Method of determining vancomycin concentration in biological fluids in newborns
SUBSTANCE: blood serum is diluted with physiologic saline in the ratio 1:2, 1:5, 1:10, 1:20 and 1:50, and tracheobronchial aspirates are diluted with physiologic saline in the ratio 1:2, 1:4, 1:8, 1:16 and 1:32. A solid nutrient medium of the following composition is prepared: a nutrient microbiological dry agar 26.5 g, a nutrient yeast extract 1.22 g, lactose 10.7 g, disodium phosphate 0.48 g, anhydrous sodium sulphite 0.83 g, sodium carbonate 0.03 g, sodium hydroxide 5.0 g, distilled water 1000 ml, pH 8.0 which contains the test strain Micrococcus lysodeiktikus 2665 in the concentration 50 million microbial cells in 1 ml of the medium. Simultaneously with the material being analysed, a reference concentration 5 mcg/ml is introduced in the wells of the diameter 8 mm on each Petri dish. In 18 hours of the incubation procedure at 37°C, a diameter of test strain growth retardation zone surrounding the wells are measured. The vancomycin concentration, mcg/ml is determined by a calibration curve of growth retardation zone diameter to the reference vancomycin concentration.
EFFECT: use of the declared method allows determining the vancomycin concentration in the biological fluids and ensuring higher clinical effectiveness.
The invention relates to medicine, in particular to Pediatrics and neonatology.
This is based on the idea of developing a standard schemes and individual selection of antibiotic therapy in newborn infants with severe perinatal pathology sepsis, pneumonia, tracheobronchitis, developed on the background of artificial lung ventilation (ALV) in children with the syndrome of respiratory disorders, on the basis of the study, the concentration of the antibiotic vancomycin in the blood and tracheobronchial aspirate and calculation of pharmacokinetic parameters.
In recent years significantly increased the incidence of severe hospital infections (sepsis, pneumonia, tracheobronchitis) in infants, caused by gram-positive strains of staphylococci (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus), multiresistant to antibiotics of group of beta-lactams (penicillins, cephalosporins, macrolides, fusidin, aminoglycosides, lincomycin, and chloramphenicol. High frequency of neonatal infectious-inflammatory diseases caused by gram - positive organisms Enterococcus, possess a natural resistance to these antibiotics [Margolia oil on canvas, Kushnaryov M.V., Dementieva G.M., Shahinian I.A., Chernukha M., Alekseev, G.V., Sahakyants E.A., Gerasimov, A., Chernikov I.N. Etiology ventilation is associirowannyh pneumonia in preterm infants. // Pediatrics. - 2005. No. 3. - P.36-39; Dementieva G.M., Kushnaryov M.V., Frolova, M.I., Margolia oil on canvas, Vetrova E.V., Chursina Y.S. Antimicrobial treatment of nosocomial pneumonia in premature infants. // Journal of pediatric pharmacology and nutrition. - 2006. - Vol. 3, No. 5. - Ñ.38-42; Kushnaryov M.V., Rumin I.I., Margolia oil on canvas, Antibiotic therapy in the neonatal period. // Russian journal of Perinatology and Pediatrics. 2007. - Volume 52, No. 5. - P.12-20; Kushnaryov M.V., Dementieva G.M. Pneumonia in newborns due to gram-positive microorganisms, and modern methods of their treatment. // Journal of pediatric pharmacology and nutrition. - 2008. Vol 5, No. 2. - P.46-50]. In this regard, in neonatal practice for the treatment of serious infectious and inflammatory diseases (sepsis, pneumonia)due to gram-positive cocci, were more likely to apply the antibiotic vancomycin, which is often the only available drug to which retains the sensitivity of the pathogen, especially nosocomial infections [Form of antibiotics for children's intensive care units and intensive care. // Edited Nowbelieve. - Moscow, 2002. - 16 S.]. However, despite the rapid and good microbiological and clinical effects of vancomycin at the given pathology, its application is limited due to high toxicity. So you know, that, in its application may thrombophlebitis and seal the vein during the injection. In 5% of cases, observed the development of allergic reactions, accompanied by fever, urticaria, asanteman and eosinophilia. Nephro - and ototoxic (including deafness) reactions occur mainly in violation of renal excretory function [Navashin S.M., Fomin I.P. // Rational antibiotic therapy. (Handbook). - M.: Medicine. - 1982. - P.56-72]. With long-term use (more than 3 weeks) possible hypotension and neutropenia [Tsaregorodtsev A.D., Tabolin VA Guide to pharmacotherapy in Pediatrics and pediatric surgery. Volume 4. Neonatology. - M.: "Malpractice-M" - 2004. - 260 S.]. Perhaps the syndrome of the "red neck" (transient reddening of the face, neck, upper body, skin itching), pseudomembranous enterocolitis [Shukhov B.C., Volodin N.N., Ryumin I.I. Antibacterial therapy in neonatology and Pediatrics. // Publisher: LLC "Center for biotechnology, medicine and pharmacy", Moscow. - 2004. - 166 C.].
