A method for predicting reconvalescence salmonella bacteria carrier

 

(57) Abstract:

The invention relates to medicine, in particular for the diagnosis of infectious diseases. For this purpose, during the height of the disease by a pure culture of Salmonella isolated from feces of a patient with salmonellosis, determine antilactoferrin activity, and koprofilia determine the content of lactoferrin and when values of the first metric 7,8-13,0 ng, and the second 460-1400 ng/ml predict reconvalescence Salmonella hosting. The method allows high accuracy to predict the formation of Salmonella reconvalescence bacteria carrier in the early stages of the disease salmonellosis, which allows an individual approach to treatment of patients with the aim of reducing the number bakterionositelej, to correct the deficiency of factors of natural resistance of the mucosa of the digestive tract. 4 table.

The invention relates to medicine, in particular for the diagnosis of infectious diseases, and can be used in clinical practice to predict reconvalescence bacteria carrier in salmonellosis.

There is a method of forecasting the Salmonella bacteria is rnga with standard group O-diagnosticum [1]. But in this way are not taken into account the biological properties of Salmonella, which largely depends on the nature of the course and outcome of infection, and that local antibody subjected to intensive exposure to proteolytic enzymes, which complicates the assessment of the results [2].

Known prediction of Salmonella bacteria carrier on the level of circulating lymphocytes sensitized to the O-antigen of Salmonella and determined in the reaction of antigen-specific rosethorne [2]. If in the acute period of the disease and during convalescence sharply reduced the number of antigen specific lymphocytes, the predicted probability of completion of the acute form of the disease by hosting. Similarly predicted Salmonella hosting at low index of completion of phagocytosis in patients with acute salmonellosis [2]. However, these methods do not take into account the biological properties of Salmonella affecting the course and outcome of infection.

Known methods of predicting Salmonella bacteria carrier, taking into account the dynamics of the rise of antibodies [3], the decrease in lytic activity of serum [4] is about 3-4 weeks of the disease.

Closest to the claimed method according to purpose and essential features is a method for predicting reconvalescence Salmonella bacteria carrier [6], according to which isolated from feces of a patient with salmonellosis pure culture of Salmonella determine the antilysocyme activity (ALA), and if antilysocyme activity of Salmonella is 8 mcg and above that in 81% of cases predict the development of bacteria carrier.

However, the known method has a number of disadvantages. Antilysocyme activity of Salmonella assessed in this way allows them to inactivate lysozyme, which is one of the factors of natural resistance and contained all the secrets of the human body. However, colonization by Salmonella of the intestinal mucosa inactivation of lysozyme for Salmonella does not play a significant role, as the antimicrobial property of lysozyme associated with hydrolytic cleavage peptidoglycan layer of the cell wall mainly gram-positive bacteria [7], and Salmonella are resistant to the action of lysozyme, as are gram-negative bacteria.

At the same time in antimicrob is Elista, participate other more effective compared to lysozyme, bactericidal proteins (lactoferrin, secretory antibodies and other), acting not only on gram-positive, and gram-negative microorganisms [2, 8], the level of which is not included in the method prototype.

The objective of the proposed technical solution is to improve the accuracy of forecasting reconvalescence Salmonella bacteria carrier.

To solve this problem in the present method of forecasting reconvalescence Salmonella bacteria carrier in pure cultures of Salmonella isolated from feces of a patient with salmonellosis, determine antilactoferrin activity, and koprofilia determine the content of lactoferrin and when values of the first metric 7,8-13,0 ng, and the second 460-1400 ng/ml predict reconvalescence Salmonella hosting.

The main features of the proposed method are

- definition antilactoferrin activity in pure cultures of Salmonella isolated from feces of a patient with salmonellosis;

- definition of the content of lactoferrin in coprofilia;

prognozirovaniya 7,8-13,0 ng, and the content of lactoferrin in coprofilia - 460-1400 ng/ml.

A new set of features allows for the implementation of the claimed invention to obtain a new technical result, which is that the new method allows high accuracy to predict the formation of Salmonella reconvalescence bacteria carrier in the early stages of the disease salmonellosis that allows an individual approach to treatment of patients with the aim of reducing the number bakterionositelej, to correct the deficiency of factors of natural resistance barrier of the epithelium of the digestive tract, for example, immunomodulators, and use of drugs that suppress the ability of enterobacteria to the degradation of these factors.

