A method of evaluating the effectiveness of leprosy medicines

 

The invention relates to medicine, namely to laboratory methods, and relates, in particular, the method of evaluating the efficiency leprosy medicines. The method is carried out by microscopic examination of blood serum, in which the serum intact and infected with M. leprae animals treated with the investigational medicinal product, in the amount of 20 µl applied to the surface of glass slides, dried at room temperature, and then, looking through the samples in the light microscope at low magnification, conduct a comparative analysis of their morphological paintings and in the presence of experimental paintings structureborne similar to that of the intact animals, judged on the effectiveness of leprosy therapy, and at sharp differences in terms of the violation of the radial symmetry of the cracks, their chaotic location the formation of a large number of polygonal separateness, the asymmetric location of mono - and polymorphic nodules, the presence of markers of inflammation, is judged on its inefficiency. Effect: simplified method. 5 Il.

The invention Rel is but to assess the effectiveness of leprosy medicines.

There is a method of evaluating the effectiveness leprosy drugs, based on the method of serial dilutions of medicinal substance in a liquid nutrient medium Shkolnikova using as a test strain cultures of M. lufu (irtuganova O. A., Urlaeva N. G. The use of M. lufu for primary selection leprosy drugs. // Actual problems of leprology. - Astrakhan, 1984. - S. 147-150).

However, the method has significant drawbacks that limit its use in practice laboratory - complexity, the need for expensive biological environments, equipment, dyes, duration of execution (not less than 20 days), which does not allow to obtain a specific technical result is a simplification of the method.

Also known a method of evaluating the effectiveness of leprosy medicines, based on the study of the saturation of macrophages culture leprosy granulation tissue (Anokhin centuries the Cultivation in vitro leprosy granulation tissue and the effect on her leprosy drugs. // Abstract. Diss...Kida. the honey. Sciences. - M., 1989. - 21 S.).

The disadvantages of this method are the complexity of the technical implementation, the need for specially about littelest version (14 days) which does not allow to obtain a specific technical result is a simplification of the method.

Closest to the proposed method is a method of assessing the effectiveness of leprosy medicines, which consists in determining the number of M. leprae (Shepard, C. C., McRae D. H. A method of counting acid-fast bacteria. - Int. J. Lepr. - 1968. - Vol.36. - P. 78-82) in the soft tissues of the paws of mice infected by the method of C. C. Shepard (Shepard C. C. The experimental disease that follows the infection of human leprosy bacilli into the footpads of mice. - J. Exp. Med. - 1960. - Vol.112. - P. 445-454). The similarity of this method to offer is that they both relate to laboratory research methods based on experimental reproduction leprosy infection by C. C. Shepard (1960), and microscopic examination of morphological parameters of the biological material.

The disadvantages of this method are:

- a lot of stages (preparation of suspensions of soft tissue pads of the paws of mice, centrifugation, smear preparation with the use of special glass slides coated with 3 circles with an area of 1 cm, the drying stroke, fixing, painting, determining the number of M. leprae using an immersion objective);

- complexity (rubbing his paws animal is in manual gomogenizirovannogo paper, heated over the flame of the burner to vapour, followed by washing with running water, discoloration sulfuric acid staining methylene blue, the final fresh water rinse; to determine the number of M. leprae is the view up to 20 fields of view along the diameter of each circle is calculated from the number of them in 1 ml of suspension by a special formula, if it is impossible to count the number of mycobacterial suspension is diluted 10, 100, 1000, etc. once, in this case, the smear preparation, staining repeated);

- direct contact with infected material;

- the need to use expensive dyes;

- the duration of the execution (not less than 48 hours).

Thus, these shortcomings do not allow to obtain a specific technical result is a simplification of the method.

The present invention solves the main task is to facilitate the method. The task was to eliminate these disadvantages and the creation of such a method of determining the effectiveness of leprosy drugs, which would in a short time, small volumes of biological fluids with minimal material costs to determine the adequacy PST, including microscopic examination of the dried serum, serum intact and infected with M. leprae animals treated with the investigational medicinal product, in the amount of 20 µl applied to the surface of glass slides, dried at room temperature, and then, looking through the samples in the light microscope at low magnification, conduct a comparative analysis of their morphological paintings and in the presence of experimental paintings structureborne similar to that of the intact animals, judged on the effectiveness of leprosy therapy, and at sharp differences in terms of the violation of the radial symmetry of the cracks, their chaotic arrangement, the formation of a large number of polygonal separateness, the asymmetric location of mono - and polymorphic nodules, the presence of markers of inflammation, is judged on its inefficiency.