Currently, neonatologists prescribe vancomycin, based on the General information of the pharmacokinetics of this drug in children, and used single doses of 5 to 20 mg/kg every 8 hours, increasing the interval between the introduction of the antibiotic until 12-24 hours in extremely premature and children first week of life [Tsaregorodtsev A.D., Tabolin VA Lead the tion of pharmacotherapy in Pediatrics and pediatric surgery. Volume 4. Neonatology. - M.: "Malpractice-M" - 2004. - 260 S.; Shukhov B.C., Volodin N.N., Ryumin I.I. Antibacterial therapy in neonatology and Pediatrics. // Publisher: LLC "Center for biotechnology, medicine and pharmacy", Moscow. - 2004. - 166 C.]. Standard schemes have been developed only on the basis of the dynamics of the concentration of antibiotic in the blood without regard to its distribution in organs and tissues, including the source of infection.
However, the use of vancomycin in intensive care and intensive therapy in infants with severe infection and in critical condition by using the recommended standard medium doses, generally ineffective. This is because the pharmacokinetics of vancomycin is characterized by a wide range of concentrations in biological liquids and depends on many factors present in the newborn (disorder of microcirculation of blood and renal excretory function, General edema syndrome, isolated pulmonary edema, decrease the concentration of free albumin and violation of the binding and transport of vancomycin, hyperbilirubinemia, intrauterine infection, parenterale introduction of large volumes of fluid). These factors can substantially prevent the penetration of vancomycin into the inflammatory focus (particularly in the respiratory tract pneumonia and tracheobronchitis), where his concentric who I may not even reach the level of the minimum overwhelming concentration (MIC) of the pathogen. When the reorganization of the respiratory tract in children on mechanical ventilation and pneumonia antibiotic partially excreted from the body together with lavagno fluid that reduces its concentration in inflammation.
Thus, the scheme of treatment of newborns with this drug when nosocomial ventilator-associated pneumonia are empirical and often do not provide sufficient bactericidal concentration in inflammation, which reduces its clinical efficacy.
However, the study of the pharmacokinetics of vancomycin in neonates is important when developing tactics antibiotic therapy. The concentration of antibiotics in biological fluids (blood, urine, cerebrospinal fluid, bile, synovial fluid and tissues of the organism and antimicrobial activity of drugs are the main parameters determining the efficiency of antibiotic therapy. The results of the study of distribution and excretion of antibiotics in their intravenously are the basis for the development of treatment regimens, ensuring the safety and therapeutic effect of drugs.
The most important pharmacokinetic parameters of antibiotic intravenously are: the value of the maximum concentration Cmax in tissues, blood and other biological liquids, the half-life T1/2 of antibiotic, i.e. the time for which its concentration in plasma is reduced by half; the residual concentration "With", i.e. the concentration of the antibiotic in biological liquids and tissues before the next introduction, as well as the apparent volume of distribution of antibiotic "Vd", which shows the percentage of drug released from the vasculature into the tissue.
Based on the study of pharmacokinetics of vancomycin can be developed with the standard scheme of its use in neonates with pneumonia, is designed for normal unmodified function of the bodies responsible for the excretion and metabolic transformation. Pharmacokinetic parameters (primarily its concentration in biological liquids and tissues) in comparison with the values of the IPC antibiotic is selected pathogen give the opportunity to justify individual treatment plan of the newborn, and to predict the effectiveness of this treatment. It should be noted that to ensure a therapeutic effect in severe diseases, including pneumonia, it is necessary to maintain the concentration of antibiotics, including vancomycin in the blood at the level of 2-4 multiple values of the IPC. Bactericidal effect depends not only on the maximum concentration, but from time maintain the level of antibiotic in the blood above the MIC for the pathogen. Therefore it is important as a ratio in which edenia antibiotic, and the value of a single dose [Navashin S.M., Fomin I.P. // Rational antibiotic therapy. (Handbook). - M.: Medicine. - 1982. - 496 S.; Sanford D., Gilbert, D., Gerberding D., Sande M. antimicrobial therapy. (Handbook). - Lane. from English. - M.: "Practice". - 1996. - 224 S.].
In infants with severe infectious-inflammatory diseases (sepsis, pneumonia), a condition which is rated as very severe or critical, developing multiple organ failure with impaired liver and kidney [Form of antibiotics for children's intensive care units and intensive care. - Moscow, 2002. - 16 S.; Tsaregorodtsev A.D., Tabolin VA Guide to pharmacotherapy in Pediatrics and pediatric surgery. Volume 4. Neonatology. - M.: "Malpractice-M" - 2004. - 260 S.]. For these children, the use of antibiotics is advisable to carry out on the basis of individual dosage according to the level of creatinine in the blood and its clearance. This will increase the effectiveness of antibiotic therapy and to prevent the development of adverse reactions, the risk of which increases in connection with violations of the basic mechanisms of excretion and metabolism of the antibiotic.
