Method for determining antibacterial resistance of human body

FIELD: medicine.

SUBSTANCE: invention describes a method for determining antibacterial resistance of a human body in diseases caused by a staphylococcus infection, based on blood neutrophil analysis; functional activity of neutrophilic granulocytes of patient's peripheral blood is determined by chemoluminescence with calculating a bacterial activation index (the BAI-index) of neutrophils representing the relation of an area under a curve of neutrophilic granulocyte chemoluminescence induced by Staphylococcus epidermidis bacteria to an area under a curve of spontaneous neutrophilic granulocyte chemoluminescence, and if the derived value is less than 1.47, a high level of antibacterial resistance is stated, while the derived values 1.47 and more shows a low level of antibacterial body resistance.

EFFECT: method is informative, precise, complies with the modern requirements for laboratory diagnostic techniques and allows predicting a nature, a clinical course period and bacterial complications in the patients of various groups.

1 ex, 2 tbl

 

Invention refers to medicine, in particular to immunology, and can be used to assess nonspecific antibacterial resistance of the human body.

A known method for the determination of nonspecific antibacterial resistance [EN 2068997 C1, publ. 10.11.1996]. The essence of the invention lies in the fact that prior to the preparation of the smear preparing a culture of the bacteria Serratia marcescens to use it as the object of phagocytosis, opsonized bacterial suspension serum of healthy individuals, incubated opsonization and Papanicolaou culture bacteria lacokanostra patients, examine the total cytochemical activity of myeloperoxidase and cationic proteins before and after incubation, and then calculate the stimulation index of neutrophils. When the index value of 0.5 and below define the reduction of antibiotic resistance of the organism. The disadvantage of this method is the duration of its execution and the need for microscopic evaluation of the activity of enzymes, which reduces the accuracy and objectivity of the method.

The objective of the invention is to create a new informative method for determining the antibiotic resistance of the organism.

The task is achieved by using a chemiluminescent analysis examined the functional activity of neutrophilic granulocyte the peripheral blood of the patient, determine the index of the bacterial activation of neutrophilic granulocytes (IBA-index), which represents the ratio of the area under the curve chemiluminescence of neutrophils induced by a bacterial suspension of Staphylococcus epidermidis to the area under the curve of spontaneous chemiluminescence of neutrophils. When the value of this index is below 1,47 determine the high level of antibiotic resistance, and when the amount equal to 1.47 and above, the low level of antibiotic resistance of the organism.

Low antibacterial resistance is characterized by increased susceptibility of the organism to bacterial infections; increasing duration of the disease and the accession opportunistic bacterial infections, as well as the weighting of the acute period and the rapid transition of the disease into a chronic form. Figure 1,47 obtained empirically based on the mapping data, a chemiluminescent analysis of the results of the medical examination and the subsequent exacerbations of chronic bacterial infections.

Activated neutrophilic granulocytes are potent effectors and trigger mechanisms of cascading reactions, ensuring the development of inflammation. Anti-infective action of neutrophils is associated mainly with the gene is a situation of active forms of oxygen, and one of the methods allowing to estimate oxygendependent biocenose neutrophilic granulocytes, is a chemiluminescent analysis. Neutrophilic granulocytes bear on their surface a wide range of receptors, some of which can interact with Neopleistocene bacteria (CR - and Toll-receptors). Phagocytosis without opsonization is associated with the presence of microbes specific virulence factors with which they penetrate into the cells. Phagocytosis is one of the main systems protect the body from foreign agents and plays an important role in inflammatory diseases, especially in staphylococcal infections. The undoubted advantage of this method is used as the inductor bacteria Staphylococcus epidermidis, which can cause severe inflammatory diseases.