Serum is the tissue of the body, but cloth highly mobile due to the weak intermolecular bonds between its elements. The structure of these relations can be identified by their fixation, which is achieved in the process of phase transition from liquid to solid (fixed).

The simplified method gives the possibility to exclude mnogoetapnaya, dyes, immersion lenses), the elapsed time (18-24 hours).

Summary of the invention illustrated by photographs, where:

Fig.1 - morphological picture of the blood of intact animals;

Fig.2 - morphological picture of the blood serum of infected animals not receiving treatment;

Fig.3 - morphological picture of the blood serum of infected animals treated with primary leprosy drug Dapsone;

Fig.4 - morphological picture of the blood serum of infected animals treated with investigational compound D 069;

Fig.5 - morphological picture of the blood serum of infected animals treated with investigational compound D 127.

The method is as follows.

20 μl of serum intact animals and animals treated with intragastric investigational drug is applied to the surface of a standard glass slides 75×25 mm and dried at room temperature 18-25°C at a relative humidity of 50-70% for 18-24 hours. Pre-glass soak for 24-48 hours in a solution of detergent, then washed in running water for 10 minutes and placed in a mixture Nikiforova, consisting of equal proportions of alcohol and ether, 30 minutes Before on the e at low magnification, evaluate their morphological picture, then make a comparative analysis of experimental and control of drugs.

If the morphological picture of a blood serum sample experimental (infected, receiving medication) animal differs from that of the control sample (intact) animal violation of radialnet cracks, their messy location, asymmetric localization of mono - and polymorphic nodules, a high level of structure (splitting areas into smaller), the presence of pathological formations in the form of linguistic structures (marker of inflammation), splitting the sample (marker intoxication), this indicates the ineffectiveness of therapy.

If the morphological picture of a blood serum sample experimental animal was not significantly different from control sample (intact) animal and is characterized by the preservation of the radial symmetry of cracks, uniformity of shapes sectors, separateness, nodules, the absence of pathological marker of inflammation (language field), this indicates the normalization of intermolecular elements of the blood serum of infected animals through effective action leprosy pharmaceutical preparations is ishah line CBA.

Below are the results of testing.

Example 1.

The experiment is set at 60 mice CBA. 20 mice infected with M. leprae by the method of C. C. Shepard, intragastric through the probe at a dose of 25 mg/kg of body weight was administered to the main leprosy drug diaminodiphenylsulfone (DDS, Dapsone). As control was used intact and infected animals (40 PCs). At the end of the experiment at the feet of infected animals was carried out by counting the number of M. leprae by the method of C. C. Shepard, D. N. McRae. Morphostructural analysis of the blood serum of experimental animals was proposed method.

The number of bacilli of leprosy in the paws of infected animals not receiving treatment, reached(2,71±0,49)×107microbial cells/ml. a Similar rate of mice treated with Dapsone, was(0,025±0,005)×107microbial cells/ml, which was significantly lower compared to untreated controls (p<0.05) and testified about the effectiveness of the test tools.

Morphological picture of a blood serum sample intact animals, which was adopted by the us as the norm, was characterized by radial symmetry cracks, monotony forms the main structural elements (sectors, separateness, nodules), lack's animals not receiving treatment, was characterized by the violation of the radial symmetry of the cracks, their chaotic arrangement, the formation of a large number of polygonal individualities, as well as the presence of a marker of inflammation - local language fields. In some cases there was a tendency of forming a double facies is a sign of intoxication (Fig.2).

Morphological picture samples of blood serum of infected animals treated with Dapsone, differed minor violation of the radial symmetry of the cracks. There was a trend towards uniformity of shapes sectors, separateness, nodules. Pathological markers of inflammation, intoxication was absent (Fig.3).

Thus, the characteristic features of morphograms infected animals are a violation of sectoral rhythm, the randomness of the location of the cracks, the formation of a large number of polygonal separateness, the presence of pathological structures of the type “the Arnold tongues”. In turn, the identity of morphograms serum intact animals and animals treated with Dapsone, testifies to the effectiveness of DDS-therapy, which is confirmed by a significant decrease in the number of M. leprae in the soft tissues of the pads of the paw.