As the closest prototype is proposed a method of determining the concentration of benzylpenicillin sodium or potassium salt in biological fluids using microbial the policy diffusion method antibiotic in the agar [Navashin S.M., Fomin I.P. // Rational antibiotic therapy. (Handbook). - M.: Medicine. - 1982. - P.56-72].
The disadvantages of this method: 1) Staphylococcus aureus 209P has a moderate sensitivity to vancomycin with optimal zone of growth inhibition at a concentration of 10 μg/ml and, therefore, does not provide sufficient sensitivity of the method. In many cases, the IPC agents of severe infection in infants less than 5 μg/ml Therefore, the use of this test strain is not possible to accurately determine the time during which the antibiotic concentration exceeds the IPC of the pathogen. 2) In a nutrient medium for benzylpenicillin with a pH of 6.8-7.0 vancomycin partially destroyed during incubation at 37°C for 18 hours. It is known that the optimum bactericidal action of vancomycin occurs when pH 8.0, and at lower pH antimicrobial effect decreases sharply [Navashin S.M., Fomin I.P. // Rational antibiotic therapy. (Handbook). - M.: Medicine. - 1982. - C.155].
The above circumstances require the development of new methods of determining the concentration of vancomycin in biological liquids with the selection of a new highly sensitive test strain and the conditions of its cultivation with vancomycin.
The technical result of the invention. The proposed method has the advantage over the prototype. We used a more sensitive test ø the AMM Micrococcus lysodeiktikus 2665 and special nutrient medium new members for its cultivation. This enabled us to determine lower concentrations of vancomycin (1 mg/ml) in biological fluids, while the method specified in the closest prototype using the test strain Staphylococcus aureus 209P, allows to determine the concentration of vancomycin 5 mg/ml and above.
The purpose of the invention: development of a new method for determining the concentration of vancomycin in biological fluids. This goal is achieved by using a microbiological method of diffusion in agar with the use of the test strain Micrcoccus lysodeiktikus 2665 and nutrient medium new members for its cultivation.
Disclosure of inventions
Description of method for determining the concentration of vancomycin in biological fluids.
1. The nutrient medium composition:
|nutrient agar microbiological dry||26,5 g|
|EKD (fodder yeast extract)||1.22 g|
|sugar milk||10.7 g|
|disodium phosphate||0,48 g|
|sodium sulfite anhydrous||0,83 g|
|sodium carbonate||0.03 g|
|sodium hydroxide||5.0 g|
|distilled water||1000 ml|
Nutrient medium autoclave 30 min at 0.5 ATM
the pH of the medium after autoclaving - 8,0
2. The test strain: Micrococcus lysodeiktikus 2665. Sowing dose Micrococcus lysodeiktikus 2665 - 50 million microbial cells in 1 ml of culture medium.
3. Preparation of Petri dishes with medium and test microbe.
Determining the concentration of vancomycin in the biological substrates is carried out in a single layer medium, poured into Petri dishes in 10 ml into each Cup. In a pre-cooled to 48-50°C. nutrient agar above composition contribute suspended microbial cells of the test strain rate of 10 ml microbial suspension in 90 ml of medium. Suspended microbial cells producing physiological solution at a concentration of 500 million to 1 ml immediately before entering into the agar. In a frozen agar containing the test strain, make holes with a diameter of 8 mm
4. Preparation of working standard solutions of the antibiotic to construct the calibration curve.
For preparation of working solutions of standard antibiotic make arbitrary, but an accurately weighed sample on an analytical balance. The sample dissolved in distilled water at the rate of 1 mg in 1 ml (basic solution). Further breeding is prepared by diluted the I of the basic solution in phosphate buffer pH 7.2 to the desired concentrations: 1,0; 2,0; 5,0; 10,0; to 12.0 µg/ml.
5. Preparation of biological material.
To determine the antibiotic in the serum last collected in a dry centrifuge tube and centrifuged at 700 g for 15 minutes Serum is sucked off and do cultivation in saline 1:2, 1:5, 1:10, 1:20, 1:50.
To determine the antibiotic in the tracheobronchial aspirate the last collected through an endotracheal tube in the redevelopment of the respiratory tract of the newborn, which is on artificial lung ventilation.
In endotracheal tube injected with 1 ml of sterile saline solution and then aspiritual content of the respiratory tract in sterile plastic container. Pre-washed twice tracheobronchial tube the same amount of saline.
Tracheobronchial aspirate was diluted with saline 1:2, then homogenized in a manual glass homogenizer and centrifuged mode 1000 g for 15 minutes. The obtained supernatant represents the soluble fraction of tracheobronchial secretions, diluted further with saline 1:2, 1:4, 1:8 1:16 and used for subsequent determination of the concentration of antibiotic.
6. The setting reaction.
Prepared solutions of standard antibiotic and tested solutions (native serum and again in the Denia the supernatant tracheobronchial aspirate and its cultivation) add to the wells of agar using pipettes in the volume of 0.1 ml, alternating between standard and tested solutions. For each test, use at least 3 cups. Cup incubated at 37°C for 18 hours.
