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Niyazmatov A. A.

EXPRESS DIAGNOSTIC SETS FOR DEFINITION OF COMMON, GENUS AND SPECIES OF ENDOTOXINS OF GRAM-NEGATIVE BACTERIA IN BLOOD SERUM . PROSPECT, CLINICAL IMPORTANCE AND NOVELTY. Niyazmatov A. A. - Russian Academy of Medical Sciences, Bakulev Scientific Centre of Cardiovascular Surgery.

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Niyazmatov A. A.

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  1. EXPRESS DIAGNOSTIC SETS FOR DEFINITION OF COMMON, GENUS AND SPECIES OF ENDOTOXINS OFGRAM-NEGATIVE BACTERIA IN BLOOD SERUM. PROSPECT, CLINICAL IMPORTANCE AND NOVELTY. Niyazmatov A. A. - Russian Academy of Medical Sciences, Bakulev Scientific Centre ofCardiovascular Surgery

  2. I. Actuality • Express diagnostics of infection and infectious complications in the postoperative period is still actual nowadays. Early diagnostics of endotoxin in blood serum of the operated patients is necessary for preventive maintenance and timely adequate therapy of the postoperative infectious complications caused by gram-negative bacteria. Diagnostic sets which will provide approximate solving of this problem are sets for definition of common, genus and species of endotoxins of gram-negative bacteriawithin 10 minutes in blood serum of patients in number of 17 sets. The sets were developed by NPF Rohat OOO and State Institution, Bakulev Scientific Centre of Cardiovascular Surgery Russian Academy of Medical Sciences.

  3. Range of derived products includes: • APM-Endotox spp.(defines endotoxin for all gram-negative bacteria); • APM-Pseudo. spp.(defines common endotoxin of Pseudomonas); • APM-Pseudo. aeru(defines endotoxin for Pseudomonas aeruginosa); • APM-Pseudo. сера(defines endotoxin for Pseudomonas cepacia); • APM-Pseudo. fluor.(defines endotoxin for Pseudomonas fluorescens); • APM-Pseudo. malto.(defines endotoxin for Pseudomonas maltophilia); • APM-Pseudo. stutz.(defines endotoxin for Pseudomonas stutzeri); • APM-Enterobac. spp.(defines common endotoxin Enterobacter); • APM-Enterobac. cloa.(defines endotoxin for Enterobacter cloacae); • APM-Enterobac. aero(defines endotoxin for Enterobacter aerogenes); • APM-Enterobac. alpha(defines endotoxin for Enterobacter alpha); • APM-Kleb. spp. (defines common endotoxin Klebsiella); • APM-Kleb. pneu(defines endotoxin for Klebsiella pneumoniae); • APM-Kleb. оху(defines endotoxin for Klebsiella oxytoca); • APM-Serra. spp.(defines common endotoxin Serratia); • APM-Serra. Marc.(defines endotoxin for Serratia marcescens); • APM-Serra. & Enterobac.(defines endotoxin for Serratia & Enterobacter).

  4. Diagnostic set consists of the following elements: • Solution of polystyrene chemical microspheres (PCM) connected with monoclonal antibodies (diagnosticum), positive control (C +), negative control (C-), buffered solution (BS), 17 planchettes to 100 researches and application sheet. • Irritability - to 4 pq/ml LPS Е. Coli or Sal. typhi; • Specificity - to 98,7 - 99 %.

  5. II. Goals and Objectives • Large scale introduction of the sets to practice of health-care institutions of the Russian Federation and EU countries. II. 1.Material and methods • As test material serum of patients udergone cardiac operation on heart employing extracorporeal circulationand serum of patientswith other pathology as well as donor serum and plasmas were used. • Appraisal of reaction results was performed by means of Activated Particles Method (APM) with applied diagnostic sets based on immobilization on a surface of polymeric chemical microspheres (PCM) of particles at the rate of 0,60 – 0,68 μm monoclonalantibodies of a subclass JgG3 and JgG2a of high-avid to endotoxins various gram-negative bacteria.