Conclusion: ispytuemym set at 60 mice CBA. 20 mice infected with M. leprae by the method of C. C. Shepard, intragastric through the probe at a dose of 6 mg/kg body weight was administered the investigational medicinal product D 069. As control was used intact and infected animals (40 PCs). At the end of the experiment at the feet of infected animals was carried out by counting the number M lr according to the method of C. C. Shepard, D. H. McRae. Morphostructural analysis of the blood serum of experimental animals was proposed method.

The number of bacilli of leprosy in the paws of infected animals not receiving treatment, reached(2,71±0,49)×107microbial cells/ml the Number of bacilli of leprosy in the paws of mice treated with the drug D 069, was significantly (p<0,05) lower than in controls and was(0,02±0,006)×107microbial cells/ml, indicative of the efficiency of the investigated tools.

Morphological picture of a blood serum sample intact animals, which was adopted by the us as the norm, was characterized by radial symmetry cracks, monotony forms the main structural elements (sectors, separateness, nodules), the absence of markers of inflammation and toxicity (Fig.1).

For morphological patterns of blood serum sample ZAR the location, the formation of a large number of polygonal individualities, as well as the presence of a marker of inflammation - local language fields. In some cases there was a tendency of forming a double facies is a sign of intoxication (Fig.2).

For morphological patterns of the samples of blood serum of infected animals treated with the investigational medicinal product, was the tendency of the formation of sectors seasoned regularity, monomorphic separateness, nodules. Markers of inflammation, intoxication was absent (Fig.4).

Thus, the picture of structureborne serum of animals of the experimental group was as close as possible to that of the intact animals that were correlated with the results of counting the number of M. leprae by C. C. Shepard, D. H. McRae and testified about the effectiveness of the drug D 069 as leprosy medicines.

Conclusion: the tested drug on the proposed evaluation method is effective.

Example 3.

The experiment is set at 60 mice CBA. 20 mice infected with M. leprae by the method of C. C. Shepard, by intragastric probe was administered the investigational medicinal product D 127. As control was used intact and infected W is the method of S. C. Shepard, D. H. McRae. Morphostructural analysis of the blood serum of experimental animals was proposed method.

The number of bacilli of leprosy in the paws of infected animals not receiving treatment, reached(2,71±0,49)×107microbial cells/ml the Number of bacilli of leprosy in the paws of mice treated with the drug D 127, amounted to(2,14±0,3)×107microbial cells/ml, which was close to those in untreated control (p>0.05) and showed the absence of leprosy activity of the studied tools.

Morphological picture of a blood serum sample intact animals, which was adopted by the us as the norm, was characterized by radial symmetry cracks, monotony forms the main structural elements (sectors, separateness, nodules), the absence of markers of inflammation and toxicity (Fig.1).

For morphological patterns of a sample of the blood serum of infected animals not receiving treatment, was characterized by the violation of the radial symmetry of the cracks, their chaotic arrangement, the formation of a large number of polygonal individualities, as well as the presence of a marker of inflammation - local language fields. In some cases there was a tendency of forming a double Cerisoles asymmetric location of the radial cracks, the lack of trend uniformity of shapes sectors, separateness, nodules. There was a high level of structure (large number of polygonal separateness), and the presence of markers of inflammation (Fig.5).

Thus, the picture of structureborne facies of the blood serum of animals of the experimental group was similar to the one observed in infected animals not receiving funds from an antibacterial effect that was correlated with the results of counting the number of M. leprae by the method of C. C. Shepard, D. H. McRae and testified about the inefficiency of the investigational medicinal product.

Conclusion: the tested drug on the proposed evaluation method is inefficient.

The application of the proposed method compared with the method of the prototype achieved a positive result, which consists in the simplification of the way. The method is simple in technical execution and results analysis, missing a lot of stages of research. The duration of the proposed method in comparison with the prototype (48 hours) requires 18-24 hours. The advantage of the proposed method is also need small quantities of blood serum, which is important for experimental includepicture, reagents, dyes.

The proposed method can be recommended for use in morphological, biochemical, microbiological laboratories.