7. The calculation of activity of the tested antibiotic.
The calculation of activity of the tested antibiotics in biological liquids is conducted according to generally accepted standard scheme [Navashin S.M., Fomin I.P. // Rational antibiotic therapy. (Handbook). - M.: Medicine. - 1982. - P.56-72].
The concentration of antibiotic in the test substrate is determined according to a standard calibration curve. To plot a standard curve using 5 concentrations of the standard solution of the drug vancomycin: 1,0; 2,0; 5,0; 10,0; 12,0 µg/ml.
One of the concentrations at which amend, is a control (for vancomycin installed a concentration of 5.0 μg/ml). For each concentration, except for the control, use 3 cups (12 cups). In 3 wells of each Cup make the solution control a concentration of 3 other wells - one taken from the concentrations of the standard. After measuring the zones of growth inhibition for each concentration derive the average value of three cups, then find the average value for the reference concentration for all cups (12 cups ×3 area =36 zones).
The difference between the average led the particular area, reference concentration, bred to 12 cups, and the average value reference concentration determined from 3 cups each concentration, find the correction to the value of this concentration. The amendment added to the average value of this concentration, if it is positive, subtract, if the amendment is negative.
A standard curve is built on a semi-log grid at fixed values of the zones taken concentrations and average zone reference concentration standard. The abscissa axis lay the diameter of the zone of growth inhibition of the test strain and the y - axis values of concentrations of vancomycin.
When determining the antibiotic concentration in biological fluids, depending on the amount of the substrate using one or more cups for the substrate and each of its cultivation. In parallel with the test material in the hole of each Cup make controlling the concentration of a standard solution. After incubation at 37°C for 18 hours measure the zone of growth inhibition of the test organism, formed by controlling the concentration of the standard and test solution. The difference found between the mean values of the zones of the test sample and reference concentration is added to the value of the control concentration on the standard curve. Then on the curve to find the concentration, according to stuudy found the value in μg/ml The resulting concentration is multiplied by the degree of cultivation and, thus, determine the content of vancomycin in 1 ml of the test material. The accuracy of the method is ±10%. The sensitivity of the method is 0.5 µg/ml.
Conditions of implementation of action
1. The use of reagents:
1) the Standard of vancomycin.
2) the Ingredients for the culture medium (nutrient agar microbiological dry, EKD, sugar, milk, disodium phosphate, sodium sulfite anhydrous, sodium carbonate, sodium hydroxide, distilled water).
3) saline solution.
2. The test strain Micrococcus lysodeiktikus 2665.
3. Using device:
2) Laboratory centrifuge.
3) Laboratory glassware (pipettes, test tubes, Petri dishes with a diameter of 10 mm).
The efficiency of use of inventions
Clinical application of the method.
Determining the concentration of vancomycin in the blood and tracheobronchial aspirate (TBA) was performed in 10 preterm infants, who was on a ventilator. Body weight at birth ranged from 1230 g up to 2250 g, gestational age 28-35 weeks. All children have developed a "ventilator-associated pneumonia. Investigation of the pharmacokinetics of vancomycin was performed after a single intravenous injection at a dose of 25 mg/kg over 30 min, 1, 2, 4, 6, 10, 12 hours after the infusion. In children the material is on the study was taken from 1 to 3 times.
The results of pharmacokinetic studies have shown that 30 min after injection of vancomycin concentration in the blood was very high, averaging 95 mcg/ml (80 to 130 µg/ml). Already after 1 hour, it ranged from 50 to 122 µg/ml half-life of vancomycin was about 3 hours. After 6 hours, the antibiotic concentration was in the range of from 6 to 25 μg/ml, and 12 hours from 0.05 to 2.5 µg/ml Excess IPC pathogens in blood was observed in all children over 6 hours and the majority of them (7 of 10) within 10 hours after infusion. This suggests that the dosage of vancomycin ensures its high bactericidal effect.
In TBA after 60 min after injection, the concentration of vancomycin reached high numbers and were widely from 2 to 20 mcg/ml At this level, the concentration of vancomycin was kept for 4-6 hours, and then gradually decreased to 12 hours and was by this time an average of 1.5 mcg/ml After 12 hours vancomycin was detected only half of the surveyed children. It should be noted that the concentration of antibiotic in the blood was higher than that of TBA for 4 hours and was approximately equal to the concentration of TBA in the range of from 6 to 10 hours and several times (from 2 to 10 times) exceeded the IPC of the pathogen.
Clinical examples of specific you is filling up method.
Example 1. Child Aivazov. Case history No. 217.
Diagnosis: bilateral pneumonia on a background of pneumopathy (atelectasis), hypoxic-ischemic lesion of the Central nervous system (brain edema). General edematous syndrome. The conjugation jaundice 1-2 degrees. Prematurity 8 weeks. The 2nd of the twins.