It is known that one of the factors that determine the possibility of development and duration of Salmonella bacteria carrier, are relationships in the system “lysozyme owner-antilysocyme activity of the pathogen and Salmonella with high ALA inactivate more lysozyme in cells and tissues of the body and can long persist in the body of patients [9]. However, lysozyme has no direct lytic on the potential bacteriolytic activity of lysozyme against microorganisms and, in particular, gram-negative bacteria is implemented due to the presence of other antimicrobial factors, including potentiate the action of lactoferrin [8, 10]. In addition, lactoferrin exerts a bacteriostatic effect on the growth of bacteria on the surface of the barrier epithelia and in phagolysosome neutrophilic granulocytes associated with its ability to create and maintain deficient in iron and other cations of metals of variable valency environment, i.e. between bacteria and lactoferrin there is competition for iron, which increases the virulence of the latter [8]. When the deficit lactoferrin significantly reduced ability to inhibit the microorganisms present on the surface of the barrier epithelium, or already favoritemovie bacteria [11].

However, it is known that the level of lactoferrin as an acute phase protein family transferring largely regulated by proinflammatory cytokines [12], and its content varies considerably in inflammation. Therefore, in laboratory and clinical practice, the values of the content of lactoferrin used to assess the degree of activity of the inflammatory response [13] as a criterion for the effectiveness of anti-inflammatory therapy [14]. Gramicidine action of lactoferrin, providing conditions for their survival in the host [15].

The use of quantitative assessment of the content of lactoferrin in coprofilia and antilactoferrin activity of Salmonella to predict reconvalescence bacteria carrier in the sources of patent and scientific literature is not found.

The authors experimentally determined relationship between reconvalescent Salmonella hosting and contribute to its formation content of lactoferrin in coprofilia patient with salmonellosis and ALA exciter, also defined intervals changes in these indicators.

Was conducted the following study.

In the experimental series was prepared coprophiliac patients with salmonellosis. For this purpose, 4 g of faeces taken in the midst of disease and during convalescence, homogenized with 8 ml of buffered saline solution (0.15 M, pH 7,2), which was preliminarily added to inactivate intestinal proteases 1 mg of trypsin inhibitor from soybean (Sigma, catalog number 9003) [16]; 0.5 ml of 50 mm EDTA; 0.5 ml of 0.025% solution of tween-20 (Sigma, catalog number 1379) [16]; 1 ml kontrikala (AWD, Germany) at a concentration of 500 u/ml l solution kontrikala at a concentration of 500 u/ml and re-centrifuged in the same mode. In the resulting supernatant, which coprofilia, determined the content of lactoferrin enzyme-linked immunosorbent assay (ELISA) using reagents JSC “Russia” (Novosibirsk region, Koltsovo) in accordance with the instruction attached to the kit reagents ELISA, and the concentration of lysozyme on methodological recommendations [17].

Conducted by the authors showed (the results are shown in table 1) that the content of lysozyme in coprofilia patients in the midst of illness increases compared with apropertyname healthy subjects only 1.6 times, in the stage of convalescence 1.5 times, while the content of lactoferrin, respectively 12 and 6.6 times.

This confirms that in all periods of Salmonella infections, especially in the midst of illness, revealed significantly higher, compared with lysozyme, the rate of increase in coprofilia lactoferrin (an integral element of the molecular system to protect against infection, amplifying the effects of other factors of natural resistance) that allows you to more accurately detect any deviation of the quantitative values of this indicator. The content of lactoferrin in coprofilia can be used as signal marker-infringement of abnoy protect the body of the patient at different periods of Salmonella infection.

Thus, changes in the content of lactoferrin in coprofilia salmonellosis cases are more sensitive signaling marker for disturbances in the natural resistance of the intestinal mucosa compared with lysozyme.

Considering that the factors contributing to the formation of bacteria carrier are deficiencies in the system nonspecific defense against infection [18], the authors have conducted a comparative study of the content of lactoferrin in coprofilia patients with salmonellosis, which was formed reconvalescence hosting and which hosting was not observed. To this end we examined 30 patients with salmonellosis in the midst of disease and during convalescence. The survey included bacteriological examination of faeces and determination in coprofilia concentration of lactoferrin ELISA analysis. In the bacteriological examination of faeces was found that in 8 out of 30 surveyed in the control crops faeces after treatment before discharge from hospital (stage of convalescence) was detected growth of Salmonella, i.e. they spar are shown in table 2.