Claims

A method of evaluating the effectiveness of leprosy medicines, including microscopic examination of blood serum, characterized in that the serum intact and infected with M. leprae animals treated with the investigational medicinal product, in the amount of 20 µl applied to the surface of glass slides, dried at room temperature, and then, looking through the samples in the light microscope at low magnification, conduct a comparative analysis of their morphological paintings and in the presence of experimental paintings structureborne similar to that of the intact animals are judged on the effectiveness of leprosy therapy, and at sharp differences in terms of the violation of the radial symmetry of the cracks, their chaotic location the formation of a large number of polygonal separateness, the asymmetric location of mono - and polymorphic nodules, the presence of markers of inflammation judge of its ineffectiveness.



 

Same patents:

The invention relates to medicine, namely to laboratory diagnosis

The invention relates to medicine, namely to Hematology

The invention relates to medicine and biology, in particular to physical therapy and immunology for the correction of immunodeficiency and associated diseases when exposed to light different areas of a person

The invention relates to medicine and can be used to determine the magnitude of blood loss in clinical practice, extreme and military surgery

The invention relates to medicine, namely to laboratory methods of research

The invention relates to medicine, namely to obstetrics and Perinatology, and can be used to predict infectious disease in full-term newborns

The invention relates to medicine and is intended to determine the composition of fatty acids in biological fluids

FIELD: medicine.

SUBSTANCE: invention relates to laboratory methods for blood analysis. Plasma is dropped in copper sulfate solution with density 1.023 g/cm3, not above, and time for drop falling on bottom of graduated cylinder with column height 243 mm is measured. The blood plasma density value is calculated by the formula:

wherein is the unknown blood plasma density (g/cm3); is copper sulfate solution density measured by areometer (g/cm3); t is average falling time of plasma drop in the copper sulfate solution (as seconds); 0.260130126 and 0.00290695 are correction coefficients. Temperature of plasma and copper sulfate solution is 20oC. Method is simple and suitable and allows carrying out analysis of small volumes of blood plasma and to reduce analysis time.

EFFECT: improved assay method.

2 ex

FIELD: medicine.

SUBSTANCE: method involves carrying out microscopic examination of blood serum samples taken from femoral vein and cubital vein. Femoral vein sample is taken on injured side. The examination is carried out before and after treatment. The blood serum samples are placed on fat-free glass slide in the amount of 0.01-0.02 ml as drops, dried at 18-30°C for 18-24 h. The set of pathological symptoms becoming larger or not changed after the treatment in comparison to sample taken before treatment, and morphological picture of samples under comparison taken from the cubital vein showing no changes or being changed to worse, the treatment is considered to be effective.

EFFECT: enabled medicamentous treatment evaluation in course of treatment to allow the treatment mode to be changed in due time; avoided surgical intervention (amputation); retained active life-style of aged patients.

4 dwg

FIELD: medicine, obstetrics, gynecology.

SUBSTANCE: in the first trimester of pregnancy one should study the content of CD8+CD11b lymphocytes and at their values being either equal or above 2% it is possible to predict gestosis. The present innovation enables to choose correct tactics of treating pregnant women that, in its turn, leads to decreased frequency of this complication of pregnancy and the risk for the development of fetal and neonatal pathology.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with blood sampling, separating plasma against erythrocytes, moreover, in plasma on should detect activity of antithrombin III, proteins C and S, XIIa-dependent fibrinolysis and concentration of plasminogen obtained results should be expressed as relative units followed by calculating integral parameter that characterizes the state of anticoagulant-fibrinolytic potential (IPAFP) by the following formula: IPAFP = [(C1 + C2)/(C3 + C4)] x 100, where C1 - the ratio of observed value of antithrombin III activity to the value of inferior border of the range of analogous parameter norm; C2 - the ratio of observed value for the activity of proteins C and S system to the value of inferior border of the range of this parameter norm; C3 - the ratio of the value of inferior border of plasminogen concentration under normal conditions to observed value of analyzed parameter; C4 - coefficient calculated with the help of regression equation: C4 = 0.9 + (0.01 x X), where X - terms of lysis of patient's euglobulin clot/min, and at IPAFP value of 101.4 U and higher one should state anticoagulant-fibrinolytic blood potential to be in norm, in interval of 64.8 - 101.3 -as insufficient, and at 64.7 and below - as critical. The present method simplifies the procedure of evaluating the state of endogenous anticoagulants and activity of XIIa-dependent fibrinolysis.