The baby was born 24.01.2009, weighing 1760, length 42 cm, head circumference 31 cm and chest circumference 29 cm at 32 weeks of gestation in a very serious condition (score on scale Apgar 5/6 points). From birth was on oxygen subsidies regarding the syndrome of respiratory disorders. The first 5 days of life the child received empirical antibiotic therapy with oxacillin, metrogram and gentamicin.
Pneumonia developed on the 6th day of life and was caused by the Association of pathogens sensitive to vancomycin (Streptococcus haemolyticus, IPC=0.5 μg/ml, Staphylococcus haemolyticus IPC=0.5 μg/ml) and resistant to the use of antibiotics, as well as .li sensitive to gentamicin. The child was assigned vancomycin intravenously at a dose of 25 mg/kg 2 times a day in combination with gentamicin and metrogram. Pharmacokinetic parameters of vancomycin this child were the following: the concentration of vancomycin in serum after 2, 6 and 12 hours was equal to 30; 6 and 2 µg/ml, respectively, and in TBA - 5; 5 and 1.5 µg/ml, respectively. Would the and recommended individual scheme is the introduction of vancomycin at a dose of 25 mg/kg 2 times per day. When stabilized on the 8th day of life was noted positive dynamics in the clinical analysis of blood with decreased leukocytosis with 18×109/l to 12.6×109/HP Recovered spontaneous breathing. The child was removed from the ventilator. Course vancomycin was 12 days with positive clinical and microbiological effect. When re-microbiological study TBA on the 11th and sputum on the 18th day of life the growth of pathogens of pneumonia were found.
Example 2. Child Zhigalov. Case history No. 257.
The diagnosis of Hypoxic-ischemic lesion of the Central nervous system (intraventricular hemorrhage grade 3 full tamponade). Postemergency occlusive hydrocephalus (hypertensive-hydrocephalic syndrome). Ventriculo-subgaleal shunt. Bilateral pneumonia. Tracheobronchitis. The conjugation jaundice. Bronchopulmonary dysplasia. Prematurity 12 weeks.
The baby was born 28.01.2009, weighing 1253, length 36 cm, head circumference : 27 cm and chest circumference of 23 cm at 27 to 28 weeks of gestation in a very serious condition (score on scale Apgar 4/5 points). From birth was on a ventilator for about a severe syndrome of respiratory disorders. To the 4th day of life was growing phenomenon of bacterial pneumonia, etiology which was presented Streptococcus haemolyticus group, overall sensitivity is output to vancomycin (MIC=0.2 µ g/ml). There was a significant leukocytosis (30,0×109/l). Previous treatment with antibiotics (Ceftriaxone, metrogylum) did not give a positive result due to the resistance of the pathogen and the child was assigned to intravenous vancomycin 25 mg/kg two times per day. Pharmacokinetic parameters of vancomycin in a child presented in the table.
|Pharmacokinetics of vancomycin in REB. Zhigalov (concentration in µg/ml)|
|The period of the survey||Serum||TBA|
|after 1 hour||50||20|
|after 6 hours||10||6|
|after 12 hours||less than 1||1,5|
As can be seen from the table, within 12 hours the child was observed concentration of vancomycin in the TBA, significantly exceeding the MIC of the pathogen. The concentration in the blood after 12 hours was already very low. Therefore, to maintain sufficient levels of the antibiotic in the blood, providing its penetration into the respiratory tract, b is l recommended 12-hour interval between doses of the drug (25 mg/kg 2 times per day). The course of treatment was 10 days, after which there was a significant positive changes in the clinical condition of the child and the rehabilitation of the respiratory tract.
Thus, the proposed method of determining the concentration of vancomycin in biological fluids in infants with severe infectious-inflammatory diseases allows the use of individual schemes antibacterial treatment taking into account the pharmacokinetics of the drug and sensitivity of pathogens, and to develop a standard scheme for its application in newborn infants with varying degrees of severity of infection with intact and impaired renal function.
The method of determining the concentration of vancomycin in biological fluids in infants microbiological hole by the method of diffusion in agar, characterized by introducing the biological fluid in the wells of agar containing the test strain Micrococcus lysodeiktikus 2665 at a concentration of 50 million microbial cells in 1 ml, cultured at 37°C for 18 hours in a nutrient medium of the following composition: nutrient agar microbiological dry 26,5 g, EKD (fodder yeast extract) 1.22 g, sugar milk 10.7 g, disodium phosphate of 0.48 g, sodium sulfite anhydrous 0,83 g sodium carbonate 0.03 g, sodium hydroxide, 5.0 g distilled water 1000 ml, pH to 8.0 with subsequent measure is m of diameter of zone of growth inhibition of the test strain around the hole and determining the concentration of vancomycin in μg/ml from the calibration curve according to the size of the diameter of the growth inhibition concentration standard of vancomycin.
SUBSTANCE: haemoglobin concentration in patient's peripheral blood, a systolic pressure value of a pulmonary artery, and an end-diastolic volume of a right ventricle of heart are evaluated. Then the value F is calculated by formula: F=175-27.3×RVt-4.35×SPPA+0.5×H, where H is the peripheral blood haemoglobin concentration, g/l; RVt is the end-diastolic volume of the right ventricle of heart, cm; SPPA is the systolic pressure value of the pulmonary artery, mm Hg. If the value F>0 is observed, a positive clinical effect is predicted, and the value F <0 means a negative clinical effect or its absence.