As can be seen from table 2, patients with established reconvalescent by hosting in coprofilia the content of lactoferrin was 2.3 times lower than in the midst of illness and 3.7 times lower than in the period of convalescence compared with those in the control crops fecal Salmonella was not sown.

Statistical processing of the results on the content of lactoferrin in coprofilia and antilactoferrin activity of Salmonella carried out using student's criterion (t), test of differences secondary to the significance level of p=0.01, t=2,83 for groups of subjects, consisting of 22 people, and t=3,50 for groups of subjects, consisting of 8 people, has allowed to establish a valid interval values [19] the content of lactoferrin in the midst of illness, which in the period of convalescence is formed reconvalescence Salmonella hosting constituting 460-1400 ng/ml (table 2).

Thus, the authors conducted studies showed that no significant increase in coprofilia lactoferrin in the midst of illness with Salmonella infection and persistent low levels of lactoferrin during the reaction.

Since it is known that enterobacteria capable of survival in the human body, including the hosting, the inactivation of many factors of natural resistance, including lactoferrin [15], the authors have conducted experimental studies to assess the relative level antilactoferrin activity (alpha) of Salmonella isolated from faeces of patients with salmonellosis, which formed the hosting and without bacteria carrier.

Antilactoferrin activity of Salmonella was studied immunoferritin analysis. From daily agar cultures of Salmonella were preparing a suspension in physiological solution (109CFU/ml) and incubated in equal volumes with a solution of lactoferrin (Sigma, catalog number 0520) at a concentration of 100 ng/ml for 24 h at 37°C. a Control sample contained instead of the microbial suspensions buffered saline. After incubation of control and test samples were centrifuged for 20 min (8000 rpm; 5°C), supernatant was collected and it was determined the residual concentration of lactoferrin ELISA analysis using reagents JSC “Vector-best” in accordance with the instructions use the line wavelength 492 nm, the content of lactoferrin was determined by constructing a calibration charts and was expressed in ng. The results of the study are given in table 3.

As can be seen from table 3, the amount of lactoferrin, inactivating Salmonella isolated from patients with established further by hosting, significantly higher compared with Salmonella isolated from patients in whom the hosting is not formed. Statistical processing of the results of research antilactoferrin activity of Salmonella carried out using student's criterion (t), test of differences secondary to the significance level of p=0.01, t=2,83 for groups of subjects, consisting of 22 people, and t=3,50 for groups of subjects, consisting of 8 people, has allowed to establish the valid interval of [16] for antilactoferrin activity of Salmonella isolated from patients without subsequent formation of bacteria carriage amounting to 4.3 to 7.7 ng (table 3).

For Salmonella isolated from patients who have been hosting, valid interval was 6,0-13,0 ng. Excluding alpha values common to Salmonella, selected from patients without formylbenzeneboronic activity which is formed hosting, amounted to 7.8-13,0 ng.

This confirms that isolated from patients with Salmonella, with alpha values in the range of 7.8-13,0 ng, capable of long-term stay in the body, causing the development of reconvalescence Salmonella bacteria carrier.

The study of the content of lactoferrin in coprofilia patients with salmonellosis, antilactoferrin activity of Salmonella and data on the nature of the flow Salmonella infection are shown in table 4.

As can be seen from table 4, reconvalescence hosting was formed only in subjects R., 39 years of age, whose acute salmonellosis both indicators, including lactoferrin in coprofilia and antilactoferrin activity of Salmonella, entered in the valid interval of the data change indicators (460-1400 ng/ml for the first indicator and 7,8-13,0 ng - for the second indicator). All other subjects, in whose valid interval was any single measure or did not get both indicators, acute infection is over the recovery.

The method is as follows.

1. From the feces of a patient gotovim previously added 0.5 ml of 50 mm EDTA, 0.5 ml of 0.025% solution of tween-20 (Sigma, catalog number 1379) [16]. 1 ml kontrikala (AWD, Germany) at a concentration of 500 u/ml, 1 mg of trypsin inhibitor from soybean (Sigma, catalog number 9003) [16].

