, and, therefore, the activity of this enzyme will be key for the sensitivity of MBT to pyrazinamide.

The method is based on the fact that pyrazic acid, an active metabolite of pyrazinamide, formed in MBT strains sensitive to pyrazinamide under the influence of pyrazinamidase, is an oxidizing agent and, in particular, is capable of oxidizing the Fe2+ ion to Fe3+ and, therefore, the use of Mohr's salt (ammonium sulfate - iron(), FeSO ₄ ⋅ (NH ₄ ) ₂ SO ₄ ⋅ 6H ₂ O) allows a color reaction in which, in the presence of pyrazic acid, the solution of Mohr's salt is stained brown-pink, which indicates the activity of the pyrazinamidase enzyme. If pyrazinamidase is inactive (in pyrazinamide-resistant strains), pyrazinamide will not be converted to pyrazic acid, and hence addition of Mohr's salt to the culture will not result in staining.

Briefly, the closest technical solution is as follows.

To obtain a sufficient amount of bacterial mass, the MBT strain is pre-cultivated on solid or liquid nutrient media. Prepare a special agar medium containing pyrazinamide at a concentration of 100 µg/ml or 200 µg/ml. Next, the MBT culture is seeded in glass tubes on agar medium with pyrazinamide and incubated at 37°C for 7 days. Then 1 ml of 1% Mohr's salt is added to the surface of the medium. The result is read visually immediately or, if staining has not occurred, after 4 days of incubation at 37°C. A red band on the surface of the agar medium indicates positive pyrazinamidase activity.

In a later modification of the Wayne LG method, pyrazinamide is added to the medium at a concentration of 400 µg/mL to increase the sensitivity of the reaction [8].

This modification of the method for determining the activity of pyrazinamidase, as a marker of sensitivity to pyrazinamide, is used in the commercial Pyrazinamidase Test Kit for Mycobacteria (Pyrazinamidase Test Kit for Mycobacteria), HiMedia, India (RU FSS 2012/12473 of 02.11.2012).

The disadvantage of the closest technical solution is its relatively high complexity and relatively low sensitivity and reliability associated with the visualization and interpretation of the result, since the colorimetric reaction is estimated by a narrow band on the surface of the agar medium, which results in low staining intensity. This reduces the efficiency of the known method and narrows the arsenal of technical means that can be used to determine the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide.

The objective of the invention is to create a highly efficient and easy-to-use method for determining the sensitivity to pyrazinamide of Mycobacterium tuberculosis, with high sensitivity and reliability, as well as expanding the arsenal of technical tools that can be used to determine the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide.

The required technical result is to increase the sensitivity and reliability of the detection of the result while reducing material and labor costs while expanding the arsenal of technical means that can be used to determine the sensitivity of Mycobacterium tuberculosis cultures to pyrazinamide.

The problem is solved, and the required technical result is achieved by the fact that to determine the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide, a color reaction is carried out for the oxidation of ferrous iron to ferric iron in the presence of pyrazic acid synthesized from pyrazinamide under the action of the mycobacterial enzyme pyrazinamidase, according to the invention, the color reaction is carried out directly in in a liquid nutrient medium for cultivation while providing a culture incubation time of 1 day, moreover, the concentration of the culture is carried out by precipitation, and the registration of the result of the color reaction over the entire reaction volume is carried out for 15 minutes.

The proposed method is implemented as follows.

Development and optimization of the conditions of the proposed method were carried out on 20 MBT strains with known phenotypic sensitivity to pyrazinamide, determined in the BASTEC MGIT 960 system (10 pyrazinamide sensitive, 10 pyrazinamide resistant).

The method can be implemented in two versions, depending on the equipment of bacteriological laboratories.

Option 1 (for laboratories using the VASTEC MGIT 960 system for culture diagnosis of tuberculosis).