  6. III. Account of reaction results (endotoxin activity) Account of reaction result by application of diagnostic sets for definition of common, genus and species of endotoxins of gram-negative bacteria: 4 Activity grade (AG) – Grade 4 of activated particles - large flocculus at the transparent background. (Abrupt-positive result - 500–125 pg/ml) 3 AG - Grade 3 of activated particles- flocculus or large seeds at the slightly blurred background. (Positive result- 125–30 pg/ml) 2 AG - Grade 2 of activated particles– lots of seeds at the blurred background. (Lightly positive result- 30–7.5 pg/ml) 1 AG - Grade 1 of activated particles- the blurred backgroundor hardly visible seeds. (Negative result) Irritability - to 4 pq/ml LPS Е. Coli or Sal. typhi; Specificity - to 98,7 - 100%.

  7. IV. Scientific data received by application of diagnostic sets for various pathologies of patients. Definition of endotoxin for cardiovascular pathologies (n >2000) of patients undergone cardiac operation employing extracorporeal circulation (EC).

  8. Table 1 IV.1. Dependence of endotoxin level of patient age (n=420) % Endotoxin аctivity

  9. Table 2 IV.2. Correlation of endotoxin level with duration of EC (n=1420) % Endotoxin аctivity минуты These patients had endotoxin level making in the mean 500 – 7,5 pg/ml

  10. Table 3 IV.3. Frequency of occurence of positive results subject to examination stage (n=320) % Endotoxin аctivity

  11. V. Examination of endotoxinof clinically healthy donors 1. For examination of endotoxin of donors analysis of 1264 serums was performed. As a result endotoxin of 12 % donor serums was found: 2. 105 samples of donor plasma were examined 23 samples of whichrepresenteddonor plasmas before and after virus inactivation by methylene blue photo processing method. A positive reaction was observed in 3 samples with endotoxin level not decreasing significantly before and after virus inactivation . Endotoxin level in these samples made 15-7,5 pg/ml up. For the first time presence of endotoxin in serum and plasma of donors is shown Without endotoxin -1112 tests; Serums with endotoxin -152 tests.

  12. VI.CYTOKINE AND ENDOTOXIN LEVELS IN BLOOD SERUM OF PUERPERANTS WITH COMPLICATED PUERPERAL PERIOD 1.Researchobjective was examination ofendotoxin and antiinflammatory cytokines in blood serum of clinically healthy puerperants and women with postnatal infectious complications. For goal achievement 66 puerperants, 32 of whom with a physiological postnatal period (control group) and 34 women with infectious diseases (basic group) wereexamined. Beside general clinical methods examination included endotoxindefinitionof blood serum by activated particles method with application of standard sets developed in a Bakulev Scientific Centre of Cardiovascular Surgery of the Russian Academy of Medical Sciences. Cytokine level Interleukin-1, Interleukin-6 and TNF in blood serum was defined with test system produced by Tsitokiny OOO (Saint Petersburg) by solid-phaseenzyme multiplied analysis.

  13. 2. Examination results reveal endotoxin absence and cytokine low level of clinically healthy puerperants. control group Pg/ml Endotoxin level

  14. 3. By infectious complications cytokinelevel firmly increasesand the overwhelming majority of peuperants have revealed endotoxin authentically increases, moreover a direct correlation dependence between severity of inflammatory process and presence of endotoxin was revealed. Basic group Pg/ml TNF IL-6 IL-1 4. Undertaken researches allow to consider endotoxin in blood serum as marker of inflammatory response, subject to presence of endotoxin and cytokines to estimate severity of desease and efficiency of taken therapy (Article in the magazine of Volgogradsky State Medical Institution, Department of Obstetrics and Gynecology. 2007.).

  15. VII. Examination of endotoxin activity of extracorporal condition patients 1. For endotoxin examinationof dialysis patients with chronic renal insufficiency (Nephrology and Dialysis Centre of Rostock, Germany)and various etiologies 64 serumswere examined. Examination was performed before dialysis procedure and after dialysis. As a result patients had before dialysis revealed endotoxin at the rate of 92%, and after dialysis –25 %: 2. Dynamics of endotoxin circulation was studied by selective hemosorption “LPS adsorber” (ALTECO, Sweden) of 10 patients with gram-negative abdominal sepsis, proved clinically, bacteriologically and by means of biochemical markers. Endotoxin level was defined by means of our set (APM-Endotox spp). It was revealed that the LPS-SORBENT preserved sorption efficiency during 6 hours of extracorporal blood clearance instead of recommended 2 hour haemocorrection by LPS-sorbtion. The examination was performed before hemosorption and every hour within 6 hours of extracorporal endotoxin elimination (Semashko Region Clinical Hospital, Nizhny Novgorod) Endotoxin revealed before dialysis -49 tests; Endotoxin revealed after dialysis –15 tests;