EFFECT: increased diagnostic value of obtained results.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnosis.

SUBSTANCE: method involves determination of the patient blood content of globulin-alpha 1, globulin-beta, globulin-gamma and the total bilirubin content followed by calculation of diagnosis indices for the patient (Y1, Y2, Y3) by using the computer program "Statistica 1.5" and introducing values X1, X2, X3 and X4 in computer wherein X1 means globulin-alpha 1 value; X2 means globulin-beta value; X3 means globulin-gamma value; X4 means total bilirubin value. Obtained values of diagnosis indices for the individual patient (Y1, Y2, Y3) are compared with average values of diagnosis indices (Y1', Y2', Y3') for different urogenital infections followed by comparison by sign and value. By the maximal coincidence of diagnosis index values for the individual patient with average diagnosis index values urogenital disease is diagnosed and the following diagnosis index average values are used: for chlamydiosis: Y1' = -2; Y2' = -0.1; Y3' = -0.2; for mycoplasmosis: Y1' = 2; Y2' = 0.8; Y3' = -0.04; for ureaplasmosis: Y1' = 2; Y2' = -1; Y3' = 0.02; for health persons: Y1' = -2; Y2' = 0.1; Y3' = 0.2. Invention provides the development of a method for express-diagnosis of infection at initial stage and diagnosis of atypical forms that occur in these diseases, and differential diagnosis of chlamydiosis, mycoplasmosis and ureaplasmosis. Invention can be used for carrying out the differential diagnosis of chlamydiosis, mycoplasmosis and ureaplasmosis.

EFFECT: improved method for express-diagnosis.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves determining absolute value of ratio between lymphocyte number and absolute value of monocyte number in peripheral blood at the end of combine radiation therapy. The ratio is divided by 4.05. The result value being greater than 1, no disease relapse occurrence is predicted during the first observation year. The value being less than 1, tumor growth progress is stated and carcinoma relapse is predicted at the first year after treatment.

EFFECT: enhanced accuracy in detecting pathological process progress before observing clinical manifestations.

1 tbl

FIELD: medicine.

SUBSTANCE: method involves determining infrared radiation absorption coefficient in blood plasma in bandwidth of 1543-1396 cm-1. The infrared radiation absorption coefficient is determined in %. The value being equal to 29.7±1.1%, catarrhal cholecystitis is diagnosed. The value being 26.4±1.4%, phlegmonous cholecystitis is diagnosed. The value being 21.2±1.8%, gangrenous cholecystitis is diagnosed. The value being equal to 18.6±0.5%, gangrenous perforated cholecystitis case is diagnosed. The value in norm is equal to 32.4±0.8%.

EFFECT: high accuracy and specificity of diagnosis.

FIELD: biomedicine.

SUBSTANCE: the present innovation deals with biomedical measuring technologies, in particular, to those to detect bactericide activity of blood serum according to the level of its inhibiting impact upon luminescence intensity of sulfur-sensitive luminescent bacteria (ΣimpO) against control - luminescence intensity the same sulfur-sensitive luminescent bacteria that had no contact with blood serum (ΣimpK), then one should calculate the value of bactericide activity of blood serum by the following formula:

As sulfur-sensitive luminescent bacteria one should apply either natural or recombinant microorganisms being characterized by direct proportionality between intensity of decreased spontaneous bioluminescence level and degree of bactericide effect. For example, it is possible to apply Escherichia coli strain with genes of Photobacterium leiognathi luminescent system. The suggested method enables to shorten the duration for detecting bactericide activity of blood serum and decrease its labor intensity.

EFFECT: higher efficiency of detection.

1 cl, 1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves determining blood insulin I and thyroxin T content and phagocytic leukocyte activity (PLA). Activity coefficient is calculated on the basis of formula KA=IxPLA/T. KA value being found greater than 2.8 units, considerable amelioration treatment effect is predicted. The value being from 1.4 to 2.8 units, amelioration is predicted. KA being less than 1.4 units, lower treatment efficiency is predicted.

EFFECT: high reliability of prognosis.

Up!