2. The homogenate was transferred into plastic tubes and centrifuged for 20 min (8000 rpm; 5°C), to nadeshiko re-add 1 ml kontrikala at a concentration of 500 u/ml and again centrifuged for 15 min (8000 rpm; 5°C).

3. Select adosados and determine the content of lactoferrin ELISA using reagents JSC “Vector-best” in accordance with the instructions for use of these reagents. Measurement of the optical density of the samples is carried out on the photometer, the recalculation of the content of lactoferrin carried out through construction of calibration graphs and expressed in ng/ml.

4. From daily agar culture of Salmonella isolated from the patient, preparing a microbial suspension in physiological solution (109CFU/ml).

5. Sterile buffered saline solution (0.15 M; pH 7,2) prepare a solution of lactoferrin (Sigma, catalog number 0520) [16] at a concentration of 100 ng/ml In the wells of a sterile polystyrene tablet make 50 ál of the bacterial suspension and solution of lactoferrin.

7. Samples incubated for 24 h at 37°C.

8. After incubation, the samples are centrifuged for 20 min (8000 rpm; 5°C).

9. Carry out the determination of the residual content of lactoferrin in the supernatant experimental and control samples using ELISA using reagents JSC “Vector-best” in accordance with the instructions for use of these reagents. Measurement of the optical density of the test and control samples is carried out on the photometer, the recalculation of the content of lactoferrin carried out through construction of calibration graphs and get the content of lactoferrin in ng.

10. Expect antilactoferrin activity by the difference in the concentration of lactoferrin in the experimental and control samples and expressed in ng lactoferrin, inactivated Salmonella.

11. Predict reconvalescence Salmonella hosting, if antilactoferrin the activity of the pathogen and the content of lactoferrin in coprofilia included in the intervals of admissible values change, components for antilactoferrin activity of the pathogen - 7,8-13,0 ng for the content of lactoferrin in coprofilia - 460-1400 ng/ml, characterizing the formation reconvalescence of Salmonella coprofilia and values antilactoferrin activity of Salmonella, isolated from faeces of patients with salmonellosis, allows us to predict the formation of reconvalescence Salmonella bacteria carrier.

Example 1. Patient K., 36 years old, undergoing treatment in the Orenburg municipal clinical hospital of infectious diseases, from the feces during the height of the disease selected Salmonella enteritidis, antilactoferrin activity of this culture was 8.7 ng. The content of lactoferrin in coprofilia was $ 1098 ng/ml Obtained concentrations of lactoferrin and antilactoferrin activity are included in the intervals of admissible values change lactoferrin and antilactoferrin activity characterizing the formation reconvalescence bacteria carrier. This forecast is confirmed by the repeated allocation of the pathogen bacteriological examination of the patient after treatment before discharge from the hospital: three times in one day from feces were inoculated culture of Salmonella.

Example 2. Patient L., 42 years, in the midst of a disease selected from the feces of S. enteritidis. Antilactoferrin the activity of the pathogen was 9.5 ng, and the content of lactoferrin in coprofilia - 1690 ng/ml is Obtained antilactoferrin is on coprofilia not included in set intervals of change of this index, therefore, it is predicted recovery without recurrence of the carrier state. This forecast is confirmed triple negative results of bacteriological examination after 3, 5, 7 days after clinical recovery.

Example 3. Patient I., 19 years old, from the feces in the midst of illness allocated S. enteritidis, values antilactoferrin activity which was 3.4 ng. The content of lactoferrin in coprofilia was 980 ng/ml is Obtained antilactoferrin activity of Salmonella is not included in the interval of admissible changes of this indicator, and the value of the content of lactoferrin in coprofilia, by contrast, is at set intervals changes lactoferrin, characterizing the formation reconvalescence bacteria carrier. Projected recovery without recurrence of the carrier state. This forecast is confirmed by repeated negative results of bacteriological examination of the patient before discharge from hospital.

To establish the effectiveness of the proposed prediction method reconvalescence Salmonella bacteria carrier was examined 64 patients with salmonellosis, the diagnosis was confirmed clinically and baccante hosting was formed in 16 patients, as confirmed by positive culture results. The effectiveness of the proposed method in this group of patients was 93.8%.