EFFECT: application of the method allows prescribing the IGCSs only to those patients showing the positive clinical effect to be reached, promoting the improvement of medical aid quality to the COPD patient, and reduced undesired steroid load on the patient.
SUBSTANCE: patient's blood serum in volume 100-200 mcl is put on a slide, and a preparation is dehydrogenated in a warm air flow at temperature 40-50°C and humidity 20-30% for 15-20 minutes in a horizontal position. A facies is matched with a burn toxemia pattern by the values: a structural property index, crystallisability, a type of crystal and amorphous structures interaction, a degree of facies desctruction, a uniformity of crystalline and amorphous facies elements distribution, a degree of facies porosity manifestation, manifestation of separate crystallisation zones, manifestation of a boundary protein band and structural relief. If observing at least six of nine criteria values matched with the pattern, burn toxemia is diagnosed.
EFFECT: increased diagnostic accuracy, reduced analysis time.
2 tbl, 1 ex, 2 dwg
SUBSTANCE: diagnostic process activity value is calculated by formula: Y=1.045×X1+0.357×X2+0.027×X3-2.590 where Y is a diagnostic value of active/inactive tuberculous arthritis; X1 is a blood serum acidic α-glycoprotein orosomucoid level; X2 is a value of α1-protease inhibitor activity; X3 is a value of erythrocyte sedimentation rate. The formula is filled in, the diagnostic value is calculated, and Y>0 enables diagnosing the active process, while Y<0 - inactive.
EFFECT: use of the technique allows higher diagnostic accuracy and reduced length of diagnosing the tuberculous arthritis activity.
1 tbl, 3 ex
SUBSTANCE: cervical mucus is examined. The mucus is sampled at an internal orifice of uterus at 4 cm from an external orifice of uterus. The sampling procedure is followed with microscopy of the sampled materials, and if observing the leukocytes count exceeding 30 in a microscopic field with magnification 10/40, the presence of protozoa and the other microscopic factors, an inflammatory process is detected in a cervical canal of uterus in sterile women.
EFFECT: higher accuracy of endocervicitis detection.
SUBSTANCE: diagnostic technique for atypical endometrial hyperplasia consists in a indirect immunofluorescence method for lysyl oxidase cell count with the use of specific polyclonal lysyl oxidase antibodies, and if observing these cells increased by more than 67% with reference to norm, the presence of atypical endometrial hyperplasia is concluded.
EFFECT: improved diagnostic accuracy.
SUBSTANCE: transmission coefficient of the reference and analysed mixed saliva is determined in each of the specified ranges. Further, the related transmission coefficient values are compared. If the transmission coefficient of the analysed mixed saliva makes 63%-76.5% from the transmission coefficient of the reference mixed saliva for the range 1636-1660 cm-1, 96.0-100% from the transmission coefficient of the reference mixed saliva for the range 2100-2200 cm-1 and 13.7-34.8% from the transmission coefficient of the reference mixed saliva for the range 3200-3600 cm-1, an oncological disease is diagnosed. It is followed with determining a transmission bandwidth of the mixed saliva nearby the range 3200-3600 cm-1. The wave length range with a lower boundary 2900 to 3100 cm-1 and an upper boundary 3550 to 3650 cm-1 corresponds to the mixed saliva parameters of a healthy person, and the wave length range with the lower boundary 3150 to 3250 cm-1 and the upper boundary 3390 to 3410 cm-1 corresponds to the mixed saliva parameters of an oncological patient.
EFFECT: higher diagnostic accuracy of the oncological disease at any stage, enabled continuous tracing and following of a course of the disease.
1 ex, 1 dwg
SUBSTANCE: for selective recovery of a viable cell population, a biological fluid sample is placed in a microfluid device cell containing a silicon microchannel matrix with through holes of size 3-30 mcm and channel length 50-300 mcm as a filter medium, and passed through the matrix at rate 0.1-4 ml/min. In case of cell size separation, a cell fraction of the size less than that of the through holes in the matrix, are collected after passing of the microchannel matrix, while the larger cells are eluated from the microchannel matrix channels by eluent backflow. In case of receptor-specific cell separation, a matrix surface is pre-modified by specific cell receptor antibodies, while the target cells are eluated from the matrix channels by isotypic antibodies or haptens.
EFFECT: more efficient and faster selective recovery of the viable cell population from biological fluids.
3 cl, 2 dwg, 4 tbl, 4 ex
SUBSTANCE: for detecting thrombinemia associated with large joint replacement, on the 10th-14th postoperative day, patient's blood plasma is examined for the hemostasis system values: the concentrations of D-dimer, plasminogen and antithrombin III. A discriminator (Z) is calculated by formula: Z=4.3936-0.001×D-0.029×P+0.017×A, where D is the concentration of D-dimer (ng/ml FEU), P is the concentration of plasminogen (percentage of normal concentration), A is the concentration of antithrombin (percentage of normal concentration). If observing the value Z <0, high probability of preserving thrombinemia is diagnosed, and pharmacological antithrombotic prevention is recommended to be continued.
