Transcript (APM-Endotox spp).

EXPRESS DIAGNOSTIC SETS FOR
DEFINITION OF COMMON, GENUS AND
SPECIES OF ENDOTOXINS OF GRAMNEGATIVE BACTERIA IN BLOOD
SERUM.
PROSPECT, CLINICAL IMPORTANCE
AND NOVELTY.
Niyazmatov A. A.
-
Russian Academy of Medical Sciences, Bakulev Scientific Centre of Cardiovascular Surgery
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 gramnegative 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
bacteria within 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.
2
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).
3
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.
a)Irritability - to 4 pq/ml LPS Е. Coli or Sal.
typhi;
b)Specificity - to 98,7 - 99 %.
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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 circulation and
serum of patients with 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 monoclonal antibodies of a subclass JgG3 and
JgG2a of high-avid to endotoxins various gram-negative
bacteria.
5
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 background or hardly
visible seeds.
(Negative result)
Irritability - to 4 pq/ml LPS Е. Coli or Sal. typhi;
Specificity - to 98,7 - 100%.
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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).
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Table 1
IV.1. Dependence of endotoxin level
of patient age
(n=420)
100
%
80
60
40
20
0
20 y/o
45 y/o
60 y/o
elder than 60
Endotoxin аctivity
Positive result
Negative result
8
Table 2
IV.2. Correlation of endotoxin level
with duration of EC
(n=1420)
100
%
80
60
40
20
0
Less than 60
60 -120
120 - 180
Endotoxin аctivity
180 - 240
More than 240
минуты
Positive result
Negative result
These patients had endotoxin level making in the mean
500 – 7,5 pg/ml
9
Table 3
IV.3. Frequency of occurence of positive
results subject to examination stage
(n=320)
100
%
80
60
40
20
0
Before the operation
After the operation
Antibiotic therapy
Before release
Endotoxin аctivity
Positive result
Negative result
10
V. Examination of endotoxin
of 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:
1112
152
Without endotoxin - 1112 tests;
Serums with endotoxin - 152 tests.
2. 105 samples of donor plasma were examined 23 samples of which represented donor
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
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VI. CYTOKINE AND ENDOTOXIN LEVELS IN BLOOD SERUM OF
PUERPERANTS WITH COMPLICATED PUERPERAL PERIOD
1.
Research objective was examination of endotoxin 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) were
examined.
Beside general clinical methods examination included endotoxin definition of 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-phase
enzyme multiplied analysis.
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2. Examination results reveal endotoxin absence and cytokine low level of
clinically healthy puerperants.
Pg/ml
control group
12
10
8
6
4
2
0
Interleukin-1
Interleukin-6
Cytokine level
Endotoxin level
TNF
13
3. By infectious complications cytokine level firmly increases and 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
60
40
20
IL-6
IL-1
TNF
0
Cytokine level
Endotoxin level
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
14
(Article in the magazine of Volgogradsky State Medical Institution, Department of Obstetrics
and Gynecology. 2007.).
VII. Examination of endotoxin activity of
extracorporal condition patients
1. For endotoxin examination of dialysis patients with chronic renal insufficiency
(Nephrology and Dialysis Centre of Rostock, Germany) and various etiologies 64 serums
were 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 %:
49
Endotoxin revealed before dialysis - 49 tests;
15
Endotoxin revealed after dialysis
– 15 tests;
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)
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VIII. Endotoxin examination of
patients with a gynecological pathology
 At the first stage we compared endotoxin level increase by hysteroscopy,
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 sampled before the beginnig of the
operation, in 30 mines and 1 hour after the beginning (if operation exceeded 60
mines) as well as at the end of intervention (table 1).
 At the second stage we dicovered a possibility of endotoxin level decrease
in blood of patients undergone the operation subject to performed antibiotic
therapy. Seven examinations were 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).