  16. VIII. Endotoxin examination of patients with a gynecological pathology • At the first stage we compared endotoxinlevel increase byhysteroscopy, laparotomy, vaginal operations, gas-free and traditional laparoscopy. The age of patients varied from 23 to 72 years. Main types of operative interventions by laparoscopic access were: adnexectomy, ovarioectomy, hysterectomy; laparotomic access – amputation of a uterus without appendages. 43 patients were examined total. The blood was sampledbefore the beginnig of the operation, in 30 mines and 1 hour after the beginning (if operation exceeded 60 mines) as well asat the end of intervention (table 1). • At the second stage we dicovered a possibility of endotoxinlevel decrease in blood of patients undergone the operationsubject toperformed antibiotic therapy. Sevenexaminationswere spent. The following scheme of blood sampling was selected: at the end of operation, in the morning of next day and on the third day. The age of patients varied within 54-60 years. In all the cases the operation presented was Pfannenstiel incision, low amputation of a uterus without appendages (table 2).

  17. Dependence of endotoxin level from the type of operative intervention Table 1

  18. Dependence of endotoxin level from antibiotik therapy scheme Table 2

  19. IX. Introduction of diagnostic sets to practice of medical institutions of Russia and EU countries. Explanation of outlet volume. • 1.1. The Russian market. Level of demand by domestic health care sector of diagnostic sets for definition of common, genus and species of endotoxin of gram-negative bacteria is defined by number of operations in the Russian Federation. At present new government of Ministry of Healthcare and Social Development of the Russian Federation takes radical actions regarding rendering of surgical services to the population of Russia by medical institutions of the country. • If in 2008about 4 million patientswithgeneral sirgical disorderscardiovascular diseaseswere operated so in 2009 the number of patientsdoubled and made about 9 million operations up. Therefore the number of diagnosed sets of these patients also doubles in comparison with 2008. • On domestic and foreign medical evidencea share of gram-negative infections makes 60 % i.e. 6.3 million patients of 9 million operationsper annum. Therefore outlet volume of diagnostic sets depends pro rata on the number number of operations performedper annum. To take into account that our diagnostic sets are included in standards of medical aid for patients with general surgical, disorders and cardiovascular surgery. The price of our sets includes the following: • Standard definitions of endotoxin of gram-negative bacteria (APM-Endotox spp). Cost of one analysis - 110 rbl., including 10 % VAT. • Expanded definitions of endotoxin of gram-negative bacteria (APM - Endotox spp., APM-genus and Pseudomonas. species, APM - genus and Enterobacter. species, APM-genus and Klebsiella. species, APM-genus and Serratiaspecies). There are 17 various diagnostic sets only. Cost of 17 analyses is 1870 rbl. including 10 % VAT.

  20. Data comparison of application of diagnostic sets and microbiological analysis (bacteriological inoculation),data are shown in the table. Table of products rates in comparison with analogs

  21. As we can seeapplication of our setsfor surgical patients is more effective in 12 times, than bacteriological innoculation. To account patients with gram-negative infection for 2009 the number will make 6.3 million patients up. Bacteriological inoculation expenses for6.3 million patients subject to one analysis with price 1200 rbl. will make 7.5 billion rbl., and whentest is carried out with diagnostic sets for the gram-negative infection of this number of patients (6.3 million) with price of one testmaking 110 rbl. budget expenses will make 693 million rbl. • Budgetary funds with application of ourdiagnostic sets will make 6.8 billion rbl.per annum. • Sale of sets in the territory of the Russian Federation • At present our sets are included to the classifier of consumables and in case of financial filling the profit from the sale of our sets will make: • 2011 more than 200 million rbl.; • 2012 more than 300 million rbl.; • 2013 more than 500 million rbl.