EFFECT: use of the declared method allows high-efficient determination of appropriateness of prolongation of a pharmacological antithrombotic prevention course following the large joint replacement.
SUBSTANCE: instant diagnostic technique for diabetic nephropathy stages involving detecting a renal structure consists that in the fact that a glass plate 12×6 cm is coated with staggered strips 4-5 mm each of 0.01-0.02 ml of a biological test system containing 0.2% aqueous amino acids: glutamic acid, valine, tyrosine, serine, tryptophan, leucine, arginine taken in equal proportions; 0.25% DNA sodium salt; 25% aqueous magnesia sulphate in the ratio 2:1:2; the plate is placed in a projection of a kidney along a spinal column between the XII thoracic and III lumbar vertebras, kept for 4-5 minutes, then the preparation is dried at temperature +18-20°C for 2-3 minutes, analysed under a microscope in polarised light with a quartz compensator, and if observing: single coarse-grained cells and/or fine-grained cells, rarefied subparallel aggregates with spiral-form inclusions in the sample, the stage I of diabetic nephropathy is stated; the presence of fine-grained cells, rounded aggregates with crystalloid inclusions, rarefied subparallel aggregates with relic spiral-form inclusions shows the stage II of diabetic nephropathy, while the presence of undifferentiated rarefication and relict sites of spiral-form aggregates enables to diagnose the stage III of diabetic nephropathy.
EFFECT: technique is easy to implement, high-quality, eliminates invasiveness.
8 dwg, 3 ex
SUBSTANCE: biopsy materials of an ileum are analysed. Sections are analysed for solitary lymphoid follicles, a myoplasty, and lymphatic vessels, and n evident myoplasty dissociation by the solitary lymphoid follicles and the presence of lymphostasis, a manifested severity of a pathological process is stated.
EFFECT: use of the method allows higher accuracy of determining the severity of Crohn's disease in children if observing a colonic localisation of the pathological process.
SUBSTANCE: method involves cultivation of recombinant koji mycelial fungi in a fluid medium, collection of recombinant protein from the prepared product. The culture medium contains as a raw substance, at least one selected from a group consisting of barley and wheat surface of which is completely or partially coated at least in the concentration 1 to 20% (weight/volume) and as nutrients, at least one inorganic salt. Recombinant koji mycelial fungi is produced by a procedure consisting in the fact that a gene coding a target protein is ligatured below a promotor of a gene coding an enzyme exposed to catabolite repression due to concentration of the nutrients, such as saccharides and amino acids, for preparing thereby the ligation product, and the ligation product is introduced in koji mycelial fungi as a host.
EFFECT: invention allows higher yield protein.
4 cl, 3 tbl, 1 ex
SUBSTANCE: invention refers to a method of producing a mutant lactobacillus Streptococcus thermophilus, to a milk ferment, a method of producing a fermented milk product and to the fermented milk product. The offered invention can be used for producing the fermented milk products with improved storage characteristics. A method of producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation, than a parent strain is implemented by introducing in DNA a genome of said parent strain of mutation codon 552 coding histidine, of teh domain HA of lactose permease. Said mutation induces replacement of said histidine by amino acid which is distinct from serine, tyrosine, histidine and threonine.
EFFECT: invention allows producing the mutant lactobacillus Streptococcus thermophilus characterised by weaker postoxidation and suitable particularly for producing the fermented milk products.
10 cl, 5 dwg, 3 tbl, 2 ex
SUBSTANCE: Streptomyces caespitosus 3810/OE strain is a mitomycin C producer prepared from a stock strain recovered from soil by processing with chemical mutagens followed by stage selection, and deposited in the Collection of Cultures of State Institution G.F.Gauze Research Institute for Investigation of New Antibiotics of the Russian Academy of Medical Science, No. INA 01082. For producing mitomycin C, the strain is grown on a sowing medium of the following compoisiton (%): glucose - 0.6-1.4, starch - 0.6-1.4, soya flour - 2-5, corn steep - 0.6-1.0, KH2PO4 - 0.01-0.03, chalk - 0.05-0.12, (NH4)2SO4 - 0.06-0.14, main water, pH before sterilisation 6.8-7.2 and then is transferred to an enzyme medium of the following composition (%): starch - 0.6-1.4, sucrose - 1-3, soya flour - 2-4, corn steep - 0.6-1.0, CoCl2•6H2O - 0.007-0.012, KH2PO4 - 0.02, chalk - 0.05-0.12, (NH4)2SO4 - 0/06-0.04, FeSO4•7H2O - 0.01-0.02, MgSO4•7H2O - 0.01-0.03, NaCl - 0.2-0.6, main water, pH before sterilisation 6.8-7.2. The strain is grown in an aeration environment for 100-120 h, then mycelium is removed by centrifugation or filtration, and a supernatant containing an end product is extracted by mixed acetonitrile and methyl or ethyl alcohol. A level of strain produced mitomycin C is 85 to 100 mcg/ml.