The method is as follows. From the venous blood of the patient allocate neutrophilic granulocytes. For this to 5 ml of blood with heparin was added 1 ml of poliglyukina. The mixture is incubated for 30 min at 37°C to accelerate the sedimentation of erythrocytes. Received leukocyte supernatant washed twice in Hanks solution without phenol red for 10 min at 400 g. The supernatant is drained, the remaining neutrophilic granulocytes diluted in 1 ml of Hanks solution and get suspended. Count the number of natref is selected granulocytes in the camera Goryaeva. To conduct chemiluminescent analysis using the following reagents: donor serum (blood group AB, RH factor negative), Hanks solution (without phenol red), luminal concentration of 100 µg/ml Prepared sample: 200 ál suspension of neutrophils, 20 ál of donor serum, 240 μl of Hanks solution, 50 μl of lyuminola and 40 µl of bacterial suspension of Staphylococcus epidermidis in a concentration of 106CFU/ml Chemiluminescent analysis is performed in two cells: spontaneous chemiluminescence is carried out without adding the inductor, the second cell type bacterial suspension of Staphylococcus epidermidis. Measuring the chemiluminescent response exercise using a chemiluminescent analyzer, for example "CL3604"within 90 minutes recording the results and management of the chemiluminescent analyzer is performed via computer. Get the curves of spontaneous and induced bacteria chemiluminescence. The index of the bacterial activation of neutrophils (IBA-index) is determined by the formula:

IBA=Sinduced/Sspontaneous,

where:

Sinduced- the value of the area under the curve chemiluminescence induced bacterial suspension of Staphylococcus epidermidis;

Sspontaneous- the value of the area under the curve of spontaneous chemiluminescence.

When IBA-index below 1,47 diagnostics which can be carried on a high level of antibiotic resistance, when IBA-index equal to 1.47 or higher, the low level of antibiotic resistance of the organism.

The method was tested on 12 patients with chronic sinusitis, received treatment in the ENT Department of the Noosa heads "Road clinical hospital" on secretness JSC Russian Railways, and 45 healthy individuals. The survey results presented in table 1.

The results of the study found that all of 45 healthy individuals value IBA-index below 1,47 (100% match). In 11 patients also noted the coincidence of the results of the analysis. In 1 patient index value below 1,47. Thus, the coincidence method for determination of antibiotic resistance in patients with chronic sinusitis 91.7%. In the statistical analysis (with count median (Me) and the interquartile spread (C25-C75)) statistically significant differences (P<0.001) in the level of IBA in the groups of healthy and sick people (table 2).

Table 2
The level of IBA in the groups of healthy and sick persons
GroupNMeC25C75
Healthy450,9300000,6300001,330000
Sick121,7000001,6400002,055000

Clinical example 1. Patient I., 43, case History No. 2149. Received in-patient treatment in the ENT Department of the Noosa heads "Road clinical hospital at the station Krasnoyarsk OJSC Russian Railways with a diagnosis of chronic rhinosinusitis. At admission the patient's condition moderate, concerned about the weakness and headaches. A study by the claimed method. IBA equal to 1.96 (above 1,47). Therefore, we examined the low level of antibiotic resistance. Clinical example 2. Patient S., 41, was surveyed all major medical specialists, including physician specialist physician, immunologist, when carrying out routine inspection of employees of JSC "Gorelektrosvyaz" on the basis of clinical research Institute of medical problems of the North SB RAMS. Deemed healthy. A study by the claimed method.

IBA 1.15 (below 1,47). Therefore, in subjects with a high level of antibiotic resistance.

The technical result of the proposed method:

- automated determination of the functionality of the activity of neutrophilic granulocytes;

- evaluation of the results does not depend on subjective factors.

Thus, the way of informative, accurate, meets all modern requirements for laboratory diagnostic techniques, allows to considerably simplify the process of determining factors of nonspecific defense, its functional activity and reserve capacity, makes it possible to identify the failure of phagocytic protection and its reactive or pathological activation.

The method of determining antibiotic resistance of the organism when the diseases caused staphyloccocal infection, based on the study of blood neutrophils, characterized in that the functional activity of neutrophilic granulocytes in the peripheral blood of the patient are examined using chemiluminescent analysis, determine the index of the bacterial activation of neutrophils (IBA-index), which represents the ratio of the area under the curve induced by the bacteria Staphylococcus epidermidis chemiluminescence of neutrophils to the area under the curve of spontaneous chemiluminescence of neutrophils, and when the value of this index is below 1,47 determine the high level of antibiotic resistance, and when the value is equal to 1.47 and up - low level of antibiotic resistance of the organism.



 

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2 ex

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4 ex, 2 tbl

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5 ex, 1 tbl

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2 ex, 1 tbl

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2 ex, 2 tbl

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1 tbl, 1 ex

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2 ex

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1 tbl, 3 ex

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2 ex

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3 ex, 1 tbl

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3 ex, 1 tbl

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2 tbl, 4 ex

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1 tbl

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1 cl, 1 ex, 1 tbl

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