A MGIT control tube is taken into the study, which remains after the drug sensitivity test in the VASTEC MGIT 960 system to 1st-line anti-drugs, which does not contain drugs, with the number of MBT growth units of at least 400 GU (about 10 ⁷ CFU/m).

The culture tube is additionally incubated for 1 week in a thermostat at 37°C to increase the bacterial mass. After incubation, the culture is concentrated by settling. To do this, carefully, trying not to agitate the mycobacterial cells from the bottom, carefully take 7.5 ml of the medium that does not contain mycobacteria with a sterile disposable Pasteur pipette from the tube. 100 µl of the diluted pure pyrazinamide substance is added to the MBT culture remaining in the volume of 800 µl to a final concentration of 800 µg/ml. Next, tubes with MBT and pyrazinamide incubated for 1 day at a temperature of 37°C in a thermostat. After a day of incubation, 200 μl of Mohr's salt solution with a concentration of 0.04 g/ml is added to the test tube. The result is evaluated not earlier than after 1 minute and not later than after 15 minutes of incubation at room temperature.

The appearance of a pinkish-brown color of the medium indicates the sensitivity of the MBT isolate to pyrazinamide, the absence of color indicates resistance to pyrazinamide.

Option 2 (for laboratories conducting culture diagnostics of tuberculosis on dense nutrient media).

800 µl of Middlebrook 7H9 liquid nutrient medium is poured into a glass or transparent plastic test tube, into which 2-3 complete loops of 3 mm diameter of a fresh MBT culture grown on a solid egg nutrient medium (for example, Lowenstein-Jensen or Finn-II) are added and incubated in a thermostat at 37°C for 7 days to adapt the MBT to new cultivation conditions. Then, 100 µl of diluted pure pyrazinamide substance is added to the MBT culture to a final concentration of 800 µg/ml.

Next, tubes with MBT and pyrazinamide incubated for 1 day at a temperature of 37°C in a thermostat. After a day of incubation, 200 μl of Mohr's salt solution with a concentration of 0.04 g/ml is added to the test tube. The result is evaluated not earlier than after 1 minute and not later than after 15 minutes of incubation at room temperature.

The appearance of a pinkish-brown color of the medium indicates the sensitivity of the MBT isolate to pyrazinamide, the absence of color indicates resistance to pyrazinamide.

Examples of the implementation of the method.

Example 1

Determination of sensitivity to pyrazinamide of MBT cultures grown in MGIT tubes in the VASTEC MGIT 960 system.

The presented method (variant 1) was used to study 230 MBT cultures grown in the BASTEC MGIT 960 system. For the same cultures, the phenotypic sensitivity to pyrazinamide was determined in the BASTEC MGIT 960 system with the PZA-kit (BD, USA) (reference method).

The addition of Mohr's salt to a concentrated MBT culture incubated for 1 week with pyrazinamide resulted in staining of the medium in 117 out of 124 test tubes with pyrazinamide-sensitive MBT isolates. Staining of the medium in test tubes with pyrazinamide-resistant MBT isolates was observed in 1 case out of 106. Therefore, in 222 out of 230 cases (96.52%), the result of determining the sensitivity to pyrazinamide in MBT isolates by the presented method coincided with the reference one.

Example 2

Determination of sensitivity to pyrazinamide of MBT cultures grown on solid nutrient medium.

The presented method (option 2) was used to study 50 MBT cultures grown on a dense nutrient medium of Levenshtein-Jensen. For the same cultures, phenotypic sensitivity to pyrazinamide was determined using the BASTEC MGIT 960 system with the PZA-kit (reference method). The addition of Mohr's salt to an MBT culture re-cultivated on Middlebrook 7H9 liquid medium and incubated for 1 week with pyrazinamide resulted in staining of the medium in 22 out of 25 tubes with pyrazinamide-sensitive MBT isolates. Staining of the medium in test tubes with pyrazinamide-resistant MBT isolates was not observed. Therefore, in 47 out of 50 cases (94%), the result of determining the sensitivity to pyrazinamide in MBT isolates by the presented method coincided with the reference.