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Table 1
Dependence of endotoxin level
from the type of operative intervention
250
125
30
7,5
time
30 minutes
1 hour
Laparotomy
Hysteroscopy
Laparoscopy
Vaginal operation
Gas-free laparoscopy
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Table 2
Dependence of endotoxin level
from antibiotik therapy scheme
Endotoxin level
Pg/ml
500
250
125
30
7,5
time
operation end
1 day
2 days
3 days
Ampicillin 1.0 х four times a day, intramuscularly
Тrichopol 0.25 х three times a day
Ciprofloxacin 100.0 / metrogyl 100.0 intravenous capsula
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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 gramnegative 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 2008 about 4 million patients with general sirgical disorders cardiovascular
diseases were operated so in 2009 the number of patients doubled 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 evidence a 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:
a) Standard definitions of endotoxin of gram-negative bacteria (APM-Endotox spp).
Cost of one analysis - 110 rbl., including 10 % VAT.
b) 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 Serratia species). There are 17
various diagnostic sets only . Cost of 17 analyses is 1870 rbl. including 10 % VAT.
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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
Rate value
To the project end
(2012)
Current
Rate name
Project object (product)
Express diagnostic sets for
definition of gram-negative
bacteria
Domestic analog
Bacteriological innoculation
Foreign analog
Bacteriological
innoculation,
Project oject
Express diagnostic sets for
definition of gram-negative
bacteria
The best analog
(prognosis)
Bacteriological
innoculation
Specificity
99-100
15-20
30-35
99-100
30-35
Irritability
4 pg/ml
Reveals microbic colonies
Reveals microbic
colonies
4 pg/ml
Reveals microbic
colonies
10 minutes
14-20 days
14 days
2 minutes
14 days
100 rubles per 1 analysis,
without VAT
6 EUR
600 rubles
1200 rubles
1200 rubles
------
100 EUR
100 rubles per 1 analysis,
without VAT
6 EUR
80 rubles
No data
No data
80 rubles
30-40 EUR
------
------
------
------
------
1 year
3 months
6 months
20 years
unknown
Analusis beginning
Russian market cost, rubles
Foreign currency
Prime cost, rubles
Operation expenses, rubles
per year
(when applicable)
Warranty period
(when applicable)
50 EUR
20
As we can see application of our sets for surgical patients is more effective
in 12 times, than bacteriological innoculation. To account patients with gramnegative infection for 2009 the number will make 6.3 million patients up.
Bacteriological inoculation expenses for 6.3 million patients subject to one
analysis with price 1200 rbl. will make 7.5 billion rbl., and when test is carried
out with diagnostic sets for the gram-negative infection of this number of
patients (6.3 million) with price of one test making 110 rbl. budget expenses
will make 693 million rbl.
Budgetary funds with application of our diagnostic 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:
a) 2011 more than 200 million rbl.;
b) 2012 more than 300 million rbl.;
c) 2013 more than 500 million rbl.
21
XI. Comparison of economic effectiveness of diagnostic sets
use and bacteriological inoculation of patients at hospital.
Bacteriological inoculation detects only undamaged gram-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
diagnostic because it will allow properly institute therapy tactics 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 effective in 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
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
XIII. Electron microscopy of PCM with
monoclonal antibodies (mAb)
and antigen
PCM with mAb
PCM with mAb and antigen
24
XIV. Diagnostic sets for definition of endotoxin of
gram-negative bacteria
(17 sets)
МАЧ-Endotox ssp
Ps.spp
APM-genus of
Pseudomonas
Ps.ar
Ps.сер
Ps.flu
Ps.mal
Ps.stu
APM-genus of
Enterobacter
En.spp
En.aer
En.clo
En.al
APM-genus of Klebsiella
Kleb.spp
Kleb.oxy
Kleb.pney
МАЧ- kind of Serratia
Ser.spp
Ser.marc
Ser.&Enter
25
XV. GENERAL VIEW OF DIAGNOSTIC
SETS
26
XVI. USE OF THE DIADNOSTIC SETS IN
MEDICAL PRACTICE FOR:
а) definition of antibiotic-sensitivity of patients with gramnegative 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 of
gram-negative infections in amounts of 17 sets - fourfold.
27
XVII.PUBLICATIONS
 Articles in domestic magazines
-6
 Theses and reports at the conferences in Bakulev
Scientific Centre of Cardiovascular Surgery, Russian
Academy of Medical Sciences
 Article in foreign magazine
-3
-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: [email protected]
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