Thus, the proposed method can accurately predict the probability of formation reconvalescence bacteria carrier in the early stages of disease development. Identified at an early stage of the disease entity with a deficit of lactoferrin in coprofilia and high values antilactoferrin activity of Salmonella are at risk of developing Salmonella bacteria carrier, as the shortage of lactoferrin as a factor nonspecific antimicrobial protection of the intestinal mucosa contributes to the selection of enterobacteria with a high potential for degradation of lactoferrin and keeping them in the host organism. The inventive method allows for early stages of disease development individual correction of deficiency of factors of natural resistance of the mucosa of the digestive tract, for example, immunomodulators and use of drugs that suppress the ability of enterobacteria to the degradation of these factors, which will reduce the number bakterionositelej and reduce papasena E. C. Free and bound to the immune complex coproantigen in salmonellosis and their diagnostic and prognostic value. Dis.... Kida. the honey. Sciences. Leningrad. 1986. - 156 S.

2. Hasenson L. B., Gull N. A. Immunological basis for diagnosis and epidemiological analysis of intestinal infections. - Leningrad. 1987. - S. 49-50.

3. Tandetnik Y. Y., Usuk N. D., Trushina centuries Serological method for the diagnosis of various forms of Salmonella bacteria carrier // Act. the matters. epidemiology and infections. diseases. - M. 1975. - Vol.5. H 1 - S. 74-77.

4. Nikolaev N. G. Indicators lytic activity of the blood serum of patients with typhoid fever, chronic bakterionositelej and salmonellosis cases //the Second all-Union Congress of infectious diseases. - Tashkent. 1985. - S. 23-24.

5. Perekopskaya T. I., Kolmogortseva centuries the Definition of mononuclear index patients OCIS in order to predict the continuing numbers // the Second all-Union Congress of infectious diseases. - Tashkent. 1985. - S. 375-376.

6. SU 1303163 IPC A1, And 61 To 37/48, 15.04.87 (prototype).

7. Bukharin, O. C., Vasilyev N. In. Lysozyme and its role in biology and medicine. - Tomsk. 1974. - S. 44-50.

8. Kokryakov Century. N. Biology of antibiotics animal origin. - Saint-Petersbu. Kokryakov Century. N. and other Synergistic antimicrobial activity of cationic proteins during phagocytosis // Modeling and clinical characteristics of phagocytic reactions / Sat. scient. works. Ed. by A. N. Mayansky. - Bitter. 1989. - S. 98-103.

11. Ellison, R. T., T. J. Giehl Killling of Gram-negative bacteria by lactoferrin and lysozyme // J. Clin. Invest. - 1991. - Vol.88. - P. 1080-1091.

12. Kashkin K. P. cytokines of the immune system: basic properties and immunobiological activity // Clinical laboratory diagnostics. - 1998. No. 11. - S. 21-32.

13. Nemtseva E. R. and other Immunoassay method for the detection of human lactoferrin and its use for the diagnosis of purulent-septic complications // Questions of medical chemistry. - 1995. - T. 41, No. 3. - S. 58-61.

14. Sukharev A. E., Nikolaev A., Vasiliev M. Y. serum lactoferrin in normal and pathological conditions // Problems of medical chemistry. - 1990. No. 3. - S. 81-83.

15. EN 2156807, IPC 27 12 Q 1/02, 1/04, 27.09.2000.

16. Catalogue “Sigma” (reagents for biochemistry and research in the field of natural Sciences). - 1999. - S. 621.

17. Bukharin, O. C., Volkov, A. N., Spikes M. C., Vyatkina L. A. application of the signal method for the diagnosis of acute dysentery by determining the amount of lysozyme in coprofilia. Method. recom. - Orenburg. 1979. the. 1996. - S. 106-113.

19. Lakin, F. Biometrics. - M.: Higher school. 1990. - S. 108.

A method for predicting reconvalescence Salmonella bacteria carrier by determining the ability of the pathogen to inactivate factors of natural resistance of a person, characterized in that a pure culture of Salmonella isolated from feces of a patient with salmonellosis, determine antilactoferrin activity, and koprofilia determine the content of lactoferrin and when values of the first metric 7,8-13,0 ng, and the second 460-1400 ng/ml predict reconvalescence Salmonella hosting.



 

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