EFFECT: improved method of producing antibiotic mitomycin.
2 cl, 1 ex
SUBSTANCE: method provides introducing a preparation of active dry yeast Saccharomyces cerevisiae in water in the ratio 1:10. Then the mixture is exposed for 4-6 h at temperature 23-27°C. The produced suspension is layered to reactivated yeast and a supernatant. The supernatant is removed.
EFFECT: method allows producing reactivated yeast with more complete living function recovery.
2 tbl, 3 ex
FIELD: food industry.
SUBSTANCE: nutrient medium contains soya flour and distilled water at preset quantitative ratios.
EFFECT: Deinococcus radiodurans yield increase.
2 tbl, 8 ex
FIELD: food industry.
SUBSTANCE: lysine-producing gram-positive bacterial strain for biologically active compounds delivery to ruminant animals is grown by way of at least single passage through the growth medium containing a quantity of lysozyme which is effective for bacteria cell walls growth induction; these bacteria are resistant to protozoal intake. The bacterial strain is extracted from the medium containing lysozyme and used for preparation of a feed supplement for ruminant animals passing through the ruminant animal paunch. The ruminant animals feeding method involves addition of an effective quantity of the feed supplements (passing through the paunch) to the feed ration.
EFFECT: increase in resistance of the lysine-producing gram-positive bacterial strain to inactivation in the paunch which strain is used for biologically active compounds delivery to ruminant animals.
17 cl, 5 dwg, 2 tbl, 13 ex
SUBSTANCE: culture medium contains dry nutrient agar, glucose, 5- aminosalicylic acid, an extract of nutrient yeast, paraaminobenzoic acid, bromthymol blue, tris-buffer, sodium carbonate, brilliant green and microbiological agar.
EFFECT: invention shortens duration of identifying klebsiell.
SUBSTANCE: method provides collection of an investigated material from a surface with a tampon made of an elastic finely porous material wetted in a solution inhibiting the development of other microorganisms. The collected investigated material with tampons is kept in the same solution for 18-24 hours. The investigated material is separated from a tampon and centrifuged. The precipitation is neutralised with 1% citric acid (in the ratio 1:1). No more than one-third of the investigated material is separate from the precipitation. The separated precipitation portion is placed on a phase-contrast slide to conduct phase-contrast microscopy. The residual precipitation is placed on a nutrient medium to be cultivated thereon if more exact quantitative assessment of mycobacterial pollution of the investigated surface is required.
EFFECT: invention allows reducing mycobacteria detection time.
2 tbl, 1 ex
SUBSTANCE: strain is obtained by insertion into genome of Escherichia coli, isolated from clinically healthy representative of canine family, of genes, determining synthesis of microcin C51, B-subunit of toxin, as well as vaccine version of elt-operon and A-subunit, enhancing immune response. Strain possesses expressed adhesion to mucous membrane of intestine of animals of canine family. Frozen dried culture of Escherichia coli EB387 represents probiotic medication for protection of animals of canine family against toxicoses, induced by cytotonic toxins of A1B5 type.
EFFECT: normalisation and stabilisation of qualitative and quantitative composition of gastrointestinal tract microflora in representatives of canine family.
2 cl, 3 ex
SUBSTANCE: nutritional medium contains liquid phase, content of liquid phase is determined by biological peculiarities of particular microorganism and solid phase. Solid phase contains coagulated serum and agarose in specified ratio of components.
EFFECT: invention allows to increase reliability of estimation of results of microorganism culture growth and development.
SUBSTANCE: nanobacteria are counted in a human nephrolith. A fixed mass is separated from the latter, mechanically powdered and divided into j=5 weight fractions pj. The powder is poured into j=5 sterile cells, water infiltrate at pore size not exceeding 0.05 mcm is added. The concentrations of nanobacteria is set between 102 to 106 cells in 1 ml by varying the water volume Vj or weight fractions pj of a powder mineral mass in each cell with using the formula. It is followed with mixing poured into j measuring cells. A nutrient medium - calves' fetal serum is added in the ratio 1:9. Two electrodes are inserted in each cell, then the measuring cells with the mixture is placed in an autoclave wherein constant temperature within 30°C≤T≤40°C is maintained. A mixture impedance (R) is periodically measured, and a point of measurement time (t) is determined until a mixture impedance slump is observed. A calibration diagram of an impedance variation time (timpj) to the concentration of nanobacteria in an initial sample (timpj) is presented. Thereafter, the above-stated stages of the method are conducted for analysed water as well. The derived impedance time (timpj) values are projected on the calibration diagram on the axis (timpj), then on the axis (lgnj).
EFFECT: invention allows evaluating the water concentration of nanobacteria.
3 dwg, 1 ex