The advantages of the proposed method in comparison with existing prototypes are:

- minimization of sample contamination and laboratory error due to the fact that the reaction is carried out directly in the mycobacteria cultivation medium;

- increasing the sensitivity of the analysis by concentrating the culture of mycobacteria, which leads to an increase in the content of the enzyme and, as a result, pyrazic acid in the reaction volume, which increases the intensity of staining;

- increasing the objectivity of the evaluation of the result due to the detection of color in the entire reaction volume.

Thus, thanks to the improvement of the known method, the required technical result is achieved, which consists in increasing the sensitivity and reliability of the detection of the result while reducing material and labor costs while expanding the arsenal of technical means that can be used to determine the sensitivity of Mycobacterium tuberculosis cultures to pyrazinamide.

Information sources

1. Steele MA, Des Prez RM. The role of pyrazinamide in tuberculosis chemotherapy. Chest 1988; 94:845-50.

2. Zhang Y., Mitchison D. The curious characteristics of pyrazinamide: a review. Int. J. Tuberc. lung dis. 2003; 7:6-21.

3. World Health Organization. 2019. WHO consolidated guidelines on drug-resistant tuberculosis treatment. Geneva: World Health Organization; 2019. WHO/CDS/TB/2019.7

4. Order of the Ministry of Health of the Russian Federation No. 109 dated March 21, 2003 (as amended on June 5, 2017) "On the improvement of anti-tuberculosis measures in the Russian Federation"

5. World Health Organization. 2009. Guidelines for surveillance of drug resistance in tuberculosis, 4th ed. WHO/HTM/TB/2009.422. World Health Organization, Geneva, Switzerland.

6. Siddiqi S.H., Rusch-Gerdes S. MGIT Procedure Manual for VASTEC MGIT 960TB System. - Guide to working with the VASTES MGIT 960 system. 2006. 89 p.

7 Wayne L.G. Simple pyrazinamidase and urease tests for routine identification of mycobacteria. Am Rev Respire Dis. 1974 Jan; 109(1): 147-51.

8. Singh P, Wesley C, Jadaun GP, Malonia SK, Das R, Upadhyay P, Faujdar J, Sharma P, Gupta P, Mishra AK, Singh K, Chauhan DS, Sharma VD, Gupta UD, Venkatesan K, Katoch VM. Comparative evaluation of Lowenstein-Jensen proportion method, BacT/ALERT 3D system, and enzymatic pyrazinamidase assay for pyrazinamide susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol. 2007;45(l):76-80.

9. Aono A, Chikamatsu K, Yamada H, Igarashi Y, Murase Y, Takaki A, Mitarai S. A simplified pyrazinamidase test for pyrazinamide drug susceptibility in Mycobacterium tuberculosis. J Microbiol Methods. 2018;154:52-54.

Claims (2)

1. A method for determining the sensitivity of cultures of Mycobacterium tuberculosis to pyrazinamide, which involves carrying out a color reaction of oxidation of ferrous iron to ferric iron in the presence of pyrazinamide synthesized from pyrazinamide under the action of the mycobacterial enzyme pyrazinamidase, adding Mohr's salt, characterized in that to 800 μl of MBT culture in liquid nutrient 100 µl of diluted pure pyrazinamide substance is added to the medium to a final concentration of 800 µl/ml, test tubes with MBT and pyrazinamide are incubated for 1 day, the color reaction is carried out directly in the liquid cultivation medium, after a day of incubation, 200 µl of Mohr's salt solution with a concentration of 0.04 are added to the test tube g / ml, the result is evaluated no earlier than 1 minute and no later than 15 minutes. 2. The method according to p. 1, characterized in that before making 100 μl of the diluted pure substance of pyrazinamide, the culture is concentrated by precipitation.