  22. XI. Comparison of economic effectiveness of diagnostic sets use and bacteriological inoculation of patients at hospital. Bacteriological inoculation detects only undamagedgram-negative bacteria but not damaged bacteria. Intercellular endotoxin is active infectious agent as in undamaged bacterial cell, so and in damaged bacteria. If bacteria is undamaged bacteriological inoculation defines it and if it damaged then does not detect. The percent of undetected endotoxin of bacteriological inoculation is 65 %. Our method detects the endotoxin not only in undamaged gram-negative bacteria but also in damaged cells of gram-negative bacteria within 98-99%. Timely detection of infection latent form is very important moment of the medical diagnosticbecause it will allow properly institutetherapytactics of a patient and early discharge a patient from hospital. Cost of one of bacteriological inoculation analysis includes : а) flora test-720 rubles. б) blood sterility test -750 rubles. в) bacteriological test of clinical material-180 rubes. г) microbiological urinoscopy-720 rubles. The total cost of test by bacteriological inoculation of one patient at hospital is 2370 rubles and test of one patient at hospital with diagnostic sets according to standard method is 110 rubles including VAT 10%. It is obvious that the use of diagnostic sets is more effectivein 21,5 times than the use of bacteriological inoculation (2370 rubles) Application of diagnostic sets according to advanced method where the cost of one test for 17 sets is 1870 rubles including VAT 10% and in case of bacteriological inoculation this amount is 2370 rubles. Our method is more effective than bacteriological inoculation in 1.3 times. 22

  23. XII. Antibiotic-sensitivity-use of diagnostic sets and bacteriological inoculation. Screening cost of antibiotics comparative activity by bacteriological inoculation of one patient at hospital is 1100 rubles. These antibiotics are ordered in resuscitation departments one or two times and then after transferring a patient to department also one or two times, if a patient does not lie too long in department because of complications then the quantity of the ordered antibiotics increases. Therefore the average quantity of ordering antibiotics to a patient at hospital shall be about three times. In terms of money it shall be 3300 rubles, in use of bacteriological analysis method of antibiotic-sensitivity.Application of our diagnostic sets according to standard scheme shall make 110 rubles (cost of one patient analysis), in case of three times order of antibiotic then its cost shall be 330 rubles. Use of our diagnostic sets for definition of antibiotic-sensitivity of a patient at hospital is more effective in ten times than use of bacteriological inoculation (3300 rubles). We define this antibiotic-sensitivity of patient during 10 minutes and in case of bacteriological analysis method it takes 48 hours or 2880 minutes. Our method is more effective in 288 times according to time of analysis than bacteriological method. 23

  24. XIII. Electron microscopy of PCM with monoclonal antibodies (mAb)and antigen PCM with mAb PCM with mAb and antigen 24

  25. XIV. Diagnostic sets for definition of endotoxin of gram-negative bacteria(17 sets) МАЧ-Endotox ssp APM-genus ofPseudomonas Ps.spp Ps.ar Ps.сер Ps.flu Ps.mal Ps.stu APM-genus ofEnterobacter En.clo En.aer En.al En.spp APM-genus ofKlebsiella Kleb.spp Kleb.oxy Kleb.pney МАЧ- kind of Serratia Ser.marc Ser.&Enter Ser.spp 25

  26. XV.GENERAL VIEW OF DIAGNOSTIC SETS 26

  27. XVI. USE OF THE DIADNOSTIC SETS IN MEDICAL PRACTICE FOR: а) definition ofantibiotic-sensitivity of patients with gram-negative pathology with the help of set «МАЧ-Endotox spp», - dualfold; b) selective hemosorption of patients with gram-negative sepsis with the help of set «МАЧ-Endotox spp» – dualfold; c) definition of general, kind and type of endotoxin ofgram-negative infections in amounts of 17 sets - fourfold. 27

  28. PUBLICATIONS • Articles in domestic magazines -6 • Theses and reports at the conferences in Bakulev Scientific CentreofCardiovascular Surgery, Russian Academy of Medical Sciences -3 • Article in foreign magazine -1 • Report at the German and Russian conference «Nanoscience-2009» - 1 • Publication of International Patent in the countries of European Union(№ 2204653 dated 07.07.2010) • Patents registered in the Russian Federation • Tel/Fax 007 (495) 414-76-96. E-mail: san51@mail.ru 28

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